ES2346281B1 - USE OF LEISHMANIA DHSP70-II STRAIN AS A VACCINE. - Google Patents

Abstract

Use of Leishmania \ DeltaHSP70-II strains as a vaccine.

The present invention relates to the use of organisms
of the genus Leishmania that have deleted the gene
HSP70-II, for the preparation of a medicament for the treatment or prevention of leishmaniasis in a mammal, more preferably in a dog, and more preferably in a human. It also refers to pharmaceutical compositions containing such organisms, and their use in the preparation of vaccines against leishmaniasis.

Description

Use of strains of Leishmania \ Deltahsp70-II as a vaccine.

The present invention is within the field of molecular biology and medicine, and refers to a defective Leishmania line for the two alleles of the HSP70-II gene (line \ Deltahsp70-II ), to a composition comprising organisms of said line, and the use of said composition as a vaccine.

Prior art

Protestant organisms of the genus Leishmania are the causative agents of a broad group of diseases known as leishmaniasis or more correctly according to SNOAPAD ( Standard Nomenciature of Animal Parasites and Diseases ), leishmaniasis. This disease presents diverse clinical and epidemiological conditions, affects about 12-14 million people worldwide, and every year between 1.5 and 2 million new cases appear (Murray et al ., 2005. Lancet 366: 1561- 1577). According to the WHO, it currently threatens 350 million people in 88 different countries, mainly in tropical and subtropical areas, but the sick are mainly in developing countries (Sudan, India, Bangladesh, Nepal and Brazil). These organisms have a digenetic life cycle, alternating between a free life phase, flagellated promastigotes in mosquitoes of the genera Phlebotomus (in the old world) and Lutzomyia (in America), and intracellular non-flagellated amastigotes that multiply inside macrophages of host mammals. Transmission from insect ectodermal (poikilothermal) vectors to mammalian hosts implies a thermal "shock", which seems to act as a signal of cellular differentiation (Zilberstein & Shapira, 1994. Annu Rev Microbiol 48: 449-470). In this context, the study of the response to thermal shock, and the regulatory mechanisms controlled by HSP proteins, can elucidate the differentiation mechanisms of these protozoa.

Living organisms respond to heat stress, and other types of stress, by synthesizing a set of highly conserved proteins, heat-shock proteins (HSPs) (Lindquist & Craig, 1988. Annu Rev Genet 22: 631-677). However, HSPs also play an important role in non-stressed cells, being involved in the folding, assembly, intracellular localization, secretion, activation, and degradation of many proteins (Feder & Hofmann, 1999. Annu Rev Physiol 61 : 243-282). Both HSP70 and HSP90 interact with many other components of signaling pathways that regulate growth and development. In this sense, the molecular relationship between HSPs and signaling proteins is critical for the proper functioning of these pathways: for example, the relative levels of these proteins have proven to be important, since a deficiency or an excess of HSPs has resulted in aberrant growth, development of malformations and cell death (Mayer & Bukau, 2005. Cell Mol Life Sci 62: 670-684).

To date HSP70 is the most conserved protein present in all organisms. Among its numerous functions, HSP70 protects cells from the deleterious effects of incorrect folding and protein aggregation. The authors of the present invention have shown that loci of the HSP70-II gene are conserved in several Leishmania species ( L. infantum, L. major, L. tropica, L mexicana, L amazonensis ) (Folgueira, et al ., 2007. Parasitology 134, 369-377).

Currently there has been an increase in cases of human leishmaniasis in immunosuppressed individuals, in an advanced stage of HIV infection, whose immune defenses are very weakened. In Spain, and other countries in the Mediterranean basin, the predominant species is L. infantum , whose infection mainly causes visceral conditions. The domestic dog is considered the main reservoir of L. infantum in the Mediterranean region, playing a key role in the transmission of the parasite to humans through the sandfly. In countries like Spain, where the disease is endemic in dogs, almost everyone suffers a latent infection, so that when a person suffers a decrease in defenses, as in the case of AIDS and other immunodeficiencies, the Leishmania hitherto latent It begins to thrive in the body. In Europe, 25% of those infected with the AIDS virus suffer both a leishmania infection. In addition, canine leishmaniasis is a problem in itself, since about 50% of infected dogs develop a clinical symptomatology that in most cases leads to the death of the animal, due to the ineffectiveness of treatment methods and The toxicity of its side effects.

Based on the clinical symptoms of the disease, leishmaniasis have been classified into 5 types: visceral or kala-azar leishmaniasis, cutaneous or East Button, cutaneous-diffuse, mucocutaneous or spundia, and post - dermal leishmaniasis kala-azar . These forms have been classified based on clinical criteria and are caused by at least 15 different species.

There is currently no effective prophylactic method and chemotherapeutic treatments do not have the desirable efficacy. Since the middle of the last century, drugs that require parenteral administration and prolonged treatments have been used, also presenting a series of adverse reactions that hinder their use (cardiotoxicity with abnormal electrocardiograms, pancreatitis, etc.). Among the drugs used as the first line of treatment are pentavalent antimonials (SSG ( Sodium stibogluconate ) and Meglumina antimoniato). As a second line (when the patient does not respond to the first) there is Amphotericin B and Aminosidine. An additional problem, since patients are mainly in developing countries, is the high price of treatments, which sometimes leads to non-compliance and the emergence of resistance.

