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Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

ABSTRACT: We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.

SUBMITTER: Sun H

PROVIDER: S-EPMC8970388 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

Sun He H Wu Guangmou G Zhang Jiyuan J Wang Yu Y Qiu Yue Y Man Hongyang H Zhang Guoli G Li Zehong Z Yue Yuhuan Y Tian Yuan Y

PloS one 20220331 3

We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an an ...[more]

PMID: 35358257

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Project description:BackgroundDue to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.ResultsWe describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed.ConclusionsOur method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.

Project description:Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.IMPORTANCE The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

Project description:UnlabelledHighly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro, they were neutralized by the antibodies in vivo, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses.ImportanceHighly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.

Dataset Information

Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

Publications

Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

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