Therapeutic composition for protecting canine, feline, ovine or ruminant animals against the nematode dirofilaria immitis and haemonchus contortus, use of the composition and method for preventing infection caused by nematodes

Abstract

The present invention relates to use of the Sm14 protein as a vaccine antigen against parasites of the phylum Nematoda, also known as nemathelminths or nematodes. In addition to its use against flatworm infections, such as Schistosoma sp, Fasciola sp. and Haemonchus contortus, the present invention also includes compositions containing Sm14 protein, FhFABP-3 protein, and LBP-4 to protect canine, feline, ovine, and ruminant animals from diseases caused by nematodes such as Dirofilaria immitis and Haemonchus contortus.

Description

Therapeutic composition to protect canines, felines, sheep or ruminants against the nematodes Dirofilaria immitis and Haemonchus contortus, use of the composition and METHOD FOR PREVENTING INFECTION CAUSED BY NEMATODES Field of Invention

[0001] This application relates to the use of the Sm 14 protein as a vaccine antigen against parasites of the phylum Nematoda, also known as nematodes. In addition to its use against infections caused by platyhelminth helminths, such as Schistosoma sp., Fasciola sp., and Haemonchus contortus, the present invention also includes compositions containing Sm 14 protein, FhFABP-3 protein, and LBP-4 protein to protect canine, feline, ovine, and ruminant animals from diseases caused by nematodes such as Dirofilaria immitis and Haemonchus contortus. Background

[0002] Nematoda, nemathelminthes, or simply nematodes, comprise a taxonomic group (Phylum) of elongated, cylindrical animals. They are triploblastic, protostome, pseudocoelomate animals. Their cylindrical, elongated, and unsegmented body exhibits bilateral symmetry.

[0003] As a representative of the Onchocercidae family, the species Dirofilaria immitis stands out, whose definitive hosts include dogs, cats, wild carnivores, equines, primates, and, rarely, humans.

[0004] Dirofilariasis is a serious and potentially fatal disease in dogs caused by a parasitic worm called Dirofilaria immitis. The disease is transmitted through the bite of infected mosquitoes, which inject larvae into the dog's bloodstream while feeding. These larvae then migrate to the heart and lungs, where they develop into adult worms that can cause serious damage to these organs.

[0005] The importance of heartworm disease in dogs lies in the fact that it can be fatal if left untreated. As the worms grow, they can cause inflammation and damage to the heart and lungs, leading to symptoms such as coughing, lethargy, exercise intolerance, and difficulty breathing. In severe cases, heartworm disease can lead to heart failure and even death.

[0006] Nematode infections in animals, including humans, are typically treated with drugs. However, the disadvantage of chemical drugs is that they must be administered frequently, as they do not prevent new infections. For example, dogs susceptible to heartworm disease are usually treated monthly. Repeated administration of medication, however, often leads to the development of resistant helminth populations that no longer respond to treatment. Furthermore, many drugs cause harmful side effects in treated animals, and as higher doses become necessary due to the buildup of resistance, the side effects become even more pronounced. Additionally, several medications only treat the symptoms of a parasitic disease but are unable to prevent infection by the parasitic helminth. Drugs that only treat symptoms are unable to eliminate the parasites.

[0007] We can cite as examples of state-of-the-art documents that use drugs for the treatment of helminthic infections in animals, such as Dirofilaria, the patent application BR112022012130-0 which refers to anthelmintic compounds with an azaindolic structure.

[0008] It has also been observed in the patent literature that the search for vaccines against heartworm disease utilizes antigens derived from Dirofilaria immitis and related nematodes. For example, document WO9323077 reports that hosts susceptible to nematode parasite infections can be easily protected from such infections when the parasites are not adapted to a parasite / host relationship with that host. In particular, feline hosts have been immunized against heartworm disease using a variety of antigens derived from Dirofilaria immitis and related nematodes.

[0009] US patent document US4842999 describes a vaccine usable for the prevention of canine heartworm disease, comprising an immunologically active polypeptide associated with D. immitis, the polypeptide being substantially free of natural contaminants and having selected molecular weights of approximately 14 kDa, approximately 58 kDa, approximately 66 kDa and approximately 90 kDa, together with an associated carrier.

[0010] Patent application BR112013002177, filed on behalf of CEVA SANTE ANIMALE, refers to formulations of the drug ivermectin for administration to domestic animals. Preferred formulations are collars, collar bands or medallions, collar accessories, ear tags, bands that are affixed to limbs or body parts, adhesive tapes and films, and peel-off films, or in the form of ready-to-use topical solutions that are administered at least twice a year up to about once a year. Researchers found in the state-of-the-art description that the product of patent application BR112013002177 is related to formulations of Moxidectin, an endectocidal drug (commercially available under the brand name ProHeart-12 from Zoetis).According to a study by the U.S. Food and Drug Administration (FDA) in “FREEDOM OF INFORMATION SUMMARY ORIGINAL NEW ANIMAL DRUG APPLICATION NADA 141-519 ProHeart® 12 (moxidectin) Dogs, 2 July 2019,” studies have shown that the product is 100% effective in preventing heartworm disease caused by *Dirofilaria immitis* for twelve months. However, several adverse events were observed in the field study, including vomiting, diarrhea, anorexia, and seizures. It is important to clarify that moxidectin is an endectocidal chemical drug whose supposed "protective" action is not based on inducing an immune response like true vaccines and depends on continued use by the target hosts. Other D. immitis antigens

[0011] Genome and transcriptome studies of Dirofilaria immitis have identified antigens with potential for use in vaccine development. The following table reveals these potential antigens. (Table 4, Godel et al., The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets. FASEB J. 2012 Nov; 26(11):4650-61. VC = Vaccine, IM = Immunomodulator).

[0012] These proteins, as well as others with partial identity to Sm 14 and nematodes, were not detected during the identification of Dirofilaria immitis immunoreactive proteins recognized by sera from infected cats (Javier González-Miguela Molecular & Biochemical Parasitology 174 (2010) 78-82). In this proteome study, glycolytic pathway proteins, heat shock proteins, glutathione transferase, thioredoxin, and galectin-1 were detected as antigens recognized by the serum of infected animals.

[0013] Some of the antigens found in the proteome study described above have already been tested as vaccines. In the case of Glutathione-S-Transferase (GST), the GST from Setaria digitata (a cattle parasite) was used, which has 100% identity with the GST from Dirofilaria immitis. Immunization with this antigen was able to induce the production of antibodies that neutralize GST activity (Bal M, et al., Asian Pacific Journal of Tropical Medicine (2011) 185-191). Regarding the GST from Dirofilaria immitis, there is a US patent: Dirofilaria immitis GP29 proteins and uses thereof US 5,618,532 (1997).

[0014] Another antigen from the proteome study mentioned above and with a patent is Thioredoxin: Dirofilaria and Brugia Thioredoxin Peroxidase Type-2 proteins and uses thereof, US 6,352,836 B1 (2002).

[0015] The development and availability of a heartworm vaccine for dogs is an ongoing area of ​​research, but currently there is no safe and commercially available vaccine for heartworm. Challenges in developing a safe heartworm vaccine include the complex life cycle of the parasite, the variability in the clinical manifestations of the disease, and the need for long-term protection. Furthermore, it is essential to develop a safe and effective vaccine that does not produce adverse side effects in dogs.

[0016] Therefore, the need remains to identify an effective product that protects animals against diseases caused by parasites, such as heartworm disease. Summary of the Invention

[0017] The present invention relates to the use of the Sm 14 protein as a vaccine antigen against parasites of the Phylum Nematoda, also known as nematodes. In addition to its use against infections caused by platyhelminth helminths, such as Schistosoma sp., Fasciola sp., and Haemonchus contortus, the present invention also includes compositions containing Sm 14 protein, FhFABP-3 protein, and LBP-4 protein to protect canine, feline, ovine, and ruminant animals from diseases caused by nematodes such as Dirofilaria immitis and Haemonchus contortus.

