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Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses

Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses MA JO R A R T IC LE Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses 1 1 1 2 3,b 1,a 1 Adrian J. Reber, Jin Hyang Kim, Renata Biber, H. Keipp Talbot, Laura A. Coleman, Tatiana Chirkova, F. Liaini Gross, 1 1 1 1 1 3 1 Evelene Steward-Clark, Weiping Cao, Stacie Jefferson, Vic Veguilla, Eric Gillis, Jennifer Meece, Yaohui Bai, 1 1 1 1 1 1 1,c Heather Tatum, Kathy Hancock, James Stevens, Sarah Spencer, Jufu Chen, Paul Gargiullo, Elise Braun, 2,4 3 3 1 1 Marie R. Griffin, Maria Sundaram, Edward A. Belongia, David K. Shay, Jacqueline M. Katz, and Suryaprakash Sambhara 1 2 Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Vanderbilt 3 4 University Medical Center, Nashville, Tennessee; Marshfield Clinic Research Foundation, Wisconsin; and Mid-South Geriatric Research Education and Clinical Center, VA TN Valley Healthcare System, Nashville, Tennessee Background. Influenza disproportionately impacts older adults while current vaccines have reduced effective- ness in the older population. Methods. We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008–2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results. Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of sub- jects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of post- vaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older + + adults still demonstrated a significant increase in antigen-specific IgG and IgA memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50–59. Conclusions. Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest neg- ative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age. Keywords. aging; immune response; influenza; older adults; vaccine. Seasonal influenza is responsible for an estimated 23 000 influenza-associated illness than younger adults and lon- deaths (range, 3349–48 614) annually in the United ger hospital stays [2–6]; these effects are even more pro- States [1, 2]. The majority of these deaths occur among nounced with more advanced age [2]. adults aged 65 years and older [1]. Adults age 65+ have Although vaccination remains the most cost-effective 10 to 30 times more hospitalizations annually due to method to reduce influenza-associated morbidity and mortality, immunosenescence renders vaccines less ef- fective in older adults [7, 8]. Contributing factors include Received 26 January 2015; accepted 14 April 2015. impaired T and B cell functionality and narrowing of the Present Affiliation: Emory Children’s Center, Division of Pediatric Infectious Dis- T and B cell repertoire [9–15]. Although it is recognized eases, Emory University, Atlanta, Georgia. Present Affiliation: Abbott Nutrition, Columbus, Ohio. that more effective vaccines are needed for older adults, Present Affiliation: Yale School of Public Health, Yale University, New Haven, the capacity of their immune systems to respond and the Connecticut. Correspondence: Suryaprakash Sambhara, DVM, PhD, Centers for Disease components of the immune system that should be tar- Control and Prevention, Influenza Division, Immunology and Pathogenesis Branch, geted for improved efficacy are not well understood. 1600 Clifton Road, Atlanta, GA 30333 (ssambhara@cdc.gov). The hemagglutination inhibition (HI) assay is currently Open Forum Infectious Diseases Published by Oxford University Press on behalf of the Infectious Diseases Society of used to evaluate vaccine immunogenicity, in part because America 2015. This work is written by (a) US Government employee(s) and is in the an HI antibody titer ≥32 or 40 is generally considered a public domain in the US. laboratory correlate of protection, having been associated DOI: 10.1093/ofid/ofv052 Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 1 � � with protection against influenza infection in 50% of the popu- Table 1. Participant Characteristics lation in a number of studies in younger adults [16–19]. Findings from recent studies also suggest that cell-mediated immune re- Age Group Overall 50–59 60–69 70–79 80+ sponses may correlate better with protection in older individuals Subject no. 90 26 23 27 14 [20, 21]. However, alternative correlates to HI have not yet been Sex (%) Male 44.4 23.1 52.2 55.6 50.0 established. Although multiple studies have compared immune Female 55.6 76.9 47.8 44.4 50.0 responses of older adults to young healthy adults, relatively Race (%) fewer have examined the progression in later life. This study White 94.4 84.6 95.7 100.0 100.0 examines factors influencing immune responses to seasonal tri- Black 4.4 11.5 4.3 0.0 0.0 valent inactivated influenza vaccine (TIV) progressing from Other 1.1 3.8 0.0 0.0 0.0 middle-age (age 50–59 years) through advanced age (age 80+) Comorbidity (%) using a comprehensive set of assessments for antibody and Heart 1.1 3.8 17.4 7.4 0.0 cell-mediated immune responses. Lung 7.8 19.2 4.3 7.4 7.1 Immune 10.0 0.0 0.0 7.4 7.1 HI ≥40 (%) METHODS H1N1 Day 0 10.2 8.0 13.0 7.7 14.3 Methods are presented in brief and described in detail in Day 28 33.7 40.7 39.1 28.0 21.4 Supplementary Figure 1. H3N2 Day 0 29.5 32.0 30.4 26.9 28.6 Study Design Day 28 77.5 74.1 82.6 92.0 50.0 Approximately 590 subjects were enrolled during September ≥4-fold rise (%) through October 2008 at Vanderbilt University Medical Center H1N1 22.7 20.0 17.4 26.9 28.6 (Nashville, TN) and Marshfield Clinic Research Foundation H3N2 64.8 72.0 47.8 76.9 57.1 (Marshfield, WI) [22]. Subjects ≥50 years old were eligible to Abbreviations: HI, hemagglutination inhibition; HIV, human immunodeficiency participate at Vanderbilt, and subjects ≥65 years old were eligi- virus. ble to participate at Marshfield. Subjects were grouped by age; a Comorbid conditions include heart and lung disease, and immunosuppression (Immune). Immunosuppression includes immune dysfunction (including HIV), 50–59 (middle-aged), 60–69, 70–79, and 80+ years. Informed transplant, chemotherapy, steroid, or other immune-modulating medications. consent was obtained from all participants. Human experimen- tation guidelines of the Department of Health and Human Ser- vices were followed in the conduct of this trial. Procedures, assays, serial 2-fold dilutions were tested in duplicate and ex- informed consent documents, and data collection forms were pressed as the reciprocal of the highest dilutions giving 50% reviewed and approved by Institutional Review Boards at each neutralization or complete inhibition of agglutination. Anti- site. Participant characteristics are summarized in Table 1. body binding to hemagglutinin (HA) was analyzed by biolayer Participants received TIV from their usual caregiver, at vaccine interferometry on an Octet Red instrument (Fortebio, Inc., clinics, or at the study site by study staff. The 2008–2009 TIV was Menlo Park, CA). H3N2 and H1N1 recombinant HA was syn- composed of A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 thesized and purified as described previously [24]. (H3N2), and B/Florida/4/2006 strains. Although HI titers were determined for all subjects, for this study, a subset of 130 rando- Cell-Mediated Responses mly selected subjects underwent a more comprehensive evalua- Analysis of T-cell responses was performed on a subset of 60 sub- tion which included HI and microneutralization (MN) assays, jects, randomly selected from each age group. Cells were stimu- antibody binding by biolayer interferometry, and T and B cell lated overnight with live H3N2 or H1N1 virus. Brefeldin A (Golgi analysis. Due to limited sample volumes and the number of as- Plug; BD Biosciences, San Diego, CA) was added to cultures for sessments performed, only the influenza A strains were examined the last 6 hours to block Golgi transport. Cells were stained with in this study. Evaluations and analysis were done by staff blinded monoclonal antibodies recognizing CD4, CD8, tumor necrosis to participant age. Serum samples were collected from all subjects factor (TNF)-α, and interferon (IFN)-γ (BD Biosciences) and an- and peripheral blood mononuclear cells (PBMCs) from a subset alyzed using an LSRII Flow Cytometer (BD Biosciences). of subjects prior to vaccination and 21–28 days postvaccination. For assessment of antigen-specific memory B cells, PBMCs Serological Assays were cultured with poke weed mitogen (Sigma-Aldrich, Microneutralization and HI assays were performed as previous- St.Louis,MO),TypeBCpGoligonucleotide (Invivogen,San ly described [23]. Hemagglutination inhibition testing was Diego, CA), and purified protein A (Sigma-Aldrich) for 5–6 performed by Focus Diagnostics, Inc. (Cypress, CA). For both days to induce polyclonal activation. A standard memory B cell 2 OFID Reber et al � � enzyme-linked immunospot (ELISPOT) was performed [25]with groups ranging from 2.3- to 3.4-fold. Hemagglutination inhibi- minor modifications (Supplementary Figure 1). Spot-forming tion titers of older adults to H1N1 were not significantly different units were counted using ImmunoSpot (Cellular Techno- than titers from middle-aged adults (Figure 1A). However, sub- logy Ltd., Cleveland, OH) and expressed as percentage anti- jects age 80+ had lower MN titers (GMT ratio < 1) than middle- gen-specific IgG or IgA B cells of total IgG or IgA-secreting aged subjects postvaccination (Figure 1B). cells. Monovalent-inactivated vaccines were provided by Sanofi In contrast to humoral responses to H1N1, subjects mounted Pasteur to assess the frequency of influenza-specific antibody- more robust responses to H3N2. More subjects (29.5% vs secreting cells (ASCs). 