WO2017163171A1

WO2017163171A1 - Chimeric protein, vaccine composition against leishmaniasis, and uses thereof

Info

Publication number: WO2017163171A1
Application number: PCT/IB2017/051610
Authority: WO
Inventors: Eduardo ANTONIO FERRAZ COELHO; Carlos ALBERTO PEREIRA TAVARES; Vivian TAMIETTI MARTINS; Tiago Antônio De Oliveira MENDES; Mariana COSTA DUARTE; Daniel Menezes SOUZA; Bruno MENDES ROATT; Daniela PAGLIARA LAGE
Original assignee: Universidade Federal De Minas Gerais - Ufmg
Priority date: 2016-03-21
Filing date: 2017-03-20
Publication date: 2017-09-28
Also published as: BR102016006121A2

Abstract

The present invention relates to a vaccine composition based on a recombinant polypeptide chimera composed of specific epitopes of human and mice CD4+ and CD8+ T lymphocytes derived from four Leishmania proteins (LiHypl, LiHyp6, LiHyV and HRF), capable of inducing a protection against visceral and cutaneous leishmaniasis, and to the use thereof.

Description

CHEMICAL PROTEIN, VACINAL COMPOSITION AGAINST

Leishmaniasis and Uses

The present invention relates to a vaccine composition based on a recombinant polypeptide chimera composed of human and mouse CD4 + and CD8 + T-lymphocyte specific epitopes derived from four proteins (LiHypl, LiHyp6, LiHyV and HRF) from Leishmania, which was able to induce protection against visceral and cutaneous leishmaniasis and its use.

Leishmaniasis are endemic infectious diseases in various tropical and subtropical regions on different continents. It is estimated that 380 million people are at risk of contracting the disease and there is an approximate incidence of 0.5 to 1, 0 and 1, 5 to 2.0 million new cases of visceral leishmaniasis per year ( VL) and cutaneous leishmaniasis (LT), respectively, worldwide. Brazil is the leading VL incidence site in the Americas, accounting for 95% of cases (Alvar J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 7: e35671, 2012).

The severity of the disease in the mammalian host, in which both man and dog fall, ranges from a single cutaneous lesion at the stinging site to the visceral form of the disease, which can be fatal when acute and not. (Grimaldi, G. and Tesh, RB Leishmaniasis of the New World: current concepts and implications for future research. Clin. Microbiol. Rev. 6; 230 ^" 50, 1993). Due to the ineffectiveness of the control measures used, the difficulty For a correct diagnosis and toxicity problems related to conventional treatments, the development of vaccines that are capable of inducing protective immunity against the disease becomes desirable (Tesh RB. Control of zoonotic visceral leishmaniasis: J Trop Med Hyg. 52; 287-92, 1995).

[004] In murine models, after the cure of leishmaniasis caused by the species Leishmania major, animals acquire lasting immunity against reinfection (Afonso LC and Scott P. Immune responses associated with susceptibility of C57BL / 10 mice to Leishmania amazonensis. Infect. & Immun 61;. 2952 , ^"59, 1993). This fact has stimulated the development of research aiming at obtaining vaccine antigens that can be used as an effective disease control measure. There are antigens that have been evaluated and partially meet requirements to be used as vaccine candidates (Stager S, et al. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol. 165; 7064 ^" 71, 2000; Dondji B, et al. Heterologous prime ^" boost vaccination with the LACK antigen protects against murine visceral leishmaniasis, // 7 / ecf Immun 73; 5286 ^" 89, 2005).

Preferably, an effective vaccine should contain immunogenic antigens conserved on different Leishmania species, and have an affordable cost to the population and their governments. However, most studies performed use unique antigens, which are expressed in only one of the morphological forms of the parasite (Fernandes AP, et al. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives. Curr Opin Microbiol. 15 : 1-10, 2012). However, due to the difficulties related to obtaining antigens that are effective against more than one species of parasites, which are easily and rapidly produced and which do not stimulate the production of parasite-specific antibodies in immunized animals, as for other reasons; A completely effective vaccine against the different species of Leishmania parasite is not yet available on the market.

