Herpes simplex virus type 2 (HSV-2) causes genital lesions, meningitis and occasionally, severe neonatal infections. Globally HSV-2 infection is one of the most common sexually transmitted diseases. As HSV-2 is frequently acquired and sustained asymptomatically, diagnosis during this phase of infection is essential both for the patient, and for preventive and epidemiological work. Type-specific serology is the diagnostic method of choice for this situation. The viral envelope glycoprotein G-2 (gG-2) exhibits two features unique in HSV-2 proteins. First, precursor gG-2 is cleaved to a secreted portion (sgG-2) and to a cell- and virion-associated, highly O-glycosylated mature portion (mgG-2). Second, mgG-2 is the only known HSV-2 glycoprotein which can be used as an antigen for type-discriminating serodiagnosis. In the present work we produced monoclonal antibodies (MAbs) against sgG-2 and mgG-2, and localized linear epitopes to the carboxy-terminal part of both proteins by a pepscan technique. Human anti-mgG-2 antibodies also recognized linear epitopes within mgG-2, localized to three regions of the protein of which one was found to be immunodominant. A synthetic peptide representing the immunodominant region was used successfully as antigen for detection of human anti-mgG-2 antibodies. The anti-mgG-2 MAbs as well as the human anti-mgG-2 antibodies identified type-specific epitopes and showed no cross-reactivity to HSV-1 antigen.One of the anti-mgG-2 MAbs was used for typing of 2,400 clinical HSV-2 isolates. The antibody was reactive with all except 13 of the HSV-2 isolates, and displayed no reactivity with clinical HSV-1 isolates indicating that the MAb was suitable for typing of HSV-2. Five of these 13 isolates presented a single frameshift mutation within the gG-2 gene with subsequent inactivation of the protein expression in four of the five isolates. The other eight isolates harbored a single missense mutation localized within the epitope which explained the loss of binding. These mutations did not diminish the antibody response to mgG-2 in the respective patients. Nucleotide sequencing of the gG-2 gene segment coding for mgG-2 among clinical HSV-2 isolates revealed that the epitope regions in mgG-2 were highly conserved.In conclusion, we have shown that mgG-2 is highly immunogenic for the human host and is a suitable antigen for type-discriminating serology, as well as for typing of HSV-2 isolates. The sgG-2 protein exhibited exclusively type-specific epitopes and may prove to be a novel antigen with serodiagnostic potential. The anti-gG-2 MAbs and HSV-2 mutants described here provide promising tools for further study on the function of the gG-2 proteins
