Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (Ka) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.

The complex formation between glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) was studied by using five recombinant baculoviruses expressing open reading frames that contain deletions in the coding region of the extracellular domain of gH. In addition, the gH-deletion mutants contained a C-terminal tag. Complex formation of gL and the gH-deletion mutants was studied by immunoprecipitations with anti-tag monoclonal antibody (MAb) A16 and with the gH-specific MAbs 37S, 46S, and 52S. All gH-deletion mutants were complexed to gL when analyzed by MAb A16. MAb 37S precipitated complexes between gL and the two gH-deletion mutants that contain the epitope of this MAb. When the gH conformation-dependent MAbs 46S and 52S were used, gL was coprecipitated together with the gH-deletion mutant lacking amino acids 31-299, but gL was not coprecipitated with the gH-deletion mutant lacking amino acids 31-473. The data from the precipitation studies do allow at least two interpretations. There is either one site for gL binding on gH (residue 300-473) or gL contacts multiple regions of gH. We were unable to demonstrate gL-dependent cell surface expression of either of the gH-deletion mutants. This suggests that the coassociation of gH with gL is necessary but not sufficient for transport of gH to the cell surface.

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.