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Cellular Immune Responses of Older Adults to Four Influenza Vaccines : Results of a Randomized, Controlled… Kumar, Arun; McElhaney, Janet E.; Kollmann, Tobias R.; Halperin, Scott A.; Scheifele, David W. Jun 30, 2017

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1   Cellular Immune Responses of Older Adults to Four Influenza Vaccines: Results of a Randomized, Controlled Comparison Arun Kumar1, Janet E. McElhaney1, 2, 3,4*, Tobias R. Kollmann4, 5, Scott A. Halperin4, 6, David W. Scheifele4, 5  1Health Sciences North Research Institute, 2Northern Ontario School of Medicine, Sudbury, Ontario, Canada; 3VITALITY Research Center, Vancouver Coastal Health Research Institute, Vancouver, BC, Canada; 4Public Health Agency of Canada/Canadian Institutes of Health Research Influenza Research Network (PCIRN), Dalhousie University, Halifax, NS, Canada; 5Vaccine Evaluation Center, University of British Columbia, Vancouver, BC, Canada; 6Canadian Center for Vaccinology, Dalhousie University, Halifax, NS, Canada.  *Address for correspondence: Dr. Janet E. McElhaney   Email: jmcelhaney@hsnri.ca   Health Sciences North Research Institute   41 Ramsey Lake Road   Sudbury, ON   P3E 5J1   Telephone: (705) 523-7300 (Ext 2725)   Fax: (705) 523-7325  Short title: Cellular Immune Responses of Seniors to Influenza Vaccines Keywords: immunization, adults, influenza, immune response, cellular immunity Clinical Trial registration number: NCT01368796  2   Abstract Cellular immunity is important for protection against the serious complications of influenza in older adults. As it is unclear if newer influenza vaccines elicit greater cellular responses than standard vaccines, we compared responses to two standard and two newer licensed trivalent inactivated vaccines (TIVs) in a randomized trial in older adults. Non-frail adults ≥65 years old were randomly assigned to receive standard subunit, MF59-adjuvanted subunit, standard split-virus or intradermal split-virus TIV. Peripheral blood mononuclear cells (PBMC) harvested pre- and 3-weeks post-vaccination were stimulated with live A/H3N2 virus. PBMC supernatants were tested for interleukin 10 (IL-10) and interferon gamma (IFN-), and lysates for granzyme B (GrB). Flow cytometry identified CD4+ and CD8+ T- cells expressing intracellular IL-2, IL-10, IFN-, GrB, or perforin. Differences following immunization were assessed for paired subjects and among groups. 120 seniors participated, 29-31 per group, which were well matched demographically.  Virus-stimulated PBMCs were GrB-rich before and after vaccination, with minimal increases evident. Immunization did not increase secretion of IFN- or IL-10. However, cytolytic effector T-cells (CD8+GrB+perforin+) increased in proportion post-vaccination in all groups (significantly in 2), to similar mean values across groups. CD4+GrB+perforin+ T-cells also increased slightly after  each vaccine, to similar mean values per group. Vaccinations did not increase the low baseline percentages of IFN-+, IL-2+ or IL-10+ CD4+ or CD8+ T-cells. In conclusion, participants had pre-existing cellular immunity to H3N2 virus. All four vaccines boosted cellular responses to a similar but limited extent, including cytolytic effector CD8+  T-cells associated with clinical protection against influenza.    Abstract word count: 249 Manuscript word count: 3569   3   Introduction Influenza virus is responsible for a substantial disease burden in older adults. The need for more effective vaccines in the older population is well recognized but there have been significant challenges in optimizing vaccine-mediated protection in this age group. Antibody responses have been used to evaluate new vaccines but have limitations as a sole predictor of vaccine efficacy 1–4.  For example, we showed that serum antibody titers against different influenza strains did not differentiate between those older individuals who subsequently developed influenza illness and those who did not 5,6. Age-related changes in T-cell responses are associated with a decline in the antibody response to influenza vaccination 7,8.  In fact, T-cell mediated mechanisms of protection are increasingly being recognized as relevant for influenza vaccine efficacy in older adults 9. Specifically, levels of the cytolytic mediator granzyme B (GrB) and the interferon-gamma:interleukin-10 (IFN-:IL-10) ratio predict protection against influenza illness 5,6 and are inversely correlated with influenza illness severity 10 in older adults. T cell correlates of protection using flow cytometric methods have been established in young adults based on experimental influenza A/H3N2 challenge 11 and natural infection with pdmH1N1 12. These studies showed that in the absence of protective levels of antibody, correlates of protection in experimental influenza A/H3N2 challenge or natural pdmH1N1 infection in young adults were, respectively, based on the IFN-+ CD4 T cell response 11 or IFN-+IL-2- CD8 T cell response 12 to matrix (M)- and nucleoprotein (NP)-derived peptides.  