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Amaral, Kyle
Master of Science - MSc
University of British Columbia
Pertussis disease is most severe in young infants. Tetanus-diphtheria-acellular pertussis (Tdap) vaccine given in pregnancy increases anti-pertussis antibodies transferred to the fetus, protecting young infants from severe pertussis disease. However, infants born to Tdap-vaccinated mothers may experience immunomodulation, leading to decreased antibody responses to their own pertussis vaccines. Many low-to-middle income countries have a high incidence of pertussis disease and deaths in young infants, but Tdap is not recommended due to the lack of data, including data on the use in pregnant women living with human immunodeficiency virus (HIV). I evaluated anti-pertussis immunogenicity and antibody avidity in pregnant people living with and without HIV in Uganda that received Tdap in pregnancy, and their newborns. I hypothesized that Tdap-vaccinated pregnant people would have higher anti-pertussis antibodies compared with those that received tetanus-diphtheria (Td) vaccine, and infants born to Tdap-vaccinated mothers would have a modulated antibody response to their pertussis vaccines. Additionally, the impact of different diphtheria-tetanus-acellular pertussis (DTaP) vaccine schedules and products on immunomodulation in infants have not been explored. I evaluated 2+1 and 3+1 dose schedules with different vaccine products as a booster in children from Quebec. I hypothesized that children that received a 3+1 dose schedule would have higher antibodies and lower immunomodulation compared with children that received 2+1 doses of DTaP vaccine. Anti-pertussis toxin (PT), -filamentous hemagglutinin (FHA), and -pertactin (PRN) immunoglobulin G (IgG) concentrations were quantified by an enzyme-linked immunosorbent assay (ELISA), and anti-PT IgG avidity was measured by a modified ELISA. I found that people that received Tdap in pregnancy had higher total anti-PT and anti-FHA IgG concentrations compared to Td-vaccinated pregnant people, regardless of HIV-status. Infants from Tdap-vaccinated mothers had lower anti-PT IgG concentration after their primary series compared with infants from Td-vaccinated mothers, regardless of HIV-exposure. The proportion of antibodies with different avidities was similar between those that received the same vaccine, regardless of HIV-status. Receiving 3+1 doses compared to 2+1 doses of DTaP led to significantly higher total anti-PT, FHA, and PRN IgG concentrations with similar avidity between all groups. Overall, pertussis vaccines increase anti-pertussis antibodies, regardless of HIV-status and age.
EVALUATION OF ANTI-PERTUSSIS IMMUNOGENICITY IN INFANTS AND CHILDREN AFTER IMMUNIZATION IN PREGNANCY WITH TETANUS-DIPHTHERIA-ACELLULAR PERTUSSIS VACCINE by Kyle Amaral B.Sc., McMaster University, 2020 B.A., McMaster University, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Kyle Amaral, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Evaluation of anti-pertussis immunogenicity in infants and children after immunization in pregnancy with tetanus-diphtheria-acellular pertussis vaccine submitted by Kyle Amaral in partial fulfilment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Dr. Manish Sadarangani, Associate Professor, Department of Pediatrics, UBC Supervisor Dr. Agatha Jassem, Associate Professor, Department of Pathology and Laboratory Medicine, UBC Supervisory Committee Member Dr. Laura Sly, Professor, Department of Experimental Medicine, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Rachel Fernandez, Professor, Department of Microbiology and Immunology, UBC Supervisory Committee Member iii Abstract Pertussis disease is most severe in young infants. Tetanus-diphtheria-acellular pertussis (Tdap) vaccine given in pregnancy increases anti-pertussis antibodies transferred to the fetus, protecting young infants from severe pertussis disease. However, infants born to Tdap-vaccinated mothers may experience immunomodulation, leading to decreased antibody responses to their own pertussis vaccines. Many low-to-middle income countries have a high incidence of pertussis disease and deaths in young infants, but Tdap is not recommended due to the lack of data, including data on the use in pregnant women living with human immunodeficiency virus (HIV). I evaluated anti-pertussis immunogenicity and antibody avidity in pregnant people living with and without HIV in Uganda that received Tdap in pregnancy, and their newborns. I hypothesized that Tdap-vaccinated pregnant people would have higher anti-pertussis antibodies compared with those that received tetanus-diphtheria (Td) vaccine, and infants born to Tdap-vaccinated mothers would have a modulated antibody response to their pertussis vaccines. Additionally, the impact of different diphtheria-tetanus-acellular pertussis (DTaP) vaccine schedules and products on immunomodulation in infants have not been explored. I evaluated 2+1 and 3+1 dose schedules with different vaccine products as a booster in children from Quebec. I hypothesized that children that received a 3+1 dose schedule would have higher antibodies and lower immunomodulation compared with children that received 2+1 doses of DTaP vaccine. Anti-pertussis toxin (PT), -filamentous hemagglutinin (FHA), and -pertactin (PRN) immunoglobulin G (IgG) concentrations were quantified by an enzyme-linked immunosorbent assay (ELISA), and anti-PT IgG avidity was measured by a modified ELISA. I found that people that received Tdap in pregnancy had higher total anti-PT and anti-FHA IgG concentrations compared to Td-vaccinated pregnant people, regardless of HIV-status. Infants from Tdap-vaccinated mothers had lower anti-PT IgG concentration after their primary series compared with infants from Td-vaccinated mothers, regardless of HIV-exposure. The proportion of antibodies with different avidities was similar between those that received the same vaccine, regardless of HIV-status. Receiving 3+1 doses compared to 2+1 doses of DTaP led to significantly higher total anti-PT, FHA, and PRN IgG concentrations with similar avidity between all groups. Overall, pertussis vaccines increase anti-pertussis antibodies, regardless of HIV-status and age. iv Lay Summary Young infants are most at risk of having severe whooping cough. To protect these young infants from severe disease before they can be vaccinated or complete their vaccine series, the whooping cough vaccine is recommended to be given during pregnancy. I found that the whooping cough vaccine given in pregnancy can increase antibodies against the pertussis bacterium, regardless of the pregnant person’s HIV status. These antibodies can cross the placenta and are present in the infant during the first few months after birth. Infants from mothers that received a whooping cough vaccine in pregnancy have lower antibody responses to their own infant whooping cough vaccines. In Quebec, the whooping cough vaccine schedule changed to a 2+1 dose schedule compared to the original 3+1 dose schedule. I found that a 3+1 doses of whooping cough vaccine led to significantly higher antibodies against pertussis compared to the 2+1 doses schedule. v Preface All pertussis immunology lab work presented in this thesis had ethics approval from the University of British Columbia under certificate number H20-00693. Chapter 1 is a literature review written solely for this thesis and is not published. Chapter 2 contains original, unpublished work. I developed the three assays with guidance and support from our lab post-doctoral fellow Dr. Gabrielle Gaultier, our lab manager Dr. Bing Cai, our lab technicians Mandy Lo and Tony Harn, a summer medical student Olivia Hunter, and my supervisor Dr. Manish Sadarangani. I was responsible with planning, laboratory work, data analysis, and writing our internal optimization and validation report. Chapter 3 contains data from a phase II, randomized controlled trial that was designed by Dr. Kirsty Le Doare and Dr. Manish Sadarangani in conjunction with experts in the field of obstetrics and neonatal medicine, including Dr. Annettee Nakimuli and Dr. Musa Sekikubo. The study was led by Dr. Eve Nakabembe, a doctoral candidate from Makerere University College of Health Sciences and St. George’s University of London. Sample collection was performed by Dr. Eve Nakabembe. I performed all the laboratory work, and performed the data analysis for the avidity data. Data analysis for the immunogenicity data was performed by Melanie Greenland, a statistician with the Oxford Vaccine Group. Melanie Greenland also generated figures and tables 3.1 and 3.2. This is part of an ongoing study that will be submitted for publication. The immunogenicity data (section 4.3.1) from Chapter 4 includes data from a published article: Febriani Y, Mansour T, Sadarangani M, Ulanova M, Amaral K, Halperin SA, De Serres G, Racine É, Brousseau N. Tdap vaccine in pregnancy and immunogenicity of pertussis and pneumococcal vaccines in children: What is the impact of different immunization schedules? Vaccine. 2023 Oct 26;41(45):6745-6753. doi: 10.1016/j.vaccine.2023.09.063. Epub 2023 Oct 8. PMID: 37816653. The work that was published was designed by Dr. Nicholas Brousseau. I performed all pertussis related laboratory work, data analysis was performed by Yossi Febriani and Thowiba Mansour from the Laval University Research Center in Quebec City, Quebec. Tables 4.1, 4.2, and 4.3 were produced by Yossi and Thowiba. The unpublished portion of this chapter includes the anti-PT avidity work which I was responsible with the design, laboratory work, data analysis, and generation of the figures and tables. vi Table of Contents Abstract ...................................................................................................................................................... iii Lay Summary .............................................................................................................................................. iv Preface .......................................................................................................................................................... v Table of Contents ........................................................................................................................................ vi List of Tables ............................................................................................................................................... ix List of Figures ............................................................................................................................................. xi List of Abbreviations ............................................................................................................................... xiii Acknowledgements ................................................................................................................................... xvi Dedication ............................................................................................................................................... xviii 1. Introduction ......................................................................................................................................... 1 1.1 Epidemiology of pertussis disease ............................................................................................... 1 1.1.1 Incidence of pertussis disease in Canada ................................................................................. 1 1.1.2 Age-specific pertussis disease burden ..................................................................................... 2 1.1.3 Prevalence of pertussis disease in Africa ................................................................................. 3 1.2 Microbiology of Bordetella pertussis .......................................................................................... 3 1.2.1 Virulence factors ...................................................................................................................... 4 1.2.2 Pathogenesis ............................................................................................................................. 6 1.2.3 Use of animal models in pertussis research ............................................................................. 7 1.2.4 Natural infection ...................................................................................................................... 8 1.3 Pertussis disease ......................................................................................................................... 12 1.3.1 Clinical manifestations........................................................................................................... 12 1.3.2 Diagnosis................................................................................................................................ 13 1.3.3 Treatment ............................................................................................................................... 14 1.3.4 Complications and Outcomes ................................................................................................ 14 1.4 Vaccines to prevent pertussis disease ......................................................................................... 15 1.4.1 Vaccine development ............................................................................................................. 15 1.4.2 Recommended vaccine schedule in Canada .......................................................................... 16 1.4.3 Effectiveness of Immunization in pregnancy ........................................................................ 17 1.4.4 Recommended vaccine schedule in Uganda .......................................................................... 17 vii 1.5 Immune responses to vaccination against pertussis ................................................................... 17 1.5.1 Immunization ......................................................................................................................... 17 1.5.2 Correlate of protection ........................................................................................................... 18 1.5.3 Immunogenicity of Tdap in pregnant individuals .................................................................. 19 1.5.4 Transplacental transfer of pertussis specific antibodies......................................................... 20 1.5.5 Infants during and after infant vaccine series ........................................................................ 21 1.6 Rational and thesis objectives .................................................................................................... 23 2. Optimization and validation of three enzyme-linked immunosorbent assays to quantify anti-pertussis toxin, -filamentous hemagglutinin, and -pertactin IgG concentrations................................ 25 2.1 Introduction ....................................................................................................................................... 25 2.2 Methods ............................................................................................................................................. 25 2.2.1 Serum samples ....................................................................................................................... 25 2.2.2 Antigens ................................................................................................................................. 27 2.2.3 Secondary antibody ................................................................................................................ 27 2.2.4 Enzyme-linked immunosorbent assays used ......................................................................... 28 2.3. Results .............................................................................................................................................. 30 2.3.1 Optimization............................................................................................................................... 30 2.3.3 Analyzing samples on the standard curve .................................................................................. 43 2.3.4 ELISA acceptance criteria for accepting a plate or a sample .................................................... 45 2.4 Discussion ......................................................................................................................................... 45 3. The immunogenicity of combined Pertussis-cOntaining vaccine (TdaP) for HIV-infected pregnant WomEn and their newboRns – A Randomized Clinical Trial (WoMANPOWER) ........... 49 3.1 Introduction ....................................................................................................................................... 49 3.2 Methods ............................................................................................................................................. 51 3.2.1 Study Design .............................................................................................................................. 51 3.2.2 Samples ...................................................................................................................................... 51 3.2.3 Vaccines ..................................................................................................................................... 52 3.2.4 ELISA protocol .......................................................................................................................... 53 3.2.5 Avidity protocol ......................................................................................................................... 53 3.2.6 Data Analysis ............................................................................................................................. 54 viii 3.3 Results ............................................................................................................................................... 55 3.3.1 Immunogenicity of TdaP versus Td in pregnant women living with and without HIV ............ 55 3.3.2 Immunogenicity of TdaP and Td in pregnant women living with HIV versus without HIV .... 59 3.3.3 Transplacental antibody transfer ratio of total anti-pertussis toxin and anti-filamentous hemagglutinin IgG in TdaP- versus Td-vaccinated pregnant women living with and without HIV .. 60 3.3.4 Transplacental antibody transfer ratio of anti-pertussis toxin and anti-filamentous hemagglutinin IgG in pregnant women living with or without HIV that received the same vaccine in pregnancy ... 62 3.3.5 Immunogenicity of DTwP in HIV-exposed uninfected and HIV-unexposed uninfected infants from TdaP versus Td-vaccinated mothers .......................................................................................... 62 3.3.6 Immunogenicity of DTwP in HIV-exposed uninfected versus HIV-unexposed uninfected infants from TdaP or Td vaccinated mothers .................................................................................................. 65 3.3.7 Anti-pertussis toxin IgG avidity in pregnant women living with and without HIV .................. 65 3.3.8 Anti-pertussis toxin IgG avidity in DTwP-vaccinated HIV-exposed uninfected and HIV-unexposed uninfected infants from TdaP or Td-vaccinated mothers ................................................. 80 3.4 Discussion ......................................................................................................................................... 92 4. Evaluation of the immunogenicity of a booster dose of DTaP in children that received different vaccine schedules and vaccine formulations ........................................................................................... 97 4.1 Introduction ....................................................................................................................................... 97 4.2 Methods ............................................................................................................................................. 99 4.2.1 Study design ............................................................................................................................... 99 4.2.2 Samples ...................................................................................................................................... 99 4.2.3 Vaccines ................................................................................................................................... 100 4.2.4 ELISA protocols ...................................................................................................................... 101 4.2.5 Anti-pertussis toxin IgG avidity protocol ................................................................................ 101 4.2.6 Statistical analysis .................................................................................................................... 101 4.3 Results ............................................................................................................................................. 103 4.3.1 Anti-PT, Anti-FHA, and Anti-PRN IgG concentrations.......................................................... 103 4.3.2 Anti-PT IgG avidity ................................................................................................................. 106 4.4 Discussion ....................................................................................................................................... 116 5. Overall conclusions and future directions .................................................................................... 123 References ................................................................................................................................................. 126 ix List of Tables Table 1.1. Antigen content in a DTaP versus a Tdap vaccine.......................................................16 Table 2.1. Acceptance criteria used in the validation of the ELISAs. .......................................... 35 Table 2.2. Results from assay variation and cross-validation experiments for all three antigens. 37 Table 2.3. Summarized results from analyzing the performance of the standard curve for each of the three antigens .......................................................................................................................... 42 Table 2.4. Summary of testing sample dilutions and sample stability.......................................... 44 Table 2.5. Criteria required to be met to accept results from a plate and for a sample. ............... 45 Table 3.1. GMCs of total anti-PT and anti-FHA IgG concentrations stratified by each group .... 58 Table 3.2. GMRs of total anti-PT and anti-FHA IgG concentrations stratified by each group .... 59 Table 3.3. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at baseline. ............................................................................ 70 Table 3.4. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at baseline + 4 weeks. .......................................................... 71 Table 3.5. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at maternal delivery. ............................................................. 72 Table 3.6. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at baseline. ......................................................................... 77 Table 3.7. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at baseline + 4 weeks. ........................................................ 78 Table 3.8. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at maternal delivery. .......................................................... 79 Table 3.9. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants and HIV-unexposed uninfected infants from Tdap- versus Td-vaccinated pregnant women at delivery.... 84 Table 3.10. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants and HIV-unexposed uninfected infants from Tdap- versus Td-vaccinated pregnant women 4-weeks post-primary series with DTwP vaccine (baby 18 weeks).................................................................... 85 Table 3.11. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants versus HIV-unexposed uninfected infants from Tdap- or Td-vaccinated pregnant women at delivery. 90 x Table 3.12. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants versus HIV-unexposed uninfected infants from Tdap- or Td-vaccinated pregnant women 4-weeks post-primary series with DTwP vaccine (baby 18 weeks).................................................................... 91 Table 4.1. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis antibodies levels at 13 (2+1 schedule) or 19 (3+1 schedule) months of age of children whose mothers did or did not receive Tdap vaccine during pregnancy. ................................................................................................................................... 104 Table 4.2. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis IgG antibodies at 13 months of age for children who received DTaP3-IPV-Hib-HB vaccine compared to children who received DTaP5-IPV-Hib® DTaP vaccine at 12 months old. ................................................................................................. 105 Table 4.3. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis IgG antibodies for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. ........ 106 xi List of Figures Figure 2.1. Determining antigen coating concentration of pertussis toxin, filamentous hemagglutinin, and pertactin. ........................................................................................................ 32 Figure 2.2. Determining the optimal secondary antibody concentration. ..................................... 34 Figure 2.3. Standard curve generated using NIBSC 06/140 anti-pertussis standard for all three antigens. ........................................................................................................................................ 41 Figure 3.1. Immunogenicity in pregnant women stratified by vaccine received in pregnancy and HIV-status. .................................................................................................................................... 57 Figure 3.2. Transplacental transfer ratios of total anti-PT and anti-FHA IgG concentrations stratified by vaccine received in pregnancy and HIV-status of the mother. ................................. 61 Figure 3.3. Immunogenicity in infants stratified by the vaccine mother received in pregnancy and HIV-exposure in utero. ................................................................................................................. 64 Figure 3.4. GMC of total anti-PT IgG with 95% confidence intervals, anti-PT TRAI, and anti-PT TAA in pregnant women stratified by vaccine received and HIV-status of a subset of samples. 69 Figure 3.5. Anti-PT fractional absolute avidity (FAA) of pregnant women at baseline, 4 weeks post Td or Tdap vaccination in pregnancy, and at delivery stratified by vaccine received in pregnancy and HIV-status............................................................................................................. 75 Figure 3.6. GMC of total anti-PT IgG with 95% confidence intervals, anti-PT TRAI, and anti-PT TAA in infants stratified by the vaccine their mother received in pregnancy and HIV-exposure in utero of a subset of samples. ......................................................................................................... 83 Figure 3.7. Anti-PT fractional absolute avidity (FAA) of infants at delivery and 4 weeks post-completion of primary series with DTwP, stratified by the vaccine their mother received in pregnancy and HIV-exposure in utero. ......................................................................................... 88 Figure 4.1. Geometric mean antibody concentrations (GMCs) of anti-pertussis toxin (PT) IgG antibodies and avidity of each group stratified by mothers’ Tdap vaccination status. ............... 107 Figure 4.2. Geometric mean concentrations (GMCs) of fractional absolute avidity levels of anti-pertussis toxin (PT) IgG of each group stratified by mothers’ Tdap vaccination status............. 110 Figure 4.3. Geometric mean antibody concentrations (GMCs) of anti-pertussis toxin (PT) IgG antibodies and avidity for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. ................................................................... 112 xii Figure 4.4. Geometric mean concentrations (GMCs) of fractional absolute avidity levels of anti-pertussis toxin (PT) IgG for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. ................................................................... 115 xiii List of Abbreviations AAU: Absolute Avidity Units aP: Acellular pertussis ACT: Adenylate cyclase toxin ART: Antiretroviral therapy AU: Avidity Units BCR: B cell receptor BrKA: Bordetella resistant to killing BSC: Biological safety cabinet CNDSS: Canadian Notifiable Disease Surveillance System CHO: Chinese hamster ovary CFU: Colony forming units CI: Confidence interval cAMP: Cyclic adenosine monophosphate DNT: Dermonecrotic toxin DTaP: Diphtheria-tetanus-acellular pertussis DTwP: Diphtheria-tetanus-whole-cell pertussis DTP: Diphtheria-tetanus-whole-cell pertussis EMA: European Medical Agency ER: Endoplasmic reticulum ELISA: Enzyme-linked immunosorbent assay EU/mL: ELISA units per milliliter FAA: Fractional absolute avidity FDA: Food and Drug Administration FHA: Filamentous hemagglutinin FIM2/3: Fimbriae 2/3 Fc: Fragment crystallizable FcR: Fc receptors FcRn: neonatal FC receptors GMC: Geometric mean concentration GMR: Geometric mean ratio xiv HB: Hepatitis B HEU: HIV-exposed uninfected Hib: Haemophilus influenzae type b HIV: Human immunodeficiency virus HUU: HIV-unexposed uninfected IAP: Integrin-associated protein IFN-γ: Interferon-γ Ig: Immunoglobulin IL: Interleukin IMPACT: Immunization Monitoring Program, ACTive IPV: Inactivated poliomyelitis vaccine IU/mL: International units per millilitre LLOD: Lower limit of detection LLOQ: Lower limit of quantification LMICs: Low-to-middle income countries LOS: Lipooligosaccharide LPS: Lipopolysaccharide LRI: Leukocyte-response integral MIP2: Macrophage inflammatory protein 2 MHC: Major histocompatibility complex NACI: National Advisory Committee on Immunization NAD: Nicotinamide adenine dinucleotide NIBSC: National Institute for Biological Standards and Controls NK: Natural killer OD: Optical density OPD: o-phenylenediamine dihydrochloride PBS: Phosphate-buffered saline PCR: Polymerase chain reaction PWLWH: Pregnant women living with HIV PWWH: Pregnant women without HIV PT: Pertussis toxin xv PRN: Pertactin RGD: Arginine-glycine-aspartate SD: Standard deviation TCT: Tracheal cytotoxin Td: Tetanus-diphtheria Tdap: Tetanus-diphtheria-acellular pertussis Th: T helper TLR: Toll-like receptor Tregs: T regulatory cells TRM: T resident memory ULOQ: Upper limit of quantification WoMANPOWER: The safety and imMunogeNicity of combined Pertussis-cOntaining vaccine (Tdap) for HIV-infected pregnant WomEn and their newboRns WHO: World Health Organization wP: Whole-cell pertussis %CV: Percent coefficient of variation xvi Acknowledgements I am extremely grateful to Dr. Sadarangani for his supervision throughout my MSc. I have learned a lot through our discussions and would not be the researcher that I am today without your guidance. I would like to thank my supervisory committee members Dr. Jassem and Dr. Fernandez for their time and providing feedback and questions that encouraged me to think about different aspects of my research. To the Sadarangani lab team, Dr. Bing Cai, Dr. Gabrielle Gaultier, Mandy Lo, Tony Harn, and Brynn McMillan, thank you for all your support, guidance, and encouragement throughout my MSc journey. Beyond research, all of you have be involved in many moments of laughter and fun and I am very grateful. To the investigators of the Vaccine Evaluation Center, Dr. Julie Bettinger, Dr. Hana Mitchell, Dr. Michael Hawkes, and Dr. Devon Greyson, thank you for always asking thought provoking questions that pushed me to think beyond my results. I would also like to thank Mistin and Ivor for always encouraging a fun and collaborative work environment. To my family and friends, thank you for your support and continued encouragement. To Dr. Le Doare, Dr. Nakabembe, Melanie Greenland, and the rest of the Ugandan and London team, thank you for the opportunity to work on such an impactful study and for analysing the immunogenicity data in Chapter 3. To Dr. Brousseau, Yossi, and Thowiba, thank you for the opportunity to work on the project in Chapter 4 and for your analysis of the immunogenicity data. To the Women’s Health Research Institute (WHRI), BC Ministry of Health, and BC Women’s Health Foundation, thank you for providing salary support during my research for Chapter 3. Further thanks to the Canadian Health Research Institute (CIHR), the Department of Health and xvii Social Care (DHSC), the Foreign, Commonwealth & Development Office (FCDO), the Medical Research Council (MRC) and Wellcome for their funding of the clinical trial included in Chapter 3. The UK funded award is part of the EDCTP2 programme supported by the European Union. xviii Dedication For my parents Jose and Lucia Amaral, and my late avô Gil Amaral, thank you for all your sacrifices that led to providing me with a world of opportunities. 1 1. Introduction 1.1 Epidemiology of pertussis disease 1.1.1 Incidence of pertussis disease in Canada Pertussis is endemic globally and is a cyclical disease with peaks every two to five years [1]. In Canada, pertussis is a nationally reportable disease, with health care professionals being responsible for reporting cases to the Public Health Agency of Canada. This is done through the Canadian Notifiable Disease Surveillance System (CNDSS). When Canada started reporting pertussis cases in 1924, incidence of pertussis disease was high, but then declined mostly in part to the introduction of vaccination in 1943 [1, 2]. Annual incidence of pertussis disease in Canada during the pre-vaccine era peaked in 1934 with 182 reported cases per 100,000 persons [2]. After the introduction of a whole cell pertussis (wP) vaccine in 1943, the incidence rate of pertussis disease declined by approximately 66% over five years from 162 to 55 reported cases per 100,000 persons [2]. Pertussis incidence continued to decrease until 1988, reaching a reported rate as low as 4.9 per 100,000 persons [2]. With declining annual pertussis cases, focus shifted from vaccination to the rare severe vaccine side effects associated with wP vaccines; more details on these side effects are discussed in section 1.4.1. These rare side effects led to vaccine hesitancy among parents and a decrease in vaccine coverage [3-5]. Between 1981 and 1985, an adsorbed whole cell vaccine that contained lower lipopolysaccharide (LPS) content was introduced in Canada [1]. Pertussis incidence began to increase in 1989 with an average annual incidence rate of 21 reported cases per 100,000 from 1989 to 2000 [2]. Potential reasons for an increase in pertussis cases included waning vaccine immunity, decrease in vaccine coverage, change in circulating strains of Bordetella pertussis, low efficacy of the adsorbed whole cell vaccine, and increased reporting [6]. In 1997, Canada replaced the adsorbed whole cell pertussis vaccine with an acellular pertussis (aP) vaccine that utilized purified bacterial protein [6-8]. In the early 2000s, incidence of pertussis disease began to increase in adolescents, leading to the implementation of a booster in this age group [9-11]. In 2010, there was a large increase in pertussis incidence again in many higher income countries that adopted aP vaccines [12]. With 2 severe cases mostly occurring in infants that were not old enough to be immunized, immunization in pregnancy with a booster of tetanus-diphtheria-acellular pertussis (Tdap) vaccine was introduced to decrease pertussis in regions where the burden was high. Over the last decade, the incidence of pertussis has peaked a few times in different geographical regions across Canada. Canada-wide incidence rates were highest in 2012 with 13 pertussis cases per 100,000 persons, followed by a three-year period of elevated pertussis cases reported between 2015 and 2017 averaging 10 cases per 100,000 persons each year [2]. Based on the most recent available data from the Public Health Agency of Canada for 2021, reported pertussis cases have been at an all-time low since the inception of pertussis reporting in Canada. In 2021, the incidence of pertussis was 0.07 cases per 100,000 persons [2]. This low incidence rate could be due to the cyclic nature of pertussis, or possibly due to public health measures that were implemented during the COVID-19 pandemic, including masking, social distancing, and the closure of child daycares, schools, and workplaces. Furthermore, immunization in pregnancy with a Tdap vaccine began being recommended in Canada in 2018 and could be responsible for a decrease in cases as well. 1.1.2 Age-specific pertussis disease burden Beyond examining the total reported pertussis incidence across Canada, surveillance of age-specific burden can provide a better understanding of populations most at-risk of severe outcomes. Based on reported cases across Canada from 1991 to 2021, infants less than one year of age have consistently had the highest incidence of pertussis compared with all other age groups, ranging from as high as 270 cases per 100,000 persons in 1994 to as low as 1.7 cases per 100,000 persons in 2021 [13]. Children aged between one and four, five and nine, and ten and fourteen have the second highest incidence of pertussis disease, with similar incidences between all three age groups [13]. This could be due to higher interactions between children in daycare, pre-school, and school settings allowing for easier transmission of B. pertussis between infected and uninfected children. Lastly, reported pertussis cases in individuals 15 or older continue to be the lowest [13]. These low reported cases could be a result of teenagers and adults not seeking medical care when sick and therefore not being tested for a B. pertussis infection. Infants less than one year of age have the highest incidence of pertussis and typically have the most severe 3 cases, especially infants less than six months of age. More details on severe outcomes of pertussis disease are discussed in section 1.3.4. 