Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.

The transmembrane and cytosolic domains of equine herpesvirus type 1 glycoprotein D determine Golgi retention by regulating vesicle formation.

Accumulating evidence underscores the pivotal role of envelope proteins in viral secondary envelopment. However, the intricate molecular mechanisms governing this phenomenon remain elusive. To shed light on these mechanisms, we investigated a Golgi-retained gD of EHV-1 (gDEHV-1), distinguishing it from its counterparts in Herpes Simplex Virus-1 (HSV-1) and Pseudorabies Virus (PRV). To unravel the specific sequences responsible for the Golgi retention phenotype, we employed a gene truncation and replacement strategy. The results suggested that Golgi retention signals in gDEHV-1 exhibiting a multi-domain character. The extracellular domain of gDEHV-1 was identified as an endoplasmic reticulum (ER)-resident domain, the transmembrane domain and cytoplasmic tail (TM-CT) of gDEHV-1 were integral in facilitating the protein's residence within the Golgi complex. Deletion or replacement of either of these dual domains consistently resulted in the mutant gDEHV-1 being retained in an ER-like structure. Moreover, (TM-CT)EHV-1 demonstrated a preference for binding to endomembranes, inducing the generation of a substantial number of vesicles, potentially originate from the Golgi complex or the ER-Golgi intermediate compartment. In conclusion, our findings provide insights into the intricate molecular mechanisms governing the Golgi retention of gDEHV-1, facilitating the comprehension of the processes underlying viral secondary envelopment.

Development and evaluation of recombinant gD protein based ELISA for sero-surveillance of BoHV-1 in India.

Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.

Greater Durability and Protection against Herpes Simplex Viral Disease following Immunization of Mice with Single-Cycle ΔgD-2 Compared to an Adjuvanted Glycoprotein D Protein Vaccine.

Herpes simplex viruses (HSV) cause chronic infections with significant morbidity. Prior vaccines, designed to generate neutralizing antibodies (nAbs) targeting glycoprotein D (gD), failed to provide durable protection. We adopted a different strategy and evaluated a single-cycle virus deleted in gD (ΔgD-2). ΔgD-2elicits antibodies that primarily mediate antibody-dependent cell mediated cytolysis (ADCC) and provides complete protection against clinical isolates of HSV in multiple lethal mouse models. To assess durability, we vaccinated mice (2 doses administered intramuscularly) with ΔgD-2, adjuvanted recombinant gD-2 (rgD-2/Alum-MPL), or uninfected cells as a control, and quantified antibody responses over one year. Mice (n = 5/group) were lethally challenged at 2, 4, 6, 8, and 10-months post-boost. ΔgD-2-vaccinated mice elicited a durable ADCC-mediating response, which provided complete protection against challenge at all timepoints. In contrast, rgD-2/Alum-MPL elicited only nAbs, which declined significantly within 6 months, provided only partial protection at early timepoints, and no protection after 6 months. Serum sampling after viral challenge showed that infection elicited low levels of ADCC-mediating antibodies in rgD-2/Alum-MPL-vaccinated mice and boosted the nAb response, but only after 6 months. Conversely, infection significantly and consistently boosted both the ADCC and nAbs responses in ΔgD-2-vaccinated mice. Results recapitulate clinical trial outcomes with gD vaccines, highlight the importance of ADCC, and predict that ΔgD-2 will elicit durable responses in humans.

A self-assembled nanoparticle vaccine based on pseudorabies virus glycoprotein D induces potent protective immunity against pseudorabies virus infection.

Pseudorabies virus (PRV) mainly causes pseudorabies (PR) or Aujeszky's disease in pigs and can infect humans, raising public health concerns about zoonotic and interspecies transmission of PR. With the emergence of PRV variants in 2011, the classic attenuated PRV vaccine strains have failed to protect many swine herds against PR. Herein, we developed a self-assembled nanoparticle vaccine that induces potent protective immunity against PRV infection. PRV glycoprotein D (gD) was expressed using the baculovirus expression system and further presented on the lumazine synthase (LS) 60-meric protein scaffolds via the SpyTag003/SpyCatcher003 covalent coupling system. In mouse and piglet models, LSgD nanoparticles emulsified with the ISA 201VG adjuvant elicited robust humoral and cellular immune responses. Furthermore, LSgD nanoparticles provided effective protection against PRV infection and eliminated pathological symptoms in the brain and lungs. Collectively, the gD-based nanoparticle vaccine design appears to be a promising candidate for potent protection against PRV infection.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

Introduction: It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. Materials and methods: In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. Results: gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. Discussion: This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I.