Prior exposure to influenza through infection or vaccination affects antibody titers and antibody responses to vaccination more so than aging does, while the decline in cell-mediated immune responses to influenza is related more to aging than to virus exposure 13.  Standard trivalent inactivated influenza vaccines (TIVs) provide a weak stimulus to T cell mediated immunity, especially in CD8+ T cells.   Although T cell memory from previous exposure to natural infection with influenza virus can be re-stimulated by vaccination, virtually all of the epitopes that drive the CD8 T cell response in humans are 4   contained within the internal proteins of the virus 14,15 not specifically included in current vaccines.  Influenza-specific CD8+ T-cells cannot protect against influenza infection but are critical for virus clearance, particularly from the lungs, and for preventing serious complications 16. These cells are highly specific for protein sequences conserved across different influenza strains and do not depend on an exact match of the vaccine strain with the circulating strain of influenza virus. Since the content of influenza internal proteins such as matrix and nucleoprotein varies widely among the available split-virus influenza vaccines and are virtually absent in subunit vaccines, differences between these vaccine formulations in the CD8 vs. CD4 T cell response to influenza vaccination would be predicted 17. Specifically, in vitro studies have suggested that a greater cellular response is elicited by split-virus vaccines due to higher matrix and nucleoprotein concentrations 17. Likewise, use of an adjuvant or the intradermal route of immunization might engage cellular immune responses to a greater extent than after standard vaccines 18–20. Given these potential differences to elicit immune responses, we evaluated the antibody 21 and cell-mediated immune responses to vaccination in a randomized study of four seasonal influenza vaccines available in Canada including standard  subunit, MF59 adjuvanted subunit and spilt-virus vaccines given intramuscularly or intradermally. This report presents the results for cell-mediated immune responses.      5   Results  In total, 120 participants were enrolled and immunized, 29-31 individuals per group. All subjects provided blood samples at both visits but one baseline and 3 post-immunization samples were of insufficient volume for all intended assays (Figure 1). All post-immunization samples were obtained per protocol.  The 4 groups were well-matched in terms of sex and age distribution, ethnicity, body mass index and frequency of co-morbid conditions (Table 1). Nearly all participants had received TIV in both pre-study years. The frequency of HAI titers ≥40 to the H3N2 virus in the 2011-2012 vaccine was similar across the 4 groups, averaging 33% (Table 1).  Global assessment of secreted effectors  GrB production by freshly isolated, virus-stimulated PBMCs was substantial prior to vaccination, with similar geometric mean concentrations (GMCs) evident among the study groups (Table 2). Three weeks after vaccine administration, a marginal increase in GMC was evident in all vaccine groups except in the TIV split-virus group, which had a marginally higher pre-vaccination GMC.  However, post-vaccination GMCs were not significantly different among the four groups. When individual responses were explored, considerable diversity was evident within and among the groups. The proportion of participants who had an increase in GrB concentration ≥1.5 fold after vaccination varied by group: split-virus 16.1% (5/31), split-virus intradermal 24.1% (7/29), subunit 38.7% (12/31) and adjuvanted subunit  46.4% (13/28). More participants had a GrB response to the subunit vaccines than to the split-virus vaccines (25/59 vs 12/60, respectively; p=0.022, chi-square test). However, our outcome measure was the group GMC of inducible GrB activity following influenza vaccination 6, rather than individual GrB response rates, the significance of which are uncertain.  Baseline production of IFN- by virus-stimulated PBMCs was substantial in each vaccine group, with no significant intergroup differences (Table 2). No post-immunization increase in IFN- production 6   by stimulated PBMCs was evident in any group, nor did post-immunization values differ significantly among the four groups. A similar phenomenon was evident with IL-10 production by stimulated PBMCs: baseline GMCs were similar among groups and did not increase significantly after vaccination (Table 2). Likewise, geometric mean ratios of IFN-:IL-10 did not increase after immunization or differ among groups (Table 2).  