DNA vaccines against leishmaniasis

Abstract

The present invention relates to the fields of veterinary medicine and virology and provides a vaccine against leishmaniasis. In particular, the present invention relates to an isolated polynucleotide comprising (i) a first expression cassette comprising a nucleic acid encoding heat shock protein (hsp) 65 and (ii) a second expression cassette comprising a nucleic acid encoding LACK (Leishmania homolog of receptor for activated C kinase). In other embodiments, the present invention relates to a DNA plasmid comprising said polynucleotide, a DNA vaccine for use in the treatment of leishmaniasis, and a method of immunizing a subject against infection with leishmaniasis.

Description

[Technical field]

[0001] The present invention relates to the field of veterinary medicine and parasitology and provides a vaccine against leishmaniasis. In particular, the present invention relates to an isolated polynucleotide comprising (i) a first expression cassette comprising a nucleic acid encoding mycobacterial heat shock protein (hsp) 65 and (ii) a second expression cassette comprising a nucleic acid encoding LACK (Leishmania homolog of receptor for activated C kinase). In other embodiments, the present invention relates to a DNA plasmid comprising said polynucleotide, a DNA vaccine for use in the prophylactic treatment of leishmaniasis, and a method of immunizing a subject against infection with leishmaniasis. [Background technology]

[0002] 2. Background of the Invention Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. It is transmitted by the bite of female sandflies. There are many species of sandflies, only a small proportion of which are possible vectors of Leishmania. Dogs, with or without clinical symptoms, are susceptible to sandflies and can transmit the Leishmania parasite.

[0003] More than 23 species of Leishmania have been described, most of which are zoonotic. The most important Leishmania protozoan that affects livestock is Leishmania infantum (also known as L. chagasi in Latin America). Dogs are the primary reservoir for human visceral leishmaniasis, caused by Leishmania infantum, a disease that is potentially fatal in dogs and humans. Because the dog's internal organs and skin are affected, the dog disease is called viscerocutaneous leishmaniasis or canine leishmaniasis. Cats, horses, or other mammals can be infected with Leishmania infantum or other Leishmania species. Leishmania braziliensis, the causative agent of tegumentary canine leishmaniasis, is widely distributed in South America.

[0004] Canine leishmaniasis is a major zoonotic disease endemic in over 89 countries. It is widespread in Europe, Africa, Asia and Central and South America, and is transmitted vertically from dog to dog in the United States. It is also of concern in non-endemic countries where imported disease is a veterinary and public health problem. Canine leishmaniasis is a multi-organ disease with highly variable immune responses and clinical symptoms. In endemic areas, the prevalence of infected dogs is much higher than the prevalence of dogs with clinical disease. Clinical disease is associated with a significant antibody response that does not provide protection. In fact, immune-mediated mechanisms are responsible for much of the pathology of canine leishmaniasis. In most symptomatic dogs, the first signs of disease due to Leishmania infection appear approximately 2-4 months after initial infection. Symptoms may include skin sores, peeling skin, ulcers, weight loss, hair loss, conjunctivitis, blindness, runny nose, muscle atrophy, inflammation, swelling and organ failure (including mild heart attacks).

[0005] Diagnosis and treatment of affected individuals are particularly complex, which hampers infection control in endemic areas. The main chemotherapy protocols used to treat canine leishmaniasis include N-methylglucamine antimoniate. Treatment often does not result in a cure that eradicates the pathogen. Treated dogs may remain carriers of infection and may relapse. They may remain infective to sandflies.

[0006] Methods for preventing canine leishmaniasis include the use of topical insecticides, prophylactic immunotherapy and vaccination. Since 2004, four vaccines against canine leishmaniasis have been licensed: two in Brazil [Leishmune® (whose manufacturing and marketing authorization was revoked in 2014) and Leish-Tec®] and two in Europe [CaniLeish® and LetiFend®]. Leish-Tec® is a recombinant vaccine based on the Leishmania A2 antigen against canine visceral leishmaniasis (CVL). After several years on the market, doubts remain regarding the efficacy and effectiveness of the vaccine, the potential infectivity of vaccinated and infected animals, or the interference of vaccine-induced antibodies in the serological diagnosis of Leishmania infantum (L. infantum).

[0007] Therefore, preventive treatment by vaccination against leishmaniasis is urgently needed.

[0008] Research on potential preventive vaccines against leishmaniasis has progressed in recent years, and several vaccination methods and several antigens have been tested. First generation vaccines based on various inactivated or attenuated parasites, second generation vaccines based on antigenic proteins or recombinant proteins, and third generation vaccines derived from antigen-encoding DNA plasmids, including heterologous prime-boost leishmania vaccines, have been tested for the control and prevention of leishmaniasis. It has been shown that CD4+ and CD8+ T lymphocytes play an important role in providing protection and cure for visceral leishmaniasis. Naked DNA immunization (DNA vaccination) is a novel approach that promotes both CD4+ and CD8+ mediated responses and helps induce protective responses against infection.

[0009] A number of DNA vaccines have already been approved for commercial use in both companion and food animals. Their mechanism of action makes DNA vaccines attractive for the control of leishmaniasis. In the past few years, research has focused on several antigens such as GP63, CP, TSA, GP64, LmSTI1, LeIf and P8, p4 and LACK (Jain K and Jain NK. J. Immun. Methods, 422 (2015)). LACK (Leishmania homolog of receptor for activated C kinase) is a 36 kDa protein that is localized in the cytosol and the outer surface of the membrane. It is expressed in both promastigote and amastigote forms of the parasite. Genes involved in host-gene interactions in Leishmania species have been identified and reported in Peacock et al., Nat Genet., 2007.

[0010] Mycobacterial HSPs, mainly Hsp65 and Hsp70, are known to regulate both innate and adaptive immunity (cellular and humoral) of the immune system. DNA constructs encoding mycobacterial antigens as 65 kDa heat shock proteins (hsp65) have been shown to induce significant protective immunity. A DNA vaccine containing mycobacterial hsp65 (DNAhsp65) has recently been developed. DNAhsp65 has been shown to exhibit broad immunotherapeutic properties against various diseases (Silva CL, Malardo T and Tahyra ASC, Front. Med. Technol. 2:603-690 (2020)). Summary of the Invention

[0011] Overview of the Invention In the present invention, it was surprisingly found that vaccination with the 65 kDa heat shock protein hsp65 and LACK (Leishmania homolog of receptor for activated C kinase) using a DNA vaccine containing hsp65 and LACK achieves an effective immune response and a reduction in parasite load. This finding is particularly surprising, since it has been recently shown in the art that immune responses induced by LACK antigens do not provide protection against experimental Leishmania infection (Coelho et al., Infection and Immunity, 71(7), 2003).

[0012] Thus, in a first aspect, the present invention provides an isolated polynucleotide comprising (i) a first expression cassette comprising a nucleic acid encoding heat shock protein (hsp) 65 or a functional fragment thereof, and (ii) a second expression cassette comprising a nucleic acid encoding LACK (Leishmania homolog of receptor for activated C kinase) or an immunogenic fragment thereof.

