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. 2022 Nov 15:13:1042916.
doi: 10.3389/fmicb.2022.1042916. eCollection 2022.

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

Weigang Ren  1 , Shuli Pei  2 , Wenming Jiang  3 , Meixia Zhao  4 , Le Jiang  4 , Honggang Liu  4 , Yongxiang Yi  5   6 , Mizhou Hui  1 , Junwei Li  5   6
Affiliations

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

Weigang Ren et al. Front Microbiol. .

Abstract

H5N1 and H9N2 influenza viruses have been reported to cause human infections and are believed to have pandemic potential. The vaccine is an effective tool to prevent influenza virus infection. However, inactivated influenza vaccines sometimes result in low antigenicity as result leads to generating of incomplete immune protection in the form of low cellular and humoral immunity. While the low temperature adapted, traditional live attenuated influenza vaccine (LAIV) is associated with the potential risk to revert to a virulent phenotype, there appears an essential need for an alternative potent methodology to design and develop influenza vaccines with substantial safety and efficacy which may confer solid protection against H9N2 or H5N1 influenza virus infections. In the present study, a replication-deficient recombinant influenza virus, WM01ma-HA(H5), expressing hemagglutinin (HA) of both H9N2 and H5N1 subtypes was developed. The chimeric gene segment expressing HA(H5), was designed using the sequence of an open reading frame (ORF) of HA adopted from A/wild duck/Hunan/021/2005(H5N1)(HN021ma) which was flanked by the NA packaging signals of mouse-adapted strain A/Mink/Shandong/WM01/2014(H9N2)(WM01ma). Due to the absence of ORF of structural protein NA, the replication of this engineered H9N2 influenza viruses WM01ma-HA(H5) was hampered in vitro and in vivo but was well competent in MDCK cells stably expressing the NA protein of WM01ma. Intranasal vaccination of mice with WM01ma-HA(H5) stimulated robust immune response without any clinical signs and conferred complete protection from infection by H5N1 or H9N2 subtype influenza viruses.

Keywords: H5N1; H9N2; influenza A virus; recombination; replication-deficient virus vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
RT-PCR and IFA were performed to verify the integration and expression of the NA in MDCK cells stably expressing NA protein. (A) The NA gene was amplified by RT-PCR with mRNA of β-Actin as internal control. (B) Expression of the NA protein was tested by IFA.
Figure 2
Figure 2
Generation of replication-deficient recombinant WM01ma-HA (H5). (A) Schematic representation of chimeric HA gene segment. The open reading frame (ORF) of HA (HN01ma, H5N1) is flanked by the NA segment-specific packaging sequences derived from WM01ma (H9N2). (B) The eight segments for WM01ma-HA (H5) recombinant viruses. (C) Rescue of recombinant WM01ma-HA (H5) influenza virus by the reverse genetic system.
Figure 3
Figure 3
Characterization of the recombinant WM01ma-HA (H5) influenza virus. Comparison of the morphologies of WM01ma (A) and the recombinant WM01ma-HA (H5) influenza virus (B) by scanning electron microscopy (scale bar 100 nm), plaque formed by the recombinant WM01ma-HA (H5) influenza virus on wild-type MDCK cells (C) and MDCK cells stably expressing the NA protein (D).
Figure 4
Figure 4
HAs of H9 and H5 were verified in the recombinant WM01ma-HA (H5) influenza virus by RT-PCR (A) and western blotting (B).
Figure 5
Figure 5
Growth curve of the recombinant WM01ma-HA (H5) influenza virus on MDCK cells stably expressing NA protein. The viral titers in each time point were calculated in triplicate. The values represent the means ± standard deviations (SD).
Figure 6
Figure 6
Evaluation of safety of the recombinant WM01ma-HA (H5) influenza virus as indicated by weight changes of experimental mice (A) and HAI titers against the HN021ma influenza virus (B) and the WM01ma influenza virus (C). Each symbol represents an individual mouse, and the horizontal lines indicate mean values. The differences between the two groups were analyzed by a one-way ANOVA with Tukey's multiple-comparison test. ns indicates not significant. (p > 0.05), *(p ≤ 0.05), **(p ≤ 0.01), ****(p ≤ 0.0001).
Figure 7
Figure 7
Cytokine secretion in intranasally vaccinated mice. Mice serum was collected at 3 days postinfection for analysis by ELISA to determine cytokine levels (A). The number of antigen-specific splenocytes secreting IFN-γ (B) or IL-5 (C) was determined by ELISpot. ND, nondetected. Each sample was tested in triplicate. The values represent the means ± standard deviations (SD). The dots represent the individual mice. ns indicates not significant. (p > 0.05), *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001),****(p ≤ 0.0001).
Figure 8
Figure 8
Vaccination with the recombinant WM01ma-HA (H5) virus conferred protection against HN021ma and WM01ma. (A) weight changes and (B) survival rates of mice vaccinated with the recombinant WM01ma-HA (H5) influenza virus and challenged with H9N2 (WM01ma) virus, (C) weight changes and (D) survival rates of mice vaccinated with the recombinant WM01ma-HA (H5) influenza virus and challenged with H5N1 (HN01ma) virus.
Figure 9
Figure 9
The viral titration in the lung tissues of WM01ma (A) or HN021ma (B) challenged mice at 4 d.p.i. Each experiment was performed in quadruplicate. The dots correspond to the values obtained from the individual mouse values and are represented as the means ± SD.
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