Exploration of individual responses did not identify differences in the small proportions of subjects in each group who had an appreciable increase in amounts of these cytokines following vaccination (data not shown).  Cell-specific assessment of intracellular effectors  Similar to the above observations, GrB expression was frequently detectable in CD8+ T lymphocytes obtained pre-vaccination: group geometric mean (GM) percentages ranged from 43.9-46.5, with no significant intergroup differences (Figure 2). No significant increases in GrB expression in CD8+ T- cells were evident post-immunization based on GM percentages, nor were differences evident among the vaccine groups. Within the CD4+ T-cell population, the proportions positive for GrB before vaccination were much smaller, with group GMPs ranging from 5.5%-9.9%. After vaccination, the proportion of CD4+ T lymphocytes positive for GrB increased slightly, the increase being statistically significant after both split-virus vaccines (p<0.001); ratio of pre- and post-immunization GMP was 1.44 with IDV, 1.58 with TIV2. However, the geometric mean proportions of CD4+GrB+ lymphocytes observed post-vaccination did not differ significantly among the four vaccine groups (Figure 2).   Perforin expression was infrequently present at baseline within CD8+ or CD4+ T lymphocytes, with no intergroup differences (Figures 3 and 4, p-value > 0.01). Following vaccination, the percentage of CD8+ T-cells positive for perforin increased in each group, reaching statistical significance with both subunit vaccines (p<0.001)(Figure 3). Ratio of pre- and post-immunization GMP was 1.71 for TIV1 and 1.95 for ADV. However, GM percentages post-vaccination did not differ significantly among the vaccine 7   groups. Among CD4+ T cells (Figure 4), the proportion of perforin+ cells increased significantly after each vaccine (2.15 to 2.61 fold) but the GM post-vaccination values remained <0.78% and did not differ among the vaccine groups.   Prior to vaccination, the GM percentage of polyfunctional CD8+ T cells expressing both GrB and perforin was <2% in all four groups (Figure 3B). After vaccination, the GM proportion of cells expressing both effectors increased significantly after both subunit vaccines (p< 0.001) but the GM percentages achieved did not differ among the vaccine groups and remained <3% in all. A similar pattern was evident with CD4+ T lymphocytes (Figure 4B): the GM percentage of cells with dual GrB and perforin expression increased significantly in each study group but reached statistical significance only with ADV and IDV vaccines (ratios of pre- and post-immunization GMPs were 1.7 and 2.05, respectively). The achieved GM proportions remained ≤0.5% in all groups, with no intergroup differences.    With respect to CD4+ T cells expressing IFN- or IL-2, the GM proportions of each remained <0.2% at both time points, with no increase following vaccination or differences among groups. Likewise, within the corresponding CD8+ sub-populations, GMPs were <0.22% for IFN-+ or IL-2+  expression at baseline, with no increase after vaccination or differences among groups. These results are consistent with previous studies of the response to a similar TIV vaccine preparation albeit with PR8 influenza virus used to stimulate PBMC 22.       8    Discussion This randomized clinical trial was designed to compare antibody and cell-mediated responses to four influenza vaccines available in Canada for the 2011-2012 influenza season. We previously published the immunogenicity, safety and tolerability results showing modestly enhanced antibody responses after  MF59-adjuvanted subunit TIV compared to the other three vaccines, with no between-group differences in safety or tolerability 21. Current reliance on antibody responses for licensing new influenza vaccines may fail to recognize important contributions of the cell-mediated immune response to protecting older adults against the serious complications of influenza. In addition, adjuvants such as MF59 and AS03 23 may further enhance protection by broadening the antibody response to neutralize drift variants of circulating influenza viruses not covered by the seasonal vaccine strains. Thus, measures of both humoral and cell-mediated immunity are needed to predict protection against influenza in older adults. We used previously established measures of the T cell response to influenza virus challenge in vitro as correlates of protection to assess differences in cell-mediated immunity in subjects recruited at the Vancouver site of this multi-centre trial.    The principal findings of this study were that seniors had pre-existing cellular immunity to H3N2 influenza virus that was boosted to a similar extent by each of the four study vaccines. It was expected that seniors would have established cellular immunity to H3N2 influenza virus from previous infections and immunizations.  