[0013] In a second aspect, the present invention provides a DNA plasmid comprising a polynucleotide described herein.

[0014] In a third aspect, the present invention provides a DNA vaccine comprising a polynucleotide described herein.

[0015] In a fourth embodiment, the DNA vaccine is used in the protection of a subject against infection with leishmaniasis.

[0016] In a fifth aspect, there is provided a method of immunizing a subject against infection with Leishmaniasis comprising administering to the subject an immunogenetically effective amount of a DNA vaccine as described herein. [Brief description of the drawings]

[0017] [Figure 1] DNAHSP-LACK: Schematic diagram of the plasmid DNA vector described in Example 1 containing the hsp65 and LACK genes. [Diagram 2] Experimental design for evaluation of immunogenicity and efficacy of saline resuspended vaccines and combinations in a mouse model. [Figure 3A] Representative graphs from immunogenicity and efficacy results in Example 3. 3A - IFN-γ response, 3B - parasite burden in liver by limiting dilution assay. [Figure 3B] Representative graphs from immunogenicity and efficacy results in Example 3. 3A - IFN-γ response, 3B - parasite burden in liver by limiting dilution assay. [Figure 4A] Representative graphs of parasite burden by qPCR in lymph nodes in Example 4: 4A - 43 days post-challenge, 4B - 190 days post-challenge, 4C - 302 days post-challenge. [Figure 4B] Representative graphs of parasite burden by qPCR in lymph nodes in Example 4: 4A - 43 days post-challenge, 4B - 190 days post-challenge, 4C - 302 days post-challenge. [Figure 4C]Representative graphs of parasite burden by qPCR in lymph nodes in Example 4: 4A - 43 days post-challenge, 4B - 190 days post-challenge, 4C - 302 days post-challenge. [Figure 5A] Representative graphs of parasite burden by qPCR in bone marrow in Example 4: 5A - 43 days post-challenge, 5B - 190 days post-challenge, 5C - 302 days post-challenge. [Figure 5B] Representative graphs of parasite burden by qPCR in bone marrow in Example 4: 5A - 43 days post-challenge, 5B - 190 days post-challenge, 5C - 302 days post-challenge. [Figure 5C] Representative graphs of parasite burden by qPCR in bone marrow in Example 4: 5A - 43 days post-challenge, 5B - 190 days post-challenge, 5C - 302 days post-challenge.

[0018] Definition of Terms The term "nucleic acid (sequence)" or "polynucleic acid (sequence)" includes RNA or DNA sequences. It can be single-stranded or double-stranded. It can be, for example, genomic, recombinant, mRNA or cDNA. In the present invention, polynucleic acids are typically double-stranded DNA.

[0019] The term "isolated" should be construed as separated from its natural environment by deliberate action or human intervention, such as by in vitro manipulation for biochemical purification.

[0020] Typically, a "(poly)nucleic acid" or "(poly)nucleic acid molecule" or "gene" encoding a protein (here the antigen hsp65 or LACK according to the invention) is an open reading frame (ORF), indicating the absence of undesired stop codons that would prematurely terminate translation into a protein. In the present invention, the nucleic acid molecule typically encodes the complete amino acid sequence of hsp65 or LACK. Alternatively, the "nucleic acid" or "nucleic acid molecule" may encode only one or more parts of the hsp65 or LACK protein, in particular only the epitope region, for recombinant expression of the corresponding one or more functional fragments (hsp65) or immunogenic fragments (LACK).

[0021] The exact nucleotide sequence of the nucleic acid molecule of the present invention is not critical, provided that the nucleotide sequence allows for expression of the desired amino acid sequence (here, the desired hsp65 and LACK proteins). However, as is well known in the art, due to the "degeneracy of the genetic code," different nucleic acids may code for the same protein.

[0022] In the present invention, nucleic acid molecule can be DNA or RNA molecule, but typically is DNA.This depends on the raw material used for its isolation and its intended use.The methods for isolating one type or another type of molecule from various starting materials and the methods for converting one type to another type are well known to those skilled in the art.

[0023] The "isolated (poly)nucleic acid" according to the invention, when it is in DNA form, can be conveniently manipulated in the context of a vector, such as a DNA plasmid. In order for the isolated (poly)nucleic acid molecule according to the invention to be able to actually express the hsp65 and LACK according to the invention, it requires appropriate expression control signals and an appropriate environment, which are part of an "expression cassette", which is defined herein as a (poly)nucleic acid that contains all the elements necessary for recombinant gene expression, such as recombinant gene expression from a DNA plasmid for the production of a protein of interest (POI).

[0024] For example, the nucleic acid molecule must be operably linked to an upstream promoter element and must contain a translation start point at the beginning of the coding sequence and a translation stop point at the end of the coding sequence. Thus, expression cassettes for recombinant expression of hsp65 and LACK proteins typically contain at least the hsp65 or LACK gene under the control of a functional promoter. To increase expression levels, translation enhancers can also be included upstream and / or downstream of the coding region, which can also be part of the expression cassette. Typically, the plasmids and vectors used in the context of a particular expression system provide such elements and enhancers. Also, the biomolecular machinery for transcription and translation is typically provided by the cells of the vaccinated subject. By modifying the various elements and enhancers, expression of the antigen according to the invention can be optimized, for example, in terms of timing, level and quality, all of which are within the ordinary capabilities of a person skilled in the art. Thus, in a preferred embodiment, the isolated nucleic acid molecule according to the invention further comprises an expression control signal.

[0025] A "translational enhancer" is a nucleotide sequence that constitutes an element that increases the production of a protein by promoting translation. Typically, translational enhancers can be found in the 5' and 3' untranslated regions (UTRs) of mRNA. In particular, the nucleotides in the 5'-UTR immediately upstream of the initiation ATG codon of a gene of interest (GOI) can have a significant effect on the level of translation initiation.

[0026] An "expression vector" (synonym "expression construct") is usually a plasmid, typically a DNA plasmid, designed for recombinant gene expression in a cell. Vectors are used to introduce a particular gene into a target cell and may use the cell's protein synthesis machinery to produce a protein of interest (POI) encoded by the gene. To express a recombinant gene to produce a POI, an expression vector typically contains at least a promoter to drive expression of the GOI and may further contain one or more translation enhancers to increase the yield of the POI.

[0027] The term "vaccine" as used herein refers to a preparation that induces or stimulates a protective immune response when administered to a subject. A vaccine can immunize an organism against a particular disease. In the context of the present invention, the vaccine can be or contain a DNA plasmid, and thus is also referred to as a "DNA vaccine", which can further contain a suitable carrier and / or adjuvant as described herein.

[0028] "DNA vaccination" involves immunization with DNA plasmids that encode antigens or immunomodulators of pathogens, ie, "DNA vaccines."

