Abstract
Background
Herpes simplex virus 2 (HSV2) causes genital herpes in >400 million persons worldwide.
Methods
We conducted a randomized, double-blinded, placebo-controlled trial of a replication-defective HSV2 vaccine, HSV529. Twenty adults were enrolled in each of 3 serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1−/HSV2−), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). Sixty participants received vaccine or placebo at 0, 1, and 6 months. The primary end point was the frequency of solicited local and systemic reactions to vaccination.
Results
Eighty-nine percent of vaccinees experienced mild-to-moderate solicited injection site reactions, compared with 47% of placebo recipients (95% confidence interval [CI], 12.9%–67.6%; P = .006). Sixty-four percent of vaccinees experienced systemic reactions, compared with 53% of placebo recipients (95% CI, −17.9% to 40.2%; P = .44). Seventy-eight percent of HSV1−/HSV2− vaccine recipients had a ≥4-fold increase in neutralizing antibody titer after 3 doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36%, 46%, and 27% of HSV1−/HSV2−, HSV1±/HSV2+, and HSV1+/HSV2− participants, respectively, 1 month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants, respectively.
Conclusions
HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV-seronegative vaccinees.
Clinical Trials Registration
Keywords: Herpes simplex, HSV2, vaccine, genital herpes
A replication-defective herpes simplex virus type 2 (HSV2) vaccine is safe, well tolerated, and immunogenic in HSV naive and previously HSV-infected subjects.
Worldwide, about 400 million people are infected with herpes simplex virus type 2 (HSV2), the predominant cause of genital herpes [1]. HSV2 can also cause encephalitis or disseminated infection in neonates and severe disease in immunocompromised patients. Moreover, HSV2 genital herpes enhances the risk of acquisition and transmission of human immunodeficiency virus [2, 3]. For these reasons, an HSV2 vaccine is needed to prevent infection or disease. While numerous prophylactic HSV vaccines have advanced to clinical trials, none have been licensed [4].
Prior studies to prevent HSV infection have focused on subunit vaccines with a goal to induce neutralizing antibodies. HSV2 dl5-29 is a replication-defective HSV2 vaccine that can infect cells and should result in a broader immune response [5]. Based on animal studies demonstrating that HSV2 dl5-29 was superior to a HSV2 glycoprotein D (gD2) subunit vaccine [6–8], we tested a clinical grade formulation of dl5-29, HSV529, in a phase 1 study of healthy volunteers.
METHODS
Trial Design and Participants
We conducted a randomized, double-blinded, placebo-controlled trial to determine the safety and immunogenicity of HSV529 in 60 healthy volunteers 18–40 years old. Participants were enrolled in 3 HSV serogroups of individuals: those negative for both HSV1 and HSV2 (HSV1−/HSV2−), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). The serotypes of participants were determined using an HSV type–specific antibody test for glycoprotein G (BioPlex 2200 System HSV1 and HSV2 IgG, Bio-Rad). Participants within each serogroup were randomized so that 15 participants received vaccine and 5 received placebo (normal saline).
Vaccine
The vaccine, derived from HSV2 strain 186, was produced in Vero cells expressing HSV2 UL5 and UL29 and manufactured by Sanofi Pasteur. Each dose was a 0.5-mL solution containing 1 × 107 plaque-forming units of HSV529. The trial was conducted at the National Institutes of Health Clinical Center after approval by the National Institutes of Allergy and Infectious Diseases Institutional Review Board. Each volunteer provided written informed consent. The study was conducted in accordance with the International Council for Harmonization for Good Clinical Practice and the ethical principles of the Declaration of Helsinki (2008).
Study Procedures
HSV529 or placebo was administered by intramuscular injection on days 0, 30, and 180. The primary end point was the frequency of local and systemic reactogenic events during the first 7 days after each injection plus adverse events occurring throughout the trial. Participants recorded symptoms on diary cards, which were reviewed at study visits before dose 2 and dose 3; on days 7 and 30 after each dose; and at 6 months after the third dose. A complete blood count (CBC) and mineral, hepatic, and acute care panels were obtained prior to each dose; a CBC was obtained 7 days after each dose. Adverse events and laboratory abnormalities were graded according to a toxicity table [9]. Participants self-collected 20 anogenital swab specimens twice daily for 10 consecutive days between 7 and 30 days after the last dose, to detect HSV shedding by qualitative, real-time polymerase chain reaction (PCR) amplification with differentiation of HSV1 and HSV2 [10–12]. Blood specimens were collected to assess immunogenicity before and 1 month after each dose and 6 months after the third dose.
