Vaccine composition comprising a chimeric protein against rhipicephalus microplus

Abstract

The present invention relates to a vaccine composition comprising one or more tick proteins or antigens, preferably selected from the list consisting of the Bm86 protein, Subolesin protein, and / or P0 protein. The present invention also relates to uses of said vaccine compositions in order to generate an immune response against ticks, and kits comprising same.

Description

VACCINE COMPOSITION COMPRISING A CHIMERIC PROTEIN AGAINST RHIPICEPHALUS MICROPLUS

[0001] TECHNICAL FIELD

[0002] The present invention relates to the field of vaccinology. In particular, the present invention relates to the field of tick vaccines. BACKGROUND OF THE TECHNIQUE

[0003] Ticks, mites of the suborder Ixodida, are ectoparasites that cause the greatest losses in the agricultural sector worldwide, affecting 80% of the global livestock population.

[0004] The economic impact is directly related to the epidemiology of the infestation and can result in direct or indirect losses. Its direct effects on production include skin damage from bites, blood loss associated with high parasite loads, severe immune reactions due to toxin injection, persistent stress affecting animal behavior and welfare, and energy loss associated with constant movement in response to the infestation [1,2].

[0005] Indirect losses are related to the effects of hemoparasites and other tick-borne diseases [2]. Examples include the cost of treating clinical cases, expenses for parasite control, loss of profits, the use of genetically tick-resistant but less productive breeds, loss of meat or milk due to acaricide residues, and restrictions on trade in animals between regions or countries.

[0006] Some authors have estimated that the losses caused by infestation with Rhipicephalus microplus and its control are between 13.9 and 18.7 billion US dollars per year worldwide.

[0007] In Uruguay, the tick species Rhipicephalus microplus has the greatest economic impact, resulting in annual losses of US$32 to US$45 million due to acaricide expenses, weight loss, treatment costs, deaths from tick-borne diseases, and campaign costs. Additionally, there may be commercial losses due to the presence of insecticide residues in meat and milk.

[0008] Over time, tick control has relied on acaricides and herd management. However, these methods have not achieved the desired success, primarily due to the high proliferation of the parasite and the development of resistance to various acaricides. Acaricides are expensive, can leave residues in meat or dairy products, can cause environmental contamination, and frequently lead to resistance. As a result, alternative tick control methods are currently being explored.

[0009] A potentially more effective and sustainable alternative to the use of chemicals is the biotechnological approach through vaccination. Since the 1990s, two recombinant vaccines have been commercially available worldwide (Gavac® from Heber Biotec, Havana, Cuba, and Tick Gard® from Hoechst Animal Health, Australia) that use a protein called Bm86 as an immunogen. This protein is located on the luminal surface of the tick's midgut, and when blocked by antibodies generated through vaccination in cattle, it causes lysis of the tick's intestinal wall, interfering with blood digestion and egg production [2].

[0010] However, the efficacy of these vaccines has been moderate, and even poor, when administered in countries where they were not developed, leading to low levels of vaccine acceptance in the market. Tick Gard® is no longer commercially available, and Gavac® [2,4] has limited availability. In recent years, research efforts have focused on identifying and developing new antigens that provide more effective protection.

[0011] The failure of the efficacy of these vaccines, both in Uruguay and in other countries in the region, varies between 51% and 91%, and these results have been associated with amino acid divergences in the Bm86 protein [4].

[0012] These results have led to the production of the recombinant antigen specific to the Bm86 region and the search for new antigenic combinations to develop effective immunity.

[0013] Bm86 is not the only antigen described that functions in protection against tick infestation. The exposed subolesin antigen is located in the salivary glands and is associated with functions such as transcription, immunogenicity, gene expression, development, and physiology [3,5]. On the other hand, the P0 antigen is a protein located in the cell cytoplasm and is essential for the assembly of the 60S ribosomal subunit. Blocking this protein is associated with the inhibition of protein synthesis and subsequent cell death.

[0014] The poor performance of current vaccines has prompted researchers and technicians to seek alternatives for developing new vaccines. One more recent approach to improving protection in vaccines in general, including tick vaccines, is the use of chimeric proteins, which are non-natural proteins formed from sections of other proteins that fuse different epitopes [4]. Antigenic nucleotide sequences are fused using a ligand sequence and inserted into an expression plasmid. Although this technology appears promising, reports in the scientific literature on the use of chimeric vaccines are scarce, and no approaches have been identified that could lead to commercialization [6].

[0015] The use of targeted antigen combinations to combat tick infestations has been reported in the academic literature as a strategy to enhance the immune response [4,2]. Compared to other vectors and pathogens, ticks have very large genomes, ranging from 2.1 to 7.1 Gb. Additionally, these genomes are highly repetitive, which can be crucial for tick survival. Recent literature reviews indicate that the use of antigen cocktails has shown improved efficacy [3,4].However, the generation of vaccines comprising antigenic cocktails entails several drawbacks, such as 1) the different antigens will compete for the stimulation of B and T cells, with some antigens being more immunogenic or immunodominant than others, 2) higher production costs, 3) need for stronger adjuvants, 4) cross-reactivity between antigens, 5) evolution of pathogens, 6) longer manufacturing time, 7) impacts on storage and stability.

[0016] In the present invention, a multivalent recombinant vaccine has been generated, composed of several antigens: BM86s, subolesin, P0, and a de novo synthesized chimeric protein based on other proteins with potential antigenicity (subolesin-P0, SP0S), with amino acid sequences that best match the population of Uruguay.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Purified recombinant BM86s and SP0S produced in E. coli separated on 12% polyacrylamide gels. A) BM86s; B) SP0S. Target antigens present are indicated by black arrows. Abbreviation: MW, molecular weight marker (Bio-Rad, cat. no. 1610374S, see key annotated to the left for size in kDa), BSA, bovine serum albumin protein control (66.5 kDa).

[0019] Visualization of proteins with Coomassie staining. SDS-PAGE (12%) analysis of recombinant expression and partial purification of the BM86s antigen. Molecular weight marker (AccuRuler RGB PLUS 02102-250 molecular weight marker of previously stained protein): 1- BM86s inclusion bodies 1 / 10, 2- Inclusion bodies applied to the IDA-Ni column, 3- Granule after solubilization with lysis buffer (1 / 10), 4- Unretained fraction from IDA-Ni, 5- Previous elution of IDA-Ni (1 / 10), 6- Washing, 7- Elution with 250 mM imidazole, 8- Elution with 500 mM imidazole. The target antigen is highlighted with an arrow.

[0020] Protein visualization was achieved using Coomassie staining. Analysis by SDS-PAGE (12%) of recombinant expression and partial purification of recombinant SP0S. Molecular weight marker (molecular size marker of previously stained protein AccuRuler RGB PLUS 02102-250), 1- BM86s inclusion bodies 1 / 10, 2- Inclusion bodies applied to the IDA-Ni column, 3- Granule after solubilization with lysis buffer (1 / 10), 4- Unretained fraction from IDA-Ni, 5- Previous elution of IDA-Ni (1 / 10), 6- Washing, 7- Elution with 250 mM imidazole, 8- Elution with 500 mM imidazole. The target antigen is highlighted with a box.

[0021] Protein visualization was achieved using silver staining. SDS-PAGE (12%) analysis of A) SP0S and B) final purified recombinant BM86s. 1) MWM - molecular size marker of previously stained protein AccuRuler RGB PLUS 02102-250, 2 - Eluted pool from the IDA-Ni column using 250 mM and 500 mM imidazole. The following lanes correspond to purified and gel-filtered samples of the antigens.

[0022] Detection of A) recombinant BM86s and B) SP0S by Western blot analysis using anti-His mouse monoclonal antibodies (GenScript). The BM86s and SP0S antigens are indicated by black arrows. Abbreviation: MW, molecular weight marker (GenScript, cat. no. M00673, see key annotated to the left for size in kDa).

[0023] Evaluation of the immune response in serum from vaccinated cattle after 1 dose + 2 boosters using the indirect ELISA assay. Antibody titers in immunized cattle were expressed as OD405 nm values ​​as a function of serial dilutions of serum samples (decimal logarithm of the serum dilution factor). Values ​​were plotted using an asymmetric sigmoidal fit. A) Immune response corresponding to the subolesin antigen, B) Immune response corresponding to the BM86s antigen, C) Immune response corresponding to the SP0S antigen, and D) Immune response corresponding to the BM86s-SP0S antigen.

[0024] Statistical analysis of the response obtained from the sera group in the ELISA test for the antigen cocktail (BM86s and SP0S) in black, BM86s in gray and SP0S in white at a serum dilution of 1 / 500. The bar chart represents the mean ± of, n=2. t-test for independent data (**p<0.01). Two-tailed ANOVA, followed by Tukey's multiple comparisons test (p<0.05, **p<0.01, ***p<0.0001).

