Efficacy of the Herpes Simplex Virus 2 (HSV-2) Glycoprotein D/AS04 Vaccine against Genital HSV-2 and HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus Model

ABSTRACT

Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection.

IMPORTANCE In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK HSV-2 vaccine Simplirix (gD/AS04) to protect humans against HSV-2 and the surprising finding that the vaccine protected against HSV-1 genital herpes instead. In this study, we report that gD/AS04 has higher efficacy against HSV-1 compared to HSV-2 genital herpes in the novel DMPA-synchronized cotton rat model of HSV-1 and HSV-2 infection. The findings help explain the results of the Simplirix trial.

INTRODUCTION

The disease burden associated with herpes simplex virus 2 (HSV-2) infection, an important cause of genital herpes worldwide, is high and presents an additional threat due to its association with an increased risk of HIV acquisition and transmission (1–3). Globally, HSV-2 is a major health threat, with 16% of the U.S. population infected by age 30 and 50 to 90% of the population in sub-Saharan Africa infected (3–5). Recent reports emphasize the importance of HSV-1 as an important etiologic agent of genital herpes particularly in the United States and other developed countries (6–8). Seroprevalence of HSV-1 among 14- to 49-year-olds in the United States reached 53.9% in 2005 to 2010 (5). No effective vaccine is available to prevent the acquisition or spread of either HSV serotype.

Vaccine efforts focus on subunit vaccines that include viral envelope glycoproteins alone or in combination with other structural and nonstructural viral proteins, replication-defective viruses, and vectored and peptide-based vaccines (9–11). Subunit vaccines based on the HSV-2 glycoprotein D (gD-2) were advanced to clinical trials based on efficacy in small animal models (12–14). However, the results of the most recent gD-2 subunit vaccine clinical trials were unexpected (9, 15). Two double-blind randomized phase 2 studies of the gD-2 vaccine Simplirix (GlaxoSmithKline), containing aluminum salt and MPL adjuvants (gD/AS04), found 73 and 74% efficacies, respectively, against genital disease in HSV-discordant couples in women who were seronegative for both HSV-1 and HSV-2 but no protection in women who were seropositive for HSV-1 at enrollment or in men (16). The subsequent phase 3 trial, which was conducted among 8,323 women 18 to 30 years of age who were seronegative for both HSV-1 and HSV-2 at enrollment, revealed that gD/AS04 vaccine was not effective against HSV-2 genital herpes disease despite inducing HSV-specific enzyme-linked immunosorbent assay (ELISA) and neutralizing antibodies (15). However, the vaccine did provide 58% (95% confidence interval, 12 to 80%) protection against HSV-1 genital disease (15). These findings highlight the need for new preclinical models that may prove more predictive of vaccine trial outcomes.

The majority of preclinical HSV-2 gD-2 vaccine-challenge studies were conducted in Hartley guinea pigs (12–14). These studies found that the vaccine elicits strong gD-2-specific ELISA and neutralizing antibody responses in the serum (15). However, although vaccination reduced acute, recurrent, and latent HSV-2 infection in guinea pigs (12–14), these outcomes did not translate into protection in the clinical trial. To address this limitation, we evaluated the gD/AS04 vaccine in a cotton rat Sigmodon hispidus model of HSV-1 and HSV-2 genital tract disease. We selected this model because studies with other pathogens, including poliovirus, adenovirus, respiratory syncytial virus, influenza virus, measles virus, and rhinovirus indicate that the cotton rat may closely recapitulate human disease (17–20). Although the vaccine induced gD-2-specific and HSV-2 neutralizing antibodies in the serum of cotton rats, it provided only partial protection against HSV-2 disease and better protected animals against HSV-1-induced genital herpes with respect to both viral replication and disease. Better protection against HSV-1 compared to HSV-2 disease could also be conferred by passive transfer of serum from gD/AS04-immunized animals.

MATERIALS AND METHODS

Cells and virus.

HSV-2(G) and HSV-1(17) were grown on Vero cells and stored at −80°C at a concentration of ∼10⁸ PFU/ml. Viruses were diluted in phosphate-buffered saline (PBS; pH 7.4) to the appropriate concentration within an hour of infection and maintained on ice.

Reagents and vaccines.

Depot medroxyprogesterone acetate (DMPA) as an injectable suspension (DepoProvera, 150 mg/ml; GreenStone NDC 59762-4537-1) was obtained from Blue Door Pharma. gD/AS04 vaccine (each 0.5-ml vaccine dose contains 20 μg of gD-2 antigen, 50 μg of MPL, and 500 μg of alum), FENDrix vaccine (each 0.5-ml dose contains 20 μg of hepatitis B surface antigen, 50 μg of MPL, and 500 μg of alum), and recombinant gD-2 antigen for ELISA (375 μg/ml) were kindly provided by GlaxoSmithKline Vaccines. Partially inactivated HSV-2 was prepared by exposing 10⁸ PFU of HSV-2(G)/ml to UV light for 2 min (UV-HSV). Completely inactivated HSV-2 was prepared by exposing the virus to a 63°C water bath for 30 min (heat-inactivated HSV-2 [HI-HSV]). The UV-treated virus had a reduction of infectious titer from 10⁸ PFU/ml prior to inactivation to ∼10⁶ PFU/ml, and the heat-inactivated virus had no detectable infectivity in a 50% tissue culture infective dose assay on Vero cells.

Animals.

Inbred S. hispidus cotton rats were obtained from a colony maintained at Sigmovir Biosystems, Inc. Six- to eight-week-old female cotton rats were used for the studies. Animals were housed in large polycarbonate cages and fed a standard diet of rodent chow and water. The colony was monitored for antibodies to adventitious respiratory viruses and other common rodent pathogens, and no such antibodies were found. All studies were conducted under applicable laws and guidelines and after obtaining approval from the Sigmovir Biosystems Institutional Animal Care and Use Committee.

Animal studies. (i) Establishing DMPA-synchronized model of HSV-1 and HSV-2 genital infection.

