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Vaccine-specific immune responses in HIV-exposed uninfected infants Ruck, Candice 2020

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VACCINE-SPECIFIC IMMUNE RESPONSES IN HIV-EXPOSED UNINFECTED INFANTS  by  Candice Ruck  B.Sc., Trinity Western University, 2006  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  August 2020  © Candice Ruck, 2020   ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  The vaccine-specific immune response in HIV-exposed uninfected infants  submitted by Candice Ruck in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine  Examining Committee: Dr. Tobias Kollmann, Pediatrics Supervisor  Dr. David Scheifele, Pediatrics Supervisory Committee Member  Dr. Theodore Steiner, Infectious Diseases Supervisory Committee Member Dr Katie Flanagan, Immunology and Pathology, University of Tasmania Additional Examiner   Additional Supervisory Committee Members: Dr. David Speert, Pediatrics Supervisory Committee Member    iii  Abstract  The advent of antiretroviral (ARV) therapy has resulted in a steep decline in mother-to-child transmission of HIV and a corresponding increase in infants who are born HIV-exposed but uninfected (HEU). There is ample evidence to suggest that infants who have been vertically exposed to HIV are at higher risk of infectious morbidity and mortality. The cause of this increased risk remains unknown and is likely due to multiple factors, one of which may be deficiencies in immune development resulting from in utero exposure to HIV and /or antiretrovirals. The purpose of this study was to interrogate the adaptive immune response in HIV-exposed, uninfected infants to determine if they differ in a significant way from their unexposed (UE) counterparts. We assembled a cohort of HEU infants and UE control infants in Cape Town, South Africa between 2008-2011 and compared their humoral and cell-mediated immune responses to the Hepatitis B virus vaccine. The results presented here reveal that HEU infants develop a robust humoral response to vaccination that does not considerably differ from that of UE infants. We also determined that in both arms of the cohort, the cellular response to HBV vaccination was primarily Th2-biased, as measured by levels of plasma IL-5 and IL-13. This response was significantly greater in UE infants at 6 months but not 12 months of age. HBsAg stimulation did not induce a strong Th1 response in either group of infants, as assessed by secretion of IFNγ and TNFα. We concluded that HEU infants have a well-developed response to the HBV vaccine that provides sufficient immunity and differs only transiently from that of UE infants.  iv  Lay Summary  Infants who are born HIV-exposed but uninfected (HEU) consistently experience higher rates of infectious morbidity and mortality, although it is not known why. It is possible that in utero exposure to HIV causes immune deficiencies. We sought to determine if the vaccine-specific response of HEU infants is impaired compared to unexposed (UE) infants. We assembled a cohort in Cape Town, South Africa between 2008-2011 and compared the antibody and cell-mediated immune responses to the Hepatitis B vaccine between HEU and UE infants. We found that HEU infants develop an antibody response comparable to that of UE infants. We also determined that UE infants produced higher levels of the cytokines IL-5 and IL-13, although the difference was not significant by 12 months of age. We concluded that HEU infants have a functional response to the HBV vaccine that provides sufficient immunity and differs only transiently from UE infants.   v  Preface The design of the overall study and initial recruitment of the cohort were carried out by T. Kollmann, M. Esser, C. de Beer and B. Reikie. The design of the experiments included in this manuscript were carried out by T. Kollmann and myself. Patient visits and sample acquisition were organized by R. Adams and sample processing was carried out by R. Adams and myself. I conducted all experimental assays and data analyses related to the contents of this manuscript.  Chapter One: This chapter contains material that has been published as: Ruck CE, Reikie BA, Marchant A, Kollmann TR, Kakkar F. Linking Susceptibility to Infectious Diseases to Immune System Abnormalities among HIV-Exposed Uninfected Infants. Front Immunol. 2016;7:310.   I contributed to the conception of the paper, the gathering and reviewing of the data and the drafting of the initial manuscript, as well as the editing of the final draft. F Kakkar was also involved in the conception of this manuscript. BA Reikie and F Kakkar contributed to the gathering and review of data and the writing of the initial draft. A Marchand and T Kollmann contributed to the design and made key contributions. F Kakkar oversaw the final construction of the manuscript.  Chapter Three: This chapter contains material that forms part of the following manuscript: Reikie BA, Naidoo S, Ruck CE, Slogrove A, de Beer C, la Grange H, Adams RC, Ho K, Smolen K, Speert DP, Cotton MF, Preiser W, Esser M, Kollmann TR. Antibody responses to vaccination among South African HIV-exposed and unexposed uninfected infants during the first 2 years of life. Clin Vaccine Immunol. 2013;20:33-38.  vi  I conducted experiments and analyzed data that comprised a portion of this manuscript, and reviewed and edited the final draft.   I also made contributions to the following manuscripts that are not specifically included in this work: • Reikie BA, Adams RC, Ruck CE, Ho K, Leligdowicz A, Pillay S, Naidoo S, Fortuno ES 3rd, de Beer C, Preiser W, Cotton MF, Speert DP, Esser M, Kollmann TR. Ontogeny of Toll-like receptor mediated cytokine responses of South African infants throughout the first year of life. PLoS One. 2012;7:e44763 • Smolen K, Ruck C, Fortuno ES 3rd, Ho K, Dimitriu P, Mohn W, Speert D, Cooper P, Esser M, Goetghebuer T, Marchant A, Kollmann T. Innate immune responses differ in children from four different continents. J Alergy Clin Immunol. 2013;133:818-26 • Reikie BA, Adams RCM, Leligdowicz A, Ho K, Naidoo S, Ruck CE, Pillay S, de Beer C, Preiser W, Cotton MF, Speert DP, Esser M, Kollmann TR. 2014. Altered innate immune ontogeny defines window of vulnerability to infectious morbidity in HIV exposed but uninfected infants. J Acquir Immune Defic Syndr. 2014;66:245-55 • Sherrid AM, Ruck CE, Sutherland D, Cai B, Kollmann TR. Lack of Broad Functional Differences in Immunity in Fully vaccinated vs. Unvaccinated Children. Pediatr Res. 2017;81:601-608 • Gelinas L, Abu-Raya B, Ruck C, Cai B, Kollmann TR. Hepatitis B Virus Vaccine–Induced Cell-Mediated Immunity Correlates with Humoral Immune Response Following Primary Vaccination During Infancy. Immunohorizons. 2017;1:42-52  vii  Ethical Considerations: This study was performed in accordance with Good Clinical Practice and was approved by the Institutional Review Board of the University of British Columbia (Protocol H11-01947) and the Research Ethics Committee of Stellenbosch University (Protocol H09-02064). Written informed consent from the next of kin or guardian was obtained for all study participants in the language of their choice.  viii  Table of Contents  Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................. iv Preface..............................................................................................................................................v Table of Contents ......................................................................................................................... viii List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Abbreviations ................................................................................................................... xiv Acknowledgements ...................................................................................................................... xvi Dedication ................................................................................................................................... xvii Chapter 1: Literature Review……………………………………………………………..…….1 1.1  HIV-Exposed Uninfected Infants .............................................................................................1 1.2  Clinical Outcomes in HEU Infants ...........................................................................................2     1.2.1  Increased Infectious Mortality in HEU Infants ..................................................................2     1.2.2  Increased Infectious Morbidity in HEU Infants .................................................................2   1.2.3  Growth Abnormalities in HEU Infants…..……………..…………..………...……….…3    1.2.4  Pre-term Birth in HEU Infants….…………...……………...…..………………...……...4 1.3  Pathogenic Agents in HEU Infants…………………………....……………….…….…….....4 1.4  Proposed Mechanisms of Infectious Susceptibility…………………………..….…………...6   1.4.1  Effects of Antiretroviral Therapy…………………..…….………………….…………..7   1.4.2  Mode of Feeding……………………………...……….……...…………………………8 1.5  Immunity in HEU Infants………………..…………………………………………..….……9 ix       1.5.1  Innate Immunity………………..…………………….………………………………9   1.5.2  Adaptive Immunity………………..………..………………….……………...……11  Cellular Immunity………………………………………………............…….11  CD4 T-cell Counts………………………………………………...…....11  CD8 T-cell Counts……………..………………………………….…....12  T Lymphocyte Function……………..…………………………….…....12  B Lymphocytes………………………..……………….…………….…13  Humoral Immunity………..….………….………………..…….………...….13  Maternally Derived Antibodies...………………………………………13  Vaccine Induced Antibodies……….………………………………...…14 1.6  Hepatitis B……………………………………………………………………....……….….14   1.6.1  Hepatitis B Prevalence…………..………………………………………………..….15   1.6.2  Co-infection with HIV…………..…………………………………………………...15   1.6.3  HBV Vaccine……………………..………………………………………………….15   1.6.4  Immunity to HBV Vaccine……………..……………………………………………16 1.7  Study Overview………………………………………..………………………..….……….17   1.7.1  Specific Aims………………………………….……….…………….……...……….17 Chapter 2: Cohort Demographics……………………………………………………………..18 2.1  Hypothesis ...............................................................................................................................18 2.2  Specific Aims ..........................................................................................................................18 2.3  Introduction………………..…..………………………….……………………….………...18        2.4  Methods…………………… ..……………………………………………………………...20   2.4.1  Cohort Description……………………………………………….………..…….......21 x    2.4.2  Final Cohort……………………………………………………………………..…...23       2.4.3  Statistical Analysis…………………………….…………………..……………........24 2.5  Results……………..……………………………………………………………………...…24 2.6  Discussion…………..……………………………………………………………………….28 2.7  Conclusion………………………………………………..……………………………...….33 Chapter 3: Humoral Immunity in HEU Infants……………………………………………...34 3.1  Hypothesis ..............................................................................................................................34 3.2  Specific Aims ..........................................................................................................................34 3.3  Introduction…………………………………….…………………………………………....34 3.4  Methods……………………………………………………………………………..…........36   3.4.1  Clinic Visits………………………….…….………………………………………..36   3.4.2  Vaccination Schedule…………...………….……………………….….…………...37   3.4.3  Blood Draw………………….………………......…………………….……………37   3.4.4  Sample Collection & Storage……….……………..……………………….….........38   3.4.5  Determination of HBsAg-specific Antibody Titers………………….……..............38   3.4.6  Statistical Analysis………………………………..……..…………………….........38 3.5  Results……………………………………………………………………………………….39   3.5.1  Maternally Transferred anti-HBsAg Titers……..……………………..…….……...39   3.5.2  Vaccine-induced anti-HBsAg Titers……..………….………………....……...........40 3.6  Discussion……………………………………………………………………………...........42   3.6.1  Maternally Transferred Antibodies…………...……………………..….…..………42   3.6.2  Vaccine-induced Antibodies-6 Month Timepoin…….…………………………….45   3.6.3  Vaccine-induced Antibodies-12 Month Timepoint………………….……..............47 xi  3.7  Conclusions…………………………………………………….………………...………….49 Chapter 4: Cellular Immunity in HEU Infants…………………………………………….…50 4.1  Hypothesis…………………………………………………………………………………..50 4.2  Specific Aims…………………….……………………………………………………........50 4.3  Introduction……………………………..…………………………………………………..50 4.4  Methods………………………………………………………………………………..........52   4.4.1  Sample Acquisition & Processing…………….……………………..……….……...52   4.4.2  PBMC Stimulation Assay…………………………………………………….……..52   4.4.3  Cytokine Measurement…………………………………………………….…..........53   4.4.4  Statistical Analysis…………………………………………………………….........54 4.5  Results………………………….……………………………………………………………54   4.5.1  Cytokine Production at 6 months of Age………………………...…………………54   4.5.2  Cytokine Production at 12 Months of Age………………………………................57 4.6  Discussion…………………………………………………………………………………...60 4.7  Conclusion…………………………...……………………………………………………...65 Chapter 5: Summary…………………………………………………………………………...67 5.1  Summary of Results……………………………...……………………...…………………..67 5.2  Limitations…………………...……………………………………………………………...71 5.3  Future Directions………...………………………………………………………………….72 5.4  Conclusions………………...………………………………………………………………..73 Bibliography .................................................................................................................................75 xii  List of Tables  Table 2.1 Number of samples assayed at each timepoint………………………………………..23 Table 2.2 Cohort demographics at birth ........................................................................................25 Table 2.3 Measures of infant anthopometry  .................................................................................27 Table 3.1 Expanded programme on immunization schedule .........................................................37 Table 3.2 Time from vaccination to blood draw…………………………………………………41 Table 3.3 Levels of protection at pre-vaccine timepoints………………………………………..42  Table 4.1. Cytokine production at 6 month timepoint (pg/ml)………………………………….55 Table 4.2. Cytokine production at 12 month timepoint (pg/ml)…………………………………58  xiii  List of Figures  Figure 1.1 Mechanisms contributing to poor health and survival in HEU infants……………..…6 Figure 2.1 Overall study design of immune development cohort in South Africa………………21 Figure 3.1 HBsAg antibody titers at pre-vaccine timepoints .............................................................. 40 Figure 3.2 HBsAg antibody titers at post-vaccine timepoints…...………………………………….……41 Figure 4.1 Cytokine values of stimulated PBMC culture supernatants at 6 months of age ……….……...56 Figure 4.2 Cytokine values of stimulated PBMC culture supernatants at 12 months of age ………...…...59   xiv  List of Abbreviations  AIDS Acquired Immune Deficiency Syndrome APC Antigen Presenting Cell ART Antiretroviral Therapy BCG Bacillus Calmette Guerin BMI Body Mass Index BSID Bayley Scales of Infant Development cART Combination Antiretroviral Therapy CBMC Cord Blood Mononuclear Cells CD Cluster of Differentiation CMV Cytomegalovirus CRP C Reactive Protein DNA Deoxyribonucleic Acid EPI Expanded Program on Immunization GBS Group B Streptococcus HAART Highly Active Antiretroviral Therapy HAZ Height for Age HBsAg Hepatitis B Surface Antigen HBV Hepatitis B Virus HEU HIV-Exposed Uninfected Hib Hemophilus influenzae type b HIV Human Immunodeficiency Virus hMPV Human Metapneumovirus HR Hazard Ratio IFN Interferon Ig Immunoglobulin IL Interleukin IP-10 Interferon gamma-induced protein-10 IPD Invasive Pneumococcal Disease IRR Incidence Rate Ratio LAZ Length for Age LBW Low Birth Rate LPS Lipopolysaccharide LRTI Lower Respiratory Tract Infection MHC Major Histocompatibility Complex MTCT Mother to Child Transmission OPV Oral Polio Vaccine OR Odds Ratio PAMP Pathogen Associated Molecular Pattern PBMC Peripheral Blood Mononuclear Cells PCP Pneumocystis Pneumonia PCR Polymerase Chain Reaction xv  PMTCT Prevention of Mother to Child Transmission Therapy RR Risk Ratio RSV Respiratory syncytial virus SEB Staphylococcus enterotoxin B SGA Small for Gestational Age                                                                                  TB Tuberculosis TH T-helper TLR Toll-like Receptor TNF Tumor Necrosis Factor TNF-RI soluble Tumor Necrosis Factor Alpha Receptor I UNAIDS Joint United Nations Programme on HIV/AIDS                                                                      WAZ Weight for Age WHO World Health Organization WLZ Weight for Length   xvi  Acknowledgements  I would like to thank Dr. Kollmann for providing me with this opportunity and for your continuous support and guidance. I remain inspired by your passion for and dedication to not only the ‘what’ of research but also to the ‘why’.  I am also grateful to the members of my supervisory committee, Dr. D. Speert, Dr. T. Steiner and Dr. D Scheifele, for their encouragement and guidance that helped direct both my research and this thesis.  To the members of the Kollmann lab, particularly the ‘human studies’ side, I am thankful for your camaraderie and support. I am particularly grateful to Kinga Smolen, Bing Cai and Laura Gelinas for giving me the benefit of your knowledge, your technical expertise and your encouragement in the face of adversity. I could not have done this without you.  To my family, your unfailing support and encouragement of all of my endeavors, academic and otherwise, mean more to me than I can ever say. You are the reason for everything that I have accomplished, and you continue to motivate me every day. Thank you from the bottom of my heart.   