A New Cage-Like Particle Adjuvant Enhances Protection of Foot-and-Mouth Disease Vaccine

Foot-and-Mouth Disease (FMD) is an acute viral disease that causes important economy losses. Vaccines with new low-cost adjuvants that stimulate protective immune responses are needed and can be assayed in a mouse model to predict their effectiveness in cattle. Immunostimulant Particle Adjuvant (ISPA), also known as cage-like particle adjuvant, consisting of lipid boxes of dipalmitoyl-phosphatidylcholine, cholesterol, sterylamine, alpha-tocopherol, and QuilA saponin, was shown to enhance protection of a recombinant vaccine against Trypanosoma cruzi in a mouse model. Thus, in the present work, we studied the effects on the magnitude and type of immunity elicited in mice and cattle in response to a vaccine based on inactivated FMD virus (iFMDV) formulated with ISPA. It was demonstrated that iFMDV–ISPA induced protection in mice against challenge and elicited a specific antibody response in sera, characterized by a balanced Th1/Th2 profile. In cattle, the antibody titers reached corresponded to an expected percentage of protection (EPP) higher than 80%. EPP calculates the probability that livestock would be protected against a 10,000 bovine infectious doses challenge after vaccination. Moreover, in comparison with the non-adjuvanted iFMDV vaccine, iFMDV–ISPA elicited an increased specific T-cell response against the virus, including higher interferon gamma (IFNγ)+/CD8+ lymphocyte production in cattle. In this work, we report for first time that an inactivated FMDV serotype A vaccine adjuvanted with ISPA is capable of inducing protection against challenge in a murine model and of improving the specific immune responses against the virus in cattle.Fil: Bidart, Juan Esteban. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Kornuta, Claudia Alejandra. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Gammella, Mariela Vanesa. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Gnazzo, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; ArgentinaFil: Soria, Ivana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Langellotti, Cecilia Ana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Mongini, Claudia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Galarza, Roxana. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Buenos Aires Sur. Estacion Experimental Agropecuaria Cuenca del Salado. Agencia de Extension Rural Chascomus.; ArgentinaFil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Lupi, Giuliana Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Quattrocchi, Valeria. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Marcipar, Iván Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentin

Modelo animal alternativo para el control de eficacia de vacuna antiaftosa y su correlación con métodos indirectos

