Class-Specific Antibody Response in Acyclovir- Treated and Adenine Arabinoside-Treated Patients with Primary Genital Herpes Simplex Virus Infection

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1 Microbiol. Immunol., 39(10), , 1995 Class-Specific Antibody Response in Acyclovir- Treated and Adenine Arabinoside-Treated Patients with Primary Genital Herpes Simplex Virus Infection Takashi Kawana*,1, Madoka Hashido2, and Yoshio Koizumi1 1 Department of Obstetrics and Gynecology, Tokyo University Branch Hospital, Bunkyo-ku, Tokyo 112, Japan, and 2Department of Epidemiology, the National Institute of Health, Shinjuku-ku, Tokyo 162, Japan Received June 7, Accepted July 13, 1995 ônh ô Abstract ôns ô: Herpes simplex virus (HSV) class-specific antibody responses after primary genital herpes infection were evaluated with an enzyme-linked immunosorbent assay (ELISA) in 16 patients treated with acyclovir (ACV), given orally, and 17 patients treated with adenine arabinoside (Ara-A), given topically. ACV significantly suppressed the levels of IgM, IgA, and IgG. In the ACV-treated patients, IgM and IgG were not detected in 4 of the 16 and in 1 of the 16 patients, respectively. We must take into account this sup pressiveeffect of ACV on antibody responses, especially on the IgM response, when serodiagnosis of HSV infection is made. NH ô Key words ôns ô: Antibody, Genital herpes, Acyclovir While the antiviral agent acyclovir (ACV) has been shown to be clinically effective for the treatment of pri marygenital herpes simplex virus (HSV) infections, some concern has been expressed regarding the altered antibody response to HSV in these patients. Investigators have observed that neutralizing antibody titers were diminished (2), or that antibody responses to major HSV proteins were delayed (1), when patients with primary genital HSV infection were treated with ACV. Howev er,the kinetics of class-specific antibody responses in pri maryhsv infection are still not well defined in patients who have been treated systemically with ACV. To elucidate these responses, we employed an enzyme-linked immunosorbent assay (ELISA) to com parethe IgM, IgA and IgG antibody responses to HSV in ACV-treated and adenine arabinoside (Ara-A)-treated patients for 6 months after infection. Materials and Methods Patient population and sera. Thirty-three female patients with acute genital herpes who were seen at the Department of Obstetrics and Gynecology of Tokyo University Branch Hospital were evaluated. All patients presented to the clinic within 8 days after developing *Address correspondence to Dr. Takashi Kawana, Department of Obstetrics and Gynecology, Tokyo University Branch Hospi tal, Mejirodai, Bunkyo-ku, Tokyo 112, Japan. genital lesions. HSV cultures were positive in all cases. Identification and typing of isolated viruses were per formedwith type-specific mouse monoclonal antibodies labeled with fluorescein isothiocyanate (MicroTrak, Syva Co., Calif., U.S.A.). Sixteen (HSV-1, 9; HSV-2, 7) of the 33 patients received oral ACV (200mg tablet) 5 times a day for 5 days (ACV-treated patients), while 17 (HSV- 1, 8; HSV-2, 9) were treated topically with 3% Ara-A ointment (Ara-A-treated patients). There was no sig nificantdifference in the age of the 2 groups; the ACV treated group were aged years, and the Ara-A - treated group were aged years. All the patients were diagnosed as having primary genital HSV infection, based on their history and on neutralizing antibody responses. Most of them had severe vulvar pain and ten derness,as well as systemic symptoms such as fever and general malaise (6). Eighty-six and 97 serum samples, respectively, were collected from the ACV-treated and Ara-A-treated patients over a 2-month to 2-year period after infection. The first sample was collected on day 5.3 }1.5 after diagnosis in the ACV-treated patients with HSV-1 and on day 5.7 }2.4 in those with HSV-2; in the Ara-A-treated patients with HSV-1 infection the serum was collected on Abbreviations: ACV, acyclovir; Ara-A, adenine arabinoside; ELISA, enzyme-linked immunosorbent assay; HSV, herpes sim plexvirus; HSV-1, herpes simplex virus type 1; HSV-2, herpes simplex virus type 2; OD, optical density; SD, standard deviation. 795