[0018] This application also includes therapeutic compositions containing the proteins Sm 14, FhFABP-3 and LBP-4 to protect animals from diseases caused by nematodes.

[0019] In a more preferred embodiment, the invention relates to a therapeutic composition for protecting canine, feline, ovine, or ruminant animals against the nematodes Dirofilaria immitis and Haemonchus contortus.

[0020] In an even more preferred embodiment, the invention further contemplates the use of the therapeutic composition and a method for preventing infection caused in canine, feline, ovine or ruminant animals by the nematode Dirofilaria immitis or Haemonchus contortus. Brief Description of the Figures

[0021] Figure 1 shows the structure of proteins homologous to Sm 14: As-p18 from A. suum, structure with oleic acid obtained by NMR (PDB 6I9F), CRE-LBP-4 from H. contortus and FABP from D. immitis, modeled by Alpha Fold2. An artificial intelligence program developed by Google's DeepMind that makes predictions about the structure of proteins.

[0022] Figure 2 shows the structure of the ABA-1 protein from A. suum, showing the fatty acid binding sites (PDB 2XV9).

[0023] Figure 3 shows the Western Blot analysis of the presence of Sm 14 homologous proteins in Dirofilaria immitis, using serum from an animal immunized with the Sm 14 antigen formulated with GLA (synthetic Monophosphoryl Lipid A).

[0024] Figure 4 shows the Western Blot analysis of the presence of Sm 14 homologous proteins in Dirofilaria immitis, using serum from an animal immunized with the Sm 14 antigen and saponin in a water-in-oil emulsion.

[0025] Figure 5 shows the titration of anti-Sm14 antibodies present in sera from animals infected with D. immitis, by ELISA.

[0026] Figure 6 shows the Western Blot analysis for the recognition of the Sm 14 protein by serum from an animal infected with D. immitis.

[0027] Figure 7 shows a nitrocellulose membrane containing the Sm 14 protein (lot 12), identified with the name of each animal.

[0028] Figure 8 shows, on the left, the nitrocellulose membranes with the presence of the Sm 14 band after development, and on the right, the two serological tests confirming infection by D. immitis.

[0029] Figure 9 shows membranes relating to sera collected pre-bloodletting (D-1) before vaccination with Sm14+GLA.

[0030] Figure 10 shows membranes incubated with sera collected 28 days after the first (1 a ) vaccination dose, the moment when the second dose is administered.

[0031] Figure 11 shows membranes incubated with sera collected 56 days after the first dose, at which time the third dose is administered. This result demonstrates the increased immune response induced by vaccination and after the administration of the second dose, where it is possible to observe the recognition of the Sm 14 protein as an antigen.

[0032] Figure 12 represents the densitometric analysis of the Western Blotting bands, where the bars determine the mean and standard deviation for on=10 and the (*) represents the p-value<0.05 for the T-test comparing 28 with 56 days.

[0033] Figure 13 is a graph showing the body weight of the animals in kilograms (Kg) during the experimental period, where Gr1 is the Sm14 protein + adjuvant 1 and Gr2 is the Sm14 protein and adjuvant 2.

[0034] Figure 14 is a graph showing the individual values ​​of heart and respiratory rate from D-1 to D+84, where 1: Gr1 is the Sm14+adjuvant protein 1; and, 2: Gr2 is the Sm14 and adjuvant protein 2.

[0035] Figure 15 is a graph showing the evaluation of immunogenicity induced in dogs by vaccination with Sm14 + GLA-SE / MPL adjuvant by ELISA.

[0036] Figures 16A and 16B show the SDS PAGE 15% and Western Blot analysis of the Sm 14 + GLA-SE formulation, kept refrigerated at domestic refrigerator temperature, i.e., (+2 and +8 degrees Celsius) and at room temperature.

[0037] Figure 17 shows the amino acid sequence alignment between the LBP-4 proteins of Haemonchus contortus and Sm 14 of Schistosoma mansoni. Detailed Description of the Invention

[0038] The inventors found that the Sm 14 protein from Schistosoma mansoni, or homologs or sequences listed SEQ ID NO. 1 to 7, is safe and induces a significant immune response that may be the basis for protection against nematode parasites. Furthermore, the LBP-4 protein from Haemonchus contortus (SEQ ID NO: 8) was also found to be safe and induces an immune response in the animals highlighted here.

[0039] Canine dirofilariasis is a parasitic disease of medical and veterinary importance, caused by the nematode *Dirofilaria immitis*, transmitted by hematophagous mosquitoes, especially those of the Culicidae family, such as *Aedes*, *Culex*, and *Anopheles*. After inoculation of infective larvae (L3) into the vertebrate host during hematophagy, larval development occurs until the adult stage, with preferential location in the pulmonary arteries and, in advanced infections, in the right ventricle of the heart. This location results in vascular obstruction, pulmonary hypertension, and eventually, congestive heart failure. Currently, prophylactic chemotherapy is used to control the disease, but this has resulted in resistance of the worm to the medications used, highlighting the need for effective vaccines.

[0040] As already mentioned here in the state of the art, representative of the Onchocercidae family, the species Dirofilaria immitis stands out, which has as definitive hosts the dog, the cat, wild carnivores, equines, primates and, rarely, humans. Considering that the parasite's life cycle is the same for both dogs and cats, the present invention aims to develop a vaccine product for use in the target species.