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

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.

HSV-1 Glycoprotein D and Its Surface Receptors: Evaluation of Protein-Protein Interaction and Targeting by Triazole-Based Compounds through In Silico Approaches.

Protein-protein interactions (PPI) represent attractive targets for drug design. Thus, aiming at a deeper insight into the HSV-1 envelope glycoprotein D (gD), protein-protein docking and dynamic simulations of gD-HVEM and gD-Nectin-1 complexes were performed. The most stable complexes and the pivotal key residues useful for gD to anchor human receptors were identified and used as starting points for a structure-based virtual screening on a library of both synthetic and designed 1,2,3-triazole-based compounds. Their binding properties versus gD interface with HVEM and Nectin-1 along with their structure-activity relationships (SARs) were evaluated. Four [1,2,3]triazolo[4,5-b]pyridines were identified as potential HSV-1 gD inhibitors, for their good theoretical affinity towards all conformations of HSV-1 gD. Overall, this study suggests promising basis for the design of new antiviral agents targeting gD as a valuable strategy to prevent viral attachment and penetration into the host cell.

Molecular determinants of the interaction between HSV-1 glycoprotein D and heparan sulfate.

Literature has well-established the importance of 3-O-sulfation of neuronal cell surface glycan heparan sulfate (HS) to its interaction with herpes simplex virus type 1 glycoprotein D (gD). Previous investigations of gD to its viral receptors HVEM and nectin-1 also highlighted the conformational dynamics of gD's N- and C-termini, necessary for viral membrane fusion. However, little is known on the structural interactions of gD with HS. Here, we present our findings on this interface from both the glycan and the protein perspective. We used C-terminal and N-terminal gD variants to probe the role of their respective regions in gD/HS binding. The N-terminal truncation mutants (with Δ1-22) demonstrate equivalent or stronger binding to heparin than their intact glycoproteins, indicating that the first 22 amino acids are disposable for heparin binding. Characterization of the conformational differences between C-terminal truncated mutants by sedimentation velocity analytical ultracentrifugation distinguished between the "open" and "closed" conformations of the glycoprotein D, highlighting the region's modulation of receptor binding. From the glycan perspective, we investigated gD interacting with heparin, heparan sulfate, and other de-sulfated and chemically defined oligosaccharides using surface plasmon resonance and glycan microarray. The results show a strong preference of gD for 6-O-sulfate, with 2-O-sulfation becoming more important in the presence of 6-O-S. Additionally, 3-O-sulfation shifted the chain length preference of gD from longer chain to mid-chain length, reaffirming the sulfation site's importance to the gD/HS interface. Our results shed new light on the molecular details of one of seven known protein-glycan interactions with 3-O-sulfated heparan sulfate.

Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.

A potent neutralizing and protective antibody against a conserved continuous epitope on HSV glycoprotein D.

Infections caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) remain a serious global health issue, and the medical countermeasures available thus far are limited. Virus-neutralizing monoclonal antibodies (NAbs) are crucial tools for studying host-virus interactions and designing effective vaccines, and the discovery and development of these NAbs could be one approach to treat or prevent HSV infection. Here, we report the isolation of five HSV NAbs from mice immunized with both HSV-1 and HSV-2. Among these were two antibodies that potently cross-neutralized both HSV-1 and HSV-2 with the 50% virus-inhibitory concentrations (IC50) below 200 ng/ml, one of which (4A3) exhibited high potency against HSV-2, with an IC50 of 59.88 ng/ml. 4A3 neutralized HSV at the prebinding stage and prevented HSV infection and cell-to-cell spread. Significantly, administration of 4A3 completely prevented weight loss and improved survival of mice challenged with a lethal dose of HSV-2. Using structure-guided molecular modeling combined with alanine-scanning mutagenesis, we observed that 4A3 bound to a highly conserved continuous epitope (residues 216 to 220) within the receptor-binding domain of glycoprotein D (gD) that is essential for viral infection and the triggering of membrane fusion. Our results provide guidance for developing NAb drugs and vaccines against HSV.

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.

Identifying HSV-1 Inhibitors from Natural Compounds via Virtual Screening Targeting Surface Glycoprotein D.