HSV2 Neutralizing Antibodies and gD-Specific Binding Antibodies
Sera underwent heat inactivation and were assayed for HSV2 neutralizing activity by a plaque-reduction assay in Vero cells, using HSV2 strain 333 [13]. Results are reported as the fold dilution of serum that inhibited 50% of plaque formation (IC50) and were calculated by nonlinear regression. A positive neutralizing antibody response was defined as a ≥4-fold increase in titer from baseline to 30 days after the third dose. The HSV gD–specific antibody titer was measured in light units (LU), using a luciferase immunoprecipitation assay [14].
HSV2-Specific T-Cell Responses
Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed and rested overnight, and CD8+ T-cell responses for all serogroups were determined by interferon γ (IFN-γ) enzyme-linked immunospot (ELISPOT) analysis (TrueBlue substrate IFN-γ kit; Cellular Technology). Peripheral blood mononuclear cells (PBMCs) were stimulated with 3 pools of CD8+ T-cell HSV2 antigenic peptides (1 μg/mL each; Biosynthesis) containing 26–38 peptides per pool. Negative controls were 0.3% dimethyl sulfoxide (DMSO) and medium. Spots were counted using a CTL S6 reader with Immunospot software. The CD8+ T-cell response was considered positive if the sum of net responses to the 3 HSV2 peptide pools was ≥30 spot-forming units (SFUs)/106 PBMCs (relative to DMSO) and ≥2-fold over the baseline value. Some PBMC samples with high ELISPOT background staining were traced to a discrete PBMC processing period, and 15 specimens from 12 participants were excluded because of bacterial contamination. The number of participant samples evaluated in each immunogenicity assay is specified in Supplementary Table 1.
CD4+ T-cell responses in HSV1−/HSV2− participants were determined by ELISPOT, because responses were presumed to be below the limits of detection of the intracellular cytokine staining (ICS) assay. PBMCs were stimulated with UV-inactivated HSV2 (strain 186; dilution, 1:1000), UV-treated mock lysate, or medium alone. Participants were considered to have a positive CD4+ T-cell response if the net response to whole HSV2 antigen was ≥30 SFUs/106 PBMCs, relative to mock lysate, and ≥2-fold over the baseline value.
CD4+ T-cell responses in the HSV1±/HSV2+ and HSV1+/HSV2− groups were evaluated by ICS [15]. PBMCs were stimulated with UV-inactivated HSV2 or mock antigen with anti-CD28 and anti-CD49d antibodies at 37°C. Brefeldin A was added after 5 hours, and cells were incubated overnight. Cells were stained with Live/Dead (Invitrogen), treated with BD FACS lyse/perm2 solutions, and stained with fluorochrome-labeled monoclonal antibodies to CD3, CD4, CD8, and the activation markers CD40L, IFN-γ, interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α). Events were recorded with BD FACSCanto II and analyzed with FlowJo (v10; TreeStar). A positive CD4+ T-cell vaccine response was defined as a net response to UV-inactivated HSV2 (relative to mock) of ≥0.05% of CD4+ T cells with ≥2 activation markers and a ≥2-fold increase over the baseline value. A functionality score based on analysis of all 16 possible activation marker combinations in CD4+ T-cell ICS data from seropositive participants was determined using OpenCyto and COMPASS [16, 17]. Samples with <25 000 CD3+ T cells were excluded.
Three participants in the HSV1±/HSV2+ group had serum neutralizing titers below the limit of detection, tested negative for gD-specific antibody, and had no net HSV2-specific CD4+ T-cell responses detected by ICS at baseline. Western blot results using baseline sera were negative for HSV1 and indeterminate for HSV2 (data not shown). We included these participants in the HSV1±/HSV2+ group per-protocol analysis, based on the results of the HSV1/HSV2 serologic assay (BioPlex 2200) used to classify subjects into serogroups.
Statistical Analysis
Adverse event data for each serogroup and for the total vaccine and placebo groups was analyzed with 95% confidence intervals (CIs), using the exact binomial method (Clopper-Pearson method). Analysis of humoral and cellular responses to HSV2 was performed after separation of subjects into the 3 serogroups, followed by separation into treatment arms (HSV529 and placebo). Assuming that log10 transformation of neutralizing and gD-specific antibody titers follows a normal distribution, the means and their 95% CIs were calculated using the usual calculation for normal distribution (Student t distribution with n − 1 degree of freedom). Pairwise tests comparing baseline T-cell responses to day 210 responses were performed using the Wilcoxon matched-pairs signed rank test; missing data at day 210 were assumed to be missing at random and were dropped from the analyses. To evaluate changes within a time series and to account for missing data, we converted T-cell immune responses to the log10 scale for parametric testing using linear mixed models to test the hypothesis that any postvaccine time point was different from baseline, followed by day-level testing when the global P value was significant. The α level of significance was set at .05. All volunteers who received at least 1 dose of vaccine or placebo were included in the safety and immunogenicity analyses. For COMPASS analyses, pairwise tests comparing baseline T-cell responses to day 210 responses were performed using a 1-sided Wilcoxon matched-pairs signed rank test.
