Impact of pre-existing infection immunity on heterologous prime-boost vaccination with H3N2 influenza viruses

We are using the pig model of influenza and experimental, inactivated swine influenza virus (SwIV) vaccines to explore heterologous prime-boost vaccination strategies. In a previous study, pigs were primed with a European H3N2 SwIV (sw/Gent/2008, G08) and boosted with an antigenically distinct North American H3N2 SwIV (sw/Pennsylvania/2010, PA10). This strategy induced antibodies and protection against both virus strains, unlike matched homologous prime-boost vaccinations. It also induced cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of a panel of 17 antigenically distinct H3N2 strains. It failed, however, to induce antibodies against the conserved hemagglutinin (HA) stalk or to other HA subtypes. While this study was performed in influenza naïve pigs, most humans have pre-existing immunity to influenza due to previous infections. Here we have examined the effect of infection-immunity to the human A/Nanchang/1995 (NC95) H3N2 strain on the former vaccination strategy. Pigs were first inoculated intranasally with live NC95. Six weeks later they were vaccinated intramuscularly with G08 followed by PA10, or vice versa, at a 4-week interval. Control groups were mock-vaccinated or only inoculated with NC95. Six weeks after the booster vaccination, pigs were challenged with an H3N2 SwIV strain from a novel clade, sw/Missouri/2015 (MO15), and euthanized 3 days later for virus titrations of the respiratory tract. Pre-challenge sera were examined in serological assays as in the previous study, and virus-specific antibody secreting cells in the circulation and nasal mucosa were determined by ELISPOT assay. The infection-only group developed seroprotective HI titers (≥40) against NC95 only. Heterologous prime-boost vaccination of NC95 infection-immune pigs induced HI antibodies against slightly more H3N2 strains (13 and 14 of 17, including MO15) than the same vaccine regimen in influenza naïve pigs (previous study) and antibody titers were up to 10-fold higher. Replication of the MO15 challenge virus was reduced in the infection-only group when compared to the mock-vaccinated controls, but all pigs tested virus-positive. Three of 5 pigs of the NC95-G08-PA10 group and all 5 pigs of the NC95- PA10-G08 group, in contrast, did not have detectable challenge virus in the upper or lower respiratory tract. Other serological assays and analyses of ELISPOT assays are pending. In this study, prior infection with an antigenically distant H3N2 strain enhanced antibody titers and protection upon heterologous prime-boost vaccination. We are now studying the mechanisms of this enhanced protection and the effects of pre-existing immunity against strains other than NC95