[0041] The present invention utilizes the Sm 14 protein as defined, for example, in Brazilian patent applications PP1100551, PI0303266, PI1005855, BR102017001309 and BR102021012953. SEQ ID NO: 1 - corresponds to the Sm14M20C62 protein Met Ser Ser Phe Leu Gly Lys Trp Lys Leu Ser Glu Ser His Asn Phe Asp Ala Vai Met Ser Lys Leu Gly Vai Ser Trp Ala Thr Arg Gin lie Gly Asn Thr Vai Thr Pro Thr Vai Thr Phe Thr Met Asp Gly Asp Lys Met Thr Met Leu Thr Glu Ser Thr Phe Lys Asn Leu Ser Cys Thr Phe Lys Phe Gly Glu Glu Phe Asp Glu Lys Thr Ser Asp Gly Arg Asn Vai Lys Ser Vai Vai Glu Lys Asn Ser Glu Ser Lys Leu Thr Gin Thr Gin Vai Asp Pro Lys Asn Thr Thr Vai lie Vai Arg Glu Vai Asp Gly Asp Thr Met Lys Thr Thr Vai Thr Vai Gly Asp Vai Thr Ala lie Arg Asn Tyr Lys Arg Leu Ser SEQ ID NO:2 - corresponds to the protein Sm14T20C62 Met Ser Ser Phe Leu Gly Lys Trp Lys Leu Ser Glu Ser His Asn Phe Asp Ala Vai Thr Ser Lys Leu Gly Vai Ser Trp Ala Thr Arg Gin lie Gly Asn Thr Vai Thr Pro Thr Vai Thr Phe Thr Met Asp Gly Asp Lys Met Thr Met Leu Thr Glu Ser Thr Phe Lys Asn Leu Ser Cys Thr Phe Lys Phe Gly Glu Glu Phe Asp Glu Lys Thr Ser Asp Gly Arg Asn Vai Lys Ser Vai Vai Glu Lys Asn Ser Glu Ser Lys Leu Thr Gin Thr Gin Vai Asp Pro Lys Asn Thr Thr Vai lie Vai Arg Glu Vai Asp Gly Asp Thr Met Lys Thr Thr Vai Thr Vai Gly Asp Vai Thr Ala lie Arg Asn Tyr Lys Arg Leu Ser SEQ ID NO:3 - corresponde à proteína Sm14A20C62 Met Ser Ser Phe Leu Gly Lys Trp Lys Leu Ser Glu Ser His Asn Phe Asp Ala Vai Ala Ser Lys Leu Gly Vai Ser Trp Ala Thr Arg Gin lie Gly Asn Thr Vai Thr Pro Thr Vai Thr Phe Thr Met Asp Gly Asp Lys Met Thr Met Leu Thr Glu Ser Thr Phe Lys Asn Leu Ser Cys Thr Phe Lys Phe Gly Glu Glu Phe Asp Glu Lys Thr Ser Asp Gly Arg Asn Vai Lys Ser Vai Vai Glu Lys Asn Ser Glu Ser Lys Leu Thr Gin Thr Gin Vai Asp Pro Lys Asn Thr Thr Vai lie Vai Arg Glu Vai Asp Gly Asp Thr Met Lys Thr Thr Vai Thr Vai Gly Asp Vai Thr Ala lie Arg Asn Tyr Lys Arg Leu Ser SEQ ID NO:4 - corresponds to protein Sm14M20S62 Met Ser Ser Phe Leu Gly Lys Trp Lys Leu Ser Glu Ser His Asn Phe Asp Ala Vai Met Ser Lys Leu Gly Vai Ser Trp Ala Thr Arg Gin lie Gly Asn Thr Vai Thr Pro Thr Vai Thr Phe Thr Met Asp Gly Asp Lys Met Thr Met Leu Thr Glu Ser Thr Phe Lys Asn Leu Ser Ser Thr Phe Lys Phe Gly Glu Glu Phe Asp Glu Lys Thr Ser Asp Gly Arg Asn Vai Lys Ser Vai Vai Glu Lys Asn Ser Glu Ser Lys Leu Thr Gin Thr Gin Vai Asp Pro Lys Asn Thr Thr Vai lie Vai Arg Glu Vai Asp Gly Asp Thr Met Lys Thr Thr Vai Thr Vai Gly Asp Vai Thr Ala lie Arg Asn Tyr Lys Arg Leu Ser SEQ ID NO:5 - Sm14M20V62 (PI1005855) Met Ser Ser Phe Leu Gly Lys Trp Lys Leu Ser Glu Ser His Asn Phe Asp Ala Vai Met Ser Lys Leu Gly Vai Ser Trp Ala Thr Arg Gin lie Gly Asn Thr Vai Thr Pro Thr Vai Thr Phe Thr Met Asp Gly Asp Lys Met Thr Met Leu Thr Glu Ser Thr Phe Lys Asn Leu Ser Vai Thr Phe Lys Phe Gly Glu Glu Phe Asp Glu Lys Thr Ser Asp Gly Arg Asn Vai Lys Ser Vai Vai Glu Lys Asn Ser Glu Ser Lys Leu Thr Gin Thr Gin Vai Asp Pro Lys Asn Thr Thr Vai lie Vai Arg Glu Vai Asp Gly Asp Thr Met Lys Thr Thr Vai Thr Vai Gly Asp Vai Thr Ala lie Arg Asn Tyr Lys Arg Leu Ser SEQ ID NO:6 - FhFABP-3 protein - Fatty acid-binding protein type 3 - Fasciola hepatica (GenBank Q9U1 G6.1). SEQ ID NO:6 has the following sequence: Met Ala Asn Phe Vai Gly Ser Trp Lys Leu Glu Gin Ser Glu Asn Met Asp Ala Vai Leu Gin Lys Leu Gly lie Asn Vai lie Lys Arg Lys Leu lie Thr Ser Ser Lys Pro Glu lie Thr Phe Thr Leu Glu Gly Asn Lys Met Thr Met Lys Thr Vai Ser Ala Leu Lys Thr Thr Vai lie Ser Phe Thr Phe Gly Glu Glu Phe Lys Glu Glu Thr Ala Asp Gly Arg Thr Vai Met Thr Thr Phe Thr Lys Asp Ser Asp Ser Lys lie Ser Gin Vai Gin Lys Cys Pro Glu Asn Thr Thr His Vai Vai Arg Glu Vai Thr Gly Gly Lys Met lie Ala Thr Vai Thr Vai Gly Asp Vai Lys Ala Vai Asn Asn Tyr His Lys Vai SEQ ID NO:7 - FhFABP-V protein - fatty acid-binding protein type V - Fasciola hepatica (GenBank AJO53793.1). SEQ ID NO:7 has the following sequence: Met Ser Gly Phe lie Gly Lys Trp Lys Leu Vai Asp Ser Arg Asp Phe Asp Lys Vai Met Vai Glu Leu Gly Vai Gly Tyr Met Thr Arg Lys lie Ala Glu Asn Thr Lys Pro Thr Vai Thr lie Thr Lys Phe Gly Glu Asp Gly Leu Thr Met Lys Thr Glu Ser Thr Phe Lys Thr Ser Glu lie Ser Phe Gin Phe Gly Vai Glu Phe Asp Glu Thr Thr Ala Asp Gly Arg Gin Vai Lys Ser Thr Vai Thr Lys Asp Ser Asp Tyr Arg lie Thr Gin Vai Gin Lys His Pro Asn Ala Asp Thr His lie Vai Arg Gin Vai Glu Asn Asp lie Met Asp Thr Thr Vai Thr Vai Arg Asp Vai Vai Ser His Arg Arg Tyr Gin Arg lie Lys

[0042] Among the patent applications and patents mentioned above, the Sm14M20V62 protein was used to demonstrate the effectiveness of the objectives contemplated by the invention.

[0043] The invention utilizes the LBP-4 protein from Haemonchus contortus, as its modeled structure possesses conserved residues between LBP-4 and Sm 14. The Sm 14 protein from Schistosoma mansoni is used as an antigen for dirofilariasis.

[0044] The Sm14 antigen of Schistosoma mansoni has shown cross-reactivity with sera from animals infected with Platyhelminthes (Fasciola hepatica, Echinostoma paraensei and Taenia saginata) as well as the nematode Ascaris suum. In the latter case, the protein recognized by the anti-Sm14 serum has a slightly higher molecular mass than the Sm14 protein (Thaumaturgo N, Vilar MM, Edelenyi R, Tendler M. Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis. Mem Inst Oswaldo Cruz. 2002. 97 Suppl 1:115-6).

[0045] Two worms of veterinary importance belong to the nematodes: Dirofilaria immitis and Haemonchus contortus. Thus, the inventors found that the Sm 14 protein functions as a vaccine antigen against parasites of the Phylum Nematoda.

[0046] NCBI BLASTP analysis identified several nematode proteins that have homology with the Sm 14 sequence, such as: • As-p18 Fatty acid-binding protein homolog [Ascaris suum] (PDB 6I9F) (26% identity) • CRE-LBP-4 protein [Haemonchus contortus] (CDJ82504.1 ) (27% identity) Fatty acid-binding protein [Dirofilaria immitis] (MCP9263057.1 ) (27% identity)

[0047] Figure 17 shows that identical residues are marked in red, similar residues are highlighted in yellow, and the consensus line below indicates positions with >50% conservation. Several conserved residues were identified, corroborating the structural and functional similarity between the LBP-4 proteins of Haemonchus contortus and the lipid-binding Sm14 proteins.

[0048] The SEQ ID NO. 8 sequence for LBP-4 is shown below. METVIPDKFF GRFVLERSEN FDEFLAAKGV NWFVRKMIQF ASVTKVISKN ENSGYNLDNL TSKKNTLYHG WKLGETFEAD GMDDVRHNIT FNFENDTLTE KHVRLNDPND KGETYYYNID ADDKLVLKME NHGITCRRWF KREKK

[0049] Despite not having very high sequence identity, the three proteins listed above have typical FABP folding patterns, indicating that they belong to this family and are true homologs of Sm 14, as can be seen in Figure 1. The modeled structure of LBP-4 (Haemonchus contortus) has conserved residues between LBP-4 and Sm 14.

[0050] Despite their identical sequence, both LBP-4 and Sm14 proteins exhibit a conserved barrel fold |3, characteristic of lipid-binding proteins, indicating evolutionary convergence and functional conservation in their lipid-binding roles. These conserved regions are considered relevant for epitope grafting and cross-reactivity analysis. Cross-reactivity data

[0051] In an initial preparation, adult Dirofilaria immitis worms of both sexes were used to prepare an extract of adult Dirofilaria immitis worms.