Although there are active ingredients of origin Vegetable and synthetic with promising and low leishmanicidal activity toxicity (peptides, organic synthesis molecules etc), but so far alone, they are not able to totally eliminate the parasite. And although several vaccines are in different stages of development, there is currently no approved vaccine Against the disease There is, therefore, the need to develop a vaccine capable of generating a response that integrates activation cellular and antibody production with a certain balance towards a type 1 response.

Description of the invention

The authors of the present invention have developed a line of L. infantum deficient for the HSP70-II gene (line \ Deltahsp70-II ), characterized in that its virulence is attenuated, not presenting variations in it, and why it is capable of generating immunity in a mammal, both against leishmaniasis caused by L. infantum and L. major .

As stated previously, the loci of the HSP70-II gene are conserved in other Leishmania species ( L. infantum, L. chagasi, L. major, L. tropica, L. mexicana, L. braziiiensis and L. amazonensis ) , and, therefore, mutants of these species that have the HSP70-II gene deleted could be used as vaccines against different leishmaniasis.

Leishmania organisms that have the HSP70-II gene deleted, can be used to generate immunity in a mammal against leishmaniasis. Thus, a first aspect of the invention relates to the use of Leishmania organisms that have deleted the HSP70-II gene, hereinafter Leishmania organisms of the invention, for the preparation of a medicament for the treatment or prevention of leishmaniasis

The organisms of the genus Leishmania belong to the Superuk Eukaryota , Order Kinetoplastida , Family Trypanosomatidae .

The methods for deleting a certain gene in the genome of an organism are known in the state of the art. Generally, the deletion methods involve several cloning steps, and comprise the construction of a deletion cassette containing an expression cassette for a marker gene flanked with base pair sequences corresponding to the promoter and terminator of the gene to be deleted. Subsequently, deletion cassettes are amplified by PCR and used to transform selected Leishmania strains. Next, the transformants are selected based on said marker gene. A " cassette " or "cassette" is a coding region of a gene from a prokaryotic or eukaryotic organism flanked by the regulatory elements necessary for its expression in vivo or in vitro . Although expression cassettes can have very varied configurations, they must contain at least one promoter ( promoter ), a coding region (eukaryotic cDNA or prokaryotic gene) and a transcription terminator ( terminator ) or a polyadenylation site, as appropriate of a gene derived from a prokaryotic organism or a cDNA from a eukaryotic organism. To this basic configuration is added, if necessary, a sequence with regulatory function for the natural expression of the gene in the chosen system.

In the context of the present invention, HSP70-II is defined by a nucleotide or polynucleotide sequence, which constitutes the HSP70 protein coding sequence (SEQ ID NO: 1), or other homologous sequences, and which has a 3 'region Characteristic UTR, which is included in SEQ ID NO: 2, as already described (Quijada et al ., 1997. J Biol Chem 272: 4493-4499). The term "homology", as used herein, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between two or more nucleotide or amino acid sequences. Since two sequences are considered homologous if they have the same evolutionary origin, in general, it is assumed that higher values of similarity or identity of 50% would indicate homology. We can consider, therefore, that identity percentages of at least 50% could maintain the function of the orthologous amino acid sequence collected in SEQ ID NO: 1, and the nucleotide sequence encoding said sequence and presenting a characteristic 3'UTR region as described (3'UTR-II) could be referred to as HSP70-II gene.

In this report, "leishmaniasis" is understood as a zoonotic disease caused by different species of protozoa of the genus Leishmania . The clinical manifestations of the disease range from skin ulcers that heal spontaneously to fatal forms in which severe inflammation of the liver and spleen occurs. The disease by its zoonotic nature, affects both dogs and humans. However, wild animals such as opossums, coatis and anteaters among others, are asymptomatic carriers of the parasite, so they are considered as reservoir animals.

In the genome of L. infantum there are 6 copies of the HSP70 gene that are arranged in a direct tandem (Quijada et al ., 1997. J Biol Chem 272: 4493-4499). The main difference between the different copies lies in their 3'UTR regions: the 3'UTR region of the HSP70 gene located at the 3 'end of the locus (3'UTR-II) is highly divergent in sequence with respect to region 3 'UTR of the remaining 5 genes (3'UTR-I). Given this sequence divergence of the 3'UTR regions, the first five HSP70 genes, those that carry the 3'UTRI region, have been called HSP70-I genes, while the sixth gene, the carrier of the 3 'region UTR-II, has been called HSP70-II. In addition to the sequence divergence of their 3'UTR regions, the HSP70-I and HSP70-II genes show clear differences in their expression during thermal shock. In other Leishmania species ( L. major, L. tropica, L. mexicana and L. amazonensis ) the arrangement of these loci, and the characteristic region 3'UTR-II, is preserved (six tandem copies for L. infantum and L. major , and 7 tandem copies for L. tropica, L. mexicana and L. amazonensis ), as described in Folgueira et al ., 2007 ( Parasitology 134: 369-377).

Therefore, in a preferred embodiment of this aspect of the invention, the organisms in which the HSP70-II gene is deleted belong to a species of Leishmania that is selected from the list comprising: Leishmania infantum, L. chagasi, L. major, L. tropica, L. mexicana, L. braziliensis and / or L. amazonensis . In another even more preferred embodiment, the organisms of the strain belong to the species L. infantum .