Cotton rats were inoculated subcutaneously (s.c.) under isoflurane anesthesia with 50 μl of DMPA solution in different concentrations. After 4 days, the animals were anesthetized with a ketamine-xylazine by intramuscular (i.m.) injection a mixture of five parts ketamine at 100 mg/ml and one part xylazine at 33 mg/ml (100 μl/100 g of body weight). The vaginal vaults of the animals were cleaned with sequential infusions and aspirations of PBS and wiped with the cotton tip applicator (infection with washing). Vaginal smears were prepared, stained with Diff-Quik, and evaluated by microscopy to define estrous cycle stage at the time of viral challenge. A 100-μl portion of virus solution was instilled intravaginally. For studies on the protective effect of vaginal wash microenvironment, the animals were infected without vaginal vault precleaning. Lesion formation and mortality after infection were monitored for several weeks. Animals of morbid appearance were euthanized. Samples for viral titrations were collected by washing the vaginal tract of animals under isoflurane anesthesia with 400 μl Eagle minimal essential medium (EMEM) supplemented with gentamicin, amphotericin B (Fungizone), and a 0.2 M sucrose solution. Animals for study of the effect of natural estrus versus diestrus were individually selected from age-matched cohort based on results of vaginal smear Diff-Quik staining on the day of the experiment.

(ii) Vaccine studies.

Cotton rats were anesthetized with isoflurane, prebled, and inoculated i.m. into a hind leg with 100 μl of gD/AS04 vaccine at 2, 0.3, or 0.06 μg (corresponding to 1:2, 1:12, and 1:72 dilutions, respectively, of gD/AS04 for both antigen and adjuvants) per animal (∼100 g of body weight). Control groups of animals were immunized i.m. with 100 μl of FENDrix (1:2 dilution of vaccine) or 100 μl of PBS (pH 7.4) per 100 g of body weight. UV-HSV or HI-HSV was administered as an i.m. injection of virus solution corresponding to an infectious dose of 10⁵ PFU per animal prior to inactivation. Three weeks later, the animals were eyebled and boosted with the same vaccine formulation as given on day 0. Three weeks after the booster vaccination, the animals were eyebled, and vaginal washes were collected using 400 μl of PBS and stored at −80°C. The animals were inoculated s.c. with DMPA (50 μl of 15 mg/ml per 100-g animal) and 4 days later inoculated intravaginally with 5 × 10⁴ PFU of HSV-2 or 2 × 10⁶ PFU of HSV-1 per animal under ketamine-xylazine anesthesia. Lesion formation and mortality after infection were monitored for several weeks, with differences between groups evaluated by log-rank test. Animals with a morbid appearance were euthanized. Samples for viral titrations were collected by washing the vaginal tracts of animals with EMEM-gentamicin-amphotericin B-sucrose as described above. Follow-up studies with HI and UV-treated virus were conducted according to the same immunization protocol except that the interval between primary vaccination and boost was 2 weeks, followed by 2.5 weeks between boost and HSV-2 challenge to accommodate a shorter experimental frame.

(iii) Passive-transfer studies.

Serum for passive-transfer studies was prepared by immunizing cotton rats with gD/AS04 (i.m. 100 μl containing 2 μg of gD-2), boosting animals twice with the same preparation at 3 and 5 weeks after the initial immunization, and terminally bleeding the animals 1 week after the last immunization for serum collection. Control serum was obtained from age-matched unmanipulated cotton rats. Control or gD/AS04 serum was inoculated i.m. into DMPA-treated animals via two injections in both hind legs, 200 μl per injection site, 24 h prior to intravaginal infection with HSV-1 (2 × 10⁵ PFU per animal) or HSV-2 (2 × 10⁴ PFU per animal). Lesion formation and mortality were monitored for 2.5 weeks after infection. Vaginal washes were collected on day 2 postinfection for HSV-1 and HSV-2 titration by plaque assay.

All animal studies were repeated at least once, and the results of representative experiments are shown.

Virus titrations.

Vaginal washes were assayed for the presence of infectious HSV by titering them on Vero cells by plaque assay. In brief, vaginal washes were clarified by centrifugation and diluted in EMEM. Confluent Vero monolayers were infected in duplicate with diluted homogenates in 24-well plates. After 1 h of incubation at 37°C in a 5% CO₂ incubator, the cells were overlaid with 0.75% methylcellulose medium. After 2 days of incubation, the overlay was removed, and the cells were fixed with 0.1% crystal violet stain for 1 h and then rinsed and air dried. Plaques were counted, and the virus titer was expressed as the PFU/ml of vaginal wash, with differences between groups evaluated by using the Student t test.

Preparation of extracts of HSV-2-infected cells for ELISA.

Vero cells were propagated in EMEM supplemented with 10% fetal bovine serum (FBS), l-glutamine, gentamicin, and amphotericin B and infected at subconfluency with HSV-2 at a multiplicity of infection of 0.01. After 18 h of infection, the cells were washed with 10 ml of Tris-NaCl buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl) with 2 mM EDTA and lysed on ice in-flask with 1% IGEPAL in Tris-NaCl buffer supplemented with 0.2 mM phenylmethylsulfonyl fluoride. The cell solution was collected into a prechilled 50-ml conical tube, subjected to one cycle of freeze-thawing, and centrifuged for 15 min at 4,000 × g at 4°C. Supernatants were filtered through the 70-μm-pore-size nylon sieve and centrifuged for 30 min at 4,500 rpm at 4°C. The protein solution was transferred to SnakeSkin pleated dialysis tubing (Pierce; molecular-weight cutoff, 10,000) and dialyzed against two changes of PBS (pH 7.4) at 4°C. The dialyzed solution was cleared by an additional centrifugation for 30 min at 4,000 × g at 4°C, divided into aliquots, and stored at −80°C until use. Extracts of mock-infected Vero cells were prepared according to the same protocol, except that no virus was used during the infection step.

ELISA for gD-2 and total HSV-2 antigens.

