AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 2:217-221 (19821 0271-7352/82/0204-0217$02.00 © 1982 ALAN R. LISS, INC. Epstein-Barr Virus in Normal Pregnant Women* KIYOSHI SAKAMOTO, JOHN GREALLY, ROBERT F. GILFILLAN, JULIANNE SEXTON, VANESSA BARNABEI, JOANNE YETZ, THOMAS BECHTOLD, JANET K. SEELEY, EAMON O'DWYER, AND DAVID T. PURTILO Departments of Pathology, University of Massachusetts, Worcester (K.S., J.S., VB., J.Y., T B., JK.S., D.T P.) and University of Nebraska Medical Center, Omaha (K.S., J.Y., T B., D.T P.); Department of Pathology and Obstetrics and Gynecology, Galway Medical College, Republic of Ireland (J.G., E.O'D.); Virology Laboratory, Massachusetts Department of Public Health, Boston (RF.G.); and Department of Pediatrics, University of Connecticut Health Center, Farmington (J K.S.) ABSTRACT: Acquired immune suppression accom- panying normal pregnancy may be associated with reactivation of Epstein-Barr virus (EBV). Pregnant women with reactivated EBV having anti-EA anti- bodies show high titers of antiviral capsid antigen (VCA) geometric mean titers (GMT) of 522 versus 170 in those lacking anti--early antigen (EA). Among twenty-seven seropositive women at parturition, 17 (63%) had generated antibody to EA, and all 27 (100%) demonstrated significant increases in antibody to VCA (p < 0.01). In contrast, antibody titers to cytomega- lovirus, herpes hominis, varicella-zoster, and rubella viruses in the pregnant women were comparable to those found in nonpregnant controls. (Am J Reprod Immunol. 1982; 2:217-221.) Key words: Epstein-Barr virus, reactivation, pregnancy, immune suppression. INTRODUCTION Cellular immunity in pregnant women is depressed as is manifested by impaired lymphocyte responses to plant mitogens and most microorganisms. 1 Multiple mecha- nisms prevent rejection of the conceptus by the mother.l-" Among these adaptive mechanisms, immune suppression is a two-edged sword, protecting the fetus from immune rejection but occasionally rendering the pregnant woman vulnerable to infection. An increased morbidity and mor- tality is observed in normal pregnant women to primary infection by many viruses." Cytomegalovirus (CMV) and herpes hominis can be associated with birth defects when pregnant women become infected.v" The extent of another herpesvirus, the ubiquitous Epstein-Barr virus (EBV) has not been thoroughly studied during pregnancy. EBV can: 1) infect and transform human B cells; 2) induce lymphoma and mononucleosis in cotton-topped marmosets; 3) cause infectious mononucleosis (1M)in hu- mans; 4) persist in a chronically infected host in a latent state; 5) produce life-threatening lymphoproliferative dis- eases in immune--compromised patients; and 6) be asso- ciated with Burkitt lymphoma and nasopharyngeal car- cinoma (NPC).7-12 Our laboratory has demonstrated how inherited and acquired immunodeficiency renders individ- uals vulnerable to EBV-induced diseases.9-12 In a contin- Submitted for publication February 11, 1982; accepted May 15, 1982. "This work was supported in part by grant CA 23561 from the National Institutes of Health, by the Lymphoproliferative Research Fund, and by a Burroughs-Wellcome Travelling Grant. Address reprint requests to David T. Purtilo, MD, Department of Pathology and Laboratory Medicine, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105. uation of these studies, we investigated the virus-host interaction in apparently healthy pregnant women. In studies described here, we provide evidence for reactiva- tion of EBV during pregnancy by demonstration of anti- body to early antigen (EA). MATERIALS AND METHODS To test the hypothesis that the immune suppression of pregnancy can reactivate latent EBV infection in normal pregnant women, we obtained with informed consent 15 ml of peripheral venous blood from 28 Irish women at parturition and 5 to 10 ml of blood from the corresponding umbilical cord of 27 of their newborns. Frozen sera ( - 20°C) from 14 nonpregnant females of from 17 to 32 years of age and 17 healthy males 19 to 41 years of age were assessed for EBV antibody titers while 40 premarital women from Massachusetts, ages 18 to 25, were used as controls for antibody titers against cytomegalovirus, herpes hominis, varicella-zoster, and rubella. Samplesfrom pregnant women were frozen at -70°C until tested for EBV-specific anti- bodies to EA, EB nuclear-associated antigen (EBNA), and viral capsid antigen (VCA). EBV-Specific Serology Antibody titers of IgG anti-VCA,13 IgM anti-VCA,14 IgA anti-VCA,15 and anti-EA,16 including diffuse (D) and restricted (R) components."? were determined by indirect immunofluorescence. For detection of antibodies to VCA, the P3HR-l cell line was maintained at 33°C for two weeks in RPMI medium with 10% fetal calf serum (Microbiol- ogical Associates, Bethesda, Md), 100 IUlml penicillin, and 100 umg/ml streptomycin (GlBCO, Grand Island, NY); harvested; and fixed in acetone." Smears of these cultures contained about 10% VCA-positive cells. Fluores- cein--conjugated (FITC) antihuman IgG, IgA, and IgM (heavy chain-specific) were obtained from Hyland Diag- nostic Laboratories (Costa Mesa, Calif). Raji cells, exposed to a high multiplicity of infection with P3HR-I-EBV, were used for the anti-EA determinations: The virus concen- tration was adjusted to yield 10% antigen-positive cells after 48 h at 37°C in the presence or 20 ug/ml cytosine arabinoside (Sigma, St. Louis, Mo), Raji cells, grown at 37°Cfor 3 to 4 days, were harvested and used for anti-EBNA assays." FITC--conjugated caprine'antibody to human B1a/ B 1c globulin (Hyland Laboratories) was used as a second antibody and diluted 1:40 before use. Viral Cultures of Throat Washings A deep throat gargle was obtained from each mother, who each gargled with 35 ml of RPMI-1640 for 3 min. The