Single-dose vaccination of a recombinant parainfluenza virus 5 expressing NP from H5N1 virus provides broad immunity against influenza A viruses

Abstract

Influenza viruses often evade host immunity via antigenic drift and shift despite previous influenza virus infection and/or vaccination. Vaccines that match circulating virus strains are needed for optimal protection. Development of a universal influenza virus vaccine providing broadly cross-protective immunity will be of great importance. The nucleoprotein (NP) of influenza A virus is highly conserved among all strains of influenza A viruses and has been explored as an antigen for developing a universal influenza virus vaccine. In this work, we generated a recombinant parainfluenza virus 5 (PIV5) containing NP from H5N1 (A/Vietnam/1203/2004), a highly pathogenic avian influenza (HPAI) virus, between HN and L (PIV5-NP-HN/L) and tested its efficacy. PIV5-NP-HN/L induced humoral and T cell responses in mice. A single inoculation of PIV5-NP-HN/L provided complete protection against lethal heterosubtypic H1N1 challenge and 50% protection against lethal H5N1 HPAI virus challenge. To improve efficacy, NP was inserted into different locations within the PIV5 genome. Recombinant PIV5 containing NP between F and SH (PIV5-NP-F/SH) or between SH and HN (PIV5-NP-SH/HN) provided better protection against H5N1 HPAI virus challenge than did PIV5-NP-HN/L. These results suggest that PIV5 expressing NP from H5N1 has the potential to be utilized as a universal influenza virus vaccine.

Fig 1

Schematic of PIV5-NP viruses. The PIV5 genome contains seven genes in the order of 3′-NP-V/P-M-F-SH-HN-L-5′, with leader and trailer regions located at the ends of the genome. The H5N1-NP gene was inserted into the PIV5 genome at the indicated gene junctions.

Fig 2

Expression of H5-NP and PIV5 proteins. (A) IF assay to detect H5N1-NP and PIV5-V/P protein expression in MDBK cells. MDBK cells in 24-well plates were mock infected or infected with PIV5 or PIV5-NP-HN/L 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-NP antibodies. The cells were photographed by using a fluorescence microscope (Advanced Microscopy Group). (B) An IP assay was used to detect H5N1-NP and PIV5 protein expression in MDBK cells. MDBK cells were mock infected or infected with PIV5 or PIV5-NP-HN/L at an MOI of 5. At 22 hpi, the cells were labeled with [³⁵S]Met-Cys Promix, lysed, and immunoprecipitated with monoclonal anti-PIV5-V/P or anti-H5N1-NP antibodies. The precipitated proteins were resolved by 15% SDS-PAGE and examined by autoradiography using a Storm Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA).

Fig 3

Growth of PIV5 and PIV5-NP-HN/L in vitro and in vivo. (A). Multiple-step growth curves of PIV5 and PIV5-NP-HN/L in tissue-cultured cells. MDBK cells were infected with PIV5 or PIV5-NP-HN/L at an MOI of 0.1, and the media were collected at 24-h intervals. Virus titers were determined by plaque assays on BHK cells. All P values were calculated by using a t test. (B). Mice were vaccinated with 10⁵ PFU of PIV5 or PIV5-NP-HN/L intranasally. Mice were euthanized on day 3 postvaccination to determine lung virus titers.

Fig 4

Induction of humoral and cellular responses by PIV5-NP-HN/L. (A). NP antibody levels induced by PIV5-NP-HN/L in mice. Mice were vaccinated with 10⁶ PFU of PIV5 or PIV5-NP-HN/L or 10⁵ PFU of X31 intranasally. At day 21 postvaccination, blood samples were collected, and sera were prepared. An ELISA was performed according to the manufacturer's instructions (KPL, Inc.), using purified H5N1-NP. (B). T cell response induced by PIV5-NP-HN/L in mice. Mice were vaccinated with PBS, 10⁷ PFU of PIV5 or PIV5-NP-HN/L, or 0.1 LD₅₀ of PR8 intranasally (n = 5 per group). At day 21 postvaccination, mice were sacrificed, and spleens were collected. Splenocytes were restimulated with Flu-NP, Ebola virus GP P2 as a negative control, or PMA-ionomycin as a positive control. Results are presented as the mean number of cytokine-secreting cells per 10⁶ splenocytes. The P value is 0.11 between PIV5 and PIV5-NP-F/SH in Flu-NP stimulation.