1.1.3 Prevalence of pertussis disease in Africa Unlike high-income countries, many low-to-middle income countries (LMICs) lack data on the incidence of pertussis disease due to suboptimal surveillance [14]. However, there is growing importance in characterizing the epidemiology, burden, and mortality of pertussis disease in LMICs, especially within Africa. Most studies available from LMICs in Africa report on the prevalence of pertussis disease and not the incidence. However, in a modelling study performed by Yeung et al. (2017), it was estimated that in 2014 there were 24.1 million cases of pertussis disease and 160,700 deaths in children less than five years of age globally. Of these cases and deaths, 7.8 million cases and 92, 500 deaths were in Africa [15]. Additionally, the prevalence of pertussis disease in LMICs was evaluated in a systematic review and meta-analysis by Muloiwa et al. (2020). The median prevalence of pertussis disease within each of the 16 studies from countries across Africa ranged from 0.6 to 14.9%, with the average prevalence of pertussis being ~5% across all studies [16]. This range in prevalence could be explained by the sample population that was analyzed in each study. One study enrolled children with a persistent cough, whereas another enrolled mother-infant pairs to evaluate community circulation of pertussis [14, 17]. One main finding consistent among studies in Africa that evaluated the prevalence of pertussis is that pertussis is circulating freely within communities and hospital settings [18]. To protect infants aged less than six months, implementation of Tdap vaccine in pregnancy may help reduce the burden of pertussis disease. 1.2 Microbiology of Bordetella pertussis Pertussis disease is caused by Bordetella pertussis, which is a Gram-negative, coccobacillus bacterium that is non-motile and strictly aerobic [19, 20]. B. pertussis is a human-specific pathogen with no known non-human reservoirs [21]. Survival of B. pertussis organisms must be maintained in humans through the continuous transmission of the organism from infected to naïve individuals [21]. This transmission is a result of aerosolized respiratory droplets [21]. B. 4 pertussis has many virulence factors that assist in its survival and pathogenesis in a host, including toxins, adhesions, and autotransporters. 1.2.1 Virulence factors There are many virulence factors that B. pertussis utilizes to adhere, colonize, and infect its hosts. In this section pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) will be focused on as these antigens are important in colonization and pathogenesis, they are in wP vaccines and most aP vaccines, and were used in my research. Pertussis toxin PT is an exotoxin that is secreted exclusively from B. pertussis [22]. PT is an A-B bacterial protein toxin that contains an enzymatically active A subunit (S1) and five binding B subunits (S2-S5) [23, 24]. This makes PT a multimeric protein with six subunits comprised of one copy of S1-S3, and S5, and two copies of S4 [24]. PT is secreted from B. pertussis using a specialized type IV secretion system called the Ptl system [25, 26]. This Ptl system consists of nine proteins, PtlA-PtlI, and is essential for protein secretion [26, 27]. Once secreted, the B oligomer of PT can bind to glycoconjugate molecules on the surface of target cells [28, 29]. PT is then endocytosed into the target cell where it then gets transported to the endoplasmic reticulum (ER) via a pathway that is not fully understood but is hypothesized to utilize the retrograde pathway [30, 31]. Once in the ER, the S1 subunit can translocate across the membrane into the cytosol where it then acts as an ADP-ribosylating toxin and transfers an ADP-ribose from nicotinamide adenine dinucleotide (NAD) to the alpha subunit of Gi/Go protein [32]. This inactivates the G protein making it unable to regulate adenylate cyclase, and therefore cyclic adenosine monophosphate (cAMP) levels increase and leads to the dysfunction of host signalling pathways [33]. PT is thought to be the main virulence factor responsible for most of the clinical manifestations of a severe pertussis infection [34]. The clinical manifestations of pertussis disease will be discussed in section 1.3.1. The involvement of PT on the clinical manifestations of pertussis disease was supported by a study in the Netherlands that found an association between increased virulence and strains with increased PT production [35]. Some effects of PT include 5 leukocytosis, hyperinsulinemia, histamine sensitivity, airway macrophage function, and leukocyte trafficking [36-40]. With PT being associated with systemic reactions, it has been one of the main antigens of interest in aP vaccines and in studies. Filamentous hemagglutinin FHA is a large filamentous protein that is produced by B. pertussis and is both exported to the bacterial cell surface and secreted [41, 42]. FHA is considered the main adhesin of B. pertussis and with fimbriae (another adhesion but in the pili family) are responsible for the initial attachment of B. pertussis to the upper respiratory tract [41, 43, 44]. There are several binding domains within FHA that facilitates binding to host cells. These include a heparin-binding domain to bind to sulfated polysaccharides, a carbohydrate-recognition domain to bind to ciliated respiratory epithelial cells and macrophages, and an arginine-glycine-aspartate (RGD) motif which binds to the leukocyte-response integrin and integrin-associated protein (LRI/IAP) and upregulates binding to complement receptor 3 on monocytes [45-48]. The importance of FHA in adherence to host cells is supported by lower efficiency of adhesion when B. pertussis strains are FHA-deficient [49]. Pertactin PRN is a cell-surface autotransporter that is also involved in the adhesion of B. pertussis to host cells [50, 51]. Like FHA, PRN contains an RGD motif that is a binding site for mammalian proteins, including fibronectin, vitronectin, and fibrinogen [52-54]. There are also data suggesting that PRN can bind to integrins as it was able to mediate adhesion to Chinese hamster ovary (CHO) cells [53]. The involvement of PRN in adhesion is supported by a 30-40% decrease in efficiency of B. pertussis to adhere to CHO cells and human HeLa cells when using a PRN-deficient strain [53]. This also suggests that PRN is not essential for adhesion [55]. PRN is also involved in evading host immune responses by decreasing the production of pro-inflammatory cytokines by dendritic cells [56]. Other virulence factors There are numerous other virulence factors that are involved in the colonization, persistence, and evasion of host immune responses. These include toxins, auto-transporters, and adhesions. 6 Toxins include adenylate cyclase toxin (ACT), tracheal cytotoxin (TCT), lipooligosaccharide (LOS) and dermonecrotic toxin (DNT). ACT is secreted by B. pertussis early in the infection and can mediate adhesion to integrins on myeloid phagocytic cells and natural killer cells [57, 58]. It is also able to enter the cytosol of host cells and inhibit superoxide generation, immune effector cell chemotaxis, phagocytosis, and bacterial killing [59]. TCT is a component of peptidoglycan in the bacterial cell wall and is released into the external milieu where it then damages ciliated cells and inhibits some neutrophil functions including migration [60-62]. LOS is an endotoxin that is like lipopolysaccharide (LPS), except it lacks the O-specific polysaccharide [63]. In conjunction with PT and TCT, LOS is involved in damaging ciliated cells in the mucosa of the respiratory tract [63].The endotoxin DNT is able to indirectly increase stress fibres, inhibit cytokinesis, interrupt cell differentiation, and inhibit DNA and protein synthesis [64]. Auto-transporters includes Bordetella resistant to killing (BrkA) protein, Vag8, and BapC. BrkA assists with adhesion to host cells and evading killing by the classical complement pathway [65]. Vag8 and BapC also assists with adhesion to host cells and evading killing by complement pathways [66, 67]. Lastly, an additional virulence factor that works as an adhesin is fimbriae serotype 2 (Fim2) and serotype 3 (Fim3). Fimbriae assists with adherence to monocytes [68]. These virulence factors do not represent an exhaustive list of all the virulence factors of B. pertussis. However, they provide possible targets of next generation pertussis vaccines. 1.2.2 Pathogenesis The sequence of how B. pertussis utilizes the virulence factors discussed above in section 1.2.1 to colonize and infect a human’s respiratory tract begins with B. pertussis being transmitted from an infected individual to another individual through the inhalation of respiratory droplets carrying B. pertussis [21]. B. pertussis then adheres to ciliated epithelial cells in the upper respiratory tract (nasopharynx and trachea) via virulence factors such as FHA and FIM2/3 [41, 43, 44, 68]. PRN can also be involved in the adhesion but is not necessary [53, 55]. Once in the upper respiratory tract other virulence factors like PT, ACT, and TCT are secreted and interact with host cells to disrupt the cellular environment and evade host immune responses as described in section 1.2.1 [33, 59-62]. After the clearance of B. pertussis, patients are left with a persistent cough for more than 2-3 months. The reasoning behind this long-lasting cough is not fully 7 understood. However, a recent publication by Hiramatsu et al. (2022) found that virulence factors PT, LOS, and Vag8 are able to produce a cough by inducing the production of a known cough inducer, bradykinin, in C57BL/6 mice [69, 70]. Whether this explains the long-term cough is not known as the relevance of these findings to human pertussis disease is questionable, but hopefully ongoing human challenge series can provide more information and lead to better understanding of the cause of the long-lasting cough. 1.2.3 Use of animal models in pertussis research As discussed above in sections 1.2.1 and 1.2.2 B. pertussis can evade host immune responses, but these immune responses to B. pertussis exposure in humans is not fully understood. Most experiments that have aimed to elucidate these mechanisms have been performed using murine and non-human primate models. The utilization of animal models in studying an obligate human pathogen has its advantages and limitations. Some animal models that have been used in pertussis research include mice and baboons. Each animal model provides their value in pertussis research. Mice are similar to humans anatomically, physiologically, and genetically allowing for them to be used in understanding internal disease progression, immune responses, and can also be used in testing vaccines [71, 72]. However, mice are typically non-symptomatic and therefore progression of symptoms cannot be easily analyzed which is a big limitation given pertussis’ characteristic symptom of a paroxysmal cough [73]. Baboons are a valuable animal model for pertussis research because a B. pertussis infection manifests similarly to humans [73]. Delivery of B. pertussis to the nasopharynx of weaning baboons resulted in low-grade fever, lymphocytosis, PT-specific antibodies, protection from subsequent challenge, and the characteristic paroxysmal cough [74]. Baboons were also essential in understanding how pertussis transmission occurs [21]. The use of baboon models is however difficult as it requires facility space, training, approval, and it is very expensive. Overall, animal models in pertussis research provides valuable information, however, the most accurate information of natural infection with B. pertussis is using human challenge series. 8 1.2.4 Natural infection Since B. pertussis is mainly an upper respiratory pathogen that has little evidence of disseminating to other tissues unless in severely immunocompromised individuals, most immune responses must occur in the mucosa of the respiratory tract to eliminate the bacteria [60, 75]. Initial interaction with the host’s innate immune system acts as the primary response to B. pertussis, which is then followed by the adaptive immune system that produces a more pathogen specific response and memory cells for a rapid immune response to re-exposures. Macrophages Alveolar macrophages along with dendritic cells are two of the first immune cells that B. pertussis interacts with [76]. Macrophages play two competing roles after infection. They provide a niche environment where the bacteria can persist and evade host immune responses in nonacidic compartments, but they also kill intracellular B. pertussis through acidic compartments with lysosome-associated membrane proteins [39, 77-79]. Macrophages can also be activated via cytokines such as interferon-γ (IFN-γ) and interleukin (IL) -17 which are derived from T cells [80, 81]. Dendritic Cells Dendritic cells have two main purposes when exposed to pathogens. They are tasked with acting as antigen presenting cells to T cells, and to promote differentiation of naïve T cells through the secretion of cytokines [82]. Upon interaction with virulence factors, a proinflammatory response is initiated [82]. Through interactions with different virulence factors, different T cell populations can be induced/generated. When LPS or LOS binds to toll-like receptor 4 (TLR4) it promotes secretion of IL-12 and IFN-γ which induces the differentiation of T-helper type 1 (Th1) cells [83, 84]. When ACT binds to dendritic cells, pro-IL-1ß is processed to IL-1ß. IL-1ß with IL-23 promotes induction of Th17 cells [85]. The expansion of Th17 cells is further promoted by IL-1 and IL-23 [86]. Regulatory T cells (Tregs) are also generated through the interaction with ACT and FHA which leads to dendritic cells producing IL-10, promoting an anti-inflammatory response [83, 87]. 9 Neutrophils Neutrophil recruitment after infection with B. pertussis is inhibited by virulence factors PT and ACT, delaying recruitment [88, 89]. IL-6 is required to produce macrophage inflammatory protein 2 (MIP2), a neutrophil-attracting chemokine, and to induce Th1 and Th17 responses [90]. The involvement of neutrophils in the clearance of B. pertussis is not fully defined. After infection with B. pertussis in naïve mice, neutrophils played a small role in the clearance of bacteria [88]. However, when using immune mice, neutrophils were shown to be more important [88]. This could potentiality be because of the presence of opsonizing antibodies that can promote phagocytosis [88]. Natural killer cells Natural killer (NK) cells are the initial source of IFN-γ which is needed for promoting a Th1 response [91]. NK cells are required to contain the bacteria within the respiratory tract [92]. Removal of NK cells leads to the dissemination of the bacteria to the liver in mice and leads to an elevated Th2 response [92]. Humoral responses Antibodies play a big role in both preventing and clearing infections by B. pertussis. In unvaccinated individuals that have had no exposure to B. pertussis or its antigens, the generation of anti-pertussis antibodies are slow and can take two weeks with peak responses being around one to two months after initial detection [93]. This slow response is due to the lack of memory B cells that have already been primed for B. pertussis specific antigens and can be activated rapidly upon detection of the pathogen. In a naïve individual, naïve B cells expressing IgM B cell receptors (BCRs) must recognize B. pertussis in the draining lymph nodes [94, 95]. B. pertussis is then taken up, processed, and antigenic peptides are then presented on MHC class II molecules [94, 95]. These activated B cells then interact with follicular helper T cells at germinal centres where the BCRs can undergo class switching and affinity maturation through somatic hypermutation [94]. These activated B cells can then leave the germinal centres as long-lived plasma cells or memory B cells [94]. Each long-lived B cells produces antibodies against a specific antigenic peptide of B. pertussis, for example, but not limited to, peptides from PT, FHA, PRN, FIM2/3, ACT, and TCT, and these antibodies circulate looking for this peptide [94]. 10 Antibodies that bind to their antigen can signal a cascade of pathways to neutralize and target the pathogen for killing. Including phagocytosis and complement-mediated pathways [95]. The B cells that turn to memory B cells can rapidly respond to reinfections and their BCRs are further refined [95]. There are five different immunoglobulin isotypes: IgA, IgD, IgE, IgG, and IgM [95]. I will be focusing primarily on IgG as it is the predominant antibody produced in response to infection and is involved in many processes to neutralize and eliminate B. pertussis [95]. IgA will be discussed briefly in relation to natural infection as it is not produced after immunization. Immunoglobulin A After natural infection with B. pertussis, both secretory immunoglobulin (Ig) A (sIgA) and serum IgA are produced [96]. Both sIgA and serum IgA can inhibit B. pertussis adherence to ciliated cells in the respiratory tract [97]. Serum IgA can also lead to bacterial killing through the promotion of B. pertussis uptake by polymorphonuclear leukocytes [98]. However, studies analyzing B. pertussis infection in IgA-deficient mice saw no differences in colony-forming units (CFU) suggesting IgA is not required or necessary for containment of the infection [99]. Immunoglobulin G In a study by Heininger et al. (2004) that investigated serologic response and antibody titre-decay after infection, they found anti-PT IgG levels peaked two months after disease [93]. These antibodies declined rapidly within two to three years with a median half-life for anti-PT IgG being 221 days [93, 94, 100]. This further shows the lack of long-term protection after infection. There are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. Of these four subclasses, IgG1 is the predominant subclass that is produced after infection [93]. IgG is involved in numerous functions. After binding of the antibody to their antigen, the Fc portion of the antibody can interact with complement and other cells to promote the killing of the bacterium. Before effector functions of Fc receptors (FcRs) can occur, antibodies must be able to bind to their target antigen. Understanding how these antibodies function is important as it is not just the quantity, but also the quality of the antibodies that matters. Two functions that can be evaluated 11 regarding antibody quality includes affinity and avidity. Affinity measures the strength of binding between a single antibody Fab fragment and an antigen, whereas avidity measures the total binding strength of the Fab region of an antibody to their antigen [101]. Antibodies with higher avidity would have undergone affinity maturation to produce antibodies with a Fab region that is more specific to an epitope of its target antigen, leading to higher affinity of these antibodies to their target antigen [101]. Once these antibodies are bound to their target antigen, the Fc portion is then recognized by FcRs and a cascade of effector functions can take place that ultimately lead to the killing/degradation of the bacteria/protein. Some effector functions of FcRs includes complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-dependent cellular cytotoxicity [101]. T lymphocytes As mentioned in the above sections on macrophages and dendritic cells, a Th1 and Th17 response occurs after infection with B. pertussis [102, 103]. Studies have suggested the importance of cell mediated responses on the clearance of B. pertussis. One study inoculated T cell deficient mice with B. pertussis and observed a persistent infection [104]. In this study they also found that transferring T cells with macrophages and B-cells led to higher protection than transferring serum from convalescent mice [104]. This suggests the importance of T cells on protection. In a separate study by Ross et al. (2013), polarized B. pertussis specific Th1 or Th17 cells were transferred into mice prior to challenge [102]. They found both Th1 and Th17 were able to reduce CFU counts by 10-fold, and when both were transferred together the CFU counts reduced 50-100-fold [102]. Besides responses to an initial infection, the production of memory T cells that can rapidly respond to additional exposures are valuable. In a study by Warfel and Merkel (2013), B. pertussis specific Th17 and Th1 cells were found circulating in a convalescent baboon two years after infection, suggesting immunological memory [103]. The importance of immunological memory for B. pertussis reinfection was further investigated by Wilk et al. (2017). In this study they focused on resident memory T (TRM) cells and found that convalescent mice were able to rapidly clear B. pertussis after reinfection [105]. 12 1.3 Pertussis disease Pertussis disease, commonly known as whooping cough, is a highly contagious respiratory disease caused by B. pertussis. Pertussis disease can also be caused by other species of Bordetella such as Bordetella parapertussis, but cases are less common and tend to cause a milder infection [106]. Most infections in fully vaccinated individuals with no co-morbidities will lead to a mild infection. However, infants less than three months of age are most at risk of severe disease and death [107, 108]. This is because they have not reached the age to begin their infant vaccine series, or have not completed their infant vaccine series [109]. 1.3.1 Clinical manifestations The usual incubation period of B. pertussis lasts from seven to ten days [110]. Clinical presentation of an infection with B. pertussis follows three stages: catarrhal, paroxysmal, and convalescence stages [111]. In the catarrhal stage, infected infants and children present with symptoms that are common with other upper respiratory illnesses. This is one of the main reasons many pertussis infections are not identified until the paroxysmal coughs begin [112, 113]. Most common symptoms at this stage include mild fever, malaise, sore throat, nasal congestion, rhinorrhea, lacrimation, sneezing, and a dry cough [60, 111, 114]. This stage lasts up to two weeks as the cough worsens [60]. Next is the paroxysmal stage and is where the inspiratory whoops begin [111]. This stage lasts two to eight weeks and includes coughing that lasts several minutes, cyanosis, eye proptosis, lacrimation, distention of neck veins, mucus production, and tongue protrusion [60, 115]. After paroxysmal coughs, post-tussive vomiting is also possible [60]. Towards the end of this stage, the frequency of paroxysms begins to decline, and the patient enters the last stage, convalescence. In this final stage the paroxysms continue to decline, and the patient begins to feel better [60]. This stage lasts one to two weeks, but the cough can remain for up to three months [111]. Adolescents and adults with pertussis experience similar symptoms but are milder [60]. 13 1.3.2 Diagnosis Prior to 2009, the case definition for a confirmed case of an infection with B. pertussis in Canada required at least one of three outcomes [116]. These were defined as a positive culture of B. pertussis, positive polymerase chain reaction (PCR) assay, and/or an epidemiological link to a confirmed case in addition to either a paroxysmal cough, vomiting or apnea from coughing, or an inspiratory whoop after coughing [116]. Furthermore, the aforementioned symptoms must not be due to another cause [116]. In 2009, the case definition changed slightly to require at least one of four symptoms in addition to a positive PCR test [117]. These four possible symptoms include a cough lasting 2 weeks or more, paroxysmal cough, vomiting or apnea from coughing, or an inspiratory whoop after coughing [117]. Detecting B. pertussis Detection of B. pertussis can be done through culture or PCR. First the collection of a specimen is performed by either collecting a nasopharyngeal aspirate or a nasopharyngeal swab [118]. For culture, swabs must be transported in medium to prevent the bacteria from drying [118]. PCR has become more common in detecting B. pertussis over culture due to it being more sensitive in detecting the bacteria, but culture has very high specificity (~100%) [119]. Furthermore, results can be collected within several hours using PCR compared to several days using a culture [118]. Isolation by culture To culture B. pertussis, a specimen is streaked on either charcoal-horseblood agar (Regan-Lowe) or Bordet-Gengou medium, supplemented with cephalexin to inhibit contaminating bacteria [118, 120]. The agar plate is then incubated for a week between 35 and 37 degrees Celsius [118]. B. pertussis colonies begin to be visible after three to seven days and appear as small, greyish-white, round, and shiny colonies [118]. Direct detection by polymerase chain reaction Unlike culturing B pertussis where it requires visually determining the correct species of Bordetella, PCR utilizes the amplification of specific targets to determine a positive result for Bordetella and to distinguish between species. Common targets used in PCR include IS481, 14 IS1001, IS1002, ptxP, and recA [119, 121-123]. These targets were chosen to be able to separate Bordetella species that are clinically relevant: IS481; target is found in B. pertussis, B. holmesii, and B. bronchiseptica, IS1001; target found in B. parapertussis and B. bronchiseptica, and IS1002; target found in B. pertussis and B. parapertussis [119, 121]. Targets ptxP and recA are monogenic targets that distinguish between B. pertussis and B. holmesii, respectively [122, 123]. 1.3.3 Treatment Upon confirmed pertussis diagnosis or a suspected case, antibiotics should be used right away [124]. A macrolide antibiotic such as azithromycin, erythromycin, or clarithromycin are the preferred antimicrobial treatment [124]. Antibiotics should be given during the catarrhal phase to decrease duration and severity of symptoms [124]. Antibiotics can still be given during the paroxysmal phase; however, this would have no impact on the duration or severity of symptoms [124]. Instead, it would help shorten the time of bacterial shedding, therefore decreasing the time to infect others [124]. 1.3.4 Complications and Outcomes In some cases where treatment is not given early enough in the disease progression or if the treatment does not work, patients can experience complications and severe outcomes. These severe cases include outcomes such as pneumonia, febrile seizures, encephalopathy, pulmonary hypertension, hypoxia, leukocytosis, hyperinsulinemia, coinfections, and death [125-129]. A study recently published by Bhaget et al. (2023) aimed to describe the burden of pertussis in Canada from 2005 to 2019. This study observed that infants less than one year of age accounted for 80% of all hospitalizations, and those aged less than four months had a hospitalization rate 8 times greater than those aged four months to 11 months [130]. Furthermore, in this period there were 17 deaths reported with zero to three deaths per year, of which all deaths were in infants less than one year of age [130]. Similarly, in another Canadian study conducted by Abu-Raya et al. (2020) which utilized IMPACT data from 1999 to 2015, infants aged less than two months had the highest mean annual incidence of pertussis hospitalizations and intensive care unit admissions with 116 and 33 per 100,000 population, respectively. In this period, there were 21 15 reported deaths of which all were less than one year of age [108]. It is evident that those less than one year of age are most impacted by severe pertussis disease. 1.4 Vaccines to prevent pertussis disease 1.4.1 Vaccine development The first pertussis vaccine utilized killed suspensions of B. pertussis and was licensed in 1914 in the United States; however, this vaccine was used more for treatment rather than as a preventative measure [131, 132]. In the 1940s, a new wP vaccine that took advantage of new methods of isolating bacteria and contained chemically inactivated B. pertussis was created [132]. This vaccine was soon after combined with diphtheria and tetanus vaccines and was commonly used [132]. The diphtheria-tetanus-whole-cell pertussis (DTwP) vaccine was effective at reducing the incidence of pertussis by approximately 99% in the United States, but soon after became subject to parent’s fears of the vaccine side effects [132, 133]. Side effects included typical local and systemic reactions such as pain at the injection site and fever, but also included more significant reactions such as prolonged crying and febrile convulsions, and acute encephalopathy [133]. These fears from the parents led to a decrease in the uptake of the DTwP vaccine and countries such as Sweden and Japan to suspend their pertussis vaccination programs in the 1970s and 1980s [132]. To address these concerns, an aP vaccine was created by Sato which contained purified antigens of B. pertussis, first only containing PT and FHA [7]. Later vaccines were created with additional pertussis antigens such as PRN and FIM2/3 and was also combined with diphtheria and tetanus toxoids. The use of a diphtheria-tetanus-acellular pertussis (DTaP) vaccine became common in the 1990s and soon after fully replaced the use of DTwP in many countries including Canada and the United States [132]. The creation and use of aP vaccines were able to address the fear of vaccine side effects as it was associated with fewer adverse reactions [133]. Furthermore, the production of aP vaccines can be better standardized compared to wP vaccines which tend to have higher variation between available products due to bacterial clones used, the methods of purification and inactivation, and the formulation, therefore making it difficult to compare between studies [133]. In Canada, aP vaccines continue to be the only type of pertussis vaccine offered throughout their pertussis vaccination program. 16 1.4.2 Recommended vaccine schedule in Canada In Canada there are routine pertussis immunizations in infants, children, adolescents, and adults. Since there are no vaccines with just aP antigens, all aP vaccines are in combination with other antigens. At minimum they contain tetanus toxoid and diphtheria toxoid. For infant and childhood vaccines they can also contain recombinant hepatitis B (HB), inactivated poliomyelitis (IPV), and/or conjugated Haemophilus influenzae type b (Hib) depending on where in the schedule they are. In Canada, it is recommended to receive three doses of DTaP-IPV-Hib as part of the infant vaccine series at 2, 4, and 6 months of age. This series is followed by a booster dose with DTaP-IPV-Hib between 12 and 23 months of age (typically received at 18 months), then followed by an additional booster of either DTaP-IPV or Tdap-IPV between four and six years of age before starting school. Both DTaP and Tdap are pertussis-containing vaccines, however Tdap contains lower amounts of diphtheria and pertussis antigens compared with DTaP (Table 1.1). Another booster is recommended between 14 and 16 years of age, and in adults 18 years of age and older with a Tdap vaccine [134]. Lastly, in 2018 NACI began recommending pregnant individuals to receive a dose of Tdap vaccine during every pregnancy between 27 and 32 weeks of gestation for optimal transfer of maternal antibodies into the fetus [135]. By immunizing in pregnancy, the maternal antibodies circulating in the infant during their first few months after birth will provide them protection from severe disease before they are able to receive their own infant vaccine series. Table 1.1. Antigen content in a DTaP versus a Tdap vaccine. Additional antigens of IPV and Hib was not included when presenting the antigen formulations for the DTaP vaccine. Abbreviations: Lf, limits of flocculation. Antigen Amount of Antigen DTaP Tdap Tetanus toxoid 5 Lf 5 Lf Diphtheria toxoid 15 Lf 5 Lf Pertussis toxoid 20 μg 2.5 μg Filamentous hemagglutinin 20 μg 5 μg Pertactin 3 μg 3 μg Fimbriae types 2 and 3 5 μg 5 μg 17 1.4.3 Effectiveness of Immunization in pregnancy Multiple studies have evaluated vaccine effectiveness in different countries after the implementation of immunization in pregnancy programs. These have shown a vaccine effectiveness of 90% to 91.4% in infants aged less than two months. Unfortunately, there are currently no studies in LMICs in Africa that have evaluated vaccine effectiveness of immunization in pregnancy with Tdap as it is not currently implemented [136-138]. 1.4.4 Recommended vaccine schedule in Uganda Although Uganda does not currently recommend the use of Tdap vaccine in pregnancy, administration of a pertussis containing vaccine begins at six weeks of life [139]. Infants receive three doses of DTwP vaccine combined with Hib and HB at 6, 10, and 14 weeks of age [139]. There are no booster doses of pertussis containing vaccines currently recommended in Uganda. The only vaccine currently recommended to be given in older children and adults is a booster dose of tetanus-diphtheria (Td) vaccine in women of reproductive age [139]. 1.5 Immune responses to vaccination against pertussis 1.5.1 Immunization T lymphocytes The resurgence of pertussis in many countries has been thought to be associated with the switch from wP to aP vaccines [140]. This is believed to be due to the difference in immune responses induced by the two different types of vaccines [140]. wP vaccines induce an immune response similar to infection [140]. This is most likely because the wP vaccine contains the whole bacteria and leads to the immune system developing a broad response to many antigens whereas with the aP vaccine responses are only generated against up to five antigens [140]. One big difference between the two types of pertussis vaccine are the T cell populations induced. Like natural infection, wP immunization leads to the production of IFN-γ and IL-17, inducing a Th1 and Th17 response [141, 142]. Immunization with aP vaccine produced IL-4, suggesting the 18 induction of a Th2 response [141, 143, 144]. However, these are not consistent in all studies, some studies have found that aP leads to a mixed Th1 and Th2 response [145]. IgG subclass distributions These differences in T cell populations also changes the IgG subclass distribution and could also impact protection against B. pertussis. The wP vaccine leads to a predominantly IgG1 response, whereas the aP vaccine leads to an IgG1 response with higher levels of IgG4 too [141, 146-148]. IgG4 binding to FcγR is reduced and thought to be poor at inducing antibody-dependent complement deposition, antibody-dependent cellular phagocytosis, and antibody-dependent cellular cytotoxicity [149-151]. Suggesting another reason why aP vaccines cannot prevent colonization and transmission as well as the wP vaccine [152]. B lymphocytes Memory B cells and long-lived plasma cells are important in the immune response to secondary infections, however there have not been much research in this area in regards to pertussis vaccines [152]. Furthermore, in studies that have investigated this area, there is no consensus. In one study they observed a stronger and broader memory B cell and plasma cell response in wP primed compared to aP primed mice [152]. In contrast to T cell populations where the vaccine type you are primed with determines your T cell polarization regardless of boosters, they found that a mixed vaccine combination was able to induce a broader B cell effector response [152]. These findings were challenged by other studies that found poor maintenance and reactivation of memory B cells and supported by others that also saw efficient memory B cell reactivations [153, 154]. However, these different findings could be a result of using different mouse models. 