CD8+ T Cells in Leishmania Infections: Friends or Foes?

Host protection against several intracellular pathogens requires the induction of CD8+ T cell responses. CD8+ T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8+ T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8+ T cells during various types of Leishmania infections, following vaccination, and as potential immunotherapeutic targets

Improvement of different vaccine delivery systems for cancer therapy

Cancer vaccines are the promising tools in the hands of the clinical oncologist. Many tumor-associated antigens are excellent targets for immune therapy and vaccine design. Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and/or delivery strategies to achieve positive clinical results. Various vaccine delivery systems such as different routes of immunization and physical/chemical delivery methods have been used in cancer therapy with the goal to induce immunity against tumor-associated antigens. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target antigen-presenting cells (APCs) have demonstrated to be effective in animal models. New developments in vaccine delivery systems will improve the efficiency of clinical trials in the near future. Among them, nanoparticles (NPs) such as dendrimers, polymeric NPs, metallic NPs, magnetic NPs and quantum dots have emerged as effective vaccine adjuvants for infectious diseases and cancer therapy. Furthermore, cell-penetrating peptides (CPP) have been known as attractive carrier having applications in drug delivery, gene transfer and DNA vaccination. This review will focus on the utilization of different vaccine delivery systems for prevention or treatment of cancer. We will discuss their clinical applications and the future prospects for cancer vaccine development

Biomarkers of Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is an immune-mediated skin pathology caused mainly by Leishmania (L.) major, Leishmania tropica, Leishmania braziliensis, L. mexicana, and L. amazonensis. The burden of CL in terms of morbidity and social stigmas are concentrated on certain developing countries in Asia, Africa, and South America. People with asymptomatic CL represent a large proportion of the infected individuals in the endemic areas who exhibit no lesion and can control the infection by as yet not fully understood mechanisms. Currently, there is no approved prophylactic control measure for CL. Discovery of biomarkers of CL infection and immunity can inform the development of more precise diagnostics tools as well as curative or preventive strategies to control CL. Herein, we provide a brief overview of the state-of-the-art for the biomarkers of CL with a special emphasis on the asymptomatic CL biomarkers. Among the identified CL biomarkers so far, direct biomarkers which indicate the actual presence of the infection as well as indirect biomarkers which reflect the host's reaction to the infection, such as alterations in delayed type hypersensitivity, T-cell subpopulations and cytokines, adenosine deaminase, and antibodies against the sand fly saliva proteins are discussed in detail. The future avenues such as the use of systems analysis to identify and characterize novel CL biomarkers are also discussed

HPV16 E7-CT (gp96) fusion protein: Molecular cloning, expression and purification of a recombinant 6xHis-tagged protein in E. coli

The development of a therapeutic vaccine against human papillomavirus (HPV) is important for the control of cervical cancer. E7 is the major transforming protein produced in cervical cancers, and therefore represents potential tumor-specific antigen that could be the target of immunotherapy for cervical cancer. Among different vaccine strategies, protein-based vaccines are capable of generating CD8+ T cell responses in vaccinated animals and humans. Recently, development of novel strategies that enhance protein vaccine potency is important for generation of effective cancer vaccines and immunotherapies. Heat shock proteins (HSPs) including Gp96 have been shown to act as potent immuno-adjuvant to enhance antigen-specific tumor immunity. Therefore, the HSP-based protein vaccines can be administered by fusing antigens to HSPs, in vitro. It has been known that the HSP fragments (e.g., N-/or C-terminal regions) as mini-chaperones are better choice for immunization. The most straightforward method to produce large amounts of recombinant protein suitable for a vaccine is to clone the gene into a prokaryotic expression vector and produce the protein in Escherichia coli. In current study, we describe cloning of the HPV16 E7 gene linked to C-terminal fragment of gp96, identification and purification of the resultant E7-CT (gp96) fusion protein for next usage as a potential vaccine candidate protein against HPV in a pre-clinical trial. The recombinant E7-CT (gp96) migrated as a 51 kDa protein in SDS-PAGE. In Western blot experiment, the existence of a 51 kDa band for rE7-CT (gp96) was confirmed by rabbit anti-His as well as mouse anti-HPV16 E7 monoclonal antibodies. The protein of interest was both in the insoluble and the soluble fraction; therefore, purification was performed under denaturating and native conditions by affinity chromatography on Ni-NTA resin using 6xHis-tag

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Abstract Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major EGFP or L. major EGFP-LUC ) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wildtypes that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study

In Silico Analysis of Six Known Leishmania major Antigens and In Vitro Evaluation of Specific Epitopes Eliciting HLA-A2 Restricted CD8 T Cell Response

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

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

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