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

Abstract

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against 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) previously identified as immunogenic [Virology (1998) 243, 208–217]. Full-length 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 expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS− homosexual men, and 84 age-matched HIV−/KS− men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-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 differential reactivities reflecting different titers of antibodies against HHV-8 proteins, 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 were reactive with both latent and lytic recombinant antigens. Western blot reactions 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 sensitive than the ORF 73 Western blots and LANA–IFA. With an exception of 2 sera 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 Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.