First evaluation of the impact of a targeted subunit vaccine against bovine viral diarrhea virus in feedlot cattle.

Bovine respiratory disease (BRD) is a serious health and economic problem in the beef industry, which is often associated with transportation and caused by different pathogens. In this study, we evaluated the effect of a novel subunit targeted vaccine against bovine viral diarrhea virus (BVDV) in feedlot cattle, a major viral agent of BRD. The core of this novel vaccine is the fusion of the BVDV structural glycoprotein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present in antigen-presenting cells. To evaluate the vaccine, 2,992 animals were randomly allocated into two groups, control group (N = 1,491) and treatment group (N = 1,501). Animals of both groups received the routine sanitary plan: two doses of clostridial, respiratory, and rabies vaccines. Animals within the treatment group also received two doses of a targeted subunit vaccine against BVDV. Serum samples were taken on the day of the first inoculation (T0) and 90 d later (T90). Viral circulation was monitored using an anti-P80 ELISA (virus-specific) and immune response was evaluated by anti-E2 ELISA (detects virus and vaccine immune responses). Only animals treated for respiratory disease were considered positive cases of BRD. Results demonstrate that the control group had significantly more animals treated for BRD cases compared to the treatment group (5.9% vs. 3.7%, P = 0.02). The control group had a greater number of animals positive for anti-P80 antibodies and significantly fewer animals positive for anti-E2 antibodies compared to the treatment group (69% vs. 61% and 71% vs. 99%, respectively, P = 0.003), consistent with natural viral circulation within this group. The treatment group, conversely, had fewer animals positive for anti-P80 antibodies and a greater number of animals positive for anti-E2 antibodies, consistent with a robust vaccine-induced antibody response and a reduction of the BVDV circulation within this group. The data indicate the new subunit targeted vaccine induced greater anti-E2 antibodies and reduced the amount of BVD virus circulation within the treatment group leading to a fewer number of animals needing to be treated for BRD.

A novel MHC-II targeted BVDV subunit vaccine induces a neutralizing immunological response in guinea pigs and cattle.

Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle.

Desarrollo de una vacuna mejorada de la subunidad del virus de la diarrea viral bovina basada en la glicoproteína E2 fusionada con un anticuerpo de cadena única que se dirige a las células presentadoras de antígenos / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold for Antigen Production in Tobacco Chloroplasts.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells. / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

Evaluation of a bovine rotavirus VP6 vaccine efficacy in the calf model of infection and disease.

Group A bovine rotavirus (BRV) is the major cause of acute viral gastroenteritis in neonatal calves worldwide. Due to the early susceptibility to the infection prevention strategies are based on the improvement of passive immunity levels through cow vaccination in the last third of gestation. The major capsid antigen (VP6) of BRV is the most immunogenic viral protein and it is highly conserved among group A BRV. In this work, VP6 protein from BRV C-486 strain (P[1]G6) was expressed in insect cells using the baculovirus expression vector system. Recombinant VP6 was used to immunize cows and vaccine's efficacy was assessed in a colostrum-deprived calf model of BRV infection and disease. Immune colostrum pool was generated using first and second milking of the immunized cows. Calves receiving one dose of immune colostrum within the first 6h of life, or colostrum-deprived calves were orally inoculated with virulent BRV at 2 days of age. The animals were monitored for diarrhea, virus shedding and isotype-specific antibodies responses to BRV in both feces and serum. Calves receiving VP6-immune colostrum showed a reduction of both diarrhea and virus shedding (in terms of viral titer and excretion period) in comparison with the colostrum-deprived calves.

Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system.

Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previously evaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. VP8 is a sialic acid binding domain of rotavirus external capsid protein VP4, which is involved in virus adhesion to host cells. In this work, the C486 bovine rotavirus (BRV) VP8 core protein (VP8d) was fused to the structure of BLS with the aim to produce an enhancement of the immune response against BRV VP8 and to evaluate the possible use of this antigen for vaccine development. The feasibility of using BLS as an antigen delivery system of polypeptides larger in size than those previously tested was also evaluated. Groups of female mice were immunized with BLS-VP8d fusion protein, VP8d or an equimolar mixture of purified VP8d and BLS (BLS+VP8d). Dams immunized with BLS-VP8 induced 97.5-100% protection against homologous challenge with C486 BRV; while pups born to dams immunized either with VP8d or BLS+VP8d presented a significant lower level of protection. The neutralizing antibody pattern was also significantly different among these experimental groups, and in concordance with challenge experiment. Overall, these results demonstrate that the BLS-VP8d chimeric protein is properly folded and stable, and that the BLS scaffold is a potent antigen delivery system that enhances the antibody response against BRV and elicits complete homotypic passive protection in a suckling mouse model.