Scolotoxin protein ssd14 from tick or peptides derived and use as vaccine against mites

Description

[001] The present invention relates to the identification and characterization of a protein from the cattle tick, Rhipicephalus microplus. The antigen, named Rm-escolotoxin SSD14, is characterized by comprising the amino acid sequence SEQ ID NO:1, with possible platelet aggregation activity, but without a known biological function. The use of this protein, obtained by chemical synthesis or produced in other organisms through recombinant DNA techniques, in animals, as an immunogen is capable of generating a protective response against the cattle tick, so that the antigen can be used as a vaccine to prevent infestation, alone or in conjunction with other antigens. The present invention falls within the fields of bioprocess engineering and biotechnology, veterinary medicine, animal management, and public health. Background of the Invention

[002] The main method of control for ticks is the use of acaricides. However, the products are Petition 870260065020, dated 02 / 07 / 2026, page 6 / 53 2 / 45 are frequently used incorrectly, accelerating the selection of tick populations resistant to the products used. This control method presents other disadvantages, such as contamination of meat, milk, and the environment. Because of the problems associated with the use of acaricides, identifying other forms of tick control is important to achieve greater productivity in the field.

[003] To develop new vaccines, it is essential to use different methods that help identify potential vaccine antigens from the proteins present in the parasite. Experiments vaccinating cattle with proteins show that immunizing animals with tick proteins (such as BYC, VTDCE, and GST) can interfere with tick biology, altering metabolism and hindering biological functions related to feeding and reproduction. However, the level of protection achieved by the different vaccines tested is not sufficient to eliminate the tick. Therefore, increased vaccine efficacy can be achieved by identifying new additional antigens. The number of antigens with protective effects has grown, but can still be explored with the identification of new molecules, enabling increased levels of protection conferred by vaccines containing more than one antigen in their formulation. Petition 870260065020, dated 02 / 07 / 2026, page 7 / 53 3 / 45 vaccine. Therefore, the use of vaccines with more than one antigen could increase the protection conferred by vaccination.

[004] To date, the commercialization of vaccines based on Bm8 6, such as Gavac® and TickGARD®, has demonstrated its effectiveness against Rhipicephalus microplus, as well as other tick species. However, genetic variations among different tick populations cause variations in the effectiveness of these vaccines in different regions. Currently, several immunogenic molecules have been explored as potential vaccine candidates, with subolesin being particularly noteworthy. This protein has shown a role in reducing the survival and reproductive capacity of R. microplus when used in the immunization of cattle.

[005] Metalloproteinases and tick proteins located in the midgut, ovaries, and saliva play crucial roles in digestion and immunology. While essential to the parasite's feeding mechanism, further exploration is needed to determine whether metalloproteinases could potentially be leveraged as candidate vaccine antigens in the future. Research on the potential iron-binding antigenic protein ferritin 2 in R. sanguineus showed that antibodies against ferritin 2, produced in rabbits vaccinated with recombinant ferritin, Petition 870260065020, dated 02 / 07 / 2026, page 8 / 53 4 / 45 decrease the tick's body weight, oviposition, and hatching.

[006] A wealth of data is being generated by numerous in silico and drug design methods, which can reduce the time and cost involved in trial-and-error studies during drug or vaccine development. One of the first steps in locating a new treatment target or a potential vaccine candidate is the identification of target proteins. Although several techniques exist for identifying B-cell epitopes, methods based on protein structure are considered the most reliable. However, X-ray crystallographic data on the structure of most tick antigens are still lacking. Despite this, several algorithms have been used to identify epitopes. In silico algorithms for epitope prediction are more accessible, faster, and easier to implement than crystallography.

[007] Tick mortality caused by vaccine use is mediated by cells and, primarily, by antibodies. Determining the B-cell epitopes of a tick protein to be used as a vaccine is important and aids in vaccine development. Based on their spatial structure, B-cell epitopes can be categorized as discontinuous (non-linear or Petition 870260065020, dated 02 / 07 / 2026, page 9 / 53 5 / 45 conformational) or continuous (linear or sequential). Conformational epitopes are a collection of amino acids brought together by protein folding, but not contiguous in the primary sequence, while linear epitopes are peptide sequences continuous in the primary amino acid sequence of the protein.

[008] One of the most significant developments for vaccine development is the development of several algorithms for predicting linear B-cell epitopes, which would be able to induce an antibody response capable of reacting against the native full protein. Currently, several linear B-cell prediction tools are available online, such as BepiPred, ABCpred, BEEP, and LAPtop. In all these methods, the average amino acid propensity value is determined using a moving window through a protein sequence.

[009] Due to the high costs of using the tick's natural hosts, many studies examine potential vaccine proteins by conducting experiments in animal models, primarily rabbits.

[0010] Using rabbits as immunization models, the immunogenic potential of 57 proteins from different tick species was examined. Certain proteins including Bm86, GST-Hl, CoAQP, OeAQP, OeAQP1, 64TRP, voraxin and serpins, Petition 870260065020, dated 02 / 07 / 2026, page 10 / 53 6 / 45 can induce immune responses that provide protection against different tick species. Furthermore, in certain cases, the vaccine efficacy in rabbits was found to be comparable to studies conducted in natural hosts (e.g., Bm86, IrFER2, RmFER2, serpins, and serine protease inhibitor). Overall, the results demonstrated that rabbits can be used as appropriate experimental models for this investigation before conducting anti-tick vaccination experiments using natural hosts. The set of new strategies for identifying and validating new antigens allowed for the identification and testing of a new tick protein with potential for use in a vaccine. Summary of the Invention

[0011] The data presented here aim to advance the development of an anti-tick vaccine. This comprehensive investigation involves the identification of potential vaccine candidates based on in silico analysis of different tick proteins, selecting the Rm-scolotoxin SSD14 protein and conducting an in vivo immunization experiment. The vaccine induces protection against tick infestation. Brief Description of the Figures Petition 870260065020, dated 02 / 07 / 2026, page 11 / 53 7 / 45

[0012] Figure 1 presents the prediction of phosphorylation sites of the Rm-scolotoxin SSD14 protein using the Netphos 3.1 web server.

[0013] Figure 2 presents the prediction of the presence of the signal peptide of the Rm-scolotoxin SSD14 protein using the Signalp 6.0 web server.

[0014] Figure 3 shows the secondary structure of the Rm-scolotoxin SSD14 protein predicted using PSIPRED (A), the predicted amino acids of the Rm-scolotoxin SSD14 protein using PSIPRED (B), and the schematic diagram of the protein's secondary structure by the PDBsum tool (C).

