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Herein, a 28-year-old man with hoarseness, skin and oral lesions is presented. At the time of admission, the patient had an erythematous plaque on his chin near his lower lip and an erythematous-violaceous plaque on his palate near the opening of the pharynx and 20 kg weight lost in last one year. The biopsy of his skin lesions by hematoxylin and eosin staining revealed an infiltration of the dermis by lymphoplasma and histiocytic cells with a loose granuloma formation suggestive of leishmaniasis. Biopsy of mucosal lesions revealed Leishman bodies in dermis. PCR was performed on the specimens of skin, bone marrow, mucosa, and saliva, the results were positive. The pathogenic agent was identified as Leishmania major by the nested PCR. Serologic tests including direct agglutination test (DAT) and indirect immunofluorescence test (IFAT) were positive with high titers of anti-L. infantum antibodies (1:102400 versus 1:800, respectively), indicative of visceral involvement. The patient responded to a combination of miltefosine and meglumine antimoniate (Glucantime®). Visceral involvement due to L. major is rarely reported. To the best of our knowledge, probably hoarseness due to L. major has not been previously reported from Iran.

Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Hypervariability of HCV proteins is an important obstacle to design an efficient vaccine for HCV infection. Multi-epitope vaccines containing conserved epitopes of the virus could be a promising approach for protection against HCV., Objectives: Cellular and humoral immune responses against multi-epitope DNA and peptide vaccines were evaluated in BALB/c mice., Materials and Methods: In this experimental study, multi-epitope DNA- and peptide-based vaccines for HCV infection harboring immunodominant CD8+ T cell epitopes (HLA-A2 and H2-Dd) from Core (132-142), NS3 (1073-1081) and NS5B (2727-2735), a Th CD4+ epitope from NS3 (1248-1262) and a B-cell epitope from E2 (412-426) were designed. Multi-epitope DNA and peptide vaccines were tested in two regimens as heterologous DNA/peptide (group 1) and homologous peptide/peptide (group 2) prime/boost vaccine in BALB/c mice model. Electroporation was used for delivery of the DNA vaccine. Peptide vaccine was formulated with Montanide ISA 720 (M720) as adjuvant. Cytokine assay and antibody detection were performed to analyze the immune responses., Results: Mice immunized with multi-epitope peptide formulated with M720 developed higher HCV-specific levels of total IgG, IgG1 and IgG2a than those immunized with multi-epitope DNA vaccine. IFN-γ levels in group 2 were significantly higher than group 1 (i.e. 3 weeks after the last immunization; 37.61 ± 2.39 vs. 14.43 ± 0.43, P < 0.05). Moreover, group 2 had a higher IFN-γ/IL-4 ratio compared to group 1, suggesting a shift toward Th1 response. In addition, in the present study, induced immune responses were long lasting and stable after 9 weeks of the last immunization., Conclusions: Evaluation of multi-epitope DNA and peptide-vaccines confirmed their specific immunogenicity in BALB/c mice. However, lower Th1 immune responses in mice immunized with DNA vaccine suggests further investigations to improve the immunogenicity of the multi-epitope DNA vaccine through immune enhancers.

Vaccination with durable immunity is the main goal and fundamental to control leishmaniasis. To stimulate the immune response, small numbers of parasites are necessary to be presented in the mammalian host. Similar to natural course of infection, strategy using live vaccine is more attractive when compared to other approaches. Live vaccines present the whole spectrum of antigens to the host immune system in the absence of any adjuvant. Leishmanization was the first effort for live vaccination and currently used in a few countries against cutaneous leishmaniasis, in spite of their obstacle and safety. Then, live attenuated vaccines developed with similar promotion of creating long-term immunity in the host with lower side effect. Different examples of attenuated strains are generated through long-term in vitro culturing, culturing under drug pressure, temperature sensitivity, and chemical mutagenesis, but none is safe enough and their revision to virulent form is possible. Attenuation through genetic manipulation and disruption of virulence factors or essential enzymes for intracellular survival are among other approaches that are intensively under study. Other designs to develop live vaccines for visceral form of leishmaniasis are utilization of live avirulent microorganisms such as Lactococcus lactis, Salmonella enterica, and Leishmania tarentolae called as vectored vaccine. Apparently, these vaccines are intrinsically safer and can harbor the candidate antigens in their genome through different genetic manipulation and create more potential to control Leishmania parasite as an intracellular pathogen.

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.

The use of an appropriate delivery system has recently emerged as a promising approach for the development of effective vaccination against visceral leishmaniasis (VL). Here, we compare two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine harbouring the L. donovani A2 antigen along with L. infantum cysteine proteinases [CPA and CPB without its unusual C-terminal extension (CPB(-CTE) )] and evaluate their potential against L. infantum challenge. Prime-boost administration of the pcDNA-A2-CPA-CPB(-CTE) delivered by either electroporation or cSLN formulation protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ and lower levels of IL-10 production, leading to a strong Th1 immune response. At all time points, the ratio of IFN-γ: IL-10 induced upon restimulation with rA2-rCPA-rCPB and F/T antigens was significantly higher in vaccinated animals. Moreover, Th2-efficient protection was elicited through a high humoral immune response. Nitric oxide production, parasite burden and histopathological analysis were also in concordance with other findings. Overall, these data indicate that similar to the electroporation delivery system, cSLNs as a nanoscale vehicle of Leishmania antigens could improve immune response, hence indicating the promise of these strategies against visceral leishmaniasis., (© 2013 John Wiley & Sons Ltd.)

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.

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.

The design of efficient cancer treatments is one of the major challenges of medical science. Therapeutic vaccines of cancer have been emerged as an attractive approach for their capacity of breaking the immune tolerance and invoking long-term immune response targeting cancer cells without autoimmunity. An efficient antigen delivery system is the key issue of developing an effective cancer vaccine. In this regard, live vaccination strategies including various live bacterial and viral vectors have attracted a great attention. Several bacterial strains such as Salmonella, Listeria monocytogenes and Lactococcus lactis effectively colonize solid tumors and act as antitumor therapeutics. On the other hand, the use of viruses as vaccine vectors such as Vaccinia, Adenovirus, Herpes simplex virus, Paramyxovirus and Retroviruses utilizes mechanisms that evolved in these microbes for entering cells and capturing the cellular machinery to express viral proteins. Viral/bacterial-vectored vaccines induce systemic T-cell responses including polyfunctional cytokine-secreting CD4+ and CD8+ T-cells. However, there is an urgent need for the development of new safe live vaccine vectors that are capable of enhancing antigen presentation and eliciting potent immune responses without the risk of development of disease in humans. Recently, nonpathogenic parasites including Leishmania tarentolae, Toxoplasma gondii and Trypanosoma cruzi have emerged to be a novel candidate for gene delivery and heterologous genes expression. In this review, recent researches on cancer therapy using genetically modified bacteria and virus are summarized. In addition, live parasite-based vectors will be discussed as a novel anticancer therapeutic approach., (Copyright © 2012 UICC.)

To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-γ response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Cell penetrating peptides (CPPs) are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargo, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In addition, the orientation of the peptide and cargo and the type of linkage are likely important. In gene therapy, the designed cationic peptides must be able to 1) tightly condense DNA into small, compact particles; 2) target the condensate to specific cell surface receptors; 3) induce endosomal escape; and 4) target the DNA cargo to the nucleus for gene expression. The other studies have demonstrated that these small peptides can be conjugated to tumor homing peptides in order to achieve tumor-targeted delivery in vivo. On the other hand, one of the major aims in molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical agents of malignant transformation. For example, cell penetrating peptide aptamers might disrupt protein-protein interactions crucial for cancer cell growth or survival. In this review, we discuss potential functions of CPPs especially for drug and gene delivery in cancer and indicate their powerful promise for clinical efficacy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions., (© 2011 Blackwell Publishing Ltd.)

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.

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 © 2011 Elsevier B.V. All rights reserved.)

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.

Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C-terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL/6 mice. Therefore, SLN-CPB and SLN-CPB(-CTE) formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen-specific T-helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN-γ, IgG2a and lower IL-4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN-γ/IL-4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN-CPB(-CTE) vaccinated group, post-challenge. Thus, SLN-CPB(-CTE) was shown to induce specific Th1 immune responses to control L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells., (© 2011 Blackwell Publishing Ltd.)

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 © 2011 Elsevier Inc. All rights reserved.)

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 © 2010 Elsevier Inc. All rights reserved.)

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.

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-gamma 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.

Human papillomaviruses (HPVs) are simple, non-enveloped, double-stranded DNA viruses and responsible for an enormous global burden of genital disease. HPV is annually associated with 500,000 new cases of cervical cancer and 250,000 cervical cancer deaths worldwide. The association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. A novel approach for primary prevention of cervical cancer has become available by the discovery of efficient prophylactic HPV vaccines based on virus-like particles. Therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Different therapeutic strategies have been developed including peptide immunization-based therapies, DNA vector-based therapies, viral/bacterial vector-based therapies, immune response modifiers, photodynamic therapy (PDT) and T cell receptor based therapy. At present, the design of therapeutic vaccines to control the growth of HPV-induced tumors has focused on utilization of E6 and E7 proteins or peptides as vaccine antigens. Human trials are the most important test for the efficacy of HPV16/18 E6 and E7 proteins as immunotherapy for cervical cancer. This review attempts to describe different therapeutic vaccinations against HPV infections.

The field of vaccinology provides excellent promises to control different infectious and non-infectious diseases. Genetic immunization as a new tool in this area by using naked DNA has been shown to induce humoral as well as cellular immune responses with high efficiency. This demonstrates the enormous potential of this strategy for vaccination purposes. DNA vaccines have been widely used to develop vaccines against various pathogens as well as cancer, autoimmune diseases and allergy. However, despite their successful application in many pre-clinical disease models, their potency in human clinical trials has been insufficient to provide protective immunity. Several strategies have been applied to increase the potency of DNA vaccine. Among these strategies, the linkage of antigens to Heat Shock Proteins (HSPs) and the utilization of different delivery systems have been demonstrated as efficient approaches for increasing the potency of DNA vaccines. The uptake of DNA plasmids by cells upon injection is inefficient. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target Antigen-Presenting Cells (APCs) are effective in animal models. Alternatively, different regimens called prime-boost vaccination are also effective. In this regimen, naked DNA is utilized to prime the immune system and either recombinant viral vector or purified recombinant protein with proper adjuvant is used for boosting. In this review, we discuss recent advances in upgrading the efficiency of DNA vaccination in animal models.

Leishmania protozoa are obligate intracellular parasites that reside in the phagolysosome of host macrophages and cause a large spectrum of pathologies to humans known as leishmaniases. The outcome of the disease is highly dependent on the parasite species and on its ascribed virulence factors and the immune status of the host. Characterization of the genome composition of non-pathogenic species could ultimately open new horizons in Leishmania developmental biology and also the disease monitoring. Here, we provide evidence that the lizard non-pathogenic to humans Leishmania tarentolae species expresses an Amastin-like gene, cysteine protease B (CPB), lipophosphoglycan LPG3 and the leishmanolysin GP63, genes well-known for their potential role in the parasite virulence. These genes were expressed at levels comparable to those in L. major and L. infantum both at the level of mRNA and protein. Alignment of the L. tarentolae proteins with their counterparts in the pathogenic species demonstrated that the degree of similarity varied from 59% and 60% for Amastin, 89% for LPG3 and 71% and 68% for CPB, in L. major and L. infantum, respectively. Interestingly, the A2 gene, expressed specifically by the L. donovani complex which promotes visceralization, was absent in L. tarentolae. These findings suggest that the lack of pathogenicity in L. tarentolae is not associated with known virulence genes such as LPG3, CPB, GP63 and Amastin, and that other factors either unique to L. tarentolae or missing from this species may be responsible for the non-pathogenic potential of this lizard parasite.

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-gamma 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.

Heat-shock proteins (HSPs) have been known as multifunctional proteins. They facilitate the folding and unfolding of proteins, participate in vesicular transport processes, prevent protein aggregation in the densely packed cytosol and are involved in signaling processes. HSPs have been involved in different fields, including autoimmunity, immunity to infections and tumor immunology. Although there are many different kinds of HSPs, only some HSPs, including HSP70 and Gp96, have immunological properties. HSP molecules have been applied into DNA- or protein (peptide)-based vaccines as antigens, chaperones or adjuvants. HSP-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. The immunogenicity of HSPs results from two different properties: a peptide-dependent capacity to chaperone and elicit adaptive cytotoxic T-lymphocyte responses against antigenic peptides and a peptide-independent immunomodulatory capacity. Furthermore, HSPs could be immunoregulatory agents with potent and widely applicable therapeutic uses. Accordingly, certain HSPs, such as HSP70 and Gp96, are highly effective carrier molecules for cross-presentation. Their ability in eliciting immune responses against different pathogens (parasite and virus) and their role in cancer immunity will be discussed in this review.

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.

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 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg 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.

Amastin surface proteins belong to a large family of developmentally regulated proteins comprising up to 45 members that have recently been discovered in the genus Leishmania and are highly similar to the amastin proteins in Trypanosoma cruzi. All members of the amastin gene family contain a highly conserved 11-amino-acid (aa) signature at the N terminus, which is unique to the amastin proteins and to the Trypanosomatidae family. Recent studies have demonstrated that this region is highly protective in a mouse model. The goal of the present study was to evaluate the potential of the 50-aa N-terminal domain of amastin proteins harboring the conserved 11-aa amastin signature peptide as a relevant immune biomarker of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We report here the amastin-binding total immunoglobulins (IgG) and/or IgG subclasses in the sera of patients at different stages of CL (n=90) and VL (n=41). In CL cases, there is no significant difference in seroreactivities between active, recovered, and nonhealed cases. However, the amastin peptide-reactive antibodies were present at high titers in 19 of 20 sera collected from patients with active VL compared to sera from patients recovered from VL and asymptomatic cases of VL. These data suggest that the amastin signature peptide could represent a relevant biomarker for the serodiagnosis of VL and, most importantly, that it could permit differentiation among the different stages of the disease.