Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic(ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells

The development of reliable, sensitive, and specific serological methods fo r the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate t he potential usefulness of HHV-8 proteins in measuring the responses agains t both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) pr eviously identified as immunogenic [Virology (1998) 243, 208-217]. Full-len gth genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expr ession systems, and purified as GST fusion proteins. These recombinant prot eins were used in Western blot reactions to test sera from 104 human immuno deficiency virus (HIV)(+)/Kaposi's sarcoma (KS)(+) homosexual men, 77 HIV+/ KS- homosexual men, and 84 age-matched HIV-/KS- men. These Sera were also t ested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradeca noylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differentia l reactivities reflecting different titers of antibodies against HHV-8 prot eins, and Variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS ( +) sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera we re reactive with both latent and lytic recombinant antigens. Western blot r eactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sens itive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 se ra from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Weste rn blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These resul ts suggest that IFA followed by confirmation with the Western blot reaction s with a panel of latent and lytic immunogenic antigens would provide a rel iable, sensitive, and specific method for the detection of HHV-8 antibodies . (C) 1999 Academic Press.