Leishmania is obligate interacellular parasite , they are transmitted by the bite of female sand fly , there are over 20 species(table 1) and subspecies that IFN-γ ect mammals and humans causing different spectra of symptoms and clinical signs collectively known as leishmaniasis. Leishmaniases are endemic in 88 countries , mostly in the tropics and subtropics with a majority of the cases happening in developing countries .2 Currently, 350 million are at risk of IFN-γ ection and about 12 million people are believed to be afflicted with the disease worldwide . (1,2,5).
The life cycle of this kinetoplastid parasite involve alternating life form in the vector , and the mammalian host. during a blood meal the vector takes up the amastigote from the host ,where it transforms to the motile promastigote,, and it became multiplies in the sand fly digestive tract, to be regurgitated with the following blood meal. The injected promastigotes come into macrophages in the mammalian host, where they transform into amastigote, escape macrophage killing mechanisms and multiply to enormous number.
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The manifestations of leishmaniasis range from a self-healing cutaneous lesion (cutaneous leishmaniasis, CL) to a lethal visceral form of the disease (visceral leishmaniasis, VL; or kala azar) and moco cutaneous leishmania(MCL). VL is a main public health problem in the East Africa and Indian subcontinent . on the other hand, VL also occurs in the Mediterranean region and Latin America, where dogs are the primary reservoir of IFN-γ ection. Parasites are transmitted by the bite of sand flies to humans from humans or IFN-γ ected dogs. . More than 90% of cutaneous leishmaniasis, which first appears as papules on the exposed skin followed by ulceration and scab formation, take place mostly in seven countries, namely Algeria, Brazil, Iran Afghanistan, , Peru, Saudi Arabia and Syria. (3)
Immunology of leishmaniasis
The responses of the immune system to Leishmania IFN-γ ection are highly complex. They can accelerate cure or make worse the disease, depending on the particular conditions . This is partly bevause of genetic variation in the parasites among types and strains,partially due to the effects of genetic difference in the mammalian host , and partly because of chance factors like the inoculum size, location, and number of IFN-γ ective bites received (4)
in leishmaniasis, Acquired resistance is mediated by T cells(6). CD4+ lymphocytes are vital for resistance and CD8+ are more affected in memory than as effector cells, CD25+ and CD4+ regulatory T cells are affected in insistence of L. major IFN-γ ection . In humans, a association between TH1 cell responses and resistance and healing of CL was explaned with a majority of cells producing IFN -as a mixed TH1/TH2 response with IL-10 and IL- 4 characterized chronic cutaneous and mucocutaneous lesions . Other studies showed this mixed cytokine profile in CL and in natural resistance, with IL-10 and IL- 4 predominating in early IFN-γ ection and a Type-1 immune profile in patients with a main exacerbated or older lesions (9),
TH1 response with high levels of TNF- α and IFN-γ in mucosal patients( 10). In visceral leishmaniasis, no relationship of IL-4 with active disease was explained; but
constant levels of IFN-γ and a direct association between increase in IL-10 and active disease by Leishmania donovani were observed (11,12) .
Human kala-azar is cdetected by high titers of Leishmania-specific antibodies appearing soon following IFN-γ ection and before the improvement of cellular immunological abnormalities. The task of these antibodies in disease protection or resolution is largely unidentified. in humans ,The TH1 cytokine
IFN-γ perhaps upregulates IgG1 and IgG3, whereas the TH2 cytokines IL-5 and IL-4 stimulate the generation of elevated levels of, IgE, IgM and IgG isotypes like IgG4. Analysis of the Leishmania specific Ig isotypes in CL and IgG subclasses in VL patients serum showed elevated levels of IgG, IgM, IgE and IgG subclasses through disease (13,14).
fitst generation vaccine
An perfect vaccine against leishmaniasis should have some properties, include (i) it must be safe; (ii) it must make long-standing protection against most human pathogens that reason leishmaniasis by a minimum number of immunizations ; (iii) it should be free of animal products that are used to manufacture the product; (iv) it must be produced as cost-effectively as possible; and (v) it should be effective in both preventing and treating leishmaniasis. . To build up such a vaccine, it is important to distinguish protective antigens and to send them in creative systems that are optimized to meet both regulatory and scientific standards(15).
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like for many other diseases, use of killed leishmania was the first approach to developing a leishmaniasis vaccine . The simplicity of growing Leishmania in culture media enabled it possible to use promastigotes grown in vitro. in the 1930s and 1940s several studies reported moderate success and are summarized in table1 (1,3) but in the1980, Mayrink and colleagues were conducted several controlled trials in Brazil . this study showed 53.3% efficiency in those who responded to the vaccination by cellular immunity measured by the leishmanin skin test(16) . in Colombia ,The vaccine was finally tested in a phase 3 trial (17)and was secure but not satisfactorily efficacious (Table 1). Other trials were done in Latin America but they could not be repeated (18)or were inconclusive, either because of the low incidence of the disease during the trials or due to flooding and ELnino, which made follow-up impossible. Razi Serum and Vaccine Institute produced vaccine from L. major , was tested in several phase 1-3 trials against CL in Iran caused by L. tropica or L. major (19) .
in countries that have a undeveloped Biotechnology industry and where a cold-chain for distribution is not feasible Autoclaving of the killed parasite vaccine was introduced (20)as the best form of sterilization and preservation of vaccines. The work of de Luca et al. (21) showed though, that, as expected for a protozoa parasite, the vaccine became loose immunogenicity due to autoclaving destroys most of the proteins of the parasite . on the other hand, the LPG complexes resists autoclaving (22) and have been concerned in immunogenicity and immunosuppression (23)in the mice model. While some protocols have used no adjuvant (24)(Table 2), most prophylactic vaccines used BCG for human and dog assays(25) (Table 2). The most important aspect of the first-generation human
vaccines is that a leishmanin skin test is used for candidate selection and for proof of immunogenicity . every time the leishmanin skin test is performed, vaccine efficiency value is obtained among the those whose skin tested positive , a study in Sudan that achieved about 43.3% of vaccine efficacy against VL is striking considering the high mortality and virulence of VL or kala-azar there. (26)
Table 2 vaccine effecacies of first generation vaccine
Second generation vaccines
The improvement of a defined vaccine candidate against leishmaniasis has been made feasible by our understanding of mechanisms of immunology that mediate defense in animal models and to a lesser degree by supporting data from the characterization of immune responses in leishmaniasis patients.
In addition, right now, genome sequencing of L. major is concluded and one of the heavy forces behind the genome project is to recognize genes that are expressed in the IFN-γ ectious stages of the parasite and in particular, in amastigotes. Access to abundant DNA sequences would be favour the development of genetic vaccines over the conventional ones, considering its simple use, low price of production and flexibility of combining various genes in a single construct(27). In contrast with the recombinant protein vaccines and attenuated organisms, DNA vaccines are comparatively simple and economical to produce(28) .
the purified gp63 has been tested in some experimental models using different strains and adjuvants, giving rise to contradictory results(29). in
Monkeys, A small scale vaccine study of rgp63 against L. major IFN-γ ection was tested
(30). Three doses of the recombinant antigen were administrated mixed with BCG as an adjuvant. After vaccination, peripheral blood mononuclear cells from these animals neither multipied nor formed IFN- áµžfollowing stimulation with antigen, after confront with virulent leishmania major promastigotes only partial protection was achieved.
. consequent to this work, the protective efficiency of LACK DNA was compared with IL-12 and of LACK
protein (31). when LACK protein plus recombinant IL-12 was administered ,It was shown that the LACK gene construct induced a strong defensive response comparable to that , and was better than protection seen with LACK protein alone . (32) The L. major parasite surface antigen-2 (PSA-2) is in both amastigote and promastigote . vaccination with PSA-2 with Corynebacterium parvum like adjuvant, It has been shown that protects mice from Leishmania through a Th1 mediated response, (33). The recombinant hydrophilic acylated surface protein B1 (rHASPB1) is capable to give protection against experimental challenge with L. donovani and Protection induced by rHASPB1 is not require adjuvant and since vaccine induced protection correlates with the presence of rHASPB1 specific IFN-γ producing CD8+ T cells, it appears that the mechanism of protection is like to DNA vaccination(34). The A2 genes are amastigote stage specific. A2 used like a recombinant protein with IL-12, or as a DNA vaccine confirmed considerable protection against VL in mice .(35)
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along with the subunit vaccine candidates the kinetoplastid membrane protein-11 (KMP-11) appears highly conserved in all Leishmania species tested and has been explained to get potent lymphoproliferative and antibody responses in experimentally IFN-γ ected mice or leishmaniasis patients and a significant protection in hamsters against leishmania donovani IFN-γ ection (36-37).
