Clinical and Serological Outcome of Genital Herpes Simplex Virus (HSV) Type 2 Inoculation following Oral HSV Type 1 Infection in Guinea-pigs

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1 J. gen. Virol. (1989), 70, Printed in Great Britain 2365 Key words: HSV/genital disease~immune response Clinical and Serological Outcome of Genital Herpes Simplex Virus (HSV) Type 2 Inoculation following Oral HSV Type 1 Infection in Guinea-pigs By DAVID I. BERNSTEIN, 1. FRANCIS K. LEE, 2 GARY ECHLER 1 AND ANDRE J. NAHMIAS 2 1James N. Gamble Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, Ohio and 2Division of Pediatric Infectious Disease, Emory University, Atlanta, Georgia 30322, U.S.A. (Accepted 8 May 1989) SUMMARY The clinical and serological outcome of genital herpes simplex virus type 2 (HSV-2) inoculation in animals previously orally infected with HSV type 1 was evaluated. A prior HSV-1 oral infection modified the genital HSV-2 infection so that only four of 18 (22~) animals were initially symptomatic although all but one animal shed HSV-2 from the cervicovaginal area for at least 5 days following inoculation. Three of four animals with symptomatic initial disease also developed recurrences, as did an additional six animals that did not manifest acute genital disease. Anti-glycoprotein gg-1 antibody was found in 17 of 18 animals with only an HSV-1 infection and antigg-2 antibody in all of nine animals with only an HSV-2 infection. Anti-gG-2 antibody was detected in eight of 17 animals with a prior HSV-1 infection following HSV-2 inoculation and one had an indeterminate response. Eight of these nine animals developed recurrent genital disease compared to one of eight that did not respond to gg-2 (P<0.006). Thus a prior oral HSV-1 infection modified both the initial presentation of HSV-2 infection and the HSV type-specific serological response. INTRODUCTION Genital herpes simplex virus type 2 (HSV-2) infection is one of the most commonly diagnosed sexually transmitted diseases. Patients can be divided into those with true primary infection and those with pre-existing HSV antibody, who have what is termed first episode, non-primary or initial infection (Nahmias et al., 1981 ; Bryson et al., 1983; Straus et al., 1985). Approximately 40 ~ of patients with their first clinically apparent episode of genital disease due to HSV-2 have pre-existing HSV antibody (Bryson et al., 1983; Reeves et al., 1981 ; Corey et al., 1983). These patients appear to have milder primary infection than those with true primary HSV-2 infections (Bryson et al., 1983 ; Corey et al., 1983; Whitley, 1985), although the proportion of patients who remain asymptomatic or develop such mild symptoms that they do not seek medical attention is unknown. A large proportion of HSV-2-infected patients are unaware that they have been infected (Mertz et al., 1985; Prober et al., 1988). Most if not all of these patients have HSV-2 latent in dorsal root ganglia; the virus could reactivate and cause recurrent symptomatic or asymptomatic disease (Mertz et al., 1985; Prober et al., D88, Mertz etal., 1988). it is possible that such patients are largely those who have pre-existing HSV-1 antibody at the time of exposure to HSV-2 and that the antibody modifies the genital disease to the extent that it is not recognized. It is also possible that in some cases a prior oral HSV-1 infection could provide complete protection against exposure to HSV-2. Detection of HSV type-specific antibody has been difficult because of the extensive crossreactivity that exists between HSV-I and HSV-2 (Spear, 1975; Eberle & Courtney, 1981; Bemstein et al., 1985). Recent methods have been based on the antibody response to typespecific HSV-1 glycoprotein G (gg-1) and HSV-2 glycoprotein G (gg-2) (Lee et al., 1985, 1986). The antibody response to gg-1 and gg-2 has proved reliable in diagnosing patients with SGM