10.2%) had preexisting HI titers ≥40 to H3N2 than H1N1 (Table 1), and H3N2 HI and MN titers increased in all age Statistical Analysis groups postvaccination (Figure 1A and B). A total of 77.5% of Log -transformed HI and MN titers were used as dependent 2 subjects achieved HI titers ≥40 to H3N2 postvaccination with variables and summarized as geometric mean titers (GMT) individual age groups ranging from 50.0% to 92.0% (Table 1). after back-transformation. Means and differences in means The percentage of subjects demonstrating a 4-fold or greater were estimated using repeated measures linear mixed models rise in HI titer was within a similar range (Table 1). All older [26, 27]. Back-transforming model-estimated means yielded age groups showed postvaccination titers comparable to mid- GMTs and back-transforming differences between day 28 and dle-aged counterparts (Figure 1A and B). day 0 means yielded GMT ratios (day 0 to day 28 fold-rise). Serum antibody binding was assessed by biolayer interferom- Models contained indicator (1,0) variables representing serum etry. Antibody binding generally followed a similar trend to HI draw day 28 vs day 0, indicator variables representing age and MN titers. Changes in antibody binding induced by H1N1 groups, and product terms representing interaction between were modest and variable by age group, with older subjects serum draw and age group. Interaction terms allowed us to exhibiting lower antibody binding capacity than middle-aged (1) estimate fold-rises having separate slopes by age group subjects (Figure 2A). In contrast, subjects in all age groups in- and (2) test for differences in fold-rises between groups. creased antibody binding capacity to H3N2 postvaccination Linear regression models used continuous year of age as an (Figure 2A). Adults in older age groups showed postvaccination explanatory variable. Dependent variables were log titers and 2 binding comparable to middle-aged counterparts, although log fold-rises for individual subjects. This allowed visualization 2 subjects in the 2 oldest age groups showed slightly lower bind- of trends in the strength of the fold-rise across age. Postvaccina- ing, which approached significance (P = .06; Figure 2A). tion log titers were regressed against prevaccination titers. Pre- and postvaccination serum responses were also regressed Effect of Age on Antibody Titers against T-cell responses. We found a modest and gradual decline in immune responses Antibody binding and T- and B-cell responses used models from middle-age through age 80+ years. The effect of age on similar to serology data; however, because these data are ex- humoral responses to TIV was not readily apparent with all pressed as percentages, we used log transformation similar 10 assays and exhibited differences between strains. When correla- to He et al [28]. Back-transforming model-estimated means tions with age did achieve statistical significance, the correla- yielded geometric mean percentages (GMPs), and back- tions were relatively low with correlation coefficients ranging transforming differences between day 28 and day 0 means yield- between −0.11 and −0.27 (Figures 2Band 3A–D). Postvacci- ed GMP ratios. Analyses were performed using SAS software, nation HI responses to H3N2 demonstrated a modest reduc- version 9.3 (SAS Institute Inc., Cary, NC). tion in HI titers to H3N2 with increasing age (R = −0.21; Figure 3A), although the reduction demonstrated by MN assay did not reach significance (Figure 3C). Despite the re- RESULTS duced responses exhibited by all subjects to H1N1, MN titers Serological Responses to H1N1 were decreased modestly with increasing age Hemagglutination inhibition responses for the entire cohort were (R = −0.25; Figure 3D). Postvaccination antibody binding ca- previously reported [22]. Within the subset of 130 subjects that pacity for both H1N1 and H3N2 also were negatively correlat- underwent comprehensive evaluation, 10.2% had HI titers ≥40 ed with increasing age (Figure 2B). Furthermore, although to H1N1 prior to vaccination with individual age groups ranging postvaccination titers correlated with age, no effect of age from 8.0% to 14.3% (Table 1). Subjects in this study mounted was observed on prevaccination HI and MN titer or antibody only modest humoral responses to H1N1. Although postvaccina- binding (Figures 2Band 3A–D). tion HI and MN titers were marginally higher in each age group The best predictor of postvaccination response was pre- (ie, GMT ratio > 1; Figure 1A and B), the majority of subjects vaccination titers. All age groups significantly increased anti- (66.3%; Table 1) did not reach HI titers ≥40 for H1N1. On aver- body titers in response to TIV (Figure 1A and B), but subjects age, subjects increased their HI titers 2.8-fold with individual age with higher cross-reactive prevaccination HI and MN titers Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 3 � � Figure 1. Assessment of antibody responses to 2008–2009 trivalent inactivated influenza vaccine. A, Serum hemagglutination inhibition (HI) antibody (n = 88) and B, microneutralization titers (n = 90) to H3N2 and H1N1 were assessed on day 0 prior to vaccination and day 28 after vaccination. The geometric mean titer (GMT) ratio (day 28 GMT/day 0 GMT) was calculated to determine the vaccine-associated change in antibody within each age group. A GMT ratio of 1 (dotted line) is indicative of no change. Day 28 GMT ratios were used to compare responses of older adult groups to middle-aged controls (day 28 GMT of older adult group/day 28 GMT of 50–59 age group). A GMT ratio less than 1 (dotted line) is indicative of a lower postvaccination response. Error bars represent 1 standard error. Significance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001. demonstrated higher postvaccination titers to H1N1 and H3N2 Vaccine-Specific Memory B Cells (Figure 3E) with correlation coefficients ranging from 0.62 to Frequencies of vaccine-specific memory B cells were evaluated 0.69. Higher prevaccination binding capacity also correlated by ELISPOT for 10 randomly selected individuals from each age with higher postvaccination binding (Figure 2B) with correla- group. Memory B cells were stimulated to selectively proliferate tion coefficients of 0.75 and 0.82 for H3N2 and H1N1, and differentiate into ASCs (Supplementary Figure 1), and iso- respectively. type-specific ASCs were measured (Figure 4A–D, left panels). 4 OFID Reber et al � � Figure 2. Assessment of antibody binding capacity by biolayer interferometry. A, Antibody binding capacity (n = 90) to H3N2 and H1N1 was assessed on day 0 prior to vaccination and day 28 after vaccination. The geometric mean percent (GMP) ratio (day 28 GMP/day 0 GMP) was calculated to determine the vaccine-associated change in antibody binding within each age group. A GMP ratio of 1 (dotted line) is indicative of no change. Day 28 GMP ratios were used to compare responses of older adult groups to middle-aged controls (day 28 GMP of older adult group/day 28 GMP of 50–59 age group). A GMT ratio less than 1 (dotted line) is indicative of lower postvaccination antibody binding. B, Antibody binding capacity was plotted by age and correlations calculated to determine the effect of age on preexisting ( ) and postvaccination antibody binding ( ). The correlation coefficient and P value are presented for preexisting ( ) and postvaccination ( ) correlations with age. The influence of preexisting, cross-reactive antibody on the day 28 postvaccination response to H1N1 ( ) and H3N2 ( ) was also determined. The correlation of pre- and postvaccination antibody responses to H1N1 ( ) and H3N2 ( ) are depicted. The correlation coefficient and P value are presented for prevaccination H1N1 ( ) and H3N2 ( ) correlations with the postvaccination antibody binding capacity. Error bars represent 1 standard error. Significance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001. Subjects in the older age groups generally showed the lowest lev- response to TIV vaccination (Figure 4). The frequencies of els of preexisting memory B cells, but they also tended to dem- preexisting (d0) memory B cells in subjects aged 70+ years onstrate the largest expansion of memory B-cell populations in were lower than in middle-aged subjects (GMP ratio < 1) for Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 5 � � Figure 3. Correlations with vaccine antibody response. Antibody to A and C, H3N2 and B and D, H1N1 were assessed from serum samples taken at prevaccination and 28 days postvaccination by A and B, HI (n = 88) and C and D, microneutralization ([MN] n = 90). Antibody levels were plotted by age, and correlations were calculated to determine the effect of age on preexisting and postvaccination antibody titers. The correlation coefficient and P value are presented for correlations with age. E, The influence of preexisting, cross-reactive antibody to H3N2 ( ) and H1N1 ( ) on the postvaccination, day 28 re- sponse was also determined. The correlation of pre- and postvaccination antibody responses to H1N1 ( ) and H3N2 ( ) are depicted. The correlation coefficient and P value are presented for prevaccination H1N1 ( ) and H3N2 ( ) correlations with the postvaccination titers. HI, hemagglutination inhibition. 