Recombinant parainfluenza virus 5 expressing hemagglutinin of influenza A virus H5N1 protected mice against lethal highly pathogenic avian influenza virus H5N1 challenge

Abstract

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.

Fig 1

Schematics of recombinant PIV5 expressing H5. (A) Schematics of recombinant PIV5 expressing H5N1 HA. The cleavage site that contains polybasic amino acid residues is deleted and the sequences are shown. (B) Titers of recombinant PIV5 expressing H5N1 HA stocks. The plaque-purified viruses were grown in MDBK cells and titrated in BHK cells.

Fig 2

Generation and analysis of recombinant PIV5 expressing H5N1 HA between HN and L of the PIV5 genome. (A) Confirmation of H5N1 HA expression using immunoblotting. MDBK cells were infected with ZL48 and lysed at 24 hpi. The lysates were run on an SDS-PAGE gel and immunoblotted with anti-H5N1 HA. (B) Confirmation of H5N1 HA expression using immunofluorescence (IF). MDBK cells were infected with ZL48 and stained with anti-H5N1 HA. Antibodies used for IF are shown on the left side of the panel. (C) Growth rate of rPIV5-H5. MDBK cells were infected with PIV5 or ZL48 at an MOI of 0.1. Media were collected at 24-h intervals. The titers of viruses in the media were determined using plaque assay.

Fig 3

Immune responses in mice inoculated with rPIV5-H5. Mice were infected with PIV5 or ZL48 at a dose of 10⁶ PFU via the intranasal route. (A) ELISA titers of anti-HA. Mice were infected with 10⁶ PFU of ZL48 or PIV5. At 21 dpi, mice were bled. Titers of anti-HA were determined using ELISA. (B) Boost of anti-HA titers. The mice depicted in panel A were boosted at 28 dpi and bled at 35 dpi. Anti-HA titers were measured using ELISA. (C) Neutralization titers. Titers of NAbs in sera of mice vaccinated with PIV5 or ZL48 against H5N1 were determined as described in Materials and Methods. (D) Cell-mediated responses. IFN-γ-producing lymphocytes (pools of n = 3 mice per group) in the mediastinal lymph nodes on day 12 postvaccination as determined by ELISpot analysis. Data are presented as means ± standard errors of the means.

Fig 4

Efficacy of rPIV5-H5 against rgVN-PR8 (H5N1) challenge in mice. The mice were inoculated with PBS, PIV5, or ZL48 (n = 10 per group) at a dose of 10⁶ PFU per mouse. At 21 dpi, the mice were challenged with rgVN-PR8 (H5N1) at a dose of 1,000 TCID₅₀. The lungs were collected at 4 days postchallenge. Titers of rgPR8H5N1 in the lungs of mice were determined using a plaque assay.

Fig 5

Efficacy of rPIV5-H5 against HPAI H5N1 challenge in mice. The mice were inoculated with PBS, PIV5, or ZL48 (n = 15 per group) at a dose of 10⁶ PFU per mouse. At 21 dpi, the mice were challenged with HPAI H5N1 at a dose of 10 LD₅₀. (A) Weights of mice challenged with H5N1. Weights were monitored daily after challenge for 15 days. Weight is graphed as the average percentages of original weight (the day of challenge). (B) Survival rate. (C) Lung titers of mice challenged with H5N1. Mice (n = 5) were sacrificed at 4 days after H5N1 challenge. The titers were determined using plaque assays in MDCK cells.

Fig 6

Analysis of recombinant PIV5 expressing H5N1 HA. (A) Analysis of H5N1 HA expression in cells infected with recombinant PIV5 expressing H5N1 HA. Cells were infected with ZL46, ZL47, and ZL48 as well as PIV5 at an MOI of 1. The expression levels of H5N1 HA in infected cells were determined using flow cytometry as described in Materials and Methods. (B) Growth rate of the recombinant viruses in tissue culture cells. Cells were infected with viruses at an MOI of 0.1. The media from infected cells were collected at 24-h intervals and used for plaque assays to determine titers of viruses.

Fig 7

Growth of the recombinant viruses in vivo. Mice (n = 5) were infected with viruses at a dose of 10⁶ PFU via the intranasal route. At 4 dpi, the lungs were collected and used for plaque assays to determine titers of viruses.

Fig 8

Efficacy of recombinant PIV5 expressing H5N1 HA against HPAI H5N1 challenge. Mice were infected with PIV5 or ZL48 at a dose of 10³, 10⁴, or 10⁵ PFU via the intranasal route. At 24 dpi, the mice were challenged with 10 LD₅₀ of H5N1. The weights of the mice were monitored daily. (A) Log-rank survival analysis of mice challenged with 10 LD₅₀ of A/Vietnam/1203/04. Relative weights of mice vaccinated with 10³ PFU (B), 10⁴ PFU (C), or 10⁵ PFU (D) and challenged with 10 LD₅₀ of HPAI H5N1. Mice vaccinated with rgVN-PR8 received 2,000 PFU of virus.