Effect of Primary Epstein-Barr Virus Infection on Human Herpesvirus 6, Cytomegalovirus, and Measles Virus Immunoglobulin G Titers

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 28, No. 2 Effect of Primary Epstein-Barr Virus Infection on Human Herpesvirus 6, Cytomegalovirus, and Measles Virus Immunoglobulin G iters ANNIKA LINDE,'* EVA FRIDELL,1 HELENA DAHL,' JAN ANDERSSON,2 PEER BIBERFELD,3 AND BRIA WAHREN' Department of Virology, National Bacteriological Laboratory, S Stockholm,' Department of Infectious Diseases, Danderyd Hospital, S Danderyd3 and Immunopathology Laboratory, Department of Pathology, Karolinska Institute, S Stockholm,3 Sweden Received 17 July 1989/Accepted 23 October 1989 Immunoglobulin G antibody titers to human herpesvirus 6 (HHV-6), measles virus, and cytomegalovirus (CMV) were examined in serum samples from 31 patients with Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM). Sera were drawn sequentially from the same patients c7 days until 3 years after onset of IM. In seropositive patients, there was a significant decrease with time after IM of the immunoglobulin G titers to the three viruses in the majority of patients; HHV-6 IgG titers decreased in 80%, measles virus IgG titers decreased in 75%, and CMV IgG titers decreased in 67%. Four patients contracted CMV infection during the observation period after IM. In these, HHV-6 IgG titers increased, while EBV and measles virus IgG titers remained essentially stationary. Polyclonal B-cell stimulation during IM is suggested to augment antiviral titers in general, but the increases of HHV-6 IgG titers during EBV and CMV infections may also be due to selective stimulation of memory B cells by related antigens or to reactivation of HHV-6 during infection with these herpesviruses. he interpretation of herpesvirus serology is sometimes complicated by increases in immunoglobulin G (IgG) to heterologous viruses. his is well known for varicella-zoster and herpes simplex virus infections, and increased titers of IgG to human herpesvirus 6 (HHV-6) during infections with other herpesviruses have also been noted (10, 11). In Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections, measurable IgM to both viruses is also found (13). hese reactivities may emerge from truly cross-reactive antibodies, reactivation of the heterologous virus, selective stimulation of memory B cells by related antigens, or polyclonal B-cell stimulation during the viral infection. EBV is a potent B-cell-stimulating agent. Appearance of IgM antibodies with various specificities during primary EBV infection is well known, but the effect of EBV infection on levels of specific IgG antibodies has not been thoroughly studied. At the onset of infectious mononucleosis (IM), antibody levels to the viral capsid antigen (VCA) normally have reached their peak. It is likely that other antiviral IgG increases due to EBV-induced polyclonal B-cell stimulation likewise have taken place. Increases in IgG between acuteand convalescent-phase samples as the result of a polyclonal stimulation are in that case difficult to measure. We therefore chose to study antiviral IgG titers in serum samples drawn sequentially from the same patients <7 days to 3 years after IM. IgG antibodies to two human herpesviruses, HHV-6 and CMV, and to the unrelated measles virus were measured to detect significant changes in specific titers with time. MAERIALS AND MEHODS Serum samples from 31 patients with IM (21 males and 10 females) between 15 and 23 years of age (mean, 18.8) were collected during a previous study on the effect of intravenous * Corresponding author. acyclovir (ACV) treatment of IM (1). For 7 days, fifteen patients received ACV and sixteen were placebo treated. In all patients, heterophile antibodies were detected. Serologically, all patients had EBV VCA IgG and IgM but no EBV nuclear antigen antibodies in their acute serum samples. he serological assays were performed at the National Bacteriological Laboratory (Stockholm, Sweden) and by the late Werner Henle, Children's Hospital of Philadelphia and School of Medicine, University of Pennsylvania, Philadelphia. Spontaneous outgrowth of EBV-transformed cells and high titers of EBV in saliva were found in all patients admitted to the study. Serum samples were drawn at c7 days, 28 days, 90 days, 180 days, 2 years, and 3 years after onset of IM. Of the 31 patients, 7 