We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Six clinical isolates of HSV-2 and two clinical isolates of HSV-1 were almost completely inactivated when TF-3 (100 μM) was present with LA over the pH range of 4.5 to 5.7, whereas four additional HSV-1 clinical isolates required TF-3 concentrations of 250 to 500 μM for comparable virus titer reduction. We evaluated sera from 30 women seronegative for HSV-1 and HSV-2 who were immunized with gD2 in the Herpevac Trial. Similar titers of HSV-1 were found in the eyes, ganglia, and brains of treated animals. However, I have seen many different titer results varying from 1.0 – 10.0. The higher neutralizing antibody titers to HSV-1 offer an explanation for the Herpevac results, and shielding neutralizing domains provides a potential mechanism. Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency.
Semen, but not cervical vaginal fluid, decreased LA-dependent antiviral activity at pH 4.0, but adding TF-3 to the mixture reduced HSV titers by 4 to 5 log10. In a subset analysis, the gD2 vaccine provided approximately 70% protection against HSV-2 genital disease in women seronegative for both HSV-1 and HSV-2 but not in women seropositive for HSV-1 or in men of any serostatus . Currently, there is no vaccine or cure for HSV infections; however, treatments are available that diminish the severity of the disease. 4) Having a higher titer 10.0 vs. Unexpectedly, the gD2 vaccine provided significant protection against HSV-1 but not HSV-2 genital infection and disease . Sixty percent of genital disease in control subjects was caused by HSV-1, which has emerged as the leading cause of primary genital disease [1, 3]. Few HSV-1 infections were detected in the first report, owing to selection of HSV-2–discordant couples.
HSV-2 gD2 is required for virus entry into cells . Previous reports showed that penciclovir has a higher affinity than acyclovir for the viral thymidine kinase, a faster rate of phosphorylation, and a longer intracellular half-life [10, 11]; however, penciclovir has a lower affinity than acyclovir for the HSV DNA polymerase. Enzyme-linked immunosorbent assay (ELISA) antibody titers but not cellular immune responses to gD2 were highly correlated with protection in the Herpevac Trial . A possible explanation for protection against HSV-1 but not HSV-2 may relate to the relative ease of blocking entry, as measured by neutralization of the 2 viruses. Therefore, we evaluated the hypothesis that neutralizing antibody titers were higher to HSV-1 than to HSV-2 in the Herpevac Trial. We measured neutralizing antibodies by 2 methods. One approach evaluated the 50% end point neutralizing titer by incubating serial 2-fold dilutions of heat-inactivated serum with 100 plaque-forming units (PFU) of virus for 1 hour at 37°C, inoculating the virus-antibody mix onto Vero cells, overlaying with methylcellulose, and counting plaques at 72 hours .
The neurovirulent HSV-1 strain McKrae was grown in Vero (African green monkey kidney) cells in EMEM:199 medium (Quality Biological, Gaithersburg, MD) with 10% fetal bovine serum (Quality Biological) and 1% glutamine-streptomycin-penicillin (Life Technologies Gibco BRL, Gaithersburg). The log10 neutralization was calculated as the difference in titer, using serum from immunized individuals and nonimmune human serum as a control. ELISA was performed at a 1:500 serum dilution, using baculovirus-expressed gD1 (bac-gD1[306t]) or gD2 (bac-gD2[306t]) as antigen [6, 8]. Subjects were immunized at 0, 1, and 6 months [1, 2]. Paired sera were obtained before the first immunization and at month 7 from 35 Herpevac Trials subjects; 30 had received 20 µg of gD2 with MPL/alum, whereas 5 received hepatitis A vaccine as a control . An additional 13 sera were obtained at month 7 from subjects seronegative for both HSV-1 and HSV-2 who were enrolled in GSK gD2 vaccine trials between 1995 and 1997. Drug-treated animals received either famciclovir or valacyclovir (both from Midwest Medical Supply, St.
