ES2415516B2 - CHEMERA MULTICOMPONENT FOR USE AS A VACCINE AGAINST INFECTION BY LEISHMANIA SPP. IN MAMMALS. - Google Patents

Abstract

The present invention relates to a multicomponent chimera, HISA70, comprising 6 genes from Leishmania infantum: H2A, H2B, H3, H4, A2 and HSP70. Likewise, it is related to an immunogenic composition comprising said multicomponent chimera and the use of said composition in the preparation of a vaccine against Leishmania spp., To confer protection against leishmaniasis, including cutaneous leishmaniasis and visceral leishmaniosis, to mammals

Description

Multicomponent chimera for use as a vaccine against infection by

Leishmania spp. in mammals

FIELD T ~ CNICO

The present invention falls within the field of Animal Health.

More specifically, the invention relates to a genetic construct,

called HISA70, which contains 6 genes that encode 6 antigens from

Leishmania infanlum involved in the appearance of clinical symptoms of

leishmaniosis, and the use of such construction in the elaboration of a

vaccine for use against infection with Leishmania spp.

STATE OF THE TECHNIQUE

Leishmaniasis constitute a group of diseases with worldwide distribution and caused by intracellular protozoan parasites of the genus

Leishmania, which are transmitted by a vector insect known as

sandfly This disease presents various clinical and epidemiological conditions. According to data from WHO, the prevalence of the disease is 12 million cases and currently threatens 350 million people in 88 different countries (hllp: //www.who.inVtopics/leishmaniasis/en/index.hlmll.principalmente in underdeveloped countries in tropical and sub-tropical areas (Sudan, India, Bangladesh, Nepal and Brazil), but also in the Mediterranean area (700 cases a year in southern Europe). In response to the clinical symptoms of the disease, leishmaniasis have They have been classified into several types: visceral leishmaniasis (LV) and the rest of leishmaniosis with cutaneous manifestations: cutaneous (Le), cutaneous-diffuse leishmaniosis (LCD) and mucocutaneous (LMC or spundia). Leishmania infanlum is the main causative agent of LV in the Mediterranean region where, in addition, the domestic dog is considered as the main reservoir, playing a key role in the transmission of the parasite to humans.On the other hand, L. major is one of

the species with the greatest impact in the Old World causing Le

(Gramiccia M., Gradoni L., The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol. 2005. 35: 1169-1180).

At present, there is no completely effective prophylactic method against leishmaniasis. Chemotherapeutic treatment, in particular therapy with pentavalent antimonial compounds (Glucantime® and Pentostam®) that interfere with the parasite's metabolic pathways, is the most widely used for its ability to accelerate healing, decrease scar size and prevent possible Recall in patients with LC. In

In the case of LV, antimonial therapy reaches a clinical cure rate of 90%. But the use of antimonial compounds is not without effects

secondary. Amphotericin B has been shown to have high efficacy, and

its adverse effects can be avoided by administration in

liposomes, however its high cost limits its use in developing countries.

On the other hand, there is clinical and experimental evidence that demonstrates that vaccination can prevent leishmaniasis. In fact, in recent years, laboratory efforts focus on the search for new

antigens and adjuvants, live-attenuated vaccines, recombinant antigens

in the form of a protein or in the form of ONA, bacteria that express leishmanial antigens and vaccine strategies with dendritic cells (CO). Among the antigens that have been tested are: amastigote-specific A2 protein, sterol-24-c-methyltransferase (SMT), several histones, ribosomal PO protein, LmSTI1 stress-inducible protein and homologous receptor-like protein mammalian activated kinase C (LACK) (from Oliveira CI, Nascimento IP, Barral A., Soto M., Barral-Netto M., Challenges and perspectives in vaccination against leishmaniasis, Parasitol Int. 2009.

58: 319-324). In reference to canine leishmaniasis, a vaccine, Leishmune®, consisting of

Fucose-Mannose ligand that is administered in the presence or absence of

QuilA adjuvant (W02006122382A2). Also in Brazil, already registered, it

find another vaccine, LeishTeC®, composed of the recombinant antigen

A2 from L. donovani (W02009089605A1). However, in the case of vaccines developed in Brazil, they do not exceed 43-75% protection

5 and, in addition, the immunological mechanisms are not yet known in detail. Finally, it is worth mentioning another vaccine (CaniLesh) composed of secreted and excreted anigens of promastigotes of L. infanfum (ES2260440T3), whose marketing has been authorized by the European Commission throughout the European Union on March 14, 2011.