1.5.2 Correlate of protection Although pertussis vaccines have been in use since the 1940s with the wP vaccine and the 1990s with the aP vaccine, there is no widely accepted correlate of protection against pertussis disease. Like other correlates of protection, researchers originally thought that an antibody concentration against PT would suffice as a correlate of protection, however, this has been argued to not be enough and that cell-mediated responses are also required. One study found that an anti-PRN 19 IgG concentration greater or equal to 7 EU/mL and anti-PT IgG concentration of greater or equal to 66 EU/mL resulted in no cases after household exposure [155]. Others have suggested that anti-PT IgG concentrations of 5 to 10 EU/mL would suffice in protection [156]. It is thought that a correlate of protection will include both an antibody concentration cut-off and involvement of cellular-mediated responses [157]. Therefore, it may be important to analyze not only immunogenicity, but also antibody function and cell-mediated responses. 1.5.3 Immunogenicity of Tdap in pregnant individuals Over the last decade, there has been an increase in studies that aimed to determine whether the Tdap vaccine is immunogenic and beneficial for both the mother and their newborn. To analyze whether this is the case, studies utilized enzyme-linked immunosorbent assays (ELISAs) to measure antibody concentrations against aP vaccine antigens. These studies have been conducted in many countries including Belgium, Thailand, Argentina, the US, Spain, the UK, and the Netherlands. Halperin et al. (2018) conducted a study where they analyzed the immunogenicity and safety of Tdap in pregnancy. In their study they found that women that received Tdap in pregnancy had significantly higher antibody concentrations against PT, FHA, PRN, and FIM compared with Td-vaccinated pregnant women, and that the vaccine was safe [158]. Tdap administered in pregnancy was also observed to be immunogenic and safe in other studies [159, 160]. Some studies aimed to analyze the antibody responses without evaluating safety and they all had similar findings that Tdap given in pregnancy led to a significant increase in antibodies against pertussis vaccine antigens when compared to a control group of pregnant women that did not receive Tdap [161-165]. Furthermore, with changes that occur to a pregnant person’s immune system, some studies aimed to evaluate whether there was a difference in immune responses to Tdap in pregnant and non-pregnant people. In two studies that aimed to examine whether responses to Tdap differed when administered to a pregnant person or non-pregnant person, the studies found that antibody responses were similar between the two groups [159, 166]. These studies have supported the introduction of immunization in pregnancy with Tdap in countries globally. 20 1.5.4 Transplacental transfer of pertussis specific antibodies As mentioned previously, infants less than three months of age are at greatest risk of developing a severe case of pertussis disease due to the functional immaturity of their immune system and not receiving their own pertussis containing vaccines until 6-8 weeks after birth [167]. One way to provide protection to this group is through receiving a pertussis containing vaccine in pregnancy. Through immunization, circulating antibodies against pertussis vaccine antigens increases and allows for greater concentrations of these antibodies to transfer through the placenta and into the fetus. Antibodies can then cross the placenta through syncytiotrophoblasts by being internalized in endosomes that have neonatal Fc receptors (FcRn) [168-170]. Maternal IgG binds to the FcRn with high affinity at pH 6 and then undergoes transcytosis to the membrane on the fetal side of the syncytiotrophoblast [171]. A change to a more basic pH (~7.4) on the fetal side leads to the dissociation of IgG from the FcRn as it has 100x lower affinity and is released into the fetal circulation [172]. The FcRn can then be recycled and used again to transport more maternal antibodies [171]. IgG subclasses transferred through the placenta Each of the four IgG subclasses transfer across the placenta at different efficiencies. The most accepted order of the efficiency of IgG subclass transfer is IgG1>IgG4>IgG3>IgG2, however, others have reported a transfer efficiency order of IgG1>IgG3>IgG4=IgG2, and even IgG4>IgG3>IgG2>IgG1 [173, 174]. In a study by Clements et al. (2020), the mean ratio for each of the transferred four IgG subclasses were calculated from 17 papers. The mean placental transfer ratios for IgG1, IgG2, IgG3, and IgG4 were calculated to be 1.36, 0.97, 1.12, and 1.12, respectively [173]. Based on these findings, the consensus between these papers would result in a placental transfer efficiency of IgG1>IgG4=IgG3>IgG2. It is also important to note that when evaluating these efficiencies, the transplacental transfer ratios are being analyzed, not the total concentrations of these antibodies. Based on this evaluation, there are greater concentrations of IgG1, IgG3, and IgG4 in cord blood than in the mother, whereas the concentration of IgG2 is higher in the mother compared to the cord blood. 21 Factors that affect transplacental transport of antibodies Increased transfer of maternal antibodies is beneficial for protecting the infant during their first few months, however, there are factors that can affect the amount transferred. These factors include gestation, concentration of antibodies in the mother, hypergammaglobulinemia, and placental/maternal infections. Premature babies tend to have lower IgG transfers than full term infants [167, 175, 176]. This is thought to be because the efficiency of IgG transport increases as the pregnancy progresses and FcRn expression increases in the third trimester [167, 177, 178]. Mothers with higher antibody concentrations in pregnancy had greater placental transfer of antibodies compared to mothers with lower concentrations of antibodies, however, the overall ratio of maternal antibodies in the infant compared to the antibodies in the mother at birth do not differ [179]. Hypergammaglobulinemia is also thought to decrease the transplacental transfer ratio due to saturation of the FcRn [167, 180]. Lastly, placental and maternal infections such as malaria and human immunodeficiency virus (HIV) have been shown in one study to reduce the levels of these antibodies against measles in newborns [181]. 1.5.5 Infants during and after infant vaccine series An infant’s immune response to their own primary vaccine series can be dependent on the type of vaccine used, acellular (DTaP) or whole cell (DTwP), as well as whether their mother was immunized in pregnancy with Tdap. Furthermore, infants born to mothers immunized in pregnancy with Tdap can experience immunomodulation which can hamper the infant’s humoral responses to their own vaccines. Immunomodulation associated with vaccination in pregnancy Although maternal antibodies have been able to provide protection to infants prior to their own primary vaccine series, there has been evidence of immunomodulation in these cohorts. The maternal antibodies circulating in the infants can lead to an increase or decrease in their own antibody production after receiving their own vaccines early in life [182]. The mechanism of how this happens is not well understood. A hypothesized mechanism is that the maternal antibodies are able to bind to Fcγ receptor FcγIIB on the B cells and cross-link with the antigen from the vaccine along with the BCR, leading to an inhibitory response and prevention of the B 22 cell to differentiate to a plasma cell [183-185]. Another hypothesis is through epitope masking where the maternal antibodies can cover the epitopes of an antigen and mask it from the infant’s B cells [185, 186]. Although lower antibody concentrations are observed in infants from Tdap-vaccinated mothers, it appears to not be clinically significant as there have not been any significant increases in severe illness [187]. However, in a transmission model generated by Bento and Rohani (2016), it was predicted that there would be a 20% increase in the incidence of pertussis in children aged five to ten years [182]. Studies that have analyzed anti-pertussis antibody concentration in children have conflicting results regarding anti-pertussis specific antibodies where they either remain lower or no longer had a difference to infants whose mothers were not immunized in pregnancy [158, 159]. Although there is no consensus on the impact of immunization in pregnancy on older children’s anti-pertussis antibody concentration, it is important to remember that lower concentrations of antibodies do not imply lower protection [188]. Not only is the concentration of antibodies important, but also their ability to function, such as their avidity profiles and their neutralizing capabilities Furthermore, cell-mediated immunity can play an essential role in responses to an infection with B. pertussis. Primary series with whole cell pertussis vaccine There are not many studies that aimed to evaluate the effect of immunization in pregnancy with Tdap on an infant’s immune responses to a primary vaccine series with wP vaccine. Only two studies have investigated this question, one from Brazil and the other from Thailand [189, 190]. Both studies utilized the same Tdap booster, but the whole cell vaccine used had different bacterial content which could explain differences in the antibody responses that were observed between the two studies. One study found that infants whose mothers were not immunized in pregnancy with Tdap had significantly higher anti-PT and anti-FHA IgG concentrations compared to infants from Tdap immunized mothers one month after the completion of their infant vaccine series with wP [190]. The other study found similar results regarding anti-PT IgG concentrations, but when analyzing anti-FHA IgG concentrations, there was no significant difference between the two infant groups [189]. With these studies it suggests there is some level of immunomodulation that happens even when the mother and infants receive different vaccine types. 23 Primary series with acellular pertussis vaccine Immunomodulation is also observed when both the mother and their infant received the same type of vaccine. However, there is a lot of variability in the results. As mentioned in section 1.4.3, at two months of age prior to their own vaccine series, antibodies against all pertussis vaccine antigens remain significantly higher in infants that were born from mothers that received Tdap in pregnancy compared to infants whose mothers did not receive Tdap in pregnancy [158, 159, 162-165]. However, at seven months of age (one month after the completion of their primary series), evidence of immunomodulation is observed in multiple studies. Halperin et al. (2018) found that infants born from Td-immunized mothers now had significantly higher anti-PT, anti-FHA, anti-PRN, and anti-FIM IgG concentrations than infants from Tdap-immunized mothers. This is not the consensus in the literature. There are studies that found only anti-PRN IgG experienced immunomodulation [164, 165], only anti-FHA IgG [159], only PT IgG [163], or that no immunomodulation was observed with both infant groups having no significant differences in the concentration of antibodies against pertussis vaccines antigens [162]. 1.6 Rational and thesis objectives Despite high vaccination rates, pertussis continues to be a public health concern. With the cyclic nature of pertussis incidence and waning immunity in adolescents and adults, this increase in pertussis disease provides an increased risk in those most vulnerable to severe pertussis disease – infants less than three months of age. The implementation of a Tdap booster during every pregnancy in many higher income countries has been a way to decrease the burden of pertussis disease in this young population. Transplacentally transferred maternal antibodies can provide protection to their infants during their first few months after birth prior to receiving their own primary series of pertussis containing vaccines. In LMICs where severe outcomes of pertussis disease in young infants is more common, Tdap in pregnancy has not yet been implemented. This is due to the lack of data of Tdap given in pregnancy in these countries, and the higher cost associated with implementing an acellular vaccine. Furthermore, immunization in pregnancy with Tdap has been associated with immunomodulation in infants. However, since implementation of immunization in pregnancy is relatively new, there have not been many studies that investigated whether this immunomodulation is also observed with antibody 24 functionality, and whether this immunomodulation dissipates with additional boosters. In this thesis I aimed to evaluate both immunogenicity and antibody functionality in mothers living with and without HIV and their infants in Uganda using a newly approved Tdap vaccine. Furthermore, I aimed to demonstrate whether immunomodulation is overcome after receiving boosters or if the primary vaccine schedule was modified to a 2+1 schedules rather than a 3+1 schedule. The first aim of my thesis was to optimize and develop three enzyme-linked immunosorbent assays to measure antibody concentrations against three common pertussis vaccine antigens – PT, FHA, and PRN. The process and data supporting this optimization and validation is presented in chapter 2. The second aim was to evaluate immunogenicity and antibody functionality in women living with and without HIV, and their infants to provide data that could be used to implement Tdap in Uganda’s pregnancy vaccine schedule. Results from this aim are presented in chapter 3. Aim three was to demonstrate whether a primary series with two doses and an earlier booster performed similarly to the originally three doses and a booster, and whether immunomodulation dissipated in both antibody concentration and antibody functionality. Results from this aim are presented in chapter 4. Lastly, chapter 5 summarizes these findings and provides input on potential future studies/experiments in immunization in pregnancy and immunomodulation. 25 2. Optimization and validation of three enzyme-linked immunosorbent assays to quantify anti-pertussis toxin, -filamentous hemagglutinin, and -pertactin IgG concentrations 2.1 Introduction Due to continued burden of pertussis disease and a lack of a known correlate of protection, development of next-generation vaccines continues to be a need within pertussis research [140]. As such, laboratory-based methods to test vaccine immunogenicity, such as enzyme linked immunosorbent assays (ELISAs) are required [191]. This chapter describes the methodology used to develop an ELISA that could be used in-house and that performed at comparable or better accuracy to commercially available kits. Using an assay developed in-house has several benefits over reliance on commercial kits, including a possibly decreased cost per assay, reduced delays in completing projects due to no extended lead times to receive kits, and increased comparability with other assays that are created in-house (e.g., antibody avidity) as many commercial kits utilize different units than the available WHO standard [193]. I developed three ELISAs that measure IgG responses to pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN). These antigens were selected due to their inclusion in acellular pertussis vaccines [22, 194, 195]. IgG was selected as the measured antibody as IgG is the predominant antibody developed in response to vaccination [196]. Furthermore, since these antigens of interest are protein based, the predominant antibodies produced are also IgG. This chapter describes the methodology used to optimize and validate our assay. The process followed was the same for each of the three antigens, therefore, the results section will be predominantly discussed regarding PT, but FHA and PRN results will also be shown. 2.2 Methods 2.2.1 Serum samples Reference material The standard curve was used to determine the total IgG concentration in samples within the OD range of the standard curve for each respective ELISA protocol. The WHO international standard pertussis antiserum (human) 1st from the NIBSC was prepared fresh for each 26 experiment and was used to create the standard curve for each assay. The standard curve for each assay was created using five dilutions of the NIBSC 06/140 standard diluted using dilution buffer (1% skim milk in 1X PBS and 0.1% Tween-20). The standard was diluted to 1:1200, 1:1300, 1:5000, 1:10000, and 1:60000 for the PT ELISA, 1:600, 1:700, 1:1000, 1:5000, and 1:20000 for the FHA ELISA, and 1:300, 1:400, 1:700, 1:3000, and 1:15000 for the PRN ELISA. Lyophilized NIBSC standard was stored in glass ampoules at -20C prior to reconstitution. An ampoule was placed on ice then brought into a biological safety cabinet (BSC) prior to reconstitution. Using a 100mL pipette, 1mL of UltraPure Distilled Water (Invitrogen; Grand Island, NY) was added to the lyophilized powder and the ampoule was rotated gently around to ensure all powder was dissolved in the water solution, then placed back on ice to dissolve for ten minutes. The ampoule underwent centrifugation at 500 xg for five minutes and brought back into the BSC and divided into 10 μL aliquots. The aliquots were placed in a PCR tube rack, labelled, and stored at -80C. When an aliquot was used for assay optimization or sample analysis, the aliquot was thawed on ice prior to use. Experimental samples Experimental samples were collected from three sources. First, serum samples were obtained from a vaccine study that consisted of children that were vaccinated with a booster dose of an acellular pertussis vaccine a month prior at 12 months or 18 months of age, refer to section 4.2.2 for a more detailed description of these samples. Second, serum-plasma cross-validation was performed using paired serum and plasma samples from the safety and imMunogeNicity of combined Pertussis-cOntaining vaccine (Tdap) for HIV-infected pregnant WomEn and their newboRns (WoMANPOWER) study. This study included pregnant women that received either Tdap or Td in pregnancy and their infants who received whole cell pertussis vaccines, refer to section 3.2.2 for a more detailed description of these samples. Lastly, serum was collected from an adult following an internal healthy control protocol and was used as the positive control for the ELISAs. All serum samples were aliquoted and stored in -80C. Prior to use, samples were placed on ice to thaw. 27 2.2.2 Antigens Purified PT and FHA were purchased from Sigma Aldrich (P7208 and F5551, respectively: Oakville, Ontario, Canada) as lyophilized powder. The PT used in this assay consists of all five subunits (S1-5) and remained in its preactivated state. Purified PRN was purchased from Creative Diagnostics (DAGB124: Shirley, New York, USA). All antigens were purchased in lyophilized form with 50 µg of protein and stored at 4C prior to reconstitution. Vials with lyophilized PT, FHA, or PRN were placed on ice and brought into a BSC for reconstitution. Each rubber topper was slightly lifted to break the vacuum seal and prevent any powder from escaping, then 500 µL of UltraPure Distilled Water (Invitrogen; Grand Island, NY) was added as per manufacturers guidelines, resulting in a 1:10 diluted solution (1 µg/10 µL). The vial was gently rotated to ensure all powder on the walls of the vial were dissolved, then placed on ice and was left to dissolve for 10 minutes. The dissolved antigen was then centrifuged for five minutes at 500 xg, then brought back into the BSC. If multiple vials of the same antigen were reconstituted, the reconstituted antigen was pooled into a single vial then aliquoted into 350 µL aliquots. All aliquots were stored at 4C for short term storage (approximately two weeks). 2.2.3 Secondary antibody Horseradish peroxidase conjugated to purified mouse monoclonal anti-human specific IgG that recognizes the Fc fragment of human IgG was used in the lab-based ELISA protocol (Millipore, 411550: Oakville, Ontario, Canada). Secondary antibody was received frozen from distributors and stored at -20C. Aliquots of 20 µL were prepared to prevent multiple freeze thaws of the stock. The stock was placed on ice to thaw. Once thawed, the stock was vortexed then centrifuged briefly for approximately two minutes to remove any bubbles or solution on the lid. A volume of 20 µL was then placed in labelled PCR tubes using a mechanical pipette. The stock was then centrifuged briefly again to collect any remaining solution on the sides of the tube and the remaining was aliquoted. The aliquots were then placed on a PCR tube rack, labelled, and stored at -20C for future use. Prior to use an aliquot was placed on ice to thaw. 28 2.2.4 Enzyme-linked immunosorbent assays used The lab-based ELISAs that were optimized were validated against standardized commercial kits (PT: Euroimmun, 2050-9601 G; FHA: Euroimmun, 2050-9601-3 G; PRN: Alpha Diagnostic, 960-250-PHG). Each commercial kit was used according to the manufacturer’s protocol. The internal ELISA protocols were adapted from a protocol provided by Dr. Halperin from his lab at the Canadian Center for Vaccinology at Dalhousie University, Halifax, Nova Scotia, and from an ELISA protocol developed in the Sadarangani lab at the Vaccine Evaluation Center by Dr. Gabrielle Gaultier to detect SARS-CoV-2 spike protein specific IgG in human serum [158]. PT ELISA protocol First, antigen coating buffer was made by adding 0.3975g of sodium carbonate (Na2CO3, fisher, S263-500) and 0.7325g of sodium bicarbonate (NaHCO3, EMD Chemicals, SX0320-1) into a glass jar with 250 mL of MilliQ Water. The solution was then mixed by inverting the glass jar a few times until the chemicals were fully dissolved. The pH of the solution was then measured using a pH meter with a target pH of 9.60  0.2. Hydrochloric acid was added to the solution if the pH was too high and sodium hydroxide was added if the pH was too low. The 0.05M carbonate buffer was stored at 4C for up to two weeks. To make the antigen coating solution, an aliquot of the reconstituted PT was placed on ice then brought into a BSC along with the 0.05M carbonate buffer. In a 15 mL conical tube, 9.65 mL of 0.05M carbonate buffer and 350 µL of PT were added to create a 3.5 µg/mL PT solution, then it was vortexed. The antigen coating solution was poured into a reservoir, then using a multi-channel pipette 100 µL of the antigen coating solution was added to each well of a 96-well flat-bottomed 2HB plate (Thermofisher Immunlon, 3455). The coated plate was then sealed using an adhesive seal and was centrifuged for two minutes at 500 xg to remove any droplets along the sides of the walls. The coating was applied for 16-18 hours at 28C. After the plate was coated, the excess coating solution was emptied into beaker with diluted bleach and the plates were then blocked by adding 200 µL of blocking buffer (3% skim milk in 1x PBS with 0.1% Tween-20) in each well and then incubated for one hour at room temperature to prevent further antigen binding to the plate. While the plate was incubating with blocking buffer, the serum samples, standard, and the positive control were diluted in dilution buffer (1% skim milk in 1x PBS with 0.1% Tween-20). After the blocking incubation 29 was complete, the excess blocking buffer was emptied into the sink and the plate was tapped dry onto paper towels. Next, 100 µL of the serum samples, standard, positive control, and the blank (dilution buffer) were added in duplicate to the plate and incubated for 3 hours at 28C to facilitate antibody binding to the coated antigen. Next, plates were washed with wash buffer (1x PBS with 0.1% Tween-20) using a plate washer to remove any unbound sera, standard, or positive control. The plate washer removed the solution from each well, then added 200 µL of wash buffer and let sit for 30 seconds, then removed the wash buffer and repeated four more times for a total of five washes. After the wash cycle was completed, the plate was tapped dry on a paper towel then 100 µL of secondary antibody diluted to 1:1500 in dilution buffer was added to each well and incubated for one hour at 28C. This allowed for the secondary antibody to bind to the Fc region of any IgG bound to the antigen. The plate was then washed following the previous wash cycle, then tapped dry on a paper towel. 100 µL of o-phenylenediamine dihydrochloride (OPD) was added according to manufacturer’s instructions and left to incubate for 10 minutes at room temperature to induce a color reaction with horseradish peroxidase conjugated to the secondary antibody. After 10 minutes, 50 µL of 3M HCl was added to stop the reaction. The optical density (OD) of each well was then measured with the Tecan infinite 200 at 490 nm. In Excel, antibody concentrations in IU/mL were calculated by plotting the blank subtracted ODs of the standard against their known IgG concentrations to generate a standard curve. Blank subtracted sample ODs were then inputted into the equation of the standard curve and multiplied by the dilution factor to determine the final anti-PT IgG concentration in IU/mL. FHA ELISA The FHA ELISA protocol followed the same protocol as the PT ELISA, except a 1X PBS buffer (100 mL of 10X PBS in 900 mL of MilliQ Water) was used as the antigen coating buffer, and the FHA coating solution was made by mixing 9.85 mL of 1X PBS buffer and 150 µL of FHA to create a 1.5 µg/mL FHA solution. 30 PRN ELISA The PRN ELISA protocol followed the same protocol as the PT ELISA, except to make the antigen coating solution, 9.75 mL of 0.05M carbonate buffer and 250 µL of PRN were added to create a 2.5 µg/mL PRN solution. 2.3. Results 2.3.1 Optimization Two parameters were tested to optimize the ELISAs: antigen coating concentration and secondary antibody concentrations. The antigen coating concentration was tested to ensure each well was coated thoroughly and produced consistent results. Secondary antibody concentration was tested to ensure it was added in excess so that every bound antibody to antigen was bound by the secondary antibody and can therefore be detected. Antigen Coating Concentration The optimal antigen coating concentration for a given antigen was defined as the antigen coating concentration at which the OD of the primary antibody (NIBSC 06/140) is highest prior to where it begins to plateau [197]. The NIBSC 06/140 standard was first diluted to two different dilutions. For all three antigens, an initial experiment was performed with a broad range of antigen coating concentrations with the goal of identifying a narrower range to investigate further. The concentrations used were 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 μg/mL. A 1:2000 and 1:10000 dilution of the NIBSC 06/140 standard was added in duplicate as the primary antibody for the optimization of the PT ELISA. The optimal coating concentration for PT was determined to be between 3.0 and 5.0 g/mL (Figure 2.1a). To narrow this range for PT, the experiment was repeated using coating concentrations of 0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 g/mL. Both the 1:2000 and 1:10000 dilutions of the NIBSC 06/140 standard indicated that the OD plateau was reached at an antigen coating concentration of 3.5 g/mL, therefore, this concentration was considered the optimal antigen coating concentration for PT and was chosen for all experiments (Figure 2.1b). The 31 optimal antigen coating concentration for FHA and PRN was determined to be 1.5 μg/mL and 2.5 μg/mL, respectively (Figures 2.1c-f). 0.5 1 2 3 4 5 60.00.51.01.5Pertussis toxin coating concentration (µg/mL)Optical Density0.5 1 2 3 4 5 60.00.51.01.5Filamentous hemagglutinin coating concentration (µg/mL)Optical Density0.5 1.5 2.5 3 3.5 4 4.50.00.51.01.5Pertussis toxin coating concentration (µg/mL)Optical Density0.5 1 1.5 2 2.5 3 3.50.00.51.01.5Filamentous hemagglutinin coating concentration (µg/mL)Optical DensityA B D C 32 FIGURE 2.1. Determining antigen coating concentration of pertussis toxin, filamentous hemagglutinin, and pertactin. (A) Pertussis toxin was initally tested at broad concentrations – 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 g/mL using standard as a sera sample at 1:2000 (orange) and 1:10000 (blue). (B) Filamentous hemagglutinin was initially tested at broad concentration - 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 g/mL using standard as a sera sample at 1:1000 (orange) and 1:5000 (blue). (C) Pertactin was initially tested at broad concentration - 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 g/mL using standard as a sera sample at 1:500 (orange) and 1:2500 (blue). (D) Pertussis toxin was then analyzed at narrower concentrations – 0.5, 1.5, 2.5, 3.0, 3.5, 4.0, and 4.5 g/mL and the final concentration was determined to be 3.5 g/mL. (E) Filamentous hemagglutinin was then analyzed at narrower concentrations – 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 g/mL and the final concentration was determined to be 1.5 g/mL. (F) Pertactin was then analyzed at narrower concentrations – 0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 g/mL and the final concentration was determined to be 2.5 g/mL. 0.5 1 2 3 4 5 60.00.51.01.5Pertactin coating concentration (µg/mL)Optical Density0.5 1.5 2 2.5 3 3.5 4 4.50.00.51.01.5Pertactin coating concentration (µg/mL)Optical DensityE F 33 Secondary Antibody Concentration The optimal concentration of secondary antibody was defined as the dilution that resulted in, (1) blank wells with an OD of <0.1 and (2) the highest OD for the lowest dilution of the standard [197]. However, for our purposes, a maximum OD of 2.0 was chosen because it ensured the full working range of the plate reader could be used as the plate reader can only reliably measures ODs up to 2.0. The wells were coated with antigen as per the optimal coating concentration defined above. For PT, the standard was diluted to 1:1000, 1:2000, and 1:10000 and was used as the primary antibody. Mouse anti-human IgG secondary antibody concentrations of either 1:750, 1:1500, 1:3000, or 1:6000 was added in duplicate. All secondary antibody dilutions resulted in a blank OD < 0.1, but a dilution of 1:1500 resulted in the lowest dilution of the standard curve having an OD closest to 2 (Figure 2.2a). Therefore, it was determined that the optimal secondary antibody dilution for the PT ELISA was 1:1500. A secondary antigen dilution of 1:1500 was also determined to be the optimal concentration for both the FHA and PRN ELISA (Figures 2.2bc). A dilution of 1:1500 was chosen over 1:750 because it reached an OD of 2.0 and would also use less resources as it was more diluted. 34 FIGURE 2.2. Determining the optimal secondary antibody concentration. Secondary antibody concentrations tested for each antigen were 1:750 (blue), 1:1500 (orange), 1:3000 (grey), and 1:6000 (yellow). (A) Secondary antibody concentration for pertussis toxin was tested using standard treated as serum samples at 1:1000, 1:2000, and 1:10000 dilutions. Optimal secondary antibody concentration was determined to be 1:1500. (B) Secondary antibody concentration for filamentous hemagglutinin was tested using standard treated as serum samples at 1:500, 1:1000, and 1:5000 dilutions. Optimal secondary antibody concentration was determined to be 1:1500. (C) Secondary antibody concentration for pertactin was tested using standard treated as serum samples at 1:250, 1:500, and 1:2500 dilutions. Optimal secondary antibody concentration was determined to be 1:1500. 1:500 1:1000 1:5000 Blank0123Sample dilutionOptical Density1:1000 1:2000 1:10000 Blank0123Sample dilutionOptical Density1:250 1:500 1:2500 Blank0123Sample dilutionOptical DensityPTPRNFHAA B C 35 2.3.2 Validation The purpose of validating our assays was to ensure that reproducible and accurate results were determined every time. Multiple parameters were analyzed, including intra-assay variation, inter-assay variation, cross-validation against commercial kits, serum-plasma cross-validation, analysis of the standard curve including linear range, blank analysis, dilutional linearity, and serum stability regarding freeze-thaws. The next few sections go into detail on the process and results for each of these parameters. TABLE 2.2. Acceptance criteria used in the validation of the ELISAs. Acceptance criteria as suggested by the FDA and EMA on the validation of bioanalytical methods. European Medicines Agency (EMA)[198] Food and Drug Administration (FDA)[199] Criteria used for this assay Intra-assay variation (precision) • at least five samples at each concentration level (LLOQ, low, medium, and high), in duplicate • %CV must be <15% • at least five QC levels analyzed in triplicate per re-analyzed (LLOQ, low, medium, high, and ULOQ) • samples should be repeated at least six times over several days • %CV must be <20% • nine samples were repeated six times over nine days, in duplicate • %CV must be <15% Inter-assay variation (precision) • LLOQ, low, medium, and high samples analyzed at least three times over at least two different days • %CV must be <15% • %CV must be <20% • ≥80% of samples must meet acceptance criteria Cross-validation (accuracy) • +/-15% of the nominal concentration • ≥67% of samples must meet acceptance criteria • +/-20% of the nominal concentration • +/-20% of the nominal concentration • ≥67% of samples must meet acceptance criteria 36 Intra-assay variation Intra-assay precision aims to analyze how well a single assay reproduces a measurement on the same sample [197]. The precision of this assay was assessed using nine samples with an unknown total anti-PT IgG concentration, analyzed in duplicate, on six plates over nine days. The within-run percent coefficient of variation (%CV) for each sample was then calculated by dividing the standard deviation of the duplicates by the arithmetic mean of the duplicates, multiplied by 100%. The criteria that needed to be met to validate the assay can be found in Table 2.1. According to the criteria set by the European Medicine Agency (EMA) on guidelines for bioanalytical method validation, all samples analyzed on the PT ELISA met the criterion for intra-assay precision with a %CV of <15% (Table 2.2) [198]. All samples analyzed on the FHA and PRN ELISA also met this EMA criterion (Table 2.2) [198]. 