[0029] A "DNA vaccine" is a type of vaccine that introduces a specific antigen-encoding DNA sequence onto the cells of a species to be immunized. DNA vaccines work by injecting a genetically engineered plasmid containing a DNA sequence that codes for the antigen against which an immune response is directed, so that the cells directly produce the antigen and elicit a protective immune response. The GOI is inserted into the DNA plasmid along with the appropriate genetic elements, such as a eukaryotic promoter for transcriptional control, a polyadenylation signal sequence for stable and efficient translation, and a bacterial origin of replication. The plasmid is transfected into the host cell, for example, by direct injection or by injection with a suitable delivery system. The GOI is then subjected to transcription and translation by the host cell machinery to produce an antigenic protein that can induce innate and adaptive immune responses (cellular and humoral).

[0030] DNA vaccines have a number of advantages over other vaccination techniques that are of particular interest in veterinary medicine. They may not be as expensive as other commercial vaccines because they can be produced in large quantities by bacteria and typically do not require expensive facilities with high biosafety levels. They are temperature stable and can be transported safely, which is important for farms located in remote areas or for wild animal vaccines that must be left outdoors for long periods of time. Different genes can be combined simultaneously, which allows for the development of vaccines against multiple strains of pathogens and combinatorial approaches against multiple pathogens. Also, because the relevant proteins are produced and presented intracellularly, both cellular and humoral immune responses are induced, which results in a more efficient immune response when the animal encounters a natural infection at a later time.

[0031] DNA vaccines can be administered by several routes and techniques that can alter the immune response and protein distribution. Typically, DNA vaccines are administered by subcutaneous or intramuscular injection.

[0032] Thus, in the context of the present invention, the term "DNA vaccine" refers to DNA sequences encoding the nucleic acid sequences of the immunoregulatory factor hsp65 and the antigen LACK, which are recombinantly expressed in a vaccinated subject to induce an immune response.

[0033] "Protecting an animal against Leishmania infection" generally means preventing, ameliorating or helping to cure a pathogenic infection by Leishmania or preventing, ameliorating or helping to cure a disorder resulting from that infection, for example preventing or reducing one or more clinical signs resulting from Leishmania infection after treatment (i.e. after vaccination). Typical clinical signs that may be reduced by vaccination in the present invention may include one or more of the following: skin sores, peeling skin, ulcers, weight loss, hair loss, conjunctivitis, blindness, runny nose, muscle atrophy, inflammation, swelling and organ failure (including mild heart attacks).

[0034] In particular, the vaccine of the present invention helps to achieve antibody and cellular immune responses in a subject, which results in a reduction in parasite burden after Leishmania infection and a reduction or prevention of clinical signs of the disease. In a preferred embodiment, the vaccine of the present invention is used to prevent or ameliorate Leishmania infection. In this embodiment, vaccination with the vaccine of the present invention achieves one or more of a reduction in clinical signs after infection and a reduction in parasite burden. Most preferably, vaccination achieves the prevention of Leishmania infection.

[0035] The term "prevention" or "preventing" is intended to mean the avoidance, delay, prevention or hindrance of Leishmania infection by prophylactic treatment. A vaccine may, for example, prevent or reduce the likelihood of infectious Leishmania invading a subject.

[0036] Detailed Description of the Preferred Embodiments Polynucleic acids and DNA plasmids The polynucleotide (or synonymously nucleic acid) of the first aspect is typically in the form of or part of a DNA plasmid (hereinafter also referred to as a DNA vector or simply a vector), such as a circular DNA plasmid. Thus, in another aspect, the present invention provides a DNA plasmid comprising a polynucleotide as described herein.

[0037] The polynucleotide of the invention comprises at least two expression cassettes for the recombinant expression of genes encoding the mycobacterial heat shock protein hsp65 and the Leishmania LACK protein.

[0038] FIG. 1 shows an example of a construct of a DNA plasmid (DNAHSP-LACK) according to the invention, which contains two expression cassettes described herein, one for expression of an immune regulator (hsp65) and one for expression of an antigen (LACK).

[0039] The first expression cassette (i) comprises a nucleic acid encoding heat shock protein (hsp) 65 or an immunogenic fragment thereof.

[0040] Hsp65 as used herein refers to the mycobacterial hsp65 gene, which has already shown the ability to modulate and / or regulate immune responses in experimental models of several diseases with distinct Th1 / Th2 immune response patterns, including leishmaniasis (Silva CL, Malardo T and Tahyra ASC, Front. Med. Technol. 2:603690 (2020)). Thus, in the polynucleotide constructs of the present invention, the mycobacterial heat shock protein hsp65 functions as an immune regulator.

[0041] The amino acid sequence of hsp65 can be represented by SEQ ID NO: 1, which refers to the amino acid sequence of hsp65 of the mycobacterial strain Mycobacterium leprae from GenBank Accession No. AAA25354.1, which can be encoded by the nucleic acid sequence provided under GenBank Accession No. M14341.1 (M. leprae 65 kd antigen). The present invention also includes functional fragments of the hsp65 protein, which contain the relevant parts of the protein essential for achieving an immunomodulatory effect. In particular, a functional fragment comprising amino acids 48-583 of SEQ ID NO: 1 is preferably used, since amino acids 1-47 of SEQ ID NO: 1 are not relevant for the coding and expression of the hsp65 protein.

[0042] Due to natural sequence variations between mycobacterial strains, the present invention also encompasses hsp65 amino acid sequences having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, or functional fragments thereof, within the conserved regions of the protein, which preferably have at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, and most preferably have at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, e.g., 91%, 92%, 93%, 94% or 95%, 97%, 98% or 99% sequence identity to the amino acid sequence of Gene Bank Accession No. AAA25354.1, or functional fragments thereof.

[0043] The second expression cassette (ii) comprises a nucleic acid encoding a LACK protein or an immunogenic fragment thereof.

[0044] LACK (Leishmania homolog of receptor for activated C kinase) as referred to herein is a 36 kDa protein that is naturally present in the cytosol and on the outer membrane surface of Leishmania infantum and is expressed in both the promastigote and amastigote forms of the parasite. In the vaccine of the present invention, LACK is expressed from the polynucleotide of the present invention and serves as an antigen to achieve an immune response against the LACK protein in a vaccinated subject. Thus, in the polynucleotide construct of the present invention, the Leishmania LACK protein serves as an antigen to induce an immune response.

[0045] The amino acid sequence of LACK can be represented by SEQ ID NO:2, which refers to the amino acid sequence of GenBank Accession No. ALP73427.1, which can be encoded by the nucleic acid sequence provided as GenBank Accession No. KT184317.1 (Leishmania infantum strain MHOM / TR / 2009 / EP174 receptor for activated protein kinase C (LACK) gene). The present invention also includes immunogenic fragments of the LACK protein that contain relevant epitopes to affect antigen binding and achieve an immunogenic response in a vaccinated subject.

[0046] Due to natural sequence variations between Leishmania strains, the present invention also encompasses LACK amino acid sequences or immunogenic fragments thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2, preferably having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:2, most preferably having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, e.g. having 91%, 92%, 93%, 94% or 95%, 97%, 98% or 99% sequence identity to the amino acid sequence of Gene Bank Accession No. ALP73427.1.