In fact, the development of vaccines against parasitic diseases, not

just in front of leishmaniosis, you have to overcome major obstacles due to

different reasons. Among them, the following can be mentioned: it is about

infections that tend to become chronic depending on the state

Immune of the host and the species of parasite involved; the

parasites display a huge variety of evasion mechanisms from the

immune response; and the intracellular location of the parasite often hinders the degree of effectiveness of effector responses by the host.

20 There is therefore a need to continue working on the development of new vaccination strategies focused on the Mediterranean distribution area of L. infanfum. It is about inducing a proper balance between Leishmania-specific immune responses of cellular and humoral type,

as well as the generation of effective memory cells against possible

25 reinfections. Among the various immunization methodologies, genetic vaccination (third generation vaccines) is one of the most efficient. It is capable of inducing the synthesis of the protein it encodes and of generating a specific immune response in the long-term host and, in addition, does not generate adverse reactions at the site of inoculation,

30 unlike protein administrations. Another alternative of great interest that has begun to be analyzed recently is CD-based vaccination to induce protective immune responses against leishmaniasis.

Many of these strategies are based on adoptive transfer in mice

BALB / c of CDs loaded with leishmanial antigens. Another alternative that has

proven effective is the use of various vaccine vehicles

recombinants of an attenuated or non-pathogenic nature in humans; for example, Salmonefla attenuated against LC or a recombinant species of

Le; shman; a non-pathogenic in humans in which the

A2 antigen from L. donovani (Costa CH, Peters NC, Maruyama SR, from Brito EC Jr, Santos IK. Vaccines for the leishmaniases: proposals for a research agenda. PLoS Negl Trap Dis. 2011 Mar 29; 5 (3): e943 .).

In the process of infection, various parasitic components of Leishmania can trigger an immune response as determinants of

surface or as intracellular determinants. The first get to be

invisible to the host's immune system to establish the infection. Intracellular determinants, initially, are also not visible to the immune system, and thus, manage to enter the host, without

However, they are exposed at the moment in which the lysis of

The parasites These determinants are highly conserved throughout evolution and contain specific epitopes of T cells and B cells.

20 intracellular localization makes them inaccessible to the antibodies produced and, in addition, they generate a virulent phenotype that causes pathology, so they are called "pathogens" (Chan9 KP, Reed SG, McGwire BS, Soon9 L. Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicily Acta Trop. 2003 Mar; 85 (3): 375-90.)

There is an urgent need to develop vaccination strategies

genetics either using CD or using live attenuated bacteria such as

Vaccine vehicle, which are constituted as potential tools of the first order in the fight against the different forms of leishmaniasis.

DESCRIPTION OF THE INVENTION

The invention faces the problem of developing new elements and

strategies with which to develop vaccines capable of conferring protection

against infection caused by Leishmania spp.

The solution provided by this invention is based on a chimera,

HISA70, which can be inserted into a eukaryotic expression plasmid or in

a prokaryotic expression plasmid, and whose administration confers

protection against subsequent infection with Leishmania spp. In

experiments performed with mice immunized with this chimera, after a

infection with L. major, which causes him, animals develop the ability to control the appearance of lesions at the point of infection (pad

plantar), maintain a low parasitic load in popliteal lymph node and

prevent parasitic visceralization. This last fact is of a great

relevance considering the extrapolation of its protective capacity against

the visceral form of leishmaniosis, where the target organs are spleen, liver and bone marrow. In fact, other tests conducted in mice vaccinated in the same way but infected with L. infantum, responsible for l V, show how half of the animals are able to reduce visceral parasitic load (both in liver and spleen) .

In the present invention, "multi-component chimera" is understood as a

Recombinant DNA molecule that comprises the fusion of different genes that encode the pathogens of interest. In the same sense, the expression of said recombinant molecule gives rise to what, in the present invention, is called "chimeric protein". "Genes in phase" means the arrangement of genes so that continuity in the reading phase of the genetic sequence is guaranteed, which in turn guarantees their

functionality, ie the expected protein translation.

