Herpes Simplex Virus (HSV) Type 2 Glycoprotein D Subunit Vaccines and Protection against Genital HSV-1 or HSV-2 Disease in Guinea Pigs
summary: In two recent clinical trials, a vaccine containing herpes simplex virus (HSV) type 2 glycoprotein D (gD2) and a novel adjuvant AS04 comprising alum (Al) and 3-deactylated monophosphoryl lipid A (3-dMPL) afforded HSV-seronegative women significant protection against HSV-2 genital disease (vaccine efficacy, 73% in study 1 and 74% in study 2) and limited protection against infection (46% in study 1 and 39% in study 2). In the present report, studies in the guinea pig model investigated the protection afforded by gD2/AS04 against HSV-1 and HSV-2 genital herpes and investigated whether immunization could prevent or reduce recurrent disease in guinea pigs that developed mucosal infection. Immunization with gD2/AS04 conveyed nearly complete protection against primary disease with either virus but did not prevent mucosal infection. Guinea pigs immunized with gD2/AS04 were significantly better protected against recurrent disease than were guinea pigs immunized with a gD2/Al vaccine, which suggests that inclusion of 3-dMPL improved protection against latent infection.
..the incidence of recurrent disease was significantly lower in the gD2/AS04 group than in control guinea pigs... immunization with either vaccine resulted in a reduction in the duration of HSV-2 shedding in the genital tract..
The Journal of Infectious Diseases 2003;187:542-549
Nigel Bourne,1,a Fernando J. Bravo,1 Myriam Francotte,2 David I. Bernstein,1 Martin G. Myers,1,aMoncef Slaoui,2 and Lawrence R. Stanberry1,a
1Division of Infectious Diseases, Children's Hospital Medical Center, Cincinnati, Ohio; 2GlaxoSmithKline Biologicals, Rixensart, Belgium
Genital herpes is a common sexually transmitted disease [1, 2]. Although herpes simplex virus (HSV) type 2 is the most common cause of genital herpes in the United States , HSV-1 accounts for about one-third of new cases annually and is the most common cause of genital herpes in some countries . Initial anogenital HSV infection can be a painful illness characterized by vesicular and ulcerative skin lesions and neurologic and urologic complications. During the initial infection, a lifelong latent infection of sacral ganglion neurons is established, reactivation of which causes recurrent genital disease that can also be painful . In addition to the pain and discomfort associated with initial and recurrent infections, genital herpes causes substantial psychological morbidity [8, 9], may increase the risk of acquiring human immunodeficiency virus infection [10, 11], and can be spread to susceptible sex partners and to fetuses or newborn infants [12, 13]. Despite the availability of effective antiviral drug therapy , the prevalence of genital herpes in the United States continues to increase , with the estimated direct costs of genital herpes now >$200 million annually . Given the magnitude of the problem, effective vaccines represent the best strategy for control of genital herpes.
However, despite >60 years of research, the development of an effective HSV vaccine has remained elusive. In the recent past, considerable attention has been focused on subunit vaccines produced by means of recombinant genetic techniques . Two highly immunogenic HSV envelope proteins, glycoproteins B (gB) and D (gD), have been selected for commercial development. A vaccine containing HSV-2 gB and gD with MF59, an oil-in-water emulsion adjuvant system, was developed by Chiron. This vaccine was highly immunogenic in humans but failed to protect volunteers against genital HSV-2 infection in phase 3 trials [16, 17]. A second vaccine has been developed by GlaxoSmithKline Biologicals; this vaccine contains HSV-2 gD (gD2) and a novel adjuvant system, AS04 , which contains aluminum hydroxide (Al) and 3-deactylated monophosphoryl lipid A (3-dMPL), a modified form of the lipid A component of the lipopolysaccharide of gram-negative bacteria. In 2 phase 3 trials, the gD2/AS04 vaccine had an efficacy of 73% and 74% against development of symptomatic HSV-2 genital herpes in HSV-seronegative women and limited efficacy (46% and 39%) against HSV-2 infection .