and 15 could not be reached or were not willing to donate samples at 2 and 3 years after IM, respectively. No differences in specific EBV titers were noted at any time point when the ACV- and placebo-treated groups were compared. Since ACV has little inhibitory effect on HHV-6 in vivo (17), it seems unlikely that the HHV-6 titers should be affected by ACV treatment. Serum samples from 37 healthy EBV-seropositive individuals without a history of IM within 5 years before sampling were examined concomitantly with the samples from the IM patients as a control. Methods for titrations of specific antibodies. Immunofluorescence assays (IFA) were used for EBV VCA and HHV-6 IgG (9, 10). For EBV VCA, slides with acetone-fixed cells from an EBV-producing P3HR-1 cell line were used. he HHV-6 virus was propagated in the HSB-2 continuous -cell line. Both the HHV-6 virus and the HSB-2 cell fine were kindly donated by S. Z. Salahuddin and R. C. Gallo of the National Institutes of Health, Bethesda, Md. (16). HSB-2 cells were infected for 3 days with medium from 50 to 90% HHV-6-infected HSB-2 cells, after which smears were made and the slides were fixed in acetone. Five to ten percent of 211

2 212 LINDE E AL. ABLE 1. Changes in HHV-6, measles virus, and CMV titers between samples drawn during (<7 to 28 days) and after (6 months to 3 years) IM No. with significant No. with significant Antigen Antigen increase/total (%)a decrease/total (%)l EBV 0/31 (0) 23/31 (74) HHV-6 1/30 (3) 24/30 (80) MEb 0/28 (0) 21/28 (75) CMV 1/9 (11) 6/9 (67) a Changes of fourfold or more in IFA and of twofold or more in ELISA were considered significant. b ME, Measles virus. the cells in the preparations were infected with HHV-6. he uninfected HSB-2 cells in the preparations served as controls for nonspecific staining. Serum samples were diluted fourfold from 1:20 to 1:1,280 in enzyme-linked immunosorbent assay (ELISA) buffer (phosphate-buffered saline without calcium and magnesium with 0.5% bovine serum albumin and 0.05% ween 20) and used for the EBV VCA and HHV-6 IgG titrations (9, 10). Sera and conjugate (fluorescein-labeled sheep anti-human immunoglobulin; National Bacteriological Laboratory, Stockholm, Sweden) were incubated for 1 h at 37 C. he preparations were counterstained in Evans blue. he slides were coded before being read to avoid subjective evaluation. ELISAs were used for CMV and measles virus antibodies. Microplates (Nunc, Aarhus, Denmark) were coated with purified measles virions (strain LEC, propagated in Vero cells) or CMV nucleocapsid antigen (18). he 1:20 serum dilutions, prepared for the IFA, were further diluted in ELISA buffer from 10-2 to One hundred microliters of each dilution was incubated per well at 37 C for 2 h. After rinsing, alkaline phosphatase goat anti-human IgG (Sigma Chemical Co., St. Louis, Mo.) was incubated overnight at 37 C. p-nitrophenyl phosphate (Sigma) was used as the substrate. Plates were read at 410 nm. iters were calculated as the reciprocal of the serum dilution giving an A410 of 0.2. Adsorption of EBV and CMV antibodies. EBV antibodies were adsorbed from 12 serum samples in two IM patients and CMV antibodies were adsorbed from two acute IM samples and from the four patients who contracted CMV infection during the observation period. A total of 2 x 108 rinsed and freeze-thawed cells of the EBV-producing P3HR- 1 cell line or 25,ug of CMV antigen (18) were placed in glass tubes. wo hundred microliters of serum diluted 1:20 in ELISA buffer was rocked with the antigen preparations for 2 h at 37 C. After centrifugation for 20 min at 1,500 x g, the supernatants were titrated as previously described for the IgG IFA and ELISAs. Another 200.tl of the serum dilutions was treated as for adsorption, but without addition of antigen. he adsorbed and unadsorbed samples were examined in parallel for IgG antibodies to EBV VCA, HHV-6, CMV, and measles virus. RESULS Antiviral IgG titers during and after IM. he titers of IgG to EBV VCA, HHV-6, measles virus, and CMV during and after IM are shown in Fig. 1. he numbers of patients in which significant titer changes occurred between samples drawn during and after acute IM are presented in able 1. Only patients who were seropositive for the relevant antigen in the acute IM sample and who remained seropositive during the whole observation period were included. All sera J. CLIN. MICROBIOL. contained EBV