Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12

PhoE protein is an abundant transmembrane protein from the Escherichia coli K-12 outer membrane. A synthetic oligodeoxynucleotide corresponding to an antigenic determinant of the C-terminal part of the VP1 protein of foot-and-mouth disease virus was inserted into the phoE gene in an area corresponding to a cell surface-exposed region of the PhoE protein. The level of expression of the hybrid protein was normal and the protein was incorporated into the outer membrane. The VP1-epitope was exposed at the cell surface since intact cells were recognized by a monoclonal antibody which was raised against the virus.     

Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.     

Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong (2002)

The nucleotide sequence of the VP1 coding region of foot-and-mouth disease virus (FMDV) strain HKN/2002, isolated from a disease outbreak occurring in Hong Kong in February 2002, was determined and compared with the sequences of other FMDVs. The VP1 coding region was 639 nucleotides in length and encoded a protein of 213 amino acid residues. Comparison of the VP1 nucleotide sequence with those of other isolates indicated that HKN/2002 belonged to serotype O. A VP1-based sequence similarity tree of several South-east Asian FMDV-O isolates showed that HKN/2002 was most closely related to FMDV isolates found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Comparison of the amino acid sequence of the major immunogenic region of HKN/2002 with that of the serotype O vaccine strain, O1/Manisa/Turkey/69, reveals significant similarity, indicating that current serotype O vaccines may offer some degree of protection against HKN/2002.     

The Solution Conformational Features of Two Highly Homologous Antigenic Peptides of Foot-and-mouth Disease Virus Serotype A,Variants A and USA,Correlate with their Serological Properties

The solution structure of a peptide corresponding to the VP1 region 141–160 of foot-and-mouth disease virus (FMDV) serotype A variant USA has been studied by NMR and computer calculations and compared with the results from a study on a highly homologous peptide deriving from serotype A, variant A. The two peptides differ in their serological behaviour and contain the immunodominant epitope of the virus which partly overlaps with its receptor binding region. Distance constraints, derived both from 2D and 3D homonuclear NMR and 2D-heteronuclear NMR experiments, were combined with DG calculations to yield 50 structures. After refinement through EM and restrained molecular dynamics simulations the selected structures shared several general features. In particular the 151–158 region was a helix in all cases while a large loop similar to that found in peptide A but comprising less residues and stabilized by an H-bond between the side chains of D147 and S150 was found in the majority of structures. A further loop, common to all structures, was identified around the RGD sequence (145–147). This was different from that found in the corresponding region of peptide A as were the conformations of the individual residues within the RGDX sequence. The different structural features shown by the two peptides were rationalized in terms of the S148 (peptide A) to F148 (peptide USA) mutation. The second mutation, that at position 153 (L in A, P in USA) did not appear to affect the structure of the peptide significantly although the different dimensions of the loop in the central region and the type of H-bond stabilizing it could be potentially ascribed to this second mutation. All criteria used pointed to different structural features for the two peptides consistent with their serological behaviour.     

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.     

Antigenic structure of the foot-and-mouth virus. V. Protection of naturally susceptible animals from foot-and-mouth disease using a synthetic peptide

We have synthesized the peptide representing 135-159 VP1 sequence of A22 strain of the foot-and-mouth disease virus (FMDV). The synthetic peptide induced 100% protection of guinea pigs against the disease. Two-fold immunization of cuttle with the peptide and single immunization of sheep induced full protection of the animals against A22 strain of FMDV.     

Antigenic sites on foot-and-mouth disease virus type A10.

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.     

The VP1 N-terminal sequence of canine parvovirus affects nuclear transport of capsids and efficient cell infection.

The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75 degrees C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.     

Synthesis and immunogenic properties of peptides--fragments of the immunodominant regions of the VP1 protein of the Asia-1 type of foot- and-mouth disease virus

Potential immunodominant epitopes were predicted on the basis of a theoretical analysis of the antigenic structure of the VP1 protein of the type Asia-1 foot-and-mouth disease virus. Peptides corresponding to the 140-153, 136-153, 132-153, 143-157, 137-157, and 193-208 fragments of the VP1 protein sequence were synthesized by the solid phase method, and the immunogenic properties of the peptides were studied on guinea pigs. The shortest peptide exhibiting the protective effect was found to correspond to the, 140-153 fragment of the VP1 sequence. The Plm-(Gly)3-(140-153)-(Gly)2-Lys(Plm)-Leu and [Ac-(140-153)-(Gly)3]8-(Lys)7-Gly synthetic constructions in combination with adjuvants provided up to 80% protection of immunized animals against infection with the foot-and-mouth disease virus.     

Peptides as viral vaccines: lessons from experiments with foot-and-mouth disease virus

The highly variable sequence encompassing amino acids 141–160 of VP1, one of the four capsid proteins of foot-and-mouth disease virus, elicits neutralizing antibodies in a range of experimental animals. The sequence has been synthesized chemically by Merrifield's solid phase method and biochemically as part of different fusion proteins. By incorporating the peptide into the core protein of hepatitis B virus, particles are produced which elicit levels of neutralizing antibody approaching those obtained with the intact virus particle.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.