Potent vesicular stomatitis virus-based avian influenza vaccines provide long-term sterilizing immunity against heterologous challenge

Abstract

The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV (J. A. Schwartz et al., Virology 366:166-173, 2007). Here we have generated and characterized VSV-based vaccines that express the A/Hong Kong/156/1997 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Postchallenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates.

FIG. 1.

Construction and characterization of recombinant VSVs expressing an H5 HA gene from the first genomic position of VSV. (A) Schematic diagram of the rVSV genomes showing the insertion of the HK/156 H5 HA gene (H5) into the first genomic position upstream of the VSV N gene in a recombinant wild-type (rWT) background [Indiana strain; VSV-H5(1)] or a rWT background where the G gene was replaced with the G gene from another VSV serotype [New Jersey (NJ); VSV-NJG-H5(1)]. The numbers in parentheses indicate the VSV genomic position at which the H5 HA gene is inserted. (B) Western blot analysis of whole-cell extracts from cells mock infected or infected with virus using a polyclonal antibody to the HK/156 H5 HA. The cells were mock infected or infected with the following viruses: rVSV (Indiana) (WT); first position vectors VSV-H5(1) [H5(1)] and VSV-NJG-H5(1) [NJG-H5(1)]; and fifth position vectors VSV-H5 HA [H5(5)] and VSV-NJG-H5 HA [NJG-H5(5)] (38). The full-length HA (HA₀) and cleaved isoforms of HA (HA₁ and HA₂) are indicated by arrows to the right of the gel. The positions (molecular mass [in kilodaltons]) of molecular size markers are indicated to the left of the gel.

FIG. 2.

Induction of heterologous cross-protective immune responses in mice after vaccination with rVSVs expressing H5 HA from the optimal first genomic position. (A and B) Mice (n = 8) were intramuscularly primed and boosted at 2.5 months postprime with 10⁷ PFU of VSV-H5(1) and VSV-NJG-H5(1), respectively. Sera collected from vaccinated mice at 2.5 months postprime (preboost; white bars) or 1 month postboost (3.5 months postprime; gray bars) were subjected to a microneutralization assay (39) against the challenge virus (HK/483[A] and VN/1203 [B]). All mice vaccinated with the negative-control vectors (n = 8) had undetectable neutralizing activity that corresponds to a titer of 10 in this assay (data not shown). Asterisks indicate sera from H5 HA-vaccinated animals with undetectable nAb. (C to F) At 2 months postboost (4.5 months postprime), vaccinated mice were intranasally challenged with either HK/483 (clade 0; n = 8) (C and E) or VN/1203 (clade 1; n = 8) (D and F). (C and D) Pathogenesis was assessed by weight loss and is shown as the average percentage of original prechallenge weight. (E and F) Kaplan-Meier plots depict the percent survival of negative-control mice (controls) or H5 HA-vaccinated mice (vaccinees).

FIG. 3.

VSV-based vaccines provide sterilizing immunity against heterologous AIVs. Mice (n = 4) were vaccinated with the first position H5 HA vectors (vaccinees) (A and C) or negative-control vectors (controls) (A and C) and challenged with either HK/483 (A and B) or VN/1203 (C and D) as in Fig. 2. (C to F) At 3 days postchallenge, the lungs were harvested, and the virus titer was determined by plaque assay. No virus was detected in any vaccinee homogenate. The limit of detection for this assay was ≤50 PFU/g (dashed line) where the average lung mass was 100 mg. (B and D) Sera collected from vaccinated mice (mouse 5 to mouse 8) at 2.5 months postprime (preboost; white bars) or 1 month postboost (3.5 months postprime; gray bars) were subjected to a microneutralization assay (39) against the challenge virus (HK/483 [B] and VN/1203 [D]). All mice vaccinated with the negative-control vectors (mouse 1 to mouse 4) had undetectable nAb activity, which corresponds to a titer of 10 in this assay (data not shown). Asterisks indicate sera from H5 HA-vaccinated animals with no detectable nAb.

FIG. 4.

Vaccination induces a broad cross-neutralizing antibody response. Sera collected at 2.5 months postprime (preboost; white bars) or 1 month postboost (3.5 months postprime; gray bars) from vaccinated mice (n = 8) that were challenged with VN/1203 (described in the legend to Fig. 2B, D, and F) were subjected to microneutralization assays (39) against the indicated viruses (HK/156 [A] and INA/5 [B]). All mice vaccinated with the negative-control vectors (n = 8) had undetectable neutralizing activity, which corresponds to a titer of 10 in this assay (data not shown). Asterisks indicate sera from H5 HA-vaccinated animals with undetectable nAb.

FIG. 5.

Long-term protective heterologous immunity in vaccinated mice. Mice (n = 5) were vaccinated and challenged at 1 year postprime with VN/1203, as described in the legend to Fig. 2. (A) Sera collected from vaccinated mice at 11 months postprime (9 months postboost) were subjected to microneutralization assays (39) against the indicated viruses (HK/156, HK/483, VN/1203, and INA/5). A titer of 10 in this assay is negative. (B and C) At 1 year postprime (10 months postboost), vaccinated mice were challenged intranasally with VN/1203 (clade 1). (B) Morbidity was assessed by weight loss and is shown as the average original prechallenge weight. (C) A Kaplan-Meier plot depicts the percent survival of negative-control or H5 HA-vaccinated mice. The control animals shown in panels B and C are the same animals as those in Fig. 2 and are shown here for comparison.