Herpes simplex virus (HSV) infections are a worldwide health problem in need of new effective treatments. Of particular interest is the identification of antiviral agents that act via different mechanisms compared to current drugs, as these could interact synergistically with first-line antiherpetic agents to accelerate the resolution of HSV-1-associated lesions. For this study, we applied a structure-based molecular docking approach targeting the nectin-1 and herpesvirus entry mediator (HVEM) binding interfaces of the viral glycoprotein D (gD). More than 527,000 natural compounds were virtually screened using Autodock Vina and then filtered for favorable ADMET profiles. Eight top hits were evaluated experimentally in African green monkey kidney cell line (VERO) cells, which yielded two compounds with potential antiherpetic activity. One active compound (1-(1-benzofuran-2-yl)-2-[(5Z)-2H,6H,7H,8H-[1,3] dioxolo[4,5-g]isoquinoline-5-ylidene]ethenone) showed weak but significant antiviral activity. Although less potent than antiherpetic agents, such as acyclovir, it acted at the viral inactivation stage in a dose-dependent manner, suggesting a novel mode of action. These results highlight the feasibility of in silico approaches for identifying new antiviral compounds, which may be further optimized by medicinal chemistry approaches.

Protective effects of fusion recombinant DNA vaccine of herpes simplex virus type 2 glycoprotein D and adjuvant CCL19 on mice / 中华微生物学和免疫学杂志

Objective:To compare the immune responses to simply mixed and fused recombinant DNA vaccines of herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and molecular adjuvant CCL19 in mice and to evaluate the protective effects.Methods:Gene recombination technology was used to construct recombinant DNA vaccines expressing HSV-2 gD and CCL19 alone or fused together. After verification by sequencing, Western blot and ELISA, BALB/c mice were immunized twice by intramuscular injection. Serum samples and vaginal lavage fluids were collected regularly after immunization. Splenocytes, mesenteric lymph node cells and rectal tissues were collected after immunization. Differences in humoral and cellular immune responses to the two forms of vaccines and their protective effects in mice were analyzed using end-point ELISA, in vitro neutralization assay, immunohistochemical staining, chemotaxis assay, vaginal virus challenge, fluorescence quantitative PCR, weighing and disease severity assessment. Results:The fused recombinant pgD-IZ-CCL19 plasmid could express gD protein and CCL19 protein in vitro, but the level of expressed CCL19 protein by pCCL19-IZ-gD plasmid was less than that by pgD-IZ-CCL19. The mice immunized with pgD-IZ-CCL19 showed higher levels of IgG in sera and IgA in vaginal lavage fluids ( P<0.01) and stronger neutralization ability than the mice vaccinated with pgD+ pCCL19. Compared with other groups, more lymphocytes were recruited in the rectal mucosa, the spleen and mesenteric lymph nodes of mice immunized with pgD-IZ-CCL19. Weight loss or disease symptoms were not observed in the pgD-IZ-CCL19 group after virus challenge. In addition, the positive rate of HSV-2 in vaginal mucosa and the mortality rate in the pgD-IZ-CCL19 group were the lowest. However, pCCL19-IZ-gD turned out ineffective in preventing HSV-2 infection. Conclusions:The fused recombinant DNA vaccine pgD-IZ-CCL19 could induce stronger immune responses in mice and provide better protective effects, which was superior to the simply mixed DNA vaccine.

Protective effects of bicistronic DNA vaccines of herpes simplex virus type 2 glycoprotein D and adjuvant CCL28 on mice / 中华微生物学和免疫学杂志