It was reassuring to observe increased numbers of cytolytic CD8+ T cells following administration of each vaccine although the increase reached statistical significance (p<0.001) only with subunit TIV and adjuvanted vaccines. Differences in the magnitude of responses among the vaccine groups resulting from compositional differences in the vaccine products were not evident.   9   Strengths of this study design included substantial uniformity among the volunteers, who were typical of seniors living independently and receiving annual influenza immunization. Such a study population was considered most likely to reveal any important differences in immune responses to the various vaccines, unlike frail seniors with reduced response capacity. Limiting participation to one center favoured high compliance with study procedures and rapid and uniform processing of PBMCs, including their consistent stimulation with live influenza virus. Measuring cellular responses to include both secreted effectors from PBMCs and intracellular effectors in CD4+ and CD8+ T lymphocytes increased the chances of observing significant differences among the vaccinated groups.  Our study had several limitations. For example, group sizes were small; larger group sizes might have revealed small response differences among the products tested. Other limitations included the use of a single virus (A H3N2 strain) as the response probe, so results may not reflect responses to H1N1 and B strains of influenza virus.  Results may not be broadly generalizable given the ethnic uniformity and general good health of the study population. The threshold set for statistical significance (p<0.001) of most comparisons was arbitrary but adopted to achieve a Bonferroni-like corrections for multiple comparisons. A lower threshold would have increased the similarity of the four vaccines to elicit a rise in examined responses following immunization.   In this study, pre-immunization PBMCs from older adults showed high levels of GrB+Perf- CD8+ T cells. Recently, we showed that GrB activity in unstimulated T cells is associated with CMV seropositivity and accumulation of late or terminally differentiated CD8+ T cells 24. This activity contributes to the level of GrB activity measured in influenza-stimulated PBMC but has demonstrated toxicity in the extracellular environment when produced in the absence of perforin 25. Thus, GrB activity in influenza-stimulated PBMC adjusted for the CMV effect 24, or the frequency of GrB+ CD8+ T cells that are also Perf+ 10   26 are the more reliable measures of the CD8 T cell response to influenza vaccination, the latter being the measure selected for this study 26. We were not able to include measurement of cytomegalovirus seropositivity in the present study. In addition, GrB, IFN- and IL-10 production in influenza-stimulated PBMC prior to vaccination reflects previous exposure to influenza virus through natural infection and immunization, and previously established T-cell memory that can be re-stimulated with vaccination. Unexpectedly, no post immunization increase in IFN- and IL-10 levels in influenza-stimulated PBMC was evident for any of these vaccines. Our results contrast with our previous studies showing a significant increase in both IFN- and IL-10 following vaccination such that the IFN-:IL-10 ratio did not change in response to vaccination 5,10 but does decline with aging 27 and is a correlate of protection 5,10. Interestingly, Co et al. evaluated the ability of 3 commercial TIVs from the 2007-2008 season in the US to elicit T-cell responses in healthy individuals 17. All three vaccines showed a significant difference in IFN- producing activated CD4+ and CD8+ T-cells that varied according to the internal protein content of the vaccines, consistent with the role of these virus proteins in activating different T-cell responses. Our results may be consistent with low levels of internal proteins in all of the vaccines used in our study.   We found that all four vaccines induced a slight increase (not significant) in GrB activity at 21 days post-vaccination, when levels measured in PBMC lysates were similar across all vaccine groups. In contrast, the geometric mean percentage of CD4+ T lymphocytes positive for GrB increased significantly after both split-virus vaccines (p<0.001) but again there was no difference in the mean percentages of CD4+GrB+ lymphocytes observed post-vaccination across the four vaccine groups. Our results are in line with a previous study in which Couch et al compared CD4+ T-cell mediated responses with or without AS03 (similar to MF59) adjuvant in TIV vaccine 18. The study concluded that inclusion of AS03 in TIV significantly enhanced antigen-specific CD4+ T cell responses in older adults on day 21 after vaccination. A positive effect of AS03 adjuvant system on the CD4 T-cell response to vaccination and protection 11   against A/H3N2 influenza in older adults were demonstrated in a large randomized clinical trial 28. Other measures of the cell-mediated immune response in our study using flow cytometry and ICC to measure T cell frequencies and contents were similar across the vaccine groups, particularly within the CD8 T cell subset depending upon which cytokine or cytolytic mediator was measured. Overall the response to vaccination was modest compared to our previous studies and may reflect low levels of influenza internal proteins in the vaccines used in this study.    