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

Abstract

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. Despite the widespread implementation of annual influenza vaccination, the effect of repeated vaccinations on the immune response in subsequent seasons is poorly understood. A team led by Rebecca Jane Cox at the University of Bergen examined the humoral and T-cell response elicited by 2009's H1N1pdm09 seasonal vaccine. Since the H1N1pdm09 strain continued to circulate in subsequent years it was included in later vaccine formulations and the authors could therefore examine the effects of repeated annual vaccination over multiple seasons. They observed that H1N1pdm09-specific polyfunctional T-cell responses and antibodies were enhanced by multiple annual vaccinations. In particular, T cells showed progressive skewing to IFN-γ+TNF+ double producers, but the magnitude of the T-cell response tended to plateau after 3-4 repeated vaccinations. The findings suggest that including the same component in subsequent annual vaccines can significantly impact the influenza immune response.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1

The study flow chart. Healthcare workers (HCWs) were vaccinated with a single dose of the AS03-adjuvanted monovalent pandemic H1N1pdm09 vaccine (European Clinical Trials Database, EudraCT 2009-016456-43; www.clinicaltrials.gov, NCT01003288). During 2010–2013 seasons, HCWs were vaccinated every year with the trivalent seasonal inactivated vaccines, containing the H1N1pdm09 virus as the A/H1N1 component during the whole study period. The A/H3N2 and B strains included in the seasonal vaccines changed between seasons during 2010–2013. The figure shows the inclusion criteria for this study, which were repeated annual vaccination between 2010 and 2013, and provision of peripheral blood monocular cells (PBMC) prevaccination (D0) and 21 days postvaccination (D21) in 2012 and 2013 seasons

Fig. 2

The magnitude of H1N1pdm09-specific T-cell responses during seasons 2012 and 2013. The single IFN-γ- or IL-2-, or double-cytokine IFN-γIL-2-secreting T cells against a the split H1N1pdm09 virus, the H1N1-specific viral internal proteins: c M1, e NP, or g PB1 prevaccination (D0) (light) and at 21 days postvaccination (D21) (dark) were measured in the T-cell FluoroSpot assay. Each symbol represents one individual’s response with the horizontal lines representing the mean magnitudes of cytokine-secreting cells per 106 PBMC with standard error of the mean (s.e.m.). The IFN-γ to IL-2 ratio was calculated for each participant at each time point and the mean ratios with s.e.m. are shown against b the split H1N1pdm09 virus, the viral internal proteins: d M1, f NP, or h PB1. The dotted line indicates the IFN-γ/IL-2 ratio of 1 showing a balanced IFN-γ and IL-2 response. An IFN-γ/IL-2 ratio above or below 1 shows a predominant IFN-γ or IL-2 response, respectively. nsp > 0.05, *p < 0.05, **p < 0.01

Fig. 3

The magnitude of cross-reactive CD4+ T-cell responses during seasons 2012 and 2013. The single IFN-γ- or IL-2-, or double-cytokine IFN-γIL-2-secreting T cells against the CD4-specific conserved epitopes from viral external/internal proteins were measured in the T-cell FluoroSpot assay a and e prevaccination (D0) (light) and at 21 days postvaccination (D21) (dark) in HCWs with annual repeated vaccination (round) or c and g prior to the influenza season in 2012 (light) and 2013 (dark) in a control group of HCWs who were only vaccinated with the AS03-adjuvanted pandemic H1N1pdm09 vaccine in 2009 (square). Each symbol represents one individual’s response with the horizontal lines representing the mean magnitudes of cytokine-secreting cells per 106 PBMC with standard error of the mean (s.e.m.). The IFN-γ to IL-2 ratio was calculated for each participant at each time point and the mean ratios with s.e.m. are shown against the CD4-specific external/internal epitopes b and f in HCWs with repeated vaccination or d and h a control group. The dotted line indicates the IFN-γ to IL-2 ratio of 1 showing a balanced IFN-γ and IL-2 response. An IFN-γ to IL-2 ratio above or below 1 shows a predominant IFN-γ or IL-2 response, respectively. nsp > 0.05, *p < 0.05, **p < 0.01