37 TABLE 2.3. Results from assay variation and cross-validation experiments for all three antigens. Intra-assay variation, inter-assay variation, cross-validation, and serum-plasma cross-validation summarized results are presented. Parameter Pertussis toxin Filamentous hemagglutinin Pertactin Intra-assay variation Count (n) Range of %CV Percentage passed (%) 9 0.4 – 11.7 100 10 0.08 – 8.72 100 10 0.19 – 13.25 100 Inter-assay variation Inter-plate Count (n) Range of %CV Percentage passed (%) Inter-operator Count (n) Range of %CV Percentage passed (%) Inter-day Count (n) Range of %CV Percentage passed (%) 9 13.0 – 20.2 88.9 9 1.2 – 11.0 100 9 1.1 – 15.9 100 10 4.7 – 17.7 100 10 2.6 – 12.9 100 10 1.6 – 10.8 100 10 9.3 – 23.7 90 10 1.9 – 15.5 100 10 1.2 – 15.2 100 Cross-validation Count (n) Range of percent nominal concentration (%) Median percent nominal concentration (%) Percentage passed (%) 28 85.32 - 220.68 112.56 78.6 30 81.4 – 153.5 103.40 90.0 30 75.94 – 176.05 103.39 83.3 Serum-plasma cross-validation Count (n) Range of percent nominal concentration (%) Percentage passed (%) 26 47 - 114 92.3 27 46 – 120 96.3 N/A Abbreviation: %CV; percent coefficient of variation 38 Inter-assay variation Inter-assay precision aims to measure how well the assay is able to be reproduced under different conditions [197]. The precision of the assay was examined using the same sample across six plates, three lab personnel, and three days. The %CV for inter-plate, inter-operator, and inter-day variation was calculated by using the arithmetic mean of the same sample measured on different plates, finding the standard deviation, then calculating the %CV as described in the inter-assay variation section. There were no universally published acceptance criteria for this metric. Therefore, it was determined that at least 80% of samples must meet the %CV criteria for the assay to be considered reproducible. Additional criteria that were met can be found in Table 2.1. Three different inter-assay conditions were analysed for precision across different analyses: inter-plate, inter-operator, and inter-day. Inter-plate. This condition focused on the precision of the total anti-PT IgG concentrations from individual plates over multiple days. The %CV for this condition was determined using the arithmetic mean of the anti-PT IgG concentrations for the same sample over nine plates analysed over a week. The standard deviation was then calculated using the same anti-PT IgG concentrations used to calculate the arithmetic mean. Only 8/9 or 89% of the samples analyzed on the PT ELISA met the FDA %CV criterion of less than 20% (Table 2.2) [199]. Both the FHA and PRN ELISA had 9/9 or 100% of the samples that met this criterion (Table 2.2). Inter-operator. This condition focused on the inter-assay variation between different operators using the same assay. Each operator analyzed three plates over three days that were analyzed over a week. The arithmetic mean of anti-PT IgG concentrations for each sample by each operator were determined and then comparisons were made between two operators at a time, as well as all three operators at once. All 9 samples (100%) analyzed on the PT ELISA met the criterion (Table 2.2). Both the FHA and PRN ELISA also had all 9 samples (100%) meet this criterion (Table 2.2). Inter-day. This condition focused on the inter-assay variation between different days of analyzing the ELISAs to evaluate if the assay produced precise results from day to day. Each operator analyzed plates on the same day, therefore, each day consists of data from each 39 operator. The arithmetic mean of each day was calculated and used to determine the %CV from day to day. When the samples analyzed on the PT ELISA from day to day were compared, all 9 samples (100%) met the criterion (Table 2.2). Both the FHA and PRN ELISA also had all 9 samples (100%) meet this criterion (Table 2.2). In conclusion, according to the FDA criterion where the %CV must be less than 20% and the internal criterion on the number of samples that must pass, all three ELISAs met these criteria [199]. Cross-validation The accuracy of the assays is a measure to determine how close the values from the assay being validated is to the true value or also called the nominal concentration [197]. To assess the accuracy of these assays, 28 samples were analyzed on both the lab-developed protocol and on a standardized commercial kit following the kit protocol. For PT, the lab-developed PT ELISA protocol was compared to the anti-Bordetella PT ELISA kit from Euroimmun, whereby the kit results were treated as the nominal concentration (i.e., the “true value” of antibody present). To determine the nominal concentration, the arithmetic mean anti-PT IgG concentration of a sample obtained from the lab-developed PT ELISA protocol was divided by the corresponding nominal concentration from the commercial kit, and then the quotient was multiplied by 100%. The resulting percentage represented the proximity of the IgG concentrations determined on the lab-developed PT ELISA protocol to the nominal concentration. According to the FDA criterion of a percent nominal concentration within 20% and the EMA criterion that 67% of samples must pass, 22/28 or 78.6% of the samples met these criteria (Table 2.2) [198, 199]. Both the FHA and PRN ELISA had 27/30 (90%) samples and 25/30 (83.3%) samples that met these criteria (Table 2.2). Serum-plasma cross-validation Serum-plasma cross-validation was performed to be able to use both serum and plasma samples within the same study and still be able to make comparisons between groups. To determine if serum and plasma samples could be directly compared using these ELISAs, 26 paired serum and plasma samples that reflected a range of antibody concentrations (4 – 2037 IU/mL and 4 – 1750 IU/mL for the PT and FHA assays, respectively) were analyzed on our internal ELISA protocol. 40 The criteria used for cross-validation was followed, but instead the serum samples were used as the nominal concentration. A total of 24/26 (92.3%) of the samples analyzed on the anti-PT ELISA met these criteria (Table 2.2). For the anti-FHA ELISA, 26/27 (96.3%) of the samples met these criteria (Table 2.2). Serum-plasma cross-validation was not analyzed for the anti-PRN ELISA due to anti-PRN IgG concentrations analyses not being an outcome for the study. Performance of standard curve The standard curve is an important aspect to an ELISA. It is used to infer the IgG concentration of unknown samples as the curve is generated using a sample with a known concentration of IgG specific to the antigen of interest. Without a proper standard curve, the IgG concentrations of the unknown samples cannot be determined accurately and therefore must meet criteria to ensure a good standard curve. These criteria include the fit of the curve, and quantification cut-offs such as upper limit of quantification, lower limit of quantification, and lower limit of detection. The standard curve was generated by plotting the optical densities (OD) of each standard point against their respective known concentrations, and log transforming both axes before a polynomial trendline was added. Linear range and curve fit First, the linear range was determined. To do this, the standard with known concentration of the analyte was serially diluted and analyzed in duplicate on a plate. The results were then plotted with the ODs on the x-axis and the calculated concentration of anti-PT IgG on the y-axis. Both axes were then log-transformed. The standard points that produced a linear range with a high end of an OD ≤2.0 was selected [200]. Five standard points were selected in total, two were at the high end, one was at the low end, and the remaining two were spread along the linear range. The expected linear range for the PT ELISA was from a blank subtracted OD of ~1.80 to ~0.07. The curve fit for the standard curve of each plate needed to have an R2 value ≥ 0.98, or the plate must be repeated as the curve could not be used to infer the antibody concentration in a sample [197]. Figure 2.3a shows an example of the standard curve produced by the anti-PT ELISA. The final standard curve for PT was made by analyzing the WHO international standard 06/140 at 1:1200, 1:1300, 1:5000, 1:10000, and 1:60000, in duplicate. All criteria for the linear range and curve fit were met. The standard curve was generated for FHA and PRN using dilutions of 41 1:600, 1:700, 1:1000, 1:5000, and 1:20000, and 1:300, 1:400, 1:700, 1:3000, and 1:15000, respectively (Figure 2.3bc). FIGURE 2.3. Standard curve generated using NIBSC 06/140 anti-pertussis standard for all three antigens. (A) The standard curve for pertussis toxin was generated by diluting the standard to 1:1200, 1:1300, 1:5000, 1:10000, and 1:60000, in duplicate and plotting the resulting optical density and IU/mL concentration on log-transformed axis and adding a polynomial trend line. (B) The standard curve for filamentous hemagglutinin was generated by diluting the standard to 1:1200, 1:600, 1:700, 1:1000, 1:5000, and 1:20000, in duplicate and plotting the resulting optical density and IU/mL concentration on log-transformed axis and adding a polynomial trend line. (C) The standard curve for pertactin was generated by diluting the standard to 1:300, 1:400, 1:700, 1:3000, and 1:15000, in duplicate and plotting the resulting optical density and IU/mL concentration on log-transformed axis and adding a polynomial trend line. y = 0.124x1.1686R² = 0.99860.0010.0100.1001.0000.010 0.100 1.000 10.000Concentration of anti-PT IgG (IU/mL)Optical Density1.00.1000. 1Concentration of anti-PT IgG (IU/mL)Opti l Density0.010 0.100 1.000 10.000y = 0.12 x1.1686R2 = 0.9 860.y = 0.0987x1.1322R² = 0.99920.0010.010.110.01 0.1 1 10Concentration of anti-FHA IgG (IU/mL)Optical density1.0000.1000.010Concentration of anti-FHA IgG (IU/mL)Optical Den0.010 0.100 1.000 10.000y = 0.0987x .13 2R2 = 0.9 20.y = 7.5432x0.8847R² = 0.99830.010.11100.001 0.01 0.1 1Concentration of anti-PRN IgG (IU/mL)Optical density10.0001.0000.100Concentration of anti-PRN IgG (IU/mL)Optical Density0.001 0.010 0.100 1.000y = 7.5432x0.8847R2 = 0.99830.010A C B 42 The lower limit of quantification (LLOQ) and lower limit of detection (LLOD) The lower limit of quantification represents the lowest IgG concentration that can be accurately determined using the assay [197]. The LLOQ was determined by finding the arithmetic mean of all ODs from the most diluted standard point, then adding two standard deviations of the ODs to this arithmetic mean. This value was then inputted into the equation of the line. For the PT ELISA, this equated to 0.0096 IU/mL. This value was then multiplied by the most concentrated sample dilution that was used in this series of experiments which was 200. The result was 1.9 IU/mL, and this was rounded up to a final LLOQ for the PT ELISA being 2.0 IU/mL (Table 2.3). Any sample that measured below this value on the PT ELISA was given the value of half the LLQ, 1.0 IU/mL. For the FHA and PRN ELISAs, the LLOQ was determined to be 3.5 IU/mL and 1.0 IU/mL, and samples were assigned a value of 1.75 IU/mL and 0.5 IU/mL, respectively, if they measured below the LLOQ (Table 2.3). The lower limit of detection was also determined as it represents the lowest concentration of IgG that can be distinguished from assay background, but unlike LLOQ, the value is not reliable at this concentration [197]. This value cannot be used as a quantity for a sample as the LLOD is lower than the LLOQ. The LLOD was determined as being the result of adding two times the standard deviation of the ODs of the lowest standard point to the arithmetic mean of the same ODs. This was determined to be 0.0096 IU/mL for the PT ELISA, and 0.008 IU/mL and 0.009 IU/mL for the FHA and PRN ELISAs, respectively (Table 2.3). TABLE 2.4. Summarized results from analyzing the performance of the standard curve for each of the three antigens. Parameter Pertussis toxin Filamentous hemagglutinin Pertactin Median R2 of standard curve 0.9961 0.9948 0.9940 ULOQ 3375 IU/mL 3144 IU/mL 2397 IU/mL LLOQ 2 IU/mL 3.5 IU/mL 1 IU/mL LLOD 0.0096 IU/mL 0.008 IU/mL 0.009 IU/mL Range of quantification 2 – 3375 IU/mL 3.5 – 3144 IU/mL 1 – 2397 IU/mL Abbreviations: ULOQ; upper limit of quantification, LLOQ; lower limit of quantification, LLOD; lower limit of detection 43 The upper limit of quantification (ULOQ) The upper limit of quantification represents the highest IgG concentration that can be accurately determined using this assay [197]. If the OD of a sample is greater than the highest OD of the standard curve, the sample was re-analyzed at a more dilute concentration. For samples that were exhausted and had an OD greater than the standard curve, a value of two times the ULOQ was assigned. The ULOQ for the anti-PT ELISA was calculated to be 3375 IU/mL (Table 2.3). A value of 6750 IU/mL was assigned to values measured above this value. The ULQ for the anti-FHA and anti-PRN ELISA were determined to be 3144 IU/mL and 2397 IU/mL, respectively, and samples were assigned a value of 6288 IU/mL and 4794 IU/mL (Table 2.3). Range of Quantification The range of quantification for the anti-PT, anti-FHA, and anti-PRN ELISAs were from 2.0 IU/ml to 3375 IU/ml, 3.5 IU/mL to 3144 IU/mL, and 1 IU/mL to 2397 IU/mL, respectively (Table 2.3). Blank analysis One of the criteria for an ELISA is to have a blank which consists of all reagents except the primary antibody (participant serum) to measure non-specific binding. These blanks were prepared by adding solely dilution buffer during the addition of primary antibody. Non-specific binding in each well was accounted for by subtracting the arithmetic mean of the blank ODs from the rest of the plate ODs. It was determined that the arithmetic mean OD of the blanks for the anti-PT, anti-FHA, and anti-PRN ELISAs must be <0.15, <0.10, and <0.10, respectively. PT having a higher blank criterion was supported by blank requirements from other papers [201, 202]. 2.3.3 Analyzing samples on the standard curve Dilutional linearity Dilutional linearity or parallelism ensures that a sample can be analyzed at different concentrations and as long as the OD is within the linear range, a similar concentrations of the test samples is inferred [197]. To do this, the standard curve was analyzed alongside nine samples with different concentrations of anti-PT IgG ranging from 42 to 205 IU/mL. Each 44 sample was serially diluted seven times to get a total of eight dilutions as follows; 1:298, 1:596, 1:1192, 1:2384, 1:4768, 1:9536, 1:19072, and 1:38144. The OD values measured for each point were plotted against the sample dilution, then both axes were log transformed. When comparing the %CV between dilutions within a sample, the %CV should be less than 20%. This criterion is met with each of the test samples, suggesting that dilutional linearity and parallelism is exhibited, and that the standard curve can be used for direct inference of IgG concentrations (Table 2.4). This was also found to be true for both the FHA and PRN ELISAs (Table 2.4). TABLE 2.5. Summary of testing sample dilutions and sample stability. Parameter Pertussis toxin Filamentous hemagglutinin Pertactin Dilutional linearity Count (n) Median %CV Range of %CV 9 7.9 5.9 – 9.6 9 5.2 2.5 – 8.4 9 4.9 2.2 – 9.5 Serum stability Count (n) Median %CV Range of %CV 9 8.4 3.7 – 20.2 9 8.2 1.6 – 12.4 9 2.5 0.6 – 6.0 Abbreviations: %CV; percent coefficient of variation Serum Stability (Freeze-thaw) Serum stability analyzes how many freeze-thaw cycles a sample can undergo until the results are no longer within an acceptable range of its nominal value. To determine how many freeze-thaws cycles the samples can undergo, the same nine samples with varying IgG concentrations were analysed over three days. Once the samples were used, they were placed in the -80℃ freezer. Overtime, the IgG concentrations decreased as the number of freeze-thaw cycles increased. For the anti-PT ELISA, the %CV for all except one of the samples were less than the criteria of a %CV of less than 20% when analyzing inter-assay precision (Table 2.4). The anti-FHA and anti-PRN ELISAs had all nine samples meet the criteria (Table 2.4). According to these results, the serum remains stable for at least three freeze-thaws. 45 2.3.4 ELISA acceptance criteria for accepting a plate or a sample The following criteria had to be met for each plate and sample that was analyzed. If one of the criteria for the assay was not met, the whole plate had to be rerun. If one of the sample criteria were not met, the sample had to be rerun. TABLE 2.6. Criteria required to be met to accept results from a plate and for a sample. Criteria Plate • Standard curve fit – R2 ≥ 0.98 • %CV between duplicates of each standard curve point must be <15% • OD of standard curve at the highest dilution (1:20000) must be higher than the blank, and the lowest dilution (1:600) must have an OD of ≤ 2.0 • Each standard point must be back calculated and be within 20% of the nominal concentration • The %CV of the arithmetic means of each standard point must be <15% • OD of blank wells must be <0.15 for the anti-PT ELISA and <0.10 for the anti-FHA and anti-PRN ELISA • Positive control must be within 20% of its nominal concentration Sample • %CV between duplicates must be <15%18. • Both duplicates must be within the linear range of the standard set by the plate that they were analysed on. 2.4 Discussion Although there is widespread coverage of pertussis vaccination worldwide, pertussis incidence continues to increase, especially amongst those less than 2 months of age [11, 194]. As new vaccines are developed to hopefully generate a more robust protective immune response, assays must be available to quantify these immune responses. ELISAs continue to be the predominant method of measuring these antibody responses due to their easy set-up, efficiency, and relatively low cost to use [203]. However, there are several disadvantages to utilizing commercially 46 available standardized kits, including cost, a requirement for ample storage space (usually at 4°C), limited applicability outside of determining seropositivity, and the non-standard units to measure antibody concentrations (IU/mL, EU/mL, or AU/mL) [203]. As such, we found it important to develop and validate our ELISAs using a widely accepted WHO standard produced by the NIBSC. The purpose of this paper was to provide the process used to develop and validate our three ELISAs that measure pertussis vaccine antigen specific IgG (PT, FHA, and PRN) and to provide evidence to support the quality of our assays. This paper can be used to follow the ELISA protocols mentioned or can be used as a reference when developing ELISAs against other antigens. There were two main steps in the ELISA creation process: optimization and validation. Optimization. There were several conditions that needed to be optimized for the development of these assays, including coating concentration and secondary antibody concentration. We chose the antigen coating concentration where the curve began to plateau as it represented the point at which a higher concentration of antigen would lead to no difference in measured antibody concentration [197]. The secondary antibody concentration was chosen to be 1:1500 because this captured the linear range of the assay by measuring up to an OD of 2.0 and had a low OD for the blanks. Furthermore, 1:750 would have also worked but it would not be necessary and just use additional resources. Validation. With the conditions of the assays determined, it was then important to test the precision and accuracy. This was completed following the guidelines set out by the FDA and EMA on developing and validating bioanalytical methods [198, 199]. Intra-assay and inter-assay precision provided confidence in the reproducibility of the ELISAs [197]. Inter-assay accuracy provided confidence that the true sample value was being measured each time. The standard curve allows for the interpolation of anti-PT, anti-FHA, and anti-PRN IgG concentrations. The LLOQ and LLOD of the assays are low, meaning they are sensitive and can measure samples with low concentrations of anti-PT, FHA, PRN specific IgG. Most studies have higher LLOQs, and cannot analyze functional properties of antibodies against PT, FHA, and 47 PRN in very low samples [159, 160, 204]. Since this cut-off for further analysis is larger than our assay’s LLOQ, these ELISAs can be used. Although it has been demonstrated that serum and plasma are comparable, it is important to note that there are factors that can affect the analysis of samples. One factor would be that the sample underwent hemolysis. This is more common for plasma samples and consists of the red blood cells rupturing, releasing their intracellular contents into the serum or plasma [205]. The released contents can cross-react with antibodies and may lead to a result that is lower than its true value [206]. In an ideal scenario, a new sample should be requested, but with clinical trials this is not possible as timepoints would have passed [207]. To account for this issue, it is recommended to either not analyze those samples if they are not critical, or to analyze them and make a note so they are not interpreted incorrectly [207]. Therefore, an initial check of sample quality was implemented to note any samples that have indications of improper sample collection, processing, or storage. This includes hemolysis which can be seen by a red tinted sample, as well as the presence of a precipitate. No samples used in the serum-plasma cross-validation showed evidence of hemolysis or the presence of a precipitate. A few areas of validation were not analyzed for these assays, including sensitivity and specificity. This was because the purpose of developing these assays were to measure a specific antibody concentration against the antigens of interest to allow for comparisons between different treatment groups, for example vaccinated and unvaccinated with a pertussis antigen containing vaccine. Since we were not determining if a participant had a pertussis infection, it did not warrant further testing. Some limitations of these ELISAs are access to the antigens. There were world shortages during the pandemic, making it difficult to acquire the antigens needed. However, this appears to have resolved as there is currently a steady availability of all three antigens: PT, FHA, and PRN. Another limitation is it can take more time to analyze samples as compared with an alternative method to quantify antibody concentration such as a multiplex serological assay. However, these assays are costly and difficult to set up a protocol unless purchasing a commercial kit. 48 In conclusion, we have optimized and validated three separate ELISAs to industry standard, to quantify IgG concentrations against three pertussis antigens that are commonly in pertussis vaccines. As there is no widely accepted correlate of protection, assays such as ELISAs continue to be one of the simpler methods to analyze humoral immune responses against vaccination and natural infection to better understand potential correlates of protection. These ELISAs provide an alternative to standardized commercial kits than can be costly, and can also be modified to analyze antibody functionality, such as avidity, which can also play a role in protection. 49 3. The immunogenicity of combined Pertussis-cOntaining vaccine (TdaP) for HIV-infected pregnant WomEn and their newboRns – A Randomized Clinical Trial (WoMANPOWER) 3.1 Introduction Pertussis, also known as whooping cough, is a highly infectious disease that is caused by the bacterium Bordetella pertussis. It is transmitted from an infected human to another human via aerosolized respiratory droplets, which can then infect the upper respiratory track [21, 73]. In 2014, the World Health Organization (WHO) estimated global pertussis cases reaching 24.1 million, with 160,700 deaths in children less than five years of age [15]. From this estimate, 7.8 million cases and 92,500 deaths were from Africa [15]. Furthermore, it was estimated that 5.1 million cases and 85,900 deaths were in infants less than one year of age [15]. To decrease the burden of pertussis disease where the burden is high, the WHO recommends receiving a booster dose of Tdap during every pregnancy [208]. This is to increase the concentration of anti-pertussis maternal antibodies that transfer through the placenta during gestation and can provide protection to these young infants during their first few months of life [183]. In LMICs and low resource settings such as Uganda, receiving a booster dose of Tdap during pregnancy has not been implemented as part of the pregnancy vaccine schedules. This is due to the lack of immunogenicity data of immunization in pregnancy with Tdap in these populations, and due to the cost associated with adding an acellular vaccine to their schedule [209]. Furthermore, HIV has a high prevalence in Africa, adding an additional layer that should be investigated to ensure vaccine effectiveness in different populations. Based on the Global HIV and AIDS statistics fact sheet from 2023, there are currently approximately 39 million people globally that were living with HIV in 2022 [210]. Of these 39 million people, 53% accounted for women and girls [210]. In Africa, it is estimated 25.6 million people live with HIV, of which 63% of all new infections in sub-Saharan Africa are in women and girls [211]. With the prevalence of women and girls living with HIV continuing to grow, it is important to investigate vaccine effectiveness in these populations as immune responses may be hampered. Although no studies have investigated Tdap immunization in women living with HIV in a controlled study, few studies have investigated the administration of influenza, group B Streptococcus, and 50 pneumococcal conjugate vaccines in these populations. In all studies, humoral immune responses to immunization in pregnant women living with HIV (PWLWH) had lower antibody responses than pregnant women without HIV (PWWH) [212-215]. In addition to HIV infection, HIV exposure and antiretroviral (ART) drug exposure can also impact the infants’ immune systems with greater susceptibility to severe infection and pre-term birth [216-218]. In addition to the mother’s immune response to Tdap vaccination, the infant’s immune response to their own primary series must also be investigated. The presence of a high concentration of circulating maternal antibodies in the infant at the time of receiving their own vaccines has been associated with the occurrence of immunomodulation [219]. Immunomodulation is when something, in this case maternal antibodies, interfere with the ability of the infant’s immune system to produce an immune response to their own vaccines [182]. The mechanism of immunomodulation is not well elucidated, but it is thought that the maternal antibodies are able to bind to the antigen and Fcγ receptors on the infant’s B cells and inhibit the B cell from producing a response to the exposure of this antigen [183-185]. Immunomodulation has been documented to occur in infants born to mothers that received Tdap in pregnancy, regardless of whether the infant received an aP or wP vaccine as their primary vaccine series [158, 190]. In this study, we aimed to investigate the immunogenicity of Tdap given in pregnancy to women living with and without HIV, and their infants. Additionally, this study uses a newly approved TdaP vaccine provided by BioNet-Asia that contains tetanus and diphtheria toxoids, genetically detoxified PT, and FHA, and has been shown to provide similar or even superior humoral responses compared to other Tdap vaccines. This vaccine was first approved in 2019 for the use in adults and has not yet been thoroughly investigated for the use in pregnant women and whether immunomodulation still occurs at a high level. Therefore, we also aimed to investigate whether immunomodulation occurs when following Uganda’s infant standard of care vaccines which utilizes a wP vaccine. 51 3.2 Methods 3.2.1 Study Design Samples were collected from a phase II, randomized controlled observer blind clinical trial based in Kampala, Uganda. A total of 181 pregnant women that attended antenatal care at Kawempe National Referral Hospital or Kisenyi Health Centre were enrolled, with 81 of them also living with HIV, but all were taking ART in pregnancy to control the infection. A participant was eligible for enrollment if they were a pregnant woman aged 18 years or older, between 16 and 26 weeks of gestation, with a low risk, singleton pregnancy. A rapid test for detecting antigens against HIV was required at screening to confirm HIV status and sample group assignment. Only pregnant women with a low-level HIV infection were enrolled in the HIV group. Prospective participants must not have had a history of being treated for pertussis, previous anaphylaxis to Td or Tdap vaccines, previous pre-eclampsia or eclampsia in pregnancy, gestational diabetes in current pregnancy, any significant maternal chronic illnesses, and history of any significant pregnancy or neonatal complications. Every enrolled pregnant woman received an initial Td vaccine between 16 and 26 weeks of gestation. They were then randomized using Research Electronic Data Capture (REDCap) software within each HIV group to receive a second dose of the standard of care vaccine, Td, or the trial vaccine, TdaP, four weeks after the initial Td administration. Infants then received wP vaccines according to Ugandan routine vaccine schedule at six, ten, and fourteen weeks of age. 3.2.2 Samples Serum and heparin plasma samples were collected from pregnant women and their infants. Samples from pregnant women were collected pre-vaccine (baseline), four weeks post Td or Tdap study vaccine (baseline + 4 weeks), and at delivery. Samples were collected from HIV-exposed uninfected (HEU) and HIV-unexposed uninfected (HUU) infants at delivery from cord blood and four weeks after the completion of their own infant vaccine series with wP vaccine (baby 18 weeks). There was no vertical transmission of HIV to any of the infants in this study. 52 Antibody responses A total of 181 pregnant women were enrolled and randomised to one of four study groups. There were 90 pregnant women randomized to receive the TdaP vaccine, of which 40 were HIV-positive and 50 were HIV-negative. The remaining 91 pregnant women were randomized to receive Td vaccine. Of which 41 were HIV-positive and 50 were HIV-negative. Anti-pertussis toxin IgG Avidity A total of 79 paired samples from five timepoints were analyzed on the anti-PT avidity assay: baseline, four weeks post Td or Tdap study vaccine, maternal delivery, cord blood, and four weeks after the completion of their own infant vaccine series with wP vaccine. A total of 20 samples from each of the four groups were randomly selected and analyzed at each timepoint, providing paired data from each mother-infant dyad over five timepoints. This sample size was determined based on a convenience sampling as we only had that number of paired samples across each timepoint. Only samples with an anti-PT IgG concentration determined previously from an anti-PT IgG ELISA, of 10 IU/mL or greater were analyzed on the anti-PT avidity assay. 3.2.3 Vaccines Two vaccines were administered to the pregnant women. The control vaccine (Td) used was donated by BioNet-Asia. Each 0.5 mL dose contained 5.0 limits of flocculation (Lf) of diphtheria toxoid and 5 Lf of tetanus toxoid, adsorbed on aluminum hydroxide. The intervention vaccine was also donated by BioNet-Asia. Each 0.5 mL dose contained 5 μg of recombinant pertussis toxin and filamentous hemagglutinin, 2.0 Lf of diphtheria toxoid, and 7.5 Lf of tetanus toxoids, adsorbed on aluminum hydroxide. Infants followed the standard of care for Uganda which was a dose of DTwP at 6, 10, and 14 weeks of age. The whole cell vaccine used in this trial was from the serum institute of India and each 0.5 mL dose contained 25 Lf of diphtheria toxoids, 2.5 Lf of tetanus toxoid, 16 opacity units (OU) of B. pertussis (whole cell), and 10 µg of Hepatitis B surface antigen, adsorbed on aluminium phosphate. 53 3.2.4 ELISA protocol Please refer to sections 2.2.4.2. for a detailed description of the anti-PT and anti-FHA ELISA protocols used. Calculation of transplacental antibody transfer ratio Transplacental antibody transfer ratios were calculated by dividing the infant anti-PT or -FHA IgG concentrations at birth (from cord blood) by the mother’s anti-PT or-FHA IgG concentrations at delivery. 3.2.5 Avidity protocol Anti-PT IgG avidity was performed by following a published protocol that has been developed and extensively used in our lab. The avidity assay utilizes a chaotropic agent (ammonium thiocyanate) at various concentrations (0.25M, 0.50M, 1.0M, 1.5M, 2.0M, and 3.0M) to disrupt the binding of the antibody to the antigen complex. To measure anti-PT IgG avidity the PT ELISA protocol from section 2.2.4 was followed with three differences. Firstly, seven successive wells in a column had the same sample added, in triplicate. This allowed for the addition of each ammonium thiocyanate concentration within each column. The triplicates were necessary due to increased variation between replicates. If a triplicate did not meet the sample acceptance criteria because of a high coefficient of variation (%CV) between replicates, the replicate when removed that resulted in the lowest %CV was not included in the analysis. Secondly, ammonium thiocyanate at different concentrations were added after the incubation of primary antibody before the secondary antibody. Lastly, the development time with OPD was increased to 12 minutes. The ammonium thiocyanate concentrations were made by first making the 3.0M concentration by dissolving ammonium thiocyanate into 1X PBS. The 3.0M ammonium thiocyanate solution was diluted using 1X PBS into separate 50 mL conical tubes to make the rest of the ammonium thiocyanate concentrations. In a deep well plate, approximately 1 mL of 1X PBS followed by 1mL of each of the ammonium thiocyanate concentrations in ascending concentrations were added to each well along a single column. This made it easier to add the different concentrations and minimized the potential for human error when adding the solution to the plate. The 1X PBS was added to wells to provide the anti-PT IgG concentrations in the absence of chaotrope which was 54 necessary to calculate the different avidity measurements. Using a multichannel pipette, 100 µL of 1X PBS and the different ammonium thiocyanate concentrations in the deep well plate were added to the plate after the primary antibody was washed off using the plate washer. The plate was then incubated for 20 minutes at 37C. Prior to the addition of the secondary antibody the plate was tapped over a waste beaker to collect the ammonium thiocyanate, then the plate was washed using the plate washer. The remainder of the protocol followed the PT ELISA protocol in section 2.2.4 with the additional development time. Avidity measurements/calculations The various concentrations used in the avidity protocol measured very low, low, medium-low, medium, medium-high, high, and very high antibody avidity levels. The concentration of antibodies with different binding strength was then measured and three different results were determined: total relative antibody index (TRAI), fractional absolute avidity (FAA) levels, and total absolute avidity (TAA) levels. TRAI considers the differing binding strengths of antibodies to the antibody-antigen complex by giving a higher weight to antibodies with higher avidity, this generates a single output per sample in avidity units (AU). FAA levels breaks down the total anti-PT IgG concentration of a sample into seven different avidity levels, where it determines what amount of the total anti-PT IgG falls into each of the seven different avidity levels. This measure generates seven values per sample as an anti-PT IgG concentration in international units per mL (IU/mL – same as ELISA unit) per avidity level (very low, low, medium-low, medium, medium-high, high, and very high). TAA level goes further to not only consider the different avidity levels of the antibodies, but also the quantity of the antibodies. This measure has a single output per sample in absolute avidity units per mL (AAU/mL). 3.2.6 Data Analysis Antibody responses Immunogenicity outcomes were summarised using geometric mean concentrations (GMCs) and 95% confidence intervals (CIs). Geometric mean ratios (GMRs), calculated as the antilogarithm of the difference between the mean of the log10 transformed titre in the linear regression model, two-sided 95% CIs were conducted to compare groups. Individual transplacental ratios were calculated by dividing infant by mother IgG concentrations at delivery and summarised using 55 geometric means. No statistical analyses were performed yet on the immunogenicity data presented in this chapter. The immunogenicity data is from a regulated clinical trial and therefore must be done by a statistician. The sample size calculation assumed a standard deviation of the antibody concentration outcomes to be 0.4 on log10 scale and the true GMR between HIV-positive versus HIV-negative groups or TdaP vaccinated versus Td vaccinated groups to be one. The study needed to recruit 50 pregnant women in each group (200 in total) to achieve 80% power at a two-sided 5% significance level, after adjusting for a 15% drop out rate to achieve 40 participants in each study arm. Statistical analyses were performed using R 4.3.1, RStudio 2023.06.2+561, and SAS 9.4. Anti-PT IgG Avidity Samples that had anti-PT IgG concentrations below 10 IU/mL were not included in any avidity analyses. All data was log-transformed (log10) prior to statistical analyses to normalize data and allow for parametric tests to be used. GraphPad Prism version 9.5.1 was used to perform statistical analysis on the anti-PT IgG avidity data. Descriptive statistics were used to determine the geometric mean concentrations (GMCs) with 95% confidence intervals at each measurement within each group, at each timepoint. An unpaired Welch’s t test was used to determine any statistical significance between groups within a timepoint. Significance was set at p <0.05. 