[0052] In the laboratory, Dirofilaria immitis worms were placed in a mortar, frozen with liquid nitrogen, and pulverized with a pestle until a homogeneous powder was obtained. The crushed material was resuspended in PBS and transferred to a Potter-Elvehjem tissue homogenizer. It was homogenized for 5 minutes. Finally, the lysate was centrifuged at 12,000 rpm for 30 minutes. After separating the supernatant, the pellet was resuspended in PBS + 1% SDS buffer to resuspend the insoluble proteins. Extracts of soluble and insoluble proteins were obtained.

[0053] The soluble and insoluble protein extracts obtained were analyzed by denaturing SDS-PAGE electrophoresis, in parallel and comparatively with the recombinant proteins Sm 14, FhFABP03 (Sm14 homologue in Fasciola hepatica) and PR21, a recombinant protein containing select epitopes of Sm 14, described by Vilar et al. (2003) (Vilar MM, Barrientos F, Almeida M, Thaumaturgo N, Simpson A, Garratt R, Tendler M. An experimental bivalent peptide vaccine against schistosomiasis and fascioliasis. Vaccine. 2003. 8;22(1): 137-44).

[0054] For the Western blot experiments, sera from sheep immunized with two doses (0 and 28 days) were used with the following formulations: (a) an aqueous formulation of the antigens (10 to 50 pg of rSm14, and 2.5 to 5.0 pg of GLA in emulsion with squalene), where GLA is a synthetic monophosphoryl lipid, agonist of Toll-Like Receptor type 4 (TLR4) and the antigen is one of the Sm14 proteins selected from sequences SEQ ID NO: 1 to 7; (b) with an oily formulation (water-in-oil emulsion - 40% / 60%) (The aqueous phase contained 10 - 50 pg of rSm14 and 1 mg of saponin, while the oily phase was Marcol 52, Cetyl-PEG was used as a surfactant).

[0055] With regard to formulation (a) above, the GLA is with Squalene Emulsion, i.e., GLA-SE. Alternatively, it is also possible to use Sm 14 or any of the sequences 1 to 7 formulated from synthetic MPL which is analogous to GLA (SyMPL), which has already been used as an adjuvant in vaccination against other infections.

[0056] The present invention also proposes, as another embodiment of the invention, the selection and use of adjuvants, as follows: Adjuvant 1: GLA + squalene; Adjuvant 2: MPL + squalene

[0057] The proteins with potential use in the composition of this invention are those mentioned and related as SEQ ID NO: 1 through SEQ ID NO: 8.

[0058] For the preparation of the alternative vaccine composition, the amount of adjuvant 1 (GLA-SE) was in the range of 10µg to 20µg per dose. The amount of adjuvant 2 (MPL-SE) used was also within the range of 10µg to 20µg. Thus, approximately 250µl of Sm14 plus 470µl of GLA were used, resulting in a volume of 720µl of the vaccine preparation. This dose resulted in a satisfactory immune response in the vaccination of the dogs, as further confirmed by ELISA and Western Blot.

[0059] With reference to Figure 3 (Aqueous formulation), we have: A. - gel colored with Coomasie blue. B. Western blot with rabbit anti-Sm14 serum (1 / 700). C - Western blot with pooled sera from animals immunized with the aqueous formulation, 35 days incubation and 1 / 200 dilution. D. Western blot with pooled pre-immune sera, time 0 days, and dilution 1 / 200. M. - Molecular mass marker; 1. - Soluble protein extract of Dirofilaria immitis in PBS; 2. - Total protein extract of Dirofilaria immitis; 3. - Sm14 protein, Lot 04; 4. - PR21 protein, Lot 02; 5. - FABP family homolog protein cloned from Fasciola hepatica, FABP03

[0060] With reference to Figure 4 (Oily formulation with saponin), we have that: A.- gel tinted with Coomasie blue. B. Western blot with pools of pre-immune sera (time 0), 1 / 200 dilution. C - Western blot with pooled sera from animals immunized with the oil formulation, 35 days incubation period and 1 / 10000 dilution. M. - Molecular mass marker; Sm14 - Sm14 protein Lot 04; FhFABP03 - Fasciola hepatica protein FABP03; PR21 - PR21 protein lot 02; Dirofilaria - Total protein extract of Dirofilaria immitis.

[0061] The results show that immunization with an oil-based formulation containing saponin can induce the production of antibodies that recognize the FhFABP03 protein of Fasciola hepatica and, more weakly, PR21.

[0062] It is important to point out that the dilution used in the case of the formulation with adjuvant described in b-formulation was much greater than that used for the formula with GLA-SE (1:10000 and 1:200, respectively).

[0063] At a 1:200 dilution, there is nonspecific recognition of high molecular weight proteins (Figure 3), which are not observed in Western Blot with a serum dilution of 1:10000. It is possible that with lower dilutions of serum from animals immunized with the Sm 14 protein in an oil formulation with saponin, D. immintis antigens may be recognized.

[0064] Sera from six female dogs at different stages of infection were also tested against the Sm14 protein. Figure 5 shows the result of the titration of anti-Sm14 antibodies present in sera from animals infected with D. immitis.

[0065] This result was corroborated by Western blot testing, a control protein unrelated to Sm14 with a molecular mass similar to Sm14 (recombinant myoglobin), and the serum of one of the animals that They showed antibodies that recognize the Sm 14 protein in the ELISA experiment (animal named “Princess”, Figure 5). For the Western blot, serum was used at a 1 / 800 dilution.

[0066] Figure 6 shows the Western blot analysis for the recognition of the Sm14 protein by serum from an animal infected with D. immitis, where: M - molecular mass marker, 1 - Sm 14 protein, 2 - negative control (unrelated protein). Ponceau - membrane stained with Ponceau dye after transfer of proteins separated by SDS-PAGE, Western blot - ECL revelation of the presence of anti-Sm 14 antibodies.

[0067] In summary, the results clearly show cross-reactivity of the immune response induced by D. immitis infection with the Sm 14 protein.

[0068] Therefore, the present invention described herein shows that the Sm 14 protein is relevant as a vaccine antigen against parasites also of the Phylum Nematoda, as previously demonstrated for human and animal helminth parasites of the class Trematoda, such as Schistosoma and Fasciola.

[0069] Through Experiment A, the inventors demonstrate the immunogenicity potentially indicative of the efficacy of the therapeutic composition to protect canine animals against the nematode Dirofilaria immitis, the use of the composition, and the method for preventing infection caused by the nematode Dirofilaria immitis in dogs. Experiment A Capacity of serum from dogs infected with Dirofilaria immitis to recognize the Sm 14 protein as an antigen.

[0070] The animals came from a veterinary clinic and had been naturally infected with the causative agent of canine dirofilariasis, where the infection was confirmed by means of two serological tests for D. immitis antigens.

[0071] Through antibody binding analysis to specific antigens, the Western blotting technique was used to evaluate the ability of D. immitis infection to produce antibodies that recognize a protein with immunogenic properties belonging to the FAB (fatty acid-binding protein) family, such as... Sm 14 protein, found in the parasite that causes schistosomiasis, Schistosoma mansoni.

[0072] For this test, a sample of purified Sm 14 (Development Lot 12) was used, which was transferred to a nitrocellulose membrane, indicating that it only contains this protein in the experiment. The membranes were cut and incubated separately with the sera from each dog individually, as shown in Figure 7.

[0073] Figure 8 shows the results, where the arrows indicate the presence of the Sm14 band being recognized as an antigen by the antibodies present in the sera of animals infected with dirofilariasis, demonstrating that the infection generated an immunogenic potential by recognizing a protein from the FABP family, from another helminth, which is the case of Sm14 from S. mansoni, probably because D. immitis also possesses a similar protein that is part of this protein family, and it is responsible for generating this antibody induction due to the presence of the parasite infecting these animals. Experiment B Monitoring the humoral immune response induced in dogs by vaccination with 3 doses of the Sm14+GLA-SE anthelmintic vaccine.