Fig. 4

The quality of CD4+ T-cell responses during seasons 2012 and 2013. The cytokine profile of H1N1pdm09-specific CD4+ T cells was assessed by IFN-γ/IL-2/TNF-α intracellular cytokine-staining (ICS) assay prevaccination (D0) (light) and at 21 days postvaccination (D21) (dark) in the 2012 and 2013 seasons. a The mean frequencies (%) of the triple producer (IFN-γ+IL-2+TNF-α+), double producers (IFN-γ+IL-2+, IFN-γ+TNF-α+, IL-2+TNF-α+), and single producers (IFN-γ+, IL-2+, TNF-α+) with standard error of the mean (s.e.m.) are shown as bars. b The mean frequencies with s.e.m. are presented for each of the three double-producer subsets. The memory CD4+ T-cell responses were assessed by differentiating the three memory subsets CD45RA−CCR7+ central memory (CM), CD45RA−CCR7−effector memory (EM), and CD45RA+CCR7− late effector memory (EMRA), from CD45RA+CCR7+ naïve T cells within the CD4+ population, then IFN-γ+ or IL-2+ cells were gated within CD4+ CM or EM population (see gating strategy in Supplementary-Fig. 4). The mean frequencies with s.e.m. of the total IFN-γ- or IL-2-secreting CD4+c CM or e EM T cells pre- and postvaccination in seasons 2012 and 2013 are shown. The mean frequencies with s.e.m. are presented for the IFN-γ+IL-2−TNF-α+ subset of the CD4+d CM or f EM T cells. *p < 0.05, **p < 0.01

Fig. 5

The impact of repeated annual vaccinations on the magnitude and the quality of H1N1pdm09-specific T cells over 5 years. a The magnitude of H1N1pdm09-specific IFN-γ-secreting T cells prevaccination (D0) (light) and at 21 days postvaccination (D21) (dark) were enumerated in the IFN-γ T-cell ELISpot assay in 2009 (n = 9), and the IFN-γ/IL-2 FluoroSpot assay in 2012 and 2013. Each symbol represents one individual’s response with the horizontal lines representing the mean magnitudes of IFN-γ-secreting cells per 106 PBMC with standard error (s.e.m.). b The cytokine profile of H1N1pdm09-specific CD4+ T cells was assessed by the IFN-γ/IL-2/TNF-α intracellular cytokine-staining (ICS) assay pre- and postvaccination in 2009, 2012, and 2013. The mean frequencies (%) with s.e.m. of each of the seven cytokine-combination subsets are shown as bars. c Changes in the prevaccination cytokine profile of CD4+ T cells after 3–4 years of repeated vaccination. The fold-changes of responses between 2009 (reference) and 2012 or 2013 were calculated for each participant and the log-transformed means of fold-changes are presented in a radar chart. Value above or below 0 represents an increase or decline of response, respectively. *p < 0.05, **p < 0.01

Fig. 6

The overall impact of repeated annual vaccinations on T-cell and humoral responses. a The 5-year H1N1pdm09-specific antibody responses following pandemic and repeated seasonal vaccinations were measured in the hemagglutination inhibition (HI) assay. Each symbol represents one individual’s response with the horizontal lines representing the geometric mean HI titer with 95% confidence interval (CI) prevaccination (D0) (light) and at 21 days postvaccination (D21) (dark) each year. The dotted line indicates the 50% protective threshold defined as HI titer ≥ 40. b The HI antibody fold-induction between pre- and postvaccination (D21/D0) titers was calculated for each participant at each time point. The horizontal lines represent the geometric mean HI antibody fold-induction with 95% CI. c The H1N1pdm09-specific memory B-cell (MBC) responses pre- and postvaccination in seasons 2009, 2012, and 2013 were measured in the MBC ELISpot assay. The horizontal lines represent the mean magnitudes of the H1N1pdm09-specific MBCs per 106 PBMC with standard error of the mean (s.e.m.). d The MBC fold-induction between pre- and postvaccination (D21/D0) was calculated for each participant at each time point. The horizontal lines represent the mean MBC fold-induction with s.e.m. e The fold-changes of prevaccination antibodies, MBCs, and T cells between 2009 and 2012 or 2013 were calculated for each participant and the log-transformed means of fold-changes are presented in a radar chart. Value above or below 0 represents an increase or decline of response, respectively. f The log-transformed mean fold-induction of T-cell and humoral responses between pre- and postvaccination (D21/D0) each year (2009, 2012, and 2013) is presented in a radar chart. The higher the value is above 0, the higher the vaccine-induced response is after vaccination. *p < 0.05, **p < 0.01, ***p < 0.001