Prophylactic properties of a Leishmania‐specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads’ draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti‐Leishmania IgG2a antibodies and a predominant IL‐12‐driven IFN‐γ production (mainly produced by CD4⁺ T cells) against parasite proteins, whereas unprotected controls showed anti‐Leishmania IgG1 antibodies and parasite‐mediated IL‐4 and IL‐10 responses. Vaccinated mice showed an anti‐LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD‐specific IFN‐γ, IL‐12 and GM‐CSF cytokines before and after infection. The protection was correlated with the Leishmania‐specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.     

AIM2 inflammasome is associated with disease severity in tegumentary leishmaniasis caused by Leishmania (V.) braziliensis

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis‐associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real‐time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2‐producing cells was higher in poor responders than in good responders. Although not quite significant, IL‐1β+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions.     

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice

Toxoplasmosis is a world‐wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL‐12‐inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1‐TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1‐TgCyP developed a high response with TgCyP‐specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1‐TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL‐2 and IFN‐γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1‐TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.     

Leishmania braziliensis amastigotes stimulate production of IL‐1β, IL‐6, IL‐10 and TGF‐β by peripheral blood mononuclear cells from nonendemic area healthy residents

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL‐1β, IL‐6, TGF‐β and IL‐23 production, whereas IL‐10 and TGF‐β are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon‐γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL‐10 production in PBMCs; however, only amastigotes induced IL‐1β, IL‐6 and TGF‐β. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.     

Th1 and Th2 immunological profile induced by cysteine proteinase in murine leishmaniasis

This study evaluated the immune response to three synthetic peptides (pI, VMVEQVICFD; pII, VGGGLCFE; pIII, PYFLGSIMNTCHYT) from the COOH-terminal region of Leishmania amazonensis cysteine proteinases, in BALB/c- and CBA-infected mice with this parasite. Only BALB/c mice, previously inoculated with pI, showed a distinct exacerbation of the lesion. Blastogenesis assays were performed with lymph node cells from the group of mice infected with L. amazonensis, but not inoculated with the peptides, and we detected lymphoproliferative responses in BALB/c and CBA mice with a 5.0x and 2.5x stimulation index, respectively. Cell phenotypes were evaluated and in both mouse strains CD8(+)cells proliferated more extensively than CD4(+)cells. INF-gamma and nitric oxide were detected only in supernatants obtained from CBA mouse lymph node cell cultures, whereas IL-4 was detected in supernatant cultures from both strains of mice. Our results indicate a preferential stimulation of CD8(+)T-cell subsets by the pI cysteine proteinase peptide and the induction of both Th1 and Th2 phenotypes during L. amazonensis infections in both BALB/c and CBA mice. We suggest that the pI peptide from the COOH-terminal region of the cysteine proteinase induces immune responses different from those elicited by the whole molecule.     

Codelivery of DNA vaccination encoding LeIF gene and IL‐12 increases protection against Leishmania major infection in BALB/c mice

Previous studies have shown that Leishmania elongation initiation factor (LeIF) antigen causes a partial immunity against leishmaniasis. The antigen develops type I immunity by overexpression of inflammatory cytokines such as interleukin‐12 (IL‐12), IFN‐γ and TNF‐α. Therefore, We evaluated immune responses induced by the LeIF gene against Leishmania major infection. Immunization with LeIF gene alone or with IL‐12 induced Th1 response and produced higher IFN‐γ and lower IL‐4 levels by splenocytes than control groups (P < 0·05) and also ratios of IFN‐γ/IL‐4 were 11·68 to 18·53 times more in immunized groups than control groups after challenge. In addition, analysis of humoral immune response revealed that immunized mice had more IgG2a levels than both control groups (P < 0·05). On the other hand, lesion size was less for immunized animals than control groups from 4th week after challenge (P < 0·05). The percentage reduction in lesion size was 29·30% for LeIF and 51·98% for LeIF + IL‐12 than PBS at 12th week post‐infection. Spleen parasite burden decreased in all immunized groups in comparison with control groups (P < 0·05). The results indicated that LeIF gene induced partial immunity against L. major infection in BALB/c mice. However, LeIF plus IL‐12 group showed more potent immunity with smaller lesions than other groups.     

Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post‐infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6‐8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti‐L. amazonensis IgG and increased production of IL‐10 and IL‐17. Because IL‐10 is a mediator of susceptibility to Leishmania infection, we blocked IL‐10 signalling in neutrophil‐depleted mice using anti‐IL‐10R. Interestingly, inhibition of IL‐10 signalling abrogated the increase in parasite loads observed in neutrophil‐depleted mice, suggesting that parasite proliferation is at least partially mediated by IL‐10. Additionally, we tested the effect of IL‐17 in inflammatory macrophages and observed that IL‐17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.     

