Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies

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Abstract
Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced of polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected human embryo lung cell extracts. Seven hybridomas producing antibodies specific for gC, 1 for gB and 1 for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, 1 specific for gB and 1 specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 .times. 10-4 in nonmutagenized stocks to 1 .times. 10-2 in mutagenized stocks. Four gC and 4 gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The 2 other gC antigenic variants and the 4 gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The 2 mar C mutants were tested for resistance to neutralization with a panel of 7 gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least 2 antigenic sites on glycoprotein gC.