Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

Abstract

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (T_RM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69⁺CD103^- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M2₈₂ epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 T_RM markers. Surprisingly, the CD69⁺CD103⁺ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to T_RM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.

Keywords: Live attenuated influenza vaccine; Respiratory syncytial virus; T cell immunity; Tissue-resident memory T cells; Viral vector.

Fig. 1

Systemic RSV M282-specific effector memory CD8 T-cell responses, measured in mouse spleen on day 7 after the second immunization. A. Representative flow cytometry plots for IFNγ and TNFα expression by Tem cells. B. Percentages of CD8 Tem cells expressing one or both cytokines. Data were analyzed with multiple t tests with Holm-Sidak's correction.

Fig. 2

CD4 and CD8 T cells in the lungs of immunized mice. A. Total number of CD4 and CD8 T cells in the lungs on day 7 after the second immunization. Data were analyzed with two-way ANOVA. B. Ratio of CD8 to CD4 T cells in the lungs. Data were analyzed with one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

Expression of CD69 and CD103 TRM markers by CD4 and CD8 T cells in the lungs of immunized mice. A. Representative flow cytometry plots for CD69 and CD103 expression by CD4 and CD8 Tem cells. B. Percentages of CD69⁺ (left panel) and CD69⁺CD103+ (right panel) CD4 and CD8 Tem cells. Data were analyzed with one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4

Influenza-specific IFNγ-producing T cells in the lungs. A. Representative flow cytometry plots for IFNγ expression by CD4 and CD8 Tem cells. B. Percentages of IFNγ-positive T cells among total CD4 or CD8 T-cell populations in the lungs (left panel) or among the corresponding effector memory subset (right panel). Data were analyzed with one-way ANOVA with Holm-Sidak's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 5

RSV-specific IFNγ-producing T cells in the lungs. A. Representative flow cytometry plots for IFNγ expression by CD8 Tem cells. B. Percentages of IFNγ-positive T cells among total CD8 T-cell population in the lungs (left graph) or among the corresponding effector memory subset (right graph). Data were analyzed with one-way ANOVA with Holm-Sidak's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6

The expression of TRM markers by LAIV virus-specific and RSV M282 epitope-specific Tem cells in the lungs of mice immunized with LAIV or LAIV-RSV candidates. A. Expression of CD69 and CD103 markers by influenza-specific CD4 and CD8 Tem cells induced by immunization with indicated vaccine. B. Expression of CD69 and CD103 markers by RSV-specific CD8 Tem cells induced by immunization with indicated vaccine. Data were analyzed by 2-way ANOVA with Holm-Sidak's multiple comparison test (*p < 0.05, ****p < 0.0001).

Fig. 7

Assessment of RSV replication and histopathological examination of lungs of mice five days after challenge with RSV. A. Representative photographs of the lung sections simultaneously stained with Congo Red, haematoxylin and Alcian blue. B. Replication of RSV A2 strain in the lungs of immunized mice at day 5 after RSV challenge. C. Scores of pathological changes. Data were analyzed with one-way ANOVA with Holm-Sidak's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. S1

Gating strategy for measuring IFNγ- and/or TNFα-secreting CD8 Tem cells in mouse spleens using an ICS assay.

Fig. S2

Gating strategy for phenotyping IFNγ-secreting CD4 and CD8 T cells in the lungs.