RESUMO

Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.

Assuntos

Células Apresentadoras de Antígenos , Antígenos Virais , Linfócitos B , Vesículas Extracelulares , Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Vírus da Febre Aftosa/imunologia , Vesículas Extracelulares/imunologia , Linfócitos B/imunologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos Virais/imunologia , Vacinas Virais/imunologia , Proteínas Virais/imunologia , Ativação Linfocitária/imunologia , Células Dendríticas/imunologia , Apresentação de Antígeno/imunologia

RESUMO

Foot-and-mouth disease is a highly contagious disease affecting cloven-hoofed animals, resulting in considerable economic losses. Its causal agent is foot-and-mouth disease virus (FMDV), a picornavirus. Due to its error-prone replication and rapid evolution, the transmission and evolutionary dynamics of FMDV can be studied using genomic epidemiological approaches. To analyze FMDV evolution and identify possible transmission routes in an Argentinean region, field samples that tested positive for FMDV by PCR were obtained from 21 farms located in the Mar Chiquita district. Whole FMDV genome sequences were obtained by PCR amplification in seven fragments and sequencing using the Sanger technique. The genome sequences obtained from these samples were then analyzed using phylogenetic, phylogeographic, and evolutionary approaches. Three local transmission clusters were detected among the sampled viruses. The dataset was analyzed using Bayesian phylodynamic methods with appropriate coalescent and relaxed molecular clock models. The estimated mean viral evolutionary rate was 1.17 × 10- 2 substitutions/site/year. No significant differences in the rate of viral evolution were observed between farms with vaccinated animals and those with unvaccinated animals. The most recent common ancestor of the sampled sequences was dated to approximately one month before the first reported case in the outbreak. Virus transmission started in the south of the district and later dispersed to the west, and finally arrived in the east. Different transmission routes among the studied herds, such as non-replicating vectors and close contact contagion (i.e., aerosols), may be responsible for viral spread.

Assuntos

Vírus da Febre Aftosa , Picornaviridae , Animais , Vírus da Febre Aftosa/genética , Argentina/epidemiologia , Teorema de Bayes , Filogenia

RESUMO

The role of water buffaloes in foot-and-mouth disease (FMD) epidemiology as one of the major hosts of the virus that can develop persistent asymptomatic infection highlights the importance of sustaining surveillance on the antibody response elicited by vaccination in these animals. There is gap in the knowledge on how serological assays that measure antibodies against capsid proteins perform with buffalo samples and which would be the most reliable test to substitute the virus neutralization test (VNT) a cumbersome and low-throughput tool for field surveillance. Alternatively, the liquid-phase blocking sandwich ELISA (LPBE) is commonly used. Previous data from our laboratory demonstrated that the vaccine-induced antibodies assessed by the LPBE yielded low specificity with buffaloes' samples. In contrast, a single-dilution avidity ELISA (AE) aimed to detect high-avidity antibodies against exposed epitopes, combined with an indirect ELISA (IE) to assess IgG levels, produced more reliable results. Here we analyzed for the first time the kinetics of the antibodies induced by vaccination in two different buffalo herds (n = 91) over 120 days using AE, IE, LPBE, and the VNT. Kinetics were similar in the different assays, with an increase of antibodies between 0- and 14-days post-vaccination (dpv) which were maintained thereafter. VNT and AE results were concordant (Kappa value = 0.76), and both assays revealed a decay in the antibody response in calves with maternal antibodies at 90 and 120 dpv, which was not evidenced by the LPBE. These results show that kinetics of antibody responses to FMD vaccination are similar in buffalo and cattle, and support the use of indirect ELISA assays, in particular Avidity ELISA, as alternatives to the VNT for vaccine-immunity monitoring irrespectively of the animal's passive or active immune status.

