Chimeric vaccine antigen with triple action against ehrlichiosis, anaplasmosis and canine babesiosis, expression vector, recombinant cell, vaccine composition, diagnostic kit, treatment method and use of chimeric protein

Abstract

The present invention relates to the development of vaccine formulations obtained from a chimeric recombinant protein (rTripEAB) with epitopes of T CD8+ and T CD4+ cells and built in an optimized form for bioinformatic tools, the sequence of which has conserved regions. The invention likewise relates to composition of the vaccine formulation with the chimeric antigen together with adjuvants, as well as to the evaluation of protective efficacy against infection by Ehrlichia canis, Anaplasma platys and Babesia canis. The technology is directly applicable for protection against infectious diseases in dogs, pigs, cows, horses, and humans, directly impacting the health and well-being of individuals.

Description

“CHIMERIC VACCINE ANTIGEN WITH TRIPLE ACTION AGAINST CANINE EHRLICHIOSIS, ANAPLASMOSIS AND BABESIOSIS, EXPRESSION VECTOR, RECOMBINANT CELL, VACCINE COMPOSITION, DIAGNOSTIC KIT, TREATMENT METHOD AND USE OF CHIMERIC PROTEIN” Field of Invention

[0001] The present invention relates to a recombinant protein (rTripEAB) with epitopes derived from CD8+ and CD4+ T cells related to the pathogens Ehrlichia canis, Anaplasma platys and Babesia canis, having been constructed in an optimized manner using bioinformatics tools. The present invention also contemplates vaccine compositions (veterinary or for human health), containing the chimeric antigen (rTripEAB), together with vaccine adjuvant(s); diagnostic kits, containing the antigen for determining diseases caused by infection by Ehrlichia canis, Anaplasma platys and / or Babesia canis', use of the recombinant protein for the treatment and / or diagnosis of diseases, preferably in dogs, swine, cattle, horses and humans, caused by any of the three pathogenic agents or by cross-protection or detection (in swine we find B. trautmann and B. perroncitoi. Babesiosis is also described in horses through B. equi, B.caballi); and method of treating diseases by stimulating the production of antibodies aimed at the three pathogens, accelerating healing and protecting against new infections, beneficially impacting the health and well-being of the individuals treated. Description of the State of the Art

[0002] Ticks are blood-sucking arthropods of great global importance, second only to mosquitoes as vectors of infectious diseases. This importance stems from the fact that these parasites are capable reservoirs and vectors of pathogens, responsible for causing mortality and morbidity in humans, wild animals, and domestic animals. Pathogens transmitted by this vector are common occurrences. in veterinary medicine, especially in dogs, and the incidence and geographic reach of these pathogens in urban and rural areas in Brazil has increased significantly, and they are currently considered a threat to public health. This concern is a result of limitations related to the prevention, control, and combat of these organisms. Among the species of pathogens to be controlled and combatted, different studies highlight Ehrlichia canis, Anaplasma platys, and Babesia canis [GHOSH, S.; NAGAR, G. Problem of ticks and tick-borne diseases in India with special emphasis on progress in tick control research: a review. Journal of vector-borne diseases 51(4):259-270. 2014; DANTAS-TORRES, F.; DA SILVA, YY; DE OLIVEIRA MIRANDA, DE et al. Ehrlichia spp. infection in rural dogs from remote indigenous villages in northeastern Brazil. Parasites & Vectors 11 (1):139. 2018; RECK, J. et al. Records of ticks on humans in Rio Grande do Sul state, Brazil. Ticks Tick Borne Diseases, v. 9, no. 5, p.1296-1301, 2018; OJEDA-CHI, M.M.; RODRIGUEZ-VIVAS, R.I.; ESTEVE- GASENT, M.D. et al. Ehrlichia canis in dogs of Mexico: Prevalence, incidence, co-infection and factors associated. Comparative immunology, microbiology and infectious diseases, v 67:101351. 2019]. .

[0003] Canine Monocytic Ehrlichiosis (CME) is a disease that affects dogs and is caused by Ehrlichia canis, a Gram-negative, obligate intracellular bacterium belonging to the order Rickettsiales, family Anaplasmataceae, and genus Ehrlichia, transmitted by the tick Rhipicephalus sanguineus. In Brazil, CME is considered the disease most commonly transmitted by this vector, with prevalence rates ranging from 15% to 30%, depending on the region, season, animal population studied, and diagnostic method used [RUDOLER N., et al. Evaluation of an attenuated strain of Erlichia canis as a vaccine for canine monocytic ehrlichiosis. Vaccine, v. 31, p. 226-233, 2012; COSTA, RL DA et al. Molecular characterization of Ehrlichia canis from naturally infected dogs from the state of Rio de Janeiro. Brazilian Journal of Microbiology, v. 50, n. 1, p. 1-12, January 23, 2019].

[0004] E. canis reproduces in the tick's salivary glands and intestines and is transmitted to dogs through contaminated saliva. The pathogen primarily infects macrophages of the vertebrate host, multiplying by binary fission. Transmission can occur when an infected tick, in this case nymphs or adults, feeds on a healthy dog. During infection, dogs present with late, nonspecific clinical manifestations and hematological and biochemical alterations, making diagnosis difficult. The infection manifests in three phases and can lead to altered immune response and hemorrhages, and in the most severe cases, death if the disease is treated late. In asymptomatic cases, the animal becomes a reservoir and source of infection, spreading the pathogen to other animals without clinical suspicion.Treatment is with antibiotic therapy associated with corticosteroids and the prognosis depends on the severity of the disease [MALHEIROS, J., COSTA, MM, DO AMARAL, RB, DE SOUSA, KCM, ANDRÉ, MR, MACHADO, RZ, & VIEIRA, MIB Identification of vector-borne pathogens in dogs and cats from Southern Brazil. Ticks and Tick-Borne Diseases, v.7, n.5, p.893-900, 2016; ATTIPA, C. et al. Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections. Ticks and Tick-Borne Diseases, v. 8, n.3, p.341-346, Mar., 2017; KAEWMONGKOL, G. et al. Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand. Veterinary Microbiology, Thailand, v.201, p. 195-200, 2017; NAMBOOPPHA, B. et al. Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant genes. Infection, Genetics and Evolution, v. 63, p. 116-125, 2018].

[0005] Another infectious disease caused by the bacterium Anaplasma platys is canine anaplasmosis, transmitted in a similar way to ehrlichiosis during the blood meal of ticks. A. platys is a gram- negative, obligately intracellular, belonging to the order Rickettssiales, family Anaplasmataceae, and genus Anaplasma. A. platys also causes thrombocytopenia in dogs throughout Brazil, and coinfection with E. canis and B. vogeli is common, and the tick R. sanguineus is the same vector for all three species. [COSTA-JUNIOR, LM, et al. Factors associated with epidemiology of Anaplasma platys in dogs in rural and urban areas of Minas Gerais State, Brazil. Medicina Veterinária Preventiva, v.1, n. 109, p. 321-326, 2013; PIANTEDOSI, D., NEOLA, B.; D'ALESSIO, et al. Seroprevalence and risk factors associated with Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and D. immitis in hunting dogs from southern Italy. Parasitology Research, v. 116, n. 10, p. 2651-2660, 2017]

[0006] The frequency of A. platys infection in Brazil ranges from 5% to 19%, with milder symptoms, except in cases of coinfection, which can worsen the animals' clinical condition. Anaplasmosis causes parasitemia and thrombocytopenia, resulting in a sudden reduction in platelet count. Symptoms of canine anaplasmosis include fever, loss of appetite, lethargy, anemia, nosebleeds, and difficulty breathing. Canine anaplasmosis is diagnosed through blood tests that detect the presence of antibodies against the bacterium Anaplasma platys. Treatment is with antibiotics and can last from a few weeks to several months, depending on the severity of the infection [HAMIANI KHATAT, S., et al. Epidemiological and clinicopathological features of Anaplasma phagocytophilum infection in dogs: a systematic review. Frontiers in Veterinary Science, v. 8, p. 664-686, 2021].