Regarding the immune response, resistance to infections of Leishmania species is related to a Th1 response profile, which is dominated by IFN-γ production by activated macrophages. On the other hand, the Th2 response profile with IL-4 and IL-10 production is associated with susceptibility to disease (Scott P. Immunol Res. 27; 489- 98, 2003). Given this, Leishmania species can evade the immune mechanism by inducing IL-4 and inhibiting IFN-γ in the early stages of infection. Thus, CD4 + and CD8 + T cells are important not only for fighting and eliminating the parasite from the host organism, but also for generating an immune memory focused on combating future parasite infections (Souza VL, et al. Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human Leishmaniasis in CBA mice. Mem Inst Oswaldo Cruz, v106 (1): 23- 31, 201 1).

Through the use of immunoproteomics recently, proteins of Leishmania infantum have been recognized by antibodies present in the sera of dogs with symptomatic and / or asymptomatic VL (Coelho VT, et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach (PLoS Negl Trop Dis. 6 (1): e1430, 2012). Among them, new diagnostic, vaccine and / or therapeutic targets were identified. Three of these proteins, called LiHypl (XP_001468941 .1), LiHyp6 (XP_001568689.1) and HRF (CAJ05086.1), when used in recombinant form and associated with saponin as an adjuvant, were able to induce protection in BALB / c mice. against experimental L. infantum infection (Martins VT, et al. Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis. PLoS One. 10 (9): e0137683, 2015) . Also, in another study, LiHyV protein (XP_001462854.1) offered recombinant and saponin-associated protection against infection with this same parasite species (Martins VT, et al. Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis (Parasit. Vectors, 8; 363, 2015); thus demonstrating the feasibility of using such antigens as candidate vaccines against LV.

[008] Currently, some documents describing the development of antigens for treatment of leishmaniasis are found in the state of the art. Patent document BR132013001 271, entitled Chimeric Protein, Vaccine Composition and Visceral Leishmaniasis Immunodiagnostic Test Kit, refers to a chimeric protein for use in canine and human LV immunodiagnostic vaccine and test composition developed through selection, identification , production and testing of new antigens by analysis proteomics, bioinformatics, peptide synthesis, immunoassay, synthetic gene construction and protein production and purification. Such technology differs from the present invention in that it comprises other immunogenic components which are different from those presented herein.

[009] The patent document P 10,609,847, entitled ^{^} Composition Comprising the N-terminal region of Leishmania histone H2B, use thereof for inducing an immune response _ is an immunogenic composition comprising an antigenic peptide consisting of a fragment of histone protein H2B Leishmania is an adjuvant that stimulates the immune response. This technology differs from the present invention in that it comprises only one antigenic compound. Moreover, the antigen in question differs from any of those described in the present invention.

[0010] Patent document WO20131 10824, entitled Multi-component ^{^} Chimera for use as a vaccine against infection by Leishmania spp. In mammals refers to a chimera, HISA70, composed of different proteins (H2A, H2B, H3, H4, A2 and HSP70) from Leishmania infantum in the composition of a vaccine against Leishmania spp. Such technology differs from the present invention in that it comprises proteins other than those listed in the present invention.

Thus, the present invention relates to a chimeric polypeptide vaccine composed of human and mouse CD4 + and CD8 + T-lymphocyte specific epitopes derived from the four proteins (LiHypl, LiHyp6, LiHyV and HRF) that have been described as capable. to induce protection, when administered alone, against L. infantum infection, to induce protection against infection by L. infantum and L. amazonensis, species causing visceral and cutaneous leishmaniasis, respectively, worldwide. The polypeptide chimera has shown promising results in inducing protection against challenge infection with both parasite species and, thus, is a new candidate for protection against distinct Leishmania species. The protection afforded by the chimeric vaccine was superior to that induced by proteins administered alone in the experimental animals with respect to the immunogenicity induced before and after experimental infection, as well as reducing the parasitic load in different organs of the evaluated animals.

In the state of the art, no technology developed for vaccine composition comprising the epitopes described in the present invention has been found, which makes it innovative and possible for industrial application. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 represents the antigenicity of recombinant chimera. The recombinant chimera (10 μg, indicated by the black arrow in the figure) was electrophoresed on a 12% SDS-PAGE denaturing gel, stained with bright Coomassie blue G-250 and transferred to a nitrocellulose membrane, which was placed. incubation with the different serum pools. For this, a molecular weight standard was used as marker (A). Reactions with the serum pools of animals immunized with recombinant proteins LiHypl (B), LiHyp6 (C), LiHyV (D), HRF (E) or sera from animals immunized with the chimera itself (F) are shown. Serum pools of mice experimentally infected with L. infantum (G) or L. amazonensis (H) were also used, as well as sera pool from uninfected and unimmunized animals (I), used as negative reaction control.