[0074] Knowing that infection with D. immitis was able to induce, in dogs, an immune response of antibodies capable of recognizing a protein from the FABP family, Sm14, the experiment aims to monitor the dynamics of antibodies induced by vaccination with Sm14+GLA.

[0075] For this purpose, blood was collected from 10 previously vaccinated animals, and serum was separated before and after different doses of the vaccine. The information obtained from the laboratory (Brazilian Animal Research Center: CPABR) where the vaccination was performed is shown below in Table 2: Information about the animals. Beagle puppies aged 4 to 6 months from CPABR. - Total number of animals tested: 10 - Blood collection dates: October 3, 2024

[0076] The same Western blotting technique was used, using Sm 14 (Lot 12) as the antigen; the membranes were cut and incubated individually with the sera from each animal. D-1: pre-bloodletting - before vaccination D+27 / D+28: application date of the 1st a dose D+55 / D+56: post 1 a dose (date of application of the 2nd a dose)

[0077] The results are shown in Figure 9, which shows the membranes corresponding to the pre-bloodletting sera (D-1) before vaccination with Sm14+GLA. The red arrow indicates the location where Sm14, if recognized by an antibody, should appear. These serum samples are negative controls of the experiment.

[0078] Figure 9 shows the membranes incubated with the sera obtained 28 days after the 1st aThe vaccination dose, at which point the second doses were administered. The administration of the second dose of the vaccine, as expected, increased the immune response, with a strong band being observed, characterized by the formation of antibodies that recognize proteins of the FABP family, as has already been seen, which infection by different nematodes is also capable of inducing, and the same was observed previously with the effect of infection by D. immitis.

[0079] Figure 10 shows the membranes incubated with the sera obtained 56 days after the first dose, at which point the third doses were administered. This result demonstrates the increased immune response (or immunogenicity) after the second dose, where recognition of Sm 14 as an antigen can be observed.

[0080] Figure 11 shows the densitometric analysis of the Western Blotting bands. The bars determine the mean and standard deviation for on=10 and the (*) represents the p-value<0.05 for the T-test comparing 28 with 56 days.

[0081] Densitometric analysis of Western blotting bands resulting from the recognition of Sm14 as an antigen by antibodies present in the sera of vaccinated dogs revealed an increase in immunogenicity after the second dose of vaccination with Sm14+GLA.

[0082] The following are experiments evaluating the immunogenicity and safety induced in dogs by vaccination with Sm 14 plus specific adjuvant. In eight (8) animals GLA-SE was used and in two (2) a synthetic equivalent to GLA, i.e., MPL. Experiment C

[0083] The aim of this experiment was to evaluate the immunogenicity induced by vaccination with Sm 14 plus a specific adjuvant, administered intramuscularly in the quadriceps of puppies, at doses of 0.72 mL of Sm14 + adjuvant 1 for group Gr1 and 0.43 mL of Sm14 + adjuvant 2 for group Gr2. Ten animals (5 males and 5 females), beagle breed, aged 4 to 6 months and weighing between 5.0 kg, 10.0 kg and 15.6 kg, were used. The selected animals were randomized, according to weight, into two groups with the same number of males and females per group: Groups Gr1 and Gr2. Table 3 contains information about the animals.

[0084] To assess the safety of the vaccine product, general health observations were carried out throughout the experimental period, in addition to clinical, hematological, and biochemical examinations at different time points. In parallel, the immunogenicity induced by vaccination was evaluated. Clinical and laboratory results demonstrated that there were no clinical or laboratory alterations. No adverse reactions were observed during or after the application of the Sm 14 vaccine. The animals showed a seroconversion rate of 90%, demonstrating a high immune response and indicating potentially high vaccine efficacy. Table 3. Information about the animals used in the study.

[0085] The animals were kept in the experimental kennel of the Brazilian Animal Research Center (CPABR), housed in pairs in covered pens, which had space for sunbathing and an automatic waterer. The cleaning of the pens was carried out daily in accordance with POP-CGS-003 (Cleaning of the Kennel, feeders and waterers).

[0086] The animals were released for approximately one hour each experimental group at a time into the recreation area adjacent to the enclosure, and were collected at the end of the recreation activities.

[0087] The animals were kept under the same environmental conditions throughout the trial period, with natural light and temperature.

[0088] The animals were fed premium feed with 23% protein in the morning and afternoon, the amount offered was adequate for the species, in order to meet the daily recommendations. The feed was stored on pallets in the CPABR feed storage area. This environment has a vector control system.

[0089] The animals had ad libitum access to water from an artesian well, through automatic waterers installed in the pens.

[0090] The dogs belong to the breeding stock of the Brazilian Animal Research Center and live in appropriate facilities. After the study ended, the animals returned to the CPABR breeding stock. Inclusion, exclusion, and post-inclusion removal criteria Inclusion criteria

[0091] Beagle dogs, both male and female, aged between 4 and 6 months, were included.

[0092] Animals that are healthy at the time of inclusion, dewormed, with a body condition score of 5.

[0093] Animals that showed physical integrity of the area where the investigational veterinary product was administered. Exclusion criteria

[0094] Animals that showed any clinical signs of disease or were not in good general condition, at the investigator's discretion. Post-inclusion removal criteria

[0095] No animals needed to be removed after being included in the study. Acclimatization

[0096] The animals were acclimatized for 7 days prior to treatment (D-7 to D0) and during this period, they were observed at least once a day to check their health.

[0097] On D-7, the animals were identified and weighed.

[0098] On day D-1, selection and randomization by sex were performed. At this time, the animals also underwent clinical examinations, weighing, and blood collection for laboratory tests.

[0099] After the acclimation period began, no changes were made to the animals' management or feeding practices. Selection of animals

[0100] On day D-1, the 10 dogs that participated in the study were selected according to the inclusion / exclusion criteria. The formation of the experimental groups is presented in Table 4.

[0101] Vaccination occurred on days D0, D+28, and D+56 of the study, and the record was kept on the appropriate form. Table 4. Information on dividing animals into experimental groups and treatment.

[0102] The experiment consisted of two experimental groups, composed of Beagle dogs aged between 4 and 6 months. Group Gr1 consisted of 8 animals (4 males and 4 females) and Gr2 of 2 animals (1 male and 1 female).

[0103] Groups Gr1 (Sm 14 + adjuvant 1) and Gr2 (Sm 14 + adjuvant 2) were vaccinated with 3 doses of the product + specific adjuvant, via intramuscular injection (quadriceps), at 28-day intervals, at times D0, D+28 and D+56.

[0104] At time points D-1, D+27, D+55, and D+84, blood samples (approximately 5 ml) were collected from the external jugular vein to obtain serum. Analysis was also performed at time points D-1 and D+84. Hematological (complete blood count, including red blood cell count and white blood cell count) and biochemical (creatinine, urea, and liver enzymes: AST and ALT).

[0105] At times D-1 and D+84, the animals underwent clinical evaluation, which consisted of assessing the parameters of respiratory rate (RR), heart rate (HR), rectal temperature (T), lymph node evaluation (LYMPH), capillary refill time (CRT). Additionally, the animals were evaluated for oral mucosa color, appetite, stool consistency, and possible alterations such as prostration, lethargy, sedation, incoordination, sialorrhea, vomiting, tremors, and abdominal pain. Table 5. Data on the animals selected for the Experiment Dosage and treatment

[0106] The animals received the Sm 14 + adjuvant 1 (Gr1) and Sm 14 + adjuvant 2 (Gr2) vaccine formulations. The vaccine preparation was carried out under strict aseptic conditions, using sterile surgical gloves and adequate asepsis at the application site.

[0107] The Sm 14 + specific adjuvant formulation was administered intramuscularly in the quadriceps every 28 days (DO, D+28 and D+56). Vaccination times were standardized for all three doses, and vaccination records were kept on the specific CPABR form (086).