[0007] Canine babesiosis is caused by protozoa of the genus Babesia, primarily the species Babesia canis canis, B. c. rossi, B. c. vogeli, and Babesia gibsoni. Transmission from the invertebrate host occurs through the ingestion of infected blood by the tick R. sanguineus, which is transmitted during the blood meal to the vertebrate host. can cause red blood cell lysis due to the multiplication of the parasite inside the erythrocytes, causing them to rupture. Clinical signs of infection are related to the host's immune status. Some dogs show no clinical signs, while others present with a severe form of the disease, which can lead to death. Therefore, diagnosis based on clinical findings can be difficult. Microscopic examination to visualize the parasite in a blood smear and serology are the best ways to obtain a definitive diagnosis of babesiosis. Treatment also involves antibiotic therapy and may vary in duration depending on the severity of the infection [SOLANO-GALLEGO, L., et al. A review of canine babesiosis: the European perspective. Parasitic Vectors, v. 9, n. 1, p. 336, 2016; DANTAS-TORRES, F., ALVES, LC, UILENBERG, G. BABESIOSIS CB Marcondes (Ed.), Arthropod Borne Diseases, Springer International Publishing p. 645, 2017; BARASH NR, et al. Prevalence of Babesia spp. and clinical characteristics of Babesia vu / pesinfections in north American dogs. Journal of Veterinary Internal Medicine, v.33, p. 2075-2081, 2019].

[0008] The prevention and control of infectious diseases in animals involves a variety of measures, including vaccination, vector control, health management, responsible use of antibiotics, and epidemiological surveillance [HOLMES AH, et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet, v. 387, p. 176-187, 2016]. Vaccination is an important tool in the prevention of infectious diseases in animals, especially farmed animals [CENTER FOR DISEASE CONTROL AND PREVENTION - CDC. Influenza planning and response. Atlanta: CDC, 2021 <https: / / www.cdc.gov / flu / pandemic- resources / 1918-commemoration / pandemic-preparedness.htm.].

[0009] Vaccine production technology has evolved over the years, with vaccines being developed to protect a variety of animal species against infectious diseases [PULENDRAN, B.; AHMED, R. Immunological mechanisms of vaccination. Nature Immunology, v. 12, p. 509-517, 2011]. More recently, recombinant vaccines have become an important tool in animal immunization, allowing the production of specific antigens for protection against specific diseases [LUNG, P.; YANG, J.; LI, Q. Nanoparticle formulated vaccines: opportunities and challenges. Nanoscale, v. 12, p. 5746-5763, 2020]

[0010] In this regard, it is possible to find references in the prior art that address the use of proteins for vaccination or antigen development solely against ehrlichiosis. For example, document BR 112022000347-1 A2 refers to the development of immunogenic compositions that can be used, in some aspects, to induce an immune response against Ehrlichia, such as Ehrlichia canis. In some embodiments, the immunogenic composition comprises an E. canis bacterin and / or an adjuvant, such as an emulsion or a liposomal adjuvant. Related methods, such as for diagnosis or vaccination against ehrlichiosis, are also provided.

[0011] Document PI 9916141-9 A2 refers to the molecular cloning and characterization of the Ehrlichia canis 120-kDa immunoreactive protein gene. It provides a PCR-amplified Ehrlichia canis 120-kDa protein gene using primers derived from the DNA sequences flanking the Ehrlichia chaffeensis 120-kDa protein gene. The recombinant Ehrlichia canis 120-kDa protein contains 14 tandem repeat units of 36 amino acids each. The described technology presents the amino acid sequence of the construct. The recombinant E. canis 120-kDa protein is antigenic and reacts with sera from dogs convalescent with canine ehrlichiosis.

[0012] Document PI 0508655-8 A2 refers to a vaccine composition comprising an amount of immunizing agent effective against Ehrlichia canis. It depicts an immunogenically stimulating amount of A combination of antigen and adjuvant as an antibody response-inducing agent and a cell-mediated immune response-inducing agent. The technology describes a method for preventing or mitigating canine ehrlichiosis in dogs.

[0013] We can still observe a lack of technology that includes the development of a recombinant protein, which has triple action, aimed at the aforementioned pathogens in a single protein and a single vaccine composition (E. canis, A. platys and B. canis) and its application in vaccine formulations and diagnostic kits.

[0014] There are documents that anticipate dual-action vaccines, encompassing E. canis and B. canis, including the use of bioinformatics techniques to obtain the epitopes of interest. As well as the use of this protein for concomitant use in diagnostics [Guy, AJ, Irani, V., MacRaild, CA, Anders, RF, Norton, RS, Beeson, ... & Ramsland, PA (2015). Insights into the immunological properties of intrinsically disordered malaria proteins using proteome scale predictions. PLoS One, 10(10), e0141729; Luo T, Zhang X, McBride JW. Major species-specific antibody epitopes of the Ehrlichia chaffeensis p120 and E. canis p140 orthologs in surface-exposed tandem repeat regions. Clin Vaccine Immunol. 2009 Jul;16(7):982-90. doi: 10.1128 / CVI.00048-09. Epub 2009 May 6. PMID: 19420187; PMCID: PMC2708412; Martins, VT (2016). Evaluation of recombinant leishmania proteins in serological diagnosis and as candidates for a vaccine against leishmaniasis.Thesis of the Graduate Program in Biochemistry and Immunology of the Institute of Biological Sciences of UFMG; Miles, S., Navatta, M., Dematteis, S., & Mourglia-Ettlin, G. (2017). Identification of universal diagnostic peptide candidates for neglected tropical diseases caused by cestodes through the integration of multi-genome-wide analyses and immunoinformatic predictions. Infection, Genetics and Evolution, 54, 338-346; Tomazic, M. L., Marugan-Hernandez, V., &. Rodriguez, A.E. (2022). Systems vaccinology for the design of rational vaccines against protozoan parasites. System Vaccinology, 297-334]. However, these documents do not teach the production of a triple-action vaccine, which would encompass epitopes of the three aforementioned pathogens—E. canis, A. platys, and B. canis—for a vaccine formulation or diagnostic kit.

[0015] Even using bioinformatics techniques, adding a new epitope to a chimeric protein is not trivial. With each new insertion of sequences corresponding to a new epitope, the final immune response may not be the same. A dual-action antigen must be equivalent to or better than immunization with isolated epitopes. And a triple-action antigen must be better than the antigens administered alone or in pairs. In other words, predicting the effect of chimeric antigens is not straightforward, and efficacy studies are required to confirm this.