[0047] Both Hsp65 and LACK may be expressed under the control of the same or separate promoters suitable for recombinant expression of the proteins in the vaccinated subject. Typically, hsp65 and LACK are expressed under the control of separate promoters. Suitable promoters that can be used for recombinant expression of hsp65 and LACK are well known in the art and are described, for example, in Poulain A. et al., J Biotechnol. 2017 255:16-27; Kim SY et al., J Biotechnol. 2002; 93(2):183-7; JR Deer, DS Allison, Biotech Progress 2004, 20(3):880-889; RV Gopalkrishnan et al., Nucleic Acids Research, 1999 27(24):4775-4782; Wang X. et al., J. Cell. Mol: Med. 2017 21(11):3044-3054, the entirety of which is incorporated herein by reference.

[0048] A promoter called EF1α / HTLV is preferably used in the construction of the vaccine of the present invention. This promoter contains NotI and EcoRI restriction sites that can be used to insert genes for recombinant gene expression. The EF1α-HTLV promoter is a composite promoter that contains the human elongation factor 1α (EF-1α) core promoter and the R segment of the human T-cell leukemia virus (HTLV) type 1 long terminal repeat and a part of the U5 sequence (R-U5'). The EF-1α promoter shows strong activity and leads to long-term sustained expression of transgenes in vivo. The R-U5' is linked to the EF-1α core promoter to enhance RNA stability (Kim SY et al., J Biotechnol. 2002; 93(2):183-7).

[0049] Thus, in a preferred embodiment, hsp65 is expressed under the control of the cytomegalovirus CMV promoter and LACK is expressed under the control of the EF1α / HTLV promoter.

[0050] The expression cassette may also contain one or more elements used to regulate or enhance expression of hsp65 and / or LACK, including expression enhancers or other cis-acting elements.

[0051] In a preferred embodiment, the expression cassette contains one or more CpG motifs. CpG sequences in the plasmid backbone play an important adjuvant role in DNA vaccines. These sequences correspond to pairs of unmethylated CpG dinucleotides on the same DNA strand. For example, to enhance the immunogenicity of the plasmid system, 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 2 to 6, e.g. 3, 4 or 5, most preferably 4, CpG motifs can be added, typically after the SV40 polyadenylation site of one or both expression cassettes. In a particularly preferred embodiment, the expression cassette leading to LACK expression contains 4 CpG motifs.

[0052] In another embodiment, the polynucleotides or DNA plasmids described herein are used for the recombinant expression of hsp65 and LACK, or immunogenic fragments thereof, which may be used as recombinant proteins in vaccines for protection against Leishmania infection.

[0053] In another aspect, the present invention provides a DNA construct, such as a DNA plasmid, containing a mycobacterial heat shock protein hsp65 gene and a Leishmania LACK gene for use in the manufacture of a pharmaceutical composition or medicament for the prevention of Leishmania infection.

[0054] In another aspect, the present invention provides a DNA vaccine comprising a polynucleotide or a DNA plasmid described herein.

[0055] Carriers and Adjuvants In another aspect, the present invention provides a DNA vaccine as described herein, further comprising a pharma- ceutically acceptable carrier and / or an adjuvant.

[0056] Pharmaceutically acceptable carriers are well known in the art. By way of example only, such a carrier can be as simple as sterile water, saline or a buffer solution (buffer), such as PBS. The vaccine can contain a single carrier or a combination of two or more carriers. In a preferred embodiment, the pharma-ceutically acceptable carrier is water or saline, most preferably saline.

[0057] Suitable carriers for vaccines and their uses or methods according to the invention and / or its embodiments are well known in the art and include diluents, adjuvants, antimicrobial agents, stabilizers, preservatives, inactivating agents or combinations thereof. Suitable diluents may include distilled water, salts, media components, sterile water, buffers, stabilizers, preservatives, bactericides, antibiotics, and chemicals that aid in solubility.

[0058] Carriers may include NaCl, sodium carbonate, thiomersal, buffered saline, sucrose, disodium adipate, disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate.

[0059] The vaccines may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0060] Suitable excipients in vaccines according to the invention include hydrolyzed gelatin, casein, sorbitol and disodium phosphate dihydrate.

[0061] Stabilizers may be used, for example, to extend the shelf life of the vaccine, or to improve freeze-drying efficiency, or to improve the appearance of the product. Useful stabilizers include, inter alia, SPGA, carbohydrates such as sorbitol, mannitol, trehalose, starch, sugars such as sucrose, dextran, lactose or glucose, amino acids such as glycine and monosodium glutamate (salts of amino acids), proteins such as (recombinant) albumin, gelatin, hydrolyzed collagen, or casein or its degradation products, and buffers such as alkali metal phosphates.

[0062] In another aspect, the invention relates to a liquid composition of a pharma- ceutically acceptable carrier and a vaccine of the invention, where the carrier is a natural deep eutectic solvent (NADES), preferably one having a water activity of less than about 0.8. NADES carriers are described in WO 2019 / 122329, which is incorporated herein by reference in its entirety.

[0063] Furthermore, vaccines can contain adjuvants or can be free of adjuvants (non-adjuvanted vaccines). Adjuvants can be classified according to the origin of their components, their physicochemical properties or their mechanism of action. Molecular adjuvants can act as immune stimulants (e.g., TLR ligands, cytokines, saponins and bacterial exotoxins that stimulate immune responses) and act directly on the immune system to enhance the immune response to antigens.

[0064] Since the vaccine of the present invention contains hsp65 as an immunostimulatory protein expressed from the polynucleotide of the present invention and acting as an adjuvant, the addition of a (further) adjuvant is not necessarily required in the present invention, however, the addition of a further adjuvant may be desirable to provide an enhanced or more rapid immune response in the vaccinated subject.

[0065] Suitably, the adjuvant in the vaccine composition according to the invention and / or any embodiment thereof may include aluminium hydroxide, aluminium phosphate, saponin, saponin complexed with cholesterol, saponin complexed with cholesterol and lipid, non-metabolisable oil, mineral oil and / or plant / vegetable oil and / or animal oil, polymer, carbomer, surfactant, natural organic compound, plant extract, carbohydrate, cholesterol, lipid, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, acrylic acid polymer, acrylic acid sugar cross-linked polymer, acrylic acid polyol cross-linked polymer.

[0066] Suitably, the adjuvant in the vaccine composition according to the invention and / or any embodiment thereof may comprise aluminium hydroxide, aluminium phosphate, saponin, Quil A, QS-21, GPI-0100, non-metabolisable oil, mineral oil and / or plant / vegetable oil and / or animal oil, polymer, carbomer, surfactant, natural organic compound, plant extract, carbohydrate, cholesterol, lipid, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid sugar cross-linked polymer), HRA-3 with cottonseed oil (CSO), or HRA-5 (acrylic acid polyol cross-linked polymer), ISCOMATRIX. Suitably, the adjuvant is saponin, acrylic acid polymer and / or aluminium hydroxide. Suitably the saponin is Quil A, ISCOM, QS21, ISCOPREP 703, ISCOMATRIX, AS series, ISCOPREP saponin, GPI0100, AbISCO, Matrix M, Matrix C, Posintro. Suitably the acrylic acid polymer is Carbopol, e.g. HRA-3, HRA-5, Carbopol 940, Carbopol 941, Carbopol 934, Carbopol 971, Havlogen, CARBIGEN.

