A major antigenic region localized to the C-terminal part of the 55K glycoprotein of human cytomegalovirus (HCMV) was mapped using synthetic peptides. Analysis of the region with six sera from healthy anti-HCMV seropositive blood donors showed that the length of the reactive sequence varied between four and eight amino acids (aa). The shortest sequence recognized was VTSG (aa 793 to 801 of the 130K precursor protein), but most sera required the three or four residues C-terminal to this to react, giving a major site ofVTSGSTKD (aa 798 to 805). Using peptides immobilized on polyethylene pins, the importance of both interior (between aa essential for the binding of an antibody) and extension spacer residues was evident. Free peptides containing the reactive sequence were prepared for use in a conventional ELISA and optimal pH conditions for coating were determined. The best results were achieved at pH 2 to 3, which is in agreement with the advantageous net charge of these peptides at this low pH. Anti-HCMV positive sera showed a sensitivity of approximately 50% for both peptides on polyethylene pins and peptides coated onto plates.
An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying quantitative analytical assessments.
An investigation was performed into the heparin‐binding properties of a synthetic peptide deduced from the sequence of human cytomegalovirus glycoprotein B. The peptide, T7‐13:3, amino acids 69–78, which was previously shown to contain a neutralization epitope was able to bind heparin coated onto microtitre plates as well as immobilized on agarose beads. Conversely, labelled heparin could be used to specifically detect the immobilized peptide. The peptide bound to human cells in a manner which suggested an interaction with extracellular matrix, and binding of the peptide to human fibroblasts could be inhibited both by adding soluble heparin and by enzymatic pretreatment of the cells with heparinase. The sequence of T7‐13:3 shows similarity to several proteins with known or supposed ability to bind heparin, e.g. basic fibroblast growth factor, the heparin‐binding capacity of which could also be inhibited by T7‐13:3. The peptide was also found to bind DNA, probably due to the similarities between DNA and heparin in terms of structure and charge. Because heparin is a chemical homologue of heparan sulfate, the results strongly indicate that the sequence represented by T7‐13:3 is involved in the binding of virus to cell surface heparan sulfate. The described region of gB may have the potential to contribute to a subunit vaccine although possible hazards, such as the induction of auto‐antibodies to heparin, and thus also to DNA, need to be considered.
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