A recombinant chimeric protein composed of human and mice‐specific CD4+ and CD8+ T‐cell epitopes protects against visceral leishmaniasis

In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4⁺ and CD8⁺ T‐cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin‐vaccinated mice presented significantly higher levels of antileishmanial IFN‐γ, IL‐12 and GM‐CSF before and after challenge, which were associated with the reduction of IL‐4 and IL‐10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real‐time PCR techniques were used. In addition, the protected animals presented higher levels of parasite‐specific nitrite, as well as the presence of anti‐Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL.     

Sambucus ebulus extract stimulates cellular responses in cutaneous leishmaniasis

This is the first study aiming to determine the therapeutic effects of the Sambucus ebulus aquatic extract as an antileishmanial herbal drug and evaluate the immune responses in Leishmania major major infected BALB/c mice. The antileishmanial activity of S ebulus aquatic extract was evaluated using MTT test as well as parasite rescue and transformation assay. Footpad swelling and parasite load of infected mice were measured by several techniques. The immune responses were evaluated by measuring the levels of IFN‐γ, IL‐4, nitric oxide and arginase. The results indicated that S. ebulus can significantly decrease L. major promastigotes and amastigotes viability, but it was not toxic to macrophages. The lesion size, parasite burden and the level of ARG decreased in the treated infected mice, while the IFN‐γ‐to‐IL‐4 ratio and the level of NO increased significantly. Altogether, the S. ebulus extract is an effective compound for killing Leishmania parasite without excessive toxicity to the host cells and can cure the CL by switching the host immune responses towards Th1 response. Thus, it may be a perfect therapeutic option for CL treatment.     

Leishmania tarentolae expressing CXCL‐10 as an efficient immunotherapy approach against Leishmania major‐infected BALB/c mice

Recent findings have demonstrated the suitability of interferon‐gamma‐induced protein 10 (IP‐10) or CXCL‐10 as an immunotherapy tool in treatment of leishmaniasis. This chemokine can overcome Leishmania (L.) infection through inducing nitric oxide (NO) production for parasite elimination. This study was undertaken to investigate the therapeutic effects of recombinant Leishmania tarentolae expressing CXCL‐10 and an expression vector encoding CXCL‐10 (pcDNA‐CXCL‐10‐EGFP) in a model of BALB/c mice susceptible to infection by Leishmania major. The outcome of intervention was examined at 3 weeks post‐treatment by evaluating the parameters of parasite burden (PB), arginase activity, NO and various cytokines such as IFN‐γ, IL‐4, IL‐6 and IL‐10. The results have shown that despite the efficacy of CXCL‐10 expression vector as gene therapy, the live therapy strategy using L. tarentolae expressing CXCL‐10 was more effective in terms of decreasing PB. Nitric oxide production increased, especially in the live therapy approaches. Arginase activity also decreased in all regimens, which demonstrates the potency of the treatment. The overall cytokine production shifted in favour of Th1 responses in the treated mice. Altogether, recombinant L. tarentolae expressing CXCL‐10 represents a promising therapeutic strategy to improve treatment of cutaneous leishmaniasis.     

Efficacy of Withania somnifera chemotypes NMITLI – 101R, 118R and Withaferin A against experimental visceral leishmaniasis

The immunoprophylactic and therapeutic potentials of root extracts of Withania somnifera chemotypes (NMITLI‐118, NMITLI‐101) and pure withanolide–withaferin A was investigated against Leishmania donovani infection in hamsters. The naive animals, fed orally with immunostimulatory doses of chemotypes 101R, 118R (10 and 3 mg/kg) and withaferin A (9 and 3 mg/kg) for five consecutive days and challenged with Leishmania parasites on day 6, were euthanized on days 30 and 45 p.c. for the assessment of parasite clearance, real‐time analysis of mRNAs of Th1/Th2 cytokines (IFN‐γ, IL‐12, TNF‐α, iNOS/IL‐4, IL‐10 and TGF‐β), NO production, reactive oxygen species (ROS) generation, lymphocyte transformation test and antibody responses. By day 45 p.c., there was a significant increase in the mRNA expression of iNOS, IFN‐γ, IL‐12 and TNF‐α but decrease in IL‐4, IL‐10 and TGF‐β, an enhanced Leishmania‐specific LTT response as well as ROS, NO and antileishmanial IgG2 levels in 101R‐treated hamsters followed by 118R‐ and withaferin A‐treated ones, respectively. When these chemotypes were given to L. donovani‐infected hamsters at different doses, there was moderate therapeutic efficacy of chemotype 101R (~50%) at 30 mg/kg × 5 followed by the other two. The results established that the 101R is the most potential chemotype and can be evaluated for combination therapy along with available antileishmanials.     

Evaluation of a novel lentiviral vaccine expressing KMP11‐HASPB fusion protein against Leishmania infantum in BALB/c mice

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania protein against which antibodies are produced in the sera of cutaneous and visceral Leishmaniasis (VL) patients. Kinetoplastid membrane protein 11 (KMP11) is another protein antigen of Leishmania which is reported as a promising candidate for vaccination of VL. It is a highly conserved surface protein present in all members of kinetoplastid family and is expressed in both promastigotes and amastigotes. In this study, the coding sequence of KMP11 and HASPB was cloned into a pCDH‐cGFP lentiviral vector as a fusion protein. The gene expression was confirmed using RT‐PCR and Western blot methods. After injection of the recombinant KMP11‐HASPB‐expressing lentiviruses to BALB/c mice, using ELISA technique, a significant increase in IFN‐γ and IL‐4 as well as IgG1 and IgG2a was observed compared to the control group. Furthermore, the number of parasites in the liver and spleen of vaccinated mice decreased significantly compared with the control group.     

Enhanced acquired antibodies to a chimeric Plasmodium falciparum antigen; UB05‐09 is associated with protective immunity against malaria

It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of UB05 and UB09 antigens (UB05‐09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross‐sectional study, recombinant UB05‐09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria‐endemic zone, using the enzyme‐linked immunosorbent assay (ELISA). Anti‐UB05‐09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05‐09‐specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B‐cell epitope prediction tools confirmed the presence of B‐cell epitopes in the study antigens. UB05‐09 chimera is a marker of protective immunity against malaria that needs to be studied further.     

HCV E1E2‐MF59 vaccine in chronic hepatitis C patients treated with PEG‐IFNα2a and Ribavirin: a randomized controlled trial

Hepatitis C virus (HCV) vaccines may be able to increase viral clearance in combination with antiviral therapy. We analysed viral dynamics and HCV‐specific immune response during retreatment for experienced patients in a phase Ib study with E1E2MF59 vaccine. Seventy‐eight genotype 1a/1b patients [relapsers (30), partial responders (16) and nonresponders (32) to interferon‐(IFN)/ribavirin‐(RBV)] were randomly assigned to vaccine (V:23), Peg‐IFNα2a‐180‐ug/qw and ribavirin 1000–1200‐mg/qd for 48 weeks (P/R:25), or their combination (P/R + V:30). Vaccine (100 μg/0.5 mL) was administered intramuscularly at week 0‐4‐8‐12‐24‐28‐32‐36. Neutralizing of binding (NOB) antibodies and lymphocyte proliferation assay (LPA) for E1E2‐specific‐CD4 + T cells were performed at week 0‐12‐16‐48. Viral kinetics were analysed up to week 16. The vaccine was safe, and a sustained virological response (SVR) was achieved in 4 P/R + V and 2 P/R patients. Higher SVR rates were observed in prior relapsers (P/R + V = 27.3%; P/R = 12.5%). Higher NOB titres and LPA indexes were found at week 12 and 16 in P/R + V as compared to P/R patients (P = 0.023 and 0.025, P = 0.019 and <0.001, respectively). Among the 22 patients with the strongest direct antiviral effects of IFN (ε ≥ 0.800), those treated with P/R + V (10) reached lower HCV‐RNA levels (P = 0.026) at week 16. HCV E1E2MF59 vaccine in combination with Peg‐IFNα2a + RBV was safe and elicited E1E2 neutralizing antibodies and specific CD4 + T cell proliferation. Upon early response to IFN, vaccinations were associated with an enhanced second phase viral load decline. These results prompt phase II trials in combination with new antiviral therapies.     

Tick saliva increases production of three chemokines including monocyte chemoattractant protein‐1, a histamine‐releasing cytokine

The effect of Ixodes ricinus tick saliva on the production of various cytokines and chemokines by mouse splenocytes was tested by a cytokine array. We demonstrated a strong upregulation of three chemokines, monocyte chemoattractant protein‐1 (MCP‐1), thymus‐derived chemotactic agent 3 (TCA‐3) and macrophage inflammatory protein 2 (MIP‐2). MCP‐1 could be induced by tick saliva itself. While TCA‐3 and MIP‐2 are engaged in Th2 polarization of the host immune response associated with tick feeding, MCP‐1 may act as a histamine release factor, increasing blood flow into the feeding lesion thus facilitating tick engorgement in the late, rapid feeding phase.