[0015] Figure 4 shows the prediction, refinement and validation of the protein, the Ramachandran plot of the refined 3D model generated by PROCHECK (A), the three-dimensional (3D) structure of the protein (B), the Z-score plot of the refined 3D model generated by ProSA-web (C,D), the ERRAT model of the protein (E), the graph generated by the verification of the 3D module of SAVES v6.0 (F).

[0016] Figure 5 shows the B cell epitopes predicted in the protein based on linear epitopes Bepipred (A), Chou and Fasman Beta, B-Turn prediction (B), Emini surface accessibility prediction (C), Karplus & Schulz flexibility prediction (D), Kolaskar & Tongaonkar antigenicity prediction (E), Parker hydrophilicity prediction (F). Petition 870260065020, dated 02 / 07 / 2026, page 12 / 53 8 / 45

[0017] Figure 6 shows the screening of potential epitopes of the Rm-scolotoxin SSD14 protein using different online / web bioinformatics tools.

[0018] Figure 7 shows the predicted tertiary structure of the Rm-scolotoxin SSD14 protein using I-Tasser; helices, loops, and strands are shown in 3D structure with different colors.

[0019] Figure 8 shows the predicted epitopes of the Rm-scolotoxin SSD14 protein using the Pymol software.

[0020] Figure 9 shows the phylogenetic analysis of sequences similar to the Rm-scolotoxin SSD14 protein from different organisms.

[0021] Figure 10 presents the SDS-PAGE (12%) showing the result of the expressed Rm-scolotoxin SSD14 protein. 1: MV (IgG), 2-5: SSD 14 expressed at 4 hours and 37 °C. The arrows indicate the bands corresponding to the proteins, along with the molecular mass of the proteins. The 50 kDa and 25 kDa bands correspond to the molecular mass pattern of the heavy and light chains of bovine immunoglobulin G, used as a reference for the mass of the other proteins. The 76 kDa band corresponds to the band of the Rm-scolotoxin SSD14 protein.

[0022] Figure 11 shows the SDS-PAGE (12%) showing the result of the purified SSD14 scolotoxin Rm protein. 1: MV (IgG), 2: BSA, 3-9: SSD 14 purified by chromatography. Petition 870260065020, dated 02 / 07 / 2026, page 13 / 53 9 / 45 affinity with nickel, eluting with 100 mM imidazole. The arrows indicate the bands corresponding to the proteins, along with the molecular mass of the proteins. The 50 kDa and 25 kDa bands correspond to the molecular mass pattern of the heavy and light chains of bovine immunoglobulin G, used as a reference for the mass of the other proteins. The 76 kDa band corresponds to the band of the Rm-scolotoxin SSD14 protein.

[0023] Figure 12 shows the western blot using antihistidine antibody, showing the result of the expressed Rm-scolotoxin SSD14 protein. P (positive control), SSD (Rm-scolotoxin SSD14). The arrows indicate the bands corresponding to the proteins, along with the molecular mass of the proteins. The 50 kDa and 25 kDa bands correspond to the molecular mass pattern of the heavy and light chains of bovine immunoglobulin G, used as a reference for the mass of the other proteins. The 73 kDa band corresponds to the band of the Rm-scolotoxin SSD14 protein.

[0024] Figure 13 shows the dot blot result of serum from rabbits immunized and control with Rm-scolotoxin SSD14 protein. Top row (indications 1, 2 and 3) sera from the three control rabbits, bottom row (indications 1, 2, 3) sera from the three rabbits after immunization. Detailed Description of the Invention Petition 870260065020, dated 02 / 07 / 2026, page 14 / 53 10 / 45

[0025] Ticks are chelicerate arthropod ectoparasites that infest vertebrate hosts, parasitizing almost all classes, including amphibians, reptiles, birds, mammals, and even humans. In addition to sucking blood, these ectoparasites also serve as vectors for a wide range of pathogens, including protozoa, bacteria, and viruses. Ticks exhibit a global distribution ranging from the Arctic to the Antarctic, with primary concentrations in tropical and subtropical habitats. Their presence depends on specific climatic conditions, namely temperature and humidity levels, which are fundamental to sustaining their populations.

[0026] Ticks cause losses in the Australian production chain that reach approximately US$42 million annually. For Mexico, the estimated losses are US$3 billion annually. In Brazil, losses attributed to ticks are close to US$3 billion per year. Research examining the financial effects of ectoparasite activity in South America found that losses in Brazil amounted to approximately 2.5 million head of cattle, resulting in 75 million kg of meat, 1.5 billion liters of milk, US$8.6 million in secondary damages, and US$25 million in chemical acaricides to combat tick infestations. Throughout South America, Central America and Petition 870260065020, dated 02 / 07 / 2026, page 15 / 53 11 / 45 In Australia, R. microplus has the greatest economic impact on cattle production.

[0027] The tick Rhipicephalus microplus is a monoxenic parasite whose main host is cattle. However, it can also occasionally parasitize other animals such as buffalo, sheep, equines, and cervids. Parasitism causes significant losses in milk and meat production, damages the hide due to lesions, and causes inflammatory reactions at the tick's attachment points. In addition, R. microplus is a vector of protozoa of the genus Babesia and the rickettsia Anaplasma marginale. It is estimated that annual economic losses due to R. microplus infestation in Brazil reach 3 billion US dollars.

[0028] Ticks have been controlled with acaricides, but their use has presented challenges due to cost, environmental impact, residues in dairy products, and tick resistance. Several types of acaricides have been used over time, including arsenic compounds, organochlorines, organophosphates, carbamates, formamidines, ivermectin, pyrethroids, macrocyclic lactones, fipronil, and fluazuron. Although biological and immunological controls are now integrated into ectoparasite management programs, chemical products are still needed. Petition 870260065020, dated 02 / 07 / 2026, p. 16 / 53 12 / 45

[0029] Biological control, which involves the conservation of natural predators and the improvement of biocontrol agents, plays an important role in tick management. Several strategies have been explored to influence tick survival, such as selection of specific breeds, pasture rotation, herd management, consideration of climatic conditions, and the use of pathogenic elements such as bacteria, fungi, nematodes, and parasitoids. For example, the pathogens Beauveria bassiana and Metarhizium anisopliae have demonstrated mortality rates of approximately 40% in Rhipicephalus appendiculatus and Amblyomma variegatum. This suggests the potential for improved biological control through a combination of control methods. In addition, bacteria such as Escherichia coli, Cedecea lapagei, and Enterobacter agglomerans are also employed in biological control.

[0030] The concept of developing tick vaccines dates back to 1939 and has been subjected to extensive experimentation. Compared to acaricides, the vaccination approach is considered sustainable, environmentally friendly, more precise, and less prone to resistance problems. However, the main challenge lies in identifying antigens that can effectively confer protection against these parasites. Vaccination is one way Petition 870260065020, dated 02 / 07 / 2026, page 17 / 53 13 / 45 reliable and long-term solution to reduce tick numbers and the transmission of disease-causing agents.

