Vaccination with Vesicular Stomatitis Virus-Vectored Chimeric Hemagglutinins Protects Mice against Divergent Influenza Virus Challenge Strains

Abstract

Seasonal influenza virus infections continue to cause significant disease each year, and there is a constant threat of the emergence of reassortant influenza strains causing a new pandemic. Available influenza vaccines are variably effective each season, are of limited scope at protecting against viruses that have undergone significant antigenic drift, and offer low protection against newly emergent pandemic strains. "Universal" influenza vaccine strategies that focus on the development of humoral immunity directed against the stalk domains of the viral hemagglutinin (HA) show promise for protecting against diverse influenza viruses. Here, we describe such a strategy that utilizes vesicular stomatitis virus (VSV) as a vector for chimeric hemagglutinin (cHA) antigens. This vaccination strategy is effective at generating HA stalk-specific, broadly cross-reactive serum antibodies by both intramuscular and intranasal routes of vaccination. We show that prime-boost vaccination strategies provide protection against both lethal homologous and heterosubtypic influenza challenge and that protection is significantly improved with intranasal vaccine administration. Additionally, we show that vaccination with VSV-cHAs generates greater stalk-specific and cross-reactive serum antibodies than does vaccination with VSV-vectored full-length HAs, confirming that cHA-based vaccination strategies are superior at generating stalk-specific humoral immunity. VSV-vectored influenza vaccines that express chimeric hemagglutinin antigens offer a novel means for protecting against widely diverging influenza viruses.

Importance: Universal influenza vaccination strategies should be capable of protecting against a wide array of influenza viruses, and we have developed such an approach utilizing a single viral vector system. The potent antibody responses that these vaccines generate are shown to protect mice against lethal influenza challenges with highly divergent viruses. Notably, intranasal vaccination offers significantly better protection than intramuscular vaccination in a lethal virus challenge model. The results described in this study offer insights into the mechanisms by which chimeric hemagglutinin (HA)-based vaccines confer immunity, namely, that the invariant stalk of cHA antigens is superior to full-length HA antigens at inducing cross-reactive humoral immune responses and that VSV-cHA vaccine-induced protection varies by site of inoculation, and contribute to the further development of universal influenza virus vaccines.

FIG 1

VSV-cH9/1 and VSV-cH5/1 vaccination and H1N1 challenge experiment. (A) Schematic of VSV-cHA prime-boost-challenge vaccination experiments: i.m. and i.n. vaccinations, PR8 homologous influenza challenge. Intranasal vaccinations were performed with 2 × 10⁵ PFU, and i.m. vaccinations were performed with 4.5 × 10⁵ PFU. (B) Postprime (VSV-cH9/1) and postboost (VSV-cH5/1) sera were tested in ELISAs against a panel of HA substrates, which are listed on the x axis. Blue bars represent the reciprocal endpoint ELISA titer of pooled postprime serums, and red bars represent the total reciprocal endpoint ELISA titer after prime and boosting vaccination. Intranasal vaccination results in greater postprime titers. The overall serum antibody titers postboost are similar between i.m.- and i.n.-vaccinated animals. (C) Vaccinated mice were challenged i.n. 1 month postboost with 10 LD₅₀ of homologous H1N1 (PR8) influenza virus from which the stalk of the chimeric HA antigens was derived. Weights were recorded daily for 14 days postchallenge and are graphically displayed as the mean percentage of the prevaccination weight. Error bars represent the 95% CI of the mean weight. All control mice died or required euthanasia per protocol by day 7 postchallenge. Significant differences between groups are indicated in the graph. (D) Kaplan-Meier survival curves. Significant differences between groups, identified by a log-rank (Mantel-Cox) test, are indicated on the graph. ns, not significant; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

FIG 2

VSV-H1 and VSV-cH9/1 vaccination and H5N1 challenge experiment. (A) Schematic of the prime-boost-challenge vaccination experiments: i.m. and i.n. vaccinations, H5N1 heterologous influenza challenge. Intranasal vaccinations were performed with 2 × 10⁵ PFU, and i.m. vaccinations were performed with 4.5 × 10⁵ PFU. (B) Serum reciprocal endpoint ELISA titers of mice vaccinated with VSV-H1 (prime) and VSV-cH9/1 (boost). Intramuscular vaccination results in greater boost, but overall serum antibody titers postboost are greater for i.n.-vaccinated animals. (C) Vaccinated mice were challenged i.n. 1 month postboost with 10 LD₅₀ of heterosubtypic H5N1 (HALo) influenza virus. Weights were recorded daily for 14 days postchallenge and are graphically displayed as the mean percentage of the prevaccination weight. Error bars represent the 95% CI of the mean weight. All control mice died or required euthanasia per protocol by day 8 postchallenge. Significant differences between groups are indicated in the graph. (D) Kaplan-Meier survival curves. Significant differences between groups, as identified by a log-rank (Mantel-Cox) test, are indicated in the graph. Two mice from the i.m.-vaccinated group challenged with HALo H5N1 influenza virus died on day 6 postchallenge (66% overall survival). ns, not significant; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

FIG 3

Vaccination with VSV-cHAs is superior to VSV-HAs at generating broadly reactive and stalk-specific serum HA antibodies. (A) Schematic of VSV-cHA versus VSV-HA prime-boost-boost experiment. Vaccinations were performed with 2 × 10⁶ PFU in each case. (B) Groups of BALB/c mice were primed (i.m.) and boosted (combined i.m. and i.n.) after 3 weeks, and again after another 3 weeks, with either VSV-cHAs or VSV-HAs. Serum was collected prior to each boost and 1 month after the final boost and then subjected to ELISA against a panel of HA substrates. Blue bars represent the reciprocal endpoint ELISA titer of pooled postprime serums, and red bars represent the total reciprocal endpoint ELISA titer after prime two boosting vaccinations. Both VSV-cHA and VSV-HA vaccination results in stalk-specific and cross-reactive serum antibody, although VSV-cHAs generate higher titer responses and improved boosting of these responses.