Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine

Abstract

Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an in silico antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-luc, negative control) in mice and big brown bats (Eptesicus fuscus). Mice vaccinated and boosted intradermally with 1 x 107 plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x107 PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x108 PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-luc and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, in silico designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife.

Fig 1. Antigenic coverage of putative T cell epitopes by the designed mosaic phylogroup I lyssavirus glycoprotein.

A) Antigenic coverage with the epitope length set to 12 amino acids. B) Antigenic coverage with the epitope length set to 9 amino acids. C) Comparison of 12-mer epitope coverage between the mosaic sequence and all input sequences.

Fig 2. In vitro assessment of rabies glycoprotein expression in novel RCN-vectored rabies vaccines.

A) Immunofluorescence of RCN expressing in silico designed lyssavirus phylogroup I glycoprotein (MoG) with and without an internal ribosomal entry site (IRES). A previously described RCN construct expressing the glycoprotein from rabies CVS-11 (RCN-G) was used as a positive control, and RCN expressing green fluorescent protein (GFP) was used as a negative control. B) Western blot of supernatant (Sup.) or pellet collected from Vero cells infected with RCN-MoG, RCN-IRES-MoG, RCN-G (positive control) or RCN-luc (negative control), The rabies glycoprotein is expected to be around 62 kDa.

Fig 3. Rabies neutralizing antibody levels in mice following vaccination with various RCN-vectored rabies vaccines.

Serum titers of rabies neutralizing antibodies (IU/ml) in mice 45 days post vaccination with RCN-MoG, RCN-IRES-MoG, or RCN-G. No significant differences were detected between groups (P = 0.399).

Fig 4. Survival after rabies challenge in mice.

Efficacies of raccoon poxvirus (RCN) vectored rabies vaccines in mice after intracerebral challenge with the CVS-11 strain of rabies virus. Every mouse (6/6) in the RCN-MoG group survived challenge to day 14 compared to 3 of 6 in the RCN-IRES-MoG group, and 4 of 5 in the RCN-G group. All (5/5) negative controls (RCN-luc) succumbed by day 9 post challenge. A chart of p-values associated with the survival curve is also provided. Survival of all vaccinated mice was significantly higher (P < 0.05) than negative controls, but there was no significant difference (P > 0.05) between vaccine treated groups.

Fig 5. Rabies virus neutralizing antibodies in bats following oronasal vaccination with RCN-based rabies vaccine constructs.

Serum rabies neutralizing antibody titers at various time-points as determined by rapid fluorescence focus inhibition test (RFFIT). Day 22 represents levels after initial vaccination, and day 65 represents levels after boost and immediately prior to challenge.

Fig 6. Survival after rabies challenge in E. fuscus bats.

Percent survival of E. fuscus bats is shown over time after experimental infection. Bats were vaccinated oronasally with RCN-MoG, RCN-G, or RCN-luc (negative control). A fourth group was given RCN-MoG topically in a glycerin jelly vehicle. Vaccinated bats had significantly greater survival than negative controls (P = 0.002).