xvii  Dedication  For my parents 1  Chapter 1: Literature Review 1.1. HIV-Exposed Uninfected Infants In 2018, there were 37.9 million people living with HIV worldwide.1 Among women living with HIV, approximately 1.5 million become pregnant each year.2 South Africa is the most affected nation, with over 7 million HIV-infected individuals as of 2018, and a prevalence rate among adults (aged 15-49) of 20.4%.3 In 2017, HIV prevalence among antenatal women was 30.7%.4 Thus, approximately 30% of infants born in South Africa are vertically exposed to HIV.  In the absence of antiretroviral therapy (ART), the risk of mother to child transmission (MTCT) ranges from 14-48%.5 The risk of transmission can be influenced by a number of factors, including but not limited to maternal disease status, mode of delivery and infant feeding practices.5 Prevention of mother to child transmission therapy (PMTCT) can significantly reduce the risk of vertical transmission. Globally, approximately 82% of HIV-infected pregnant women now have access to PMTCT.1 South Africa launched a nationwide PMTCT program in 2002 and by 2010, 91.7% of HIV-infected pregnant women were receiving either highly active antiretroviral therapy (HAART) or a PMTCT regimen consisting of ARV prophylaxis for mother and infant.6 The high rate of uptake of PMTCT in South Africa has resulted in a steep decline in rates of vertical transmission, which had fallen to 3.5% by 2010.6 The success of PMTCT has resulted in a sharp rise in the number of infants who are born HIV-exposed, but uninfected (HEU). As this population has expanded, both in South Africa and globally, evidence has accumulated that HEU infants are more susceptible to infectious morbidity and mortality compared to their unexposed (UE) counterparts.  2  1.2. Clinical Outcomes in HEU Infants The earliest reports of increased morbidity and mortality among HEU infants came from MTCT trials.7 Subsequent cohorts of HEU infants consistently reported a similar pattern across a wide geographic range. Studies from both the pre-PMTCT8,9 and post-PMTCT10,11 eras reported increased mortality that was predominantly due to infectious causes. 1.2.1. Increased Infectious Mortality in HEU Infants The infections that HEU infants experience are primarily pneumonia, diarrheal disease and sepsis, consistent with the leading causes of illness and death among infants regardless of HIV exposure.12 However, the HEU population suffers increased frequency and severity of these infectious events compared to UE infants, although where longitudinal data is available, this disparity has largely normalized by 2 years of age.12  HEU mortality increased from 2- to 4-fold above that of their UE counterparts in cohorts located in Zimbabwe (9.2% vs. 2.9%),13 Botswana (6.7% vs. 1.7%),14 Tanzania (5.7% vs. 1.3%),15 Zambia (5% vs. 1%),8 Malawi (18.7% vs. 4.3%),1 and Uganda (16.5% vs. 12.8%).9 Higher mortality rates from invasive pneumococcal disease (IPD),16 lower respiratory tract infection (LRTI),17 and pneumonia18 have all been reported in HEU infants.  In addition to in utero HIV exposure, reported risk factors for mortality among HEU infants include low birth weight (LBW),19 cessation of breastfeeding14 and advanced maternal disease.13 A pooled analysis of 7 MTCT trials over a range of follow-up periods identified maternal mortality and low maternal CD4 count as risk factors for infant mortality.20  1.2.2. Increased Infectious Morbidity in HEU Infants Higher rates of infectious morbidity have also been reported in the HEU population across multiple settings. HEU infants experienced higher rates of hospitalization in India,21 South 3  Africa,16 and Zimbabwe.22 As with mortality, the cause is predominantly infectious. HEU neonates in a large Caribbean cohort had a significantly higher incidence of infections compared to UE neonates (26/1000 vs. 8.1/1000),23 while HEU infants in a South African cohort were 3.6 times more likely to be hospitalized with a severe infection over the first year of life than were UE infants.24 Risk of cough, fever, and hospitalizations was significantly increased among HEU infants in a cohort in Tanzania,15 and HEU infants had a higher risk of all-cause lower respiratory tract infection (LRTI) (IRR: 1.4, CI 1.3-1.5) in a South African surveillance study as well as having longer hospital stays and being more likely to die in-hospital.17  1.2.3. Growth Abnormalities in HEU Infants Multiple studies have described that HEU infants are deficient in growth across numerous metrics. These include geographically diverse populations including Kenya,25 Tanzania,26 Malawi,27 Cameroon,28 Uganda,29 Nigeria,30 South Africa,31 Brazil,32 and the U.S.33,34 While few studies have examined longitudinal patterns of growth in this population, a large cohort study in Zimbabwe reported reduced growth in HEU infants compared to UE infants over the first 12 months of life.35 Similarly, a longitudinal cohort study in South Africa reported lower mean weight-for-age (WAZ) scores at all timepoints over the first 12 months, as well as lower mean length-for-age (LAZ) scores and a higher proportion of stunting in HEU compared to UE infants.36  There is evidence that this trend towards suppressed growth is transient. Studies of longitudinal cohorts that have described reduced early growth in HEU infants have also reported that these disparities either lessened or disappeared altogether by later timepoints.15,37,38 However, results have been inconsistent, and few studies have extended into childhood. A Zambian study reported that HEU infants had lower weight-for-age, height-for-age and body 4  mass index (BMI)-for-age Z-scores at 18 months, and that these differences were still present when children were re-examined at 7.5 yrs,39 while a large European study that followed participants to 10 years of age found that HEU children displayed normal growth patterns consistent with the reference standards.40 In HEU infants, growth deficiencies are associated with increased morbidity and mortality, a trend which mirrors that of the general population. Low birth weight (LBW) was associated with a 6-fold increase in the incidence rate (IR) for neonatal mortality, while small-for-gestational-age (SGA) infants had a 7-fold higher neonatal mortality IR compared to HEU infants of average weight.41 In a South African birth cohort, lower WAZ was associated with an increased risk of severe pneumonia requiring hospitalization in HEU infants.42 Higher WAZ scores were associated with a 28% decreased risk of infectious disease hospitalization in a Kenyan HEU cohort,43 and low birth weight was significantly associated with 24 month mortality in an HEU cohort in Botswana.10  1.2.4. Pre-term Birth in HEU Infants Multiple studies have reported lower gestational age in HEU infants.31,32,44 Pre-term birth (PTB) is associated with lower birth weight and could be related to the growth deficiencies observed in HEU infants. One cohort of HEU infants reported that 61% of LBW cases were attributable to PTB.41  PTB is also associated with an increased risk of mortality, with a 7-fold increase in neonatal mortality incidence risk reported in HEU infants that were born pre-term compared to term births.41  1.3. Pathogenic Agents in HEU Infants Identifying susceptibilities to particular pathogens could be indicative of specific immune deficiencies. Thus far, the most telling infectious finding in HEU infants has been a series of case 5  reports of HEU infants infected with the fungus Pneumocystis jirovecii (previously carinii). A case series in 1997 described P. jirovecii pneumonia (PJP) in 2 HEU infants in Texas.45 PJP was subsequently diagnosed in HEU infants in 3 separate cohorts in South Africa.46-48 As this organism is typically an opportunistic pathogen primarily identified in immunocompromised individuals, these findings in HIV-negative infants are suggestive of an immune deficiency, particularly with respect to the function of T lymphocytes.49 Unfortunately, a shortcoming of many of the HEU studies is that the causative organism was not identified, often due to constraints in testing associated with resource-poor settings. Where testing has been performed, HEU infants have demonstrated an increased susceptibility to encapsulated organisms such as Streptococcus pneumoniae and Group B Streptococcus (GBS). Rates of invasive pneumococcal disease caused by S. pneumoniae were higher in HEU infants in South Africa16 and Belgium, where a 4-fold higher incidence of IPD was observed.50 A 13-fold higher incidence of group GBS infection was also observed in this cohort,51 while a 2.25-fold greater incidence of GBS was reported in a hospital-based study in South Africa.52 In a large cohort of HEU infants in France encapsulated organisms (Hemophilus influenzae and S. pneumoniae) were responsible for 56.7% of bacterial infections.53        While increased viral infections could also point towards specific immune deficiencies, the difficulty of testing for viral pathogens in resource-limited settings has resulted in a dearth of information on viral prevalence in HEU infants. In a South African study of infants hospitalized with LRTIs, an increased incidence of Respiratory Syncytial Virus (RSV) and Human Metapneumovirus was reported among HEU infants, while case fatality ratios were increased in HEU infants with RSV compared to UE infants.17  6  1.4. Proposed Mechanisms of Infectious Susceptibility The existence of a range of clinical and developmental deficiencies in HEU infants is well described in the literature; however, heterogeneity between studies is a confounding factor when attempting to draw conclusions from the available data. Differences between studies in administration of maternal and infant antiretroviral therapy, inconsistencies in mode of feeding, and differences in maternal immune status and socioeconomic status all complicate any attempts to draw conclusion about the effects of in utero HIV exposure.  Thus, the mechanism underlying the increased clinical severity remains unknown and is likely due to multiple factors, potentially including exposure to antiretrovirals, an increased infectious environment, a more inflammatory maternal environment, reduced maternal care due to illness and differences in mode of feeding.54   Figure 1.1: Mechanisms contributing to poor health and survival in HEU infants From: Ruck C et al. “Linking Susceptibility to Infectious Diseases to Immune System Abnormalities among HIV-Exposed Uninfected Infants”. Front Immunol. 2016;7:310. Licensed under CC-BY v. 4.0.    7  1.4.1. Effects of Antiretroviral Therapy The enormous benefits of antiretrovirals (ARVs) in preventing vertical transmission of HIV are undeniable. However, the long-term effects of ARV exposure on infant development are incompletely understood, although it has been cited as a potential factor in growth deficiencies.55 This is far from conclusive, as growth abnormalities among HEU infants have been reported from the pre-ARV era,39 and a meta-analysis found that maternal ARV use did not significantly worsen the risk of PTB or LBW in HEU infants,56 although there is evidence that timing of ARV initiation and type of therapy (combination vs. monotherapy) may influence infant outcomes.57 In utero exposure to maternal HAART was associated with a greater risk of severe infant anemia compared to infants exposed to zidovudine alone.58 ARV exposure has also been linked to immune abnormalities in infants, although these results are also inconsistent. Lower total lymphocyte and neutrophil counts and reduced absolute CD4 counts were observed in ARV-exposed (HEU) infants at 0-2 months of age.59 These disparities in lymphocyte and CD4 counts persisted at 6-25 months of age.59 Similarly, ARV exposure was associated with reductions in CD4 and CD8 counts in the French Perinatal Cohort.60  No significant difference in T-cell subsets was found between neonates born to ARV-treated and untreated mothers.55 Plasma levels of IL-10 were lower in the treated group, while IL-1b and TNF-a were higher compared to the untreated arm. A similar trend was also observed in the maternal arm of this cohort.55 Upon stimulation of T-cell cultures with anti-CD3, levels of TNF-a and IFN-g were significantly higher in the ARV-exposed neonates, while levels of IL-10 were lower. Again, this trend was mirrored in the maternally derived cultures.55 Both increased maternal IL-10 levels and decreased maternal TNF-a levels were associated with higher birth weight.55 Maternal HAART was associated with a reduction in TNF-a levels in cord blood.61 8  Increased neutropenia was associated with combination therapy compared to monotherapy.62 HEU infants demonstrated increased CD4 and CD8 proliferation in response to both SEB and BCG, a distinction that was not diminished when duration of ARV use was factored into the analysis.63 These findings, although of interest, are limited by heterogeneity between study designs and difficulties in controlling for study variables including drug use, geography and socioeconomic indicators.    1.4.2. Mode of Feeding Mode of feeding is an additional factor that could contribute to poor clinical outcomes in HEU infants. Breastfeeding has convincingly been shown to have both nutritional and immunological benefits. The immune components of breastmilk, including immunoglobulins, cytokines, antimicrobial proteins/peptides, and oligosaccharides, promote protection of the infant during the early period of immune vulnerability.54 Numerous studies have reported that lack of breastfeeding is associated with increased malnutrition, as well as increased infections including RTIs and diarrhea.64  Prior to 2010, WHO guidelines recommended that HIV-positive women practice formula feeding for the first 6 months of life, with breastfeeding recommended only when formula feeding was not feasible, affordable, sustainable or safe.65 The widespread adoption of replacement feeding among HIV-positive women was associated with a rise in morbidity and mortality among their HEU infants,64 driven largely by increased rates of infections66 and malnutrition.67 Use of replacement feeding has also been linked to an increased risk of mortality in both UE68 and HEU infants.69-72 9  1.5. Immunity in HEU Infants HEU infants experience in utero exposure to HIV and /or antiretrovirals at a time when their immune system is still undergoing development and is susceptible to external influence. It has therefore been hypothesized that the increased infectious susceptibility experienced by HEU infants may be related to deficiencies in immune development resulting from this in utero exposure. To this end, the innate and adaptive immune system of HEU infants has been extensively interrogated, from both a phenotypic and a functional perspective. A comprehensive review of these studies reveals that the immunologic development of HEU infants is broadly similar to that of unexposed infants, although several differences have been reported, as described below.  The majority of studies have been cross-sectional and performed within the first 12 months of life and are therefore unable to determine whether the disparities are transient or fixed. Increased immune activation has been reported in HEU children between 6-12 years of age.73 In contrast, differences in innate immunity detected in the longitudinal cohort presented here were no longer detectable by 12 months of age.74 This is consistent with the picture that has emerged from a compilation of the infectious morbidity and mortality studies, which is that the increased infectious risk is largely confined to the first 2 years of life. 1.5.1. Innate Immunity Thus far, few studies have focused on the innate arm of the HEU immune response, and inconsistencies abound. Antigen presenting cells (APC) of HEU infants have been variously reported as being either hypo-or hyper-responsive. In the South African cohort presented here, pathogen associated molecular pattern (PAMP)-stimulated APCs of HEU infants displayed a higher proportion of responder cells and greater magnitude of cytokine production per cell 10  compared to matched UE infants.74 This hyper-responsive innate profile was observed at 2 & 6 weeks of age and had normalized by 12 months of age.  Increased expression of MHCII on unstimulated APCs,75 as well as higher monocyte- and cDC-derived production of IL-12 in response to stimulation with multiple PAMPs74 has been reported in HEU infants. Conversely, reduced CBMC-derived IL-12 production was reported in a US-based cohort.76  IL-12 is an important inducer of Th1 cells, and a reduction in this cytokine, if verified in the HEU population, could explain an impaired memory/effector Th1 response to intracellular pathogens.77  An anergic TLR4 response to LPS stimulation was observed in 7/12 HEU infants in a study investigating the ability of HEU infants to recognize and respond to bacterial pathogens,78 findings that could indicate an impaired ability to respond to gram-negative bacteria among a sub-set of HEU infants, although the mechanism remains unclear. However, these findings conflict with the observation that HEU infants had a higher comparative proportion of monocytes and cDCs responding to LPS stimulation at 6 weeks of age.74   The most consistent finding across studies is that of a more hyper-inflammatory profile among the HEU population. HEU neonates had increased levels of systemic inflammatory markers CRP,32,79 IL-6,74,80 IL-8,80 IP-10,32 sCD14,32 and sTNF-RI.32 While some longitudinal studies reported that inflammation present at birth subsided over time,32,74 CRP remained elevated in a Zimbabwean cohort at 6 months of age.79 The presence of a hyper-inflammatory state in HEU infants could induce immune paralysis,78 blunting the ability of APCs to stimulate the cell mediated immune (CMI) response and resulting in increased infectious susceptibility.  