Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope.

BACKGROUND: Leishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1-28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67-81 and 122-135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%). CONCLUSIONS/SIGNIFICANCE: Our work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.

Concomitant helminth infection downmodulates the Vaccinia virus-specific immune response and potentiates virus-associated pathology.

The aim of this work was to elucidate the immunopathological mechanisms of how helminths may influence the course of a viral infection, using a murine model. Severe virulence, a relevant increase in the virus titres in the lung and a higher mortality rate were observed in Ascaris and Vaccinia virus (VACV) co-infected mice, compared with VACV mono-infected mice. Immunopathological analysis suggested that the ablation of CD8+ T cells, the marked reduction of circulating CD4+ T cells producing IFN-γ, and the robust pulmonary inflammation were associated with the increase of morbidity/mortality in co-infection and subsequently with the negative impact of concomitant pulmonary ascariasis and respiratory VACV infection for the host. On the other hand, when evaluating the impact of the co-infection on the parasitic burden, co-infected mice presented a marked decrease in the total number of migrating Ascaris lung-stage larvae in comparison with Ascaris mono-infection. Taken together, our major findings suggest that Ascaris and VACV co-infection may potentiate the virus-associated pathology by the downmodulation of the VACV-specific immune response. Moreover, this study provides new evidence of how helminth parasites may influence the course of a coincident viral infection.

Parasitological and immunological aspects of early Ascaris spp. infection in mice.

Studies related to the immunobiological aspects of an Ascaris spp. infection are still scarce, especially those that aim to elucidate the early events of the immune response. In this study, we demonstrated a novel standardized method for early experimental Ascaris infection, providing additional information about the infectivity of eggs embryonated in vitro as well as the influence of host age on development of the infection. Finally, we characterised the immunopathology of early infection, focusing on the tissue and systemic cytokine profiles and the histopathology of infection in the lungs of BALB/c mice. Our results demonstrated that the highest egg infectivity occurred on the 100th and 200th days of in vitro embryonation and that 8 week-old BALB/c mice were more susceptible to infection than 16 week-old mice. Ascaris-infected mice showed an early, significant level of IL-5 production in the lungs 4 days p.i., followed by an increase in the level of neutrophils in the inflammatory infiltrate at 8 days p.i, which was correlated with the peak of larval migration in the tissue and a significant level of IL-6 production. The inflammatory infiltrate in the lungs was gradually replaced by mononuclear cells and eosinophils on the 10th and 12th days p.i., respectively, and an increase in TNF levels was observed. The downmodulation of systemic TCD4(+) cell numbers might suggest that T cell hyporesponsiveness was induced by the Ascaris spp. larvae, contributing to safeguarding parasite survival during larval migration. Taken together, the novel aspects of Ascaris infection presented here enabled a better understanding of the immunopathological events during larval migration, providing insight for further studies focused on immunisation and immunoprophylatic assays.