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.

Immunogenicity of foot-and-mouth disease virus type O1 replicated in either monolayer or suspended BHK cell system.

The efficacy of vaccines formulated from the 10th passage of foot-and-mouth disease virus (FMDV) type O1 in monolayer baby hamster kidney (BHK) cells and the 8th passage in suspension BHK cells was compared in steers. The vaccines were inactivated with ethylenimine, contained an equal amount of antigen and were emulsified in oil-adjuvant. Six animals were vaccinated with each vaccine. During the challenge of immunity (91 days post-vaccination, DPV), one out of the six steers from the monolayer vaccine group became infected with the challenge virus while none of the six steers from the suspension vaccine group contracted the disease during the test period. The neutralizing antibody titers (means) of the serum samples taken at different DPV also did not suggest significant variation between these vaccines. In addition exposure to FMDV infected animals demonstrated that both vaccines elicited an immune state in the vaccinates.

Histological and histochemical characterisation of mammary gland tissue of cows infected with foot-and-mouth disease by contact exposure.

Foot-and-mouth disease virus was observed to replicate in secretory epithelial cells of bovine mammary gland alveoli as a result of systemic infection initiated by exposure to infected animals. Viral antigens were demonstrated using fluorescent antibody and immunoperoxidase labelling techniques before the development of signs of clinical disease. In addition, labelled antigens were observed associated with cytoplasmic-like fragments in luminal membrane limited structures. Histologically, lesions of the alveolar secretory epithelium consisted of focal necrosis of these cells which eventually sloughed into the lumen.

Secretory antibody responses in cattle infected with foot-and-mouth disease virus.

Antibody responses in serum, saliva, nasal secretions, or esophageal-pharyngeal fluid of foot-and-mouth disease virus-infected steers were examined by single radial immunodiffusion and mouse-neutralization tests. In steers infected with type O foot-and-mouth disease virus, high serum antibody titers were detected within 10 days after infection. Antibody was first detected in saliva at 30 days and gradually increased to a plateau at about 90 days. Small amounts of antibody continued to be secreted in saliva and in nasal secretions for at least 6 months. Antibody was not detected in esophageal-pharyngeal fluid. The major antibody activity in secretions was due to secretory immunoglobulin A as revealed by radioimmunoelectrophoresis.

Brain and spinal cord lesions in pigs inoculated with swine vesicular disease (UKG strain) virus and coxsackievirus B5.

Pigs inoculated intravenously with swine vesicular disease virus (UKG strain), those inoculated with coxsackievirus B5, and other pigs exposed by pen contact to the same viruses developed diffuse encephalomyelitis. Perivascular cuffing, with lymphocytes and formation of neuroglia cell foci, were most prominent in telencephalon, diencephalon, and mesencephalon. Encephalitis was of mild to severe intensity. Severity of lesions was more extensive and severe in the pigs exposed to swine vesicular disease virus. Pen contact exposure to either of the 2 viruses caused a more severe central nervous system reaction than did intravenous inoculation. The type and the distribution of lesions produced by the 2 viruses indicate that they may be related.

Pathogenesis of swine vesicular disease in pigs.

Pigs exposed to swine vesicular disease virus developed vesicular lesions by postinoculation day 2. Lesions first appeared on the coronary band and then on the dewclaw, tongue, snout, lips, and bulbs of the heels. The onset of viremia coincided with febrile response and the appearance of vesicles. Virus was isolated from the nasal discharge, esophageal-pharyngeal fluid, and feces as early as postinoculation day 1. Greater amounts of virus were isolated from samples collected during the first week of infection, and lesser amounts from samples collected during the second week. The appearance and the distribution of specific fluorescence in various tissues indicated that during the development of swine vesicular disease virus infection, the epithelial tissues were initially involved, followed by a generalized infection of lymph tissues, and subsequently, a primary viremia. Seroconversion was detectable as early as postinoculation day 4. A mild nonsuppurative meningoencephalomyelitis throughout the CNS was observed in both inoculated and contact-exposed pigs. The olfactory bulbs were most severely and were frequently affected, particularly in contact pigs. The most severe brain lesions were found in pigs 3 to 4 days after the onset of viremia; contact pigs showed more severe brain lesions than inoculated pigs. Microscopic changes were also found in the coronary band, snout, tongue, and heart.

Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle.

Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant. The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection. The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected. The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines.

A review of the current status of oil adjuvants in foot--and--mouth disease vaccines.

Review of lipovaccines since 1916, including oil adjuvants (1935-1943) and the two Freund's adjuvants. The first oil-adjuvanted vaccines appeared in 1961. Criticism of the water-in-oil and oil-in-water vaccines and description of the mineral oil adjuvants which are available today. The authors set forth the advantages of oil adjuvants and secondary reactions which might occur; they are particularly interested in the foot-and-mouth disease vaccine intended for use in swine and express their regret that no method of standardization has yet been adopted for oil-adjuvanted vaccines which are promising but which necessitate still further study.

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.

Residual viruses in pork products.

Partly cooked canned hams and dried pepperoni and salami sausages were prepared from the carcasses of pigs infected with African swine fever virus and pigs infected with hog cholera virus. Virus was not recovered from the partly cooked canned hams; however, virus was recovered in the hams before heating in both instances. Both African swine fever virus and hog cholera virus were recovered from the dried salami and pepperoni sausages, but not after the required curing period.

Antigenic and morphologic comparisons of Ibaraki and bluetongue viruses.

Bluetongue disease virus, type 10, and Ibaraki disease virus, which causes a bluetongue-like disease of cattle, were compared to determine whether they are the same or different viruses. They were similar in morphology, but neutralization tests, complement-fixation tests, and ferritin tagging indicated that they have antigenic differences. Therefore, they should be considered as different viruses. Two other viruses of this group, African horsesickness and equine encephalosis, were included to show that Ibaraki and bluetongue had developmental morphological features that could be used to differentiate them from the two equine viruses.

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.

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.