2 796 T. KAWANA ET AL day 5.9 }2.4 and in those with HSV-2 it was collected on day 5.1 }2.1(no significant difference). The sera were stored at -40C until use. Enzyme-linked immunosorbent assay for determina tionof HSV class-specific antibodies. To determine the levels of IgM, IgA and IgG antibodies to HSV, we employed ELISA as described previously (5). In brief, NP40-lysed extracts of HEL-R66 cells infected with HSV-1 (TVK171; clinical isolate) or HSV-2 (THH54; clinical isolate) were coated on microplates as antigen. Patient sera (diluted 1:100), affinity-purified goat F(ab')2 to human IgM, IgA or IgG labeled with horseradish peroxidase (Tago Inc., Burlingame, Calif., U.S.A.) (dilut ed1:2,000) and finally the substrate (ortho-phenylene diamine) were sequentially added to each well and the plates were incubated at 37C for 90min, 60min and 20 min, respectively. The HSV-specific antibody activity was calculated as: optical density (OD) of HSV antigen -OD of control antigen. The cut-off value was deter minedas the mean OD +3 SD of 30 negative speci mens. Statistical analysis. Student's t-test and the chi-square test were used for statistical analysis. P values of <0.05 were regarded as significant. Results IgM Antibody Response The IgM antibody response in ACV-treated and Ara- A-treated patients is shown in Fig. 1. In Ara-A-treated patients, the IgM antibody appeared within 1 week after the onset of the disease in 9 out of 16 patients (56.2%), with its level being highest in the 2- to 4-week period after onset. In HSV-1-infected patients it declined to the initial levels within 16 weeks after onset (Fig. 1A), while in HSV-2-infected patients it was still detected at low levels after 16 weeks (Fig. 1B). In patients with primary HSV-1 genital infection, the average IgM antibody levels were lower in ACV-treated than in Ara-A-treated patients. The difference was sig nificantat 2 to 4 weeks (P<0.01) and at 8 to 12 weeks (P<0.05). In patients with primary HSV-2 genital infection, the average IgM antibody levels were signifi cantlylower in ACV-treated than in Ara-A-treated patients after the second week of infection. Four out of 7 ACV-treated patients with primary HSV-2 infection showed very low or undetectable levels of IgM anti body,whereas all Ara-A-treated patients had detectable levels. There were no significant differences in IgM anti bodylevel between HSV-1-infected and HSV-2-infected patients in either the ACV-treated or the Ara-A-treated group. IgA Antibody Response The IgA antibody response in ACV-treated and Ara- A-treated patients is shown in Fig. 2. In the Ara-Atreated patients, IgA antibody appeared within 1 week after the onset of the disease in 9 out of 16 patients (56.2%). Although the IgA antibody levels showed a pattern similar to that of IgM, the elevation of IgA seemed to be more pronounced, reaching peak levels in Fig. 1. A: IgM antibody response in ACV-treated and Ara-A-treated patients with primary HSV-1 infection. B: IgM antibody

3 ANTIBODY RESPONSE OF GENITAL HERPES INFECTION 797 Fig. 2. A: IgA antibody response in ACV-treated and Ara-A-treated patients with primary HSV-1 infection. B: IgA antibody Fig. 3. A: IgG antibody response in ACV treated and Ara-A-treated patients with primary HSV-1 infection. B: IgG antibody the 1- to 2-week period after onset, earlier than the IgM response. After reaching the peak level, IgA antibody declined until 8 to 12 weeks after onset. In patients with primary HSV-1 genital infection, IgA antibody levels in ACV-treated and Ara-A-treated patients were similar. ACV-treated patients with HSV-2 infection, on the other hand, had significantly lower IgA antibody levels than Ara-A-treated patients after the second week of infection, although the antibody peak was observed at almost the same time. IgG Antibody Response The IgG antibody response in ACV-treated and Ara- A-treated patients is shown in Fig. 3. In Ara-A-treated patients, the IgG antibody appeared within 1 week after the onset of the disease in 3 out of 8 HSV-1-infected (Fig. 3A) and in all 8 HSV-2-infected patients (Fig. 3B). It continued to increase thereafter. In ACV-treated patients,