In this report, we examined how immunization with the gD2/AS04 vaccine affected the natural history of genital HSV infection in a guinea pig model that closely mimics human disease. Intravaginal inoculation with HSV is followed by viral replication in the genital mucosa and the development of a self-limiting primary vesiculoulcerative genital skin disease , which is followed by episodic spontaneous recurrent lesions . Both HSV-1 and HSV-2 can cause genital skin disease, producing similar primary infections, but the incidence and frequency of recurrent disease is higher in HSV-2infected guinea pigs , as it is in humans . Thus, the model allows for the assessment of a number of outcome measures relevant to vaccine efficacy, including the ability to protect against mucosal infection, genital tract disease resulting from the initial infection, and the subsequent development of recurrent HSV infections, which is an indirect measure of latency . The studies described here addressed 2 questions of clinical importance: does the HSV-2 vaccine protect equally against genital HSV-1 and HSV-2 infection and disease and, if the vaccine does not protect against mucosal infection, does it affect the natural history of recurrent disease?
Vaccine immunogenicity. Figure 1 shows the gD2-specific ELISA and HSV-2neutralizing antibody titers present in the serum of immunized guinea pigs 1 day before virus challenge. All guinea pigs that were administered vaccine formulations containing gD2 developed HSV-specific humoral immune responses following immunization. Unimmunized and ADJ-immunized control guinea pigs did not develop detectable humoral immune responses.
Genital tract infection. Guinea pigs were defined as infected if virus was isolated by plaque titration assay from cervicovaginal secretions collected 2448 h after intravaginal HSV challenge. By this definition, 21 (91%) of 23 HSV-1 and 22 (92%) of 24 HSV-2inoculated control guinea pigs (i.e., unimmunized and ADJ-immunized groups) became infected. The virus titers in vaginal secretions of HSV-1 or HSV-2challenged ADJ-immunized control guinea pigs were similar to those of the unimmunized control guinea pigs on both day 1 and day 2 after virus inoculation (table 1). In addition, the duration of HSV-1 or HSV-2 replication in the genital tract was similar for both control groups (figure 2). Thus, immunization with adjuvant alone did not affect the magnitude or duration of viral replication in the genital mucosa.
Among guinea pigs challenged with HSV-1, immunization with gD2-containing subunit vaccines protected a small proportion against mucosal infection; 12 (40%) of 30 immunized guinea pigs were uninfected, compared with 2 (9%) of 23 control guinea pigs (P < .05). When the 2 vaccine formulations were analyzed separately, only the gD2/Al vaccine provided significant protection, compared with the combined control guinea pigs (P < .05; table 1). Although immunization only marginally affected the HSV-1 infection rate, both vaccine formulations significantly reduced virus titers in the genital tracts of infected guinea pigs on days 1 and 2 after inoculation (P < .01 for each; table 1), resulting in a shortened duration of viral shedding (figure 2).
In guinea pigs challenged with HSV-2, gD2-containing vaccines afforded no protection against mucosal infection; 6 (20%) of 30 immunized guinea pigs were uninfected, compared with 2 (8%) of 24 control guinea pigs (P > .05). Furthermore, when analyzed separately, neither formulation protected against mucosal infection. However, guinea pigs immunized with gD2/Al had significantly lower virus titers in cervicovaginal swab samples on day 1 after inoculation (P < .01), whereas those immunized with gD2/AS04 had significantly lower titers on both days 1 and 2 after inoculation (P < .001, day 1; P < .01, day 2). As with HSV-1challenged guinea pigs, immunization with either vaccine resulted in a reduction in the duration of HSV-2 shedding in the genital tract (figure 2).
Disease resulting from primary infection. Table 2 summarizes the effect of immunization on the incidence and severity of primary genital herpes. Although immunization afforded little or no protection against mucosal infection, it did provide significant protection against disease resulting from the infection. Twenty (95%) of the 21 unimmunized and ADJ-immunized control guinea pigs infected with HSV-1 developed characteristic herpetic skin lesions during primary infection (table 2). The 2 control groups were comparable with regard to the incidence of hindlimb paralysis, mortality, and the incidence and severity of primary genital skin disease. Immunization with either gD2 formulation provided complete protection from the cutaneous and neurologic manifestations of disease resulting from HSV-1 genital tract infection.
With regard to disease resulting from HSV-2 challenge, 21 (95%) of 22 control guinea pigs developed genital skin disease during the primary infection (table 2). There was no difference between the 2 control groups in the incidence of disease, hindlimb paralysis, or death. However, ADJ-immunized guinea pigs did experience less-severe skin disease than did unimmunized control guinea pigs (P < .005). Immunization with either gD2 formulation significantly reduced the incidence of primary skin disease resulting from HSV-2 genital tract infection, compared with either control group (P < .001), with only a single guinea pig in each of the 2 vaccine groups developing evidence of mild herpetic skin disease.
