Antigenic structure of the foot-and-mouth disease virus. III. Immunogenic properties of synthetic peptides of the sequence of the immunodominant region of VP1 proteins of the O1K and A22 strains of foot-and-mouth virus

Immunogenic and protective properties of uncoupled and KLH-conjugated peptides covering the sequence of the immunodominant region of VP1 proteins of the O1K and A22 strains of foot-and-mouth disease virus have been studied. The uncoupled peptides 136-148 O1K, 136-152 O1K, 131-149 A22 and 140-149 A22 were shown to be immunogenic in guinea pigs and induced 50-100% protection against homologous virus. On the other hand, the A22 specific peptides, in contrast to the O1K peptides, were not immunogenic in rabbits. Immunization of nonresponders with the A22 specific peptides containing the O1K peptide can bypass nonresponsiveness to the A22 peptide in terms of the antibody production. The induced antibodies showed virus-neutralizing activity in vitro.     

Antigenic structure of the foot-and-mouth virus. VI. Functional segments of the immunodominant region of the VP1 protein of foot-and-mouth virus strains O1K and A22]

B- and T-epitopes have been localized within the protective fragments of VP1 protein, viz., 136-152 of the O1K strain and 135-159 of the A22 strain of the foot-and-mouth disease virus (FMDV). Antibodies eliciting after immunization of various animals with the 135-159 A22 peptide are directed to different sites of the peptide. Immunogenicity of fragments of the 135-159 A22 peptide on mice correlates with their activity on T-cells of the same animals and protective activity on guinea pigs. The investigations were carried out using synthetic fragments of the 136-152-O1K and 135-159-A22 peptides.     

Synthetic peptides simulating the protective epitopes of VP1 protein of foot-and-mouth disease virus type O and A

In a search of novel approaches to cattle protection from foot-and-mouth disease we have prepared a series of peptides from the major antigenic region 130-160 of the VP1 protein. The 144-159 peptide as well as 141-152, 141-148, 148-159 segments (strain O1K) were inactive in all in vitro and in vivo experiments on virus inhibiting. On the other band, synthetic 136-152, 136-148 O1K sequences as well as 131-149, 140-149 A22 sequences afforded 50 to 100% protection, both in the free state and conjugated with keyhole limpet hemocyanin. Therefore the 136-145 region should be considered as an essential part of the major sequential epitope, necessary for full-scale antiviral immune response. We also believe that the 136-152 segment is so far the smallest peptide capable of eliciting virus neutralizing antibodies and antiviral protection without conjugation with a high-molecular carrier.     

Antigenic structure of the foot-and-mouth-disease virus. I. Synthesis of protective peptides from the major immunogenic region of VP1 protein of foot-and-mouth virus type O1K

A series of overlapping peptides with the sequence of the immunodominant region of VP1 protein of FMDV strain O1K have been synthesized by the classical solution method. Peptides were purified by standard methods and used for immunization of guinea pigs. It is shown that the 136-152 and 136-148 segments provide antiviral protection in guinea pigs, both in the free state and conjugated with an immunogenic carrier. Results with uncoupled peptides indicated that these segments may form not only B-, but also T-cell affecting sites.     

Synthetic peptide designs based on immunoactive fragments of the VP1 protein of the foot-and-mouth disease virus strain A22

Peptide constructs consisting of 44-53 aa were synthesized on the basis of sequences 135-159, 170-190 and 197-213 of VP1 from the foot-and-mouth disease A22 strain. Immunogenic and protective properties of the peptide constructs were studied in guinea pigs and mice of three lines. The constructs were shown to induce higher levels of antibodies and exhibit higher protective effects than the separate peptides. The most active among the peptides studied was the construct involving the VP1 fragments 135-160 and 170-190: it protected pigs from the experimental infection by the foot-and-mouth disease virus.     

Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.     

Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]

Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.     

Identification of an exposed region of the immunogenic capsid polypeptide VP1 on foot-and-mouth disease virus

Iodination of intact foot-and-mouth disease virus results in the selective labeling of VP1, substantiating its exposed location on the virion. A comparison of tryptic peptides revealed that a single tyrosine-containing peptide was labeled with iodine on intact or protease-cleaved virus. The labeled peptide from intact and protease-cleaved virus was characterized by molecular weight sizing and sequence analysis. Carboxypeptidase digestion of intact VP1, limited trypsin-cleaved VP1, and VP1 purified from bacterially contaminated tissue cultures yielded carboxyterminal residues of leucine, valine-arginine, and serine-alanine, respectively. The correlation of these findings with previous data on the amino acid sequence derived from nucleotide sequencing of serotypes A12 and O1 of foot-and-mouth disease virus VP1 places the probable exposed antigenic region of VP1 in a serotype-variable region including residues 136 through 144.     