[0025] Longitudinal ELISA response obtained for cattle immunized with an antigen cocktail over time. The X-axis corresponds to serum dilutions. Black columns correspond to the first dose; gray columns correspond to the first dose and one booster; white columns correspond to the first dose and two boosters.

[0026] Individual efficacy of the cocktail vaccine in cattle (n=6, housed in boxes 7-12) after one dose and two boosters of BM86s and SP0S. The correlation with the serum response was assessed by indirect ELISA.

[0027] : Correlation of potency (percentage of efficacy) and antibody titers as measured by ELISA (Abs 405 nm) for individual cows (n=6).

[0028] The longitudinal immune response was quantified at 21, 42, and 60 days after the first dose in pooled sera from groups of animals vaccinated with formulations containing separate antigens or combined antigens and compared with sera from the DILAVE trial (60 days after the first dose).

[0029] Results of ELISA in adult cattle vaccinated with purified BM86 and SP0 (group X) compared to animals showing 70% protection against ticks (DILAVE 2022). Left: Longitudinal analysis of the immune response of sera pooled by treatment group at 4 time points: untreated, 21 days after the first dose, 42 days after the first dose, and 60 days after the first dose. Right: Comparison of individual levels of untreated versus treated after the third dose (day 60) in animals vaccinated with purified BM86 and SP0S compared to animals showing 70% protection against ticks (DILAVE 2022). X: group dosed with 200 μg of BM86 and 200 μg of SP0S; Z: group dosed with placebo. C+: serum from animals showing 70% protection against ticks from DILAVE 2022 (positive control); C-: untreated serum from DILAVE (negative control).

[0030] Tick ​​load distribution by treatment group in adult cattle vaccinated after 3 doses. The group vaccinated with the BM86 and SP0S antigens showed a 93% lower tick load compared to the placebo control group.

[0031] GENERAL DEFINITIONS

[0032] It should be noted that, as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Furthermore, unless otherwise indicated, the term “at least” preceding a series of items should be understood to refer to each item in the series. Those skilled in the art will recognize, or will be able to determine through routine experimentation alone, many equivalents to the specific embodiments of the invention described herein. It is intended that the present invention encompasses such equivalents.

[0033] The term “approximately” when referring to a given quantity indicates that a number may vary by ±20% around its stated value. Preferably “approximately” means ±15% around its value, more preferably “approximately” means ±10%, 8%, 6%, 5%, 4%, 3%, or 2% around its value, or even “approximately” means ±1% around its value, in that order of preference.

[0034] As used herein, the conjunction “and / or” between multiple enumerated items is understood to encompass both individual and combined options. For example, when two items are joined by “and / or,” a first option refers to the applicability of the first item without the second. A second option refers to the applicability of the second item without the first. A third option refers to the applicability of the first and second items together. Any one of these options is understood to be within the meaning and thus satisfy the requirement of the term “and / or” as used herein. It is also understood that the concurrent applicability of more than one of the options is within the meaning and thus satisfies the requirement of the term “and / or.”

[0035] Throughout this specification and the claims that follow, unless the context otherwise requires, the word “comprising,” and variations such as “comprising” and “comprising,” shall be understood to imply the inclusion of a specified integer or stage or group of integers or stages, but not the exclusion of any other integer or stage or group of integers or stages. When used herein, the term “comprising” may be substituted for the term “containing” or “including,” or sometimes, when used herein, for the term “having.” Any of the foregoing terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the present invention, may be substituted for the term “consisting of,” although the latter is less preferred.

[0036] When used herein, “consisting of” excludes any element, step, or component not specified in the claim. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel features of the claim.

[0037] The terms “sequence identity” or “percent identity” in the context of two or more nucleotide sequences, polypeptide sequences, or protein sequences refer to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of nucleotide or amino acid residues that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by BLAST alignment, or any other algorithm known to experts), or alternatively, by visual inspection. “Sequence identity” or “percent identity” can be determined by calculating the number of identical nucleotides or amino acids at the same positions in a nucleic acid, polypeptide, or protein.Calculating the percent identity involves determining the optimal alignment between two or more sequences. Alignment for determining the percent identity of amino acid sequences can be achieved in various ways within the scope of the technique, for example, using publicly available software such as BLAST, ALIGN, or Megalign (DNASTAR). Those skilled in the technique can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment along the entire length of the sequences being compared. Preferably, the algorithm used to calculate the percent identity of two or more sequences is a local alignment algorithm, such as the one used by BLAST.

[0038] An “antigen” or “immunogen” is a substance that induces a specific immune response in a host animal. The presence of antigens in the body normally triggers an immune response. Thus, antigens are “targeted” by antibodies. An “epitope” refers to the specific antigenic determinant of an antigen. An epitope can comprise three amino acids in a spatial conformation that is unique to that epitope. Generally, an epitope consists of at least five such amino acids, and more commonly, at least 8–10. Methods for determining the spatial conformation of these amino acids are known in the art.

[0039] A subunit vaccine is a vaccine that presents one or more antigens to the immune system without introducing whole or other pathogenic particles. In this document, a “protein subunit vaccine” refers to isolated antigens specific to a pathogen, preferably a tick. In this document, a “protein subunit vaccine” also refers to recombinant antigens specific to a pathogen, preferably a tick.

[0040] An “immunogenic fragment” of an antigen according to the present invention is a partial amino acid sequence of the antigen or a functional equivalent of such a fragment that also acts as an antigen, which is detected by and binds to an antigen-specific antibody or a B-cell receptor. An immunogenic fragment of an antigen is shorter than the complete antigen and preferably has a length of between approximately 10, 50 or 100 and approximately 1000 amino acids, more preferably a length of between approximately 10, 50 or 30 and approximately 500 amino acids, even more preferably a length of between approximately 50 and approximately 250 amino acids.

[0041] “Antibodies,” as used herein, are polyclonal and / or monoclonal antibodies or fragments thereof, including recombinant antibody fragments, as well as immunological binding equivalents thereof, that are capable of specifically binding to pathogenic proteins and / or fragments thereof. The term “antibody” is used to refer either to a homogeneous molecular entity or to a mixture such as a serum product consisting of a plurality of different molecular entities. For example, recombinant antibody fragments may be derived from a monoclonal antibody or may be isolated from libraries constructed from an immunized non-human animal.

[0042] The term “treatment,” as used herein, refers to medical care provided to a patient for an illness or injury. This term includes curing the illness, but also improving, mitigating, or reducing the symptoms of that illness. Therefore, the term “therapeutic treatment” or “treatment,” as used herein, refers to bringing a body from a pathological state or disease back to its normal, healthy state. The term “therapeutic treatment” or “treatment,” as used herein, also refers to mitigating, improving, or reducing the harmful effects of radiation on a subject, or to reducing in some way the symptoms associated with such effects. The term “prophylactic treatment,” as used herein, refers to preventing a pathological condition.

[0043] The terms “fusion protein” and “chimeric protein” are considered synonymous and are therefore used interchangeably herein. They refer to artificial proteins (i.e., not naturally occurring) created by joining two or more genes that originally encode for separate or identical proteins. They thus comprise two or more domains that are originally encoded by different genes in nature. The term “domain” refers to distinct functional and / or structural units in a protein, responsible for a particular function or interaction, contributing to the overall role of the protein. Preferably, in the context of the present invention, a domain is a region of a protein polypeptide chain comprising a tick protein or a fragment thereof. DESCRIPTION OF THE ACHIEVEMENTS

[0044] In a first aspect, the present invention provides a vaccine composition, also referred to herein as “the vaccine composition of the invention”, comprising at least one of the following antigens or proteins: the tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof,

[0045] -the tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof, and / or the tick P0 protein, an immunogenic fragment, or a functional equivalent thereof.

[0046] The vaccine composition of the invention can generate an immune response against ticks when the vaccine composition is inoculated into a mammal.

[0047] The term “tick Bm86 protein or antigen” refers hereto to a protein located on the luminal surface of the tick midgut that, when blocked, causes lysis of the tick's intestinal wall, interfering with blood digestion and egg production. Preferably, the tick Bm86 protein comprises, consists of, or essentially consists of SEQ ID NO: 5, or a sequence with at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5.Preferably, the tick Bm86 protein is encoded by a nucleotide sequence comprising, consisting of, or essentially consisting of SEQ ID NO: 6, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6. In the context of the present invention, "immunogenic fragment or functional equivalent of the tick Bm86 protein" refers to a protein or polypeptide capable of eliciting an immune response against the tick Bm86 protein.The said protein or polypeptide may be longer or shorter than the tick Bm86 protein as defined herein, or may contain part of the complete sequence of the tick Bm86 protein as defined herein, provided that it is capable of eliciting an immune response against said Bm86 protein.

