scholarly journals Using Split Luciferase Assay and anti-HSV Glycoprotein Monoclonal Antibodies to Predict a Functional Binding Site Between gD and gH/gL

2021 ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Tina M Cairns ◽  
Roselyn J Eisenberg ◽  
Gary H Cohen
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Cell Fusion ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Active Form ◽  
The Other ◽  
Luciferase Assay ◽  
Functional Sites ◽  
Cell Cell

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. HSV entry begins with gD binding its receptor (nectin-1), which then activates gH/gL to enable the conversion of pre-fusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps and localization of their epitopes identifies functional sites on each protein. Utilizing this approach, we have identified the gH/gL binding face on gD and the corresponding gD binding site on gH/gL. Here, we used combinations of these Mabs to define the orientation of gD and gH/gL relative to each other. We reasoned that if two Mabs, one directed at gD and the other at gH/gL, block fusion more effectively than when either were used alone (additive), then their epitopes would be spatially distanced and binding of one would not directly interfere with binding of the other during fusion. However, if the two Mabs blocked fusion with equal or lesser efficacy that when either were used alone (indifferent), we propose that their epitopes would be in close proximity in the complex. Using a live cell fusion assay, we found that some Mab pairings blocked the fusion with different mechanisms while other had a similar mechanisms of action. Grouping the different combinations of antibodies into indifferent and additive groups, we present a model for the orientation of gD vis-à-vis gH/gL in the complex. Importance: Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB and a gD receptor. Virus-neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with essential steps in the fusion pathway. Thus, the epitopes of these Mabs overlap and point to critical, functional sites on their target proteins. Here, we combined gD and gH/gL antibodies to determine whether they work in an additive or non-additive (indifferent) fashion to block specific events in glycoprotein-driven cell-cell fusion. Identifying combinations of antibodies that have additive effects will help in the rational design of an effective therapeutic “polyclonal antibody” to treat HSV disease. In addition, identification of the exact contact regions between gD and gH/gL can inform the design of small molecules that would interfere with the gD-gH/gL complex formation, thus preventing the virus from entering the host cell.

scholarly journals Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Eric Lazear ◽  
J. Charles Whitbeck ◽  
Tina M. Cairns ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Protein Interactions ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Epitope Mapping ◽  
Virus Entry ◽  
Cellular Receptor ◽  
Functional Sites ◽  
Cell Cell

ABSTRACTHSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two “faces” on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.IMPORTANCEVirus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

scholarly journals The Impact on Infectivity and Neutralization Efficiency of SARS-CoV-2 Lineage B.1.351 Pseudovirus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 633
Author(s):  
Yeong Jun Kim ◽  
Ui Soon Jang ◽  
Sandrine M. Soh ◽  
Joo-Youn Lee ◽  
Hye-Ra Lee
Keyword(s):  
South Africa ◽  
Monoclonal Antibodies ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Viral Spread ◽  
New Variant ◽  
Global Concern ◽  
The Impact

A new variant of SARS-CoV-2 B.1.351 lineage (first found in South Africa) has been raising global concern due to its harboring of multiple mutations in the spike that potentially increase transmissibility and yield resistance to neutralizing antibodies. We here tested infectivity and neutralization efficiency of SARS-CoV-2 spike pseudoviruses bearing particular mutations of the receptor-binding domain (RBD) derived either from the Wuhan strains (referred to as D614G or with other sites) or the B.1.351 lineage (referred to as N501Y, K417N, and E484K). The three different pseudoviruses B.1.351 lineage related significantly increased infectivity compared with other mutants that indicated Wuhan strains. Interestingly, K417N and E484K mutations dramatically enhanced cell–cell fusion than N501Y even though their infectivity were similar, suggesting that K417N and E484K mutations harboring SARS-CoV-2 variant might be more transmissible than N501Y mutation containing SARS-CoV-2 variant. We also investigated the efficacy of two different monoclonal antibodies, Casirivimab and Imdevimab that neutralized SARS-CoV-2, against several kinds of pseudoviruses which indicated Wuhan or B.1.351 lineage. Remarkably, Imdevimab effectively neutralized B.1.351 lineage pseudoviruses containing N501Y, K417N, and E484K mutations, while Casirivimab partially affected them. Overall, our results underscore the importance of B.1.351 lineage SARS-CoV-2 in the viral spread and its implication for antibody efficacy.

Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants

2021 ◽  
Author(s):  
Shanan N. Emmanuel ◽  
J. Kennon Smith ◽  
Jane Hsi ◽  
Yu-Shan Tseng ◽  
Matias Kaplan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Gene Delivery ◽  
General Population ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Transduction Efficiency ◽  
Viral Neutralization ◽  
Patient Cohort ◽  
Antigenic Epitopes

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.

scholarly journals Immunogenetic and structural analysis of a class of HCV broadly neutralizing antibodies and their precursors

2018 ◽  
Vol 115 (29) ◽  
pp. 7569-7574 ◽  
Author(s):  
Fernando Aleman ◽  
Netanel Tzarum ◽  
Leopold Kong ◽  
Kenna Nagy ◽  
Jiang Zhu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Analysis ◽  
Neutralizing Antibodies ◽  
Antigenic Site ◽  
The Other ◽  
Maturation Process ◽  
Broadly Neutralizing Antibodies ◽  
Vaccine Candidates ◽  
Rational Vaccine Design ◽  
Hairpin Conformation

Elicitation of broadly neutralizing antibodies (bnAbs) is a leading strategy in rational vaccine design against antigenically diverse pathogens. Here, we studied a panel of monoclonal antibodies (mAbs) from mice immunized with the hepatitis C virus (HCV) envelope glycoproteins E1E2. Six of the mAbs recognize the conserved E2 antigenic site 412–423 (AS412) and cross-neutralize diverse HCV genotypes. Immunogenetic and structural analysis revealed that the antibodies originated from two different germline (GL) precursors and bind AS412 in a β-hairpin conformation. Intriguingly, the anti-HCV activity of one antibody lineage is associated with maturation of the light chain (LC), whereas the other lineage is dependent on heavy-chain (HC) maturation. Crystal structures of GL precursors of the LC-dependent lineage in complex with AS412 offer critical insights into the maturation process of bnAbs to HCV, providing a scientific foundation for utilizing the mouse model to study AS412-targeting vaccine candidates.

Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

2001 ◽  
Vol 85 (05) ◽  
pp. 866-874 ◽  
Author(s):  
A. P. Bijnens ◽  
T. H. Ngo ◽  
A. Gils ◽  
J. Dewaele ◽  
I. Knockaert ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Biochemical Characterization ◽  
Tissue Type ◽  
Binding Region ◽  
Plasminogen Activator Inhibitor 1 ◽  
Inhibitory Antibodies ◽  
Pai 1

SummaryIncreased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore, eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising -helices hD, hE and hF, -strands s4C, s3A, s2A and s1A and the loops connecting these elements), (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data, together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.

scholarly journals BROADLY NEUTRALIZING ANTI- HIV-1 ANTIBODIES DO NOT INHIBIT HIV-1 ENV-MEDIATED CELL-CELL FUSION

2021 ◽  
Author(s):  
Nejat Düzgüneş ◽  
Michael Yee ◽  
Deborah Chau
Keyword(s):  
Cell Fusion ◽  
Neutralizing Antibodies ◽  
Culture Media ◽  
Genetic Material ◽  
Inhibitory Effect ◽  
Hippeastrum Hybrid Agglutinin ◽  
Calcein Am ◽  
Antibodies Monoclonal ◽  
Hiv 1 ◽  
Cell Cell

PG9, PG16, PGT121, and PGT145 antibodies were identified from culture media of activated memory B-cells of an infected donor and shown to neutralize many HIV-1 strains. Since HIV-1 spreads via both free virions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 Env-mediated cell-cell fusion. Clone69TRevEnv cells that express Env in the absence of tetracycline were labeled with Calcein-AM Green, and incubated with CD4+ SupT1 cells labeled with CellTrace Calcein Red-Orange, with or without antibodies. Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at up to 50 μg/mL) had little or no inhibitory effect on fusion between HIV-Env and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin completely inhibited fusion. Our results indicate that transmission of the virus or viral genetic material would not be inhibited by these broadly neutralizing antibodies. Thus, antibodies generated by HIV-1 vaccines should be screened for their inhibitory effect on Env-mediated cell-cell fusion.

scholarly journals Using a split luciferase assay (SLA) to measure the kinetics of cell–cell fusion mediated by herpes simplex virus glycoproteins

Methods ◽  
2015 ◽  
Vol 90 ◽  
pp. 68-75 ◽  
Author(s):  
Wan Ting Saw ◽  
Zene Matsuda ◽  
Roselyn J. Eisenberg ◽  
Gary H. Cohen ◽  
Doina Atanasiu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Luciferase Assay ◽  
Simplex Virus ◽  
Kinetics Of ◽  
Split Luciferase ◽  
Cell Cell

scholarly journals Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Rachel B. Brouillette ◽  
Elisabeth K. Phillips ◽  
Natarajan Ayithan ◽  
Wendy Maury
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
New World ◽  
Cross Reactivity ◽  
Receptor Binding Site ◽  
Glycoprotein Complex ◽  
Machupo Virus ◽  
Cross Neutralization

ABSTRACT The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies.

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