ELISA for gD-2-specific IgG was performed on 96-well Immulon-B microplates coated with recombinant gD-2 (provided by GSK) at 1 μg/ml. Serum IgG was detected with chicken anti-cotton rat IgG (Immunology Consultants Laboratory) at 0.4 μg/ml, followed by horseradish peroxidase (HRP)-conjugated goat anti-chicken IgG (KPL) at 50 ng/ml. The signal was detected with SureBlue TMB substrate (KPL). The amount of anti-gD-2 IgG in each sample was calculated based on the standard curve of serially diluted serum collected from animals immunized with 2 μg of D/AS04 twice (gD-2 ELISA serum standard), with an amount of anti-gD-2 IgG in a 1:500 dilution of standard serum sample equal to 1,000 U.

For total HSV-2 protein ELISA, the plates were coated with the extracts from HSV-2-infected Vero cells diluted 1:500 in coating buffer (KPL). Chicken anti-cotton rat IgG and HRP-conjugated goat anti-chicken IgG were used at 0.4 μg/ml and 50 ng/ml, respectively. Signal was detected using SureBlue TMB substrate. The amount of anti-HSV IgG in each sample was calculated based on the standard curve obtained with sera from animals vaccinated twice with UV-HSV i.m. (total HSV-2 protein ELISA serum standard), with the amount of anti-HSV IgG in a 1:500 dilution of standard serum sample equal to 1,000 U. Assay conditions for total HSV-2 protein ELISA were optimized by testing total HSV-2 protein ELISA serum standard sera on plates coated with extracts of HSV-2- or mock-infected Vero cells. Assay conditions minimizing background reading arising from wells coated with mock-infected cells were selected. All experimental sera or vaginal wash samples were tested in duplicate.

Neutralizing antibody assay.

Serial dilutions of serum or vaginal washes were mixed with 10⁵ PFU of HSV-2/ml, followed by incubation for 1 h at 24°C, and then inoculated onto subconfluent monolayers of Vero cells in 96-well plates. After 1 h of incubation, the inoculum was removed, and the cells were washed with PBS, overlaid with EMEM supplemented with 10% FBS, l-glutamine, gentamicin, and amphotericin B, and returned to the incubator for 24 h. The neutralizing titer was expressed as the reciprocal of the highest dilution of serum at which the infectivity was completely neutralized (no cytopathic effect visible) in 50% of the wells.

RESULTS

Establishing HSV-2 genital herpes model in DPMA-synchronized cotton rats.

Hormonal fluctuations during normal menstrual cycle cause changes in mucosal immunity that may predispose to genital viral infections. Increased susceptibility to infection with human immunodeficiency virus (HIV) or simian HIV (SHIV) was noted to occur during the time after ovulation corresponding to the diestrus stage of the reproductive cycle in rodents (21, 22). The injectable medroxyprogesterone acetate formulation DepoProvera (DMPA) modulates menstrual cycle in humans and was shown to induce diestrus and to predispose small animals to more severe HSV-2 infection (23). DMPA has not been tested in cotton rats before. To investigate the efficacy of DMPA in cotton rats and to define the optimal dose of the drug inducing diestrus, groups of animals in the proestrus/estrus stage (referred to as estrus) were identified and treated with different doses of DMPA. The stage of the estrus cycle was monitored by cytological analysis of vaginal swabs from each individual animal (Fig. 1). DMPA in the amount of 7.5 and 1.5 mg per animal induced diestrus in all cotton rats within a day of administration, but the effect was too long-lasting at 4 and 3 weeks, respectively. A DMPA dose of 0.3 mg per animal induced diestrus for 3 to 6 days. The DMPA dose of 0.75 mg per animal, or ∼7.5 mg/kg, was selected for all subsequent studies. The 7.5-mg/kg dose is slightly larger than that used in humans (∼2 mg/kg) but is experimentally relevant since it induced diestrus in cotton rats for at least 1 week, the period needed to reliably accommodate the 4-day time window between DMPA treatment and HSV infection.

FIG 1.

Induction of diestrus in cotton rats (S. hispidus) by DMPA. Young female cotton rats were inoculated with DMPA s.c. at 0.3, 1.5, or 7.5 mg per animal. (A) The stage of the estrus cycle was monitored at various times after treatment by cytological analysis of vaginal swabs from each individual animal. E, estrus; D, diestrus. (B) Photomicrographs of vaginal smears stained with Diff-Quik, exemplifying estrus and diestrus. The inset image on the diestrus panel provides a close-up of neutrophils, which are abundant in the vaginal vault secretions during diestrus.

To define the range of HSV-2 doses capable of inducing genital disease in DMPA-treated animals, the animals were inoculated with DMPA and infected 4 days later with various doses of HSV-2, ranging from 10¹ to 5 × 10⁴ PFU per animal. The vaginal vaults of all animals were washed prior to virus inoculation to remove any excess mucus that could have interfered with the infection. Infection with 5 × 10⁴ PFU of HSV-2 resulted in lesions forming on days 4 to 7 postinfection and 100% mortality within a week of infection (Fig. 2A). Lesions usually appeared as the inflamed areas of the skin with occasional groups of blisters close to the opening of the vaginal vault. Self-scarification commonly ensued, followed by the spontaneous death of the animal or the need for it to be euthanized. Mortality decreased and the time to the onset of lesions increased progressively with the dose reduction. No mortality was seen in animals infected with 10¹ PFU of HSV-2. In all cases, lesion formation was associated with ensuing mortality. More than 3 log₁₀ PFU of HSV-2/ml was detected in vaginal secretions collected 2 days after infection from groups of animals with no survival (animals infected with 5 × 10⁴ and 10³ PFU/animal) (Fig. 2B). An ∼10-fold-smaller amount of virus was recovered from animals with ∼20% survival (animals infected with 10² PFU/animal), and no virus was detected in vaginal washes of the group that survived HSV-2 infection (10¹ PFU per animal infectious dose). The viral load was lower on day 4 postinfection and was undetectable on day 7. HSV-2 was recovered from vaginal washes of all animals with clinical signs of disease but not from cotton rats without disease signs.

FIG 2.

