Identification of amino acids in H9N2 influenza virus neuraminidase that are critical for the binding of two mouse monoclonal antibodies

Gao

et al. 2018

J Virol

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The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses, and the emergence of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be important contributors to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. The loop formed in the region of residue 250 (250 loop) and the domain formed by the loops containing residues 370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 10F4, and 11B2, which recognize these two antigenic domains, were characterized in depth. These four MAbs differ in their abilities to inhibit cleavage of small and large substrates (methyl-umbelliferylacetyl neuraminic acid [MU-NANA] and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced the in vitro spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations but to different degrees. These MAbs have different in vivo levels of effectiveness: 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection and suggests that in vitro properties can be used to rank antibodies with therapeutic potential. IMPORTANCEThe novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in a larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and the emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity, and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies in vitro measures that can be used to predict the in vivo efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.

Section: Methodsmentioning

confidence: 99%

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

Gao

et al. 2018

J Virol

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“…To identify if 1G8 targets the same epitope in NA of H3N2 HSIV as previously reported for H9N2 AIV ( Wan et al, 2016 ), escape mutant of rgH1N2(PUMCH06) selected by 1G8 was characterized. The K199R (N2 numbering) mutation in NA was found in selected escape mutant of rgH1N2(PUMCH06) virus.…”

Section: Resultsmentioning

confidence: 99%

“…The inhibition of NA activity by 1G8 was measured with enzyme-linked lectin assay (ELLA) and 4-(methylumbelliferyl)- N -acetylneuraminic acid (Mu-NANA) assay as previously described ( Wan et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

A Cross-Reactive Monoclonal Antibody Against Neuraminidases of Both H9N2 and H3N2 Influenza Viruses Shows Protection in Mice Challenging Models

Wang

et al. 2021

Front. Microbiol.

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Neuraminidases (NAs) of H9N2 avian influenza virus (AIV) and H3N2 human seasonal influenza virus (HSIV) share similar antigenic structures. However, there are few reports on epitopes shared by these two NAs. We previously reported a monoclonal antibody (mAb) 1G8 against the NA of H9N2 AIV with neuraminidase inhibition (NI) ability. In this study, 1G8 was shown to cross-react with and inhibit the NA of H3N2 HSIV. In a passive transfer experiment, 1G8 provided protection to mice challenged with rescued H1N2 viruses carrying H9N2 NA or H3N2 NA. Mutation at amino acid position 199 was also selected and proved to be crucial for H3N2 HSIV to escape from mAb 1G8. Moreover, we found that residue 199 contributed to inducing broad protective antibodies without the influence of the N-linked glycosylation at amino acid position 200 in NAs. Residues as residue 199, which are not shielded by glycosylation modification, would form ideal epitopes for developing universal vaccine and protective antibodies.

“…The virus was propagated in 9-day-old embryonated chicken eggs and preserved at − 70 °C. 1G8 hybridoma cells were screened from sp2/0 cells fused with spleen cells from BALB/C mice immunized with X1 virus, as previously reported (Wan et al 2016 ). Madin-Darby canine kidney (MDCK) cells (ATCC, CCL-34) and COS-1 cells (ATCC, CRL-1650) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Massachusetts, USA).…”

Section: Methodsmentioning

confidence: 99%

“…However, few antibodies were chickenized for passive immunization of chickens (Roh et al 2015 ). We previously reported a mouse-derived mAb 1G8, that has a significant inhibitory effect on the NA enzyme activity of H9N2 AIV (Wan et al 2016 ). Additionally, mAb 1G8 could react with all H9N2 AIV strains isolated in eastern China.…”

Section: Introductionmentioning

confidence: 99%

Generation of a recombinant chickenized monoclonal antibody against the neuraminidase of H9N2 avian influenza virus

Wang

et al. 2020

AMB Expr

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We previously reported a monoclonal antibody (mAb), 1G8, against the neuraminidase (NA) of H9N2 avian influenza virus (AIV) with significant NA inhibitory activity. To generate a recombinant chickenized mAb (RCmAb) against the NA of H9N2 AIV for passive immunization in poultry, the gene of the fragment of antigen binding (Fab) of mAb 1G8 was cloned and fused with the fragment crystallizable (Fc) gene of chicken IgY. The RCmAb 1G8 was expressed in COS-1 cells and could be detected in cell culture supernatant. The results of NA inhibitory activity tests of the RCmAb 1G8 in an enzyme-linked lectin assay (ELLA) and a microneutralization (MN) assay showed that the RCmAb 1G8 maintained significant NA inhibitory activity and neutralizing ability. This is the first chickenized antibody against AIV, which would be a good candidate for passive immunization in poultry.