[0067] Suitable adjuvants are saponin-based adjuvants, more suitable are Quillajasaponin-based adjuvants.Saponin-based examples include Quil A, ISCOM, QS21, ISCOMATRIX, ISCOPREP 703, AS series, GPI0100, AbISCO, Posintro.Suitable adjuvants are cholesterol-complexed saponins.Suitable cholesterol-complexed saponins include AS01, AS15, AS02, ISCOM, ISCOMATRIX, Matrix-M, AbISCO.

[0068] Suitably, the adjuvant is present at a concentration of about 0.01 to about 50%, or about 2% to about 45%, or about 5% to about 40%, or about 7% to about 35%, or about 10% to about 30%, by volume, relative to the volume of the final product. Suitably, the adjuvant is present at a concentration of about 0.01 to about 10%, or about 0.02 to about 5%, or about 0.05 to about 2.5%, or about 0.1 to about 2%, or about 0.15 to about 1.5%, or about 0.2 to about 1.2%, or about 0.3 to about 1%, or about 0.4 to about 0.8%, or about 0.5 to about 0.7%.

[0069] Thus, preferably, the vaccine according to the invention is in freeze-dried form, ie provided as a lyophilisate.

[0070] The vaccine may contain or be capable of expressing another active substance, for example an active substance that may stimulate early protection before the LACK-induced adaptive immune response. The active substance may also be another antigen that may induce an immune response against another disease in the vaccinated subject (combination vaccine).

[0071] Vaccines and their production To produce the polynucleotide molecule according to the invention, comprising the two expression cassettes (i) and (ii) for the recombinant expression of hsp65 and LACK, in vitro recombinant DNA methods known to those skilled in the art can be used. Conveniently, this can be done by generating PCR fragments and subcloning, or by de novo gene synthesis techniques.

[0072] Alternatively, the polynucleotide molecules according to the invention may be produced by cell-based in vitro expression systems, since this offers advantages in terms of yield and safety. The expression systems may be prokaryotic or eukaryotic, and in the case of eukaryotic cells, may be based on host cells of yeast, mammalian, insect or plant origin, all of which are described in the prior art.

[0073] Therefore, the present invention further relates to the preparation of said polynucleotide, such as DNA plasmid, for use in the preparation of a vaccine.In particular, the DNA plasmid of the present invention can be used for vaccination of a subject.Preferably, the DNA plasmid is formulated in a composition comprising the DNA plasmid and one or more pharma- ceutically acceptable carriers and / or adjuvants.

[0074] In another aspect, the present invention provides a DNA vaccine for use in protecting a subject against Leishmania infection. The subject is not particularly limited and can be an animal or a human. Preferably, the subject is a dog, which is an animal species susceptible to infection with canine Leishmania. Thus, a preferred utility of the present embodiment is in veterinary applications, particularly for vaccination of dogs against Leishmaniasis.

[0075] Infection may be caused by more than 20 of the approximately 30 known Leishmania species that infect mammals. The vaccine of the present invention may provide protection against one or more Leishmania species. In particular, since the LACK region is conserved among Leishmania species, the vaccine of the present invention may achieve broad protection against a wide range of Leishmania species. Thus, in a preferred embodiment, the vaccine of the present invention provides protection against infection by the following Leishmania species: Leishmania donovani complex, which includes three species (Leishmania donovani, Leishmania infantum and Leishmania chagasi); Leishmania spp., which includes three major species (Leishmania mexicana, Leishmania amazonensis and Leishmania venezuelensis); The L. mexicana complex; L. tropica; L. major; L. aethiopica; and the Viannia subgenus, which includes four major species [Leishmania (V.) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) panamensis, and Leishmania (V.) peruviana]. The majority of infections are caused by Leishmania infantum. The different species are morphologically indistinguishable, but they can be distinguished by isoenzyme analysis, molecular methods, or monoclonal antibodies.

[0076] The vaccine may be used therapeutically to treat an existing Leishmania infection, but is preferably used prophylactically to prevent or reduce the likelihood of infection and / or to ameliorate the clinical signs of infection and / or to prevent or reduce the likelihood of the spread of the disease.

[0077] The present invention also provides a method of protecting a subject against Leishmania infection by administering an effective amount of the vaccine of the present invention, which comprises the steps of preparing a DNA plasmid as described above, optionally adding a pharma- ceutically acceptable carrier and / or adjuvant, and administering the vaccine to a subject.

[0078] The present invention further provides a polynucleotide as described herein for use in the manufacture of a DNA vaccine for protecting a subject against Leishmania infection.

[0079] Administration The present invention contemplates administering an effective amount of the DNA vaccine according to the present invention to an animal at least once. The DNA vaccine may be administered by any method known in the art, including any local or systemic administration method. Administration may be performed, for example, by administering the antigen into muscle tissue (intramuscular, IM), into the dermis (intradermal, ID), under the skin (subcutaneous, SC), under the mucous membrane (submucosal, SM), intravenously (intravenous, IV), into a body cavity (intraperitoneal, IP), orally, anally, etc. For current vaccines, IM, ID and SC administration are preferred.

[0080] The present invention relates to the following embodiments.

[0081] In a first embodiment, the present invention provides a method for producing a composition comprising the steps of: (i) a first expression cassette comprising a nucleic acid encoding heat shock protein (hsp) 65 or a functional fragment thereof; and (ii) a second expression cassette comprising a nucleic acid encoding LACK (Leishmania homolog of receptor for activated C kinase) or an immunogenic fragment thereof; The present invention provides an isolated polynucleotide comprising:

[0082] In embodiment 2, the present invention relates to the polynucleotide according to embodiment 1, wherein hsp65 comprises amino acids 48 to 583 of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having at least 90% sequence identity thereto.

[0083] In embodiment 3, the present invention relates to the polynucleotide according to embodiment 1 or 2, wherein LACK comprises the amino acid sequence of SEQ ID NO: 2 or comprises an amino acid sequence having at least 90% sequence identity thereto.

[0084] In an embodiment 4, the invention relates to the polynucleotide according to any one of the previous embodiments, wherein the nucleic acids encoding hsp65 and LACK are under the control of different promoters.

[0085] In embodiment 5, the invention relates to the polynucleotide according to any one of the previous embodiments, wherein the nucleic acid encoding hsp65 is under the control of a cytomegalovirus (CMV) promoter.

[0086] In embodiment 6, the invention relates to the polynucleotide according to any one of the previous embodiments, wherein the nucleic acid encoding LACK is under the control of the EF1α-HTLV promoter.

[0087] In embodiment 7, the invention relates to the polynucleotide according to any one of the previous embodiments, wherein the second expression cassette further comprises one or more CpG motifs.

