Live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse T-cell responses in young children

Abstract

Background: Two doses of either trivalent live attenuated or inactivated influenza vaccines (LAIV and TIV, respectively) are approved for young children (≥ 24 months old for LAIV and ≥ 6 months old for TIV) and induce protective antibody responses. However, whether combinations of LAIV and TIV are safe and equally immunogenic is unknown. Furthermore, LAIV is more protective than TIV in children for unclear reasons.

Methods: Children 6-35 months old were administered, 1 month apart, 2 doses of either TIV or LAIV, or combinations of LAIV and TIV in both prime/boost sequences. Influenza-specific antibodies were measured by hemagglutination inhibition (HAI), and T cells were studied in flow cytometric and functional assays. Highly conserved M1, M2, and NP peptides predicted to be presented by common HLA class I and II were used to stimulate interferon-γ enzyme-linked immunospot responses.

Results: All LAIV and/or TIV combinations were well tolerated and induced similar HAI responses. In contrast, only regimens containing LAIV induced influenza-specific CD4(+), CD8(+), and γδ T cells, including T cells specific for highly conserved influenza peptides.

Conclusions: Prime/boost combinations of LAIV and TIV in young children were safe and induced similar protective antibodies. Only LAIV induced CD4(+), CD8(+), and γδ T cells relevant for broadly protective heterosubtypic immunity.

Clinical trials registration: NCT00231907.

Figure 1.

Induction of influenza-specific CD4⁺, CD8⁺, and γδTCR⁺ T cells capable of antigen-specific proliferative and effector cytokine responses by live attenuated influenza vaccination. Dot plots from 1 subject were gated on CD4⁺ (A), CD8⁺ (B), and γδTCR⁺ (C), CD3⁺ T cells after rest (left column) or live H3N2 influenza virus stimulation (right column) for 7 days in vitro. The top and bottom rows present cellular responses before and 60 days after LAIV vaccination, respectively. The percentages given in the upper left quadrants are the percentages of T cells that both proliferated (became CFSE^low) and produced interferon γ (IFN-γ).

Figure 2.

Live attenuated influenza vaccines (LAIV) but not trivalent inactivated vaccines (TIV) induced significantly increased postvaccination influenza-specific T-cell responses. A, Overall results for influenza-specific CD4 ⁺, CD8⁺, and γδTCR⁺ T-cell responses induced in all prime/boosted vaccination groups. Shown are all subjects’ peripheral blood mononuclear cell (PBMC) responses after live influenza virus stimulation for 7 days in vitro (matching unstimulated background responses have been subtracted). Paired responses detected in prevaccination and 1-month post-booster-vaccination PBMCs are presented (clear and shaded bars, respectively) for influenza-specific CD4⁺, CD8⁺, and γδTCR⁺ T cells capable of both antigen-specific proliferation and effector cytokine responses (as in Figure 1; n = 10–13 subjects per group). *P < .05 by Wilcoxon matched pairs test comparing prevaccination and postvaccination responses; **P < .01 by Wilcoxon matched pairs test comparing prevaccination and postvaccination responses. B, Induction of universally relevant influenza-specific T-cell responses by LAIV and not TIV vaccination. PBMCs from 10 subjects given TIV/TIV (TIV group) and 10 subjects given LAIV at least once (LAIV group) were stimulated with pools of highly conserved predicted HLA class I (peptide pool 1 [PP1]) and class II (peptide pool 2 [PP2]) peptide pools before and after vaccination and were studied by interferon γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (Spot Ferming Cells, SFC). *P < .05 by Mann–Whitney U test comparing LAIV-alone responses with TIV-alone responses; **P < .05 by Wilcoxon matched pairs test comparing responses before and after vaccination.

Figure 3.

Inhibition of influenza viral replication by T cells reactive with highly conserved, universally relevant influenza-specific epitopes in human target cells. Peripheral blood mononuclear cells were stimulated with highly conserved, universally relevant pools of influenza peptides predicted to be CD4⁺ and CD8⁺ T-cell epitopes restricted by common HLA class II (PP2) and HLA class I (PP1), respectively (Ag, antigen). Live H3N2 influenza virus was used as a positive control stimulus of influenza immunity. One week later, expanded T cells were co-cultured with autologous monocyte and macrophage targets (MΦ) infected with H3N2 influenza (FLU). Twenty-four hours later, total RNA was extracted and influenza genome copies were quantified by quantitative reverse-transcription polymerase chain reaction. Shown are the percentages of influenza replication inhibition mediated by peptide-stimulated T cells compared with matching rested T cells for 2 representative adult volunteers (V1 and V2).