RESUMO

FMD remains endemic in many Asian and African countries where multiple variants of serotypes O and A, among others, currently circulate. Due to lack of cross-protection between serotypes and incomplete protection between some strains even within a serotype, an important challenge for the application of effective vaccination programs is to select highly immunogenic and widely cross-reactive vaccine strains. Adaptation of a candidate field virus for use as a vaccine can be quite complex, so that whenever possible, the use of well-established vaccine viruses could have enormous advantages. FMD vaccine strains harmonized for use in South America have shown excellent results in FMD control, not only in the region, where it is still used systematically as a preventive measure, but also more recently in some Asian countries. To gain further insight into the immunogenic spectrum of these strains, VN tests (VNT) were performed with sera from cattle and/or pigs vaccinated with monovalent (type O) or trivalent (types O and A) formulations against 122 type O and 32 type A field viruses isolated from 35 countries in Asia and Africa, belonging to different lineages. Almost all VNT titers obtained were within the expected protective level, indicating the wide immunogenic spectrum of high potency FMD vaccines formulated with O1 Campos, A24 Cruzeiro and A Argentina 2001 South American vaccine strains belonging to EURO-SA topotypes against currently active viruses from other topotypes. These in vitro results are in line with previously reported in vivo challenge tests in pigs against three A/ASIA/Sea-97 isolates and two isolates belonging to type O lineages O/SEA/Mya-98 and O/ME-SA/Ind-2001e.

Assuntos

Doenças dos Bovinos , Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Bovinos , Animais , Suínos , Febre Aftosa/epidemiologia , Argentina/epidemiologia , Antígenos Virais , Sorogrupo , Anticorpos Antivirais

RESUMO

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. KEY POINTS: • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.

Assuntos

Vírus da Febre Aftosa , Febre Aftosa , Raiva , Vacinas , Animais , Anticorpos Antivirais , Vírus da Febre Aftosa/genética , Glicoproteínas/genética

RESUMO

The foot-and-mouth disease virus (FMDV) causes a highly infectious disease of all cloven-footed animals. The RNA genome of the virus continuously evolves, leading to the generation of new strains; this necessitates the selection of new vaccine strains to ensure complete protection. Infection with one FMDV serotype does not provide cross-protection against the other FMDV serotypes. Many of the recovered animals may become carriers of the FMDV, but they still remain susceptible to the other serotypes. Coinfection with multiple FMDV serotypes has been reported and studied to understand the virus evolution. Isolation and characterization of all the involved serotypes in the mixed infection case is essential to understand the molecular evolution of the virus. In this study, two cases of coinfection were studied by selective isolation of each of the FMDV serotypes under the cross-serotype-specific immune pressure. It was estimated that the virus present in a minimum of 10-0.92 TCID50 could be isolated from the mixed population containing other serotypes in infective doses of 100.25 TCID50 or less. All involved serotypes present in the mixed infection cases were isolated, without any cross-contamination. Virus characterization revealed that genotype 2 was of serotype A virus from a sample collected in 1995, which was last reported in 1986, indicating a possible subdued prevalence of the genetic group even after vanishing from the field.

Assuntos

Coinfecção , Vírus da Febre Aftosa , Febre Aftosa , Animais , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/isolamento & purificação , Filogenia , Sorogrupo

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious viral disease, which has been reported for over 100 years, and against which the struggle has lasted for the same amount of time. It affects individuals from the order Artiodactyla, such as cattle, swine, sheep, wild animals from this order, and a few non-cloven hoofed species, such as mice and elephants. FMD causes large-scale economic losses for agricultural production systems; morbidity is almost 100% in an affected population, accompanied by a high mortality rate in young animals due to myocarditis or an inability to suckle if a mother is ill. The aetiological agent is an Aphthovirus from the family Picornaviridae, having seven serotypes: A, O, C, SAT1, SAT2, SAT3, and Asia 1. Serotype variability means that an immune response is serospecific and vaccines are thus designed to protect against each serotype independently. A host's adaptive immune response is key in defence against pathogens; however, this virus uses successful strategies (along with most microorganisms) enabling it to evade a host's immune system to rapidly and efficiently establish itself within such host, and thus remain there. This review has been aimed at an in-depth analysis of the immune response in cattle and swine regarding FMD virus, the possible evasion mechanisms used by the virus and describing some immunological differences regarding these species. Such aspects can provide pertinent knowledge for developing new FMD control and prevention strategies.