RESULTS
Study Population
Sixty participants—30 women and 30 men, with a mean age of 31 years (range, 21–40 years)—were enrolled and vaccinated (Figure 1 and Supplementary Table 2). Fifteen participants in the HSV1±/HSV2+ group received 3 doses of vaccine. Fourteen of 15 participants each in the HSV1+/HSV2− and HSV1−/HSV2− groups received 3 doses of vaccine, and 1 participant in each group received only the first 2 doses of vaccine. Five of 5 participants in each serogroup received 3 doses of placebo.
Figure 1.
Screening, enrollment, vaccination, and follow-up. Participants in each serogroup were randomized to receive vaccine or placebo in a 3:1 ratio. Initially, 4 participants eligible for the group that was positive or negative for herpes simplex virus type 1 (HSV1) and positive for HSV2 (HSV1±/HSV2+) were enrolled and dosed with the first dose. After a 4-week safety assessment period, enrollment was open to all 3 serogroups. After dosing 4 participants each in the group positive for HSV1 and negative for HSV2 (HSV1+/HSV2−) and the group negative for both HSV1 and HSV2 (HSV1−/HSV2−) and a 7-day safety assessment period, enrollment continued into all 3 groups. Overall, 97% of participants received all 3 doses of vaccine or placebo. Blood specimens were collected on the day before each injection, 1 day after the first dose, 7 days after each dose, 30 days after the third dose, and 360 days after day 0 for evaluation of immune responses. PFU, plaque-forming units. aTwo participants did not receive the third dose of vaccine and missed several study visits because of unplanned pregnancy.
Vaccine Safety
Vaccinations were safe and well tolerated, with the majority of local and systemic reactogenic events of mild-to-moderate severity (Table 1). Overall, 89% of vaccine recipients experienced injection site reactions, compared with 47% of placebo recipients (95% CI, 12.9%–67.6%; P = .006); 64% of vaccine recipients experienced systemic reactions, compared with 53% of placebo recipients (95% CI, −17.9% to 40.2%; P = .44). Tenderness (ie, discomfort to touch or with movement) or pain at the injection site were the most frequent local events. Six of 15 vaccine recipients (40%) in each serogroup experienced pain at the injection site. Twelve of 15 vaccine recipients (80%) each in the HSV1±/HSV2+ and HSV1+/HSV2− groups and 15 of 15 vaccine recipients (100%) in the HSV1−/HSV2− group experienced tenderness at the injection site. Headache and malaise were the most frequent systemic events, with 7 of 15 (47%), 5 of 15 (33%), and 10 of 15 (67%) in the HSV1±/HSV2+, HSV1+/HSV2−, and HSV1−/HSV2− groups, respectively, experiencing headache and 4 of 15 (27%), 7 of 15 (47%), and 5 of 15 (33%), respectively, experiencing malaise after vaccination. One participant in the HSV1±/HSV2+ group developed grade 3 fever with chills, malaise, myalgia, and neck pain on the day after HSV529 vaccination, which resolved without therapy. One other participant in this group had grade 2 fever, and 1 had grade 1 fever. There was no significant difference in the frequency of unsolicited adverse events in vaccine versus placebo recipients, and none were related to HSV529 administration. Two serious adverse events were reported during the trial; 2 participants became pregnant, of whom one had a child born with ankyloglossia and the other experienced a miscarriage. Both events were deemed unlikely related to vaccination.
Table 1.