[0088] In an eighth embodiment, the present invention relates to a DNA plasmid comprising the polynucleotide according to any one of the first to seventh embodiments.

[0089] In embodiment 9, the present invention relates to the use of a polynucleotide according to any one of embodiments 1 to 7 or of a DNA plasmid according to embodiment 8 for the recombinant expression of hsp65 and LACK or an immunogenic fragment thereof.

[0090] In embodiment 10, the present invention relates to a DNA vaccine comprising the polynucleotide according to any one of embodiments 1 to 7, or comprising the DNA plasmid according to embodiment 8.

[0091] In embodiment 11, the present invention relates to the DNA vaccine according to embodiment 10, further comprising a pharma- ceutically acceptable carrier.

[0092] In embodiment 12, the present invention relates to a DNA vaccine according to embodiment 10 or 11, which is a non-adjuvanted vaccine.

[0093] In embodiment 13, the present invention relates to a DNA vaccine according to embodiment 10 or 11, which comprises an adjuvant.

[0094] In embodiment 14, the present invention relates to the DNA vaccine according to embodiment 13, wherein the adjuvant is a saponin.

[0095] In embodiment 15, the present invention relates to a DNA vaccine according to any one of embodiments 10 to 14 for use in protecting a subject against infection with leishmaniasis.

[0096] In embodiment 16, the present invention relates to a DNA vaccine for use according to embodiment 15, wherein the subject is a dog.

[0097] In embodiment 17, the present invention relates to a DNA vaccine for use according to embodiment 15 or 16, wherein the infection is due to canine leishmaniasis.

[0098] In embodiment 18, the present invention relates to a method for immunizing a subject against infection with Leishmaniasis, comprising administering to the subject an immunogenetically effective amount of the DNA vaccine according to any one of embodiments 10 to 14.

[0099] It should be understood that combinations of the embodiments described herein are anticipated and, for brevity, only some exemplary combinations are explicitly described, but this should not be construed as being limited to the embodiments described. [ka] TIFF2025500350000003.tif154114TIFF2025500350000004.tif173116TIFF2025500350000005.tif173113TIFF2025500350000006.tif170115TIFF2025500350000007.tif192128TIFF2025500350000008.tif173115TIFF2025500350000009.tif182114TIFF2025500350000010.tif82124Example Example 1: Construction of plasmid pVAX_hsp65_lack The pVAX_hsp65 plasmid containing the hsp65 gene was constructed from the pVAX vector (Invitrogen, Carlsbad, CA, USA). This plasmid was digested with BamHI and NotI (Gibco BRL, Gaithersburg, MD, USA), and then the M. leprae hsp65 gene of SEQ ID NO:4, which contains amino acids 48-483 of SEQ ID NO:1, and the CMV intron A were inserted. DH5α E. coli transformed with pVAX or pVAXhsp65 were cultured in LB liquid medium (Gibco BRL, Gaithersburg, MD, USA) containing kanamycin (100 μg / ml). The plasmid was purified using the Concert High Purity Maxiprep System (Gibco BRL, Gaithersburg, MD, USA). Plasmid concentrations were measured by spectrophotometry at λ=260 and 280 nm using a Gene Quant II apparatus (Pharmacia Biotech, Buckinghamshire, UK).

[0100] For the construction of the Leishmania DNA vaccine based on the plasmid pVAX_hsp65, a promoter called EF1a / HTLV was inserted. In this case, the Lack gene could be inserted with the sequence of the NotI and EcoRI enzymes (GCGGCCGC-NotI enzyme site / GAATTC-EcoRI) as restriction sites. The EF1a-HTLV promoter is a composite promoter that contains the human elongation factor 1 alpha (EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5') of the human T-cell leukemia virus (HTLV) type 1 long terminal repeat. Thus, the final construction of the DNA plasmid was based on the insertion of the hsp65 and Lack genes into different promoters (CMV promoter for hsp65 and EF1a / HTLV for Lack).

[0101] The great advantage of these plasmid systems is that they make it possible to maintain the immunomodulatory activity of hsp65 and to consider the insertion of the LACK immunogenic gene for use in the specific protection of dogs against infections caused by Leishmania.

[0102] To enhance the immunogenicity of the multi-purpose plasmid system, four CpG motifs were also added after the SV40 polyadenylation site of the LACK gene expression cassette.

[0103] A DNA plasmid construct containing only the Lack gene in the absence of the hsp65 gene was also made and used as a control in immunogenicity and vaccine efficacy studies. Thus, the following plasmid DNA was constructed: (i) pVAX_hsp65 (control) (ii) pVAX_lack (control) (iii) pVAX_hsp65_lack (iv) pVAX_A2 (reference control) (v) pVAX_hsp65_A2 (reference control).

[0104] To obtain the recombinant protein (LACK) and specific antibody (anti-LACK), plasmids pET28a_lack and pET28a_A2 were produced by standard methods. These plasmids were used to transform E. coli strain BL21 for expression of Lack protein. The proteins were used in the production of anti-Lack polyclonal antibodies and in immunogenicity evaluation experiments of DNA vaccines in mice and dogs. The production of mouse anti-Lack polyclonal antibodies was performed in mice by immunization with each of the proteins administered with Freud's incomplete adjuvant. Plasmids pVAX_hsp65, pVAX_hsp65_lack, pVAX_lack were transformed into E. coli strain TOP10 for purification and characterization of the cloned genes in each plasmid by polyacrylamide gel analysis after restriction enzyme digestion.

[0105] After laboratory scale production, transfection of HEK293 cells with each of the plasmids and demonstration of endogenous hsp65 and lack proteins in each vaccine construct were performed. Protein expression was characterized by Western blot technique using anti-hsp65 and anti-LACK polyclonal antibodies.

[0106] mRNA was detected by RT-PCR using specific primers. pVAX_hsp65, pVAX_hsp65_lack and pVAX_lack vaccines were produced in laboratory scale in amounts of approximately 10 mg using standard methods. After bench scale production, DNA plasmids were characterized for the following: identity by restriction enzyme digestion; appearance, concentration, purity (A260 / 280 between 1.7 and 2.0); homogeneity (>90% in supercoiled form); genomic DNA (<5%); and endotoxin (<40 EU / mg as per industry guideline - FDA-2007). After characterization of each of the vaccines, they were used for immunogenicity and efficacy studies in mice. After laboratory scale production and quality control testing, vaccine constructs pVAX_hsp65, pVAX_hsp65_lack and pVAX_lack were ready to be tested for immunogenicity and efficacy in an experimental model of leishmaniasis in mice.

[0107] Cloning of the lack gene on the plasmid pVAX_hsp65 was performed by GeneScript, resulting in the plasmid pVAX_hsp65_lack. Together with the lack gene, the promoter region (double EF1α / HTL promoter), polyadenylation signal (SV40) and CPG sequence were also cloned as described above using standard techniques.

[0108] The resulting plasmid, pVAX_hsp65_lack, is that of SEQ ID NO:3 and is depicted diagrammatically in FIG.