11  1.5.2. Adaptive Immunity As noted above, the findings of PJP in HEU infants suggests the presence of an immune deficiency and  implicates T lymphocytes as an area of particular focus. Cellular Immunity Investigations into the cell-mediated compartment of the immune response in HEU infants have frequently reported a profile of increased inflammation and activation. At 3-6 months of age, both the T cell and B cell profiles of HEU infants was more similar to that of HIV-infected infants than to UE infants.81 Results have been inconsistent, likely due to heterogeneity between studies and populations, and therefore the mechanism underlying these observations has yet to be elucidated.  A more thorough examination of how the CMI response functions in the HEU population, and how it influences the response to intracellular pathogens, is required to develop clinical guidelines to protect this vulnerable population. CD4 T-cell Counts HIV specifically targets CD4 T cells, therefore the quantity and function of this subset of the CMI response is of particular interest. Over the first year of life, reduced CD4 T cell counts were present in HEU compared to UE infants, both with82,84 and without ARV prophylaxis.83 These disparities appear to be transient and confined to early life, as they had resolved by 12 months of age in one study,82 while another study reported a higher absolute count of CD4 T cells in HEU infants at 6-12 months of age.85  Few studies have thus far undertaken comparable investigations in older children. Frequencies of CD38+HLA-DR+CD4+T cells were increased compared to controls when measured at 12 months of age, and this difference persisted at follow-up between 6-12 years of age, suggesting a state of increased immune activation that persists into childhood.73 Comparison 12  of total cell numbers in HEU and UE children aged 6-18 years revealed a persistent reduction in CD4 subsets in HEU children; however, when stratified by ARV use this difference was only significant for HEU infants exposed to ARVs.86 Where differences in lymphocyte subsets are detected, it remains elusive whether the variances described are primarily attributable to HIV exposure, ARV exposure or some combination of these. CD8 T-cell Counts A lower percentage of naïve CD8 cells and a higher percentage of effector memory CD8 cells in HEU compared to UE infants at 6 months of age was reported in a Malawian cohort; however, these differences were no longer detectable by 12 months of age.82 Percentages of CD8 T cells were slightly reduced in HEU infants at 3-6 months of age; again, by 12 months the difference was no longer statistically significant.85 T Lymphocyte Function Functional capacity of T cell subsets in HEU infants appears to vary by age and stimulus. While cytokine polyfunctionality was diminished in response to stimulation with Staphylococcal enterotoxin B (SEB), Bacillus Calmette-Guerin (BCG), and Bordetella pertussis,63 CD4 and CD8 proliferation increased in response to SEB and BCG but not to B pertussis.63 No difference in TNF-a, IFN-g or IL-2 production was detected in response to tetanus toxoid at 3 months of age, however at 12 months of age IL-2 production decreased significantly in the HEU arm of the cohort.87 The degree of HIV exposure may be a contributing factor. When cellular responses to polyclonal stimuli were analyzed by maternal viral load, lymphoproliferation as well as IFN-g and TNF-a were detected at higher levels in neonates whose mothers did not control the virus compared to those who did.88   13 B Lymphocytes Thus far, few studies have investigated B cells beyond the measure of antibodies as a surrogate for B cell function, and results have been inconsistent. Proportions of memory B cells were reduced in HEU infants, suggesting that memory B cell responses could be delayed in HEU infants, although the antibody response was not affected by these findings.89 No difference in B cell subsets was detected at 6 and 12 months of age,82 while an increased CD19+ B cell count was observed in HEU infants from 3 to 12 months of age in a separate cohort.85 As with the T cell compartment, B cells may be quantitatively affected by maternal viral load. An increased percentage of CD19+ B cells was only observed in infants born to mothers with viral load >1000 copies/ml.90 Humoral Immunity Decreased levels of pathogen-specific antibodies are frequently associated with HIV infection.91-93 Levels of  anti-GBS antibodies were lower in HIV-positive women compared to HIV-negative women.91,92 HIV-positive women also demonstrated reduced levels of specific antibodies to Hemophilus influenzae type B (Hib) and pneumococcus.93 Therefore, deficits in maternally derived humoral immunity have been investigated as contributing factors to the increased infectious susceptibility among HEU infants. Humoral responses have also been extensively investigated in HEU infants to determine whether they receive adequate levels of vaccine-mediated protection. Maternally Derived Antibodies The immaturity of the immune system in early life leaves infants vulnerable to infections. During this time, protection is conferred by maternally acquired antibodies, transferred both transplacentally and by breastfeeding.94 Levels of maternally derived antibodies are frequently 14  diminished in HEU infants. Reduced levels of placentally transferred antibodies to tetanus,93,95-97 measles,98,99 pertussis,93 Hib,93,100 polio,101 pneumococcus,93 and GBS91 have all been reported in HEU infants.  Due to the risk of MTCT associated with breastfeeding, rates and duration of exclusive breastfeeding are frequently reduced among HIV-infected women, depriving HEU infants of protective post-natal antibodies during the early period of immune vulnerability prior to vaccination. Vaccine Induced Antibodies Despite frequently receiving reduced humoral protection at birth, a robust antibody response following vaccination has been reported in multiple HEU cohorts, although, as with the CMI response, there are numerous inconsistencies. Compared to UE infants, HEU infants displayed higher titers of antibodies following vaccination to pertussis (134.3 IU/ml vs. 261.3 IU/ml, p < 0.001)102 and pneumococcus (47.32 mg/l vs 14.77 mg/l, p = .001).93 In contrast, HEU infants had lower geometric mean anti-tetanus tires compared to UE infants (1.52 IU/ml vs. 2.71 IU/ml, p=0.01).103  1.6. Hepatitis B Hepatitis B virus (HBV) infects the liver and may cause acute or chronic infections. Although fewer than 10% of adults become chronically infected, this number is far higher in infants who have been perinatally exposed.104 HBV is a risk factor for the development of cirrhosis of the liver and hepatocellular carcinoma,104 and is associated with approximately 600,000 deaths per year globally.105  15  1.6.1. Hepatitis B Prevalence Globally, seroprevalence of Hepatitis B is estimated at 3.7%, as of 2005.104 Endemicity varies widely by region, with much of Africa being highly endemic, with a seroprevalence greater than 8%.105 In South Africa, HBV prevalence rates of 9.7% have been reported.106 South African prevalence varies greatly between rural and urban populations, with estimates ranging from approximately 1% in urban areas to 10% in rural areas.107  1.6.2. Co-infection with HIV In common with HIV, HBV is transmitted via blood or other bodily fluids.104 Thus, co-infection with both viruses is not uncommon, particularly in regions such as sub-Saharan Africa where both are endemic. The co-infection rate was 4.8% in an urban HIV-infected cohort in Johannesburg,107 and 6.2% in a large urban cohort in Tanzania,108 while cohorts in Mozambique and Zambia reported co-infection rates of 7.6% and 11.3%, respectively.109 A rural HIV-positive cohort in the Eastern Cape region of South Africa showed a prevalence rate of 7.1% for HIV-HBV co-infection, although rates were notably higher in males than females, at 12.1% compared to 5.5%.110 A South African antenatal cohort study of HIV-positive women reported that 7.4% were diagnosed with chronic hepatitis B.111  1.6.3. HBV Vaccine The HBV vaccine is a recombinant DNA vaccine consisting of Hepatitis B surface antigen (HBsAg).105 It typically contains alum as an adjuvant and is administered via intramuscular injection in a 3-dose series in early infancy. At the time this study was implemented, the Expanded Program on Immunization (EPI) schedule in South Africa called for the administration of the vaccine at 6, 10, and 14 weeks of age. In addition to reducing morbidity and mortality due 16  to acute infection, the Hepatitis B vaccine has also contributed to a drastic reduction in the rate of liver cancer worldwide.112 The vaccine is considered highly effective, and produces a protective response in >90% of vaccinees.105 Anti-HBs levels ≥10 mIU/ml are considered to be a reliable correlate of protection, as this level of response was protective against HBV infection for 90-100% of individuals in vaccine efficacy studies.105 Five years after it was introduced into the EPI schedule in South Africa, an assessment of the efficacy reported 86.8% of vaccinated infants had HBsAg-specific antibody titers ≥10 mIU/ml.113  This study compares the development of the pathogen-specific adaptive immune response between infants who are HIV-exposed and those who are not. To ensure that the type, dose and timing of the pathogenic exposure are consistent amongst all participants, the investigation is focused on the development of the adaptive immune response to the HBV vaccine. 1.6.4. Immunity to HBV Vaccine Although anti-HBsAg titers have typically been used as a measure of HBV vaccine efficacy, there is evidence that the CMI response also plays an important role in the vaccine-specific response. Reports of long-term cellular responses even in the absence of protective antibody titres114,115 indicate that serology is not the only measure of protection. Cytokines also play an essential role in vaccine-induced immunity. Non-responses to the HBV-vaccine may be related to cytokine deficiencies,116,117 suggesting that assessment of cytokine production may be an effective measure of vaccine-specific immunity. 17  1.7. Study Overview The hypothesis of this study is that HEU infants experience impaired immune development related to in utero exposure to HIV, and that this impairment contributes to an increased infectious susceptibility. 1.7.1. Specific Aims The specific aims of this study are as follows: 1) To compare the levels of maternal antibodies received by HEU infants prior to vaccination with that of UE infants. 2) To compare the HBsAg-specific response, both humoral and cellular, between HEU and UE infants at 6 and 12 months of age and determine if HEU infants are impaired in their ability to develop an anamnestic response to the HBV vaccine.  18  Chapter 2: Cohort Demographics 2.1. Hypothesis The hypothesis of this chapter is that HEU infants experience demographic differences compared to UE infants, and that these differences contribute to an increased infectious morbidity and mortality.  2.2. Specific Aims The specific aims for this chapter are: 1) To compare maternal health demographics between HEU and UE infants 2) To compare socio-economic and environmental determinants of health between HEU and UE infants 3) To compare growth metrics and other infant health indices between HEU and UE infants 2.3. Introduction The past two decades have seen a surge in the availability of ARV use in the developing world, where the burden of HIV is heaviest. In South Africa, nearly 30% of women attending antenatal clinics are HIV-positive.118 By 2011, the expansion of the national PMTCT program had reduced the rate of vertical HIV transmission to 2.7% of infants born to HIV-infected mothers,119 resulting in massive expansion of the population of HEU infants. A growing body of research has revealed that in utero exposure to HIV is associated with a worsening of clinical outcomes for these infants, specifically an increase in infectious morbidity15-17,22,23 and mortality.8,9,11,13-15  Although the mechanisms underlying this increased infectious susceptibility have not been definitively determined, it is likely that the causes are multifactorial and may be influenced by a range of maternal, infant, and environmental factors. 19  The environment exerts a strong influence on the development of the infant immune system. The earliest developmental influences are derived from the mother, and therefore disruptions to the maternal environment may affect infant development. Maternal infections,120 a pro-inflammatory maternal milieu,121 and disturbance of the maternal microbiome122 all influence early immune ontogeny. HEU infants also experience increased exposure to opportunistic pathogens associated with HIV infection. Complications associated with maternal HIV infection including increased rates of maternal illness and mortality, and the associated reduction in maternal care, may also increase infant susceptibility to infections, as does exposure, both antenatal and neonatal, to the ARVs that comprise the PMTCT regimen.   Disparities in mode of feeding further contribute to the increased infectious susceptibility of HEU infants. Breastmilk contains components that have immunomodulatory properties that contribute to infant immune development,123 as well as maternally derived antibodies that provide the infant with additional protection during the period of immune vulnerability associated with early life. Despite the highly beneficial effects of breastfeeding, the increased risk of HIV transmission has resulted in higher rates of formula feeding among HEU infants, as well as reduced duration of exclusive breastfeeding, as a means of reducing the risks of MTCT.36,124 Replacement feeding has been linked to an increased risk of infectious morbidity and mortality, in both HEU64,69,71,72 and healthy infants.68  A pooled analysis found an association between never breastfeeding and an increased risk of mortality in HEU infants.125 It is probable that the increased infectious morbidity and mortality experienced by HEU infants is in fact due to a complex interplay between a number of these factors. However, it remains an open question as to whether impairment in the immune response is the mechanism via which these factors exert their influence. This investigation represents one component of a 20  prospective cohort study formed for the purpose of providing a comprehensive comparison of the longitudinal development of the immune system between HEU and UE infants. The specific focus of this study is to detect the existence of variations in the development of the adaptive immune response of the HBV vaccine. As part of this study, demographic data was collected for multiple maternal and infant parameters, the analysis of which is presented in this chapter. 2.4. Methods This is a sub-study of a prospective longitudinal pilot study formed for the comparison of immune development and function between HEU and UE infants. The study recruited infants at birth and followed them at regular intervals over the first 2 years of life (Fig. 2.1). The overarching purpose of the larger study was to perform a comprehensive assessment of the innate and adaptive immune development in HEU infants and compare it to that of UE infants. The study was a collaborative effort between the University of British Columbia in Canada and Stellenbosch University in South Africa and ethics approval was obtained from both institutions.   21   Figure 2.1. Overall study design of immune development cohort in South Africa        BCG, bacille Calmette-Guerin; OPV, oral polio vaccine; IPV, inactivated polio vaccine* (from July 2009);  DTP, diptheria-tetanus-pertussis; HBV, hepatitis B virus; Hib, hemophilus influenzae type B;  PCV, pneumococcal conjugate vaccine; PBMC, peripheral blood mononuclear cells  2.4.1. Cohort Description Study participants were recruited from the maternity ward of Tygerberg Hospital over the period of March-June 2009. Mothers were recruited following delivery and informed consent was obtained by a qualified study nurse in the preferred language of the mother/caregiver. All participants were informed that they could withdraw from the study at any time.  Infants were required to be clinically healthy with no congenital abnormalities, and to be of appropriate gestational age and birth weight to be eligible for the study. Maternal exclusion criteria included treatment for current infection or TB, untreated hypertension, anemia or malnutrition.  Infant HIV status was tested by HIV DNA-PCR Amplicore HIV-1 DNA test kit 22  version 1.5 (Roche Diagnostics) at 2 and 6 weeks of age, with 4 infants testing positive at the 2-week timepoint. These infants were subsequently excluded from analysis.  