Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-? (IFN-?), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-? (TNF-?), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-? and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-?, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-? production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease

Efficacy of an Immunotherapy Combining Immunogenic Chimeric Protein Plus Adjuvant and Amphotericin B against Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) in the Americas is a chronic systemic disease caused by infection with Leishmania infantum parasites. The toxicity of antileishmanial drugs, long treatment course and limited efficacy are significant concerns that hamper adequate treatment against the disease. Studies have shown the promise of an immunotherapeutics approach, combining antileishmanial drugs to reduce the parasitism and vaccine immunogens to activate the host immune system. In the current study, we developed an immunotherapy using a recombinant T cell epitope-based chimeric protein, ChimT, previously shown to be protective against Leishmania infantum, with the adjuvant monophosphoryl lipid A (MPLA) and amphotericin B (AmpB) as the antileishmanial drug. BALB/c mice were infected with L. infantum stationary promastigotes and later they received saline or were treated with AmpB, MPLA, ChimT/Amp, ChimT/MPLA or ChimT/MPLA/AmpB. The combination of ChimT/MPLA/AmpB significantly reduced the parasite load in mouse organs (p p Leishmania parasites and to produce Th1-type cytokines into the culture supernatants. To conclude, our data suggest that the combination of ChimT/MPLA/AmpB could be considered for further studies as an immunotherapy for L. infantum infection

Recombinant endonuclease III protein from <i>Leishmania infantum</i> associated with Th1-type adjuvants is immunogenic and induces protection against visceral leishmaniasis

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL

Immunization with recombinant LiHyp1 protein plus adjuvant is protective against tegumentary leishmaniasis

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease

Immunogenicity and protective efficacy of a new Leishmania hypothetical protein applied as a DNA vaccine or in a recombinant form against Leishmania infantum infection.

Vaccination is one the most important strategies for the prevention of visceral leishmaniasis (VL). In the current study, a new Leishmania hypothetical protein, LiHyP, which was previously showed as antigenic in an immunoproteomic search in canine VL, was evaluated regarding its immunogenicity and protective efficacy against Leishmania infantum infection. The effects of the immunization using LiHyP were evaluated when administered as a DNA plasmid (DNA LiHyP) or recombinant protein (rLiHyP) associated with saponin. The immunity elicited by both vaccination regimens reduced the parasitism in liver, spleen, bone marrow and draining lymph nodes, being associated with high levels of IFN-?, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD4+ T cells contributed more significantly to IFN-? production in the rLiHyP/saponin group, while CD8+ T cells were more important in the production of this cytokine in the DNA LiHyP group. In addition, increased IFN-? secretion, along with low levels of IL-10, were found when PBMCs from treated VL subject and healthy individuals were stimulated with the recombinant protein. In conclusion, when administered either as a DNA plasmid or recombinant protein, LiHyP can direct the immune response towards a Th1 immune profile, protecting animals against L. infantum infection; therefore, it can be seen as a promising immunogen against human VL

Canine visceral leishmaniasis biomarkers and their employment in vaccines.

The natural history of canine visceral leishmaniasis (CVL) has been well described, particularly with respect to the parasite load in different tissues and immunopathological changes according to the progression of clinical forms. The biomarkers evaluated in these studies provide support for the improvement of the tools used in developing vaccines against CVL. Thus, we describe the major studies using the dog model that supplies the rationale for including different biomarkers (tissue parasitism, histopathology, hematological changes, leucocytes immunophenotyping, cytokines patterns, and in vitro co-culture systems using purified T-cells subsets and macrophages infected with L. infantum) for immunogenicity and protection evaluations in phases I and II applied to pre-clinical and clinical vaccine trials against CVL. The search for biomarkers related to resistance or susceptibility has revealed a mixed cytokine profile with a prominent proinflammatory immune response as relevant for Leishmania replication at low levels as observed in asymptomatic dogs (highlighted by high levels of IFN-? and TNF-? and decreased levels in IL-4, TGF-? and IL-10). Furthermore, increased levels in CD4+ and CD8+ T-cell subsets, presenting intracytoplasmic proinflammatory cytokine balance, have been associated with a resistance profile against CVL. In contrast, a polyclonal B-cell expansion towards plasma cell differentiation contributes to high antibody production, which is the hallmark of symptomatic dogs associated with high susceptibility in CVL. Finally, the different studies used to analyze biomarkers have been incorporated into vaccine immunogenicity and protection evaluations. Those biomarkers identified as resistance or susceptibility markers in CVL have been used to evaluate the vaccine performance against L. infantum in a kennel trial conducted before the field trial in an area known to be endemic for visceral leishmaniasis. This rationale has been a guiding force in the testing and selection of the best vaccine candidates against CVL and provides a way for the veterinary industry to register commercial immunobiological products