[0048] The term “tick subolesin protein or antigen” refers hereto to a protein located in the salivary glands of ticks and associated with functions such as transcription, immunogenicity, gene expression, development, and physiology. Preferably, the tick subolesin protein comprises, consists of, or essentially consists of SEQ ID NO: 1, or a sequence with at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1.Preferably, the tick subolesin protein is encoded by a nucleotide sequence comprising, consisting of, or essentially consisting of SEQ ID NO: 7, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7. In the context of the present invention, "immunogenic fragment or functional equivalent of the tick subolesin protein" refers to a protein or polypeptide capable of eliciting an immune response against the tick subolesin protein.The said protein or polypeptide may be longer or shorter than the tick subolesin protein as defined herein, or may contain part of the complete sequence of the tick subolesin protein, provided that it is capable of eliciting an immune response against the tick subolesin protein.

[0049] The term “tick P0 protein or antigen” refers hereto to a protein located in the cell cytoplasm of tick cells and is essential for the assembly of the 60s ribosomal subunit. Preferably, the tick P0 protein comprises, consists of, or essentially consists of SEQ ID NO: 2, or a sequence with at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2. Preferably, the tick P0 protein is encoded by a nucleotide sequence comprising, consists of, or essentially consists of SEQ ID NO: 10, or a sequence with at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10.In the context of the present invention, "immunogenic fragment or functional equivalent of tick P0 protein" refers to a protein or polypeptide capable of eliciting an immune response against tick P0 protein. Such protein or polypeptide may be longer or shorter than tick P0 protein as defined herein, or may contain part of the complete sequence of tick P0 protein, provided that it is capable of eliciting an immune response against tick P0 protein.

[0050] In one embodiment, the vaccine composition comprises tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof, and tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof. In one embodiment, the vaccine composition comprises tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof, and tick P0 protein, an immunogenic fragment, or a functional equivalent thereof. In one embodiment, the vaccine composition comprises tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof, and tick P0 protein, an immunogenic fragment, or a functional equivalent thereof.

[0051] The antigens may be present in the vaccine composition of the invention as separate, i.e., independent, proteins or polypeptides. Alternatively, the antigens may be present in the vaccine composition of the invention as part of a single polypeptide. "Part of a single polypeptide" herein refers to antigens linked by peptide bonds, thus forming part of a fusion protein or a chimeric protein.

[0052] In a preferred embodiment, the vaccine composition comprises tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof, and tick P0 protein, an immunogenic fragment, or a functional equivalent thereof, wherein said subolesin and P0 proteins, fragments, or functional equivalents are part of a single polypeptide, forming a fusion protein. Preferably, the vaccine composition comprises a chimeric protein comprising or consisting of a first and a second domain, wherein the first domain comprises or consists of tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof; and the second domain comprises or consists of tick P0 protein, an immunogenic fragment, or a functional equivalent thereof.It is worth noting that the first domain can be located at either the N-terminal or C-terminal end of the chimeric protein, and the same applies to the second domain. However, it is preferred that the first domain be located in the N-terminal region (i.e., the 5' region, preferably the 5' end region) of the chimeric protein, and that the second domain be located in the 3' direction of the first domain. Preferably, the second domain is located in the 3' direction, i.e., towards the C-terminal region, of the chimeric protein.

[0053] Therefore, in a preferred embodiment, the vaccine composition comprises a chimeric protein antigen comprising: a first domain comprising or consisting of SEQ ID NO: 1, or an immunogenic fragment or functional equivalent thereof having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1, and a second domain comprising or consisting of SEQ ID NO: 2, or an immunogenic fragment or functional equivalent thereof having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2.

[0054] In a more preferred embodiment, the vaccine composition comprises a chimeric protein antigen comprising or consisting of a first and a second domain, wherein the first domain comprises or consists of SEQ ID NO: 1, and the second domain comprises or consists of SEQ ID NO: 2.

[0055] In one embodiment, the first and second domains of the chimeric protein antigen are part of a single polypeptide and are linked, preferably covalently, by a linker. A “linker,” as used herein, is a short peptide sequence located between the two domains of the chimeric protein. The linker peptides are positioned to provide flexibility of movement to the two domains. In the context of the present invention, the linker has at least one amino acid residue, preferably at least two consecutive amino acid residues, and optionally 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues. The linker peptide includes flexible linkers, rigid linkers, and linkers that are cleaved in vivo. Preferably, the binder comprises one or more amino acids glycine and / or serine.Most preferably, the linker comprises, consists of, or essentially consists of SEQ ID NO: 4, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4. Accordingly, a preferred embodiment of the invention is that the vaccine composition comprises a chimeric protein comprising or consisting of a first and a second domain, wherein the first domain comprises or consists of SEQ ID NO: 1, an immunogenic fragment, or a functional equivalent thereof; wherein the second domain comprises or consists of SEQ ID NO: 2, an immunogenic fragment, or a functional equivalent thereof; and wherein the first and second domains are joined by a linker, preferably a flexible linker, most preferably a linker comprising or consisting of SEQ ID NO: 4.

[0056] In one embodiment, the chimeric protein comprises an additional peptide, preferably at its N-terminus. Preferably, the additional peptide comprises, consists of, or essentially consists of SEQ ID NO: 9, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9.

[0057] In a more preferred embodiment, the vaccine composition comprises a chimeric protein antigen comprising or consisting of SEQ ID NO: 3, or an immunogenic fragment or functional equivalent thereof having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3. In a more preferred embodiment, the vaccine composition comprises a chimeric protein antigen comprising SEQ ID NO: 3, wherein an additional peptide is located at the N-terminal end of SEQ ID NO: 3, and wherein, preferably, said additional peptide comprises or consists of SEQ ID NO: 9.

[0058] In a more preferred embodiment, the vaccine composition comprises a chimeric protein antigen comprising or consisting of SEQ ID NO: 11, or an immunogenic fragment or functional equivalent thereof having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11. Preferably, the vaccine composition comprises a chimeric protein antigen encoded by a nucleotide sequence comprising, consisting of, or essentially consisting of SEQ ID NO: 8, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8.

[0059] In one embodiment, the vaccine composition comprises the three proteins described herein, namely, tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof; tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof; and tick P0 protein, an immunogenic fragment, or a functional equivalent thereof. In one embodiment, the vaccine composition of the invention comprises tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof; tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof; and tick P0 protein, an immunogenic fragment, or a functional equivalent thereof, wherein the subolesin protein and the P0 protein are in the form of a chimeric protein, i.e., comprised in a single polypeptide, and wherein the Bm86 protein is provided as a separate protein.Therefore, preferably, the vaccine composition of the invention comprises at least two proteins: a first protein comprising or consisting of the tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof, and a second protein that is a chimeric protein comprising or consisting of a first and a second domain, wherein the first domain comprises or consists of the tick subolesin protein, an immunogenic fragment, or a functional equivalent thereof; the second domain comprises or consists of the tick P0 protein, an immunogenic fragment, or a functional equivalent thereof; and wherein, preferably, the first and second domains are connected or linked by a linker, preferably a flexible polypeptide linker.

[0060] In one embodiment, the vaccine composition comprises at least two proteins: a first protein comprising or consisting of the tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof, wherein the tick Bm86 protein comprises, consists of, or essentially consists of SEQ ID NO: 5, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and a second protein that is a chimeric protein comprising or consisting of a first and a second domain, wherein: the first domain comprises or consists of the tick subolesin protein, an immunogenic fragment, or a functional equivalent of the same;wherein the tick subolesin protein comprises, consists of, or essentially consists of SEQ ID NO: 1, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, the second domain comprises or consists of the tick P0 protein, an immunogenic fragment, or a functional equivalent thereof;wherein the tick P0 protein comprises, consists of, or essentially consists of SEQ ID NO: 2, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and optionally, wherein the first and second domains are connected or linked by a linker, preferably a flexible polypeptide linker, preferably a linker comprising, consists of, or essentially consists of SEQ ID NO: 4, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4.;

[0061] In one embodiment, the vaccine composition comprises at least two proteins: a first protein comprising or consisting of tick Bm86 protein, wherein the tick Bm86 protein comprises or consists of SEQ ID NO: 5, and a second protein that is a chimeric protein comprising or consisting of a first and a second domain, wherein: the first domain comprises or consists of tick subolesin protein, wherein the tick subolesin protein comprises or consists of SEQ ID NO: 1, the second domain comprises or consists of tick P0 protein, wherein the tick P0 protein comprises or consists of SEQ ID NO: 2, and optionally, wherein the first and second domains are connected or linked by a linker, preferably a flexible polypeptide linker, preferably a linker comprising or consisting of SEQ ID NO: 4.