6 OFID Reber et al � � + + Figure 4. Age-associated changes in preexisting memory B cells. The number of H3N2 (H3)-specific A, immunoglobulin (Ig)G and B, IgA as well as + + H1N1 (H1)-specific C, IgG and D, IgA memory B cells were assessed by B cell enzyme-linked immunospot at day 0 before vaccination and on day 28 postvaccination. The day 0 geometric mean percent (GMP) of memory B cells of older adults were compared with middle-aged controls by generating day 0 GMP ratios (day 0 GMP of older adult group/day 0 GMP of 50–59 age group). A d0 GMP ratio less than 1 (dotted line) is indicative of fewer antigen-specific, preexisting memory B cells. The GMP ratio (day 28/day 0% antigen-specific cells) was calculated to determine changes in the number of memory B cells within each age group associated with vaccination. A GMP ratio of 1 (dotted line) is indicative of no change. Error bars represent 1 standard error. Sig- nificance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001; n = 42. Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 7 � � H3-specific IgG and IgA memory B cells (Figure 4A and B). Al- factor to the observed reduced effectiveness of seasonal influen- though preexisting (d0) H1-specific IgG memory B cells tended za vaccines in persons 65 years and older. Decreased T and B to be lower in subjects over age 60, there were no significant dif- cell functionality and narrowing of the T and B cell repertoire ferences over time in any of the age groups (Figure 4C). Postvac- results in reduced antibody titers, lower antibody affinity, and cination, H3- and H1-specific IgG memory B cells increased in narrowing of the antibody repertoire [29–32]. the 70–79 age group as well as the 60–69 age group in the case of All 2008–2009 TIV strains changed from the previous season’s H1 (Figure 4A and C). It is interesting to note that middle-aged vaccine, resulting in the majority of the population studied here subjects had decreased numbers of H3-specific IgG memory B having a relatively low level of preexisting cross-reactive, neutral- cells postvaccination (Figure 4A). All age groups exhibited in- izing antibodies to the influenza A virus vaccine components. In creased numbers of H3-specific IgA memory B cells in response our study, only 10.2% and 29.5% of subjects had preexisting HI to vaccination. However, H1-specific IgA memory B cells in- titers ≥40 to H1N1 and H3N2, respectively (Table 1). During the creased only among subjects in the 2 oldest age groups (Fig- 8influenza seasons preceding the 2008–2009 season, there was ure 4B and D). only 1 change in the predominant H1N1 strain; the vaccine an- tigen changed from A/New Caledonia/20/1999 to A/Solomon Is- T-Cell Responses lands/3/2006 for the 2007–2008 season. In contrast, 4 different Peripheral blood mononuclear cells were stimulated with live H3N2 strains predominated in the same period. In addition, virus, and the proportions of IFN-γ and TNF-α-producing the previous season was dominated by H3N2, with 60% of T cells were assessed. Although sampling closer to the time of H3N2 samples tested being more antigenically similar to A/Bris- vaccination would have been more beneficial for assessment of bane/10 than to A/Wisconsin/67, that season’s vaccine strain T-cell responses, it would not have been optimal for detection of [33]. Thus, vaccination and/or natural infection with more anti- changes in antibody titers. T-cell responses were highly variable, genically diverse H3N2 strains in the recent past may have led to and the small number of subjects meant detection of potentially more cross-protective prevaccination titers for H3N2. Indeed, meaningful differences in T-cell responses was difficult (Sup- our memory B cell ELISPOT indicated that subjects had a larger plementary Figure 2A–H). Similar to what was observed for number of preexisting H3N2-specific, IgG memory B cells than memory B cells, subjects in the 50–59 year old age category gen- those against H1N1 (Figure 4A and C). erally had higher preexisting H1N1- and H3N2-specific CD4 Three assays were used to comprehensively assess the effect of T-cell responses (Supplementary Figure 2I–L). However, after age on antibody responses. The HI assay has historically been vaccination, CD4 T-cell responses among older subjects ap- used to assess protection from influenza and is the best under- peared to be either comparable or lower but not statistically dif- stood correlate of protection against influenza infection in ferent from their middle aged counterparts (Supplementary younger adults. However, HI is a surrogate assay and does not Figure 2M–P). No significant CD8 T-cell responses were directly measure virus neutralization. The MN assay measures found (data not shown); however, TIV is not known to effec- functional neutralization and has been suggested to be more tively induce CD8 T-cell responses. sensitive than HI [34, 35], but a threshold MN titer associated Postvaccination HI, MN, and antibody binding capacity in this with protection has not been described. Biolayer interferometry study all correlated with IFN-γ-producing CD4 T cells with cor- detects all antibody capable of binding to HA, not just those relation coefficients ranging from 0.31 to 0.46 (Figure 5A). Only that neutralize virus infectivity, presumably including antibody antibody binding capacity for H1N1, which had demonstrated with broader functionality such as opsonization, initiation of only low and variable changes to H1N1 (Figure 2A), did not cor- the complement cascade, and/or antibody-mediated cellular cy- relate with IFN-γ-producing CD4 T cells (Figure 5A). Tumor totoxicity. Thus, biolayer interferometry may give an indication necrosis factor-α producing CD4 T cells did not correlate with of broader protection beyond simple virus neutralization, pro- any of the serological responses, nor did CD8 T cells demon- viding some indication of the quality of the antibody response. strate any correlation (data not shown). It is interesting to note Subjects in all age groups increased HI and MN titers postvac- that preexisting IFN-γ responses to H3N2 by CD4 T cells cor- cination (Figure 1A and B); however, many did not exhibit HI ti- related with postvaccination antibody binding (R = 0.35; Fig- ters ≥40. This was especially true for H1N1; only 33.7% of subjects ure 5B), but not with HI or MN titers (data not shown). As reached titers ≥40, compared with 77.5% for H3N2 (Table 1). with the postvaccination response, preexisting CD4 T cell re- Subjects in all age groups demonstrated increased binding capacity sponses to H1N1 showed no correlation with antibody binding. to H3N2; however, only subjects age 50–59 and 70–79 increased H1N1 binding (Figure 2A). It should be noted, however, that H3N2 virus activity has been found consistently to be associated DISCUSSION with more severe morbidity and mortality among older adults Immunosenescence is considered an impediment to protection than H1N1 [2, 5]. Furthermore, as previously stated, cell-mediated of older adultsfrominfectiousdiseasesand a contributing immune responses may correlate better with protection in older 8 OFID Reber et al � � + Figure 5. Correlations with T-cell responses. A, Postvaccination interferon (IFN)-γ production by H1N1- or H3N2-stimulated CD4 T cells correlated with postvaccination hemagglutination inhibition (HI), microneutralization (MN), and antibody binding capacity. B, correlations were also calculated between + + + + prevaccination IFN-γ CD4 T cells and postvaccination antibody binding capacity to determine the influence of preexisting IFN-γ CD4 T cells on antibody response to vaccination. The correlation coefficient and P values are presented for each comparison. Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 9 � � adults than HI titers [20, 21]. Protection from influenza in older seemingly contradictory to recent studies by Sasaki et al [38] adults may be more complex than increased HI titers. that showed frequencies of vaccine-specific plasmablasts and In general, we confirmed that antibody responses decreased concentration of plasmablast-derived polyclonal antibody with increasing age, although results varied by assay type and were lower in older adults than young adults, whereas yields virus strain. Antibody binding capacity was negatively correlat- of secreted IgG per plasmablast and overall vaccine-specific ed with age for both strains, consistent with a potential impact avidity or affinity of polyclonal antibodies were not different be- on affinity maturation and possible narrowing of the antibody tween age groups. In their study, subjects age 70–100 were com- repertoire (Figure 2B). However, this effect was relatively mod- pared with those aged 18–30, whereas our subjects were all 50 est; correlation coefficients ranged from −0.11 to −0.27. Prevac- years and older. Although the quantity of the antibody response cination titers were the best predictors of postvaccination HI, decreases in advanced age compared with young adults, this MN, and antibody binding, with correlation coefficients be- change could be gradual and subtle during transition from mid- tween 0.62 and 0.82 (Figures 2B and 3E). Although postvacci- dle-age to older adults (60–79) to elderly adults (80+). Instead, nation titers were impacted by age, prevaccination titers, the qualitative changes (eg, affinity maturation, class switching, etc.) best predictors of postvaccination response, showed no correla- may start to compromise antibody responses. Consistent with tion with age (Figures 2B and 3A–D). A meta-analysis by Voor- this idea, the age-associated decline in HI titers was significant douw et al [36] analyzing