Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris.

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.

Antiviral and antiluteolytic activity of recombinant bovine IFN-omega1 obtained from Pichia pastoris.

The gene coding for bovine interferon-omega1 (BoIFN-omega1) was recently cloned and expressed at high levels in the yeast Pichia pastoris. The recombinant BoIFN-omega1 protein shows antiviral activity in different cell lines and has an antiluteolytic effect in cyclic ewes. In this article, we describe a method for purification of BoIFN-omega1 expressed in the methylotrophic yeast P. pastoris and characterization of its activity in vivo. The recombinant protein secreted to the culture medium had low activity because of self-aggregation. BoIFN-omega1 was solubilized using urea and desalting and finally purified by ion exchange chromatography on Q-Sepharose Fast Flow. The yield of purified product was approximately 300 mg/L of fermentation culture, with a specific antiviral activity of 10(8) IU/mg. Its purity was at least 80%. The biologic characterization of purified BoIFN-omega1 was determined by induction of an antiviral state on ewes challenged with 100 lethal doses (LD) of Aujeszky virus and by the extension of the corpus luteum life span and interestrous interval in cyclic cows. Ewes treated with 2 x 106 IU/kg BoIFN-omega1 were protected from Aujeszky virus infection. In cows receiving an intrauterine infusion of 1 mg BoIFN-omega1, equally distributed between the two uterine horns, twice daily from day 14 to day 22 of the experimental estrous cycle, the lifespan of the corpus luteum (25 vs. 19 days) and the interestrous intervals (26 vs. 21 days) were extended when compared with a control group (p < 0.05). We show that recombinant BoIFN-omega1 purified from P. pastoris has high antiviral activity and is an effective antiluteolytic agent in cattle.

Control of ticks resistant to immunization with Bm86 in cattle vaccinated with the recombinant antigen Bm95 isolated from the cattle tick, Boophilus microplus.

The recombinant Bm86-containing vaccine Gavac(TM) against the cattle tick Boophilus microplus has proved its efficacy in a number of experiments, especially when combined with acaricides in an integrated manner. However, tick isolates such as the Argentinean strain A, show low susceptibility to this vaccine. In this paper we report on the isolation of the Bm95 gene from the B. microplus strain A, which was cloned and expressed in the yeast Pichia pastoris producing a glycosylated and particulated recombinant protein. This new antigen was effective against different tick strains in a pen trial, including the B. microplus strain A, resistant to vaccination with Bm86. A Bm95-based vaccine was used to protect cattle against tick infestations under production conditions, lowering the number of ticks on vaccinated animals and, therefore, reducing the frequency of acaricide treatments. The Bm95 antigen from strain A was able to protect against infestations with Bm86-sensitive and Bm86-resistant tick strains, thus suggesting that Bm95 could be a more universal antigen to protect cattle against infestations by B. microplus strains from different geographical areas.

A model to simulate the effect of vaccination against Boophilus ticks on cattle.

This paper describes a vaccination model to simulate the effect of cattle vaccination with concealed antigens on Boophilus tick spp. The model considers the vaccination effect in three parts: antibody titer, accumulation of damaging vaccination effects by parasite stages, and the effect of accumulated damage on all tick life stages. Biological parameters for ticks and hosts, as well as parameters describing tick-host interaction, were included. The validity of this model, integrated with the TICKSIM simulation program, was demonstrated for the Bm86-containing vaccine Gavac by comparing simulated and real data for several geographic locations in the Americas. All model parameters were estimated using field data collected in the different geographic locations. The model sensitivity to changes in antibody titer level and titer half-life was studied, and the impact on tick population density of changes in these parameters was evaluated. Simulation results showed that to achieve a higher level of tick control, an increase in the maximum antibody titer levels was more important than extending titer half-life in geographical locations with short seasonal peaks of tick infestation. The TICKSIM program, integrated with the new vaccination model, proved to be a framework for designing and evaluating tick control strategies, including vaccination with GavacTM.