2 2366 D. I. BERNSTEIN AND OTHERS documented infection to one or the other serotype (Lee et al., 1985, 1986; Ashley et al., 1988; Sullender et al., 1988). The identification of patients infected with both viruses is, however, most difficult (McClung et al., 1976; Nahmias et al., 1970; Plummer, 1973) and only limited data are available from them (Ashley et al., 1988). Thus, the sensitivity at which HSV-2 antibodies can be identified in patients with a prior HSV-1 infection is unknown and will be difficult to assess. We have therefore investigated the outcome of genital HSV-2 inoculation in guinea-pigs previously infected with HSV-1 orally. Vaginal inoculation of guinea-pigs with HSV-2 produces a disease pathophysiologically similar to human disease including symptomatic recurrences (Stanberry et al., 1982; Bernstein et al., 1986). We were thus able to determine the effects of prior oral HSV-1 infection on the clinical, virological and serological response of animals inoculated intravaginally with HSV-2. METHODS Animalinoculation. Thirty-one female Hartley guinea-pigs (Charles River Breeding Laboratory) weighing 300 to 350 g were used. To infect animals orally the upper lip was scarified with a 27-gauge needle or with a multiple puncture apparatus (Bignell) and then inoculated with 20 ~tl of medium containing 2 x 105 p.f.u, of HSV-1 strain Shealy (18 animals) (obtained originally from Dr Kurt Bubel, University of Cincinnati, Ohio, U.S.A.) or 2 x l0 s p.f.u. HSV-2 strain MS (four animals) (ATCC VR-540). Vaginal inoculation with 5 x 105 p.f.u, of HSV-2 strain MS was performed as previously described (Stanberry et al, 1982; Bernstein et al., 1986). In experiment 1, 12 animals were inoculated with HSV-2 6 months after oral infection (four with prior HSV-1 oral infections, four with prior oral HSV-2 infection and four controls, no prior oral infection). In experiment 2, 19 animals were inoculated 10 months after oral infection (14 with prior HSV-1 oral infection and five controls). Genital HSV-2 disease. Animals were examined daily for external skin lesions. The acute disease, during the defined period of days 0 to 14 (when all lesions had healed), was quantified on a scale of 0 to 4 as previously described (Stanberry et al., 1982). Recurrent disease was scored from day 15 until day 60 (Bernstein et al., 1986). Cervicovaginal HSV-2 cultures were obtained daily for the first 10 days after vaginal inoculation by insertion of a moistened calcium alginate swab into the vagina (Stanberry et al., 1982; Bernstein et al., 1986). Samples were frozen at - 70 C until virus was titrated by plaque assay in rabbit kidney cells (Bernstein et al., 1986). To compare the differences in mean viral titres we computed the area under the log10 virus titre-day curve (viral shedding curve) (Kern et al., 1978). Immunodot assay. Serotype-specific antibodies were identified by detection of antibody to gg-1 and gg-2 as described previously (Lee et al., 1985, 1986). Briefly, immunoaffinity column-purified gg-1 and gg-2 were diluted in Tris-buffered saline (TBS) ph 7.2, dotted onto nitrocellulose discs and placed into 96-well plates. After drying and blocking with 5% bovine serum albumin (BSA) in TBS (TBS-BSA) the coded test sera were diluted 1:50 in TBS-BSA and added to duplicate wells. Following overnight incubation the discs were washed and reblocked with TBS-BSA prior to the addition of goat anti-guinea-pig IgG-peroxidase conjugate (Sigma). After incubation for 1 h, the substrate 4-chloro-l-naphthol was added for 15 min. Discs were then rinsed with distilled water and read after overnight drying. The immunodot assay was interpreted by two observers without knowledge of the inoculation history. RESULTS Oral HSV-1 infection Following abrasion of the upper lip and HSV-1 inoculation, vesicular lesions were observed beginning on day 2 at the inoculation site with occasional satellite lesions noted by day 5 near the inoculation site and under the ipsilateral nares (Fig. 1). Lesions healed by days 7 to 9. The use of the multiple puncture apparatus resulted in more distinct vesicles. Genital HSV-2 infection Typical genital HSV-2 disease developed in all control animals from which HSV-2 was recovered (Fig. 2). Genital lesions peaked on day 6 or 7 and resolved by day 14. Urinary retention developed in all animals and hind limb paralysis was noted in 63 %. Prior oral infection with HSV-1 10 weeks or 6 months prior to genital inoculation reduced clinical disease~so that only three of 18 (17%) HSV-2 vaginally inoculated animals developed any genital e~ions and one other developed urinary retention without lesions. No other animal developed urinary

3 HSI,'-2 following oral HSI,'-I infection 2367 Fig. 1. Oral HSV-I infection of Hartley guinea-pigs 5 days after inoculation. (a) Animals were inoculated by abrading the upper lip with a 27-gauge needle prior to HSV-1 inoculation. (b) Animals were inoculated with the use of a multiple puncture apparatus. Arrowheads indicate HSV lesions \+ A T A\+ 1 i i zx--a~ Time after inoculation (days) Fig. 2. The clinical course of external genital skin disease in female guinea-pigs inoculated with HSV-2 after a prior oral HSV-1 infection (11) or in controls with no prior HSV infection (z~x). Values are expressed as mean lesion score + s.d. retention or hind limb paralysis. In experiment 1 (genital inoculation 6 months after oral infection) one of four animals with a prior oral HSV-1 infection developed a single genital lesion on day 13 (animal 4, Table 1). In experiment 2 (genital inoculation 10 weeks after oral HSV-1 infection) two of 14 animals developed single genital lesions on day 4 and 5 (animals 12 and 14). None of the four animals given HSV-2 orally 6 months prior to genital inoculation (animals 5 to 7 plus one animal not included in Table 1) developed any clinical genital disease. In experiment 1, no significant differences were noted in recovery of cervicovaginal HSV-2 from control (area under the viral shedding curve ) compared to animals with prior HSV-1 infection (area under the viral shedding curve ) although shedding was decreased from days 2 to 6 in HSV-l-infected animals (Fig. 3 a). Animals inoculated orally with HSV-2 6+months prior to vaginal challenge had an area under the viral shedding curve of compared to for control (P = 0-06, two-tailed Student's t-test) (Fig. 3b). In