[0062] In one embodiment, the vaccine composition comprises two proteins: a first protein comprising or consisting of the tick Bm86 protein, an immunogenic fragment, or a functional equivalent thereof, wherein the tick Bm86 protein comprises or consists of SEQ ID NO: 5, or a sequence having at least 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and a second protein that is a chimeric protein comprising or consisting of SEQ ID NO: 3, or an immunogenic fragment or functional equivalent thereof having at least 80%, 82%, 84%, 85%, or 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3.

[0063] In one embodiment, the vaccine composition comprises two proteins: a first protein comprising the tick Bm86 protein, wherein the tick Bm86 protein comprises or consists of SEQ ID NO: 5, and a second protein that is a chimeric protein comprising SEQ ID NO: 3.

[0064] In one embodiment, the vaccine composition comprises two proteins: a first protein consisting of the tick Bm86 protein SEQ ID NO: 5, and a second protein that is a chimeric protein consisting of SEQ ID NO: 3.

[0065] In one embodiment, the proteins comprising the vaccine composition are isolated antigens, preferably recombinant antigens. Preferably, the vaccine composition of the invention is a protein subunit vaccine. Preferably, the vaccine composition does not comprise other tick-derived immunogens, such as attenuated ticks or killed ticks.

[0066] Preferably, the tick protein(s) comprising the vaccine composition of the invention can generate an immune response against ticks when inoculated into a mammal. An “immune response” against an antigen or composition is the development in a subject of an innate, humoral, and / or cellular immune response against an antigen present in the composition of interest. Preferably, such immune response is an enhanced immune response.The term “enhanced immune response,” when used for an immune response against tick antigens, such as an antibody response (e.g., neutralizing antigen-specific antibody response), a cytokine response, a CD8 T cell response (e.g., immunodominant CD8 T cell response), or a CD4 T cell response, refers to an increase in the immune response in a subject administered a vaccine comprising at least one tick antigen (treated mammal) relative to the corresponding immune response observed from a mammal administered a vaccine not comprising any tick antigens (control mammal).

[0067] Ideally, the immune response against ticks is a protective immune response. The term “protective immune response” means that the vaccinated individual can control an infection caused by the ticks against which the vaccination was administered. Typically, an individual who has developed a protective immune response develops only mild to moderate clinical symptoms or no symptoms at all due to tick infection, and / or the number of ticks present on the vaccinated individual is reduced compared to a control or unvaccinated individual. Typically, an individual with a protective immune response or protective immunity against a tick will not die as a result of infection with that tick.The term “infection” is understood in this document to mean, but is not limited to, the presence of tick replication on the subject's body when ticks feed on the host at different stages of development (nymphs and adults).

[0068] The expert in the technique can measure the presence of an immune response in various ways, whether it is an enhanced or protective immune response. The examples provided below show one way to measure or detect the presence of an immune response: by performing ELISA assays or by conducting tick provocation tests on vaccinated animals.However, in the context of the present invention, it is preferable that the presence of the immune response be measured by analyzing one or more of the following parameters: a reduction in the number of ticks on the inoculated mammal compared to a control mammal, a reduction in the reproductive capacity of the ticks on the inoculated mammal compared to a control mammal, a reduction in the teleogyne decomposition coefficient (CRT) on the inoculated mammal compared to a control mammal, a reduction in the oviposition coefficient (CRO) on the inoculated mammal compared to a control mammal, and / or a reduction in the hatching ratio (CRE) on the inoculated mammal compared to a control mammal.

[0069] A “control mammal” is a mammal of the same species as the treated mammal, but which has not been treated with the vaccine composition of the invention. In one embodiment, the increase / decrease in these parameters is preferably a statistically significant increase / decrease. “Statistically significant increase” or “statistically significant decrease” refers hereto to the determination by an analyst that the increase or decrease, respectively, in any of the above parameters cannot be explained by chance alone and is therefore influenced or caused by treatment with the composition of the invention. Statistical hypothesis testing is the method by which the expert makes this determination. This test provides a p-value, which is the probability of observing results as extreme as those in the data, assuming that the results are indeed due to chance alone.In this document, a p-value of 0.1 or lower (preferably 0.05, 0.01, 0.001, or lower) is considered statistically significant. For example, an increase or decrease in any of the parameters defined above is statistically significant when a statistical test is performed to compare it with the baseline level in a reference or untreated cell or subject, and the resulting p-value from that statistical test is 0.1 or lower, preferably 0.05, 0.01, 0.001, or lower.

[0070] Preferably, the presence of an immune response is measured by analyzing the efficacy of the vaccine composition. In this context, efficacy is measured as follows:

[0071] % effectiveness = 100 x [(1-CRT*CRO*CRE)].

[0072] In one embodiment, the vaccine composition has an efficacy of at least 50%, 60%, 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0073] In a preferred embodiment, the animal subject treated with the vaccine composition is a mammal. Preferably, the mammal is an ungulate or a domestic animal. “Ungulate” refers to hoofed animals such as cows, horses, sheep, and goats. “Domestic animal” includes, without limitation, canids, felids, and bovids, such as dogs, cats, guinea pigs, rabbits, horses, goats, wild boars, cattle, ferrets, porcine species such as pigs, piglets, sows or gilts, and sheep. Preferably, the mammal is a bovine. “Bovine” refers to cattle and sheep. Most preferably, the mammal is selected from the list consisting of cows, bulls, oxen, and calves.

[0074] Preferably, the tick against which the immune response is generated by administering the vaccine composition of the invention is of the family Ixodidae, preferably of the genus Rhipicephalus. Most preferably, the tick is a Uruguayan variant belonging to the genus Rhipicephalus microplus.

[0075] The vaccine composition of the invention may further comprise a pharmaceutically acceptable carrier. The carrier suitable for preparing the vaccine in liquid form may include water, an isotonic saline solution (i.e., with a salt concentration equal to that of physiological cell culture medium), oil, the culture medium in which the bacteria are grown, or mixtures thereof. The proteins comprising the vaccine composition, which are the principal active ingredient, may be incorporated into liposomes or other vehicles, such as nanoparticles, for use in a vaccine formulation, or may be conjugated with polysaccharides or other polymers.

[0076] Additionally, if desired, the vaccine composition may include other pharmaceutically acceptable auxiliary substances or excipients such as, for example, wetting agents, dispersing agents, emulsifying agents, buffering agents (e.g., phosphate buffer), stabilizing agents such as carbohydrates (e.g., glucose, sucrose, mannitol, sorbitol, starch or dextrans), or proteins (e.g., albumin, casein, bovine serum or skimmed milk).

[0077] In addition, the vaccine composition may include a pharmaceutically acceptable adjuvant. An “adjuvant” is a substance used to enhance the immune response. Pharmaceutically acceptable adjuvants include, but are not limited to: aluminum hydroxide, aluminum phosphate, aluminum oxide, muramyl-dipeptides, vitamin E, squalene, saponins (e.g., Quil A, QS-21), ginseng, zymosan, glucans, nonionic block polymers, monophosphoryl-lipid A, vegetable oils, Freund's complete or incomplete adjuvant, incomplete Freund's adjuvant, emulsions of the W / O, O / W, and W / O / W type, Ribi adjuvant system (Ribi Inc.), heat-labile enterotoxin of E. coli (recombinant or otherwise), cholera toxin, dimethylaminoethyl dextran, dextrans or analogues or mixtures thereof. Preferably, the adjuvant is Freund's incomplete adjuvant.

[0078] The vaccines of the invention are normally prepared as parenteral vaccines in the form of liquid solutions, emulsions, or suspensions. They may also be prepared in a solid form suitable for dissolving or suspending in a liquid vehicle prior to injection.

[0079] Specifically, the vaccine is prepared in liquid form or as a dry powder, lyophilized, freeze-dried, spray-dried, or foam-dried. Liquid vehicles that may be used to prepare the vaccine of the invention include, for example, water (particularly water for injection), saline solution with a physiological salt concentration, or the culture medium in which the bacteria are grown.

[0080] In a first alternative aspect, the present invention provides a vaccine composition comprising an antibody or antiserum that is reactive with the proteins defined above. This antibody or antiserum can be obtained by vaccinating an animal with the vaccine composition of the first aspect or any embodiment thereof. This antibody or antiserum can also be used as a vaccine or as a medicament.

[0081] In a second aspect, the present invention relates to the use of the vaccine of the first aspect (including the alternative first aspect), or any embodiment thereof, in therapy or as a medicament. In one embodiment, such use comprises generating an immune response against ticks in a mammal, preferably a bovine. Furthermore, the medicinal use may also involve the treatment and prevention of tick infestations, or diseases or symptoms associated with or caused by a tick infestation. For example, the use may be to reduce the number of ticks infesting the mammal, or to reduce the risk of such mammal becoming infested with ticks. Preferably, the vaccine of the invention is for use in a method of preventing and / or treating diseases in a mammal, preferably ungulates, preferably bovines.