Recurrent disease. After recovery from the initial infection, all surviving guinea pigs were evaluated for episodic recurrent disease. The evaluation of recurrent infection was complicated by the severity of primary disease in control groups (particularly in HSV-2challenged guinea pigs). Many guinea pigs either died or had extensive damage to the perineum, which precluded evaluation for recurrent lesions. Because only guinea pigs that could be accurately assessed from days 22 to 63 after inoculation were included, the number of guinea pigs was small, even after appropriate control guinea pigs were combined. Results are shown in table 3. Recurrent disease developed in 5 (42%) of 12 HSV-1 and all 3 HSV-2infected control guinea pigs, with HSV-2infected guinea pigs experiencing significantly more recurrent disease (P < .01).
Only 1 HSV-1infected guinea pig from each vaccine immunized group developed recurrent disease, and both of these guinea pigs experienced only a single lesion-day during the 42-day observation period. HSV-2infected guinea pigs immunized with gD2/Al developed significantly fewer recurrences than did control guinea pigs (P < .02). However, although only 1 (7%) of 14 gD2/Al-immunized guinea pigs exhibited signs of primary genital herpes, recurrences were observed in 9 guinea pigs, which indicates that the subclinical genital tract infection had resulted in the establishment of a latent infection that was sufficiently robust to produce recurrent infections. In contrast, only 1 gD2/AS04-immunized guinea pig developed recurrences, the same guinea pig that had previously experienced symptomatic primary infection. Thus, the incidence of recurrent disease was significantly lower in the gD2/AS04 group than in control guinea pigs (P < .05) and in gD2/Al-immunized guinea pigs (P < .01).
In recent phase 3 clinical trials of HSV-2discordant couples, a gD2/AS04 vaccine provided HSV-seronegative women with significant protection against HSV-2 genital herpes disease and limited protection against HSV-2 infection . Among questions that were not addressed in the clinical trials were whether the vaccine could protect against HSV-1 genital herpes and whether vaccine recipients who became infected were afforded some protection against latency and subsequent recurrent infections. We have used a guinea pig model of genital HSV infection to explore these questions and to compare the gD2/AS04 formulation used in the human clinical trials to a formulation that lacked 3-dMPL.
Because gD is highly conserved between HSV-1 and HSV-2 [26, 27], we hypothesized that the gD2 vaccines would engender cross-protective immune responses that would afford protection against HSV-1 challenge. The present study confirmed this hypothesis, with both gD2 vaccines providing female guinea pigs with complete protection against primary disease resulting from intravaginal HSV-1 challenge. These results suggest that the gD2/AS04 vaccine that protected women against HSV-2 genital disease will also provide protection against HSV-1 genital herpes, a finding with clinical relevance given epidemiologic data indicating the increasing importance of HSV-1 as a cause of genital herpes [1, 46].
Both vaccine formulations also provided excellent protection against primary disease after HSV-2 challenge, with only 1 guinea pig in each vaccine group developing mild symptoms. However, in contrast to their efficacy against primary disease caused by either virus, the vaccines provided only modest protection against HSV infection, reducing but not preventing viral replication in the vaginal mucosa of the majority of guinea pigs. This finding is similar to that seen with the gD2/AS04 vaccine in recent clinical trials, in which protection against infection occurred in 39%46% of HSV-seronegative females . Furthermore, studies with a variety of other experimental HSV vaccines in the guinea pig model and experiments in which guinea pigs latently infected with HSV-2 received a second vaginal virus challenge also have failed to prevent viral replication in the genital tract, suggesting that it may be very difficult to provide immunologic protection of mucous membranes against challenge with high virus inocula [24, 2832].