Vaccination against ticks (Boophilus spp.): the experience with the Bm86-based vaccine Gavac.

The control of tick infestations and the transmission of tick-borne diseases remain a challenge for the cattle industry in tropical and subtropical areas of the world. Traditional control methods have been only partially successful and the parasites continue to result in significant losses for the cattle industry. Recently, vaccines containing the recombinant B. microplus gut antigen Bm86 have been developed. Our vaccine formulation (Gavac, Heber Biotec S.A., Havana, Cuba) has been registered and is commercially available in Cuba, Colombia, Dominican Republic, Brazil and Mexico. In controlled pen trials, Gavac has been effective for the control of artificial infestations of B. annulatus, B. decoloratus and chemical-sensitive and resistant B. microplus strains from Australia, Africa, America and Iran. In controlled field trials in Cuba, Brazil, Argentina and Mexico, Gavac has shown a 55-100% efficacy in the control of B. microplus infestations in grazing cattle 12-36 weeks after the first vaccination. Field trials under production conditions have been conducted in Cuba, Colombia, Brazil and Mexico in pure and cross-bred cattle herds. The application of Gavac has increased the time between acaricide treatments by an average of 32 /-21 days (P = 0.0005) resulting in important savings for the cattle industry. In Cuba, a cost-effectiveness analysis was conducted in more than 260000 animals. The cost-effectiveness analysis showed a 60% reduction in the number of acaricide treatments, together with the control of tick infestations and transmission of babesiosis, which resulted in savings of 23.4 dollars animal(-1) year (-1). These results clearly demonstrate the advantage of vaccination and support the application of Gavac for the control of Boophilus spp. infestations.

Reproductive and safety assessment of vaccination with Gavac against the cattle tick (Boophilus microplus).

Recent developments in cattle tick control have incorporated the use of recombinant Bm86 vaccines against this ectoparasite. The vaccine developed by our group (Gavac) contains an antigen expressed in Pichia pastoris, and has been successfully employed for the control of tick infestations and transmission of tick-borne diseases. Here, we examined the safety and effect of the Gavac vaccine on reproductive parameters in cattle. Toxicity tests in mice and guinea pigs demonstrated the safety of Gavac. To study the adverse effects of vaccination on reproduction, a field trial involving 9,500 animals in Cuba was conducted. The cattle at 3 farms were vaccinated while those on a fourth farm were left unvaccinated and served as the control. Following vaccination, the control of tick infestation and the transmission of babesiosis were used to demonstrate the efficacy of the vaccine. No adverse effects were observed in any of the reproductive parameters studied when comparing the data before and after vaccination with Gavac and between the vaccinated farms and the control farm. These results demonstrate that under the conditions of our study vaccination with Gavac is safe for use on cattle.

Field studies and cost-effectiveness analysis of vaccination with Gavac against the cattle tick Boophilus microplus.

The control of tick infestations and the transmission of tick-borne diseases remains a challenge for the cattle industry in tropical and subtropical areas of the world. Traditional control methods have been only partially successful and the parasites continue to result in significant losses for the cattle industry. Recently, vaccines containing the recombinant Boophilus microplus gut antigen Bm86 have been developed. These vaccines have been shown to control tick infestations in the field. However, extensive field studies investigating the efficacy and cost-effectiveness of vaccination have not been reported and are needed to appraise the effect of this new approach for tick control. Here is reported the results of the application of Gavac in a field trial including more than 260,000 animals in Cuba. In this study the correlation between the antibody response to vaccination and the effect on ticks fertility is determined. Physiological status of the animals was found to affect the primary response to vaccination but not the antibody titers after revaccination. A cost-effectiveness analysis showed a 60% reduction in the number acaricide treatments, together with the control of tick infestations and transmission of babesiosis, which resulted in savings of $23.4 animal-1 year-1. These results clearly demonstrate the advantage of vaccination and support the application of Gavac for tick control.