Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine.

A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

Foot-and-mouth disease virus: immunogenicity and structure of fragments derived from capsid protein VP and of virus containing cleaved VP.

Peptide fragments were obtained from the immunogenic capsid protein VP3, ca. 24 kilodaltons (kd), of foot-and-mouth disease virus type A12 119ab by three procedures: (1) spontaneous proteolysis of in virion VP3 in tissue cultures to produce a 15 kd peptide, designated S fragment; (2) trypsin treatment of purified virus to produce a 16 kg peptide, designated T fragment; and (3) cyanogen bromide cleavage of purified VP3 to produce a 13 kd fragment. Following isolation and purification by gel electrophoresis, VP3 and each of the three fragments were immunogenic for livestock. Lyophilization appeared to impair the immunogenicity of VP3. In addition, viruses containing VP3 fragments produced either by the spontaneous- or trypsin-induced proteolysis were as immunogenic as virus with its VP3 intact. Amino acid sequencing of N-terminal regions revealed that the S fragment was homologous with the N-terminus of VP3, whereas the 13 kd fragment possessed a unique N-terminus. Thus, putative common immunogenic amino acid sequences would appear to reside within an overlap region of the 15 kd S and 13 kd fragments. Sequencing of cDNA prepared to viral genome RNA provided three kinds of information: it (1) placed the above overlap region in the second and third quarters of VP3; (2) demonstrated that the codons for the C-terminus of VP1 and N-terminus of VP3 are contiguous; and (3) supported earlier evidence that these same codons program a chain reversal where VP1 and VP3 are joined in the precursor polyprotein.

Foot-and-mouth disease and its antigens.

Many factors combine to make foot-and-mouth disease (FMD) one of the most damaging and intractable disease of animals. These include its extreme contagion, wide geographic distribution, great multiplicity of both susceptible animal hosts and viral serotypes, a relatively short duration of immunity to a given serotype and a post-recovery carrier state of the virus in many animal species (e.g., cattle, sheep and goats). Nevertheless, import restrictions and other actions of the U.S. Government have kept the United States free of FMD since 1929, even during outbreaks of the disease in Mexico and Canada in the early 1950's. Beginning in the late 1940's, the systematic vaccination of cattle has been practiced in several areas of the world with varying degrees of success. While this procedure has succeeded in Western Europe in greatly reducing the incidence of FMD, the presence of live virus in some batches of vaccine and the escape of virus from vaccine from manufacturing facilities are now responsible for a large proportion of the outbreaks that still occur there. A vaccine is needed that has no possibility of producing the disease. During the last few years, it has been demonstrated that capsid protein VP1, isolated from type A and C virions or biosynthesized in E. coli transformed with the gene for VP1, can be used to immunize livestock against FMD. Immunization of livestock has also been achieved with a 13 kd fragment (amino acid residues 55 through 179) cleaved with CNBr from the 213 amino acid long VP1 chain of type A virions. Immunogenic sites on intact virions, 12 S subunit particles and isolated VP1 chains have been studied by a combination of methods, including: assessment of the immunogenicity of VP1-specific fragments and synthetic peptides and mapping monoclonal antibodies (Mabs) generated with virus, VP1 and the 13 kd fragment to virus, 12 S subunits, VP1, VP1-specific fragments and a biosynthetic 32mer. Correlation of these results with sites having variant and serotype sequence variability indicates that the 136-179 region of type A12 VP1 possesses four putative neutralization-specific epitopes (ca. 137-143, 146-151, 152-157 and 170-175). Of three neutralization-specific epitopes on type A12 virus, two are also present on 12 S subunits and isolated VP1 chains. Mabs to the three epitopes appear to neutralize virus by different mechanisms: by viral aggregation, by blocking the site on viral VP1 that binds to cell receptors or by interfering with a postreceptor attachment step, possibly penetration or uncoating.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant DNA technology for the preparation of subunit vaccines.