It is uncertain whether a vaccine that prevented primary herpes disease but not infection would affect the spread of genital herpes. The likelihood that this would occur would be increased if vaccine recipients who became infected had fewer recurrent infections than did unimmunized persons with genital herpes. By use of a nucleic acid vaccine in the guinea pig model, we have shown elsewhere  that prophylactic immunization can reduce the magnitude of latent viral infection and that this reduction correlates with a decrease in recurrent infections. Consequently, a second aim of these studies was to determine the ability of these vaccines to modify recurrent disease. We found that guinea pigs immunized with the gD2/Al vaccine experienced significantly fewer recurrences than did HSV-2infected control guinea pigs; however, recurrences were observed in 9 of 15 guinea pigs, including 8 that had experienced only subclinical primary infection. Substantially greater protection was seen with the gD2/AS04 vaccine, in which the only guinea pig to develop recurrent HSV-2 infections was the guinea pig that had disease associated with primary infection. None of the gD2/AS04-immunized guinea pigs that experienced subclinical primary infection developed recurrent disease. These results suggest that inclusion of 3-dMPL engendered immune responses that provided greater protection against latent infection and subsequent recurrent HSV-2 disease than was observed with alum alone. The use of MPL as an adjuvant has been reported to stimulate Th1-type responses to immunization in vaccinated guinea pigs and humans [33, 34] (authors' unpublished data). Th1-type, rather than Th2-type, immune responses may be needed to control HSV infections and, in particular, to protect the sensory ganglia from acute infection .
Regarding the impact of immunization on HSV-1 recurrences, both vaccine formulations afforded excellent protection against recurrent disease in HSV-1infected guinea pigs, with only 1 guinea pig from each immunized group experiencing a single recurrent episode. This apparent protection against recurrent HSV-1 genital herpes might have been facilitated by the natural history of HSV-1 genital infection, which typically results in a lower incidence and frequency of recurrent infections than does HSV-2 [22, 23].
As with all animal models of human disease, it is important to remember that the guinea pig model of genital herpes is indeed a model. The immune responses to immunization and the mechanism of protection may differ from those in humans, and so care must be taken in interpreting results. With these caveats, the results of these and other studies have implications for establishing expectations for HSV vaccines. They predict that HSV vaccines may not be capable of inducing a sterilizing immunity that will completely protect the genital mucosa from infection [24, 2832]. However, it is realistic to expect that effective vaccines could protect the subject from developing symptomatic primary disease. If HSV vaccines cannot prevent infection of the genital mucosa, both symptomatic and asymptomatically infected subjects might establish latent infection and later experience recurrent genital herpes. Immunized persons who become infected might be expected to have fewer recurrences than do those who did not receive a vaccine. A major factor contributing to the spread of genital herpes is the ability of HSV to be shed from the genital tract in the absence of recognizable signs or symptoms of infection [39, 40]. Thus, it will be important in future vaccine studies to establish their ability to affect asymptomatic shedding as well as recurrent disease. If the frequency or magnitude of this asymptomatic virus shedding is also reduced in immunized subjects, it will increase the likelihood that immunization would reduce the spread of genital herpes.
If immunization does not affect the spread of infection, the major benefits of the vaccine would be to protect the immunized host from experiencing severe initial genital herpes and frequent recurrent infections. Although less than ideal, a vaccine that protected against severe primary and frequent recurrent genital herpes would reduce the substantial physiologic and psychological morbidity associated with symptomatic genital herpes and health care costs associated with treatment of the disease and would be preferable to no vaccine at all. The possibility that people immunized with such a vaccine could subsequently become subclinically infected and inadvertently spread infection to susceptible partners might be addressed by a policy of universal immunization so that, even if exposed, the partner might become infected but would not experience clinical disease. Because HSV vaccines may provide only partial protection against genital infection, it will also be important to examine whether immunization reduces the risk of vertical transmission for women who become infected during pregnancy.
In summary, studies using a guinea pig model of genital herpes showed that a subunit gD2 vaccine that, in clinical trials, provided women significant protection against HSV-2 genital disease but limited protection against infection also provided significant protection against genital HSV-2 disease in female guinea pigs. The experiments further showed that gD2 vaccines provided cross-protection against disease resulting from genital HSV-1 challenge. The inclusion of 3-dMPL significantly increased protection against recurrences of genital herpes in infected guinea pigs. These results suggest that the gD2/AS04 vaccine may protect women against both HSV-1 and HSV-2 genital herpes and that women who experience breakthrough infection may experience fewer recurrent infections and, therefore, may be less likely to spread infection to susceptible sex partners or to their fetuses or newborns. Recent mathematical modeling data suggest that widespread use of the vaccine by seronegative women could reduce the spread of genital herpes among both men and women . Collectively, these results support the further development of the gD2/AS04 vaccine.