3.3 Results 3.3.1 Immunogenicity of TdaP versus Td in pregnant women living with and without HIV Total anti-PT IgG GMCs were similar across all groups at baseline (Figure 3.1a, Table 3.1). After PWLWH received a second vaccination in pregnancy, total anti-PT IgG concentrations in TdaP-vaccinated pregnant women were 11.4-fold higher than Td-vaccinated pregnant women (GMC, 134 IU/mL [95% CI, 91 – 196] vs. 12 IU/mL [95% CI, 9 – 16]) (Table 3.2). Similarly, TdaP-vaccinated pregnant women living without HIV had 26.5-fold higher total anti-PT IgG concentrations than Td-vaccinated women living without HIV (GMC, 245 IU/mL [95% CI, 168 – 358] vs. 9 IU/mL [95% CI, 7 – 13]). At delivery, total anti-PT IgG concentrations in TdaP- 56 vaccinated women remained higher than Td-vaccinated pregnant women, irrespective of HIV-status (HIV+; GMC, 84 IU/mL [95% CI, 53 – 134] vs. 11 IU/mL [95% CI, 7 – 16], HIV-; GMC, 130 IU/mL [95% CI, 85 – 197] vs. 7 IU/mL [95% CI, 5 – 10]). Similar trends were observed for total anti-FHA IgG concentrations. Total anti-FHA IgG GMCs were similar across all groups at baseline (Figure 3.1b, Table 3.1). One month after the second vaccination in pregnancy, total anti-FHA IgG concentrations in TdaP-vaccinated PWLWH were 5.7-fold higher than Td-vaccinated PWLWH (GMC, 102 IU/mL [95% CI, 65 – 158] vs. 18 IU/mL [95% CI, 13 – 24]) (Table 3.2). Similarly, TdaP-vaccinated PWWH had 19.8-fold higher total anti-FHA IgG concentrations than Td-vaccinated PWWH (GMC, 314 IU/mL [95% CI, 236 – 416] vs. 16 IU/mL [95% CI, 12 – 20]). At delivery, total anti-FHA IgG concentrations in TdaP-vaccinated women remained higher than Td-vaccinated pregnant women, irrespective of HIV-status (HIV+; GMC, 64 IU/mL [95% CI, 40 – 103] vs. 20 IU/mL [95% CI, 14 – 28], HIV-; GMC, 223 IU/mL [95% CI, 170 – 293] vs. 16 IU/mL [95% CI, 12 – 22]). 57 FIGURE 3.1. Immunogenicity in pregnant women stratified by vaccine received in pregnancy and HIV-status. Data is presented as GMCs with 95% confidence intervals. A) Total anti-PT IgG concentrations B) Total anti-FHA IgG concentrations Baseline Baseline + 4 weeks Maternal DeliveryTotalanti-PT IgG (IU/mL)1000100101LLOQ = 2 IU/mLTd vaccine, HIV positiveTdaP vaccine, HIV positiveTd vaccine, HIV negativeTdaP vaccine, HIV negativeBaseline Baseline + 4 weeks Maternal DeliveryTotalanti-FHA IgG (IU/mL)1000100101LLOQ = 3.5 IU/mLA B 58 TABLE 3.1. GMCs of total anti-PT and anti-FHA IgG concentrations stratified by each group TdaP Td HIV positive (N=40) HIV negative (N=50) HIV positive (N=41) HIV negative (N=50) Anti-PT IgG concentrations Baseline 11 (7, 15) [n=39] 11 (8, 14) [n=48] 12 (9, 17) [n=37] 11 (8, 15) [n=48] Baseline + 4 weeks 134 (91, 196) [n=40] 245 (168, 358) [n=46] 12 (9, 16) [n=38] 9 (7, 13) [n=48] Maternal delivery 84 (53, 134) [n=36] 130 (85, 197) [n=41] 11 (7, 16) [n=34] 7 (5, 10) [n=38] Baby delivery 115 (70, 187) [n=36] 185 (112, 305) [n=33] 12 (8, 17) [n=33] 8 (5, 12) [n=40] Baby 18 weeks 17 (10, 29) [n=20] 29 (19, 44) [n=36] 101 (48, 209) [n=25] 136 (67, 278) [n=32] Transplacental ratio 1.3 (1.2, 1.5) [n=36] 1.2 (0.8, 1.8) [n=35] 1.2 (1, 1.5) [n=31] 1.1 (0.9, 1.4) [n=38] Delivery to 18-week fold-change 0.2 (0.1, 0.3) [n=19] 0.1 (0.1, 0.2) [n=25] 6 (1.8, 19.9) [n=24] 19.5 (5.8, 65.2) [n=27] Anti-FHA IgG concentrations Baseline 14 (10, 22) [n=39] 20 (15, 26) [n=48] 19 (13, 26) [n=37] 20 (16, 27) [n=48] Baseline + 4 weeks 102 (65, 158) [n=40] 314 (236, 416) [n=46] 18 (13, 24) [n=38] 16 (12, 20) [n=48] Maternal delivery 64 (40, 103) [n=36] 223 (170, 293) [n=41] 20 (14, 28) [n=34] 16 (12, 22) [n=38] Baby delivery 74 (44, 124) [n=36] 283 (196, 409) [n=33] 18 (12, 26) [n=33] 18 (12, 25) [n=40] Baby 18 weeks 18 (12, 25) [n=20] 24 (18, 33) [n=36] 18 (13, 25) [n=25] 17 (12, 23) [n=32] Transplacental ratio 1.1 (1, 1.3) [n=36] 1 (0.7, 1.5) [n=35] 0.9 (0.8, 1.2) [n=31] 1 (0.8, 1.2) [n=38] Delivery to 18-week fold-change 0.2 (0.1, 0.4) [n=19] 0.1 (0.1, 0.1) [n=25] 1 (0.5, 1.9) [n=24] 1 (0.6, 1.8) [n=27] 59 TABLE 3.2. GMRs of total anti-PT and anti-FHA IgG concentrations stratified by each group TdaP versus Td HIV-positive versus HIV-negative HIV-positive HIV-negative TdaP Td Anti-PT IgG concentrations Baseline 0.87 (0.54, 1.39) 1 (0.65, 1.53) 1 (0.64, 1.57) 1.15 (0.74, 1.8) Baseline + 4 weeks 11.35 (6.96, 18.49) 26.46 (16.22, 43.14) 0.55 (0.32, 0.93) 1.27 (0.81, 2) Maternal delivery 7.9 (4.32, 14.46) 19.59 (11.23, 34.17) 0.65 (0.35, 1.2) 1.61 (0.94, 2.75) Baby delivery 9.61 (5.21, 17.74) 23.52 (12.27, 45.06) 0.62 (0.31, 1.23) 1.52 (0.85, 2.71) Baby 18 weeks 0.17 (0.07, 0.43) 0.21 (0.1, 0.46) 0.6 (0.31, 1.15) 0.74 (0.27, 2.03) Transplacental ratio 1.08 (0.85, 1.38) 1.14 (0.74, 1.75) 1.06 (0.72, 1.57) 1.12 (0.81, 1.54) Delivery to 18-week fold-change 0.03 (0.01, 0.11) 0.01 (0, 0.02) 1.3 (0.51, 3.32) 0.31 (0.06, 1.63) Anti-FHA IgG concentrations Baseline 0.78 (0.46, 1.3) 0.97 (0.68, 1.39) 0.73 (0.46, 1.15) 0.91 (0.61, 1.36) Baseline + 4 weeks 5.68 (3.34, 9.67) 19.75 (13.65, 28.57) 0.32 (0.2, 0.54) 1.13 (0.77, 1.64) Maternal delivery 3.23 (1.82, 5.73) 13.56 (9.14, 20.13) 0.29 (0.17, 0.48) 1.21 (0.78, 1.86) Baby delivery 4.23 (2.2, 8.14) 16.14 (9.86, 26.42) 0.26 (0.14, 0.49) 1 (0.59, 1.68) Baby 18 weeks 0.97 (0.61, 1.53) 1.47 (0.97, 2.23) 0.72 (0.46, 1.14) 1.1 (0.7, 1.71) Transplacental ratio 1.23 (0.97, 1.55) 0.99 (0.63, 1.55) 1.14 (0.74, 1.75) 0.92 (0.69, 1.23) Delivery to 18-week fold-change 0.2 (0.08, 0.51) 0.09 (0.05, 0.16) 2.36 (1.23, 4.52) 1.02 (0.44, 2.36) 3.3.2 Immunogenicity of TdaP and Td in pregnant women living with HIV versus without HIV Pregnant women living with or without HIV that received a Td vaccine as a second vaccination in pregnancy had similar total anti-PT IgG concentrations 4-weeks post-vaccination (GMC, 12 IU/mL [95% CI, 9 – 16] vs. 9 IU/mL [95% CI, 7 – 13]) (Figure 3.1a, Table 3.1). However, PWWH that received TdaP vaccine as a second vaccination in pregnancy had higher total anti-PT IgG concentrations 4-weeks post-vaccination than PWLWH that also received TdaP (GMC, 60 245 IU/mL [95% CI, 168 – 358] vs. 134 IU/mL [95% CI, 91 – 196]). Similar trends were observed at delivery with no difference in total anti-PT IgG concentrations between Td-vaccinated women living with or without HIV (GMC, 11 IU/mL [95% CI, 7 – 16] vs. 7 IU/mL [95% CI, 5 – 10]), and TdaP-vaccinated PWWH had higher total anti-PT concentrations than TdaP-vaccinated PWLWH (GMC, 130 IU/mL [95% CI, 85 – 197] vs. 84 IU/mL [95% CI, 53 – 134]). Similar trends were observed for total anti-FHA IgG concentrations. Pregnant women living with or without HIV that received a Td vaccine as a second vaccination in pregnancy had similar total anti-FHA IgG concentrations 4-weeks post-vaccination (GMC, 18 IU/mL [95% CI, 13 – 24] vs. 16 IU/mL [95% CI, 12 – 20]) (Figure 3.1b, Table 3.1). However, PWWH that received TdaP vaccine as a second vaccination in pregnancy had higher total anti-FHA IgG concentrations 4-weeks post-vaccination than PWLWH that also received TdaP (GMC, 314 IU/mL [95% CI, 236 – 416] vs. 102 IU/mL [95% CI, 65 – 158]). Similar trends were observed at delivery with no difference in total anti-FHA IgG concentrations between Td-vaccinated women living with or without HIV (GMC, 20 IU/mL [95% CI, 14 – 28] vs. 16 IU/mL [95% CI, 12 – 22]), and TdaP-vaccinated PWWH had higher total anti-FHA IgG concentrations than TdaP-vaccinated PWLWH (GMC, 223 IU/mL [95% CI, 170 – 293] vs. 64 IU/mL [95% CI, 40 – 103]). 3.3.3 Transplacental antibody transfer ratio of total anti-pertussis toxin and anti-filamentous hemagglutinin IgG in TdaP- versus Td-vaccinated pregnant women living with and without HIV Tdap-vaccinated pregnant women had slightly higher transplacental transfer ratios of total anti-PT IgG antibodies than Td-vaccinated pregnant women, regardless of HIV status (HIV+; transplacental ratio 1.3 [95% CI, 1.2 – 1.5] vs. 1.2 [95% CI, 1 – 1.5], HIV-; transplacental ratio 1.2 [95% CI, 0.8 – 1.8] vs. 1.1 [95% CI, 0.9 – 1.4]) (Figure 3.2a, Table 3.1). Similar trends were observed when comparing transplacental transfer ratios of total anti-FHA IgG in PWLWH, however PWWH had no differences in transplacental transfer ratios of total anti-FHA IgG (HIV+; transplacental ratio 1.1 [95% CI, 1 – 1.3] vs. 0.9 [95% CI, 0.8 – 1.2], HIV-; transplacental ratio 1 [95% CI, 0.7 – 1.5] vs. 1 [95% CI, 0.8 – 1.2]) (Figure 3.2b, Table 3.1). 61 FIGURE 3.2. Transplacental transfer ratios of total anti-PT and anti-FHA IgG concentrations stratified by vaccine received in pregnancy and HIV-status of the mother. Transplacental transfer ratios were calculated by dividing infant by mother IgG concentrations at delivery and summarised using geometric means. A) Transplacental transfer ratios of anti-PT IgG. B) Transplacental transfer ratios of anti-FHA IgG. 0.0 0.5 1.0 1.5 2.01111Tranplacental transfer ratio of anti-PT IgGFor groupsTd vaccine, HIV positiveTdaP vaccine, HIV positiveTd vaccine, HIV negativeTdaP vaccine, HIV negativeTd vaccine, HIV positiveTdaP vaccine, HIV negativeTd vaccine, HIV negativeTdaP vaccine, HIV positiveTransplacental transfer ratio of anti-PT IgG0. 0. 1. 1. 2.0.0 0.5 1.0 1.5 2.01111Tranplacental transfer ratio of anti-FHA IgGFor groupsTd vaccine, HIV positiveTdaP vaccine, HIV positiveTd vaccine, HIV negativeTdaP vaccine, HIV negativeTd vaccine, HIV positiveTdaP vaccine, HIV negativeTd vaccine, HIV negativeTdaP vaccine, HIV positiveTransplacental transfer ratio of anti-FHA IgG0.0 0.5 1. 1. 2.0A B 62 3.3.4 Transplacental antibody transfer ratio of anti-pertussis toxin and anti-filamentous hemagglutinin IgG in pregnant women living with or without HIV that received the same vaccine in pregnancy PWLWH had slightly higher transplacental transfer ratios of total anti-PT IgG compared with PWWH within each vaccine group (TdaP; transplacental ratio, 1.3 [95% CI, 1.2 – 1.5] vs. 1.2 [95% CI, 0.8 – 1.8], Td; transplacental ratio, 1.2 [95% CI, 1 – 1.5] vs. 1.1 [95% CI, 0.9 – 1.4]) (Figure 3.2a, Table 3.1). This was similar for transplacental transfer ratios of total anti-FHA IgG between PWLWH and PWWH when looking within the TdaP vaccine group (transplacental ratio, 1.1 [95% CI, 1 – 1.3] vs. 1 [95% CI, 0.7 – 1.5]). However, PWWH had slightly higher transplacental transfer ratios of total anti-FHA IgG than PWLWH in the Td vaccine group (transplacental ratio, 1 [95% CI, 0.8 – 1.2] vs. 0.9 [95% CI, 0.8 – 1.2]) (Figure 3.2b, Table 3.1). 3.3.5 Immunogenicity of DTwP in HIV-exposed uninfected and HIV-unexposed uninfected infants from TdaP versus Td-vaccinated mothers Infants from TdaP-vaccinated mothers in pregnancy had higher total anti-PT IgG concentrations at delivery than infants from Td-vaccinated mothers, irrespective of fetal HIV exposure (Figure 3.3a, Table 3.1, Table 3.2). HEU infants from TdaP-vaccinated mothers living with HIV had 9.6-fold higher total anti-PT IgG than HEU infants from Td-vaccinated mothers living with HIV (GMC, 115 IU/mL [95% CI, 70 – 187] vs. 12 IU/mL [95% CI, 8 – 17]). Similarly, HUU infants from TdaP-vaccinated mothers living without HIV had 23.5-fold higher total anti-PT IgG than HUU infants from Td-vaccinated mothers living without HIV (GMC, 185 IU/mL [95% CI, 112 – 305] vs. 8 IU/mL [95% CI, 5 – 12]). Inversely, 4-weeks after completion of infant primary series with DTwP (baby 18 weeks timepoint), infants from Td-vaccinated mothers now had higher total anti-PT IgG concentrations than infants from TdaP-vaccinated mothers, irrespective of fetal HIV-exposure (HEU infants; GMC, 101 IU/mL [95% CI, 48 – 209] vs. 17 IU/mL [95% CI, 10 – 29], HUU infants; GMC, 136 IU/mL [95% CI, 67 – 278] vs. 29 IU/mL [95% CI, 19 – 44]). Infant total anti-FHA IgG concentrations at delivery followed similar trends to total anti-PT IgG concentrations in infants at delivery (Figure 3.3b, Table 3.1, Table 3.2). HEU infants from TdaP-vaccinated mothers living with HIV had 4.2-fold higher total anti-FHA IgG than HEU infants from Td-vaccinated mothers living with HIV (GMC, 74 IU/mL [95% CI, 44 – 124] vs. 18 IU/mL 63 [95% CI, 12 – 26]). Similarly, HUU infants from TdaP-vaccinated mothers living without HIV had 16.1-fold higher total anti-FHA IgG than HUU infants from Td-vaccinated mothers living without HIV (GMC, 283 IU/mL [95% CI, 196 – 409] vs. 18 IU/mL [95% CI, 12 – 25]). Differently to the trend observed for total anti-PT IgG concentrations in infants’ 4-weeks post-primary series with DTwP, total anti-FHA IgG concentrations in infants’ post-primary series with DTwP were similar between infants from TdaP- and Td-vaccinated mothers, regardless of fetal HIV-exposure (HEU infants; GMC, 18 IU/mL [95% CI, 12 – 25] vs. 18 IU/mL [95% CI, 13 – 25], HUU infants; GMC, 24 IU/mL [95% CI, 18 – 33] vs. 17 IU/mL [95% CI, 12 – 23]). 64 FIGURE 3.3. Immunogenicity in infants stratified by the vaccine mother received in pregnancy and HIV-exposure in utero. Data is presented as GMCs with 95% confidence intervals. A) Total anti-PT IgG concentrations B) Total anti-FHA IgG concentrations Baby Delivery Baby 18 weeksTotalanti-PT IgG (IU/mL)1000100101LLOQ = 2 IU/mLTd vaccine, HIV positiveTdaP vaccine, HIV positiveTd vaccine, HIV negativeTdaP vaccine, HIV negativeTotalanti-FHA IgG (IU/mL)1000100101LLOQ = 3.5 IU/mLBaby Delivery Baby 18 weeksTd vaccine, HIV positiveTdaP vaccine, HIV positiveTd vaccine, HIV negativeTdaP vaccine, HIV negativeA B 65 3.3.6 Immunogenicity of DTwP in HIV-exposed uninfected versus HIV-unexposed uninfected infants from TdaP or Td vaccinated mothers No differences in total anti-PT IgG concentrations were observed between HEU and HUU infants from Td-vaccinated mothers at delivery (GMC, 12 IU/mL [95% CI, 8 – 17] vs. 8 IU mL [95% CI, 5 – 12]) (Figure 3.3a, Table 3.1). HUU infants from TdaP-vaccinated mothers had higher total anti-PT IgG concentrations at delivery than HEU infants from TdaP-vaccinated mothers (GMC, 185 IU/mL [95% CI, 112 – 305] vs. 115 IU/mL [95% CI, 70 – 187]). After completion of infant primary series with DTwP (baby 18 weeks timepoint), HUU infants had slightly higher total anti-PT IgG concentrations than HEU infants when compared between infants whose mothers received the same second vaccine in pregnancy (TdaP; GMC, 29 IU/mL [95% CI, 19 – 44] vs. 17 IU/mL [95% CI, 10 – 29], Td; GMC, 136 IU/mL [95% CI, 67 – 278] vs. 101 IU/mL [95% CI, 48 – 209]). Similar trends were observed for total anti-FHA IgG concentrations at delivery. No differences in total anti-FHA IgG concentrations were observed between HEU and HUU infants from Td-vaccinated mothers at delivery (GMC, 18 IU/mL [95% CI, 12– 26] vs. 18 IU mL [95% CI, 12 – 25]) (Figure 3.3b, Table 3.1). HUU infants from TdaP-vaccinated mothers had higher total anti-PT IgG concentrations at delivery than HEU infants from TdaP-vaccinated mothers (GMC, 283 IU/mL [95% CI, 196 - 409] vs. 74 IU/mL [95% CI, 44 – 124]). After completion of infant primary series with DTwP (baby 18 weeks timepoint), no differences in total anti-FHA IgG concentrations were observed between HEU and HUU infants when compared between infants whose mothers received the same second vaccine in pregnancy (TdaP; GMC, 18 IU/mL [95% CI, 12 – 25] vs. 24 IU/mL [95% CI, 18 – 33], Td; GMC, 18 IU/mL [95% CI, 13 – 25] vs. 18 IU/mL [95% CI, 12 – 23]). 3.3.7 Anti-pertussis toxin IgG avidity in pregnant women living with and without HIV Comparison between Tdap versus Td-vaccinated pregnant women within the same HIV-status Total anti-PT IgG concentrations were compared in the subset of samples randomly selected for anti-PT IgG avidity analyses. In the subset of samples, there were no significant differences in total anti-PT IgG concentrations across all groups at baseline (p >0.05) (Figure 3.4a, Table 3.3). 66 Tdap-vaccinated pregnant women had significantly higher total anti-PT IgG concentrations 4-weeks post-vaccination than Td-vaccinated pregnant women, irrespective of HIV status (HIV+; GMC, 217 IU/mL [95% CI, 117 – 405] vs. 6 IU/mL [95% CI, 3 – 10], p<0.0001, HIV-; GMC, 122 IU/mL [95% CI, 78 – 191] vs. 12 IU/mL [95% CI, 7 – 19], p<0.0001) (Table 3.4). Total anti-PT IgG concentrations in TdaP-vaccinated pregnant women remained significantly higher than Td-vaccinated pregnant women at delivery, regardless of HIV status (HIV+; GMC, 107 IU/mL [95% CI, 55 – 209] vs. 5 IU/mL [95% CI, 3 – 9], p<0.0001, HIV-; GMC, 76 IU/mL [95% CI, 41 – 141] vs. 11 IU/mL [95% CI, 6 – 18], p<0.0001) (Table 3.5). 67 A 68 B 69 FIGURE 3.4. GMC of total anti-PT IgG with 95% confidence intervals, anti-PT TRAI, and anti-PT TAA in pregnant women stratified by vaccine received and HIV-status of a subset of samples. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05, *** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. A) Total anti-PT IgG concentrations (IU/mL). B) Total relative avidity index of anti-PT IgG (AU). C) Total absolute avidity levels of anti-PT IgG (AAU/mL). C 70 TABLE 3.3. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at baseline. TdaP Td P value HIV-positive Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 11.0 (6.7 – 17.9) 7.4 (4.7 – 11.7) 0.1997 TRAI of anti-PT IgG (AU), mean (SD) 127.8 (14.6) 132.8 (21.5) 0.6572 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 23.1 (16.0 – 33.4) 35.0 (21.9 – 55.8) 0.1255 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 1.3 (0.3 – 6.6) 1.0 (0.2 – 4.5) 1.4 (0.3 -7.2) 3.6 (2.6 – 4.8) 1.6 (0.3 – 8.4) 2.2 (0.4 – 13.0) 1.5 (0.5 – 4.9) 2.7 (0.7 – 9.6) 1.2 (0.3 – 5.3) 2.5 (0.8 – 8.4) 4.2 (2.5 – 7.0) 4.0 (2.6 – 6.2) 4.6 (2.6 – 8.0) 2.8 (0.9 – 8.6) 0.4257 0.8684 0.4980 0.5258 0.2255 0.3723 0.3637 HIV-negative Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 8.0 (5.1 – 12.7) 12.1 (7.6 – 19.2) 0.2294 TRAI of anti-PT IgG (AU), mean (SD) 119.7 (24.2) 113.8 (31.1) 0.6853 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 24.5 (14.3 – 42.1) 22.9 (8.4 – 62.5) 0.8875 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.8 (2.4 – 6.0) 0.6 (0.2 – 2.5) 2.0 (0.7 – 5.9) 3.0 (1.8 – 5.2) 3.4 (2.1 – 5.4) 2.0 (0.5 – 7.5) 1.2 (0.3 – 5.1) 1.7 (0.2 – 15.6) 0.5 (0.05 – 4.8) 3.9 (1.5 – 10.3) 2.8 (1.5 – 5.2) 3.5 (1.8 – 1.0) 1.8 (0.2 – 17.4) 0.9 (0.08 – 9.6) 0.4342 0.7791 0.2750 0.8206 0.9068 0.9186 0.7837 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 71 TABLE 3.4. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at baseline + 4 weeks. TdaP Td P value HIV-positive Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 217.4 (116.7 – 405.1) 5.8 (3.4 – 10.0) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 137.6 (21.1) 125.8 (20.5) 0.1729 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 153.4 (95.8 – 245.8) 32.7 (21.8 – 49.0) <0.0001 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.5 (1.0 – 12.5) 1.6 (0.4 – 6.5) 12.5 (7.4 – 21.1) 20.1 (12.5 – 32.1) 21.8 (13.3 – 35.6) 27.2 (16.6 – 44.5) 10.7 (6.4 – 17.9) 0.8 (0.1 – 4.1) 1.7 (0.6 – 5.0) 2.7 (0.7 – 9.9) 4.0 (3.1 – 5.1) 4.8 (2.6 – 8.6) 5.3 (3.1 – 8.9) 1.4 (0.5 – 4.0) 0.1347 0.9526 0.0297 <0.0001 0.0003 <0.0001 0.0015 HIV-negative Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 122.2 (78.2 – 190.9) 11.9 (7.3 – 19.2) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 128.1 (22.1) 109.1 (13.5) 0.0365 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 243.7 (126.1 – 471.0) 20.9 (10.5 – 41.5) <0.0001 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 9.1 (2.5 – 33.4) 2.4 (0.5 – 10.6) 33.4 (19.0 – 58.7) 34.3 (17.2 – 68.2) 36.5 (18.4 – 72.4) 41.8 (22.1 – 78.9) 11.7 (4.5 – 30.6) 3.1 (1.8 – 5.4) 0.8 (0.1 – 4.7) 3.2 (1.4 – 7.4) 2.8 (1.3 – 6.1) 3.2 (1.3 – 7.7) 3.1 (1.6 – 6.4) 0.5 (0.06 – 4. 0.1542 0.3194 0.0012 0.0003 0.0010 <0.0001 0.0285 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 72 TABLE 3.5. Anti-PT IgG avidity comparisons between Tdap- versus Td-vaccinated pregnant women living with or without HIV at maternal delivery. TdaP Td P value HIV-positive Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 107.3 (55.0 – 209.1) 5.3 (3.2 – 9.0) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 135.4 (25.2) 128.7 (16.8) 0.4725 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 112.8 (61.3 – 207.7) 35.0 (23.0 – 53.1) 0.0012 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.7 (1.4 – 10.2) 1.0 (0.2 – 4.7) 7.8 (2.6 – 24.0) 13.0 (6.9 – 24.6) 16.4 (8.6 – 31.4) 19.5 (10.4 – 36.7) 7.2 (3.4 – 15.4) 3.2 (1.7 – 5.9) 0.6 (0.1 – 3.0) 2.2 (0.7 – 7.2) 4.6 (3.3 – 6.6) 4.7 (3.5 – 6.5) 6.1 (4.2 – 8.7) 1.1 (0.3 – 4.2) 0.7672 0.6308 0.1042 0.0054 0.0012 0.0021 0.0155 HIV-negative Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 75.6 (40.6 – 140.9) 10.9 (6.5 – 18.4) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 120.9 (20.2) 127.6 (48.8) 0.9114 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 137.0 (74.2 – 253.2) 16.7 (7.7 – 36.5) <0.0001 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 4.0 (1.0 – 15.3) 7.4 (3.1 – 17.4) 20.0 (11.2 – 35.5) 20.1 (11.3 – 35.7) 23.7 (12.3 – 45.8) 28.1 (14.7 – 53.5) 7.0 (2.8 – 17.4) 0.6 (0.05 – 7.0) 0.2 (0.02 – 2.5) 2.2 (1.3 – 3.9) 1.0 (0.2 – 6.5) 2.7 (1.2 – 5.9) 3.5 (1.6 – 7.5) 0.1 (0.02 – 1.9) 0.1312 0.0113 <0.0001 0.0071 0.0001 0.0002 0.0034 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 73 There were no significant differences in TRAI of anti-PT IgG across all groups at baseline (p>0.05) (Figure 3.4b, Table 3.3). TdaP-vaccinated PWLWH had significantly higher TRAI of anti-PT IgG 4-weeks post-vaccination than Td-vaccinated PWLWH (arithmetic mean, 128 AU [SD, 22] vs. 109 IU/mL [SD, 14], p=0.0365) (Table 3.4). No significant difference in TRAI of anti-PT IgG was observed between TdaP- and Td-vaccinated PWLWH (arithmetic mean, 138 AU [SD, 21] vs. 126 AU [SD, 21], p>0.05). There were also no significant differences in TRAI of anti-PT IgG at delivery between TdaP- and Td-vaccinated pregnant women, regardless of HIV status (HIV+; arithmetic mean, 135 AU [SD, 25] vs. 129 AU [SD, 17], p>0.05, HIV-; arithmetic mean, 121 AU [SD, 20] vs. 128 AU [SD, 49], p>0.05) (Table 3.5). No significant differences were observed in TAA levels of anti-PT IgG across all groups at baseline (p>0.05) (Figure 3.4c, Table 3.3). Tdap-vaccinated pregnant women had significantly higher TAA levels of anti-PT IgG 4-weeks post-vaccination than Td-vaccinated pregnant women, irrespective of HIV status (HIV+; GMC, 153 AAU/mL [95% CI, 96 – 246] vs. 33 AAU/mL [95% CI, 22 – 49], p<0.0001, HIV-; GMC, 244 AAU/mL [95% CI, 126 – 471] vs. 21 AAU/mL [95% CI, 11 – 41], p<0.0001) (Table 3.4). TAA levels of anti-PT IgG concentrations in TdaP-vaccinated pregnant women remained significantly higher than Td-vaccinated pregnant women at delivery, regardless of HIV status (HIV+; GMC, 113 AAU/mL [95% CI, 61 – 208] vs. 35 AAU/mL [95% CI, 23 - 53], p=0.0012, HIV-; GMC, 137 AAU/mL [95% CI, 74 – 253] vs. 17 AAU/mL [95% CI, 8 – 37], p<0.0001) (Table 3.5). Similar to TRAI and TAA, there were no significant differences in FAA levels of anti-PT IgG across all groups at baseline (p>0.05) (Figure 3.5a-g, Table 3.3). Tdap-vaccinated pregnant women had significantly higher FAA levels of anti-PT IgG with medium-low, medium, medium-high, high, and very high avidities 4-weeks post-vaccination than Td-vaccinated pregnant women, irrespective of HIV status (Table 3.4). At delivery, TdaP-vaccinated PWLWH had significantly higher FAA levels of anti-PT IgG with medium, medium-high, high, and very high avidities than Td-vaccinated PWLWH (Table 3.5). Tdap-vaccinated PWWH had significantly higher FAA levels of anti-PT IgG with low, low-medium, medium, medium-high, high, and very high avidities than Td-vaccinated PWWH. 74 Very low fractional absolute anti-PT IgG avidity (IU/mL)1000 1000Low fractional absolute anti-PT IgG avidity (IU/mL)Low-medium fractional absolute anti-PT IgG avidity (IU/mL)Mediumfractional absolute anti-PT IgG avidity (IU/mL)1100100.010.11100100.010.110001100100.010.110001100100.010.1Baseline Delivery4 weeks post Td or Tdap in pregnancyBaseline Delivery4 weeks post Td or Tdap in pregnancyBaseline Delivery4 weeks post Td or Tdap in pregnancyBaseline Delivery4 weeks post Td or Tdap in pregnancyVery low fractional absolute anti-PT IgG avidity (IU/mL)1000 1000Low fractional absolute anti-PT IgG avidity (IU/mL)Low-medium fractional absolute anti-PT IgG avidity (IU/mL)Mediumfractional absolute anti-PT IgG avidity (IU/mL)1100100.010.11100100.010.110001100100.010.110001100100.010.1Bas line D livery4 weeks post Td or Tdap in pregnancyBas line D livery4 weeks post Td or Tdap in pregnancyBas line D livery4 weeks post Td or Tdap in pregnancyBas line D livery4 weeks post Td or Tdap in pregnancyB D C A 75 FIGURE 3.5. Anti-PT fractional absolute avidity (FAA) of pregnant women at baseline, 4 weeks post Td or Tdap vaccination in pregnancy, and at delivery stratified by vaccine received in pregnancy and HIV-status. (3.5) All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05, *** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. A) very-low FAA. B) low FAA C) low-medium FAA D) medium FAA E) medium-high FAA F) high FAA G) very-high FAA Medium-high fractional absolute anti-PT IgG avidity (IU/mL)Highfractional absolute anti-PT IgG avidity (IU/mL)Baseline Delivery4 weeks post Td or Tdap in pregnancyBaseline Delivery4 weeks post Td or Tdap in pregnancyBaseline Delivery4 weeks post Td or Tdap in pregnancyVery high fractional absolute anti-PT IgG avidity (IU/mL)10001100100.010.110001100100.010.110001100100.010.1Medium-high fractional absolute anti-PT IgG avidity (IU/mL)Highfractional absolute anti-PT IgG avidity (IU/mL)Baseline Delivery4 weeks p st Td or Tdap in pregnancyBaseline Delivery4 weeks p st Td or Tdap in pregnancyBaseline Delivery4 weeks p st Td or Tdap in pregnancyVery high fractional absolute anti-PT IgG avidity (IU/mL)10001100100.010.110001100100.010.110001100100.010.1E F G Td vaccine, HIV negativeTdap vaccine, HIV negativeTd vaccine, HIV positiveTdap vaccine, HIV positive 76 Comparisons between pregnant women living with HIV versus without HIV that received the same vaccine There were no significant differences in total anti-PT IgG concentrations between pregnant women with different HIV statuses that received the same vaccine (p>0.05) (Figure 3.4, Table 3.6). In this subset of samples, Td-vaccinated PWLWH had significantly higher anti-PT IgG concentrations than Td-vaccinated PWWH, 4-weeks post-vaccination and at delivery (4-weeks post-vaccination; GMC, 12 IU/mL [95% CI, 7 – 19] vs. 6 IU/mL [95% CI, 3 – 10], p=0.0467, delivery; GMC, 11 IU/mL [95% CI, 7 – 18] vs. 5 IU/mL [95% CI, 3 – 9], p=0.0486) (Figure 3.4, Table 3.7, Table 3.8). Unlike Td-vaccinated pregnant women, there were no significant differences in total anti-PT IgG concentrations between TdaP-vaccinated pregnant women living with or without HIV, 4-weeks post-vaccination and at delivery (4-weeks post-vaccination; GMC, 217 IU/mL [95% CI, 117 – 405] vs. 122 IU/mL [95% CI, 78 – 191], p=0.1240, delivery; GMC, 107 IU/mL [95% CI, 55 – 209] vs. 76 IU/mL [95% CI, 41 – 141], p=0.4265)). Furthermore, there were no significant differences in TRAI of anti-PT IgG and TAA levels of anti-PT IgG between pregnant women with different HIV statuses that received the same vaccine (P>0.05) (Figure 3.4, Table 3.6, Table 3.7, Table 3.8). Most FAA levels of anti-PT IgG were similar between pregnant women with different HIV statuses that received the same vaccine across all three timepoints: baseline, 4-weeks post-vaccination, and delivery (p>0.05) (Figure 3.4, Table 3.6, Table 3.7, Table 3.8). The only significant differences were observed in Tdap-vaccinated pregnant women. TdaP-vaccinated PWLWH had significantly higher FAA level of anti-PT IgG with medium-low avidity 4-weeks post-vaccination and low avidity at delivery compared with TdaP-vaccinated PWLWH (medium-low; GMC, 33 IU/mL [95% CI, 19 – 59] vs. 13 IU/mL [95% CI, 7 – 21], p=0.0109, low; GMC, 7 IU/mL [95% CI, 3 – 17] vs. 1 IU/mL [0 – 5], p=0.0248). 77 TABLE 3.6. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at baseline. HIV-positive HIV-negative P value Tdap Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 11.0 (6.7 – 17.9) 8.0 (5.1 – 12.7) 0.3403 TRAI of anti-PT IgG (AU), mean (SD) 127.8 (14.6) 119.7 (24.2) 0.3507 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 23.1 (16.0 – 33.4) 24.5 (14.3 – 42.1) 0.8378 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 1.3 (0.3 – 6.6) 1.0 (0.2 – 4.5) 1.4 (0.3 -7.2) 3.6 (2.6 – 4.8) 1.6 (0.3 – 8.4) 2.2 (0.4 – 13.0) 1.5 (0.5 – 4.9) 3.9 (2.4 – 6.0) 0.6 (0.2 – 2.5) 2.0 (0.7 – 5.9) 3.0 (1.8 – 5.2) 3.4 (2.1 – 5.4) 2.0 (0.5 – 7.5) 1.2 (0.3 – 5.1) 0.1708 0.6069 0.6819 0.5585 0.3182 0.9122 0.8193 Td Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 7.4 (4.7 – 11.7) 12.1 (7.6 – 19.2) 0.1255 TRAI of anti-PT IgG (AU), mean (SD) 132.8 (21.5) 113.8 (31.1) 0.2088 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 35.0 (21.9 – 55.8) 22.9 (8.4 – 62.5) 0.3680 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 2.7 (0.7 – 9.6) 1.2 (0.3 – 5.3) 2.5 (0.8 – 8.4) 4.2 (2.5 – 7.0) 4.0 (2.6 – 6.2) 4.6 (2.6 – 8.0) 2.8 (0.9 – 8.6) 1.8 (0.2 – 15.6) 0.5 (0.05 – 4.8) 3.9 (1.5 – 10.3) 2.8 (1.5 – 5.2) 3.5 (1.8 – 1.0) 1.8 (0.2 – 17.4) 0.9 (0.08 – 9.6) 0.7110 0.4304 0.4972 0.2416 0.6868 0.3465 0.3024 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 78 TABLE 3.7. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at baseline + 4 weeks. HIV-positive HIV-negative P value Tdap Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 217.4 (116.7 – 405.1) 122.2 (78.2 – 190.9) 0.1240 TRAI of anti-PT IgG (AU), mean (SD) 137.6 (21.1) 128.1 (22.1) 0.1626 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 153.4 (95.8 – 245.8) 243.7 (126.1 – 471.0) 0.2396 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.5 (1.0 – 12.5) 1.6 (0.4 – 6.5) 12.5 (7.4 – 21.1) 20.1 (12.5 – 32.1) 21.8 (13.3 – 35.6) 27.2 (16.6 – 44.5) 10.7 (6.4 – 17.9) 9.1 (2.5 – 33.4) 2.4 (0.5 – 10.6) 33.4 (19.0 – 58.7) 34.3 (17.2 – 68.2) 36.5 (18.4 – 72.4) 41.8 (22.1 – 78.9) 11.7 (4.5 – 30.6) 0.2783 0.6864 0.0109 0.1875 0.2079 0.2710 0.8611 Td Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 5.8 (3.4 – 10.0) 11.9 (7.3 – 19.2) 0.0467 TRAI of anti-PT IgG (AU), mean (SD) 125.8 (20.5) 109.1 (13.5) 0.0906 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 32.7 (21.8 – 49.0) 20.9 (10.5 – 41.5) 0.1745 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 0.8 (0.1 – 4.1) 1.7 (0.6 – 5.0) 2.7 (0.7 – 9.9) 4.0 (3.1 – 5.1) 4.8 (2.6 – 8.6) 5.3 (3.1 – 8.9) 1.4 (0.5 – 4.0) 3.1 (1.8 – 5.4) 0.8 (0.1 – 4.7) 3.2 (1.4 – 7.4) 2.8 (1.3 – 6.1) 3.2 (1.3 – 7.7) 3.1 (1.6 – 6.4) 0.5 (0.06 – 4. 0.0859 0.4353 0.7991 0.3989 0.5476 0.3165 0.5093 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 79 TABLE 3.8. Anti-PT IgG avidity comparisons between Tdap- or Td-vaccinated pregnant women living with HIV versus without HIV at maternal delivery. HIV-positive HIV-negative P value Tdap Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 107.3 (55.0 – 209.1) 75.6 (40.6 – 140.9) 0.4265 TRAI of anti-PT IgG (AU), mean (SD) 135.4 (25.2) 120.9 (20.2) 0.0680 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 112.8 (61.3 – 207.7) 137.0 (74.2 – 253.2) 0.4458 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.7 (1.4 – 10.2) 1.0 (0.2 – 4.7) 7.8 (2.6 – 24.0) 13.0 (6.9 – 24.6) 16.4 (8.6 – 31.4) 19.5 (10.4 – 36.7) 7.2 (3.4 – 15.4) 4.0 (1.0 – 15.3) 7.4 (3.1 – 17.4) 20.0 (11.2 – 35.5) 20.1 (11.3 – 35.7) 23.7 (12.3 – 45.8) 28.1 (14.7 – 53.5) 7.0 (2.8 – 17.4) 0.9434 0.0248 0.1299 0.2983 0.4057 0.4020 0.9523 Td Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 5.3 (3.2 – 9.0) 10.9 (6.5 – 18.4) 0.0486 TRAI of anti-PT IgG (AU), mean (SD) 128.7 (16.8) 127.6 (48.8) 0.7226 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 35.0 (23.0 – 53.1) 16.7 (7.7 – 36.5) 0.0915 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 3.2 (1.7 – 5.9) 0.6 (0.1 – 3.0) 2.2 (0.7 – 7.2) 4.6 (3.3 – 6.6) 4.7 (3.5 – 6.5) 6.1 (4.2 – 8.7) 1.1 (0.3 – 4.2) 0.6 (0.05 – 7.0) 0.2 (0.02 – 2.5) 2.2 (1.3 – 3.9) 1.0 (0.2 – 6.5) 2.7 (1.2 – 5.9) 3.5 (1.6 – 7.5) 0.1 (0.02 – 1.9) 0.1449 0.3953 0.9855 0.0901 0.1338 0.1436 0.0715 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 80 3.3.8 Anti-pertussis toxin IgG avidity in DTwP-vaccinated HIV-exposed uninfected and HIV-unexposed uninfected infants from TdaP or Td-vaccinated mothers Comparisons between infants from Tdap- versus Td-vaccinated mothers with the same HIV exposure status In this subset of samples, similar trends from maternal delivery were observed in their infants at delivery (Figure 3.6, Table 3.9, Table 3.10). At delivery, infants from TdaP-vaccinated mothers had significantly higher total anti-PT IgG concentrations than infants from Td-vaccinated mothers, irrespective of HIV exposure (HEU infants; GMC 169 IU/mL [95% CI, 82 – 345] vs. 6 IU/mL [95% CI, 3 - 10], p<0.0001, HUU infants; GMC, 97 IU/mL [95% CI, 53 – 177] vs. 13 IU/mL [95% CI, 8 – 23], p<0.0001). In contrast, infants from Td-vaccinated mothers had significantly higher total anti-PT IgG concentrations than infants from TdaP-vaccinated mothers 4-weeks post-primary series with DTwP, regardless of HIV exposure (HEU infants; GMC, 181 IU/mL [95% CI, 76 – 430] vs. 24 IU/mL [95% CI, 14 – 40], p=0.0071, HUU infants; GMC, 71 IU/mL [95% CI, 27 – 186] vs. 15 IU/mL [95% CI, 9 – 27], p=0.0002). 81 A 82 B 83 FIGURE 3.6. GMC of total anti-PT IgG with 95% confidence intervals, anti-PT TRAI, and anti-PT TAA in infants stratified by the vaccine their mother received in pregnancy and HIV-exposure in utero of a subset of samples. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05, *** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. A) Total anti-PT IgG concentrations (IU/mL). B) Total relative avidity index of anti-PT IgG (AU). C) Total absolute avidity levels of anti-PT IgG (AAU/mL). C 84 TABLE 3.9. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants and HIV-unexposed uninfected infants from Tdap- versus Td-vaccinated pregnant women at delivery. TdaP Td P value HIV-positive Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 168.6 (82.4 – 345.0) 6.0 (3.4 – 10.4) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 150.6 (20.5) 143.5 (27.5) 0.4007 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 134.2 (73.1 – 246.5) 36.2 (21.4 – 61.4) 0.0016 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 4.0 (1.2 – 13.1) 1.1 (0.2 – 4.5) 10.3 (5.6 – 18.9) 13.3 (7.5 – 23.6) 15.8 (8.5 – 29.5) 23.9 (12.8 – 44.3) 11.1 (4.8 – 25.3) 1.0 (0.2 – 4.5) 1.0 (0.3 – 3.1) 1.6 (0.4 – 5.7) 2.8 (1.0 – 8.1) 5.1 (3.2 – 8.1) 5.1 (2.5 – 10.3) 2.9 (1.2 – 7.0) 0.1346 0.9416 0.0108 0.0119 0.0042 0.0015 0.0242 HIV-negative Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 96.8 (52.9 – 177.3) 13.3 (7.9 – 22.5) <0.0001 TRAI of anti-PT IgG (AU), mean (SD) 123.3 (24.3) 126.2 (26.2) 0.7844 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 241.9 (126.3 – 463.1) 24.8 (11.2 – 55.2) <0.0001 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 10.5 (2.5 – 43.4) 9.0 (3.7 – 22.0) 15.8 (4.7 – 53.8) 29.6 (13.5 – 65.0) 32.8 (17.5 – 61.6) 40.8 (20.7 – 80.3) 15.5 (7.1 – 33.6) 2.9 (1.6 – 5.3) 1.1 (0.2 – 5.4) 1.8 (0.3 – 10.9) 2.1 (1.2 – 3.6) 3.5 (1.7 – 7.1) 4.1 (1.9 – 8.9) 1.1 (0.1 – 8.8) 0.0871 0.0199 0.0360 <0.0001 <0.0001 <0.0001 0.0202 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 85 TABLE 3.10. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants and HIV-unexposed uninfected infants from Tdap- versus Td-vaccinated pregnant women 4-weeks post-primary series with DTwP vaccine (baby 18 weeks). TdaP Td P value HIV-positive Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 24.0 (14.4 – 39.9) 180.5 (75.8 – 429.9) 0.0071 TRAI of anti-PT IgG (AU), mean (SD) 104.4 (29.9) 94.0 (14.7) 0.5505 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 31.3 (19.4 – 50.6) 146.4 (66.8 – 321.1) 0.0015 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 4.2 (1.1 – 16.3) 0.6 (0.1 – 3.2) 6.1 (3.1 – 12.1) 4.3 (2.6 – 7.1) 5.1 (3.1 – 8.4) 3.9 (1.6 – 9.6) 0.8 (0.2 – 2.6) 13.8 (3.5 – 54.2) 15.0 (5.6 – 40.0) 36.4 (16.2 – 81.6) 25.5 (11.6 – 55.7) 19.9 (8.8 – 45.2) 20.3 (9.4 – 43.9) 4.3 (1.2 – 15.4) 0.1871 0.0019 0.0012 0.0004 0.0055 0.0060 0.0474 HIV-negative Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 15.2 (8.7 – 26.6) 70.8 (27.0 – 186.0) 0.0002 TRAI of anti-PT IgG (AU), mean (SD) 98.9 (29.2) 87.1 (11.1) 0.2881 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 30.6 (18.4 – 51.1) 238.2 (144.2 – 393.4) <0.0001 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 5.8 (3.2 – 10.7) 2.5 (0.8 – 7.6) 4.4 (2.5 – 7.8) 5.1 (2.9 – 8.9) 4.7 (2.8 – 8.0) 4.8 (3.0 – 7.7) 0.6 (0.2 – 1.7) 44.8 (25.8 – 77.7) 34.3 (21.5 – 54.6) 66.9 (41.0 – 108.9) 45.3 (28.0 – 73.3) 35.4 (21.2 – 59.0) 28.9 (17.5 – 47.7) 8.8 (4.3 – 18.1) <0.0001 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 0.0002 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 86 There were no differences in TRAI of anti-PT IgG between infants from TdaP- and Td-vaccinated mothers, irrespective of HIV-status (p>0.05) (Figure 3.6, Table 3.9, Table 3.10). However, TAA levels of anti-PT IgG followed the same trends as total anti-PT IgG concentrations. At delivery, infants from TdaP-vaccinated mothers had significantly higher TAA levels of anti-PT IgG than infants from Td-vaccinated mothers, irrespective of HIV exposure (HEU infants; GMC, 134 IU/mL [95% CI, 73 - 247] vs. 36 IU/mL [95% CI, 21 – 61], p=0.0016, HUU infants; GMC, 242 IU/mL [95% CI, 126 - 463] vs. 25 IU/mL [95% CI, 11 – 55], p<0.0001). In contrast, infants from Td-vaccinated mothers had significantly higher TAA levels of anti-PT IgG than infants from TdaP-vaccinated mothers 4-weeks post-primary series with DTwP, regardless of HIV exposure (HEU infants; GMC, 146 IU/mL [95% CI, 67 – 321] vs. 31 IU/mL [95% CI, 19 – 51], p=0.0015, HUU infants; GMC, 238 IU/mL [95% CI, 144 – 393] vs. 31 IU/mL [95% CI, 18 – 51], p<0.0001). Similar trends to TAA levels of anti-PT IgG were observed when FAA levels of anti-PT IgG were analyzed (Figure 3.7, Table 3.9, Table 3.10). HEU infants from TdaP-vaccianted mothers had significantly higher FAA levels of anti-PT IgG with medium-low, medium, medium-high, high, and very high avidities at delivery. Infants unexposed to HIV from Tdap-vaccinated mothers followed the same trends as HEU infants from Tdap-vaccinated mothers at delivery, with the addition of having significantly higher antibodies with low avidity compare with HUU infants from Td-vaccinated mothers. In contrast, 4-weeks after the completion of a primary series with DTwP, HEU infants from Tdap-vaccinated mothers had significantly higher FAA levels of anti-PT IgG with low, medium-low, medium, medium-high, high, and very high avidities. Same trends were observed for HUU infants, except with the addition of having significantly higher antibodies with very low avidity as well, 87 10001100100.010.110001100100.010.110001100100.010.110001100100.010.1Very low fractional absolute anti-PT IgG avidity (IU/mL)Low fractional absolute anti-PT IgG avidity (IU/mL)Low-medium fractional absolute anti-PT IgG avidity (IU/mL)Mediumfractional absolute anti-PT IgG avidity (IU/mL)Cord blood 4 weeks post infant vaccine series (wP)Cord blood 4 weeks post infant vaccine series (wP)Cord blood 4 weeks post infant vaccine series (wP)Cord blood 4 weeks post infant vaccine series (wP)A D C B 88 FIGURE 3.7. Anti-PT fractional absolute avidity (FAA) of infants at delivery and 4 weeks post-completion of primary series with DTwP, stratified by the vaccine their mother received in pregnancy and HIV-exposure in utero. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05, *** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. A) very-low FAA. B) low FAA C) low-medium FAA D) medium FAA E) medium-high FAA F) high FAA G) very-high FAA 10001100100.010.110001100100.010.110001100100.010.1Medium-high fractional absolute anti-PT IgG avidity (IU/mL)Very high fractional absolute anti-PT IgG avidity (IU/mL)High fractional absolute anti-PT IgG avidity (IU/mL)Cord blood 4 weeks post infant vaccine series (wP)Cord blood 4 weeks post infant vaccine series (wP)Cord blood 4 weeks post infant vaccine series (wP)E G F Td vaccine, HIV negativeTdap vaccine, HIV negativeTd vaccine, HIV positiveTdap vaccine, HIV positive 89 Comparisons between HEU versus HUU infants with the same maternal vaccination status HEU infants from Td-vaccinated mothers had significantly higher total anti-PT IgG concentrations at delivery than HUU infants from Td-vaccinated mothers (GMC, 13 IU/mL [95% CI, 8 – 23] vs. 6 IU/mL [95% CI, 3 – 10], p=0.0347) (Figure 3.6, Table 3.11, Table 3.12). There were no other significant differences in total anti-PT IgG concentrations observed between HEU and HUU infants from mothers that received the same vaccine in pregnancy (p>0.05). TRAI of anti-PT IgG was significantly higher at delivery in HEU infants from TdaP-vaccinated mothers than HUU infants from TdaP-vaccinated mothers (arithmetic mean, 151 AU [SD, 21] vs. 123 AU [SD, 24], p =0.0015). There were no other significant differences in TRAI of anti-PT IgG between HEU and HUU infants from mothers that received the same vaccine in pregnancy (p>0.05). TAA and FAA levels of anti-PT IgG were similar between HEU and HUU infants from mothers that received the same vaccine in pregnancy, at delivery and 4-weeks post-primary vaccination series (p>0.05), except at delivery in infants from Tdap-vaccinated mothers where HUU infants had significantly higher antibodies with low avidity than HEU infants (GMC, 9 IU/mL [95% CI, 4 – 22] vs. 1 IU/mL [95% CI, 0 – 5], p=0.131). 90 TABLE 3.11. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants versus HIV-unexposed uninfected infants from Tdap- or Td-vaccinated pregnant women at delivery. HIV-positive HIV-negative P value Tdap Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 168.6 (82.4 – 345.0) 96.8 (52.9 – 177.3) 0.2229 TRAI of anti-PT IgG (AU), mean (SD) 150.6 (20.5) 123.3 (24.3) 0.0015 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 134.2 (73.1 – 246.5) 241.9 (126.3 – 463.1) 0.1728 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 4.0 (1.2 – 13.1) 1.1 (0.2 – 4.5) 10.3 (5.6 – 18.9) 13.3 (7.5 – 23.6) 15.8 (8.5 – 29.5) 23.9 (12.8 – 44.3) 11.1 (4.8 – 25.3) 10.5 (2.5 – 43.4) 9.0 (3.7 – 22.0) 15.8 (4.7 – 53.8) 29.6 (13.5 – 65.0) 32.8 (17.5 – 61.6) 40.8 (20.7 – 80.3) 15.5 (7.1 – 33.6) 0.2794 0.0131 0.5109 0.0923 0.0919 0.2281 0.5362 Td Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 6.0 (3.4 – 10.4) 13.3 (7.9 – 22.5) 0.0347 TRAI of anti-PT IgG (AU), mean (SD) 143.5 (27.5) 126.2 (26.2) 0.1955 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 36.2 (21.4 – 61.4) 24.8 (11.2 – 55.2) 0.3705 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 1.0 (0.2 – 4.5) 1.0 (0.3 – 3.1) 1.6 (0.4 – 5.7) 2.8 (1.0 – 8.1) 5.1 (3.2 – 8.1) 5.1 (2.5 – 10.3) 2.9 (1.2 – 7.0) 2.9 (1.6 – 5.3) 1.1 (0.2 – 5.4) 1.8 (0.3 – 10.9) 2.1 (1.2 – 3.6) 3.5 (1.7 – 7.1) 4.1 (1.9 – 8.9) 1.1 (0.1 – 8.8) 0.1685 0.9103 0.8890 0.5646 0.3282 0.6525 0.3314 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 91 TABLE 3.12. Anti-PT IgG avidity comparisons between HIV-exposed uninfected infants versus HIV-unexposed uninfected infants from Tdap- or Td-vaccinated pregnant women 4-weeks post-primary series with DTwP vaccine (baby 18 weeks). HIV-positive HIV-negative P value Tdap Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 24.0 (14.4 – 39.9) 15.2 (8.7 – 26.6) 0.2136 TRAI of anti-PT IgG (AU), mean (SD) 104.4 (29.9) 98.9 (29.2) 0.7087 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 31.3 (19.4 – 50.6) 30.6 (18.4 – 51.1) 0.9463 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 4.2 (1.1 – 16.3) 0.6 (0.1 – 3.2) 6.1 (3.1 – 12.1) 4.3 (2.6 – 7.1) 5.1 (3.1 – 8.4) 3.9 (1.6 – 9.6) 0.8 (0.2 – 2.6) 5.8 (3.2 – 10.7) 2.5 (0.8 – 7.6) 4.4 (2.5 – 7.8) 5.1 (2.9 – 8.9) 4.7 (2.8 – 8.0) 4.8 (3.0 – 7.7) 0.6 (0.2 – 1.7) 0.6324 0.1397 0.4207 0.6306 0.8248 0.6543 0.6640 Td Total anti-PT IgG concentrations (IU/mL), GMC (95% CI) 180.5 (75.8 – 429.9) 70.8 (27.0 – 186.0) 0.1398 TRAI of anti-PT IgG (AU), mean (SD) 94.0 (14.7) 87.1 (11.1) 0.2043 TAA levels of anti-PT IgG (AAU/mL), GMC (95% CI) 146.4 (66.8 – 321.1) 238.2 (144.2 – 393.4) 0.2734 FAA levels of anti-PT IgG (IU/mL), GMC (95% CI): Very low Low Medium-low Medium Medium-high High Very high 13.8 (3.5 – 54.2) 15.0 (5.6 – 40.0) 36.4 (16.2 – 81.6) 25.5 (11.6 – 55.7) 19.9 (8.8 – 45.2) 20.3 (9.4 – 43.9) 4.3 (1.2 – 15.4) 44.8 (25.8 – 77.7) 34.3 (21.5 – 54.6) 66.9 (41.0 – 108.9) 45.3 (28.0 – 73.3) 35.4 (21.2 – 59.0) 28.9 (17.5 – 47.7) 8.8 (4.3 – 18.1) 0.1034 0.1191 0.1794 0.1906 0.2143 0.4186 0.3031 Abbreviations: Td, tetanus-diphtheria; TdaP, tetanus-diphtheria-acellular pertussis; HIV, human immunodeficiency virus; PT, pertussis toxin; IgG, immunoglobulin G; IU, international units; GMC, geometric mean concentration; CI, confidence interval; TRAI, total relative avidity index; AU, avidity units; SD, standard deviation; TAA, total absolute avidity; AAU, absolute avidity units; FAA, fractional absolute avidity. 92 3.4 Discussion This study filled a significant gap in knowledge and provided data supporting maternal vaccination strategies in LMICs by evaluating the immunogenicity of maternal immunisation with TdaP in HIV-positive and HIV-negative pregnant women and their infants. TdaP vaccination induced higher anti-PT and anti-FHA responses compared to Td vaccination for both HIV-positive and HIV-negative pregnant women four weeks following second vaccination, at delivery, and for their infants at delivery. Marginally higher antibody concentrations in infants at delivery compared to their mothers indicated active transplacental antibody transfer in HIV-positive and HIV-negative TdaP recipients for anti-PT. However, concentrations were similar between mother/infant pairs in the Td groups and for anti-FHA concentrations. Anti-PT IgG concentrations reduced for infants of TdaP recipients and increased for infants of Td recipients by 18 weeks post-delivery. Anti-FHA IgG reduced to similar concentrations across the four groups by 18 weeks post-delivery comparable to mother baseline concentrations. Anti-PT and anti-FHA IgG concentrations in HIV-positive and HIV-negative pregnant women at baseline were comparable to existing studies in maternal immunization with TdaP in non-immunocompromised populations in the United States, Canada, and Vietnam [158, 159, 165, 220]. However, concentrations following TdaP vaccination were higher in our study for both HIV-negative and HIV-positive recipients in comparison [158, 159, 162, 163, 165, 220]. Our study utilised a TdaP vaccine containing genetically detoxified PT which has been shown to be more immunogenic in pregnant women compared to Tdap with chemically modified PT and may explain these differences [221]. PWWH had higher anti-PT and anti-FHA IgG concentrations than PWLWH post-vaccination and at delivery. This trend was consistent with findings from studies that compared immunogenicity between PWLWH and PWWH that received influenza, pneumococcal, or investigational group B streptococcal vaccines [213, 215, 222]. The clinical significance of the reduced concentrations of anti-PT and anti-FHA IgG in PWLWH compared to PWWH remains unknown as there is no widely accepted correlate of protection against pertussis disease. These differences were also apparent in infants at delivery for both anti-PT and anti-FHA IgG. Newborns from TdaP-vaccinated mothers had higher anti-PT and anti-FHA IgG concentrations, 93 regardless of HIV-exposure, with HUU having the highest. This reduced concentration of maternal antibodies in the cord blood of HEU infants is possibly due to lower pre-vaccination baseline antibodies in the PWLWH. Although maternal antibodies are lower in HEU infants compared with HUU infants, clinical significance of these reduced concentrations are unknown. Maternal antibodies in HEU infants may increase if their mothers had received a second dose of TdaP in pregnancy. After infants completed a primary series containing pertussis, these trends were no longer apparent. Anti-PT IgG concentrations were higher in infants whose mothers received Td in pregnancy than infants whose mothers received TdaP in pregnancy, irrespective of fetal HIV-exposure. This was consistent with studies in LMICs investigating Tdap immunization in pregnancy and used DTwP vaccine as an infant primary series [189, 190]. These two studies in addition to ours provides evidence of immunomodulation following DTwP vaccine in infants from Tdap-vaccinated mothers. Immunomodulation has been well documented in infants from Tdap-vaccinated mothers that received DTaP vaccines as a primary series, suggesting that immunomodulation is not a result of vaccine type. Although immunomodulation was observed, clinical significance is not fully understood. In a study from Australia, maternally vaccinated infants had lower vaccine effectiveness following their third infant pertussis vaccination compared to infants from unvaccinated mothers, however this did not translate to a greater risk of pertussis disease. Unlike anti-PT IgG concentrations post-infant primary series, anti-FHA IgG concentrations were low and comparable across all four groups. This was consistent with one study from Brazil but was different from a study in Thailand which saw higher concentrations in infants from unvaccinated mothers. These differences could be due to different whole cell pertussis vaccines used in the studies. Furthermore, low humoral immune responses to FHA content in whole cell pertussis vaccines is consistent in literature with no maternal immunization as well. This suggests immunogenic responses to DTwP vaccines are low regardless of maternal immunization. 94 Furthermore, we evaluated anti-PT avidity in both the mothers and newborns to assess antibody functionality. Generally, there were no differences in anti-PT TRAI except that TdaP-vaccinated PWWH have higher anti-PT TRAI than Td-vaccinated PWWH. This was unexpected as an additional exposure to Tdap would have been predicted to lead to further affinity maturation and therefore larger proportions of antibodies with higher avidities. However, a possible explanation for no differences in anti-PT TRAI between Tdap- and Td-vaccinated pregnant women could be due to recent repeated exposures through the participants life. Although anti-PT TRAI was similar, when assessing the anti-PT TAA index, similar trends to the anti-PT IgG concentrations are observed. This is expected as TAA is a measure of avidity that considers antibody concentration. Since we have established that Tdap-vaccinated pregnant women had higher anti-PT concentrations, it makes sense that they would have higher overall avidity as they have more antibodies. Lastly, we can further break down anti-PT TAA into anti-PT FAA to analyze the concentrations of antibodies with different avidity strengths. Similarly to anti-PT TAA, Tdap-vaccinated pregnant women had higher concentrations of antibodies with each of the different avidities. This again was expected as these pregnant women received the Tdap vaccine which was able to induce the production of more anti-PT IgG. Overall, this data suggests that HIV-status of the pregnant women does not affect maturation of these antibodies. However, caution should be used as only pregnant women with well-controlled HIV infection were enrolled. Similar trends were observed in infant cord blood as seen in the pregnant women at delivery. There were no differences in anti-PT TRAI at cord blood between infants from Tdap- and Td-vaccinated with the same HIV-exposure. As mentioned for their mothers, this was not expected as I expected an additional exposure to pertussis antigens would further shift the avidity profile to have higher strength avidities. Anti-PT TAA and anti-PT FAA being higher at infant cord blood in infants from Tdap-vaccinated mothers compared to Td-vaccinated mothers was as expected. These mothers had higher concentrations of anti-PT IgG; therefore, more antibodies were able to transfer into the fetus. There were generally no significant differences between HIV-exposures suggesting no impact from any potential HIV-related morphological changes to the placenta. 95 When evaluating anti-PT IgG avidity in infants four weeks after the completion of their primary series, the results show immunomodulation. When evaluating anti-PT TRAI, no differences were observed. This was expected as the maternal antibodies would have mostly subsided and what was measured was mostly the infants’ own antibodies. Therefore, their own pertussis vaccines would have acted as their own priming and re-stimulation events. Interestingly, when comparing anti-PT TAA and anti-PT FAA between infants from Tdap-vaccinated and Td-vaccinated mothers, I observed evidence of immunomodulation of anti-PT IgG avidity. This was evident by infants from Tdap-vaccinated mothers having lower anti-PT TAA and different anti-PT FAA levels compared with infants from Td-vaccinated mothers. This was not expected because even though the maternal antibodies are preventing a full humoral immune response by the infant’s immune system to their own vaccines, we did not know whether this would also impact the activation of B cells and subsequent induction of further affinity maturation. However, as mentioned previously, TAA and FAA both consider antibody concentration so since the antibody concentrations are being immunomodulated, we would see this in these measurements. These results show that immunomodulation does not only impact the concentration of the antibodies, but also their function. Furthermore, these results also show that HIV-exposure does not significantly impact the level immunomodulation observed. This study is the first to evaluate immunization in pregnancy with TdaP in pregnant women living with and without HIV, and their newborns. A strength of this study is that it was conducted in a low-resource setting where the results will help inform local governments on the ability of this vaccine to induce the production of antibodies in pregnancy which can transfer to the fetus and provide protection. This study does have a couple of limitations, firstly being that only women with low-level HIV infections were enrolled, making generalizing to all HIV infections not possible. Furthermore, this study was conducted during the COVID-19 pandemic. This made it more difficult to enroll participants and led to lower enrollment than what the study was powered for. Another limitation is that no statistical tests were performed on the immunogenicity data. This prevents any conclusions from being made based on statistically significant differences from our findings. Statistics will be performed and included in the final published article. Furthermore, no adjustments were performed to account for CD4 counts, viral loads, and other co-variables such as interval between vaccination in pregnancy and birth, and 96 intervals between doses of infant vaccinations. Additionally, the sample sizes for the avidity analyses were not calculated based on powering. This makes formulating conclusions of p-values slightly above our pre-set level of significance (0.5) difficult as it could be trending to be significantly different, but the comparisons were not properly powered to detect one. Lastly, no tests were performed to exclude anyone with anti-PT concentrations that were suggestive of colonization or a recent infection with B. pertussis. In conclusion, the TdaP vaccine used in this study was able to induce a humoral immune response in the pregnant women, irrespective of their HIV-status. These antibodies were able to transfer into the fetus and were measurable in the infant’s cord blood. Immunomodulation of both anti-PT IgG concentrations and anti-PT avidity were observed, however the clinical significance of this is not known. This TdaP vaccine should be added into Uganda’s vaccine schedule to be received in pregnancy to protect young infants prior to when they can start and/or complete their primary series of their own pertussis vaccine. 97 4. Evaluation of the immunogenicity of a booster dose of DTaP in children that received different vaccine schedules and vaccine formulations 4.1 Introduction Pertussis cases have been increasing over the last decade in many high-income countries [224]. This is thought to be in part due to waning immunity of vaccines, the decreased protection associated with a switch from whole cell pertussis (wP) vaccines to acellular pertussis (aP) vaccines, and improvements in the detection of pertussis infections [11, 225-227]. From 2005 to 2019, there were 33,481 cases of pertussis disease, 1,593 hospitalizations, and 17 deaths reported in Canada [130]. Of the 1,593 hospitalization and 17 deaths, 80% of hospitalizations and all deaths were in infants less than one year of age, and infants less than four months had a hospital rate 8.2 times higher than infants four months old to one year of age [130]. Due to the increasing incidence of pertussis disease and the disproportionate impact on infants less than four months of age, the National Advisory Committee on Immunization (NACI) in Canada updated their recommendations for Tdap in pregnancy in 2018, stating that a Tdap booster should be administered to all pregnant individuals at every pregnancy between 27 and 32 weeks of gestation [135]. This is to allow for anti-pertussis maternal antibodies to transfer across the placenta and provide protection to the young infant prior to their ability to receive and/or complete their own primary series [183]. Immunization in pregnancy with a Tdap vaccine has been previously associated with immunological modulation in infants [182]. This has been proposed to be due to the maternal antibodies binding to the antigen and Fcγ receptors on the infant’s B cells, diminishing the ability of the infant’s immune system to generate a response to the DTaP vaccine they receive as infants [183-185]. Another hypothesis is that the maternal antibodies can bind to epitopes of the infant pertussis vaccine and mask it, making it difficult for the infant’s immune system to recognize it and generate a response [185, 186]. Immunological modulation has been shown to occur in infants born to mothers that received Tdap in pregnancy, once the infants begin their own primary series with DTaP [158, 219]. However, few studies have followed through with their cohorts to when they receive their first booster at approximately 18 months of age to see if this immunomodulation has dissipated. Furthermore, few studies have investigated whether a 98 different vaccine schedule or a different formulation of the DTaP vaccine may decrease the level of immunological modulation. In Quebec, Canada, immunization with Tdap in pregnancy began being recommended in May 2018 [228]. A few months later, the Quebec Immunization Committee also recommended to change their vaccine schedule for DTaP from a 3+1 schedule with primary series received at two, four, and 6 months of age followed by a booster at 18 months of age, to a 2+1 schedule with primary series at two and four months of age followed by a booster at 12 months of age [229]. This switch was implemented in June of 2019 and continues to be the only province that has shifted from the 3+1 schedule [230]. This change in vaccine schedule and formulation provided a unique opportunity to be able to investigate whether immunological modulation decreases with the new schedule and/or vaccine formulations, and if receiving a booster dissipates any evidence of immunological modulation. There are also few studies that investigated antibody functionality in these cohorts to see if antibody function is also undergoing immunological modulation. In this study, we aimed to evaluate whether vaccine schedule and/or vaccine formulations used in the infant’s primary series has an impact on the level of immunomodulation in antibody concentration and antibody functionality in the infant, and whether a booster dose of DTaP can dissipate this immunomodulation. To do this, samples were collected from a prospective clinical trial on hepatitis B vaccination in children that was completed in Quebec during the time of implementing the change in immunization in pregnancy and the infant vaccine schedule. Samples were analyzed on anti-pertussis toxin (PT), anti-filamentous hemagglutinin (FHA), and anti-pertactin (PRN) IgG specific enzyme-linked immunosorbent assays to analyze pertussis specific antibodies. A subset of samples were then analyzed on an anti-PT IgG avidity assay to assess antibody functionality. I hypothesized that children from Tdap-vaccinated mothers would experience immunomodulation and have lower total IgG antibodies against PT, FHA, and PRN than children from Tdap-unvaccinated mothers. This was based on literature where after a booster dose of DTaP, children from Tdap-vaccinated mothers continued to have lower 99 concentrations of one or more anti-pertussis vaccine antigens (anti-PT, anti-FHA, and anti-PRN IgG) than children from Tdap-unvaccinated mothers [158, 232-234]. 4.2 Methods 4.2.1 Study design This was an observational study that used data collected from a prospective clinical trial on hepatitis B vaccine schedules in children in Quebec City, Canada. In the original trial, the aim was to compare immunogenicity of pediatric hepatitis B vaccination schedules with a two or three vaccine dose schedule. Children in the two-dose hepatitis B group received the reduced 2+1 DTaP vaccine schedule (2, 4, and 12 months of age), whereas children assigned to the three-dose hepatitis B group received the original 3+1 DTaP vaccine schedule (2, 4, 6, and 18 months of age). The 2+1 DTaP vaccine group received DTaP3-IPV-Hib-HB as their first dose, followed by DTaP5-IPV-Hib as their second dose to complete their primary series. Children were then randomized to receive DTaP3-IPV-Hib-HB (group 1) or DTaP5-IPV-Hib (group 2) for their booster dose. Children in the 3+1 DTaP vaccine schedule received DTaP3-IPV-Hib-HB, DTaP3-IPV-Hib-HB, and DTaP5-IPV-Hib as their first, second, and third doses of their primary series, respectively. All children in the 3+1 DTaP group received DTaP3-IPV-Hib-HB as their booster dose (group 3). Each group was further stratified based on whether their mother was immunized in pregnancy with Tdap. Mothers were contacted to confirm reception of Tdap in pregnancy. The mother was considered Tdap-vaccinated if they had received the Tdap vaccine at any point during pregnancy, except from 0 to 13 days before delivery. 4.2.2 Samples Samples for immunogenicity analysis From the original clinical trial, 360 participants were eligible for immunogenicity analysis. Of the 360 participants, 139 were in group 1 (2+1 vaccine schedule; DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB), 133 were in group 2 (2+1 vaccine schedule; DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP5-IPV-Hib), and 88 were in group 3 (3+1 vaccine schedule; DTaP3-IPV-Hib-HB - DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib - DTaP3-IPV-Hib-HB). Children from Tdap-vaccinated mothers made up 91, 85, and 18 participants from groups 1, 2, and 3, respectively. Samples for children that followed a 2+1 vaccine schedule (Groups 1 and 2), had 100 their serum sample collected at 13 months of age, one month after receiving their booster dose of DTaP vaccine. Children that followed the 3+1 vaccine schedule (group 3) had their serum sample collected at 19 months of age, one month after receiving their booster dose of DTaP vaccine. Samples for avidity analysis After analyzing immunogenicity, a follow-up study was designed that utilized a subset of participants that were randomly selected to analyse anti-PT IgG avidity. The required sample size to make comparisons between groups was calculated using a power calculation. Using previously published data on the difference in total relative avidity index between infants from Tdap-vaccinated and unvaccinated mothers, it was determined that a sample size of 15 per group would give the analysis 90% power. Since the Tdap-vaccinated in group 3 only had 18 participants, it was decided to analyze 36 samples from each of the three groups, of which would include 18 samples from children whose mother was Tdap-vaccinated and 18 samples from children whose mother was not Tdap vaccinated in pregnancy. A total of 108 samples were analyzed following the avidity protocol. 4.2.3 Vaccines Vaccine used in pregnancy Mothers received a Tdap5 booster in pregnancy. Each 0.5 mL dose contained 5.0 limits of flocculation (Lf) of tetanus toxoid, 2.0 Lf of diphtheria toxoid, 2.5 μg of pertussis toxin, 5.0 μg of filamentous hemagglutinin, 3.0 μg of pertactin, and 5 μg of fimbriae types 2 and 3, adsorbed on aluminum phosphate. Vaccines used in infant’s primary series Infants received both DTaP5-IPV-Hib and DTaP3-IPV-Hib-HB as part of their primary series. Each 0.5 mL dose of DTaP5-IPV-Hib contained 5 Lf of tetanus toxoid, 15 Lf of diphtheria toxoid, 20 μg of pertussis toxin, 20 μg of filamentous hemagglutinin, 3 μg of pertactin, 5 μg of fimbriae types 2 and 3, 10 μg of purified capsular polysaccharide of Haemophilus influenzae type b, and 40.0, 8.0, and 32.0 D-antigen units of type 1, 2, and 3 inactivated poliomyelitis vaccine, respectively, adsorbed on aluminum phosphate. A single 0.5 mL dose of DTaP3-IPV- 101 Hib-HB contained 10 Lf of tetanus toxoid, 25 Lf of diphtheria toxoid, 25 μg of pertussis toxin, 25 μg of filamentous hemagglutinin, 8 μg of pertactin, 10 μg of hepatitis B surface antigen, 10 μg of purified capsular polysaccharide of Haemophilus influenzae type b, and 40, 8, and 32 D-antigen units of type 1, 2, and 3 inactivated poliomyelitis vaccine, respectively, adsorbed on aluminum. 4.2.4 ELISA protocols Refer to section 2.2.4. for a detailed description of the anti-PT, anti-FHA, and anti-PRN IgG ELISA protocols used. 4.2.5 Anti-pertussis toxin IgG avidity protocol Refer to section 3.2.5. for a detailed description of the anti-PT IgG avidity assay protocol used. Avidity measurements/calculations Refer to section 3.2.5. for a detailed description of the measurements that were calculated using the avidity protocol. 4.2.6 Statistical analysis Anti-PT, anti-FHA, and anti-PRN IgG concentration analyses We had three outcomes to investigate. First, whether total IgG geometric mean concentrations (GMCs) against PT, FHA, and PRN were different between children from Tdap-vaccinated and Td-vaccinated mothers within each group. Second, whether total IgG GMCs against PT, FHA, and PRN differed between children following the 2+1 DTaP schedule with different vaccine products for their booster dose, irrespective of whether their mother received Tdap in pregnancy. Lastly, whether total IgG GMCs against PT, FHA, and PRN were different in children following a 3+1 or 2+1 DTaP vaccine schedule, irrespective of if their mothers received Tdap in pregnancy. To make these comparisons, GMCs and 95% confidence intervals (CI) of anti-PT, anti-FHA, and anti-PRN IgG were calculated at 13 months of age in children following a 2+1 DTaP vaccine 102 schedule, and at 19 months of age in children following a 3+1 DTaP vaccine schedule. Comparisons for all three outcomes were performed using geometric mean ratios (GMRs) with 95% CIs that were derived from a generalized linear regression model with log-concentrations as the outcome, and Tdap-vaccination in pregnancy status of mother as a covariate. GMR for each antigen was analyzed separately and adjusted for pre-specified factors that could potentially influence children’s immune response (aGMR), including the mother's age (<35 years old, 35+ years old), the interval between vaccine doses (days), gestational age at birth (<37 weeks, ≥37 weeks), number of siblings (0, 1, 2+), and breastfeeding status (yes, no). All analyses were performed using SAS software (SAS Institute, Cary, North Carolina, v. 9.4). P-values ≤0.05 were considered statistically significant. Anti-PT IgG avidity analyses The main outcome for the anti-PT IgG avidity analysis was to compare avidity profiles within each group, between children from Tdap-vaccinated and Tdap-unvaccinated mothers. This comparison was what the follow-up study was powered to detect a difference in. Re-powering was performed using the data from the main outcome to determine whether we are powered to compare avidity profiles between different vaccine product and vaccine schedules, but it was determined that there were not enough samples and that it was not feasible to test the additional samples required. Samples that had an anti-PT IgG concentration determined by an ELISA to be less than 10 IU/mL was not included in any avidity analyses. Anti-PT IgG avidity measurements, including total relative avidity index (TRAI), total absolute avidity (TAA), and fractional absolute avidity (FAA) were calculated using R software. All data were then log-transformed (log10) prior to statistical analyses to normalize data and allow for parametric tests to be used. GraphPad Prism version 9.5.1 was used to perform statistical analysis on the anti-PT IgG avidity data. Descriptive statistics were used to determine the geometric mean concentrations (GMCs) with 95% confidence intervals at each measurement within each group. An unpaired Welch’s t test was used to determine any statistical significance within groups between children of Tdap-vaccinated and Tdap-unvaccinated mothers, between groups with different vaccine product, and between 103 groups with different vaccine schedules. Anti-PT IgG concentration data of the subset of samples used in the avidity analyses were compared using as well. Significance was set at p <0.05. 4.3 Results 4.3.1 Anti-PT, Anti-FHA, and Anti-PRN IgG concentrations Comparison of GMCs within groups between mothers’ Tdap vaccination status To make comparisons between mothers’ Tdap vaccination status, groups 1 and 2 were combined to not stratify the comparison based on booster vaccine product. GMCs of anti-PT, anti-FHA, and anti-PRN were higher in children following the 2+1 vaccine schedule whose mothers were Tdap-unvaccinated in pregnancy: 109.9 IU/mL vs. 82.9 IU/mL, 133.3 IU/mL vs. 87.2 IU/mL, and 149.6 IU/mL vs. 105.9 IU/mL, respectively. These differences were statistically significant: anti-PT aGMR of 0.77 (95%CI: 0.65-1.00, p<0.05); anti-FHA aGMR of 0.66 (95%CI: 0.55-0.79, p<0.05); and anti-PRN aGMR of 0.72 (95%CI: 0.52-0.99, p<0.05) (Table 4.1). Children in the 3+1 vaccine schedule whose mothers were Tdap-unvaccinated had higher anti-PT IgG GMCs than children from Tdap-vaccinated mothers (130.2 IU/mL vs. 113.0 IU/mL). There was no difference in anti-FHA IgG, but children from Tdap-vaccinated mothers had higher anti-PRN IgG than children from Tdap-unvaccinated mothers (385.1 IU/mL vs. 237.5 IU/mL). However, these differences were not statistically significant (P>0.05) (Table 4.1). 104 TABLE 4.1. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis antibodies levels at 13 (2+1 schedule) or 19 (3+1 schedule) months of age of children whose mothers did or did not receive Tdap vaccine during pregnancy. Table from a published journal article that I was co-author on. Reprinted with permission of Vaccine [231]. Vaccination schedule Vaccine antigen Tdap-vaccinated mothers Unvaccinated mothers Unadjusted GMR (95% CI) Adjusted GMR1 (95% CI) p-value N GMC (95% CI) N GMC (95% CI) 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB PT 91 87.7 (77.1-99.9) 48 122.9 (102.8-147.0) 0.71 (0.57-0.89) 0.76 (0.61-0.95) 0.017 FHA 93.0 (80.5-107.4) 154.7 (126.8-188.7) 0.60 (0.47-0.77) 0.62 (0.48-0.8) <0.001 PRN 123.9 (95.0-161.6) 147.6 (102.4-212.9) 0.84 (0.53-1.32) 0.85 (0.53-1.35) 0.487 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP5-IPV-Hib PT 85 78.1 (67.8-89.9) 48 98.3 (81.5-118.6) 0.79 (0.63-1.00) 0.78 (0.62-1.00) 0.047 FHA 81.3 (70.3-94.1) 114.9 (94.7-139.5) 0.71 (0.56-0.90) 0.70 (0.54-0.90) 0.006 PRN 89.6 (68.8-116.7) 151.7 (106.7-215.7) 0.59 (0.38-0.92) 0.60 (0.38-0.94) 0.026 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB or DTaP5-IPV-Hib2 PT 176 82.9 (75.4-91.2) 96 109.9 (96.6-125.1) 0.75 (0.64-0.89) 0.77 (0.65-0.90) 0.001 FHA 87.2 (78.7-96.6) 133.3 (116.0-153.3) 0.65 (0.55-0.78) 0.66 (0.55-0.79) <0.001 PRN 105.9 (87.9-127.7) 149.6 (116.2-192.8) 0.71 (0.52-0.97) 0.72 (0.52-0.99) 0.042 3+1 DTaP3-IPV-Hib-HB – DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB PT 18 113.0 (84.9-150.4) 70 130.2 (112.6-150.5) 0.87 (0.63-1.20) 0.88 (0.64-1.23) 0.454 FHA 239.3 (171.3-334.2) 240.1 (202.6-284.4) 1.00 (0.69-1.45) 0.96 (0.64-1.42) 0.826 PRN 385.1 (242.6-611.1) 237.5 (187.9-300.2) 1.62 (0.97-2.72) 1.62 (0.94-2.79) 0.081 1 GMR adjusted for mother's age (<35 year-olds, 35+ year-olds), interval between 2-month dose and 4-month dose (all study group), interval between 4-month dose and 12-month dose (2+1 schedule), interval between 4-month dose and 6-month dose (3+1 schedule), interval between 6-month dose and 18-month dose (3+1 schedule), interval between booster dose and blood sampling (12-month dose and blood sampling for the 2+1 schedule; 18-month dose and blood sampling for the 3+1 schedule), gestational week at birth (<37w, ≥37w), number of siblings (0, 1, 2+), breastfeeding status (yes, no). 2 This category was the pooled of the two previous categories (2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB and 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP5-IPV-Hib). 105 Comparison of GMCs between vaccine products in the 2+1 vaccine schedule groups irrespective of mothers’ Tdap vaccination status To make comparisons between vaccine product, groups were no longer stratified on mothers’ vaccine status, but rather the GMRs were adjusted to account for the mothers’ Tdap vaccination status. Children that received DTaP3-IPV-Hib-HB as their 12-month booster dose had higher GMCs of anti-PT, anti-FHA, and anti-PRN IgG: 98.6 IU/mL vs. 84.8 IU/mL, 110.9 IU/mL vs. 92.1 IU/mL, and 131.6 IU/mL vs. 108.3 IU/mL, respectively. However, only the difference in anti-FHA IgG GMCs was statistically significant (aGMR 1.21 (95%CI: 1.02-1.43, p<0.05). (Table 4.2). TABLE 4.2. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis IgG antibodies at 13 months of age for children who received DTaP3-IPV-Hib-HB vaccine compared to children who received DTaP5-IPV-Hib® DTaP vaccine at 12 months old. Table from a published journal article that I was co-author on. Reprinted with permission of Vaccine [231]. Vaccine antigen 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB schedule 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib –DTaP5-IPV-Hib schedule Unadjusted GMR (95% CI) Adjusted GMR1 (95% CI) p-value N GMC (95% CI) N GMC (95% CI) PT 139 98.6 (88.4-110) 133 84.8 (75.9-94.9) 1.16 (0.99-1.36) 1.15 (0.99-1.34) 0.076 FHA 110.9 (98.4-125) 92.1 (81.5-104.1) 1.20 (1.01-1.43) 1.21 (1.02. 