Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD.IMPORTANCEAntibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD, gB, and, to a lesser extent, gC. While several key HSV-neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

Herpes simplex virus 1 (HSV-1) and HSV-2 infect many humans and establish a latent infection in sensory ganglia. Although some infected people suffer periodic recurrences, others do not. Infected people mount both cell-mediated and humoral responses, including the production of virus-neutralizing antibodies (Abs) directed at viral entry glycoproteins. Previously, we examined IgGs from 10 HSV-seropositive individuals; all neutralized virus and were directed primarily against gD or gD+gB. Here, we expand our studies and examine 32 additional sera from HSV-infected individuals, 23 of whom had no recurrent disease. Using an Octet RED96 system, we screened all 32 serum samples directly for both glycoprotein binding and competition with known neutralizing anti-gD and -gB monoclonal Abs (MAbs). On average, the recurrent cohort exhibited higher binding to gD and gB and had higher neutralization titers. There were similar trends in the blocking of MAbs to critical gD and gB epitopes. When we depleted six sera of Abs to specific glycoproteins, we found different types of responses, but always directed primarily at gD and/or gB. Interestingly, in one dual-infected person, the neutralizing response to HSV-2 was due to gD2 and gB2, whereas HSV-1 neutralization was due to gD1 and gB1. In another case, virus neutralization was HSV-1 specific, with the Ab response directed entirely at gB1, despite this serum blocking type-common anti-gD and -gB neutralizing MAbs. These data are pertinent in the design of future HSV vaccines since they demonstrate the importance of both serotypes of gD and gB as immunogens. IMPORTANCE We previously showed that people infected with HSV produce neutralizing Abs directed against gD or a combination of gD+gB (and in one case, gD+gB+gC, which was HSV-1 specific). In this more extensive study, we again found that gD or gD+gB can account for the virus neutralizing response and critical epitopes of one or both of these proteins are represented in sera of naturally infected humans. However, we also found that some individuals produced a strong response against gB alone. In addition, we identified type-specific contributions to HSV neutralization from both gD and gB. Contributions from the other entry glycoproteins, gC and gH/gL, were minimal and limited to HSV-1 neutralization. Knowing the variations in how humans see and mount a response to HSV will be important to vaccine development.

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. Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) mediates virus entry and cell-to-cell spread. We hypothesized that an effective gD2 vaccine needs to block these activities. Neutralizing titers, which assess blocking virus entry, correlated with protection against HSV-2 genital lesions in gD2-immunized guinea pigs. We used a high throughput biosensor assay to evaluate whether gD2-immunized guinea pigs produce antibodies that block the activities of gD2 epitopes involved in virus entry and cell-to-cell spread. We detected a strong correlation between the number of epitopes blocked by immune serum and protection against genital lesions. Passive transfer of monoclonal antibodies in mice infected intravaginally with HSV-2 confirmed the in vivo importance of antibodies to these epitopes. We re-evaluated our prior report of epitope-specific antibody responses in the gD2 Herpevac Trial, a trial that failed to protect subjects against HSV-2 genital infection. We detected antibody responses to significantly fewer epitopes in immunized humans than guinea pigs. We conclude that neutralizing antibodies and antibodies to multiple gD2 epitopes involved in virus entry and cell-to-cell spread represent important immune correlates of protection. Future human trials that contain gD2 as an immunogen need to assess these responses and correct deficiencies prior to embarking on large trials.

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.

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. Understanding the complex steps of herpes simplex virus (HSV) entry into susceptible cells is key for developing an effective vaccine to prevent clinical disease and control recurrences of this important human pathogen. For either serotype (HSV-1 or HSV-2), it is the interaction of glycoprotein D (gD) with receptor that initiates the cascade of events to begin infection. Importantly, gD stimulates high titers of virus-neutralizing antibodies that predict its importance in a protective response. Many HSV vaccines under development center around gD. In one recent clinical trial, subjects vaccinated with gD2 were protected against HSV-1 genital infection but not HSV-2, leading us to address how type specificity affects the antigenic topography of gD. Using a large panel of monoclonal antibodies (mAbs), we employed a new high-throughput, array-based approach to organize the mAbs into communities. Relationships within and between communities differed depending on the isotype and length of gD, suggesting significant differences in the epitope topography. We found that a subset of mAbs bound both gD1 and gD2 yet only neutralized HSV-1, possibly accounting for the anomalous results of the clinical trial. This new technology has proven to yield significant insights about how different regions of gD contribute to immune responses.

Similar to phosphatidylinositol bisphosphate, sphingomyelin breakdown generates several lipids, including sphingosylphosphorylcholine (SPC), that are putative signaling molecules. The present study was undertaken to evaluate the involvement of SPC in transmitter release process. Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of SPC on neurosecretion in the form of miniature endplate potentials (MEPPs) were assessed. Extracellular application of SPC mixture (d,l-SPC) at 1, 10, and 25 μM increased the MEPP frequency by 68, 96, and 127%, respectively. d-erythro-SPC (dissolved in dimethyl sulfoxide but not coupled to bovine serum albumin), but not l-threo-SPC, was active extracellular; the former (at 10 μM) increased the MEPP frequency by 143%. d-erythro-SPC treatment did not significantly change the median amplitude or frequency-distribution of MEPPs. Intracellular delivery via liposomes, in which 10, 100, or 1000 μM SPC mixture was entrapped in liposomal aqueous phase, induced a concentration-dependent increase in MEPP frequency of 45, 91, and 100%, respectively. d-erythro-SPC and l-threo-SPC at the concentration of 100 μM increased the MEPP frequency by 117 and 67%, respectively, or 91 and 61%, respectively, when coupled to bovine serum albumin. Pretreatment with thapsigargin significantly reduced but did not abolish the effects of extracellular d-erythro-SPC (10 μM) or liposomes containing 100 μM d-erythro-SPC. Liposomes filled with 100 μM d-myo-inositol 1,4,5-trisphosphate (IP₃) enhanced the MEPP frequency to the same magnitude as 100 μM d-erythro-SPC entrapped in liposomes. However, administration of 100 μM d-erythro-SPC and IP₃ entrapped in the same liposomes enhanced the MEPP frequency by 70%, which was less than that produced by these two compounds alone. The result provides the first electrophysiological evidence that SPC can modulate transmitter release by an extra- or intracellular action at the frog motor nerve ending.