influenza clinical trial data from the only for H3N2 (but not H1N1) and MN titer only for H1N1 European Union showed similar influences of age and prevac- (but not H3N2), yet antibody binding capacity for both anti- cination titers on postvaccination responses. gens declined with age (Figure 2B). For a typical influenza season, some proportion of the popula- Serological responses in this older adult population correlat- tion has at least partial immunity to circulating strains through ed with IFN-γ-producing CD4 T cells, with subjects exhibiting + + prior vaccination or natural infection. Preexisting, antigen- higher numbers of IFN-γ CD4 T cells also exhibiting higher specific or cross-reactive memory B cells are frequently the HI, MN, and antibody binding capacity (Figure 5A). The role of main source for protective antibody. Replacement of all 3 vac- IFN-γ in protection of all age groups against viral infection has cine strains for the 2008–2009 influenza season provided a long been recognized. McElhaney et al [20] previously demon- unique opportunity to assess changes in antigen-specific mem- strated that older adults with higher IFN-γ/interleukin-4 ratios ory B cell frequency in association with age. Indeed, 2008–2009 were less likely to exhibit influenza-like illness than those with + + TIV boosted the frequencies of class-switched memory B cells lower ratios. Furthermore, preexisting IFN-γ CD4 T cells cor- (antigen-specific IgG and IgA) in almost all age groups (Fig- related with postvaccination antibody binding capacity, whereas ure 4A–D). Subjects in all age groups significantly expanded HI and MN titers did not (Figure 5B), suggesting that preexist- + + H3-specific IgA memory B cells (Figure 4B), and despite the over- ing or cross-reactive IFN-γ CD4 T cells may play a role in en- all lower antibody titers to H1, there were significant expansions hancement of antibody quality. of H1-specific memory B cells in most age groups (Figure 4Cand Preexisting immunity in this study had a significant effect on D). Although it is normally thought that HI activity and virus the ensuing immune response to vaccination. The constant neutralization are primarily mediated by serum IgG, it has been evolution of the influenza virus makes yearly vaccine strains dif- speculated that IgA memory B cells may home to mucosal surfac- ficult to predict. Yet, despite the change in all 3 influenza vac- es upon infection, providing protection of mucosal surface cine strains for the 2008–2009 season, we found cross-reactivity through local production of IgA [37]. Assessment of both IgA to these strains in our study population. Although T cell epi- and IgG memory B cells may provide a more comprehensive pic- topes are recognized as being much more highly conserved, ture of protection upon infection. we were also able to demonstrate low levels of preexisting or It is noteworthy that despite a reduced frequency of preexist- cross-reactive antibody. During the 2009 H1N1 pandemic, ing memory B cells associated with increasing age, fold-rises in older adults were believed to be relatively more protected to memory B cell frequency among subjects aged 60+ was either the virus due to exposure to related viruses earlier in life [23]. comparable to or better than that found among subjects in This exemplifies the impact that prior immunity may have in the middle-age group (Figure 4A–D). These findings suggest providing protection from influenza and the potential longitude that the gross function of B cells pertaining to memory B-cell of this impact. The history of a person’sinfluenza vaccination/ generation was not drastically compromised. More importantly, infection likely influences their current responses, and the im- however, binding capacity of post-vaccine serum antibody de- mune system of older adults may have an extensive lifetime of clined for both strains with increasing age (Figure 2B), indicat- exposure from which to draw. ing that the capacity of somatic hypermutation in antibody This study was limited by several factors. First, for several as- variable regions is affected by advancing age. Our data suggest says, it was feasible to test only a small number of samples, thus that immunosenescence in advanced age is manifested more by limiting the number of statistical comparisons that could be qualitative changes than quantitative changes. This result is made and reducing the power to detect differences by age 10 OFID Reber et al � � 2. Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with groups. Second, populations at the 2 sites were different. Partic- influenza and respiratory syncytial virus in the United States. JAMA ipants at Vanderbilt were typically younger and more likely to 2003; 289:179–86. be employed, whereas those at Marshfield were older and had 3. World Health Organization (WHO). Fact sheet N°211. April 2009. previously received influenza vaccination. Differences observed Available at: http://www.who.int/mediacentre/factsheets/fs211/en/ index.html. Accessed 3 March 2014. between groups may not have been solely due to age but instead 4. Falsey AR, Hennessey PA, Formica MA, et al. Respiratory syncytial affected by differences in methods of recruitment or other site- virus infection in elderly and high-risk adults. N Engl J Med 2005; specific differences. Finally, chronic preexisting medical condi- 352:1749–59. 5. Thompson WW, Shay DK, Weintraub E, et al. Influenza-associated tions, sex, and race are all known to affect immune function, but hospitalizations in the United States. JAMA 2004; 292:1333–40. variables representing these factors were not included in the 6. Mullooly JP, Bridges CB, Thompson WW, et al. Influenza- and RSV- analyses performed because of limited sample sizes. associated hospitalizations among adults. Vaccine 2007; 25:846–55. 7. Targonski PV, Jacobson RM, Poland GA. Immunosenescence: role and measurement in influenza vaccine response among the elderly. Vaccine CONCLUSIONS 2007; 25:3066–9. 8. Aspinall R, Del Giudice G, Effros RB, et al. Challenges for vaccination in the elderly. Immun Ageing 2007; 4:9. Our findings underscore the importance of understanding not 9. Whisler RL, Grants IS. Age-related alterations in the activation and ex- only immunosenescence as an obstacle for influenza vaccination pression of phosphotyrosine kinases and protein kinase C (PKC) of older adults but also other factors influencing vaccine respons- among human B cells. Mech Ageing Dev 1993; 71:31–46. es. Older adults in this study were able to mount significant hu- 10. Weksler ME. Changes in the B-cell repertoire with age. Vaccine 2000; 18:1624–8. moral and cell-mediated immune responses to TIV. Although age 11. Naylor K, Li G, Vallejo AN, et al. The influence of age on T cell gener- negatively affected these postvaccination responses, age was not ation and TCR diversity. J Immunol 2005; 174:7446–52. the biggest influence. Prevaccination responses had a much larger 12. Fulop T, Larbi A, Wikby A, et al. Dysregulation of T-cell function in the elderly: scientific basis and clinical implications. Drugs Aging 2005; impact, irrespective of age at vaccination. A better understanding 22:589–603. of factors influencing preexisting, cross-reactive immunity would 13. Gibson KL, Wu YC, Barnett Y, et al. B-cell diversity decreases in old age provide better insight into mechanisms for achieving better pro- and is correlated with poor health status. Aging Cell 2009; 8:18–25. 14. Siegrist CA, Aspinall R. B-cell responses to vaccination at the extremes tection of older adults from yearly influenza epidemics. of age. Nat Rev Immunol 2009; 9:185–94. 15. Caraux A, Klein B, Paiva B, et al. Circulating human B and plasma cells. Supplementary Material Age-associated changes in counts and detailed characterization of circu- lating normal CD138− and CD138+ plasma cells. Haematologica 2010; 95:1016–20. Supplementary material is available online at Open Forum Infectious Diseases 16. Hobson D, Curry RL, Beare AS, Ward-Gardner A. The role of serum (http://OpenForumInfectiousDiseases.oxfordjournals.org/). haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J Hyg (Lond) 1972;70: Acknowledgments 767–77. 17. Potter CW, Oxford JS. Determinants of immunity to influenza infection We thank Dr. Michael Decker of Sanofi Pasteur (Swiftwater, PA) for pro- in man. Br Med Bull 1979; 35:69–75. viding TIV and monovalent influenza vaccines to assess cell-mediated im- 18. Beare AS, Hobson D, Reed SE, Tyrrell DA. A comparison of live and mune responses. killed influenza-virus vaccines. Report to the Medical Research Coun- Disclaimer. The findings and conclusions in this report are those of the cil’s Committee on Influenza and other Respiratory Virus Vaccines. authors and do not necessarily represent the views of the Centers for Disease Lancet 1968; 2:418–22. Control and Prevention or the Agency for Toxic Substances and Disease 19. Beare AS, Tyrrell DA, Hobson D, et al. Live influenza B vaccine in Registry. volunteers. A report to the Medical Research Council by their Committee Financial support. This work was supported by the Centers for Disease on Influenza and Other Respiratory Virus Vaccines. J Hyg (Lond) 1969; Control and Prevention (CDC 1 U18 IP000184-01 and CDC 5 U18 67:1–11. IP000183-02) and in part by an appointment to the Research Participation 20. McElhaney JE, Xie D, Hager WD, et al. T cell responses are better Program at the Centers for Disease Control and Prevention administered by correlates of vaccine protection in the elderly. J Immunol 2006; 176: the Oak Ridge Institute for Science and Education through an interagency 6333–9. agreement between the US Department of Energy and Centers for Disease 21. McElhaney JE, Ewen C, Zhou X, et al. Granzyme B: correlates with pro- Control and Prevention. tection and enhanced CTL response to influenza vaccination in older Potential conflicts of interest. H. K. T. has received research funding adults. Vaccine 2009; 27:2418–25. from Sanofi Pasteur and MedImmune. M. R. G. and M. S. have received re- 22. Talbot HK, Coleman LA, Crimin K, et al. Association between obesity search funding from MedImmune. J. M. K. has received research funding and vulnerability and serologic response to influenza vaccination in from GlaxoSmithKline and Juvaris, Inc. (now Colby Pharmaceuticals). older adults. Vaccine 2012; 30:3937–43. All authors have submitted the ICMJE Form for Disclosure of Potential 23. Hancock K, Veguilla V, Lu X, et al. Cross-reactive antibody responses to the Conflicts of Interest. Conflicts that the editors consider relevant to the con- 2009 pandemic H1N1 influenza virus. N Engl J Med 2009; 361:1945–52. tent of the manuscript have been disclosed. 