In summary, our study demonstrated similar in-vitro T-cell responses following administration of four commercially available influenza vaccines despite differences in composition (split-virus vs subunit}, presence of adjuvant and route of administration. A randomized controlled comparison was important for reducing the chance of confounding in the interpretation of the results and highlighted the variability of the response to vaccination depending on the selected measure of cell-mediated immunity.  A variety of mechanisms can be postulated for our observed results but larger studies including surveillance for laboratory-confirmed influenza in older adults are needed to understand how these measures of the cell-mediated immune response translate to clinically relevant protection and the proposed mechanism of protection for each of these vaccines.           12    Participants and Methods/Materials Study design and participants  This single center study was nested within a larger, prospective, multicenter trial 21 comparing the safety and immunogenicity of influenza vaccines in seniors and was conducted between September, 2011, and January, 2012. The institutional research review board of each center provided ethics approval. The nested study added investigations of cellular immune responses to the standard protocol, previously described 21. The study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and Good Clinical Practice Guidelines of the International Committee on Harmonization.  Eligible subjects were non-frail adults ≥65 years of age, in good health or with stable health conditions, living independently either in the community or in seniors’ residences. Volunteers were required to have had TIV vaccination in at least one of the two previous seasons. Exclusion criteria were previously reported 21. Written informed consent was obtained prior to enrolment.  Participants in the nested study were centrally randomized to receive one of 4 study vaccines, with stratification of the process by age sub-group and sex. Assignments were arranged in balanced blocks of 6, determined by computer-generated random number lists. Following enrolment, detailed information was obtained from participants regarding their health, prior TIV vaccinations, medication use and general fitness. A baseline blood sample (20 mL) was obtained, following which the assigned vaccine was administered with subject and evaluator blinding. Follow-up blood samples (20 mL) were obtained 3 weeks (window 20-28 days) after vaccination. Study vaccines  Single commercial lots of 4 TIVs for 2011-2012 were obtained: a subunit vaccine (Agriflu, Novartis Vaccines, lot #112104)(TIV1); a formulation of the subunit vaccine with MF59 adjuvant (Fluad, 13   Novartis Vaccines, lot #117703)(ADV); a split-virus vaccine (Vaxigrip, Sanofi Pasteur, lot # C4109AA)(TIV2); and an intradermal preparation of the split-virus vaccine (Intanza 15, Sanofi Pasteur, lot #H8187-1)(IDV). Each formulation delivered 15 µg of hemagglutinin of each component strain per dose. Each product was approved for use in adults ≥65 years of age and was supplied in pre-filled syringes. Constituent strains in these products were A/California/7/2009 (H1N1)-like, A/Perth/16/2009 (H3N2)-like and B/Brisbane/60/2008. Vaccines were stored and transported at 2-8 °C, avoiding freezing.  Injections were given in the deltoid area, using a 1” safety needle for IM injections or the micro-needle supplied for intradermal injections. Immunoassays  The heparinized venous blood samples were promptly (<4 hours) processed to harvest peripheral blood mononuclear cells (PBMCs) by Ficoll gradient purification 29. A portion of the unstimulated cells were cryopreserved 30 and stored in liquid nitrogen for subsequent simultaneous analysis of paired pre- and post-immunization samples (see below). Aliquots (3.0 x 106 PBMC/mL) of fresh PBMC were stimulated with influenza A/H3N2 virus at a MOI of 2 in AIM V medium (Gibco, Grand Island, NY) as previously described 31. The specific virus was sucrose-gradient purified, live influenza A/Victoria/3/75, which we have shown stimulates equivalent T cell responses to H3N2 vaccine strains related to the cross-reactivity of the T-cell epitopes among the different strains of influenza virus. PBMC were harvested after 20 hours of culture, with cells and medium then frozen at -80 °C. Unstimulated aliquots of PBMCs were processed in parallel as negative controls.  