Local and Systemic Solicited Reactogenicity After Any Vaccine Injection, by Maximum Intensity
| Symptom, Maximum Intensity | HSV1±/HSV2+ | HSV1+/HSV2− | HSV1−/HSV2− | All Participants | HSV529 vs Placebo | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HSV529 (n = 15) | Placebo (n = 5) | HSV529 (n = 15) | Placebo (n = 5) | HSV529 (n = 15) | Placebo (n = 5) | HSV529 (n = 45) | Placebo (n = 15) | |||||
| No. | % (95% CI) | No. | % (95% CI) | P | 95% CI for Difference in Proportions, % | |||||||
| At injection site | ||||||||||||
| Pain | ||||||||||||
| Any | 6 (40) | 1 (20) | 6 (40) | 0 | 6 (40) | 2 (40) | 18 | 40 (26–56) | 3 | 20 (5–49) | .27 | −9 to 49 |
| Grade 1 | 6 (40) | 1 (20) | 6 (40) | 0 | 6 (40) | 2 (40) | … | … | … | … | … | |
| Grade 2 | 0 | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Tenderness | ||||||||||||
| Any | 12 (80) | 0 | 12 (80) | 4 (80) | 15 (100) | 1 (20) | 39 | 87 (73–94) | 5 | 33 (13–61) | .00021 | 23–84 |
| Grade 1 | 3 (20) | 0 | 3 (20) | 2 (40) | 6 (40) | 1 (20) | … | … | … | … | … | |
| Grade 2 | 9 (60) | 0 | 9 (60) | 2 (40) | 9 (60) | 0 | … | … | … | … | … | |
| Erythema | ||||||||||||
| Any | 1 (6.7) | 0 | 1 (7) | 0 | 1 (7) | 0 | 3 | 7 (2–19) | 0 | 0 (0–25) | .73 | −5 to 18 |
| Grade 1 | 1 (7) | 0 | 1 (7) | 0 | 1 (7) | 0 | … | … | … | … | … | |
| Grade 2 | 0 | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Swelling | ||||||||||||
| Any | 1 (7) | 0 | 0 | 0 | 0 | 0 | 1 | 2 (0–13) | 0 | 0 (0–25) | 1 | −4 to 9 |
| Grade 1 | 1 (7) | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Grade 2 | 0 | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Systemic | ||||||||||||
| Fever | ||||||||||||
| Any | 3 (20) | 0 | 0 | 0 | 0 | 0 | 3 | 7 (1.7–19) | 0 | 0 (0–25) | .73 | −5 to 18 |
| Grade 1 | 1 (7) | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Grade 2 | 1 (7) | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Grade 3/4 | 1 (7) | 0 | 0 | 0 | 0 | 0 | … | … | … | … | … | |
| Headache | ||||||||||||
| Any | 7 (47) | 3 (60) | 5 (33) | 3 (60) | 10 (67) | 1(20) | 22 | 49 (34–64) | 7 | 47 (22–73) | 1 | −29 to 34 |
| Grade 1 | 5 (33) | 3 (60) | 4 (27) | 3 (60) | 7 (47) | 1 (20) | … | … | … | … | … | |
| Grade 2 | 2 (13) | 0 | 0 | 0 | 3 (12) | 0 | … | … | … | … | … | |
| Grade 3/4 | 0 | 0 | 1 (7) | 0 | 0 | 0 | … | … | … | … | … | |
| Malaise | ||||||||||||
| Any | 4 (27) | 2 (40) | 7 (47) | 2 (40) | 5 (33) | 1 (20) | 16 | 36 (22–51) | 5 | 33 (13–61) | 1 | −28 to 32 |
| Grade 1 | 4 (27) | 1 (20) | 6 (40) | 1 (20) | 3 (20) | 1 (20) | … | … | … | … | … | |
| Grade 2 | 0 | 1 (20) | 0 | 1 (20) | 2 (13) | 0 | … | … | … | … | … | |
| Grade 3/4 | 0 | 0 | 1 (7) | 0 | 0 | 0 | … | … | … | … | … | |
| Myalgia | ||||||||||||
| Any | 3 (20) | 0 | 3 (20) | 1 (20) | 4 (27) | 0 | 10 | 22 (12–37) | 1 | 7 (0–34) | .34 | −6 to 38 |
| Grade 1 | 3 (20) | 0 | 2 (13) | 0 | 4 (27) | 0 | … | … | … | … | … | |
| Grade 2 | 0 | 0 | 0 | 1 (20) | 0 | 0 | … | … | … | … | … | |
| Grade 3/4 | 0 | 0 | 1 (7) | 0 | 0 | 0 | … | … | … | … | … |
Data are no., no. (%), or % of subjects with at least 1 solicited event, unless otherwise indicated.
Abbreviations: −, negative; +, positive; ±, positive or negative.