[0016] It is worth noting that bioinformatics techniques combined with artificial intelligence now facilitate antigen development by predicting epitopes derived from CD4+ and CD8+ cells. For example, document WO2019147925A1, "Compositions and methods for combination cancer vaccine and immunologic adjuvant therapy," reports the use of bioinformatics tools to identify new tumor antigens (NeoAntigens) and new epitopes (Neo-Epitopes) for use in adenoviral vectors as vaccine adjuvants. The epitopes can be obtained using the same bioinformatics tool as the present invention: NetMHC, NetMHCII. The epitopes formed can be targeted for immunization against various pathogens, including bacteria and their combinations. However, the vaccine is intended for the development of cancer treatments, not canine diseases.

[0017] On the other hand, document WO2011119484A1 “Bioinformatic processes for determination of peptide bond”, which describes methods for the in silico identification of peptides and sets of peptides (epitopes) internal to or on the surface of microorganisms and mammals, with a high probability of stimulating humoral and cell-mediated immune responses. The method combines several predictive tools to create a composite of both the topology and the binding characteristics or affinity of specific sets of peptides within an entire proteome, identifying regions with a high probability of being B-cell binding sites or MHC binding sites containing T-cell epitopes on the surface of microorganisms or cells, or MHC binding sites containing T-cell epitopes internal to microorganisms or cells. However, WO2011119484A1 does not demonstrate the use of these tools for the pathogens described above, nor how the three would be combined into a single antigen.

[0018] It's worth emphasizing again that the aforementioned computer programs do not guarantee the efficacy of these epitopes in the final protection of the treated animal. They need to be tested, especially since the programs only provide the sequence of the suggested epitope. These epitopes need to be optimized, linked to a recombinant protein production construct, among other adjustments that call their efficacy into question.

[0019] In other words, simply having the sequences doesn't mean you have the complete invention, as a viable construct needs to be developed. The links used or even the adjuvants can compromise the immunizing power of the final recombinant protein, and the antigens are not ready for use. This is especially true when combining more than one epitope (chimera), or even a triple protein, which would immunize the animal against three pathogens simultaneously. This can lead to cross-reactivity, compromising or increasing the scope of immunization.

[0020] Therefore, the development of a recombinant protein and its application in a triple-action vaccine formulation with high immunogenic potential would be highly desirable. It would be necessary to develop proteins recombinant vaccines composed of different peptide segments from different pathogens. This approach could lead to broader and more effective vaccines that protect against multiple pathogens.

[0021] The three aforementioned diseases are hematological pathologies that can be controlled and treated with medication, but there is currently no vaccine for their prevention and control. However, vaccine strategies to control the infection are showing increasing promise.

[0022] Because these are recurrent canine pathologies and recombinant protein technology presents itself as a promising alternative, inventions based on the design of triple chimeric proteins become interesting as vaccine candidates for the control and treatment of these diseases and the development of vaccine formulations, which would be of great importance and biotechnological contribution.

[0023] Given the above scenario, it is clear that it is necessary to develop new triple-action chimeric vaccine antigens and their respective vaccine formulations, which can protect the treated individual against ehrlichiosis, anaplasmosis and canine babesiosis simultaneously.

[0024] It would be even more interesting to use bioinformatics tools, such as NetMHCII 4.0 and NetCTLPan 1.1, to predict an amino acid sequence with the highest chance of success in a triple vaccine, for human or veterinary use, protecting against diseases caused by Ehrlichia canis, Anaplasma platys, and Babesia canis. This could serve both to immunize the vaccinated individual and, furthermore, it would be desirable for the same chimeric protein to be capable of composing a diagnostic kit for detecting these pathogens in individuals suspected of having these infestations. Brief Description of the Invention

[0025] The present invention, therefore, relates to obtaining a recombinant chimeric protein (rTripEAB), by means of bioinformatics and pharmaceutical and recombinant DNA techniques, which comprises amino acid sequences of 3 epitopes derived from CD8+ T and CD4+ T cells related to the pathogens Ehrlichia canis, Anaplasma platys and Babesia canis, having a diagnostic function and stimulating immunogenic protection.

[0026] This recombinant chimeric protein was initially constructed in an optimized manner using bioinformatics tools such as predictive algorithms (NetMHCII 4.0 and NetCTLPan 1.1), and codon optimization programs (e.g., BepiPred 3.0).

[0027] Therefore, this technology is based on the expression of the recombinant rTripEAB protein in E. coli BL21 (DE3), followed by purification, for use in adjuvanted vaccine formulations against canine ehrlichiosis, anaplasmosis, and babesiosis. Through biological assays, the recombinant antigen's potential to induce protection against the pathogens E. canis, A. platys, and B. canis in an experimental model was evaluated. This contributes to the development of a new vaccine of veterinary and potentially human interest.

[0028] However, the development of a triple vaccine against Ehrlichia canis, Anaplasma platys and Babesia canis will allow immunological protection against three pathogens that commonly affect dogs, reducing or eliminating symptoms, physiological damage to the animals and the transmission capacity of the pathogens.

[0029] In this first embodiment of the invention, therefore, it is contemplated to obtain 9 polypeptides (SEQ ID NO: 1 to 9) for the formation of a final optimized chimeric polypeptide / protein (SEQ ID NO: 10 or 11), capable of having an antigenic function, when inserted as the active principle of a vaccine, and a diagnostic function, when used in antibody detection kits and techniques. The protective or diagnostic effect is aimed at the three selected pathogens Ehrlichia canis, Anaplasma platys and Babesia canis, but could also have a function in a range of non- specific, due to cross-reactions, having an effect on pathogens of the same family, order or class of the 3 selected pathogens.

[0030] Therefore, in some aspects of this invention we have a SEQ ID NO: 10, referring to the amino acid sequence of the chimeric protein, and SEQ ID NO: 11, referring to the nucleotide sequence of the synthetic gene capable of encoding the amino acid sequence of the synthetic or recombinant chimeric protein, formed by the junction of the polypeptides of SEQ ID NO: 1 to 9 (amino acid sequences). As well as nucleotide sequences similar to SEQ ID NO: 11, containing mutations, deletions, insertions, neutral substitutions or alterations that maintain a genetic identity that allow the generation of immunizing power equal to or equivalent to that of SEQ ID NO: 10.

[0031] In some aspects, the amino acid sequence or nucleotide sequence encoding the antigen(s) of the present invention also falls within the scope of protection, even if it exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0032] In a second embodiment of the invention, we have a process for producing the chimeric protein that can be produced by recombination or by chemical synthesis of the protein. The process has the following general steps: a) Obtaining the epitopes: prediction of the peptides related to the epitopes of CD4 T lymphocytes + and CD8 T +of the pathogens of interest by bioinformatics techniques; b) Construction of the chimeric sequence: joining of the 9 epitopes with linkers and codon optimization; and c) Obtaining the chimeric protein: by chemical synthesis of the chimeric amino acid sequence obtained in “b” or the insertion of the corresponding nucleotide sequence in a recombinant cell and subsequent isolation and purification.

[0033] In a third embodiment of the invention, we have the expression vector (construct) containing SEQ ID NO: 11 or its equivalents, forming a vector capable of being inserted into a cell and providing the production of proteins or polypeptides according to SEQ ID NO: 10. More specifically, the preferred expression vector is constructed from the junction of the pet28a vector and SEQ ID NO: 11 or equivalent sequence capable of producing SEQ ID NO: 10 or its functional equivalents.

[0034] In a fourth embodiment of the invention, if production occurs by recombination techniques (and not by chemical synthesis), we have the recombinant cell that has the expression vector produced, which contains SEQ ID NO: 11 or an equivalent sequence capable of producing SEQ ID NO: 10 or its functional equivalents.

