Cellugyrin (synaptogyrin-2) dependent pathways are used by bacterial cytolethal distending toxin and SARS-CoV-2 virus to gain cell entry.

Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the region of membrane lipid rafts, the CdtB and CdtC subunits are internalized and traffic to intracellular compartments. These events are dependent upon, cellugyrin, a critical component of synaptic like microvesicles (SLMVCg+). Target cells, such as Jurkat cells, rendered unable to express cellugyrin are resistant to Cdt-induced toxicity. Similar to Cdt, SARS-CoV-2 entry into host cells is initiated by binding to cell surface receptors, ACE-2, also associated with cholesterol-rich lipid rafts; this association leads to fusion and/or endocytosis of viral and host cell membranes and intracellular trafficking. The similarity in internalization pathways for both Cdt and SARS-CoV-2 led us to consider the possibility that cellugyrin was a critical component in both processes. Cellugyrin deficient Calu-3 cells (Calu-3Cg-) were prepared using Lentiviral particles containing shRNA; these cells were resistant to infection by VSV/SARS-CoV-2-spike pseudotype virus and partially resistant to VSV/VSV-G pseudotype virus. Synthetic peptides representing various regions of the cellugyrin protein were prepared and assessed for their ability to bind to Cdt subunits using surface plasmon resonance. Cdt was capable of binding to a region designated the middle outer loop (MOL) which corresponds to a region extending into the cytoplasmic surface of the SLMVCg+. SARS-CoV-2 spike proteins were assessed for their ability to bind to cellugyrin peptides; SARS-CoV-2 full length spike protein preferentially binds to a region within the SLMVCg+ lumen, designated intraluminal loop 1A. SARS-CoV-2-spike protein domain S1, which contains the receptor binding domains, binds to cellugyrin N-terminus which extends out from the cytoplasmic surface of SLMV. Binding specificity was further analyzed using cellugyrin scrambled peptide mutants. We propose that SLMVCg+ represent a component of a common pathway that facilitates pathogen and/or pathogen-derived toxins to gain host cell entry.

A Trivalent HSV-2 gC2, gD2, gE2 Nucleoside-Modified mRNA-LNP Vaccine Provides Outstanding Protection in Mice against Genital and Non-Genital HSV-1 Infection, Comparable to the Same Antigens Derived from HSV-1.

HSV-1 disease is a significant public health burden causing orofacial, genital, cornea, and brain infection. We previously reported that a trivalent HSV-2 gC2, gD2, gE2 nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccine provides excellent protection against vaginal HSV-1 infection in mice. Here, we evaluated whether this HSV-2 gC2, gD2, gE2 vaccine is as effective as a similar HSV-1 mRNA LNP vaccine containing gC1, gD1, and gE1 in the murine lip and genital infection models. Mice were immunized twice with a total mRNA dose of 1 or 10 µg. The two vaccines produced comparable HSV-1 neutralizing antibody titers, and surprisingly, the HSV-2 vaccine stimulated more potent CD8+ T-cell responses to gE1 peptides than the HSV-1 vaccine. Both vaccines provided complete protection from clinical disease in the lip model, while in the genital model, both vaccines prevented death and genital disease, but the HSV-1 vaccine reduced day two vaginal titers slightly better at the 1 µg dose. Both vaccines prevented HSV-1 DNA from reaching the trigeminal or dorsal root ganglia to a similar extent. We conclude that the trivalent HSV-2 mRNA vaccine provides outstanding protection against HSV-1 challenge at two sites and may serve as a universal prophylactic vaccine for HSV-1 and HSV-2.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

Receptor Binding-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein D Permit Interaction with the gH/gL Complex to Activate Fusion.