Recombinant DNA technology appears to be on the verge of producing safe and effective protein vaccines for animal and human diseases. The procedure is applicable to most viruses because their isolated surface proteins generally possess immunogenic activity. Strategies used for the preparation and cloning of the appropriate genes depend on the characteristics of the viral genomes: whether DNA or RNA; their size, strandedness, and segmentation; and whether messenger RNA are monocistronic or polycistronic. Cloned surface proteins of foot-and-mouth disease and hepatitis B viruses are being tested for possible use as practical vaccines. Two doses of the cloned foot-and-mouth disease viral protein have elicited large amounts of neutralizing antibody and have protected cattle and swine against challenge exposure with the virus. Surface proteins have also been cloned for the viruses of fowl plague, influenza, vesicular stomatitis, rabies, and herpes simplex. Cloning is in progress for surface proteins of viruses causing canine parvovirus gastroenteritis, human papillomas, infectious bovine rhinotracheitis, Rift Valley fever, and paramyxovirus diseases. In addition, advances in recombinant DNA and other facilitating technologies have rekindled interest in the chemical synthesis of polypeptide vaccines for viral diseases. The bioengineering of bacterial vaccines is also under way. Proteinaceous pili of enterotoxigenic Escherichia coli are being produced in E coli K-12 strains for use as vaccines against neonatal diarrheal diseases of livestock.

N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.

A foot-and-mouth disease vaccine for swine.

An inactivated vaccine containing purified foot-and-mouth disease virus type O1, strain Brugge, emulsified with incomplete Freund's adjuvant was studied in swine. The antigen mass ranged from 0.02 to 416 mug in 0.25 ml of vaccine. At 90 days postinoculation (DPI) 33 to 100% of the swine which had been inoculated with 0.72% mug or larger amounts of antigen were protected against challenge. There was little protection at 182 DPI although the neutralizing titers obtained with 2.9, 34.6 and 416 mug doses of antigen were similar to those observed at 90 DPI. The 50% protective dose for swine was approximately 2.3 mug of antigen whether used in a freshly prepared state or after storage at 4 degrees C for 105 or 259 days. Significant protection was afforded when small volumes (0.1 and 0.5 ml) of vaccine were applied with a jet injector gun to the ear or neck of swine. Initial tissue reactions at the site of inoculation were minimal with these small doses of vaccine and generally disappeared ny 90 DPI.

Effect of zinc and other chemical agents on foot-and-mouth-disease virus replication.

Chemical agents reported to inhibit the growth of various ribonucleic acid and deoxyribonucleic acid viruses were tested against foot-and-mouth disease virus in cell culture. These included Zn(2+), aurintricarboxylic acid, polyribocytidylic acid, polyriboinosinic acid, phosphonoacetic acid, and the viral contact inactivator N-methyl isatin beta-thiosemicarbazone alone and with CuSO(4). The most effective agent, Zn(2+), inhibited foot-and-mouth disease virus production in primary calf kidney cells by 1 log unit at 0.05 mM Zn(2+) and completely at 0.50 mM. Zinc was inhibitory even when added late in infection and was nontoxic to uninfected cells as measured by protein and nucleic acid syntheses. Polyacrylamide gel patterns of [(35)S]methionine-labeled, virus-specific proteins showed increasing amounts of higher-molecular-weight material, in accord with reports that Zn(2+) inhibits post-translational cleavages of other picornavirus precursor polypeptides.

Quantitation of the antigenicity and immunogenicity of purified foot-and-mouth disease virus vaccine for swine and steers.

The antigenicity and immunogenicity of a purified preparation of foot-and-mouth disease virus [type A(12), strain 119 (FMDV A-119)] inactivated with 6.0 mmN-acetylethylenimine at 37 C were compared in swine and steers. Three antigen doses were tested, 640, 160, and 40 ng. In accordance with findings for guinea pigs, as previously determined by dose-response curves, as little as fourfold changes in antigen in the region of the minimum effective dose produced marked differences in the serological and immune responses of swine. The minimum effective dose of antigen for antibody formation in swine and guinea pigs, as determined by mouse median protective dose (PD(50)) values, was 160 ng. The minimum immunogenic dose for swine was also 160 ng. The vaccinated swine were challenged with either FMDV A-119 or with heterologous subtype A(24) strain Cruzeiro or type A strain A-CANEFA-1. Those immunized with 640 ng of antigen were about equally immune to the three challenge viruses; most swine having a mouse PD(50) value of 2.0 or greater were immune regardless of which strain was used for challenge. In steers, the smallest dose tested, 40 ng, was satisfactory in eliciting circulating antibodies and immunity. Physical and biological tests indicated that the antigen used in the vaccine is stable for at least 9 months at 4 C.

Inhibition by rifampin of African swine fever virus replication in tissue culture.

