Augmented CD4+ T-cell and humoral responses after repeated annual influenza vaccination with the same vaccine component A/H1N1pdm09 over 5 years

Annual seasonal influenza vaccination is recommended for high-risk populations and often occupational groups such as healthcare workers (HCWs). Repeated annual vaccination has been reported to either have no impact or reduce antibody responses or protection. However, whether repeated vaccination influences T-cell responses has not been sufficiently studied, despite the increasing evidence of the protective roles of T-cell immunity. Here, we explored the impact of repeated annual vaccination with the same vaccine strain (H1N1pdm09) over multiple seasons in the post-2009 pandemic era and showed that repeated vaccination increased both T-cell and humoral responses. Using the T-cell FluroSpot and intracellular cytokine-staining, the hemagglutination inhibition (HI), and the memory B-cell (MBC) ELISpot assays, we investigated pre- and postvaccination T cells, antibodies, and MBCs in a cohort of HCWs repeatedly vaccinated with H1N1pdm09 for 5 years (pandemic vaccination in 2009 and subsequently annual seasonal vaccination containing H1N1pdm09 during 2010–2013). We found that the prevaccination H1N1pdm09-specific T cells, antibodies, and MBCs were significantly increased after 3–4 repeated vaccinations and maintained at high levels throughout seasons 2012 and 2013. The cross-reactive IFN-γ-secreting CD4+ cells recognizing conserved viral external or internal epitopes were also maintained throughout 2012 and 2013. Repeated vaccination improved the multifunctional memory CD4+ responses. Particularly, the IFN-γ+TNF-α+CD4+ T cells were boosted following each vaccination. HI antibodies were significantly induced after each vaccination over 5 years. Our findings indicate a broad impact of repeated annual vaccination, even with the same vaccine component, on the influenza-specific T-cell and humoral immunity and support the continuing recommendation of annual influenza vaccination.


INTRODUCTION
Influenza virus remains a major health challenge due to its continuous ability to evade the hosts' immunity. Annual seasonal influenza vaccination is the main method of prophylaxis for highrisk populations and healthcare workers (HCWs) providing protection against influenza A/H1N1, A/H3N2, and B viruses. 1 In 2009, a novel H1N1 virus (H1N1pdm09) emerged and caused the first pandemic of the twenty-first century. HCWs were prioritized for pandemic vaccination to protect their patients and themselves, and maintain the integrity of the healthcare system. 2 The AS03adjuvanted monovalent H1N1pdm09 vaccine was used during the pandemic in Norway and provided protection against laboratoryconfirmed influenza infection and hospitalization. 3 The H1N1pdm09 virus continued to circulate after 2009 replacing earlier H1N1 strains and was therefore included in the seasonal vaccines as the A/H1N1 component during seasons 2010−2016.
Antibodies directed against the main viral surface glycoprotein, hemagglutinin (HA), can neutralize the influenza virus. The hemagglutination inhibition (HI) assay has been widely used to evaluate the HA-specific antibody responses. An HI titer of 40 is established as a surrogate correlate of protection against influenza at a 50% protective threshold. 4 Inactivated influenza vaccines are standardized by the quantity of HA of each strain and induce HI antibodies after vaccination. Moreover, T cells have recently gained more recognition for their protective roles. Preexisting influenza-specific interferon (IFN)-γ-secreting CD4 + or CD8 + T cells can recognize conserved viral epitopes and provide crossprotection from heterosubtypic influenza A viruses, even in the absence of protective antibodies. [5][6][7][8] Importantly, influenza vaccines have been used for decades; however, the long-term impact of repeated annual vaccination on antibody responses is not fully understood [9][10][11] and there are limitations of our knowledge of its impact on T-cell responses. The emergence of the H1N1pdm09 virus and its inclusion as the A/ H1N1 component in the seasonal vaccines for multiple years provided a unique opportunity to investigate the impact of repeated vaccination. Previously, we investigated the impact of repeated annual vaccination upon preexisting influenza-specific CD8 + and CD4 + T cells prior to two consecutive influenza seasons in HCWs who were either repeatedly vaccinated or only received a pandemic vaccination. 12 In the current study, we further explored the impact of annual vaccination on T cells, particularly CD4 + T cells, and humoral immunity by assessing paired pre-and postvaccination T cell, antibody, and memory B-cell (MBC) responses in repeatedly vaccinated HCWs over 5 years. We have extended our previous findings to show that repeated annual vaccination with the same strain augmented both humoral and CD4 T-cell responses, maintained the cross-reactive IFN-γ-secreting CD4 + T cells recognizing viral external and internal epitopes, while increasing multifunctional memory CD4 + responses. Our findings have implications for the seasonal influenza vaccination strategy and vaccine development.

