An adjuvanted herpes simplex virus 2 subunit vaccine elicits a T cell response in mice and is an effective therapeutic vaccine in Guinea pigs

Abstract

Immunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2ΔTMR340-363) and a truncated form of infected cell polypeptide 4 (ICP4383-766), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4(+) and CD8(+) T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003- or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease.

Fig 1

SDS-PAGE analysis of purified recombinant proteins. After purification from baculovirus-infected cell cultures, antigens were electrophoresed through a 4 to 20% Tris-glycine polyacrylamide gel under reducing conditions and visualized by Coomassie blue staining (A) or Western blot analysis with either an ICP4-specific polyclonal antiserum (B) or a gD-specific monoclonal antibody (C). Prestained molecular mass markers were run on the same gel. Lane 1, ICP4_383-766 (300 ng); lane 2, gD2ΔTMR (300 ng); lane 3, ICP4_383-766 mixed with gD2ΔTMR (300 ng each).

Fig 2

Antibody response to GEN-003/MM-2 vaccine formulation. C57BL/6 mice (n = 15) were immunized s.c. three times at three-week intervals with the GEN-003/MM-2 vaccine formulation (2 μg of each antigen plus 20 μg MM-2). Five mice per group received a fourth immunization 44 days after the third immunization. Serum samples were taken from five mice by cardiac puncture seven and 55 days after the third immunization and four days after the fourth immunization. (A) Serum samples were serially diluted 5-fold, and antibody titers to ICP4_383-766 and gD2ΔTMR protein were measured by endpoint ELISA. IgG1 and IgG2c endpoint titers are shown as the mean ± the standard error of the mean (SEM) per group. (B) Neutralizing antibody titers to virus were determined using a colorimetric assay as described in Materials and Methods. Titers are shown as the mean ± SEM titer that resulted in 50% neutralizing activity per GEN-003/MM-2 vaccine-immunized group. No ELISA or neutralizing antibody titers were detected in serum from naïve mice or MM-2 adjuvant-immunized mice. P values were calculated by Student's t test. Data are representative of two experiments. NS, not significant.

Fig 3

Cellular responses elicited in mice following immunization with HSV-2 antigens plus MM-2 adjuvant. (A) ELISPOT analysis of IFN-γ secretion by CD8⁺ T cells elicited in C57BL/6 mice (n = 3) vaccinated twice, nine days apart, with 10 μg of ICP4_383-766 and 5 μg of gD2ΔTMR with 12 μg of MM-2 (GEN-003/MM-2) or 10 μg of ICP5 plus 12 μg of MM-2. One week after the second immunization, sorted CD8⁺ T cells were restimulated with naïve splenocytes pulsed with the indicated protein plus MM-2 and washed extensively. (B) ELISPOT analysis of IFN-γ secretion by CD4⁺ and CD8⁺ T cells from mice vaccinated twice, three weeks apart, with 2 μg of GEN-003 plus 24 μg of MM-2 adjuvant. Seven days after the second immunization, spleens were harvested, and sorted T cells were restimulated via naïve splenocytes pulsed with the indicated overlapping peptide (OLP) pools. IFN-γ production is represented by the mean number of spot-forming units (SFU) per 2 × 10⁵ T cells from three assay replicates ± SEM. (C) Supernatants obtained in the ELISPOT assay in panel B were evaluated for the presence of TNF-α, IL-2, IL-4, and IL-6 by cytometric bead array (CBA). CD4⁺ and CD8⁺ T cell responses to antigen-specific and irrelevant OLPs, respectively, are shown as the mean cytokine concentration (pg/ml) of triplicate wells ± the standard deviation (SD). Data are representative of three replicate experiments.