[0108] A veterinarian with experience in dog handling and familiar with animal health and behavior performed OGS (Organic Glucose Testing) on ​​all animals at least once a day throughout the study. The OGS consisted of... The study observed the animals' general physical appearance and behavior, abnormalities in food and water consumption, urine and feces appearance, and other parameters, according to the species, that are indicative of their health. During the experimental period, the animals did not present any behavioral changes and / or alterations in their health.

[0109] The animals underwent clinical examination at times D-1 and D+84. The clinical examination assessed the animals' parameters according to the following methodology: Respiratory Rate (RR): This parameter was obtained in breaths per minute (bpm), measured by observing the movements of the animals' rib cage for one minute. Heart Rate (HR): This parameter was obtained in beats per minute (bpm) using stethoscopy. Rectal temperature (T): was measured using a digital thermometer, inserted into the rectal ampulla of the animals. Lymph Node Assessment (LYMPH): assessments were performed by palpation to evaluate size (0 = normal; 1 = enlarged) and sensitivity (pain - scores: 0 = absent; 1 = mild; 2 = moderate; 3 = intense). The lymph nodes evaluated were: popliteal, submandibular, and prescapular. Capillary refill time (CRT): was recorded in seconds by pressing the gingival mucosa with the thumb and, upon releasing the pressure, recording the time elapsed until the characteristic color of the tissue was restored.

[0110] Furthermore, the animals were inspected for oral mucosa color (scores: 0 = normal color; 1 = pale; 2 = pale), appetite (scores: 0 = normal; 1 = absent; 2 = decreased; 3 = increased), stool consistency (0 = normal; 1 = pasty; 2 = liquid; 3 = bloody); and possible alterations such as prostration, lethargy, sedation, incoordination, sialorrhea, vomiting, tremors, and abdominal pain were evaluated, recording the following scores: 0 = absent; 1 = mild; 2 = moderate; 3 = intense. Blood collection

[0111] Blood samples were collected using the appropriate form. Table 6 details the days and procedures for which the blood samples were intended. Table 6. Blood collection. Complete Blood Count and Biochemistry - Blood Serum

[0112] Blood samples were collected for complete blood count and serum biochemistry analysis at time points D-1 and D+84.

[0113] The blood sample was obtained by puncturing the external jugular vein (approximately 5 ml per collection) using disposable hypodermic needles. After collection, the blood was carefully transferred to the respective vials. The total amount was distributed into two separate Vacutainer® tubes, one containing anticoagulant (EDTA - 0.5 mL of blood) for complete blood count tests and the other without additives, for biochemical tests and obtaining serum for immunological analyses. Each tube was identified with the animal's microchip number, study number, study day, and sample collection date. Subsequently, the samples were processed at the CPABR Clinical Analysis Laboratory.

[0114] For the complete blood count, the following were analyzed: red blood cell count (RBC) and hemoglobin count (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), differential white blood cell count (segmented neutrophils, band neutrophils, lymphocytes, eosinophils, basophils, and monocytes), in addition to the platelet count (PQT).

[0115] For the biochemical profile to assess liver and kidney function, the following serum levels were measured: urea (UR), creatinine (CR), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Immunology

[0116] For the immunological analyses, blood samples were collected to obtain serum at the following times: D-1, D+27, D+55, and D+84.

[0117] A blood volume of approximately 5 mL was collected in a tube without EDTA (without anticoagulant) through puncture of the external jugular vein, using disposable hypodermic needles.

[0118] Serum collection: After collection, the blood was kept at rest for a maximum of 2 hours at 4°C for complete clot retraction. Subsequently, each tube was centrifuged (5000 rpm / 5 min) and the serum was carefully collected and aliquoted into 3 plastic, polypropylene or polystyrene tubes (never glass). Eppendorf tubes were used. Each tube was labeled with the following information: CPABR-PEC-4915-24 Immunogenicity Animal identification number Date / time of the study Serum - Test, counter-test 1 and 2

[0119] The serum samples were stored in cryogenic boxes, labeled with a map of the samples and identified with the following information: CPABR-PEC-4915-24 Immunogenicity Date / time of the study Serum - Test, counter-test 1 and 2 Researcher contact information: Contact information for the sponsoring company:

[0120] The tubes were placed in small boxes with internal grids, so that they remained in an upright position and in the order in which they were identified. The tubes were identified on the lid (for example: numbering them from 1 to 100), thus facilitating verification by the laboratory teams without unnecessarily thawing the samples.

[0121] The samples were placed in the aforementioned boxes. The box was then placed in secondary packaging such as a ziploc plastic bag. During Throughout the entire period that the material was stored at the research center, the freezer temperature was duly recorded. For this purpose, the values ​​obtained daily from the temperature monitoring were recorded on an appropriate form, thus characterizing the control of the cold chain and the quality of the experiment. VACCINE SAFETY ASSESSMENT: CLINICAL ANALYSIS:

[0122] Immediately after administration of the product and daily until the end of the study, all animals were evaluated and no animal presented any signs indicative of toxicity from the administered vaccine, including excitement, convulsions, dysphonia, cough, depression, prostration, sedation, lethargy, apathy, ataxia, hyperesthesia, motor incoordination, lack of balance, sweating, edema of the limbs or face, urticaria, jaundice, head tremors, skin tremors, muscle tremors, vomiting, diarrhea, dyspnea, decreased appetite, or anorexia / sialorrhea. The information obtained during these evaluations was recorded on a specific CPABR form, where the absence of any signs of vaccine toxicity and its safety were identified. LABORATORY ANALYSIS Blood samples

[0123] The complete blood count was performed using a BC-2800-Vet-Mindray® analyzer. The differential diagnosis was made using a blood smear and optical microscopy.

[0124] All biochemical determinations (UR, AST, ALT, UR) were performed using the Labtest® Kit and the Mindray® BS-120 automated biochemical analyzer.

[0125] Serum biochemistry analysis was performed according to the standard clinical laboratory procedure (POP-LAB-002 - Biochemical Analyses).

[0126] The CPABR clinical laboratory provided the hematological test results, which were attached to the final report. The external laboratory, Labcare - Veterinary Analysis Laboratory, also provided the reports, which were attached to the final report. INTERPRETATION OF RESULTS

[0127] The results of the evaluation parameters and OGS were reported in a descriptive manner. ADVERSE EVENT

[0128] An adverse event is any harmful, unintentional response that may manifest as illness, abnormality, or injury observed in animals participating in a clinical study after administration of the test products. The adverse event may or may not be related to the use of the test product.

[0129] During the experimental period, no animal presented any adverse event; therefore, form 005 was not completed for this purpose. EUTHANASIA OR MORTALITY OF ANIMALS

[0130] No animals died or needed to be euthanized (for animal welfare reasons). Amendments, Protocol Deviations and Notes

[0131] There were no deviations from protocol.

[0128] An amendment to the protocol was necessary, recorded on the appropriate form, duly dated and approved. Amendment 01 - Date of occurrence: 30SEP24 Description:

[0132] The current protocol - Dosage and Treatment - establishes that the animals included in the study would receive the Sm 14 vaccine formulation associated with different adjuvants, namely: Group 1 (Gr1): Sm14 + Adjuvant 1, at a dose of 0.5 mL; Group 2 (Gr2): Sm14 + Adjuvant 2, at a dose of 0.5 mL. Change made: Adjustments were made to the doses administered to the experimental groups, as follows: • Group 1 (Gr1): Sm14 + Adjuvant 1, at a total dose of 0.72 mL; • Group 2 (Gr2): Sm14 + Adjuvant 2, at a total dose of 0.43 mL. Impact on the study: No impact. RESULTS AND DISCUSSION Weight of animals

[0133] Body weight is the best parameter to assess the overall condition of a puppy (HOSKINS, 1997; BARRETO, 2003; DOMINGOS et al., 2008).

[0134] The animals that were screened, selected, and randomized presented a body condition score classified as 5, according to the scale proposed by LAFLAMME (1997), a parameter widely used as a reference for animals in ideal body condition and compatible with an adequate nutritional status.