One aspect of the present invention therefore relates to a chimera.

multicomponent, which we have called HISA70, which includes the phase fusion of genes from L. infantum that encode 6

pathogens involved in the parasitic virulence phenotype and, as a consequence, in the appearance of the clinical symptoms of leishmaniasis. Specifically, the multi-component chimera HISA70, comprises the genes that

encode nucleosomal histone proteins (H2A, H2B, H3, H4), A2 antigen and HSP70 thermal shock protein, from L. infantum, as described in SEQ ID NO: 1. The invention is also

refers to a plasmid, eukaryotic expression or prokaryotic expression, in the

that HISA70 is inserted to vehicle it. In this sense, if necessary, it

include the ends of the multicomponent chimera target sequences of

the restriction enzymes that facilitate the cloning of said chimera in the

selected vector.

In another embodiment of the invention, the multi-component chimera comprises a sequence with a percentage of identity with SEQ ID NO: 1 of at least 75%, the percentage of sequence identity being understood as the

percentage of matches of the same nucleotides between two sequences

aligned, along the entire length of both sequences.

Along the sequence of the multicomponent chimera, nucleotide triplets encoding glycins can be introduced, to give more structural flexibility to the protein resulting from the expression of the chimera. Compared to any other amino acid, glycine has the simplest structure (thanks to its group Hydrophobic aliphatic apolar side chain that joins the -NH2 and -COOH groups, respectively) that imposes the minimum impediment to be rich, so It is the amino acid of choice to achieve this effect. Between 1 and 7 nucleotide triplets that encode glycins can be introduced, placing them at the beginning and / or end of the

multicomponent chimera and / or at the beginning of each of the genes that the

make up in such a way that, in the resulting chimeric protein, glycines

30 will be located at the beginning and / or at the end of the chimeric protein, and / or at the beginning of the peptide resulting from the translation of each gene.

The invention also relates to an immunogenic composition comprising a multicomponent chimera as described herein, or a eukaryotic or prokaryotic expression plasmid containing a multicomponent chimera as described herein.

Another aspect of the invention relates to an immunogenic composition that

comprises the chemical protein HISA70, characterized by SEO ID NO: 2, or a protein with a percentage of identity with SEO ID NO: 2 from, to

less, 85%, for use in the development of a vaccine against

Leishmania spp. It is understood by percentage of sequence identity

amino acid the percentage of matches of the same amino acids between

the two sequences aligned, along the full length of both

sequences

In this sense, the vaccine made with any of the immunogenic compositions of the invention is used in the protection of mammals, more specifically of dogs and humans, against any of the

forms of leishmaniosis (cutaneous or visceral).

An advantage of the invention is the antigenic character and high specificity of each of its components (tool of great diagnostic value), so that the specific antibodies generated are directed towards the antigenic determinants of the divergent regions of the nucleosomal histones of the virulence factor A2, and of the HSP70 protein of the parasite, thus avoiding cross-reactivity with microorganisms causing other diseases, as well as autoimmunity processes.

DESCRIPTION OF THE DRAWINGS To facilitate the understanding of the main features of the invention and as an integral part of this specification, a

series of figures. For illustrative purposes, the following has been represented:

5 Figure 1. Schematic representation of the multi-component chimera

HISA70, with indication of the target sequences of the insertion enzymes

used for the cloning described in Example 1, and of the triplets encoding glycines introduced along the sequence, according to Example 1.

10 Figure 2. Progression of plantar lesion in the 3 groups of mice after infection with L. majar (Fig. 2A). Appearance of the lesions after 7 weeks of infection (Fig. 2B). The asterisks indicate the existence of significant differences with respect to the control groups (PBS and empty plasmid) (p <0.05).

15 Figure 3. Parasitic load in popliteal lymph node (0) and spleen (e) after 7 weeks of infection with L. majar. The average values are represented with a horizontal stroke. The asterisks indicate the existence of significant differences with respect to the control groups (PBS and plasmid 20 vacuo) (p <0.05).

Figure 4. Arginase activity (mU) in plantar lesion at 7 weeks of infection with L. majar. The average values are represented with a horizontal stroke. Asterisks indicate the existence of significant differences

25 with respect to the control groups (PBS and empty plasmid) (p <0.05).