[0082] Preferably, the use is in the treatment, prevention, enhancement, or reduction of a tick-borne infection or diseases that occur when a mammal becomes infected with ticks. “Prevention” means preventing the disease from occurring. “Enhancement” means an improvement in a patient's condition, or the activity of making an effort to correct, or at least make more acceptable, that condition. “Reduction” or “mitigation” means lessening the severity, seriousness, or pain of the disease. The second aspect also involves methods for the treatment or prevention of a tick-borne infection in a mammal, preferably an ungulate, most preferably a bovine.

[0083] In some embodiments, the vaccine composition defined herein is administered to a mammal, preferably an ungulate, most preferably a bovine, susceptible to or at risk of infection, to induce an immune response against the antigens present in said vaccine composition and thereby enhance the animal's own immune response capabilities. Such an amount is defined as an "immunogenically effective amount." Therefore, in one embodiment, the vaccine composition of the first aspect (including the alternative first aspect), or any embodiment thereof, is administered to a mammal, preferably an ungulate, most preferably a bovine, in an immunogenically effective amount to prevent or treat a tick infection. Preferably, the immunogenically effective amount is approximately 50-200 μg, preferably 100 μg, per antigen per dose.

[0084] In one embodiment, the vaccine composition is used to generate an immune response, preferably an enhanced and / or protective response against ticks. Preferably, this immune response is capable of producing one or more of: a reduction in the number of ticks on the inoculated mammal compared to a control mammal, a reduction in the reproductive capacity of ticks on the inoculated mammal compared to a control mammal, a reduction in the teleogyne decomposition coefficient (CRT) in the inoculated mammal compared to a control mammal, a reduction in the oviposition coefficient (CRO) in the inoculated mammal compared to a control mammal, and / or a reduction in the hatching ratio (CRE) in the inoculated mammal compared to a control mammal.

[0085] when administered to the mammal. Preferably, the vaccine composition, when used according to the second aspect, achieves efficacy against a tick infestation, as explained above.

[0086] The vaccine composition of the invention can be administered by various routes. These routes include, but are not limited to, oral, transdermal, transmucosal, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous administration. Specifically, it is administered subcutaneously. Depending on the desired duration and efficacy of treatment, the compositions according to the invention can be administered once or multiple times, even intermittently, for example, daily for several days, several weeks, or several months, and at different dosages. Specifically, the vaccine compositions of the invention are administered multiple times. More particularly, the vaccination schedule comprises two doses, with the second dose administered three weeks after the first.

[0087] A variety of vaccination regimens can be effective in immunizing mammals, preferably cattle and other bovine animals. For example, both young and adult ungulates, preferably cows and calves, can be vaccinated. The first immunization with an antigen is commonly called sensitization immunization. Preferably, the immune response, with a single dose, is capable of, or is characterized as being sufficient to, prevent a vaccinated subject from contracting an infection that leads to morbidity and / or mortality in that subject when infected with ticks.

[0088] As explained, the vaccine composition of the first aspect (including the alternative first aspect) may be used according to the second aspect as sensitization immunization. However, a second immunization, also called a booster immunization, may be provided after the first immunization to reinforce it. Booster immunizations are generally administered once or multiple times weeks or months after administration of the sensitization composition, for example, approximately 1 or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks, or one to two years. Preferably, the initial booster inoculation is administered 1–12 weeks or 2–12 weeks after sensitization, more preferably 1, 2, 4, or 8 weeks after sensitization.In a preferred embodiment, the initial booster inoculation is administered 4 or 8 weeks after sensitization. In further preferred embodiments, the initial booster is administered at least 2 weeks or at least 4 weeks after sensitization. In yet another preferred embodiment, the initial booster is administered 4–12 weeks or 4–8 weeks after sensitization. Therefore, the vaccine composition of the invention can be used for sensitization, as a booster, or both (sensitization and booster). Preferably, the vaccine composition is administered at least twice.

[0089] The proteins present in vaccine compositions may be administered together or separately in one or more immunizations. Preferably, the proteins or antigens are administered together, or at least simultaneously.

[0090] The second aspect also provides methods for preventing and / or treating diseases in a mammal, preferably bovine, preferably cattle, most preferably selected from the list consisting of cows, bulls, oxen, or calves. More particularly, for preventing and / or treating tick-borne infections or diseases, preferably caused by Rhipicephalus microplus. Therefore, the vaccine of the first aspect can be administered to a mammal in need in an immunologically effective quantity as a method for preventing and / or treating a tick-borne infection.

[0091] In a third aspect, the present invention also provides methods for detecting the presence or absence of ticks in a biological sample. This can be accomplished using antibodies or antisera against tick proteins, as defined in the first alternative aspect of the invention, since they will bind to and thus detect ticks and / or tick proteins present in a biological sample.

[0092] Furthermore, in a third aspect, the present invention provides methods for purifying and detecting antibodies against the proteins comprising the vaccine composition of the invention. This can be accomplished by using the proteins comprising the vaccine composition, as defined in the first aspect or any embodiment thereof, to bind to and thereby detect the antibodies against ticks. Examples of useful assays are described below:

[0093] For example, the pure proteins and antibodies disclosed in this case can be useful in immunoassays such as ELISA, immunoblotting, and agglutination assays. Western blot (immunoblotting) analysis can also be used to detect the presence of tick antibodies in the sample. This technique is a reliable method for confirming the presence of antibodies against a particular protein in the sample. Additionally, both antibodies and antigens can be used for affinity chromatography.

[0094] The third aspect also includes methods for purifying or isolating the antigens comprising the vaccine composition of the invention.

[0095] It is understood that each embodiment disclosed herein is applicable to each of the other disclosed embodiments. Therefore, all combinations of the various elements described herein are within the scope of the invention. It should also be understood that, unless clearly stated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are listed.

[0096] In a fourth aspect, the invention also provides a vaccination kit characterized in that it comprises a container containing the vaccine composition or vaccine of the invention. This vaccination kit may be further characterized in that it comprises an information leaflet or manual containing information on the administration of said vaccine composition.

[0097] In a fifth aspect, the present invention provides a diagnostic kit comprising the proteins included in the vaccine of the invention. Preferably, the kit comprises an isolated chimeric protein comprising: a first domain comprising SEQ ID NO: 1, or a functional equivalent thereof having at least 90% identity with SEQ ID NO: 1, and a second domain comprising SEQ ID NO: 2, or a functional equivalent thereof having at least 90% identity with SEQ ID NO: 2.

[0098] More preferably, the kit comprises a chimeric protein, wherein the chimeric protein comprises or consists of a first domain consisting of SEQ ID NO: 1 and a second domain consisting of SEQ ID NO: 2. More preferably, the kit comprises a chimeric protein, wherein the chimeric protein comprises or consists of SEQ ID NO: 3. More preferably, the kit comprises a chimeric protein, wherein the chimeric protein comprises or consists of SEQ ID NO: 3, and an additional protein, comprising or consisting of SEQ ID NO: 5.

[0099] This diagnostic kit may be useful for identifying antibodies against tick proteins defined in SEQ ID NO: 1, 2 or 5.

[0100] The present invention also covers the following points.

[0101] A vaccine composition comprising an isolated chimeric protein comprising: a first domain comprising SEQ ID NO: 1, or a functional equivalent thereof having at least 90% identity with SEQ ID NO: 1, a second domain comprising SEQ ID NO: 2, or a functional equivalent thereof having at least 90% identity with SEQ ID NO: 2.

[0102] The vaccine composition according to point 1, where the first domain consists of SEQ ID NO: 1 and the second domain consists of SEQ ID NO: 2.

[0103] The vaccine composition according to any one of points 1 or 2, wherein the first and second domains are connected by a peptide linker, preferably a linker comprising or consisting of SEQ ID NO: 4.

[0104] The vaccine composition according to any one of points 1 to 3, wherein the chimeric protein consists of SEQ ID NO 3.

[0105] The vaccine composition according to any one of points 1 to 4, further comprising a protein comprising SEQ ID NO: 5, or a functional equivalent thereof having at least 90% identity with SEQ ID NO: 5.

[0106] The vaccine composition according to any one of points 1 to 5, comprising the proteins consisting of SEQ ID NO: 3 and 5.

[0107] The vaccine composition according to any one of points 1 to 6, wherein the immune response against ticks results in one or more of: a reduction in the number of ticks on the inoculated mammal compared to a control mammal, a reduction in the reproductive capacity of ticks on the inoculated mammal compared to a control mammal, a reduction in the teleogyne decomposition coefficient (CRT) on the inoculated mammal compared to a control mammal, a reduction in the oviposition coefficient (CRO) on the inoculated mammal compared to a control mammal, and / or a reduction in the hatching ratio (CRE) on the inoculated mammal compared to a control mammal.

[0108] The vaccine composition according to any one of points 1 to 7, where the tick is Rhipicephalus microplus.

[0109] The vaccine composition according to any one of points 1 to 8, wherein the mammal is a bovine, preferably cattle, most preferably selected from the list consisting of cows, bulls, oxen, or calves.

[0110] The vaccine composition according to any one of points 1 to 9, for use in a method of generating an immune response against ticks in a mammal.

[0111] The vaccine composition for use according to point 10, where the tick is Rhipicephalus microplus.

[0112] The vaccine composition for use according to any one of points 10 or 11, wherein the mammal is a bovine, preferably cattle, most preferably selected from the list consisting of cows, bulls, oxen, or calves.

[0113] The vaccine composition for use according to any one of points 10 to 12, where the vaccine is administered as sensitization and / or as a booster.

[0114] A vaccination kit comprising the vaccine composition according to any one of points 1 to 9.

[0115] An antibody or antiserum that is reactive with the immunogenic proteins of any one of points 1 to 9.

[0116] LIST OF SEQUENCES

[0117] SEQ ID NO: 1: subolesin (amino acid sequence)

[0118] MACATLKRTHDWDPLHSPSGRSPKRRRCMPLSPPPTRAHQIDPSPFGDVPP KLTSEEIAANIREEMRRLQRRKQLCFQGADPESQHTSGLSSPVHRDQPLFTFR QVGLICERMMKERESKIREEYDHVLSTKLAEQYDTFVKFTYDQIQKRFEGATP SYLS

[0119] SEQ ID NO: 2: p0 (amino acid sequence)

[0120] AAGGGAAAAKPEESKKEEAK

[0121] SEQ ID NO: 3: subolesin-p0, also called SP0S (amino acid sequence)

[0122] MACATLKRTHDWDPLHSPSGRSPKRRRCMPLSPPPTRAHQIDPSPFGDVPP KLTSEEIAANIREEMRRLQRRKQLCFQGADPESQHTSGLSSPVHRDQPLFTFR QVGLICERMMKERESKIREEYDHVLSTKLAEQYDTFVKFTYDQIQKRFEGATP SYLSGGGGSGGGGSGGGGSAAGGGAAAAKPEESKKEEAK

[0123] SEQ ID NO: 4: ligador (secuencia de aminoácidos)

[0124] GGGGSGGGGSGGGGS

[0125] SEQ ID NO: 5: BM86s (secuencia de aminoácidos)

[0126] ESNPSKGSCVCEASDDLTLQCKIKNDFATDCRNRGGTAKLRTDGFIGATCDC GEWGAMNKTTRNCVPTTCLRPDLTCKDLCEKNLLQRDSRCCQGWNTANCS AAPPADSYCSPGSPKGPDGQCKNACRTKEAGFVCKHGCRSTDKAYECTCPS GSTVAEDGITCKSISYTVSCTVEQKQTCRPTEDCRVQKGTVLCECPWNQHLV GDTCISDCVDKKCHEEFMDCGVYMNRQSCYCPWKSRKPGPNVNINECLLNE YYYTVSFT

[0127] SEQ ID NO: 6: BM86s (secuencia de ácido nucleico)

[0128] TGAAAGTAACCCGAGCAAGGGTAGCTGCGTCTGCGAAGCATCCGGACGATC TAACGCTACAATGCAAAATTAAATGACTTCGCAACTGACTGCCGAAACC GAGGTGGCACTGCTAAGTTGCGCACGGATGGGTTTATTGGCGCAACGTGT TACCACGTGTCTTCGTCCCGACTTGACCTGCAAAGACCTCTGCGAGAAAAA CCTGCTTCAAAGGGATTCTCGTTGTTGTCAGGGGTGGAACACAGCAAACT GTTCAGCCGCTCCTCCAGCTGACTCCTATTGCTCTCCTGGGAGCCCCAAA GGACCGGACGGACAGTGTTAAAATGAGAGGAGGATTGATTGATTGATTGAGAG TGTCTGCAAGCATGGATGCAGGTCCCACCGACAAGGCGTACGAGTGCACGT GCCCGAGTGGCTCTACCGTCGCCGAAGATGGCATTACCTGCAAAAGTATT TCGTACACAGTCAGCTGCACTGTTGAGCAAAAACAGACCTGCCGCCCAAC CGAAGACTGTCGTGTGTGCAGGAAGGAGGAGGGGGGGGGGACT ATCAACATCTAGTGGGGGACACGTGCATAAGTGATTGCGTCGACAAGAAA TGTCACGAAGAATTTGGACTGTGGCGTATATGAATCGACAAAGCTGC TATTGTCCATGGAAATCAAGGAAGCCGGGCCCAAATGTCAACATCAATGAA TGCCTACTGAATGAGTTACTACTACCATCATTCAATGAA

[0129] SEQ ID NO: 7: subolesin (nucleic acid sequence)

[0130] ATGGCTTGCGCAACATTAAAGCGGACACATGACTGGGACCCCTTGCACAG TCCGAGTGGCAGATCGCCGAAGCGACGCCGATGTATGCCTCTGTCCCCG CCTCCTACAAGGGCGCACCAGATCGATCCCTCGCCCTTCGGAGACGTGCC ACCAAAGTTGACTTCAGAGGAGATAGCAGCCAACATCCGTGAGGAGATGC GACGGCTACAACGGCGCAAGCAGCTCTGTTTCCAGGGCGCTGACCCTGA ATCCCAGCATACCAGTGGTCTCTCGTCACCAGTGCATCGAGACCAGCCCC TGTTCACGTTCCGCCAGGTTGGGCTCATTTGCGAACGAATGATGAAGGAG CGAGAGAGCAAGATACGGGAGGAGTATGACCATGTGCTATCTACCAAACT CGCAGAACAGTACGACACATTTGTTAAATTTACCTACGACCAAATTCAGAA GCGGTTTGAAGGTGCCACGCCCAGCTATTTGTCGTAA

[0131] SEQ ID NO: 8: SP0S (secuencia de ácido nucleico)

[0132] ATGGCTTGCGCAACATTAAAGCGGACACATGACTGGGACCCCTTGCACAG TCCGAGTGGCAGATCGCCGAAGCGACGCCGATGCCTCTGTCCCCG CCTCCTACAAGGGCGCACCAGATCGATCCCTCGCCCTTCGGAGACGTGCC ACCAAAGTTGACTTCAGAGGAGATACCAGGGACCAGGATGGA GACGGCTACAACGGCGCAAGCAGCTGTTTCCAGGGCGCTGACCCTGA ATCCCAGCATACCAGTGGTCTCTCGTCACCAGTGCATCGAGACCAGCCCC TGTTCACGTTCCGCCAGGTTGGGCTCATTTGCGAACGAATGATGAAGGAG CGAGAGAGCAAGATACGGGAGGAGTGAGTGACCATCAGCCATCA CGCAGAACAGTACGACACATTTGTTAAATTTACCTACGACCAAATTCAGAA GCGGTTTGAAGGTGCCACGCCCAGCTATTTGTCGggcggcggcggcggcggc ggcggcagcggcggcggcggcgcggcggcggcggcgcggcggcggcgcggcggcggcgcggcggcggcgaaaaaaaaaaaaaaaaa

[0133] SEQ ID NO: 9: N-terminal peptide

[0134] MGSSHHHHHHSSGLVPRGSH

[0135] SEQ ID NO: 10: nucleic acid sequence of P0

[0136] gcggcgggcggcgcggcggcgaaccggaagaaagcaaaaagaagaagcgaaataa

[0137] SEQ ID NO: 11: subolesin-p0, also referred to as SP0S, with N-terminal peptide (amino acid sequence)

[0138] MGSSHHHHHHSSGLVPRGSHMACATLKRTHDWDPLHSPSGRSPKRRRCMP LSPPPTRAHQIDPSPFGDVPPKLTSEEIAANIREEMRRLQRRKQLCFQGADPE SQHTSGLSSPVHRDQPLFTFRQVGLICERMMKERESKIREEYDHVLSTKLAEQ YDTFVKFTYDQIQKRFEGATPSILSGGGGSGGGGSGGGGSAAGGGAAAAKP EESKKEEAK

[0139] The invention is then described by means of the following examples, which should be considered as merely illustrative and in no case limiting of the scope of the present invention. EXAMPLES

[0140] Materials and methods

[0141] A sampling of native ticks in Uruguay was conducted, including specimens from the following departments: Tacuarembó, Treinta y Tres, Flores, Lavalleja, and Cerro Largo. In all studies, the Mozo strain in the telogen phase was used as the reference strain. This reference strain was provided by the Parasitology Department of the Veterinary Laboratories Division (DILAVE) and has been the reference strain for Uruguay since 1973.

[0142] Collected ticks were washed with 70% ethanol and rinsed with 1X PBS before tissue dissection under a microscope. The tissues obtained were the salivary gland, intestine, and ovary. Those used for RNA extraction were promptly removed and stored at -80°C until further use. RNA extraction was performed using TRIzol reagent and the Quick-RNA fungal / bacterial micropreparation kit. The integrity of the extracted RNA was assessed by electrophoresis on a 1% agarose gel.

[0143] Reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA extracted from the intestine (Bm86 expression) and salivary glands (subolesin expression) using the RevertAid reverse transcriptase kit (200 U / μl). This kit contains a DNA polymerase that synthesizes complementary DNA (cDNA) from single-stranded RNA. This technology allowed the generation of mRNA from different antigens along with their respective cDNA. In all cases, the actin gene (a constitutive gene) was used as a control.

[0144] Using the generated cDNA, a polymerase chain reaction (PCR) was performed with primers previously designed to amplify the corresponding antigens. Amplifications were carried out in the SimpliAmp thermocycler. The cycling conditions consisted of 2 minutes at 94 °C, followed by 35 cycles of: 30 seconds at 94 °C, 30 seconds at the hybridization temperature of 53 °C, and 1 minute at 72 °C. Finally, there was a 7-minute extension step at 72 °C.

[0145] The PCR products were analyzed by agarose gel electrophoresis (1.5%) using a molecular weight marker (Gene Ruler DNA molecular size marker mixture). The resulting amplicons were sent to Macrogen (Seoul, Korea) for sequencing. Once the sequences of the fragments corresponding to the Bm86 and subolesin antigens were obtained, they were edited using SnapGene software. This procedure involved removing unreliable base sequence ends. Bioinformatics analysis confirmed the presence of a degree of polymorphism in Bm86, consistent with expected results. Furthermore, the absence of polymorphism in subolesin was confirmed.

[0146] The gene sequences were used to construct three plasmids: a) one for the expression of what is referred to herein as BM86s, with a BM86-based sequence containing mutations corresponding to the polymorphism found in the Uruguayan tick species, b) another for the recombinant expression of S0, and c) a final one for a chimeric protein composed of a DNA fragment from the gene encoding the subolesin protein and the DNA encoding a 20-amino-acid peptide from the P0 protein. The two antigens are held together by a flexible linker with a (GGGGGS)3 sequence. This protein is called SP0S.

[0147] Synthesis of recombinant plasmids

[0148] For the recombinant expression of BM86, subolesin, and SP0S, the pet28a(+) expression plasmid was used. The three recombinant plasmids were generated using GenScript. The chosen expression vector is characterized by having a promoter region recognized by the T7 RNA polymerase regulated by the T7 promoter. Thus, after the addition of IPTG, the T7 RNA polymerase is expressed from the bacterial chromosome and induces protein expression from the vector. The vector includes a sequence encoding a six-histidine tag, which fuses with the N-terminal region of the recombinant protein after translation, facilitating its localization by Western blot and purification by affinity chromatography using Ni resins. Additionally, it contains the gene that confers resistance to kanamycin.

[0149] Laboratory and pilot scale expression

[0150] The expression of recombinant antigens in E. coli bacteria (BL21 DE3) was carried out both on laboratory scale (flasks) and on pilot scale (bioreactor).

[0151] Laboratory-scale antigen production was carried out in 1-liter flasks containing 250 ml of Luria-Bertani (LB) medium. The expression conditions were as follows: growth at 37 °C and shaking at 200 rpm. Bacterial growth was monitored by spectrophotometric measurements at an optical density (OD) of 600 nm, and culture induction was performed by adding the inducing agent IPTG (final concentration of 1 mM) at an OD of 0.6–0.8. The induction time was 3 hours at 37 °C.

[0152] The fermentation scale-up was performed in a 5 L Sartorius biostatic bioreactor. An alternative culture medium, ZYM-5052, was used, which allowed for growth and autoinduction. The expression conditions for the three antigens were as follows: agitation at 250 rpm, temperature at 37 °C, and pH at 6.8. Bacterial growth curves were constructed by measuring optical density (OD) at 1-hour intervals to determine the optimal induction time. In all cases, the culture was harvested by centrifugation at 5000 rpm.

[0153] Antigen purification

[0154] To obtain the present target antigens in purified form, the generated bacterial sediment was weighed and subjected to lysis using the following lysis buffer: phosphate-buffered saline (PBS) containing 1 mg / ml lysozyme and 1 mM PMSF (10 ml / gram of sediment).

[0155] The mixture was incubated for 30 minutes at 4 °C, followed by sonication of the sample on ice for 10 minutes (amplitude capacity of 210 μm, vibration amplitude of 20%, 1 min on and 1 min off). The sample was then centrifuged at 14,000 rpm for 1 hour at 4 °C.

[0156] The sediment containing inclusion bodies was prepared to verify the correct expression of the antigens using 12% SDS-PAGE gels.

[0157] Purification was performed as follows. 1.4 g of inclusion bodies containing the antigen were weighed and resuspended in 10 mL of lysis buffer. The mixture was incubated on a shaker at 37 °C for 1 hour with minimal shaking (50 rpm). Subsequently, the mixture was centrifuged at 15,000 rpm for 30 minutes. The insoluble fraction was solubilized in lysis buffer and loaded onto a column containing 1 g of iminodiacetic acid-activated agarose loaded with 100 mM NiSO₄ (IDA-Ni), and equilibrated with the same lysis buffer without imidazole.

[0158] The column loaded with 10 mL of sample was shaken on a roller shaker for 1 hour at 25 °C. The percolate was then collected, and 10 mL of wash buffer (lysis buffer containing 10 mM imidazole) was added and collected. Elution buffer 1 (250 mM imidazole) was added, the column was shaken for 30 minutes at 25 °C, and the sample was collected. Elution buffer 2 (500 mM imidazole) was added, the column was shaken for 30 minutes at 25 °C, and the sample was collected.

[0159] Samples eluted with 250 mM and 500 mM imidazole were pooled and used for gel filtration via FPLC using a 200 pg (120 ml) HiLoad Superdex column. A flow rate of 1 ml / min and detection at 280 nm were used. The column was equilibrated with two volumes of PBS. Two ml of the applied sample, previously filtered through 0.45 µm filters and then 0.22 µm filters, were injected into the column. Elution was performed in PBS, collecting the first 40 ml (dead volume) and then the subsequent 80 ml in 1 ml increments.

[0160] An alternative purification method that obtains antigens from inclusion bodies followed by nickel columns produced BM86s and SP0S with a purity of over 84% according to SDS-PAGE analyses (Figure 1).

[0161] Evaluation of the immune response

[0162] In the initial stage, mice were used as a model organism to test the immune response. The immunization protocol for the next objective involved using 6 mice for each antigen type. Two 100 μg doses of antigen, along with Freund's incomplete adjuvant (IFA), were injected 15 days apart.

[0163] The humoral response was analyzed using Western blot. For this analysis, antigens were separated using 12% SDS-PAGE and transferred to a nitrocellulose membrane. The resulting membranes were cut into strips and blocked overnight with PBS-TWEEN and 5% skim milk. Subsequently, they were incubated for 1 hour at 25°C with a 1 / 500 dilution of the obtained mouse sera (antigens and control). They were then washed with 0.5% PBS-TWEEN and incubated with the alkaline phosphatase-conjugated anti-IgG secondary antibody. Visualization was performed using NBT BCIP.

[0164] Subsequently, direct experimentation was conducted in cattle. The immunization protocol for this objective involved the use of 15 animals (3 animals per formulation) to evaluate the immunogenicity of the antigens. A single dose and two boosters were administered over a 60-day period, with 2 ml of 100 μg of antigen along with Freund's incomplete adjuvant (IFA). The formulations used were: 1) BM86s, 2) subolesin, 3) SP0S, 4) BM86s and SP0S, and 5) control (PBS with adjuvant).

[0165] The humoral response was evaluated using an indirect ELISA. For this analysis, the plate was sensitized with 100 μl of the antigens to be immunized and incubated at 4 °C for 16 hours. A blocking solution of 0.5% PBS-TWEEN with 5% milk was added to each well, and 300 μl were added per well for 1 hour at 37 °C.

[0166] Subsequently, the bovine sera obtained were incubated at 37°C for 1 hour, in a dilution range of 1 / 500 to 1 / 32000. Then, an alkaline phosphatase-conjugated anti-IgG secondary antibody was added at a dilution of 1 / 4000 and incubated for 45 minutes at 37°C.

[0167] The indirect ELISA was developed using 10 mg of p-nitrophenyl phosphate (PNPP) in 10 ml of 100 mM Tris buffer, 5 mM MgCl2, and 100 mM NaCl at pH 9.5.

[0168] Three washes with 0.5% PBS-TWEEN were performed between each ELISA stage.

[0169] A second assay was conducted to determine the percentage of biological protection in vaccinated animals, correlating it with the antibody titer generated by the immune response. The antibody titer was determined using the ELISA technique mentioned above.

[0170] The immunization phase took place at a DILAVE facility located in the department of Lavalleja, km 8 of Route 90, Aguas Blancas area (Uruguay). The immunization protocol was the same as previously mentioned, inoculating a total of 12 cattle animals, 6 with BM86s and SP0S (antigen cocktail), and 6 as a control (PBS and adjuvant).

[0171] Subsequently, 10 cattle were selected for the barn trial. The efficacy evaluation stage was carried out in barns at the “Miguel C. Rubino” facility of DILAVE, km 17 of Route 8. The experimental cattle came from the DILAVE field, free of ectoparasites and infectious diseases, without prior treatment with ectoparasiticidal agents.

[0172] The animals were subjected to provocation tests twice a week for three weeks with 100 mg of R. microplus larvae. The methodology was based on the technical procedure PR-PAR-02 “Determination of efficacy and residual activity of acaricidal products”.

[0173] In the barn trial, collected ticks were counted, weighed, and 20 were randomly selected from each animal per day. They were incubated in an oven at 27°C and 90% relative humidity to study their reproductive behavior, recording the weight of the incubated ticks, the eggs, and the hatching percentage.

[0174] To determine the percentage of biological protection, three parameters were analyzed:

[0175] Swelling weight reduction coefficient (SWR)

[0176] CRT = Average weight of swollen ticks in the vaccinated group / Average weight of swollen ticks in the control group

[0177] Coefficient of reduction in oviposition (CRO)

[0178] CRO = Average weight of eggs laid by swollen ticks in the vaccinated group / Average weight of eggs laid by swollen ticks in the control group

[0179] Hatching reduction coefficient (CRC)

[0180] CRE = Percentage of egg hatching in the vaccinated group / Percentage of egg hatching in the control group

[0181] The coefficients were calculated by comparing the animals in the treated and control groups. The approval criterion was based on the following indicators: the overall efficacy in the parasite's life cycle must be equal to or greater than 50%.

[0182] A third trial was conducted to determine the percentage of biological protection in fields infested with endemic ticks in endemic areas (Maldonado, Uruguay). Animals were vaccinated with 200 μg of each column-purified nickel antigen (BM86S and SP0S), correlating the dose with the antibody titer generated by the immune response. Two groups of 10 female cattle each (36–60 months of age), matched for age, weight, and health status, were used. The antibody titer was determined using a modified ELISA technique (Abs 450 nm). Efficacy was validated by quantifying the number of adult ticks per animal in the trial three weeks after the third dose, comparing the groups vaccinated with either antigen or placebo.

[0183] Results and discussion

[0184] The information obtained from the antigenic sequences allowed the present inventors to conclude the following: the degree of polymorphism present in Bm86, which is consistent with the expected results, and the absence of polymorphism in subolesin.

[0185] The generation of the recombinant antigen BM86s, whose sequence matches that of indigenous populations in this country, increases the levels of immunoprotection, thus reducing failures in the percentages of protection when foreign vaccines are used.

[0186] Expression of recombinant antigens

[0187] Bacterial fermentations were carried out at both laboratory (flask) and pilot (bioreactor) scales for the expression of recombinant antigens. In all cases, overexpression of the target antigens was observed compared to other proteins.

[0188] The antigens used in the formulation were expressed in the insoluble fraction as inclusion bodies. Presenting antigens in this form allows for a greater degree of protection, avoiding costly and complex procedures such as purification, solubilization, and refolding.

[0189] One innovative aspect is the generation of a chimeric protein as an immunization antigen. The following genetic construct involves a DNA fragment from the gene encoding the subolesin protein and the DNA encoding a 20-amino-acid peptide from the P0 protein. The two antigens are held together by a flexible linker.

[0190] Evaluation of the immune response

[0191] Analysis of sera from subsequently immunized mice revealed bands corresponding to the expected molecular weights of the recombinant antigens. No recognition was detected in the control samples (PBS with Freund's incomplete adjuvant). Therefore, it is concluded that immunization with different formulations elicits a robust immune response, as assessed by Western blot, against the target antigens (Figure 5). The humoral response in cattle was evaluated using the ELISA technique. Figure 6 shows the results obtained for the ELISA tests for each antigen and for the BM86s and SP0S mixture (antigen cocktail). Good immune response values ​​were obtained with all formulations tested. These results allowed the inventors to conclude that immunization with different formulations triggers an immune response as a protective mechanism.The response to the antigen cocktail (BM86s and SP0S) was shown to be significantly higher than that of the isolated recombinant proteins under the studied conditions (Figure 7). The response increased with the two consecutive boosters, as shown in Figure 8.

[0192] The efficacy of the antigenic cocktail in cows was evaluated after the immunization protocol, yielding an average of 70% protection (Figure 9). These results correlate with the immune response measured by ELISA (Figure 10A), in which cows with an increased antibody titer (Abs 450 nm) showed better protection. Furthermore, the average antibody titer, as assessed by pooling the sera from vaccinated cows, was significantly higher than the ELISA response of control cows vaccinated with PBS plus adjuvant.

[0193] Additional purification steps result in stronger immune responses compared to the initial method used to immunize cattle in the DILAVE trial. Figure 11 shows ELISA tests quantifying the immune response of cattle vaccinated with 3 doses of nickel column-purified antigens (100 μg per dose of BM86s, 100 μg per dose of SP0S) and for the mixture of BM86s and SP0S (100 μg each), compared to animals vaccinated with the original purification method (DILAVE).

[0194] Furthermore, when cattle living in tick-infested fields in endemic areas (Maldonado, Uruguay) were vaccinated with 3 doses of nickel column-purified antigens (200 μg of each antigen combined), they developed a robust immune response, compared to the DILAVE trial group vaccinated with a cocktail of 100 μg of each antigen (Figure 12). Additionally, the group vaccinated with the antigen cocktail had a 93% lower tick burden compared to the placebo group (Figure 13).

[0195] Conclusions and innovations

[0196] The combination of BM86s, subolesin, and SP0S in an antigenic cocktail presents a promising alternative for the generation of new and more effective formulations for the control of tick infestations in cattle.

[0197] The current innovations are as follows:

[0198] The use of a cocktail of recombinant antigens as a tick vaccine with sequences adapted to the target species.

[0199] The formulation of a recombinant vaccine composed of multiple antigens (multivalent vaccine) generates different antibody populations in the host organism as a result of immunization, which may be associated with better levels of protection.

[0200] The use of a chimeric protein antigen with a completely novel design. The sequence consists of selected sections of two known antigens fused by a flexible loop sequence designed by the present inventors.

[0201] References

[0202] 1. Jaime Betancur Hurtado, O. and Giraldo-Ríos, C. Economic and Health Impact of the Ticks in Production Animals. in Ticks and Tick-Borne Pathogens (IntechOpen, 2019). doi:10.5772 / intechopen.81167

[0203] 2. Fuente, J. de la et al. A ten-year review of commercial vaccine performance for control of tick infestations on cattle. Anim. Heal. Res. Rev. 8, 23–28 (2007).

[0204] 3. Trentelman, J. J. A. et al. A combination of antibodies against Bm86 and Subolesin inhibits engorgement of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae in vitro. Parasit. Vectors 12, 362 (2019).

[0205] 4. Pereira, D. F. S. et al. Rhipicephalus microplus: An overview of vaccine antigens against the cattle tick. Ticks Tick. Borne. Dis. 13, 101828 (2022).

[0206] 5. Parthasarathi, B. C. et al. Analysis of Genetic Diversity in Indian Isolates of Rhipicephalus microplus Based on Bm86 Gene Sequence. Vaccines 9, 194 (2021).

[0207] 6. Contreras, M., Kasaija, P. D., Kabi, F., Mugerwa, S. y De la Fuente, J. The Correlation between Subolesin-Reactive Epitopes and Vaccine Efficacy. Vaccines 10, 1327 (2022).

Claims

Vaccine composition comprising two proteins, wherein: a first protein comprises SEQ ID NO: 5, and a second protein comprises SEQ ID NO:

3. Vaccine composition according to claim 1, for use in a method of generating a protective immune response against ticks in a mammal. Vaccine composition for use according to claim 2, wherein the tick is Rhipicephalus microplus. Vaccine composition for use according to any one of claims 2 or 3, wherein the mammal is a bovine, preferably cattle, most preferably selected from the list consisting of cows, bulls, oxen, or calves. Vaccine composition for use according to any one of claims 2 to 4, wherein the vaccine is administered as a sensitizer and / or as a booster. Vaccination kit comprising the vaccine composition according to claim 1. Diagnostic kit comprising two proteins, wherein: a first protein comprises SEQ ID NO: 5, and a second protein comprises SEQ ID NO:

3. Antibody or antiserum that is reactive with the immunogenic proteins according to claim 1.

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