[0035] In a fifth embodiment of the invention, we have vaccine compositions (veterinary or for human health) containing the chimeric antigen (rTripEAB), together with vaccine adjuvant(s) and other pharmaceutically acceptable carriers and excipients. More specifically, the preferred concentration of the active ingredient (chimeric protein SEQ ID NO: 10) will be between 0.5 and 100 pg / mL, in doses with a volume between 0.5 ml and 2 ml.

[0036] In some aspects of these compositions, quantities of antigens (0.5 to 10 pg of purified chimeric protein / ml) and their adjuvants, vehicles and excipients are used in concentrations, allowing the manufacture of vaccines that contain at least 0.5 to 100 pg per dose administered, being capable of generating immune responses against antigens that have similarity with, for example, the chimeric antigen used, in particular, at least one antigen that is associated or related to infectious diseases such as ehrlichiosis, anaplasmosis and babesiosis.

[0037] In a sixth embodiment of the invention, we have the process for producing the vaccine composition, which has the following steps: a) use of an amount of 0.5 to 100 pg per individual dose of the purified chimeric protein obtained in the previous process as the active ingredient of the vaccine formulation; b) use of the vaccine adjuvant Aluminum Hydroxide at a concentration of 0.2%, added with Saponin adjuvant (Quil-A™) at a concentration of 1% in the final dose volume.

[0038] In a seventh embodiment of the invention, we have a diagnostic kit containing the chimeric antigen or its respective antibody in a device capable of detecting the presence of antigens / antibodies corresponding to the pathogens Ehrlichia canis, Anaplasma platys and / or Babesia canis in the collected biological material.

[0039] In the eighth embodiment of the invention, we have the use of recombinant protein for the manufacture of diagnostic kits and vaccines for the prevention or treatment of diseases caused by any of the three pathogenic agents in dogs, pigs, cattle and humans.

[0040] And finally, in the ninth and final modality of the invention, we have the method of treating diseases through the administration of therapeutic doses of vaccine compositions to stimulate the production of antibodies aimed at the three pathogens, accelerating healing and protecting against new infections, beneficially impacting the health and well-being of the individuals treated.

[0041] In this sense, the target protein of this invention was designed based on conserved peptides from E. canis, A. platys, and B. canis pathogens, which act as CD4+ or CD8+ T lymphocyte epitopes. The T cell epitopes were selected based on the highest scores visualized by the predictive algorithms. The recombinant chimeric antigen (rTripEAB) was optimized by bioinformatics tools. This protein was expressed in E. coli and purified with satisfactory yield and purity for application in the development of a triple-action vaccine. Therefore, the vaccine potential of the recombinant antigen was evaluated in mice, and the data from this study demonstrate the protective efficacy and vaccine potential of the recombinant antigen against triple infection. Brief Description of the Figures

[0042] FIGURE 1 - Expression and purification of the recombinant antigen rTripEAB in E. coli BL21 (DE3). Item (A) of FIGURE 1 shows the SDS-PAGE gel (12.5%) using the soluble and insoluble fractions obtained after expression of the recombinant protein and lysis of the bacterial cells. Item (B) of FIGURE 1 shows the SDS-PAGE gel (12.5%) of the fractions obtained after purification of the insoluble fraction, highlighting the rTripEAB protein with a theoretical molecular mass of 22 kDa. Item (C) of FIGURE 1 shows the SDS-PAGE gel and Western blotting of the collected fractions, confirming the size, expression, and purification of the recombinant protein.

[0043] FIGURE 2 - Production of specific antibodies against the rTRipEAB protein. An enzyme-linked immunosorbent assay (ELISA) was performed with blood obtained from immunized animals after three doses of the vaccine and before challenge with E. muris (item (A) in FIGURE 2), A. platys (item (B) in FIGURE 2), B. canis (item (C) in FIGURE 2), confirming immunoconversion by the protein.

[0044] FIGURE 3 - Parasite load of E. muris (item (A) in FIGURE 3), A. platys (item (B) in FIGURE 3) or B. canis (item (C) in FIGURE 3) in the blood of mice, quantified by real-time PCR and expressed in copies of the pathogen genome per 100 ng of DNA extracted from the blood after euthanasia. The mice were immunized and challenged 2 weeks after the last immunization with 1 x 10 5 of pathogen intraperitoneally. The result shows that the groups immunized with adjuvant combined with the recombinant protein showed a significant reduction of parasite load, when compared to the control (non-immunized animals).

[0045] FIGURE 4 - Percentage of CD3 T cells + CD4 + and CD3 T + CD8 + in the spleen of immunized mice and after challenge. Analysis of spleen cell populations from animals challenged with E. muris (item (A) in FIGURE 4), A. platys (item (B) in FIGURE 4) or B. canis (item (C) in FIGURE 4) showed that all treatments induced expansion of CD4 T lymphocytes + (memory) and CD8 + (identification of elimination of intracellular pathogens), when compared to the non-immunized control.

[0046] FIGURE 5 - Production of IL-4 and IL-10 by spleen cells after stimulation with the recombinant protein rTripEAB. After 15 days of challenge with (item (A) of FIGURE 5) E. muris, (item (B) of FIGURE 5) A. platys and (item (C) of FIGURE 5) B. canis, spleen cells were cultured for 24 h in the presence of protein stimulation. The cytokine concentration was determined in the supernatants. It can be identified that the groups previously stimulated with the antigen showed significant cytokine production compared to the non-immunized control group. Detailed Description of the Invention

[0047] The present invention describes the production of antigens and vaccine formulations from a recombinant protein (rTripEAB) containing CD8+ and CD4+ T cell epitopes, optimized using bioinformatics tools. Therefore, the invention encompasses the composition of the vaccine formulation that combines the chimeric antigen with adjuvants. The protective efficacy of the vaccine was evaluated, demonstrating its ability to prevent infection by Ehrlichia canis, Anaplasma platys, and Babesia canis. This technology has direct application in preferential protection against infectious diseases in dogs, positively impacting their health and well-being.

[0048] The analysis pipeline was developed using Bash scripts and PERL. All epitopes were identified using a conservative approach to minimize false-positive epitope selection. Initially, linear B-cell epitopes were predicted across the entire proteome of E. canis, B. canis, and A. platys using the BepiPred 3.0 program (https: / / services.healthtech.dtu.dk / services / BepiPred-3.0 / ) [CLIFFORD, JN et al. BepiPred-3.0: Improved B-cell epitope prediction using protein language models. Protein Science, v. 31, n. 12, 1 Dec. 2022]. This program assigns to each amino acid in the sequence a prediction score that positively correlates with the probability of that amino acid participating in a linear B-cell epitope. In this analysis, a cutoff value of 1.3 was used, meaning that only amino acids with a prediction score above 1.3 were considered as likely epitope participants.This cutoff value provides 96% specificity and 13% sensitivity.