Leishmaniasis is especially important not only in developing countries, but also in developed countries, as it is a zoonosis in which the dog is considered the main reservoir of the L. infantum parasite. In this way, the cases registered in many countries, including Spain, are due to contact with dogs. It has been observed that cats and rats can also act as reservoirs. Therefore, in a preferred embodiment of this aspect of the invention, the mammal is a dog. In another preferred embodiment of this aspect of the invention, the mammal is human. In an even more preferred embodiment, leishmaniasis is caused by the parasite L. infantum .

Dogs belong to the species Canis lupus subsp. familiarize yourself . Organisms of the species Canis lupus subsp. familiaris belong to Superuk Eukaryota , ( Metazoa / Fungi group ), Metazoa Kingdom, Phylum Chordata , Craniata Subphylum, Mammalia Class, Carnivorous Order, Canidae Family and Canis Genus. Man is a human mammal that belongs to the species Homo sapiens . The organisms of the species Homo sapiens belong to Superuk Eukaryota , ( Metazoa / Fungi group ), Metazoa Kingdom, Phylum Chordata , Subphylum Craniata , Mammalia Class, Primates Order, Hominidae Family and Genus Homo .

The use of Leishmania organisms of the invention, in which the HSP70-II gene is deleted, as immunogens, means that they can be used in compositions such as vaccines in mammals, including humans, or produce a response in the production of antibodies in animals . For the formulation of such compositions, an immunologically effective amount of at least one of the Leishmania organisms of the invention is mixed with a suitable physiologically acceptable carrier for administration to mammals including humans. The immunogens may be incorporated into liposomes or other similar vesicles, and / or mixed with an adjuvant or absorbent as is known in the field of vaccines. For example, they can be mixed with other immunostimulatory complexes. Alternatively, the immunogens are not coupled and merely mixed with a physiologically acceptable carrier such as a normal buffered or saline compound suitable for administration to mammals including humans.

Thus, another aspect of the invention relates to a pharmaceutical composition, hereinafter referred to as the composition of the invention, comprising Lehismania organisms of the invention, that is, having the HSP70-II gene deleted. In a preferred embodiment of this aspect of the invention, the pharmaceutical composition comprises promastigotes of the Leishmania strains of the invention. In another preferred embodiment the composition of the invention further comprises pharmacologically acceptable excipients.

The term "promastigote" refers to the flagellar state of a trypanosomatid protozoan in which the scourge arises from a kinetoplast in front of the nucleus and emerges from the anterior termination of the organism (apical pole). It is generally the extracellular phase, in the intermediate invertebrate host or in culture, of Leishmania parasites.

Therefore, and as described above, the immunogens (the Leishmania organisms of the invention) of the invention would exhibit protective antigenic sequences. These protective antigens are capable of generating a protective (immunogenic) host immune response, that is, a host response that leads to the generation of immune effector molecules, antibodies or cells that damage, inhibit or kill the invading biological entity, " thus protecting "the host from a clinical or sub-clinical disease and a loss of productivity. Such a protective immune response can commonly be manifested by the generation of antibodies.

Thus, another aspect relates to antibodies, hereinafter antibodies of the invention, produced after immunization of an animal with Lehismania organisms of the invention or the composition of the invention. In a preferred embodiment of this aspect of the invention, the animal used for immunization is a non-human mammal.

In another aspect of the invention, the antibodies produced after immunization of the animal, hereafter Antibodies of the invention are used as medicament.

The antibodies of the present invention can formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways. So, the antibodies may be in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions can be buffered or unbuffered and have active components or additional inactive Additional components include salts to modulate ionic strength, preservatives including, but not limited to antimicrobial agents, antioxidants, chelators, and similar, and nutrients including glucose, dextrose, vitamins and minerals Alternatively, antibodies can be prepared for its administration in solid form. The antibodies can be combined with several inert vehicles or excipients, including but without limit yourself to; binders such as microcrystalline cellulose, gum tragacanth, or jelly; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.

Antibodies or their formulations can administered to an animal, including a mammal and, therefore, to Man, in a variety of ways. Such means include, but without limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.

The dosage of antibodies to obtain a pharmaceutically effective amount depends on a variety of factors, such as age, weight, sex, tolerance, ... animal.

Another aspect refers to the composition of the invention, for use as a medicine. Another aspect of the invention refers to the use of the composition of the invention, for The preparation of a medicine. Another aspect of the invention is refers to the use of the composition of the invention, for the development of a medicine for the treatment or prevention of leishmaniasis In a preferred embodiment of this aspect of the invention, the drug is a vaccine, hereinafter vaccine of the invention. In a more preferred embodiment, the vaccine also It comprises pharmacologically acceptable excipients. In other preferred embodiment of this aspect of the invention, the composition (or the vaccine) of the invention further comprises a adjuvant In another preferred embodiment the vaccine is polyvalent.

In the context of the present invention the term "vaccine" refers to an antigenic preparation used to establish the immune system response to a disease. They are antigen preparations that once inside the organism provoke the response of the immune system, by means of antibody production, and generate immune memory producing permanent or transient immunity.

The term "antigen" herein is refers to a molecule (usually a protein or a polysaccharide), which can induce the formation of antibodies. There is many different types of molecules that can act as antigens, such as proteins or peptides, polysaccharides and, more rarely, Other molecules such as nucleic acids.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, it also refers to the organisms of the invention, or to the composition of the invention comprising organisms of the invention in a therapeutically effective amount, which is capable of generating an immune response against an organism of the genus Leishmania , which is causing such disease in man or animals. It includes, therefore, what is known as a vaccine, as previously defined herein.

