Efficacy of parainfluenza virus 5 mutants expressing hemagglutinin from H5N1 influenza A virus in mice

Abstract

Parainfluenza virus 5 (PIV5) is a promising viral vector for vaccine development. PIV5 is safe, stable, efficacious, cost-effective to produce and, most interestingly, it overcomes preexisting antivector immunity. We have recently reported that PIV5 expressing the hemagglutinin (HA) from highly pathogenic avian influenza (HPAI) virus H5N1 (PIV5-H5) provides sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. It is thought that induction of apoptosis can lead to enhanced antigen presentation. Previously, we have shown that deleting the SH gene and the conserved C terminus of the V gene in PIV5 results in mutant viruses (PIV5ΔSH and PIV5VΔC) that enhance induction of apoptosis. In this study, we inserted the HA gene of H5N1 into PIV5ΔSH (PIV5ΔSH-H5) or PIV5VΔC (PIV5VΔC-H5) and compared their efficacies as vaccine candidates to PIV5-H5. We have found that PIV5ΔSH-H5 induced the highest levels of anti-HA antibodies, the strongest T cell responses, and the best protection against an H5N1 lethal challenge in mice. These results suggest that PIV5ΔSH is a better vaccine vector than wild-type PIV5.

Fig 1

Schematics of recombinant PIV5 expressing H5-HA. PIV5 genome contains seven genes in the order of 3′-NP-V/P-M-F-SH-HN-l-5′ with leader (Le) and trailer (Tr) regions located at the ends of the genome. PIV5ΔSH lacking the SH gene and PIV5VΔC lacking the conserved C-terminal of the V protein have been described before (21, 24). The H5N1 HA gene was inserted into PIV5, PIV5ΔSH, or PIV5VΔC between the HN and L genes. The additional mutation sites within two clones of PIV5VΔC-H5 genome were as indicated.

Fig 2

Confirmation of H5-HA expression. (A) HA expression in PIV5-HA virus-infected MDBK cells. MDBK cells were mock infected or infected with PIV5, PIV5-H5, PIV5ΔSH-H5, PIV5VΔC-H5-3, or PIV5VΔC-H5-4 at an MOI of 0.1. At 2 dpi, the cells were fixed, permeabilized, and then incubated with monoclonal anti-PIV5-V/P or anti-H5N1-H5 antibodies. The cells were photographed using a fluorescence microscope (Advanced Microscopy Group). (B) Comparison of H5-HA protein expression levels in virus-infected MDBK cells by flow cytometer. MDBK cells were mock infected or infected with PIV5, PIV5-H5, PIV5ΔSH-H5, PIV5VΔC-H5-3, or PIV5VΔC-H5-4 at an MOI of 5. The mean fluorescence intensity (MFI) of H5-HA was examined by flow cytometer at 2 dpi. *, P < 0.05 between PIV5ΔSH-H5 and PIV5-H5.

Fig 3

Growth of PIV5-HA viruses in vitro. (A) MBDK cells were infected with PIV5, PIV5-H5, PIV5ΔSH-H5, or PIV5ΔSH. (B) Vero cells were infected with PIV5, PIV5-H5, PIV5VΔC-H5-3, PIV5VΔC-H5-4, or PIV5VΔC at an MOI of 0.1. Media were collected at 24-h intervals, and virus titers were determined by plaque assay on BHK cells. p.i., postinfection.

Fig 4

Growth of PIV5-HA viruses in vitro and in vivo. Mice were vaccinated with 10⁵ PFU of PIV5, PIV5-H5, PIV5ΔSH-H5, PIV5VΔC-H5-3, or PIV5VΔC-H5-4 intranasally. Mice were euthanized on day 3 postvaccination to determine lung virus titers.

Fig 5

Induction of humoral and cellular responses by PIV5-HA viruses. (A) HA-antibody levels in mice induced by PIV5-HA viruses. Mice were vaccinated with 10³ PFU of PIV5, PIV5-H5, PIV5ΔSH-H5, PIV5VΔC-H5-3, or PIV5VΔC-H5-4 intranasally. At day 21 postvaccination, blood samples were collected, sera were prepared, and an ELISA was performed to detect H5-HA-specific IgG. *, P < 0.05 between PIV5ΔSH-H5 and PIV5-H5. (B). T cell response in mice induced by PIV5-HA viruses. Mice were vaccinated with PBS or 10³ PFU of PIV5, PIV5-H5, or PIV5ΔSH-H5 intranasally. At day 21 postvaccination, the mice were sacrificed, and the spleens were collected. Splenocytes were restimulated with HA, Ebola GP P2 as a negative control, or PMA-ionomycin as a positive control. The results are presented as the mean number of cytokine-secreting cells subtracted from the number of mock-stimulated cells per 10⁶ splenocytes.

Fig 6

Protection of PIV5-HA viruses against H5N1 HPAI challenge. (A). Mice were vaccinated with PBS (n = 9), 10³ PFU of PIV5 (n = 7), PIV5-H5 (n = 10), PIV5ΔSH-H5 (n = 8), PIV5VΔC-H5-3 (n = 10), or PIV5VΔC-H5-4 (n = 10) or with 2,000 PFU of rgA/VN-PR8 (n = 10). At day 21 postvaccination, the mice were challenged with 10 LD₅₀ H5N1 HPAI. Weight loss (A) and survival (B) were monitored for 16 days after H5N1 challenge. The P value of peak weight loss between PIV5-H5 and PIV5ΔSH-H5 is 0.28.