RESUMO

Although replication-defective human adenovirus type 5 (Ad5) vectors that express in situ the capsid-encoding region of foot-and-mouth disease virus (FMDV) have been proven to be effective as vaccines in relevant species for several viral strains, the same result was not consistently achieved for the O1/Campos/Brazil/58 strain. In the present study, an optimization of the Ad5 system was explored and was proven to enhance the expression of FMDV capsid proteins and their association into virus-like particles (VLPs). Particularly, we engineered a novel Ad5 vector (Ad5[PVP2]OP) which harbors the foreign transcription unit in a leftward orientation relative to the Ad5 genome, and drives the expression of the FMDV sequences from an optimized cytomegalovirus (CMV) enhancer-promoter as well. The Ad5[PVP2]OP vaccine candidate also contains the amino acid substitutions S93F/Y98F in the VP2 protein coding sequence, predicted to stabilize FMD virus particles. Cells infected with the optimized vector showed an ∼14-fold increase in protein expression as compared to cells infected with an unmodified Ad5 vector tested in previous works. Furthermore, amino acid substitutions in VP2 protein allowed the assembly of FMDV O1/Campos/Brazil/58 VLPs. Evaluation of several serological parameters in inoculated mice with the optimized Ad5[PVP2]OP candidate revealed an enhanced vaccine performance, characterized by significant higher titers of neutralizing antibodies, as compared to our previous unmodified Ad5 vector. Moreover, 94% of the mice vaccinated with the Ad5[PVP2]OP candidate were protected from homologous challenge. These results indicate that both the optimized protein expression and the stabilization of the in situ generated VLPs improved the performance of Ad5-vectored vaccines against the FMDV O1/Campos/Brazil/58 strain and open optimistic expectations to be tested in target animals.

RESUMO

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.

Assuntos

Anticorpos Antivirais/imunologia , Doenças dos Bovinos/prevenção & controle , Modelos Animais de Doenças , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Imunoglobulina G/sangue , Vacinas Virais/imunologia , Animais , Argentina , Bovinos , Doenças dos Bovinos/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Febre Aftosa/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/veterinária

RESUMO

Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides' ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins' conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides' structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation's immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides' in vitro protection-inducing ability.

Assuntos

Vírus da Febre Aftosa , Febre Aftosa , Peptídeos , Vacinas Virais , Animais , Anticorpos Antivirais , Proteínas do Capsídeo/genética , Bovinos , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/imunologia

RESUMO

To know the epidemiological context of hand-foot-and-mouth disease (HFMD) in a region of Uruguay and to identify the Enterovirus responsible for an outbreak in a rural childcare center in 2018. Swab samples from skin lesions and/or stools samples were collected from children suffering HFMD during an outbreak in a rural childcare center. Samples were subject to viral RNA extraction and reverse-transcription polymerase chain reaction towards VP1 coding segment, to identify the Enterovirus type by sequencing and phylogenetic analysis. Total of 149 cases of HFMD affecting 98 boys and 51 girls were reported in Salto Province-Uruguay in 2018. Total 60% of the cases were originated from outbreaks, which occurred in ten educative and childcare institutions from both urban and rural areas. Coxsackievirus-6 (CV-A6) was identified as responsible for one of the rural outbreaks. Uruguayan strains were more related to strains reported in Russia, Turkey, and Germany (2014-2017) than to strains reported in Brazil and Argentina from 2015 to 2016. This is the first report of CV-A6-associated HFMD in Uruguay, evidencing a wide geographic range of the virus in the Latin American region. Our report also warns about CV-A6-associated HFMD during winter, contrarily to most reports that register HFMD during summer and fall seasons.

Assuntos

Enterovirus Humano A/classificação , Enterovirus Humano A/isolamento & purificação , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/virologia , Criança , Creches , Pré-Escolar , Surtos de Doenças , Enterovirus Humano A/genética , Fezes/virologia , Feminino , Genótipo , Humanos , Lactente , Masculino , Epidemiologia Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , População Rural , Análise de Sequência de DNA , Pele/virologia , Uruguai/epidemiologia

RESUMO

Las proteínas no capsidales del virus de la fiebre aftosa se utilizan como marcadoras en la evaluación de animales que han estado en contacto con el virus, a diferencia de los inmunizados, ya que la vacuna no debe tener estas proteínas, por lo tanto los animales no deben presentar anticuerpos contra ellas. El objetivo de esta investigación fue la caracterización de la proteína no capsidal 3D y la producción de anticuerpos policlonales in vivo. La proteína se purificó del cultivo de virus inactivo, por cromatografía de intercambio iónico. La elución de los picos fue sometida a electroforesis uni-bidimensional; demostrándose un alto grado de pureza (>90%) en el pico tres, donde se identifico la proteína 3D, por la técnica de MALDI-TOF y electroespray de trampa iónica. La proteína purificada, se inoculó en cabras y el suero hiperinmune fue precipitado y sometido a cromatografía de afinidad para la obtención de inmunoglobulinas; la reacción inmunitaria se confirmó por medio de inmunodifusión y Western blot. El proceso de purificación demostró ser eficiente y útil para la obtención de anticuerpos específicos, los cuales tendrán utilidad en la elaboración de un ensayo inmunoenzimático que mida la pureza de la vacuna frente al contenido de estas proteínas.