DETAILED DESCRIPTION OF TECHNOLOGY

The present invention relates to a polypeptide chimera-containing vaccine composition composed of human and mouse CD4 + and CD8 + T lymphocyte specific epitopes from four Leishmania immunogenic proteins and their use for the protection and / or treatment against leishmaniasis cutaneous and visceral.

More specifically, the chimeric protein proposed in the present invention is defined by SEQ ID NO: 1. The leishmaniasis vaccine composition comprises the chimeric protein defined by SEQ ID NO: 1, in addition to the association of pharmaceutically acceptable adjuvants.

Pharmaceutically acceptable adjuvants according to the present invention may be selected, without limitation, from those cited in the following literature: Remington's Pharmaceutical Sciences, Mack Publishing, European or Brazilian Pharmacopoeia or new excipients to be preferably saponins. Preferably, adjuvants should be Th1 immune response inducers, such as saponins, or compounds that are capable of stimulating a cellular immune response prime by the production of IFN-γ and IL-12.

The present invention further relates to the use of the chimeric protein in tegumentary and visceral leishmaniasis vaccine compositions.

The proposed vaccine composition may be administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous and / or as a device that can be implanted and / or injected.

The present invention may be better understood by the following non-limiting examples.

Example 1 - Construction of recombinant chimera.

For construction of the polypeptide chimera, the proteins LiHypl, LiHyp6, LiHyV and HRF were subjected to bioinformatics analysis for selection of immunogenic regions rich in murine and human T lymphocyte epitopes. The Net CTL Pan program was used, so that the best epitopes that bound to the human MHC class I alleles A2, A3 and B7, which together represent over 90% of the human population of any ethnicity, were selected. The same program was used so that epitopes binding to the Η-2-Kd, Η-2-Ld and Η-2-Dd alleles of BALB / c mice were also selected. The epitopes specific to the class I major histocompatibility complex (MHC) molecules that were selected for chimera construction are shown in Tables 1 and 2.

In order to identify MHC class II binding epitopes, the Net MHC II 2.2 program was used to assess the binding affinity of 15 amino acid peptides to the 26 human alleles. Only epitopes with the ability to interact with binding affinity of less than 500 nM and in at least 30% of alleles were selected. The program was also used to select epitopes that bound to the 1-Ad and 1-Ed alleles of BALB / c mice. The epitopes specific to MHC class II molecules that have been selected for chimera construction are shown in Tables 3 and 4. Table 1. In silico prediction of specific epitopes to MHC class I T-lymphocyte molecules from humans selected for recombinant chimera construction.

MHC class I of human

Protein Number Sequence Allele Net CTL Score

1 69-ILNDGRFQL-77 A2 1,125

2 156-MVPDRSVYI-164 A2 1,069

LiHypl 3 83 - ASFMPLLER-91 A3 1,090

4 77-LPPLPPASF-85 B7 1,398

5 80-LPPASFMPL-88 B7 1,390

6 37-SLATAFGLV-45 A2 1,0202

LiHypó

7 52-LLYRSTFRH-60 A3 1,2444

8 87-YMAHIRSYM-95 A2 1.0595

9 l 1 l-FQTNAAAFV-119 A2 1.0653

HRF 10 88-MAHIRSYMK-96 A3 1, 1722

11 112-QTNAAAFVK-120 A3 1, 1752

12 115 - A A AF VKKVL- 123 B7 1,121

13 90-SMSMArTTV-98 A2 1,322

14 69-VSGNGLTIK-77 A3 1.0395

15 83-TPSSARLSM-91 B7 1,7256

LiHyV 16 97-TVAQSArTL-105 B7 1, 1648

17 109-MPANSDIRI-117 B7 1, 1793

18 116-RIVATTSSL-124 B7 1.3978

19 125 - AP AQSLFDF- 133 B7 1.5079

Table 2. In silico prediction of specific epitopes to mouse T lymphocyte MHC class I molecules selected for recombinant chimera construction.

MHC Class I Mouse Protein Number Sequence Allele Net CTL Score

1 77-LPPLPPASF-85 Η-2-Ld 0.27186

2 79-PLPPASFMP-87 Η-2-Ld 0.08248

LiHypl 3 165-MSGPARYV Y-173 Η-2-Dd 0.23280

4 59-DVYTRASDR-67 Η-2-Kd 0.07271

5 83 - ASFMPLLER-91 Η-2-Kd 0.20710

LiHypó 7 29-LTYAETVVS-37 Η-2-Kd -

LiHyV 8 82-STPSSARLS-90 Η-2-Ld 0, 11316

Table 3. In silico prediction of epitopes specific to MHC class II T-lymphocyte molecules from humans selected for recombinant chimera construction.

