A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

Abstract

Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCERecurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza virus besides vaccination. It is important to develop a humanized neutralizing monoclonal antibody (MAb) against all of the clades of H5N1. For the first time, we report in this study that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza virus neutralization. R ecurrence of highly pathogenic avian influenza (HPAI) virus subtype H5N1 in humans and poultry continues to be a serious public health concern because of its unabated and widespread geographic circulation (1). Since their emergence in Asia over a decade ago, HPAI H5N1 viruses have spread to Ͼ60 countries on three continents and are endemic in poultry in Southeast Asia and Africa (2). They have caused disease in several mammals, including humans, often with lethal consequences. To date, H5N1 has resulted in 667 human cases worldwide, including 393 deaths (3). Although no sustained human-to-human transmission of the virus h...

Converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play

“…[3][4][5][6][7] Illustrating this trend, the number of antiviral mAbs developed and tested in preclinical and clinical trials has grown exponentially in the past 10 years and includes mAbs directed against life-threatening agents, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza virus, dengue virus, Ebola virus and severe acute respiratory syndrome virus coronavirus, among others. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] Importantly, recent clinical data have also demonstrated the efficacy of anti-HIV mAbs in controlling viremia, when administered to HIV-infected patients, lending strong support to the idea that mAbs could broaden the therapeutic arsenal against severe viral infections. 23,24 Their use as antiviral agents is all the more likely to be considered given that multiple biological activities may account for their therapeutic effects.…”

Generation of neutralizing and non-neutralizing monoclonal antibodies against H7N9 influenza virus

“…MAbs were produced as described previously [28]. Briefly, 6-week-old, female, BALB/c mice (Shanghai Laboratory Animal Center) were immunized with two intramuscular injection of H7N9 virus vaccine in Quick Antibody adjuvant (Biodragon, China) with 2week interval.…”

“…The viruses were titrated using HA of 1% chicken red blood cells (RBCs) before the HI assay was performed [33]. Control negative and positive sera were treated with receptor destroying enzyme (RDE) and inactivated at 56°C for 30 min [28]. The mAbs and inactivated sera were 2-fold serially diluted in PBS in a 96-well V-bottom plate and were mixed with 4 HA units (HAU) of virus per well.…”

“…Two murine mAbs (1H10 and 2D1) were produced in our study using hybridoma technology [28]. The isotypes of the two mAbs were detected and listed in Table 1.…”

Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

“…Most of the characterized mAbs against HA from H5N1 virus recognize the epitopes localized in the globular head of HA, most of them overlap the RBS and are isolate-specific, (e.g. Cao et al, 2012b;Du et al, 2013;Wu et al, 2014), while antibodies binding to the stem region have often a broad activity (Tan et al, 2015;Zuo et al, 2015). Extensive review of H5 HA antigenic sites was provided by (Velkov et al, 2013) who described the anti-HA antibodies divided into 3 groups: (i) RBS selective, (ii) non-RBS, membrane-distal globular domain selective and (iii) HA2 selective.…”