24. Carney PJ, Lipatov AS, Monto AS, et al. Flexible label-free quantitative assay for antibodies to influenza virus hemagglutinins. Clin Vaccine Im- References munol 2010; 17:1407–16. 1. Thompson MG, Shay DK, Zhou H, et al. Estimates of deaths associated 25. Crotty S, Aubert RD, Glidewell J, Ahmed R. Tracking human antigen- with seasonal influenza—United States, 1976–2007. MMWR 2010; specific memory B cells: a sensitive and generalized ELISPOT system. 59:1057–62. J Immunol Methods 2004; 286:111–22. Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 11 � � 26. Brown H, Prescott R. Applied Mixed Models in Medicine. 2nd ed. Chich- 34. Gitelman AK, Kaverin NV, Kharitonenkov IG, et al. Dissociation of the ester, UK: J. Wiley & Sons; 2006. haemagglutination inhibition and the infectivity neutralization in the 27. Littell RC, Milliken GA, Stroup WW, et al. SAS for Mixed Models. reactions of influenza A/USSR/90/77 (H1N1) virus variants with mono- 2nd ed. Cary, NC: SAS Institute, Inc.; 2006. clonal antibodies. J Gen Virol 1986; 67(Pt 10):2247–51. 28. He XS, Holmes TH, Zhang C, et al. Cellular immune responses in chil- 35. Rowe T, Abernathy RA, Hu-Primmer J, et al. Detection of antibody to dren and adults receiving inactivated or live attenuated influenza vac- avian influenza A (H5N1) virus in human serum by using a combina- cines. J Virol 2006; 80:11756–66. tion of serologic assays. J Clin Microbiol 1999; 37:937–43. 29. Kogut I, Scholz JL, Cancro MP, Cambier JC. B cell maintenance and 36. Voordouw AC, Beyer WE, Smith DJ, et al. Evaluation of serological tri- function in aging. Semin Immunol 2012; 24:342–9. als submitted for annual re-licensure of influenza vaccines to regulatory 30. Lefebvre JS, Haynes L. Aging of the CD4 T cell compartment. Open authorities between 1992 and 2002. Vaccine 2009; 28:392–7. Longevity Science 2012; 6:83–91. 37. Czerkinsky C, Prince SJ, Michalek SM, et al. IgA antibody-producing 31. Goodwin K, Viboud C, Simonsen L. Antibody response to influenza cells in peripheral blood after antigen ingestion: Evidence for a common vaccination in the elderly: a quantitative review. Vaccine 2006;24: mucosal immune system in humans. Proc Natl Acad Sci U S A 1987; 1159–69. 84:2449–53. 32. Allman D, Miller JP. B cell development and receptor diversity during 38. Sasaki S, Sullivan M, Narvaez CF, et al. Limited efficacy of inacti- aging. Curr Opin Immunol 2005; 17:463–7. vated influenza vaccine in elderly individuals is associated with 33. Epperson S, Blanton L, Dhara R, et al. Influenza activity—United States decreased production of vaccine-specific antibodies. J Clin Invest and Worldwide, 2007–2008 season. MMWR 2008; 57:692–7. 2011; 121:3109–19. 12 OFID Reber et al � � http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

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Abstract

MA JO R A R T IC LE Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses 1 1 1 2 3,b 1,a 1 Adrian J. Reber, Jin Hyang Kim, Renata Biber, H. Keipp Talbot, Laura A. Coleman, Tatiana Chirkova, F. Liaini Gross, 1 1 1 1 1 3 1 Evelene Steward-Clark, Weiping Cao, Stacie Jefferson, Vic Veguilla, Eric Gillis, Jennifer Meece, Yaohui Bai, 1 1 1 1 1 1 1,c Heather Tatum, Kathy Hancock, James Stevens, Sarah Spencer, Jufu Chen, Paul Gargiullo, Elise Braun, 2,4 3 3 1 1 Marie R. Griffin, Maria Sundaram, Edward A. Belongia, David K. Shay, Jacqueline M. Katz, and Suryaprakash Sambhara 1 2 Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Vanderbilt 3 4 University Medical Center, Nashville, Tennessee; Marshfield Clinic Research Foundation, Wisconsin; and Mid-South Geriatric Research Education and Clinical Center, VA TN Valley Healthcare System, Nashville, Tennessee Background. Influenza disproportionately impacts older adults while current vaccines have reduced effective- ness in the older population. Methods. We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008–2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results. Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of sub- jects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of post- vaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older + + adults still demonstrated a significant increase in antigen-specific IgG and IgA memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50–59. Conclusions. Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest neg- ative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age. Keywords. aging; immune response; influenza; older adults; vaccine. Seasonal influenza is responsible for an estimated 23 000 influenza-associated illness than younger adults and lon- deaths (range, 3349–48 614) annually in the United ger hospital stays [2–6]; these effects are even more pro- States [1, 2]. The majority of these deaths occur among nounced with more advanced age [2]. adults aged 65 years and older [1]. Adults age 65+ have Although vaccination remains the most cost-effective 10 to 30 times more hospitalizations annually due to method to reduce influenza-associated morbidity and mortality, immunosenescence renders vaccines less ef- fective in older adults [7, 8]. Contributing factors include Received 26 January 2015; accepted 14 April 2015. impaired T and B cell functionality and narrowing of the Present Affiliation: Emory Children’s Center, Division of Pediatric Infectious Dis- T and B cell repertoire [9–15]. Although it is recognized eases, Emory University, Atlanta, Georgia. Present Affiliation: Abbott Nutrition, Columbus, Ohio. that more effective vaccines are needed for older adults, Present Affiliation: Yale School of Public Health, Yale University, New Haven, the capacity of their immune systems to respond and the Connecticut. Correspondence: Suryaprakash Sambhara, DVM, PhD, Centers for Disease components of the immune system that should be tar- Control and Prevention, Influenza Division, Immunology and Pathogenesis Branch, geted for improved efficacy are not well understood. 1600 Clifton Road, Atlanta, GA 30333 (ssambhara@cdc.gov). The hemagglutination inhibition (HI) assay is currently Open Forum Infectious Diseases Published by Oxford University Press on behalf of the Infectious Diseases Society of used to evaluate vaccine immunogenicity, in part because America 2015. This work is written by (a) US Government employee(s) and is in the an HI antibody titer ≥32 or 40 is generally considered a public domain in the US. laboratory correlate of protection, having been associated DOI: 10.1093/ofid/ofv052 Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 1 � � with protection against influenza infection in 50% of the popu- Table 1. Participant Characteristics lation in a number of studies in younger adults [16–19]. Findings from recent studies also suggest that cell-mediated immune re- Age Group Overall 50–59 60–69 70–79 80+ sponses may correlate better with protection in older individuals Subject no. 90 26 23 27 14 [20, 21]. However, alternative correlates to HI have not yet been Sex (%) Male 44.4 23.1 52.2 55.6 50.0 established. Although multiple studies have compared immune Female 55.6 76.9 47.8 44.4 50.0 responses of older adults to young healthy adults, relatively Race (%) fewer have examined the progression in later life. This study White 94.4 84.6 95.7 100.0 100.0 examines factors influencing immune responses to seasonal tri- Black 4.4 11.5 4.3 0.0 0.0 valent inactivated influenza vaccine (TIV) progressing from Other 1.1 3.8 0.0 0.0 0.0 middle-age (age 50–59 years) through advanced age (age 80+) Comorbidity (%) using a comprehensive set of assessments for antibody and Heart 1.1 3.8 17.4 7.4 0.0 cell-mediated immune responses. Lung 7.8 19.2 4.3 7.4 7.1 Immune 10.0 0.0 0.0 7.4 7.1 HI ≥40 (%) METHODS H1N1 Day 0 10.2 8.0 13.0 7.7 14.3 Methods are presented in brief and described in detail in Day 28 33.7 40.7 39.1 28.0 21.4 Supplementary Figure 1. H3N2 Day 0 29.5 32.0 30.4 26.9 28.6 Study Design Day 28 77.5 74.1 82.6 92.0 50.0 Approximately 590 subjects were enrolled during September ≥4-fold rise (%) through October 2008 at Vanderbilt University Medical Center H1N1 22.7 20.0 17.4 26.9 28.6 (Nashville, TN) and Marshfield Clinic Research Foundation H3N2 64.8 72.0 47.8 76.9 57.1 (Marshfield, WI) [22]. Subjects ≥50 years old were eligible to Abbreviations: HI, hemagglutination inhibition; HIV, human immunodeficiency participate at Vanderbilt, and