Fig 4

Antigen-specific in vivo cytotoxicity in GEN-003/MM-2 vaccine-immunized mice. (A) Splenocytes derived from naïve mice were pulsed with gD2 or ICP4_383-766 OLPs and then separately labeled with CFSE at 1 μM and 4 μM CFSE, respectively. As negative controls, unpulsed naïve splenocytes were labeled with 250 nM CFSE. CFSE-labeled splenocytes were analyzed by flow cytometry to ensure appropriate separation of peaks. (Left) Dot plot shows the live gate (R1) in which 1 × 10⁵ events were acquired. (Right) Histogram shows gated events evaluated for fluorescence intensity of the respective CFSE-labeled populations. (B) Five C57BL/6 mice per group were immunized three times on days 0, 21, and 42 with 2 μg of each antigen and 10 μg or 20 μg of MM-2. On day 48, equal mixtures of each of the pulsed and CFSE-labeled target populations were adoptively transferred into the GEN-003/MM-2 vaccine-immunized mice. After 16 h, mice were euthanized and splenocytes were analyzed by flow cytometry. (Left) Dot plot shows the live gate (R1) in which 1 × 10⁵ events were acquired. (Right) Histogram shows gated events evaluated for fluorescence intensity of the respective CFSE-labeled populations. Peaks represent unpulsed cells (a), gD2 OLP-pulsed cells (b), and ICP4_383-766 OLP-pulsed cells (c). Representative data from one mouse are shown. (C) In vivo cytolytic activity for individual mice against each of the OLP-pulsed targets relative to the unpulsed controls is shown as the mean percentage (%) of specific lysis ± SEM. Data are representative of 2 similar experiments. (D) One group of mice (n = 6) was immunized subcutaneously (s.c.) three times, three weeks apart, with 2 μg of each antigen and 20 μg of MM-2; the other group (control; n = 5) did not receive vaccinations. The mice were rested until day 86, when they all received a recall immunization. Five days later, CFSE-labeled and OLP-pulsed target cells were generated as described above and adoptively transferred into all mice. After 16 h, mice were euthanized and splenocytes were analyzed for the presence of CFSE-positive (CFSE⁺) cells by flow cytometry as previously described. Values represent specific lysis ± SEM of target cells in control and immunized mice. P values were calculated by comparing immunized and control groups using two-way Student's t test.

Fig 5

Antibody responses of HSV-2-infected guinea pigs vaccinated with GEN-003 or GEN-003/MM-2 vaccine. Guinea pigs were infected intravaginally with 5 × 10⁵ PFU of HSV-2 strain MS, allowed to recover from primary disease, and then vaccinated s.c. three times on days 14, 21, and 36 postinfection with PBS, 15 μg of each antigen only (GEN-003), or 15 μg of each antigen plus 50 μg MM-2 adjuvant (GEN-003/MM-2). (A) Antibody titers in plasma samples (n = 4) to ICP4_383-766 or gD2ΔTMR were measured by endpoint ELISA at day 42 postinfection (six days after the third immunization). Data are shown as the mean ELISA endpoint titer of each group ± SEM. (B) Neutralizing antibody titers were determined by a colorimetric assay as described in Materials and Methods. The titer was defined as the reciprocal of the serum dilution required to decrease the OD₅₉₀ obtained using the virus control by 50%. Data are shown as the mean neutralizing antibody titer of each group ± SEM. P values compare the GEN-003-immunized group to the GEN-003/MM-2-immunized group or to the PBS control group using Student's t test. (C) Antibody titers in plasma samples (n = 4) to ICP4_383-766 or gD2ΔTMR were measured by endpoint ELISA at day 42 postinfection (six days after the third immunization). P values compare the GEN-003/MM-2-vaccinated group to the MM-2 control group using Student's t test.

Fig 6

Therapeutic treatment of infected guinea pigs with HSV-2 antigens or GEN-003 vaccine. Guinea pigs were infected intravaginally with 5 × 10⁵ PFU of HSV-2 strain MS, allowed to recover from primary disease, and then vaccinated s.c. three times on days 14, 21, and 36 postinfection. (A and B) PBS (n = 9), 50 μg of MM-2 alone (n = 12), and 15 μg of each antigen alone plus 50 μg of MM-2 adjuvant (n = 9 to 11) (Table 1, experiment 1). (A) Cumulative mean lesion severity scores (as described in Materials and Methods) are shown for each group. (B) The number of days with a recurrent lesion is shown. Each symbol indicates an individual animal from the same group. The P value compares the PBS control group to each antigen-immunized group using unpaired, two-tailed Student's t test. (C and D) Fifteen micrograms of each antigen (GEN-003) or 15 μg of each antigen plus 50 μg of MM-2 (GEN-003/MM-2) (Table 1, experiment 2). (C) Cumulative mean lesion severity scores (as described in Materials and Methods) are shown for each group: PBS (n = 13), GEN-003 (n = 14), and GEN-003/MM-2 (n = 11). (D) The number of days with a recurrent lesion is shown. Each symbol indicates individual animals from the same group. The P value compares the PBS control group to the GEN-003 or GEN-003/MM-2 vaccine-immunized group using unpaired, two-tailed Student's t test. (E and F) Fifty micrograms of MM-2 or 15 μg of gD2ΔTMR plus 15 μg of ICP4_383-766 with 50 μg of MM-2 (GEN-003/MM-2). (E) Cumulative mean lesion severity scores are shown for each group: MM-2 (n = 19) and GEN-003/MM-2 (n = 20). (F) The number of days with a recurrent lesion is shown. Each symbol indicates an individual animal from the same group. P values compare the GEN-003/MM-2-vaccinated group to the MM-2 control group using unpaired, two-tailed Student's t test.