These sera were stored at −80°C. Seven of 13 sera were from subjects enrolled at the University of Pennsylvania in HSV-007 (3 controls and 4 subjects immunized with 20 µg gD2 with MPL/alum) . Five of 13 sera were from subjects enrolled in a dose-ranging study that used 20, 40, or 80 µg of gD2 with MPL/alum (HSV-014), and 1 subject was immunized with 20 µg gD2 with MPL/alum to evaluate diluent volumes (HSV-015). Four low-passage HSV-1 and 3 low-passage HSV-2 clinical isolates were used for neutralization assays. The gC and gE deletion strains were derived from low-passage HSV-1 and HSV-2 wild-type (WT) viruses NS and 2.12, respectively [9–11]. The swab was placed in 1 mL of EMEM medium + 1.2% amphotericin B (Quality Biological). A, Fifty percent end point neutralizing antibody titers of herpes simplex virus type 1 (HSV-1) and HSV-2 Herpevac Trial sera obtained at months 0 and 7 from hepatitis A (HepA; n = 5) and glycoprotein D2 (gD2; n = 30) immunized subjects.
B, Neutralization … A, Log10 neutralization of wild-type (WT), gCnull, and gEnull mutant viruses by sera from gD2 HSV-014 and 015 vaccinees (n = 6). Error bars represent standard errors of the mean. B, Enzyme-linked immunosorbent assay (ELISA) titers at a 1:500 dilution … Plaques were identified and counted as indicated earlier. Selected subjects were not infected with HSV before month 7. The geometric mean 50% end point neutralization titer of sera taken at month 7 was 1:101 for HSV-1, compared with 1:29 for HSV-2 (3.5-fold difference; Figure A).
The mean percentage neutralization at each serum dilution was higher against HSV-1 than against HSV-2 (Figure B). A significant correlation was detected between gD2 ELISA titers (provided by the Herpevac investigators) and neutralizing antibody titers to HSV-1 and HSV-2 for the 30 subjects immunized with gD2 (Figure C) . We evaluated neutralizing antibody titers of 13 additional sera from subjects enrolled in GSK gD2 vaccine trials between 1995 and 1997, including 3 sera from placebo-immunized subjects and 10 from subjects immunized with gD2 with MPL/alum. Animals were anesthetized with a combination of ketamine/xylazine in PBS and placed onto cardboard resting on top of a 60 Hz/115V TM-20 transilluminator (UVP, Upland, CA) emitting a peak wavelength of 302 nm. Six of the 10 sera (those from HSV-014 and 015) were tested for neutralizing antibody titers against 3 additional low-passage HSV-1 strains and 2 additional low-passage HSV-2 strains. The geometric mean neutralizing antibody titer was 1:242 against HSV-1 and 1:106 against HSV-2 (2.3-fold difference; Figure E). As a potential explanation for the greater neutralization of HSV-1 than HSV-2, we hypothesized that HSV-2 gC2 and gE2 may shield neutralizing domains on HSV-2 gD2 more effectively than gC1 and gE1 shield domains on HSV-1 gD1.
This hypothesis is based on our prior studies demonstrating that gC1 and gE1 block access of neutralizing antibodies to HSV-1 glycoproteins involved in virus entry . We compared neutralization of WT HSV-1 and HSV-2 with gC or gE mutant strains derived from the WT viruses. Monolayers were checked daily for CPEs. Neutralization was significantly greater against WT HSV-1 (2.2 log10) than against WT HSV-2 (1.0 log10; Figure A), confirming results using the 50% end point neutralizing titer (Figure ). The gD2 vaccine sera reduced the titer of HSV-1 gCnull (NS-gCnull) virus by 2.2 log10 and HSV-1 gEnull (NS-gEnull) virus by 1.8 log10, which are not statistically different from findings for WT HSV-1 strain NS, indicating that gC1 and gE1 do not block neutralization by gD2 antibodies (Figure A). Strikingly, gC2 and gE2 had a significantly greater effect on blocking HSV-2 strain 2.12 neutralization, since the gD2 vaccine sera reduced the titer of HSV-2 gCnull (2.12-gC2null) virus by 3.3 log10 and HSV-2 gEnull (gE2-del) virus by 4.4 log10 (Figure A). Importantly, ELISA revealed that antibody produced by immunization with the gD2 vaccine reacted equally well to soluble, purified gD1 and gD2 proteins (Figure B), suggesting that antibody bound comparably to both glycoproteins.