In this report, the term "adjuvant" is refers to an agent, as long as it does not have an antigenic effect in case same, which can stimulate the immune system by increasing its vaccine response. Although not limited to them, the salts of aluminum "aluminum phosphate" and "aluminum hydroxide" They are the two adjuvants most commonly used in vaccines. Other substances, such as squalene, can also be Use as adjuvants.

As defined herein, the term "polyvalent" is used to refer to the vaccine comprising the combination of two or more antigens in total, including one or more of any of the species, types, varieties or mutants that are included in the classification of the genus Leishmania .

In the sense used in this description, the term "therapeutically effective amount" refers to the amount of Leishmania organisms of the invention that allow to produce the desired effect. Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.

The compositions provided by this invention (comprising organisms the Leishmania strains of the invention, or antibodies of the invention) can be facilitated by any route of administration, for which said composition will be formulated in the appropriate pharmaceutical form and with pharmacologically excipients. acceptable to the route of administration chosen.

The terms "polynucleotide" and "acid nucleic "are used interchangeably here, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) as deoxyribonucleotides (DNA or DNA)

The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" is used interchangeably here, and refers to a polymeric form of amino acids of any length, which they may or may not be chemically or biochemically modified.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the figures

Fig. 1. (A). Growth rates of the wild-type Leishmania infantum line, the \ Deltahsp70-II mutant line and the HSP70-II re-expressing line. For the δ Deltahsp70-II and reexpressing lines HSP70-II (p70II-Pac-transfected mutant) were cultured in the presence (+) or absence of selection antibiotics. Data represent the mean and standard deviation of at least three independent experiments. (B) Proliferation of promastigotes of the wild type, of the δ Deltahsp70-II mutant and of the HSP70-II re-expressing line was determined by CFSE staining.

Fig. 2. Morphology of the wild-type promastigotes of Leishmania infantum , of the mutant line \ Deltahsp70-II and of the reexpressing line HSP70-II. At different times along the growth curve (cell densities are shown in Fig. 1A), promastigotes were recovered from the culture and stained with Giemsa. Promastigote morphology was recorded by microscopy. The images were taken under immersion oil using the 63x objective. The arrows indicate cells with two flagella.

Fig. 3. Analysis by flow cytometry of the interaction of promishigotes of the wild type of Leishmania infantum , of the mutant line \ Deltahsp70-II and of the reexpressing line HSP70-II with human monocytes U937: (A) Uninfected macrophages ; (B) macrophages infected with wild-type promastigotes; (C) macrophages infected with promastigotes \ Deltahsp70-II; (D) macrophages infected with HSP70-II reexpressing promastigotes (tah Deltahsp70-II mutants transfected with p70II-Pac). Data represent percentages of labeled CFSE macrophages, at least 30,000 were counted. (E, F) Rates of macrophage infection by promastigotes of the wild type of Leishmania infantum , of the mutant line \ Deltahsp70-II and of the reexpressing line HSP70-II; (E) percentage of infected macrophages; (F) number of parasites per infected cell. The data are means and standard deviations of two
experiments

Fig. 4. Determination of the presence of nitrites in the culture medium through the use of Griess reagent four weeks after infection by stationary promastigotes of L. infantum , in control mice and mice vaccinated with stationary promastigotes ( infectives) of the \ Deltahsp70-II line intravenously.

Fig. 5. Evolution of the lesions caused by the infection of mice with L. major in the control mice (A) and in the vaccinated mice (B) with stationary phase (infective) promastigotes of the line? Deltahsp70-II via intrave-
nosa.

       \ vskip1.000000 \ baselineskip
    

Examples

The invention will be illustrated by tests performed by the inventors which reveals the specificity and effectiveness of the strain of L. infantum deficient for hsp70-II gene (line \ Deltahsp70-II) for the preparation of a vaccine against to leishmaniasis.

       \ vskip1.000000 \ baselineskip
    

Example 1 Generation of a defective Leishmania infantum mutant line for the hsp70-II gene Parasites

Promastigotes of Leishmania infantum from strain MCAN / ES / 96 / BCN / 150, MON-1 in Vitro at 26 ° C were grown in RPMI 1640 medium (Invitrogen), supplemented with 10% (v / v) fetal bovine serum (FCS, heat inactivated for 30 min. At 56 ° C), 0.1 mg / ml streptomycin and 100 U / ml penicillin.

Clones used

The constructs were first made on the pBICAT vector (Larreta et al ., 2004. BMC Mol. Biol. 5: 3), a derivative of pBluescript containing the CAT gene, encoding the chloramphenicol acetyl transferase enzyme of E. coli . Finally, the constructs were transferred to the expression vector for trypanosomatids pX63Neo (LeBowitz et al ., 1991. Gene 103: 119-123).

To obtain the regulatory regions of the HSP70 genes, the clones pUCB2C, which contains the 5.72 kb Sal I fragment from the genomic clone B2g3, and pUCB2F, which contains the 3.81 kb Sal I fragment were started from from the B2g6 genomic clone (Quijada et al ., 1997. J Biol Chem 272, 4493-4499).