Herpes simplex virus (HSV) requires four essential virion glycoproteins-gD, gH, gL, and gB-for virus entry and cell fusion. To initiate fusion, the receptor binding protein gD interacts with one of two major cell receptors, HVEM or nectin-1. Once gD binds to a receptor, fusion is carried out by the gH/gL heterodimer and gB. A comparison of free and receptor-bound gD crystal structures revealed that receptor binding domains are located within residues in the N-terminus and core of gD. Problematically, the C-terminus lies across and occludes these binding sites. Consequentially, the C-terminus must relocate to allow for both receptor binding and the subsequent gD interaction with the regulatory complex gH/gL. We previously constructed a disulfide bonded (K190C/A277C) protein that locked the C-terminus to the gD core. Importantly, this mutant protein bound receptor but failed to trigger fusion, effectively separating receptor binding and gH/gL interaction. Here, we show that "unlocking" gD by reducing the disulfide bond restored not only gH/gL interaction but fusion activity as well, confirming the importance of C-terminal movement in triggering the fusion cascade. We characterize these changes, showing that the C-terminus region exposed by unlocking is: (1) a gH/gL binding site; (2) contains epitopes for a group (competition community) of monoclonal antibodies (Mabs) that block gH/gL binding to gD and cell-cell fusion. Here, we generated 14 mutations within the gD C-terminus to identify residues important for the interaction with gH/gL and the key conformational changes involved in fusion. As one example, we found that gD L268N was antigenically correct in that it bound most Mabs but was impaired in fusion, exhibited compromised binding of MC14 (a Mab that blocks both gD-gH/gL interaction and fusion), and failed to bind truncated gH/gL, all events that are associated with the inhibition of C-terminus movement. We conclude that, within the C-terminus, residue 268 is essential for gH/gL binding and induction of conformational changes and serves as a flexible inflection point in the critical movement of the gD C-terminus.

Rapid and accurate detection of herpes simplex virus type 2 using a low-cost electrochemical biosensor.

Herpes simplex virus type 2 (HSV-2) infection, which is almost exclusively sexually transmitted, causes genital herpes. Although this lifelong and incurable infection is extremely widespread, currently there is no readily available diagnostic device that accurately detects HSV-2 antigens to a satisfactory degree. Here, we report an ultrasensitive electrochemical device that detects HSV-2 antigens within 9 min and costs just $1 (USD) to manufacture. The electrochemical biosensor is biofunctionalized with the human cellular receptor nectin-1 and detects the glycoprotein gD2, which is present within the HSV-2 viral envelope. The performance of the device is tested in a guinea pig model that mimics human biofluids, yielding 88.9% sensitivity, 100.0% specificity, and 95.0% accuracy under these conditions, with a limit of detection of 0.019 fg mL-1 for gD2 protein and 0.057 PFU mL-1 for titered viral samples. Importantly, no cross-reactions with other viruses were detected, indicating the adequate robustness and selectivity of the sensor. Our low-cost technology could facilitate more frequent testing for HSV-2.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 µg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 µg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 µg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 µg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 µg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-µg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 µg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

Trivalent nucleoside-modified mRNA vaccine yields durable memory B cell protection against genital herpes in preclinical models.

Nucleoside-modified mRNA vaccines have gained global attention because of COVID-19. We evaluated a similar vaccine approach for preventing a chronic, latent genital infection rather than an acute respiratory infection. We used animal models to compare an HSV-2 trivalent nucleoside-modified mRNA vaccine with the same antigens prepared as proteins, with an emphasis on antigen-specific memory B cell responses and immune correlates of protection. In guinea pigs, serum neutralizing-antibody titers were higher at 1 month and declined far less by 8 months in mRNA- compared with protein-immunized animals. Both vaccines protected against death and genital lesions when infected 1 month after immunization; however, protection was more durable in the mRNA group compared with the protein group when infected after 8 months, an interval representing greater than 15% of the animal's lifespan. Serum and vaginal neutralizing-antibody titers correlated with protection against infection, as measured by genital lesions and vaginal virus titers 2 days after infection. In mice, the mRNA vaccine generated more antigen-specific memory B cells than the protein vaccine at early times after immunization that persisted for up to 1 year. High neutralizing titers and robust B cell immune memory likely explain the more durable protection by the HSV-2 mRNA vaccine.

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

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.

