Glycoprotein B of human cytomegalovirus : target for neutralising antibodies, ligand for cellular receptors, inducer of autoimmunity?

Abstract

Human cytomegalovirus (HCMV), a member of the Herpesviridae, is widespread in humans, causing persistent infections mostly without any clinical symptoms. However in immunosuppressed individuals HCMV still represents a significant factor in morbidity, why there is clearly a need for effective therapy and prophylaxis against HCMV infection. It has been stated that HCMV as a primary step of entry binds to cell-surface heparan sulphate (HS) and that the infectivity of HCMV in vitro may be inhibited by heparin, a carbohydrate polymer which is closely related to HS. The glycoprotein gB is suggested as being in part responsible for the interaction with cell surface HS. Moreover, gB is also regarded as the prime candidate for a subunit vaccine against HCMV infection. In the present study investigative tools such as synthetic peptides and a recombinant fusion protein were designed and manufactured in order to study the gB molecule from several perspectives. Epitopes recognised by human HCMV seropositive sera were for the first time identified in detail within the sequences amino acids (aa) 67-86 (peptide T7-13) and 798-806 (peptide gB51) of gB. Using optimised coating conditions for enzyme-linked immunosorbent assay (ELISA) the peptides T7-13 and gB51 showed a sensitivity of more than 50% respectively with high specificity. These results suggest that such peptides might be useful for diagnostic purposes in a peptide mixture as an alternative to native antigen preparations.

The immunodominant domain of gB, recognised by neutralising antibodies, is located within aa 541-640 (Neutralisation-related domain 1; NRD1), However, due to its conformational complexity antibody reactivity cannot be clearly demonstrated with synthetic peptides. Therefore a fusion protein designated NRD1-BCCP, containing the region of interest, was constructed. The fusion partner, biotin carboxyl carrier protein (BCCP), contributed with an in vivo biotinylation of the molecule. This would facilitate immobilisation and detection. NRD 1 BCCP was successfully used to monitor antibody titre increases in renal allograft recipients suffering from acute HCMV infection. Nonetheless, by further studies antibody crossreactivity to the NRD1 in HCMV seronegative individuals was observed. The most likely explanation for this is the highly conserved sequences within this part of the gB homologues of all members of the Herpesviridae. Such cross-reactivities might affect the protective immune responses towards a certain member of the Herpesviridae either by contributing to the neutralising capacity or on the other hand blocking it. Sequence analysis suggested that NRD2 contains a heparin-binding motif. Since this region constitutes a neutralisation epitope it is likely that it contributes to the interaction with target cells. An investigation into the heparin-binding properties of this domain showed that the peptide T7-13:3 (aa 69-78), which had previously been shown to be recognised by HCMV neutralising antibodies, indeed was able to bind heparin, HS and interestingly also DNA. Others have suggested that HCMV infection may be associated with the induction of autoantibodies, for instance against heparin and DNA, Therefore the last part of this thesis was dedicated to monitoring anti-heparin reactivity in renal allograft recipients. In patients with acute primary HCMV infection, a correlation was seen between the serological responses against HCMV, NRD1 and heparin. Furthermore, anti-heparin antibodies were isolated from serum and showed to be cross-reactive with DNA. These results suggest that acute HCMV infection may induce an autoantibody response and it is plausible that gB has a role in this process.