To clone the 5'-UTR and upstream regulatory sequences, contained in plasmid pUCB2F, the oligonucleotides collected in the sequences (SEQ ID NO: 3 and SEQ ID NO: 4) which include the included regions recognized by restriction enzymes to facilitate cloning. Digestion of the PCR product with the enzymes Xba I and Eco RV, resulted in a 1664 bp fragment that was cloned into the pBICAT vector to give rise to clone pCATC3 '. pBICAT is a derivative of pBluescript containing the coding region of the CAT gene (Larreta et al ., 2004. BMC Mol. Biol. 5, 3).

To clone 3'-UTR_II together with the downstream sequence, clone pUCB2C was used as a template for amplification with specific primers. This clone is a derivative of pUC18 that contains the 5.72-kb SalI fragment of the genomic clone pB2g3 (Quijada et al ., 1997. J. Biol. Chem. 272, 4493-4499). The specific primers were, the direct (SEQ ID NO: 5), and the reverse (SEQ ID NO: 6). The amplified DNA fragment was digested with HindIII plus SalI, and the 1685pb fragment was cloned into CATC3 'to obtain CATC3'C6. This plasmid was digested with BglII, and the chimeric gene CAT-HSP70-II was cloned into pX63NEO to generate pXcat70-II. Both constructs and PCR amplified fragments were checked by sequencing. The construction of plasmid pX5HisCAT3His, which contains the CAT gene flanked by UTRs derived from the histone H2A gene, has already been described (Larreta et al ., 2004. BMC Mol. Biol. 5: 3).

       \ vskip1.000000 \ baselineskip
    

Null mutant generation \ Deltahsp70-II

The HSP70-II alleles were sequentially replaced with the ORFs of the selected marker genes comprising either the NEO gene or the HYG gene. The constructs were prepared and the marker genes were flanked by specific regions located upstream and downstream of the ORF of the HSP70-II gene. For this purpose, the CATC3'C6 clone was chosen, which contains the CAT gene flanked by the upstream region plus the 5'-UTR and 3'-UTR plus the downstream sequences of the HSP70-II gene. The CAT gene in CATC3'C6 was replaced by double digestion with BstBI plus HindIII with the appropriate cassettes containing either the NEO gene or the HYG gene. For amplification of the NEO cassette, the cloning vector pcDNA3.1 (Invitrogen) was used as a template and the following primers, direct (SEQ ID NO: 7) and reverse (SEQ ID NO: 8). For the amplification of the HYG cassette, the cloning vector pIRES1hyg (Clontech) was used as a template and the following primers, direct (SEQ ID NO: 9) and reverse (SEQ ID NO: 10). The resulting constructions were called Neo70-II and Hyg70-II, respectively. 2 µg of these constructs were cut with BglII, and the linearized DNAs were used individually for the transfection of the HSP70-II gene in L. infantum . After transfection and selection, the resulting null mutant HSP70-II was designated as \ Deltahsp70-II :: NEO / \ Deltahsp70-II :: HYG , following the genetic nomenclature of Clayton et al. 1998. ( Mol. Biochem. Parasitol. 97: 221-224).

       \ vskip1.000000 \ baselineskip
    

Transfection of Leishmania promastigotes

DNA for transfection was prepared using the Maxi Kit system (Qiagen, Hilden, Germany). The promastigotes of L. infantum were collected in the late logarithmic phase by centrifugation, and were suspended at 2x10 8 parasites / ml in Cytomix buffer (van den Hoff, 1992. Nucleic Acids Res. 20: 2902), using 10 ^ 8} parasites by transfection. Electroporation was carried out by means of the Bio-Rad GenePulser using the conditions described in Robinson & Beverley, 2003 ( Mol Biochem Parasitol 128, 217-228). For clonal selection, the transfected parasites were cultured in blood agar plates (Quijada et al ., 2003. Mol. Biochem. Parasitol. 130, 139-141) supplemented with 20 µg / ml G418 (Roche Applied Science) or with 50 µg / ml hygromycin B (Sigma).

Therefore, promastigotes of both the L. infantum wild line (MCAN / ES / 96 / BCN150) and the mutant line ? Deltahsp70-II , were grown in vitro as described in Larreta et al ., (2004). BMC Mol. Biol. 5: 3). To culture the mutants, 20 µg G418 (Roche Diagnostics, Mannheim, Germany) / ml, and 50 µg hygromycin B (Sigma-Aldrich, St. Louis, MO) / ml were added to the medium.

       \ vskip1.000000 \ baselineskip
    

Example 2 Study of growth kinetics, microscopic examination and cell cycle analysis by line flow cytometry mutant \ Deltahsp70-II

Growth kinetics : Promastigotes were collected from stationary phase cultures and diluted to a concentration of 1 x 10 6 / ml in 10 ml of fresh medium. At 24-hour intervals, cell density was determined by enumeration in the hemocytometer. For fluorescent staining, promastigotes were collected, washed twice with phosphate buffered saline (PBS), and resuspended to a concentration of 1 x 10 7 cells / ml in PBS. CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester; Invitrogen) was added to a final concentration of 5 µM and the cells were incubated at 26 ° C for 10 minutes in the dark. The staining reaction was stopped by adding 5 volumes of frozen culture medium. Finally, the promastigotes were washed twice with the medium, resuspended at 1 x 10 6 cells / ml, and then cultured at 26 ° C. The average CFSE fluorescence was determined after staining at 1, 2, 3, 4, and 7 days of culture, by flow cytometry in a FACSCalibur cytometer (Becton Dickinson, San Jose, CA) using CellQuest software.