VCA IgG antibodies. here was a continuous decrease of the mean titers during the observation period (Fig. 1A). IgG titers of.160 to HHV-6 were found in 30 of 31 serum samples drawn at c7 days after the onset of IM, and the patients remained seropositive to HHV-6 in all samples examined. One patient was HHV-6 seronegative. Of 31 patients, 28 had measles virus IgG titers above the limit for seropositivity (.1,000) in all samples. Measles virus and HHV-6 IgG titers were significantly (P c 0.001; Wilcoxon signed-rank test) higher in serum samples drawn at c7 and 28 days after the onset of IM than in those drawn at 2 and 3 years after IM. here was a gradual decrease of titers from 90 days until 3 years after the EBV infection (Fig. 1B and C). Nine patients were initially CMV seropositive, with titers of >1,000 in their first sample, and they retained their CMV antibodies during the observation period. he CMV IgG titers were significantly lower at 2 or 3 years than at c7 days after IM in six of nine patients (able 1). Because of great individual variation and a low number of seropositive patients, a significant decrease in mean IgG titers was not seen for CMV (Fig. 1D). Our limit for IgG seropositivity to CMV is a titer of 100. Of 31 patients, 3 had CMV titers of <100 in the samples drawn s7 days after the onset of IM and in subsequent serum samples. In 19 of 31 patients, low titers of 100 to 1,000 were noted in the acute IM samples. In these patients, titers became <100 in one or more samples drawn at 6 months and later after IM. he positive reactions in the acute IM samples were probably nonspecific. hree patients seroconverted from a CMV IgG titer of <100 to >5,000 between 6 months and 2 years, and one patient did so between 2 and 3 years, after their primary IM. hese patients did not report any clinical illness, and the exact date of CMV infection cannot be determined. he IgG titers to EBV VCA, HHV-6, and measles virus before and after CMV seroconversion are shown in Fig. 2. EBV VCA IgG titers continued to decrease as expected late after IM (two patients) or remained stationary after CMV seroconversion (two patients). HHV-6 IgG titers increased after the CMV seroconversion in all patients. he increases were significant (fourfold or more) in three of four patients. A significant increase in measles virus IgG titers was seen in one of four patients. One of thirty patients apart from the CMV-infected ones had a significant increase in HHV-6 IgG titer between 6 months and 2 years after IM; otherwise, no significant increases in HHV-6 IgG titers were noted between 6 months and 3 years after EBV infection (able 1). Antiviral IgG titers after adsorption of EBV antibodies. o examine whether the high titers of IgG to heterologous viruses seen during primary EBV infection and of IgG to HHV-6 in connection with CMV infection were due to cross-reactive antibodies, EBV and CMV antibodies were adsorbed with EBV VCA-expressing P3HR-1 cells and CMV antigen. In 10 of 12 samples, EBV VCA IgG antibodies were not detectable after adsorption with P3HR-1 cells. In two samples, titers were reduced from.1,280 to 80. In one sample drawn 28 days after IM, the HHV-6 IgG titer was reduced from.1,280 to 320. In the rest of the serum samples, the HHV-6 IgG titers remained constant. Measles virus and CMV IgG titers were unaffected by adsorption of EBV antibodies. Low CMV titers between 100 and 1,000 noted in early serum samples from one patient, who at 6 months and later after IM lacked CMV IgG antibodies, remained unchanged after adsorption. he results after adsorption with CMV antigen were similar. CMV IgG titers measured by ELISA were reduced

3 VOL. 28, 1990 EFFEC OF EBV ON HHV-6, CMV, AND MEASLES VIRUS IgG 213 A 1280, B 1280' ' c s7 day. 28 day. G0 daly 180 day. 2 y.as3 jeu haly N-S S7 days 28 days 90 da" 180 duy 2 y.. 3 yen hoeiy N D Downloaded from , 20000' on May 12, 2018 by guest ' S7 day. 28 day. 90 day. 180 day 2 y.. 3 ym h.al y s7 dwy. 28 âwy 90 day. 180 de" 2 y s3 ym haiy Nm2S N FIG. 1. Antiviral IgG titers in patients monitored from IM onset until 3 years after IM. Data are means plus or minus standard deviations (indicated by bars). (A) EBV VCA IgG IFA titers. (B) HHV-6 IgG IFA titers. (C) Measles virus IgG ELISA titers. (D) CMV IgG ELISA titers. N, Number of patients initially seropositive, from whom samples were drawn at the respective time points.