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The noncapsid proteins of the foot and mouth disease are used as markers in the evaluation of animals that have been in contact with the virus, to discriminated the immunized animals, because the vaccine should not have these proteins, therefore animals should not present antibodies against them. The aim of this investigation was the characterization of the 3D non-capsid protein and the production of polyclonal antibodies in vivo. The protein was purified from the culture of inactivated virus, by ion exchange chromatography. The elution of the peaks were submit an one-two-dimensional electrophoresis; Demonstrated a high degree of purity (> 90%) in peak three, where the 3D protein was identified, by the MALDI-TOF technique and ion trap electrospray. The purified protein, inoculated in goats and the hyperimmune serum, was precipitate out and submitted to affinity chromatography to obtain immunoglobulins; the immune reaction was confirmed by means of immunodiffusion and Western blot. The purification process proved to be efficient and useful for obtaining specific antibodies, which will be useful in the preparation of an immunoenzymatic assay that measures the purity of the vaccine against to the content of these proteins.

Assuntos

Humanos , Proteínas do Capsídeo , Febre Aftosa , Vírus , Eletroforese , Doenças dos Animais , Anticorpos

RESUMO

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.

Assuntos

Proteínas do Capsídeo/genética , Febre Aftosa/tratamento farmacológico , Proteínas Recombinantes/genética , Vacinas/genética , Animais , Capsídeo/química , Capsídeo/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/uso terapêutico , Febre Aftosa/imunologia , Febre Aftosa/virologia , Vírus da Febre Aftosa/efeitos dos fármacos , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/patogenicidade , Humanos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Vacinas/uso terapêutico , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia

RESUMO

Foot-and-mouth disease (FMD) remains one of the most feared viral diseases affecting cloven-hoofed animals, and results in severe economic losses. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of recombinant FMDV-like particles (VLPs) as subunit vaccines has gained importance because of their immunogenic properties and safety. We evaluated the production of FMD VLPs, via Agrobacterium-mediated transient expression, and the immunogenicity of these structures in mice. Leaves were infiltrated with pEAQ-HT and pRIC 3.0 vectors encoding the capsid precursor P1-2A and the protease 3C. The recombinant protein yield was 3-4 mg/kg of fresh leaf tissue. Both groups of mice immunized with purified VLPs and mice immunized with the crude leaf extract elicited a specific humoral response with similar antibody titers. Thus, minimally processed plant material containing transiently expressed FMD VLPs could be a scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

RESUMO

Galectin-8 (Gal-8) is a mammalian ß-galactoside-binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen-specific immune responses in vivo, we investigated whether Gal-8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow-derived DCs (BMDCs) treated with exogenous Gal-8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal-8-treated BMDCs (Gal-8-BMDCs) stimulated antigen-specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL-3, IL-2, IL-6, TNF, MCP-1, and MCP-5, as well as growth factor G-CSF, were augmented in Gal-8-BMDC conditioned media, with IL-6 as the most prominent. Remarkably, BMDCs from Gal-8-deficient mice (Lgals8-/- BMDC) displayed reduced CD86 and IL-6 expression and an impaired ability to promote antigen-specific CD4 T cell activation. To test if Gal-8-induced activation correlates with the elicitation of an effective immune response, soluble Gal-8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot-and-mouth disease virus (FMDV) model. When a single dose of Gal-8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL-6 and IFN-γ, as well as lymphoproliferative responses, were also incremented in Gal-8/antigen-immunized animals only at 48 h after immunization, suggesting that Gal-8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal-8 as an immune-stimulator molecule to enhance the adaptive immune response.