HSV-2 genital herpes in the cotton rat model: dose dependency study. Animals were pretreated with DMPA and 4 days later inoculated intravaginally with different doses of HSV-2 after vaginal vault precleaning (“washing”). (A) Lesion formation and mortality were monitored for several weeks after infection as described above. “0” indicates no lesion, “1” indicates a lesion(s) was detected, and a shaded box indicates animal death. The same notation applies to all subsequent figures. (B) Vaginal washes were collected on days 2 and 4 postinfection, and the HSV-2 load was quantified by plaque assay. The results represent means ± the standard errors (SE) for five animals per group. The asterisk indicates P < 0.05 compared to the uninfected group.

Establishing HSV-1 genital herpes model in DMPA-synchronized cotton rats.

A dose-response study was conducted for HSV-1 by infecting animals intravaginally with 2 × 10⁴ to 2 × 10⁶ PFU of HSV-1(17) after DMPA pretreatment and vaginal vault washing. HSV-1 in the dose of 2 × 10⁶ PFU/animal induced 100% mortality in cotton rats (Fig. 3A). Disease severity, however, appeared to be less than that seen for animals infected with HSV-2 at 5 × 10⁴ PFU, since lesions in this group of animals persisted for up to 2 weeks before animal death. Less self-scarification was seen in HSV-1-infected animals compared to HSV-2-infected animals, and the lesions showed a tendency for being located more in the anogenital area. Clinical manifestation was proportional to the infectious virus dose, with doses of 2 × 10⁵ and 2 × 10⁴ PFU/animal inducing less mortality. Lesion formation was not always associated with lethal outcome, as was seen for HSV-2 genital herpes. Recovery of some animals infected with the lower doses of HSV-1 was seen between the first and the second month postinfection, with some fresh new lesions detected 2 months after the initial HSV-1 challenge indicative of reactivation. Reactivation had milder disease manifestation and did not lead to mortality. HSV-1 load in vaginal washes of infected animals with disease of comparable severity was ∼2 log higher for HSV-1 than for HSV-2 (compare Fig. 3B and 2B). Similar to HSV-2, the viral load was lower on day 4 than on day 2 postinfection but was still significantly elevated. Clearance was seen by day 7 postinfection.

FIG 3.

HSV-1 genital herpes in the cotton rat model: dose dependency study. Animals were pretreated with DMPA and 4 days later washed and inoculated intravaginally with different doses of HSV-1. (A) Lesion formation and mortality were monitored for 1 month after infection as described above. Additional clinical observation was made ∼1.5 month later (day 77). “N1” designates a “new lesion.” (B) Vaginal washes were collected on days 2 and 4 postinfection, and the HSV-1 load was quantified by plaque assay. The results represent the means ± the SE for four to eight animals per group. The asterisk indicates P < 0.05 compared to uninfected group.

Importance of DMPA for susceptibility of cotton rats to HSV-1 and HSV-2 infection.

To verify the importance of DMPA treatment for supporting uniform and consistent genital infection and disease in cotton rats, animals in DMPA-induced diestrus were infected with HSV-1 or HSV-2 and compared to animals infected with the same dose of the virus in their natural diestrus or estrus. For HSV-2, inoculation with 5 × 10⁴ PFU of virus per animal resulted in 100% infection and mortality of animals in diestrus, both natural and DMPA induced (Fig. 4). Infection of animals in natural estrus was less efficient, with two of eight animals succumbing to the disease. Viral loads in vaginal washes of animals in natural diestrus and DMPA-induced diestrus were comparable but were reduced in animals infected in estrus (Fig. 4B).

FIG 4.

Effect of estrus cycle stage and DMPA pretreatment on HSV-2 infection in the cotton rat model. Animals were inoculated with 0.75 mg of DMPA s.c. and 4 days later washed and infected with HSV-2 at 5 × 10⁴ PFU per animal. Cotton rats in their natural estrus or diestrus stage were identified and infected with the same dose of HSV-2. (A) Lesion formation and mortality were monitored for several weeks after infection. The asterisk indicates P < 0.05 compared to the group infected in DMPA-induced diestrus. (B) Vaginal washes were collected on days 2 and 4 postinfection, and the HSV-2 load was quantified by plaque assay. The results represent the means ± the SE for eight animals per group. The asterisk indicates P < 0.05 compared to the group infected in DMPA-induced diestrus.

HSV-1 genital herpes in cotton rats was also dependent on the reproductive cycle stage at the time of infection. Animals in diestrus were more susceptible to HSV-1 genital infection than animals in estrus (Fig. 5). Both natural and DMPA-induced diestrus predisposed to HSV-1 genital herpes, although significantly higher disease mortality was seen in animals pretreated with DMPA (88% versus 33% mortality in groups with DMPA-induced versus natural diestrus). This was accompanied by significantly elevated HSV-1 titers in vaginal washes. In contrast to HSV-2, no animals infected during natural estrus stage were susceptible to genital HSV-1 infection, and no virus was detected in vaginal wash samples from any of the animals infected with HSV-1 in estrus. Thus, HSV-1 genital infection appears to be more dependent on the DMPA treatment and the correct stage of the estrous cycle than HSV-2 infection.

FIG 5.

Effect of estrus cycle stage DMPA pretreatment on HSV-1 infection in the cotton rat model. Cotton rats identified to be in their natural estrus or diestrus or treated 4 days earlier with DMPA to induce diestrus were washed and infected intravaginally with HSV-1 at 2 × 10⁶ PFU per animal. (A) Lesion formation and mortality were monitored for a month after infection as described above. The asterisk indicates P < 0.05 compared to the group infected in DMPA-induced diestrus. (B) Vaginal washes were collected on days 2 and 4 postinfection, and the HSV-1 load was quantified by plaque assay. The results represent the means ± the SE for six to eight animals per group. The asterisk indicates P < 0.05 compared to the group infected in DMPA-induced diestrus.

HSV-1 and HSV-2 genital herpes in animals infected without vaginal vault washing.