Microscopic examination : Promastigotes cultured at 26 ° C were taken for 4, 7, 9, or 12 days, washed twice with PBS, and placed on microscope slides. The preparations were fixed in methanol for 5 minutes, air dried for 24 hours, and stained with azur-eosin-methylene blue in solution according to Giemsa (Merck, Darmstadt, Germany) diluted 1/20 in distilled water and washed five times with distilled water. Images were taken using an Axioskop2 plus microscope and a Coolsnap FX color camera.

       \ newpage
    

Analysis of the cell cycle by flow cytometry : four million promastigotes were taken by centrifugation, washed twice with PBS, resuspended in 1 ml of fixative solution (30% PBS / 70% methanol) and incubated at 4 ° C for 1 hour. The fixed cells were collected by centrifugation and resuspended in 500 µl of pH 7 citrate buffer (45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, and 0.1% Triton X-100) containing 20 µg of RNase A (Roche Diagnostics) / ml and 50 µg of propidium iodide (Sigma-Aldrich) / ml. Samples were incubated at 37 ° C for 20 min in the dark and then fluorescence was measured by flow cytometry with a FACSCalibur cytometer.

Results

The delegated mutant δ Delsp70-II was viable, but its growth properties in axenic medium were different from those of the parental line, mainly during the late exponential phase (Fig. 1 A). The densities of the deletion mutant (12-14 x 10 6 per ml) during the stationary phase were lower than those of the parental line (25-27 x 10 6 per ml). Specifically, coinciding with the entry into the stationary phase, the stability of the \ Deltahsp70-II culture gradually decreases, while the number of microorganisms in the wild crop was maintained (and even slightly increased). Similar growth curves were obtained when the δ Deltahsp70-II mutant was grown in the absence or presence of hygromycin B and G418. Growth was quantitatively measured with the stable intracytoplasmic dye CFSE. When the CFSE stained cell divides, the dye is distributed between the daughter cells and the fluorescence intensity decreases correlatively. After staining of the Δ Promastigotes and wild with CFSE, the cultures were started at an initial density of 10 6 cells / ml. The CFSE fluorescence intensity was determined at 0, 1, 2, 3, 4 and 7 days of culture. Notably, until day 4, there was a similar decrease in fluorescence intensity in promastigotes \ Deltahsp70-II and wild. However, the distribution of the fluorescence peaks was wider in the \ Deltahsp70-II population than in the wild. Again, the growth of promastigotes \ Deltahsp70-II expressing HSP70-II in an episomal manner was more homogeneous than the growth of the mutant line (Fig. 1B).

Examination of the cultures under the immersion microscope revealed that the morphologies of the cells in the \ Deltahsp70-II cultures were remarkably heterogeneous. This was documented by Giemsa staining of the promastigotes of wild crops and Δ Delsp70-II at different times (Fig. 2). In the Del Deltahsp70-II cultures, a significant number of cells were smaller than in the wild promastigotes. In addition, rounded forms, promastigotes with two flagella, and dividing forms were observed more frequently in the \ Deltahsp70-II culture. The number of aberrant forms increased with time in the crop. In contrast, morphological alterations were rare after the introduction of an episomal copy of HSP70-II into the mutant line (Fig. 2).

The distribution of the cell cycle showed that the percentage of cells in the G2 / M phase was higher in the \ Deltahsp70-II cultures than in the wild cultures, a model that was maintained throughout the culture period. Similarly, the data showed that on days 4 and 5, the number of cells in the S phase was higher in the Del Deltahsp70-II culture than in the wild culture, indicating a delay in the S phase of the mutant cells.

       \ vskip1.000000 \ baselineskip
    

Example 3 In vitro macrophage invasion analysis, promastigote-macrophage interaction, experimental infection in BALB / c mice and antibody response analysis

The promonocytes of human cell lines of histocytic lymphoma U937 were induced to differentiate in macrophages by the addiction of 1 x 10-8 M forbol-myristate-acetate (PMA) at culture medium. These macrophages were then incubated in RPMI culture medium at 37 ° C / 5% CO2 with one phase stationary promastigotes with a protozoan ratio: 5: 1 cells. After two hours of incubation, the non-internalized promastigotes were removed and the remaining cells were incubated in a medium of RPMI culture at 37 ° C / 5% CO2 for more than 4 days. To the days 1, 2, 3 and 4 the cells were fixed with methanol and stained with Giemsa as described above. At least 600 were counted macrophages by preparation; the percentage of macrophages was determined infected and the number of intracellular promastigotes per cell infected.

To evaluate the interactions promastigote-macrophage, promastigotes were stained with CFSE (as previously described) and then incubated with macrophages U937 (multiplicity 5: 1 macrophage protozoan) during 24 hours at 37 ° C / 5% CO 2. CFSE flouorescence associated with macrophage was determined on a FACSCalibur cytometer.

Groups of 7-week-old female mice BALB / c (n = 4) were inoculated intravenously into the vein side of tail with 10 7 promastigotes in stationary phase in 100 µl of PBS. The serum obtained from the mouse was stored in 50% of glycerol at -20 ° C. Four weeks after infection, it evaluated the parasitic load in the spleen and liver by dilution limiting The reciprocal of the highest dilution that was positive for promastigote growth was considered the number of viable microorganisms per organ.