4 214 LINDE E AL. EBV VCA Ige IFA titer 1280 HHV6 IgG IFA titer 1280 Measles IgG EUSA titer ' during IM lowest value after CMV-inf during IM lowest value after CMV-inf during IM lowest value after CMV-inf. FIG. 2. EBV, HHV-6, and measles virus IgG titers in four patients who seroconverted to CMV 6 months to 3 years after IM. he highest IgG titer at the onset of IM or 28 days after onset (during IM), the titers in the serum samples drawn before CMV seroconversion (lowest value), and the titers in the first samples containing CMV IgG antibodies (after CMV inf.) are presented for each patient. to <100 (two samples) or reduced by eightfold or more (four samples). he EBV, HHV-6, and measles virus IgG titers remained completely unaffected by the CMV adsorption. DISCUSSION J. CLIN. MICROBIOL. In this study, successive, significant decreases of HHV-6 and measles virus IgG titers were demonstrated with time after primary EBV infections. Not until after 2 to 3 years did the titers reach the levels found in healthy, seropositive individuals. CMV IgG titers appeared to be less affected during IM than were measles virus and HHV-6 titers. Fewer patients were, however, seropositive for CMV, which prevented a reliable evaluation of titer changes. In the majority of the CMV IgG-seropositive patients (six of nine), there were significant titer decreases during the observation period. he HHV-6, measles virus, and CMV IgG titers were largely unaffected by adsorption of EBV-specific IgG antibodies and cellular antibodies reactive with P3HR-1 cells. he high levels of antibody to heterologous viruses found during and after primary EBV infections probably result from polyclonal B-cell activation of previously primed B-cell clones. his polyclonal stimulation is not necessarily an effect of EBV itself. Lymphokine production induced by the infection may also mediate the stimulation of B cells to increased IgG production. he finding of increased IgG3 production from EBV-infected B cells in vitro (19) in contrast to elevation of total serum IgGl during primary EBV infection in vivo (7) points to different mechanisms of B-cell stimulation by EBV in vivo and in vitro. Reactivation of HHV-6 and CMV during the EBV infection or stimulation of primed memory B cells by related antigens as alternative explanations for the elevated titers cannot be excluded. he parallel development of titers to the nonlatent measles virus, however, argues for a polyclonal stimulation as the main reason for the increase in IgG to the heterologous herpesviruses. he presence of EBV antibodies or cellular antibodies that cross-react with epitopes on any of the examined viruses to an extent that influences the titers of IgG to the other viruses seems unlikely. As is the case in previously described findings (16), elimination of EBV antibodies by adsorption generally left the titers of IgG to the heterologous viruses unaffected. We have previously shown that HHV-6 IgG titers are higher both in primary EBV- and CMV-infected persons than in healthy persons (10). Similar findings have also been reported recently (11, 12). Of 31 patients, 4 seroconverted to CMV at some time during the 3 years of follow-up after IM. he HHV-6 IgG titers increased in all patients after CMV seroconversion, while measles virus and EBV VCA IgG titers did not change significantly. An isolated IgG increase makes polyclonal B-cell stimulation less likely. DNA homologies between HHV-6 and CMV have been described previously (4), but the existence of truly cross-reacting antibodies seems excluded also for these viruses. In accordance with previous findings (16), the HHV-6 IgG titers were unaffected by adsorption with CMV antigen. Related but not necessarily cross-adsorbable antigens, activation of HHV- 6-specific memory cells during CMV infection, or reactivation of HHV-6 by CMV infection seem to be probable explanations for the elevation of HHV-6 IgG titers during CMV infection. he true frequency of seropositivity to HHV-6 is still not settled (3, 8, 14). When we examined 76 healthy Swedish adults, we found IgG antibodies in 97% when we considered weak-positive reactions as truly positive (10). When sera

5 VOL. 28, 1990 EFFEC OF EBV ON HHV-6, CMV, AND MEASLES VIRUS IgG 215 from patients with primary EBV infections were examined in this study, 31 (97%) of the 32 samples contained HHV-6 IgG antibody titers of It is likely that the high titers were the result of EBV-induced stimulation of B cells already primed to produce HHV-6 antibodies. If so, a true seropositivity to HHV-6 of 97% in the examined age group was revealed. In chronic fatigue syndrome, elevated levels of antibody to EBV are frequently found. In some studies, elevated titers of antibodies to other viruses, such as CMV, measles virus, and rubella virus, have also been noted (6). Our study underlines the danger in drawing conclusions regarding viral activity from levels of specific IgG antibodies, since these can be affected by stimulators other than the appropriate antigen. EBV is a well-known nonspecific stimulator. It is tempting to speculate that elevated titers to viruses other than EBV in chronic fatigue syndrome may be the result of polyclonal B-cell activation due to EBV activity. However, in vitro studies have proven that other agents (measles virus, rubella virus, varicella-zoster virus, and purified protein derivative) can cause augmented antibody production to heterologous viruses (2). It may even be possible that stimulation of memory B cells by various agents is necessary for the maintenance of specific antibody production by primed B cells throughout life. ACKNOWLEDGMEN his study was supported by the Swedish Medical Research Council. LIERAURE CIED 1. Andersson, J., S. Britton, I. Ernberg, U. Andersson, W. Henle, B. Skoldenberg, and A. isell Effect of acyclovir on infectious mononucleosis: a double-blind, placebo-controlled study. J. Infect. Dis. 153: Arneborn, P., G. Biberfeld, M. Forsgren, and L. V. von Stedingk Specific and non-specific B cell activation in measles and varicella. 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