Challenge studies were conducted next without vaginal vault precleaning (“washing”) immediately prior to infection with HSV-2 or HSV-1. The experiments described above were conducted using such washing to establish robust, uniform models of HSV-2 and HSV-1 genital herpes. However, washing was contraindicated for vaccine studies, since it could mask mucosal immunity induced by vaccination.

For experiments on infection without washing, challenge doses inducing 100% mortality in washed animals (5 × 10⁴ PFU of HSV-2 and 2 × 10⁶ PFU of HSV-1 per animal) were selected. HSV-2 genital herpes was only moderately affected by washing. In both cases (infection with or without washing), 100% mortality was seen by day 11 postinfection (Fig. 6A). The onset of mortality was slightly earlier for animals infected after washing compared to animals infected without washing (5/8 versus 3/8 dead within a week, respectively). A slightly higher viral load was detected in the vaginal washes of animals infected with HSV-2 after washing (Fig. 6B), but the difference failed to reach statistical significance. For HSV-1, washing of the vaginal tract prior to infection had a more profound effect on ensuing disease. All animals infected after washing were dead within a month postinfection. In contrast, 50% of the animals infected without washing survived infection within the same time frame (P < 0.05). Moreover, an ∼2-log₁₀-higher viral load was detected in animals infected with HSV-1 after washing than in animals infected without washing (P < 0.05). Thus, it appears that vaginal vault secretions are more important for guarding against HSV-1 than HSV-2 genital herpes and that more severe HSV-1 infection and disease is seen when these secretions are removed prior to infection. Although having a strong HSV-1 disease would be beneficial for evaluation of vaccine efficacy, all subsequent vaccine studies were performed without washing of the vaginal vault prior to infection in order to preserve mucosal immunity.

FIG 6.

Effect of vaginal vault precleaning on HSV-1 and HSV-2 genital herpes. DMPA-treated cotton rats were intravaginally inoculated with HSV-2 (5 × 10⁴ PFU per animal) or HSV-1 (2 × 10⁶ PFU per animal) with or without vaginal vault washing immediately prior to infection. (A) Lesion formation and mortality were monitored for 1 month after infection as described above. The asterisk indicates P < 0.05 compared to the group infected with the same virus after washing. (B) Virus titers were quantified in vaginal washes collected on days 2 and 4 postinfection and expressed as the means ± the SE for eight animals per group. The asterisk indicates P < 0.05 compared to the group infected with the same virus after washing.

Efficacy of gD/AS04 vaccine against HSV-2 genital herpes in DMPA-synchronized cotton rats S. hispidus.

To investigate gD/AS04 efficacy in the HSV-2 challenge model, female cotton rats were immunized i.m. with different doses of gD/AS04 twice with an interval of 21 days between the doses and between the second dose and DMPA treatment and then 4 days later were intravaginally challenged with 5 × 10⁴ PFU of HSV-2 per animal. gD/AS04 vaccine doses ranged from 0.06 to 2 μg per animal. PBS- or FENDrix-immunized animals served as a negative control.

All of the PBS- or FENDrix-treated animals succumbed to HSV-2 disease, whereas gD/AS04 provided no protection at the lowest doses (0.06 and 0.3 μg/ml) tested but provided partial protection (2/5 cotton rats had no lesions) from disease when administered at the highest dose of 2 μg per animal (Fig. 7A). This high dose of vaccine protected ca. 50 to 60% of the immunized animals against mortality in two independent studies. Notably, two of the gD/AS04-immunized animals (90644 and 90651) developed lesions but then recovered. In contrast, all of the PBS- or FENDrix-immunized animals died shortly after lesion onset. For comparison to the efficacy against HSV-1 genital herpes, the efficacy against HSV-2 disease was expressed as the percentage of disease-free animals that were alive and not showing any lesions at the time of analysis (Fig. 7B), and it was found to be significant only for the 2-μg vaccine dose. There were no differences in the amount of HSV-2 shed in vaginal washes in the gD/AS04-immunized cotton rats (Fig. 7C).

FIG 7.

Efficacy and immunogenicity of gD/AS04 vaccine in the cotton rat HSV-2 genital herpes model. Cotton rats were inoculated i.m. with the indicated doses of gD/AS04 vaccine Simplirix (“gD”) with an interval of 21 days and challenged 6 weeks after the initial immunization with HSV-2 at 5 × 10⁴ PFU per animal after pretreatment of animals with DMPA 4 days earlier. Control groups included HSV-2-challenged cotton rats inoculated i.m. with PBS, FENDrix, or HSV-2 partially inactivated with UV (“UV-HSV”) and PBS-immunized uninfected animals. (A) Lesion formation and mortality were monitored for 4 weeks after the challenge. The results shown are those of a representative experiment. (B) Percent disease-free plot of gD/AS04-immunized animals from the experiment described in panel A. The results are cumulative from two independent experiments, with 6 to 10 animals in all per group. Some animals in group vaccinated with gD/AS04 at 0.06 μg recovered from lesions by day 21 of the study, resulting in an apparent increase in percentage of disease-free animals. The asterisk indicates P < 0.05 compared to the group vaccinated with Fendrix. (C) The HSV-2 viral load was quantified by plaque assay on vaginal wash samples collected on day 2 postinfection. The results represent the means ± the SE for four to five animals per group. The asterisk indicates P < 0.05 compared to HSV-2-challenged animals immunized with FENDrix. For immunogenicity evaluation, serum was collected 21 days after initial immunization (prior to boost), 2.5 weeks after the boost (D38), and 7 days (select groups) after HSV-2 infection and analyzed for neutralizing activity (D), gD specific antibody titers by ELISA (E), or reactivity against crude cell extracts from HSV-2-infected cells (F). Panel F includes an inset that shows the reactivity of day 38 sera against extracts of control, uninfected Vero cells for the groups shown in the main panel, labeled 1 to 6 in the order of their appearance. The results are representative of two independent experiments with 4 to 10 samples per group. The asterisk indicates P < 0.05 compared to PBS-immunized animals.