1.Ashley RL, Wald A. Genital herpes: review of the epidemic and potential use of type-specific serology. Clin Microbiol Rev 1999; 12:18. First citation in article | PubMed
2.Stanberry L, Cunningham A, Mertz G, et al. New developments in the epidemiology, natural history, and management of genital herpes. Antiviral Res 1999; 42:114. First citation in article | PubMed
3.Fleming DT, McQuillen GM, Johnson RE, et al. Herpes simplex virus type 2 in the United States, 1976 to 1994. N Engl J Med 1997; 337:110511. First citation in article | PubMed
4.Ribes JA, Steele AD, Seabolt JP, Baker DJ. Six-year study of the incidence of herpes in genital and nongenital cultures in a central Kentucky medical center patient population. J Clin Microbiol 2001; 39:33215. First citation in article | PubMed
5.Tayal SC, Pattman RS. High prevalence of herpes simplex virus type 1 in female anogenital herpes simplex in Newcastle-upon-Tyne 198392. Int J STD AIDS 1994; 5:35961. First citation in article | PubMed
6.Thompson C. Genital herpes simplex typing in genitourinary medicine: 19951999. Int J STD AIDS 2000; 11:5012. First citation in article | PubMed
7.Whitley RJ, Roizman B. Herpes simplex virus infections. Lancet 2001; 357:15138. First citation in article | PubMed
8.Patel R, Boseli F, Cairo I, Barnett G, Price M, Wulf HC. Patients' perspectives on the burden of recurrent genital herpes. Int J STD AIDS 2001; 12:6405. First citation in article | PubMed
9.Mindel A. Psychological and psychosexual implications of herpes simplex virus infections. Scand J Infect Dis Suppl 1996; 100:2732. First citation in article | PubMed
10.Perez G, Skurnick JH, Denny TN, et al. Herpes simplex type II and Mycoplasma genitalium as risk factors for heterosexual HIV transmission: report from the heterosexual HIV transmission study. Int J Infect Dis 1998; 3:511. First citation in article | PubMed
11.Chen CY, Ballard RC, Beck-Sague CM, et al. Human immunodeficiency virus infection and genital ulcer disease in South Africa: the herpetic connection. Sex Transm Dis 2000; 27:219. First citation in article | PubMed
12.Mertz GJ, Benedetti J, Ashley R, Selke SA, Corey L. Risk factors for the sexual transmission of genital herpes. Ann Intern Med 1992; 116:197202. First citation in article | PubMed
13.Brown ZA, Selkes S, Zeh J, et al. The acquisition of herpes simplex virus during pregnancy. N Engl J Med 1997; 337:50915. First citation in article | PubMed
14.Tao G, Kassler WJ, Rein DB. Medical care expenditures for genital herpes in the United States. Sex Transm Dis 2000; 27:328. First citation in article | PubMed
15.Bernstein DI, Stanberry LR. Herpes simplex virus vaccines. Vaccine 1999; 17:16819. First citation in article | PubMed
16.Langenberg AGM, Burke RL, Adair SF, et al. A recombinant glycoprotein vaccine for herpes simplex type 2: safety and efficacy. Ann Intern Med 1995; 122:88998. First citation in article | PubMed
17.Corey L, Langenberg AGM, Ashley R, et al. Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infectiontwo randomized controlled trials. JAMA 1999; 282:33140. First citation in article | PubMed
18.Thoelen S, De Clercq N, Tornieporth N. A prophylactic hepatitis vaccine with a novel adjuvant system. Vaccine 2001; 19:24003. First citation in article | PubMed
19.Bernstein DI. Efficacy of a prophylactic herpes simplex type 2 glycoprotein D vaccine: results of two clinical efficacy trials. SmithKline Beecham Herpes Vaccine Efficacy Study Group. Pediatr Res 2001; 49(Suppl):256a. First citation in article
20.Stanberry LR, Kern ER, Abbott TM, Overall JC Jr. Genital herpes in guinea pigs: pathogenesis of the primary infection and the description of recurrent disease. J Infect Dis 1982; 146:397404. First citation in article | PubMed
21.Stanberry LR, Kern ER, Richards JT, Overall JC Jr. Recurrent genital herpes simplex virus infections in guinea pigs. Intervirology 1985; 24:22631. First citation in article | PubMed
