Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.

Purification of the integral membrane glycoproteins D of herpes simplex virus types 1 and 2, produced in the recombinant baculovirus expression system, by ion-exchange high-performance liquid chromatography.

Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Freijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr., 646 (1993) 37]. In the present study this novel approach was applied to the purification of the integral membrane glycoprotein D of Herpes simplex virus type 1 and 2. The glycoproteins D of types 1 (gD-1) and 2 (gD-2) were cloned into the baculovirus expression system and produced in protein-free cultured insect cells. Detergent extracts of recombinant baculovirus-infected insect cells containing gD-1 or gD-2 were prepared using pentaethyleneglycol monodecyl ether, for extraction (final concentration 2%, w/v). The same detergent was used as additive in the elution buffers for HPIEC on a Mono Q HR 5/5 column. At low (0.005%) detergent concentration, most of the proteins present in the extract including part of gD were eluted with the sodium chloride gradient whereas a subsequent blank run using the same gradient at higher detergent concentration (0.1%) resulted in selective elution of pure gD.

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals.

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.

A ten-residue fragment of an antibody (mini-antibody) directed against lysozyme as ligand in immunoaffinity chromatography.

The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule interact with amino acids present in an epitope of a protein. A ten-residue peptide was synthesized with an amino acid sequence analogous to the hypervariable L3 segment of a monoclonal antibody directed against lysozyme. The peptide was immobilized on CH-Sepharose 4B and the affinity adsorbent was used to purify lysozyme added to a detergent extract of insect cells infected with a recombinant baculovirus. This methodology may also be applicable to other antigen-antibody combinations, in immunoaffinity chromatography for selective purification of a protein or in an immunosensor for detection of a protein.

Synthetic antibody fragment as ligand in immunoaffinity chromatography.

The possibility that a fragment of an antibody molecule may interact with a protein antigen was tested by studying the binding properties of a thirteen-residue synthetic peptide with an amino acid sequence similar to part of a hypervariable segment of a monoclonal antibody directed against lysozyme. Affinity adsorbents were prepared with this peptide and with non-related peptides as ligand. Non-specific interactions could be abolished by washing the column with 0.05 M sodium thiocyanate in 20 mM tris-HCl (pH 7.4). Lysozyme was only bound to the antilysozyme adsorbent and could be eluted with 1 M sodium thiocyanate. The results show that immunoaffinity chromatography with synthetic peptide ligands which mimic the antigen-binding site may be a useful tool in the selective purification of proteins.