Figure 5. Anti-Leishmania humoral title at 7 weeks of infection with L. majar. IgGl []) and IgG2a (11) isotypes. The asterisks indicate the existence of significant differences with respect to the control groups (PBS and plasmid

30 vacuum) (p <0.05).

Figure 6. Parasitic load in spleen (O), in liver (.), And their respective statistical means (-) at 8 weeks of infection with L. infantum.

MODE OF CARRYING OUT THE INVENTION.

Having described the present invention, it is further illustrated by the following examples.

Example 1. Cloning of genes. The coding sequences of H2A, H2B, H3, H4, A2 and HSP70 of L. infanlum were obtained from the GeneDB and GenBank sequence databases with the following access numbers: H2A (A J21.v3.1160), H2B (Un.J09.v3.1410), H3 (UnJl0.v3.0920), H4 (Un J31.v3.3320), A2 (GenBank S69693) and HSP70 (GenBank CAA69282.1). The corresponding coding regions were optimized for expression in mouse cells using the GeneOptimizer® expert software system (Geneart AG). Stop codons of the genes located in the first position and in the intermediate positions of the chimera were removed, and several glycine coding nucleotide triplets were introduced: at the beginning of H2A, H3 and A2, and at the end of HSP70, just in front of the stop codon of the multi-component chimera (Fig. 1). Finally, the sequences were chemically synthesized by GeneArt (http://www.geneart.com) giving rise to H / SA70, characterized by SEO ID NO: 1, which encodes the multi-component chimera HISA70, characterized by SEO ID NO: 2. In addition, recognition sites for restriction enzymes Xhol and Nofi were included to enable subsequent plasmid constructs. The HISA70 sequence was included in a model eukaryotic expression plasmid, which in this example was pCMVpm2A.

Example 2. Analysis of the protective capacity of HISA70. The protective capacity of HISA70 was tested in groups of 8-week-old BALB / c female mice (n = 6) that were immunized three times, at 2-week intervals, subcutaneously in the left plantar pad, with 100 ~ g of the eukaryotic expression plasmid used for cloning in example 1 as a control (pControl or plasmid control) or with the chimera

cloned into the eukaryotic expression plasmid (pHISA70). Another control group was inoculated with PBS following the same scheme. After 4 weeks

since the last immunization, all groups were infected in the

right plantar pad with 1 x 10 '5 L. majar metaclinical promastigotes (clan V1: MHOM / IU80 / Friedlin). The metacyclic, infective forms,

were previously isolated from parasitic cultures in phase

stationary using PNA peanut agglutinin (Vector Laboratories, Burlingame, CA). Following the current ethical regulations regarding animals of

experimentation, at the time when plantar lesions appeared

10 necrosed (at 7 'week of infection) in unprotected animals, all groups were euthanized. At the time of sacrifice, it

obtained sera, popliteal lymph nodes and spleens in order to perform

parasitic cultures and corresponding determinations

immunological All trials with mice followed the conditions

15 conventional approved by the ethical committee of animal experimentation of the Complutense University of Madrid.

Example 3: Evaluation of the ability to control the appearance of the skin lesion. In order to evaluate the capacity conferred by the vaccine 20 on the control of plantar inflammation at the point of infection, a weekly monitoring was made. For this, the degree of inflammation in the infected plantar pad (left) was measured with a caliber compared to the uninfected plantar pad (right). The clinical signs of ulceration were also evaluated. Figure 2A shows the

25 plantar inflammation size during 7 weeks post-infection and it can be observed that mice vaccinated with pHISA70 did not present lesions, or were very small, and, in addition, did not develop ulcerations (Fig. 2B) after 7 weeks of infection, unlike the control groups (PBS and peontrol).

Example 4: Assessment of the ability to control the parasite load

after an infection with L. major. At the time of sacrifice, they were obtained

Popllite lymph nodes, draining to the point of infection, and spleens of all mice in order to perform parasitic cultures in Schneider medium (Gibco, BRL) supplemented with 20% (v / v) inactivated FCS, streptomycin 0, 1 mg / ml and penicillin 100 U / ml. This medium was used both for the cultivation of the Leishmania major parasites until their stationary phase (2 x 107 parasites / mi) and for the differentiation of amastigotes to promastigotes, at 26 ° C in both cases, to determine the parasite load by dilution limit. For this, a cell suspension was prepared by macerating the organs in 1 ml of Schneider medium. 10 Subsequently, serial dilutions 1: 4 of the homogenized tissue suspension were made in a 96-well plate and incubated at 26 ° C

for 10 days. The wells were examined for promastigotes.