[0031] From in silico analyses, we identified a protein with potentially immunogenic characteristics. The protein is named SSD14-like scolotoxin (Rmescolotoxin SSD14). The physicochemical characteristics of SSD14-like scolotoxin were evaluated using Expasy ProtP to analyze the following parameters: total number of positively and negatively charged residues, overall mean hydrophilicity, aliphatic index, instability index, estimated half-life, extinction coefficients, and number of amino acids. The antigenicity and allergenicity of the proteins were determined using the Antigenpro and Vaxijen 2.0 web servers. Vaxijen was used to estimate allergenicity, and Antigenpro was employed for microarray analysis to predict antigenicity. Vaxijen was developed to allow the classification of antigens based solely on the physicochemical characteristics of the proteins, without the need for sequence matching.The Allertop website was used to assess the allergenicity of proteins based on their fundamental physicochemical properties. Using the Signal P 6.0 website, signaling peptides in amino acid sequences were verified to predict the secondary structure of Rm. Petition 870260065020, dated 02 / 07 / 2026, page 18 / 53 For the 14 / 45 scolotoxin SSD14, the PSIPRED online server was used. NovaFold was used to predict the tertiary structure. The Ellipro Immunological Epitope Database (IEDB) website was used to predict linear B-cell epitopes based on the 3D structure of a protein and default settings. The default threshold for the minimum epitope prediction after protein ID submission was 0.5 for minimum score and 6 for maximum distance. Other web servers were used to study the toxicity, antigenicity, and allergenicity of linear B-cell epitopes. The Toxinpred website was used with a threshold of 0.5 to assess epitope toxicity. Vaxijen was used to test non-toxic linear epitopes for antigenicity at a threshold of 0.5. Following investigations into toxicity and antigenicity, a threshold was applied to determine the allergenicity of a subset of selected epitopes using the Allertop internet server.

[0032] Cloning of the coding regions of the Rmescolotoxin SSD14 protein was conducted using RT-PCR methodology. For this purpose, primers designed based on known regions of the coding region of this R. microplus gene, obtained from a transcript bank, were used. These primers were used in the PCR reaction to amplify the protein coding sequence from cDNAs of R. microplus tissues. The PCR product resulted in fragments of 2139 bp, Petition 870260065020, dated 02 / 07 / 2026, page 19 / 53 15 / 45 corresponding to the cDNA coding sequence of the Rm-scolotoxin SSD14 protein, plus the addition of an 18-nucleotide sequence encoding 6 histidine amino acids. Subsequently, the fragment was inserted into the pET-30a (+) vector and E. coli bacteria were transformed (DE3 Star strain) to obtain plasmids. The recombinant plasmids were then extracted by minipreparation and their nucleic acid sequences were determined. The predicted amino acid sequences for the Rm-scolotoxin SSD14 protein were confirmed by comparative analysis with sequences available in GenBank and the transcript database.

[0033] For protein expression, E. coli DE3 Star bacteria were transformed by electroporation with the recombinant plasmid pET-30a-SSD14. The transformed bacteria were plated on LB agar plates containing kanamycin (50 μg / mL) and incubated for 18 hours at 37°C in an incubator. One isolated colony from each transformant was selected and inoculated into 25 mL of LB medium supplemented with kanamycin (50 μg / mL), growing for 16 hours at 37°C with agitation at 180 rpm. After growth, the cells were collected by centrifugation at 12,000 g for 5 minutes at 4°C, resuspended in fresh antibiotic medium, and used to inoculate 500 mL of LB medium. The flasks were then incubated at 37°C with agitation at 180 rpm until a Petition 870260065020, dated 02 / 07 / 2026, page 20 / 53 16 / 45 optical density of 0.6 at 600 nm. To induce protein expression, isopropyl-εD-galactoside (IPTG) was added at a final concentration of 1 mM, followed by incubation of the culture for 4 hours at 37°C. The cultured cells were then centrifuged at 12,000 g for 5 minutes at 4°C, and the pellet was resuspended in 10 mM sodium phosphate buffer containing 300 mM NaCl at pH 7.2. For cell lysis, the cells were subjected to sonication. Sonication was performed by exposing the bacterial extract to sonic waves 5 times with a 30-second pulse duration and 25% intensity. The soluble and insoluble fractions were separated by centrifugation at 10,000 g for 5 minutes at 4°C. The samples were analyzed by SDS-PAGE (12%) and Western blot to confirm the expression of the recombinant protein.

[0034] The recombinant protein was obtained in the form of inclusion particles, therefore insoluble. For protein solubilization, 0.5 M, 1 M, 2 M, and 4 M urea solutions were used. Solutions of increasing concentration were added to the sediment, and between each step, the material was centrifuged at 10,000 g for 10 minutes at 4°C, the supernatant was removed, and a more concentrated urea solution was added. This process was repeated for all urea solutions. At each step, a sample was removed for subsequent analysis by SDS-PAGE-12%. Petition 870260065020, dated 02 / 07 / 2026, page 21 / 53 17 / 45

[0035] After solubilization, the fraction containing Rmescolotoxin SSD14 was subjected to Ni2+ metal ion affinity chromatography for purification of the target protein. Elutions were performed with phosphate buffer (20 mM Sodium Phosphate, 0.5 M NaCl, pH 7.4) containing increasing concentrations of imidazole ranging from 20 mM to 500 mM. Samples were analyzed by 12% SDS-PAGE.

[0036] To test the ability of Rm-scolotoxin SSD14 to induce a protective immune response, four rabbits were immunized with the recombinant protein. The purified Rmescolotoxin SSD14 sample was mixed with the adjuvant Montanide and inoculated into the rabbits. Two groups of rabbits, each containing four animals, were used (vaccinated group and control group). Each rabbit was vaccinated subcutaneously by injecting 100 μg of purified Rmescolotoxin SSD14 protein in 0.5 ml of PBS along with 0.5 ml of adjuvant (Montanide ISA 61 VG). The rabbits in the control group received an inoculation of 0.5 ml of PBS along with 0.5 ml of adjuvant. Three vaccine doses were administered to each rabbit at 14-day intervals. Before vaccination, after each dose, and 28 days after tick infestation, blood was collected from the marginal vein of the left ear of rabbits. To validate the humoral response, Western blot analysis was performed on post-vaccination rabbit serum. Petition 870260065020, dated 02 / 07 / 2026, page 22 / 53 18 / 45 immunization. In addition, ELISA was used to determine antibody titers. For the challenge of rabbits after immunization, each rabbit was subjected, two weeks after the last antigen dose, to infestation by 20 adults (10 males and 10 females) and 30 nymphs of R. sanguineus. The protective effect of Rm-scolotoxin SSD14 immunization against R. sanguineus was analyzed based on biological parameters. The number of nymphs that completed feeding, the weight of the engorged adult female, the weight of the eggs produced, and the hatched larvae were analyzed. The values ​​of the vaccinated and control groups were compared to each other.

