Efficacy of Heterologous Prime-Boost Vaccination with H3N2 Influenza Viruses in Pre-Immune Individuals: Studies in the Pig Model

Abstract

In a previous study in influenza-naïve pigs, heterologous prime-boost vaccination with monovalent, adjuvanted whole inactivated vaccines (WIV) based on the European swine influenza A virus (SwIAV) strain, A/swine/Gent/172/2008 (G08), followed by the US SwIAV strain, A/swine/Pennsylvania/A01076777/2010 (PA10), was shown to induce broadly cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of 15 antigenically distinct H3N2 influenza strains. Here, we used the pig model to examine the efficacy of that particular heterologous prime-boost vaccination regimen, in individuals with pre-existing infection-immunity. Pigs were first inoculated intranasally with the human H3N2 strain, A/Nanchang/933/1995. Seven weeks later, they were vaccinated intramuscularly with G08 followed by PA10 or vice versa. We examined serum antibody responses against the hemagglutinin and neuraminidase, and antibody-secreting cell (ASC) responses in peripheral blood, draining lymph nodes, and nasal mucosa (NMC), in ELISPOT assays. Vaccination induced up to 10-fold higher HI antibody titers than in naïve pigs, with broader cross-reactivity, and protection against challenge with an antigenically distant H3N2 strain. It also boosted ASC responses in lymph nodes and NMC. Our results show that intramuscular administration of WIV can lead to enhanced antibody responses and cross-reactivity in pre-immune subjects, and recall of ASC responses in lymph nodes and NMC.

Keywords: H3N2; antibody cross-reactivity; heterologous prime-boost; humans; influenza; pre-existing immunity; swine; vaccination.

Figure 1

Evolution of hemagglutination inhibition (HI) antibody titers against the strains used for infection, (A) NC95, and vaccination, (B) G08 and (C) PA10. The titers were determined at weeks 0, 4, 7, 11, and 15 following infection with NC95. The interval between infection (Inf.) and the first vaccination (1°) was 7 weeks. The second vaccination (2°) was administered 4 weeks later. The different groups are shown on the x-axis. The dotted lines indicate the detection limit, which in this case was a titer of 10. Asterisks denote statistical differences between groups that received heterologous (NC95-PA10-G08) and homologous (NC95-G08-G08) vaccines in Mann–Whitney U test (p < 0.05).

Figure 2

Virus titers in nasal swabs, nasal mucosa, trachea, and lungs, 3 days after challenge with 10⁷ TCID₅₀ of MO15. The bars represent mean virus titers ± SD. The dotted line indicates the detection limit. Each color of the bar represents a different vaccine group as shown by the legend at the top right corner. The numbers above the bars indicate the number of pigs with detectable virus titers. Significant reductions of virus titers as compared with the naïve challenge control group (PBS-PBS-PBS, black asterisks) and infection-immune challenge control group (NC95-PBS-PBS, pink asterisks) in ANOVA are shown above the bars. * p < 0.05, ** p < 0.01.

Figure 3

Numbers of virus-specific IgG and IgA antibody-secreting cell (ASC) in peripheral blood mononuclear cells (PBMC) (A,B), tracheobronchial lymph nodes (TBLN) (C,D), lymph nodes of the head (LNH) (E,F), and nasal mucosa (NMC) (G,H) for each individual pig after infection with NC95 and vaccination with PA10 followed by G08. Responses in the different tissues were examined at weeks 1, 2, 7, 8, 11, and 12 after infection. The first and second vaccinations were performed at weeks 7 and 11, respectively. Symbols represent responses of four individual pigs, which were examined at each time point. The mean numbers of ASC are shown by the bars. The different colors represent responses against the five different viruses (● NC95; ▲ PA10; ⯀ G08; ▼ VIC75; ◆ HK14). The asterisk (*) means that ASC responses were not examined at that particular time point.

Figure 4

Numbers of virus-specific interferon-secreting cell (ISC) in PBMC for each individual pig after infection with NC95 and vaccination with PA10 followed by G08. Responses in PBMC were examined at weeks 1, 1.5 (day 11), 2, 7, 8, 11, and 12 after infection. The first and second vaccinations were performed at weeks 7 and 11, respectively. Symbols represent responses of four individual pigs which were examined at each time point. The mean numbers of ISC are shown by the bars. The different colors represent responses against the five different viruses (● NC95; ▲ PA10; ⯀ G08; ▼ VIC75; ◆ HK14).