As part of routine care, CD4 counts were obtained for HIV-positive mothers following confirmation of infection. All available maternal CD4 counts were extracted from hospital records and included in study documentation. ARV regimens were determined according to CD4 count, as per national guidelines. Mothers with CD4 >200 cells/ul were assigned to the standard dual therapy PMTCT regime consisting of zidovudine from 28 weeks’ gestation for the mother and for the first month of life for the infant, in addition to single-dose nevirapine at birth for both mother and infant. Mothers with a CD4 count <200 cells/ul were eligible to receive combination antiretroviral therapy (cART). Following enrollment, the initial post-natal study visit to the Tygerberg Hospital Children’s Infectious Diseases Clinical Research Unit (KID-CRU) occurred at 2 weeks of age, with follow up visits at 6 weeks, 12 weeks, 6 months, 12 months, and 24 months of age. Study visits included a blood draw (4.5 ml) performed by a qualified phlebotomist and a full clinical assessment by a qualified pediatrician. Additional information on feeding, nutrition, health and medication history, and household demographics were documented at each visit. The study design and details of each timepoint are outlined in Figure 2.1.  Mothers who were HIV-positive were counselled to exclusively formula feed for the first 6 months of life, in accordance with the national guidelines of South Africa at that time. To facilitate formula as a safe feeding option, infant formula was provided free to HIV-positive mothers only from birth to 6 months. 23  2.4.2. Final Cohort       A total of 95 mothers were initially recruited at birth. Prior to the first timepoint (2 weeks), 28 of these refused further participation. An additional 5 subjects were either lost to follow-up or to a post-natal maternal diagnosis that was among the exclusion criteria, including hypertension and anemia.  A further 4 infants were excluded from analysis after testing positive for HIV at the 2-week timepoint. The final cohort at 2 weeks consisted of 27 HEU and 30 UE infants. At 6 weeks, 27 HEU (100%) infants and 28 UE (93%) infants were retained. At the 6-month timepoint, 25 HEU (93%) infants and 22 UE (73%) infants remained enrolled in the study, while 23 HEU (85%) and 21 UE (70%) infants remained at the 12-month timepoint. Due to variables such as missed visits, as well as limitations in sample variables such as number of cells, the number of samples assayed at each timepoint was not necessarily representative of the study retention figures. Table 2.1 summarizes the number of infant samples available for each assay at each timepoint.  Table 2.1. Number of samples assayed at each timepoint   Humoral 2 wks Humoral 6 wks Humoral 6 months Humoral 12 months CMI 6 months CMI 12 months HEU 19 20 19 20 18 13 UE 10 21 18 17 13 11   2.4.3. Statistical Analysis Qualitative data was analyzed by Fisher’s exact test. Quantitative data was normally distributed and was therefore analyzed by unpaired student’s t-test, with two-sided alpha set at p 24  ≤ 0.05. Data was compiled with Microsoft Excel 2016 and analysis was performed using GraphPad Prism v. 8.3.1 (La Jolla, CA USA). 2.5.  Results Maternal demographics were similar between the two groups. Measures of maternal health did not differ by HIV infection status, as number of genital infections and receipt of antenatal care were not significantly different between groups (Table 1). There was no significant difference in maternal age or alcohol consumption, although the HIV-negative mothers were significantly more likely to smoke during gestation (p= 0.03) (Table 1). While levels of maternal education were similar between HIV-infected and HIV-negative mothers, greater disparities were observed in housing, with fewer HEU infants residing in formal housing or having indoor access to water compared to UE infants, although the latter group had a higher number of people per household (Table 1). The HEU arm had a higher number of female infants than the UE arm (73% vs. 50%, p=0.10), although this was not statistically significant. Gestational age did not differ between the two groups. At birth, infant demographics differed only with respect to ethnicity, with the majority of HEU infants being of African descent and the majority of UE infants being mixed race (p=0.0001) (Table 3). The number of caregiver-reported infectious events did not differ; however, as previously published, the HEU infants had a 2.74 times higher risk of hospitalization compared to the UE infants (RR 2.74: 0.85-8.78).24 The number of infectious events up to 12 months of age did not vary significantly between groups (3.7 vs. 2.9, p= 0.16). All HEU infants and all but 1 UE infant were positive for cytomegalovirus (CMV) IgG 2 at 12 months of age.    25  Table 2.2: Cohort demographics at birth  Maternal Demographics  HEU  UE p-value Mat. Age (mean/years) (SD)    25.7 (6.8) 27.3 (5.7)   0.3 Multigravida (%) 20 (77) 20 (74) >0.99 Received Antenatal Care (%) 27 (100) 24 (86) 0.11 Smoked During Pregnancy (%) 3 (11) 11 (39) 0.03 Drank During Pregnancy (%) 0 (0) 4 (14) 0.11 Genital infections during  pregnancy (%) 4 (15) 6 (25) 0.49      Antenatal CD 4 count  mean - cells/ul (range) 335.4 (131-673) Diag. prior to pregnancy (%) 13 (48) Gest. Age at start of therapy (mean in wks) (SD)  27.2 (4.7) PMTCT Received (%):  4 (15) 19 (73) 3 (12) HAART Dual therapy None             26  Social & Environmental Determinants         HEU         UE  p-value Completed Secondary Ed (%)  6 (22)  9 (32) 0.55 Formal Housing  (%) 11 (42) 23 (82) 0.004 Access to Indoor Water (%) 10 (38) 24 (86) 0.001 Mean # of people in household (SD) 4.0 (2.4) 5.5 (2.2) 0.02  Infant Demographics   HEU UE  p-value Gestational age-mean in wks (range) 37.6  (30-41) 37.9  (33-42) 0.7 Sex – Female (%) 19 (73) 14 (50) 0.10 Birth Length-mean in cm (range) 47.4  (34.5-54.0) 49.8  (38.0-59.0) 0.07 Birth Weight-mean in g (range) 2963  (1900-3820) 2986  (2080-4100) 0.8 Head Circumference-mean in cm (range)   33.5  (28.0-48.0) 33.0  (28.0-38.0) 0.6 Number of infectious events – to 12 mos (SD) 3.7 (2.4) 2.9 (1.6) 0.16 CMV IgG +:12 mos (%) 20 (100%)                18 (95)                                         0.49             Ethnicity:   0.0001 1African 21 8 2Mixed 5 20       1African =Xhosa-speaking South African, Malawian (n=2) and Zimbabwean(n=1) descent.      2Mixed = Colored and Caucasian (n=1)   27  Among HIV-positive women, mean antenatal CD4 counts ranged considerably (131-673 cells/ul), although only 4 women met the <200 cells/ul criteria to qualify for cART, with the majority receiving dual therapy (73%) (Table 1). Less than half of women (48%) were diagnosed prior to pregnancy.   Table 2.3: Measures of infant anthropometry Infant Anthropometry – 6 Months     HEU   UE  p-value Number of infants in  in follow-up a 6 mos                               25   22  WAZ (SD) +0.21 (0.98) -0.73 (1.18) 0.01 LAZ (SD) -0.45 (1.07) -0.87 (1.61) 0.30 WLZ (SD) +0.74 (1.03) -0.09 (1.17) 0.01          Infant Anthropometry – 12 Months                 HEU     UE  p-value Number of infants in follow-up at 12 months                  23     21  WAZ(SD) +0.3 (1.15) -0.47 (1.09) 0.03 LAZ (SD) +0.12 (1.11) -0.53 (1.43) 0.1 WLZ (SD) +0.36 (1.21) -0.26 (1.05) 0.08               WAZ=weight for age; LAZ=length for age; WLZ=weight for length  Measures of anthropometry, based on the WHO international reference standards,  did not differ significantly at birth by weight, length or head circumference (Table 1), although by 6 months of age HEU infants had significantly higher WAZ (+0.21 vs. -0.73, p= 0.01) and WLZ (+ 28  0.74 vs. -0.09, p= 0.01) scores. Only WAZ scores remained significantly higher at the 12-month timepoint (+0.3 vs. -0.47, p=0.03) (Table 2). Mode of feeding was not comparable between groups as all unexposed infants were exclusively breastfed and all but one of the HEU infants were exclusively formula fed, in accordance with WHO guidelines at the time of study. 2.6.  Discussion Data on clinical outcomes were collected during the study and have been previously published.24 The higher risk of hospitalization experienced by the HEU infants indicates an increase in infection severity but not frequency. 24 The similarity in overall number of infections suggests that exposure to a more infectious environment is not a significant factor, at least in the context of controlled maternal HIV infection, while the increase in severity of infection could indicate the presence of an immune deficiency, although the lack of breastfeeding in the HEU arm cannot be ruled out as a contributing factor.  Maternal characteristics did not vary considerably between groups. No significant difference was observed in maternal age or multigravidity. Receipt of antenatal care and number of genital infections were likewise similar. The absence of opportunistic infections suggests that maternal health was not significantly compromised in the HIV-positive mothers of this cohort. While rates of alcohol consumption were similar, HIV-negative mothers reported higher rates of smoking than HIV-positive women, representing a risk factor for infant health impairment among UE infants that HEU infants did not experience.  Maternal educational levels were also similar between groups. Reduced maternal education has been associated with increased infectious morbidity in infants;126 however the mechanism for this remains unclear. Educational attainment could represent a surrogate for socioeconomic status whereby increased levels of poverty are the real driver of poor infant health outcomes. 29  Further evidence to support this theoretical link can be found in the multiple studies that have reported an association between increased maternal education and improved anthropometric measures.126-129 Although maternal educational attainment did not differ in this study, other socioeconomic measures did vary by maternal HIV status. HEU infants were more likely to live in informal housing or lack access to indoor water, indicating that mothers who were HIV-positive experienced reduced socioeconomic circumstances, a risk factor for worse health outcomes among their infants. However, they did report fewer average people per household, suggesting that conditions of crowding, another risk factor for poor infant health outcomes, were not a factor for the HEU infants of this cohort. Infants in the HEU group were more likely to be female, although this difference was not significant. There was significant difference in ethnicities, with the majority of HEU infants being of African descent, while the majority of UE infants were of mixed ethnicity, a trend that is likely related to the distribution patterns of HIV in South Africa.  Possibly due to the small size of the cohort, many of the disparities that have distinguished HEU infants in previous studies were not observed in this cohort. Gestational age, frequently reported to be reduced in HEU infants, was similar between groups in this cohort. However, this may be due to the designation of pre-term birth as part of the exclusion criteria in this cohort and thus may not accurately represent the larger HEU population in South Africa. The infants of both groups were of similar size at birth, although a non-significant decline in birth length was observed in HEU infants. These findings indicate that the trends towards low birthweight and nutritional deficiencies that are frequently observed in HEU infants were not present in this cohort. Again, given the link between pre-term birth and low birth weight, the exclusion of infants born pre-term could have had the effect of removing these infants from the final cohort. 30  Maternal malnutrition, another factor that contributes to infant growth deficiencies, was also among the exclusion criteria for this study.   Among the maternal infections to which HEU infants may experience increased exposure is CMV.  Prevalence of CMV is high among adults although rates vary by geographic region, ranging from 36-77% in developed nations to nearly 100% in the developing world.130 Among children, CMV rates are significantly higher in the developing world,131,132 although prevalence was not higher in HEU infants at 6 weeks of age in a Zimbabwean cohort.133 In this cohort, nearly all infants of both groups were positive for CMV IgG at 12 months of age, indicating a much higher early CMV prevalence among South African infants generally, but not among HEU infants specifically. Both groups likely had active CMV infections, consistent with onset early in infancy.134 Some differences in anthropometry were observed between groups at the post-vaccine timepoints. HEU infants had a significantly higher WAZ and WLZ compared to UE at 6 months of age, although the two groups did not differ significantly in LAZ. At 12 months of age, HEU infants still had significantly higher WAZ compared to UE infants. In this cohort, the trend towards increased anthropometric measures in HEU infants at later timepoints could be attributable to the formula feeding practiced by HIV-positive mothers. Formula feeding was associated with reductions in WAZ, LAZ, and WLZ in HEU infants at 6 months of age;135 however, these results were inconsistent, with breastfed HEU infants displaying slower longitudinal growth compared to those who were formula fed.136 Replacement feeding is associated with higher rates of growth up to at least 1 year of age,137-139 likely as a result of excessive protein consumption associated with formula feeding.138  31  The disparities in feeding mode between the two arms present a confounding factor. Among HEU infants in other studies, formula feeding was associated with a greater than 6-fold increased risk of mortality compared to breastfeeding71,72 In this cohort, despite UE infants being almost exclusively breastfed, the similar rates of infections between the two groups, as well as the lack of growth deficiencies among the HEU infants, suggest that it is possible to mitigate the risks associated with a lack of breastfeeding.  The available evidence indicates that growth deficiencies in the HEU population cannot be attributed solely to differences in mode of feeding. In a birth cohort in Mozambique where feeding methods were similar between the HEU and UE arms until 6 months of age, HEU infants had lower WAZ,84 while a Kenyan study reported that at 2 years of age, HEU infants demonstrated rates of underweight (29%), wasting (18%) and stunting (58%) that were above that of the general population and that did not differ between the formula-fed and breast-fed arms of the cohort.127 At 12-24 months of age, the risk of severe malnutrition was significantly higher in non-breastfeeding HEU infants compared to both breastfeeding and non-breastfeeding UE infants.139 The results of these studies suggest that some mechanism associated with in utero HIV exposure transiently affects the growth of HEU infants, although whether the relevant exposure is HIV, ARVs, or a pro-inflammatory maternal milieu, remains to be determined.  A high maternal viral load was significantly associated with infant stunting127 and lower infant weight.141 Reductions in WAZ,135,136 and LAZ,135 increased intrauterine growth restriction (IUGR),142 as well as reduced weight, length and head circumference143 were all significant in infants born to mothers with diminished CD4 counts.  Increased risk of LBW was associated with higher maternal cervical HIV RNA41 and low maternal CD4.144 On average, the HIV-positive mothers in the present cohort did not experience advanced disease as measured by CD4 32  count, and this could account for the comparatively stable health and nutritional status of their infants, despite not receiving the immune modulating factors associated with breastfeeding.   All but 3 (11%) of the HIV-infected women in this study received some form of antiretroviral therapy. In 2008, when the study described here began enrolling participants, the PMTCT guidelines were for single dose nevirapine as described above, with the addition of AZT for the mother from 28 weeks’ gestation.118 Combination antiretroviral therapy (cART) was provided to women with CD4 count <200 cells/ mm3. Most of the HIV-positive mothers in the present study (70%) received PMTCT prophylaxis, while only 15% met the CD4 threshold for cART. The effects of both vertical and direct ARV exposure on infant development have been the subject of much scrutiny in recent years, particularly with the expansion of PMTCT as standard of care across the world. There has been some evidence that ARV exposure influences anthropometric development, although the evidence for this has been inconsistent. Lower birth weight has been reported in ARV-exposed infants,55 although it is difficult to distinguish between the effects of HIV exposure and ARV-exposure. In contrast, birth WAZ was reduced in HEU infants whose mothers did not receive ARV therapy.136 This is consistent with the data associating advanced maternal disease with reduced anthropometric measures.127,135,141,143 A meta-analysis found that maternal ARV use did not significantly worsen the risk of PTB or LBW in HEU infants.56 Evidence of growth deficiencies reported in HEU infants in the pre-ARV era,25,127 as well as the well-documented association between maternal disease severity and infant anthropometry, indicates that HIV exposure has a greater effect than does exposure to ARVs, and that the beneficial effects of ARVs vastly outweigh any potential negative effects that may result from exposure to the drugs. 33  2.7.   Conclusion The similar health and growth measures between the 2 groups are encouraging. Despite HEU households having reduced socioeconomic measures, as well as ARV exposure and a lack of breastfeeding, the clinical and growth parameters of HEU infants in this cohort were comparable to those of UE infants, indicating that maternal disease control has significant potential for the management of this continually growing HEU population.    