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ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. Neospora caninum is an Apicomplexa parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with economic impact on the livestock industry. Hence, we evaluated the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.

Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N. caninum to survive at the maternal-fetal interface.

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neospora lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA + CpG, NcESA, NcESA + CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA or NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA + CpG demonstrated higher IFN- levels and IFN-/IL-10 ratio. After lethal challenge, mice immunized with NLA + CpG or NLA had lower morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA + CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN- and inflammation, rendering mice more susceptible to parasite challenge. © 2009 Published by Elsevier Ltd.

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.