For analysis of the antibody response, the Leishmania crude antigen was prepared from L. infantum promastigotes by incubating the microorganism in a lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl pH 8 , and 1 mM PMSF) for 15 min. The suspension, stored on ice, was sonicated until a decrease in viscosity was observed. The insoluble material was centrifuged to form a pellet at 10,000 xg for 5 min, and the supernatant was stored immediately at -70 ° C until use.

Serum samples were analyzed for specific antibodies against total Leishmania antigens by a standard ELISA. Briefly, standard plates (NUNC A / S, Roskilde, Denmark) were stored overnight at 4 ° C with 100 µL of Leishmania crude antigen (2 µg / ml in PBS). Mouse serum was tested at 1: 100 dilution. As secondary antibodies, the following peroxidase conjugates (Nordic Immunology Laboratories, Tilburg, Netherlands) were used: goat anti-mouse IgG (1: 1000 dilution), goat anti-mouse IgG1 (1: 1000 dilution), and anti IgG2a - goat mouse (dilution 1: 1000). The peroxidase substrate was orthophenylenediamine dihydrochlorhydra (DAKO A / S, Glostrup, Denmark). After 30 minutes, the reaction was stopped by the addition of 100 µL of 1M H 2 SO 4, and the absorbance was read at 450 nm. The significance of the differences was examined by the Student t test; P <0.05 was considered statistically significant.

       \ vskip1.000000 \ baselineskip
    

Results

Continuous cultures and passes in Leishmania culture result in a gradual loss of infectivity. Therefore, since the generation of a δ Deltahsp70-II mutant by double replacement requires repeated transfection and drug selection, the possibility that the lack of infectivity of the mutant line is a consequence of prolonged in vitro culture should not be excluded. To test this possibility, BALB / c mice were inoculated with high doses of mutant microorganisms. At 28 days after infection, Leishmania cells were recovered from the spleen of the infected mouse, which provided a direct demonstration that the Del Deltahsp70-II mutant maintained its infectivity.

Flow cytometry was used to analyze the interaction between macrophages U937 and promastigotes \ Deltahsp70-II (Fig. 3). Although the percentage of fluorescent macrophages was comparable when these cells were cultured with both promastigotes \ Deltahsp70-II (76.54%) and wild (92.22%), there were marked differences in fluorescence intensities. The increased fluorescence shown by macrophages incubated with wild promastigotes was an indication that several protozoa were interacting with a single macrophage (Fig. 3B). Transfection of the δ Deltahsp70-II mutant with the p70II-Pac construct expressing HSP70-II markedly improved its ability to interact with macrophages (Figs. 3C, D).

Microscopic analysis of the Leishmania-macrophage interaction after 1, 2, 3, and 4 days of protozoan-macrophage incubation showed that mutant and wild promastigotes infected a similar number of macrophages, suggesting that HSP70-II deficiency does not affect penetration in the macrophages. However, after 48 hours, the number of amastigotes present in the macrophages differed significantly between the \ Deltahsp70-II and wild promastigotes, and the difference increased even more on days 3 and 4. Moreover, the re-expression of HSP70 -II in the deleted mutant was not enough to recover its limited ability to multiply within the macrophage (Fig. 3F).

The ability of the deletion mutant to cause visceral leishmaniasis in BALB / c mice was also investigated. Four weeks after infection, high numbers of microorganisms were present in the liver and spleen of the mouse infected with the wild promastigotes of L. infantum (Table 1). However, in mice infected with the δ Deltahsp70-II mutant, only protozoa were recovered in the spleen, and not in the liver. Based on the cell count, it was determined that the null mutant ? Deltahsp70-II was about 1000 times less infective than the parental strain in the spleen of the BALB / c and avirulent mice in the mouse liver.

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

(Table goes to page next)

       \ newpage
    

one

       \ vskip1.000000 \ baselineskip
    

Low numbers of protozoa were found in the spleen of mice infected with cells re - expressing HSP70-II (the δ Deltahsp70-II mutant transfected with p70II-Pac). After the reintroduction of the gene into the mutant ? Deltahsp70-II , the levels of HSP70-II mRNA were lower than in wild promastigotes. Analysis of the humoral response induced in mice infected with Leishmania showed that animals infected with wild promastigotes had a pronounced reactivity against total Leishmania antigens. Serum from mice infected with the Ahsp70-II mutant (or the mutant transfected with p70II-Pac) also reacted positively against Leishmania antigens, but to a lesser extent than sera from animals infected with wild culture. The analysis of specific IgG isotypes showed a significantly higher IgG2a / IgG1 ratio in animals infected with the Del Deltahsp70-II mutant (or the re-expressing mutant), indicating that there are qualitative differences in the humoral response of the mouse infected with the wild culture vs. The mutant

       \ vskip1.000000 \ baselineskip
    

Example 4 Study of virulence reversal

For the study of virulence reversal, sequential mouse infections were performed, as has been done in other cases (Spath et al ., 2004. Infect Immun 72 , 3622-3627). When infections were performed sequentially, parasites recovered from the spleen of mice infected with the \ Deltahsp70-II mutant were used to perform successive infections. The mutant line tah Deltahsp70-II produced very low parasitemias in the spleen, which were between one thousand and one million times lower than that produced with an equal dose of wild promastigotes of L. infantum . In no case, parasites were detected in the liver of mice infected with the Del Deltahsp70-II line, while this organ was highly infected with the parasites of the wild-type L. infantum line . Throughout the process no variation was detected in the virulence of the mutant line, which is a demonstration of the stability of the altered genotype of the \ Deltahsp70-II line.

