Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection

Abstract

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4(+)IL-17A(+)TNFα(+)). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development.

Figure 1. Chemical structure of CRX-601.

Figure 2. Induction of Th17 cells following intranasal vaccination with liposomal CRX-601 and split flu antigen.

Antigen-specific T cell responses in mice vaccinated intranasally with split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) and various concentrations of CRX-601 liposomes were evaluated in the spleen at day 5 post-secondary vaccination. Expression of IL-17A/IFNγ (A), IFNγ/TNFα (B) and IL-17A/TNFα (C) were examined in CD4⁺ T cells. Data shown is representative of 3 independent experiments. Data in B and C are means ± SEM for three replicates analyzed by two-way ANOVA, Bonferroni post test analysis (* p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001 are significantly different from naive controls).

Figure 3. Intranasal vaccination induces both mucosal and systemic immune responses.

Anti-influenza total IgG and IgA titers in vaginal (A) and tracheal wash (B) as well as total IgG, IgG1, IgG2a titers in serum samples from mice vaccinated intranasally with split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) and CRX-601 liposomes were examined 14 days post boost vaccination. Percent survival (D) and percentage weight change relative to starting weight (E) following challenge with A/Hong Kong/1968 (H3N2) influenza virus is shown. Data is representative of two independent experiments with 8 mice per group for antibody titers and 10 mice per group for influenza challenge. Data in A, B and C are means ± SEM from 8 mice and E is means ± SEM for 10 mice analyzed by one-way ANOVA, ** p<0.01 denotes significance compared to vehicle control (Dunnet post-test).

Figure 4. Enhanced vaccine efficacy following subcutaneous immunization.

Balb/c mice were vaccinated with CRX-601 liposome (1 µg/mouse) and split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) either via an intranasal (intrapul) or subcutaneous (subcut) route. Splenic T cell responses (A) and serum influenza-specific IgG titers (B) were both examined 5 days post boost vaccination. Percentage survival (C) and percentage weight change (D) following challenge with a lethal dose of A/Hong Kong/1968 (H3N2) influenza virus. Lung influenza specific CD4⁺ T cells responses were evaluated at 5 days post challenge (E, F), in addition to changes in frequency (G) and cell number (H) of Lung Gr-1^hi neutrophils. Data from 5 days post boost are means ± SEM for 3 (T-cell) or 8 (Antibody) replicates and is representative of two independent experiments. Data from 5 day post challenge are means ± SEM for three replicates and is representative of two independent experiments. * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001 denote significance when compared to vehicle treated controls (Figure 4A,F (two-way ANOVA, Bonferroni post test), Figure 4B,C, D,G,H (One-way ANOVA Dunnet post-test)).

Figure 5. IL-17A neutralization reduces augmented neutrophil response.

Mice vaccinated intranasally with split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) and CRX-601 were administered with anti-Ly-6G or anti-IL-17A (100 µg) i.p. one day prior to challenge with influenza virus and then daily for a further 6 days post challenge. Neutrophil depletion efficiency was monitored in peripheral blood 24 h post antibody treatment (A,B). Confirmation of neutrophil depletion (C) as well as enumeration of Gr-1⁺ subsets (D) was examined in the lung 5 days post infection. Data in B and D are means ± SEM for three biological replicates and is representative of two independent experiments. * p<0.05, ** p<0.01, ***p<0.001, are significantly different from control, Rat IgG2a (One-way ANOVA, Dunnet post-test).

Figure 6. Adaptive immunity is unaffected by IL-17A blockade or neutrophil depletion following viral challenge.

Mice vaccinated intranasally with split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) and CRX-601 were administered with anti-Ly-6G (IA8) or anti-IL-17A (100 µg) i.p. one day prior to challenge with influenza and then daily for a further 6 days post challenge. Serum influenza virus specific antibody titers (A) and lung T cell responses (B) were evaluated 5 days post influenza challenge. **** in Figure 6A denotes significance of IgG subtypes compared to unvaccinated IgG2a control using a One-way ANOVA, Dunnet post test. In Figure 6B, **** denotes significance compared to the unvaccinated IgG2a control group using a two-way ANOVA- (Bonferroni- Post-test). Data are means ± SEM for five (A) or three (B) replicates analyzed.

Figure 7. Neutralization of IL-17A and neutrophil depletion ameliorates enhanced weight loss.

Mice vaccinated intranasally with split influenza virus antigen (A/Uruguay/716/2007 (H3N2)) and CRX-601 were administered with anti-Ly-6G (IA8) or anti-IL-17A (100 µg) i.p. one day prior to 5LD₅₀ challenge with influenza A/Hong Kong/1968 (H3N2) and then daily for a further 6 days post challenge. Control mice were vaccinated with split flu plus vehicle and treated with anti-Ly-6G or isotype control antibodies as indicated. Survival (A) and percentage weight loss (B) were examined following influenza virus challenge (10 mice/group). Black lines indicate weight loss in control (split-flu+vehicle) mice and red lines indicate weight loss in adjuvanted groups treated with the indicated antibody. At day 5 post influenza challenge, (C) viral titers in the lung relative to control mice were evaluated (Rat IgG2a) by RT-PCR. This data is representative of two independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 are significantly different from control, Rat IgG2a unless otherwise stated (One-way ANOVA, Dunnet post-test).

Figure 8. Heterogeneous expression of CCR6 on vaccine primed Th17 cells.

5 days post influenza challenge, lung CD4⁺ T cells from vaccinated mice were examined for antigen-specific IL-17A or IFNγ expression (A), then gated subsets were examined for transcription factors RORγt and T-bet levels (B) as well as CCR6 and CXCR3 expression (C).

Figure 9. Influenza virus-primed mice maintain a Th1 profile following subsequent intranasal vaccination.

Control mice or mice primed with a non-lethal dose of Philippines/2/82/X-79, were subsequently vaccinated intranasally (d28) with split influenza virus antigens derived from either A/Uruguay/716/2007 (H3N2) or A/New Caledonia/20/1999 (H1N1) strains in the presence or absence of CRX-601 liposomes (1 µg/mouse). All groups (10 mice per group) were then challenged with a lethal dose (5LD₅₀) of A/Hong Kong/1968 (H3N2) or A/Puerto Rico/8/1934 (H1N1) (A). Antigen-specific lung T cell responses (B,C) and neutrophil numbers (D,E) were evaluated 5 days post influenza challenge with either A/Hong Kong/1968 (H3N2) (B,D) or A/Puerto Rico/8/1934 (H1N1) (C,E). Percent survival for groups challenged with A/Hong Kong/1968 (H3N2) (F) or A/Puerto Rico/8/1934 (H1N1) (G) are also shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 are significantly different from no vaccination control (two-way ANOVA, Bonferroni post-test (B,C) and one-way ANOVA, Dunnet post test (D,E).