34  Chapter 3: Humoral Immunity in HEU Infants 3.1.  Hypothesis The hypothesis of this chapter is that HEU infants experience impaired production of specific antibodies compared to UE infants, leaving them potentially vulnerable to infectious diseases. 3.2.  Specific Aims The specific aims for this chapter are: 4) To compare levels of maternally derived anti-HBs antibodies between HEU and UE infants prior to vaccination 5) To compare HBV vaccine-specific antibody production following vaccination between HEU and UE infants 3.3.  Introduction Infants are immunologically naïve at birth and therefore are at increased risk of infection. Antibodies transferred across the placenta provide the neonate with protection in early life while the immune system is still developing. Multiple studies have reported that HEU infants have decreased levels of maternally transferred specific antibodies.93,95 These findings suggest that HEU infants may be more susceptible to infections in the neonatal period as a result of reduced maternal protection.  The possibility that in utero HIV exposure could impair immune development has raised concerns that HEU infants may not develop sufficiently protective responses following vaccination. Several studies have examined the anamnestic response to a range of childhood vaccines in HEU infants to determine if they mounted protective responses equivalent to that of UE infants, with varied results. Among HEU infants, comparatively increased antibody titers 35  have been reported by some studies93,102 while decreased responses have been reported by others.124 Despite these disparities in overall titers, most HEU infants do mount a humoral response sufficient to reach protective antibody levels.93 South Africa introduced the Hepatitis B vaccine into their EPI in 1995. The primary series of recombinant HBsAg was administered as a monovalent vaccine at 6, 10 and 14 weeks of age at the time of study initiation. The vaccine is considered highly effective, with a seroconversion rate of approximately 95%,145 although this can be affected by a number of factors, including the age at which the primary series is administered.146 Anti-HBs titers >10mIU/ml are considered a reliable correlate of protection.147  The development of a protective response following vaccination involves the coordinated efforts of both the humoral and cellular arms of the adaptive immune system. The relative contribution of each arm varies according to the type of vaccine and the nature of the pathogen. Vaccines administered at too young an age may fail to elicit sufficient protection. Interference from high levels of maternal antibodies may also interfere with development of an adequate primary response.93 The dose and timing of the immunizations given to infants are therefore optimized to achieve protection at the earliest age possible that will also produce  durable immune memory. Despite the inconsistent trends with respect to antibody titers, when considering vaccine-specific protection, it is relevant to examine not only overall antibody titers, but also the proportion of the population that achieves protective levels. Five years after it was introduced into the EPI schedule in South Africa, an assessment of the efficacy of the HBV vaccine reported that 86.8% of vaccinated infants had protective levels of anti-HBs titers, while 13.2% had antibody levels below the threshold of protection.113  36  Thus far investigations into the vaccine specific immune response in the HEU population have focused on the development of antigen specific immunity immediately following vaccination, and very little is known about the longer-term maintenance of protective immunity in this population. The longitudinal design of the study presented here allows for the examination of both the vaccine specific response following the primary series of vaccines as well as an assessment of the durability of those responses over time. 3.4. Methods 3.4.1.  Clinic Visits Study recruitment and inclusion/exclusion criteria are described in Chapter 2. Infants were evaluated at the Children’s Infectious Diseases Clinical Research Unit (KID- CRU) of Tygerberg Hospital at 2 and 6 weeks, and 6,12 and 24 months of age. In addition to a blood draw, each study visit included a complete clinical assessment by a pediatrician.  The 2- and 6-week blood draws took place prior to immunization. These timepoints represent an assessment of maternally transferred antibody levels. The 6- and 12-month timepoints represent a longitudinal analysis of the antibody response following completion of the primary series of HBV vaccination, administered at 6, 10 and 14 weeks. Immunization was performed during scheduled study visits and confirmation of immunization was documented by clinic nurses on individual Road to Health Cards. Immunization status was verified by referencing the Road to Health Card for each infant. Only infants with confirmed vaccination records were included in the analysis. Timing of vaccination in relation to blood draw was recorded and infants who received their 14-week vaccinations less than 1 month prior to the 6 month blood draw were removed from analysis of the 6-month timepoint, but were included in the 12-month analysis.  37  3.4.2. Vaccination Schedule The Expanded Program on Immunization in South Africa recommended that childhood vaccinations be administered according to the following schedule:  Table 3.1: Expanded programme on immunization schedule  BIRTH 1.5 MOS 2.5 MOS 3.5 MOS 9 MOS 18 MOS BCG         X      OPV         X         X           X         X           IPV          X*         X*         X*          X DTP          X         X         X          X HBV          X         X         X   Hib          X         X         X   Measles             X         X BCG, bacille Calmette-Guerin; OPV, oral polio vaccine; IPV, inactivated polio vaccine* (from July 2009); DTP, diptheria-tetanus-pertussis; HBV, hepatitis B virus; Hib, hemophilus influenzae type B          All infants received the oral polio vaccine (Sanofi Pasteur, Lyon, France) and the bacillus Calmette Guérin (BCG; Danish strain 1331, Statens Serum Institute) vaccine at birth. They received OPV plus a quadravalent vaccine containing diphtheria, tetanus toxoid, pertussis and Hib (DTP-Hib; Sanofi Pasteur) at 6, 10 and 14 weeks and 18 months of age. From July 2009, the DTP-Hib vaccine was replaced with the pentavalent diphtheria, tetanus toxoid, acellular pertussis (aP), inactivated polio (IPV), and Hib (DTaP-IPV/Hib; Sanofi Pasteur) vaccine, in accordance with the updated EPI. Infants also received Hepatitis (Hep) B (Heber Biotec, Havana, Cuba) at 6, 10 and 14 weeks of age.  3.4.3. Blood Draw At each study visit, a maximum of 4.5 ml of blood was collected via peripheral venipuncture of the jugular vein into pre-primed syringes containing 250 ul of sodium heparin. Plasma was extracted by centrifugation at 3200 rpm for 5 minutes. Plasma was collected in Eppendorf tubes in 400ul aliquots and frozen at -80o C. 38  3.4.4. Sample Collection & Storage Samples were shipped by air freight from Cape Town to the Child and Family Research Institute (CFRI) laboratory in Vancouver.  Plasma samples were shipped on dry ice and upon arrival stored in a -80o C freezer.  3.4.5. Determination of HBsAg-specific Antibody Titers Titers of HBsAg-specific IgG were measured using a quantitative human anti-hepatitis B surface antigen ELISA kit (4220-AHB; Alpha Diagnostic International Inc., San Antonio, TX). Plasma samples were undiluted, and tests were performed as per manufacturer’s instructions.  Measures of anti-HBsAg IgG were expressed in mIU/ml. An anti-HBs level of 10mIU/ml was taken as the cut-off for vaccine-mediated protection.146 Samples that fell below the lower limit of detection of the assay were assigned a value of 0 mIU/ml for the purpose of analysis. Control samples obtained from a single blood draw of an adult subject were run in duplicate on each plate to ensure consistency and accuracy.  3.4.6. Statistical Analysis Data was compiled using Microsoft Excel 2016 and analysis was performed using GraphPad Prism version 8.3.1 (La Jolla CA USA). Fisher’s exact test was used for analysis of categorical data. Quantitative data was not normally distributed as assessed by the Shapiro-Wilk test and was therefore analyzed by the non-parametric Mann-Whitney test. Significance was set at p ≤0.05.   39  3.5. Results 3.5.1. Maternally Transferred anti-HBsAg Titers At the pre-vaccine timepoints, sufficient plasma to test for vaccine-specific antibodies was only available for 19 of the HEU infants and 10 of the UE infants at 2 weeks of age, while at 6 weeks plasma titers were obtained for 20 HEU infants and 21 UE infants.  Levels of maternally transferred anti-HBsAg antibodies were low for UE infants prior to vaccination. The majority of HEU infants had similarly low titers at the pre-vaccine study visits, with the exception of a small number of individuals who expressed antibody levels far in excess of that of the majority of the HEU arm (data not shown). This trend was consistent at both the 2- and 6-week timepoints. At 2 weeks of age, the mean levels of anti-HBsAg antibodies were 102.6 pg/ml in the HEU infants, compared to 0.10 pg/ml in the UE infants, while at 6 weeks of age, the levels were 87.25 pg/ml in the HEU infants compared to 0.29 pg/ml in the UE infants.   40  HEU UE HEU UE0.11101001000Anti-HBsAg titres (mIU/mL)  2 weeks                    6 weeks*p=0.05                     *p=0.04 Figure 3.1: Anti-HBsAg antibody titers at pre-vaccine timepoints  Anti-HBsAg IgG titers at 2 and 6 weeks of age plotted in logscale. Horizontal line = mean antibody titers, vertical bars = standard error of the mean (SEM) * denotes statistical significance by Mann-Whitney U-test.   3.5.2. Vaccine-induced anti-HBsAg titers      At the 6-month timepoint, 19 HEU and 18 UE infants were retained in the study and included in the analysis based on timing of vaccination in relation to blood draw. At the 12-month timepoint, 20 HEU infants and 17 UE infants were retained for analysis. Elapsed time from the final vaccination in the primary series, scheduled for 14 weeks of age, and the 6-month blood draw was a median of 76.5 days for HEU infants and 86.0 days for UE infants (p= 0.03). At the 12-month timepoint, the median of 258.5 days for HEU infants compared to 261.0 days for UE infants did not reach statistical significance (p= 0.29).       At the 6-month timepoint, anti-HBsAg IgG responses were similarly robust in both groups following completion of the primary series of vaccinations. Mean antibody concentration for  41  Table 3.2: Time from vaccination to blood draw  Median # of days   6 Month (median in days) p-value 12 Month (median in days) p-value HEU 76.5 0.03 258.5 0.29 UE 86.0 261.0  the HEU infants was 206.0 mIU/ml, and 194.8 mIU/ml for the UE infants. Despite having a mean antibody response at 6 months that was slightly more robust than that of the UE infants, the HEU infants demonstrated a steeper decline in mean HBV-specific antibodies over the interval. The mean concentration at 12 months was 110.4 mIU/ml, compared to 153.8 mIU/ml in the UE arm, although this difference did not reach the level of statistical significance.  HEU UE HEU UE0100200300400Anti-HBsAg titres (mIU/mL)6 months                  12 months p=0.97                       p=0.14 Figure 3.2: Anti-HBsAg Antibody levels at Post-Vaccine Timepoints  Anti-HBsAg IgG titers at 6 and 12 months of age. Horizontal line = mean antibody titers,  vertical bars = standard error of the mean (SEM). Differences were not statistically significant  by Mann-Whitney U-test.  42  Following vaccination, both arms of the cohort achieved close to 100% protection from HBV, with all 19 (100%) of HEU infants and all but 1 (6%) UE infant exceeding the 10 mIU/ml  threshold of protection at 6 months of age. This high level of protection was retained through 12 months of age, at which time 19 (95%) of HEU and 16 (94%) of UE infants exhibited anti-HBsAg titers above 10 mIU/ml. There was no significant difference in the mean level of protection achieved between the two cohort arms at either timepoint.  Table 3.3: Rates of protection at post-vaccine timepoints  6 Month 12 Month # protected        (%) # not protected (%) p-value # protected # not protected (%) p-value HEU 19 (100) 0 (0) 0.49 19 (95) 1 (5) 1.00 UE 17 (94) 1 (6) 16 (94) 1 (6)   3.6. Discussion 3.6.1. Maternally Transferred Antibodies Reduced levels of maternally derived antibodies have frequently been observed in HEU infants and have been cited as a potential reason for the increased infectious susceptibility of HEU infants. In this cohort, at 2 weeks of age only 36 % of HEU infants had anti-HBsAg titers that met the protective level of 10mIU/ml, and only 30 % met this threshold at 6 wks. Among the UE infants, maternally derived titers were even more diminished/absent, with none of the UE infants meeting the minimum level of protection at either 2 or 6 weeks of age, despite being exclusively breastfed over this time. Although the majority of HEU infants were indeed vulnerable prior to vaccination, their level of protection exceeded that of UE infants, and 43  therefore does not support the hypothesis that increased infectious susceptibility is the result of deficiencies in maternal antibody transfer. It has previously been established that maternal HIV infection may be associated with decreased transfer of specific antibodies. The mechanism driving the diminished transfer of maternal antibodies has not been determined. Transplacental transfer of IgG begins early in the second trimester and increases as gestation progresses, with the majority of IgG transfer occurring in the last 4 weeks of gestation.148 By 40 weeks of gestation fetal IgG levels surpass maternal levels.149 Efficiency of IgG transfer and decay rates of maternal antibodies vary widely by antigen-specificity.94 A South African study reported reduced maternal levels of antibodies to Hib and pneumococcus in HIV-positive women93 while decreased levels of antibodies to GBS have also been described in HIV-infected women.91,92 HIV-positive women were 16.27 times more likely to be seronegative for anti-tetanus antibody than HIV-negative women in a Nigerian cohort, while their infants were 33.75 times more likely to be seronegative.95 Increased viral load was determined to be a significant predictor for reduced maternal specific antibody levels, implicating disease severity as a potential factor, although viral load was not independently associated with a reduction in transplacental transfer.150  Reduced placental transfer of antibodies has been reported in the context of maternal HIV infection,151-153 and could be a mechanism by which antibody transfer to HIV-exposed infants is impaired. The HBsAg-specific response is primarily comprised of IgG1,154,155 and this subclass is typically the most efficiently transported across the placenta.94,156 However, placental integrity may be compromised as a result of maternal HIV infection,149 a phenomenon that could result in impairment in placental transfer of antibodies. Further, the extent of placental disruption and the corresponding effect on antibody transfer may be linked to disease severity or immune 44  activation. High maternal viral load was found to be associated with reduction in placental transfer of measles-specific IgG,157 even when maternal antibody titers were comparable with uninfected mothers.152 Transfer of anti-pneumococcal capsular polysaccharide (PCP) IgG was reduced in infants of mothers with high viral load and low CD4 count.158 Likewise, transplacental transfer of all subclasses of IgG was inversely correlated with levels of soluble CD14 (sCD14).159  There is evidence that transplacental antibody transfer may also be influenced by use of PMTCT, with higher levels of specific antibodies present in the cord blood of infants born to women on triple therapy compared to infants born to women on short-course ZDV.150 Low birth weight has also been associated with impaired placental transfer of all IgG subclasses, with IgG1 and IgG2 being the most affected.160  It has not been established that reduced maternal antibody levels are directly correlated with lower infant levels. Despite describing a reduction in maternal GBS antibodies, le Doare et al. reported no statistically significant difference in the transplacental transfer ratio of IgG2 for any GBS serotype.161 Additionally, multiple studies have described an association between increased maternal IgG levels and a reduction in transfer of both total and specific IgG.153,159 The mechanism behind this is not definitively known, although competition among IgG antibodies for a limited number of placental Fc receptors could be involved.149  In the cohort presented here, in utero exposure to HIV was associated with increased titers of maternally transferred HBsAg antibodies at the pre-vaccine timepoints, compared to the UE infants.  