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The beta-chemokine receptors CCR5 and CCR2b are the cofactors for entry of M-tropic viruses into cells. Previous studies have reported that M-tropic trains are more sensitive to substitutions in the regions of CCR5 than CCR2b. To understand the molecular basis of CCR5 usage in viral tropism, chimeric molecules of both CCR5 and CCR2b were constructed and regions involved in cofactor utilization by HIV-1 strains were mapped. The chimeras showed that the amino terminus and the first extracellular loop of CCR5 contribute to enhancing the entry of HIV-1 cofactors into the cell.

HIV-1 isolate 89.6 uses Fusin and CKR-5 as entry cofactors indicating that the evolution from a M-tropic to T-tropic phenotype probably occurs due to changes in HIV-1 env that allow the use of dual cofactors. The beta-chemokine receptors CKR-3 and CHR-2b allow the formation of syncytia mediated by HIV-1 89.6 env proteins but fail to allow fusion by T-tropic and M-tropic strains. Expression of CKR-5 with CD4 allows nonpermissive cells to form syncytia with cells expressing M-tropic proteins. However, CKR-5 fails to allow entry of cells expressing T-tropic and HIV-1 env proteins.

Summary Drug combinations are synergistic when their combined efficacy exceeds the sum of the individual actions, but they rarely include ineffective drugs that become effective only in combination. We identified several “enabling pairs” of neutralizing and non-neutralizing anti-ebolavirus monoclonal antibodies, whose combination exhibited new functional profiles, including transforming a non-neutralizing antibody to a neutralizer. Sub-neutralizing concentrations of antibodies 2G4 or m8C4 enabled non-neutralizing antibody FVM09 (IC 50 >1 μM) to exhibit potent neutralization (IC 50 1–10 nM). While FVM09 or m8C4 alone failed to protect Ebola-virus-infected mice, a combination of the two antibodies provided 100% protection. Furthermore, non-neutralizers FVM09 and FVM02 exponentially enhanced the potency of two neutralizing antibodies against both Ebola and Sudan viruses. We identified a hotspot for the binding of these enabling antibody pairs near the interface of the glycan cap and GP2. Enabling cooperativity may be an underappreciated phenomenon for viruses, with implications for the design and development of immunotherapeutics and vaccines. [ABSTRACT FROM AUTHOR]

Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics. [Display omitted] • The estimated size of an EBOV-GP-specific repertoire is about 20,000 clonal families • The clonally expanded repertoire in response to EBOV is functionally diverse • EBOV mAbs have germline-encoded cross-reactivity and neutralization characteristics • A high prevalence of public clonotypes with neutralizing properties is identified Chen et al. investigate the complexity of the human antibody response to a single viral protein, the EBOV GP, using paired single-cell sequencing. The study shows an immense diversity in the clonally expanded B cell repertoire and a high prevalence of public clonotypes. [ABSTRACT FROM AUTHOR]

Summary We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value. [ABSTRACT FROM AUTHOR]

The E1 and E2 envelope proteins of hepatitis C virus (HCV) form a heterodimer that drives virus-host membrane fusion. Here, we analyze the role of each amino acid in E1E2 function, expressing 545 individual alanine mutants of E1E2 in human cells, incorporating them into infectious viral pseudoparticles, and testing them against 37 different monoclonal antibodies (MAbs) to ascertain full-length translation, folding, heterodimer assembly, CD81 binding, viral pseudoparticle incorporation, and infectivity. We propose a model describing the role of each critical residue in E1E2 functionality and use it to examine how MAbs neutralize infection by exploiting functionally critical sites of vulnerability on E1E2. Our results suggest that E1E2 is a surprisingly fragile protein complex where even a single alanine mutation at 92% of positions disrupts its function. The amino-acid-level targets identified are highly conserved and functionally critical and can be exploited for improved therapies and vaccines. [Display omitted] • Test 545 hepatis C virus (HCV) E1E2 envelope mutants for infectivity, antibody binding • Identify residues important for HCV E1 and E2 folding, E1E2 interaction, infectivity • HCV E1E2 is a fragile protein complex where most mutations compromise function • Functional residues of E1E2 are highly conserved across genotypes Pfaff-Kilgore et al. describe the role of individual amino acids in hepatis C virus E1E2 protein function by generating a comprehensive E1E2 mutation library and testing 545 clones for E1E2 folding, assembly, and infectivity. Their model describes the role of residues in E1E2 functionality and examines how antibodies neutralize infection. [ABSTRACT FROM AUTHOR]

