A prime-boost combination of a three-protein cocktail and multiepitopic MVA as a vaccine against Babesia bigemina elicits neutralizing antibodies and a Th1 cellular immune response in mice.

In the intraerythrocytic protozoan parasites of the genus Babesia both innate and adaptive immune responses are necessary to confer protection against clinical disease. In particular, the adaptive immune response involves the production of neutralizing antibodies as well as the presentation of parasite antigens to CD4+ T lymphocytes by professional antigen-presenting cells. Therefore, the development of alternative vaccines that replace the use of live attenuated strains should include relevant epitopes targeting both B and T cell responses. The aim of this study was to design new Babesia bigemina immunogens and evaluate the humoral and cellular responses in mice. To achieve this, three B. bigemina recombinant antigens called Apical Membrane Antigen 1 (AMA-1), Rhoptry Associated Protein 1 (RAP-1) and the Thrombospondin Related Anonymous Protein 1 (TRAP-1) were obtained. Besides, two recombinant modified vaccinia virus Ankara vectors coding for chimeric constructs containing bioinformatically predicted B and T cell epitopes from the same three antigens were generated. These immunogens were evaluated in prime-boost heterologous schemes. Among the combinations tested, priming with a cocktail of the three proteins followed by a booster immunization with a mix of both viruses induced the highest activation of IFN-γ+ CD4+ and CD8+ antigen-specific T cell responses. Remarkably, all vaccine schemes containing antigen cocktails also induced antibodies that were capable of neutralizing merozoite invasion of bovine erythrocytes in vitro at a level comparable to an anti B. bigemina hyperimmune bovine serum. Our results offer a new perspective for vaccines against B. bigemina combining bioinformatics predictions and prime-boost immunization regimes for future control measures against bovine babesiosis.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Novel biotechnological platform based on baculovirus occlusion bodies carrying Babesia bovis small antigenic peptides for the design of a diagnostic enzyme-linked immunosorbent assay (ELISA).

Baculoviruses are large DNA virus of insects principally employed in recombinant protein expression. Its ability to form occlusion bodies (OBs), which are composed mainly of polyhedrin protein (POLH), makes them biotechnologically attractive, as these crystals (polyhedra) can incorporate foreign peptides and can be easily isolated. On the other hand, peptide microarrays allow rapid and inexpensive high-throughput serological screening of new candidates to be incorporated to OBs. To integrate these 2 biotechnological approaches, we worked on Babesia bovis, one of the causative agents of bovine babesiosis. Current molecular diagnosis of infection with B. bovis includes enzyme-linked immunosorbent assay (ELISA) techniques, which use merozoite lysate obtained from infected bovine erythrocytes. However, it is important to produce recombinant antigens that replace the use of crude antigens. Here, we describe a new biotechnological platform for the design of indirect ELISAs based on 5 antigenic peptides of 15 amino acid residues of B. bovis (ApBb), selected from a peptide microarray and expressed as a fusion to POLH. An Sf9POLHE44G packaging cell line infected with recombinant baculoviruses carrying POLH-ApBb fusions yielded higher levels of chimeric polyhedra, highlighting the advantage of a trans-contribution of a mutant copy of polyhedrin. Finally, the use of dissolved recombinant polyhedra as antigens was successful in an ELISA assay, as B. bovis-positive sera recognized the fusion POLH-ApBb. Thus, the use of this platform resulted in a promising alternative for molecular diagnosis of relevant infectious diseases.

The dynamics of erythrocyte infection in bovine anaplasmosis: a flow cytometry-based analysis.

Anaplasma marginale (A. marginale) is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia. In the present work, we combine SYTO16 labeling of parasitized cells with the statistical power of flow cytometry to study the evolution of erythrocyte infection during bovine anaplasmosis.