These results contrast with the findings of most previous studies. Despite some inconsistencies, reduced levels of maternally transferred antibodies in HEU infants have been reported for tetanus,95,97,102 Hib,162 GBS,91 and measles.98,152  Consistent with the results reported here, Simani et al. reported higher pre-vaccination GMCs for anti-HBsAg in HEU infants.102 45  Even among the infants in the cohort represented here, these findings represent an aberration, as pre-vaccine levels of tetanus, pertussis and Hib were all reduced in the HEU arm, although only tetanus reached statistical significance.96 Upon closer examination of the data, it was observed that anti-HBsAg titers were highly variable among the HEU infants, with a small subset having levels that far exceeded that of the rest of the cohort, regardless of exposure status. We therefore hypothesized that these infants were likely born to HBV-infected mothers, and that correspondingly high maternal levels of anti-HBsAg were therefore passed on to their infants. It was not possible to test this theory as we did not test the mothers for Hepatitis B; however, it is highly plausible given the specificity of the antibody assay and the fact that HIV/Hepatitis B co-infection rates approach 36% in some regions of Sub-Saharan Africa.106 At the 6-wk timepoint, the mean anti-HBsAg titer of the HEU infants displayed a decrease from the 2 week-timepoint, while the UE infants increased slightly over the same period. Maternal antibodies transferred via breastmilk provide infants with protection during the pre-vaccine period, and the duration and frequency of feeding positively influence breastmilk antibody levels.163 At the time of this study, formula feeding was the recommended practice for HIV-infected women. Therefore, natural deterioration in the levels of placentally transferred antibodies could explain the decrease in titers demonstrated by the HEU infants over this interval. 3.6.2. Vaccine-induced Antibodies – 6-month timepoint As with most vaccines, measurement of the humoral immune response is the current standard for assessment of the efficacy of the HBV vaccine, primarily due to the comparative ease with which antibody titers could be measured. The currently accepted correlate of protection for the 46  Hepatitis B vaccine is ≥10 mIU/ml.147 However, there is some evidence of protection at titers below this value,164 and complete protection may not be conferred at any titre.165  After completion of the primary series of vaccinations, the percentage of HEU infants that met or exceeded protective anti-HBsAg titers was not significantly different from that of UE infants. Mean antibody levels were also higher in the infants of the HEU group. This is consistent with the findings of Manicelli et al, who reported protective anti-HBsAg titers in 93.2 % of HEU infants at 6 months of age.166 These results support the conclusion that in utero HIV exposure does not impair the development of vaccine-specific immunity, and even suggests that HEU infants are able to develop a more robust humoral response than that of unexposed infants. There is precedent for these findings in the literature as higher levels of post-vaccination antibodies have been reported in HEU infants in response to pertussis,93,102 and pneumococcus.93  The decreased transfer of maternal antibody titers frequently associated with HIV infection could render HEU infants at increased risk in the neonatal period but subsequently could benefit the infant in the development of a vaccine-specific response. Levels of maternal antibodies and the magnitude of the vaccine-specific antibody response are often inversely related. Higher levels of maternal antibodies have been associated with a reduction in infant vaccine-specific responses,93,167-171 although protective titers were obtained in the majority of subjects. Increased maternal antibody titers could interfere with the development of the infant immune response to vaccines, by an indeterminate mechanism. Hypothetical models include a vaccine antigen/maternal IgG complex cross-linking the B-cell receptor (BCR) with FcγIIB receptor, inhibiting B-cell activation and thus B-cell proliferation and antibody production.172 Other hypotheses include neutralization of live vaccine virus by maternal antibodies, FC-dependent phagocytosis of vaccine antigens coated by maternal IgG, or masking of vaccine epitopes by 47  maternal IgG preventing binding by infant B cells.148 At least one study has provided evidence that transplacental transfer of antibodies do not interfere with vaccine responses, reporting no significant difference in the vaccine response of infants born to anti-HBsAg positive mothers compared to those born to anti-HBsAg negative mothers.173 As well, long-term (>5yrs) immunogenicity was conferred by the Hepatitis B vaccine at similar levels regardless of maternal antibody titers at time of vaccination.174   In contrast to this trend, the HEU infants described here had higher titers of anti-HBsAg antibodies both at the pre-and post-vaccination time points, although the relatively small subset of HEU infants described above with high titers could have skewed the dataset away from following the more typically observed pattern, and is therefore not sufficient grounds for rejecting this hypothesis. Due to the small number of infants in this subset, we did not analyze them separately to determine if these infants had lower responses to vaccination.  Previous investigations reported a non-responder rate ranging from 6.7-11.5% in HEU infants.103,176-178 The reported rate of non-responders in a healthy population is 5-10%, so the response rate of HEU infants is within range of what would be expected outside of the context of in utero HIV exposure. Non-responder rates in the cohort described here were 0% among HEU infants and 6% among UE infants at 6 months of age. Abramczuk et al. reported an increase in non-responses in HEU infants compared to UE infants103. In contrast, responder rates of >99% were reported in both the HEU and UE infant populations of a South African cohort, with no reported disparity by exposure status.102  3.6.3. Vaccine-induced Antibodies – 12-month timepoint The success of vaccines is dependent upon the maintenance of an effective memory response over the duration of many years. Vaccine responses display large interindividual variability and 48  may be influenced in both magnitude and duration by a multitude of factors. Higher initial titers following vaccination are associated with increased rates of long-term protection and an increased response following a booster dose.178-181 Other factors include age, sex, genetics, nutritional status and environment. Vaccine-specific elements including type, dose, adjuvant and mode of administration can also influence the immune outcome. Some of these factors are well established while others are thus far inconsistent or poorly understood.182 In response to the HBV vaccine, females have demonstrated higher antibody responses in multiple studies,183-186 while variations in response have also been reported between ethnic groups living in the same geographic location.187 Anti-HBs titers drop in the first year following administration of the primary series of vaccinations178 and frequently fall below the level deemed protective in the decades after vaccination. This is particularly true when the primary series is administered in infancy, with a significant proportion of childhood vaccinees losing protective antibody titers by adulthood.114 Individuals who reach a lower peak response following primary vaccination will experience more rapid decline in antibody level.188 In this cohort, 95% of HEU infants and 94% of UE infants retained protective anti-HBsAg titers at 12 months of age, similar to the 87.5% reported in a Malawian HEU cohort at 12 months of age,166 although several infants had titers that had dropped below the level of 100 mIU/ul that is considered to confer long-lasting protection without additional boosting.188 Additionally, it is noteworthy that although HEU infants had an anti-HBsAg response that was slightly more robust than that of the UE infants at 6 months of age, they also experienced a greater decline over the ensuing 6 months. Few previous studies have performed a longitudinal comparison, therefore the consistency of this trend in the larger population is yet to be determined. Although this difference did not reach statistical significance, 49  and all but one of the HEU infants retained protective titers, a greater number of these infants had antibody levels that fell below 100 mIU/ml, warranting increased long-term monitoring of this group to determine if boosting is required in the future.   Pre-term birth also occurs more frequently among HEU infants.31,44 Deficiencies in both innate and adaptive immunity have been reported in pre-term infants including reduced pathogen recognition by APCs, reduced interaction between B cells and T cells, and diminished activity of Th1 cells.189 Decreased vaccine responses have been reported in pre-term infants, including lower geometric mean concentrations (GMC)190 and reduced rates of seroprotection191 following the primary series of HBV vaccine. These results may be notable given that many studies of HEU infants, including the study described here, listed pre-term birth among their study exclusion criteria. Thus, given the increased rate of pre-term birth among HEU infants and the immunological deficiencies recorded in these populations, it is plausible that these studies have not captured some of the immunodeficiencies that may exist in pre-term HEU infants.  Despite the overall high immunogenicity of the HBV vaccine, reduced seroconversion has been reported in infants who are LBW or SGA, a status that is increased in HEU infants. Given that these are often associated with pre-term delivery, disruption of the peak period of maternal antibody transfer during the third trimester likely contributes to this trend.  3.7. Conclusion Although immunologic disparities between HEU and UE infants exist, there is little evidence that HEU infants have substantially lower responses to the Hepatitis B vaccine.  This conclusion is supported by the relative absence of Hepatitis B as a cause of the increased morbidity and mortality associated with HIV exposure. 50  Chapter 4: Cellular Immunity in HEU Infants 4.1.  Hypothesis The hypothesis of this chapter is that HEU infants experience impaired vaccine-specific cellular immune responses compared to UE infants, leaving them potentially vulnerable to infectious diseases 4.2.  Specific Aims The specific aims for this chapter are: 1) To assess cellular immunity to the HBV vaccine by measuring production of secreted cytokines following stimulation of peripheral blood mononuclear cells (PBMCs) with HBsAg.  2) To compare HBsAg-specific cellular immunity between HEU and UE infants 4.3.   Introduction Upon pathogenic exposure, cells of the innate immune system recognize pathogen associated molecular patterns (PAMPs).192 These APCs migrate to the lymphoid tissues where they present antigen to T cells on either major histocompatability complex (MHC) -I (CD8) or -II (CD4) molecules, thereby activating the adaptive system to proliferate and produce cytokines and antibodies.193 The role of CD4 cells in supporting the activities of B cells and CD8 cells is particularly integral to the development of a pathogen-specific memory response. Th1 CD4 cells produce IFN-γ, TNFα and IL-2,194 and feature prominently in the immune response to intracellular pathogens. Th2 cells produce IL-4, IL-5, IL-10, and IL-13194 and may be the predominant response against extracellular pathogens. The balance between Th1 and Th2 responses is determined through an antagonistic mechanism whereby cytokines produced by one response will inhibit the magnitude of the opposing response.194 Other, less well-characterized T-51  cell subsets include Th17, Th22, T regulatory (Tregs) and T follicular helper (Tfh) cells.195 The importance of cell mediated immunity has increasingly been recognized in the development of vaccine-specific immunity. Cell-specific memory responses can develop and persist even in the absence of protective antibody titers.114 Evaluation of cytokine production therefore represents one of the most relevant measures with which to assess immune performance.116 However, there remains a lack of information on what constitutes an efficacious cellular immune response to HBV vaccination in infants. Reports of opportunistic infections such as P. jirovecii has raised concerns that HEU infants experience deficits in T-cell quantity or function.45-48 Thus far, investigations into cellular immune function in the HEU population have yielded results that were inconsistent and inconclusive. Increased markers of immune activation and inflammation32,74,79 have been reported, although they are frequently transient and confined to early life.32,74 It therefore remains unclear whether this more hyper-active immune profile has the potential to influence the development of long-term vaccine-specific immunity.  Previous measures of vaccine-specific immunity in HEU infants have been largely restricted to assessment of the antibody response, with minimal investigation into the development of vaccine-specific cell-mediated immunity. As well, most have been cross-sectional in design, which does not provide the opportunity to observe the development of the immune response, or how this response is sustained over time.  This study seeks to investigate the development of the adaptive immune response in a cohort of HEU infants, and to determine if this response differs qualitatively or quantitatively from that of an unexposed control group. To ensure consistency of the type, dose and timing of pathogenic exposure, cellular immune responses to the HBV vaccine were assessed. Evaluations of HBV-52  specific immunity were performed at 6 months of age, following receipt of the primary series of vaccinations, to assess the development of vaccine-specific immunity, and again at 12 months of age to assess the durability of this response over time.  4.4.  Methods 4.4.1. Sample Acquisition & Processing Infants were evaluated at the Children’s Infectious Diseases Clinical Research Unit (KID- CRU) of Tygerberg Hospital at 2 and 6 weeks, and 6, 12 and 24 months. At each study visit, a maximum of 4.5 ml of blood was collected into pre-prepared syringes containing 250 ul of sodium heparin. Following separation of plasma, the remaining blood was diluted with phosphate buffered saline in a 1:1 ratio, layered over Ficoll-Hypaque, and centrifuged for 25 minutes with no brake. After extraction, PBMC’s were aliquoted into cryovials in 500 ul aliquots and frozen overnight at -80o C. The following day they were transferred to a liquid nitrogen tank. Samples were shipped by air freight from Cape Town to the CFRI laboratory in Vancouver.  PBMCs were shipped in liquid nitrogen containers and upon arrival stored in a liquid nitrogen tank until processed. 4.4.2. PBMC Stimulation Assay         Prior to assaying the cohort samples, a series of experiments to optimize the PBMC stimulation assay were performed. Optimal cell numbers were identified following a series of titration experiments (data not shown). Stimulant concentrations were chosen according to a previously established in-lab protocol.  The 7-day duration was selected to allow for the assessment of cellular proliferation from the same experiment. Following optimization, the assay was validated by performing 3 separate repetitions of 3 adult control samples to ensure consistency and accuracy (data not shown). During assay of cohort samples, control samples 53  obtained from a single blood draw of an adult subject were run in duplicate on each plate to ensure consistency.  Cryopreserved PBMC were thawed and resuspended in R10 (RPMI with 10% fetal bovine serum (Thermo Fisher, Waltham, MA), beta-mercaptoethanol (50 µmol/l, Sigma-Aldrich), and 1X penicillin/streptomycin (Life Technologies). Cells were then washed twice with R10, treated with DNAse (60 µg/ml, Sigma-Aldrich), and incubated overnight at 37°C in 5% CO2. The next day, 2 mmol/l EDTA (Sigma-Aldrich) was added, and cells were washed twice before being passed through a 70 µm filter, stained with trypan blue, and counted. This was followed by the addition of Oregon Green staining buffer (Life Technologies) at a concentration of 5 µmol/L for 5 min, and the reaction was then stopped by the addition of FBS. Cells were then washed twice and inoculated into 96-well plates at 5 × 105 cells/well for the unstimulated and HBsAg wells and 2 × 105 cells/well for the positive control well. Cells were then stimulated with HBsAg (5 µg/mlL, Aldevron, Fargo, ND) plus costimulatory factors anti-CD49d and anti-CD28 (1 µg/mL each, CD49/28, eBioscience, San Diego, CA). The positive controls were stimulated with Staphylococcal enterotoxin B (SEB, 0.25 µg/ml, Sigma-Aldrich), and the unstimulated controls was plated with anti-CD49d and anti-CD28 (1 µg/ml each, CD49/28, eBioscience, San Diego, CA). After 7 days, cells were centrifuged and 50 µl supernatant was removed and frozen for bulk cytokine analysis. 4.4.3. Cytokine Measurement Supernatants were assayed by multiplex assay (Procarta, Norwich UK) using the high-biotin protocol with overnight 4°C incubation. During the optimization phase, stimulated control samples were assayed with a panel of 50 cytokines to determine the representative cytokines that showed the best expression. Levels of the following cytokines were chosen to be measured for 54  the adaptive assay: IL-10, IL-5, IL-13, TNF-α, and IFN-γ. Samples were diluted 1:2 to fall within the standard curve. Data were within the linear range for all analytes. Beadlytes, biotin, and streptavidin-phycoerythrin were used at half the manufacturer’s recommended concentrations. Adult control samples were included with each plate to ensure consistency. Assays were read using Luminex 200 Total System (Luminex, Austin, TX) running MasterPlex (MiraiBio, San Bruno, CA) software. 