MHC class II of human

Protein Number Sequence Net MHCII Score

1 52-LLYRSTFRHAMLLRV-66 38.46

2 53-LYRSTFRHAMLLRVQ-67 38.46

LiHypó

3 54-YRSTFRHAMLLRVQR-68 38.46

4 55-RSTFRHAMLLRVQRE-69 42.31

5 56-STFRHAMLLRVQRET-70 38.46

HRF 6 108 -RKAFQTN A A AF VKKV - 122 30.77

Table 4. In silico prediction of specific epitopes to mouse T lymphocyte MHC class II molecules selected for recombinant chimera construction.

MHC Class II Mouse

Protein Number Sequence Allele Net MHCII Score

1 169- ARYV YFHMVLPVEAQ- 183 Η-2-IAd 394.4

LiHypl

2 170-RYV YFHMVLPVEAQR-184 Η-2-IAd 446.9 3 171 - YV YFHMVLPVEAQRF- 185 Η-2-IAd 161,2

4 172- V YFHMVLPVEAQRFS - 186 Η-2-IAd 160.5

5 173- YFHMVLPVEAQRFSL-187 Η-2-IAd 169.2

6 174-FHMVLPVEAQRFSLV-188 Η-2-IAd 255.3

7 55-RSTFRHAMLLRVQRE-69 Η-2-IAd 204.8

8 56-STFRHAMLLRVQRET-70 Η-2-IAd 182.5

LiHypó 9 57-TFRHAMLLRVQRETR-71 Η-2-IAd 221.1

10 58 -FRH AMLLR VQRETRF-72 Η-2-IAd 252.0

11 59-RHAMLLRVQRETRFD-73 Η-2-IAd 484.1

12 83-TPSSARLSMSMArfT-97 Η-2-IAd 411.1

13 84-PSSARLSMSMArfTV-98 Η-2-IAd 347.9

14 85-SSARLSMSMArTTVA-99 Η-2-IAd 193.3

15 86-SARLSMSMArrTVAQ- 100 Η-2-IAd 145.8

16 87-ARLSMSMArTTV AQS-101 Η-2-IAd 113.9

LiHyV 17 88-RLSMSMArTTVAQSA-102 Η-2-IAd 93.5

18 89-LSMSMAnTVAQSAI-103 Η-2-IAd 165.8

19 90-SMSMArTTVAQSArr-104 Η-2-IAd 231.5

20 97-TVAQSArTLSGVMPA-l 11 Η-2-IAd 382.5

21 98-VAQSAITLSGVMPAN-l 12 Η-2-IAd 391.0

Example 2 - Validation of chimera antigenicity.

In order to validate the antigenicity of the polypeptide chimera, Western blot experiments were performed using BALB / c mouse sera that were subcutaneously immunized with the rLiHypl, rl_iHyp6, rLiHyV or rHRF plus saponin proteins. sera from animals chronically infected with L. infantum or L. amazonensis. In the results, it can be observed that all serum pools used (n = 8, per group), except for the negative control, which was composed of sera from animals that were not immunized or infected; were able to recognize polypeptide chimera (-30.0 kDa), thus demonstrating the antigenicity of the recombinant protein in vaccinated and / or infected animals (Figure 1). Example 3- Assessment of chimera-induced cellular immune response prior to challenge infection.

The immunogenicity of recombinant chimera was evaluated in immunized BALB / c mice by assaying IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 cytokines produced by animal splenocytes, 30 days after administration of the last dose of the immunogen. Following in vitro stimulation with specific proteins or a mixture of extracts of the two parasites (SLA), significantly elevated levels of IFN-γ, IL-12 and GM-CSF were observed in the vaccinated group of animals. regarding the levels produced by the splenocytes of the control groups, consisting of animals that received saponin or saline (Table 5). Also, the chimera plus saponin immunized group produced higher levels of IFN-γ, IL-12 and GM-CSF than the levels of these cytokines obtained in the groups immunized with rLiHypl, rLiHyp6, rLiHyV or rHRF plus saponin proteins. No increase was observed in IL-4 and IL-10 production in any of the groups evaluated.

