Mechanisms of virus neutralization by antibody

Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection.

However, how neutralizing antibodies inhibit infection in vitro remains unclear. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. One binding domain, VNAR-3B4, was capable of neutralizing coronaviruses across multiple phylogenetic lineages class 2B and class 2C , owing to partial sequence conservation of the 3B4 epitope.

The small size and protruding CDR3 makes 3B4 uniquely effective at accessing this conserved epitope, underscoring the utility of neutralizing VNAR domains. Our data also suggest that the three VNARs we identified would be effective at neutralizing the existing Alpha, Beta, and Delta variants as well as variants yet to emerge.

Should vaccination fail due to the emergence of a viral variant, alternative therapies like our VNARS, alone or in combination, are essential to maintaining control over the spread of the virus.

After the initial pseudovirus screen to identify potent neutralizers from our libraries, none of the candidate VNARs underwent affinity maturation and were subsequently characterized as purely monomers. In pseudotype and authentic virus assays, our three lead VNARs performed as well or better than other neutralizing antibodies reported in the literature Our VNARs were more effective at neutralization compared to other monovalent single-domain antibodies derived from camelids.

In a previous study, investigators found that tethering the affinity matured camelid antibody mNb6 in triplicate to form a trimer mNb-tri resulted in a highly potent construct with low picomolar neutralization IC 50 values against both pseudo and authentic SARS-CoV-2 virus. Though not effective at neutralizing pseudovirus as monomer, VHHFc had a low nanomolar potency 1. These findings highlight the ability and versatility of the diminutive VNAR scaffold for the development of highly specific and effective agents against a given target.

Interestingly, only two of the interactions in VNAR-3B4 were dependent on RBD residue identity, as the majority of hydrogen bonding occurred in direct contact with the peptide backbone of the spike RBD. This feature also makes the 2C02 interaction residue independent to the extent that residue identity of this interface maintains its non-polar features. The partial residue independence of each interaction indicates that VNARs 3B4 or 2C02 as a treatment could maintain potency for binding the RBD in emerging spike mutants or other coronaviruses.

Comparison of the VNARs to other NAbs in the literature reveals distinct differences about their epitopes and mechanisms of action. Importantly, the separate epitopes of 3B4 and 2C02 suggest that they would be therapeutically effective as a multi-NAb cocktail, similar to the REGEN-COV antibody cocktail of casirivimab and imdevimab, which bind to closely located epitopes.

Taken together, our VNARs occupy a unique molecular space that have potential to completely alter the landscape of biologics by making antibody-class drugs even smaller, yet as potent and efficacious. Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome pNL All cell culture reagents were purchased from Thermo Fisher. Phage was PEG-precipitated from the culture supernatant and used for round one of bio-panning.

Mid-log phase E. Three additional rounds of selection were conducted using the same antigen concentration, and wash stringency. An equal volume of ice-cold 2. The supernatant was filtered through a 0. Pseudotyped retroviruses expressing a luciferase reporter gene were prepared by co-transfecting HEKT cells with a plasmid encoding an Env-defective and luciferace-encoding HIV-1 genome pNL Cell infection assays were carried out as described previously The following day, cells were washed twice with DPBS and media was replaced with a complete medium.

Luminescence values are reported relative to levels measured in cells treated with virus alone, background corrected by luminescence values in cells unexposed to virus. Samples were tested in technical triplicates across three independent biological experiments. Data were processed in Microsoft Excel v Concentration-response curves and IC 50 values were generated in OriginLab b.

Vero E6 cells 2. Medium was removed from cells and the virus-antibody mixture was added to achieve a final multiplicity of infection of 0. Percent maximal infection was determined for each dilution as the ratio of the fluorescent signal to the maximal signal for non-treated controls in the same plate.

Each sample was tested with two technical replicates across three independent biological experiments. Binding affinities were determined by analysis of generated binding curves in the Octet DataAnalysis v The controls were averaged and subtracted from the data before modeling and the dissociation constant K D was calculated. Figures were generated by plotting data in GraphPad Prism v9.

The crystals grew to full size in three days. The RBD-2C02 complex was crystallized by the sitting drop vapor diffusion method, using a reservoir solution containing 0. All X-ray diffraction data were processed using XDS version The mFo-DFc map clearly showed the presence of glycans in later stages of the refinement.

In the RBD-2C02 crystal there is one complex in the asymmetric unit. The summary of data collection and model refinement statistics is shown in Supplementary Table 2. By default, SwissModel will only account for one of the protein chains in a coordinate file and will not effectively model interactions between residues of multiple chains. The sequence was renumbered in numerical order and thirty amino acids of the pattern Gly-Ala-Ser were inserted as a spacer between the proteins in the modified sequence.

In PyMol, resulting models were then separated into individual chains and segments and renumbered to match the original coordinate file. No data exclusions were made. No randomization or blinding measures were made. Further information on research design is available in the Nature Research Reporting Summary linked to this article. The data that support this study are available from the corresponding authors upon reasonable request.

Source data are provided with this paper. Yadav, P. Neutralization of variant under investigation B. Greaney, A. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe 29 , — e Liu, Y. Neutralizing activity of BNTb2-elicited serum.

Med , — Article Google Scholar. Wang, P. Nature , — Couzin-Frankel, J. Relief and worry for immune-suppressed people. Science , — Boyarsky, B. JAMA , — Huang, Y. Greenberg, A. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Stanfield, R. Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Maturation of shark single-domain IgNAR antibodies: evidence for induced-fit binding.