Preferential induction of IL-10 in APC correlates with a switch from Th1 to Th2 response following infection with a low pathogenic variant of Theiler's virus

Theiler's murine encephalomyelitis virus induces immune-mediated demyelination in susceptible mice after intracerebral inoculation. A naturally occurring, low pathogenic Theiler's murine encephalomyelitis virus variant showed a single amino acid change within a predominant Th epitope from lysine to arginine at position 244 of VP1. This substitution is the only one present in the entire viral capsid proteins. In this paper, we demonstrate that the majority of T cells specific for VP1(233-250) and VP2(74-86) from wild-type virus-infected mice are Th1 type and these VP1-specific cells poorly recognize the variant VP1 epitope (VP1(K244R)) containing the substituted arginine. In contrast, the Th2-type T cell population specific for these epitopes predominates in variant virus-infected mice. Immunization with UV-inactivated virus or VP1 epitope peptides could not duplicate the preferential Th1/Th2 responses following viral infection. Interestingly, the major APC populations, such as dendritic cells and macrophages, produce IL-12 on exposure to the pathogenic wild-type virus, whereas they preferentially produce IL-10 in response to the low pathogenic variant virus. Thus, such a spontaneous mutant virus may have a profoundly different capability to induce Th-type responses via selective production of cytokines involved in T cell differentiation and the consequent pathogenicity of virally induced immune-mediated inflammatory diseases.     

Synthesis and immunochemical properties of oligopeptides--fragments of capsid proteins of the hepatitis A virus]

The hepatitis A virus (HAV) capsid protein VP1, VP2 and VP3 are exposed at the virion surface and should therefore contain antigenic determinants. Algorithms for hydrophilicity, antigenicity and flexibility were used to predict probable antigenic sites. Synthesis of 7- to 23-membered overlapping peptides from seven sites, viz., 1-11, 1-17, 2-33, 11-25, 73-82, 76-86, 98-109, 98-112, 102-107, 102-108, 108-127, 113-123, 118-140, 276-298 from VP1, 42-62 from VP2, 76-85 from VP3, and 1-23 from VP4, was performed by various solid-phase methods. Free peptides and their conjugates with different carriers were used for immunization and study of antigenicity. The peptides did not interact with antibodies to the hepatitis A virus, whereas their conjugates did not induce the formation of anti-HAV-antibodies.     

Cross-reactive and serotype-specific antibodies against foot-and-mouth disease virus generated by different regions of the same synthetic peptide.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.     

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.     

The molecular basis of the antigenic variation of foot-and-mouth disease virus.

We have cloned and sequenced the viral protein (VP1)-coding regions of two foot-and-mouth disease virus (FMDV) serotypes (C1 and A5). Comparison of the derived amino acid sequences with the known VP1 sequence of FMDV O1K and the two FMDV A subtypes A10 and A12 shows two highly variable regions in the protein, at positions 40-60 and 130-160, as possible antigenic sites. In both variable regions, several sites could be detected where all three sequences of the A subtypes are identical but the three types A, C and O differ from each other. The second variable region overlaps with a major immunogenic determinant of the virus.     

Synthetic immunogenic complexes containing a peptide from the surface protein of the foot-and-mouth disease virus

Synthetic constructions containing a peptide antigenic determinant (C-terminal peptide 205-213 of the surface VP1 protein of the foot-and-mouth disease virus, O1K strain), glucosaminylmuramayl dipeptide (GMDP), and polyionic synthetic carriers were prepared. The polymerized peptide and peptide-BSA conjugates were synthesized as well. Among the constructions obtained only peptide-BSA conjugate proved to be highly immunogenic. Application of synthetic constructions to design immunogenic complexes is discussed.     

