Neutralizing antisera to human cytomegalovirus were produced in rabbits with alkaline-buffered extracts of infected cell cultures. The antibody activity was complement dependent and associated primarily with the 7S immunoglobulin fraction. Antisera with homologous K values greater than 10.00 were shown to be suitable for neutralization kinetic studies and were so used to examine the antigenic relatedness of strains AD169, Davis, Esp, C-87, Kerr, and Towne. Based upon the degree of relationship

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... s determined by normalized K values, antisera to the Davis or AD169 strains discriminated three antigenic groups, and an antiserum to strain Esp discriminated two groups. Common antigens of human cytomegaloviruses (CMV) have been demonstrated by complement fixation (6, 19), fluorescent-antibody (4, 11), and indirect radioimmunoassay tests (7); a common soluble antigen reactive in complement fixation and fluorescent-antibody tests has been partially characterized (20). However, antigenic variation between CMV strains has been demonstrated in neutralization tests (2, 8, 13, 22) . Currently, questions regarding the degree of variability and importance of the antigenic spectrum of the CMVs are unresolved. Studies have been hindered by the lack of a relatively simple, standardized methodology. Neutralizing antisera produced in animals to CMV (1, [7] [8] [9] 13, 15, 16, 18) generally have been low in potency, cytotoxic, or have not been studied for their capacity to discriminate antigenic differences. We report here the production of neutralizing antisera in rabbits to CMV immunogens obtained from infected cell cultures by an adaptation of the glycine buffer method of extraction (13); the antisera were characterized and used in neutralization kinetic tests to examine the antigenic relationships of six widely used strains of CMV. MATERIALS AND METMODS Cell cultures. Primary human fibroblast cultures were prepared locally from fetal lung tissue or were purchased from Microbiological Associates, Bethesda, Md. Cell cultures were grown at 360C in 1-liter Brockway bottles, roller bottles (285 by 110 mm), or wells (16 mm in diameter) of 24-well plastic tissue culture dishes and maintained in Eagle minimal essential medium supplemented with 5% fetal calf serum, 100 U of penicillin per ml, and 100 ug of streptomycin per ml. Tissue culture dishes were incubated in a humidified 5% C02 atmosphere. Viruses. The Davis, Esp, and Kerr strains of CMV have been maintained in this laboratory since isolation, as has AD169 since its receipt as an isolate from Wallace Rowe. Strain C-87 was obtained from Fred Rapp, Hershey Medical School, Hershey, Pa., and the Towne strain was obtained from Stanley Plotkin, the Wistar Institute of Anatomy and Biology, Philadelphia, Pa. Relevant information on the origin of each strain, and on the passage level when used, is summarized in Table 1. Plaque assay. Cell cultures grown in 16-mm-diameter wells of Disposotrays (Flow Laboratories, Rockville, Md.) were used for the determination of plaque-forming units by a method previously described (5). Preparation of immunogen. The medium was removed from infected cell cultures showing 100% cytopathic effect. The cells were dislodged with a rubber policeman into glycine-buffered saline (0.05 M glycine, pH 9.0) and centrifuged for 15 min at 250 x g. After two washes in glycine-buffered saline, the cell pellet was resuspended in glycine-buffered saline to make a 10% suspension (vol/vol) and disrupted mechanically with a tissue grinder or by sonic treatment. The disrupted cell suspension was extracted overnight at 40C in glycine-buffered saline and centrifuged at 250 x g for 15 min; the resulting supernatant constituted the immunogen. Preparation ofantisera. White, female, New Zealand rabbits between 3 and 6 months old were used for antibody production. Immunogen was mixed 1:1 with complete Freund adjuvant and emulsified. Initially each animal was injected with 4.0 to 6.0 ml intramuscularly and 2.0 to 4.0 ml into the subscapular region; some animals additionally received 0.2 ml of antigen in each footpad. Additional injections of antigen without adjuvant were given intramuscularly and subscapularly 2 or 3 weeks later and then at 7-to 11-151 on May 8, 2020 by guest http://iai.asm.org/ Downloaded from