[0135] During the experimental period, from D-7 to D+84, the animals showed an increase in body weight. At time D-7, the average weight was 6.79 kg in group Gr1 and 8.65 kg in group Gr2. At time D+84, group Gr1 had an average of 10.39 kg and group Gr2 11.60 kg (Table 6 and Figure 13). This is an essential indicator for evaluating the healthy development of animals, and is frequently used as a parameter in measuring animal growth and nutritional status.

[0136] When analyzing the two experimental groups, Gr1 and Gr2, it was found that both maintained a body condition score of 5 throughout the study, characterizing an adequate nutritional condition compatible with the age range of the animals. Table 7: Variation in Animal Weight during the Experimental Period Clinical Examination

[0137] The animals underwent a complete clinical examination at experimental time points D-1 and D+84 to analyze physiological parameters and possible systemic reactions resulting from the administration of the product. Investigational Sm 14 and adjuvants. The clinical evaluation included measuring heart and respiratory rate, rectal temperature, presence of lymph nodes, capillary refill time (CRT), and inspection of the oral mucosa, fundamental parameters for determining the clinical status of the animals.

[0138] Additionally, the animals were examined for the presence of systemic reactions, including signs of prostration, lethargy, sedation, motor incoordination, salivation, episodes of vomiting, abdominal pain, feces, and the occurrence of tremors. These parameters are frequently used to detect adverse effects and are essential for the evaluation of the formulated investigational product Sm14.

[0139] Heart rate and respiratory rate values ​​are higher (HOSKINS, 2008; LAREDO, 2009) for puppies than for adult dogs.

[0140] These parameters can be influenced by animal handling and ambient temperature.

[0141] The data obtained from the assessments performed at day D-1 and after administration of the three doses of the investigational product indicated that all physiological parameters for heart rate (HR), capillary refill time (CRT), mucous membranes, and body temperature remained within the normal ranges recommended for the species. The respiratory rate (RR) value obtained during days D-7 and D+84, for some animals, was above that recommended by FEITOSA (2008), and this can be explained by the fact that breathing becomes faster during moments of excitement, stress, exercise, and high ambient temperatures. However, these changes were isolated and without clinical relevance (Table 8 and Figure 14).

[0142] All animals in this study presented parameters used for evaluating systemic reaction within normal limits in all groups. Therefore, no adverse events were identified. Table 8. Individual clinical examination values ​​at times D-1 and D+84. Reference Values: Table 9: Individual values ​​of systemic reaction observations from D-1 to D+84. Laboratory tests Blood count

[0143] A complete blood count (CBC) is a fundamental hematological test that allows the quantification and characterization of blood cell elements. It is widely used in the clinical evaluation of animals due to its ability to provide comprehensive information about the physiological or pathological state of the organism. It is a first-line test in the investigation of hematopoietic function, enabling not only the absolute count of each blood cell component, but also the analysis of its morphology, contributing to the detection of anemias, infections, inflammatory processes, and various hematological disorders (GARCIA-NAVARRO, 2005).

[0144] A complete blood count (CBC) is composed of different parameters, including the red blood cell count, which evaluates the red blood cell series through erythrocyte count, hemoglobin concentration, hematocrit, and erythrocyte indices; the white blood cell count, which quantitatively and qualitatively analyzes leukocytes, allowing inferences about the body's immune response; and the platelet count, essential for evaluating hemostasis. In addition to these components, the morphological analysis of blood cells provides further information that may indicate metabolic, infectious, or immune-mediated alterations (GARCIA-NAVARRO, 2005).

[0145] In the present study, the animals evaluated presented hematological values ​​within the reference ranges for the species, with parameters of the erythrogram, leukogram and platelet count compatible with physiological normality (Table 10). Table 10. Individual values ​​of hematocrit (HT), red blood cell count (RBC), hemoglobin (HB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in dogs from D-1 to D+84. Biochemical

[0146] The results of the biochemical analyses performed on the animals did not show significant alterations in the parameters evaluated. Serum concentrations of liver enzymes, renal markers, and blood metabolites remained within the physiological reference values ​​for the species under study, indicating the absence of metabolic or organic dysfunctions resulting from the experiment (Table 11). Table 11. Individual data from biochemical examination D-1 and D+84 IMMUNOGENICITY ASSESSMENT

[0147] The evaluation of immunogenicity induced in puppies by the Sm 14+ adjuvant vaccination was analyzed using ELISA.

[0148] In assays with sera from immunized animals, readings of OD450nm greater than 0.2 relative to time 0 (cutoff) were considered positive, indicating the presence of specific antibodies against the Sm 14 protein. Values ​​below the cutoff (<0.2) were considered negative, suggesting the absence of a detectable immune response at the time of testing (Table 12 and Figure 14). Table 12. Evaluation of immunogenicity induced in dogs by vaccination with Sm 14 + GLA-SE / MPL adjuvant by ELISA.

[0149] After the second dose of vaccination, seroconversion was observed in 90% of the animals tested with Sm14+GLA-SE (N=8) or Sm14+MPL (N=2).

[0150] The vaccine formulation described here has proven to be stable, as reported below. Stability of the formulated vaccine

[0151] The antigen formulations were prepared as indicated below: Vaccine formulation (rSm14 50uQ + GLA-SE 2.5uo) It was mixed in a sterile Eppendorf tube: • 0.2 ml of GLA-SE adjuvant (lot # 21A001), with, • 0.2 ml of Adjuvant Diluent (lot #21 A002) Eppendorf tube contains 10 pg / ml of GLA-SE. Next, 0.4 ml was removed from the vial containing the rSm14 antigen and added to the Eppendorf tube containing the GLA-SE + diluent mixture. The components were mixed by inversion five times. As a result, 0.8 ml of the vaccine mixture is obtained, containing a concentration of 100 pg / ml of Sm14 and 5 pg / ml of GLA-SE. Preparation of the control antigen It was mixed in a sterile Eppendorf tube: • 0.4 ml of antigen from the vial containing the rSm14 Ag solution. • 0.4 ml of Tris Buffer (50mM Tris HCl pH 9.50. As a result, 0.8 ml of the antigen was obtained at a concentration of 100 pg / ml of Sm14.

[0152] The formulations are stored at room temperature (range of 22°C - 25°C) and under refrigeration (range of 4°C - 8°C) for a period of one (1) month. Sample preparation for SDS-PAGE During storage, 40 µl of the formulated vaccine was taken and mixed with 10 µl of 5x sample buffer (TA5X). The protein was denatured by incubation at 100°C for 5 minutes. The prepared material was then frozen at -20°C until use. 15pl of the prepared samples were seeded onto 15% SDS-PAGE gel. Western Blot

[0153] To verify the antigen identity by Western blot, a previously characterized anti-Sm14 serum was used. After electrophoresis on a 15% SDS-PAGE gel, the protein separated by electrophoresis was transferred to a nitrocellulose membrane. After verifying the transfer by staining the membrane with Ponceau, a blocking solution of 5% skim milk in TBS-T buffer was created. After blocking, the membrane was incubated with anti-Sm14 serum at a 1:1000 dilution in TBS-T buffer with 0.5% skim milk for 1 hour at room temperature with constant agitation. After washing the membrane with YBS-T buffer, the anti-rabbit secondary antibody conjugated with alkaline phosphatase, diluted 1:10000 in BS-T buffer with 0.5% skim milk, was added. Incubation was performed for 1 hour at room temperature with constant agitation. After washing the membrane, it was developed using BCIP / NBT substrate. CONCLUSION

[0154] Based on the results obtained, following the proposed methodology and under the experimental conditions of this study, it can be concluded that the administration of the vaccine containing Sm 14 associated with a specific adjuvant, administered in three doses, did not promote changes in the physiological parameters of the animals belonging to the experimental groups Gr1 and Gr2.

[0155] Clinical evaluation, encompassing the measurement of hematological, biochemical, and physiological variables, demonstrated that the values ​​remained within the normal ranges recommended for the... This indicates the absence of relevant adverse effects resulting from immunization.

