Multi-antigenic recombinant subunit vaccine against Lawsonia intracellularis: The etiological agent of porcine proliferative enteropathy.

Proliferative enteropathy, caused by Lawsonia intracellularis, represents a threat for swine industry. Current vaccines are effective but difficult to obtain and scaled up, because of demanding bacterial culture conditions. In this work, a subunit vaccine candidate against L. intracellularis was developed and its efficacy was evaluated in vivo, alone or co-formulated with pig recombinant IFN-α. The vaccine formulation contains three chimeric antigens: two outer membrane proteins and a secreted one, which were engineered by adding T epitopes using bioinformatics tools. After simultaneously expressing the three antigens in E. coli, its immunogenicity was tested in mice and pigs. Antigens co-formulated with porcine IFN-α were also assayed in the last species. Immune response was assessed by ELISA and qPCR, and histopathological studies of intestinal epithelial tissue were performed after challenge. Mice and pigs showed an increased IgG response against chimeric antigens. Particularly, there were significant differences in the antibody response when porcine IFN-α was co-administrated with L. intracellularis antigens. Besides, mRNAs from il12 and cd4 marker were detected during the first week after immunization of pigs, suggesting a Th1-type cellular immune response. The significant enhancement of oas2 gene expression indicates the effect exerted by porcine IFN-α. Post-mortem histopathological analysis post-challenge revealed damage only into epithelial cells of the gastrointestinal tract from animals of the negative control group. Injuries were related to atrophy of the intestinal villi, where a decrease of globet cells and a greater migration of lymphocytes were observed. Overall, our results demonstrated that the vaccine candidate elicited significant humoral and cellular immune responses. Besides, histopathological analysis suggested that vaccinated animals were protected against experimental L. intracellularis infection. This research constitutes a step forward to the generation of the first recombinant chimeric vaccine against L. intracellularis, representing a faster, easier and cost effective approach to counteract the porcine proliferative enteropathy.

Effectiveness of the E2-classical swine fever virus recombinant vaccine produced and formulated within whey from genetically transformed goats.

Subunit recombinant vaccines against classical swine fever virus (CSFV) are a promising alternative to overcome practical and biosafety issues with inactivated vaccines. One of the strategies in evaluation under field conditions is the use of a new marker E2-based vaccine produced in the milk of adenovirally transduced goats. Previously we had demonstrated the efficacy of this antigen, which conferred early protection and long-lasting immunity in swine against CSFV infection. Here, we have used a simpler downstream process to obtain and formulate the recombinant E2 glycoprotein expressed in the mammary gland. The expression levels reached approximately 1.7 mg/ml, and instead of chromatographic separation of the antigen, we utilized a clarification process that eliminates the fat content, retains a minor amount of caseins, and includes an adenoviral inactivation step that improves the biosafety of the final formulation. In a vaccination and challenge experiment in swine, different doses of the E2 antigen contained within the clarified whey generated an effective immune response of neutralizing antibodies that protected all of the animals against a lethal challenge with CSFV. During the immunization and after challenge, the swine were monitored for adverse reactions related to the vaccine or symptoms of CSF, respectively. No adverse reactions or clinical signs of disease were observed in vaccinated animals, in which no replication of CSFV could be detected after challenge. Overall, we consider that the simplicity of the procedures proposed here is a further step toward the introduction and implementation of a commercial subunit vaccine against CSF.

Efficacy of the Bm86 antigen against immature instars and adults of the dog tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

The Bm86 antigen has been used to control ticks of the Boophilus genera in integrated programs that also include the use of acaricides. Because of recent phylogenetic studies have lead to the inclusion of all Boophilus species within the Rhipicephalus genera, we aimed to investigate the efficacy of the Bm86 antigen on the biotic potential of Rhipicephalus sanguineus. Domestic dogs were vaccinated with Bm86 and challenged with the three instars of R. sanguineus. Male and female mongrel dogs were divided into two groups of four animals each, comprising non-vaccinated and vaccinated animals. Immunized dogs were given two doses of an experimental formulation containing 50mug of recombinant Bm86, at 21 days interval while the other group was given placebo, consisting of the same preparation without Bm86. Each dog was challenged 21 days after the last dose with 250 larvae, 100 nymphs and 55 adults (25 females and 30 males) released inside feeding chambers (one per instar) glued to their shaved flank. The effect of the vaccination was evaluated by determining biological parameters of ticks including the yield rates of larvae, nymphs and adult females. Adult females engorged weight, egg mass weight, efficiency rate of conversion to eggs (ERCE) and hatchability. In addition, sera were collected from dogs at 0, 21, 36, 45 and 75 days after the vaccination and used for the detection of specific antibodies by ELISA. Collection rates of larvae, nymphs and adult females fed on vaccinated dogs were significantly (p<0.05) reduced by 38%, 29% and 31%, respectively, as compared with non-vaccinated controls. Significant reductions were also observed in weight of engorged females and egg mass, in ERCE, but not in the hatch rate of ticks fed on immunized dogs. ELISA data revealed a marked and significant increase in optical densities of sera from vaccinated animals after the second dose of Bm86. We concluded that the Bm86 antigen used as a vaccine for dogs reduced the viability and biotic potential of the R. sanguineus.

Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype.

Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004-2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.

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.