[0049] CD4 T cell epitopes + were identified by bioinformatics, using the computer program NetMHCII 4.0 (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.0 / ) [REYNISSON, B. et al. Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass Spectrometry MHC Eluted Ligand Data. Journal of Proteome Research, vol. 19, no. 6, p. 2304-2315, 5 June. 2020]

[0050] Peptides of 9 to 15 amino acids that bound with an affinity of less than 50 nM to at least 30% of different alleles were considered as potential epitopes. CD8 T cell epitopes +were predicted using the NetCTLPan 1.1 program (https: / / services.healthtech.dtu.dk / services / NetCTLpan-1 .1 / ) [STRANZL, T. et al. NetCTLpan: Pan-specific MHC class I pathway epitope predictions. Immunogenetics, v. 62, n. 6, p. 357-368, Jun. 2010.] to select the peptides with the highest values ​​for binding to the MHC type I molecule, proteasome cleavage and TAP transport efficiency. In all analyses, coverage of a minimum of 30% of population alleles was considered. The epitopes identified with the highest values ​​for CD4 T + and CD8 T + and lowest possible values ​​for B cells in the analyses described were used to construct a synthetic chimeric protein antigen.

[0051] After the construction and synthesis of the synthetic gene, the ability of the protein and the conserved peptides contained in it to induce a protective immune response against the three diseases in an experimental model was verified.

[0052] More specifically, the characterization of the chimeric protein demonstrated that it has a total of 09 (nine) conserved peptides, which were selected to compose the final chimeric protein, for use as a diagnostic or vaccine antigen.

[0053] Table 1 summarizes the degree of conservation of these pathogen peptides and whether they act as CD4+ T lymphocyte (immune memory response) or CD8+ T lymphocyte (TH1 cellular immune response) epitopes. Table 1. Degree of conservation of peptides among the pathogens E. canis, A. platys and B. canis and performance as CD4+ T or CD8+ T lymphocyte epitopes. Epitope Peptide E. canis A. platys B. canis CD4 CD8 1 MGIAYFRGYRVRLFLIIQD 100% 100% 80% 2 LIEACLRLRPDRIIV 100% 100% 92% 3 VVRTMRSDDVVYNLAV 100% 100% 100% 4 EACLRLRPDRIIVGELRG 100% 100% 100% 5 AGIHIIMATQRPSVDVI 100% 100% 80% X 6 QRPKRFFGAARNIENGGSL 100% 100% 100% 7 MECWALQAYGAAYTLQ 100% 100% 100% 8 ALDYLVGFTLSPLLWR 100% 75% 100% X 9 GSNPLFLYGGVGLGKTHL X

[0054] The function of the proteins from which the peptides originated are described in Table 2.

[0055] Most of the source proteins are constitutive and produced in high quantities throughout the life cycle of pathogens and are essential for their maintenance. Table 2. Function of the peptide-origin proteins. n .. „ Protein Identity Identity Peptide _ . . . , „ E. canis A. platys B. canis 1 MGIAYFRGYRVRLFLII type IV type IV Negative QD secretory regulator of system system the PHO conjugative conjugative system DNA transfer DNA transfer family family protein protein 2 LIEACLRLRPDRIIV P-type DNA P-type DNA hypothetical transfer transfer protein ATPase ATPase VirB11 VirB11 3 VVRTMRSDDVVYNLAV type IV type IV hypothetical secretory secretory protein system system conjugative conjugative DNA transfer DNA transfer family family protein protein 4 EACLRLRPDRIIVGELR P-type DNA P-type DNA ABC G transfer transfer transporter ATPase ATPase VirB11 VirB11 5 AGIHIIMATQRPSVDVI DNA DNA 26S translocase translocase proteasome FtsK FtsK regulatory subunit T4 6 QRPKRFFGAARNIENG transcription transcription uncharacteriz GSL termination termination ed protein factor Rho factor Rho BXIN_2588 7 MECWALQAYGAAYTL DNA- DNA- DNA-directed directed directed RNA RNA RNA polymerase polymerase polymerase subunit beta subunit beta subunit beta 8 ALDYLVGFTLSPLLWR type I DNA NADH- splicing factor topoisomera quinone 3a protein oxidoreducta subunit L 9 GSNPLFLYGGVGLGKT chromosom chromosoma ATPase, HL al replication I replication AFG1 family initiator protein protein DnaA DNAA

[0056] The peptides were used in the construction of a chimeric protein sequence (SEQ ID NO: 10), containing 222 amino acids, molecular mass of 22.94 kDa; isoelectric point of 9.93; average degree of hydrophobicity of -0.031 (polar protein - polar reference <0); stability index of 36.63 (protein classified as stable - stability reference <60); having as preferred vector pET28a(+) - resistance to kanamycin and expression induced by IPTG and the preferred host bacterium E. coli BL21 (DE3).

[0057] Between the peptides of SEQ ID NO:10 a linker was added to increase the solubility of the protein and favor cleavage by proteasome during the processing of the cellular immune response induced by the antigen and adjuvant, as can be seen in table 1 below. TABLE 1: Details of SEQ ID NO: 10

[0058] The sequence of the synthetic gene SEQ ID NO: 11 was designed from the sequence of the chimeric protein SEQ ID NO: 10, using codons optimized for expression in E. coli. As highlighted in TABLE 2 below: TABLE 2 - Details of SEQ ID NO: 11

[0059] The methods, procedures, results, and advantages of the present invention can be better understood from the illustrative examples below. They are included for a better understanding of the invention and do not limit the scope of protection. Example 1: Obtaining the recombinant cell

[0060] After designing the chimera, the protein sequence was submitted to the Codon Optimization program (http: / / www.idtdna.com / CodonOpt), generating a viable nucleotide sequence for large-scale expression in Escherichia coli. The gene was chemically synthesized and cloned into the PET28a vector. In the transformation step, 5 pL of the vector containing the insert was added to 50 pL of competent E. coli BL21 (DE3) cells, previously stored at -80°C. The samples were then transferred to 0.1 cm electroporation cuvettes and subjected to a 2.50 kV pulse. After electroporation, 200 pL of liquid Luria Bertani (LB) culture medium was added to the cuvette, transferred to 1.5 mL tubes, and incubated for one hour at 37°C under shaking on a shaker at 180 rpm. After this period, the samples were plated on solid LB-agar medium 1.5% containing 0.1 mg / mL of Kanamycin and stored in an oven at 37°C, overnight.Plasmid DNA was extracted and a PCR reaction was performed to confirm the presence of the amplicon at the cloning site, according to the manufacturer's recommendations and sequencing. Example 2: Obtaining and purifying recombinant proteins

[0061] To induce the expression of recombinant proteins, bacterial colonies isolated in each cloning system were inoculated into 50 mL of LB culture medium (Kanamycin 0.1 mg / mL) and incubated overnight. at 37°C under shaking at 180 rpm. Then, the cultures were inoculated into 500 ml of LB medium with antibiotic (Kanamycin 0.1 mg / ml) and grown until reaching an optical density (OD eoonm) between 0.6-0.8 at 37°C under shaking at 180 rpm. After reaching the desired OD, expression was induced with the addition of 1 mM IPTG for 4 hours at 37°C under shaking at 180 rpm. After the induction period, the culture was centrifuged at 6000 xg for 30 minutes at 4°C and then stored at -80°C. Aliquots of the culture immediately before the addition of IPTG, and after the expression induction period, were also frozen to be used as expression control.

[0062] The pellet containing the culture after the expression step was resuspended in 100 ml of PBS containing 30 mM Imizadol and 100 pg / mL lysozyme and incubated for 30 minutes on ice. The extract was then lysed in a homogenizer for five homogenization cycles as recommended by the equipment manufacturer. The sample was then centrifuged at 8000 xg for 30 minutes at 4°C, and the soluble and insoluble fractions were collected. The recombinant proteins were purified by affinity chromatography using the "ÃKTAprime plus" system (GE Healthcare, Piscataway, USA). At this stage, the insoluble fraction was applied to a 5 ml "HisTrap HP" column. The column was previously washed with five times its volume of buffer A (PBS containing 30 mM Imidazole). Elution was performed by adding buffer B (PBS containing 500 mM Imidazole).