Table 5- Production levels of IFN-γ, IL-12 and GM-CSF cytokines (in pg / ml). Cytokines were quantified in animal splenocyte culture supernatants. The mean ± standard deviation of the groups is shown.

Cytokines (in pg / ml)

IFN-gamma stimulation IL-12 GM-CSF

Medium 66 + 8 55 + 5 60 + 5

Saline Protein Mix 70 + 10 67 + 8 57 + 7

SLA 58 + 6 69 + 6 59 + 8

Medium 64 + 5 55 + 4 70 + 8

Saponin Protein Mix 77 + 10 65 + 8 77 + 6

SLA 59 + 10 59 + 7 59 + 5

Middle 56 + 5 58 + 9 64 + 4 LiHyp 1 / LiHypl saponin 998 + 98 233 + 55 199 + 33

SLA 433 + 35 155 + 22 138 + 16

Medium 55 + 5 57 + 7 63 + 6

LiHypó / saponin LiHYpo 677 + 57 201 + 21 177 + 26

SLA 322 + 20 134 + 15 107 + 10

Medium 60 + 10 64 + 4 58 + 5

LiHyV / Saponin LiHyV 905 + 45 195 + 30 180 + 30

SLA 405 + 45 133 + 17 121 + 19

Medium 49 + 5 54 + 5 62 + 7

HRF / saponin HRF 877 + 55 178 + 20 160 + 22

SLA 377 + 32 125 + 20 121 + 17

Medium 59 + 9 63 + 5 58 + 6

Chimera / Saponin Chimera 1,644 + 109 425 + 40 307 + 32

SLA 766 + 30 255 + 20 170 + 15

Example 4- Evaluation of protective efficacy against challenge infection by L. infantum or L. amazonensis.

Ten weeks after challenge infection, the parasitic burden of the various groups was evaluated on the infected paw (L amazonensis), liver, spleen, draining lymph node and bone marrow of animals infected with L. infantum (Table 6) or L. amazonensis (Table 7). It can be observed that in both cases, although animals immunized with individual recombinant proteins plus saponin had lower parasitic load than control groups (saline and saponin), animals immunized with the most recombinant chimera saponin showed more significant reductions in parasitism when compared to all other experimental groups.

Table 6. Results of parasitic burden of animals immunized and challenged with L. infantum. Parasitic load was determined by a limiting dilution technique 10 weeks after challenge infection. The mean ± standard deviation of the groups is shown.

Table 7. Results of parasitic load of animals immunized and challenged with L. amazonensis. Parasitic load was determined by a limiting dilution technique 10 weeks after challenge infection. The mean ± standard deviation of the groups is shown.

Infected Paw Spleen Liver Lymph Nodes Bone Marrow

Saline 8.5 + 0.5 6.5 + 0.4 4.4 + 0.2 7.3 + 0.4 3.3 + 0.2

Saponin 8.0 + 0.3 6.1 + 0.2 4.1 + 0.4 6.8 + 0.5 3.1 + 0.3

LiHyp 1 / saponin 3.2 + 0.3 2.7 + 0.4 2.5 + 0.3 3.1 + 0.3 1.9 + 0.3

LiHypó / saponin 3.7 + 0.4 3.1 + 0.3 2.9 + 0.4 3.7 + 0.3 2.3 + 0.3

LiHyV / saponin 3.0 + 0.3 2.6 + 0.2 2.3 + 0.4 3.0 + 0.2 1.9 + 0.5

HRF / saponin 4.1 + 0.4 3.7 + 0.4 3.3 + 0.4 3.9 + 0.3 2.5 + 0.4

Chimera / saponin 1.8 + 0.3 1.6 + 0.2 1.5 + 0.3 1.6 + 0.5 0.8 + 0.3 Example 5- Evaluation of cellular immune response following challenge infection.

Evaluation of cellular immune response following challenge infection was also performed. Splenocyte cultures of the immunized and infected animals were stimulated with the specific proteins used in the immunizations or a mixture of extracts from the two parasites (SLA) and the IFN-γ, IL-12, GM-CSF, IL-4 and IL-10. Results showed significantly higher levels of IFN-γ, IL-12 and GM-CSF in the vaccinated group compared to the levels of these cytokines in the splenocytes of the control groups (Table 8). As observed before infection, the chimera plus saponin immunized group produced higher levels of IFN-γ, IL-12 and GM-CSF than the levels of these cytokines obtained in the groups immunized with rLiHypl, rLiHyp6, rLiHyV or rHRF proteins. saponin. However, animals in the control groups (saline and saponin) produced significantly higher levels of IL-4 and IL-10 than the other groups evaluated (Table 9).

Table 8- Levels of production of IFN-γ, IL-12 and GM-CSF cytokines (pg / ml). Cytokines were quantified in animal splenocyte culture supernatants. The mean ± standard deviation of the groups is shown.

Cytokines (in pg / ml)

IFN-gamma stimulation IL-12 GM-CSF

Medium 77 + 10 66 + 5 57 + 7

Saline Protein Mix 88 + 7 78 + 7 68 + 8

SLA 80 + 5 88 + 9 79 + 9

Medium 66 + 4 68 + 3 58 + 4

Saponin Mix Proteins 96 + 9 88 + 6 77 + 8

SLA 101 + 15 99 + 7 88 + 4 Medium 66 + 7 65 + 4 58 + 6

LiHyp 1 / saponin LiHyp 1 1,211 + 105 343 + 35 266 + 25

SLA 776 + 50 257 + 28 199 + 20

Medium 60 + 4 55 + 4 58 + 4

LiHypó / saponina LiHYpo 977 + 46 255 + 33 199 + 20

SLA 554 + 35 199 + 21 166 + 22

Medium 48 + 7 55 + 6 63 + 3

LiHyV / LiHyV saponin 1,101 + 90 288 + 23,203 + 26

SLA 667 + 33 255 + 20 196 + 25

Medium 52 + 2 59 + 6 55 + 4

HRF / saponin HRF 933 + 25 233 + 18 177 + 19

SLA 477 + 23 177 + 21 155 + 10

Medium 63 + 6 65 + 7 68 + 8

Chimera / Saponin Chimera 2,322 + 122,544 + 30,372 + 20

SLA 1.556 + 99 388 + 35 295 + 45

Table 9- IL-4 and IL-10 cytokine production levels (in pg / ml). Cytokines were quantified in animal splenocyte culture supernatants. The mean ± standard deviation of the groups is shown.

Cytokines (in pg / ml)

IL-4 Stimulus IL-10

Medium 59 + 7 65 + 6

Saline Protein Mix 98 + 12 86 + 5 SLA 488 + 40 455 + 25

Medium 64 + 5 55 + 4

Saponin Protein Mix 77 + 10 65 + 8

SLA 356 + 88 367 + 48

Medium 77 + 8 68 + 10

LiHyp 1 / saponin LiHyp 1 70 + 8 67 + 4

SLA 79 + 10 83 + 4

Medium 65 + 6 68 + 8

LiHypó / Saponina LiHY Powder 77 + 5 77 + 4

SLA 80 + 10 86 + 9

Medium 59 + 7 66 + 7

LiHyV / LiHyV Saponin 70 + 5 75 + 10

SLA 88 + 5 87 + 10

Medium 70 + 6 68 + 8

HRF / saponin HRF 121 + 11 133 + 25

SLA 101 + 9 106 + 12

Medium 55 + 6 59 + 4

Chimera / Saponin Chimera 66 + 5 75 + 6

SLA 73 + 7 80 + 6

Claims

1 - CHEMICAL PROTEIN, characterized in that it comprises the polypeptide sequence defined by SEQ ID NO: 1.

VACINAL COMPOSITION against cutaneous and visceral leishmaniasis, characterized in that it comprises the chimeric protein defined in claim 1 and pharmaceutically acceptable adjuvants.

VACINAL COMPOSITION against tegumentary and visceral leishmaniasis according to claim 2, characterized in that the adjuvants are preferably compounds capable of inducing the development of a Th1-type immune response, primared by the production of IFN-γ and IL-12 cytokines. , like the saponin.

VACINAL COMPOSITION against cutaneous and visceral leishmaniasis according to claim 2, characterized in that it can be administered by the intradermal, intramuscular, oral, nasal, intravenous, subcutaneous routes and / or as a device that can be implanted and / or injected.

Use of the CHEMERIC PROTEIN defined in claim 1, characterized in that it is for the treatment and / or prevention of cutaneous and visceral leishmaniasis in dogs and humans.

USE OF THE VACINAL COMPOSITION as defined in claims 2 to 4, characterized in that it is for the treatment and / or prevention of cutaneous and visceral leishmaniasis in dogs and humans.