Background/Objectives: Vaccines may improve the control and eradication of bovine tuberculosis. However, the evaluation of experimental candidates requires the assessment of the protection, excretion, transmission and biosafety. A natural transmission trial among likely infected animals was conducted. Methods: Seventy-four male heifers were randomly distributed (five groups) and vaccinated subcutaneously with attenuated strains (M. bovis Δmce2 or M. bovis Δmce2-phoP), a recombinant M. bovis BCG Pasteur (BCGr) or M. bovis BCG Pasteur. Then, they cohoused with a naturally infected bTB cohort under field conditions exposed to the infection. Results: A 23% of transmission of wild-type strains was confirmed (non-vaccinated group). Strikingly, first vaccination did not induce immune response (caudal fold test and IFN-gamma release assay). However, after 74 days of exposure to bTB, animals were re-vaccinated. Although their sensitization increased throughout the trial, the vaccines did not confer significant protection, when compared to the non-vaccinated group, as demonstrated by pathology progression of lesions and confirmatory tools. Besides, the likelihood of acquiring the infection was similar in all groups compared to the non-vaccinated group (p > 0.076). Respiratory and digestive excretion of viable vaccine candidates was undetectable. To note, the group vaccinated with M. bovis Δmce2-phoP exhibited the highest proportion of animals without macroscopic lesions, compared to the one vaccinated with BCG, although this was not statistically supported. Conclusions: This highlights that further evaluation of these vaccines would not guarantee better protection. The limitations detected during the trial are discussed regarding the transmission rate of M. bovis wild-type, the imperfect test for studying sensitization, the need for a DIVA diagnosis and management conditions of the trials performed under routine husbandry conditions. Re-vaccination of likely infected bovines did not highlight a conclusive result, even suggesting a detrimental effect on those vaccinated with M. bovis BCG.

Unlike other lentiviruses, EIAV replication can be eventually controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs. Maintenance of this carrier state is absolutely dependent on active immune responses and probably this could be extremely related with the appearance of virus specific immune response during early stage of infection. Since the virus undergoes rapid mutation following infection, the immune response must also adapt to meet this challenge. Nevertheless, more studies should be focalized in conserved regions, especially in viral enveloped proteins. Indeed, few studies have examined the cellular and humoral immune responses to the viral antigens throughout natural infection. Thus, the goal of the current study is to determine if native conserved sequences within viral glycoproteins of EIAV may induce humoral and cellular immune response from early to latent stages of natural infection. To that purpose, we analyzed 10 newly infected horses, 28 inapparent carriers and 12 EIAV uninfected (control) horses. Four synthetic peptides, gp90-A(416-444)/B(118-149) and gp45-A(522-546)/B(651-670), representing conserved regions in envelope proteins, were used to find the peptide-specific antibodies by ELISA and T cell responses by lymphoproliferation in vitro. To evaluate if memory T cells are CD4+ or CD8+ cells, lymphoproliferation assays were performed with prior blocking using anti-MHC II or anti-MHC I specific monoclonal antibodies, respectively. Mann Whitney and Kruskal–Wallis tests were used to compare data and statistical significance of differences was defined at P < 0.05. The results showed that 9/10 (90%) and 28/28 (100%) of newly infected and inapparent carrier horses respectively, had antiviral antibodies against at least one of the four regions studied, corresponding to EIAV envelope glycoproteins. Indeed, the majority of naturally infected horses had antibodies to gp90-A(416-444); gp45-A(522-546) and gp45-B(651-670), being their levels superior during carrier stage. By contrast, no antibody against gp90-B(118-149) could be detected in recently infected horses. Only four newly infected horses had lymphocytes capable of recognizing at least one synthetic peptide, being peptide gp90-B(118-149), the most recognized one, in spite of the fact that no specific antibodies were found. By contrast, we have detected proliferation of effector/memory T cells with at least one peptide in the most carrier horses, being gp90-A(416-444) the synthetic peptide more recognized. Our results indicated that these viral conserved sequences, like these synthetic peptides studied, may represent T and B epitopes from early to latent stages of natural infection. If this is so, it is vital to define other conserved regions during natural infection in order to incorporate all of them as candidate vaccine molecules.

Equine Infectious Anemia (EIA) is a retroviral disease of equids, for which there is no vac-cine particularly adapted to American viral strains. In this work we searched for possible epitope regions for the surface proteins gp90 and gp45, rationally employing the latest available bioinformatics tools that constitute the state of the art in the field. We selected eight regions that contain numerous overlapping epitopes that have a high coverage amongst American viral strains and designed a chimeric envelope protein with those proteins fused in tandem as a novel vaccine candidate. In silico predictors were used to analyze chimeric protein physicochemical and immunogenic properties, as well as its al-lergenicity and toxicity. Protein structure was predicted and validated, and its ability to trigger cytotoxic immune responses was predicted by molecular docking to ELA alleles. The proposed sequence is predicted to be highly immunogenic and sets the base for a novel EAIV vaccine that could be used to protect against several American field strains.