HSV2 Neutralizing and gD–Specific Antibody Responses
Neutralizing Antibody Responses in HSV-Seronegative Subjects
The geometric mean titer (GMT; based on the IC50) of HSV2 neutralizing antibody in HSV1–/HSV2– participants was increased 30 days after the third dose of vaccine, compared with baseline (16.4 vs 2.7; P < .0001; Figure 2A). The GMT increased to 4.7 as early as 30 days after the first vaccination (P < .05, compared with baseline) and increased further, to 8.0, after the second dose (P < .05, compared with baseline; Figure 2B), when 5 of 14 participants (36%) had a ≥4-fold rise in titer from baseline (Supplementary Table 3). The GMT peaked 30 days after the third vaccination, at 16.4, when 11 of 14 participants (78%) had a ≥4-fold rise in titer, compared with baseline (Figure 2B and Supplementary Table 3). The GMT remained significantly elevated 6 months after the last vaccination, compared with baseline (7.4 vs 2.7; P < .0001; Figure 2B and Supplementary Table 4). One of 15 vaccine recipients had no neutralizing antibody detected at all time points tested.
Figure 2.
Herpes simplex virus type 2 (HSV2)–specific neutralizing antibody responses. A, Individual participant responses in each serogroup, comparing the titer at baseline (day 0) to the titer 30 days after the third dose of vaccine or placebo (day 210). Short horizontal black bars represent the log10 geometric mean titer (GMT) in each serogroup. P values were calculated using a paired t test. B, The log10 GMT for each serogroup over time. Arrows indicate vaccine administration days. Horizontal dashed lines indicate the lower limit of detection. Serogroups comprised individuals negative for both HSV1 and HSV2 (HSV1−/HSV2−), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). *P < .05, based on a paired t test for individual subject titers, compared with day 0.
Neutralizing Antibody Responses in HSV-Seropositive Subjects
The geometric mean HSV2 neutralizing antibody titer was increased in HSV1±/HSV2+ participants 30 days after the third vaccination, compared with baseline (101.8 vs 69.5; P = .0036), but the increase from baseline was less marked among HSV1+/HSV2– participants (35.9 vs 29.8; P = .062; Figure 2A). The GMTs in the HSV1+/HSV2– group were increased after the first and second doses of vaccine (34.5 and 38.6, respectively; P < .05), compared with baseline, but did not increase further after the third dose (35.9; Figure 2B). No additional time points were evaluated in the HSV1±/HSV2+ group. None of the vaccine recipients in the seropositive groups had a ≥4-fold rise in neutralizing titer (Supplementary Table 3). The peak GMT (which occurred on day 210) for participants in the HSV1–/HSV2– group (16.4) was below the GMT before vaccination in the HSV1+/HSV2– and HSV1±/HSV2+ groups (29.8 and 69.5, respectively), indicating that 3 doses of HSV529 did not induce neutralizing titers comparable to those in persons naturally infected with HSV (Figure 2A).
HSV2 gD–Specific Antibody Responses
HSV2 gD is a primary target for neutralizing antibodies [18]. HSV529 vaccine recipients in the HSV1–/HSV2– group had the greatest increase in the geometric mean serum gD antibody responses, compared with baseline, 30 days after the third dose of HSV529 (404 576 vs 4897 LU/uL; P < .0001; Figures 3A). The increase in gD antibody responses in vaccine recipients in the HSV1±/HSV2+ group (1 737 800 vs 677 641 at baseline; P = .040) and the HSV1+/HSV2– group (1 156 112 vs 736 207 at baseline; P = .045) were less pronounced than in the HSV-seronegative group but were statistically significant (Figure 3A). The geometric mean gD antibody response was significantly increased at every time point after the second vaccination, compared with baseline, in the HSV1–/HSV2– group (Figure 3B and Supplementary Table 4).
Figure 3.
Herpes simplex virus (HSV) glycoprotein D (gD)–specific antibody responses. A, Individual participant responses, comparing the titer at baseline (day 0) to the titer at day 210, 1 month after the third dose. Short horizontal bars represent the log10 geometric mean for each serogroup. B, The log10 geometric mean for each serogroup over time. Horizontal dotted-dashed lines represent the mean of the responses of a pool of sera that were negative for HSV type 2 (HSV2) neutralizing activity. Arrows indicate vaccine administration days. Serogroups comprised individuals negative for both HSV1 and HSV2 (HSV1−/HSV2−), those positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+), and those positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). Geometric means for the HSV1–/HSV2– group were 4897 light units (LU)/uL (on day 0), 63 095 LU/uL (on day 60), 31 622 LU/uL (on day 180), 398 107 LU/uL (on day 210), and 251 188 LU/uL (on day 360). gD antibody levels were determined by luciferase immunoprecipitation systems. *P < .05, based on a paired t test for individual subject titers, compared with day 0.