Structural refinement and analysis of Mengo virus

The structure of Mengo encephalomyelitis virus was refined at 3 A resolution with a final R-factor of 0.221 and a root-mean-square deviation from idealized bond lengths of 0.019 A for 10 A to 3 A data with F greater than or equal to 3 sigma(F). The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement averaging procedure and constrained by the icosahedral symmetry of the virus. The virus consists of 60 protomers each having three major subunits, VP1, VP2 and VP3, along with one smaller internal protein, VP4. The three major subunits form similar eight-stranded beta-barrel structures. Alterations in the original sequence were found at position 45 in VP1 (Arg to Ala) and at position 58 in VP3 (Met to Val). The residues in loops I and II of VP1 (82 to 102), the "FMDV loop" in VP1 (205 to 213), the flexible loop of VP3 in the putative receptor attachment site (175 to 185) as well as the terminal regions 260 to 268 in VP1, 253 to 256 in VP2 and 13 to 15 in VP4 were built or modified in regions of weak density. The variation in temperature factors at the end of the refinement is over a wide range (from 2 to 80 A2), with the disordered outer and inner regions showing high mobility. Four cis proline residues, 105 in VP1, 85 and 152 in VP2 and 59 in VP3, have been identified. The disulfide bridge Cys86 to Cys88 in VP3 has been characterized. One phosphate ion and 233 water positions were included in the refinement. It is suggested that this phosphate is associated with the receptor attachment site. There are two major hydrogen-bonding networks involving solvent atoms; one involving only the subunits of a protomer, and the other connecting the protomers in a pentamer. The distribution of atom types around the icosahedral symmetry axes shows that the 5-fold channel is more hydrophobic than that along the 3-fold axis and that there are more charged residues around the 2-fold axis. The analysis of contacts between the different subunits supports the assignment of the protomeric unit. The five protomers that form the pentameric unit are held together by interactions involving the smaller VP4 protein and the amino termini of VP1 and VP3. The pentamers are associated by means of the amino-terminal region of the VP2 subunits, the beta F strand of the VP3 subunits, the C terminus of the VP4 subunits and the electrostatic helical (alpha A) interactions of VP2 subunits across the icosahedral 2-fold axes. The superposition of the corresponding subunits of Mengo virus, human rhinovirus 14 and southern bean mosaic virus has provided an improved sequence alignment. The largest structural similarity is between the VP3 subunits of Mengo virus and rhinovirus, while the least similarity is between the VP1 subunits. The various specialized insertions in the different subunits can be associated with specific functional requirements.     

Studies on antigenic variability of C strains of foot-and-mouth disease virus by means of synthetic peptides and monoclonal antibodies.

Peptides representing the sequence of the immunodominant loop of foot-and-mouth disease virus strain C-S8 (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1) and of several variant viruses have been prepared by solid phase methods. In addition, five peptides with single-residue replacements at Leu147 (Ile, Nle, Val, Ala, Gly) have been synthesized. Tosyl and dinitrophenyl protections for histidine have been compared, the latter being found to give better synthetic products. The peptides have been tested in an immunodot assay against a panel of monoclonal antibodies directed towards the VP1 loop. Immunochemical results are discussed on the basis of the mobility of the region reproduced by the peptides and the nature of the side chain of residue 147.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro.     

Structural proteins of polyoma virus: proteolytic degradation of virion proteins by exogenous and by virion-associated proteases.

A model has previously been proposed for the genetic relatedness of the structural proteins of polyoma virus, based upon similarities in the peptide maps of the major capsid protein VP1 with the virion proteins VP2 and VP3. Newer evidence suggests that this model is incorrect, and that protein VP1 is a product of one viral gene and that the multiple components of VP2 and VP3 are products of a second viral gene. Two-dimensional peptide maps of several preparations of polyoma purified separately from four separate infected-cell lysates has shown a variable content of VP1 peptides in proteins VP2 and VP3, with some preparations being free of detectable VP1 material in VP2 and VP3. An alternative explanation for the presence of VP1 peptides in the regions of VP2 and VP3 of some polyoma preparations involves the cleavage of proteins of polyoma virions during exposure to proteolytic enzymes in lysates of infected cells or to endogenous proteolytic activity of virions. Prolonged incubation of infected-cell lysates at 37 degrees C leads to an increase in the amount of 86,000-dalton dimer of VP1, a decrease in the relative amount of VP1, a decrease in or a loss of the lower band of VP2, and the appearance of a new major protein band of approximately 29,000 daltons. Two-dimensional peptide maps of the new 29,000-dalton protein show that it contains some VP1 peptides, indicating that this protein is derived from proteolytic cleavage of VP1. In addition, extensively purified polyoma virus contains a proteolytic activity that can be activated during disruption of the virus with 0.2 M Na2CO3-NaHCO3 (pH 10.6) in the presence of 5 X 10(-3) M dithiothreitol.