Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 x 10(7) to 5 x 10(9) CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates.

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

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.

NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein.

An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1. This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd. NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography. This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived. This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.

Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.

Engineering a peptide epitope display system on filamentous bacteriophage.

The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein-protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines.