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Pharmacophores such as hydroxyethylamine (HEA) and phthalimide (PHT) have been identified as potential synthons for the development of compounds against various parasitic infections. In order to further advance our progress, we conducted an experiment utilising a collection of PHT and HEA derivatives through phenotypic screening against a diverse set of protist parasites. This approach led to the identification of a number of compounds that exhibited significant effects on the survival of Entamoeba histolytica, Trypanosoma brucei, and multiple life-cycle stages of Leishmania spp. The Leishmania hits were pursued due to the pressing necessity to expand our repertoire of reliable, cost-effective, and efficient medications for the treatment of leishmaniases. Antileishmanials must possess the essential capability to efficiently penetrate the host cells and their compartments in the disease context, to effectively eliminate the intracellular parasite. Hence, we performed a study to assess the effectiveness of eradicating L. infantum intracellular amastigotes in a model of macrophage infection. Among eleven L. infantum growth inhibitors with low-micromolar potency, PHT-39, which carries a trifluoromethyl substitution, demonstrated the highest efficacy in the intramacrophage assay, with an EC50 of 1.2 +/- 3.2 μM. Cytotoxicity testing of PHT-39 in HepG2 cells indicated a promising selectivity of over 90-fold. A chemogenomic profiling approach was conducted using an orthology-based method to elucidate the mode of action of PHT-39. This genome-wide RNA interference library of T. brucei identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry. Notwithstanding the favourable properties and demonstrated efficacy in the Plasmodium berghei infection model, PHT-39 was unable to eradicate L. major infection in a murine infection model of cutaneous leishmaniasis. Currently, PHT-39 is undergoing derivatization to optimize its pharmacological characteristics. Author summary: Two novel compound series, based on hydroxyethylamine (HEA) and phthalimide (PHT) pharmacophores, previously led to the discovery of antiplasmodial lead compounds, both with therapeutic efficacy in a murine Plasmodium infection model. Here we employed these compound libraries, supplemented with newly designed HEA derivatives, for phenotypic screening against a wide range of protist parasites associated with unmet medical needs. We report potent hit compounds for Entamoeba histolytica, Trypanosoma brucei and Leishmania spp., with limited overlap in their structure–activity relation. PHT-39 efficiently penetrated host macrophages to effectively eliminate Leishmania intracellular amastigotes, while displaying high selectivity. Leveraging an orthology-based genome-wide RNAi library screen, we propose an uptake route for the PHT-39 that relies on a transmembrane flippase, known as crucial uptake transporter for the two frontline antileishmanials miltefosine and amphotericin. Despite favourable drug properties of PHT-39, experimental PHT-39 treatment failed to eliminate the Leishmania infection in a cutaneous leishmaniasis animal model. Considering PHT-39 high selectivity and the ability of the drug to effectively reach the intracellular stage of the parasite, we suggest PHT-39 for further optimization. [ABSTRACT FROM AUTHOR]

Seven types of Coronaviruses (CoVs) have been identified that can cause infection in humans, including HCoV‐229E, HCoV‐NL63, HCoV‐OC43, HCoV‐HKU1, SARS‐CoV, HCoV‐MERS, and SARS‐CoV‐2. In this study, we investigated the genetic structure, the homology of the structural protein sequences, as well as the investigation of the active site of structural proteins. The active site of structural proteins was determined based on the previous studies, and the homology of their amino acid sequences and structure was compared. Multiple sequence alignment of Spike protein of HCoVs showed that the receptor‐binding domain of SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV was located at a similar site to the S1 subunit. The binding motif of PDZ (postsynaptic density‐95/disks large/zona occludens‐1) of the envelope protein, was conserved in SARS‐CoV and SARS‐CoV‐2 according to multiple sequence alignment but showed different changes in the other HCoVs. Overall, spike protein showed the most variation in its active sites, but the other structural proteins were highly conserved. In this study, for the first time, the active site of all structural proteins of HCoVs as a drug target was investigated. The binding site of these proteins can be suitable targets for drugs or vaccines among HCoVs. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis is the most severe form of leishmaniasis caused by Leishmania (L.) donovani complex. Drug-resistant strains have been developed as a consequence of the current chemotherapeutic interventions, which has increased the need for advanced preventive and therapeutic strategies. A2-CPA-CPB-CTE-recombinant strain of L. tarentolae, which is non-pathogenic to humans, was shown protective in live vaccine as well as its DNA vaccine counter-part in both murine and canine models. Methods: We evaluated the effectiveness of these DNA and live vaccination harboring A2-CPA-CPB-CTEin protecting hamsters against L. infantum infection using prime-boost regimens, namely DNA/DNA and Live/Live (n=9 hamsters per group). Cationic solid lipid nanoparticles (cSLN) were utilized as an adjuvant for DNA priming and electroporation for boosting DNA. At different time points post-challenge, parasite burden and body weight as well as humoral immune responses were measured. Results: Both immunization strategies partially protect hamsters against L. infantum challenge. This protective immunity is associated with remarkable decrease in parasite load in liver and spleen of vaccinated hamsters eight weeks after challenge compared to control group. Conclusion: Both test groups (DNA/DNA and Live/Live) elicited high levels of IgG2 and total IgG as humoral immune responses and lower level of parasite propagation in both liver and spleen. [ABSTRACT FROM AUTHOR]

Leishmaniasis, as a major health problem in tropical and sub-tropical areas in the world, needs novel, safe, nontoxic and plausible therapeutic solutions for its control. As a part of innate immune system, natural antimicrobial peptides have a potential to be used as new generation of antibiotics especially after persistent resistance of conventional antimicrobial agents. Brevinin 2R, a member of Defensin families of host defense peptides, showed promising effects against bacterial and fungal infections as well as cancerous cell lines. In the current research, the anti-leishmanial effect of Brevinin 2R and its lauric acid conjugate was investigated against Leishmania major (L. major) parasite. The data revealed that, conjugation of fatty acid to Brevinin 2R, strengthen its effect on L. major promastigotes in respect to toxicity and hemolytic effect. These peptides showed anitleishmanial activity through cell membrane disruption and changes in the electrical and mitochondrial membrane potential. No signs of apoptosis induction or caspase activation were detected. Despite its hemolytic and cytotoxic effect in in vitro conditions, lauric acid- Brevinin 2R (L- Brevinin 2R) did not show site specific adverse reactions in animal model. Treatment course with L- Brevinin 2R in the L. major infected mice exhibited decreased parasite load in the lymph nodes adjacent to the infected site despite cytokine production profile and footpad swelling data. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a fatal disease caused by the intracellular protozoan parasite Leishmania infantum. Dogs are the primary reservoirs of this parasite, and vaccination of dogs could be an effective method to reduce its transfer to humans. In order to develop a vaccine against VL (apart from the choice of immunogenic candidate antigens), it is necessary to use an appropriate delivery system to promote a proper antigen-specific immune response. In this study, we compared two vaccine delivery systems, namely electroporation and cationic solid–lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine containing the Leishmania donovani A2 antigen, and L. infantum cysteine proteinases of type I (CPA) and II (CPB) without its unusual C-terminal extension. The protective potencies of these two vaccine delivery systems were evaluated against L. infantum challenge in outbred dogs. Our results show that the administration of pcDNA -A2-CPA-CPB −CTE GFP vaccine as a prime-boost by either electroporation or cSLN formulation protects the dogs against L. infantum infection. Partial protection in vaccinated dogs is associated with significantly ( p < 0.05) higher levels of IgG2, IFN-γ, and TNF-α and with low levels of IgG1 and IL-10 as compared to the control group. Protection was also correlated with a low parasite burden and a strong delayed-type hypersensitivity (DTH) response. This study demonstrates that both electroporation and cSLN formulation can be used as efficient vaccine delivery systems against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. [ABSTRACT FROM AUTHOR]

Background: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. Author Summary: More than 98 countries are reported as endemic for leishmaniasis, a vector-borne disease transmitted by sand flies. Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L. tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models. Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L. tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice. This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections. [ABSTRACT FROM AUTHOR]

Background: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings: Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion: Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis. Author Summary: Leishmaniasis is currently a serious health as well as economic problem in underdeveloped and developing countries in Africa, Asia, the Near and Middle East, Central and South America and the Mediterranean region. Cutaneous leishmaniasis is highly endemic in Iran, remarkably in Isfahan, Fars, Khorasan, Khozestan and Kerman provinces. Since effective prevention is not available and current curative therapy is expensive, often poorly tolerated and not always effective, alternative therapies including vaccination against leishmaniasis are of priority to overcome the problem. Although Th1 dominant response is so far considered as a pre-requisite for the immune system to overcome the infection, CD8+ T cell response could also be considered as a potent arm of immune system fighting against intracellular Leishmania. Polytope vaccine strategy may open up a new way in vaccine design against leishmaniasis, since they act as a potent tool to stimulate multi-CD8 T cell responses. Clearly there is a substantial need to evaluate the promising epitopes from different proteins of Leishmania parasite species. Some new immunoinformatic tools are now available to speed up this process, and we have shown here that in silico prediction can effectively evaluate HLA class I-restricted epitopes out of Leishmania proteins. [ABSTRACT FROM AUTHOR]

Background: As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings: Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2- individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2- recovered individuals. Based on cutoff scores calculated from the response of HLA-A2- recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2- recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion: Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis. [ABSTRACT FROM AUTHOR]

Abstract: Earlier generations of Leishmania vaccines have reached the third-phase of clinical trials, however none of them have shown adequate efficacy due to lack of an appropriate adjuvant. In this study, cationic solid lipid nanoparticles (cSLNs) were used to formulate three pDNAs encoding L. major cysteine proteinase type I (cpa), II (cpb) and III (cpc). BALB/c mice were immunized twice with a 3-week interval, with SLN-pcDNA-cpa/b/c, pcDNA-cpa/b/c, SLN, SLN-pcDNA and PBS. Footpad assessments, parasite burden, cytokine and antibody responses were evaluated. Mice vaccinated with SLN-pcDNA-cpa/b/c significantly (p <0.05) showed higher protection levels with specific Th1 immune response development compared to other groups. This is the first report demonstrating cSLNs as a nanoscale vehicle boosting immune response quality and quantity; in a designable trend. The nanomedical feature of this novel formulation can be applied for wide-spread use in genetic vaccination against leishmaniasis, which is currently managed only through relatively ineffectual therapeutic regimens. [Copyright &y& Elsevier]

Background: We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings: Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance: This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines. Author Summary: Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings: Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance: This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines. [ABSTRACT FROM AUTHOR]

Background & objectives: Cervical cancer is the second most frequent cancer among females worldwide, especially human papilloma viruses (HPV) types 16 and 18. In viral systems the identification of serological markers would facilitate the diagnosis of HPV infections and virus-related disease. The aim of the present investigation was to determine and search for serologic markers in cervical cancer patients associated with HPV. Methods: A total of 58 Iranian women with invasive cervical carcinoma including adenocarcinoma and squamous cell carcinoma (SCC) were included. Serum antibody response to HPV infections in patients was detected by Western blot and ELISA techniques based on recombinant HPVI 6E7 and the N-terminal and C-terminal fragments of gp96 (NT-gp96 and CT-gp96) proteins. These recombinant proteins were expressed in Escherichia coli as a His-tag protein and purified using affinity chromatography. Results: The ELISA results indicated that patients with high antibody response to HPVI6E7 had significant seroreactivity to CT-gp96 fragment. In Western blot analysis, a strong association between anti-E7, anti-NT-gp96 and anti-CT-gp96 reactivity and cervical cancer was obtained using purified recombinant proteins. In adenocarcinoma cases, no significant difference was observed in seroreactivities between normal and patients. Interpretation & conclusions: The evaluation of cervical cancer patients' seroreactivities against three recombinant proteins (rE7, rNT-gp96 and rCT-gp96) showed significantly higher levels of these markers in SCC only, but not in adenocarcinoma and control groups. Also, the usage of both techniques (ELISA and Western blotting) can provide more reliable tools for diagnosis of cervical cancer. [ABSTRACT FROM AUTHOR]

Abstract: Visceral leishmaniasis is the most acute form of leishmaniasis and vaccination is the best approach to control it. One of the major groups of virulence factors in Leishmania belongs to cysteine proteinase family. In this study, for the first time, the protective potential of Leishmania infantum cysteine proteinase type III (CPC) by using a prime-boost strategy is evaluated in BALB/c mice. The experiment was carried out in three groups of mice. Vaccinated group was primed with pcDNA-cpc and boosted with rCPC-DHFR in combination with CpG motif and Montanide 720 as adjuvant. Control groups received pcDNA and rDHFR or PBS. The ratio of IgG2a/IgG1, nitric oxide concentration and IFN-γ induction in vaccinated group is significantly higher than controls. Furthermore, the parasite load of vaccinated group is significantly lower than controls. In addition, sera reactivity of visceral leishmaniasis individuals was examined and showed considerable reactivities toward rCPC in comparison with cutaneous leishmaniasis. The achieved result is highly encouraging the use of cysteine proteinases types I, II and III as vaccine candidate against visceral leishmaniasis. [Copyright &y& Elsevier]

Abstract: Human papillomavirus, particularly type 16 (HPV16) is present in more than 99% of cervical cancers. E7 is the major oncogenic protein produced in cervical cancer-associated HPV16. An efficient vaccine against viral infection requires induction of strong humoral and cellular responses against viral proteins. Heat shock proteins (HSPs) like Gp96 have been described as potent tumor vaccines in animal models and are currently studied in human clinical trials. In this study, we investigated the utility of HPV16 E7 along with Gp96 as an adjuvant in C57BL/6 mice model. We compared the level of humoral and cellular immune responses by E7+Gp96 co-injection as DNA/DNA and prime-boost (DNA/protein) immunization strategies. In prime-boost immunization strategies, we first immunized C57BL/6 mice with the complete open-reading frame of E7 and Gp96 (pcDNA-E7 and pcDNA-Gp96) and then boosted with rE7, rNT-gp96 (N-terminal extension of Gp96) and rCT-gp96 (C-terminal extension of Gp96) mixed with Montanide 720 in different formulations. The humoral immune responses against rE7 and the different truncated forms of rGp96 suggested a mixed Th1/Th2 response with high intensity toward Th2. Assessment of lymphoproliferative and cytokine responses against rE7 and the different fragments of Gp96, showed that DNA vaccination including E7 and Gp96 induced Th1 response. We concluded that co-delivery of naked DNA E7+Gp96 plasmid was immunologically more effective than E7 alone. Our study demonstrated that co-delivery of E7+Gp96 as DNA/DNA and E7+CT-gp96 as DNA/protein could be an effective approach to induce E7-specific immune responses as a potential vaccine candidate for cervical cancer. [Copyright &y& Elsevier]

Abstract: The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100μg of pcDNA-CTE and 3 weeks later boosted with combination of 30μg rCTE, 50μg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1–8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection. [Copyright &y& Elsevier]

Abstract: We evaluated the use of a novel gene porter (Den123-a nontoxic self-assembled dendritic spheroidal nanoparticle made of biodegradable monomers), aiming to enhance and improve the desired immune response in protection from allergy. Footpad DNA immunization in Balb/c mice was done three times using the Bet v 1a gene with or without Den123 with 2-week intervals followed by sensitization with rBetv1 (5μg) in alum twice in a weekly interval. Different doses of pCMV-Betv1 were used (10μg and 100μg). The protective role of different formulations was evaluated by measuring the IgG1, IgG2a and IgE antibody production, cytokine release of isolated splenocytes and β-hexosaminidase release from the RBL cells. Higher and increasing ratios of IgG2a/IgG1 were seen in mice which received plasmids in combination with Den123. Den123 and DNA vaccine synergistically enhanced the Interferon gamma released from splenocytes. In the presence of Den123, IgE inhibition was independent of the dose and type of the injected DNA. All DNA-pre-immunized mice demonstrated low basophil degranulation. It is therefore concluded that administration of the DNA entrapped in Den123 nanoparticles results in sustained release of plasmids, Th1/Th2 balanced immune response with promising IgE inhibition. Also higher amounts of DNA contributed to stronger Th1 response. [Copyright &y& Elsevier]

Abstract: Vaccination with a cocktail of DNA encoding cysteine proteinases has been previously shown to confer protection against experimental cutaneous leishmaniasis (CL). In the present study we test the efficacy of immunization against Leishmania infantum in a murine model of infection, using a prime-boost strategy. BALB/c mice were immunized twice, in a 3 weeks interval, with cocktail of plasmids DNA encoding type I (cpb) and II (cpa) cysteine proteinases. DNA immunization was then followed by a boost with rCPA/rCPB in addition to CpG ODN and Montanide720 as adjuvant. Analysis of the immune response showed that vaccination mainly elicited antigen-specific IgG2a antibodies, suggesting the induction of a Th1 immune response. This was further confirmed by the analysis of the splenic cytokine production: at all time points the ratio of IFN-γ/IL-5 induced upon restimulation with rCPA and rCPB was always significantly higher in vaccinated group compared to both control groups. [Copyright &y& Elsevier]

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4+ Th1 and CD8+-T cell immunity is required to control the infection. Thus far multivalent subunit vaccines have met this requirement more promisingly. However several full protein sequences cannot be easily arranged in one construct. Instead, new emerging immune-informatics based epitope formulations surpass this restriction. Herein, we aimed to examine the protective potential of a dendritic cell based vaccine presenting epitopes to CD8+ and CD4+-T cells in combination with DNA vaccine encoding the same epitopes against murine cutaneous leishmaniasis. Immature DCs were loaded with epitopes (selected from parasite proteome) in vitro with or without CpG oligonucleotides and were used to immunize BALB/c mice. Peptide coding DNA was used to boost the system and immunological responses were evaluated after Leishmania (L.) major infectious challenge. The pre-challenge response to included epitopes was Th1 polarized which potentially lowered the infection at early time points post-challenge but not at later weeks. Collectively, DC prime-DNA boost was found to be a promising approach for Th1 polarization however the constituent epitopes undoubtedly make a significant contribution in the protection outcome of the vaccine. [Display omitted] • Leishmaniasis is a complex vector-borne disease without effective vaccine. • Multivalent subunit vaccines formulated as recombinant proteins or DNA constructs have generated more promising results. • Multi-epitope peptide-based vaccines have now come into focus to make multivalent vaccines more achievable. • DC prime-DNA boost strategy could be a promising formulation for robust Th1 polarization against immunogenic epitopes. • Constituent epitopes of a multi-epitope peptide-based vaccine play a significant role in vaccine efficacy. [ABSTRACT FROM AUTHOR]

Typically, antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. Discovering novel therapeutic agents is considered as an undeniable demand due to increasing microbial species with antibiotic resistance. In this direction, the unique ability of AMPs to modulate immune responses highlighted them as novel drug candidates in the field of microbiology. Patients affected by leishmaniasis; a neglected tropical disease, confront serious problems for their treatment including resistance to common drugs as well as toxicity and high cost of therapy. So, there is a need for development of new drug candidates to control the diseases. Jellein, a peptide derived from royal jelly of honeybee has been shown to have promising effect against several bacterial and fungal species. In current study, anti-leishmanial effect of Jellein and its lauric acid conjugated form was investigated against two forms of Leishmania major (L. major) parasite. Moreover, cytotoxic effect of these peptides was studied in THP1 cell line and human Red Blood Cells (RBCs). Furthermore, the mechanism of action of peptides on L. major promastigotes was assessed through different methods. The results demonstrated that, conjugation of lauric acid to Jellein not only had no effect on the elevation of antimicrobial activity but also halted it completely. Moreover, Jellein caused a limitation in the number of L. major promastigotes by pore formation as well as changing the membrane potential rather than induction of apoptosis or activation of caspases. Image 1 • Antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. • Patients affected by leishmaniasis need new therapeutic agents due to toxic effect of current drugs. • Jellein but not its lauric acid conjugated form limited promastigote proliferation. • Jellein and its lauric acid conjugated form did not affect amastigote form of the parasite. • Jellein could affect L. major promastigotes through membrane disruption rather than apoptosis pathway. [ABSTRACT FROM AUTHOR]

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat. Author summary: In the blood of their human and animal hosts, single-celled parasites of the species Trypanosoma brucei need to hide from the body's defense system to survive. These parasites have a special armor made of proteins (known as VSGs) that changes regularly, helping them stay undetected by the immune system. This armor also covers other important proteins (ISGs) that the parasites need in order to interact with their host, but must hide to avoid being attacked. However, this protective layer makes it hard for the parasites to reach outside their shield. In our study, we looked at these hidden proteins in detail and discovered that they can change shape. Some can stretch out beyond their protective layer when necessary, while others stay concealed inside. This ability to change shape lets these otherwise hidden proteins interact with their surroundings without getting caught by the immune system. Our findings provide further evidence for a multi-layered defense strategy in these parasites. [ABSTRACT FROM AUTHOR]

Human Neutrophil Peptide-1 (HNP1) produced by neutrophils, is a well-known antimicrobial peptide which plays a role both in innate as well as in adaptive immunity and is under intensive investigation as a potential therapeutic agent. Previous in vitro experiments have indicated the leishmaniacidal effect of recombinant HNP1 on Leishmania major (L. major) promastigotes and amastigotes. In the current study, we further extended the idea to explore the remedial effect of HNP1 in the two modalities of peptide therapy (folded HNP1) and gene therapy in L. major infected BALB/c mice. To this end, mice in five different groups received synthetic folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), Amphotericin B (G4) and PBS (G5), which was started three weeks after infection for three consecutive weeks. Footpad swelling was monitored weekly and a day after the therapy ended, IFN-γ, IL-4, IL-10, IL-6 and nitric oxide produced by splenocytes were analyzed together with the parasite load in draining lymph nodes. Arginase activity and dermal histopathological changes were also analyzed in the infected footpads. We demonstrated that both therapeutic approaches effectively induced Th1 polarization and restricted parasite burden. It can control disease progression in contrast to non-treated groups. However, pcDNA-HNP1-EGFP is more promising in respect to parasite control than folded HNP1, but less effective than AmB treatment. We concluded with the call for a future approach, that is, a DNA-based expression of HNP1 combined with AmB as it can improve the leishmaniacidal efficacy. [ABSTRACT FROM AUTHOR]

The characterization of protein--protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches. [ABSTRACT FROM AUTHOR]

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG–treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells. Author Summary: In Iran, cutaneous leishmaniasis (CL) is a widespread and highly endemic disease in young individuals. To date, treatment strategy is based on chemotherapy accompanied with high incidence of toxicity and drug resistance. Distinctive mode of action of defensins (members of antimicrobial peptides) with low susceptibility to resistance and low toxicity to mammalian cells makes them suitable candidates for anti-leishmanial agents. The most active human defensin is human neutrophil peptide-1 (HNP-1) produced by neutrophils; the first effector cells during Leishmania infection. In this work, we used recombinant HNP-1 (rHNP-1) against both the promastigote and amastigote forms of Leishmania (L.) major. Furthermore, immunomodulatory effect of rHNP-1 on Leishmania-infected neutrophils was investigated. Our result showed that rHNP-1 has anti-parasitic effect against L. major promastigotes and amastigotes and also reduces infectivity rate of Leishmania-infected neutrophils. Moreover, assessment of cytokine production from Leishmania-infected neutrophils reveals an increase in TNF-α and a decrease in TGF-β production after rHNP-1 treatment; a cytokine pattern anticipated to facilitate control of parasites. The immunomodulatory effect of rHNP-1 on cytokine production from parasite-infected neutrophils besides its direct effect on free parasites is considered as promising step towards developing new anti-leishmanial agents. [ABSTRACT FROM AUTHOR]

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG–treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. Author Summary: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and has emerged as an opportunistic infection in HIV-1 infected patients in many parts of the world. Drug-resistant forms have developed so emergence and increased the need for advanced preventive strategies. Using live avirulent organisms as a vaccine has been proven to be more effective than other regimens. The lizard protozoan parasite Leishmania tarentolae is considered as nonpathogenic to humans. In our previous work, a recombinant L. tarentolae strain expressing the amastigote-specific L. donovani A2 antigen as a vaccine candidate elicited protection against L. infantum challenge in mice. Furthermore, combinations of CPA/CPB cysteine proteinases were more protective against visceral and cutaneous Leishmania infections than the individual forms. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against visceral leishmaniasis in BALB/c mice consisting of the A2-CPA-CPB-CTE tri-fusion genes formulated with cationic solid lipid nanoparticles and a recombinant L. tarentolae expressing the tri-fusion. Assessments of cytokine production, humoral responses, parasite burden and histopathological studies support that the recombinant L. tarentolae A2-CPA-CPB-CTE candidate vaccine elicits a protective response against visceral leishmaniasis in mice and represents an important step forward in the development of new vaccine combinations against Leishmania infections. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual Cterminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. [ABSTRACT FROM AUTHOR]

Abstract: Although DNA vaccines represent an attractive approach for generating antigen-specific immunity, improvement of their potency is highly demanded. In the present study, three strategies including linkage to immunostimulatory molecules (N-terminal of gp96), co-administration of chemokines (IP-10 or RANTES) and PEI600-Tat as non-viral gene delivery system have been applied to enhance DNA vaccine efficacy against HPV infections. We found that C57BL/6 immunization with E7-NT-gp96 fusion gene led to increased level of IFN-γ compared to E7 alone. The fused genes showed considerable protective potency in tumor mice model. In addition, E7-NT-gp96 delivered with PEI600-Tat was more protective against E7-expressing tumors comparing with E7-NT-gp96 alone. Our results showed that co-administration of IP-10 with E7-NT-gp96 delivered by PEI600-Tat elicits significant IFN-γ production and consequently a strong preventive response against TC-1 tumor cells in contrast to increased tumor growth by RANTES co-delivery. Also in therapeutic experiment, our data showed that co-immunization of IP-10 at the same inoculation site of TC-1 along with E7-NT-gp96 delivery by PEI600-Tat is able to significantly suppress TC-1 tumor growth. The successful treatment by this immunization protocol was associated with the elevated levels of IFN-γ and IL-2 production in the lymph nodes. These data indicated that fusion of NT-gp96 to E7 in combination with IP-10 co-administration and PEI600-Tat delivery system can synergistically enhance the potency of HPV DNA vaccines. Therefore, this approach suggests a combinational therapeutic strategy against cervical and other HPV-related cancers. [Copyright &y& Elsevier]

Several species of protozoan parasites of the genus Leishmania are pathogenic to mammals and cause a wide spectrum of pathologies in human. However, the genus includes some species which infect reptiles. Leishmania tarentolae is a lizard pathogen absolutely nonpathogenic to mammals. Recent studies have shown that among some major virulence factors, A2 is absent in this species. First identified as an amastigote-specific gene in Leishmania donovani, A2 has been proved to play a major role in parasite virulence and visceralization capability. In this study, we have transfected A2 episomally into L. tarentolae and evaluated its effect on infectivity and survival of the parasites, in vitro and in vivo. During infection of in vitro-cultured intraperitoneal macrophages of BALB/c mice, A2-expressing L. tarentolae parasites demonstrated significantly higher level of infectivity in days 3 and 4 post-infection in comparison with the wild-type strain as control. Furthermore, in vivo infection showed that A2 has significantly increased the ability of L. tarentolae to survive in the liver of BALB/c mice. Altogether, our results show that A2 is functional in L. tarentolae, although through an unknown mechanism, and loss of A2 has been one of the factors partly contributing to the loss of virulence of L. tarentolae. [ABSTRACT FROM AUTHOR]

Abstract: An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency. [Copyright &y& Elsevier]

Abstract: One of the main issues in vaccine development is implementation of new adjuvants to improve the antigen presentation and eliciting the protective immune response. Heat shock protein (HSP) molecules are known as natural adjuvants. They can stimulate the innate and adaptive immune response against infectious diseases and cancer. Lipophosphoglycan 3 (LPG3), the Leishmania homologous with GRP94 (glucose regulated protein 94), a member of HSP90 family, is involved in assembly of LPG as the most abundant macromolecule on the surface of Leishmania promastigotes. In the present study as a primary step, we tested LPG3 as a vaccine candidate in two regimens, DNA/DNA and prime-boost (DNA/Protein), against Leishmania major infection in BALB/c mice model. Our results showed that LPG3 and its fragment (rNT-LPG3) are highly immunogenic in BALB/c mice and can stimulate the production of both IgG1 and IgG2a. In prime-boost immunization strategy, the level of antibody response was higher compared with DNA/DNA immunization. The levels of IFN-γ in the supernatant of splenocytes from mice immunized with DNA/DNA and prime-boost regimens were significantly higher when compared to control groups. In fact, immunization with prime-boost vaccination has higher ratio of IFN-γ/IL-5, suggesting a shift towards a Th1 response. In addition, sera reactivity against LPG3 in visceral leishmaniasis (VL) patients was significantly higher in comparison with cutaneous leishmaniasis (CL) patients. Therefore, we recommend further investigations on the usage of LPG3 co-delivery with candidate antigens for vaccine development against leishmaniasis. [Copyright &y& Elsevier]

Abstract: Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccine against this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, but none of them conferred complete long-term protection. The use of live attenuated vaccines has recently emerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovani A2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated its protective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperitoneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ production prior and after challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to a potent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, characterized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection. All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against visceral leishmaniasis. [Copyright &y& Elsevier]

Abstract: Signal Peptidase (SPase) is an essential enzyme in both prokaryotes and eukaryotes; which removes signal sequence from secretary proteins. Previously, type I SPase from Leishmania major (Lmjsp) has been isolated and characterized. The sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant SPase (rSP) and synthetic SPase (Sy-Sp) antigens. Therefore, the Leishmania SPase, albeit localized intracellularly, is a significant target of the Leishmania specific immune response and highlights its use as a candidate vaccine against cutaneous leishmaniasis (CL). In this study as a first report, the potential protection of Lmjsp was evaluated in three different vaccination strategies (DNA/DNA, Protein/Protein and DNA/Protein), against L. major infection. We demonstrated that vaccination with SPase through all three mentioned strategies induced a parasite specific Th1 response and conferred partial protection against parasite challenge. However, our results indicated that DNA/DNA strategy developed more effective protective responses than the other two approaches and induced 81% reduction in L. major parasite load. [Copyright &y& Elsevier]

The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae ( L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-γ and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy. [ABSTRACT FROM AUTHOR]

Abstract: DNA vaccines have emerged as a promising approach for generating antigen-specific immunotherapy. However, due to their low immunogenicity, there is a need to enhance DNA-based vaccine potency. Two main strategies to increase DNA-based vaccine potency are the employment of immuno-adjuvants such as heat shock proteins (HSPs) and a method of improving the delivery of naked plasmid DNA by electroporation. In the current study, we evaluated the effects of linkage of human papillomavirus (HPV) type 16 E7 as a model antigen to N-terminal and C-terminal of glycoprotein 96 (NT-/CT-gp96) on the potency of E7-specific immunity generated by DNA vaccines. We found that subcutaneous DNA injection with E7-CT (gp96) followed by electroporation generates the significant E7-specific IFN-γ immune responses as well as the best protective effects in vaccinated mice as compared to E7 or E7-NT (gp96) DNA vaccines. Therefore, our data indicate that subcutaneous administration of E7 DNA linked to CT (gp96) fragment followed by electroporation can significantly enhance the potency of DNA vaccines. Indeed, the structural domains of immuno-chaperones show the potential of generating effective immune responses against different clinical disorders such as cancer. Altogether, our results show that comparable regions of gp96 (N-/C-terminal fragments of gp96) may have qualitatively different immunological effects in vaccine design. [Copyright &y& Elsevier]

Abstract: Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host–parasite interactions at the cellular level. [Copyright &y& Elsevier]

Abstract: Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival. The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite. [ABSTRACT FROM AUTHOR]

Corrigendum to "Antileishmanial effect of rapamycin as an alternative approach to control Leishmania tropica infection" [Vet. Parasitol. [Extracted from the article]

Anthroponotic cutaneous leishmaniasis (CL) caused by Leishmania tropica (L. tropica) represents a public health challenge in several resource poor settings. We herein employed a systems analysis approach to study molecular signatures of CL caused by L. tropica in the skin lesions of ulcerative CL (UCL) and non-ulcerative CL (NUCL) patients. Results from RNA-seq analysis determined shared and unique functional transcriptional pathways in the lesions of the UCL and NUCL patients. Several transcriptional pathways involved in inflammatory response were positively enriched in the CL lesions. A multiplexed inflammatory protein analysis showed differential profiles of inflammatory cytokines and chemokines in the UCL and NUCL lesions. Transcriptional pathways for Fcγ receptor dependent phagocytosis were among shared enriched pathways. Using L. tropica specific antibody (Ab)-mediated phagocytosis assays, we could substantiate Ab-dependent cellular phagocytosis (ADCP) and Ab-dependent neutrophil phagocytosis (ADNP) activities in the lesions of the UCL and NUCL patients, which correlated with L. tropica specific IgG Abs. Interestingly, a negative correlation was observed between parasite load and L. tropica specific IgG/ADCP/ADNP in the skin lesions of CL patients. These results enhance our understanding of human skin response to CL caused by L. tropica. [ABSTRACT FROM AUTHOR]

• Rapamycin can inhibit the propagation of Leishmania tropica. • SEM and TEM showed negative effect of rapamycin on the shape of the parasites. • Clinically relevant dose of rapamycin is a promising treatment against Leishmania tropica. • Combination therapies of rapamycin with AmB or glucantime were not successful. Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 μg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica. [ABSTRACT FROM AUTHOR]

An abstract of the paper on the use of non-pathogenic Leishmania as a candidate vaccine for lesihmaniasis presented during the Institut Pasteur International Network Annual Scientific Meeting at Hong Kong, China, from November 22-23, 2010 is presented.