22.Bourne N, Stanberry LR. Animal models of herpesvirus genital infection: guinea pig. In: Zak O, Sande MA, eds. Handbook of animal models of infection. London: Academic Press, 1999:90710. First citation in article
23.Reeves WC, Corey L, Adams HG, Vontver LA, Holmes KK. Risk of recurrence after first episodes of genital herpes: relation to HSV type and antibody response. N Engl J Med 1981; 305:3159. First citation in article | PubMed
24.Bourne N, Stanberry LR, Bernstein DI, Lew D. DNA immunization against experimental genital herpes simplex virus infection. J Infect Dis 1996; 173:8007. First citation in article | PubMed
25.Stanberry LR, Kit S, Myers MG. Thymidine kinasedeficient herpes simplex virus type 2 genital infection in guinea pigs. J Virol 1985; 55:3228. First citation in article | PubMed
26.Eisenberg RJ, Ponce de Leon M, Cohen GH. Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2. J Virol 1980; 35:42835. First citation in article | PubMed
27.Watson RJ. DNA sequence of the herpes simplex virus type 2 glycoprotein D gene. Gene 1983; 26:30712. First citation in article | PubMed
28.Stanberry LR, Bernstein DI, Kit S, Myers MG. Genital reinfection after recovery from initial genital infection with herpes simplex virus type 2 in guinea pigs. J Infect Dis 1986; 153:105561. First citation in article | PubMed
29.Stanberry LR, Myers MG, Stephanopoulos DE, Burke RL. Preinfection prophylaxis with herpes simplex virus glycoprotein immunogens: factors influencing efficacy. J Gen Virol 1989; 70:317785. First citation in article | PubMed
30.Bournsell MEG, Entwhistle C, Blakeley D, et al. A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J Infect Dis 1997; 175:1625. First citation in article | PubMed
31.Da Costa XJ, Bourne N, Stanberry LR, Knipe DM. Construction and characterization of a replication-defective herpes simplex virus 2 ICP8 mutant strain and its use in immunization studies in a guinea pig model of genital disease. Virology 1997; 232:112. First citation in article | PubMed
32.Spector FC, Kern ER, Palmer J, et al. Evaluation of a live attenuated recombinant virus RAV 9395 as a herpes simplex virus type 2 vaccine in guinea pigs. J Infect Dis 1998; 177:114354. First citation in article | PubMed
33.Ulrich JT, Myers KR. Monophosphoryl lipid A as an adjuvant. In: Powell MF, Newman MJ, eds. Vaccine design: the subunit and adjuvant approach. New York: Plenum Press 1995:495524. First citation in article
34.Moore A, McCarthy L, Mills KH. The adjuvant combination monophosphoryl lipid A and QS21 switches T-cell responses induced with a soluble recombinant HIV protein from Th2 to Th1. Vaccine 1999; 17:251727. First citation in article | PubMed
35.Singh M, O'Hagan D. Advances in vaccine adjuvants. Nat Biotechnol 1999; 17:107581. First citation in article | PubMed
36.Milligan GN, Bernstein DI, Bourne N. T lymphocytes are required for protection of the vaginal mucosae and sensory ganglia of immune mice against reinfection with HSV type 2. J Immunol 1998; 160:6093100. First citation in article | PubMed
37.Deshpande SP, Kumaraguru U, Rouse BT. Why do we lack an effective vaccine against herpes simplex virus infections? Microbes Infect 2000; 2:9738. First citation in article | PubMed
38.Cunningham AL, Mikloska Z. The holy grail: immune control of human herpes simplex virus infection and disease. Herpes 2001; 8(Suppl 1):6A10. First citation in article | PubMed
39.Rooney JF, Felser JM, Ostrove JM, Straus SE. Acquisition of genital herpes from an asymptomatic sexual partner. N Engl J Med 1986; 314:15614. First citation in article | PubMed
40.Mertz GJ, Coombs RW, Ashley R, et al. Transmission of genital herpes in couples with one symptomatic and one asymptomatic partner: a prospective study. J Infect Dis 1988; 157:116977. First citation in article | PubMed
41.Garnett GP. Herpes simplex virus type 2 epidemiology in the United States and the potential impact of a vaccine. Int J STD AIDS 2001; 12(Suppl 2):149. First citation in article