Anti-idiotypic studies of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.
|Title:||Anti-idiotypic studies of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.|
|Authors:||Tackaberry, Eilleen S.|
|Abstract:||Human cytomegalovirus (HCMV) is an ubiquitous member of the herpesvirus family that is responsible for morbidity and mortality in immunocompromised individuals. Our understanding of the immune response to this complex pathogen is still incomplete, limiting our capacity to devise effective strategies for its control. The goal of the research described in this thesis was to use the idiotype (id) network of the immune response as a means of manipulating the response to HCMV infection. More specifically, the objectives were to generate anti-id antibodies that would mimic an epitope of the viral envelope glycoprotein complex B (gB), an immunodominant constituent of HCMV that is widely regarded as a potential vaccine candidate. The anti-id antibodies would then be evaluated for their ability to act as surrogate immunogens in stimulating an immune response to gB in naive hosts, as well as for their utility in developing improved HCMV diagnostics. Anti-id antibodies (Ab2) were induced against the gB-specific neutralizing murine monoclonal antibody CMVB1 (mAb1). Two different series of mAb2 were generated, as well as rabbit polyclonal Ab2. Extensive analyses indicated that the Ab2 were directed to idiotopes closely associated with the combining site of mAb CMVB1, and thus might mimic the original reference epitope of gB. Purified mAb2 and rabbit Ab2 were therefore prepared and used to immunize mice. Resulting data showed that all mice mounted an anti-Ab2 (Ab3) response whereas mice immunized with appropriate controls did not. The Ab3 mouse sera were then further evaluated to determine if they exhibited antigen-specific reactivity for the reference antigen, gB. Using assays previously developed to characterize the anti-HCMV activity of the Ab1 mAb CMVB1, we found no detectable antigen-specific activity in any of the Ab3 sera raised against the mAb2. However, the Ab3 sera raised against purified rabbit Ab2 did exhibit anti-HCMV activity, in a manner analogous to that of mAb CMVB1. This was manifested by binding to HCMV-infected cells, by immunoprecipitating viral proteins identified as gB, and by neutralizing viral activity in vitro. A comparison of the id profiles of the Ab1 and antigen-specific Ab3 showed that at least one idiotope was co-expressed on the Ab1 and antigen-specific Ab3 antibodies, suggesting some dominance of this recurrent id. However, the data also demonstrated that different subsets of B lymphocytes had been stimulated by gB compared to the rabbit Ab2. These results not only revealed mechanisms by which the id network may contribute to the generation of antibody diversity in the immune response, but also illustrated how subtle structural differences may influence the immune response that is induced, considerations of some relevance in the selection of id based vaccines and therapies. The mAb2 were used to develop an innovative ELISA for detecting a laboratory strain of HCMV, based on the ability of viral antigen to inhibit the specific interaction between Ab1 (mAb CMVB1) and complementary mAb2. A linear dose-response curve was obtained for HCMV between 20 and 0.6 $\times$ 10$\sp3$ PFU/ml, with 50% inhibition at approximately 3 $\times$ 10$\sp3$ PFU/ml. The principles of this immunoassay show promise not only for HCMV, but also for the rapid measurement of other infectious agents.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|