subjects ≥65 years old were eligi- virus. ble to participate at Marshfield. Subjects were grouped by age; a Comorbid conditions include heart and lung disease, and immunosuppression (Immune). Immunosuppression includes immune dysfunction (including HIV), 50–59 (middle-aged), 60–69, 70–79, and 80+ years. Informed transplant, chemotherapy, steroid, or other immune-modulating medications. consent was obtained from all participants. Human experimen- tation guidelines of the Department of Health and Human Ser- vices were followed in the conduct of this trial. Procedures, assays, serial 2-fold dilutions were tested in duplicate and ex- informed consent documents, and data collection forms were pressed as the reciprocal of the highest dilutions giving 50% reviewed and approved by Institutional Review Boards at each neutralization or complete inhibition of agglutination. Anti- site. Participant characteristics are summarized in Table 1. body binding to hemagglutinin (HA) was analyzed by biolayer Participants received TIV from their usual caregiver, at vaccine interferometry on an Octet Red instrument (Fortebio, Inc., clinics, or at the study site by study staff. The 2008–2009 TIV was Menlo Park, CA). H3N2 and H1N1 recombinant HA was syn- composed of A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 thesized and purified as described previously [24]. (H3N2), and B/Florida/4/2006 strains. Although HI titers were determined for all subjects, for this study, a subset of 130 rando- Cell-Mediated Responses mly selected subjects underwent a more comprehensive evalua- Analysis of T-cell responses was performed on a subset of 60 sub- tion which included HI and microneutralization (MN) assays, jects, randomly selected from each age group. Cells were stimu- antibody binding by biolayer interferometry, and T and B cell lated overnight with live H3N2 or H1N1 virus. Brefeldin A (Golgi analysis. Due to limited sample volumes and the number of as- Plug; BD Biosciences, San Diego, CA) was added to cultures for sessments performed, only the influenza A strains were examined the last 6 hours to block Golgi transport. Cells were stained with in this study. Evaluations and analysis were done by staff blinded monoclonal antibodies recognizing CD4, CD8, tumor necrosis to participant age. Serum samples were collected from all subjects factor (TNF)-α, and interferon (IFN)-γ (BD Biosciences) and an- and peripheral blood mononuclear cells (PBMCs) from a subset alyzed using an LSRII Flow Cytometer (BD Biosciences). of subjects prior to vaccination and 21–28 days postvaccination. For assessment of antigen-specific memory B cells, PBMCs Serological Assays were cultured with poke weed mitogen (Sigma-Aldrich, Microneutralization and HI assays were performed as previous- St.Louis,MO),TypeBCpGoligonucleotide (Invivogen,San ly described [23]. Hemagglutination inhibition testing was Diego, CA), and purified protein A (Sigma-Aldrich) for 5–6 performed by Focus Diagnostics, Inc. (Cypress, CA). For both days to induce polyclonal activation. A standard memory B cell 2 OFID Reber et al � � enzyme-linked immunospot (ELISPOT) was performed [25]with groups ranging from 2.3- to 3.4-fold. Hemagglutination inhibi- minor modifications (Supplementary Figure 1). Spot-forming tion titers of older adults to H1N1 were not significantly different units were counted using ImmunoSpot (Cellular Techno- than titers from middle-aged adults (Figure 1A). However, sub- logy Ltd., Cleveland, OH) and expressed as percentage anti- jects age 80+ had lower MN titers (GMT ratio < 1) than middle- gen-specific IgG or IgA B cells of total IgG or IgA-secreting aged subjects postvaccination (Figure 1B). cells. Monovalent-inactivated vaccines were provided by Sanofi In contrast to humoral responses to H1N1, subjects mounted Pasteur to assess the frequency of influenza-specific antibody- more robust responses to H3N2. More subjects (29.5% vs secreting cells (ASCs). 10.2%) had preexisting HI titers ≥40 to H3N2 than H1N1 (Table 1), and H3N2 HI and MN titers increased in all age Statistical Analysis groups postvaccination (Figure 1A and B). A total of 77.5% of Log -transformed HI and MN titers were used as dependent 2 subjects achieved HI titers ≥40 to H3N2 postvaccination with variables and summarized as geometric mean titers (GMT) individual age groups ranging from 50.0% to 92.0% (Table 1). after back-transformation. Means and differences in means The percentage of subjects demonstrating a 4-fold or greater were estimated using repeated measures linear mixed models rise in HI titer was within a similar range (Table 1). All older [26, 27]. Back-transforming model-estimated means yielded age groups showed postvaccination titers comparable to mid- GMTs and back-transforming differences between day 28 and dle-aged counterparts (Figure 1A and B). day 0 means yielded GMT ratios (day 0 to day 28 fold-rise). Serum antibody binding was assessed by biolayer interferom- Models contained indicator (1,0) variables representing serum etry. Antibody binding generally followed a similar trend to HI draw day 28 vs day 0, indicator variables representing age and MN titers. Changes in antibody binding induced by H1N1 groups, and product terms representing interaction between were modest and variable by age group, with older subjects serum draw and age group. Interaction terms allowed us to exhibiting lower antibody binding capacity than middle-aged (1) estimate fold-rises having separate slopes by age group subjects (Figure 2A). In contrast, subjects in all age groups in- and (2) test for differences in fold-rises between groups. creased antibody binding capacity to H3N2 postvaccination Linear regression models used continuous year of age as an (Figure 2A). Adults in older age groups showed postvaccination explanatory variable. Dependent variables were log titers and 2 binding comparable to middle-aged counterparts, although log fold-rises for individual subjects. This allowed visualization 2 subjects in the 2 oldest age groups showed slightly lower bind- of trends in the strength of the fold-rise across age. Postvaccina- ing, which approached significance (P = .06; Figure 2A). tion log titers were regressed against prevaccination titers. Pre- and postvaccination serum responses were also regressed Effect of Age on Antibody Titers against T-cell responses. We found a modest and gradual decline in immune responses Antibody binding and T- and B-cell responses used models from middle-age through age 80+ years. The effect of age on similar to serology data; however, because these data are ex- humoral responses to TIV was not readily apparent with all pressed as percentages, we used log transformation similar 10 assays and exhibited differences between strains. When correla- to He et al [28]. Back-transforming model-estimated means tions with age did achieve statistical significance, the correla- yielded geometric mean percentages (GMPs), and back- tions were relatively low with correlation coefficients ranging transforming differences between day 28 and day 0 means yield- between −0.11 and −0.27 (Figures 2Band 3A–D). Postvacci- ed GMP ratios. Analyses were performed using SAS software, nation HI responses to H3N2 demonstrated a modest reduc- version 9.3 (SAS Institute Inc., Cary, NC). tion in HI titers to H3N2 with increasing age (R = −0.21; Figure 3A), although the reduction demonstrated by MN assay did not reach significance (Figure 3C). Despite the re- RESULTS duced responses exhibited by all subjects to H1N1, MN titers Serological Responses to H1N1 were decreased modestly with increasing age Hemagglutination inhibition responses for the entire cohort were (R = −0.25; Figure 3D). Postvaccination antibody binding ca- previously reported [22]. Within the subset of 130 subjects that pacity for both H1N1 and H3N2 also were negatively correlat- underwent comprehensive evaluation, 10.2% had HI titers ≥40 ed with increasing age (Figure 2B). Furthermore, although to H1N1 prior to vaccination with individual age groups ranging postvaccination titers correlated with age, no effect of age from 8.0% to 14.3% (Table 1). Subjects in this study mounted was observed on prevaccination HI and MN titer or antibody only modest humoral responses to H1N1. Although postvaccina- binding (Figures 2Band 3A–D). tion HI and MN titers were marginally higher in each age group The best predictor of postvaccination response was pre- (ie, GMT ratio > 1; Figure 1A and B), the majority of subjects vaccination titers. All age groups significantly increased anti- (66.3%; Table 1) did not reach HI titers ≥40 for H1N1. On aver- body titers in response to TIV (Figure 1A and B), but subjects age, subjects increased their HI titers 2.8-fold with individual age with higher cross-reactive prevaccination HI and MN titers Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 3 � � Figure 1. Assessment of antibody responses to 2008–2009 trivalent inactivated influenza vaccine. A, Serum hemagglutination inhibition (HI) antibody (n = 88) and B, microneutralization titers (n = 90) to H3N2 and H1N1 were assessed on day 0 prior to vaccination and day 28 after vaccination. The geometric mean titer (GMT) ratio (day 28 GMT/day 0 GMT) was calculated to determine the vaccine-associated change in antibody within each age group. A GMT ratio of 1 (dotted line) is indicative of no change. Day 28 GMT ratios were used to compare responses of older adult groups to middle-aged controls (day 28 GMT of older adult group/day 28 GMT of 50–59 age group). A GMT ratio less than 1 (dotted line) is indicative of a lower postvaccination response. Error bars represent 1 standard error. Significance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001. demonstrated higher postvaccination titers to H1N1 and H3N2 Vaccine-Specific Memory B Cells (Figure 3E) with correlation coefficients ranging from 0.62 to Frequencies of vaccine-specific memory B cells were evaluated 0.69. Higher prevaccination binding capacity also correlated by ELISPOT for 10 randomly selected individuals from each age with higher postvaccination binding (Figure 2B) with correla- group. Memory B cells were stimulated to selectively proliferate tion coefficients of 0.75 and 0.82 for H3N2 and H1N1, and differentiate into ASCs (Supplementary Figure 1), and iso- respectively. type-specific ASCs were measured (Figure 4A–D, left panels). 