4 2368 D. I. BERNSTEIN AND OTHERS Table 1. Antibody response to HSV-1 glycoprotein G and HSV-2 glycoprotein G in controls and animals previously infected orally Guinea-pig no. (experiment) Animals with prior HSV-1 oral infection Sera obtained after Sera obtained oral infection and before after HSV-2 genital inoculation genital inoculation * * Acute clinical Genital gg-1 gg-2 gg-1 gg-2 genital disease recurrence 1 (1)* (1) (1) (1) (2)t + - NDII ND (2) (2) I (2) (2) (2) (2) (2) (2) (2) (2) + - I (2) (2) (2) Animals with prior HSV-2 oral infection 5 (1) (1) (1) Control animals without prior oral HSV-1 or HSV-2 infection 8 (1) (1) (2) (2)~ (2) (2) (2) * Animals 1 to 9 were used in experiment 1 in which animals were infected with HSV-2 intravaginally 6 months after the oral infection. 1" Animals 10 to 28 were used in experiment 2 in which animals were inoculated with HSV-2 intravaginally 10 weeks after the HSV-I oral infection. :~ This animal did not shed HSV-2 after intravaginal HSV-2 inoculation and was not considered infected. Did not develop external lesions but did develop urinary retention. II ND, Not determined. I, Indeterminate, a discrepancy between observers. experiment 2, animals with a prior HSV-1 oral infection shed significantly less virus than control animals (area under the viral shedding curve of compared to ; P < ) (Fig. 3 c). If all animals given HSV-1 orally are compared to controls, significantly less virus was shed on days 3 to 10 (P < 0.05 on each day) and the area under the viral shedding curve was reduced from to (P < ) (Fig. 4). All but one HSV-1 orally infected animal shed HSV-2 for at least 5 days after genital inoculation. All control animals that shed HSV-2 developed recurrent genital lesions and nine of 18 animals with a prior HSV-1 oral infection developed a clinically obvious recurrent lesion. This included three of the four with acute symptoms and six that had not developed detectable symptoms after the acute disease. HSV-2 was recovered from four of six recurrent lesions evaluated. None of the three animals (one died during a bleed) with a prior HSV-2 oral infection developed recurrences.

5 HSV-2 following oral HSV-1 infection /i i i i i i i i I I iii i I i I i I i I /I I I I I I i i I I/ ~-i~,.;~ ~ ~ 6 ~ ~ ia) 4 _/ _. (b) ~ t. _ (c)_ Time after inoculation (days) Fig. 3. The course of cervicovaginal HSV-2 replication in animals after intravaginal HSV-2 inoculation. In experiment l, animals were inoculated with HSV-2 6 months after either oral HSV-1 infection (a) or oral HSV-2 infection (b). In experiment 2, animals were inoculated with HSV-2 10 weeks after oral HSV-1 infection (c). Replication was measured by plaque titration of vaginal swab samples and expressed as the geometric mean titres + S.D. for prior oral infection (ll) and for controls with no prior infection (LX). 8 I I I I I I I i I I o~ O.o 4 * -- ;> 2 ** 0-" I I I I J 1 T ~ i 9 mm 10 Time after inoculation (days) Fig. 4. The course of cervicovaginal HSV-2 replication after intravaginal HSV-2 inoculation in all animals with a prior oral HSV-1 infection (n) or controls with no prior HSV infection (z~). Replication was measured by plaque titration of vaginal swab samples and expressed as the geometric mean titre + S.D. * indicates significant (/><0-05) difference between groups; ** indicates difference was significant at the P < 0,005 level. Area refers to area under the curve (see Methods). Antibody response Sera were available from all but three animals, two controls from experiment 1 and one animal with a prior HSV-2 oral infection (Table 1). Three sera obtained before either oral or genital HSV infection did not react to gg-1 or gg-2 (data not shown). After HSV-1 oral infection, 17 of 18 animals responded to gg-1 and none to gg-2. After HSV-2 genital inoculation all control animals evaluated (no prior HSV infection) except one (which did not shed HSV-2) responded to gg-2 but not to gg-1. Eight of 17 animals with a prior HSV-1 infection responded to gg-2 at 30 to 40 days after HSV-2 genital inoculation and one had an indeterminate response. Eight of these nine developed recurrent genital disease compared to one of eight that did not respond to gg-2 (P < by Fisher's exact test). The other animal with anti-gg-2 antibody had developed a single lesion on day 13. The one HSV-1 orally infected animal without detectable anti-gg-1 antibody (Table 1, animal 1) had gg-1 antibody when re-evaluated after HSV-2 inoculation and one gg-1-positive animal (Table 1, animal 4) lost detectable anti-gg-1 antibody when re-evaluated after HSV-2 inoculation (6 months after HSV-1 oral infection). One animal (Table 1, animal 5; oral and genital HSV-2 infection) initially responded to gg-2 then responded only to gg-1 at 2 weeks after HSV-2 inoculation (data not shown) and then to both gg-1 and gg-2. No correlation could be found between the magnitude of vaginal virus replication and either the detection of anti-gg-2 antibody or recurrent disease. The area under the log10 vaginal viral