The total number of parasites per tissue was calculated from the reciprocal of the highest dilution where promastigotes were detected, taking into account the

15 weight of the entire organ. Figure 3 shows a significant reduction of the parasitic load in the popliteal lymph node and an absence of splenic parasitic visceralization in mice vaccinated with pHISA70, unlike the two control groups.

20 Example 5: Determination of the immune resistance of mice

mediated by arginase activity at the point of infection. As i know

has described (Iniesta V., Gomez-Nieto LC, Corraliza l., The inhibition of arginase by N (omega) -hydroxy-I-arginine controls the growth of Leishmania inside macrophages, J Exp Med. 2001. 193: 777-784 ), there is an association between the multiplication of Leishmania parasites within infected phagocytic cells and the arginase activity generated in said cells. The

Arginase is an enzyme involved in the synthesis of polyamines, nutrients

essential for parasitic proliferation. Therefore, the activity was analyzed

arginase in cell suspensions resulting from the maceration of

30 sections of the plantar pads of the mice of example 2, to determine the level of multiplication of Leishmania. The following method was used: to lyse cell membranes, 50 ~ I of Triton X-100 (0.1% v / see water) was added to each sample, stirred and 50 ~ I of 200 mM Tris-HCI pH was added 7.4. In the lysates 5 (MnCI JI, 1 M (arginase coenzyme) were added and incubated at 56 ° C for 10 min. Then 25 ~ I of 0.5 M arginine, pH 9.7 (substrate of the arginase) followed by incubation at 37 ° C for 1 h. In parallel, a standard curve was made using increasing amounts (1.5-30 ~ g) of urea. Subsequently, 400 ~ I of an acid mixture consisting of H was added , SO, 18 M, H, PO, 14.7 MYH, O (in relation

1: 3: 7). To quantify the urea formed, 25 ~ I of isonitropropiophenone (3% ISPF in absolute ethanol) was added. The samples were boiled at 99 ° C for 30 min. Finally, the samples were transferred to 96-well flat bottom plates (Nunc) in duplicates of 200 ~ I and their

absorbance in the corresponding reader at 540 nrn. Figure 4 shows

how protected mice belonging to the pHISA70 vaccination group were the only ones that had significantly low levels of arginase activity at the site of infection, which correlate with a

lower parasite load compared to control groups.

Example 6: Specific parasitic humoral response. Previous studies of LC in the murine model have shown that the relative production of IgG1 and IgG2a antibodies is correlated with the induction of Th2-susceptibility and Th1-resistance to Leishmania, respectively (Scol! P. The role of TH1 and TH2 cells in experimental cutaneous leishmaniosis Exp Parasitol. (1989) 68: 369-372). In this context, the detection, in the sera of mice immunized with pHISA70 and of the two control groups at 7 weeks post-infection, of Leishmania-specific antibodies by ELlSA was performed. Standard plates were upholstered with the Leishmania soluble antigen (SLA: 2 g / ml) for 16 h at 4 ° C in PBS. All incubation periods were done under stirring conditions at room temperature. Serial dilution of the sera was performed to determine the antibody titer, which was defined as the inverse of the highest serum dilution factor with an absorbance> 0.2. As secondary antibodies the isotypes were used

IgGl and goat anti-mouse IgG2a with peroxidase (Nordic Immunology, Tilburg, The Netherlands) diluted 1/1000. After 1 h of incubation, the plates

were washed and revealed by adding the peroxidase substrate (ABTS,

Rache Diagnostics, Spain) and measuring in the corresponding plate reader at 405 nm. Protected mice that had been vaccinated with pHISA70 produced a low Leishmania-specific humoral titer, mixed type: IgGl and IgG2a (Fig. 5), compared to the high titre of IgGl parasite.

specific presented by the control groups. As a whole, this data

show the ability of the vaccine to change in the host a Th2 profile of susceptibility to Leishmania to a mixed immune balance oriented to a Thl profile of resistance against L. majar infection. Which means that pHISA70 arouses the ability to control infection in vaccinated mice.