[0156] Following administration of the second vaccine dose, the animals showed a seroconversion rate of 90%, demonstrating a high immune response and indicating high vaccine efficacy. This result suggests that the protocol and immunization were entirely safe and were able to induce antibody production, conferring protection against the target pathogen.

[0157] Therefore, the high immunogenicity and safety of the therapeutic composition for protecting canine animals against the nematode Dirofilaria immitis has been proven, as well as the use of the composition and method for preventing infection caused by the nematode Dirofilaria immitis. REFERENCES ALMEIDA, Marília S. et al. Vaccination against Fasciola hepatica infection using a Schistosoma mansoni defined recombinant antigen, Sm14. Parasite Immunology, vol. 25, no. 3, p. 135-137, 2003. BARRETO, CS. Evaluation of canine puppies. 2003. 19 pp. Monograph - Faculty of Veterinary Medicine and Zootechnics, São Paulo State University - Botucatu, SP. Available at [link to document]<http: / / www.geocities.com / andbt / semi03 / Christ ianne.pdf> BRAZIL: Arouca Law, regarding the creation and use of animals in teaching and scientific research activities within the national territory. Law No. 11,794 of October 8, 2008. BRAZIL. Decree No. 6,899, of July 15, 2009. Provides for the composition of the National Council for the Control of Animal Experimentation - CONCEA, establishes the rules for its operation and that of its Executive Secretariat, creates the Registry of Institutions for the Scientific Use of Animals - CIUCA, through the regulation of Law No. 11,794, of October 8, 2008, which provides for procedures for the scientific use of animals, and takes other measures. DOMINGOS, TCS; ROCHA, AA; CUNHA, ICN Basic care for pregnant and neonatal canine and feline females: a literature review. J. Bras. Cienc. Anim. v. 1 , n.2, p. 94-120, 2008. GARCIA-NAVARRO, CEK (2005). Manual of Veterinary Hematology (2 a (ed.). São Paulo: Varela. FEITOSA, FLF General or Routine Physical Examination. In: FEITOSA, FLF Veterinary Semiology. Roca LTDA Publisher, 1st ed., Chapter 4, p. 77-102, 2008. HARVEY, JW Veterinary Hematology: a diagnostic guide and color atlas. Missouri: Elsevier, 2012. 360p. HENRIQUE, CH et al. (2012) 'Clinical safety of recombinant Sm14 antigen in cattle', A Hora Veterinária, 32(188), pp. 40-43. HOSKINS JD. (Ed.). Veterinary Pediatrics: Dogs and Cats from Birth to Six Months. 2nd ed. Rio de Janeiro: Interlivros, 1997 HOSKINS JD. Geriatrics and Gerontology of the dog and cat. São Paulo: Editora Roca, 2008. KANEKO, J.J.; HARVEY, J.W.; BRUSS, M.L. Clinical biochemistry of domestic animals. 6.ed. New York: Academic Press, 2008. 916p. LAFLAMME, D. P. Understanding and managing obesity in dogs and cats. Veterinary Clinics of North America - Small Animal Practice, v.36, p.1283-1295, 2006. Disponível em: doi: 10.1016 / j.cvsm.2006.08.005 LAFLAMME, D. P. Evaluation of weight loss protocols for dogs, D P Laflamme 1 , G Kuhlman, D F Lawler, DOI: 10.5326 / 15473317-33-3-253 LAREDO, F. Anaesthesia in neonatal and pediatric patients. In: PROCEEDINGS OF THE SOUTHERN EUROPEAN VETERINARY CONFERENCE; CONGRESO NACIONAL AVEPA. 2009, Barcelona, Spain: [s.e], 2009. MENDES, Ricardo E. et al. Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen. Memórias do Instituto Oswaldo Cruz, v. 105, p. 698-705, 2010. RAMOS, Celso Raul Romero et al. r-Sm14-pRSETA efficacy in experimental animals. Memórias do Instituto Oswaldo Cruz, v. 96, p. 131-135, 2001. RAMOS, Henrique Roman. Development and characterization of an anti-schistosomiasis vaccine composed of the Sm 14 protein from Schistosoma mansoni. using cholera toxin type B (CTB) as an adjuvant. 2009. Doctoral thesis. University of São Paulo. SANTINI-OLIVEIRA, Marilia et al. Development of the Sm14 / GLA-SE schistosomiasis vaccine candidate: An open, non-placebo-controlled, standardized-dose immunization phase lb clinical trial targeting healthy young women. Vaccines, vol. 10, no. 10, p. 1724, 2022. SANTINI-OLIVEIRA, Marilia et al. Schistosomiasis vaccine candidate Sm14 / GLA-SE: Phase 1 safety and immunogenicity clinical trial in healthy, male adults. Vaccine, vol. 34, no. 4, p. 586-594, 2016. SANTOS, Tatiane dos et al. Pre-clinical toxicological test for the development of an anti-helminthic vaccine based on the r-Sm14 antigen of Schistosoma mansoni. 2012. Master's Dissertation, INCQS Fiocruz. TENDLER, Miriam et al. Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine. Proceedings of the National Academy of Sciences, vol. 93, no. 1, p. 269-273, 1996. TENDLER, M.; SIMPSON JG The biotechnology-value chain: development of Sm14 as a schistosomiasis vaccine. Acta Tropica, vol. 108, p. 263-266, 2008. TIMANOVA-ATANASOVA, A. et al. Fatty acid binding protein from Fasciola hepatica: purification and binding characteristics. Experimental Pathology and Parasitology, vol. 6, no. 11, 2003. VIANA, FAB Veterinary therapeutic guide. Belo Horizonte: OEM, 320p, 2003. VICH GL9 - Guidance for Industry: Good Clinical Practice. US Department of Health and Human Services Food and Drug Administration Center for Veterinary Medicine; May 9, 2001. BUSH, BM Interpretation of laboratory results for small animal clinicians. 1 a ed., São Paulo: Editora Roca LTDA, 2004.

Claims

CLAIMS 1. Therapeutic composition to protect canine, feline, ovine or ruminant animals against the nematodes Dirofilaria immitis or Haemonchus contortus, characterized by consisting of the use of an adjuvant containing GLA-SE or MPL-SE and a conformation of the Sm14 protein selected from the sequences SEQ ID NOs: 1 to 5 or a protein from the FABP family selected from the sequences SEQ ID NOs: 6 to 7 or a protein with the SEQ ID NO:

8.

2. Composition according to claim 1 characterized by comprising: (a) an aqueous formulation of the antigens (10 to 50 pg of rSm14, and 2.5 to 5.0 pg of GLA in emulsion with squalene), where GLA is a synthetic monophosphoryl lipid, an agonist of Toll-Like Receptor type 4 (TLR4) and the antigen is one of the Sm14 proteins selected from sequences SEQ ID NO: 1 to 7; (b) with an oily formulation (water-in-oil emulsion - 40% / 60%), wherein the aqueous phase contained 10 - 50 pg of rSm14 and 1 mg of saponin, while the oily phase was Marcol 52, Cetyl-PEG was used as a surfactant.

3. Composition according to claim 1 characterized by consisting of: an aqueous formulation of the Sm14 antigen, wherein the amount per dose varies from 10 to 50 mg of rSm14, and 10 to 20.0 mg of GLA in emulsion with squalene, and wherein the GLA is a synthetic monophosphoryl lipid and the antigen is one of the Sm14 proteins selected from sequences SEQ ID NO: 1 to 7, or the adjuvant MPL-SE in the same amounts.

4. Composition according to any one of claims 1 to 3 characterized by parenteral administration, by subcutaneous injection, by intramuscular injection, by subcutaneous injection, or by oral route, including sublingual, sublabial and / or buccal and / or nasal.

5. Use of any of the proteins defined by the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 characterized by being for use in the composition defined in claims 1, 2 or 3 to protect canines, felines, ovines or ruminants against nematode parasites Dirofilaria immitis or Haemonchus contortus.

6. Method for the prevention of infection caused by Dirofilaria immitis or Haemonchus contortus, characterized in that said method employs the composition defined in any one of claims 1 to 3.

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