New synthetic molecules incorporated into polymeric micelles used for treatment against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Exploring drug repositioning for leishmaniasis treatment: Ivermectin plus polymeric micelles induce immunological response and protection against tegumentary leishmaniasis.

Leishmania amazonensis can cause a wide spectrum of the clinical manifestations of leishmaniasis in humans. The development of new therapeutics is a long and expensive task; in this context, drug repositioning could be considered a strategy to identify new biological actions of known products. In the present study, ivermectin (IVE) was tested against distinct Leishmania species able to cause disease in humans. In vitro experiments showed that IVE was effective to reduce the infection degree and parasite load in Leishmania donovani- and L. amazonensis-infected macrophages that were treated with it. In addition, using the culture supernatant of treated macrophages, higher production of IFN-γ and IL-12 and lower levels of IL-4 and IL-10 were found. Then, IVE was used in a pure form or incorporated into Poloxamer 407-based polymeric micelles (IVE/M) for the treatment of L. amazonensis-infected BALB/c mice. Animals (n = 16 per group) were infected and later received saline, empty micelles, amphotericin B (AmpB), IVE, or IVE/M. They were euthanized at one (n = 8 per group) and 30 (n = 8 per group) days after treatment and, in both endpoints, immunological, parasitological, and biochemical evaluations were performed. Results showed that both IVE and IVE/M induced higher levels of IFN-γ, IL-12, GM-CSF, nitrite, and IgG2a antibodies, as well as higher IFN-γ expression evaluated by RT-qPCR in spleen cell cultures. Such animals showed low organic toxicity, as well as significant reductions in the lesion's average diameter and parasite load in their infected tissue, spleen, liver, and draining lymph node. The efficacy was maintained 30 days post-therapy, while control mice developed a polarized Th2-type response and high parasite load. In this context, IVE could be considered as a new candidate to be applied in future studies for the treatment against distinct Leishmania species.

In vitro evaluation of antileishmanial activity, mode of action and cellular response induced by vanillin synthetic derivatives against Leishmania species able to cause cutaneous and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, mainly due to their toxicity of the drugs, high cost and/or by the emergence of parasite resistant strains. In this context, new therapeutics should be searched. In this study, two novel synthetic derivatives from vanillin: [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] or 3s and [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde] or 3t, were evaluated regarding their antileishmanial activity against distinct parasite species able to cause cutaneous and visceral leishmaniasis. Results showed that compounds 3s and 3t were effective against Leishmania infantum, L. amazonensis and L. braziliensis promastigote and amastigote-like forms, showing selectivity index (SI) of 25.1, 18.2 and 22.9, respectively, when 3s was used against promastigotes, and of 45.2, 7.5 and 15.0, respectively, against amastigote-like stage. Using the compound 3t, SI values were 45.2, 53.0 and 80.0, respectively, against promastigotes, and of 35.9, 46.0 and 58.4, respectively, against amastigote-like forms. Amphotericin B (AmpB) showed SI values of 5.0, 7.5 and 15.0, respectively, against promastigotes, and of 3.8, 5.0 and 7.5, respectively, against amastigote-like stage. The treatment of infected macrophages and inhibition of the infection upon pre-incubation with the molecules showed that they were effective in reducing the infection degree and inhibiting the infection in pre-incubated parasites, respectively, as compared to data obtained using AmpB. The mechanism of action of 3s and 3t was evaluated in L. infantum, revealing that both 3s and 3t altered the parasite mitochondrial membrane potential leading to reactive oxygen species production, increase in lipid corps and changes in the cell cycle, causing the parasite' death. A preliminary assay using the cell culture supernatant from treated and infected macrophages showed that 3s and 3t induced higher IL-12 and lower IL-10 values; suggesting the development of an in vitro Th1-type response in the treated cells. In this context, data indicated that 3s and 3t could be considered therapeutic agents to be tested in future studies against leishmaniasis.

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.

Recombinant guanosine-5'-triphosphate (GTP)-binding protein associated with Poloxamer 407-based polymeric micelles protects against Leishmania infantum infection.

Leishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5'-triphosphate (GTP)-binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lymphoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL.

Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis.