Example 7: Assessment of the ability to control the parasite load after infection with L. infantum. The immunization of animals is

performed as described in example 2 but using L. intan / um in

L time to get the infection. At the time of sacrifice, the

spleens and liver of all mice in order to perform parasitic cultures in Schneider medium (Gibco, BRL) supplemented with 20% (v / v) inactivated FCS, streptomycin 0.1 mg / ml and penicillin 100 Ulml. This medium was used both for the cultivation of the Leishmania intan / um parasites until their stationary phase (2 x 107 parasites / ml) and for the differentiation of amastigotes to promastigotes, at 26 ° C in both cases, to determine the parasite load by dilution limit. For this, a cell suspension was prepared by macerating the organs in ml of

Schneider half. Subsequently, serial dilutions were made 1: 4 of the

suspension of homogenized tissue in a 96-well plate and were incubated at 26 ° C for 10 days. The wells were examined for promastigotes. The total number of parasites per tissue was calculated from the reciprocal of the highest dilution where promastigotes were detected, taking into account the weight of the entire organ. In figure 6

a significant reduction in parasitic loads in the spleen and in the liver of vaccinated mice can be observed with pHISA70, unlike the two control groups.

FREE TEXT OF THE LIST OF SEQUENCES

The free text used in the sequence list corresponds in Spanish to

The following features:

SEO ID NO: 1 Artificial DNA sequence; genetic construction that comprises genes H2A, H2B, H3, H4, A2 and HSP70 of L. infantum. Nucleotides from 7 to 402: H2A gene of L. infantum without stop codon. Nucleotides from 403 to 735: H2B gene of L. infantum without stop codon. Nucleotides from 736 to 1125: H3 gene of L. infantum without stop codon. Nucleotides from 1126 to 11425: H4 gene of L. infantum without stop codon. Nucleotides from 1426 to 2133: gene A2 of L. infantum without stop codon. Nucleotides from 2134 to 4095: HSP70 gene of L. infantum. Nucleotides 1 to 6: restriction enzyme recognition site Xhol Nucleotides from 4096 to 4103: enzyme recognition site of

Notl restriction

SEO ID NO: 2 Artificial sequence, protelna: nucleotides from 7 to 4095 of SEO ID NO: 1 CLAIMS

Claims (14)

1. Multicomponent chimera comprising a sequence with at least 75% identity with SEO ID NO: 1. 2. Multicomponent chimera according to claim 1 wherein the sequence is SEO ID NO: 1. 3. Multi-component Ouimera according to any of claims 1-2 in  that the genes included in the sequence are in phase. 4. Ouimera multi-component according to any of claims 1-3 in  that the genes come from Leishmania spp. 5. Multicomponent chimera according to claim 4 wherein the genes They come from Leishmania infanlum. 6. Ouimera multicomponente according to any of claims 1-5 which includes between 1 and 7 triplets encoding glycine, located at the beginning and / or at the end of the chimera, and / or at the beginning of each gene. 7. Multi-component chimera according to claim 6 wherein the number of Triplets encoding glycine that are included is 4. 8. Eukaryotic expression or prokaryotic expression plasmid comprising  the chimera of claims 1-7. 9. Chimeric protein comprising a sequence with at least 85% identity with SEO ID NO: 2. 10. Chimeric protein according to claim 9 wherein the sequence is SEO IDNO: 2. eleven . Chimeric protein according to claim 10 which includes between 1 and 7 glycines at the beginning and / or at the end of the chimeric protein, and / or at the beginning of peptide resulting from the translation of each gene. 12. Immunogenic composition comprising the chimera of the claims 1-7 or the plasmid of claim 8 or the chimeric protein of claims 9-11. 13. Use of the immunogenic composition of claim 12 in the preparation of a vaccine for administration to a mammal to confer protection against diseases caused by Leishmania spp. 14. Use according to claim 13 wherein the Leishmania species is 15 L. infantum or L. major. 15. Use according to any of claims 13-14 in which the disease Caused by Leishmania is visceral leishmaniasis or cutaneous leishmaniosis. Use according to any of claims 13-15 in which the mammal is the dog or the man.