6 2370 D. I. BERNSTEIN AND OTHERS shedding per day curve of animals with a previous oral HSV-1 infection that did not respond to gg-2 ( ) was similar to that of animals that did respond ( ). Similarly, the area under the log10 vaginal virus per day curve of animals with a prior oral infection that developed recurrent disease ( ) was not significantly different from those which did not develop recurrent disease ( ). DISCUSSION Although a prior HSV-1 infection can modify human genital HSV-2 disease (Corey et al., 1983) the extent to which it alters the clinical and serological outcome is undefined because patients who do not develop clinical disease would not seek medical attention. Patients who develop a latent infection after asymptomatic primary disease may later develop clinically obvious disease (Bernstein et al., 1984) or asymptomatically shed virus and expose either their sexual partner (Rooney et al, 1986) or newborns (Prober et al, 1988) to HSV. Prospective studies under way will help define the role of prior HSV-1 infection in genital HSV-2 disease. In this animal model, guinea-pigs with a prior oral HSV-1 infection developed milder initial disease with reduced but not absent cervicovaginal shedding when challenged intravaginally with HSV-2. It appeared that shortening the interval between the oral infection and genital inoculation or the use of HSV-2 to infect animals orally may be important factors in limiting genital HSV-2 replication although the number of animals on which these data were based was small. Future experiments will investigate both of these parameters further. Three of four animals with a prior oral HSV-1 infection and initial symptomatic genital infection developed recurrences and, more interestingly, an additional six animals that had not developed symptomatic disease initially, did manifest symptomatic recurrent genital disease. Thus, a total of l0 of 18 (56~) animals with a prior oral HSV-1 infection eventually developed symptomatic genital HSV-2 disease after genital inoculation. These experiments support our observation that patients presenting with their first clinical episode of genital herpes may not be experiencing a primary infection but a recurrence of a previously asymptomatic infection (Bernstein et al., 1984). An additional eight animals with a prior oral HSV-1 infection did not develop clinical disease or seroconvert to gg-2 but demonstrated local genital HSV-2 replication. Extrapolation of these results to human disease suggests that a prior oral infection would predispose a patient to develop asymptomatic or mild disease after exposure to HSV-2 and these patients may indeed make up a significant proportion who are unaware of their genital HSV-2 infection. The immunodot assay for HSV serotype-specific antibody was shown to be reliable. AntigG-1 antibody was detected in 17 of 18 acutely HSV-1-infected animals and was detected in the other initially gg-l-seronegative animal on repeat examination after the HSV-2 inoculation. Only one animal lost detectable anti-gg-1 antibody at 6 months after HSV-1 infection. AntigG-2 antibody was detected in all HSV-2-infected control animals. Before dual infections, no anti-gg-2 antibodies were detected although one animal with an HSV-2 oral infection developed anti-gg-1 antibody. It is possible but unlikely that this animal acquired an HSV-1 infection from animals in adjacent cages. In 17 animals with a prior HSV-1 infection, anti-gg-2 antibody was detected in eight animals after HSV-2 genital inoculation and one animal had an indeterminate response. Anti-gG-2 antibody was detected in eight of 10 animals that developed either acute or recurrent disease and an indeterminate response was observed in a ninth animal. Since it is not known whether the other animals developed a latent infection or recurrent asymptomatic shedding, the sensitivity of this assay cannot be stated with certainty. Nevertheless, it did identify seven of nine animals that developed a genital recurrence including five of six initially asymptomatic animals. Future studies will address the question of latency in asymptomatic animals. In summary, a prior HSV-1 oral infection modified genital HSV-2 infection so that only 22 were initially symptomatic although 56 ~o eventually developed clinical symptoms. It is not clear whether the remaining animals were latently infected although all but one shed virus from the

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