4.4.4. Statistical Analysis When cell counts permitted the plating of technical replicates, the average of the 2 samples was taken. Data was assessed for normalcy by the Shapiro-Wilk test. The majority of datasets were not normally distributed, and data was therefore analyzed by the non-parametric Mann-Whitney U-test. Significance was set at p ≤0.05. Data was compiled using Microsoft Excel 2016 and analysis was performed using Graphpad Prism version 8.3.1 (La Jolla CA USA). 4.5.  Results 4.5.1. Cytokine Production at 6 Months of Age The number of infants for whom there was a sufficient quantity of PBMCs to perform the CMI assay at this timepoint was 18 HEU and 13 UE. The unstimulated cytokine production at 6 months of age was similar between the 2 groups for all cytokines. IFNγ production in HEU infants was 16.7 pg/ml compared to 16.8 pg/ml for UE infants (p=0.98), while levels of TNFα were 26.3 pg/ml in the HEU infants vs. 55.5 pg/ml in the UE infants (p=0.06). The Th2 cytokines were also similar regardless of exposure status. IL-5 production was 36.6 pg/ml in the HEU infants compared to 46.3 pg/ml in the UE infants (p=0.13). Production of IL-13 was 38.9 g/ml in the HEU group and 65/4 pg/ml in the UE group (65.4 pg/ml) (p=0.22). Levels of IL-10, a 55  cytokine that serves a regulatory function, also did not differ between groups (HEU: 30.8 pg/ml vs. UE:22.4 pg/ml; p=0.90).  Table 4.1. Cytokine production at 6-month timepoint (pg/ml)  Unstimulated-median (IQR) SEB-median (IQR) HBsAg-median (IQR) HEU UE p-value HEU UE p-value HEU UE p-value IFNγ 16.7 (3.7-57.2) 16.8 (4.3-50.1) 0.98 254 (136-633) 258 (112-387) 0.65 29.5 (17.6-85.9) 32.3 (18.5-58.3) 0.73 TNFα 26.3 (5.4-46.3) 55.5 (12.7-101) 0.06 434.0 (176-1224) 765.2 (204-1380) 0.51 34.5 (10.5-62.8) 38.6 (16.9-70.8) 0.76 IL-5 36.6 (22.5-50.2) 46.3 (29.8-99.0) 0.13 369.5 (127-980) 517.9 (145-1223) 0.75 174.6 (83.5-601) 561.3 (343-1026) 0.03* IL-10 30.8 (5.7-64.3) 22.4 (8.0-52.6) 0.90 56.0 (23.8-130) 22.5 (9.5-82.2) 0.20 44.1 (27.8-146) 54.7 (12.4-125) 0.92 IL-13 38.9 (33.4-64.5) 65.4 (37.2-112) 0.22 566.0 (180-1564) 518.7  (91.1-1555) 0.88 184.4 (101-471) 632.8 (376-884) 0.03* *Data presented as median (IQR). Analyzed by Mann Whitney U-test, *significance set at p≤0.05.  In response to stimulation with SEB, the HEU and UE infants again responded similarly, demonstrating a cell-mediated response that did not differ at a functional level. No significant difference was observed in median production of IFNγ between HEU and UE infants (254.4 pg/ml vs. 257.5 pg/ml, p=0.65), and median levels of TNFα were likewise not significantly different between the groups (HEU: 434.0 pg/ml vs UE: 765.2 pg/ml, p=0.51). Similarly, levels of the Th2 cytokines IL-5 and IL-13 did not differ by exposure status at the 6-month timepoint.  56  IL-5 production did not differ significantly by exposure status (HEU: 369.5 pg/ml vs. UE: 517.9 pg/ml, p=0.75), while production of IL-13 was also similar between groups (HEU: 566.0 pg/ml vs. UE: 518.7 pg/ml, p=0.88). Production of the regulatory cytokine IL-10 was not substantial for either group, consistent with previous reports that SEB does not stimulate IL-10 production in PBMCs.197 HEU infants produced 56.0 pg/ml of IL-10 while UE infants produced 22.5 pg/ml at 6 months of age (p=0.20).   HEU UEHEU UEHEU UE0.1110100100010000IFN (pg/ml) Unstim          HBV            SEBp=0.98        p=0.74       p=0.65HEU UEHEU UEHEU UE110100100010000TNF (pg/ml)Unstim OHBV SEB       Unstim         HBV           SEBp=0.06        p=0.76        p=0.51HEU UEHEU UEHEU UE110100100010000IL-5 (pg/ml)      Unstim         HBV           SEB p=0.13  p=0.03*        p=0.75HEU UEHEU UEHEU UE1101001000IL-10 (pg/ml)      Unstim         HBV           SEBp=0.90        p=0.92       p=0.20HEU UEHEU UEHEU UE10100100010000IL-13 (pg/ml)      Unstim         HBV           SEBp=0.22 p=0.03*        p=0.88 Figure 4.1: Log-scale graph of PBMC secreted cytokine production at 6 months of age  HEU (n=18) infants in red, UE (n=13) infants in blue. Horizontal lines = median values, vertical bars = interquartile range (IQR). * denotes statistical significance (p≤0.05) by Mann Whitney U-test.    57  In response to stimulation with HBsAg, the infants of both groups displayed a subdued Th1 response. There was no difference in median production of IFNγ between HEU (29.5 pg/ml) and UE infants (32.3 pg/ml) (p=0.74), while levels of TNFα were similarly close between groups (HEU 34.5 pg/ml vs. UE:38.6 pg/ml; p=0.76). The Th2 response to HBsAg stimulation was far more robust.  Median IL-5 production was significantly higher in UE infants compared to HEU infants (UE: 561.3 pg/ml vs. HEU: 174.6 pg/ml, p=0.03). UE production of IL-13 was also significantly higher than that of HEU infants (HEU:184.4 pg/ml vs. UE: 632.8 pg/ml; p=0.03). The regulatory cytokine IL-10 was produced in only minimal levels in response to stimulation with HBsAg, and these levels did not differ significantly between HEU (44.1 pg/ml) and UE (54.7 pg/ml) (p=0.92).  4.5.2. Cytokine Production at 12 Months of Age The number of infants for whom there was a sufficient quantity of PBMCs to perform the CMI assay at this timepoint was 13 HEU and 11 UE. Similar to the results of the earlier timepoint, the cytokine production of the unstimulated samples at the 12-month timepoint did not differ between groups. Levels of IFNγ were 14.3 pg/ml in the HEU infants vs. 13.5 pg/ml in the UE infants (p=0.73), while TNFα was 53.9 pg/ml in the HEU group compared to 52.3 pg/ml in the UE group (p=0.90). Levels of Th2 cytokines were also similar, with the HEU infants producing 30.7 pg/ml of IL-5 compared to 36.1 pg/ml in the UE infants (p=0.55), and 32.7 pg/ml of IL-13 compared to 42.9 pg/ml in  the UE cohort (p=0.15). Consistent with these trends, production of IL-10 also did not vary between groups (HEU:57.9 pg/ml vs. UE: 45.0 pg/ml; p=0.75). In response to stimulation with SEB, the HEU and UE infants again responded similarly, demonstrating a cell-mediated response that did not differ at a functional level. A trend towards 58  higher median median production of IFNγ was observed in HEU (1258 pg/ml) compared to UE (495.3 pg/ml) infants, although this did not reach the level of statistical significance. It should also be noted that the median IFNγ levels of both groups were markedly higher at this timepoint compared to the 6-month timepoint. The median levels of TNFα were likewise higher for both   Table 4.2. Cytokine production at 12-month timepoint (pg/ml)  Unstimulated-median (IQR) SEB-median (IQR) HBsAg-median (IQR)  HEU UE p-value HEU UE p-value HEU UE p-value IFNγ 14.3  (5.1-59.3) 13.5  (1.7-59.1) 0.73 1258  (437-2866) 495  (194-1367) 0.19 41.6  (29.8-93.3) 42.4  (2.98-81.0) 0.53 TNFα 53.9 (36.6- 93.5) 52.3  (9.0-111) 0.90 1452  (705-2343) 962.6  (270-1972) 0.34 51.2  (20.3-77.8) 39.8  (5.4-72.2) 0.42 IL-5 30.7  (25.6-40.1) 36.1  (8.0-57.2) 0.55 273.7  (209-579) 383.6  (162-1366) 0.74 126.9  (34.8-277) 246.2  (115-552) 0.12 IL-10 57.9  (15.4-94.6) 45.0 (2.5-75.3) 0.75 46.5  (33.0-75.3) 34.8  (6.6-75.8) 0.34 70.3  (25.2-185) 25.7  (13.8-102) 0.21 IL-13 32.7  (31.7-86.7) 42.9  (33.9-118) 0.15 493.2  (257-1055) 516.1  (115-1071) 0.74 187.9  (77.1-344) 600.0  (162-819) 0.09 Data presented as median (IQR). Analyzed by Mann Whitney U-test, *significance set at p≤0.05.  groups at 12 months of age (HEU: 1452.0 pg/ml vs. UE: 962.6 pg/ml; p= 0.34), although again not demonstrating significant differences based on exposure status. Levels of the Th2 cytokines IL-5 and IL-13 likewise did not differ between groups at either timepoint. Reflecting the trend that was observed at 6 months of age, IL-5 production was slightly lower in the HEU infants (273.7 pg/ml) than the UE infants (383.6 pg/ml), although this disparity was no longer 59  significant (p=0.74). Production of IL-13 maintained levels that were consistent with the 6-month timepoint (HEU:493.2 pg/ml vs. UE: 516.1 pg/ml), and did not differ between groups (p= 0.74). IL-10 production likewise did not vary by exposure status (HEU: 46.5 vs. UE: 34.8; p=34). Stimulation with HBsAg again failed to elicit a robust Th1 response. Production of IFNγ did not differ between HEU infants (41.6 pg/ml) and UE infants (42.4 pg/ml) (p=0.53), and TNFα production was also similar (HEU:51.2 pg/ml vs. 39.8 pg/ml; p=0.42).  Compared to the 6-month timepoint, the median levels of TNFα at 12 months were lower than the unstimulated baseline.  HEU UEHEU UEHEU UE0.1110100100010000IFN (pg/ml)      Unstim         HBV           SEBp=0.73         p=0.53         p=0.19HEU UEHEU UEHEU UE110100100010000TNF (pg/ml)Unstim OHBV SEB  p=0.90       p=0.42          p=0.34      Unstim         HBV           SEBHEU UEHEU UEHEU UE110100100010000IL-5 (pg/ml)      Unstim         HBV           SEB p=0.55        p=0.12        p=0.74HEU UEHEU UEHEU UE0.11101001000IL-10 (pg/ml)      Unstim         HBV           SEBp=0.75        p=0.21        p=0.34HEU UEHEU UEHEU UE10100100010000IL-13 (pg/ml)      Unstim         HBV           SEBp=0.15       p=0.09        p=0.74 Figure 4.2: Log-scale graph of PBMC secreted cytokine production at 12 months of age  HEU infants (n=13) in red, UE (n=11) infants in blue. Horizontal lines = median values, vertical bars = interquartile range (IQR). * denotes statistical significance (p ≤0.05) by Mann Whitney U-test.  60  The Th2 response to HBsAg stimulation was far more robust. Similar to the trend established at the 6-month timepoint, UE infants produced higher median levels of IL-5 at 12 months of age compared to the HEU infants, although the difference was no longer significant (HEU: 126.9 pg/ml vs. UE: 246.2 pg/ml; p=0.12). UE production of IL-13 also continued to reflect the higher trends established at the 6-month timepoint (HEU:187.9 pg/ml vs. 600.0 pg/ml, p=0.09), albeit no longer at a level that reached statistical significance. The regulatory cytokine IL-10 was only minimally produced in response to stimulation with HBsAg, and these levels did not differ between HEU (46.5 pg/ml) and UE (34.8 g/ml) (p=0.34). 4.6.   Discussion This study sought to test the hypothesis that the development of the adaptive immune system in infants is negatively affected by in utero HIV exposure.  Cell mediated immunity is particularly important in the development of a durable response to intracellular pathogens. HBsAg-specific T and B cells are present in the absence of specific antibodies, indicating the persistence of T-cell memory response over time.115 Long-term T-cell responses to HBsAg were present 32 years after primary vaccination in individuals who had either maintained or lost protective antibody titers.114 The findings of these and other studies indicates that assessment of HBV-specific T cell function may provide a more sensitive measure of ongoing vaccine protection than current standards of humoral immunity.114 Thus far, there are a limited number of studies investigating the cellular response to vaccines in HEU infants. The few results that are available have been inconsistent and appear to be stimulus dependent. In response to stimulation with SEB and BCG, HEU infants displayed increased proliferation of CD4 and CD8 cells, while comparable levels of CD4 and CD8 61  proliferation between HEU and UE infants have been observed upon stimulation with Bordetella pertussis.63  At 6 months of age HBsAg stimulation failed to induce a strong Th1 response, as represented by production of IFNγ and TNFα, in either HEU or UE infants, with no significant difference based on exposure status. This trend was maintained at the 12-month timepoint. These results are comparable to those of a Kenyan HEU cohort in which no difference was detected in TNF-α or IFN-γ production at 3 months of age in response to tetanus toxoid.87 The muted overall response among South African infants is also consistent with those of  a Vancouver-based cohort of healthy 12-month-old HBV responders who had relatively low levels of IFNγ and TNFα in response to stimulation with HbsAg.196  The infants of both groups responded far more strongly in response to stimulation with the bacterial superantigen SEB (Figure 4.1), indicating that despite the HBsAg-specific findings, their Th1 response is intact. The IFNγ and TNFα responses to SEB were substantially higher in both groups at 12 months compared to the 6-month responses. This could reflect the bias towards immune tolerance that infants exhibit in early life, a feature that may explain their vulnerability to infectious disease.197 In this case, the increased production of IFNγ and TNFα induced by SEB stimulation at the 12 month timepoint would reflect a system that has developed and grown more responsive with age. In contrast to the limited Th1 response, the Th2 response to HBsAg stimulation was robust at both the 6 and 12-month timepoints. Again, these results are consistent with those of a cohort of healthy responder infants in Vancouver, who demonstrated a strong Th2 bias in response to HBsAg stimulation,196 similar to that observed in the infants described here. The longitudinal Th2 trend diverged over time from that displayed by the Th1-defining cytokines, as production 62  of IL-5 decreased between 6 months and 12 months, while production of IL-13 remained stable over this period. These findings further indicate that the initial Th2 bias of early life has developed into a more balanced profile with advancing age.  Interestingly, at the 6-month timepoint, production of the Th2-defining cytokines IL-5 and IL-13 were significantly higher in UE infants compared to HEU infants, a trend that continued at the 12-month timepoint, although the difference between the groups was no longer statistically significant. The reasons for this enhanced Th2 response are not clear. The lack of any discernible distinction in IFNγ production would indicate that the HEU infants did not experience a greater Th1-induced suppressive effect on Th2 cytokine production. Despite these findings, there is no evidence from this or other studies that a diminished Th2 response translates to a reduction in vaccine-specific antibodies among the HEU population. The transient pro-inflammatory profile depicted in HEU infants in other cohorts was not evident here, as there was no significant difference between the groups in IFNγ production in response to any stimulatory condition at any timepoint. However, this cannot be taken as conclusive evidence that such a response was not present earlier in life, as the pro-inflammatory bias that has been reported in HEU infants is often transient and is most evident during the neonatal period.74 As the earliest timepoint for this study was 6 months of age, it is possible that such a bias, if it existed, had already resolved.  No significant difference was observed between the 2 groups in production of IL-10, a cytokine produced by Th2 and regulatory T cells198 that has been shown to have a suppressive effect on the Th1 response.199,200 Alum is used as an HBV vaccine adjuvant to preferentially promote humoral immunity as it inhibits Th1 responses through enhancement of IL-10.201 IL-10 levels were not robust among the infants of either arm of this cohort, a trend that could be 63  associated with the moderate production of HBsAg-specific antibodies  that was observed in both groups.  This is consistent with reports that levels of IL-10 were significantly higher in infants classified as high responders to the HBV vaccine, compared to low responders196 or non-responders.202  HBV is T-cell dependent, therefore impairment in Th-cell function could result in a deficient response to vaccination.202 Multiple studies have reported that failure to produce an antibody response to the Hepatitis B vaccine is associated with a reduced or absent cell mediated response. The humoral response to the HBV vaccine observed in this cohort, while not robust, was adequate and supports a conclusion that the cellular immune response is functionally intact and that sufficient T cell help is being provided for T-dependent antibody generation.  Despite these encouraging results attesting to the intact immune status of HEU infants, the literature consistently reports that this population suffers from more severe infectious outcomes. A finding that has frequently emerged from the existing body of research into HEU infants is an association between more advanced maternal disease, as measured by either CD4 count, viral load or both, and increased infectious susceptibility.  HIV has been detected in fetal tissues from the first and second trimester,203,204 confirming the ability of the virus to cross the placenta. Many studies have investigated the impact of in utero HIV exposure on the subsequent immune development and function of the infant. However, only a subset of these studies has examined whether maternal disease severity, as measured by viral load or CD4 count, corresponds to the extent of immune dysfunction in the infant. This is significant as infants born to mothers with more severe HIV disease, as measured by either CD4 count or viral load, experience higher levels of in utero viral exposure and the potentially damaging effects that accompany it. High maternal viral load is a significant predictor 64  of infant mortality9 and morbidity, while reduced maternal CD4 counts have similarly been associated with increased infant mortality9,15 and morbidity.23,140  Infant immune parameters are also influenced by maternal disease status. Infants of women with a higher viral load had reduced numbers of naïve CD8 T cells and increased numbers of memory CD8 T cells at delivery205 and lower CD4 subsets.90 An increase in absolute B cell counts was associated with lower maternal CD4 count.85 Functionally, a detectable maternal viral load resulted in infant CBMCs that produced higher levels of IFNγ and TNF-α and less IL-10 compared to those with a controlled viral load.88  These findings are consistent with an increased state of immune activation possibly resulting from in utero exposure to HIV and/or a more activated maternal immune response. In the context of maternal HIV infection, viral particles may not be the only in utero exposure influencing fetal immune development. The maternal environment exerts the earliest influences on the development of the infant immune system. Exposure to a significantly activated intrauterine immune environment could be a contributing factor to dysregulation of the infant immune system.  