Study population
Fourteen HCWs (mean age 41.2 years old, range 30-63 years), who received the AS03-adjuvanted pandemic vaccine in 2009 and subsequently annual seasonal vaccination during seasons 2010-2013, were included in this study (Fig. 1). Most HCWs (12/ 14) were female, worked on a clinical ward, and had history of previous seasonal vaccination before 2009 (Supplementary- Table 1).
All cytokine-secreting T cells against the split virus were maintained at high levels throughout the two seasons, although no significant boost of these responses was observed after vaccination (Fig. 2a). The IFN-γ-secreting T cells recognizing the three internal proteins were maintained throughout 2012 and 2013 (Fig. 2c, e, g). However, we observed a decline in IL-2secreting T cells recognizing M1 and NP after 1 year and a low but stable level of double-cytokine-secreting cells, leading to an increased IFN-γ/IL-2 ratio over time (Fig. 2d, f). Whereas IFN-γ dominated the responses against PB1, as there was almost no IL-2 or double-cytokine secretion (Fig. 2g, h). The dominant IFN-γ trend was not observed in the H1N1pdm09-specific T cells against the split virus (containing mainly surface glycoproteins), which had a balanced IFN-γ/IL-2 response (Fig. 2b).
Maintenance of cross-reactive IFN-γ-secreting CD4 + T cells The influenza-specific cross-reactive CD4 + T-cell responses were investigated using separate CD4 external or internal peptide pools. We found that the cross-reactive IFN-γ-or IL-2-secreting CD4 + T cells against external epitopes were maintained, while there was a trend of increased double-cytokine responses after vaccination (p = 0.094) (Fig. 3a). The cross-reactive IFN-γ-or double-cytokine-secreting CD4 + T cells against internal epitopes were maintained throughout the two seasons, whereas the IL-2secreting cells declined after vaccination in 2012 (p = 0.088) and further in 2013 (p = 0.003) (Fig. 3e), resulting in the shift toward IFN-γ dominance over time (Fig. 3f). However, the trend of predominant IFN-γ was not observed in the cross-reactive responses to external epitopes (Fig. 3b).
To further examine this trend, T-cell responses were assessed in a control group of HCWs who were only vaccinated with the AS03adjuvanted H1N1pdm09 vaccine in 2009 and received no further seasonal vaccinations. We have previously reported a decline in all cytokine-secreting cross-reactive CD4 + T cells against internal epitopes and H1N1pdm09-specific T cells after a year in these HCWs ( Fig. 3g and Supplementary-Fig. 1a). 12 We assessed the CD4 + T cells against external epitopes and observed a decline in double-cytokine secretion after a year, although not significant (Fig. 3c). A balanced IFN-γ/IL-2 response against the CD4 external epitopes and split H1N1pdm09 virus was found in this control group ( Fig. 3d and Supplementary-Fig. 1b). However, no trend of increasing IFN-γ/IL-2 ratio in the CD4 + T cells specific for viral internal epitopes was observed, supporting the impact of repeated vaccination in favoring IFN-γ responses (Fig. 3h).

Enhanced quantity and quality of T cells after repeated vaccinations
We further investigated the long-term impact of repeated vaccination on T cells during the 5 study-years by retrospectively analyzing the H1N1pdm09-specific T-cell responses after pandemic vaccination in 2009 13  By comparing cytokine profiles across these three seasons, we found a significant increase in multifunctional CD4 + T cells (secreting 2−3 cytokines) after repeated vaccinations (Fig. 5b,  Supplementary-Fig. 2b), indicating an enhanced T-cell quality over the 5 years. 14,15 The frequencies of triple producer (p = 0.008), and double producers IFN-γ + TNF-α + (p = 0.018) and IL-2 + TNF-α + (p = 0.034) CD4 + T cells prevaccination were significantly higher in 2013 than in 2009. Interestingly, the IFN-γ + TNF-α + and IL-2 + TNFα + CD4 + responses were boosted following 2009 vaccination (p = 0.031). In contrast, the frequencies of single IL-2-secreting T cells in the last two seasons were significantly lower than in 2009 (p = 0.008). The prevaccination cytokine-profile radar chart (Fig. 5c) shows that repeated vaccinations skewed the CD4 + T-cell responses toward IFN-γ dominance and the IL-2 responses were shifted from single to multifunctional IL-2 (double IL-2 + TNF-α + or triple IFN-γ + IL-2 + TNF-α + co-producers).