A new set of molecular markers for the genotyping of Babesia bovis isolates.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constraint for the development of cattle industries worldwide. The existence of different strains and subpopulations has long been described in this hemoparasite. However, few molecular markers have been reported for strain genotyping and characterization. Minisatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis. In this work we report a set of five molecular markers containing minisatellites that showed a variable degree of polymorphism in several American strains. We have used a bioinformatics approach to search for marker sequences contained in open reading frames. Five genes were chosen and primers were designed in conserved regions flanking the repeat region. Two of the genes were the previously described Bv80/Bb-1 and TRAP. The other three genes were named p200, Antigen 3 and Desmoyokin. Amplification by PCR, sequencing and comparative analysis of 11 strains from Argentina, Brazil, Uruguay, Mexico and USA determined that the tandem repeats varied in number and sequence among the isolates. Genome analysis of the five markers revealed that they were single copy and distributed across the four B. bovis chromosomes. When the new markers were analyzed in an experimental infection, absolute sequence conservation was found, indicating the stability of these markers during the course of infection. These markers were also stable during three syringe passages through calves. The application of this panel of molecular markers could provide new molecular tools for the genotyping of B. bovis isolates and analysis of changes in parasite populations following vaccination.

Development of an indirect ELISA for the diagnosis of equine piroplasmosis.

An indirect ELISA (iELISA) for the detection of specific anti-Theileria equi antibodies in horse serum was developed. Its performance showed good concordance (K= 0.79) when compared with a competitive ELISA recommended by the World Organisation for Animal Health. Horse serum samples from two provinces located in the north and east of Argentina (Formosa and Entre Rios, respectively) were analyzed by this iELISA. A high percentage of positive horses were found in Formosa, consistent with the climatic conditions of the region that are apt for the development of tick vectors. Surprisingly, seropositive animals were also detected, although in a lower proportion, in Entre Rios, which has a temperate weather and is presumably tick free. Breeding of thoroughbred racing horses is an important economic asset of Argentina. Since equine piroplasmosis is a constraint for horse export, the epidemiological situation in different regions of the country needs to be assessed for the implementation of control measures. The iELISA developed in this work provides a convenient tool for this type of study.

Search for Babesia bovis vaccine candidates.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constrain for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated forms of the parasite, but they have several drawbacks and thus the development of alternative subunit vaccines, either based in recombinant versions of full size proteins or in recombinant or synthetic peptides containing combinations of protective B-cell and T-cell epitopes is needed. Our current strategies for the identification of vaccine candidate antigens include the identification of functionally relevant antigens, bioinformatics, and comparative genomics using the recently sequenced B. bovis genome. These led us to the functional and immunological characterization of members of the VMSA gene family, a group of well conserved putative cysteine and serine proteases, and to the definition of a surface exposed B-cell epitope present in the Merozoite Surface Antigen-2c. Work in progress is focused in defining additional epitopes, and to determine whether they are neutralization-sensitive. These approaches might unravel useful vaccine candidates for B. bovis, and will increase our understanding of the pathogenicity mechanisms of these and related hemoparasites.

Taking advantage of the polymorphism of the MSA-2 family for Babesia bovis strain characterization.

The Merozoite Surface Antigen-2 (MSA-2) family of Babesia bovis is a group of variable genes which share conserved 5' and 3' conserved ends. These genes encode membrane anchored glycoproteins, named MSA-2a1, a2, b and c, which are immunodominant antigens located on the surface of sporozoites and merozoites. In this work, we have analyzed the sequences of the msa-2a1, a2 and 2b genes in two geographically distant strains from Mexico and Argentina and detected a certain degree of genotypic diversity that can be exploited for the development of a new molecular tool for the discrimination of B. bovis field samples. Here, we describe a PCR restriction assay based on the msa2-a1, -a2 and -2b genes of B. bovis. When field strains from Argentina, Mexico and USA were analyzed, the results showed a strain-specific band pattern indicating the presence of differentially located BspMI restriction sites. This approach provides a simple method for the genotyping/strain differentiation of B. bovis field samples.

Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle.