Bovine Viral Diarrhea Virus (BVDV) belongs to the genus Pestivirus, in the family Flaviviridae. BVDV is distributed worldwide and causes important economical losses to cattle and biological industries. BVDV infection results in clinical manifestations in cattle that range from mild respiratory or gastroenteric disease to hemorrhagic syndromes and mucosal disease, depending on the virulence of the virus and the reproductive and immune status of\nthe host. The strategy to control BVDV in Argentina is based on vaccination with inactivated formulations. However, these commercial vaccines are not useful enough to protect cattle\nfrom BVDV. Given the need for updated information about circulating strains of BVDV in Argentina, it was proposed to study the phylogenetic and antigenic characteristics of 30\nrecently isolated strains obtained from ?Pampa Húmeda? region; and with that information, to develop a subunit vaccine based on a truncated form of BVDV E2 glycoprotein (tE2), expressed in Baculovirus Expression Vector System.\nIt is well-known that subunit vaccines generally evoke poor immunity so repeated and high doses of antigen are required to induce an appropriate immune response. In order to enhance the immunogenicity of tE2 glycoprotein, and to reduce the amount of antigen, it was proposed to use the single chain antibody ?APCH? to target tE2 protein to antigen\npresenting cells.\nResults from this work show the presence of three BVDV subgenotypes in the studied region and an important antigenic diversity among them, which is of great importance for the development of vaccines and diagnostic kits for BVDV. In regard to the vaccine development, it was possible to generate oily formulations based on tE2 glycoprotein and on APCH-tE2 fusion protein from Singer strain (BVDV 1a). Both immunogens were evaluated and compared in Guinea Pig model and even though both vaccines induced neutralizing antibodies, tE2 protein targeted by APCH was more efficient: evoked a higher humoral response by using a lower dose. Based on this result, the fusion protein was evaluated in adult cattle; and was able to induce high levels of neutralizing antibodies (>128) up to 360 days post vaccination. Due to this great performance, at this\nmoment the vaccine development is in SENASA register stage. With the intention of developing a vaccine capable of providing protection against\nthe three genotypes of BVDV, it was generated a combined vaccine based on tE2 proteins from BVDV1a (Singer), BVDV 1b (25366) and BVDV 2a (VS253), which are antigenically 23 different strains, fused to APCH. This combined vaccine was first evaluated in guinea pigs, which developed neutralizing antibodies against the three viral strains. Lastly, the combined vaccine was used to perform a challenge assay in colostrums-deprived calves with heterologous BVDV 1b and BVDV 2 field strains. Vaccinated animals showed elimination or\nreduction of pyrexia, nasal and ocular virus shedding, viremia, leukopenia and clinical signs of BVDV in comparison with non-vaccinated control animalsFil: Gnazzo, Victoria. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias, ArgentinaLa protección contra el Virus de la Fiebre Aftosa (VFA) ha sido relacionada con el desarrollo de la respuesta humoral, ya que coincide con la eliminación de lesiones, disminución de la viremia y con la reducción de la excreción viral. Los test controles oficiales en Argentina para evaluar la potencia de las vacunas de VFA son: la Prueba de Generalización Podal (PGP) en bovinos vacunados y desafiados y la cuantificación de\nanticuerpos (Acs) a los 60 días post-vacunación por ELISA en fase líquida con el cual se estima la Expectativa Porcentual de Protección (EPP) para la evaluación de la eficacia de las vacunas a nivel de lote.\nEn este trabajo se demuestra que el ratón BALB/c podría reemplazar al bovino en las pruebas de potencia de las vacunas y se propone que a partir de parámetros\ninmunológicos humorales evaluados en ratón se pueda estimar el porcentaje de protección en bovinos luego de la vacunación y el desafío. Se estudió la correlación de la respuesta inmune de bovinos y ratones frente a la vacunación y al desafío viral contra el serotipo O1Campos del VFA. La relación entre los niveles de Acs en bovinos y ratones a los 60 y 21 días post-vacunación respectivamente,\nfue estadísticamente significativa (90%) entre los niveles de Acs anti-VFA de ambas especies, apoyada por el cálculo del índice Kappa (0,75) en un total de 39 vacunas oleosas.\nAsimismo, se determinó que la protección en ambas especies luego de la vacunación y desafío también resultaba en una buena concordancia 88% (Kappa 0,75).Sin embargo, en algunos casos se observó que vacunas que presentaban un alto nivel de\nanticuerpos en ambas especies no protegían frente al desafío por lo que se realizó la medición de una serie de parámetros inmunológicos que podrían mejorar predicción de la protección en ratones, sin la necesidad de utilizar virus infectivo. Los parámetros analizados fueron, IgA en saliva, IFN-? secretado por los esplenocitos de ratones vacunados, el isotipo de inmunoglobulina inducida por la vacunación, la relación entre los tipos de IgG y el índice de avidez de los sueros. Con los resultados obtenidos de estas mediciones se planteó el diseño de un modelo logístico de predicción\nde la protección que incluyó un subconjunto óptimo de variables explicativas. Se realizó a partir de una base de datos que contenía las posibles variables predictoras de protección\n(variables continuas) y la respuesta de Protección o No protección (variable dicotómica).Las variables Acs ELISA FL, Relación entre isotipos (IgG2b/IgG1) e índice de Avidez fueron identificadas como buenas predictoras, obteniéndose un modelo de predicción de protección óptimo. Con este modelo se sometieron a evaluación los datos de predicción de\nprotección estimados por el modelo murino con los datos reales de protección en el bovino para una serie de vacunas, se obtuvo una concordancia del 88% asociada a un Kappa de 0,75. Esto indica que el modelo murino propuesto podría servir como alternativa rápida, económica y en concordancia con el concepto de las 3 Rs (Reemplazar, Refinar y Reducir) para la evaluación de la eficacia de vacunas contra el VFA serotipo O1Campos

Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling

The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been ongoing controversy regarding the possible adverse effects of glyphosate on the environment and on human health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity. This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline, with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. The direct effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concerns about the clinical findings from human offspring in populations exposed to GBH in agricultural fields.Fil: Paganelli, Alejandra Raquel. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gnazzo, Victoria. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acosta, Helena. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Lopez, Silvia Liliana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carrasco, Andres Eduardo. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Co-inoculation of baculovirus and FMDV vaccine in mice, elicits very early protection against foot and mouth disease virus without interfering with long lasting immunity

Baculoviruses (Bvs) potentiate the immune response against soluble antigens. We investigated whether Bv could be used as immunoactivator in foot-and-mouth disease (FMD) vaccines using the BALB/c mouse model. Mice were vaccinated with a single dose of inactivated FMDV (iFMDV), iFMDV. +. Bv, Bv, or culture medium. Humoral and cellular immune responses were higher in animals immunized with iFMDV. +. Bv than in mice vaccinated with iFMDV alone. Animals receiving iFMDV. +. Bv had significantly lower viremia at 2, 4 and 7. dpv, than those immunized with iFMDV alone. In order to prolong the immune response, iFMDV oil vaccine was co-inoculated with Bv. Animals receiving iFMDV oil vaccine. +. Bv were protected two days earlier than those receiving the iFMDV oil vaccine alone. Both formulations protected until 14. dpv, the last day of the experiment.This is the first report in which Bv is used as an adjuvant in a FMDV vaccine.Fil: Quattrocchi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Molinari, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad del Salvador; ArgentinaFil: Langellotti, Cecilia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad del Salvador; ArgentinaFil: Gnazzo, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad del Salvador; ArgentinaFil: Taboga, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Mouse model as an efficacy test for foot‐and‐mouth disease vaccines

Protection against foot‐and‐mouth disease virus (FMDV) has been linked to the development of a humoral response. In Argentina, the official control tests for assessing the potency of FMD vaccines are protection against podal generalization (PPG) and expected percentage of protection (EPP) curves built with quantitative data of antibodies determined by liquid‐phase blocking ELISA (lpELISA). The results of these tests are used to accept or discard vaccines at the batch level. In this report, a mouse model was assessed as an alternative efficacy control for FMDV vaccines. To this aim, groups of cattle (n = 18) and BALB/c mice (n = 16) were inoculated with commercial FMDV vaccines and bleedings were performed 60 days post vaccination (dpv) in cattle and 21 dpv in mice. Specific FMDV antibody titres were measured in both species by a standardized lpELISA. A statistically significant association between antibody levels in cattle and mice has already been demonstrated. However, some vaccines have been misclassified since they were considered protective based on lpELISA results but did not induce good protection in cattle upon challenge. For this reason, other immunological parameters were evaluated to improve the prediction of protection in mice, without the need of using infective virus. In addition, antibody titres by lpELISA, the IgG2b/IgG1 isotype ratio and the Avidity Index were identified as good predictors, resulting in an optimal predictive model of protection. This mouse model could be a simple and economic alternative for testing FMD vaccines since the disadvantages of high costs and facility requirements associated with the use of large animals are overcome.Instituto de VirologíaFil: Gnazzo, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Quattrocchi, Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Soria, Ivana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Pereyra, Erica Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Langellotti, Cecilia Ana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Pedemonte, Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: López, Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Marangunich, Laura An. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentin

Recombinant empty capsids recognition by sera from vaccinated bovines.

<p>Percentage of positivity of bovine sera analyzed by ELISA for the recognition of recombinant empty capsids and mock transfected cell lysates. Percentage of positivity was calculated as (A₄₉₂ from recombinant protein or mock lysate/A₄₉₂ from iFMDV) × 100. Each pair of bars represents the sera from an individual animal.</p

Antigenic reactivity of recombinant structural proteins.

<p>Lysates of cells transfected with pTT5-P12A3C, co-transfected with pTT5-VP0, pTT5-VP3 and pTT5-VP1, mock transfected cells or purified iFMDV were analyzed by ELISA with four MAbs (1–5,2–4,3–2,3–3) directed against conformational epitopes of FMDV. Error bars are standard deviation from three independent experiments.</p

Analysis of humoral response of BALB/c mice immunized with recombinant proteins.

<p>The results were obtained from the average of seven sera in each group. Mean ± SD sample is shown.</p