1.43) 0.026 PRN 131.6 (106.5-162.6) 108.3 (87.3-134.5) 1.21 (0.90-1.64) 1.25 (0.92-1.69) 0.156 1 GMR adjusted for mother's Tdap vaccination status (vaccinated, unvaccinated), mother's age (<35 year-olds, 35+ year-olds), interval between 2-month dose and 4-month dose (days), interval between 4-month dose and 12-month dose (days), interval between 12-month dose and blood sampling, gestational week at birth (<37w, ≥37w), number of siblings (0, 1, 2+), breastfeeding status (yes, no). Abbreviations: CI, confidence interval; DTaP, diphtheria-tetanus-acellular pertussis; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; GMR, geometric mean concentration ratio; PRN, pertactin; PT, pertussis toxin; Tdap, tetanus-diphtheria-acellular pertussis. Comparison of GMCs between vaccine schedule irrespective of mothers’ Tdap vaccination status Stratification within each group based on mothers’ Tdap vaccination status was removed and instead adjusted in the GMRs. Children following the 3+1 vaccine schedule has higher GMCs of anti-PT, anti-FHA, and anti-PRN IgG: 126.5 IU/mL vs. 91.6 IU/mL, 239.9 IU/mL vs. 101.3 IU/mL, and 262.2 IU/mL vs. 119.7 IU/mL, respectively. These differences were statistically significant: anti-PT aGMR of 1.27 (95%CI: 1.07-1.50, p<0.05); anti-FHA aGMR of 2.04 106 (95%CI: 1.69-2.46, p<0.05); and anti-PRN aGMR of 2.04 (95%CI: 1.48-2.80, p<0.05) (Table 4.3). TABLE 4.3. Geometric mean antibody concentrations (GMCs) and geometric mean antibody concentration ratios (GMRs) of anti-pertussis IgG antibodies for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. Table from a published journal article that I was co-author on. Reprinted with permission of Vaccine [231]. Vaccine antigen 3+1 DTaP3-IPV-Hib-HB – DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB schedule 2+1 DTaP3-IPV-Hib-HB – DTaP5-IPV-Hib – DTaP3-IPV-Hib-HB or DTaP5-IPV-Hib schedule Unadjusted GMR (95% CI) Adjusted GMR1 (95% CI) p-value N GMC (95% CI) N GMC (95% CI) PT 88 126.5 (110.4-144.8) 272 91.6 (84.8-98.9) 1.38 (1.18-1.61) 1.27 (1.07-1.50) 0.006 FHA 239.9 (206.4-278.9) 101.3 (93.0-110.3) 2.37 (1.99-2.82) 2.04 (1.69-2.46) <0.001 PRN 262.2 (203.5-337.8) 119.7 (103.6-138.2) 2.19 (1.64-2.93) 2.04 (1.48-2.80) <0.001 1 GMR adjusted for mother's Tdap vaccination status (vaccinated, unvaccinated), mother's age (<35 year-olds, 35+ year-olds), interval between booster dose and blood sampling (12-month dose and blood sampling (2+1 schedule)), 18-month dose and blood sampling (3+1 schedule)), gestational week at birth (<37w, ≥37w), number of siblings (0, 1, 2+), breastfeeding status (yes, no). Abbreviations: CI, confidence interval; DTaP, diphtheria-tetanus-acellular pertussis; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; GMR, geometric mean concentration ratio; PRN, pertactin; PT, pertussis toxin; Tdap, tetanus-diphtheria-acellular pertussis. 4.3.2 Anti-PT IgG avidity Comparison within groups between mothers’ Tdap vaccination status GMCs of anti-PT IgG for this subset of samples were higher in children following the 2+1 vaccine schedule whose mothers were Tdap-unvaccinated in pregnancy, irrespective of vaccine product used for their booster, but was not statistically significant: group 1 (115.6 IU/mL vs. 84.36 IU/mL, p=0.1251) and group 2 (100.8 IU/mL vs. 76.91 IU/mL, p=0.1915). Children following a 3+1 vaccine schedule had similar anti-PT IgG GMCs regardless of mothers’ vaccine status (127.9 IU/mL vs. 135.9 IU/mL, p=0.9121) (Figure 4.1a). There were no significant differences in TRAI within groups 1, 2 or 3: p=0.3505, p=0.1790, p=0.0770, respectively (Figure 4.1b). Children who followed a 2+1 vaccine schedule, received DTaP5-IPV-Hib as their booster, and whose mother was Tdap-unvaccinated in pregnancy had significantly higher TAA levels than children whose mothers were Tdap-vaccinated in pregnancy: 143.2 IU/mL vs. 89.33 IU/mL, p=0.0485. No significant difference in TAA levels within groups 1 (p=0.5058) and 3 (p=0.6240) (Figure 4.1c). 107 FIGURE 4.1. Geometric mean antibody concentrations (GMCs) of anti-pertussis toxin (PT) IgG antibodies and avidity of each group stratified by mothers’ Tdap vaccination status. Blue bars represent GMCs of children from Tdap-unvaccinated mothers, and red bars represent GMCs of children from Tdap-vaccinated mothers. Comparisons were made within each group to analyze effect of mothers’ Tdap vaccination status on children’s humoral responses, and comparisons were made between groups 1 and 2 of the same maternal Tdap vaccination status to compare vaccine product effect on humoral responses. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05. A) Anti-PT IgG GMCs presented on log-transformed y-axis. B) Total relative avidity index of anti-PT IgG. C) Total absolute avidity levels of anti-PT IgG. (4.1) Group 1 Group 2 Group 3101001000ELISAns ns nsnsnsTotal anti-PT IgG concentration (IU/mL)1001010Group 1 Group 2 Group 3nnsnnnA C B 108 Children in group 2 whose mothers were Tdap-unvaccinated in pregnancy had significantly higher FAA levels of low, low-medium, and high avidity anti-PT IgG compared with children from Tdap-vaccinated mothers: low (10.7 IU/mL vs. 6.4 IU/mL, p = 0.0122), low-medium (17.8 IU/mL vs. 11.4 IU/mL, p = 0.0437), and high (27.0 IU/mL vs. 14.5 IU/mL, p = 0.0218). There was no significant difference in FAA levels within groups 1 and 3 (Figure 4.2a-g). 109 A C B D 110 FIGURE 4.2. Geometric mean concentrations (GMCs) of fractional absolute avidity levels of anti-pertussis toxin (PT) IgG of each group stratified by mothers’ Tdap vaccination status. Blue bars represent GMCs of children from Tdap-unvaccinated mothers, and red bars represent GMCs of children from Tdap-vaccinated mothers. Comparisons were made within each group to analyze effect of mothers’ Tdap vaccination status on children’s humoral responses, and comparisons were made between groups 1 and 2 of the same maternal Tdap vaccination status to compare vaccine product effect on humoral responses. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05. Y-axes were all log transformed. A) GMCs of very low FAA of anti-PT IgG. B) GMCs of low FAA of anti-PT IgG. C) GMCs of low-medium FAA of anti-PT IgG. D) GMCs of medium FAA of anti-PT IgG. E) GMCs of medium-high FAA of anti-PT IgG. F) GMCs of high FAA of anti-PT IgG. G) GMCs of very high FAA of anti-PT IgG. (4.2) E G F 111 Comparison between vaccine products in the 2+1 vaccine schedule groups Comparisons between vaccine products were performed between children in groups 1 and 2 with the same maternal Tdap vaccination status. There were no significant differences in anti-PT IgG GMCs, TRAI of anti-PT IgG, or TAA levels of anti-PT IgG (p>0.05) (Figure 4.1a-c). All FAA levels of anti-PT IgG were also similar with no significant differences (Figure 4.2a-g). Comparison between 3+1 and 2+1 doses of DTaP vaccine To compare 3+1 and 2+1 dose DTaP vaccine schedules, groups 1 and 2 were combined to represent the 2+1 vaccine schedule. Stratification based on mothers’ Tdap vaccination status was kept and comparisons were made between 3+1 and 2+1 vaccine schedules between groups of children with the same maternal Tdap vaccination status. GMCs of anti-PT IgG was similar in children that received 3+1 or 2+1 doses of DTaP vaccine and whose mothers were Tdap-unvaccinated in pregnancy (110.4 IU/mL vs. 107.9 IU/mL, p=0.8949). Children of Tdap-vaccinated mothers that received 3+1 doses of DTaP had higher anti-PT IgG GMCs than children that received 2+1 doses of DTaP and had Tdap-vaccinated mothers but was not significant (113.0 IU/mL vs. 80.6 IU/mL, p=0.0632) (Figure 4.3a). There were no significant differences in TRAI between 3+1 and 2+1 doses of DTaP in children of Tdap-unvaccinated mothers (123.9 IU/mL vs. 121.3 IU/mL, p=0.5301) and children of Tdap-vaccinated mothers (115.7 IU/mL vs. 121.0 IU/mL, p=0.1781) (Figure 4.3b). Children whose mothers were Tdap-unvaccinated in pregnancy had similar TAA regardless of DTaP vaccine schedule (132.4 IU/mL vs. 135.4 IU/mL, p=0.9100), but children from Tdap-vaccinated mothers had significantly higher TAA after receiving 3+1 doses instead of 2+1 doses of DTaP vaccine (148.3 IU/mL vs. 99.6 IU/mL, p=0.0481) (Figure 4.3c). 112 FIGURE 4.3. Geometric mean antibody concentrations (GMCs) of anti-pertussis toxin (PT) IgG antibodies and avidity for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. Blue bars represent GMCs of children from Tdap-unvaccinated mothers, and red bars represent GMCs of children from Tdap-vaccinated mothers. Comparisons were made between the two groups between children with the same maternal Tdap vaccination status. All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05. A) Anti-PT IgG GMCs presented on log-transformed y-axis. B) Total relative avidity index of anti-PT IgG. C) Total absolute avidity levels of anti-PT IgG. (4.3) Group 1 + 2 Group 3101001000ELISAnsnsTotal anti-PT IgG concentration (IU/mL)100010102+1 vaccine schedule 3+1 vaccine schedulennsC B A 113 There were no differences in similar FAA levels of anti-PT at each different avidity level in children from Tdap-unvaccinated mothers regardless of whether they received 3+1 or 2+1 doses of DTaP vaccine (p>0.05). Children from Tdap-vaccinated mothers had significantly higher FAA levels of low, low-medium, medium, and medium-high anti-PT IgG avidities when they received 3+1 doses of DTaP vaccine compared to 2+1 doses of DTaP vaccine: low (11.9 IU/mL vs. 5.7 IU/mL, p=0.0013), low-medium (20.5 IU/mL vs. 11.4 IU/mL, p=0.0007), medium (22.1 IU/mL vs. 14.0 IU/mL, p=0.0423), and medium-high (18.2 IU/mL vs. 11.8 IU/mL, p=0.0386) (Figure 4.4a-g). 114 C A D B 115 FIGURE 4.4. Geometric mean concentrations (GMCs) of fractional absolute avidity levels of anti-pertussis toxin (PT) IgG for children who received 3+1 doses of DTaP vaccine compared to children who received 2+1 doses of DTaP vaccine. Blue bars represent GMCs of children from Tdap-unvaccinated mothers, and red bars represent GMCs of children from Tdap-vaccinated mothers. Comparisons were made between the two groups between children with the same maternal Tdap vaccination status All statistics performed utilized an unpaired Welch’s t test: * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. Y-axes were all log transformed. A) GMCs of very low FAA of anti-PT IgG. B) GMCs of low FAA of anti-PT IgG. C) GMCs of low-medium FAA of anti-PT IgG. D) GMCs of medium FAA of anti-PT IgG. E) GMCs of medium-high FAA of anti-PT IgG. F) GMCs of high FAA of anti-PT IgG. G) GMCs of very high FAA of anti-PT IgG. (4.4) G E F 116 4.4 Discussion In this study, we investigated the potential immunomodulation of anti-pertussis IgG concentrations and their avidity in children of mothers that were vaccinated with Tdap in pregnancy. We also examined the effect of vaccine formulation and vaccine schedule on possible immunomodulation. To investigate immunomodulation of anti-PT, anti-FHA, and anti-PRN IgG concentrations, ELISAs were used to measure antibodies against each respective antigen. When comparing anti-PT, anti-FHA, and anti-PRN IgG GMCs within the 2+1 and 3+1 vaccine schedules, our findings were not fully as expected. In children that followed a 2+1 vaccine schedule, children from Tdap-unvaccinated mothers had significantly higher GMCs against PT, FHA, and PRN than children whose mothers were Tdap-vaccinated in pregnancy. This provided evidence of immunomodulation within this group one-month post-booster dose with DTaP which was expected. However, in children that followed a 3+1 vaccine schedule, there was no evidence supporting immunomodulation as demonstrated by no significant differences between children whose mothers were Tdap-vaccinated and children whose mothers were Tdap-unvaccinated for antibodies measured against all three antigens. However, anti-PRN IgG concentrations could have potentially reached significance if we had a larger sample size for children from Tdap-vaccinated mothers as the p-value was 0.081 and the sample size was only 18 compared to 70 for children from Tdap-unvaccinated mothers. The results of children from the 3+1 vaccine schedule are consistent with a few studies, however, these studies had small sample sizes [159, 164, 235]. In studies with larger sample sizes, immunomodulation has been demonstrated to occur in children that received 3+1 doses of DTaP [158, 232]. As there are no studies that have investigated immunomodulation in children that followed a 2+1 vaccine schedule, more studies are required. However, the fact that immunomodulation occurs in children that received 2+1 doses of DTaP is not surprising. Children that received 2+1 doses of DTaP still received their primary series when maternal antibodies would have been circulating as maternal antibody half-life can range from 28 to 35 days [236]. A follow-up study is necessary to investigate whether immunomodulation in children that received 2+1 doses of DTaP is overcome after their second booster prior to starting school (between 4-5 years of age). 117 We also examined whether using two different formulations of the DTaP vaccine as a booster dose had an impact on if immunomodulation occurred, and if so to what degree. I hypothesized that a booster dose of DTaP with greater amounts of purified antigen content would have a lower degree of immunomodulation compared to a booster dose of DTaP with lower amounts of purified antigen. With increased availability of antigen, I believed it would provide a higher likelihood of the children’s immune cells to recognize and elicit a greater humoral immune response. It was found that children that received DTaP3-IPV-Hib-HB had higher GMCs of IgG against PT, FHA, and PRN than children that received DTaP5-IPV-Hib as a booster, however, only FHA was significantly higher. This supports my hypothesis that a vaccine with greater pertussis antigen content could help provide more opportunities for the infant’s immune cells to recognize and bind to the antigens as DTaP3-IPV-Hib-HB had higher amounts of PT, FHA, and PRN than DTaP5-IPV-Hib. To my understanding there are no studies that have compared immunomodulation in children from Tdap-immunized mothers that received two different formulations of DTaP vaccine. There is a study that investigated 12 different acellular pertussis vaccines and one whole cell vaccine and they found no correlation between antibody responses against PT and FHA on the quantity of antigen in the vaccine used but did find a correlation between anti-PRN IgG and quantity of PRN in the vaccine used [237]. However, this study was performed in 1997, more than a decade before the introduction of immunization in pregnancy with Tdap, and therefore did not investigate any immunomodulation and cannot be used to compare results. Another study compared the use of DTaP3-IPV-Hib-HB and DTaP5-IPV-Hib as a booster dose in toddlers between 11 and 18 months of age, but in toddlers whose mothers were Tdap-unvaccinated [238]. Results were similar to our findings for PT and PRN, but in this study, they also observed no differences in anti-FHA IgG where we did. More studies are necessary to evaluate the effect different vaccine formulation have on immunomodulation in children from Tdap-vaccinated mothers. However, both formulations used in our study resulted in a robust response against all pertussis vaccine antigens in the vaccine. Since there is no widely accepted correlate of protection against pertussis, no comment can be made on whether there is any clinical significance on protection against pertussis disease in these children. The result from our study provides assurance that both DTaP3-IPV-Hib-HB and DTaP5-IPV-Hib use as a DTaP booster dose can be used interchangeably to facilitate the generation of a robust humoral immune response against pertussis vaccine antigen. 118 The last question we had regarding anti-pertussis IgG GMCs in our study was whether there was a significant difference in anti-PT, anti-FHA, and anti-PRN GMCs between children that followed a 2+1 or a 3+1 DTaP vaccine schedule when adjusted for mothers’ Tdap vaccination status. I hypothesized that children that received 3+1 doses of DTaP had higher GMCs against PT, FHA, and PRN compared to children that received 2+1 doses of DTaP. This was thought to be because children that received 3+1 doses of DTaP would have received an extra dose than children that received 2+1 doses of DTaP which would potentially allow for a great boosting effect. As expected, in this study we found that children that received 3+1 doses of DTaP vaccine had significantly higher GMCs against all three pertussis vaccine antigens analyzed compared to children that received 2+1 doses of DTaP vaccine. To my understanding, there are no studies that have investigated a 3+1 and 2+1 DTaP vaccine schedule. However, studies have been conducted that aimed to analyze the impact of under-vaccination and delay in vaccination with DTaP and its association with pertussis risk in infants and young children [239]. Although not a direct comparison to vaccination with set timelines like our study, they found a 4.8-fold and 3.2-fold higher pertussis risk in infants and children that were under-vaccinated for their primary or booster, respectively [239]. If we use under-vaccination as a surrogate for a decreased vaccine schedule – 2+1 doses of DTaP vaccine – it suggests decreased immunity against infection and therefore potentially lower antibodies against pertussis. However, there are many factors that can be involved in pertussis infection and therefore, a direct comparison between the studies is not warranted. Overall, 3+1 doses of DTaP vaccine provides significantly greater anti-PT, anti-FHA, and anti-PRN IgG concentrations than 2+1 doses of DTaP vaccine, but although the differences are significant, it may not be clinically significant. Further longitudinal studies are required to address the clinical relevance of reduced humoral responses after receiving 2+1 doses of DTaP vaccine on its effectiveness to prevent pertussis infection in children. Beyond analyzing anti-pertussis GMCs, I also wanted to determine if anti-PT IgG avidity also underwent immunomodulation, and if immunomodulation occurred, was the degree of immunomodulation different dependent on child booster dose formulation and DTaP vaccine schedule. I hypothesized that children born from Tdap-unvaccinated mothers would have greater avidity than those whose mothers were Tdap-vaccinated in pregnancy, and there would be no 119 significant difference in avidity level between groups that received different vaccine formulations. With the reduced humoral immune responses associated with immunomodulation in infants born from Tdap-vaccinated mothers, I believed there would be a decrease in somatic hypermutation and affinity maturation, and as a result a decrease in anti-pertussis IgG with greater specificity to pertussis antigens. In this study, when comparing within each group between children whose mothers were Tdap-vaccinated or Tdap-unvaccinated in pregnancy, significant differences were only seen in TAA and FAA within group 2 (2+1 doses of DTaP; DTaP3-IPV-Hib-HB - DTaP5-IPV-Hib – DTaP5-IPV-Hib). As no differences were seen in TRAI of anti-PT IgG, this suggested that the proportions of anti-PT IgG with the seven different avidity levels were similar between children from Tdap-vaccinated and Tdap-unvaccinated mothers, regardless of vaccine formulations and schedule. TAA provides a weighted sum of anti-PT IgG with different avidity strengths. With the significant difference in TAA observed within group 2 of children that received DTaP5-IPV-Hib as their booster DTaP dose, children from Tdap-unvaccinated mothers had higher concentrations of antibodies with higher avidity than children from Tdap-vaccinated mothers. This is apparent when analyzing the FAA results as these children in group 2 from Tdap-unvaccinated mothers had higher concentrations of anti-PT IgG with low, low-medium, and high avidity levels. To my understanding, current literature has mainly focused on newborn and infant cohorts to analyze the impact of Tdap vaccination in pregnancy on immunomodulation of anti-PT IgG avidity. In these studies, they found that avidity was not mainly driven by antibody level, and cord blood from newborns born from Tdap-vaccinated mothers had an avidity profile with higher concentrations of low-medium, medium, medium-high, high, and very high avidity levels compared to newborns from Tdap-unvaccinated mothers. These results only focused on measuring the avidity profile of maternal antibodies that were transferred transplacentally from the mother to fetus, but comparing these to our results, it suggests that these differences observed in newborns can be overcome after a booster dose of DTaP, except if DTaP5-IPV-Hib is used. Although differences were found in group 2, these differences could be explained by factors such as intervals between receiving the DTaP doses, interval from booster and blood sampling, and gestational age at birth. The avidity data was not adjusted for these co-variables which may impact the results. When comparing children that received 2+1 doses of DTaP vaccine, but with a different booster, no significant differences were observed. This suggests that the anti-PT IgG avidity profiles developed is not greatly impacted 120 by the vaccine formulation used as a DTaP vaccine booster, specifically regarding the two formulations tested in this study. Overall, our results suggest that immunomodulation of anti-PT IgG avidity is not occurring at a high degree in most children after receiving a booster dose of DTaP vaccine. Lastly, I wanted to determine whether there was a difference in anti-PT IgG avidity in children that received 3+1 doses of DTaP vaccine compared to children that received 2+1 doses of DTaP vaccine. I hypothesized that children that received 3+1 doses of DTaP vaccine would have greater antibody avidity than those that received 2+1 doses of DTaP vaccine. To perform this analysis, both groups 1 and 2 were combined based on the vaccination status of their mothers. This was possible because there were no differences between these groups. Since adjustments were not made on the mothers’ vaccination status, comparisons were only made between children with the same maternal vaccination status between the 3+1 and 2+1 dose DTaP vaccine schedules. In this study, children from Tdap-vaccinated mothers that received 3+1 doses of DTaP vaccine had higher TAA and anti-PT IgG concentrations with low, low-medium, medium, and medium-high FAA levels. These results demonstrate that an additional dose of DTaP in a primary series leads to higher concentrations of antibodies with higher avidity strengths. There are no studies that have made this comparison and therefore is an area of research that requires more research. The clinical implications on a reduced TAA and FAA observed in children from Tdap-vaccinated mothers that received 2+1 doses of DTaP is unknown as there is no widely accepted correlate of protection against pertussis infection. Further studies are required to determine if avidity is an appropriate measure to be used in determining a correlate of protection. As of now, I hypothesize that antibodies with higher avidity will provide greater protection as it would provide a greater total binding strength between the antibodies and its target antigens. This study comprised of a few strengths. First, the study was designed to directly compare different vaccine schedules which had not been done previously, filling a gap in the literature. Secondly, the avidity analysis utilized a modified protocol which provided a more in-depth evaluation of antibody avidities, allowing for antibodies to be split into seven different avidity strengths. There were also a few limitations to this study. First, in the antibody concentration analyses, we only had a sample size of 18 children from Tdap-vaccinated mothers that received 121 3+1 doses of DTaP. Therefore, the lack of immunomodulation observed in this group could be due to being underpowered to detect a difference within this group. Secondly, for the avidity analyses, only the comparison within each group between children from Tdap-vaccinated and Tdap-unvaccinated mothers was properly powered to detect a significant difference if there truly was a difference. Comparison in avidity between children that received different vaccine formulation and vaccine schedules was underpowered so there may be a difference, but we did not have the sample size to detect one. Therefore, these results should be interpreted with caution and further studies are necessary. Third, for the avidity analyses, no adjustments were made for co-variables like it was performed for the antibody concentration data. This made it difficult to interpret the data in a uniform way and the differences seen could be explained by co-variables such as gestational age at birth, interval between doses of DTaP vaccine, and interval between vaccination and blood sampling. Furthermore, no participant demographics were incorporated into any analyses. Studies are required that incorporate sex, ethnicity, and socioeconomic status as potential variables that can impact humoral immune responses. Lastly, immunogenicity data prior to 12 months of age was not collected. This made it not possible to comment on immunomodulation or the lack thereof in these children prior to receiving a booster dose of DTaP. Therefore, comments on how 3+1 doses of DTaP vaccine may have been able to attenuate immunomodulation must be interpreted with caution as there may have not been any immunomodulation in this cohort prior to the booster dose of DTaP vaccine. As vaccination in pregnancy with Tdap continues to be recommended, it is important to ensure the effectiveness of such a recommendation. This study has added to the available data to provide reassurance of minimal impacts of immunomodulation after receiving a booster dose of DTaP after a 3-dose primary series. Furthermore, this study has added to an area that has not been investigated prior by incorporating comparisons of immunomodulation between vaccine formulations and schedules, and by assessing avidity. In this study, we were able to demonstrate that moderate degree of immunomodulation occurs in children following the 2+1 DTaP vaccine schedules, however, each group still led to high antibody concentrations against PT, FHA, and PRN. With no correlate of protection, the clinical significance of the relatively small differences in antibody concentrations within and between groups remains unclear. It was also evident that an additional dose of DTaP was able to minimize the immunomodulation associated with Tdap- 122 vaccination in pregnancy. I was also able to demonstrate that an additional dose of DTaP also increases the avidity of antibodies, however the clinical significance of this increase in avidity profile is unknown and requires further investigation. In conclusion, a 2+1 dose DTaP vaccine schedule, irrespective of vaccine formulation, still provided a high humoral immune response to anti-pertussis vaccine antigens. Additional studies are required to determine a correlate of protection to fully understand the significance of anti-pertussis IgG concentration and avidity levels on the ability to prevent infection. 123 5. Overall conclusions and future directions Severe pertussis disease continues to disproportionately impact infants less than two months of age, especially in LMICs where immunization in pregnancy programs have not been implemented. With no correlate of protection being widely accepted, in this thesis I aimed to evaluate and better understand how immunization in pregnancy affects the immunogenicity and antibody avidity in infants from immunized mothers compared with infants from non-immunized mothers. In this thesis, I have provided a thorough outline of how I optimized and validated three ELISAs to measure total anti-PT, anti-FHA, and anti-PRN IgG concentrations in both serum and heparin plasma samples. These ELISAs were developed to industry standard and were essential in being able to evaluate immunogenicity in my other two studies, and a modified version of the ELISA was used in measuring anti-PT avidity. Using these ELISAs, I observed that the novel TdaP vaccine given in pregnancy was effective at inducing a humoral immune response as shown by significantly higher total anti-PT and anti-FHA IgG concentrations in TdaP-vaccinated pregnant women compared to Td-vaccinated pregnant women. Although we saw lower total anti-PT and anti-FHA IgG concentrations in HIV-positive compared with HIV-negative pregnant women that both received TdaP, the concentration of these antibodies were still significantly higher than at baseline. Furthermore, pregnant women irrespective of HIV-status had similar anti-PT TRAI suggesting that B cells are being induced and are able to undergo affinity maturation through somatic hypermutation. In infants, anti-PT and anti-FHA IgG concentrations were higher at cord blood in infants from TdaP-vaccinated mothers compared with infants from Td-vaccinated mothers, suggesting passive transfer of antibodies through transplacental antibody transfer. Interestingly, infants from TdaP-vaccinated mothers had lower concentrations 4-weeks post completion of their primary series with DTwP compared with infants from Td-vaccinated mothers. No differences were seen for anti-FHA IgG concentrations 4-weeks post-primary series between any of the groups. Lastly, immunomodulation of anti-PT avidity was observed 4-weeks post-primary series when analyzing TAA and FAA data. However, no differences were observed when evaluating TRAI, suggesting immunomodulation did not impact the proportion of antibodies with different avidities but rather just the concentration of the antibodies in total. Overall, the TdaP vaccine used in this study 124 should be implemented to be used in pregnancy as it is able to generate a humoral immune response in the pregnant women and transfer into the fetus to provide protection prior to when the infants can start and/or finish their own primary series. Lastly, I found that receiving a 3+1 dose series compared with a 2+1 dose series led to higher total anti-PT, anti-FHA, and anti-PRN IgG concentrations, as well as higher avidity. Furthermore, children that received the 3+1 dose schedule had no evidence of immunomodulation 4-weeks post booster, whereas the children that received the 2+1 dose schedule had evidence of immunomodulation for all three pertussis antigens measures. However, the 2+1 dose schedule was able to still produce high concentrations and since there is no known correlate of protection, the clinical significance of this difference in unknown. To be able to make better conclusions on the results from my thesis, better understanding of a correlate of protection against pertussis or vaccine effectiveness studies in these cohorts would be helpful. Further investigation of the function of anti-pertussis antibodies is necessary as these functions may play an important role in determining a correlate of protection. Some examples of functions that could be investigated includes the effector functions of FcRs such as ADCP and ADCC. Understanding these functions will provide better understanding on how antibodies against different antigens are better at performing different functions such as binding/activating complement, promoting phagocytosis, and activating cytotoxicity pathways. Additionally, the ability of anti-pertussis antibodies to neutralize B. pertussis should be evaluated as it is known that acellular pertussis vaccines are not good at preventing colonization, suggesting lower ability to neutralize the bacteria. This could be especially important in the development of new pertussis vaccines as maybe antibodies against other pertussis antigens such as ACT could provide better neutralization. Furthermore, studies are necessary to better understand the mechanism of immunomodulation in infants from Tdap-vaccinated mothers after they receive their own pertussis vaccines. There are a few hypotheses, but it has not been fully elucidated yet. Although it is important to determine the mechanism of immunomodulation, I believe it is most important to have better understanding of the clinical significance of immunomodulation. There have been a few studies that have used 125 modelling to estimate the impacts of immunomodulation, but a large-scale longitudinal study following infants from Tdap-vaccinated and Tdap-unvaccinated mothers would be optimal. In this study I propose performing immunogenicity measurements from birth and then pre- and post- vaccinations following them up to after their pre-school booster. Evaluation of immunomodulation overtime can be evaluated to see any long-term impacts on antibody concentration, as well as tracking known pertussis exposure and pertussis infection to see if immunomodulation has any clinical impacts. Statistics can then be performed to see if there are any correlations between level of immunomodulation to pertussis infection. Regarding the two studies that I have included in this thesis, it would be necessary to test the TdaP vaccine in high-level HIV infected pregnant women to allow better generalizability. It would also be valuable to perform multi-variable analyses to understand whether other HIV measures such as viral load, CD4 count, and ART are associated with higher or lower immunogenicity or avidity. Lastly, the participants from the study comparing vaccine schedules should continue longitudinal surveillance or a new study could be started to evaluate the effect of different vaccine schedules on the effectiveness to prevent infection with pertussis. 126 References 1. Vaccine preventable disease: Surviellance report to December 31, 2019. 2022, Public Health Agency of Canada. 2. Reported cases from 1924 to 2021 in Canada - Notfiable diseases on-line. Public Health Agency of Canada. 3. Kimura, M. and H. Kuno-Sakai, Pertussis vaccines in Japan—A clue toward understanding of Japanese attitude to vaccines. 1991, Oxford University Press. 4. Romanus, V., R. Jonsell, and S.-O. Bergquist, Pertussis in Sweden after the cessation of general immunization in 1979. The Pediatric infectious disease journal, 1987. 6(4): p. 364-371. 5. Gangarosa, E.J., et al., Impact of anti-vaccine movements on pertussis control: the untold story. The Lancet, 1998. 351(9099): p. 356-361. 6. Canada communicable disease report: National consensus conference on pertussis. 2003, Health Canada. 7. Sato, Y. and H. Sato, Development of acellular pertussis vaccines. Biologicals, 1999. 27(2): p. 61-9. 8. Sato, Y. and H. Sato, Further characterization of Japanese acellular pertussis vaccine prepared in 1988 by 6 Japanese manufacturers. Tokai J Exp Clin Med, 1988. 13 Suppl: p. 79-88. 9. Skoff, T.H., et al., Early impact of the US Tdap vaccination program on pertussis trends. Archives of pediatrics & adolescent medicine, 2012. 166(4): p. 344-349. 10. Clark, T.A., N.E. Messonnier, and S.C. Hadler, Pertussis control: time for something new? Trends in microbiology, 2012. 20(5): p. 211-213. 11. Clark, T.A., Changing Pertussis Epidemiology: Everything Old is New Again. The Journal of Infectious Diseases, 2014. 209(7): p. 978-981. 12. Winter, K., et al., California pertussis epidemic, 2010. The Journal of pediatrics, 2012. 161(6): p. 1091-1096. 13. Customize your own chart - Notifiable diseases on-line. Public Health Agency of Canada. 14. Kayina, V., et al., Pertussis Prevalence and Its Determinants among Children with Persistent Cough in Urban Uganda. PLOS ONE, 2015. 10(4): p. e0123240. 15. Yeung, K.H.T., et al., An update of the global burden of pertussis in children younger than 5 years: a modelling study. The Lancet Infectious Diseases, 2017. 17(9): p. 974-980. 16. Muloiwa, R., et al., The burden of laboratory-confirmed pertussis in low- and middle-income countries since the inception of the Expanded Programme on Immunisation (EPI) in 1974: a systematic review and meta-analysis. BMC Med, 2020. 18(1): p. 233. 17. Gill, C.J., et al., Incidence of Severe and Nonsevere Pertussis Among HIV-Exposed and -Unexposed Zambian Infants Through 14 Weeks of Age: Results From the Southern Africa Mother Infant Pertussis Study (SAMIPS), a Longitudinal Birth Cohort Study. Clin Infect Dis, 2016. 63(suppl 4): p. S154-s164. 18. Ben Fraj, I., et al., Seroprevalence of pertussis among healthcare workers: A cross-sectional study from Tunisia. Vaccine, 2019. 37(1): p. 109-112. 