Vaccinia virus and African swine fever virus are deoxyribonucleic acid viruses of cytoplasmic origin. The fact that rifampin inhibits the replication of the former virus led to an investigation of its effect on African swine fever virus. The virus used was cytopathogenic to a PK-15 cell line, hemadsorbing in pig leukocyte cultures and lethal to pigs. Rifampin clearly inhibited the multiplication and cytopathogenicity of the virus in PK-15 cells. There was a 1- to 5-log reduction in virus titer depending upon the rifampin concentration, the multiplicity of infection, and the time after infection. Inhibition was greatest at a concentration of 200 mug of rifampin/ml. The drug was not viricidal per se, and the inhibition of virus replication was not due to the cell-granulating effect of rifampin since cultures which were transiently pretreated for long as 90 hr with 200 mug of drug/ml supported viral replication to the same degree as untreated cultures.

Cell-free translation of foot-and-mouth disease virus RNA into identifiable non-capsid and capsid proteins.

Foot-and-mouth disease virus (a member of the picornavirus group) RNA could be translated effectively in an S-30 extract from Ehrlich ascites tumour cells. This translation was inhibited by aurintricarboxylic acid, cycloheximide, puromycin and RNase. Cell-free products of translation were identified by disc gel electrophoresis and immunoprecipitation with specific antisera. Gel electrophoresis of the products without prior immunoprecipitation suggested the synthesis of some of the non-capsid proteins and capsid proteins VP1, VP2 and VP3 of the virus. Immunoprecipitations with antisera against whole virus and VP3 indicated the synthesis of VP3 and of at least two additional peptides of 100 000 and 56 000 daltons containing antigenic sites of VP3. Gel electrophoresis after immunoprecipitation with antiserum against virus infection-associated antigen indicated the synthesis of a different 56 000-dalton protein appearing to resemble non-capsid protein NCVP5. The amount of foot-and-mouth disease virus and VP3-specific peptides in the virus RNA-directed products were measured by immunoprecipitation.

Immunogenicity of namogram to milligram quantities of inactivated foot-and-mouth disease virus. I. Relative virus-neutralizing potency of guinea pig sera.

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.

Number and molecular weights of foot-and-mouth disease virus capsid proteins and the effects of maleylation.

Evidence was obtained by gel electrophoresis that foot-and-mouth disease virus (FMDV) type A(12) protein migrates mainly in a zone corresponding to polypeptide(s) approximately 25,000 daltons in molecular weight. Additional minor components were observed, four with molecular weights ranging from 10,000 to 22,500 daltons and one with a molecular weight of 37,500 daltons. The minor components comprised about 10% of the total protein and were present in variable amounts. The 75S empty capsids contained primarily 25,000-, 37,500- and 50,000-dalton zones. These molecular weights were estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate versus proteins of known molecular weight, including poliovirus and vesicular stomatitis virus proteins. Maleylation of the amino residues of FMDV protein solubilized it to about 5 to 10 mg/ml in aqueous, nondenaturing solvents. This permitted molecular weights to be estimated also by gel filtration. Maleylation of 70% of the available amino groups of the FMDV protein produced heat and sodium dodecyl sulfate-stable polymeric aggregates of 10 to 20% of the 25,000-dalton zone. It also resulted in an increase in the molecular weight of this zone by an amount equivalent (ca. 1,000) to that expected from the added maleyl residues.

Foot-and-mouth disease virus immunogenic capsid protein VPT: N-terminal sequences and immunogenic peptides obtained by CNBr and tryptic cleavages.

The immunogenic capsid protein (VPT), circa 30 kiladaltons (kd), of foot-and-mouth disease virus was examined for (i) its ability to induce neutralizing antibody in guinea pigs after chemical modifications and CNBr or tryptic cleavages and (ii) N-terminal amino sequence homology across three virus types. The immunogenicity of VPT was inactivated by glutaraldehyde treatment, carboxymethylation and maleylation or citraconylation. However, de-citraconylation restored part of the lost activity. Cleavage of type A12 VPT with CNBr produced an immunogenic peptide of circa 13 kd. A slightly larger (ca. 16 kd) immunogenic doublet, VPTab, was obtained by tyrptic cleavage of VPT in the virion. Sequence homologies of circa 85% were found between the first 26 amino acids at the N-terminus of VP chains from virus types A12 strain 119 (A12), C3 Resende (C3R) and O1 Brugge (O1B).

The structural polypeptides of aphthovirus are phosphoproteins.

Analysis of aphthovirus A12, strain 119ab, grown in the presence of inorganic 32P revealed that two of the major viral polypeptides, VP4 and trypsin-sensitive protein VP3, were highly phosphorylated. The other major polypeptides, VP1 and VP2, were also phosphorylated but to a much lesser extent. Polypeptides VP0 and P56, of which there are approximately one of two copies per aphthovirion, were also labeled with 32P. Phosphoserine and phosphothreonine appeared to be the amino acids labeled with 32P.

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.