The gD2 amino acid sequence included in the vaccine has 84% identity with gD1 ; therefore, cross-protection against HSV-1 is not surprising. Purity of the DNA was checked by a ratio of the optical density (OD) readings at 260/280 nm. The impressive difference in neutralizing antibody titers provides a plausible explanation for the protection against HSV-1 infection and disease reported in the Herpevac Trial. The low HSV-2 mean neutralizing titer of 1:29 at month 7 may explain the lack of protection observed against HSV-2. The neutralizing titers of Herpevac Trial sera cannot be compared with sera from the 1995–1997 studies since Herpevac samples, not the 1995–1997 samples, were intentionally selected to include subjects with high, medium, and low ELISA titers. In addition, higher antigen doses were used in some of the 1995–1997 studies. Nevertheless, the sera can be compared for neutralizing titers to HSV-1 and HSV-2, since the same serum sample was tested against both viruses.
Each 25-µL reaction of 145 ng of TG DNA was done in triplicate on a 96-well plate and then repeated for a total of 6 runs per animal. Higher neutralizing titers to HSV-1 than to HSV-2 were noted using a total of 4 HSV-1 and 3 HSV-2 low-passage isolates, which supports the generalizability of the observation. We previously reported that HSV-1 gC1 and gE1 did not block neutralization by gD antibodies; however, gC1 and gE1 blocked neutralization when gD antibodies were combined with neutralizing antibodies to other glycoproteins involved in entry, including gB and gH/gL. We interpreted these results as indicating that gC1 and gE1 block domains of interaction between gD1 and other entry molecules . Here we hypothesized that gC2 and gE2 shield gD2 from neutralizing antibodies, as a mechanism to explain the neutralizing antibody results. The ratio of gC1 to gD1 molecules on HSV-1 strain NS is 1:14, which may help explain the lack of blocking by gC1 . Geometric means and one-way analysis of variance with log-transformed numbers were used to analyze results from ocular swabs, acute-phase tissue virus titers, and quantitative PCR.
Glycosylation of envelope proteins prevents antibody access to neutralizing domains on HIV-1 gp120 and influenza hemagglutinin . Our results suggest that neighboring glycoproteins also may block antibody access. A hypothetical model depicting differences between gC and gE of HSV-1 and HSV-2 in blocking antibody access is shown in Figure C and 2D. Other potential mechanisms to explain greater neutralization of HSV-1 than HSV-2 include the possibility that epitopes recognized by antibody on gD1 may be more essential for virus entry than analogous epitopes on gD2 or that fewer gD molecules are expressed on HSV-1, making it easier to neutralize. In the Herpevac Trial, ELISA titers correlated with protection against HSV-1, while CD4+ or CD8+ T-cell responses did not [1, 5]. None of the untreated animals survived beyond pi day 5, whereas all of the mock-infected animals survived. T-cell responses that were not measured in the trial may also be important for protection.
Our results suggest that a vaccine containing gD2 antigen will likely induce higher neutralizing antibody titers to HSV-1 than to HSV-2, which is encouraging in terms of potential benefits of a vaccine that includes gD2. However, approaches are required to improve the neutralizing titers to HSV-2. One possibility is to induce antibodies to gD2 domains that are not shielded, while other possibilities include using higher concentrations of antigen, using more-potent adjuvants, or preventing antibody and complement immune evasion by the virus to enhance the efficacy of the antibodies produced .