Pratap Malik

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

NEW VECTORS FOR PEPTIDE DISPLAY ON THE SURFACE OF FILAMENTOUS BACTERIOPHAGE

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.

Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase

Many small peptides can be displayed on every copy of the major coat protein in recombinant filamentous bacteriophages but larger peptides can only be accommodated in hybrid virions mixed with wild‐type protein subunits. A peptide insert of 12 residues capable of display at high copy number in a hybrid virion was found to be incapable of supporting recombinant virion assembly, a defect that could not be overcome by over‐expressing leader peptidase in the same Escherichia coli cell. In contrast, over‐expressing leader peptidase did increase the copy number of two 9‐residue peptides that were poorly incorporated into hybrid virions. The factors that limit peptide display are varied and not restricted to the early stages of viral assembly.

ACCESSIBILITY OF PEPTIDES DISPLAYED ON FILAMENTOUS BACTERIOPHAGE VIRIONS :SUSCEPTIBILITY TO PROTEINASES

The genome of the filamentous bacteriophage fd has been engineered so that small peptides can be inserted into the exposed N-terminal segment of pVIII, the major protein of the virus capsid. Most small peptides can be displayed on all 2700 copies of pVIII (a recombinant virion), but larger peptides can be displayed only in virions in which modified and wild-type proteins are intermingled (hybrid virions). Peptides displayed in this way are highly immunogenic and capable of interacting with receptors and other ligands. The physical accessibility of the displayed peptides was tested by examining their susceptibility to digestion with proteinases. Potential cleavage sites in peptides displayed on recombinant or hybrid virions were in general found to be accessible to trypsin and chymotrypsin; and the density of incorporation of peptides in the virion had no effect on the susceptibility to cleavage. However, peptide bonds towards the C-terminal end of an insert, located approximately 47 residues or fewer from the C-terminus of the coat protein, were protected from digestion, presumably because of their proximity to the bulk viral surface. These results have important implications for the design and optimization of peptide display systems using filamentous bacteriophages.

CHAPTER 8 – Multiple Display of Foreign Peptide Epitopes on Filamentous Bacteriophage Virions

This chapter discusses multiple displays of foreign peptide epitopes on filamentous bacteriophage virions. Display of foreign peptides on the surface of filamentous bacteriophage virions has become an important technique for generating specific antipeptide antibodies and exploring vaccine design. In the bacteriophage fdH, a blunt-ended fragment of DNA encoding the peptide is inserted at an engineered HpaI restriction site in the bacteriophage gene VIII, such that the new amino acid sequence appears between residues 3 and 4 of the mature coat protein. Generating the HpaI restriction site in fdH causes two amino acid replacements, G3V and D4N, in the N-terminal region of the mature gVIIIp. In these plasmid vectors, the modified gene VIII is placed under the control of the IPTG-inducible tac promoter when grown in lacI q strains of Esherichia coli . In the vector pKfdH, a blunt-ended DNA fragment encoding the desired peptide insert is cloned into the engineered HpaI site, as for bacteriophage fdH. Inserts of up to 16 amino acids have been displayed at a density of several hundred to a thousand copies of the peptide per virion. It is found that fewer copies per virion are generally obtained with longer inserts.