The diagnosis of leishmaniasis presents problems due to the variable sensitivity and/or specificity of tests. In addition, high levels of anti-parasite antibodies can remain after treatment, making it difficult to conduct a prognostic follow-up of patients. In this context, it is necessary to identify new candidates to be examined for the sensitive and specific diagnosis of the disease. In the present study, four Leishmania proteins, previously shown as antigenic for tegumentary leishmaniasis (TL), were evaluated, and their linear specific B-cell epitopes were predicted and used to generate a new gene codifying chimeric protein called ChimB, which was cloned, and the recombinant version was expressed, purified, and evaluated in ELISA (Enzyme-Linked Immunosorbent Assay) to diagnose TL and visceral leishmaniasis (VL). A total of 220 human serum samples were used, and, when ChimB was used, results showed sensitivity, specificity, and positive and negative predictive values of 100% for the diagnosis of both diseases; however, when using peptides, the sensitivity values reached from 28.0% to 57.3% and specificity varied from 16.3% to 83.7%. A soluble Leishmania extract (SLA) showed sensitivity and specificity values of 30.7% and 45.9%, respectively. The area under the curve (AUC) value for ChimB was 1.0, while for synthetic peptides, this value reached between 0.502 and 0.635, whereas for SLA, the value was of 0.589. Serological assays using sera samples collected before and after treatment showed significant reductions in the anti-ChimB antibody levels after therapy, suggesting a prognostic role of this recombinant antigen. In conclusion, preliminary data suggest the use from ChimB as a potential candidate for the diagnosis and prognosis of leishmaniasis.

Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins.

Serological tests used for the diagnosis of tegumentary leishmaniasis (TL) presents problems, mainly related to their variable sensitivity and/or specificity, which can be caused by low levels of antileishmanial antibodies or by presence of cross-reactive diseases, respectively. In this context, the search for new antigenic candidates presenting higher sensitivity and specificity is urgently required. In the present study, the amino acid sequences of the LiHyT, LiHyD, LiHyV, and LiHyP proteins, which were previously showed to be antigenic in the visceral leishmaniasis (VL), were evaluated and eight B-cell epitopes were predicted and used for construction of gene codifying a chimeric protein called ChimLeish. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA (Enzyme-Linked Immunosorbent Assay) for the diagnosis of TL. The own B cell epitopes used to construct the chimera were synthetized and also evaluated as antigens, as well as a soluble Leishmania braziliensis antigenic extract (SLA). Results showed that ChimLeish presented 100% sensitivity and specificity to diagnose TL, while synthetic peptides showed sensitivity varying from 9.1% to 90.9%, while specificity reached from 98.3% to 99.1%. SLA showed sensitivity and specificity of 18.2% and 98.3%, respectively. A preliminary prognostic evaluation showed that anti-ChimLeish IgG antibodies declined in significant levels, when serological reactivity was compared before and six months after treatment, suggesting also a possible prognostic role of this antigen for TL.

Leishmania LiHyC protein is immunogenic and induces protection against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccination could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leishmania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expression evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC associated with adjuvants could be considered for future studies as a vaccine candidate against VL.

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease of global importance caused by parasites of the genus Leishmania, and coinfection with human immunodeficiency virus (HIV) is common in countries where both diseases are endemic. In particular, widely used immunological tests for VL diagnosis have impaired sensitivity (Se) and specificity (Sp) in VL/HIV coinfected patients and there is also cross-reactivity with other endemic diseases, e.g., Chagas disease, malaria, and tuberculosis. To develop new antigens to improve the diagnosis of VL and VL/HIV coinfection, we predicted eight specific B-cell epitopes of four Leishmania infantum antigens and constructed a recombinant polypeptide chimera antigen called ChimLeish. A serological panel of 195 serum samples was used to compare the diagnostic capabilities of ChimLeish alongside the individual synthetic peptides. ChimLeish reacted with sera from all VL and VL/HIV coinfected patients [Se = 100%; Sp = 100%; area under the curve (AUC) = 1.0]. Peptides showed lower reactivities (Se = 76.8 to 99.2%; Sp = 67.1 to 95.7%; AUC between 0.87 and 0.98) as did a L. infantum antigenic preparation used as an antigen control (Se = 56.8%; Sp = 69.5%: AUC = 0.45). Notably, ChimLeish demonstrated a significant reduction (p < 0.05) of anti-ChimLeish antibodies after treatment and cure of a small number of patients. Although only a limited serological panel was tested, preliminary data suggest that ChimLeish should be evaluated in larger sample studies for the diagnosis of VL and VL/HIV coinfection.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.

Screening diagnostic candidates from Leishmania infantum proteins for human visceral leishmaniasis using an immunoproteomics approach.

There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.

A recombinant fusion protein displaying murine and human MHC class I- and II-specific epitopes protects against Leishmania amazonensis infection.

Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

Non-invasive urine-based ELISA using a recombinant Leishmania protein to diagnose tegumentary leishmaniasis.

The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85%, 29%, 58%, and 31%, respectively, and a specificity of 97.5%, 98%, 100%, and 97.5%, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95%, 74%, 67%, and 52%, respectively, and a specificity of 100%, 99%, 98%, and 86%, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.

