Positive and negative Kaposi sarcoma-associated (KSA) individuals need an improved diagnostic assay for human herpesvirus-8 (HHV-8) detection. The selection of one or more viral epitopes that elicit strong immune responses is essential in developing this assay.

One approach to improve assay sensitivity is to use one or more viral peptides from viral envelope glycoproteins in an immunoassay. Spira et al. (2000) showed that a combination of peptides improved assay sensitivity. They looked at several variables—first, monoclonal antibody-enhanced immunofluorescence assay (MIFA) on virus-infected cells on slides. Second, an immunoblot (IB) assay detecting HHV-8 ORF65 (nucleic acids 256-519), which was expressed as a fusion protein in E. coli and partially purified. Third, an IgG ELISA assay kit from Advanced Biotechnologies, Inc. (ABI EIA), which uses an extract of purified HHV-8 whole virions isolated from the KS-1 cell line. Lastly, a peptide enzyme immunoassay (EIA), which included peptides from the ORF65 gene (amino acids 140-170) or ORF K8.1 gene (amino acids 32-62). The MIFA was the most sensitive of these assays (detecting lytic antigen in 97%) with a panel of 88 specimens from HIV-infected patients, followed closely (93% detection) by a combination of the ORF65 and the ORF K8.1 peptides.

Mortazavi, Lidenge, Tran, West, and Wood (2020) also looked at the response of neutralizing antibodies to several HHV-8 antigenic determinants. They measured immune responses to specific HHV-8 envelope proteins: ORF8 (gB), ORF28, ORF 22 (gH), ORF47 (gL), ORF39 (gM), ORF53 (gN) and gpK8.1. They concentrated on these envelope glycoproteins since the vaccination with other viruses previously showed to elicit neutralizing antibodies that target viral membrane surface proteins. These proteins play an important role in virus attachment to target cells, the fusion of the viral envelope with either cytoplasmic or endosomal membranes, and virion morphogenesis and egress. Mulama et al. (2019) showed immunization with HHV-8 envelope glycoproteins gB, gpK8.1, and gH/gL can elicit neutralizing antibody responses in animal models and play a major role in virus attachment to cells. Although most of these eight viral glycoproteins elicited neutralizing antibody responses, only the gH/gL complex yielded responses that are consistent and comparable among most individuals. Eighty percent of study participants showed high levels of neutralizing antibodies against the gH/gL complex. They speculated that gH and gL might undergo conformational changes when complexed, which then expose yet-to-be-determined epitopes that are highly immunogenic, resulting in high recognition by over 80% of the infected individuals. One possible explanation for this is that some neutralizing antibodies are targeting the carbohydrate moieties on the glycoproteins. Studies have shown the HHV-8 gB is associated with high levels of mannose, which could be targeted by HHV-8 specific neutralizing antibodies.

HHV-8 membrane glycoproteins are known to elicit strong antibody responses upon infection. Peptides from these viral proteins, either individually, or in combination with other viral peptides, would be of value in assay development for detecting either HHV-8 or Kaposi’s sarcoma in infected individuals.

By David Kilpatrick, PhD and Abbas Vafai, PhD

Spira, T. J., Lam, L., Dollard, S. C., Meng, Y-X., Pau, C. P., Black, J. B., Burns, D., Cooper, B., Hamid, M., Huong, J., Kite-Powell, K., & Pellett, P. E. (2000). Comparison of serologic assays and PCR for diagnosis of human herpesvirus 8 infection. Journal of Clinical Microbiology, 38(6), 2174-2180.

Mortazavi, Y., Lidenge, S. J., Tran, T., West, J. T., & Wood, C. (2020). The Kaposi’s sarcoma-associated herpesvirus (KSHV) gH/gL complex is the predominant neutralizing antigenic determinant in KSHV-infected individuals. Viruses, 12(3), 256.

Mulama, D. H., Mutsvungma, L. Z., Totonchy, J., Ye, P., Foley, J., Escalante, G. M., Rodriguez, E., Nabiee, R., Muniraju, M., Wussow, F., Barasa, A. K., & Ogembo, J. G. (2019). A multivalent Kaposi sarcoma-associated herpesvirus-like particle vaccine capable of eliciting high titers of neutralizing antibodies in immunized rabbits. Vaccine, 37(30), 4184-4194.