Total GrB activity was measured in PBMC lysates by cleavage of the substrate IEPDpna (Calbiochem, Billerica, MA) as previously described and validated 5,29. GrB activity was measured against a commercially available GrB standard (Biomol, Enzo Life Sciences, Ann Arbor, MI), adjusted for the amount of protein in the lysate and reported as units per mg protein (BCA assay, Pierce, Rockford, IL) in the PBMC lysates. Interferon gamma (IFN-) and interleukin 10 (IL-10) secreted by stimulated PBMCs 14   into the culture medium were measured using Bio-plex assay kits from Bio-Rad Laboratories (Mississauga, ON). Briefly, 50 µL culture medium was incubated with antibody-coupled beads, complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentrations. Human recombinant cytokine standards were provided by the vendor (Bio-Rad Laboratories). Cytokine levels were determined using a multiplex array reader and software from LuminexTM Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories). Minimum level of detection (MLD) for the cytokines (pg/mL) was 0.5 for IL-10 and 1.5 for IFN-. Undetectable cytokine levels were assigned a value of ½ of the MLD for determination of geometric mean values.  Upon completion of the study visits, paired pre- and post-vaccination samples of cryopreserved PBMCs were thawed, tested for viability (by trypan blue dye exclusion), with live cells then adjusted in number and stimulated by incubation with live A/H3N2 virus, as above. For the last 6 hours of incubation, samples were treated with the secretion inhibitor brefeldin to permit identification of effector molecule production at the single cell level by intracellular flow cytometry as previously described 29. Aliquots of stimulated and unstimulated cells from pre- and post-immunization samples of the same subject were studied side-by-side. During the cytometric analysis, CD3+ T lymphocytes were further divided on the basis of surface marker expression for CD4 and CD8. Within each of the CD4+ and CD8+ T cell subsets, the percentage of cells expressing intracellular effector molecules such as IFN-, interleukin 2 (IL-2), GrB or perforin was then identified by immunocytochemistry (ICC). We also assessed if cells were able to express more than one of the effector molecules (polyfunctionality) such as the dual presence of GrB and perforin.    15   Statistical Analysis  Group size was set at 30 participants per group (Table 3), based on locally achievable enrolment numbers and previously validated power calculations for GrB levels in influenza-challenged PMBC, which demonstrated that 12-18 subjects per group were needed to detect a 25% difference in GrB levels24.   Groups were compared for participant characteristics and completion of the relevant visits. In each study group, responses of fresh PBMCs to virus stimulation were compared between baseline and post-vaccination samples by calculating and comparing the geometric mean concentrations (GMC) and 95% confidence intervals, for each measured effector. We calculated mean GrB activity in lysates of influenza-stimulated PBMC. In addition, the IFN-:IL-10 ratio was calculated and compared among groups. In Tables 2 and 3, GMC, 95% confidence interval and standard deviation values are presented as antilog of the log-transformed data.  To analyze the effect of the pre and post-immunization time points and different vaccine groups, one-way ANOVA test was applied.  Statistical analyses were performed using SAS 9.3; graphs were created using R 3.2.1. For analysis of the flow cytometry data, the geometric mean percentage of CD4+ and CD8+ cells expressing the targeted effector molecules was calculated by paired t tests for virus-stimulated cells obtained pre- and post-immunization; statistical significance level was set as α= 0.001.         16       Conflicts of Interest  Janet McElhaney has participated on advisory boards for GlaxoSmithKline, Sanofi Pasteur and Pfizer and on data monitoring boards for Sanofi Pasteur; she has participated in clinical trials sponsored by GlaxoSmithKline and has received honoraria and travel and accommodation reimbursements for presentations sponsored by Merck, GlaxoSmithKline, Sanofi Pasteur and Pfizer, and travel reimbursement for participation on a publication steering committee for GlaxoSmithKline.  Arun Kumar, Tobias Kollmann and David Scheifele report no funding from commercial sources in the past 3 years or other commercial interests.  Scott Halperin has served on ad hoc advisory boards for Sanofi Pasteur, GlaxoSmithKline and Novartis Vaccines and has received funding for undertaking clinical trials from all three companies.   Financial Disclosure Statement This study was funded by a grant from the PHAC/CIHR Influenza Research Network (PCIRN). Additional funding for the laboratory analyses was provided by Sanofi Pasteur and Novartis Vaccines, which also supplied the vaccines used in the study.  