4 798 T. KAWANA ET AL on the other hand, the IgG antibody level was significantly lower than in Ara-A-treated patients after the second week of infection. This tendency was more prominent in patients with HSV 2 infection than in those with HSV-1. Discussion Our study demonstrated the kinetics of HSV class-specific antibody responses in patients with primary genital HSV infection, and the effect of the oral administration of ACV on these antibody responses. We employed ELISA to clarify the class-specific antibody response to HSV infection. Detergent-lysed extracts of HSV-1- infected cells were used as antigen, since in a preliminary study we found no difference in antibody response between HSV-1- and HSV 2-infected patients when we used this preparation as antigen. In Ara-A-treated patients, IgM, IgA and IgG antibodies appeared within 1 week after infection in 56.2%, 56.2% and 78.8%, respectively, and thereafter all 3 antibodies were detected in all patients. Although the IgM and IgA antibodies showed a similar pattern, the increase in IgA levels was earlier and more pronounced than that of IgM. The IgA antibody level was highest during the 1- to 2-week period after infection, while the IgM peak occurred 2 to 4 weeks after infection. The IgA antibody that appears after primary HSV infection has been shown to be polymeric IgA (5). In the Ara-A-treated group, the IgM and IgG antibodies in HSV-2-infected patients appeared earlier than in HSV 1-infected patients and the levels of IgG were significantly higher than in the HSV 1-infected patients. Since genital HSV 2 infection usually occurs in adults, its booster effect on the HSV 1 antibody established during childhood may be one possible explanation for this phenomenon. The difference in IgA antibody titer between ACV-treated and Ara-A-treated patients was observed in HSV-2-infected patients but not in HSV-1-infected patients. We think this discrepancy was due to the booster effect by HSV-2 infection, because higher IgG antibody titer was observed in Ara-A-treated patients with HSV 2 infection than in those with HSV 1 infection. By HSV type-specific antibody analysis using gg-1 and gg-2 as antigen (8, 9), about 70% of HSV 2-infected patients were shown to have dual infection of HSV-1 and HSV-2 (data not shown). The greater persistence of IgM and IgA antibodies in Ara-A-treated HSV 2-infected patients than in Ara-A-treated HSV-1-infected patients may reflect a higher recurrence rate of HSV 2 than HSV 1. For example, in this study, one patient infected with HSV-2 had recurrences on days 19, 89 and 193 after infection, and her IgM and IgA antibodies did not decline to the initial levels at any time during the study period. Our study corroborates other investigators' findings regarding the suppressive effect of oral ACV on the humoral immune response (1, 2). While previous studies have mainly demonstrated the suppressive effect of ACV on IgG response, we have demonstrated its effect on IgM and IgA, as well as on IgG. Interestingly, although ACV treatment lowered the level of each of these antibodies, it did not basically affect the time of seroconversion on antibody response patterns. However, when the serologic diagnosis of HSV infection is made, we must take into account that in some ACV treated patients IgM antibody cannot be detected. This reduced antibody response in patients who received ACV is most probably due to a reduced exposure to viral antigens, since ACV has been shown to reduce the duration of viral shedding (3). Two ACVtreated patients developed no or very low IgG, IgA and IgM antibodies to HSV 2. These patients presented very mild symptoms with small herpetic lesions that were treated successfully with ACV, which might result in weak antigenic stimulation and low antibody response. Alternatively, this diminished response could be due to an immunosuppressive effect of the drug. In this regard, Gold et al (4) reported that long-term administration of ACV did not affect levels of antibodies to cytomegalovirus and rubella virus, while it reduced levels of antibody to HSV 2-related proteins. Bernstein et al (2) also noted that an immunosuppressive effect of ACV seemed unlikely, since the drug level necessary to cause such an effect would be much greater than that detected in ACV treated patients. ACV therapy has also been shown to diminish the cellular immune response to HSV (7). From the clinical point of view, it is a matter of concern whether ACV treatment may affect the time of first recurrence. Lafferty et al (7) reported that they found no differences between 6 ACV-treated and 5 placebo-treated patients in this regard. However, more studies are necessary to clarify the effects of ACV, such as the time to first recurrence and the recurrence rate, on the long-term natural course of genital herpes. References 1) Ashley, R.L., and Corey, L Effect of acyclovir treatment of primary genital herpes on the antibody response to herpe simplex virus. J. Clin. Invest. 73: , 2) Bernstein, D.I., Lovett, M.A., and Bryson, Y.J The effects of acyclovir on antibody response to herpes simplex virus in primary genital herpetic infections. J. Infect. Dis. 150: ) Bryson, Y.J., Dillon, M., Lovett, M., Acuna, G., Taylor, S., Cherry, J.D., Johnson, L., Wiesmeier, E., Growdon, W.,

5 ANTIBODY RESPONSE OF GENITAL HERPES INFECTION 799 Creagh-Kirk, T., and Keeny, R Treatment of first episodes of genital herpes simplex virus infections with oral acyclovir: a randomized double blind controlled trial in normal subjects. N. Engl. J. Med. 308: ) Gold, D., Ashley, R., Solbery, G., Abbo, H., and Corey, L Chronic-dose acyclovir to suppress frequently recur ringgenital herpes simplex infection; effect on antibody response to herpes simplex virus type 2 proteins. J. Infect. Dis. 158: ) Hashido, M., Kawana, T., and Inouye, S Differentia tionof primary from nonprimary herpesvirus infection by detection of polymeric immunoglobulin A activity. J. Clin. Microbiol. 27: ) Kawana, T., Kawagoe, K., Takizawa, K., Cheng, J.T., Kawaguchi, T., and Sakamoto, S Clinical and virologic studies on female genital herpes. Obstet. Gynecol. 60: ) Lafferty, W., Brewer, L.A., and Corey, L Alteration of lymphocyte transformation response to herpes simplex virus infection by acyclovir therapy. Antimicrob. Agents Chemo ther.26: ) Lee, F.K., Pereira, L., Griffin, C., Reid, E., and Nahmias, A.J A novel glycoprotein for detection of herpes simplex virus type 1-specific antibodies. J. Virol. Methods 14: ) Lee, F.K., Coleman, R.M., Pereira, L., Bailey, P.D., Ta tsuno,m., and Nahmias, A.J Detection of herpes simplex virus type 2-specific antibody with glycoprotein G. J. Clin. Microbiol. 22: