RIG-I ligand enhances the immunogenicity of recombinant H7HA protein

Abstract

Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

Keywords: Antibody responses; Cell-mediated immunity; H7HA; Influenza; Mouse model; Protective immunity; RIG-I ligand.

Figure 1. RIG-I activation enhanced serum antibody responses and cell-mediated immune responses

BALB/c mice (5 animals/group) were immunized intramuscularly (i.m.) with recombinant H7HA protein (3 µg) with or without 5’pppRNA or mock-immunized with PBS as control. Mice were boosted one month later with the same immunogen formulation as they received previously. (a) Serum samples were collected three weeks post- primary and three post- booster immunizations and HI titers were measured against SH2/PR8 virus (*p<0.05 as compared to H7HA and PBS group). (b–c) Spleens were collected at 1 week after booster immunization and single cell suspensions were prepared. Cells were stimulated in vitro with SH2/PR8 virus (MOI=1) overnight with GolgiPlug™ added in the last 6 hours of incubation. The percentage of CD44⁺ activated CD4⁺ or CD8⁺ T cells were measured by surface staining of CD44 (b); the percentage of IFNγ-producing activated CD4⁺ or CD8⁺ T cells were then measured by intracellular cytokine staining (c, **p<0.01 or *p<0.05 as compared to H7HA and PBS group). (d) Spleen and bone marrow samples were collected at one week post-boost and the frequency of recombinant H7HA protein-specific IgG+ ASCs was measured using the ELISPOT assay. The percentage of HA-specific IgG⁺ ASCs in total IgG⁺ ASCs are presented (**p<0.01 or ***p<0.001 as compared to H7HA and PBS group). The error bars represent standard error of the means (SEM).

Figure 2. Addition of RIG-ligand to rH7HA protein enhanced the protective immunity against viral challenge

BALB/c mice (5 mice/group) were immunized intramuscularly (i.m.) with recombinant H7HA protein (3 µg) with or without 5’pppRNA or PBS control. Mice were boosted one month later with the same vaccine formulation they received earlier. Immunized mice were challenged with 50 LD₅₀ of wild type A(H7N9) virus and weight loss (*p<0.05 or ***p<0.001 as compared to H7HA and PBS group) (a) and survival (*p<0.05 as compared to H7HA and PBS group) (b) were monitored for 15 days post-challenge. The error bars represent standard error of the means (SEM).