A versatile H5N1-VSV platform for safe influenza virus research applications

Abstract

The H5N1 strain of influenza A virus (IAV) continues to cause severe infections in a range of avian and mammalian species, including sporadic but concerning cases in humans. There is growing concern that circulating H5N1 strains could lead to widespread human outbreaks. Research with highly pathogenic H5N1 viruses is restricted to Biosafety Level 3 (BSL-3) laboratories. Vesicular stomatitis virus (VSV)-based vaccine vectors expressing heterologous viral proteins from Ebola, SARS-CoV-2, Lassa virus, etc., have previously been shown to be safe and effective in animal models and human clinical trials. Here, we report the development of a recombinant VSV expressing the hemagglutinin (HA) and neuraminidase (NA) genes of H5N1 IAV (H5N1-VSV), which serves as a versatile platform to study various aspects of H5N1 IAV biology. H5N1-VSV replicated robustly to titers comparable to those of the full H5N1 virus in multiple cell lines. In mice, H5N1-VSV vaccination was safe, elicited strong immunity, and conferred protection against a circulating H5N1 strain. Notably, we found that polymorphisms in antigenic site Sa of circulating strains emerged under immune selection pressure in cattle, resembling the evolution of pandemic IAV in humans. These findings suggest that H5N1-VSV can serve as a safe, adaptable platform for influenza research.

Keywords: H5N1; H5N1-VSV; VSV vector; influenza virus.

Fig 1

Generation of recombinant VSV expressing HA and NA from the 2024 H5N1. (A) Genome organization of VSV-GFP and H5N1-VSV. (B) Immunofluorescence of HA and GFP in A549 cells infected with H5N1-VSV. Cells were infected with indicated viruses (MOI = 1), and at 16 hours post infection (hpi), cells were fixed and subjected to immunofluorescence analysis. (C) Replication kinetics in A549 and MDCK cells. Cells were infected with H5N1-VSV (MOI = 0.01) and viral loads in the supernatants were measured. Data are shown as mean titer (PFU/mL) of triplicate samples ± SD. (D) Western blot for HA and NA in infected MDCK cells. Cells were infected with indicated virus (MOI = 1), and at 16 hpi, cell lysates were collected for Western blot analysis. Low-pathogenic H5N1 is included as control.

Fig 2

Replication kinetics and oseltamivir sensitivity of H5N1-VSV. (A and B) Replication of H5N1-VSV, H5N1/Tx24, and VSV in A549 and MDCK cells. Cells were infected with indicated viruses (MOI = 0.01), and at various times post-infection, viral loads in the supernatants were measured. Data are shown as mean titer (PFU/mL) of triplicate samples ± SD. (C) Sensitivity of various IAV strains to oseltamivir carboxylate. A549 cells were infected with indicated viruses (MOI = 0.01) and incubated with varying concentrations of oseltamivir carboxylate. At 48 hpi, viral loads in the supernatants were measured. Data are shown as mean percentage inhibition relative to DMSO control of triplicate samples ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA). ∗ denotes P < 0.05 or lower, and ns, non-significant.

Fig 3

Protective efficacy of H5N1-VSV vaccination in mice. (A and B) Neutralization titers in the sera post-vaccination. Sera from vaccinated mice were treated with RDE-II prior to use in neutralization assays. (C through E) Protection in vaccinated mice following lethal H5N1 challenge: (C) weight loss (n = 4–5), (D) survival (n = 4–5), and (E) lung titers (n = 4–6). Statistical significance was determined by ANOVA. ∗ denotes P < 0.05, **P < 0.01, and ns, non-significant.

Fig 4

Immune selection at antigenic site Sa in cattle H5N1 isolates. (A) HA sequence comparison of currently circulating H5 isolates and neutralization escape mutants. Hu—human, Ca—cattle, and Ms Ab—mouse polyclonal sera selected variant. GeneBank sequences of indicated strains are Hu[MI]: XBE32674.1, Ca[MI]: XFE99892.1, and Ca[TX]: XAJ06472.1. (B and C) Structural modeling of site Sa mutations on HA trimer. (B) Top view. At passage 5, A172T mutation was present in four out of five independent replicates, and E142K mutation was present in one replicate. (C) Side view.