Protection against herpes simplex virus type 2 infection in a neonatal murine model using a trivalent nucleoside-modified mRNA in lipid nanoparticle vaccine.

Neonatal herpes is a dreaded complication of genital herpes infection in pregnancy. We recently compared two vaccine platforms for preventing genital herpes in female mice and guinea pigs and determined that HSV-2 glycoproteins C, D and E expressed using nucleoside-modified mRNA in lipid nanoparticles provided better protection than the same antigens produced as baculovirus proteins and administered with CpG and alum. Here we evaluated mRNA and protein immunization for protection against neonatal herpes. Female mice were immunized prior to mating and newborns were infected intranasally with HSV-2. IgG binding and neutralizing antibody levels in mothers and newborns were comparable using the mRNA or protein vaccines. Both vaccines protected first and second litter newborns against disseminated infection based on virus titers in multiple organs. We conclude that both vaccines are efficacious at preventing neonatal herpes, which leaves the mRNA vaccine as our preferred candidate based on better protection against genital herpes.

Characterizing Epitope Binding Regions of Entire Antibody Panels by Combining Experimental and Computational Analysis of Antibody: Antigen Binding Competition.

Vaccines and immunotherapies depend on the ability of antibodies to sensitively and specifically recognize particular antigens and specific epitopes on those antigens. As such, detailed characterization of antibody-antigen binding provides important information to guide development. Due to the time and expense required, high-resolution structural characterization techniques are typically used sparingly and late in a development process. Here, we show that antibody-antigen binding can be characterized early in a process for whole panels of antibodies by combining experimental and computational analyses of competition between monoclonal antibodies for binding to an antigen. Experimental "epitope binning" of monoclonal antibodies uses high-throughput surface plasmon resonance to reveal which antibodies compete, while a new complementary computational analysis that we call "dock binning" evaluates antibody-antigen docking models to identify why and where they might compete, in terms of possible binding sites on the antigen. Experimental and computational characterization of the identified antigenic hotspots then enables the refinement of the competitors and their associated epitope binding regions on the antigen. While not performed at atomic resolution, this approach allows for the group-level identification of functionally related monoclonal antibodies (i.e., communities) and identification of their general binding regions on the antigen. By leveraging extensive epitope characterization data that can be readily generated both experimentally and computationally, researchers can gain broad insights into the basis for antibody-antigen recognition in wide-ranging vaccine and immunotherapy discovery and development programs.

Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

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 antibodies 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.

An HSV-2 nucleoside-modified mRNA genital herpes vaccine containing glycoproteins gC, gD, and gE protects mice against HSV-1 genital lesions and latent infection.

HSV-1 causes 50% of first-time genital herpes infections in resource-rich countries and affects 190 million people worldwide. A prophylactic herpes vaccine is needed to protect against genital infections by both HSV-1 and HSV-2. Previously our laboratory developed a trivalent vaccine that targets glycoproteins C, D, and E present on the HSV-2 virion. We reported that this vaccine protects animals from genital disease and recurrent virus shedding following lethal HSV-2 challenge. Importantly the vaccine also generates cross-reactive antibodies that neutralize HSV-1, suggesting it may provide protection against HSV-1 infection. Here we compared the efficacy of this vaccine delivered as protein or nucleoside-modified mRNA immunogens against vaginal HSV-1 infection in mice. Both the protein and mRNA vaccines protected mice from HSV-1 disease; however, the mRNA vaccine provided better protection as measured by lower vaginal virus titers post-infection. In a second experiment, we compared protection provided by the mRNA vaccine against intravaginal challenge with HSV-1 or HSV-2. Vaccinated mice were totally protected against death, genital disease and infection of dorsal root ganglia caused by both viruses, but somewhat better protected against vaginal titers after HSV-2 infection. Overall, in the two experiments, the mRNA vaccine prevented death and genital disease in 54/54 (100%) mice infected with HSV-1 and 20/20 (100%) with HSV-2, and prevented HSV DNA from reaching the dorsal root ganglia, the site of virus latency, in 29/30 (97%) mice infected with HSV-1 and 10/10 (100%) with HSV-2. We consider the HSV-2 trivalent mRNA vaccine to be a promising candidate for clinical trials for prevention of both HSV-1 and HSV-2 genital herpes.

Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR/EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.

Effective oncolytic virotherapy may require systemic delivery, tumor targeting, and resistance to virus-neutralizing (VN) antibodies. Since herpes simplex virus (HSV) glycoprotein D (gD) is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together, revealed that both substitutions (1) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (2) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (3) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.

Dynamic organization of Herpesvirus glycoproteins on the viral envelope revealed by super-resolution microscopy.

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.

Saliva enhances infection of gingival fibroblasts by herpes simplex virus 1.

Oral herpes is a highly prevalent infection caused by herpes simplex virus 1 (HSV-1). After an initial infection of the oral cavity, HSV-1 remains latent in sensory neurons of the trigeminal ganglia. Episodic reactivation of the virus leads to the formation of mucocutaneous lesions (cold sores), but asymptomatic reactivation accompanied by viral shedding is more frequent and allows virus spread to new hosts. HSV-1 DNA has been detected in many oral tissues. In particular, HSV-1 can be found in periodontal lesions and several studies associated its presence with more severe periodontitis pathologies. Since gingival fibroblasts may become exposed to salivary components in periodontitis lesions, we analyzed the effect of saliva on HSV-1 and -2 infection of these cells. We observed that human gingival fibroblasts can be infected by HSV-1. However, pre-treatment of these cells with saliva extracts from some but not all individuals led to an increased susceptibility to infection. Furthermore, the active saliva could expand HSV-1 tropism to cells that are normally resistant to infection due to the absence of HSV entry receptors. The active factor in saliva was partially purified and comprised high molecular weight complexes of glycoproteins that included secretory Immunoglobulin A. Interestingly, we observed a broad variation in the activity of saliva between donors suggesting that this activity is selectively present in the population. The active saliva factor, has not been isolated, but may lead to the identification of a relevant biomarker for susceptibility to oral herpes. The presence of a salivary factor that enhances HSV-1 infection may influence the risk of oral herpes and/or the severity of associated oral pathologies.

Nucleoside-modified mRNA encoding HSV-2 glycoproteins C, D, and E prevents clinical and subclinical genital herpes.

The goals of a genital herpes vaccine are to prevent painful genital lesions and reduce or eliminate subclinical infection that risks transmission to partners and newborns. We evaluated a trivalent glycoprotein vaccine containing herpes simplex virus type 2 (HSV-2) entry molecule glycoprotein D (gD2) and two immune evasion molecules: glycoprotein C (gC2), which binds complement C3b, and glycoprotein E (gE2), which blocks immunoglobulin G (IgG) Fc activities. The trivalent vaccine was administered as baculovirus proteins with CpG and alum, or the identical amino acids were expressed using nucleoside-modified mRNA in lipid nanoparticles (LNPs). Both formulations completely prevented genital lesions in mice and guinea pigs. Differences emerged when evaluating subclinical infection. The trivalent protein vaccine prevented dorsal root ganglia infection, and day 2 and 4 vaginal cultures were negative in 23 of 30 (73%) mice compared with 63 of 64 (98%) in the mRNA group (P = 0.0012). In guinea pigs, 5 of 10 (50%) animals in the trivalent subunit protein group had vaginal shedding of HSV-2 DNA on 19 of 210 (9%) days compared with 2 of 10 (20%) animals in the mRNA group that shed HSV-2 DNA on 5 of 210 (2%) days (P = 0.0052). The trivalent mRNA vaccine was superior to trivalent proteins in stimulating ELISA IgG antibodies, neutralizing antibodies, antibodies that bind to crucial gD2 epitopes involved in entry and cell-to-cell spread, CD4+ T cell responses, and T follicular helper and germinal center B cell responses. The trivalent nucleoside-modified mRNA-LNP vaccine is a promising candidate for human trials.

Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion.

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants.

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.

Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD.

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.

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

HSV 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.IMPORTANCE Virus 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.