Evaluation of multivalent H2 influenza pandemic vaccines in mice

Abstract

Subtype H2 Influenza A viruses were the cause of a severe pandemic in the winter of 1957. However, this subtype no longer circulates in humans and is no longer included in seasonal vaccines. As a result, individuals under 50years of age are immunologically naïve. H2 viruses persist in aquatic birds, which were a contributing source for the 1957 pandemic, and have also been isolated from swine. Reintroduction of the H2 via zoonotic transmission has been identified as a pandemic risk, so pre-pandemic planning should include preparation and testing of vaccine candidates against this subtype. We evaluated the immunogenicity of two inactivated, whole virus influenza vaccines (IVV) in mice: a monovalent IVV containing human pandemic virus A/Singapore/1/1957 (H2N2), and a multivalent IVV containing human A/Singapore/1/1957, avian A/Duck/HongKong/319/1978 (H2N2), and swine A/Swine/Missouri/2124514/2006 (H2N3) viruses. While both vaccines induced protective immunity compared to naïve animals, the multivalent formulation was advantageous over the monovalent in terms of level and breadth of serological responses, neutralization of infectious virus, and reduction of clinical disease and respiratory tissue replication in mice. Therefore, multivalent pandemic H2 vaccines containing diverse viruses from animal reservoirs, are a potential option to improve the immune responses in a pre-pandemic scenario where antigenic identity cannot be predicted.

Keywords: H2N2; Influenza; Monovalent; Multivalent; Pandemic; Vaccine.

Figure 1. Cross-reactive, Humoral Responses to Monovalent and Multivalent H2 Vaccines

Mice were vaccinated with one dose (A, C, E) or two doses (B, D, F) of monovalent or multivalent H2 vaccine as indicated. Sera (31 days post-prime) was evaluated in the A, B) HAI assay (n=15 mice/group), C, D) vsIgG ELISA (n=30 mice/group) and E, F) NAI assay (n=14 mice/group) against the indicated test antigens. Dotted lines indicate the assay limit of detection. Statistical significance was determined by the Log₂ conversion and comparison of the GMTs. Samples below the assay cutoffs (HAI 1:20, NAI 1:10, vsIgG 1:200 serum dilutions) were assigned a value of 1 for calculations. *p≤0.05, **p≤0.01, p≤0.001, ****p≤0.0001, ns = not significant

Figure 2

Cross-reactive, Virus Neutralizing Responses to Monovalent and Multivalent H2 Vaccines. Mice were vaccinated with one dose (A) or two doses (B) of monovalent or multivalent H2 vaccine as indicated. Sera (31 days post-prime) was evaluated in microneutralization assay (n=15 mice/group) against the indicated test antigens. Dotted lines indicate the assay limit of detection. Statistical significance was determined by the Log₂ conversion and comparison of the GMTs. Samples below the assay cutoff (1:10 serum dilution) were assigned a value of 1 for calculations. *p≤0.05, **p≤0.01, p≤0.001, ****p≤0.0001, ns = not significant

Figure 3. Cross-reactive, Cell Mediated Response to Monovalent and multivalent H2 Vaccines

Mice (n=5) were vaccinated with monovalent or multivalent H2 vaccines. Splenocytes (1×10⁶, 10 days post-prime) were stimulated with the indicated vaccine antigens. IFN-γ secreting splenocytes were determined by ELISpot. Statistical significance was determined by comparison of the mean values of triplicate measures from each animal. **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = not significant.

Figure 4. Post-virus Challenge Weight Loss in Vaccinated Mice

Mice (n=10/virus group) were vaccinated with monovalent (A-C) or multivalent (D-F) with one (primed) or two vaccine doses (boosted) and challenged 31 days post-prime with Swine/MO/2006 (A,D), Singapore/1957 (B, E) or Duck/HK/1978 (C,F). Singapore/1957 and Duck/HK/1978 virus challenge did not induce weight loss. Animals were weighed daily as an indicator of morbidity and data are presented as the mean % starting weight ± SD. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Fig. 5. Post-challenge Virus Replication in Tissues from Vaccinated Mice

Mice (n=3/virus group) were vaccinated with monovalent (A, B) or multivalent (C, D) with one (primed) or two (boosted) doses of vaccine. Animals were challenged (31 days post-prime) with 5.5×10⁶ EID₅₀ units of the indicated virus. At 3 dpi, virus replication in the nasal turbinates (A, C) or lungs (B, D) was determined in the TCID₅₀. Data are presented as individual TCID₅₀ titers for each animal. Dotted lines indicate the limit of assay detection (1Log₁₀TCID₅₀); values below the cutoff are indicative of 0 and for graphical presentation only.