A Proteomic Platform for EBV and KSHV Serological Screening

Abstract 1747

Individuals with HIV infection have an increased susceptibility to EBV- and KSHV-associated malignancies. Evaluating a possible role for specific humoral responses to individual viral proteins in the control of viral load and prevention of EBV or KSHV related disease development in this population has been hampered by the limited number of viral antigens currently used for screening serum samples. We have developed a protein array platform that can be utilized for EBV and KSHV serological screening. The array consists of EBV and KSHV proteins printed onto glass slides. All eighty four EBV open reading frames and eighty six KSHV open reading frames were cloned into bacterial vectors and the inserts validated by DNA sequencing in both directions. Authenticated inserts were transferred to a yeast vector that expresses proteins as N-terminal GST-fusions. Seventy nine EBV proteins and eighty one KSHV proteins were successfully purified from yeast. First generation EBV/KSHV protein arrays have been printed with these proteins plus a variety of control proteins that include GST, and human IgG, IgM and IgA. Initial serological assays compared IgG antibody responses in HIV positive individuals, cancer patients and healthy adults. The HIV positive group (i) had a broader EBV antigenic response than the cancer patients or healthy normal's and (ii) recognized a wider range of EBV antigens compared to KSHV. This may reflect more extensive exposure to replicating EBV versus KSHV in these patients. In addition to the previously described dominant antigenic responses to EBV and KSHV, the assays identified multiple additional immunogenic viral proteins. The ability to compare serological responses to the complete repertoire of individual EBV and KSHV encoded proteins should provide new insight into B cell mediated immune regulation of these viruses.