Immunotherapy Using Immunogenic Mimotopes Selected by Phage Display plus Amphotericin B Inducing a Therapeutic Response in Mice Infected with Leishmania amazonensis.

Leishmania amazonensis can cause cutaneous and visceral clinical manifestations of leishmaniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion's average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis.

The association between rLiHyp1 protein plus adjuvant and amphotericin B is an effective immunotherapy against visceral leishmaniasis in mice.

Treatment of visceral leishmaniasis (VL) is compromised by drug toxicity, high cost and/or the emergence of resistant strains. Though canine vaccines are available, there are no licensed prophylactic human vaccines. One strategy to improve clinical outcome for infected patients is immunotherapy, which associates a chemotherapy that acts directly to reduce parasitism and the administration of an immunogen-adjuvant that activates the host protective Th1-type immune response. In this study, we evaluated an immunotherapy protocol in a murine model by combining recombinant (r)LiHyp1 (a hypothetical amastigote-specific Leishmania protein protective against Leishmania infantum infection), with monophosphoryl-lipid A (MPLA) as adjuvant and amphotericin B (AmpB) as reference antileishmanial drug. We used this protocol to treat L. infantum infected-BALB/c mice, and parasitological, immunological and toxicological evaluations were performed at 1 and 30 days after treatment. Results showed that mice treated with rLiHyp1/MPLA/AmpB presented the lowest parasite burden in all organs evaluated, when both a limiting dilution technique and qPCR were used. In addition, these animals produced higher levels of IFN-γ and IL-12 cytokines and IgG2a isotype antibody, which were associated with lower production of IL-4 and IL-10 and IgG1 isotype. Furthermore, low levels of renal and hepatic damage markers were found in animals treated with rLiHyp1/MPLA/AmpB possibly reflecting the lower parasite load, as compared to the other groups. We conclude that the rLiHyp1/MPLA/AmpB combination could be considered in future studies as an immunotherapy protocol to treat against VL.

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 < 0.05) and induced a Th1-type immune response, which was characterized by higher ratios of anti-ChimT and anti-parasite IgG2a:IgG1 antibodies, increased IFN-γ mRNA and IFN-γ and IL-12 cytokines and accompanied by lower levels of IL-4 and IL-10 cytokines, when compared to other treatments and controls (all p < 0.05). Organ toxicity was also lower with the ChimT/MPLA/AmpB immunotherapy, suggesting that the inclusion of the vaccine and adjuvant ameliorated the toxicity of AmpB to some degree. In addition, the ChimT vaccine alone stimulated in vitro murine macrophages to significantly kill three different internalized species of 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 Leishmania infantum 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.

B-Cell Epitopes-Based Chimeric Protein from SARS-CoV-2 N and S Proteins Is Recognized by Specific Antibodies in Serum and Urine Samples from Patients.

The impact of the COVID-19 pandemic caused by the SARS-CoV-2 virus underscored the crucial role of laboratorial tests as a strategy to control the disease, mainly to indicate the presence of specific antibodies in human samples from infected patients. Therefore, suitable recombinant antigens are relevant for the development of reliable tests, and so far, single recombinant proteins have been used. In this context, B-cell epitopes-based chimeric proteins can be an alternative to obtain tests with high accuracy through easier and cheaper production. The present study used bioinformatics tools to select specific B-cell epitopes from the spike (S) and the nucleocapsid (N) proteins from the SARS-CoV-2 virus, aiming to produce a novel recombinant chimeric antigen (N4S11-SC2). Eleven S and four N-derived B-cell epitopes were predicted and used to construct the N4S11-SC2 protein, which was analyzed in a recombinant format against serum and urine samples, by means of an in house-ELISA. Specific antibodies were detected in the serum and urine samples of COVID-19 patients, which were previously confirmed by qRT-PCR. Results showed that N4S11-SC2 presented 83.7% sensitivity and 100% specificity when using sera samples, and 91.1% sensitivity and 100% specificity using urine samples. Comparable findings were achieved with paired urine samples when compared to N and S recombinant proteins expressed in prokaryotic systems. However, better results were reached for N4S11-SC2 in comparison to the S recombinant protein when using paired serum samples. Anti-N4S11-SC2 antibodies were not clearly identified in Janssen Ad26.COV2.S COVID-19-vaccinated subjects, using serum or paired urine samples. In conclusion, this study presents a new chimeric recombinant antigen expressed in a prokaryotic system that could be considered as an alternative diagnostic marker for the SARS-CoV-2 infection, with the potential benefits to be used on serum or urine from infected patients.