Studies the binding and functional properties of chemokines and chemokine variants onto point mutants of CCR5, a functional receptor for inflammatory chemokines. Residues in CCR5 extracellular domains; Extracellur loops; Domains and residues important to CCR5 binding.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs. [Display omitted] • LIBRA-seq identifies 54042-4, a potently neutralizing SARS-CoV-2 antibody • 54042-4 maintains potent neutralization against Alpha, Beta, Gamma, and Delta VOCs • The epitope of 54042-4 is highly conserved among current SARS-CoV-2 isolates Kramer et al. demonstrate that antibody 54042-4 recognizes residues highly conserved across global SARS-CoV-2 isolates. Antibody 54042-4 potently neutralizes all known circulating variants of concern (VOCs) and could be developed as a clinical candidate to treat COVID-19 infection. [ABSTRACT FROM AUTHOR]

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants. [Display omitted] • Public clonotypes are shared among SARS-CoV-2-vaccinated and -infected individuals • Group 3 antibodies share the same binding site as CR3022 but neutralize variants • Germline-revertant forms of public clonotypes bind efficiently to spike protein • Public clonotype responses inform research on new viral variants of concern Chen et al. describe neutralizing and non-neutralizing public antibodies elicited to SARS-CoV-2 spike in vaccinated and naturally infected individuals. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting common germline-encoded properties for recognition. [ABSTRACT FROM AUTHOR]

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies. [Display omitted] • Glycan cap antibody-mediated GP destabilization correlates with synergy • Cryo-EM structures reveal antibodies target a highly conserved epitope • Antibodies use long CDRH3 loops to displace and mimic portions of the glycan cap • Glycan cap antibodies block cleavage events required for viral entry A rare subset of ebolavirus antibodies targeting the glycan cap are broadly neutralizing. Murin et al. report cryo-EM structures and custom in vitro assays identifying a conserved site of vulnerability in the glycan cap and detail mechanisms of action, including structural mimicry, trimer instability, and blocking cleavage. [ABSTRACT FROM AUTHOR]

Summary Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti- Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings. Graphical Abstract Highlights • DMAbs are an in vivo approach for mAb development/delivery against Zaire ebolavirus • DMAbs can be functionally equivalent to recombinant mAb • Reproducible and cost-effective mouse model for in vivo mAb evaluation • Enables evaluation of fully human mAbs rapidly in vivo Monoclonal antibodies are an important approach for emerging infectious disease prevention. Patel et al. demonstrate engineering and in vivo delivery of DNA-encoded monoclonal antibodies (DMAbs) targeting the Zaire ebolavirus (EBOV) glycoprotein. DMAbs protect against lethal mouse-adapted EBOV and are useful for rapid evaluation of fully human mAbs in live animal models. [ABSTRACT FROM AUTHOR]

The lack of structural information on hepatitis C virus (HCV) surface proteins has so far hampered the development of effective vaccines. Recently, two crystallographic structures have described the core portion (E2c) of E2 surface glycoprotein, the primary mediator of HCV entry. Despite the importance of these studies, the E2 overall structure is still unknown and, most importantly, several biochemical and functional studies are in disagreement with E2c structures. Here, the main literature will be discussed and an alternative disulfide bridge pattern will be proposed, based on unpublished human monoclonal antibody reactivity. A modeling strategy aiming at recapitulating the available structural and functional studies of E2 will also be proposed. [ABSTRACT FROM AUTHOR]