Severity of heterosubtypic influenza virus infection in ferrets is reduced by live attenuated influenza vaccine

Abstract

Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

Fig. 1. Outline of study design.

Blood samples for serum and antigen stimulation, and nasal washes, were collected as described in “Methods”.

Fig. 2. Vaccination with LAIV induces virus shedding and sero-conversion.

a Virus titres in nasal wash following intra-nasal vaccination with LAIV. Day -3 samples are plotted as day 0. Points show group mean and standard deviation (n = 6). b Serum HAI titres to H1N1 and H3N2 viruses 24 days post-vaccination. c Serum microneutralization titres to H1N1 and H3N2 viruses 24 days post-vaccination. For (b) and (c), Solid bars: tested with H1N1 virus; hatched bars: tested with H3N2 virus. Dashed lines show limits of detection.

Fig. 3. Cellular immune responses to vaccination with LAIV.

IFN-γ response in antigen-stimulated whole blood was determined by ELISA. Blood was stimulated with wild-type (a) H1N1 or (b) H3N2 viruses. Points show mean and standard error. The black line (All LAIV) shows the mean of all LAIV-vaccinated ferrets. The dotted horizontal line shows the limit of quantitation. *PBS/H3 group was significantly lower than the vaccinated groups on (a) day 8 and (b) day 11 (Mann–Whitney test, P < 0.0001, n = 6).

Fig. 4. Nasal wash virus titres following challenge with wild-type viruses.

a Mean and SD following challenge. b Area under the curve (AUC) analysis for each group. LAIV/H3 group shed significantly less virus than control group PBS/H3 (one-way ANOVA, P < 0.0001, n = 6).

Fig. 5. Nasal wash cell counts following challenge with wild-type viruses.

Points show group mean and standard deviation. The increase in cell count represents the inflammatory response in the nasal cavity following infection. Samples taken on day 26 post-vaccination are plotted as 0 dpi. The LAIV/H3 group is significantly different from the PBS/H3 group (P = 0.04), and both groups were significantly different from the LAIV/H1 group (P < 0.0001; AUC, one-way ANOVA with Tukey’s multiple comparisons test, n = 6).

Fig. 6. Protection from clinical disease conferred by LAIV vaccination.

a Loss of bodyweight, normalised to day of challenge. b Temperature post-challenge. Group means and SD are plotted (n = 6).

Fig. 7. Cellular immune responses to virus challenge in vaccinated and unvaccinated animals.

IFN-γ response in antigen-stimulated whole blood was determined by ELISA. Blood was stimulated with wild-type a H1N1 or b H3N2 viruses. Points show mean and standard error. Samples taken 2 days before the challenge (equivalent to day 26 post-vaccination) are plotted as day 0. *Group LAIV/H1 day 2 values were significantly higher than the other two groups for both H1 stimulation (a; 1-way ANOVA with Tukey’s multiple comparisons test, P = 0.003, n = 6) and H3 stimulation (b; P = 0.006).

Fig. 8. Cellular immune response in lungs, measured by IFN-γ ELISpot.

Purified lung lymphocytes were stimulated with a H1N1 or b H3N2 viruses. Lungs were collected at 14 dpi (groups LAIV/H3, LAIV/H2 and PBS/H3) or at 26 days post-vaccination (group LAIV/cull). Lines show group mean and SD. Groups were compared by one-way ANOVA with Tukey’s multiple comparisons test, n = 6. **P < 0.01; ***P < 0.001.