[0063] The purified fractions of the recombinant protein were quantified in triplicate using the Bradford method, from a standard curve of the BSA (Bovine Serum Albumin) protein, with reading in a spectrophotometer at 595 nm.

[0064] All fractions obtained were analyzed by 12.5% ​​SDS-PAGE polyacrylamide gel electrophoresis. The 12.5% ​​separation gel was prepared using 30% bis-acrylamide, 1.5 M Tris-HCl buffer pH 8.8, 0.01% SDS, 0.5% v / v ammonium persulfate and 0.05% v / v TEMED. The separation gel The concentration was prepared in a similar manner to the separation, but using 0.5 M Tris-HCI buffer, pH 6.8. The samples obtained from the induction and solubility test were added to sample buffer (10% SDS, 0.5 mM Tris-HCI, pH 6.8, 1% Bromophenol blue, 5% 2-[3-mercaptoethanol], and 10% glycerol), preheated for 10 minutes to denature the proteins, and subsequently applied to the polyacrylamide gel for electrophoretic separation. Electrophoresis was performed using running buffer (25 mM Tris-HCI, 192 mM glycine, 0.1% SDS, and pH 8.3) under a constant voltage of 120 V. After the run, the gels were stained by incubation for 12-16 hours with the Coomassie Blue solution (Coomassie Brilliant Blue G-250 0.25%, methanol 50% and acetic acid 10%) and then destained in a 25% methanol and 7.5% acetic acid solution.

[0065] To confirm the purification of the protein of interest, Western blotting analysis was performed. The proteins separated in SDS-PAGE were transferred to a nitrocellulose membrane previously incubated in transfer buffer in a semi-dry system. Subsequently, the nitrocellulose membrane was incubated in PBS plus 3% BSA for 1 h, under low agitation to block nonspecific sites. To label the protein of interest, the primary mouse anti-his antibody in PBS-T (dilution 1:3000) was used, followed by the secondary anti-mouse antibody conjugated with peroxidase (dilution 1:5000). Development was done with a DAB (3,3'-Diaminobenzidine) solution prepared in a vial protected from light and containing 1 ml of 0.3% NÍCI2 solution, 1 ml of 1 M Tris buffer pH 7.6, 8 ml of distilled water and 10 pL of H2O2.

[0066] Confirmation of protein expression and purification was performed by Western blotting. The protein band appeared on the polyacrylamide gel and on the nitrocellulose membrane between 25 kDa and 20 kDa (FIGURE 1). Thus, the SDS-PAGE and Western blotting results confirmed the theoretical size of the protein (22 kDa). The fractions of Purified protein was pooled and quantified using the Bradford method. The concentration of fraction 1 was determined to be 0.94 mg / mL, and the protein production yield was 9.4 mg per liter of LB culture medium inoculated with E. coli. Example 3: Vaccine formulations

[0067] After expression and purification, the protein was incorporated into four vaccine formulations to evaluate the protective efficacy and response with different adjuvants, namely Inject Freund's Incomplete Adjuvant (FIA), Saponin, Aluminum Hydroxide, Polygen and Emulsigen. Example 3.1 - 0.2% Aluminum Hydroxide + 1% Saponin: For the formulation with Aluminum Hydroxide and Saponin (H / S), a 0.9% saline solution was initially prepared (0.9 g of NaCl in 100 ml of distilled water or milli-q), followed by the 0.2% aluminum hydroxide solution (0.02 g of aluminum hydroxide in 10 mL of 0.9% saline solution). The pH of the aluminum hydroxide solution was corrected to 7.0 with the aid of a pH meter. Subsequently, the volume to be prepared was calculated and the protein was added to the 0.2% aluminum hydroxide solution slowly and gradually, maintaining stirring until completely homogeneous. For a final formulation, 1% saponin was added. The pH was checked and adjusted to 7, with the aid of a pH indicator strip. Example 3.2 - Poliqen 10%: For the formulation with Poligen, a 0.9% saline solution was initially prepared (0.9 g of NaCl in 100 mL of distilled or deionized water) followed by the Polygen 10% solution (10 g of Poligen in 10 mL of 0.9% saline solution). Subsequently, the volume to be prepared was calculated and the protein was added to the Poligen solution slowly and gradually, maintaining stirring until completely homogenized. Example 3.3 - Emulsigen 10%: For the formulation with Poligen, a 0.9% saline solution was initially prepared (0.9 g of NaCl in 100 mL of distilled or deionized water) followed by the 10% polygen solution (10 g of poligen in 10 mL of 0.9% saline solution). Subsequently, the volume to be prepared and the protein was added to the poligen solution slowly and gradually, maintaining agitation until completely homogenized. Example 3.4 - Incomplete Freund's Adjuvant: For the formulation with Incomplete Freund's Adjuvant, equal volumes of antigen and adjuvant were added and homogenized with a syringe until a white emulsion was formed. The formulation was homogenized for 5 minutes until a more stable emulsion was formed. Vortexing was used before use to ensure emulsion stability. Example 4: Animal efficacy studies

[0068] Adult male BALB / C mice (6 to 8 weeks of age; total number of 114 animals) were immunized according to a dose-booster protocol, with three doses (with an initial dose and two boosters) of 100 uL each containing saline (control group 1), adjuvant only (control group 2), or formulations containing 10 pg of the antigen and adjuvants, administered subcutaneously at 14-day intervals between doses. Fifteen days after the last immunization, the mice were challenged by intraperitoneal inoculation of 1 x 10 3 CFU of E. muris, B. canis or A. platys obtained from cell cultures.

[0069] After vaccination and before challenge, an indirect ELISA test was performed to verify immunoconversion. 10 pL of blood was collected from each animal by tail-bleeding before each immunization and one week before challenge with each pathogen. The blood was diluted in 90 pL of 1X PBS and stored at -20°C until the ELISA assay. Subsequently, the stored blood was further diluted in 1X PBS. The protein was diluted to 50 pg per well in carbonate buffer (pH 9.3), and the ELISA plate was sensitized and incubated at 4°C overnight. Unbound antigen (protein) was discarded, and the plate was washed four times with PBS-T. The plate was blocked by adding 100 pL of blocking solution (2% BSA solution in PBS-T) per well and incubating for 1 hour at 37°C. Excess blocking solution was removed. and 100 pL of blood diluted in PBS-T was added per well of the plate, incubating for 1 h at 37°C. The plate was washed with PBS-T and 100 pL per well of secondary antibody (anti-mouse conjugated with peroxidase) diluted in PBS-T was added, incubating for 1 h at 37°C. The excess secondary antibody was removed and the plate washed with PBS-T. For development, 100 pL per well of the OPD (o-phenylenediamine dihydrochloride) solution was used (10 ml of citrate buffer pH 5.0, 10 mg of OPD and 10 pL of H2O2). The reaction was stopped by adding 50 pL per well of 0.5 M H2SO4 solution. The plate was read in a spectrophotometer at 492 nm.