another recombinant protein is LCR1 which shares homology with Trypanosoma cruzi flagellar antigen from L. chagasi motivated proliferation of splenic T lymphocytes from L. IFN-γ antum IFN-γ ected C3H and BALB/c mice and inducedIFN-γ but not IL-5 , IL-4, or IL-10 secretion. Immunization with LCR1 can protect BALB/c mice against challenge with L. IFN-γ antum (38) . The combination of four plasmid DNAs, encoding the L. IFN-γ antum histones H2B, H3 ,H2A, and H4, were tested for protection in BALB/c mice. that was found that the immunized animals developed a specific Th1 immune response, which was associated with an antigen specific production ofIFN-γ and a limited humoral response against histones. Both CD8+ and CD4+ T cells contributed to the conflict of vaccinated mice to CL in these experiments(39)
The last advance in second-generation vaccines is the apply of recombinant proteins that were intensively tested since the 1990s (Table 2). The Leishmania recombinant vaccine candidates were assayed only
in combination (34,35), or as chimeras or poly-proteins , most of them required to be formulated with adjuvants(33,39,40), or delivered by bacteria
(35,42) (Table 3). The LmSTI1 (L. major stress inducible protein 1) andThe TSA (thiol-specific antioxidant) are protective for monkeys and mice
against CL(43). mice were protected against CL and VL by multicomponent Leish-111f fusion protein including the antigens LmSTI1, TSA, and LeIF (Leishmania elongation initiation factor), in formulation with squalene and MPL-SE9 , but, in mixture with Adju Primeor MPL-SE , was only immunogenic in dogs challenged with L. chagasi and L. IFN-γ antum .(40,41) . The H1 histone that protected mice and monkeys against CL, the HASPB1 or both in combination with Montanide(45,41), and the protein Q, a chimeric antigen composed of the genetic fusion of five of the acidic ribosomal protein Lip2a, Lip2b, P0 and the histone H2A used with BCG (42)developed partial protection against CVL at the clinical level and in dogs against IFN-γ ection (46). the plasmid DNA encoding LACK was more efficient than the L. major LACK with IL-12, the recombinant PSA2 antigen in ISCOMs or with C. parvum, induced a TH1 response but not protection against CL (33,47,48).
Table3 second generation recombinant antigens
Candidates for third-generation vaccines
DNA vaccines are more constant and have the benefit of their low cost of production, no need of cold chain for distribution, in Compared to recombinant protein vaccines, they have flexibility of combining compound genes in a simple construct. DNA vaccination can generates strong immune responses appears by the non-methylated CpG sequences of bacteria and to the intense replication in the host, leading to the expression of the recombinant proteins for longer time.
The most-studied antigens (Table 3) were those before assayed the same as recombinant proteins (49,50) The gene encoding gp63 was the first to be used as a DNA vaccine, and immunised mice developed
strong Th1 responses as well as significant resistance to IFN-γ ection with L. major ). LACK is the most extensively studied DNA vaccine against both visceral leishmaniasisand cutaneous . DNA vaccination with a plasmid harboring the LACK gene without ort with, co-administration of IL-12 induced robust, long- time protection against L. major challenge in mice, dependent on the immunoregulatory role of CD8+ T cells .(51)
The LACK, LeIF, TSA, KMP11, LmSTI1, H1, CpA + CpB, NH36 are the most talented candidates that may find a place in the future(15).
LACK DNA induced a TH1 reaction that protected against IFN-γ ection by L. major In mice .(49,52) Even reduced portions of the PSA2 gene and LACK gene were superior to p20 and GP63 against L. major IFN-γ ection(49), and mice can immunization with HPB-LACK (Table 3) against VL (53) . The immunization resulted in an raise in IL-12 and IFN-γ expression, lymphocyte proliferative response, IgG2 to IgG1 ratio while it due to decreases in number of parasites in target organ, and IL-4 expression.
mice protcted against CL by Vaccination either with the LmSTI1and the TSA or DNA vaccines, or with both as a tandem digene construct (43), protected through a CD4 + TH1 response.
mice also were protected against L. major IFN-γ ection through a TH1 response by Injection of a mixture of four histone plasmids (H2A, H2B, H3 and H4) in Balb/c (54). protected hamsters were protected against VL through a mixed cytokine TH1/TH2 response by Vaccination with 200 _g of KMP11 (55).
Leishmaniasis is a main reason of morbidity and mortality in the world. An effective vaccine need to Control this disease. although despite great effort, there is not available an effective vaccine.The major technical issues in the plan of a Leishmania vaccine are not different from those for any other vaccine. They contain specificity, the class of response induced, and the induction of durable immunological memory. Leishmania vaccine development has confirmed to be a difficult and challenging task, which is mostly hampered by insufficient IFN-γ ormation of parasite pathogenesis and the Leishmania vaccines complexity of immune responses needed for protection.The main Concerns are dependable correlates of immunity that need to be developed in order to evaluate vaccines, as well as a need for a regular testing system for new vaccine candidates. Then, the subject of delivery systems,antigen formulation and adjuvant would have to be determined. at present,there seem to be as many problems and questions as there are solutions, but given the rapid development in the vaccinology field, a successful anti-Leishmania vaccine should be possible.