Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in an aggressive timeline. In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the SARS-CoV-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity. The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV. IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibod-ies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

Herpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support of this, we have shown that gD is structurally divided into two "faces": one for the binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when (i) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip, (ii) gD is bound to either one of its receptors on a chip, and (iii) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding, and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.IMPORTANCE HSV entry is a multistep process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, until now we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen.

Broadly neutralizing antibodies (bNAbs) are rarely elicited by current human immunodeficiency virus type 1 (HIV-1) vaccine designs, but the presence of bNAbs in naturally infected individuals may be associated with high plasma viral loads, suggesting that the magnitude, duration, and diversity of viral exposure may contribute to the development of bNAbs. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) from two subjects who developed broadly neutralizing autologous antibody responses during HIV-1 infection. In both subjects, we identified collections of mAbs that exhibited specificity only to a few autologous envelopes (Envs), with some mAbs exhibiting specificity only to a subset of Envs within the quasispecies of a particular sample at one time point. Neutralizing antibodies (NAbs) isolated from these subjects mapped mostly to epi-topes in the Env V3 loop region and the CD4 binding site. None of the individual neutralizing mAbs recovered exhibited the cumulative breadth of neutralization present in the serum of the subjects. Surprisingly, however, the activity of polyclonal mixtures comprising individual mAbs that each possessed limited neutralizing activity, could achieve increased breadth of neutralizing activity against autologous isolates. While a single broadly neutralizing antibody targeting one epitope can mediate neutralization breadth, the findings presented here suggest that a cooperative polyclonal process mediated by diverse antibodies with more limited breadth targeting multiple epitopes also can achieve neutralization breadth against HIV-1.

While HSV-2 typically causes genital lesions, HSV-1 is increasingly the cause of genital herpes. In addition, neonatal HSV infections are associated with a high rate of mortality and HSV-2 may increase the risk for HIV or Zika infections, reinforcing the need to develop an effective vaccine. In the GSK Herpevac trial, doubly sero-negative women were vaccinated with a truncated form of gD2 [gD2(284t)], then examined for anti-gD serum titers and clinical manifestations of disease. Surprisingly, few vaccinees were protected against genital HSV-2 but 86% were protected from genital HSV-1. These observations suggest that subtle differences in gD structure might influence a protective response. To better understand the antigenic structure of gD and how it impacts a protective response, we previously utilized several key anti-gD monoclonal antibodies (mAbs) to dissect epitopes in vaccinee sera. Several correlations were observed but the methodology limited the number of sera and mAbs that could be tested. Here, we used array-based surface plasmon imaging (SPRi) to simultaneously measure a larger number of protein-protein interactions. We carried out cross-competition or "epitope binning" studies with 39 anti-gD mAbs and four soluble forms of gD, including a form [gD2(285t)] that resembles the Herpevac antigen. The results from these experiments allowed us to organize the mAbs into four epitope communities. Notably, relationships within and between communities differed depending on the form of gD, and off-rate analysis suggested differences in mAb-gD avidity depending on the gD serotype and length. Together, these results show that gD1 and gD2 differ in their structural topography. Consistent with the Herpevac results, several mAbs that bind both gD1 and gD2 neutralize only HSV-1. Thus, this technology provides new insights into the antigenic structure of gD and provides a rationale as to how vaccination with a gD2 subunit may lead to protection from HSV-1 infection.

The use of modern, commercial biosensors is dependent on software tools for experimental design, execution, and analysis of data. In order to appreciate the experimental outputs from surface plasmon resonance (SPR) studies, a brief discussion on the implications of assay setup is first covered. Next, a review of some common software tools for analysis is presented, followed by a description of general practices used in the processing of SPR data. Lastly, a step-by-step illustration of data processing routines for epitope binning and kinetic experiments is provided. Collectively, this demonstrates how software tools are applied to developing and understanding robust and carefully controlled SPR experiments.

Introduction: Surface plasmon resonance (SPR) for affinity/kinetics measurements of drug candidates has been a mainstay application for characterizing drug candidates for many years. Recently, with the growth of monoclonal antibodies (mAbs) as a drug class and the availability of higher-throughput biosensors, the role of label-free biosensors has evolved to include epitope characterization in the early drug discovery process through epitope binning and mapping of mAbs.Areas covered: This manuscript outlines the importance of using epitope characterization early in the drug discovery process and describes a strategy for success in discovering drug leads. Updated practices for integrating epitope characterization with other biochemical/biophysical data, cell-based functional data, and computational prediction tools are also discussed.Expert opinion: The authors propose using epitope characterization during early drug discovery by: (1) using epitope binning of mAbs following the pre-screening of clones to assure selection of mAb candidates with epitope diversity, (2) binning the maximum number of mAbs in order to fully define epitope engagement profiles, and (3) integrating epitope binning/mapping data with binding affinity, kinetics, cell-based functional assays, etc. to better describe functional epitope. This approach, together with structural and binding prediction data, will improve the quality of leads and improve the selection speed for clinical candidates.

An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it.

The use of Surface Plasmon Resonance (SPR)-based optical biosensors contributes extensively to discovery and development of therapeutic monoclonal antibodies, owing to its ability to real-time analyze interactions of an antigen with an antibody without intrinsic or extrinsic labels. SPR has been a mainstay in pharmaceutical companies for almost two decades, and its role in drug discovery has experienced significant growth with the expanded number of therapeutic antibodies. Additionally, the burgeoning field of biosimilars depends on SPR to ascertain comparability to innovator mAbs. While the promise of the technology is exciting, the full role of SPR has yet to be realized. SPR has historically been hampered by limited throughput; however, new instruments and methods have emerged that allow for the analysis of up to thousands of biomolecular interactions per day. Here, we detail the use of traditional and emerging SPR techniques for characterizing monoclonal antibodies such as antigen/antibody kinetics, epitope profiling, and immunogenicity screening. In conjunction with efforts to improve throughput and sensitivity, SPR is expected to continue in its growth as a central technique in pharmaceutical discovery and development.

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)-driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23-dependent IL-22 production from Th17 cells and cooperatively blocked IL-17-dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.