[0037] Sera collected on days 0, 14, 28, 42, and 72 from immunized rabbits and the control group were tested by ELISA to determine antibody levels. One hundred ng of Rmescolotoxin SSD14 protein diluted in 50 µl of carbonate / bicarbonate buffer (pH 9.6) were placed in ninety-six-well plates, which were then incubated at 4°C overnight. After three rounds of washing with 200 µl of PBSTween (0.05%, pH 7.4), 100 µl of the primary antibody diluted 1:1000 were applied to each well, and the mixture was incubated at 37°C for 2 hours. Rabbit anti-peroxidase conjugate antibody (diluted 1:5000) was added to each well and incubated for one hour at 37°C. The plates were revealed after being incubated at room temperature for 15-20 minutes. Petition 870260065020, dated 02 / 07 / 2026, page 23 / 53 19 / 45 in the dark, by adding 10 ml of phosphate-citrate buffer (pH 5.0) comprising 5 µl of H2O2 (30%) and 3.4 mg of OPD (O-phenylenediamine dihydrochloride). Using a microplate reader (BioTek Epoch 2), the absorbance was measured at 490 nm after the reaction stopped by adding 50 µl of H2SO4 (12.5%) to each well.

[0038] The native Rm-scolotoxin SSD14 protein has a sequence of 713 amino acids with a theoretical pI (isoelectric point) of 4.96 and a molecular mass of 76067.90 Da. The protein has 96 negatively charged residues and 61 positively charged residues. The instability index was calculated at 34.67, classifying the protein as stable. The calculated aliphatic index was 78.19 and the GRAVY index was -0.246. All these results were analyzed using the ProtParam server.

[0039] The most studied post-translational modification (PTM) is phosphorylation, which frequently occurs in the cytosol or nucleus of target proteins and is one of the fundamental forms of PTM. One of the two main mechanisms by which this modification can rapidly alter protein function is through binding to interaction domains or by allosteric effect. The Netphos 3.1 web server predicted that SSD14 has 66 phosphorylation sites, including 37 serines, 28 threonines, and 1 tyrosine. The results are shown in Figure 1. Petition 870260065020, dated 02 / 07 / 2026, page 24 / 53 20 / 45

[0040] The characteristics of the amino acids that constitute the signal peptide region of a protein play an important role in determining how that protein interacts with the protein transport system and, ultimately, where it is transported. Different cellular locations are specified by distinct classes of signal peptides. It is necessary to emphasize once again that not all proteins possess signal peptides; those that do not are retained in the cytoplasm. Upon reaching their destination, the amino acids corresponding to the signal peptide are often separated from the rest of the protein. Signal peptidases are the type of enzyme that catalyzes this cleavage. According to SigalP-6.0, the Rmescolotoxin SSD14 protein does not possess a signal peptide (Figure 2).

[0041] Determining the isoelectric point (pI) allows us to determine how basic or acidic a molecule is. The side chain of an amino acid, in particular, impacts its pI due to its chemical structure. Twenty typical amino acids have isoelectric values ​​between 5.5 and 12.0. The solubility of an amino acid can be influenced by its pI. The physicochemical properties of recombinant Rm-scolotoxin SSD14 were analyzed, revealing a hypothetical isoelectric point (pI) of 4.9, a Gravy value of -0.331, and a molecular mass of 73.5 kDa. The system demonstrated structural stability with an index Petition 870260065020, dated 02 / 07 / 2026, page 25 / 53 21 / 45 instability of 36.32 and thermostable aliphatic index of 74.65. The protein has a half-life greater than 30 hours in mammalian cells (in vitro), 20 hours in yeast cells and more than 10 hours in E. coli (Table 1). Table 1. Physicochemical properties of the recombinant protein Physicochemical properties: Molecular mass value 73.5 kDa; Number of amino acids 688; Isoelectric point (pI) 4.90; Total number of atoms 10165; Number + Lys) total of positive residues (Arg) 59; Number + Glu) total of negative residues (Asp) 96; Instability index (II) 36.32 (stable); Extinction coefficient 69370 M-1 cm-1

[0042] Predicting only the linear amino acid sequence may not be sufficient to estimate the immunogenicity of a protein; therefore, the secondary structure was determined. The secondary structure of the Rm-scolotoxin SSD14 protein was predicted and visualized using the PSIPRED web server. The Rm-scolotoxin SSD14 protein consists of 33.8% (233 / 688) alpha helix and 13.2% (91 / 688) beta sheet. Types of Petition 870260065020, dated 02 / 07 / 2026, page 26 / 53 22 / 45 amino acids of the Rm-scolotoxin SSD14 protein are shown in Figure 3.

[0043] The 3D structure of the protein was predicted and generated using I-Tasser (Iterative Threading ASSembly Refinement) (Figure 4B). In I-Tasser, energy functions incorporate statistical terms based on predicted structural features and information from the Protein Database. During protein modeling, a set of fragment substitutions and random perturbations is used to model the target protein. As a result, several models were generated using random seeds, and their energies were calculated. Finally, the model with the lowest energy was selected and then underwent refinement. The refined construct demonstrated a ProSA-web Z-score of -10.73 (Figure 4C and D). ProSAweb estimated the accuracy of the construct by analyzing energy as a role of amino acids in protein structure.For comprehensive validation, PROCHECK conducted a Ramachandran analysis, confirming that 95.1% of the residues occupied the most favorable region (red), 3.9% were in the region of additional licenses (yellow), and 0.3% were in the area of ​​limited licenses (light yellow) (Figure 4A). ERRAT assessed the overall quality of the protein's 3D structure, assigning it a score of 94.7% (Figure 4E). Graph generated by verification of the 3D module of SAVES v6.0 (Figure). Petition 870260065020, dated 02 / 07 / 2026, page 27 / 53 23 / 45 4F). These assessments collectively contribute to the validation and improvement of the protein's structural design.

[0044] B-cell epitopes play an important role in the development of epitope-based vaccines. A dominant linear B-cell epitope can be used in autoimmune diseases as a target for neutralizing antibody responses. Furthermore, they are capable of inducing an antibody that cross-reacts with the antigenic protein. In this study, using the amino acid scale-based method, we predicted the identification of the B-cell epitope. Different analytical methods were used for the prediction of the continuous B-cell epitope, as shown in Figure 5.