Acknowledgements We gratefully acknowledge the excellent support of the staff of the Vaccine Evaluation Center in Vancouver including Ed Fortuno, Carol LaJeunesse, Bing Cai and Shu Yu Fan.  We additionally want to thank the staff at the VITALiTY Research Center and the VCHRI Clinical Research Unit including Gale 17   Tedder, Lynn Cunada, and Connie Feschuk for their outstanding commitment to study coordination, recruitment, and vaccine administration; and Dominica Kwok, Stephanie Hughes, and Jee Lee for their excellent technical assistance.  Authors’ Contributions to the Study DS, JM, SH developed the protocol and secured funding. JM and DS oversaw the clinical activities. JM and TK carried out the laboratory analyses. DS oversaw data assembly and analysis. AK assisted with data analysis and interpretation. All authors contributed to development of the manuscript and approved the final version.    18   References 1.  Bernstein E, Kaye D, Abrutyn E, Gross P, Dorfman M, Murasko DM. Immune response to influenza vaccination in a large healthy elderly population. Vaccine 1999; 17:82–94; PMID: 10078611; http://dx.doi.org/10.1016/S0264-410X(98)00117-0 2.  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J Infect Dis 2012; 205:466–73; PMID:22147791; http://dx.doi.org/10.1093/infdis/jir769          23   Figure Legends Figure 1. Subject disposition summary.  Altogether 120 participants were enrolled and each group of 29 to 31 subjects was immunized with TIV1, ADV, IDV or TIV2 influenza vaccines.  TIV1 = subunit vaccine; ADV = subunit vaccine with MF59 adjuvant; TIV2= split-virus vaccine; IDV= split-virus vaccine given intradermally.  Figure 2. CD8+ and CD4+ T-cells mediated GrB responses to four commercial vaccines.  PBMCs from pre  (light circles) and post-vaccinated (dark circles) elderly individuals were stimulated with live A/H3N2 influenza virus.  Phenotype of the stimulated T-cells was measured by flow cytometry and percentage of CD8+ (panel A) and CD4+ T-cells (panel B) expressing GrB was then measured by immunocytochemistry (ICC). Individual values are expressed as percentages of CD4+ and CD8+ cells expressing GrB, while the group geometric mean percentage is denoted with a bold star (  ). Horizontal bars represent 95% confidence intervals. Paired t-tests were used to compare means of pre- and post-vaccinated individuals in each group (*significant difference p ≤ 0.001). In panel B, only IDV and TIV2 vaccines induced significant increases in proportions of GrB+CD4+ T-cells after vaccination.  TIV1 = subunit vaccine; ADV = subunit vaccine with MF59 adjuvant; TIV2= split-virus vaccine; IDV= split-virus vaccine given intradermally.  Figure 3. CD8+ T-cells mediated perforin and GrB responses to four commercial vaccines.  PBMCs from pre- (light circles) and post-vaccinated (dark circles) elderly individuals were stimulated with live A/H3N2 influenza virus. Phenotype of the stimulated T-cells was measured by flow cytometry and percentages of CD8+ T-cells expressing perforin (panel A) or both perforin and GrB (panel B) were then measured by immunocytochemistry (ICC). Individual values are expressed as percentages of CD8+ cells expressing perforin alone or with GrB, while the group geometric mean percentage is denoted with a bold star (  ). 24   Horizontal bars represents 95% confidence of intervals.  Paired t-tests were used to compare means of pre and post vaccinated individuals in each group (*statistical significance p< 0.001).  Both panels show significant increases after both subunit vaccines.  TIV1 = subunit vaccine; ADV = subunit vaccine with MF59 adjuvant; TIV2= split-virus vaccine; IDV= split-virus vaccine given intradermally.  Figure 4. CD4+ T-cells mediated perforin and GrB responses to four commercial vaccines.  PBMCs from pre (light circles) and post-vaccinated (dark circles) elderly individuals were stimulated with live A/H3N2 influenza virus.  Phenotype of the stimulated T-cells was measured by flow cytometry and percentages of CD4+ T-cells expressing perforin (panel A) or both perforin and GrB (panel B) were then measured by immunocytochemistry (ICC). individual values are expressed percentage of CD4+ cells expressing perforin alone or with GrB, while group geometric mean percentage is denoted with a bold star (  ) . Horizontal bars represent 95% confidence intervals.  Paired t-tests were used to compare means of pre- and post-vaccinated individuals in each group (*statistical significance p< 0.001).  All vaccines induced increases in perforin expression (Panel A) but only ADV and IDV induced increases in dual perforin-GrB expression.  TIV1 = subunit vaccine; ADV = subunit vaccine with MF59 adjuvant; TIV2= split-virus vaccine; IDV= split-virus vaccine given intradermally.    

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