RESUMEN
We present a comparison of methods for evaluating the potency of foot and mouth disease vaccine in the laboratory. The anti-FMDV antibodies (Ab) in vaccinated mice were tested by liquid phase (lp) ELISA, solid phase (sp) ELISA and virus neutralization (VN), and were compared with the Ab titres detected by lpELISA, which is the official test in Argentina for testing the potency of FMD vaccines and protection against a virulent challenge in cattle. The results demonstrated that it is possible to relate the Ab levels induced in vaccinated mice with both the Ab and protective responses elicited in cattle. Furthermore, it was found that the anti-FMDV Ab titres in mice detected by lpELISA 14 days after vaccination should be an accurate parameter for predicting the results of the challenge test in cattle. Thus, this test in mice appears to be an inexpensive and rapid alternative for testing FMD vaccines in cattle.
Asunto(s)
Anticuerpos Antivirales/análisis , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Enfermedades de los Roedores/prevención & control , Vacunación/veterinaria , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Fiebre Aftosa/inmunología , Ratones , Pruebas de Neutralización/veterinaria , Enfermedades de los Roedores/inmunologíaRESUMEN
The anti-foot and mouth disease virus (FMDV) serum antibody activity of protected and non protected animals immunized with inactivated FMDV originated in either bovine tongue tissue (BTTV vaccines) or BHK-21 cell suspension cultures (BHKV vaccines) was evaluated. The results show that 80-100% of the BTTV immunized and only 40-60% of the BHKV immunized animals with liquid-phase blocking sandwich ELISA (lp ELISA) serum titres of 1.5-1.7 U, were protected against the challenge with any of the four infectious FMDV argentine reference strains. This difference becomes almost marginal among BTTV and BHKV vaccinated animals with a strong anti-FMDV humoral response (i.e. lp ELISA titres > or = 1.95 U). Isotyping of the anti-FMDV response in immunized cattle with low lp ELISA titres revealed that BTTV vaccines were able to induce remarkably higher anti-FMDV IgG1 titres than their BHKV counterparts (i.e. mean titres of 1.95 and 1.35 U. respectively). This difference in specific IgG1 serum levels induced by BTTV and BHKV vaccines seems to be also limited to those animals with low anti-FMDV lp ELISA titres. These results together with the fact that the specific serum IgG1, but not the IgG2, isotype response of 219 vaccinated animals correlates almost linearly with their capacity to pass the challenge, suggests that the superior performance of BTTV vaccines is close related to their ability to raise a stronger anti-FMDV IgG1 response than BHKV vaccines.
Asunto(s)
Anticuerpos Antivirales/sangre , Aphthovirus/inmunología , Isotipos de Inmunoglobulinas , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/clasificación , Formación de Anticuerpos , Bovinos , Línea Celular , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/sangre , Lengua/citología , Vacunas de Productos Inactivados/inmunologíaRESUMEN
An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia virus recombinant vTF1-6,2 resulted in expression of chimeric proteins efficiently reactive with both anti-FMDV and anti-VSV G antibodies. However, in vitro translation of transcripts of these VSV-G/FMDV-ASA chimeric plasmids resulted in proteins that were recognized by anti-G serum but not by anti-FMDV serum, indicating a requirement for in vivo conformation to expose the ASA antigenic determinant. Insertion of DNA coding for a dimer of the ASA unidecapeptide between the VSV-NJ G gene region coding for amino acids 160 and 161 gave rise to a chimeric ASA-dimer protein designated GF2d, which reacted twice as strongly with anti-FMDV antibody as did chimeric proteins in which the ASA monomer was inserted in the same position or two other G-gene positions. For even greater expression of chimeric VSV-G/FMDV-ASA proteins, plasmid pGF2d and a deletion mutant p(delta)GF2d (G protein deleted of 324 C-terminal amino acids) were inserted into baculovirus vectors expressing chimeric proteins GF2d-bac and deltaGF2d-bac produced in Sf9 insect cells. Mice vaccinated with three booster injections of 30 microg each of partially purified GF2d-bac protein responded by enzyme-linked immunosorbent assay with FMDV antibody titers of 1,000 units, and those injected with equivalent amounts of deltaGF2d-bac protein showed serum titers of up to 10,000 units. Particularly impressive were FMDV neutralizing antibody titers in serum of mice vaccinated with deltaGF2d-bac protein, which approached those in the sera of mice vaccinated with three 1-microg doses of native FMDV virions. Despite excellent reactivity with native FMDV, the anti-deltaGF2d-bac antibody present in vaccinated mouse serum showed no capacity to bind to sodium dodecyl sulfate (SDS)-denatured FMDV virions and only minimal reactivity with VP1 protein by Western blotting (immunoblotting) after SDS-polyacrylamide gel electrophoresis. It was also shown in a competitive binding assay that a synthetic ASA unidecapeptide, up to concentrations of 200 microg/ml, was quite limited in its ability to inhibit binding of anti-deltaGF2-bac antibody to native FMDV virions. These results suggest that the chimeric VSV-G/FMDV-ASA proteins mimic the capacity of FMDV to raise and react with neutralizing antibodies to a restricted number of ASA conformations present on the surface of native FMDV particles.
