A Replication-Defective Influenza Virus Harboring H5 and H7 Hemagglutinins Provides Protection against H5N1 and H7N9 Infection in Mice

Abstract

The recent highly pathogenic avian influenza (HPAI) H5N1 and H7N9 viruses have caused hundreds of human infections with high mortality rates. Although H5N1 and H7N9 viruses have been mainly limited to avian species, there is high potential for these viruses to acquire human-to-human transmission and initiate a pandemic. A highly safe and effective vaccine is needed to protect against a potential H5N1 or H7N9 influenza pandemic. Here, we report the generation and evaluation of two reassortant influenza viruses, PR8-H5-H7NA and PR8-H7-H5NA. These viruses contain six internal segments from A/Puerto Rico/8/1934 (PR8), the HA segment from either A/Alberta/01/2014 (H5N1) [AB14 (H5N1)] or A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], and a chimeric NA segment with either the BC15 (H7N9) HA gene or the AB14 (H5N1) HA gene flanked by the NA packaging signals of PR8. These viruses expressed both H5 and H7 HAs in infected cells, replicated to high titres when exogenous NA was added to the culture medium in vitro, and were replication-defective and non-virulent when administered intranasally in mice. Moreover, intranasal vaccination with PR8-H5-H7NA elicited robust immune responses to both H5 and H7 viruses, conferring complete protection against both AB14 (H5N1) and BC15 (H7N9) challenges in mice. Conversely, vaccination with PR8-H7-H5NA only elicited robust immune responses towards the H7 virus, which conferred complete protection against BC15 (H7N9) but not against AB14 (H5N1) in mice. Therefore, PR8-H5-H7NA has strong potential to serve as a vaccine candidate against both H5 and H7 subtypes of influenza viruses. Importance Avian influenza H5N1 and H7N9 viruses infected human with high mortality rates. A highly safe and effective vaccine is needed to protect against a potential pandemic. We generated and evaluated two reassortant influenza viruses, PR8-H5-H7NA and PR8-H7-H5NA as vaccine candidates. Each virus contains one type of HA in segment 4 and the other subtype of HA in segment 6, thus expressing both H5 and H7 subtypes of HA molecule. The viruses’ replication is dependent in the addition of exogenous NA in cell culture, and are replication-defective in vivo. Vaccination of PR8-H5-H7NA virus confers protection to both H5N1 and H7N9 virus challenge; conversely, vaccination of PR8-H7-H5NA only provides protection to H7N9 virus challenge. Our data revealed when engineering such virus, the H5 or H7 HA in segment 6 affects the immunogenicity. PR8-H5-H7NA has strong potential to serve as a vaccine candidate against both H5 and H7 subtypes of influenza viruses.

A replication-deficient H9N2 influenza virus carrying H5 hemagglutinin conferred protection against H9N2 and H5N1 influenza viruses in mice

“…The research found that the 5' and 3' packaging signals of each vRNA segment of the influenza virus are crucial to assemble a progeny virion with newly synthesized vRNA segments. Previously, one group reported an approach for the generation of a reassortant chimeric replication-defective influenza virus which grew well in vitro through the addition of bacterial sialidase and provided protection against H5N1 and H7N9 infection in mice (Tian et al, 2021 ). Based on this information, it is workable to generate a genome-modified LAIV.…”

Development and application of reverse genetic technology for the influenza virus

“…Studies have found that the low-pathogenicity avian influenza virus has only one alkaline arginine at the HA cleavage site and can be cleaved by trypsin-like proteases present in the respiratory and digestive tracts; therefore, the virus is generally restricted to replication in the respiratory and digestive tracts [65,66]. The highly pathogenic H5 and H7 subtype avian influenza viruses have multiple consecutive alkaline amino acids at the HA cleavage site that can be cleaved by proteases widely present in cells, which potentially leads to systemic infection [67]. A reverse genetic system was used to remove multiple basic amino acids from the HA cleavage site of highly pathogenic avian influenza virus, and the results showed that the virus's pathogenicity in poultry and mice was reduced [68].…”

Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice

“…Newer H5N1 and H7N9 vaccine candidates are being investigated to protect against a potential pandemic. For example, two reassortant influenza vaccines expressing H5 or H7 HAs (PR8-H5-H7NA and PR8-H7-H5NA) have been shown to replicate to high titers when exposed to exogenous neuraminidase (NA) in vitro, and these vaccine candidates were shown to be replication-defective and nonvirulent when administered intranasally in mice [ 19 ]. Vaccination with PR8-H5-H7NA elicited robust immune responses to H5 and H7 viruses and conferred complete protection against H5N1 and H7N9 challenges in mice.…”