Nada S. Daifalla

Growing evidence shows that antioxidant proteins ofLeishmaniacould be used as vaccine candidates. In this study, we report the efficacy ofLeishmania donovaniiron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-αor IL-2 by antigen-stimulated T cells. Eight weeks afterL. majorinfection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γproduction by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high doseL. majorinfection.

Visceral leishmaniasis (VL) is a serious and fatal disease. Therapeutic drugs are toxic and non-sterilizing. The etiological agents Leishmania infantum and Leishmania donovani cause active and asymptomatic diseases. Effective drugs to treat VL exist but unfortunately, post-treatment relapses are common. Little is known why drugs are non-sterilizing or how these intracellular pathogens can escape treatment. Here, using a murine model of VL we found that CD271+/Sca1+ bone marrow mesenchymal stem cells (BM-MSCs) are readily infected in vitro and in vivo by L. infantum. Because BM-MSCs express potent drug efflux pumps, e.g., ABCG2 it is possible that this unique intracellular infectious niche could allow L. infantum to escape anti-parasite drugs.

We assessed the immune response against recombinant proteins of two related, albeit functionally different, peroxidoxins fromLeishmania donovani: peroxidoxin 1 (LdPxn1) and peroxidoxin 2 (LdPxn2) in BALB/c mice. We also evaluated the effect of coadministration of TLR agonists (CpG ODN and GLA-SE) on the antigen-specific immune response. Immunization with recombinant LdPxn1 alone induced a predominantly Th2 type immune response that is associated with the production of high level of IgG1 and no IgG2a isotype while rLdPxn2 resulted in a mixed Th1/Th2 response characterized by the production of antigen-specific IgG2a in addition to IgG1 isotype. Antigen-stimulated spleen cells from mice that were immunized with rLdPxn1 produced low level of IL-10 and IL-4 and no IFN-γ, whereas cells from mice immunized with rLdPxn2 secreted high level of IFN-γ, low IL-4, and no IL-10. Coadministration of CpG ODN or GLA-SE with rLdPxn1 skewed the immune response towards a Th 1 type as indicated by robust production of IgG2a isotype. Furthermore, the presence of TLR agonists together with rLdPxn1 antigen enhanced the production of IFN-γand to a lesser extent of IL-10. TLR agonists also enhanced a more polarized Th 1 type immune response against rLdPxn2.

To date, no universally effective and safe vaccine has been developed for general human use. Leishmania donovani Peroxidoxin-1 (LdPxn-1) is a member of the antioxidant family of proteins and is predominantly expressed in the amastigote stage of the parasite. The aim of this study was to evaluate the immunogenicity and protective efficacy of LdPxn-1 in BALB/c mice in heterologous DNA-Protein immunization regimen in the presence of fusion murine granulocyte-macrophage colony-stimulating factor (mGMCSF) DNA adjuvant. A fusion DNA of LdPxn1 and mGMCSF was cloned into a modified pcDNA vector. To confirm the expression in mammalian system, Chinese hamster ovary cells were transfected with the plasmid vector containing LdPxn1 gene. BALB/c mice were immunized twice with pcDNA-mGMCSF-LdPxn-1 or pcDNA-LdPxn1 DNA and boosted once with recombinant LdPxn-1 protein. Three weeks after the last immunization, mice were infected with Leishmania major promastigotes. The result showed that immunization with pcDNA-mGMCSF-LdPxn1 elicited a mixed Th-1/Th-2 immune response with significantly higher production of IFN-γ than controls. Intracellular cytokine staining of antigen-stimulated spleen cells showed that immunization with this antigen elicited significantly higher proportion of CD4⁺ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced significantly higher proportion of multipotent CD4⁺ cells that simultaneously express the three Th-1 cytokines. Moreover, a significant reduction in the footpad swelling was seen in mice immunized with pcDNA-mGMCSF-LdPxn1 antigen. Expression study in CHO cells demonstrated that pcDNA-mGMCSF-LdPxn-1 was expressed in mammalian system. The result demonstrates that immunization of BALB/c mice with a plasmid expressing LdPxn1 in the presence of mGMCSF adjuvant elicits a strong specific immune response with high level induction of multipotent CD4⁺ cells that mediate protection of the mice from Leishmania major infection. To our knowledge, this is the first study showing the vaccine potential of Leishmania peroxidoxin -1. Leishmaniasis, a disease caused by protozoan parasites under the genus Leishmania, claims the lives of thousands of people annually. It mainly affects people in poor communities in Africa, Asia and South America. Although several drugs are available for the treatment of leishmaniasis, their efficacy is limited by the emergence of drug resistant parasite strains and by the inherent side-effects of some drugs. In light of these challenges, developing effective vaccine is considered as a crucial step in the control and ultimate elimination of the disease. In this study, we have evaluated the potential of the antioxidant Leishmania Peroxidoxin-1 as a candidate vaccine for leishmaniasis. The efficacy of the candidate vaccine was assessed in DNA-Protein immunization strategy in mice. We also investigated the adjuvant role of GMCSF DNA fused with the vaccine antigen in a pcDNA plasmid vector. The result showed that Leishmania Peroxidoxin-1 together with fusion GMCSF adjuvant in a pcDNA plasmid induces a partially protective immune response in mice. Further analysis of the immune response demonstrated that the antigen-adjuvant combination elicits CD4⁺ T cells that express IFN-γ, TNF-α, or IL-2. The antigen also induced a high frequency of CD4⁺ T cells that simultaneously express all the three cytokines. The study on samples taken from leishmaniasis patients showed that the recombinant Leishmania Peroxidoxin-1 protein is recognized by and elicit immune response in humans, a crucial requirement in the development of a vaccine.