Asunto(s)
Antígenos Virales/inmunología , Aphthovirus/inmunología , Cápside/inmunología , Glicoproteínas de Membrana , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Aphthovirus/genética , Baculoviridae/genética , Cápside/genética , Proteínas de la Cápside , Línea Celular , Chlorocebus aethiops , Cricetinae , Fiebre Aftosa/inmunología , Vectores Genéticos , Inmunogenética , Pruebas de Neutralización , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Spodoptera/citología , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
The lowest expected protection (LEP) at a 95% confidence of 245 foot and mouth disease (FMD) commercial vaccines was calculated from the titres of liquid-phase blocking sandwich ELISA (lpELISA) of cattle sera obtained from 3920 animals at 60 days post-vaccination (d.p.v.) and challenged with live virus at 90 d.p.v. It was found that LEP evaluation is highly specific (i.e. it is able to predict the failure in 100% of the cases) although its ability to predict the challenge (PG test) approval (i.e. sensitivity) comprised only 65% of the vaccines that passed the trial. It was possible, nevertheless, to improve the sensitivity of the evaluation by using an alternative coefficient (Ro), exclusively dependent on the number of animals exhibiting the highest and lowest lpELISA titres in a particular vaccine trial. This coefficient was capable of predicting the PG approval of 90% of the vaccines, yet maintaining acceptable levels of safety (87% of specificity). Based on these results and as a first step towards the replacement of the challenge protocol in Argentina, we propose a swift approval for commercialization of FMD vaccines which are able to reach the highly restricting LEP passmark of 82%, and the rejection of those not reaching the 50% LEP limit. More extensive experience with this new protocol will allow a finer adjustment of the LEP and Ro values and to set more precisely the cut-off points for direct approval or disapproval of vaccines by lpELISA, eliminating the use of live FMDV in the field.
Asunto(s)
Aphthovirus/inmunología , Fiebre Aftosa/prevención & control , Vacunas Virales/uso terapéutico , Animales , Anticuerpos Antivirales/sangre , Bovinos , Ensayo de Inmunoadsorción Enzimática , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.
Asunto(s)
Anticuerpos Antivirales/sangre , Aphthovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Adyuvantes Inmunológicos , Animales , Argentina , Bovinos , Fiebre Aftosa/inmunología , Reproducibilidad de los Resultados , VacunaciónRESUMEN
A 43kDa cellular polypeptide (P43), which comigrates with host-cell actin in both SDS-PAGE and isoelectrofocusing slab gels, was found associated to 140 S aphthoviral particles purified from BHK 21 cells labeled with [35S]methionine prior to infection. Ultracentrifugation analysis of disrupted virions demonstrates that polypeptide P43 is not associated to VP1-3 containing 12 S subunits but remains, like viral polypeptide VP4, at the top of the sucrose gradients. In addition, in vitro iodination or trypsin treatment show that P43 is protected from the action of both procedures and therefore supports the hypothesis that host-cell polypeptide P43 is located within the viral particles.