UV-treated virus administered at a dose equivalent to 10³ PFU per animal (corresponding to original 10⁵ PFU per animal formulation prior to UV exposure) administered in a prime-boost regimen conferred 100% protection against lesion formation and mortality (Fig. 7A and B). All animals immunized with UV-HSV survived vaginal challenge. No virus was recovered from vaginal washes of HSV-2-infected animals immunized with UV-HSV (Fig. 7C). Completely inactivated HSV (HI-HSV) provided partial protection (Fig. 8A and B) similar to that observed with gD-2 subunit vaccine, suggesting the need for at least some active viral gene expression to achieve full protection.

FIG 8.

Defining correlates of protection in the cotton rat HSV-2 vaccine model. The efficacy and immunogenicity of heat-inactivated HSV-2 (HI-HSV), UV-treated HSV-2 (UV-HSV), and gD/AS04 (2 μg of gD-2 antigen, 5 μg of MPL, and 50 μg of alum) were compared. Animals were immunized with appropriate formulation, boosted 2 weeks later, and infected 2.5 weeks after the boost. (A and B) Survival curves (A) and viral load in genital tract secretions collected on day 2 postinfection (five to six animals per group) (B). (C and E) Serum IgG against crude HSV-2-infected cell extract (C) and serum neutralizing antibody levels (E) were quantified in samples collected immediately prior to HSV-2 challenge. (D) The vaginal wash IgG against crude HSV-2-infected cell extract was quantified in samples collected prior to challenge and on days 4 and 7 postinfection. Anti-HSV IgG results represent the fold induction over values detected in PBS-immunized, PBS-challenged animals. Day 7 data for PBS-immunized, HSV-2-challenged group were not available. Panel D includes an inset that shows the reactivity of day 7 vaginal wash samples against extracts of control, uninfected Vero cells for the groups shown in the main panel, labeled 1 to 4 in the order of their appearance. The results are cumulative of two independent experiments. The asterisk indicates P < 0.05 compared to PBS-immunized animals.

Immunogenicity of gD/AS04 vaccine in the cotton rat model.

To investigate antibody responses to gD/AS04 immunization in cotton rats, serum was collected at various time points of the experiment: 3 weeks after priming, 2 weeks after the boost, and 1 week after intravaginal challenge. gD/AS04 immunization induced a strong dose-dependent serum neutralizing antibody response and an increase in gD-2-reactive IgG in cotton rat serum (Fig. 7D). Serum anti-gD-2 IgG levels increased significantly after the first dose of gD/AS04, whereas the neutralizing antibody response to gD/AS04 required priming and boosting with gD/AS04. gD/AS04 vaccine induced significantly higher levels (an ∼5-fold difference) of gD-2-specific IgG compared to UV-HSV immunization, although the latter induced a strong neutralizing antibody response against HSV-2. The low level of ELISA binding antibodies reactive with recombinant gD-2 in animals immunized with UV-HSV was surprising and suggested that the recombinant protein used for immunization could be immunologically different from the native HSV-2 glycoprotein D and/or that antibodies to other proteins contribute to neutralizing activity. Consistent with this notion, higher levels of antibodies reactive with crude lysates of HSV-2-infected cells were observed in serum of animals immunized with UV-HSV compared to gD/AS04 (Fig. 7F).

To further investigate the correlates of protection, the immunogenicities of UV-HSV, HI-HSV, and gD/AS04 (2 μg) were compared in a follow-up study. UV-HSV again completely protected against disease and reduced the amount of virus detected in vaginal washes (Fig. 8A and B). HI virus induced an increase in IgG reactive with infected cell lysates in the serum and vaginal washes (Fig. 8C and D) but failed to induce neutralizing antibody responses (Fig. 8E). The UV-treated virus induced the highest levels of anti-HSV-2 IgG in both the serum and vaginal washes obtained prior to challenge and 4 and 7 days postinfection (Fig. 8D). No anti-gD-2 or neutralizing antibodies were detected in vaginal washes under the conditions tested (data not shown).

Efficacy of gD/AS04 vaccine against HSV-1 genital herpes in cotton rats S. hispidus.

To determine whether gD/AS04 vaccine protected against HSV-1 genital herpes, animals were vaccinated with gD/AS04 using the same experimental protocol as for HSV-2 and challenged intravaginally with HSV-1 at 2 × 10⁶ PFU/animal. All animals vaccinated with 2 μg of gD/AS04 were completely protected against HSV-1 disease, whereas all control animals developed genital herpes (Fig. 9A). Intermediate vaccine dose of 0.3 μg of gD/AS04 also induced significant protection by delaying lesion onset by ∼2 weeks and reducing disease-associated mortality, resulting in a significant increase in the number of disease-free animals compared to the FENDrix-immunized group (Fig. 9B) (significant protection against HSV-1 disease induced by vaccination with 0.3 and 2 μg of gD/AS04 was confirmed by a second independent study). Protection was accompanied by a dose-dependent reduction in viral replication in vaginal wash samples (Fig. 9C), which was also significant for the highest and intermediate vaccine doses (also confirmed by the second study). Viral load was reduced by ∼1.5 and 1 log₁₀ in animals vaccinated with 2 and 0.3 μg of gD/AS04, respectively, compared to FENDrix-vaccinated animals. Immunization with UV-HSV protected 80% of animals from signs of disease and reduced HSV-1 replication to undetectable levels (Fig. 9A to C). Overall, significant protection against HSV-1 genital herpes was achieved by the highest and intermediate gD/AS04 doses, which also caused significant reduction in vaginal viral load. For HSV-2, only the highest vaccine dose induced statistically significant (albeit partial) protection against HSV-2 disease, and no antiviral efficacy was seen for any of the vaccine doses.

FIG 9.

