Reginaldo G. Bastos

Recombinant Mycobacterium bovis BCG

The Bacillus Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that has been broadly used as a vaccine against human tuberculosis. This live bacterial vaccine is able to establish a persistent infection and induces both cellular and humoral immune responses. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis of the potential use of this attenuated mycobacterium as a recombinant vaccine. Over the years, a range of strategies has been developed to allow controlled and stable expression of viral, bacterial and parasite antigens in BCG. Herein, we review the strategies developed to express heterologous antigens in BCG and the immune response elicited by recombinant BCG constructs. In addition, the use of recombinant BCG as an immunomodulator and future perspectives of BCG as a recombinant vaccine vector are discussed.

Bovine WC1− γδT cells incubated with IL-15 express the natural cytotoxicity receptor CD335 (NKp46) and produce IFN-γ in response to exogenous IL-12 and IL-18

The gammadeltaT cells of ruminants are believed to participate in innate immunity and have been described with regulatory, inflammatory and cytotoxic functions. Here we describe a subset of CD3(+) TcR1(+) WC1(-)gammadeltaT cells expressing CD335 (NKp46), classically associated with CD3(-) natural killer (NK) cells, as a consequence of incubation with IL-15. This population, undetectable at the time of collection, developed after 2 week of IL-15 culture from splenic leukocytes (SPL) reaching greater than 50% of the total gammadeltaT cells. However, they did not grow well from peripheral blood leukocytes (PBL). Splenic gammadeltaT cells positively selected by magnetic separation prior to incubation with IL-15 and analyzed by flow cytometry, consistently yielded CD3(+) cells expressing CD335. These cells arose from the CD335(-)gammadeltaT cell population suggesting that the new population represents up-regulation of CD335 by gammadeltaT cells. CD335 mRNA expression from sorted IL-15-incubated SPL CD335(+)gammadeltaT cells or NK cells exceeded that of CD335(-)gammadeltaT cells. Incubation with IL-12 and IL-18 increased the number of CD335(+)gammadeltaT cells in both the PBL and SPL fractions as compared to controls or IL-12 or IL-18 alone. In addition, CD335(+)gammadeltaT cells demonstrated a robust ability to produce IFN-gamma in response to exogenous IL-12 and IL-18. Taken as a whole, we describe a new phenotypically distinct bovine gammadeltaT cell sub-population capable of participating in type 1 immune responses.

The Rhipicephalus (Boophilus) microplus Bm86 gene plays a critical role in the fitness of ticks fed on cattle during acute Babesia bovis infection

BackgroundRhipicephalus (Boophilus) microplus is an economically important tick of cattle involved in the transmission of Babesia bovis, the etiological agent of bovine babesiosis. Commercial anti-tick vaccines based on the R. microplus Bm86 glycoprotein have shown some effect in controlling tick infestation; however their efficacy as a stand-alone solution for tick control has been questioned. Understanding the role of the Bm86 gene product in tick biology is critical to identifying additional methods to utilize Bm86 to reduce R. microplus infestation and babesia transmission. Additionally, the role played by Bm86 in R. microplus fitness during B. bovis infection is unknown.ResultsHere we describe in two independent experiments that RNA interference-mediated silencing of Bm86 decreased the fitness of R. microplus females fed on cattle during acute B. bovis infection. Notably, Bm86 silencing decreased the number and survival of engorged females, and decreased the weight of egg masses. However, gene silencing had no significant effect on the efficiency of transovarial transmission of B. bovis from surviving female ticks to their larval offspring. The results also show that Bm86 is expressed, in addition to gut cells, in larvae, nymphs, adult males and ovaries of partially engorged adult R. microplus females, and its expression was significantly down-regulated in ovaries of ticks fed on B. bovis-infected cattle.ConclusionThe R. microplusBm86 gene plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. Therefore, the data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation particularly in B. bovis endemic areas.

Differential Expression of Three Members of the Multidomain Adhesion CCp Family in Babesia bigemina, Babesia bovis and Theileria equi

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis.

Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus) microplus increases the infection rate of Babesia bovis in larval progeny

BackgroundThe cattle tick Rhipicephalus (Boophilus) microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species.ResultsIn this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp) in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi) gene and a putative lipocalin (Lpc) gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny.ConclusionThe silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.

Bovine NK cells acquire cytotoxic activity and produce IFN-γ after stimulation by Mycobacterium bovis BCG-or Babesia bovis-exposed splenic dendritic cells

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites

OBJECTIVE The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA. METHODS Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. RESULTS RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. CONCLUSION B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.

Babesia bovis expresses a neutralization-sensitive antigen that contains a microneme adhesive repeat (MAR) domain.

A gene coding for a protein with sequence similarity to the Toxoplasma gondii micronemal 1 (MIC1) protein that contains a copy of a domain described as a sialic acid-binding micronemal adhesive repeat (MAR) was identified in the Babesia bovis genome. The single copy gene, located in chromosome 3, contains an open reading frame encoding a putative 181 amino acid protein, which is highly conserved among distinct B. bovis strains. Antibodies against both recombinant protein and synthetic peptides mimicking putative antigenic regions in the B. bovis-MIC1 (Bbo-MIC1) protein bind to the parasite in immunofluorescence assays and significantly inhibit erythrocyte invasion in in vitro B. bovis cultures. Bbo-MIC1 is recognized by antibodies in serum from B. bovis infected cattle, demonstrating expression and immunogenicity during infection. Overall, the results suggest that Bbo-MIC1 protein is a viable candidate for development of subunit vaccines.

Babesia bovis expresses Bbo-6cys-E, a member of a novel gene family that is homologous to the 6-cys family of Plasmodium

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the B. bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family contains six genes termed Bbo-6cys-A, B, C, D, E and F encoding for proteins containing an arrangement of 6 cysteine residues. The Bbo-6cys genes A, B, C, D, and E are tandemly arranged as a cluster of Chromosome 2 in the B. bovis genome, whereas gene F is located in a distal region in the same chromosome. The Bbo-6cys-E gene, with higher homology to PFS230, was selected for further examination. Immunoblot analysis using recombinant Bbo-6cys-E protein and B. bovis-positive bovine serum demonstrated expression by the parasite and immunogenicity during B. bovis infection. Immunofluorescence analysis using anti-Bbo-6cys-E antibodies confirmed expression of Bbo-6cys-E in in vitro blood stages of B. bovis. In addition, polyclonal antisera against both recombinant Bbo-6cys-E and specific synthetic peptides containing predicted B-cell epitopes of Bbo-6cys-E, significantly inhibited erythrocyte invasion by B. bovis in in vitro neutralization assays, suggesting an important functional role for this protein. Identification of this new gene family in B. bovis and further investigation on its biological significance may aid our understanding of the bovine, tick and parasite relationships and the development of improved control methods against B. bovis infection in cattle.

Targeted surface expression of an exogenous antigen in stably transfected Babesia bovis.

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine delivery platform.