To allay concerns that the antibody responses in the HSV1–/HSV2– group could be due to a new HSV infection during the study, participants collected anogenital swab specimens between study days 187 and 210. Shedding of HSV2 was not detected, but HSV1 was found by PCR in 1 of 20 swab specimens from 1 participant in the HSV1–/HSV2– group. HSV2 was detected in 8 of 19 participants (42%) in the HSV1±/HSV2+ group but in no participants in the HSV1+/HSV2– group.
HSV2-Specific T-Cell Responses
T-Cell Responses in HSV-Seronegative Subjects
Pairwise comparisons of median CD8+ T-cell ELISPOT responses on days 0 and 210 revealed that HSV2-specific CD8+ T-cell responses in the HSV1−/HSV2− group increased 4.6-fold (23 SFUs/106 PBMCs; interquartile range [IQR], 11–42 SFUs/106 PBMCs) relative to baseline (5 SFUs/106 PBMCs; IQR, 3–12 SFUs/106 PBMCs; P = .01; Figure 4A), although only 2 of 14 individuals (14%) met the definition of a positive CD8+ T-cell response. The median CD8+ T-cell responses were higher than baseline after each dose of vaccine but remained below the positive response threshold (Figure 4A and Supplementary Table 5). Pairwise analysis showed increased median HSV2-specific CD4+ T-cell responses after 3 doses of vaccine in HSV1–/HSV2– subjects on day 210 (11 SFUs/106 PBMCs; IQR, 1–127 SFUs/106 PBMCs), compared with baseline (0 SFUs/106 PBMCs; IQR, 0–3 SFUs/106 PBMCs; P = .001), but only 5 of 14 participants (36%) met the definition of a positive response (Figure 4B). The median HSV2-specific CD4+ T-cell response was increased above baseline at all time points measured after vaccination but remained below the positive response threshold (Figure 4B and Supplementary Table 6).
Figure 4.
Herpes simplex virus type 2 (HSV2)–specific T-cell responses after 3 doses of vaccine or placebo in participants negative for both HSV1 and HSV2 (HSV1–/HSV2–). A, Results of pairwise testing of interferon γ (IFN-γ) enzyme-linked immunospot (ELISPOT) findings to determine changes in CD8+ T-cell responses between baseline (day 0) and after the third dose of HSV529 or placebo (day 210; left) and throughout the study period (right). B, Results of pairwise testing of IFN-γ ELISPOT findings to determine changes in CD4+ T-cell responses between baseline (day 0) and after the third dose of HSV529 or placebo (day 210; left) and throughout the study (right). For pairwise comparisons (left panels), each line represents paired samples from 1 subject, and short horizontal bars are medians. The Wilcoxon rank sum test (2-sided) was used to calculate P values. For time course analyses (right), symbols represent median net response ± interquartile range, and parametric testing with linear mixed models was used for analysis. Horizontal dashed lines indicate the threshold for a positive response. Arrows indicate vaccine administration days. NEG, negative; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell; TNTC, too numerous to count. *P < .05.
T-Cell Responses in HSV-Seropositive Participants
Pairwise comparisons showed that individual median CD8+ T-cell responses at day 210 were not notably different from baseline for HSV1±/HSV2+ vaccinees (144 SFUs/106 PBMCs; IQR, 92–287 SFUs/106 PBMCs; P = .38) or HSV1+/HSV2− vaccinees (147 SFUs/106 PBMCs ; IQR, 20–323 SFUs/106 PBMCs; P = .41 ; Figure 5A). Only 1 of 13 HSV1±/HSV2+ participants (8%) and 2 of 11 HSV1+/HSV2− participants (18%) had a positive CD8+ T-cell response to HSV529, based on the study definition. Time course analyses (Figure 5A and Supplementary Table 7) showed steady CD8+ T-cell responses over time in both HSV-infected serogroups.
Figure 5.
Herpes simplex virus type 2 (HSV2)–specific T-cell responses after 3 doses of vaccine or placebo in HSV-seropositive participants. A, Results of pairwise testing of interferon γ (IFN-γ) enzyme-linked immunospot (ELISPOT) findings to determine changes in CD8+ T-cell responses between baseline (day 0) and after the third dose of HSV529 or placebo (day 210; left) and throughout the study period (right) among individuals who were positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+) and those who were positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). B, Results of pairwise testing of IFN-γ ELISPOT findings to determine changes in CD4+ T-cell responses to UV-inactivated HSV2 between baseline (day 0) and after the third dose of HSV529 or placebo (day 210; left) and throughout the study (right) in HSV1±/HSV2+ and HSV1+/HSV2− serogroups. For pairwise comparisons, each line represents samples from 1 subject, and short horizontal bars are medians. The rank sum test (2-sided) was used to calculate P values. For time courses, symbols represent median responses (interquartile ranges), and parametric testing with linear mixed models was used for analysis. Horizontal dashed lines indicate the threshold for a positive response. Arrows indicate vaccine administration days. NEG, negative; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell; TNTC, too numerous to count. *P < .05.
