Boosting of Cross-Reactive and Protection-Associated T Cells in Children After Live Attenuated Influenza Vaccination

Abstract

Background: Live attenuated influenza vaccines (LAIVs) stimulate a multifaceted immune response including cellular immunity, which may provide protection against newly emerging strains. This study shows proof of concept that LAIVs boost preexisting, cross-reactive T cells in children to genetically diverse influenza A virus (IAV) strains to which the children had not been exposed.

Methods: We studied the long-term cross-reactive T-cell response in 14 trivalent LAIV-vaccinated children using the fluorescent immunospot assay (FluoroSpot) with heterologous H1N1 and H3N2 IAVs and CD8+ peptides from the internal proteins (matrix protein 1 [M1], nucleoprotein [NP], polymerase basic protein 1 [PB1]). Serum antibody responses were determined by means of hemagglutination inhibition assay. Blood samples were collected before vaccination and up to 1 year after vaccination.

Results: Preexisting cross-reactive T cells to genetically diverse IAV strains were found in the majority of the children, which were further boosted in 50% of them after receipt of LAIV. Further analyses of these T cells showed significant increases in CD8+ T cells, mainly dominated by NP-specific responses. After vaccination with LAIV, the youngest children showed the highest increase in T-cell responses.

Conclusion: LAIV boosts durable, cross-reactive T-cell responses in children and may have a clinically protective effect at the population level. LAIV may be a first step toward the desired universal influenza vaccine.

Keywords: Influenza; LAIV; T-cell; cellular immune response; children; cross-reactive; heterologous; protection.; vaccine.

© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Figures

Figure 1.

Phylogenetic trees for the influenza A H1 and H3 strains. The phylogenic trees show the genetic divergence from the homologous vaccine viruses and the heterologous wild-type strains. The influenza A/H1N1 strains were the California/09(H1N1) vaccine strain and the heterologous historical Solomon Islands/06(H1N1) strain from 2006. The influenza A/H3N2 strains were the homologous Texas/12(H3N2) live attenuated influenza vaccine (LAIV) vaccine strain and the drifted wild-type Switzerland/13(H3N2) strain from 2014–2015, the year after the trial. Phylogenetic trees were built using the neighbor-joining method with Poisson correction in MEGA software, version 6.0.6 [16].

Figure 2.

Hemagglutination inhibition (HI) responses to the homologous and heterologous influenza AHN1 and H3N2 strains. The HI responses after live attenuated influenza vaccine (LAIV) are shown for homologous vaccine strains, California/09(H1N1) (A, B) and Victoria/12-like(H3N2) (equivalent to Texas/12[H3N2]) (E, F) and the heterologous wild-type influenza strains, Solomon06(H1N1) (C, D) and Switzerland/13(H3N2) (G, H). Bars represent geometric mean titers with 95% confidence intervals,* indicating a trend of an increase (P = .09). The HI responses are shown as individual responses; each symbol represents a single child, and a titer of 40, considered the protective level, is indicated by dotted lines. To compare HI responses over time, the nonparametric Kruskal-Wallis test was used, with correction for multiple comparisons; differences were considered significant at P < .05.

Figure 3.

Durability of cross-reactive T-cell cytokine responses to heterologous influenza A virus strains after live attenuated influenza vaccine (LAIV). The long-term responses up to 1 y, for influenza-specific interferon (IFN) γ +, interleukin 2 (IL-2)+, and IFN-γ +IL-2+ T-cell responses after LAIV are indicated per 106 peripheral blood mononuclear cells (PBMCs). Each symbol represents an individual child, and bars represents group means with standard errors of the mean. For statistical analysis of the heterologous T-cell response after vaccination, the nonparametric Kruskal-Wallis test was used, with correction for multiple comparisons; differences were considered significant at P < .05. * ; ** . Abbreviation: SFUs, spot-forming units.

Figure 4.

Long-term and individual influenza-specific CD8+ T-cell responses after live attenuated influenza vaccine (LAIV) vaccination. Influenza-specific interferon (IFN) γ +, interleukin 2 (IL-2)+ and IFN-γ +IL-2+ CD8+T-cell responses up to 1 y after LAIV are indicated per 106 peripheral blood mononuclear cells (PBMCs). Bars represent means with standard errors of the mean. A–C, The IFN-γ + (A), IL-2+ (B), and IFN-γ +IL-2+ (C) cytokine responses are shown for all children. D–I, The kinetics of the CD8+ T-cell response with individual responses are shown for children <10 y old (n = 10) (D–F) or ≥10 y old (n = 4) (G–I). For statistical analyses of the postvaccination response, the Wilcoxon matched-paired signed rank test was used for the 12 subjects with available samples up to day 180. * ; ** . Abbreviation: SFUs, spot-forming units.

Figure 5.

Influenza-specific individual CD8+ peptide responses after live attenuated influenza vaccine (LAIV) vaccination. A, Influenza-specific response to individual peptides covering the NP, M1, and PB1 proteins of influenza A virus, with means and standard errors of the mean. B, Because the dominant response was to the NP peptide, interferon (IFN) γ + NP-specific CD8+ peptide responses are shown according to age ≥10 or <10 y. The total response (NP, M1, and PB1) is shown in A. To compare the response over time between the 2 age groups, the nonparametric Mann-Whitney test was used, with differences considered significant at P < .05. Abbreviations: M1, matrix protein 1; NP, nucleoprotein; PB1, polymerase basic protein 1.