Maintenance of high humoral responses after repeated vaccinations Throughout the 5 years of our study, the H1N1pdm09-specific HI antibodies were significantly boosted after each vaccination (Fig.  6a). After the second vaccination, HI antibodies persisted above the 50% protective threshold (HI titers ≥ 40) for 1-year postvaccination in all HCWs, except one who had chronic respiratory and neurological conditions. The prevaccination HI titers gradually increased from 2009 to 2012, then were maintained between 2012 and 2013. The antibody fold-induction between pre-and postvaccination titers was highest after the adjuvanted pandemic vaccination in 2009 then declined after multiple vaccinations (Fig.  6b). The fold-induction after seasonal vaccination in 2012 and 2013 was significantly lower than in earlier seasons. We further assessed the H1N1pdm09-specific MBC responses in HCWs pre-and postvaccination in 2012 and 2013 (n = 5 due to limited PBMC availability), while the 2009 data were retrospectively analyzed from nine HCWs. 13 The prevaccination H1N1pdm09-specific MBCs was significantly higher in 2012 and 2013 than in 2009 (Fig. 6c). Notably, MBCs were significantly boosted following pandemic vaccination in 2009 (p = 0.031), but not after seasonal vaccination in 2012 and 2013. The foldinduction after vaccination was higher in 2009 than in 2013 (p = 0.061) (Fig. 6d).
We investigated the overall impact of repeated vaccination against H1N1pdm09 on immune responses using radar charts. An increase in both humoral and T-cell immunity specific for H1N1pdm09 prevaccination were observed after 3−4 vaccinations (Fig. 6e). Interestingly, MBCs and double producers CD4 + T cells continue to increase from 2012 to 2013. The fold-induction of the vaccine-induced responses postvaccination was highest after adjuvanted pandemic vaccination in 2009 (Fig. 6f).

DISCUSSION
There is limited knowledge about the long-term impact of repeated annual influenza vaccination on the influenza-specific T-cell immunity despite decades of use of influenza vaccine. Here, we provide unique data on humoral and T-cell responses after repeated annual vaccination with the same H1N1pdm09 strain from its introduction in 2009 and over four subsequent seasons.
Repeated vaccinations resulted in an increase in both the quantity and the quality of H1N1pdm09-specific T-cell responses. One study suggested that >20 IFN-γ-secreting T cells/10 6 PBMC as detectable protective influenza-specific T cells at a population level. 8 In our repeatedly vaccinated HCW cohort, the  -Fig.  1a). 12 Moreover, the quality of CD4 + T cells was improved with higher multi-cytokine-secreting responses, associated with superior function compared to single-cytokine producers. 14,15 The boost of IFN-γ + TNF-α + CD4 + responses after each vaccination in 2009, 2012, and 2013 suggests a continuous increase of these responses. We hypothesize that the magnitude of T cells may reach a plateau after 3−4 repeated vaccinations, like the antibodies, while the quality of T cells continues to improve. Although this study did not assess the quality of antibodies, our earlier report showed that repeated vaccination maintained the antibody avidity. 16 This implies that repeated vaccination may have a broader impact on immune responses, which are not usually assessed in conventional vaccine immunogenicity studies. 17 Future studies should therefore include diverse immunological assessments to better understand vaccine immunogenicity in populations with different influenza exposure backgrounds.
We demonstrated that the H1N1pdm09-specific HI antibodies were boosted after each vaccination during the 5 study-years. The prevaccination antibodies increased each year until 2012 and 2013 persisting above the 50% protective threshold, whereas the antibody fold-induction postvaccination declined and was lowest in the last two seasons compared to previous seasons. This suggests that repeated vaccinations sustain high protective antibody levels rather than boosting them. The long-term antibody persistence is probably due to activation of MBCs that can undergo continuous proliferation and differentiation to maintain constant levels of plasma cells and antibodies. 18 T cells also contribute to induction and maintenance of antibodies by providing help to activate naïve and MBCs. 19 This hypothesis was supported by the significantly higher H1N1pdm09-specific MBCs and T cells observed after 3−4 repeated vaccinations. Moreover, the decline in antibody fold-induction observed in later seasons may be due to the presence of high prevaccination HI antibodies, which may block the vaccine epitopes, resulting in a reduction of B-and T-cell activation, [20][21][22] and therefore limit the boosting ability of subsequent vaccinations.