[0070] The results of the ELISA analysis of the blood of the animals used in the first experiment showed that there was induction of antibody production, after the second immunization of the animals, for all groups that received adjuvant (Emulsigen, Polygen, aluminum hydroxide + saponin (H / S) or FIA) combined with the protein, which did not happen for the control groups (animals treated with 0.9% saline or animals treated only with adjuvant) (item (A) of FIGURE 3). This indicates that the dose of protein (antigen) was sufficient to perform immunoconversion (production of an immune response) by the animals.

[0071] The parasite load of E. muris, B. canis, or A. platys in the blood of animals after euthanasia was determined by real-time PCR. The analyses were performed in a StepOne™ Real-Time PCR System Thermal Cycling Block (Applied Biosystems) using GoTaq® qPCR Master Mix (Promega, Madison, WI, United States). Ten μl of reaction mixture containing 5 μl of GoTaq® qPCR Master Mix, 0.1 μl of CXR Reference Dye, 0.2 pM of each primer (forward and reverse), and 1 μl of total DNA extracted from blood were prepared.

[0072] The results of the experiment in which the animals were challenged with E. muris (item (A) in FIGURE 3) showed that H / S + protein, Emulsigen + protein and FIA + protein presented a significant reduction of parasite load, when compared to the control (non-immunized animals). For the animals challenged with A. platys, it was observed that there was significant pathogen control for the group immunized with H / S + protein in relation to the control group (item (B) in FIGURE 3). For the animals challenged with B. canis (item (C) in FIGURE 3), there was no statistical difference between the treatments and the control group; however, the parasite load detected for the group immunized with H / S + protein was the lowest among all treatments.

[0073] The average percentage of protection provided by the protein against each of the three pathogens was calculated, representing the percentage reduction in parasite load conferred by immunization with the protein. These results showed that the protein combined with aluminum hydroxide and saponin conferred the greatest protection, reducing the parasite load of E. muris or A. platys by 99.9%. Considering B. canis, the reduction was 90%. The Emulsigen + protein combination also showed a high percentage reduction in the load of E. muris (99.5%) and A. platys (95.7%). FIA + protein showed a 98.2% reduction in the parasite load of E. muris.

[0074] The spleens of the animals were aseptically removed and macerated in DMEM (Dulbecco's Modified Eagle Medium) culture medium, followed by filtration through a nylon mesh. The tubes containing the cell suspension were centrifuged at 4°C, 1200 rpm for 8 minutes. The supernatant was discarded and the pellet was homogenized in 10 mL of ammonium chloride solution diluted (1:10) in PBS-1X. After centrifugation again, the supernatant was discarded and the pellet was resuspended in DMEM. Cell counts were performed in a Neubauer chamber to obtain a corrected concentration of 1x10 5 of cells / mL in DMEM containing 10% fetal bovine serum. Spleen cells were plated using 6 wells per animal (3 protein-stimulated and 3 non-stimulated) in 200 uL of medium in 96-well plates. The remaining cells were used for Antibody labeling for flow cytometric analysis. To stimulate spleen cells, 0.5 pg of protein was added to the respective wells of the plate. The culture plate was incubated for 24 hours at 37°C in a CO2 incubator. The same volume of sterile PBS-1X was added to the unstimulated wells. After 24 hours, the supernatant was transferred to another plate and stored at -80°C for cytokine analysis.

[0075] Analysis of spleen cell populations from animals challenged with E. muris, A. platys, or B. canis showed that all treatments induced expansion of CD4 T lymphocytes. + (memory) and CD8 + (identification of elimination of intracellular pathogens), when compared to the non-immunized control (FIGURE 4). Considering the groups of animals challenged with E. muris, there was an increase in the percentage of CD4 T cells + and CD8 +for treatments with adjuvant + protein, however, when compared to the control group, the results were not statistically significant (item (A) of FIGURE 4).

[0076] Regarding the challenge of animals with A. platys or B. canis, for induction of CD4 cells + , the H / S adjuvant was the most efficient (items (B) and (C) in FIGURE 4). When analyzing the adjuvant + protein treatments, it was observed that the greatest response was for H / S + protein. For CD8 cells + The results were similar. H / S and H / S + protein induced the highest percentages of these cells. Thus, when compared with another adjuvant in combination with protein, H / S presented the best result (items (B) and (C) in FIGURE 4). These data are consistent with the average percentages of parasite load reduction for H / S + protein.

[0077] The supernatant from cultures stimulated with the recombinant protein was collected and used to measure cytokines IL-4 and IL-10, using a commercial kit (BD OptEIA™ Set Mouse, San Diego, CA, USA), according to the manufacturer's instructions. 100 pL of the capture antibody was added per well, diluted at a concentration of 1:250 in Sodium Carbonate buffer (0.1 M, pH 9.5 for IL-4) or Sodium Phosphate Buffer. (0.2M, pH 6.5 for IL-10). After overnight incubation at 4°C, the plate was washed 3 times with 0.01 M PBS buffer, pH 7, containing 0.05% Tween 20 (PBS-Tween 20) and blocked with 200 pL of diluent solution containing PBS and 10% fetal bovine serum, with incubation for 1 hour at room temperature. After blocking, the plates were washed once, followed by the addition of 100 pL of cell culture supernatant, standard and medium. The plates were incubated overnight at 4°C and washed 3 times again. 100 pL of the detection antibody combined with streptavidin (dilution 1:250) was added, incubating for 1 hour at room temperature. After the wells were washed, 100 pL of the substrate solution (10 mL of citrate-phosphate buffer + 15 mg of OPD + 4 pL of hydrogen peroxide (H2O2)) was added. After incubation for 30 minutes, 50 pL of the stopping solution (H2SO42M) was added. The absorbances were read in a spectrophotometer at a wavelength of 492 nm.

[0078] The cell suspension obtained from the spleen was incubated with anti-CD4 (FITC - Fluorescein isothiocyanate), anti-CD8b (PE - Phycoerythin) and anti-CD3 (PerCP - Peridinin Chlorophyll Protein) antibodies previously diluted in 0.01 M PBS, pH 7, containing 0.05% Tween 20 (PBS-Tween 20), protected from light. The incubation with the antibodies was carried out in the dark for 30 minutes at 4°C. Then, the material was washed with PBS to eliminate unbound antibodies, followed by centrifugation at 1300 rpm for 7 minutes. The final pellet was resuspended in 200 pL of preservative solution in polystyrene tubes, specific for flow cytometry (BD Pharmingen) and kept at 4°C, completely protected from light. The cell preparations were analyzed on a flow cytometer. Example 5: Determination of cytokines

[0079] In order to evaluate the cytokine pattern induced by immunizations, animals challenged with E. muris, A. platys, or B. canis were euthanized and spleen cells were obtained to determine the cytokines II-4 and IL-10. The cells were stimulated with the protein or remained unstimulated after 24 hours. The results demonstrate that the groups immunized with FIA and Emulsigen produced more IL-4 cytokines compared to the control group. In the groups immunized with the protein and adjuvant, no significant differences in cytokine production were observed compared to the control group (item (A) in FIGURE 5).

[0080] In the group immunized with aluminum hydroxide, IL-10 production was significant when compared to the control group and other experimental groups (item (A) in FIGURE 5). Cytokine production was not elevated, but the identified cells responded to the protein stimulus and were able to recognize the pathogen. Considering the groups of animals challenged with A. platys, IL-4 production presented a different profile from that obtained in the challenge with E. muris (items (A) and (B) in FIGURE 5). Cells in the emulsigen + protein group, stimulated with the antigen, showed significant IL-4 production compared to the control group (item (B) in FIGURE 5).

[0081] In the experimental groups challenged with B. canis, there was no significant difference (item (C) in FIGURE 5). However, IL-10 production by the aluminum hydroxide / saponin, emulsigen, and emulsigen + protein groups challenged with A. platys was significant, highlighting lower cytokine production in these groups compared to the control (item (B) in FIGURE 5). For animals challenged with B. canis, a significant difference in IL-10 was found in the emulsigen and emulsigen + protein groups, when compared to the non-immunized control group (item (C) in FIGURE 5). Cell analyses of the groups not stimulated with protein showed no significant results.

Claims

Claims 1. CHIMERIC RECOMBINATE ANTIGEN characterized by being an optimized recombinant chimeric protein (rTripEAB), containing the amino acid sequence SEQ ID NO: 10 or the nucleotide sequence SEQ ID NO 11, or even sequences capable of encoding the chimeric, synthetic or recombinant protein, SEQ ID NO: 10, which may contain mutations, deletions, insertions, neutral or equivalent substitutions, simultaneously comprising conserved peptides from pathogens of Ehrlichia canis, Anaplasma platys and Babesia canis, derived from the amino acid sequences SEQ ID NO 1 to 9, acting as CD4+ T or CD8+ T lymphocyte epitopes, containing linkers and codon optimization that promote its protective or diagnostic effect of triple action for diseases in animals such as dogs, swine, cattle, horses and humans.

2. ANTIGEN, according to claim 1, characterized in that the diseases capable of being diagnosed, prevented or treated are specifically those caused by the pathogens Ehrlichia canis, Anaplasma platys and Babesia canis and by non-specific pathogens, of the same family, order or class, which present cross-reaction, such as Babesia trautmann and Babesia perroncitoi in pigs, or even Babesia equi and Babesia caballi in horses.

3. ANTIGEN, according to claims 1 and 2, characterized in that they were formed from 09 (nine) polypeptides, SEQ ID NO: 1 to 9, obtained by bioinformatics techniques and subsequently optimized: with the addition of a linker between the peptides, increasing the solubility of the protein and favoring cleavage by the proteasome during the processing of the cellular immune response induced by the antigen and the adjuvant; and with the optimization of codons to present an antigenic function capable of serving as an active ingredient in veterinary or medicinal vaccines, as well as in disease detection methods and kits.

4. ANTIGEN, according to claims 1 to 3, characterized in that the amino acid sequence of the ANTIGEN or the nucleic acid sequence encoding the ANTIGEN(s) presents at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or at least 99% sequence identity with SEQ ID NO: 10 or SEQ ID NO:

11.

5. EXPRESSION VECTOR characterized by having as a heterologous expression gene the nucleotide sequence SEQ ID NO:11, being capable of producing a chimeric recombinant antigen as defined in claims 1 to 4.

6. VECTOR, according to claim 5, characterized in that it is preferably a pet28a expression vector aimed at producing the antigen fused to the histidine tail for purification.

7. RECOMBINANT CELL characterized by having the expression vector as defined in claims 5 and 6, being capable of producing the chimeric recombinant antigen as defined in claims 1 to 4.

8. CELL, according to claim 7, characterized in that it is preferably the E. coli BL21 cell.

9. PROCESS FOR PRODUCTION OF CHIMERIC RECOMBINANT ANTIGEN characterized by having the following steps: a) Obtaining the epitopes: prediction of the peptides referring to the epitopes of CD4+ T and CD8+ T lymphocytes of the pathogens of interest, from the proteome of E. canis, A. platys and B. canis, by bioinformatics techniques and conservative approach to minimize the selection of false-positive epitopes; b) Constructing the chimeric sequence: joining of the 09 (nine) epitopes, SEQ ID NO: 1 to 9, with linkers and codon optimization as defined in claim 3, generating SEQ ID NO: 10; and c) Obtaining the chimeric protein: by chemical synthesis of the chimeric amino acid sequence obtained in b or by inserting the expression vector, as defined in claims 5 and 6, containing the nucleotide sequence corresponding to the chimeric antigen SEQ ID NO:11, as defined in claims 1 to 4, into a recombinant cell, as defined in claims 7 and 8, and subsequent isolation and purification, and may be used as an active ingredient in vaccine compositions or in the detection of diseases in animals such as dogs, pigs, cattle, horses and humans.

10. PROCESS, according to claim 9, characterized in that the chimeric protein is preferably expressed in E. coli and purified with satisfactory yield and purity to be applied in the development of a vaccine with triple action, protecting against isolated or combined infection of the three pathogens Ehrlichia canis, Anaplasma platys and Babesia canis or even of non-specific pathogens.

11. HUMAN OR VETERINARY VACCINE COMPOSITION characterized by having triple action and presenting the following elements: a) 0.5 to 100 pg / mL of chimeric recombinant antigen, as defined in claims 1 to 4; b) 1.2% of final volume of vaccine adjuvant(s); c) pharmaceutically acceptable vehicles and excipients.

12. COMPOSITION, according to claim 11, characterized in that the amount of chimeric antigens is preferably 10 pg of purified chimeric protein per dose administered to the individual.

13. COMPOSITION, according to claim 11, characterized in that the preferred adjuvants are selected from: Inject Freund's Incomplete Adjuvant (FIA), Saponin, Aluminum Hydroxide, Polygen and Emulsigen.

14. COMPOSITION, according to claim 13, characterized in that the preferred adjuvant is Aluminum Hydroxide in a concentration of 0.2%, added with Saponin adjuvant (Quil-A™) at a concentration of 1% in final dose volume.

15. PROCESS FOR PRODUCTION OF THE VACCINE COMPOSITION characterized by having the following steps: a) Production of the purified chimeric recombinant protein, as defined in claims 9 and 10 (active ingredient); b) Insertion of 0.5 to 100 pg of chimeric recombinant protein obtained in item “a” per individual dose; c) Addition of the vaccine adjuvant Aluminum Hydroxide at a concentration of 0.2%, added with Saponin adjuvant (Quil-A™) at a concentration of 1% in the final dose volume; and d) Complete the volume to 0.5ml_, 1 ml_ or 2ml_ with pharmaceutically acceptable vehicles and excipients.

16. DIAGNOSTIC KIT characterized by having the chimeric recombinant antigen, as defined in claims 1 to 4, in a physical device capable of detecting the presence of the desired pathogens in the biological samples tested, enabling the diagnosis of diseases caused by any of the three pathogenic agents Ehrlichia canis, Anaplasma platys and / or Babesia canis and cross-reactive pathogens, in dogs, swine, cattle, horses and humans.

17. USE OF THE CHIMERIC RECOMBINANT PROTEIN characterized by being for the manufacture of vaccines or for the manufacture of diagnostic kits, capable of respectively causing: a) a protective effect in the individual treated against the diseases ehrlichiosis, anaplasmosis and canine babesiosis simultaneously; or b) detecting infection by Ehrlichia canis, Anaplasma platys and / or Babesia canis simultaneously.

18. METHOD OF TREATMENT OF CANINE EHRLICHIOSIS, ANAPLASMOSIS AND BABESIOSIS characterized by being a simultaneous treatment comprising the administration of at least 0.5 to 100 pg of the recombinant chimeric antigen, as defined in claims 1 to 4, preferably with the application of three doses every 15 days via intramuscular route.

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