4 OFID Reber et al � � Figure 2. Assessment of antibody binding capacity by biolayer interferometry. A, Antibody binding capacity (n = 90) to H3N2 and H1N1 was assessed on day 0 prior to vaccination and day 28 after vaccination. The geometric mean percent (GMP) ratio (day 28 GMP/day 0 GMP) was calculated to determine the vaccine-associated change in antibody binding within each age group. A GMP ratio of 1 (dotted line) is indicative of no change. Day 28 GMP ratios were used to compare responses of older adult groups to middle-aged controls (day 28 GMP of older adult group/day 28 GMP of 50–59 age group). A GMT ratio less than 1 (dotted line) is indicative of lower postvaccination antibody binding. B, Antibody binding capacity was plotted by age and correlations calculated to determine the effect of age on preexisting ( ) and postvaccination antibody binding ( ). The correlation coefficient and P value are presented for preexisting ( ) and postvaccination ( ) correlations with age. The influence of preexisting, cross-reactive antibody on the day 28 postvaccination response to H1N1 ( ) and H3N2 ( ) was also determined. The correlation of pre- and postvaccination antibody responses to H1N1 ( ) and H3N2 ( ) are depicted. The correlation coefficient and P value are presented for prevaccination H1N1 ( ) and H3N2 ( ) correlations with the postvaccination antibody binding capacity. Error bars represent 1 standard error. Significance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001. Subjects in the older age groups generally showed the lowest lev- response to TIV vaccination (Figure 4). The frequencies of els of preexisting memory B cells, but they also tended to dem- preexisting (d0) memory B cells in subjects aged 70+ years onstrate the largest expansion of memory B-cell populations in were lower than in middle-aged subjects (GMP ratio < 1) for Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 5 � � Figure 3. Correlations with vaccine antibody response. Antibody to A and C, H3N2 and B and D, H1N1 were assessed from serum samples taken at prevaccination and 28 days postvaccination by A and B, HI (n = 88) and C and D, microneutralization ([MN] n = 90). Antibody levels were plotted by age, and correlations were calculated to determine the effect of age on preexisting and postvaccination antibody titers. The correlation coefficient and P value are presented for correlations with age. E, The influence of preexisting, cross-reactive antibody to H3N2 ( ) and H1N1 ( ) on the postvaccination, day 28 re- sponse was also determined. The correlation of pre- and postvaccination antibody responses to H1N1 ( ) and H3N2 ( ) are depicted. The correlation coefficient and P value are presented for prevaccination H1N1 ( ) and H3N2 ( ) correlations with the postvaccination titers. HI, hemagglutination inhibition. 6 OFID Reber et al � � + + Figure 4. Age-associated changes in preexisting memory B cells. The number of H3N2 (H3)-specific A, immunoglobulin (Ig)G and B, IgA as well as + + H1N1 (H1)-specific C, IgG and D, IgA memory B cells were assessed by B cell enzyme-linked immunospot at day 0 before vaccination and on day 28 postvaccination. The day 0 geometric mean percent (GMP) of memory B cells of older adults were compared with middle-aged controls by generating day 0 GMP ratios (day 0 GMP of older adult group/day 0 GMP of 50–59 age group). A d0 GMP ratio less than 1 (dotted line) is indicative of fewer antigen-specific, preexisting memory B cells. The GMP ratio (day 28/day 0% antigen-specific cells) was calculated to determine changes in the number of memory B cells within each age group associated with vaccination. A GMP ratio of 1 (dotted line) is indicative of no change. Error bars represent 1 standard error. Sig- nificance is indicated by *P ≤ .05, **P ≤ .01, P ≤ .001; n = 42. Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 7 � � H3-specific IgG and IgA memory B cells (Figure 4A and B). Al- factor to the observed reduced effectiveness of seasonal influen- though preexisting (d0) H1-specific IgG memory B cells tended za vaccines in persons 65 years and older. Decreased T and B to be lower in subjects over age 60, there were no significant dif- cell functionality and narrowing of the T and B cell repertoire ferences over time in any of the age groups (Figure 4C). Postvac- results in reduced antibody titers, lower antibody affinity, and cination, H3- and H1-specific IgG memory B cells increased in narrowing of the antibody repertoire [29–32]. the 70–79 age group as well as the 60–69 age group in the case of All 2008–2009 TIV strains changed from the previous season’s H1 (Figure 4A and C). It is interesting to note that middle-aged vaccine, resulting in the majority of the population studied here subjects had decreased numbers of H3-specific IgG memory B having a relatively low level of preexisting cross-reactive, neutral- cells postvaccination (Figure 4A). All age groups exhibited in- izing antibodies to the influenza A virus vaccine components. In creased numbers of H3-specific IgA memory B cells in response our study, only 10.2% and 29.5% of subjects had preexisting HI to vaccination. However, H1-specific IgA memory B cells in- titers ≥40 to H1N1 and H3N2, respectively (Table 1). During the creased only among subjects in the 2 oldest age groups (Fig- 8influenza seasons preceding the 2008–2009 season, there was ure 4B and D). only 1 change in the predominant H1N1 strain; the vaccine an- tigen changed from A/New Caledonia/20/1999 to A/Solomon Is- T-Cell Responses lands/3/2006 for the 2007–2008 season. In contrast, 4 different Peripheral blood mononuclear cells were stimulated with live H3N2 strains predominated in the same period. In addition, virus, and the proportions of IFN-γ and TNF-α-producing the previous season was dominated by H3N2, with 60% of T cells were assessed. Although sampling closer to the time of H3N2 samples tested being more antigenically similar to A/Bris- vaccination would have been more beneficial for assessment of bane/10 than to A/Wisconsin/67, that season’s vaccine strain T-cell responses, it would not have been optimal for detection of [33]. Thus, vaccination and/or natural infection with more anti- changes in antibody titers. T-cell responses were highly variable, genically diverse H3N2 strains in the recent past may have led to and the small number of subjects meant detection of potentially more cross-protective prevaccination titers for H3N2. Indeed, meaningful differences in T-cell responses was difficult (Sup- our memory B cell ELISPOT indicated that subjects had a larger plementary Figure 2A–H). Similar to what was observed for number of preexisting H3N2-specific, IgG memory B cells than memory B cells, subjects in the 50–59 year old age category gen- those against H1N1 (Figure 4A and C). erally had higher preexisting H1N1- and H3N2-specific CD4 Three assays were used to comprehensively assess the effect of T-cell responses (Supplementary Figure 2I–L). However, after age on antibody responses. The HI assay has historically been vaccination, CD4 T-cell responses among older subjects ap- used to assess protection from influenza and is the best under- peared to be either comparable or lower but not statistically dif- stood correlate of protection against influenza infection in ferent from their middle aged counterparts (Supplementary younger adults. However, HI is a surrogate assay and does not Figure 2M–P). No significant CD8 T-cell responses were directly measure virus neutralization. The MN assay measures found (data not shown); however, TIV is not known to effec- functional neutralization and has been suggested to be more tively induce CD8 T-cell responses. sensitive than HI [34, 35], but a threshold MN titer associated Postvaccination HI, MN, and antibody binding capacity in this with protection has not been described. Biolayer interferometry study all correlated with IFN-γ-producing CD4 T cells with cor- detects all antibody capable of binding to HA, not just those relation coefficients ranging from 0.31 to 0.46 (Figure 5A). Only that neutralize virus infectivity, presumably including antibody antibody binding capacity for H1N1, which had demonstrated with broader functionality such as opsonization, initiation of only low and variable changes to H1N1 (Figure 2A), did not cor- the complement cascade, and/or antibody-mediated cellular cy- relate with IFN-γ-producing CD4 T cells (Figure 5A). Tumor totoxicity. Thus, biolayer interferometry may give an indication necrosis factor-α producing CD4 T cells did not correlate with of broader protection beyond simple virus neutralization, pro- any of the serological responses, nor