Efficacy of gD/AS04 vaccine in the cotton rat HSV-1 genital herpes model. Cotton rats were inoculated i.m. with the indicated doses of the gD/AS04 vaccine Simplirix (“gD”) with an interval of 21 days and challenged 6 weeks after the initial immunization with HSV-1(17) at 2 × 10⁶ PFU per animal after pretreatment with DMPA 4 days earlier. Control groups included HSV-1-challenged cotton rats immunized with FENDrix or with HSV-2 partially inactivated with UV (“UV-HSV”). (A) Lesion formation and mortality were monitored for 4 weeks after the challenge. The results shown are those of a representative experiment. (B) Percent of disease-free animals (lack of lesions and/or mortality) from the experiment described in panel A. The results are representative of two independent experiments. Some animals in group vaccinated with gD/AS04 at 0.06 μg recovered from lesions by day 28 of the study, resulting in an apparent increase in percentage of disease-free animals. The asterisk indicates P < 0.05 compared to FENDrix-vaccinated animals. (C) The HSV-1 viral load was quantified by a plaque assay on vaginal wash samples collected on day 2 postinfection. The results represent the means ± the SE for five to eight animals per group. The asterisk indicates P < 0.05 compared to HSV-1-challenged animals immunized with FENDrix.

Passive transfer of gD/AS04 hyperimmune serum confers protection against HSV-1 genital herpes in the cotton rat model.

Recent findings from the Herpevac gD/AS04 trial suggested that protection against HSV-1 in females correlated with antibodies to HSV-2 gD-2 (24). Thus, we hypothesized that the passive transfer of serum from gD/AS04-immunized cotton rats would confer protection against HSV-1 but not against HSV-2. To address this, we prepared hyperimmune serum by vaccinating cotton rats several times with 2 μg of gD/AS04, collecting the serum, and inoculating it i.m. into naive cotton rats 24 h prior to HSV-1 or HSV-2 infection. Negative-control animals were inoculated with equal volumes of control serum collected from naive cotton rats and were infected with HSV-1 or HSV-2. Passive transfer of hyperimmune serum protected animals from genital HSV-1 disease but not from HSV-2 disease. None of the HSV-1-infected animals pretreated with gD/AS04 serum exhibited signs of genital herpes, whereas 100% of the animals inoculated with control serum developed genital herpes (Fig. 10). Only modest reduction in HSV-1 titers in vaginal washes was detected: 3.25 ± 0.47 log₁₀ PFU/ml in animals treated with immune serum versus 3.80 ± 0.47 log₁₀ PFU/ml in animals inoculated with control serum (P = 0.29), suggesting that local viral replication may not alone predict disease outcome. gD/AS04-induced serum was less effective against HSV-2 genital herpes: 80% of HSV-2-infected animals pretreated with the hyperimmune serum still displayed signs of disease, and 40% of the animals died.

FIG 10.

Passive transfer of immunity via gD/AS04 hyperimmune serum. Serum for passive immunoprophylaxis was obtained from cotton rats immunized three times with gD/AS04 at 2 μg per animal. Control serum was obtained from age-matched unmanipulated cotton rats. Control or gD/AS04 serum was inoculated i.m. into DMPA-pretreated animals 24 h prior to intravaginal infection with HSV-1 (2 × 10⁵ PFU per animal) or HSV-2 (2 × 10⁴ PFU per animal). Lesion formation and mortality were monitored for 2.5 weeks after infection. The results shown are those of a representative experiment. The asterisk indicates P < 0.05 compared to the group treated with control serum and infected with the same virus.

DISCUSSION

Prior preclinical models of genital herpes failed to predict the unexpected human clinical trial outcome of partial protection against genital HSV-1, but no protection against HSV-2 following immunization with the gD/AS04 vaccine (12–15). Therefore, we developed a DMPA-synchronized cotton rat model of HSV-1 and HSV-2 genital infection and used it to evaluate gD/AS04 vaccine efficacy. DMPA treatment was used to achieve stringency, since it rendered cotton rats highly and consistently susceptible to infection and disease. The DMPA-treated animals uniformly succumbed to high-dose HSV-2 infection and displayed genital herpes disease when inoculated intravaginally with HSV-1. In contrast to an earlier described cotton rat model of HSV-2 genital herpes (25), this modified DMPA-treated vaginal challenge model does not recapitulate HSV-2 human disease, which is characterized by frequent episodes of viral reactivation and recovery but, importantly, recapitulated some of the findings from the clinical vaccine trials.

Specifically, we found that the gD/AS04 vaccine induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to reduce HSV-2 viral load. We also found that gD/AS04 provided better protection against HSV-1 than HSV-2 disease. These findings are generally consistent with the results of the Herpevac clinical trial and follow-up analysis of its participants (8, 15, 16). Moreover, although HSV-1 load was not evaluated in the Herpevac trial, our results suggest that gD/AS04 vaccination can diminish HSV-1 vaginal titers. The doses of vaccine that significantly improved HSV-1 disease outcome also caused significant decline in viral load in vaginal secretions of HSV-1-infected cotton rats.

The reported efficacy of gD/AS04 against HSV-2 genital herpes was not consistent between phase 2 and phase 3 clinical trials (15, 16). Phase 3 trial did not reveal any, whereas phase 2 studies found protection, albeit dependent on a variety of factors. Thus, it appears that a possibility of partial efficacy of Simplirix against HSV-2 disease still cannot be completely ruled out. It would be difficult to reconcile, however, with the lack of antiviral efficacy demonstrated in phase 3 studies (15). The cotton rat model suggests that gD/AS04 induces partial protection against HSV-2-induced disease but that this protection is not associated with an antiviral effect. We assessed the role of serum antibodies in mediating protection in passive-transfer experiments. The gD/AS04 immune sera significantly protected recipient naive animals against HSV-1, but not HSV-2 genital disease, confirming the clinical findings that antibodies against gD-2 correlate with protection against HSV-1. Passive transfer, however, did not significantly reduce the amount of virus detected in vaginal washes, indicating lack of additional antiviral mechanism(s) and suggesting that genital herpes disease can be reduced without dramatically inhibiting viral replication.