HIV infection is associated with an increase in serum inflammatory mediators; however it remains an open question as to whether these cytokines can cross the placenta so their potential effect on the developing fetus cannot be determined.206 A lack of correlation between maternal and infant levels of inflammation has been described by multiple studies,80,32 suggesting that the increased inflammation that has been described in HEU infants may not be due to direct transfer of maternal inflammatory mediators. The presence of inflammation in cohorts with controlled maternal viral load suggests that antigenic exposure resulting from placental transfer does not provide an adequate explanation either. 65  A consistent picture has therefore emerged from a disparate group of studies: that infants born to mothers with advanced disease exhibit greater clinical and immunological disparities, although the precise mechanism(s) underlying this are not known. Where maternal disease is more severe the infant will be exposed to quantitatively higher levels of HIV antigen and will also experience an increased infectious exposure and a maternal immune environment biased towards systemic activation and chronic inflammation. These exposures may, individually or combined, exert an influence on the development of the infant immune response that translates as an increased susceptibility to infections in early life.  Most of the infants described in this study were not exposed to advanced maternal disease.  The median maternal CD4 count was 337/mm3 (131-673/mm3). Although this does not meet the lower limits of the normal range (500/mm3), it is above the level of 200/mm3 that is the immunologic criterion for classification of stage 3 disease. Viral load was not determined; however, the administration of PMTCT or cART during pregnancy for the majority of the HIV-positive mothers would support a conclusion that the infants were not exposed to an uncontrolled viral load. Controlled maternal viral load would mitigate the development of deleterious effects on infant immune development arising from direct viral exposure. 4.7.  Conclusion While further studies are required to more clearly elucidate the mechanisms by which each of these factors contributes to the increased morbidity/mortality observed in HEU infants, practical indications can be derived from the data presented here. The HEU infants of this cohort demonstrated a transient decrease in their HBV-specific Th2 response compared to their UE counterparts.  This suggests that even exposure to moderately severe maternal disease can cause 66  perturbations in infant immunity. These findings support the expanded use of maternal ARVs to better control maternal disease.          67  Chapter 5: Summary 5.1.  Summary of Results The purpose of this investigation was to determine if HEU infants experience impaired immune development related to in utero exposure to HIV. To achieve this, we measured the humoral and cellular compartments of the immune response following HBV vaccination in HEU infants at 6 and 12 months of age, and compared these responses with an unexposed control group to determine if HEU infants experience impairment in adaptive immunity.  Determination of pre-vaccine HBV-specific antibodies revealed that, on average, both HEU and UE infants lacked sufficient levels of maternally transferred antibodies to be considered protective. This high level of susceptibility supports the recent adoption in South Africa of the vaccination schedule recommended by the WHO, whereby the first dose of HBV vaccine is administered within 24 hours after birth. The other finding of note at these early timepoints was the small subset of 6 HEU infants with specific antibody levels more than 10-fold higher than the remaining infants in the group. Maternal antibody transfer of this magnitude suggests prior maternal HBV infection, a finding that would not be unexpected given the shared mode of transmission and established rates of HBV/HIV co-infection in South Africa. These findings indicate that maternal HBV testing and/or measurement of maternal anti-HBsAg titers should be included in the study protocol of any future cohorts. Following vaccination, rather than being impaired, the antibody response of the HEU infants was similar to that of the UE infants at the 6-month timepoint. This concurs with the findings of other studies and demonstrates that the humoral arm of the immune response remains intact in HEU infants. Although the lack of statistically significant differences between groups was maintained at 12 months of age, both groups had a notable decline in titers, with the HEU 68  decline being more pronounced. Based on the rate of antibody decline in both groups, extending future studies past two years of age to assess levels of continued protection and the necessity of a booster dose is advisable. Following vaccination, the assessment of CMI revealed a subdued Th1 response in all infants in response to HBsAg. In contrast, a strong Th2 bias was observed in the infants of both groups, although this response was more robust in the UE arm. The differences between the 2 groups were no longer significant at 12 months of age, again consistent with previous studies that suggest that clinical or immune impairments in HEU infants are transient and confined to early life. Aside from HIV exposure, mode of feeding and ARV-exposure were the largest differences between the 2 groups, although the data presented here indicate that these circumstances did not have an appreciable effect on immune function. In accordance with WHO guidelines, the majority of HEU infants in this study were exposed to both maternal and infant zidovudine and also received nevirapine at birth, with only 4 infants exposed to maternal cART. Although there has been some evidence published to suggest that ARV exposure is associated with deficiencies in growth and hematological abnormalities, the lack of any long-term disparities between groups with respect to the development of an HBV-specific immune response suggests that, in this cohort at least, ARV exposure did not have a lasting inhibitory effect on either infant growth or the adaptive immune response.  HEU infants were not breastfed; a condition that has been associated in the literature with increased infection and diminished immunity.207-209 However this disparity did not appear to translate into any significant difference in the measured immune parameters between the 2 groups, suggesting that the lack of breastfeeding did not hinder the development of vaccine-69  specific immunity and may even, in the case of the humoral response, have been beneficial due to the absence of interference from maternal antibodies.  Aside from the absence of any notable and lasting difference in immune response, there are other measures by which HEU infants in this cohort did not follow the trends that are most commonly seen in this population. HEU infants were more likely to reside in informal housing and lack access to running water.  Increased measures of socioeconomic insecurity are associated in the literature with higher rates of illness; however, this was not observed in this cohort. Growth, another measure by which HEU infants have often been disadvantaged compared to their unexposed peers, was similar between the groups. Interestingly, where growth disparities did exist, they favored the HEU infants. Replacement feeding is associated with increased growth in healthy infants, and it appears that in the HEU infants of this cohort this overcame any deleterious effects on growth arising from either HIV or ARV exposure. It is plausible that the lack of notable differences between the two arms of the cohort was due in part to the beneficial effects of ARV therapy, as the HEU infants in this cohort were not exposed to high viral loads or depleted CD4 counts that accompany advanced stage disease. The theme that has consistently emerged from the literature is this: that infants born to mothers with advanced disease experience a more severe clinical and immunological phenotype, although the precise mechanism driving this trend is not known. Where maternal disease is more severe the infant will be exposed to quantitatively higher levels of HIV antigen and will also experience an increased infectious exposure and a maternal immune environment biased towards systemic activation and chronic inflammation. These exposures may, individually or combined, exert an influence on the development of the infant immune response that translates as an increased susceptibility to infections in early life.  70  Although measures of maternal viral load were not available in this study, the more stringent exclusion criteria of this cohort, combined with the fact that nearly all HIV-positive mothers were on ARV therapy and had an adequate average CD4 count, suggest that maternal disease status in this cohort was not severe enough to have a significant impact on infant outcomes. Taken together, it suggests that, if exposure to HIV or maternal inflammation is responsible for infant immune dysfunction, the level of exposure must exceed a certain threshold to exert this effect. In support of this inference, future studies enrolling a larger cohort may want to broaden the inclusion parameters and stratify the analysis by maternal disease status. Expanding the maternal parameters measured to include inflammatory markers and test for co-infections with, at a minimum, HBV and CMV, would also enhance future investigations. While further studies are required to more clearly elucidate the mechanisms by which each of these factors contributes to the increased morbidity/mortality observed in HEU infants, practical indications can be derived from the data presented here. Infants born to mothers with high CD4/low viral load fare much better than those born to mothers with advanced disease. In terms of vaccine-induced immunity, HIV-exposed infants appear to be similar to their unexposed counterparts, when examined in the context of controlled maternal disease. Therefore, increasing maternal access to ART represents an important measure to improve health outcomes of the infant. A summary of the analysis presented here reveals that similar levels of immune responsiveness were observed, regardless of HIV-exposure status. While some subtle differences were detected between the two infant groups in both the humoral and cellular analysis, the findings of this study do not support a conclusion that HEU infants experience impairment in the development of an immune response to the HBV vaccine.  71  5.2.   Limitations There are limitations to the study presented here. This data covers only 12 months of the cohort; therefore, the duration of protection conferred by the HBV vaccine cannot be known. The sample size is small, with only 28 participants in the HEU group and 27 in the control group, and the number of samples with a sufficient number of PBMCs to perform the CMI assay was even smaller. Therefore, this small cohort size did not permit a longitudinal assessment. As well, the sample size was too small to incorporate confounding factors into the analysis, or to perform comparisons within groups. Thus, we were unable to perform sub-analyses according to the presence/absence of a humoral response or the stage of maternal disease. The strength of the existing literature on the link between maternal disease status and clinical and immunological outcomes in the infant suggest that such analysis is warranted and is an avenue for future study.  As this was a hypothesis-generating pilot study, I also opted not to correct for multiple comparisons. Applying such a correction can increase the Type II error rate, which could result in failing to detect a real difference between the groups. In future studies that employed a hypothesis-testing approach, it would be advisable to apply such corrections; in the context of this analysis, it should be noted that the possibility of a false positive exists. An additional limitation was that, out of necessity due to ethical considerations, the two groups could not be evenly matched on the basis of feeding practices or ARV exposure. The HEU group was not breastfed, while the control group was, and the HEU group was exposed to ARVs, while the control group was not. These factors have both been implicated as potential contributors to immune aberrations and infectious morbidity, so this discrepancy between groups has the potential to be significant to the current study. However, given that observations were 72  relatively short-term, questions about the duration of HBV immunity in HEU infants remain unanswered.  It should also be noted that Tygerberg Hospital, where recruitment took place, is a tertiary-care unit serving the Paarl district of South Africa. Referrals to this unit come through primary or secondary care providers; therefore, births that take place there are at higher risk of complications. Since both the HEU and UE infants were recruited from Tygerberg this does not represent a source of discrepancy between arms of the cohort. However, these infants may not present an accurate representation of the larger population. The immune parameters measured in this cohort were similar between groups despite demonstrating some notable discrepancies from similar results in the literature. This may be indicative of a disparity either between the infants of at-risk pregnancies represented in this cohort, or of a larger discrepancy between South African infants compared to those of other geographical regions. Future studies should focus on recruiting from primary centers to further elucidate the nature and extent of these disparities. The preponderance of evidence that advanced maternal disease, in the form of decreased CD4 counts or increased viral load, affects the extent to which HEU infants experience immunological and/or clinical disparities should influence future studies to include this consideration in their analysis. It also underscores the importance of maternal ART, not only as a means of preventing vertical transmission but also to improve infant outcomes by controlling maternal disease. 5.3.  Future Directions There are several avenues for future research directions that are suggested by the results of this pilot study. In particular, a larger follow-up study would allow for a more layered analysis of both the cellular and humoral measures, particularly with respect to stratification by maternal disease status.  A more detailed humoral analysis should also include an assessment of maternal 73  HBV status and antibody levels. A larger cohort would also allow the analysis of antibody levels to be stratified by infant vaccine responder status. The association between prenatal and /or fetal malnutrition and impairment of immune development is well established but is not yet detailed enough to be clinically useful. There is a need for a more accurate definition of the specific immune and metabolic components involved, and an understanding of the mechanisms by which cytokines and cells of the immune system are influenced by macro- and micronutrients. Trials into supplementation that have produced deleterious outcomes have already shown that merely providing supplementation is not an adequate solution. It may be that the correct balance is as important as a sufficient supply and understanding that balance and the biological processes that are involved is an essential first step to designing effective interventions. Reports that stress and anxiety during pregnancy are associated with decreased immune function in infants underscores the delicate balance of the physiological state and the importance of understanding that balance before attempting to correct it. Given that stress has an inhibitory effect on the immune system in adults it would not be unreasonable to assume that it has also affects immune development when exposure occurs in utero or early life. However, very little is known about the effects of stress on immune development at this stage and much more investigation is required. More research is also needed into the magnitude of the effect of malnutrition on the immune response, and the corresponding nutritional depletion due to immune activation, creating a negative cycle. 5.4.  Conclusion Thus far, the differences in immune function that have been detected between HIV- exposed and unexposed infants have been more subtle than the previously reported disparities in morbidity and mortality would suggest. The data presented here as well as previously published 74  reports indicate that in utero HIV exposure more typically results in mild perturbations of immune function rather than severe deficiencies. 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