Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap -null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap -null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a Δ sap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans -infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.

Summary: Secreted aspartic proteinases (Sap) have been described as virulence factors implicated in the mechanisms of host colonization by the yeast Candida albicans in different types of candidiasis. Intraperitoneal inoculation of C. albicans into BALB/c mice rapidly leads to systemic candidiasis, with significant colonization of the kidneys measurable in the following week. In this study we assessed the potential of vaccination with C. albicans secreted aspartic proteinase 2 (Sap2) in preventing systemic candidiasis in BALB/c mice. Intradermal injection of highly purified native Sap2 protein incorporated in alum adjuvant provided efficient immune protection, as indicated by a 20‐fold decrease in the colonization of kidneys. The protective effect of Sap2 immunization with alum adjuvant was also observed in mice infected with a lethal inoculum of C. albicans. Immunization with the native Sap2 alone, as well as with a denatured recombinant form of the protein, also conferred protection, albeit to a lesser level. In all cases, protection correlated with an increase in serum antibodies to Sap2. Moreover, passive transfer of anti‐Sap2 immunoglobulin G (IgG) significantly decreased the yeast burden in kidneys of C. albicans‐infected mice. This result shows that immune protection against systemic candidiasis in mice immunized with Sap2 is antibody‐mediated. Taken together, these analyses demonstrate that Sap2 can be successfully used as a vaccination target in systemic candidiasis and reveals the potential immunomodulatory role of Sap2 on C. albicans infection.

Summary: Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite‐evasion immune‐mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69‐expressing peritoneal and splenic B cells. This early B‐cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin‐secreting splenic B cells and a rise in serum levels of N. caninum‐specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum‐infected mice. The B‐cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with γ‐irradiated parasites. Contrasting with the peripheral B‐cell expansion, a depletion of B‐lineage cells was observed in the bone‐marrow of the N. caninum‐infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite‐specific antibodies nevertheless impaired interferon‐γ (IFN‐γ) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non‐lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti‐N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite‐specific, non‐polyclonal, B‐cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN‐γ in the host protective immune mechanisms against this parasite.

It is well established that CD8⁺ T cells play an important role in protective immunity against protozoan infections. However, their role in the course of Neospora caninum infection has not been fully elucidated. Here we report that CD8-deficient mice infected with N. caninum presented higher parasitic loads in the brain and lungs and lower spleen and brain immunity-related GTPases than their wild-type counterparts. Moreover, adoptive transfer of splenic CD8⁺ T cells sorted from N. caninum-primed immunosufficient C57BL/10 ScSn mice prolonged the survival of infected IL-12-unresponsive C57BL/10 ScCr recipients. In both C57BL/6 and C57BL/10 ScSn mice CD8⁺ T cells are activated and produce interferon-γ (IFN-γ) upon challenged with N. caninum. The host protective role of IFN-γ produced by CD8⁺ T cells was confirmed in N. caninum-infected RAG2-deficient mice reconstituted with CD8⁺ T cells obtained from either IFN-γ-deficient or wild-type donors. Mice receiving IFN-γ-expressing CD8⁺ T cells presented lower parasitic burdens than counterparts having IFN-γ-deficient CD8⁺ T cells. Moreover, we observed that N. caninum-infected perforin-deficient mice presented parasitic burdens similar to those of infected wild-type controls. Altogether these results demonstrate that production of IFN-γ is a predominant protective mechanism conferred by CD8⁺ T cells in the course of neosporosis.

Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.