HSV2-specific CD4+ T-cell ICS assays showed that the HSV529 vaccine significantly increased CD4+ T-cell responses in HSV1±/HSV2+ and HSV1+/HSV2− participants at day 210, relative to baseline (P = .0005 and P = .001, respectively; Figure 5B). Only 6 of 13 (46%) and 3 of 11 (27%) in the HSV1±/HSV2+ and HSV1+/HSV2− groups, respectively, had increases meeting the positive CD4+ T-cell response definition. The median percentage of CD4+ T cells expressing ≥2 cytokines increased slightly on day 30 after the first vaccination (0.48%; IQR, .28%–.67%) relative to baseline (0.27%; IQR, .17%–.54%) in the HSV1+/HSV2– group (log10 change, 0.15; P = .01) and then remained relatively stable (Figure 5B and Supplementary Table 8). In the HSV1±/HSV2+ group, median CD4+ T-cell responses increased relative to the baseline value of 0.44% (IQR, .12%–.79%) and remained stable (Figure 5B and Supplementary Table 8).
COMPASS analysis, which measures functionality as combinations of T-cell activation markers in response to antigen stimulation, showed an increase in CD4+ T-cell functionality at day 210 (median, 0.815) as compared to baseline (median, 0.743) in 13 HSV1±/HSV2+ participants (P = .002) and a more modest increase in 10 HSV1+/HSV2− participants (0.740 vs 0.717; P = .021; Figure 6A). The median net percentage of CD4+ T cells from 13 HSV1±/HSV2+ participants that simultaneously produced IFN-γ, TNF-α, IL-2, and CD40L in response to HSV2 antigen increased between baseline (0.18%) and day 210 (0.23%; Padjusted = .009; Figure 6B). A similar increase for this combination of T-cell activation markers was not found for the HSV1+/HSV2- group (data not shown).
Figure 6.
COMPASS functionality scores of T-cell activation markers in response to antigen stimulation among individuals who were positive or negative for HSV1 and positive for HSV2 (HSV1±/HSV2+) and those who were positive for HSV1 and negative for HSV2 (HSV1+/HSV2−). A, Functionality score changes within individuals between day 0 (baseline) and day 210. Unadjusted P values were calculated using 1-sided Wilcoxon signed rank tests. B, Increase in polyfunctional CD4+ T cells (ie, those expressing CD40L, interferon γ [IFN-γ], interleukin 2 [IL-2], and tumor necrosis factor [TNF-α]) between days 0 and 210. The P value is adjusted for multiple comparisons among all 16 possible Boolean subsets, using the Bonferroni method. Short, horizontal black bars represent median values. Day 0, before vaccination (ie, baseline); day 210, 1 month after the third dose of vaccine.
DISCUSSION
We found that replication-defective HSV529 vaccine was safe and well tolerated. More injection site reactions occurred in vaccinated subjects as compared to placebo recipients; however, these reactions did not occur more frequently in a particular serogroup and did not result in refusal of additional doses of vaccine. A similar number of vaccine and placebo recipients experienced a solicited systemic reaction. Although immune correlates of protection against HSV2 genital herpes are unknown, neutralizing antibodies might be important for the prevention of primary HSV infection, as neutralizing antibodies in cord blood or blood from neonates ≤2 weeks old correlated with protection of newborns from HSV2 infection [19]. The HSV529 vaccine induced neutralizing antibody responses in HSV-seronegative individuals. Over one third of HSV-seronegative participants had a ≥4-fold rise in neutralizing antibody titer after 2 doses of HSV529, and the proportion doubled after the third dose. Three seronegative vaccine recipients did not have a ≥4-fold rise in neutralizing antibody titer. None of the participants in the other 2 groups had this level of increase in neutralizing antibody titer. There was a 6.1-fold increase in the GMT of neutralizing antibody 30 days after the third dose of HSV529 in HSV1−/HSV2− vaccine recipients (Supplementary Table 9). In comparison, sera from 30 HSV2-seronegative women who received the gD2 subunit vaccine in the Herpevac trial showed an approximately 1.45-fold increase in the GMT 30 days after the third dose [20, 21]. This comparison suggests a difference between the size of the neutralizing antibody response induced by a replication-defective vaccine versus that induced by a glycoprotein subunit vaccine, but this comparison is limited by the lack of a good clinical practice–validated neutralization assay. The HSV529 vaccine induced an increase in HSV2-specific CD8+ and CD4+ T-cell responses in a minority of HSV-seronegative subjects relative to baseline after 3 doses, and these responses were modest. Only 14% of HSV-seronegative participants had responses that met the definition of a CD8+ T-cell response, and 36% met the criteria for a CD4+ T-cell response. The median CD8+ T-cell response was near the level of the positive response threshold after 3 doses of HSV529 and did not reach the median responses measured in participants naturally infected with HSV2. The median HSV2-specific CD4+ T-cell responses were also below the positive response threshold at all time points after vaccination. We were not able to compare the CD4+ T-cell responses in the HSV-seronegative group to those of participants with natural HSV infection, because these responses were evaluated by different assays. While CD4+ T-cell responses were detected in seronegative women vaccinated with the gD2 subunit vaccine in the Herpevac trial, CD8+ T-cell responses were not detected [20, 22].
