Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.

An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle, guinea pigs and IFNAR(-/-) mice.

Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(-/-) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2.

Quantitation of cytokine gene expression by real time PCR in bovine milk and colostrum cells from cows immunized with a bovine rotavirus VP6 experimental vaccine.

In a previous work, VP6 recombinant protein was produced using baculovirus system and it was evaluated in a colostrum-deprived calf model. This vaccine was able to protect calves against viral challenge without inducing neutralizing antibodies (NAb), suggesting that another immunological effectors were involved in the protection observed. In this work, groups of cows (n=4) were immunized in the last third of gestation with a bovine rotavirus (BRV) experimental vaccine and with a VP6 subunit vaccine. At birth, colostrums from vaccinated and non-vaccinated cows were processed and viable colostral mononuclear cells were obtained. With the purpose of determining the cytokine patterns generated by cells from immune secretions (colostrums and milk), a relative quantification by real time PCR was standardized. Quantitative real time PCR (qPCR) was used to determine transcript levels of IL-4, IL-6, IL-10, IL-12, IFN-γ and IFN-α from these cells. Colostral and milk mononuclear cells expressed a different cytokine transcript expression pattern regarding the vaccine used. These results demonstrated that the colostral cellular population was active and could exert its action influencing the final immune response.

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model.

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens. In this work, the C486 BRV VP8* protein was produced in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern blot, northern blot and western blot. VP8* was highly stable in the transplastomic leaves, and formed insoluble aggregates that were partially solubilized by sonication. The recombinant protein yield was 600 µg/g of fresh tissue (FT). Both the soluble and insoluble fractions of the VP8* plant extracts were able to induce a strong immune response in female mice as measured by ELISA and virus neutralization test. Most important, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Results presented here contribute to demonstrate the feasibility of using antigens expressed in transplastomic plants for the development of subunit vaccines.

Development and validation of an ELISA for quantitation of bovine viral diarrhea virus antigen in the critical stages of vaccine production.

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.

Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV.

Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.

A simple and reliable indirect enzyme-linked immunosorbent assay for detection of antibodies directed against a major bovine viral diarrhea virus (BVDV) immunogen, the E2 glycoprotein (tE2-ELISA), has been developed using the recombinant C-terminal truncated E2 glycoprotein (tE2) expressed in a Drosophila melanogaster system. This strategy demonstrated that tE2 is secreted efficiently in the supernatant, no purification steps are necessary, it is easy to produce and carries out the post translational modifications necessary to preserve its native conformation. Preliminary analysis of 183 cattle serum samples using tE2-ELISA showed a 98% specificity and a 100% sensitivity compared with the standard homologous BVDV virus neutralization test. The results also showed that the tE2 is immunoreactive because the conformation and antigenicity of the original E2 are maintained to a large extent. To our knowledge this is the first study report of the recombinant tE2 of BVDV expressed in D. melanogaster system as an antigen for ELISA.

[Expression of foot and mouth disease virus antigens in transgenic plants]. / Expresión de antígenos del virus de la fiebre aftosa en plantas transgénicas.

Owing to its geographical distribution and its highly contagious character, the foot and mouth disease (FMD) virus is responsible for one of the most dreaded of all livestock diseases. The currently-used vaccine is polyvalent and is based on an inactivated virus. Current research on FMD vaccines focuses on the creation of vaccines that are easier and cheaper to produce, and that avoid manipulation of large quantities of virus. The use of transgenic plants to express relevant antigens has been evaluated for the purpose of vaccine production. The authors' working group has taken the FMD virus as a model to evaluate the feasibility of using transgenic plants to express viral antigens and to develop experimental vaccines. The purpose of this paper is to set forth the working group's results in the expression of FMD antigens in transgenic plants.

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector.

We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4 microg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.

Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Adjuvant effects of sulfolipo-cyclodextrin in a squalane-in-water and water-in-mineral oil emulsions for BHV-1 vaccines in cattle.

The antibody and cell mediated immune responses induced by BHV-1 were analysed in cattle after vaccination and challenge exposure to the virulent strain LA of BHV-1. Animals were vaccinated intramuscularly (IM) with inactivated virus vaccines against BHV-1 containing either a water in mineral oil adjuvant (W/O), a water in mineral oil adjuvant plus Avridine (W/O+Avridine) or sulfolipo-cyclodextrin in squalane in-water emulsion (SL-CD/S/W). No significant differences were registered in the antibody response induced by the three evaluated vaccines. However, the BHV-1 specific cell-mediated immunite response was stronger and appeared earlier when SL-CD/S/W was included in the formulation. The efficacy of the vaccines was also evaluated after intranasal challenge of the calves with a virulent BHV-1 LA strain. Animals vaccinated with SL-CD/S/W had reduced virus excretion and clinical symptoms compared with the mock-vaccinated animals. Comparison of levels of BHV-1 specific IgG2 and IgG1 with virus shedding revealed that, regardless of the adjuvant administered, animals showing BHV-1 specific IgG2/IgG1 ratios higher than 1 were those with a significant lower number of individuals shedding virus. Additionally, animals vaccinated with SL-CD/S/W presented no post-vaccinal reactions. These factors, combined with the higher efficacy and the ease of manipulation of the biodegradable oil, makes the vaccine formulated with this new adjuvant an important contribution for the veterinary vaccines industry.

A comparison of methods for measuring the antibody response in mice and cattle following vaccination against foot and mouth disease.

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.

Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1.

The utilization of transgenic plants expressing recombinant antigens to be used in the formulation of experimental immunogens has been recently communicated. We report here the development of transgenic plants of alfalfa expressing the structural protein VP1 of foot and mouth disease virus (FMDV). The presence of the transgenes in the plants was confirmed by PCR and their specific transcription was demonstrated by RT-PCR. Mice parenterally immunized using leaf extracts or receiving in their diet freshly harvested leaves from the transgenic plants developed a virus-specific immune response. Animals immunized by either method elicited a specific antibody response to a synthetic peptide representing amino acid residues 135-160 of VP1, to the structural protein VP1, and to intact FMDV particles. Additionally, the immunized mice were protected against experimental challenge with the virus. We believe this is the first report demonstrating the induction of a protective systemic antibody response in animals fed transgenic plants expressing a viral antigen. These results support the feasibility of producing edible vaccines in transgenic forage plants, such as alfalfa, commonly used in the diet of domestic animals even for those antigens for which a systemic immune response is required.