The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays

The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known OF predicted to be presented on the surface of the intracellular Mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-p-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated at-ray is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins 135 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class 11 alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro. (C) 2009 Elsevier Ltd. All rights reserved.