Fig 5

PIV5-NP-HN/L protection against H1N1 challenge. Mice were vaccinated with PBS (n = 10), 10⁶ PFU of PIV5 (n = 10) or PIV5-NP-HN/L (n = 10), or 10⁵ PFU of X31 (n = 9) intranasally. At day 21 postvaccination, mice were challenged with 10 LD₅₀ of A/Puerto Rico/8/34 (H1N1). Weight loss (A) and survival (B) were monitored daily for 14 days following challenge. Weight loss is presented as the average percentage of original weight (the day of challenge). (C) Lung titers of mice challenged with H1N1. Mice (n = 5) were sacrificed at 3 days postchallenge. The titers were determined by using a TCID₅₀ assay using MDCK cells. The P value was >0.05 for PBS versus PIV5-NP-HN/L and for PIV5-NP-HN/L versus X31; the P value was <0.05 for PBS versus X31.

Fig 6

PIV5-NP-HN/L protection against H5N1 HPAI virus challenge. Mice were vaccinated with PBS (n = 9) or 10⁷ PFU of PIV5 (n = 7), PIV5-NP-HN/L (n = 6), or rgA/VN-PR8 (n = 10). At day 21 postvaccination, mice were challenged with 10 LD₅₀ of H5N1 HPAI virus. Weight loss (A) and survival (B) were monitored for 16 days following influenza virus challenge.

Fig 7

Analysis of recombinant PIV5 expressing NP. (A) Multiple-step growth curves of PIV5 and PIV5-NP viruses. MDBK cells were infected with PIV5 or PIV5-NP viruses at an MOI of 0.1, and the media were collected at 24-h intervals. Virus titers were determined by plaque assays using BHK cells. (B) H5N1-NP expression levels in PIV5-NP-infected cells. MDBK cells were infected with PIV5 or PIV5-NP viruses at an MOI of 5. The ratios of MFI of H5-NP to that of PIV5-VP were examined by flow cytometry. (C) Growth of PIV5 and PIV5-NP viruses in vivo. Mice were vaccinated with 10⁵ PFU of PIV5 or PIV5-NP viruses intranasally. Mice were euthanized on day 3 postvaccination to determine lung virus titers.

Fig 8

PIV5-NP viruses prime T cell responses. Mice were vaccinated with PBS, 10⁷ PFU of PIV5 or PIV5-NP viruses, or 0.1 LD₅₀ of PR8 intranasally (n = 5 per group). At day 21 postvaccination, mice were sacrificed, and spleens were collected. Splenocytes were restimulated with Flu-NP, Ebola virus GP P2, or PMA-ionomycin. Results are presented as the mean number of cytokine-secreting cells per 10⁶ splenocytes (P = 0.08 for PIV5 versus PIV5-NP-F/SH; P = 0.29 for PIV5 versus PIV5-NP-SH/HN; P = 0.43 for PIV5 versus PIV5-NP-HN/L after Flu-NP stimulation).

Fig 9

PIV5-NP protection against H5N1 HPAI virus challenge. Mice were vaccinated with PBS (n = 10), 10⁷ PFU of PIV5 (n = 10) or PIV5-NP viruses (n = 10, except n = 9 for PIV5-NP-F/SH), or 2,000 PFU of rgA/VN-PR8 intranasally (n = 7). At day 21 postvaccination, mice were challenged with 20 LD₅₀ of H5N1 HPAI virus. Weight loss (A) and survival (B) were monitored for 14 days following influenza virus challenge. Weight loss is graphed as an average percentage of the original weight (the day of challenge).