19. Rivera, I., B. Linz, and E.T. Harvill, Evolution and Conservation of Bordetella Intracellular Survival in Eukaryotic Host Cells. Front Microbiol, 2020. 11: p. 557819. 127 20. Bouchez, V. and N. Guiso, Bordetella pertussis, B. parapertussis, vaccines and cycles of whooping cough. Pathogens and Disease, 2015. 73(7). 21. Warfel, J.M., J. Beren, and T.J. Merkel, Airborne transmission of Bordetella pertussis. J Infect Dis, 2012. 206(6): p. 902-6. 22. Carbonetti, N.H., Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools. Future Microbiol, 2010. 5(3): p. 455-69. 23. Burns, D.L., Secretion of Pertussis Toxin from Bordetella pertussis. Toxins (Basel), 2021. 13(8). 24. Tamura, M., et al., Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry, 1982. 21(22): p. 5516-22. 25. Llosa, M., C. Roy, and C. Dehio, Bacterial type IV secretion systems in human disease. Molecular Microbiology, 2009. 73(2): p. 141-151. 26. Weiss, A.A., F.D. Johnson, and D.L. Burns, Molecular characterization of an operon required for pertussis toxin secretion. Proceedings of the National Academy of Sciences, 1993. 90(7): p. 2970-2974. 27. Craig-Mylius, K.A. and A.A. Weiss, Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion. FEMS Microbiology Letters, 1999. 179(2): p. 479-484. 28. Witvliet, M.H., et al., Binding of pertussis toxin to eucaryotic cells and glycoproteins. Infection and Immunity, 1989. 57(11): p. 3324-3330. 29. Armstrong, G.D., L.A. Howard, and M.S. Peppler, Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin. Journal of Biological Chemistry, 1988. 263(18): p. 8677-8684. 30. Plaut, R.D. and N.H. Carbonetti, Retrograde transport of pertussis toxin in the mammalian cell. Cellular Microbiology, 2008. 10(5): p. 1130-1139. 31. el Bayâ, A., et al., Endocytosis and retrograde transport of pertussis toxin to the Golgi complex as a prerequisite for cellular intoxication. European journal of cell biology, 1997. 73(1): p. 40-48. 32. Hsia, J.A., et al., Amino acid-specific ADP-ribosylation. Sensitivity to hydroxylamine of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages. J Biol Chem, 1985. 260(30): p. 16187-91. 33. Teter, K., Intracellular Trafficking and Translocation of Pertussis Toxin. Toxins (Basel), 2019. 11(8). 34. Gregg, K.A. and T.J. Merkel, Pertussis Toxin: A Key Component in Pertussis Vaccines? Toxins (Basel), 2019. 11(10). 35. Mooi, F.R., et al., Bordetella pertussis strains with increased toxin production associated with pertussis resurgence. Emerg Infect Dis, 2009. 15(8): p. 1206-13. 36. Taub, R.N., et al., Distribution of labeled lymph node cells in mice during the lymphocytosis induced by Bordetella pertussis. J Exp Med, 1972. 136(6): p. 1581-93. 37. Yajima, M., et al., Islets-activating protein (IAP) in Bordetella pertussis that potentiates insulin secretory responses of rats. Purification and characterization. J Biochem, 1978. 83(1): p. 295-303. 128 38. Munoz, J.J., H. Arai, and R.L. Cole, Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis. Infect Immun, 1981. 32(1): p. 243-50. 39. Carbonetti, N.H., et al., Pertussis toxin targets airway macrophages to promote Bordetella pertussis infection of the respiratory tract. Infect Immun, 2007. 75(4): p. 1713-20. 40. Kirimanjeswara, G.S., et al., Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of Bordetella pertussis. J Clin Invest, 2005. 115(12): p. 3594-601. 41. Scheller, E.V. and P.A. Cotter, Bordetella filamentous hemagglutinin and fimbriae: critical adhesins with unrealized vaccine potential. Pathog Dis, 2015. 73(8): p. ftv079. 42. Alonso, S., et al., Role of ADP-ribosyltransferase activity of pertussis toxin in toxin-adhesin redundancy with filamentous hemagglutinin during Bordetella pertussis infection. Infect Immun, 2001. 69(10): p. 6038-43. 43. Locht, C., et al., The filamentous haemagglutinin, a multifaceted adhesion produced by virulent Bordetella spp. Mol Microbiol, 1993. 9(4): p. 653-60. 44. Ashworth, L.A., L.I. Irons, and A.B. Dowsett, Antigenic relationship between serotype-specific agglutinogen and fimbriae of Bordetella pertussis. Infect Immun, 1982. 37(3): p. 1278-81. 45. Relman, D., et al., Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell, 1990. 61(7): p. 1375-82. 46. Ishibashi, Y., S. Claus, and D.A. Relman, Bordetella pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/CD18). J Exp Med, 1994. 180(4): p. 1225-33. 47. Prasad, S.M., et al., Identification of a carbohydrate recognition domain in filamentous hemagglutinin from Bordetella pertussis. Infect Immun, 1993. 61(7): p. 2780-5. 48. Hannah, J.H., et al., Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA. Infect Immun, 1994. 62(11): p. 5010-9. 49. Urisu, A., J.L. Cowell, and C.R. Manclark, Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells. Infect Immun, 1986. 52(3): p. 695-701. 50. Henderson, I.R., F. Navarro-Garcia, and J.P. Nataro, The great escape: structure and function of the autotransporter proteins. Trends Microbiol, 1998. 6(9): p. 370-8. 51. Leo, J.C., I. Grin, and D. Linke, Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane. Philos Trans R Soc Lond B Biol Sci, 2012. 367(1592): p. 1088-101. 52. Leininger, E., et al., Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin. Infect Immun, 1992. 60(6): p. 2380-5. 53. Leininger, E., et al., Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells. Proc Natl Acad Sci U S A, 1991. 88(2): p. 345-9. 54. Hynes, R.O., Integrins: a family of cell surface receptors. Cell, 1987. 48(4): p. 549-54. 129 55. Khelef, N., et al., Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins. Infect Immun, 1994. 62(7): p. 2893-900. 56. Hovingh, E.S., et al., Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor. Emerg Microbes Infect, 2018. 7(1): p. 39. 57. Bumba, L., et al., Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. PLoS Pathog, 2010. 6(5): p. e1000901. 58. Fedele, G., M. Bianco, and C.M. Ausiello, The Virulence Factors of Bordetella pertussis: Talented Modulators of Host Immune Response. Archivum Immunologiae et Therapiae Experimentalis, 2013. 61(6): p. 445-457. 59. Fiser, R., et al., Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores. PLoS Pathog, 2012. 8(4): p. e1002580. 60. Mattoo, S. and J.D. Cherry, Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev, 2005. 18(2): p. 326-82. 61. Dorji, D., et al., Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance. Medical Microbiology and Immunology, 2018. 207(1): p. 3-26. 62. Luker, K.E., et al., Bordetella pertussis tracheal cytotoxin and other muramyl peptides: distinct structure-activity relationships for respiratory epithelial cytopathology. Proc Natl Acad Sci U S A, 1993. 90(6): p. 2365-9. 63. Koj, S., et al., Structure and Immunogenicity of the Bordetella pertussis LOS-Derived Oligosaccharides in the Endosomal-Like Pre-Processing Mice Model. Vaccines (Basel), 2021. 9(6). 64. Horiguchi, Y., Escherichia coli cytotoxic necrotizing factors and Bordetella dermonecrotic toxin: the dermonecrosis-inducing toxins activating Rho small GTPases. Toxicon, 2001. 39(11): p. 1619-1627. 65. Fernandez, R.C. and A.A. Weiss, Cloning and sequencing of a Bordetella pertussis serum resistance locus. Infect Immun, 1994. 62(11): p. 4727-38. 66. Marr, N., et al., Bordetella pertussis autotransporter Vag8 binds human C1 esterase inhibitor and confers serum resistance. PLoS One, 2011. 6(6): p. e20585. 67. Noofeli, M., et al., BapC autotransporter protein is a virulence determinant of Bordetella pertussis. Microb Pathog, 2011. 51(3): p. 169-77. 68. Hazenbos, W.L.W., et al., Bordetella pertussis Fimbriae Bind to Human Monocytes via the Minor Fimbria) Subunit FimD. The Journal of Infectious Diseases, 1995. 171(4): p. 924-929. 69. Hiramatsu, Y., et al., The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model. mBio, 2022. 13(2): p. e03197-21. 70. Carbonetti, N., What Causes the Cough in Whooping Cough? mBio, 2022. 13(3): p. e0091722. 71. Vandamme, T.F., Use of rodents as models of human diseases. J Pharm Bioallied Sci, 2014. 6(1): p. 2-9. 130 72. Bryda, E.C., The Mighty Mouse: the impact of rodents on advances in biomedical research. Mo Med, 2013. 110(3): p. 207-11. 73. Melvin, J.A., et al., Bordetella pertussis pathogenesis: current and future challenges. Nat Rev Microbiol, 2014. 12(4): p. 274-88. 74. Warfel, J.M., et al., Nonhuman primate model of pertussis. Infect Immun, 2012. 80(4): p. 1530-6. 75. Janda, W.M., et al., Unexpected isolation of Bordetella pertussis from a blood culture. J Clin Microbiol, 1994. 32(11): p. 2851-3. 76. Paddock, C.D., et al., Pathology and pathogenesis of fatal Bordetella pertussis infection in infants. Clinical infectious diseases, 2008. 47(3): p. 328-338. 77. Hellwig, S.M., et al., Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice. FEMS Immunology & Medical Microbiology, 1999. 26(3-4): p. 203-207. 78. Friedman, R.L., et al., Uptake and intracellular survival of Bordetella pertussis in human macrophages. Infection and immunity, 1992. 60(11): p. 4578-4585. 79. Lamberti, Y.A., et al., Intracellular trafficking of Bordetella pertussis in human macrophages. Infection and immunity, 2010. 78(3): p. 907-913. 80. Mahon, B.P. and K.H. Mills, Interferon-γ mediated immune effector mechanisms against Bordetella pertussis. Immunology letters, 1999. 68(2-3): p. 213-217. 81. Higgins, S.C., et al., TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. The Journal of Immunology, 2006. 177(11): p. 7980-7989. 82. Higgs, R., et al., Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunology, 2012. 5(5): p. 485-500. 83. Higgins, S.C., et al., Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology. The Journal of Immunology, 2003. 171(6): p. 3119-3127. 84. Banus, H.A., et al., Host genetics of Bordetella pertussis infection in mice: significance of Toll-like receptor 4 in genetic susceptibility and pathobiology. Infection and immunity, 2006. 74(5): p. 2596-2605. 85. Dunne, A., et al., Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. The Journal of Immunology, 2010. 185(3): p. 1711-1719. 86. Fedele, G., et al., Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways. PloS one, 2010. 5(1): p. e8734. 87. McGuirk, P., C. McCann, and K.H. Mills, Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. The Journal of experimental medicine, 2002. 195(2): p. 221-231. 88. Andreasen, C. and N.H. Carbonetti, Role of neutrophils in response to Bordetella pertussis infection in mice. Infect Immun, 2009. 77(3): p. 1182-8. 131 89. Confer, D. and J. Eaton, Pathogen-host jujitsu: phagocyte impotence caused by internalized bacterial adenylate cyclase. Transactions of the Association of American Physicians, 1982. 95: p. 1-7. 90. Zhang, X., et al., Decreased Leukocyte Accumulation and Delayed Bordetella pertussis Clearance in IL-6−/− Mice. The Journal of Immunology, 2011. 186(8): p. 4895-4904. 91. Barbic, J., et al., Role of gamma interferon in natural clearance of Bordetella pertussis infection. Infection and immunity, 1997. 65(12): p. 4904-4908. 92. Byrne, P., et al., Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen‐specific Th1 and enhancement of Th2, but not Tr1 cells. European journal of immunology, 2004. 34(9): p. 2579-2588. 93. Heininger, U., J.D. Cherry, and K. Stehr, Serologic Response and Antibody-Titer Decay in Adults with Pertussis. Clinical Infectious Diseases, 2004. 38(4): p. 591-594. 94. van Twillert, I., W.G.H. Han, and C.A.C.M. van Els, Waning and aging of cellular immunity to Bordetella pertussis. Pathogens and Disease, 2015. 73(8). 95. Murphy, K. and C. Weaver, Janeway's immunobiology. 2016: Garland science. 96. Goodman, Y.E., A.J. Wort, and F.L. Jackson, Enzyme-linked immunosorbent assay for detection of pertussis immunoglobulin A in nasopharyngeal secretions as an indicator of recent infection. J Clin Microbiol, 1981. 13(2): p. 286-92. 97. Tuomanen, E.I., et al., Characterization of antibody inhibiting adherence of Bordetella pertussis to human respiratory epithelial cells. J Clin Microbiol, 1984. 20(2): p. 167-70. 98. Hellwig, S.M., et al., Immunoglobulin A-mediated protection against Bordetella pertussis infection. Infect Immun, 2001. 69(8): p. 4846-50. 99. Wolfe, D.N., et al., Comparative role of immunoglobulin A in protective immunity against the Bordetellae. Infect Immun, 2007. 75(9): p. 4416-22. 100. Dalby, T., et al., Antibody responses to pertussis toxin display different kinetics after clinical Bordetella pertussis infection than after vaccination with an acellular pertussis vaccine. J Med Microbiol, 2010. 59(Pt 9): p. 1029-1036. 101. Oostindie, S.C., et al., Avidity in antibody effector functions and biotherapeutic drug design. Nature Reviews Drug Discovery, 2022. 21(10): p. 715-735. 102. Ross, P.J., et al., Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine. PLoS Pathog, 2013. 9(4): p. e1003264. 103. Warfel, J.M. and T.J. Merkel, Bordetella pertussis infection induces a mucosal IL-17 response and long-lived Th17 and Th1 immune memory cells in nonhuman primates. Mucosal Immunology, 2013. 6(4): p. 787-796. 104. Mills, K.H., et al., Cell-mediated immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance in a murine respiratory infection model. Infect Immun, 1993. 61(2): p. 399-410. 105. Wilk, M.M., et al., Lung CD4 Tissue-Resident Memory T Cells Mediate Adaptive Immunity Induced by Previous Infection of Mice with Bordetella pertussis. The Journal of Immunology, 2017. 199(1): p. 233-243. 106. HEININGER, U., et al., Clinical characteristics of illness caused by Bordetella parapertussis compared with illness caused by Bordetella pertussis. The Pediatric Infectious Disease Journal, 1994. 13(4): p. 306-309. 132 107. Mbayei, S.A., et al., Severe Pertussis Infections in the United States, 2011–2015. Clinical Infectious Diseases, 2018. 69(2): p. 218-226. 108. Abu-Raya, B., et al., Burden of Children Hospitalized With Pertussis in Canada in the Acellular Pertussis Vaccine Era, 1999-2015. J Pediatric Infect Dis Soc, 2020. 9(2): p. 118-127. 109. Mikelova, L.K., et al., Predictors of death in infants hospitalized with pertussis: a case-control study of 16 pertussis deaths in Canada. The Journal of Pediatrics, 2003. 143(5): p. 576-581. 110. Heininger, U., et al., Comparative Efficacy of the Lederle/Takeda acellular pertussis component DTP (DTaP) vaccine and Lederle whole-cell component DTP vaccine in German children after household exposure. Pertussis Vaccine Study Group. Pediatrics, 1998. 102(3 Pt 1): p. 546-53. 111. Kilgore, P.E., et al., Pertussis: Microbiology, Disease, Treatment, and Prevention. Clin Microbiol Rev, 2016. 29(3): p. 449-86. 112. Heininger, U., K. Stehr, and J.D. Cherry, Serious pertussis overlooked in infants. Eur J Pediatr, 1992. 151(5): p. 342-3. 113. Deeks, S., et al., Failure of physicians to consider the diagnosis of pertussis in children. Clin Infect Dis, 1999. 28(4): p. 840-6. 114. Nieves, D.J., et al., Clinical and Laboratory Features of Pertussis in Infants at the Onset of a California Epidemic. The Journal of Pediatrics, 2011. 159(6): p. 1044-1046. 115. Cherry, J., et al., Feigin and Cherry's Textbook of Pediatric Infectious Diseases E-Book: 2-Volume Set. 2013: Elsevier Health Sciences. 116. Case definitions for diseases under national surveillance. 2000, Public Health Agency of Canada. 117. Case definitions for communicable diseases under national surveillance. 2009, Public Health Agency of Canada. 118. Von König, C.-H.W., M. Riffelmann, and T. Coenye, Bordetella and Related Genera, in Manual of Clinical Microbiology. 2015. p. 838-850. 119. Loeffelholz, M., Towards improved accuracy of Bordetella pertussis nucleic acid amplification tests. J Clin Microbiol, 2012. 50(7): p. 2186-90. 120. van der Zee, A., J.F. Schellekens, and F.R. Mooi, Laboratory Diagnosis of Pertussis. Clin Microbiol Rev, 2015. 28(4): p. 1005-26. 121. Riffelmann, M., et al., Nucleic Acid amplification tests for diagnosis of Bordetella infections. J Clin Microbiol, 2005. 43(10): p. 4925-9. 122. Roorda, L., et al., A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements. BMC Research Notes, 2011. 4(1): p. 11. 123. Tizolova, A., N. Guiso, and S. Guillot, Insertion sequences shared by Bordetella species and implications for the biological diagnosis of pertussis syndrome. European Journal of Clinical Microbiology & Infectious Diseases, 2013. 32(1): p. 89-96. 124. Communicable Disease Control, Chapter 1 - Management of Specific Disease: Pertussis. 2010, BC Centre for Disease Control. 125. Wang, C., et al., Analysis of clinical characteristics of severe pertussis in infants and children: a retrospective study. BMC Pediatr, 2021. 21(1): p. 65. 133 126. Cherry, J.D., et al., An Observational Study of Severe Pertussis in 100 Infants ≤120 Days of Age. The Pediatric Infectious Disease Journal, 2018. 37(3): p. 202-205. 127. Rocha, G., et al., Severe Pertussis in Newborns and Young Vulnerable Infants. The Pediatric Infectious Disease Journal, 2013. 32(10): p. 1152-1154. 128. GOULIN, G.D., K.M. KAYA, and J.S. BRADLEY, Severe pulmonary hypertension associated with shock and death in infants infected with Bordetella pertussis. Critical Care Medicine, 1993. 21(11): p. 1791-1794. 129. Murray, E.L., et al., Characteristics of Severe Bordetella pertussis Infection Among Infants ≤90 Days of Age Admitted to Pediatric Intensive Care Units – Southern California, September 2009–June 2011. Journal of the Pediatric Infectious Diseases Society, 2013. 2(1): p. 1-6. 130. Bhagat, D., et al., Pertussis epidemiology in Canada, 2005–2019. Canada Communicable Disease Report, 2023. 49(1): p. 21-28. 131. Klein, N.P., Licensed pertussis vaccines in the United States. Human Vaccines & Immunotherapeutics, 2014. 10(9): p. 2684-2690. 132. Kuchar, E., et al., Pertussis: History of the Disease and Current Prevention Failure, in Pulmonary Dysfunction and Disease, M. Pokorski, Editor. 2016, Springer International Publishing: Cham. p. 77-82. 133. Pertussis. World Health Organization. 134. Pertussis (whooping cough) vaccines: Canadian Immunization Guide. Public Health Agency of Canada. 135. Update on immunization in pregnancy with tetanus toxoid, reduced diphtheria toxoid and reduced acellular pertussis (Tdap) vaccine. 2018, Public Health Agency of Canada. 136. Amirthalingam, G., et al., Effectiveness of maternal pertussis vaccination in England: an observational study. The Lancet, 2014. 384(9953): p. 1521-1528. 137. Dabrera, G., et al., A case-control study to estimate the effectiveness of maternal pertussis vaccination in protecting newborn infants in England and Wales, 2012-2013. Clin Infect Dis, 2015. 60(3): p. 333-7. 138. Baxter, R., et al., Effectiveness of Vaccination During Pregnancy to Prevent Infant Pertussis. Pediatrics, 2017. 139(5). 139. Vaccination schedule for Uganda. Available from: https://immunizationdata.who.int/pages/schedule-by-country/uga.html?DISEASECODE=&TARGETPOP_GENERAL=. 140. Kapil, P. and T.J. Merkel, Pertussis vaccines and protective immunity. Curr Opin Immunol, 2019. 59: p. 72-78. 141. da Silva Antunes, R., et al., Th1/Th17 polarization persists following whole-cell pertussis vaccination despite repeated acellular boosters. J Clin Invest, 2018. 128(9): p. 3853-3865. 142. Ausiello, C.M., et al., Vaccine- and antigen-dependent type 1 and type 2 cytokine induction after primary vaccination of infants with whole-cell or acellular pertussis vaccines. Infect Immun, 1997. 65(6): p. 2168-74. 143. White, O.J., et al., Th2-polarisation of cellular immune memory to neonatal pertussis vaccination. Vaccine, 2010. 28(14): p. 2648-2652. 144. Rowe, J., et al., Antigen-specific responses to diphtheria-tetanus-acellular pertussis vaccine in human infants are initially Th2 polarized. Infect Immun, 2000. 68(7): p. 3873-7. 134 145. Ryan, M., et al., Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children. Immunology, 1998. 93(1): p. 1-10. 146. van der Lee, S., et al., Whole-cell or acellular pertussis vaccination in infancy determines IgG subclass profiles to DTaP booster vaccination. Vaccine, 2018. 36(2): p. 220-226. 147. Schure, R.M., et al., Differential T- and B-cell responses to pertussis in acellular vaccine-primed versus whole-cell vaccine-primed children 2 years after preschool acellular booster vaccination. Clin Vaccine Immunol, 2013. 20(9): p. 1388-95. 148. Hendrikx, L.H., et al., Different IgG-subclass distributions after whole-cell and acellular pertussis infant primary vaccinations in healthy and pertussis infected children. Vaccine, 2011. 29(40): p. 6874-6880. 149. van der Zee, J.S., P. van Swieten, and R.C. Aalberse, Inhibition of complement activation by IgG4 antibodies. Clin Exp Immunol, 1986. 64(2): p. 415-22. 150. de Taeye, S.W., T. Rispens, and G. Vidarsson, The Ligands for Human IgG and Their Effector Functions. Antibodies (Basel), 2019. 8(2). 151. Rispens, T. and M.G. Huijbers, The unique properties of IgG4 and its roles in health and disease. Nature Reviews Immunology, 2023. 152. Valeri, V., et al., The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings. JCI Insight, 2022. 7(21). 153. Stenger, R.M., et al., Impaired long-term maintenance and function of Bordetella pertussis specific B cell memory. Vaccine, 2010. 28(40): p. 6637-46. 154. Versteegen, P., et al., Memory B Cell Activation Induced by Pertussis Booster Vaccination in Four Age Groups of Three Countries. Front Immunol, 2022. 13: p. 864674. 155. Cherry, J.D., et al., A search for serologic correlates of immunity to Bordetella pertussis cough illnesses. Vaccine, 1998. 16(20): p. 1901-1906. 156. Plotkin, S.A., Correlates of protection induced by vaccination. Clin Vaccine Immunol, 2010. 17(7): p. 1055-65. 157. Leef, M., et al., Protective Immunity to Bordetella pertussis Requires Both B Cells and Cd4+ T Cells for Key Functions Other than Specific Antibody Production. Journal of Experimental Medicine, 2000. 191(11): p. 1841-1852. 158. Halperin, S.A., et al., A Randomized Controlled Trial of the Safety and Immunogenicity of Tetanus, Diphtheria, and Acellular Pertussis Vaccine Immunization During Pregnancy and Subsequent Infant Immune Response. Clinical Infectious Diseases, 2018. 67(7): p. 1063-1071. 159. Munoz, F.M., et al., Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial. JAMA, 2014. 311(17): p. 1760-1769. 160. Perrett, K.P., et al., Immunogenicity, transplacental transfer of pertussis antibodies and safety following pertussis immunization during pregnancy: Evidence from a randomized, placebo-controlled trial. Vaccine, 2020. 38(8): p. 2095-2104. 161. Fallo, A.A., et al., Prevalence of Pertussis Antibodies in Maternal Blood, Cord Serum, and Infants From Mothers With and Those Without Tdap Booster Vaccination During Pregnancy in Argentina. Journal of the Pediatric Infectious Diseases Society, 2016. 7(1): p. 11-17. 135 162. Rice, T.F., et al., Antibody responses to Bordetella pertussis and other childhood vaccines in infants born to mothers who received pertussis vaccine in pregnancy – a prospective, observational cohort study from the United Kingdom. Clinical and Experimental Immunology, 2019. 197(1): p. 1-10. 163. Maertens, K., et al., Pertussis vaccination during pregnancy in Belgium: Results of a prospective controlled cohort study. Vaccine, 2016. 34(1): p. 142-150. 164. Maertens, K., et al., The Effect of Maternal Pertussis Immunization on Infant Vaccine Responses to a Booster Pertussis-Containing Vaccine in Vietnam. Clinical Infectious Diseases, 2016. 63(suppl_4): p. S197-S204. 165. Hoang, H.T.T., et al., Pertussis vaccination during pregnancy in Vietnam: Results of a randomized controlled trial Pertussis vaccination during pregnancy. Vaccine, 2016. 34(1): p. 151-159. 166. Taton, M., et al., Impact of pregnancy on polyfunctional IgG and memory B cell responses to Tdap immunization. Vaccine, 2023. 41(27): p. 4009-4018. 167. Palmeira, P., et al., IgG placental transfer in healthy and pathological pregnancies. Clin Dev Immunol, 2012. 2012: p. 985646. 168. Simister, N.E., et al., An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta. European Journal of Immunology, 1996. 26(7): p. 1527-1531. 169. Simister, N.E. and A.R. Rees, Isolation and characterization of an Fc receptor from neonatal rat small intestine. Eur J Immunol, 1985. 15(7): p. 733-8. 170. Simister, N.E. and K.E. Mostov, An Fc receptor structurally related to MHC class I antigens. Nature, 1989. 337(6203): p. 184-7. 171. Ober, R.J., et al., Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level. Proc Natl Acad Sci U S A, 2004. 101(30): p. 11076-81. 172. Vaughn, D.E. and P.J. Bjorkman, Structural basis of pH-dependent antibody binding by the neonatal Fc receptor. Structure, 1998. 6(1): p. 63-73. 173. Clements, T., et al., Update on Transplacental Transfer of IgG Subclasses: Impact of Maternal and Fetal Factors. Front Immunol, 2020. 11: p. 1920. 174. de Souza, E.G., et al., Maternal–Foetal Diabetes Modifies Neonatal Fc Receptor Expression on Human Leucocytes. Scandinavian Journal of Immunology, 2016. 84(4): p. 237-244. 175. Silveira Lessa, A.L., et al., Preterm and term neonates transplacentally acquire IgG antibodies specific to LPS from Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa. FEMS Immunol Med Microbiol, 2011. 62(2): p. 236-43. 176. van den Berg, J.P., et al., Transplacental transport of IgG antibodies specific for pertussis, diphtheria, tetanus, haemophilus influenzae type b, and Neisseria meningitidis serogroup C is lower in preterm compared with term infants. The Pediatric infectious disease journal, 2010. 29(9): p. 801-805. 177. Okoko, B.J., et al., The transplacental transfer of IgG subclasses: influence of prematurity and low birthweight in the Gambian population. Ann Trop Paediatr, 2002. 22(4): p. 325-32. 178. Saji, F., et al., Dynamics of immunoglobulins at the feto-maternal interface. Rev Reprod, 1999. 4(2): p. 81-9. 136 179. Flannery, D.D., et al., Comparison of Maternal and Neonatal Antibody Levels After COVID-19 Vaccination vs SARS-CoV-2 Infection. JAMA Network Open, 2022. 5(11): p. e2240993-e2240993. 180. Brambell, F.R., The transmission of immunity from mother to young and the catabolism of immunoglobulins. The Lancet, 1966. 288(7473): p. 1087-1093. 181. Scott, S., et al., Neonatal Measles Immunity in Rural Kenya: The Influence of HIV and Placental Malaria Infections on Placental Transfer of Antibodies and Levels of Antibody in Maternal and Cord Serum Samples. The Journal of Infectious Diseases, 2005. 191(11): p. 1854-1860. 182. Bento, A.I. and P. Rohani, Forecasting Epidemiological Consequences of Maternal Immunization. Clin Infect Dis, 2016. 63(suppl 4): p. S205-s212. 183. Niewiesk, S., Maternal Antibodies: Clinical Significance, Mechanism of Interference with Immune Responses, and Possible Vaccination Strategies. Frontiers in Immunology, 2014. 5. 184. Kim, D., et al., Insights into the regulatory mechanism controlling the inhibition of vaccine-induced seroconversion by maternal antibodies. Blood, The Journal of the American Society of Hematology, 2011. 117(23): p. 6143-6151. 185. Siegrist, C.-A., Mechanisms by which maternal antibodies influence infant vaccine responses: review of hypotheses and definition of main determinants. Vaccine, 2003. 21(24): p. 3406-3412. 186. Karlsson, M.C.I., et al., Efficient IgG-mediated suppression of primary antibody responses in Fcγ receptor-deficient mice. Proceedings of the National Academy of Sciences, 1999. 96(5): p. 2244-2249. 187. Amirthalingam, G., et al., Sustained Effectiveness of the Maternal Pertussis Immunization Program in England 3 Years Following Introduction. Clin Infect Dis, 2016. 63(suppl 4): p. S236-s243. 188. Kollmann, T.R., et al., Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from Immune Ontogeny. Immunity, 2017. 46(3): p. 350-363. 189. Vaz-de-Lima, L.R.A., et al., Effect of maternal Tdap on infant antibody response to a primary vaccination series with whole cell pertussis vaccine in São Paulo, Brazil. Vaccine: X, 2021. 7: p. 100087. 190. Wanlapakorn, N., et al., Quantity and Quality of Antibodies After Acellular Versus Whole-cell Pertussis Vaccines in Infants Born to Mothers Who Received Tetanus, Diphtheria, and Acellular Pertussis Vaccine During Pregnancy: A Randomized Trial. Clinical Infectious Diseases, 2019. 71(1): p. 72-80. 191. Guiso, N., et al., What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories. Eur J Clin Microbiol Infect Dis, 2011. 30(3): p. 307-12. 192. Mahony, J.B., et al., Performance and Cost evaluation of one commercial and six in-house conventional and real-time reverse transcription-pcr assays for detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol, 2004. 42(4): p. 1471-6. 193. Riffelmann, M., et al., Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis. J Clin Microbiol, 2010. 48(12): p. 4459-63. 137 194. Havers, F.P., Moro, P.L., Hariri, S., and Skoff, T. , Epidemiology and prevention of vaccine-preventable diseases. 14th ed. 2021: U.S. Department of Health and Human Services. 195. Dewan, K.K., et al., Acellular Pertussis Vaccine Components: Today and Tomorrow. Vaccines (Basel), 2020. 8(2). 196. Bartlett, B.L., A.J. Pellicane, and S.K. Tyring, Vaccine immunology. Dermatol Ther, 2009. 22(2): p. 104-9. 197. Rajna, M. and Z. Irena, Optimization, Validation and Standardization of ELISA, in Norovirus, M. Gyula, Editor. 2020, IntechOpen: Rijeka. p. Ch. 2. 198. Guideline on bioanalytical method validation. 2010, European Medicines Agency. 199. Bioanalytical method validation: guidance for industry. 2018, U.S. Food and Drug Administration. 200. Dixit, C.K., et al., Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays. Nat Protoc, 2011. 6(4): p. 439-45. 201. Miura, K., et al., Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines. Vaccine, 2008. 26(2): p. 193-200. 202. Guidance and protocol for the serological diagnosis of human infection with Bordetella pertussis. 2012: European centre for disease prevention and control. 203. Sakamoto, S., et al., Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of Natural Medicines, 2018. 72(1): p. 32-42. 204. Healy, C.M., M.A. Rench, and C.J. Baker, Importance of timing of maternal combined tetanus, diphtheria, and acellular pertussis (Tdap) immunization and protection of young infants. Clin Infect Dis, 2013. 56(4): p. 539-44. 205. Barnard, E.B., et al., Factors affecting blood sample haemolysis: a cross-sectional study. Eur J Emerg Med, 2016. 23(2): p. 143-6. 206. Lippi, G., et al., Haemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. Clin Chem Lab Med, 2008. 46(6): p. 764-72. 207. Simundic, A.-M., N. Nikolac, and G. Lippi, Hemolysis detection and management of hemolyzed specimens. Biochemia Medica, 2010. 20(2): p. 154-159. 208. Pertussis: Prevention. World Health Organization. 209. Dbaibo, G., V. Tatochenko, and P. Wutzler, Issues in pediatric vaccine-preventable diseases in low- to middle-income countries. Hum Vaccin Immunother, 2016. 12(9): p. 2365-77. 210. Global HIV & AIDS statistics - Fact Sheet. 2023; Available from: https://www.unaids.org/en/resources/fact-sheet. 211. Epidemiological fact sheet: HIV statistics, globally and by WHO region. 2023. 212. Nunes, M.C., et al., Kinetics of Hemagglutination-Inhibiting Antibodies Following Maternal Influenza Vaccination Among Mothers With and Those Without HIV Infection and Their Infants. The Journal of Infectious Diseases, 2015. 212(12): p. 1976-1987. 213. Heyderman, R.S., et al., Group B streptococcus vaccination in pregnant women with or without HIV in Africa: a non-randomised phase 2, open-label, multicentre trial. Lancet Infect Dis, 2016. 16(5): p. 546-555. 138 214. Nunes, M.C., et al., Neutralization and hemagglutination-inhibition antibodies following influenza vaccination of HIV-infected and HIV-uninfected pregnant women. PLoS One, 2018. 13(12): p. e0210124. 215. Almeida, V.D.C., et al., Immunogenicity of 23-valent pneumococcal polysaccharide vaccine in HIV-infected pregnant women and kinetics of passively acquired antibodies in young infants. Vaccine, 2009. 27(29): p. 3856-3861. 216. Kuhn, L., D.M. Thea, and G.M. Aldrovandi, Bystander effects: children who escape infection but not harm. Journal of acquired immune deficiency syndromes (1999), 2007. 46(5): p. 517. 217. Marinda, E., et al., Child mortality according to maternal and infant HIV status in Zimbabwe. The Pediatric infectious disease journal, 2007. 26(6): p. 519-526. 218. Cotter, A.M., et al., Is antiretroviral therapy during pregnancy associated with an increased risk of preterm delivery, low birth weight, or stillbirth? J Infect Dis, 2006. 193(9): p. 1195-201. 219. Voysey, M., et al., The Influence of Maternally Derived Antibody and Infant Age at Vaccination on Infant Vaccine Responses : An Individual Participant Meta-analysis. JAMA Pediatr, 2017. 171(7): p. 637-646. 220. Munoz, F.M., et al., Safety and immunogenicity of tetanus diphtheria and acellular pertussis (Tdap) immunization during pregnancy in mothers and infants: a randomized clinical trial. Jama, 2014. 311(17): p. 1760-9. 221. Puthanakit, T., et al., A phase 2 randomized controlled dose-ranging trial of recombinant pertussis booster vaccines containing genetically inactivated pertussis toxin in pregnant women. Vaccine, 2023. 41(31): p. 4541-4553. 222. Nunes, M.C., et al., Immunogenicity and safety of different dosing schedules of trivalent inactivated influenza vaccine in pregnant women with HIV: a randomised controlled trial. Lancet HIV, 2020. 7(2): p. e91-e103. 223. Wheatley, A.K., et al., HIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation. Sci Rep, 2016. 6: p. 26478. 224. Domenech de Cellès, M., et al., The pertussis enigma: reconciling epidemiology, immunology and evolution. Proceedings of the Royal Society B: Biological Sciences, 2016. 283(1822): p. 20152309. 225. Liko, J., S.G. Robison, and P.R. Cieslak, Priming with whole-cell versus acellular pertussis vaccine. New England Journal of Medicine, 2013. 368(6): p. 581-582. 226. Misegades, L.K., et al., Association of childhood pertussis with receipt of 5 doses of pertussis vaccine by time since last vaccine dose, California, 2010. Jama, 2012. 308(20): p. 2126-2132. 227. Cherry, J.D., The 112-year odyssey of pertussis and pertussis vaccines—mistakes made and implications for the future. Journal of the Pediatric Infectious Diseases Society, 2019. 8(4): p. 334-341. 228. Whooping Cough. [cited 2023; Available from: https://www.quebec.ca/en/health/health-issues/a-z/whooping-cough. 229. Québec, C.s.I.i.d., Optimisation du calendrier de vaccinaiton des jeunes enfants. 2018, Institute National de Santé Publique du Québec. 139 230. Québec Immunization Program. [cited 2023; Available from: https://www.quebec.ca/en/health/advice-and-prevention/vaccination/quebec-immunisation-program. 231. Febriani, Y., et al., Tdap vaccine in pregnancy and immunogenicity of pertussis and pneumococcal vaccines in children: What is the impact of different immunization schedules? Vaccine, 2023. 41(45): p. 6745-6753. 232. Martinón-Torres, F., et al., Impact of maternal diphtheria-tetanus-acellular pertussis vaccination on pertussis booster immune responses in toddlers: Follow-up of a randomized trial. Vaccine, 2021. 39(11): p. 1598-1608. 233. Abu-Raya, B., et al., The Effect of Tetanus-Diphtheria-Acellular-Pertussis Immunization During Pregnancy on Infant Antibody Responses: Individual-Participant Data Meta-Analysis. Front Immunol, 2021. 12: p. 689394. 234. Maertens, K., et al., Pertussis vaccination during pregnancy in Belgium: Follow-up of infants until 1 month after the fourth infant pertussis vaccination at 15 months of age. Vaccine, 2016. 34(31): p. 3613-3619. 235. Hardy-Fairbanks, A.J., et al., Immune Responses in Infants Whose Mothers Received Tdap Vaccine During Pregnancy. The Pediatric Infectious Disease Journal, 2013. 32(11). 236. Oguti, B., et al., The half-life of maternal transplacental antibodies against diphtheria, tetanus, and pertussis in infants: an individual participant data meta-analysis. Vaccine, 2022. 40(3): p. 450-458. 237. Pichichero, M.E., et al., A Safety and Immunogenicity Comparison of 12 Acellular Pertussis Vaccines and One Whole-Cell Pertussis Vaccine Given as a Fourth Dose in 15- to 20-Month-Old Children. Pediatrics, 1997. 100(5): p. 772-788. 238. Berner, R., et al., Safety and immunogenicity of fully liquid DTaP5-IPV-Hib pediatric combination vaccine (Pediacel®) compared to DTaP3-HBV-IPV/Hib (Infanrix® Hexa) when coadministered with heptavalent pneumococcal conjugate vaccine (PCV7) as a booster at 11–18 months of age: A phase III, modified double-blind, randomized, controlled, multicenter study. Vaccine, 2012. 30(35): p. 5270-5277. 239. Rane, M.S., P. Rohani, and M.E. Halloran, Association of Diphtheria-Tetanus-Acellular Pertussis Vaccine Timeliness and Number of Doses With Age-Specific Pertussis Risk in Infants and Young Children. JAMA Netw Open, 2021. 4(8): p. e2119118.
Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Sadarangani, Manish
Evaluation of anti-pertussis immunogenicity in infants and children after immunization in pregnancy with tetanus-diphtheria-acellular pertussis vaccine
Text