Assuntos

Apresentação de Antígeno , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Febre Aftosa/imunologia , Galectinas/imunologia , Imunidade Adaptativa , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Linfócitos T CD4-Positivos/virologia , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Células Dendríticas/virologia , Febre Aftosa/genética , Febre Aftosa/prevenção & controle , Febre Aftosa/virologia , Vírus da Febre Aftosa/crescimento & desenvolvimento , Vírus da Febre Aftosa/imunologia , Galectinas/genética , Galectinas/farmacologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunização , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-3/genética , Interleucina-3/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quimioatraentes de Monócitos/genética , Proteínas Quimioatraentes de Monócitos/imunologia , Transdução de Sinais , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia

RESUMO

Background: Foot and mouth disease (FMD) is the causative agent and one of the most transmissible livestock diseaseswhich cause important economic losses. The genome of FMD virus is a positive-sense, single-stranded RNA and it wrappedwith 60 copies of 4 structural proteins, VP1, VP2, VP3 and VP4. The VP1 is a major immunogenic antigen with criticalepitopes for inducing immune responses. The current vaccine, which successfully prevents disease, includes inactivatedwhole virus antigen. However, it is not without problems. The aim of this study is enhancement of immune responsesagainst Iranian isolate of FMD-type O/IRN/1/2010 based on VP1 and human HSP70 fusion protein in BALB/c mice.Materials, Methods & Results: In this study FMD virus type O/IRN/2010 was isolated from vesicles of the infected cattlein Qoum, and serotyped as a new linage of type O (PanAsia-linage 2) in Iran. The isolated FMD virus was propagated onIBRS2 cell line and whole RNA of the infected cells was extracted by commercial kit instruction. The extracted RNA wasamplifi ed using VP1 gene-specifi c primer pairs by means of one-step RT-PCR. The specifi c primer pair was designed byAllelID6 software. There are sequences of Kpn I and BamH I restriction enzymes and three overhanging nucleotides atthe start of forward and reverse primers, respectively. The VP1 nucleotide sequence was deposit in Genbank-NCBI database under accession number JN 676146. The purifi ed VP1 gene was sub-cloned into PTZ57R/T vector. Then digestedVP1 gene by KpnI and BamHI enzymes was ligated in pcDNA3.1+ vector as a DNA vaccine. Also, the improved DNAimmunization system was constructed using pcDNA3.1+ plasmid contains VP1 gene of Iranian isolate FMD virus typeO/IRN/1/2010 and human HSP70 gene and expression of VP1-HSP70 fusion protein confi rmed in BHKT7 cell line byGuinea pig specifi c polyclonal antibody against FMD virus type O and conjugated rabbit...

Assuntos

Animais , Camundongos , Genoma Viral , Proteínas Virais/isolamento & purificação , Vacinas de DNA/análise , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Camundongos Endogâmicos BALB C

RESUMO

Background: Foot and mouth disease (FMD) is the causative agent and one of the most transmissible livestock diseaseswhich cause important economic losses. The genome of FMD virus is a positive-sense, single-stranded RNA and it wrappedwith 60 copies of 4 structural proteins, VP1, VP2, VP3 and VP4. The VP1 is a major immunogenic antigen with criticalepitopes for inducing immune responses. The current vaccine, which successfully prevents disease, includes inactivatedwhole virus antigen. However, it is not without problems. The aim of this study is enhancement of immune responsesagainst Iranian isolate of FMD-type O/IRN/1/2010 based on VP1 and human HSP70 fusion protein in BALB/c mice.Materials, Methods & Results: In this study FMD virus type O/IRN/2010 was isolated from vesicles of the infected cattlein Qoum, and serotyped as a new linage of type O (PanAsia-linage 2) in Iran. The isolated FMD virus was propagated onIBRS2 cell line and whole RNA of the infected cells was extracted by commercial kit instruction. The extracted RNA wasamplifi ed using VP1 gene-specifi c primer pairs by means of one-step RT-PCR. The specifi c primer pair was designed byAllelID6 software. There are sequences of Kpn I and BamH I restriction enzymes and three overhanging nucleotides atthe start of forward and reverse primers, respectively. The VP1 nucleotide sequence was deposit in Genbank-NCBI database under accession number JN 676146. The purifi ed VP1 gene was sub-cloned into PTZ57R/T vector. Then digestedVP1 gene by KpnI and BamHI enzymes was ligated in pcDNA3.1+ vector as a DNA vaccine. Also, the improved DNAimmunization system was constructed using pcDNA3.1+ plasmid contains VP1 gene of Iranian isolate FMD virus typeO/IRN/1/2010 and human HSP70 gene and expression of VP1-HSP70 fusion protein confi rmed in BHKT7 cell line byGuinea pig specifi c polyclonal antibody against FMD virus type O and conjugated rabbit...(AU)