The controversy of annual influenza vaccination policy involves conflicting reports on the impact of repeated vaccination, which reported either no significant interference or a reduction in antibody responses and vaccine effectiveness (VE). [9][10][11]23,24 In studies that found that repeated vaccination led to decreased protection, this was mainly related to the H3N2 viruses. The H3N2 subtype has been associated with lower VE and undergoes more frequent antigenic changes compared to the H1N1 and B viruses included in the seasonal vaccines. 25 A recent study investigating the impact of repeated vaccination against H1N1pdm09 on VE suggests that VE was highest after 2−3 vaccinations and reduced in vaccinees immunized with >3 vaccinations. 11 Our study did not find a reduction in H1N1specific immune responses after 3−4 vaccinations, although protection was not assessed. Our findings show the maintenance of H1N1pdm09-specific antibodies, MBCs and T cells at high levels with improved quality of CD4 + T cells after 3−4 repeated vaccinations. However, enhanced T-cell responses do not prevent infection, the outcome in most studies investigating protection use, but reduce the severity of illness. [5][6][7][8] We suggest that broader outcome measures of protection, such as severity scores or time to recover, should be incorporated into future studies together with various immunological assessments to better evaluate the impact of repeated vaccination. Taken together, we support the continuation of the current recommendation of annual influenza vaccination, even with the same vaccine component, to provide protection against all circulating seasonal strains. Our findings point to the importance of inclusion of influenza vaccination history, when evaluating vaccine immunogenicity and effectiveness.
Remarkably, the long-lived H1N1pdm09-specific CD4 + CM T cells were boosted after vaccination. Since CM cells can rapidly proliferate and differentiate into effector cells upon antigen encounter, 26 the persistent CD4 + CM and EM responses following repeated vaccination may provide long-lasting protection. These findings agree with our previous report 12 and provide an explanation for the long-term maintenance of IFN-γ + and multifunctional CD4 + T cells in repeatedly vaccinated HCWs, which was not observed in HCWs immunized with only pandemic vaccination. Our findings suggest that repeated vaccination optimizes the quantitative responses while shifting the influenza-specific immunity towards long-term memory and multifunctional responses. However, this hypothesis needs to be verified with the other influenza vaccine strains, H3N2 and B viruses, which change more frequently than H1N1 viruses.
Influenza-specific CD4 + T cells can recognize conserved epitopes from heterosubtypic influenza A viruses and provide cross-protection 5 and are boosted after a single vaccination. 13,27,28 We extended these findings by separately investigating the crossreactive CD4 + responses to conserved viral external or internal epitopes following repeated vaccination, as these cells may be phenotypically different with distinct potential functions. [29][30][31][32] After 3−4 repeated vaccinations, a decline in IL-2 responses resulting in a trend of increased IFN-γ dominance against viral internal epitopes was observed, which agrees with previous reports. 33,34 Long-term exposure to influenza through vaccination or infection may shape the T-cell responses towards conserved epitopes that are repeatedly recognized by influenza-specific memory T cells. We hypothesize that these responses are multifunctional with predominantly IFN-γ and a low level of IL-2. Interestingly, analyses of the quality of H1N1pdm09-specific CD4 + T cells after repeated vaccinations provided a potential explanation supporting this hypothesis. We found the boosting of IFNγ + TNF-α + CD4 + T-cell responses following each vaccination, the increased multi-cytokine-secreting and decreased single IL-2secreting cells after 3−4 repeated vaccinations. The ratio of IFNγ to IL-2-secreting cells, calculated using the cytokine profile data showed that the H1N1pdm09-specific CD4 + T cells remained IL-2 enriched in 2009 but changed toward predominantly IFN-γ secretion in 2012 and 2013 (Supplementary-Fig. 3). However, whether this IFN-γ dominance in cross-reactive CD4 + T cells indicates a greater potential for help and/or other functions will need to elucidate in further studies.
We faced difficulties due to the long follow-up period, such as losing participants and missing samples. The strict inclusion criteria that only HCWs repeatedly vaccinated for 5 years greatly limited the sample size of our study. However, this design allows us to study the long-term impact of repeated vaccinations on immune responses while clarifying the effect of vaccination sequence without the bias of missing vaccination history. As HCWs who were repeatedly vaccinated for 5 years were identified in the last two seasons, the T-cell responses in 2009 were retrospectively evaluated. Although the low number of participants with accessible data and the lack of 2010 and 2011 information on T cells limited our observation, the immune responses after repeated vaccinations were undoubtedly enhanced. As memory Tcell responses are rapidly generated, we cannot dismiss the possibility of T-cell expansion at 7-14 days, 13 time points that were not investigated in this study. Further studies are required to confirm our findings in larger populations with documented vaccination history, and to evaluate both antibody and T-cell responses against other vaccine strains after repeated annual vaccination.