In the process of defining correlates of immunity to HSV-2, we identified a “positive control” immunization strategy using partially inactivated HSV-2 treated briefly with UV light. Intramuscular immunization of cotton rats with UV-HSV (with a remaining infectious titer of 10³ PFU per animal) protected 100% of animals against HSV-2 disease and infection. Importantly, UV-HSV immunization induced only low levels of antibodies against recombinant gD-2 but comparable to the gD/AS04 levels of neutralizing antibodies and the high levels of antibodies reactive with lysates of HSV-2-infected cells. These findings suggest that the UV-HSV elicited antibodies that recognize different neutralizing epitopes within glycoprotein D or induced neutralizing antibodies against other viral proteins.

Although subsequent clinical trials did not confirm protection against HSV-2, they did show that serum gD-2 antibodies and HSV-2 neutralizing antibodies against HSV-2 were induced by the gD/AS04 vaccine in human subjects (9, 15). The findings presented here suggest that gD-2-specific serum antibodies are not a correlate of protection against HSV-2, which is consistent with the clinical trial outcome. Antibodies against gD-2 in cotton rats, however, may provide a correlate of protection against HSV-1, similar to recently reported findings in humans (26). The results of our studies also suggest that serum neutralizing antibodies, which are commonly used as correlates of protection in preclinical and early clinical evaluations of vaccine candidates, are not the sole predictors of HSV-2 vaccine candidate efficacy. The gD/AS04 vaccine, but not HI virus, induced significant serum neutralizing antibodies, and yet both gD/AS04 and HI-HSV provided similar levels of protection. Conversely, the UV-HSV and gD/AS04 vaccines induced similar levels of serum neutralizing antibodies, but only the UV-HSV was fully protective. Notably, immunization with UV-treated virus was associated with the highest levels of HSV-specific IgG antibodies in vaginal washes. The levels increased following vaginal challenge, which may have contributed to the strong antiviral effect. Mucosal antibodies, which have not been reported in clinical vaccine trials, may provide a correlate of protection, but further studies are needed.

Immune mechanisms responsible for a higher efficacy of partially compared to completely inactivated HSV-2 also warrant further investigation. Intracellular production of proteins and nucleic acids by replicating virus may engage intracellular pattern recognition receptors and enhance immunogenicity, whereas the expression of nonstructural proteins may be required to provide important antigens. Engagement of T cell immunity by virus retaining partial replicative ability might also be of importance for effective protection against HSV-2 (27).

The studies described here were limited by the fact that genital disease severity against which gD/AS04 efficacy was evaluated is different in cotton rats infected with HSV-1 or HSV-2. The infectious dose required to induce genital disease and 100% mortality is substantially higher for HSV-1 than for HSV-2 (∼10⁶ PFU/animal versus ∼10³ PFU/animal, respectively). The progression of genital herpes is very rapid for HSV-2, with mortality typically following lesion formation in 2 to 3 days or preceding any lesion detection. In contrast, animals infected with HSV-1 could display lesions for over 2 weeks prior to succumbing to the disease despite having >2-log-higher titer in vaginal washes compared to animals infected with HSV-2. Moreover, HSV-1 disease severity was further reduced in animals infected without vaginal vault washing immediately prior to infection. It is possible that the higher efficacy of gD/AS04 against HSV-1 genital herpes was merely due to the milder disease observed in HSV-1-infected cotton rats. However, the fact that reduced HSV-1 disease was seen for the two vaccine doses which also caused significant inhibition of HSV-1 replication, whereas no antiviral efficacy was seen for HSV-2, and only partial protection was achieved by the highest vaccine dose, combined with the better efficacy of gD/AS04 serum against HSV-1 disease, argue against that possibility.

The recent emergence of HSV-1 as the major cause of genital herpes in parts of the world is surprising since for the larger portion of the 20th century HSV-2 was considered to be the primary cause of this disease. Our findings suggest that certain human practices may have been contributing to changing epidemiology of the genital herpes. Thus, we found that in the absence of DMPA treatment, cotton rats are naturally more resistant against HSV-1 than HSV-2 genital herpes but that the susceptibility to HSV-1 infection and disease significantly increase after DMPA injection. The use of injectable hormonal contraceptives in women increased from 4.5% in 1995 to 23% in 2006 to 2010 (28). A number of studies have suggested an increased risk of acquisition of HIV in association with DMPA (29–31). It is of interest that one of the highest reported rates of HSV-1 genital herpes was detected in participants of the Herpevac trial study, all of which were required to use a highly effective method of birth control that included hormonal contraceptives. Taken together, these findings suggest that the increased use of hormonal contraceptives could have contributed to the larger role HSV-1 plays in the epidemiology of genital herpes today. Our studies also indicate that vaginal secretions may inherently have a stronger protective effect against HSV-1 than HSV-2 genital disease. Removing vaginal secretions (a procedure analogous to douching in women) predisposed animals to a more lethal HSV-1 disease and dramatically increased HSV-1 replication. Douching has generally been discouraged by the American College of Obstetricians and Gynecologists because it may disrupt the mucosal microenvironment (32) and predispose to higher acquisition of sexually transmitted diseases (33). Although further studies are needed, our data suggest for the first time that HSV-1 genital herpes may be one of those diseases.

The primary goal of the present study was to establish comparable models of HSV-1 and HSV-2 genital herpes in an attempt to reproduce and to start dissecting unexpected findings from the recent gD/AS04 clinical trial. Unfortunately, we were not able to define models of identical disease severity, in part due to some of the factors described above. Nevertheless, we were able to successfully establish HSV-1 genital herpes model and used it, together with the HSV-2 model, to evaluate gD/AS04 efficacy against both viruses. We demonstrated that the vaccine has higher antiviral efficacy against HSV-1 than HSV-2 and better protects against genital disease caused by HSV-1 compared to HSV-2. We focused mainly on the correlates of immunogenicity and protection that were used in the development of previous subunit vaccines and the role of humoral immunity. Next-generation vaccines will likely target broader systemic and tissue-specific T cell memory responses (10, 22). Future cotton rat model development should include evaluation of T cell responses and mucosal responses and comparative studies of various vaccine platforms to better understand immunity of genital herpes and differences in the HSV-1 and HSV-2 genital herpes pathogenesis.