We found that some HSV-seronegative persons had HSV2-specific CD4+ and/or CD8+ T-cell responses detected by the ELISPOT assay at baseline (Figure 4A and 4B). Possible reasons for this phenomenon include cross-reactivity of memory T-cell responses to varicella zoster virus (VZV), prior exposure to HSV2 that elicited a T-cell response to the virus but did not result in infection or an antibody response, or inaccuracies in the serologic assay used to assign subjects to serogroups [23–26].
In the 2 HSV-seropositive groups, the HSV529 vaccine boosted the mean HSV2-specific CD4+ T-cell responses but not CD8+ T-cell responses. CD4+ T-cell responses increased 2.8-fold as compared to baseline after 3 doses of HSV529 in the HSV1±/HSV2+ group, and the CD4+ T-cells were polyfunctional (producing CD40L, IFN-γ, IL-2, and TNF-α), indicating that HSV529 may have potential as a therapeutic vaccine. A study evaluating whether vaccination with HSV529 increases immune responses in the skin at the site of genital lesions in volunteers with recurrent genital HSV2 lesions has been conducted, and final results are pending (clinical trials registration NCT02571166).
The only other replication-defective HSV2 vaccine evaluated in a Food and Drug Administration–approved, randomized, controlled trial was a disabled infectious single-cycle vaccine candidate with a deletion in the glycoprotein H gene. This vaccine was safe in immunocompetent patients with a history of recurrent genital herpes but had no clinical benefit, and quantitative immune responses to the vaccine were not reported [27].
This first-in-human study of the replication-defective HSV vaccine HSV529 showed that it was safe and well tolerated, with evidence of immunogenicity in HSV-naive and previously infected subjects. The most prominent antibody and T-cell responses were induced in HSV-naive subjects, with more-modest responses in subjects previously infected with HSV, indicating that this vaccine may have potential as a prophylactic or a therapeutic vaccine. Modifications of HSV529, such as increasing expression of certain viral proteins, inhibiting expression of viral immune evasion genes, or adding an adjuvant, might improve its immunogenicity.
SUPPLEMENTARY DATA
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. We thank the study volunteers; the research nurses (Carla Williams, Melissa Law, Vivien Agbakoba, and Jenny Ahn); the personnel in the Immunological Monitoring Laboratory, National Cancer Institute at Frederick (Luz Fuentes and Yanmei Wang); Gary Fahle in the National Institutes of Health Clinical Laboratory, for performing HSV PCR analysis of anogenital swabs; Amalia S. Magaret, for review of time course statistics; members of the Office of Clinical Research Policy and Regulatory Operations, for regulatory and monitoring assistance; and members of The Visual and Medical Arts Unit at Rocky Mountain Laboratories, for assistance with the figure panels.
Disclaimer. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.
Financial support. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID); the NIAID and Sanofi Pasteur (clinical trial agreement); and the National Cancer Institute, National Institutes of Health (contract HHSN261200800001E).
Potential conflicts of interest. K. J. L. holds a patent and patent applications related to HSV epitopes. D. M. K. has received research funding from Admedus and Immune Design and has served as a consultant for HSV vaccines to Biomedical Research Models. A. C. and L.-J. C. are employees of Sanofi Pasteur. S. P. was an employee of Sanofi Pasteur at the time of the study. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
Presented in part: International Herpesvirus Workshop, Ghent, Belgium, 29 July 2017; ID Week, San Diego, California, 4–8 October 2017
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