[0109] Plasmids (iv) and (v) containing the A2 antigen were constructed following the same procedure as described above for the LACK-containing antigen. DNA hsp65, whose immunomodulatory activity has already been confirmed, was used as a vaccine platform and the Leishmania A2-encoding gene was added to the plasmid, resulting in construct pVAX_hsp65_A2(v). Plasmid pVAX_A2(iv) was constructed by GeneScript Inc. by removing the hsp65 gene from plasmid pVAX_hsp65_A2.

[0110] Example 2: Characterization of plasmid pVAX_hsp65_lack Plasmid pVAX_hsp65_lack constructed by GeneScript was transformed into E. coli strain TOP10 for propagation and storage. The plasmid was characterized by restriction analysis with NotI and EcoRI enzymes, which were added to the ends of the lack gene during construction, and with BamHI and ApaI enzymes, which were used in cloning the hsp65 gene. For digestion, 1 μg of plasmid (for each digestion), 1 μL of each enzyme was used in a final 20 μL reaction, which was incubated at 37°C for 30 min to 1 h. Digestion was evaluated on a 1% agarose gel with the addition of GelRed to the samples.

[0111] Plasmid pVAX_hsp65_lack was also used to transfect HEK293 cells, evaluate lack and hsp65 protein expression by Western blot, and identify mRNA for lack and hsp65 by RT-PCR using specific primers. Transfection of HEK cells with plasmid pVAX_hsp65_lack was performed to evaluate the ability of this new plasmid construct to generate transcript (mRNA) and protein. After 24 hours of culture of HEK cells in the presence of DNA plasmid, the medium was removed, cells were washed with PBS (phosphate buffered saline) and Trizol (Invitrogen) was added. Cells were homogenized, harvested and frozen at -70°C. RNA extraction was performed according to the manufacturer's instructions. After extraction and quantification, 1 μg of RNA was treated with DNAseI and subjected to a cDNA generation reaction. RNA from all transfections (with pVAX, pVAX_hsp65 and pVAX_hsp65_lack) was treated in the same way.

[0112] Detection of the transcripts encoded by the plasmid pVAX_hsp65_lack was performed by polymerase chain reaction (PCR) using specific oligonucleotides. As a positive control reaction, PCR specific primers were used for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primers. After 24 h of culture of HEK cells in the presence of DNA plasmid, the medium was removed, cells were washed with PBS, harvested in PBS or CelLyticM (Sigma) and evaluated by Western blot.

[0113] The results showed that the plasmid pVAX_hsp65_lack was constructed correctly because the genes lack and Hsp65 were characterized in the plasmid pVAX_hsp65_lack by restriction enzymes specific to the cloning sites of the respective genes. The expression of Lack and hsp65 mRNA was characterized by PCR in HEK cells transfected with pVAX_hsp65_lack. Also, Hsp65 and lack proteins were characterized by Western blot analysis in the supernatant of HEK cells transfected with pVAX_hsp65_lack plasmid.

[0114] Example 3: Immunogenicity and efficacy of pVAX_hsp65_lack in a mouse model We tested different plasmid constructs and combinations resuspended in saline in a mouse model. To that end, we designed an experiment to evaluate their immunogenicity and efficacy after experimental infection in mice (Mus musculus-Balb / c). A total of 128 mice were randomly divided into eight experimental groups:

[0115] Group A-pVAX_hsp65 (control) Group B-pVAX_A2 (reference control) Group C - Leish-Tec® (Control) Group D-pVAX_hsp65_lack Group E-pVAX_hsp65_A2 (reference control) · Group F-pVAX_hsp65_lack+pVAX_hsp65_A2 Group G-pVAX_lack (control) Group H - Saline (negative control) The experimental groups were then divided into two (immunogenicity and efficacy) containing 8 animals each. The animals were vaccinated with 3 doses applied subcutaneously with a 15-day interval between each dose. They were then vaccinated with 10% of Leishmania infantum (L. infantum) chagasi, cepaNCL / IOCL3241 15 days after the third vaccination. 8 Animals were experimentally infected with the parasite via the intraperitoneal route (Figure 2).

[0116] Fifteen days after the third vaccination, blood samples were taken for antigen-specific (Hsp65, Lack and A2) antibody IgG1 and IgG2 quantification by ELISA test to evaluate immunogenicity, and spleen samples of animals (8 per group) were taken for IFN-g quantification in a commercial kit according to the manufacturer's instructions.

[0117] Thirty days after experimental infection, efficacy of the vaccine formulations was assessed by parasite burden in the liver and spleen using qPCR and limiting dilution assays.

[0118] result: Immunogenicity results showed antibody responses in the vaccinated groups. An increased IFN-γ response was observed in the group vaccinated with pVAX_hsp65_lack (FIG. 3A), indicating a Th1 regulatory response.

[0119] In particular, the antibody response of the pVAX_hsp65_lack vaccine was approximately twice that of the commercial vaccine Leish-Tec. Also, the IFN-γ cellular immune response was significantly higher compared to the DNA vaccine in which the same pVAX plasmid backbone was used but which contained only one of the hsp65 and Lack genes. Furthermore, Lack as an antigen achieved a high IFN-γ response when combined with the immunomodulator hsp65. On the other hand, the vaccine containing the A2 antigen achieved an antibody response similar to that of the vaccine pVAX_hsp65_lack, but did not benefit from its combination with the hsp65 immunomodulator, because the IFN-γ response was lower for the hsp65_A2 combination construct compared to the A2 construct that does not contain the immunomodulator. Interestingly, the combination of both pVAX_hsp65_lack and pVAX_hsp65_A2 combination constructs did not result in an enhanced antibody response compared to the single constructs.

[0120] In addition, in the efficacy evaluation, the parasite burden in the liver of the vaccinated group pVAX_hsp65_lack after 30 days of challenge was 10 times higher than that of the non-vaccinated group (saline). 3 was significantly lower (Figure 3B).

[0121] Notably, parasite load reduction was higher with the pVAX_hsp65_lack vaccine than with the commercial vaccine Leish-Tec, which did not achieve any significant reduction in parasite load. Parasite load was also lower compared to a DNA vaccine using the same pVAX plasmid backbone but containing only one of the hsp65 and Lack genes. Also, no significant reduction in parasite load could be observed with any of the comparative vaccines using the A2 antigen. Thus, although the pVAX_A2 vaccine achieved an antibody response similar to the vaccine pVAX_hsp65_lack, this antibody response did not result in a significant reduction in parasite load. The combination of both combination constructs pVAX_hsp65_lack and pVAX_hsp65_A2 also did not result in a significant reduction in parasite load, reflecting the measured antibody response, compared to the single constructs.

[0122] In conclusion, the mouse model experiments showed that the immune response and protection observed in the group vaccinated with pVAX_hsp65_lack antigen was superior to the other experimental groups. In particular, it was surprisingly observed that the antibody and cellular immune response as well as the parasite load were superior in the group vaccinated with pVAX_hsp65_lack compared to the group vaccinated with pVAX_hsp65_A2. This indicates that the combination of the hsp65 immunomodulator with the LACK antigen can achieve better results than the combination of hsp65 with the A2 antigen. This result is particularly surprising, since it has been recently shown in the art that the immune response induced by the LACK antigen does not provide protection against experimental Leishmania infection and is weaker than that of the A2 antigen (Coelho et al., Infection and Immunity, 71(7), 2003).