The search for vaccine candidates against bovine babesiosis caused by Babesia bovis is greatly focused on the identification of merozoite surface-exposed antigens that are widely conserved, functionally relevant and immunodominant in cattle protected against B. bovis infections. We have recently identified msa-2c, a member of the B. bovis variable merozoite surface antigen (VMSA) gene family, which in contrast to other members, appears to be highly conserved among geographically distant B. bovis strains. In this study, we further investigated the potential of the msa-2c gene product as diagnostic and vaccine candidate for bovine babesiosis. RT-PCR studies demonstrated that MSA-2c is transcribed in merozoites of the Argentine R1A strain. In addition, antibodies against R1A recombinant MSA-2c reacted in immunoblots with a single protein of approximately 30kDa in B. bovis merozoite extracts from both R1A and Australian "S" strains, demonstrating translation of this protein in these two strains and conservation of B-cell epitopes between them. These antibodies reacted with the cell surface of R1A merozoites in fixed immunofluorescence assays, indicating the surface localization of MSA-2c. This localization was confirmed by live immunofluorescence studies in two different strains, R1A and S2P. These results also demonstrate the conservation of MSA-2c surface-exposed B-cell epitopes between these two strains. Sera from cattle either naturally or experimentally infected with Argentine strains of B. bovis specifically recognized rMSA-2c in immunoblots, reinforcing the idea that B-cell epitopes in rMSA-2c are widely conserved among field strains of B. bovis. Furthermore, our results show that these B-cell epitopes are highly immunogenic, suggesting that MSA-2c may be a useful diagnostic tool for the detection of bovine babesiosis by B. bovis. Experimental vaccination of five bovines with rMSA-2c resulted in elicitation of high specific anti-rMSA-2c IgG titers, with similar amounts of IgG(1) and IgG(2) produced. Importantly, bovine anti-rMSA-2c antibodies were able to neutralize in vitro bovine erythrocyte invasion by R1A merozoites suggesting a significant functional role for MSA-2c. Taken together these results postulate MSA-2c as a candidate for the development of novel tools for improved control of bovine babesiosis.

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7.

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.

Lysosomal phospholipase A1 in Trypanosoma cruzi: an enzyme with a possible role in the pathogenesis of Chagas' disease.

We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A(1) was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T. cruzi cells in host tissues. Thus Plase A(1) emerges as a target to block pathogenesis in trypanosomal infections.

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion.

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.

Inhibition of early endosome fusion by Trypanosoma cruzi-infected macrophage cytosol.

Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote-infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock-infected cytosols (heat-killed trypomastigotes were used in the parasite-macrophage interaction assay). GTP gamma S addition potentiates the fusogenic activity driven by trypomastigote-infected macrophage cytosol-mediated assays, unlike the biphasic fusogenic effect obtained with GTP gamma S treatment of macrophage cytosol controls. Calcium-stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP-regulated factors, and not calcium-regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote-infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment-fusion of the host lysosomes upon trypomastigote infection and further survival of the parasite within its host.

Trypanosoma cruzi: involvement of intracellular calcium in multiplication and differentiation.

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Trypanosoma cruzi: participation of intracellular Ca2+ during metacyclic trypomastigote-macrophage interaction.

The interaction between Trypanosoma cruzi, the protozoan causative of Chagas's disease, and its host cell is a complex process in which multiple signals including those of Ca2+ are involved. Macrophage cytosolic Ca2+ levels were studied during the interaction of these cells with metacyclic trypomastigotes of T. cruzi, since this event is an initial step in the natural infection. In this model we detected an increase in the macrophage cytosolic Ca2+ concentration after infection, or incubation with a metacyclic lysate or with isolated membranes, suggesting that these increments could be necessary for parasite invasion. This fact was confirmed by treating macrophages with a Ca2+ chelator or a Ca2+ channel antagonist which decreased the infection percentages while parasitization levels increased after treatment with Ca2+ channel agonist.