In conclusion, we provide a unique overview of the long-term impact of repeated annual influenza vaccination against the same vaccine strain on humoral and T-cell immunity. Repeated vaccinations with H1N1pdm09 not only maintained the high magnitude of strain-specific HI antibodies and T cells, and crossreactive IFN-γ + CD4 + T cells, but also increased MBC and multifunctional CD4 + T-cell responses. This study highlights a broad immunological impact of repeated vaccination and supports the current recommendation of annual seasonal influenza vaccination. Our findings suggest that routine collection of influenza vaccination history and diverse immunological approaches should be included when evaluating vaccine immunogenicity and effectiveness.

Study population and sampling
An open-label 5-year extension of a single-arm clinical trial was conducted in HCWs (Haukeland University Hospital, Norway) vaccinated with the 2009 AS03-adjuvanted pandemic H1N1pdm09 vaccine (www.Clinicaltrials.gov, NCT01003288). The study was approved by the regional ethics committee (REKVest-2012/1772) and the Norwegian Medicines Agency. 35 All participants provided written informed consent before inclusion and new consent for the follow-up blood samples. During 2010-2013, HCWs were annually vaccinated with the non-adjuvanted seasonal trivalent inactivated vaccine (Vaxigrip, Sanofi Pasteur or Influvac, Abbott Laboratories) containing the H1N1pdm09 as the A/H1N1 component and different A/H3N2 and B viruses (Fig. 1). Serum samples were collected pre-and 21 days postvaccination for each season from 2009 to 2013. HCWs who provided additional PBMC samples pre-and 21-day postvaccination in 2012 and 2013 were included in this study. Sera were separated from clotted blood and stored at −80°C until analyzed. PBMC were isolated and cryopreserved at −150°C in 90% fetal bovine serum/10% dimethyl sulfoxide until analyzed. 12 Hemagglutination inhibition (HI) assay Receptor destroying enzyme-treated sera were tested in duplicate with 0.7% turkey red blood cells (TRBC) and eight HA units of inactivated A/ California/07/2009 (H1N1) antigen, as described previously. 35 The HI titer was the reciprocal of the highest serum dilution causing 50% inhibition of hemagglutination. Titers <10 were assigned a value of 5 for calculation purposes. Sera <40 were screened for nonspecific binding and preadsorbed with TRBC before re-analyzing.
T-cell FluoroSpot assay PBMC were stimulated with the split H1N1pdm09 antigen, the H1N1specific M1, NP, or PB1 peptide pools (BEI Resources), or the conserved CD4 internal or external peptide pools to measure the IFN-γ-and/or IL-2secreting T-cell responses, as described earlier. 12,36 The M1, NP, or PB1 peptide pools included overlapping epitopes that covered the complete sequence of the three proteins. The CD4 peptide pools were chemically synthesized and consisted of HLA−class-II-restricted T-cell epitopes from internal or external viral proteins that are conserved among influenza A subtypes with high prevalence and HLA−supertype coverage (Supplementary-Tables 2, 3). 36 Cytokine-secreting cells were counted and the background from non-stimulated cells was subtracted from stimulation responses.
Intracellular cytokine-staining (ICS) assay PBMC were stimulated with the split H1N1pdm09 antigen to measure the IFN-γ-, IL-2-and/or TNF-α-secreting CD4 + T-cell responses, as described previously. 12 Data were acquired on an LSRFortessa flow cytometer and analyzed in FlowJo version-10 (see Supplementary-Fig. 4 for the gating strategy).

Memory B-cell (MBC) ELISpot assay
The H1N1pdm09-specific IgG + MBC responses were measured by the ELISpot assay, as described elsewhere. 37

Statistical analyses
Comparisons of the T-cell responses assessed in the FluoroSpot or ICS assays in 2012 and 2013 and the 5-year log-transformed HI antibody titers were performed using the nonparametric repeated-measure Friedman test, followed by the Dunn−Bonferroni post-hoc test. The retrospective data for IFN-γ-secreting T cells, cytokine profile and MBCs in 2009 were compared to the prospective data in 2012 or 2013 using the nonparametric Kruskal−Wallis or Mann−Whitney test, as appropriate, with Bonferroni correction. Adjusted p values < 0.05 were considered statistically significant. Analyses were performed in SPSS-Statistics version-24 and visualized in Prism version-7.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.