Assuntos

Animais , Camundongos , Genoma Viral , Proteínas Virais/isolamento & purificação , Vírus da Febre Aftosa/imunologia , Vírus da Febre Aftosa/isolamento & purificação , Vacinas de DNA/análise , Camundongos Endogâmicos BALB C , Proteínas de Choque Térmico HSP70

RESUMO

Foot-and-Mouth Disease Virus serotype O has been circulating regularly throughout most provinces of Ecuador, one of the two South American countries that still remain endemic, although satisfactory vaccination coverage was reported. This study concentrates in the characterization of isolates collected during 2008-2011, focusing particularly on the antigenic and immunogenic relationships of the field viruses with the O1/Campos vaccine strain in use in the region and with an experimental vaccine formulated with a representative strain of the 2010 epidemic. The results established that antigenically divergent variants poorly protected by the vaccine in use emerged and co-circulated in a limited period of time. A monovalent vaccine formulated with the representative 2010 strain elicited high antibody titers and protected against challenge with homologous virus. In addition, cross-reactive antibodies to predominant viruses in the region were established. In overall this study indicates the ability of the virus to diversify under field conditions in which a vaccine strain with poor match is applied, and the potential of the selected 2010 field virus as a vaccine candidate for incorporation into strategic antigen banks and/or for addition to current formulations for systematic vaccination, in order to prevent the emergence of even more divergent isolates in the future.

Assuntos

Variação Antigênica , Antígenos Virais/imunologia , Vírus da Febre Aftosa/classificação , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Doenças dos Bovinos/prevenção & controle , Equador , Febre Aftosa/prevenção & controle

RESUMO

The O1Campos strain of foot and mouth disease virus (FMDV) used as inducing agent in the pleurisy model was able to trigger a pro-inflammatory effect on normal and immune guinea pigs. The proinflammatory activity which was detected at two times of the pleurisy (24 and 48 hours) on normal guinea pigs was characterized only by mononuclear (MN) cell influx, during the first interval of the reaction and by edematogenic effect, MN and polimorphonuclcar (PMN) leucocyte migration, at the last time of the reaction. The inflammatory reaction profiles recorded on immune guinea pigs (vaccinated with anti-O1Campos oil adjuvanted vaccine), both after 7 and 30 days post vaccination (pv) have showed, in both interval, lower intensities than that observed in normal guinea pigs, although in the 7 days PV guinea pigs the accumulations of total leucocytes and PMN were similar to that displayed by normal animals, after 48 hours of the reaction. Besides, on thirty days PV guinea pigs the FMDV induced a significant increase in volume of exudate and MN cell infiltration, after 24 hours, and all of the inflammatory parameters values dropped to normal levels, during the second interval of the reaction. It was found a negative association between the increase in serum neutralizing antibody titer, from 7 to 30 days PV and the intensities of pleural inflammatory parameters on the immun

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A estirpe O1Campos do vírus da febre aftosa (VFA) usada como agente indutor do teste de pleurisia foi capaz de desencadear um efeito pró-inflamatório em cobaias normais e imunes. A atividade pró-inflamatória do VFA, detectada em dois intervalos de pleurisia (24 e 48 horas) foi demonstrada, somente por quimiotaxia de leucócitos mononucleares (MN), no primeiro intervalo e por efeito edematogênico, migração de MN e polimorfonucleares (PMN), no último intervalo de reação. Os perfis de reação inflamatória induzida pelo VFA em cobaias imunes (imunizadas com vacinas oleosas anti-VFAO1Campos), aos 7 e aos 30 dias pós-vacinação (PV) apresentaram imensidades mais baixas do que as observadas em cobaias normais, embora nas cobaias com 7 dias de vacinação a quimiotaxia de leucócitos totais e de PMN tenha sido similar àquela encontrada nos animais normais, no intervalo de 48 horas de reação. Ademais, nas cobaias com 30 dias PV, o VFA induziu um aumento significante no volume de exsudato e na infiltração de MN, no intervalo de 24 horas, sendo que os valores de todos os parâmetros do exsudato inflamatório caíram a níveis normais, no segundo intervalo de reação. Nas cobaias imunes foi observada uma associação negativa entre o aumento no título de anticorpos soro-neutralizantes, de 7 para 30 dias PV e as intensidades dos parâmetros inflamatórios pleurais. O teste de pleurisia revelou-se um proce