[0045] Amino acid fragments within a protein sequence can be part of linear B-cell epitopes. Using a prediction method to identify B-cell epitopes can streamline and reduce the overall cost of the vaccine development process. The ABCPRED web tool was used to predict linear epitopes; the results are listed in Table 2. Table 2. Linear epitopes of the Rm-scolotoxin SSD14 protein predicted using the ABCPRED web tool. End Peptide Score the waste Petition 870260065020, dated 02 / 07 / 2026, page 28 / 53 24 / 45 104 119 KGTEDTGLTSTESSVD 16 0, 93 164 179 AVAT DAAP CANVTRNI 16 0.91 98 113 TCKCPSKGTEDTGLTS 16 0.91 415 430 RVGIDALERFNSTTLQ, PGMSNSYDVYPTRPNF 16 0.89 499 514 GGTAHATFWGDTGDVI 16 0.89 478 493 ESTT FDDPAHYGFLNN 16 0.89 381 396 EAEWKEPIVADLSNGN 16 0.8 825 30 ERAPGAATWDMFVGNE 16 0.88 53 68 GDTVFTGSASEAGVVS 16 0.87 523 538 HFGSKLRTSSGVILNN 16 0.87 87 102 AKSEDGTSSAATCKCP 16 0.87 613630KLIIDHPRQ 0.86 432 448 HRFIEACKHGYGGRSH 16 0.86 130 145 TTSTPLGDVEDARCGE 16 0.86 59 74 GSASEAGVVSTTLLDS 16 0.85 369 384 GGVITEHDLKS 165757 372 IGEQMAKEIRDDGGVI 16 0.85 319 334 NETTNDTLAEGDLLVQ 16 0.85 553 568 DVYPTRPNFIEPRKRP 16 0.84 178 193 NIMKKGGNLADAAVAT 18 46 154 394 EDARCGEPLADATASD 16 0.84 78 93 GHEEVEPTEAKSEDGT 16 0.84 69 84 TTLLDSTPAGHEEVEP 16 0.83 Petition 870260065020, of 02 / 07 / 2026, p. 29 / 53 25 / 45 630 645 NRVIVEPHFPQETIAE 16 0, 83 115 130 ESSVDAASENATTEQT 16 0.83 670 685 AGCLHASYDSRRGGSV 16 0.82 664 679 NGIKKEAGCLHASYDS 16 0, 8383 TTESVAALQSTSGDNG 16 0.82 493 508 NSYPEDGGTAHATFWG 16 0.82 459 474 LEQNMTTPEWAAAAVGN 16 0.82 210 223 GFMATIYLRRSKKAMT 16 0.81 186 PCANVTRNIMKKGGNL 16 0.82 606 623 RSLWGGYDIKEAIDRP 16 0.81 573 588 VPSVFVDCKGDAVLAL 16 0.81 531 546 SSGVILNNQMDDFATP 16 0.805530 AITSTINNHFGSKLRT 16 0.80 32 47 ESDLRATTESVAALQS 16 0.79 505 520 TFWGDTGDVIAITSTI 16 0.79 06 21 HEEELDLTTATPDAAT 16 0.79 438 438 438 GDSGGGGGGGH66 16 0.79 520 TFWGDTGDVIAITSTI 0.78 224 239 MTLIARERAPGAATWD 16 0.78 593 608 GPRIT SAVALVALRS L 16 0.77 24 39 PQFTGVLPESDLRATT 16 0.77 202 217 PHSMGIGGGFMATIY 1638,7 LFTVASPGS GAVLAGI 16 0.76 17 32 PDAATALPQFTGVLPE 16 0.75 363 378 KEIRDDGGVITEHDLK 16 0.75 Petition 870260065020, of 02 / 07 / 2026, p. 30 / 53 26 / 45 346 361 NKGPDYFYKGGIGEQM 16 0.75 252 267 GLSVAVPGELRGYRTL 16 0.75 187 202 ADAAVAT L CMGVVL P 16 0.75 583 598 DAVLALGGTGGPRITS 16 0.74 260 275 QKHKDDLSESLKKVFW 16 0.72 243 258 GNETLSRRGGLSVAVP 16 0,71 655 670 VNE GKGSTVNGIKKEA 16 0,70 621 636 PRLHHQLIPNRVIVE P 16 0,70 309 324 LSESLKKVFWNETTND 16 0,69 47 62 STSGDNGDTVFTGSAS 16 0.67 269 284 RKLHATLNWRDHFDDA 16 0.67 445 460 RSHLGDSDFEDVKELE 16 0.65 330 345 DLLVQADLADTLEVIA 16 0.65 649 664 KGHVFEVNEGKGSTVN 16 0.64 288 303 ARYGFPVGPHLAAALQ 16 0.64 423 438 RFNSTTLQWHRFIEAC 16 0.63 296 311 PHLAAALQKHKDDLSE 16 0.60 281 296 FDDAIRLARYGFPVGP 16 0.60 451 466 SDFEDVKELEQNMTTP 16 0.58 387 402 PIVADLSNGNTLFTVA 16 0,53 Petition: 870260065020, on 07 / 02 / 2026, page. 31 / 53 27 / 45

[0046] To predict the toxicity of epitopes, ToxinPred was used to predict toxic and non-toxic epitopes. All predicted linear epitopes are classified as non-toxic and are described in Table 3. Table 3. Screening of the toxicity of predicted epitopes in the Rm-scolotoxin SSD14 protein using ToxinPred. Sequence Pont Pred Hydrofo Hydropa Hydrofil Cob Mol peptidic uaçã ição bicidad ticidad icidade rar • oee pes Svm o KGTEDTGLT -0.6 No -0.24 -0.99 0.69 -3.162 STESSVD 9 00 6.8 7 AVATDAAPC -0.5 No -0.07 0.40 -0.17 0.0 158 ANVTRNI 8 0 6.9 9 TCKCPSKGT -0.3 No -0.24 -0.80 0.45 0.0 162 EDTGLTS 4 0 8.0 2 RVGIDALER -1.2 No -0.22 -0.29 0.12 0.0 182 FNSTTLQ 8 0 0.2 7 PGMSNSYDV -0.5 No -0.19 -1.01 -0.21 0.0 184 YPTRPNF 3 0 5.2 Petition 870260065020, dated 02 / 07 / 2026, page 32 / 53 28 / 45 3 GGTAHATFW -0.8 No 0.06 0.02 GDTGDVI 3 -0.37 -1.160 50 4.9 3 ESTTFDDPA -0.8 No -0.14 -0.94 HYGFLNN 6 -0.07 -2.182 50 8.1 0 EAEWKEPIV -1.0 No -0.15 -0.78 ADLSNGN 7 0.39 -3.177 00 2.1 3 ERAPGAATW -1.1 No -0.11 -0.51 DMFVGNE 3 0.10 -2.175 00 1.1 2 GDTVFTGSA -1.0 No 0.05 0.44 SE AG WS 7 -0.12 -2.148 00 3.7 7 HFGSKLRTS -lh3 No -0.14 -0.21 SGVILNN 0 -0.19 2.5 173 0 0.2 0 AKSEDGTSS -0.0 No -0.24 -0.64 AATCKCP 3 0.54 0.0 155 0 5.8 9 KEAIDRPRL -1.5 No -0.34 -1.13 HHQLIPN 4 0.42 2h0 193 0 7.4 Petition 870260065020, dated 02 / 07 / 2026, p. 33 / 53 29 / 45 8 HRFIEACKH -0.1 No GYGGRSH 6 -0.29 -1.11 0.17 3.5 185 0 5.3 0 TTSTPLGDV -0.6 No EDARCGE 1 -0.23 -0.72 0.58 -3.165 00 0.9 5 GSASEAGW -1.5 No STTLLDS 7 0.01 0.45 -0.07 -2.149 00 3.8 1 GGVITEHDL -1.5 No KSYEAEW 9 -0.11 -0.71 0.19 -2.183 50 4.2 1 IGEQMAKEI -0.9 No RDDGGVI 8 -0.16 -0.36 0.59 -2.173 00 1.2 0 NETTNDTLA -0.9 No EGDLLVQ 5 -0.17 -0.60 0.25 -4.173 00 3.0 6 DVYPTRPNF -0.7 No IEPRKRP 7 -0.43 -1.55 0.61 2h0 198 0 5.4 9 NIMKKGGNL -1.1 No ADAAVAT 9 -0.06 0.11 0.04 1.0 157 0 4.0 Petition 870260065020, dated 02 / 07 / 2026, page 34 / 53 30 / 45 6 EDARCGEPL -0.9 No -0.25 ADATASD 3 -0.75 0.82 -4.162 00 0.8 6 GHEEVEPTE -1.0 No -0.31 AKSEDGT 1 -1.67 1.12 -4.171 50 4.9 4 TTLLDSTPA -1.3 No -0.12 GHEEVEP 6 -0.63 0.31 -3.169 50 6.0 3 NRVIVEPHF -0.9 No -0.14 PQETIAE 5 -0.44 0.12 -1.187 50 9.3 4 ESSVDAASE -0.5 No -0.25 NATTEQT 5 -0.99 0.57 -4.163 00 9.8 1 AGCLHASYD -0.5 No -0.21 SRRGGSV 2 -0.41 0.11 1.5 163 0 5.9 9 NGIKKEAGC -0.6 No -0.18 LHASYDS 2 -0.68 0.28 0.5 169 0 3.0 9 TTESVAALQ -0.5 No -0.13 STSGDNG 8 -0.48 0.11 -2.153 00 7.7 Petition 870260065020, dated 02 / 07 / 2026, page 35 / 53 31 / 45 8 NSYPEDGGT -0.6 No -0.07 -0.91 AHATFWG 9 -0.25 -1.170 50 9.9 7 LEQNMTTPE -1.7 No -0.06 -0.41 WAAAAVGN 3 -0.23 -2.173 00 2.1 3 GEMATIYLR -1.5 No -0.20 -0.14 RSKKAMT 4 -0.03 4h0 187 0 4.5 2 PCANVTRNI -0.5 No -0.22 -0.45 MKKGGNL 1 0.08 3h0 171 0 6.2 9 RSLWGGYDI -1.0 No -0.25 -0.89 KEAIDRP 2 0.42 0.0 187 0 6.3 3 VPSVFVDCK -0.7 No 0.10 1.2 3 GDAVLAL 6 -0.30 -1.163 00 3.1 6 SSGVILNNQ -1.3 No -0.07 -0.17 MDDFATP 4 -0.16 -2.170 00 9.0 9 AITSTINNH -1.2 No -0.16 -0.33 FGSKLRT 9 -0.19 2.5 176 0 0.2 Petition 870260065020, dated 02 / 07 / 2026, page 36 / 53 32 / 45 2 ESDLRATTE -0.7 No -0.20 -0.32 0.36 -2.167 SVAALQS 1 00 8.0 0 TFWGDTGDV -1.1 No 0.12 0.62 -0.54 -2.169 IAITSTI 1 00 7.1 1 HEEELDLTT -1.0 No -0.17 -0.76 0.49 -4.171 ATPDAAT 1 50 3.9 9 CKHGYGGRS -0.6 No -0.24 -1.07 0.25 1.0 173 HLGDSDF 4 0 6.0 8 MTLIARERA -1.1 No -0.14 -0.18 0.06 0.0 175 PGAATWD 9 0 9.2 3 GPRITSAVA -1.5 No 0.00 1.03 -0.34 2h0 162 LVALRSL 0 0 4.1 9 PQFTGVLPE -1.0 No -0.13 -0.32 0.01 -1.173 SDLRATT 8 00 2.1 5 PHSMGIGGG -1.2 No 0.20 0.75 -0.87 0.5 165 FMATIYL 2 0 2.2 Petition 870260065020, dated 02 / 07 / 2026, page 37 / 53 33 / 45 1 LFTVASPGS -1.0 No 0.25 1.48 GAVLAGI 7 -0.76 0.0 145 0 9.9 4 PDAATALPQ -1.2 No 0.03 0.18 FTGVLPE 3 -0.23 -2.162 00 7.0 5 KEIRDDGGV -1.3 No -0.32 -1.09 ITEHDLK 2 1.01 -1.182 50 5.2 5 NKGPDYFYK -0.5 No -0.17 -1.15 GGIGEQM 5 0.14 0.0 180 0 4.2 5 GLSVAVPGE -1.4 No -0.08 0.22 LRGYRTL 4 -0.14 1.0 168 0 8.2 0 ADAAVATLC -0.8 No 0.21 1.72 MGWLP 7 -0.69 -1.156 00 2.1 6 DAVLALGGT -1.8 No 0.02 0.41 GGPRITS 2 -0.15 0.0 148 0 4.9 1 ELRGYRTLL -1.5 No -0.29 -0.40 RKLHATL 0 0.12 3.5 194 0 0.5 Petition 870260065020, dated 02 / 07 / 2026, page 38 / 53 34 / 45 7 PQETIAELE -0.9 No NKGHVFE 0 -0.17 -0.86 0.40 -2.184 50 1.2 5 PNFIEPRKR -0.9 No PLSSMVP 8 -0.25 -0.67 0.24 2h0 186 0 8.4 5 ASDSTMGNF -1.3 No SHWAVAT 3 -0.01 -0.06 -0.46 -0.168 50 2.0 1 QKHKDDLSE -1.2 No SLKKVFW 3 -0.34 -1.29 0.64 1.5 198 0 8.5 1 GNETLSRRG -1.3 No GLSVAVP 3 -0.17 -0.21 0.14 1.0 161 0 3.0 4 VNEGKGSTV -0.9 No NGIKKEA 8 -0.23 -0.86 0.62 1.0 163 0 1.0 6 PRLHHQLIP -1.3 No NRVIVEP 8 -0.18 -0.36 -0.11 2h0 191 0 8.5 3 LSESLKKVF -1.2 No WNETTND 7 -0.23 -0.91 0.26 -1.191 00 1.3 Petition 870260065020, dated 02 / 07 / 2026, page 39 / 53 35 / 45 3 STSGDNGDT -0.6 No -0.11 -0.51 VFTGSAS 7 0.11 -2.150 00 2.6 8 RKLHATLNW -1.0 No -0.37 -1.31 RDHFDDA 3 0.39 1.0 199 0 5.4 1 RSHLGDSDF -1.0 No -0.34 -1.25 EDVKELE 0 1.03 -3.187 50 6.1 9 DLLVQADLA -1.1 No 0.06 0.96 DTLEVIA 6 -0.11 -4.169 00 9.1 7 KGHVFEVNE -0.8 No -0.18 -0.77 GKGSTVN 2 0h30 0.5 170 0 2.1 0 ARYGFPVGP -, 97 No 0, 04 0.33 HLAAALQ -0.58 1.5 166 0 8.1 6 RFNSTTLQW -0.5 No -0.21 -0.47 HRFIEAC 8 -0.32 1.5 200 0 9.5 0 PHLAAALQK -1.1 No -0.23 -0.86 HKDDLSE 5 0.48 0.0 177 0 3.2 Petition 870260065020, dated 02 / 07 / 2026, pages 40 / 53 36 / 45 1 FDDAIRLAR -0.9 No -0.08 YGFPVGP 8 0.02 -0.09 0.0 179 0 4.2 6 SDFEDVKEL -1.0 No -0.28 EQNMTTP 9 -1.22 0.67 -4.188 00 3.2 4 PIVADLSNG -1.0 No 0.09 NTLETVA 4 0.76 -0.56 -1.163 00 2.0

[0047] The Vaxijen 2.0 algorithm was used to verify the antigenicity of non-toxic epitopes. The Rmescolotoxin SSD14 protein contains 46 probable antigens. All probable antigens were checked for allergenicity using the Allertop web server, which showed that the Rmescolotoxin SSD14 protein contains 25 likely non-allergenic epitopes. The results are shown in Table 4. Antigenicity is predicted at 0.887200 and 0.6449 for Antigenpro and Vaxijen 2.0, respectively. The Allertop web server classified the Rm-escolotoxin SSD14 protein as likely non-allergenic. Petition 870260065020, dated 02 / 07 / 2026, pp. 41 / 53 37 / 45 Table 4. Screening of the antigenicity and allergenicity of predicted epitopes of the SSD14 scolotoxin Rm protein using Vaxijen 2.0 and Allertop, respectively. Peptides Antigenicity Allergenicity KGTEDTGLTSTESSV D 0.7662 non-allergenic AVAT DAAP CANVT RN I 0.5805 non-allergenic TCKCPSKGTEDTGLT S 0.9530 allergenic GGTAHATFWGDTGDV I 1.4593 allergenic ESTTFDDPAHYGFLN N 0.8870 non-allergenic EAEWKEPIVADLSNG N 1.0184 non-allergenic GDTVFTGSASEAGW S 1.4801 allergenic HFGSKLRTSSGVILN N 0.8687 allergenic AKSEDGTSSAATCKC P 1.4341 non-allergenic HRFIEACKHGYGGRS H 1.1600 allergenic Petition 870260065020, dated 02 / 07 / 2026, pages 42 / 53 38 / 45 TTSTPLGDVEDARCG E 1.5997 allergenic GSASEAGWSTTLLD S 1.0746 allergenic GGVITEHDLKSYEAE W 1.1232 allergenic IGEQMAKEIRDDGGV I 0.7742 allergenic NETTNDTLAEGDLLV Q 0.8841 allergenic DVYPTRPNFIEPRKR P 0.6034 non-allergenic NIMKKGGNLADAAVA T 0.6653 allergenic EDARCGEPLADATAS D 1.5598 non-allergenic GHEEVEPTEAKSEDG T 0.8891 non-allergenic TTLLDSTPAGHEEVE P 1.0492 non-allergenic NRVIVEPHFPQETIA E 0.6179 allergenic Petition 870260065020, dated 02 / 07 / 2026, pages 43 / 53 39 / 45 ESSVDAASENATTEQ T 0.8056 non-allergenic AGCLHASYDSRRGGS V 1.4722 non-allergenic TTESVAALQSTSGDN G 1.1840 allergenic GFMATIYLRRSKKAM T 0.9605 non-allergenic VPSVFVDCKGDAVLA L 1.0888 allergenic SSGVILNNQMDDFAT P 0.6382 allergenic AITSTINNHFGSKLR T 0.5923 non-allergenic ESDLRATTESVAALQ S 0.9527 non-allergenic TFWGDTGDVIAITST I 0.8789 allergenic HEEELDLTTATPDAA T 0.7963 non-allergenic CKHGYGGRSHLGDSD 1.0350 allergenic Petition 870260065020, dated 02 / 07 / 2026, pp. 44 / 53 40 / 45 MTLIARERAPGAATW D 0.5670 non-allergenic PQFTGVLPESDLRAT T 1.5591 non-allergenic PHSMGIGGGFMATIY L 0.9039 non-allergenic LFTVASPGSGAVLAG I 0.7223 non-allergenic KEIRDDGGVITEHDL K 1.3156 non-allergenic GLSVAVPGELRGYRT L 0.7454 allergenic ADAAVAT L CMG WL P 0.7458 non-allergenic PQETIAELENKGHVF E 0.6554 allergenic PNFIEPRKRPLSSMV P 0.795 allergenic ASDSTMGNFSHWAVA T 0.8414 non-allergenic VNEGKGSTVNGIKKE A 0.7709 non-allergenic STSGDNGDTVFTGSA 1.8664 non-allergenic allergenic S Petition 870260065020, dated 02 / 07 / 2026, pages 45 / 53 41 / 45 RFNSTTLQWHRFIEA 0.6790 allergenic W FDDAIRLARYGFPVG 0.6735 non-allergenic

[0048] Candidate vaccines are chosen primarily for their ability to induce an adequate and protective immune response, making immunogenicity a key factor. The immunogenicity of non-allergenic linear epitopes was verified using the IEDB web server, which calculated the immunogenicity score for each epitope according to its immunogenicity. The results are shown in Table 5. Table 5. Screening of the immunogenicity of linear epitopes of the non-allergenic Rm-scolotoxin SSD14 protein using the IEDB web server. Peptides Length Score MTLIARERAPGAATWD 16 0.66413 KEIRDDGGVITEHDLK 16 0.55227 FDDAIRLARYGFPVGP 16 0.49187 ESTTFDDPAHYGFLNN 16 0.39412 HEEELDLTTATPDAAT 16 0.35071 Petition 870260065020, dated 02 / 07 / 2026, pp. 46 / 53 42 / 45 DVYPTRPNFIERPRKRP 16 0.286 TTLLDSTPAGHEEVEP 16 0.26043 AVAT DAAP CANVT RNI 16 0.2347 EAEWKEPIVADLSNGN 16 0.2094 STSGDNGDTVFTGSAS 16 0.19524 GHEEVEPTEAKSEDGT 16 0.19522 EDARCGEPLADATASD 16 0.19188 PQFTGVLPESDLRATT 16 0.18028 PHSMGIGGGFMATIYL 16 0, 159 ESSVDAASENATTEQT 16 0, 145 ESDLRATTESVAALQS 16 0.13463 ASDSTMGNFSHWAVAT 16 0.02886 LFTVASPGS GAVLAGI 16 -0.05408 KGTEDTGLTSTESSVD 16 -0.06428 ADAAVAT L CMG WL P 16 -0.13691 AITSTINNHFGSKLRT 16 -0.16379 AGCLHASYDSRRGGSV 16 -0.19862 AKSEDGTSSAATCKCP 16 -0.333 VNE GKGS TVNGIKKEA 16 -0.3637 GFMATIYLRRSKKAMT 16 -0.40751

[0049] Several tools were used to analyze the physiological properties of the Rmescolotoxin SSD14 protein epitopes using Toxinpred, Vaxigen, and AllerTop. Petition 870260065020, dated 02 / 07 / 2026, pp. 47 / 53 43 / 45 Toxinpred predicted 72 epitopes as non-toxic, among which Vaxigen classified 46 epitopes as having high antigenicity. Of these 4 epitopes, 25 were predicted as non-allergenic by AllerTop, as shown in Figure 6.

[0050] The structure of a protein influences its function and its ability to be targeted by antibodies. The I-Tasser was used to estimate that the tertiary structure of the Rmescolotoxin SSD14 protein consists of 52% (364 / 688) unorganized structure, 33.8% (233 / 688) alpha helix and 13.2% (91 / 688) beta sheet (Figure 7).

[0051] The predicted epitopes were analyzed in the tertiary structure of the Rm-scolotoxin SSD14 protein using Pymol software. In Figure 8, each epitope has a corresponding color and position in the tertiary structure.

[0052] An in silico analysis also showed a high similarity between sequences of the Rm-scolotoxin SSD14 protein and other ticks and mites (Figure 9). This data is consistent with the fact that mites and ticks belong to a single evolutionary lineage. This data is relevant because an immune response against the Rm-scolotoxin SSD14 protein has the potential to affect the biology of other tick and mite species.

[0053] The expression was performed on the selected strain of E. E. coli (BL 21 DE3 Star) (Figure 10) using conditions Petition 870260065020, dated 02 / 07 / 2026, pages 48 / 53 44 / 45 established in preliminary experiments. The soluble fraction of the expressed protein was purified by affinity chromatography on a nickel column, eluting with 100 mM imidazole (Figure 11). The predicted molecular mass of the Rmescolotoxin SSD14 protein was 73.5 kDa, which was confirmed by running the expressed and purified protein samples on SDS-PAGE and Western blot (Figure 12). After purification, the quantity was quantified and concentrated to the appropriate level for immunization, and subsequently used in immunization and vaccination assays in rabbits.

[0054] To verify the immune response induced by immunization of rabbits with the Rm-scolotoxin SSD14 protein, a dot-blot assay was performed by placing 1 μg of Rm-scolotoxin SSD14 protein as an antigen in the respective sera of the control and vaccinated groups. Anti-rabbit IgGs were used as a secondary antibody, while rabbit sera from the corresponding group acted as the primary antibody. Nitrocellulose membranes probed with sera from rabbits immunized with Rm-scolotoxin SSD14 protein showed a strong signal confirming humoral responses, while the membranes of the control sera showed weak signals. The assay results are shown in Figure 13).

[0055] New Zealand rabbits were either vaccinated with Rm-scolotoxin SSD14 or unvaccinated (control group) and Petition 870260065020, dated 02 / 07 / 2026, pages 49 / 53 45 / 45 infested with R. sanguineus to evaluate the protection induced by immunization using Rm-scolotoxin SSD14 as antigen. After vaccination with Rm-scolotoxin SSD14, egg laying by adult females decreased by 15.96% and egg fertility decreased by 37.45%, with an overall vaccine efficacy of 47.44% (Table 6). Table 6. Efficacy of the vaccine containing the Rmescolotoxin SSD14 protein for rabbits infested with R. sanguineus. Adult females Number of females3 Weight of females13 Egg laying3 Larval hatching31 Control 5.75 ± 0.96 181.79 ± 14.01 0.55 ± 0.01 0.17 ± 0.01 Vaccinated s 5.25 ± 1.71 152.52 ± 25.06 0.47 ± 0.07 0.10 ± 0.01 Reduction 8.69% 16.1% 15.96%* 37.45%* *p < 0.05 (Student's t-test).

[0056] Immunization of rabbits with the Rm-escolotoxin SSD14 protein induced the production of antibodies capable of interfering with biological parameters of ticks, allowing the characterization of the Rm-escolotoxin SSD14 protein as an antigen with vaccine potential for the development of a vaccine against mites.

Claims

1-Tick-derived Rm-escolotoxin SSD14 protein or peptides as a mite vaccine”, characterized in that the protein comprises the amino acid sequence SEQ ID NO:1, an oily or metallic adjuvant, in a physiologically acceptable vehicle, named Rm-Escolotoxin SSD14, from the cattle tick, Rhipicephalus microplus, 2-Tick scolotoxin SSD14 protein or derived peptides as a mite vaccine” according to claim 1, characterized in that the protein is obtained by chemical synthesis or produced in other organisms by means of recombinant DNA techniques, 3-Tick scolotoxin SSD14 protein or derived peptides as a mite vaccine” according to claim 1-2, characterized in that the protein is present in a concentration ranging from 0.01 to 5.0 mg / ml, 4-Use of the protein as defined in claims 1-3, characterized by being for the formulation of a vaccine.

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