[0123] It was also possible to show that the DNA vaccine of the present invention achieves an increased immune response (cellular and humoral) and a reduced parasite load compared to subjects vaccinated with the prior art vaccine Leish-Tec®, a commercially available vaccine based on the Leishmania A2 antigen, which represents a significant improvement compared to the prior art vaccine.

[0124] Example 4: Immunogenicity and efficacy of pVAX_hsp65_lack in a dog model The aim of this study was to evaluate the efficacy and safety of an experimental vaccine against Leishmania infantum in dogs.

[0125] Forty-eight previously neutered, healthy, conventionally bred puppies (male and female) aged 6-12 months, negative for Leishmania infantum by serology and bone marrow PCR, were used in the study. The study was blinded and randomized, with four groups containing 10-11 animals each. Each animal represented an experimental unit. Animals were randomly assigned to the experimental groups according to age, weight, and sex. Two experimental vaccines against Leishmania infantum were tested and their results were compared with a negative control and a positive control (a competing vaccine available in the Brazilian commercial area).

[0126] All groups were vaccinated with a three-dose protocol administered via the subcutaneous route at 21-day intervals (Table 1). [Table 1] Fourteen weeks after the administration of the third dose of vaccine, all animals were challenged with a Leishmania infantum inoculum by the intravenous route. From this point on, animals were monitored for the development of clinical changes associated with canine leishmaniasis, taking into account laboratory markers and clinical signs.

[0127] For immunogenicity assessment of humoral and cellular immune responses to the vaccine, blood samples were collected on days 21 (before administration of the second dose), 42 (before administration of the third dose), 56, 141 (just before challenge), 184, 219, 254, 331, 388, and 443.

[0128] Aspiration punctures from bone marrow and lymph nodes (popliteal or prescapular) were performed for detection of Leishmania infantum DNA to prove the absence of parasitic infection in animals before challenge or to quantify parasite burden after challenge. Bone marrow samples were taken according to the method of Paparcone et al. (2013). Bone marrow and popliteal lymph node samples were obtained on days 56 and 141 to prove the absence of L. infantum genetic material immediately prior to challenge. After this, samples from the same tissues were taken on days 184, 254, 331, 388 and 443 to quantify parasite burden by real-time qPCR.

[0129] Figures 4A, 4B and 4C show parasite burden (individual data and median) by qPCR in lymph nodes at 43, 190 and 302 days after challenge. The saline group was not vaccinated, groups Vac5 and Vac6 received the pDNA vaccine of the present invention (pVAX_hsp65_lack) and the reference group received commercially available Leish-Tec. Vac5 is a vaccine containing pDNA and saponin and Vac6 is a vaccine containing pDNA and saline. To quantify the parasite burden of Leishmania infantum (L. infantum), qPCR was used using primers [forward: 5'-GTGGGGGGGAGGGGCGTTCT-3' (SEQ ID NO: 5) and reverse: 5'-ATTTTACACCAACCCCCAGTT-3' (SEQ ID NO: 6)] targeting the circular DNA (kDNA) of the parasite kinetoplast. qPCR reactions were normalized with 10 ng of purified genomic DNA using a SYBR® Green probe.

[0130] Figures 5A, 5B, and 5C show parasite burden by qPCR in bone marrow at days 43, 190, and 302 post-challenge (individual and median values) for the same groups of animals as described in Figure 4.

[0131] Clinical examinations were performed on days 247 and 302 after Leishmania challenge. Some examples of clinical signs included ear dermatitis, onycholysis, lymphadenopathy, muscle atrophy, ear dermatitis, peeling skin, alopecia, depigmentation, skin ulcers, hyperkeratosis, and blepharitis. It is clear that the challenge model used can induce clinical changes in the animals, a situation that highlights the protective potential of the vaccine used in the study. Animals showing such changes were observed in all experimental groups, with differences in the frequency of more or less clinically affected animals in each of the groups.

[0132] Based on the findings obtained for each dog in each of the groups, Table 2 was compiled, which shows the frequency of animals per group that exhibited changes in physical examination and changes found in laboratory markers related to canine leishmaniasis, which proved to be the primary endpoint highlighting the potential protective effect of the test vaccine formulation. [Table 2] As can be seen in Table 2, the groups receiving the vaccines according to the invention (Vac5, Vac6) show the lowest number of animals with symptomatic disease and the highest number of animals with asymptomatic disease.

[0133] Although the parasite burden in lymph nodes and bone marrow was similar across all dog groups up to 302 days after challenge, comparing the clinical signs and laboratory changes obtained in the saline and reference vaccinated groups it can be concluded that the experimental vaccine based on plasmid pVAX_Lack_Hsp65 reduced the severity of the disease.

Claims

1. (i) a first expression cassette comprising a nucleic acid encoding heat shock protein (hsp) 65 or a functional fragment thereof; and (ii) a second expression cassette comprising a nucleic acid encoding LACK [Leishmania homolog of receptor for activated C kinase] or an immunogenic fragment thereof.

2. An isolated polynucleotide comprising:

2. The polynucleotide of claim 1, wherein hsp65 comprises amino acids 48 to 583 of the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90% sequence identity thereto.

3. 3. The polynucleotide of claim 1 or 2, wherein LACK comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity thereto.

4. 2. The polynucleotide of claim 1, wherein the nucleic acids encoding hsp65 and LACK are under the control of different promoters.

5. A polynucleotide according to any one of the preceding claims, wherein the nucleic acid encoding hsp65 is under the control of a cytomegalovirus (CMV) promoter.

6. The polynucleotide of any one of the preceding claims, wherein the nucleic acid encoding LACK is under the control of the EF1α-HTLV promoter.

7. 2. The polynucleotide of claim 1, wherein the second expression cassette further comprises one or more CpG motifs.

8. A DNA plasmid comprising the polynucleotide according to any one of claims 1 to 7.

9. Use of a polynucleotide according to any one of claims 1 to 7 or of a DNA plasmid according to claim 8 for the recombinant expression of hsp65 and LACK or immunogenic fragments thereof.

10. A DNA vaccine comprising the polynucleotide according to any one of claims 1 to 7, or the DNA plasmid according to claim 8.

11. The DNA vaccine of claim 10, further comprising a pharma- ceutically acceptable carrier.

12. The DNA vaccine of claim 10 or 11, which is a non-adjuvanted vaccine.

13. 12. The DNA vaccine of claim 10 or 11, comprising an adjuvant, preferably wherein the adjuvant is a saponin.

14. The DNA vaccine according to any one of claims 10 to 13 for use in the protection of a subject against infection with leishmaniasis, preferably wherein the subject is a dog.

15. The DNA vaccine for use according to claim 14, wherein the infection is due to canine leishmaniasis.

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