Immunogenicity of Influenza Vaccines: Evidence for Differential Effect of Secondary Vaccination on Humoral and Cellular Immunity

Abstract

While currently used influenza vaccines are designed to induce neutralizing antibodies, little is known on T cell responses induced by these vaccines. The 2009 pandemic provided us with the opportunity to evaluate the immune response to vaccination in a unique setting. We evaluated both antibody and T cell responses in a cohort of public health care workers (18-52 years) during two consecutive influenza seasons from 2009 to 2011 and compared the MF59-adjuvanted pandemic vaccine with the unadjuvanted seasonal subunit vaccine that included the pandemic strain [The study was registered in the Netherlands Trial Register (NTR2070)]. Antibody responses were determined in serum by a hemagglutination inhibition assay. Vaccine-specific T cell responses were evaluated by detecting IFN-γ producing peripheral blood mononuclear cells using whole influenza virus or vaccine-specific peptide pools as stimulating antigens. Mixed effects regression models were used to correct the data for influenza-specific pre-existing immunity due to previous infections or vaccinations and for age and sex. We show that one dose of the pandemic vaccine induced antibody responses sufficient for providing seroprotection and that the vaccine induced T cell responses. A second dose further increased antibody responses but not T cell responses. Nonetheless, both could be boosted by the seasonal vaccine in the subsequent season. Furthermore, we show that the seasonal vaccine alone is capable of inducing vaccine-specific T cell responses, despite the fact that the vaccine did not contain an adjuvant. In addition, residual antibody levels remained detectable for over 15 months, while T cell levels in the blood had contracted to baseline levels by that time. Hereby, we show that pandemic as well as seasonal vaccines induce both humoral and cellular responses, however, with a different profile of induction and waning, which has its implications for future vaccine design.

Keywords: T cells; cellular; humoral; influenza vaccines; pandemic.

Figure 1

Design of the clinical study. The study was performed during two consecutive influenza seasons. During season 1 (2009–2010), individuals were vaccinated at the start of the study and 3 weeks later with the MF59-adjuvanted subunit vaccine. Three weeks before the start of the study or at week 6, an optional seasonal 2009–2010 vaccination was allowed. Gray arrows depict reallocation in control and vaccine groups. During season 2 (2010–2011), individuals in the vaccine group received the unadjuvanted seasonal 2010–2011 subunit vaccine at week 52. An unvaccinated control group was included in both seasons. Study participants could change between vaccine and control group at the start of season 2, resulting in a vaccine (V₁) and control (C₁) group in season 1 and vaccine-vaccine (V₁V₂), vaccine-control (V₁C₂), control-control (C₁C₂), and control-vaccine (C₁V₂) groups in season 2.

Figure 2

Study disposition. In season 1 (2009–2010), 15 participants were excluded from the per protocol analysis: eight were lost to follow up, two due to occupational vaccination while in the control group, four only received the first dose of the vaccination, and one individual was too old. In season 2 (2010–2011), two individuals were excluded from the per protocol analysis: one individual withdrew consent, one was excluded due to use of corticosteroids. All participants of the per protocol group were included in the humoral analysis, while a subgroup was included in the cellular analysis.

Figure 3

HI titers of influenza virus-specific antibody responses. Geometric mean titer (GMT) with SD of A(H1N1)pdm09-specific antibodies in vaccinated individuals and individuals of the control group of the per protocol group during season 1 and season 2. Antibody responses were tested with paired T test for longitudinal samples of individuals in the same group and unpaired T test with Welch's correction for analysis of samples from different groups. ■ vaccinated □ controls ^… protective antibody level of 40 ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, N.A. not applicable.

Figure 4

IFN-γ-specific responses of influenza virus-stimulated and SEB-stimulated PBMCs by ELISpot. Spots per million PBMCs of A(H1N1)pdm09 virus-specific (A,B) and SEB-induced (C,D) IFN-γ responses by ELISpot in vaccinated individuals and individuals of the control group during season 1 and season 2. In red the mean and SD of each data set is depicted. ELISpot data were analyzed with Wilcoxon matched-pairs rank test. •vaccinated, ■ controls ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.000,1 N.A. not applicable.

Figure 5

GMT ratios of serological and relative response rates of cellular immune responses. GMT ratios (A) en Relative response rates (B) were calculated by mixed effects regression models for antibody titer (A) and IFN-γ spots (B) for vaccinated individuals and individuals of the control group during season 1 and season 2. Statistical analysis of a time point compared to baseline are depicted in the graph, while analysis between time points is depicted above the graphs. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 6

A(H1N1)pdm09 virus-specific cellular responses in season 1 and 2. Responses against HA (A) and NA (B) peptide pools were measured with an IFN-γ ELISpot in individuals of the vaccine group. In addition, responses against HA (C) and NA (D) were measured on controls. In red the mean and SD of each data set is depicted. ELISpot data were analyzed with Wilcoxon matched-pairs rank test ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001.

Figure 7

H3N2 virus-specific cellular responses in season 2. Responses against peptide pools of A/Perth/16/2009(H3N2) HA (A,C) and NA (B,D) were measured with an IFN-γ ELISpot in individuals of the vaccine (A,B) and control group (C,D). In red the mean and SD of each data set is depicted. ELISpot data were analyzed with Wilcoxon matched-pairs rank test. ^***P < 0.001.