did CD8 T cells demon- viding some indication of the quality of the antibody response. strate any correlation (data not shown). It is interesting to note Subjects in all age groups increased HI and MN titers postvac- that preexisting IFN-γ responses to H3N2 by CD4 T cells cor- cination (Figure 1A and B); however, many did not exhibit HI ti- related with postvaccination antibody binding (R = 0.35; Fig- ters ≥40. This was especially true for H1N1; only 33.7% of subjects ure 5B), but not with HI or MN titers (data not shown). As reached titers ≥40, compared with 77.5% for H3N2 (Table 1). with the postvaccination response, preexisting CD4 T cell re- Subjects in all age groups demonstrated increased binding capacity sponses to H1N1 showed no correlation with antibody binding. to H3N2; however, only subjects age 50–59 and 70–79 increased H1N1 binding (Figure 2A). It should be noted, however, that H3N2 virus activity has been found consistently to be associated DISCUSSION with more severe morbidity and mortality among older adults Immunosenescence is considered an impediment to protection than H1N1 [2, 5]. Furthermore, as previously stated, cell-mediated of older adultsfrominfectiousdiseasesand a contributing immune responses may correlate better with protection in older 8 OFID Reber et al � � + Figure 5. Correlations with T-cell responses. A, Postvaccination interferon (IFN)-γ production by H1N1- or H3N2-stimulated CD4 T cells correlated with postvaccination hemagglutination inhibition (HI), microneutralization (MN), and antibody binding capacity. B, correlations were also calculated between + + + + prevaccination IFN-γ CD4 T cells and postvaccination antibody binding capacity to determine the influence of preexisting IFN-γ CD4 T cells on antibody response to vaccination. The correlation coefficient and P values are presented for each comparison. Effect of Preexisting Immunity and Age on Influenza Vaccine Responses OFID 9 � � adults than HI titers [20, 21]. Protection from influenza in older seemingly contradictory to recent studies by Sasaki et al [38] adults may be more complex than increased HI titers. that showed frequencies of vaccine-specific plasmablasts and In general, we confirmed that antibody responses decreased concentration of plasmablast-derived polyclonal antibody with increasing age, although results varied by assay type and were lower in older adults than young adults, whereas yields virus strain. Antibody binding capacity was negatively correlat- of secreted IgG per plasmablast and overall vaccine-specific ed with age for both strains, consistent with a potential impact avidity or affinity of polyclonal antibodies were not different be- on affinity maturation and possible narrowing of the antibody tween age groups. In their study, subjects age 70–100 were com- repertoire (Figure 2B). However, this effect was relatively mod- pared with those aged 18–30, whereas our subjects were all 50 est; correlation coefficients ranged from −0.11 to −0.27. Prevac- years and older. Although the quantity of the antibody response cination titers were the best predictors of postvaccination HI, decreases in advanced age compared with young adults, this MN, and antibody binding, with correlation coefficients be- change could be gradual and subtle during transition from mid- tween 0.62 and 0.82 (Figures 2B and 3E). Although postvacci- dle-age to older adults (60–79) to elderly adults (80+). Instead, nation titers were impacted by age, prevaccination titers, the qualitative changes (eg, affinity maturation, class switching, etc.) best predictors of postvaccination response, showed no correla- may start to compromise antibody responses. Consistent with tion with age (Figures 2B and 3A–D). A meta-analysis by Voor- this idea, the age-associated decline in HI titers was significant douw et al [36] analyzing influenza clinical trial data from the only for H3N2 (but not H1N1) and MN titer only for H1N1 European Union showed similar influences of age and prevac- (but not H3N2), yet antibody binding capacity for both anti- cination titers on postvaccination responses. gens declined with age (Figure 2B). For a typical influenza season, some proportion of the popula- Serological responses in this older adult population correlat- tion has at least partial immunity to circulating strains through ed with IFN-γ-producing CD4 T cells, with subjects exhibiting + + prior vaccination or natural infection. Preexisting, antigen- higher numbers of IFN-γ CD4 T cells also exhibiting higher specific or cross-reactive memory B cells are frequently the HI, MN, and antibody binding capacity (Figure 5A). The role of main source for protective antibody. Replacement of all 3 vac- IFN-γ in protection of all age groups against viral infection has cine strains for the 2008–2009 influenza season provided a long been recognized. McElhaney et al [20] previously demon- unique opportunity to assess changes in antigen-specific mem- strated that older adults with higher IFN-γ/interleukin-4 ratios ory B cell frequency in association with age. Indeed, 2008–2009 were less likely to exhibit influenza-like illness than those with + + TIV boosted the frequencies of class-switched memory B cells lower ratios. Furthermore, preexisting IFN-γ CD4 T cells cor- (antigen-specific IgG and IgA) in almost all age groups (Fig- related with postvaccination antibody binding capacity, whereas ure 4A–D). Subjects in all age groups significantly expanded HI and MN titers did not (Figure 5B), suggesting that preexist- + + H3-specific IgA memory B cells (Figure 4B), and despite the over- ing or cross-reactive IFN-γ CD4 T cells may play a role in en- all lower antibody titers to H1, there were significant expansions hancement of antibody quality. of H1-specific memory B cells in most age groups (Figure 4Cand Preexisting immunity in this study had a significant effect on D). Although it is normally thought that HI activity and virus the ensuing immune response to vaccination. The constant neutralization are primarily mediated by serum IgG, it has been evolution of the influenza virus makes yearly vaccine strains dif- speculated that IgA memory B cells may home to mucosal surfac- ficult to predict. Yet, despite the change in all 3 influenza vac- es upon infection, providing protection of mucosal surface cine strains for the 2008–2009 season, we found cross-reactivity through local production of IgA [37]. Assessment of both IgA to these strains in our study population. Although T cell epi- and IgG memory B cells may provide a more comprehensive pic- topes are recognized as being much more highly conserved, ture of protection upon infection. we were also able to demonstrate low levels of preexisting or It is noteworthy that despite a reduced frequency of preexist- cross-reactive antibody. During the 2009 H1N1 pandemic, ing memory B cells associated with increasing age, fold-rises in older adults were believed to be relatively more protected to memory B cell frequency among subjects aged 60+ was either the virus due to exposure to related viruses earlier in life [23]. comparable to or better than that found among subjects in This exemplifies the impact that prior immunity may have in the middle-age group (Figure 4A–D). These findings suggest providing protection from influenza and the potential longitude that the gross function of B cells pertaining to memory B-cell of this impact. The history of a person’sinfluenza vaccination/ generation was not drastically compromised. More importantly, infection likely influences their current responses, and the im- however, binding capacity of post-vaccine serum antibody de- mune system of older adults may have an extensive lifetime of clined for both strains with increasing age (Figure 2B), indicat- exposure from which to draw. ing that the capacity of somatic hypermutation in antibody This study was limited by several factors. First, for several as- variable regions is affected by advancing age. Our data suggest says, it was feasible to test only a small number of samples, thus that immunosenescence in advanced age is manifested more by limiting the number of statistical comparisons that could be qualitative changes than quantitative changes. This result is made and reducing the power to detect differences by age 10 OFID Reber et al � � 2. Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with groups. Second, populations at the 2 sites were different. Partic- influenza and respiratory syncytial virus in the United States. JAMA ipants at Vanderbilt were typically younger and more likely to 2003; 289:179–86. be employed, whereas those at Marshfield were older and had 3. World Health Organization (WHO). Fact sheet N°211. April 2009. previously received influenza vaccination. Differences observed Available at: http://www.who.int/mediacentre/factsheets/fs211/en/ index.html. Accessed 3 March 2014. between groups may not have been solely due to age but instead 4. Falsey AR, Hennessey PA, Formica MA, et al. Respiratory syncytial affected by differences in methods of recruitment or other site- virus infection in elderly and high-risk adults. N Engl J Med 2005; specific differences. Finally, chronic preexisting medical condi- 352:1749–59. 5. Thompson WW, Shay DK, Weintraub E, et al. Influenza-associated tions, sex, and race are all known to affect immune function, but hospitalizations in the United States. 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Published: Apr 1, 2015

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