Objective:To study the protective effects of bicistronic DNA vaccines carrying herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and adjuvant CCL28 sequences that were connected by internal ribosome entry site (IRES) sequence in mouse model.Methods:The recombinant DNA vaccines, pgD-IRES-CCL28 and pCCL28-IRES-gD, encoding HSV-2 gD and adjuvant CCL28 were constructed with IRES sequence. After verified by sequencing, they were intramuscularly injected twice into BALB/c mice. Serum samples and vaginal lavage fluids were collected regularly. Splenocytes, mesenteric lymph node cells and rectal mucosa tissues were separated and collected. The titers of antigen-specific antibodies in immunized mice were analyzed with end-point ELISA. In vitro neutralization assay was used to measure neutralizing antibody titers in serum and vaginal lavage fluid after vaccination and virus challenge. CCL28-responsive immune cells in splenocytes, mesenteric lymph node cells and rectal tissues were detected by chemotaxis experiment and immunohistochemical staining. The protective effects of the bicistronic DNA vaccines were evaluated by fluorescent quantitative PCR, weighing and disease severity assessment. Humoral and cellular immune responses induced by the bicistronic DNA vaccines and their efficacy in immunoprotection were analyzed by comparing with pgD+ pCCL28 group. Results:IgG titers in serum samples and IgA antibody titers in vaginal lavage fluids of mice immunized with pCCL28-IRES-gD were similar to those in pgD+ pCCL28 group. The neutralizing ability of antibodies, the number of rectal mucosal IgA+ plasma cells and CCL28-responsive immune cells in mucosal tissues were increased in pCCL28-IRES-gD group. Serum neutralizing antibodies were not produced immediately in the mice challenged with HSV-2, but no weight loss, disease symptoms or death was observed. However, pgD+ pcDNA3.1 and pgD-IRES-CCL28 were ineffective against HSV-2 infection in mice.Conclusions:The recombinant bicistronic DNA vaccine of pCCL28-IRES-gD could induce stronger mucosal immune response in mice and provide better protective effects.

Adenovirus-mediated RNA interference against herpes simplex virus infection in vitro.

INTRODUCTION: Herpetic keratitis caused by the herpes simplex virus (HSV) is the most common form of ocular herpes that causes corneal blindness. Although treatments for herpes keratitis have improved in recent years. there is still considerable room for new treatments against viral infection that shows great promise. The aim of the study was to evaluate the effect of RNA interference on HSV Type 1 (HSV1) infection in vitro, first prophylactically then therapeutically. MATERIAL AND METHODS: The highly conserved glycoproteins D (gD) and E (gE) were chosen as targets for this study. Different small interfering RNA (siRNA) duplexes that target gD and gE were designed and chemically synthesized. The recombinant adenovirus type 5 was developed and used as the vehicle with which we delivered the siRNA into the Vero cells infected with the HSV1 KOS strain. Evaluation of the efficacy of siRNA-mediated inhibition was performed either before virus inoculation (prophylactically) or after virus inoculation at the first appearance of lesions (therapeutically). The expression of messenger RNA encoding gD and gE was detected using a real-time polymerase chain reaction (qPCR). We analyzed HSV replication in Vero cells, cytotoxicity of HSV, and cell viability. RESULTS: When used prophylactically, the siRNA-targeting gD and gE created a more marked decrease in viral titer than when used therapeutically. The transfection of cells with recombinant adenovirus containing the siRNA expression cassette was associated with very low cytotoxicity. CONCLUSIONS: Adenovirus-mediated siRNA-targeting gD and gE genes effectively inhibit the replication of the HSV in Vero cells. In addition, these findings indicate that the prophylactic use of siRNA is far more effective at inhibiting HSV replication than the therapeutic use.

Antibodies Elicited in Response to a Single Cycle Glycoprotein D Deletion Viral Vaccine Candidate Bind C1q and Activate Complement Mediated Neutralization and Cytolysis.

Herpes simplex virus (HSV) prevention is a global health priority but, despite decades of research, there is no effective vaccine. Prior efforts focused on generating glycoprotein D (gD) neutralizing antibodies, but clinical trial outcomes were disappointing. The deletion of gD yields a single-cycle candidate vaccine (∆gD-2) that elicits high titer polyantigenic non-gD antibodies that exhibit little complement-independent neutralization but mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Active or passive immunization with DgD-2 completely protects mice from lethal disease and latency following challenge with clinical isolates of either serotype. The current studies evaluated the role of complement in vaccine-elicited protection. The immune serum from the DgD-2 vaccinated mice exhibited significantly greater C1q binding compared to the serum from the gD protein vaccinated mice with infected cell lysates from either serotype as capture antigens. The C1q-binding antibodies recognized glycoprotein B. This resulted in significantly greater antibody-mediated complement-dependent cytolysis and neutralization. Notably, complete protection was preserved when the DgD-2 immune serum was passively transferred into C1q knockout mice, suggesting that ADCC and ADCP are sufficient in mice. We speculate that the polyfunctional responses elicited by DgD-2 may prove more effective in preventing HSV, compared to the more restrictive responses elicited by adjuvanted gD protein vaccines.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae.

OBJECTIVES: Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid. RESULTS: The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD. CONCLUSIONS: These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.