       \ vskip1.000000 \ baselineskip
    

Example 5 Vaccination assays with the mutant \ Deltahsp70-II against infection with L. infantum

5 mice were infected with 10 7 promastigotes in stationary (infective) phase of the Del Deltahsp70-II line intravenously. After twelve weeks of infection, the animals were infected with 10 6 stationary promastigotes of L. infantum silvestre. Four weeks after infection, mice were sacrificed and peritoneal macrophages were collected. In parallel, macrophages present in the peritoneum of control mice (infected with wild L. infantum , but not vaccinated with the mutant? Deltahsp70-II) were collected. The macrophages were deposited on 96-well plates at a concentration of 10 6 cells per ml and incubated for 48 hours in the absence or in the presence of 50 µg / ml of Leishmania soluble antigen (SLA; prepared from of promastigotes of L. major or L. infantum ).

After incubation, the presence of nitrites in the culture medium through the use of Griess reagent (used following the house protocol supplier, Promega). The results are shown in Fig. Four.

The nitrites present in the culture medium are the result of the production of nitric oxide by the macrophages in culture. The production of nitric oxide is part of the macrophage defense mechanism (the host cell for Leishmania ) against parasite infection and is considered as the best parameter associated with a protective immune response against Leishmania (Liew &O'Donnell, 1993). Adv Parasitol 32 , 161-259). These results indicate that vaccination with the Del Deltahsp70-II line is inducing protective immunity since macrophages are prepared to produce high levels of nitric oxide when the parasite is detected (either L. infantum or L. major ).

       \ vskip1.000000 \ baselineskip
    

Example 5 Vaccination assays with the mutant \ Deltahsp70-II against infection with L. major

Infection of mice with L. major is used as a model vaccine assay system for two fundamental reasons. On the one hand, by producing skin lesions at the immunization site (plantar pad), the development of the infection can be monitored without sacrificing the animal. On the other hand, there is a clear association between protection / susceptibility and the induction of a Th1 / Th2 response (Liew &O'Donnell, 1993. Adv Parasitol 32, 161-259 ).

5 mice were infected with 10 7 promastigotes in stationary (infective) phase of the Del Deltahsp70-II line intravenously. After four weeks of infection, the animals were infected with 10 4 stationary promastigotes of L. major in the right hind leg pad. In parallel, a group of control mice (not vaccinated) with the same dose of L. major was infected. The size of the lesion was monitored weekly by using a digital caliper. The results, individually represented for each mouse, are shown in Fig. 5a and 5b. As can be seen, the evolution of the lesion in vaccinated mice was much smaller, being null in three of the 5 mice.

<110> Autonomous University of Madrid

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<120> Use of strains of Leishmania \ Deltahsp70-II as a vaccine.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<130> 1595.13

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 10

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<170> PatentIn version 3.4

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 653

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Leishmania infantum

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

2

3

4

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 1119

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Leishmania infantum

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

5

6

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 7

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 8

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 25

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 9

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 10

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 28

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm eleven

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 12

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 28

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 13

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 29

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

         \ vskip1.000000 \ baselineskip
      

\ hskip1cm 14

Claims (14)

1. Use of Leishmania organisms that have the HSP70-II gene deleted, for the preparation of a drug for the treatment or prevention of leishmaniasis in a mammal. 2. Use of Leishmania organisms that have the HSP70-II gene deleted according to the preceding claim, wherein the organisms that have the HSP70-II gene deleted belong to a Leishmania species that is selected from the list comprising: Leishmania infantum, L Chagasi, L. major, L. tropica, L. mexicana, L. braziliensis and / or L. amazonensis . 3. Use of Leishmania organisms that have the HSP70-II gene deleted according to any of claims 1-2, wherein the organisms that have the HSP70-II gene deleted belong to the Leishmania infantum species. 4. Use of Leishmania organisms according to any of claims 1-3, wherein the mammal is the dog. 5. Use of Leishmania organisms according to any of claims 1-3, wherein the mammal is man. 6. Use of Leishmania organisms according to any of claims 1-5, wherein leishmaniasis is caused by parasites of the L. infantum species. 7. Pharmaceutical composition comprising a Leishmania organism that has the HSP70-II gene deleted. 8. Pharmaceutical composition according to the preceding claim, wherein the Leishmania organism is in the form of promastigote. 9. Pharmaceutical composition according to any of claims 7-8 further comprising pharmacologically acceptable excipients and / or adjuvants. 10. Pharmaceutical composition according to any of claims 7-9, for use as medicine. 11. Use of a pharmaceutical composition according to any of claims 7-9, for the development of a medicine for the treatment or prevention of leishmaniasis 12. Use of the pharmaceutical composition according to any of claims 7-9, for the development of a medicine for the treatment or prevention of Leishmaniasis in a mammal. 13. Use of the pharmaceutical composition according to claim 12, wherein the mammal is man. 14. Use of the pharmaceutical composition according to claim 12, wherein the mammal is the dog.