Ubah, O. An anti-hTNF-alpha variable new antigen receptor format demonstrates superior in vivo preclinical efficacy to Humira R in a transgenic mouse autoimmune polyarthritis disease model.

Novel, anti-hTNF-alpha variable new antigen receptor formats with enhanced neutralizing potency and multifunctionality, generated for therapeutic development. Wrapp, D. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies.

Cell , — Li, Y. Natl Acad. Ge, X. Cui, J. Origin and evolution of pathogenic coronaviruses. Wang, N. Cell Res. DNA-based and alphavirus-vectored immunisation with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus. Chung KM, et al. Antibody recognition of cell surface-associated NS1 triggers Fc-gamma receptor-mediated phagocytosis and clearance of West Nile Virus-infected cells.

Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms. Shu PY, et al. Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever. J Med Virol. Churdboonchart V, et al. Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop Med Hyg. Prospects for a virus non-structural protein as a subunit vaccine.

Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica.

Identification of epitopes on the E glycoprotein of Saint Louis encephalitis virus using monoclonal antibodies. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Mandl CW, et al. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

Sukupolvi-Petty S, et al. Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes. Throsby M, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. Induction of epitope-specific neutralizing antibodies against West Nile virus. Sanchez MD, et al. The neutralizing antibody response against West Nile virus in naturally infected horses.

A multi-hit model for the neutralization of animal viruses. A model for neutralization of viruses based on antibody coating of the virion surface. Curr Top Microbiol Immunol. Pierson TC, et al. Stoichiometric requirements for antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe. Occupancy and mechanism in antibody-mediated neutralization of animal viruses.

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III ED3 of dengue 2 virus. Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites. Morrey JD, et al. Humanized monoclonal antibody against West Nile virus envelope protein administered after neuronal infection protects against lethal encephalitis in hamsters.

Defining limits of treatment with humanized neutralizing monoclonal antibody for West Nile virus neurological infection in a hamster model. Antimicrob Agents Chemother. Nybakken GE, et al. Structural basis of West Nile virus neutralization by a therapeutic antibody. Kaufmann B, et al. West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Krishnan MN, et al. Rab 5 is required for the cellular entry of dengue and West Nile viruses.

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry. Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. Anderson R. Manipulation of cell surface macromolecules by flaviviruses. Adv Virus Res. Involvement of lipids in different steps of the flavivirus fusion mechanism.

Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH. Pantophlet R, Burton DR. GP target for neutralizing HIV-1 antibodies. Annu Rev Immunol. He RT, et al. Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. Jennings AD, et al. Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site.

A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model. Jiang WR, et al. Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence. Tassaneetrithep B, et al. J Exp Med. Navarro-Sanchez E, et al. Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses.

EMBO Rep. Sakuntabhai A, et al. Avariant in the CD promoter is associated with severity of dengue disease. Nat Genet.

Lozach PY, et al. J Biol Chem. Davis CW, et al. Pokidysheva E, et al. Subunit organization and binding to multivalent ligands. A new mechanism for the neutralization of enveloped viruses by antiviral antibody. Crystal structure of the west nile virus envelope glycoprotein. Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies. Mehlhop E, et al. Complement activation is required for induction of a protective antibody response against West Nile virus infection.

Mehlhop E, Diamond MS. Protective immune responses against West Nile virus are primed by distinct complement activation pathways.

Meyer K, et al. Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein. Complement component C1q enhances the biological activity of influenza virus hemagglutinin-specific antibodies depending on their fine antigen specificity and heavy-chain isotype. Schlesinger JJ, Chapman S. The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against YF encephalitis in mice.

Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses. Rothman AL. Immunology and immunopathogenesis of dengue disease.

Vaughn DW, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. Observations related to pathogenesis of dengue hemorrhagic fever.

Relation of disease severity to antibody response and virus recovered. Yale J Biol Med. Kliks SC, et al. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Klimstra WB, et al. Targeting Sindbis virus-based vectors to Fc receptor-positive cell types.

Iankov ID, et al. Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response. Huang KJ, et al. The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection.

Wallace MJ, et al. Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice. Takada A, et al. Antibody-dependent enhancement of Ebola virus infection.

Enhancement of coxsackievirus B3 infection by antibody to a different coxsackievirus strain. Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry. Measurement of antibody-dependent infection enhancement of four dengue virus serotypes by monoclonal and polyclonal antibodies. Profiles of antibody-dependent enhancement of dengue virus type 2 infection.

Microb Pathog. Gimenez HB, et al. Neutralizing and enhancing activities of human respiratory syncytial virus-specific antibodies. Clin Diagn Lab Immunol. Invitro enhancement of respiratory syncytial virus infection of U cells by human sera.

Antibody-dependent enhancement of respiratory syncytial virus infection by sera from young infants. Mady BJ, et al. Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. Gould EA, Buckley A. Antibody-dependent enhancement of yellow fever and Japanese encephalitis virus neurovirulence. Antibody-mediated early death in vivo after infection with yellow fever virus.

Antibody-mediated infection of macrophages and macrophage-like cell lines with 17D-yellow fever virus. Antibody-dependent enhancement of human immunodeficiency virus type 1 infection. Dengue viruses and mononuclear phagocytes. Infection enhancement by non-neutralizing antibody. Goncalvez AP, et al. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention.

In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody.