WO2009146513A1 - Immunogens for controlling bovine tick - Google Patents

Abstract

The present invention is related to the field of biotechnology of vaccines, i.e. to the development of vaccinal peptides (mimo topes) for controlling bovine tick. Said vaccine has potential application due to the fact that tick proteins are recognized by serum of mice inoculated with fusion peptide-expressing phages, and also relying on the fact that Teleogines presented a blackened color suggesting hemorrhagic damages in challenge assays. Since there was a great need for new methods for controlling Boophilus microplus, a potential vaccine could be developed by using isolated peptides, in conjunction or associate with existing antigens for an effective control of ticks.

Description

IMMUNOGENS FOR CONTROLLING BOVINE TICK FIELD OF THE INVENTION

The present invention is related to the field of biotechnology of vaccines, i.e. to the development of vaccinal peptides (mimotopes) for controlling bovine tick.

Said vaccine has potential application due to the fact that tick proteins are recognized by serum of mice inoculated with fusion peptide-expressing phages, and also relying on the fact that Teleogines presented a blackened color suggesting hemorrhagic damages in challenge assays. Since there was a great need for new methods for controlling

Boophilus microplus, a potential vaccine could be developed by using isolated peptides, in conjunction or associate with existing antigens for an effective control of ticks. BACKGROUND OF THE INVENTION

Extensive studies related to development of a method of controlling Boophilus microplus ticks rely on vaccine development, since this is one of the most important bovine ectoparasites, causing great losses to the world cattle production because of its direct and indirect effects. Application of chemical substances is nowadays the main control method. However, due to drawbacks involving this practice, use of vaccines is an alternative of better viability because vaccines are residue-free, specific and show a minor chance of developing resistance.

Main B. microplus host is a bovine, but other animals can be parasited, such as, for example, buffalos, donkeys, sheeps, goats, dog, cat, swine, deer, ounce, sloth, kangaroo, rabbit and stags. In these animals, excepting for bovines, ticks do not reach adult stage in view of many factors, amongst them immunity factor, which promote high mortality, mainly of larvae (Riek RF. Austral. J. Agric. Res. 10: 614-619, 1959) .

Boophilus microplus is one of the most important parasites found in bovines. Such a parasitism cause huge economic loss in the worldwide cattle growth business, mainly affecting tropical and subtropical countries. In Brazil, Boophilus microplus tick is found in twenty six states and 95.6% districts (Teixeira Leite N. et al. Ecosystem, 16:137-141-1991) in 66.04% of said districts, during twelve months a year. In view of climatic conditions, regions were ticks have a higher incidence are South, Southeast and Central West (Horn SC. BoI. Def. San. Ani. Brasilia: Agriculture Department, 1983) .

In accordance with information issued by Agriculture Secretaries of Brazilian States, which have evaluated economic losses caused by Boophilus microplus, which amounted to US$ 968 billion (Horn SC et al . BoI. Def. San. Ani. Brasilia: Agriculture Department, 1984), because of an increase in cattle herd of from 76 millions heads in 1983 to 169 millions heads in 2000, such losses could exceed two billions of dollars (Grisi L et al . A Hora Veterinaria, 21(125) -.8-10, 2002) . With regard to leather production only, such losses are higher than R$500 million per year.

Said losses are caused by both direct and indirect effects. As a direct effect, it can be mentioned hematophagous action that exerts negative effect on the weight gain and nutritional state, thereby affecting milk and meat production, and it may also cause animal death

(Horn SC et al . BoI. Def. San. Ani. Brasilia,_. Agriculture, BR, 1984) . Damage caused on animal skin may lead to secondary pathologies, causing loss in the leather market.

As an indirect effect, B. microplus tick also causes losses because it is an important transmitter of hemoparasitosis such as Anaplasma sp (Rickettsia) and Babesia spp (Protozoan), which form cattle parasitic sadness complex.

B. microplus tick is a monoxene parasite, i.e., it performs its changes or metamorphosis in a sole host. Its life cycle can be subdivided into two phases: parasitic phase and non-parasitic phase (Gonzales JC. Porto Alegre, Sulina, BR, 103, 1975) .

Bos indicus cattle (zebu) is more resistant to ticks than Bos Taurus (European) , wherein European cattle has an average of 10.5 times more ticks than Zebuine crossbreeds (Francis J et al . Aust . Vet. J. 40:247-53, 1964) . On the other hand, amongst European breeds it can be found those with more resistance to ticks, as in the case of Jersey breed (Utech KBW et al . Austral. J. Agric. Res. 29:885-895, 1978) . It has been noted that individual variation in relation to tick resistance also occurs within one same breed (Gomes A. Thesis, Rio de Janeiro: ϋniversidade Federal do Rio de Janeiro, 1995) . Moreover, amongst other variables that can influence a parasite-host relation, sex (Stera MJ et al . Aust. J. Exp. Biol. Med. Sci. 62:47-52, 1984), age (Sutherst RW et al . Aust. J. Agric,

30:353-68, 1979), time of the year (Gomes et al. Trop. Anim.

Health. Prod. 21:20-24, 1989), nutritional condition

(Sutherst RW et al . , Austral. J. Agric. Res., 343:329-339, 1983), color of skin and hair (Oliveira GP et al . , Pesq.

Agrop. Bras., 22 (4 ) : 433-439, 1987, can be mentioned.

In highly resistant animals, the amount of larvae that succeed and reach a complete cycle is less than 1% and for a susceptible animal, said proportion increases to about 20% or more (Willadsen et al . 1977, cited by Verissimo CJ. Thesis (Master of Science) , FCAV, Jaboticabal Campus, UNESP, 163 1990) . During parasitic phase of B. microplus, more losses in resistant animals, either larvae or nymphae, mainly before the periods of molts, occur (Bennett GF. Acarologia, 16:643-650, 1965) .

Zebuines contain more than the two folds the amount of dermal mastocytes per volume unit in the crotch region than taurines in naturally infested animals (Moraes FR. Thesis (Ph. D), FCAV, ϋnesp, Jaboticabal, SP, 106 p, 1988) . Dermal changes impair blood ingestion and cause the weight of engorged females to be less in zebuines than in taurines. Consequently, these teleogines produce a less number of eggs and larvae. Higher resistance of zebu cattle has not affected oviposition, embryogenesis and larvae hatching.

In a study about tissue changes effected during cattle parasitism by Boophilus microplus, it has been verified that the degree of eosinophil degranulation is greater in a site where larvae are fixed on resistant animals (Schleger AV et al . , Austral. J. Biol Sci., 29:399- 512, 1976) . In accordance with Schleger et al . , formation of antigen-antibody complex, complement fixation and lymphokines recruit eosinophils. Great afflux of basophils and mastocytes also occurs (Willadsen, 1977 cited by Verissimo J., Thesis (Master of Science), FCAV, Jaboticabal Campus, UNESP, p. 163, 1990) . Said cells release histamine that is the main mediator in the inflammatory response, thus increasing capillary permeability of blood vessels and the pathway of host defense elements. Enzymes released by attracted and degranulated eosinophils cause tissue damage as evidenced by epidermal vesicle observed in hosts, and local irritation. Said products are also toxic to larva, thereby blocking its feeding or forcing it to migrate to another location. Irritation also stimulates self-cleaning and epidermal vesicle formed under the site of fixation and consequently removal of larvae becomes easier. In those final phases of parasitic cycle, an intense infiltration of neutrophils occurs, as they are attracted by factors generated by fixation of complement.

Arthropods when parasiting animals inject saliva containing immunoresponse-inducing antigens. Said antigens stimulate three different types of immunoresponse : Thl-type response, Thl-type response associated with production of IgG antibodies and with basophil infiltrate; TH2-type response with production of IgE and 1-type hypersensitivity. Each of those three types of responses can modify the skin of an animal and impair feeding of parasites. Tick saliva reduces macrophage function and is immunosuppressive (Tizard

IR. Veterinary Immunology. Saunders Company, 6: 482p, 2000) .

Chemical control is the main way of controlling ticks in cattle herd, mainly by using spray or aqueous solution bath. Dorsal, injectable and gastric bolus systems have been improved in the last years, thus rendering their handling easier. New routes of administrating said products are being developed in order to facilitate handling and enhancing effectiveness of chemical products in control of ticks . Extensive use of acaricides has led to an elevated number of problems such as handling costs, dose costs and short period of protection. In addition, their use promotes selection of acaricide-resistant tick strains, reducing protective period of products and also increasing treatment costs. For chemical control, arsenic, organochlorinated, organophosphorated derivatives; carbamatos; amidines; and synthetic pyrethroids are used. On average, different active principles used for controlling ticks have presented a useful life slightly above one decade, when almost all populations of tick present in the field were resistant to the employed drug (Graf JF et al . Parasitology 129 : 427-S422, 2004) .

Most of drugs used nowadays are pyrethroid derivatives, ivermectins and benzoyl phenyl urea (Da Silva Vaz JR I. Thesis, Porto Alegre : Universidade Federal do Rio Grande do SuI, 1997) . Moreover, said control methodology is extremely disadvantageous due to the fact that chemical agents show harmful effects on an animal during the treatment and also on human health, caused by the extensive residual effect.

Alternative control forms have been studied, mainly for the purpose of avoiding the presence of chemical residues in products of animal origin for human and animal consumption. Alternative control of ticks has been encouraged in spite of its doubtful effectiveness. There are several methods, which can be or cannot be associated with each other, and the following are examples of these methods: selection of tick-resistant cattle (Davis GP. Aust. J. Agric. Res., 44:179, 1993); cultivation of pastures, where survival of larvae is very difficult (Sutherst RW et al. Nature, 295:320-321, 1982); rotation of pastures (Elder JK et al., Aust. Vet. J., 56:219-231, 1980); management of natural preys, such as Egretta ibis (Alves-Branco FPJ et al . Comunicado tecnico EMPRAPA, 1:1-4, 1983) and ants (Gonzalez JC. 2^nd Ed. Porto Alegre: Author's Edition, 1995); use of pathogens such as Beauveria bassiana fungus (Crodoves CO. 2^nd Edition Rio Grande do SuI: Farming and Cattle Raising. 176, 1997); and bacteria such as Erwinea sp (Brum JGW, International Journal of Systematic Bacteriology, 31:317- 326, 1981) .

Vaccines have an attractive alternative to the traditional chemical technology of controlling B. microplus, since they exhibit prophylactic and therapeutic control of agents that cause many diseases in humans and animals, are not chemical agents, are less expensive, and take a higher period of time for developing resistance (Willadsen P. Veterinary Parasitology. 71:209-222, 1997) .

Three commercial vaccines with moderate effectiveness have been developed. The first commercial vaccine was produced on large scale in Australia, with the trade name TickGard® (AU8700401) , comprising Bm8β antigen and developed by genetic engineering in E. coli associated with an oily adjuvant. The second commercially available vaccine against ticks, designated Gavac® (BR PI 9300625-0) , is based on Bm86 antigen, cloned in Pichia pastoris, and also associated with an oily adjuvant. The third vaccine comprising Bm86 antigen and a new adjuvant designated Vaximax was commercially registered under the trade name TickGard^Plus®. Field tests with said commercially available vaccines, TickGard^Plus and Gavac^®, conducted in Brazil, have shown a moderate effectiveness and required concomitant application with chemical products. In order to obtain certain effectiveness from said vaccines is essential their association with handling procedures favoring decontamination of pastures .

Development of vaccines against tick, a process formerly contemplated only for viruses, protozoans, and bacteria, has allowed, after the first infestations of cattle under natural conditions, to develop expressive immunoresponse against tick tissue. This finding has led many researchers to seek for tick antigens capable of protecting cattle. Most of these efforts to seek for antigens have been focused on digestive tract and salivary glands of ticks, wherein the proteins isolated to date are mainly originated from digestive tract. Nevertheless, it has been discovered that antibodies present in blood of vertebrate host passes through digestive tract and are still active in the hemolymph of a tick. By this way, there has also been proposed using as antigens in the preparation of a vaccine tick, tick proteins having functions physiologically essential for the survival thereof and with which the immune system of bovines would not normally contact. However, the search for good internal targets of an animal faces the lack of more detailed biochemical knowledge of its physiology.

Various molecules involved in physiology of ticks and interaction with host have been reported by many groups of researchers. A study of said proteins would allow for identification of new antigens for developing vaccines. Once they have been characterized, the respective genes can be cloned so -that said proteins can be produced in amounts sufficient for tests of immunoprotection. The fact that functional bovine antibodies are found in several tick tissues has expanded the range of target molecules beyond those present in intestine so that molecules found in practically any tick tissue have become a potential target

(Da Silva Vaz Junior et al . , Vet. Parasitol., 62:155-160, 1996) .

Three different approaches for developing vaccines for control of ticks are presently used: Use of exposed antigens, use of concealed antigens and, finally, combination of both approaches.

The first of them, - use of exposed antigens-, is based on the observation of naturally occurring resistance of cattle acquired after repetitive infestations with ectoparasite and allowed for identification of antigens by naturally acquired immunity (Roberts JA. Journal Parasitol. 54 (4) : 657-62, 1968; Wagland BM. Aust. J. Agric. Res. 26:1073-1080, 1975; Willadsen P. et al . , J. Immunology. , 143:1346-1351, 1989; J. Parasitol. 79 (5) : 710-715, 1993) . In a recent review, the main recombinant antigens, tested and classified as exposed antigens, were: calreticulin, immunoglobulin-binding protein, histamine-binding protein, P29, HL34, RIM36 and 64TRPs (Nuttall PA et al., Parasite Immunology, 28:155-163, 2006) . The second methodology is based on the identification of concealed agents, which are not naturally introduced into the immune system of a host, thereby requiring successive inoculations for generating an immunoresponse (Willadsen P et al . , Parasitology Today. 4:196-198, 1988) . Antigens developed as anti-tick recombinant vaccines, including antigens, which are constituents of commercial vaccines, are: Bm86, Bm91, Bm95, Vitellin, BmPRM (paramyosine) , HLSl (serpine), Voraxine, HLS2, P27/P30 (troponine 1-like protein and 4D8) (Nuttall PA et al., Parasite Immunology, 28:155-163, 2006) .

Presently, a new concept refers to identification of antigens, which, when used as vaccines, target both secreted antigenic and concealed epitopes. 64P Antigen is possibly the unique example of 64P antigen isolated from R. append±culatus . Although being a concealed antigen, 64P contains cross-linkage with antigens of intestine, hemolymph, and salivary glands of adult ticks and total extracts of JR. appendiculatus nymphs and larvae (Trimnell AR et al., Vaccine, 23:4329-4341, 2005); Trimnell AR et al . , Vaccine, 20:3560-3568, 2006) . The double action existing in this mechanism, represented by the performance of host defense mechanisms in the feeding site and intestine, provides for a self-sustaining strategy to control ectoparasites maintained by natural infestations.

In addition to Bm86 antigen, other proteins of B. microplus have been characterized as immunogens potentially constitutive parts of vaccines, but none of them is presently being used as commercial vaccine. The most important antigens are: Bm91 proteins (Riding GA et al . , J. Immunology, 153:5158-5166, 1994) and BMA7 (Mckenna RV et al., Parasite Immunol., 20:325-336, 1998), a group of proteins (molecular masses varying between 30 kDa and 200 kDa) using a QU13 monoclonal antibody (Lee RP et al . , Immunol., 72:121-126, 1991) . Another monoclonal antibody, BrBm2, recognizes an intestinal protein having 27 kDa, (Toro-Oriz RD et al . , Vet. Parasitol. , 69:297-306, 1997) .

Antigens relative to ovigenesis and embryonic development are the following: Vitellin (Logullo C et al . , Insect. Biochem. MoI. Biol., 32:1805-1811, 2002), BYC {Boophilus Yolk Catepsin) (Logullo C et al . Parasitol. 116:525-532, 1998), a vitellin-degrading cysteine endopeptidase (VTDCE) (Seixas A et al . Parasitol. 126:155- 1563, 2003) .

Antigens related to digestion, oxidative stress and immune system are the following: heme-binding proteins (HeLp) , a lipoprotein capable of transporting heme to different tissues (Maya-Monteiro CM et al . , J. Biol. Chem. , 275:36584-36589, 2000), a THAP, a heme-binding protease, related to VT degradation (Sorgine MH et al . , J. Biol. Chem., 275:28659-28665, 2000) and VT itself, which is a heme-binding protein with antioxidant function (Logullo et al . , Insect Biochemistry and Molecular Biology, 32(12) :1805- 1811, 2002) , a cysteine endopeptidase designated BmCLl (Renard G et al . , Insect Biochem. MoI. Biol. 30:1017-1026, 2000) . A few antigens have been tested in vaccination assays as effective recombinant antigens. Although many tick proteins have been proposed as specific protective antigens, there is still a need for identification and characterization of new antigens for the control of ticks (De Ia Fuente J. et al . , Expert Rev Vaccines, 2:583-593, 2003; Willadsen P. Parasitology, 129:S1-S2, 2004) .

For Rhipicephalus appendiculatus, cement protein 64P when used for host immunization, has represented a significant advance because it exhibited a systemic action against parasite (Trimnel AR et al . , Vaccine 20:3560-3582, 2002), and because it is related to immunoglobulin-binding proteins (Wang H. et al . , Cell MoI Life Sci., 56:286-295, 1999) . Said matrix protein p29 in salivary gland of Haemaphysalis longicomis (Mulenga A et al . , Infect Immun. , 67:1652-1681, 1999), protein HL34 of unknown function (Tsuda A et al., Vaccine, 19:4287-4296, 2001), serine protease- inhibiting serpin-2 protein (Imamura S et al . Vaccine, 23:1301-1311, 2005), troponin-like P27/p30 protein (You MJ. J Vet Sci 6:47-51, 2005) represent important advances in the control of Haemaphysalis longicomis.

4D8 and 4E6 proteins (unknown functions) of Ixodes scapularis and nucleotidase 4F8 are some antigens having important functions (Almazan C et al . , Vaccine, 23:4403- 4416, 2005), in addition to engorgement factor AhEF of Amblyomma hebraeum (Weiss BL et al . Proc. Natl. Acad. Sci. USA, 101:5874-5892, 2004), which have significantly affected tick infestations in various experiments.

Intestinal recombinant proteins Bm86 and Bm95, having unknown functions, are a milestone in the development of protective antigens against B. microplus (Willadsen P et al. Proc. Natl. Acad. Sci. USA, 86:9657-9661, 1989, Rodriguez M. et al . , J. Biotechnol. , 33:135-146, 1994; Garcia-Garcia JC et al . , Vaccine, 18:2275-2287, 2000), peptidase (Bm91 protein) (Willadsen P et al . , Parasite Immunol., 18:241-246, 1996) .

Other recombinant antigens have not accomplished the desired success, such as calreticulin (CRT) of B. microplus (Ferreira CA et al . , Experim. Parasitol. , 101:25- 34, 2002) and paramyosin, in which the recombinant protein is a IgG ligand and collagen (Ferreira CAS et al., Parasitology, 125:265-274, 2002) .

Results of these experimental studies have shown the viability of controlling infestations by using multiple gene products that target many physiological mechanisms of ticks .

As a result of these and more studies, vaccines and antigenic components have been patented, such as, for example: galectin (NZ 535468, BR PI 0303322-8), RhcA and RhcB genes for cysteine proteinase molecules (CN 1657617), cement protein (US 2003170257, NZ 504532, BR PI 0110278-8),

TdPI tryptase (NZ 516809) , peroxyredoxin recombinant protein

(JP 2002010785), Bm95 (ZA 9901320), polypeptide corresponding to HT-I neurotoxin (WO 97/47649), cell membrane glycoprotein (US 6,235,283, EP 0290565, CA1339466), various antigens (JP 62084029, IL 84574), synganglion (AU 5970786), carboxypeptidase (WO 95/04827), particulate antigen formulations (ZA 9302352), Kunitz-type protease inhibitor (PI 0406057-1), tick kytinase (JP 0302335), serine protease-inhibiting antigen (BR PI 0004655-8), aspartic protease (BR PI 9903530-8), anti-tick vaccines (BR PI 8707549-0) .

Various vaccine products, in addition to the commercially available vaccines for B. microplus, have been prepared by several methodologies other than the recombinant form, and their results are worthy of mention. US Patent 5,281,836 of 2005 discloses three chemically synthesized peptides (SBm4912, SBm.7462 and SBml9733) derived from BM86 glycoprotein have been constructed and used for animal immunization. Said peptides, comprising saponin as adjuvant, were inoculated into animals. The best effectiveness results, 81.05%, were obtained from the synthetic SBm7462 peptide. Subsequently, it has been verified the viability of using SBm7462 immunogen in latex microspheres for delaying immune response (Sales-Junior PA et al . Veterinary Immunology and Immunopathology, 107:281-290, 2005) .

DNA vaccines comprising vectors coding for Bm86 gene have shown promising results, although yet insufficient for an adequate protection (De Rose R et al, Immunol. Immunopathol. , 1:151-160, 1999; Ruiz LM et al . Veterinary Parasitology, 144:138-145, 2007) .

BR Patent Application PI 0004655-8 refers to the use of a serine protease-inhibiting protein (BMTI) having 72.8% protective effect, after administration of three doses to the animals (Andreotti R et al . , International Immune Immuno Pharmacology, 2:557-563, 2002) . In a further work, it has been reported a study conducted for evolution of a synthetic BmTI N-terminal fragment as antigen against bovine ticks and it presents 18.4% effectiveness level compared to the control group (Andreotti R. Experimental Parasitology, 116:166-70, 2007) .

US Priority Application Serial No. EP1749835 describes the use of ELI (expression library immunization) and analyses of cDNA sequences protective against experimental infestations with I. scapularis (Almazan C et al. Vaccine 21 (13-14) :1492-1501, 2003. This was the first case of ELI being used in arthropods and, particularly, in ticks (Alamazan C et al . Vaccine 21 (13-14 ) : 1492-1501, 2003) . These experiments allowed for characterization and use of 4F8, 4D8 and 4E6 antigens in large spectrum anti-tick vaccine formulations (Almazan C et al . , Vaccine, 23 (35) :4403-4416, 2005; Almazan C et al . , Vaccine, 23(46- 47) : 5294-5298, 2005) . WO 03/093416 and US 20040022795 firstly refer to antigens protective against Ixodides ssp. Subsequently, US 20050123554, 20060040361, EP 1655306, US 7,214,784 and EP 1749835 refer to Ixodides ssp and other species of ticks.

Use of RNAi for determining gene function has been demonstrated in various tick species, including B. microplus, where in the latter Bm86, Bm91 and subolesin antigens were silenced. Eggs generated by engorged females injected with subolesin dsRNA were abnormal, thereby suggesting that subolesin could play a role in embryonic growth. Results shown by this research have led to new possibilities of discovering new antigens (Nijhof AM et al . , International Journal for Parasitoloy, 37(6), 37 ( 6) : 653-662, 2007; Kocan KM et al . , Parasitology Research, 100 (6) : 1411- 1415, 2007) .

Up to the present time, the following patent applications referring to development of vaccinal antigens for control of bovine tick were filed in Brazil: BR PI 0110278, BR PI 0604365, BR PI 9704150, BR PI 0004655, BR PI 0406057, BR PI 9903530, BR PI 0303322, BR PI 0303321, BR PI 0504346, and BR PI 0501904.

In view of the previous discussion referring to tick control by using vaccines, the necessary characteristics for obtaining an ideal vaccine are the following: activity against all tick species, activity against all stages, prolonged immunity, tick fixation hindering, reduction in incidence of diseases, lack of resistance and effective costs (Nuttall PA et al, Parasite Immunology, 28:155-163, 2006; Tellam RL et al . , Veterinary Parasitology, 103:141-156, 2002) .

Researches in the area of recombinant vaccines for tick control have discovered that prototypes containing more than one antigen are more effective than those having only one antigen (Willadsen P et al . , Parasite Immunology 18:241- 246, 1996) . In view of that, there is a clear need for identification of new antigens, identification of important epitopes of known antigens or even improvement in the already existing vaccines for controlling bovine tick. Therefore, using concomitantly various antigens in order to enhance protective immune response is a trend nowadays .

Problems relative to development and production of conventional vaccines for tick control are challenging, as a satisfactory solution for the use of traditional culture methodologies has not yet be attained. Methods of purifying and obtaining in natura tick immunogens require a complex procedure in order to become commercially viable. On the other hand, recombinant proteins are based on one sole antigen and may not produce the same protective effect as that of the similar original protein. The costs for vaccines based on chemically synthesized antigens are superior to the former vaccines and in some cases they cannot function as the original protein.

A vaccine with real chances of substituting the use of acaricides is not yet available. It is important to study the abilities of the already available immunogenic antigens and the association of new antigens to be discovered. Identification of various proteins having potential to be used in vaccines against ticks will allow to evaluate that the two present vaccines having partial effectiveness become more effective by adding new immunogens, specific adjuvants and cytokines, leading to changes in inoculation form and introduction of immunogens into bovine immunologic system (Vaz Junior IS et al . , XIII Congresso Brasileiro de Parasitologia Veterinaria & I Simpόsio Latino-Americano of Ricketisioses , Ouro Preto, MG, 2004) .

In this context, molecular biotechnology is an important source of information in the field of parasitology and is a very promising way to attain effective vaccines. In view of scientific studies and technological development in the field of parasitic proteomics and genomics, there is a possibility of identifying and correlating data for developing and commercializing new drugs, thereby reducing costs and time spent for designing new products (Dalton JP et al. Veterinary Parasitology, 98:149-167, 2001) . According to the authors, mapping and identification of the main epitopes that confer protection are necessary. Said mapping could lead to identification of the main epitopes responsible for rendering effective immunoresponse and eliminating epitopes related to development of evasion mechanisms of immunoresponse of a parasite to a host.

Generally speaking, we may conclude that studies conducted up to now justify the confidence on the viability of developing a vaccine based on already disclosed proteins and the research for new and extremely important proteins. In view of the discussed above, a vaccine based on specific selection of tick epitopes encompassing different antigens of different phases and organs represents a widely viable possibility of constituting a polyimmunogenic vaccine compared to the remaining existing vaccines. Identification and characterization of epitopes capable of generating immune response against tick allow for the use of recombinant or even chemically synthesized peptides for vaccinal purposes on industrial scale. Therefore, working only on the immunogenic region eliminates the need for using a complete protein. Moreover, said vaccines possess some desirable characteristics such as, for example, high purity degree, known chemical characterization, absence of contaminants, production on large scale, easy storage due to high stability, absence of proteolytic enzymes, low costs, and industrial-scale manufacture (Patarroyo JH et al . , Veterinary Immunology and Immunopathology, 88 (304 ) : 163-172, 2002) .

Therefore, the present application is directed to the use of new recombinant or chemically synthesized peptides, which function as antigenic epitopes and which are potential targets for generating immune response against ticks, wherein they comprise sequences that recognize protein motifs common to the polyprotein existing in various growth phases, different organs and tissues, in addition to non-protein structures. The present application relies on the use of Phage Display process for identification and selection of specific mimetic peptide sequences of B. mlcroplus, wherein functional epitopes are determined by means of polyclonal antibodies obtained from chickens immunized with total B. mlcroplus extract. This study involved the first use of selection of peptide libraries in arthropods for developing arthropod specific mimotopes specific and, particularly, in ticks.

DETAILED DESCRITPION OF THE INVENTION Techniques applied to molecular biology are potentially important in elucidating new antigens for development of a new effective vaccine. Among these techniques, Phage Display process ("expression of biomolecules in phages") is a methodology capable of selecting peptides for several purposes, such as mapping and identification of epitopes recognized by antibodies. Said process consists of successive cycles of selection, washing, elution and amplification of filamentous phages expressing random peptide sequences that bind, with affinity, to various molecules, including immunoglobulins. Commercial peptide libraries may be used for selection of synthetic peptides that mimic natural epitopes. These clones are sequenced and said sequences are translated, and, subsequently, immunotests for reactivity against target of interest are carried out (Barbas CFIII et al . , Plain View, NY: Cold Spring Harbor Laboratory Press, 2001) .

M13 phage has widely been used in Phage Display libraries. Peptide or protein expressed on the phage surface allows for selecting sequences based on binding affinity with a target molecule (antibodies, peptides, enzymes, cell surface receptors etc.) . In said system, a gene coding for peptide or protein of interest is generally fusioned to one of the genes of said two coat proteins of phage. Therefore, peptide is expressed at N-terminal end of viral pill or pVIII proteins.

Phage Display process, or presentation of biomolecules in phages, was first developed by Smith (Smith GP, Science, 228:1315-1317, 1985) . Said process involves EcoRI restriction enzyme by fusion with PIII protein of phage capsid. Phage Display process allows for selecting peptides and proteins, including antibodies, having high affinity and specificity for several targets . The principal advantage of the referred to technology resides in a direct bond existing between the experimental phenotype and the encapsulated genotype, thus showing evolution of the selected ligands to optimized molecules (Azzazy HM et al., Clinical Biochemistry, 35:425-445, 2002) . Phage display has proven to be an extremely powerful technique for obtaining libraries containing millions or even billions of different peptides or proteins . The most widely employed methodology of libraries is based on the use of filamentous phages (Smith GP. Science, 228:1315-1317, 1985) .

A particle of filamentous phage is formed by DNA single strand surrounded by a protein coat consisting of five proteins (pill, pVI, pVII, pVIII and pIX) . Of these five proteins, approximately 2,800 copies of PVIII and five copies of pill exist. In said system, peptide-coding gene or protein of interest is generally fusioned to one of the genes from said two proteins of the phage protein coat (Brigido MM et al . , Biotechnologia Ciencia <£ Desenvolvimento, 26:44-51, 2002) . Therefore, peptide is generally expressed at the pill or pVIII N-terminal end. pill is related to the infectivity of phage through its binding to pilus F of bacterial cell. It contains three domains (Dl, D2, and D3) separated from each other by means of glycine residues. Because of the low representativity of pill in relation to pVIII, libraries of fusioned synthetic peptides in pill are more indicated for discovery of high- affinity ligands compared to bound pVIII libraries. Despite the fact that Phage Display technology was first introduced approximately fifteen years ago, applications and development of this technology are starting to be explored. Such exploration of Phage Display will bring about an enormous range of ligands, including recombinant antibodies and peptides, having predefined specificities. In addition, recent technologies relying on Phage Display will improve diagnosis due to the production of molecules that are impossible to be obtained by conventional methods. Specifically, recombinant antibodies against toxic antigens or conservative sequences and carbohydrates can be isolated, amongst others (Soderlind E et al . , Nature Biotechnology, 18:856, 2000) . US Patent 7,083,945, US Paten Application

2006068421, US Patent Application 2005202512, GB Patent 2408332, US Patent Application 20031044604, EP Patent 1452599, US Patent Application 2006035223 disclose Phage Display process for expressing recombinant polypeptides having affinity with the ligand, in addition to proposing methodologies that render the technique more effective.

Selection of libraries fusioned to phages presents high versatility to obtain monoclonal and polyclonal antibody specific peptides, which will recognize linear epitopes and also mimotopes, peptides mimicking discontinued conformational linear epitopes and even non-protein antigen epitopes. Said antigens expressed in phages are advantageously important as compared to vaccines of synthetic peptides (Huerta M et al . , Vaccine, 20:262-266, 2001; Sciutto E et al . , Microbes Infect., 2:1875-1890, 2000) . They can even be produced on large scale, at low costs, because they are secreted by bacteria. Phage surface density is 200-400 m²/g and fusion with peptides expressed in PVIII comprises more than 25% of its weight and extends along 50% of its surface area. Phages are also resistant to temperature and many organic and chemical solvents, as well as to other types of stresses. What is more important is that proteins of the phage itself are highly immunogenic, and most of time they do not require adjuvant for their use with immunogens (Petrenko VA et al . , Protein Eng. , 13:589- 592, 2000) .

Phages are commonly used as immunogenic particles for generation of antibodies against recombinant peptides expressed in amino-terminal regions of surface proteins, and cross-react with the original target, indicating that expressed mimotopes can be used as candidates for vaccinal subunits (Willis AE et al . , Gene, 128:79-83, 1993; Meola A et al., J. Immunol., 154:3162-3172, 1995; Demangel C et al . , MoI Immunol., 33:909-916, 1996; Yang WJ et al . , Journal of Immunological Methods, 276:175-183, 2003) .

Researchers have recently used Phage Display for development of vaccines against a variety of diseases: HIV-I (Scala G et al . , J Immunol., 192:6155-6161, 1999); De Berardinis PD et al . Nat. Biotech., 18:873-876, 2000), viral hepatitis C-type (Puntoriero G et al . , Embo J., 17:3521- 3533, 1998), Alzheimer disease (Frenkel D et al . , Proc. Natl. Acad. Sci. USA, 16:5675-5679, 2002) . Some examples of application of this technology in the vaccine field are disclosed in WO 2006/005943, WO 2006/071896, US Patent Application 2006094017, US Patent Application 2003082524, DE Patent 10041342, US Patent 6,939,948.

An especial application of this technology to medium size animals has been carried out by Manoutcharian et al . , Veterinary Immunology and Immunopathology, 99(1-2) :11- 24, 2004) . Manoutcharian and et al . used for the first time recombinant phages as vaccines for swines, and observed that phages containing recombinant peptides of Taeni solium, an agent responsible for causing cysticercosis in humans, had ability to induce immune response.

Such works have demonstrated that the complete antigenic profile of an organism can be obtained through selections of peptide libraries expressed in phages and reveal a potential use of phages expressing epitopes as vaccines .

Another innovation in this invention refers to the use of chicken immunoglobulin (IgY) for proteomic researches in B. microplus . This methodology designated by Zahng as IgY technology (Zhang WW. Drug Discovery Today, 8:364-371, 2003) employs serum and eggs as antibody sources (Gassmann M et al., FASEB J., 4:2528-2532, 1990) . As taught by Lemamy (Lemamy GJ. International Journal of Cancer, 80:896-902, 1999) , fowls have the ability to produce antibodies having high titer and high immune response persistence to bacterial and mammalian proteins. In addition, antibodies produced in chickens have biochemical advantages such as resistance to extreme pH (Lee et al . J Biochem MoI. Biol, 35:488-493, 2002), resistance to elevated temperature (Jensenius et al . , J. Immunol Methods, 46:63-68, 1988), high affinity (Ikemori at al., Poult Sc±. , 72:2361-2365, 1993), and avidity (Stuart et al., Anal Biochem, 173:142-150, 1988) . For this reason, they have effectively been used in immunoassays (Schade et al., ALTEX, 13:5-9, 1996) .

By this way, the great potential of methodology for a specific vaccine of antigen mimetic epitopes, - representatives of different phases and organs - , seems to be an extremely powerful process for producing a new polyimmunogenic vaccine compared to other existing methodologies. Identification and characterization of epitopes capable of generating important immune responses against ticks allow for the use of recombinant or even chemically synthesized peptides for vaccinal purposes. Work is thus made only on peptide immune regions and thus the use of complete protein is unnecessary. Moreover, said vaccines show some desirable characteristics, such as high purity degree, known chemical characterization, absence of contaminants, large scale production, easy storage due to high stability, absence of proteolytic enzymes, low cost and industrial scale production (Patarroyo JH et al . , Veterinary Immunology and Immunopathology, 88:163-172, 2002) .

By using a methodology that is fast, effective and capable of providing new protective antigens, the present invention was developed, which, by means of obtaining specific peptide epitopes of B. microplus tick, has introduced a significant advance in the field of vaccines with potential application for effectively controlling said parasite .

The improvement of the present invention consists of obtaining mimotope sequences of structures present in total extract of different tick phases, and their use as immunogens by using methodologies of recombinant expression or chemical synthesis; and a composition associated with said immunogens as a vaccine. In view of these aspects, the present application refers to the use of one hundred and ten conformational linear peptide mimotopes having various sizes and forms so that their use as immunogen expressed in viral capsid, other isolated vectors in combination or even chemically synthesized and conjugated to a hapten carrier, or other presentations, is capable of generating cross-immune response against tick during blood ingestion in order to provide for immunoprophylactic control of tick-borne disease.

The present invention proposes the use of new recombinant peptides, which function as antigenic epitopes and are potential targets for generating immune response to a variety of tick species, wherein said peptides comprise sequences that recognize common protein motifs common in parasite polyprotein found in all growth phases.

Further we should evidence the potentiality of the present invention obtained from studies with mice and bovines. Said peptides have been tested in immunization and challenge assays and exhibited the ability to produce reactivity in serum of said animals by recognizing proteins of the parasite tissues and organs.

The present invention will be better understood by the following detailed description and Examples showing experimental results listed in Tables 1 to 12 and accompanied drawings . Experimental procedures performed herein are shown in more details at the end of this specification. Methodology used for selection, characterization and use of recombinant peptides and protein motifs that mimic antigenic regions has been applied to B. microplus proteins; and it can be effectively applied to other species as well. EXAMPLES

Example 1: Production and characterization of hyperimmune serum in chickens recognizing total antigens of cattle tick (Boophilus microplus) .

The objective of this Example is to extract total proteins, analyze immune response from chicken immunized with these inocula, purify specific immunoglobulins of serum and eggs and investigate recognition of antibodies of chickens immunized with said total protein extract of larvae and adult cattle tick Boophilus microplus . For production of polyclonal serum, chickens were immunized with total proteins extracted from larvae and teleogines of cattle tick Boophilus microplus . Quality of extraction was evaluated by protein profile using SDS-PAGE - 10% (Figure 1) . Lines A and B represent total proteins extracted from larvae and adults, respectively. The result is consonant with the desired protein profile and shows various protein bands of varied intensities and molecular weights .

Immunization was performed by immunizing three- week White-Leghorn chickens. All immunized animals developed reactive antibodies to B. microplus protein extract in contrast with negative control animals (Figure 2) . A graph was plotted by placing ELISA values (OD 492 nm) versus three-folded serial dilution of the tested sera. The results were expressed as titers (Titer = Dilution of serum based on cut off value) . The legend was represented in the following form: chickens immunized with larva antigens (GLl, GL2), chickens immunized with proteins of adults (GTl and GT2), preirnmune sera (SPILl, SPIL2, SPITl and SPIT2) and reaction negative controls (BLl and BL2 ) . Titers are above 1:145,800 for larvae (arrow A) and above 1:16,200 for adults (arrow B) . These results show the ability of fowls to produce antibodies having high affinity, high titer and great persistence to immune response to bacterial and mammalian proteins. This is probably due to the fact that B. microplus is not a natural chicken ectoparasite. The great evolutionary distance existing between poultry and arthropods is a contributory factor, as well as the fact that in poultry antibody production is made by gene conversion, i.e. quite different from mammalians (somatic mutation) , although having the same antibody variability rate . After obtaining a satisfactory titer, chicken immunoglobulins (IgY) present in serum were precipitated wit ammonium sulfate and submitted to ionic exchange column chromatography. Said purified immunoglobulins were partially characterized as to purity by SDS-PAGE (Figure 3) . The method for purifying immunoglobulins from chicken serum has proven to be effective. Figure 3 shows an electrophoresis pattern of purified IgYS preparations (A) compared to total serum (B) and also to a routinely purification pattern for rabbit IgGs in our laboratory (C) under non-reductive conditions. Dithiothreitol-DTT reductive conditions (lines D and E) for determining light and heavy chains were used, respectively. A strong band of approximately 180 kDa in the serum corresponding to the IgY molecular weight (line F) was observed. Under DTT reductive conditions, wherein one band corresponding to a molecular weight of approximately 66 kDa (antibody heavy chain) and two lower bands corresponding to approximately 28 kDa (light chain) have been formed. It was possible to show a good selectivity in the purification of antibodies with ammonium sulfate associated with effective ion exchange chromatography.

An alternative method for affinity purification of antibodies was also carried out. A tick protein extract was adsorbed on a nitrocellulose membrane. Said protein- containing membrane was incubated with a partially purified IgY fraction, subsequently washed with PBST and then monitored by spectrophotometric reader at the end of washing step in order to verify release of proteins from the membrane. Antibodies were then eluted, neutralized and the eluate concentration was measured by spectrophotometry (OD 280) and quality evaluated by SDS-PAGE 10% (Figure 4) . This experiment shows the presence of lower intensity band for eluted antibodies from total proteins of larvae and adults (Lines C and D, respectively) compared to bands of a partially purified fraction of anti-larva IgY antibodies and adults in the experiment above (Lines A and B, respectively) . Finally, Western blotting (Figure 5) was carried out in order to determine the functionality of purified polyclonal antibodies . These results have shown reactivity of IgYs against the main stages of larvae (A) and adults (B) of B. microplus over several dilutions of the purified IgY antibodies (1:180, 1:360, 1:540, 1:1420) . Weak bands for lower molecular weight proteins and strong bands for higher molecular weight proteins were identified.

In conclusion, from analysis of immunoresponse in chickens immunized with protein extract of B. microplus, it was possible to show the capacity of using chicken as a source for producing specific and functionally reactive polyclonal antibodies against B. microplus. Chicken antibodies were then purified in order to eliminate any interference in subsequent experiments. Western blotting analyses have shown that purified IgY would recognize various proteins and that activity of the purified IgY had not been lost during purification process. In view of these results, works could be continued regarding the use of Phage Display library.

Example 2 : Preparation of mimetic peptides of B. microplus proteins by Phage Display process.

This Example refers to selection and characterization of mimetic recombinant peptides and protein motifs, to which the present invention is directed. Various parameters can be used for indicating whether or not a selection of each peptide is a success, as can be seen from Table 1. These same parameters have formerly been used by Rodi et al . (Rodi DJ and et al . , Journal of Molecular Biology, 322:1039-1052, 2002) . Table 1 shows enrichment of the selections by titers obtained during steps of biological selection. Said selections are indicated by letter S, wherein the total amount of seven different selections were performed. Second column shows libraries used in the referred to selections. Third column shows targets used for each selection of clones. Fourth column shows selection cycles for each selection. Further, entry and exit of phages in each selection step were determined. Titers are indicated by counting colony forming units (CFU) or Translator Units

(TU) for Ph.D.- and Fd-tet-type libraries, respectively.

Effective enrichment rate in each selection S was presented.

The higher the enrichment coefficient throughout the selection cycles the greater the enrichment. All these steps are represented by enrichment values that can be confirmed by dividing the exit titer by the entry titer and then multiplied it by 100. Finally, effective enrichment rate was determined by dividing the enrichment rate of the last cycle by the rate of the first cycle, thereby totalizing the number of times that a selection enriched.

In general, all selections have enriched during the initial phases compared to the final steps of selection, excepting for selection Sl (0.6), thus showing that selection of clones was directed towards the target. With regard to selections S3, S5 and S7, there have obtained enrichment rates in all selection steps. Selections Sl, S2, S4, and S6 have undergone impoverishment from the first to the second cycle. This can be explained by the increase in stringency, which has been altered due to reduction of the number of particles in the entry of second cycle and increase of Tween 20 detergent in washings. Subsequently, enrichment from the second to the third cycle, in all steps, occurred. It has also been observed that the titer in the exit of the third cycle was higher than that of the exit after the first washing cycle. This shows that a positive selection of clones, which represent, in each selection, a specific subpopulation of peptides for the target has also occurred. This can be exemplified herein by means of selection S6 that enriched 1.091 times from the set of clones of the initial library representing the highest rate amongst these selections. In selection Sl, impoverishment occurred (0.6 times), thus showing poor effectiveness in the selection.

In conclusion, this Example has allowed for selection of subpopulations of clones of phages containing recombinant peptides binding to a biological target represented by total B. Microplus anti-Pts Polyclonal IgY. Next procedure (Example 3) is the characterization of sequences of these peptides binding to the biological target used for selection. TABLE 1

Selection Lxbrary Biological Target Cycle input output Rate Enrichment⁵ (CFO/μL)^a (CFU/μL)- (Exit/Entry)-

Ph.D.- > l^c 4,OxIO¹⁰ 3,0x10" 3,0x10^"'

Adult antl-PTS

Sl 12mer 2° 5,OxIO⁸ 3,OxIO¹ 6,OxIO^'6

Polyclonal IgYs 0,6

3° 2,OxIO¹¹ 9,0x10" 4,OxIO^-5

Ph.D.- 1^G 4,OxIO¹⁰ 2,OxIO⁵ 5,0x10^""

Larvae anti-PTS

S2 12mer 2° 5,OxIO⁹ 1,OxIO² 2,OxIO^"6

Polyclonal IgYs

3° 2,OxIO¹¹ 5,OxIO⁵ 2,5xlO^"3

4,OxIO¹⁰ l,2xlθ⁵ 3,0x10^""

Ph.D.- Larvae antl-PTS 2^C 2,OxIO¹¹ 2,8xlO^δ l,4xlθ^"3

S3 16 C7C Polyclooonal IgYs 3⁰ 2,OxIO¹¹ 9,6xlO⁶ 4,8xlO^"3

1° 3,OxIO¹¹ 1,3x10' 4, 3xlO^"6

F88- Larvae anti-PTS 1,OxIO¹⁰ 9,3xlO² 9, 6x10^"°

S4 346

15mer Polyclonal IgYs 3" 1,OxIO¹⁰ l,5xlθ⁵ 1, 5xlO^"3

40 1,OxIO¹⁰ 3,9xlO⁵ 3, 9xlO^"3

1° 2,OxIO¹² 2,4xlO³ 1, 3xlO^"7

Larvae anti-PTS

S5 Cy s 3 2° 1,OxIO¹² 6,0x10" 6, 0x10^"" 250

Polyclonal IgYs

3° 1,OxIO¹² 3,OxIO⁵ 3, OxIO^"5

1° 2,OxIO¹² 1, IxIO⁵ 5, 5xlO^"6

Larvae anti-PTS

S6 Cys6 2° 1,OxIO¹² 2,1x10" 2, 1x10^"° 1091

Polyclonal IgYs

3° 1,OxIO¹- 6,OxIO⁷ 6, OxIO^"3

1° 2,0xl0¹² 5, 6xlO³ 2, 8xlO^"7

Fuse- Larvae anti-PTS 2° 1,OxIO¹² 1,5x10" l,5xlθ^"6

S7 393

6mer Polyclonal IgYs 1,OxIO¹² 1,IxIO⁶ 1,1x10^""

- CFU/μL: Colony forming unit per μL (Ph.D.- 12mer, Ph.D.- C7C) , TU/μL: Transduct unit per μL (F88-15 mer, Cys3, and Fuse βmer) .

- After each selection cycle, the titer of phages selected by polyclonal antibodies was determined and the elution rate was determined by dividing the phage output value divided by the input value of each cycle.

- Enrichment was determined by dividing the rate obtained from the third cycle divided by the first cycle rate for each selection.

Example 3: Analyses of selected peptide sequences. After the biological selection process, the selected clones were characterized by DNA sequencing of a region corresponding to an insert of recombinant peptides (Table 2) . Table 2 lists in numerical order all peptide sequences obtained by DNA sequencing of the region corresponding to the insert fusioned in the library constitutive phage. This representation is in conformity with the library used (SEQ ID N0:l to SEQ ID NO: 46 library Ph. D. -12 mer, SEQ ID NO:47 to SEQ ID NO:74 library Ph. D.- C7C, SEQ ID NO:75 a SEQ ID NO:79 for Library F88-15 mer, SEQ ID NO:80 a SEQ ID NO:90 for library Cys3, SEQ ID N0:91 to SEQ ID NO: 93 for library Cysβ, SEQ ID NO: 94 to SEQ ID NO:107 for library Fuse-6mer, and, finally, SEQ ID NO: 108 to SEQ ID NO-.110 referring to library Ph.D.-C7C. On the left ^' of a sequence it is represented the sequence identification number and on left, in brackets, it is represented the peptide frequency number as it appeared more than once in a selected population.

After three selection cycles, there have been obtained 110 distinct peptide sequences (SEQ ID N0:l to SEQ ID NOrIlO), which are represented by recombinant peptides expressed in filamentous bacteriophages (46 clones of library Ph.D.-12mer, 28 clones of library Ph.D.-C7C, 5 clones of library F88-15 mer, 11 clones of library Cys3, 3 clones of library Cysβ and 14 clones of library Fuse-6mer) and then 3 peptides of an experimental selection. The highest number of clones was obtained by using libraries Ph.D.-12mer and Ph.D-C7C. Library Cys 3 presented the smallest variability of sequences.

Peptide SEQ ID NO:47, SEQ ID N0:91, SEQ ID NO: 18, SEQ ID NOrI, SEQ ID NO: 2, SEQ ID NO:75, SEQ ID N0:7β, SEQ ID NO: 80 were the most frequent in those selections and some of them became the main selected mimotopes, specially when frequency is regarded as one of the principal indications in a selection in function of higher affinity with a biological target . Characterization of peptides and protein motifs obtained in accordance with the present invention has been performed by bioinformatics analysis. Firstly, it was used AAFREQ program (http://relic.bio. anl .gov/aafreqs. aspx) that evaluated frequency of amino acids within a population and also their frequency in the respective predominant position inside a recombinant insert, thereby indicating which amino acids are being super- or sub-represented (Table 3) . In this study, there have been analyzed 107 peptides having an length of from 6 to 16 amino acids per peptide, thus totalizing 1,109 amino acids. Amino acids such as Leucine

(L) , Proline (P) and Serine (S) presented a frequency of

0.1001, 0.0974 and 0.0956, respectively. In this analysis, it was possible to note increase or decrease in frequency of some critical amino acids in the peptide sequence, thus providing important indications of specificity in the process of recognizing ligands for a biological target.

Consensus sequences between peptides were determined by multiple alignments carried out by MOTIF2 program (http://relic.bio. anl . gov/motif2. aspx) . Said program does not allow for conservative substitutions of amino acids, but identical joints of three or more amino acids. A conservative motif for aligned peptides is regarded as such when it is sufficiently long to generate a partial secondary structure. MOTIF2 program identifies continuous and discontinuous motifs in a population of peptides. Table 4 represents multiple linear alignments between amino acids sequences present in selected peptides in accordance with the respective biological selection. In each aligned subgroup, consensus sequences (CS) are represented in bold and individual frequency of each peptide as well as the frequency of each consensus sequence in a subpopulation . By this way, letters in bold denote conservative residues and underlined amino acids denote aliphatic amino acids.

TABLE 2

TABELA 3

AA #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 T. Freq.

A 10 9 4 4 5 4 5 4 1 4 4 4 2 1 0 0 61 0.0550

C 2 0 1 1 3 11 0 1 0 11 1 3 0 1 0 0 35 0.0316

D 6 4 1 2 4 4 4 2 4 1 0 1 0 0 0 0 33 0.0298

E 2 2 5 2 2 3 3 3 1 3 3 1 0 0 0 0 30 0.0271

F 4 2 4 1 7 12 0 1 2 1 1 2 0 0 0 0 37 0.0334

G 11 8 2 10 2 1 5 0 0 1 1 0 0 0 0 42 0.0379

H 6 7 16 7 4 11 5 4 4 2 2 1 0 0 1 0 70 0.0631

I 4 2 1 5 2 3 3 2 3 1 2 1 0 1 1 0 31 0.0280

K 4 3 6 16 5 5 7 7 4 2 3 2 2 1 0 0 67 0.0604

L 3 23 10 9 3 10 7 4 5 8 18 8 2 0 1 0 111 0.1001

M 3 3 2 2 8 3 3 3 1 2 3 5 1 0 2 1 42 0.0379

N 7 5 5 8 4 2 1 1 3 3 4 4 1 0 0 53 0.0478

P 13 10 10 6 9 4 13 6 12 6 3 10 0 3 2 1 108 0.0974

Q 5 5 5 2 5 4 2 4 3 3 2 0 3 0 0 45 0.0406

R 2 1 6 3 3 5 4 5 1 6 1 3 2 0 0 0 42 0.0379

S 16 6 12 11 17 8 8 7 9 -> 4 3 1 1 0 1 106 0.0956

T 1 6 5 16 8 6 12 2 3 4 6 9 3 4 0 0 85 0.0766

V 3 4 2 3 1 3 4 3 2 1 2 0 3 1 0 34 0.0307

W 2 4 5 0 3 2 3 0 5 2 1 2 2 0 0 0 31 0.0280

X 0 1 0 2 0 1 0 0 0 0 0 0 0 0 0 0 4 0.0036 y 3 2 6 5 4 6 6 3 1 2 3 1 0 0 0 0 42 0.0379 total AA 1109

Consensus sequences were aligned and characterized in Table 4. Eight groups of peptides formed consensus sequences by multiple alignments, the motifs being the following: NxxxKxxL (SCl), TPDKS (SC2), PxxKxH (SC3), LHS

(SC4), LHxxL (SC5) , HTS (SC6), PxFF (SC7), and LYGS (SC8) .

The most frequent motifs in the selected subpopulations were PxxKxH, NxxxKxxL and HTS, in 68.2%, 65% and 42%, respectively. TPDKS motif, comprising the overlaid sequences TPDKS in 7 peptides, presents consensus sequences, TPD, TPxK, TPxxS, being comprised of 4 peptides each. In these sequences, TP amino acids along 7 distinct clones were kept constant. A consensus motif was taken into consideration after occurring three or more times in distinct clones. Said analyses showed peptides existing within a same subpopulation subjected to a selection by immunoaffinity with a biological target.

Similarities between consensus sequences with proteins of Boophilus microplus deposited in GenBank were determined by FASTAcom program (Table 5) . In this table, it is shown, in sequential order, identification number of similar sequence for each consensus sequence (No.)_? position of alignment of a peptide with a protein motif of the original protein (Pos) , Accession Number (Accession No.) and the protein name.

Alignment has revealed similarities of motifs to various antigenic proteins and also proteins having important cell functions . The minimum identity number was three amino acids for each consensus. For TPDKS motif, the presence of at least three exact identities, in the alignment, were considered, because the complete motif has not generated alignment. Only PxxKxH consensus sequence has not shown similarity, possibly due to the fact that peptides were selected from libraries Ph.D-C7C and, therefore, it could represent a conformational structure of tick proteins; then determination by linear alignments was not possible. Another hypothesis is that the sequence of the similar protein has not yet been determined and consequently it had not been deposited in a data bank. NXXXKXXL consensus sequence was aligned with the following proteins: Salivary gland metalloprotease, Protein- G-associated receptor; Calreticulin, Glutathione S- transferase and Glyceraldehyde-3-phosphate dehydrogenase. TPDKS consensus aligned with at least three amino acids of proteins: Egg cathepsin, Bm95, GP80 precursor, Intracellular cystatin, Intestinal protein BM86, Phospholipids, Salivary gland metalloprotease, Cathepsin-type protein precursor, Paramyosin, Calreticulin, Membrane B antigen, P450 CYP319A, Heme-binding aspartic proteinase, Reverse transcriptase-type protein and Glyceraldehyde-3-phosphate dehydrogenase. LHS sequence aligned only with a secretion Protein and a P450 CYP319A. LHXXL consensus presented the following alignments: Acetylcholinesterase, GP80 precursor, Intestinal Protein Bm86, Paramyosin, P450 CYP319A1, Esterase and Angiotensinogen conversing enzyme. HTS consensus aligned with GP80 precursor enzyme and Protective antigen 4D8. PXFF consensus aligned with Cytochrome oxidase subunit 1, NADH dehydrogenase subunit 2 and Cytochrome P450. Finally, LYGS consensus aligned with Acetylcholinesterase, Egg cathepsin, GP80 precursor, glucose 6-phosphate dehydrogenase isoform, Actin, Salivary gland metalloprotease, Ferritin, Membrane B antigen, Acetylcholinesterase, esterase, Heme-binding aspartic proteinase, Cytochrome P450, Carboxylesterase- related protein, Cytochrome P450 4Wl and Angiotensin converting enzyme. PXXKXH consensus sequence has not generated alignments with proteins deposited with data banks. This is possibly due to the fact that the molecule conformational structure does not allow for alignments only with linear structure.

In conclusion, this analysis shows that the isolated peptides have critical residues possessing identity and conservation, in which they could mimic the structure and function of original epitopes existing in B. microplus proteins .

TABLE 4

Consensus Frequency Consensus Frequency

VNYNGKEHLLIP 9/74 GLFTPDKSPAKT 2/74

SNNADYKQSLLL 2/74 SIPTYTPDKVTY 1/74

VNWNSWHKTNLS 3/74 HAWQSKTPDKTR 1/74

SPLNNFYKTQLR 3/74 TLFTPDKSPAKT 2/74

IDSNHVYKDFLT 1/74 GALIYTPEKYTI 1/74

MNSWYKFMLPDI** 7/74 GPYDTPMFSLNM 1/74

DAWKMRLSQMYD 22/74 APYDTPWPSPSL 1/74

(SCl) NxxxKxxL* 65,0% (SC2) TPDKS 12,0%

PLEKSHL 32/66 GLHSGIQ 1/66

PLSKMHI. 5/66 SLHSSVQ 1/66

PTEKKHV 2/66 SLHSHLS 3/66

PWSKMHI 1/66 (SC4) LHS 7,5%

PISKEHV 1/66 SLHSHLS 3/66

PLAKQHL 1/66 GLHANLQ 1/66

PMSKSHL 1/66 NLHLGLA 1/66

PPEKIHV 1/66 (SC5)LHxxL 7,5%

PPEKSHL 1/66

(SC3) PxxKxH 68,2%

QLHTSI 2/19 PGPRFF 2/19

QVHTSI 2/19 PAPRFF 1/19

GHTSLR 1/19 PMPRFF 1/19

MLHTSM 1/19 RGPMFF 1/19

NLHTSF 1/19 (SC7) PxFF 26% PLHTSL 1/19 RLLYGS 2/19

:sc6) HTS 42% QLLYGS 1/19 HGLYGS 1/19 :SC8) LYGS 21%

*Bold letters denote conserved residues. **Sequence reverse to peptide IDPLMFKYWSNM Underlined = Aliphatic amino acids.

TABLE 5

15 Angiotensin converting enzyme

660 |gb]AAB04998.1 tt sequence scanned = 198# aa in scanned proteins = 62584

In order to evaluate similarity between a peptide population and a group of protein sequences, analyses of multiple alignments were carried out by using FastaScan program (http : //relic-bio. anl . gov/fastaskan . aspx) . The result generated a list of proteins classified in decreasing order of similarities to peptides. Scores were generated by determining similarity of each peptide sequence along the protein sequence. In Table 6, 206 B. microplus proteins describe in GenBank were classified in accordance with the similarity to local peptide population (107) . Said Table shows only ten proteins having the highest indices of similarity. In Table 7, the same analysis was carried out, and subpopulations of selected peptides were divided in accordance with seven adopted selection strategies and classified in decreasing order, wherein, in this case, only protein with the highest index of similarity for each selection strategy is represented.

TABLE 6

48 2008/000157

TABLE 7

TABLE 8

Further, the presence of similarity between peptides and proteins, deposited in GenBank, restricted to bovine Boophilus microplus tick has been investigated. Analysis was carried out by using the function "Search for Short Nearly Exact Matches" through PAM30 matrix of BLAST software program. Amino acids similar in four or more identical AA within a peptide sequence were determined as mimotope of the corresponding protein sequences. In Table 8, alignments have been classified in decreasing order of frequencies of peptide mimotopes. Said Table, first column, illustrates a ranking, in decreasing order, of proteins, deposited in GenBank, containing motifs similar to the selected peptides . Third column represents the relation of the number of peptides similar to proteins

(mimotopes) and fourth column represents the number of alignments. Sixty six different proteins referring to B. microplus were aligned. Three proteins containing more protein motifs similar to peptides are highlighted, with the most frequent proteins being salivary gland metalloproteases, notch-like proteins and GP80 precursor. Said proteins showing the highest matches in decreasing order were notch-like protein, precursor protein of GP80 protein and salivary gland metalloproteases. As can be seen from this Example, by using bioinformatics analysis methods, phage clones and B. microplus proteins were characterized in accordance with a sequence of linear amino acids, which are constituent parts of these fusion peptides. Thus, it has been possible to demonstrate the antigenic profile of B. microplus by selecting Phage Display libraries. Said analyses revealed the potential use of this specific peptide epitopes as vaccine candidates .

Example 4 : Study of reactivity specificity of selected phages

Various studies have been carried out in order to determine clone validation by using analysis of specific interaction between the selected peptides and chicken anti- protein IgYs of B. microplus . Firstly, an analysis was conducted in order to characterize immunoreactivity of clones, before and after sequencing, by using Phage-Elisa methodology. Immunoreactivity was confirmed through Dot Blot assays. Specificity of the isolated target peptides has been confirmed by inhibition assays through Dot Blot test. Such confirmation of specificity of a recombinant insert, in recognition of an antibody against a fusioned peptide in phage proteins PIII, was determined using Western Blot. Immunogenicity was determined by using assays of immunization of phages in mice so that recognition of immune serum against total tick proteins is detected. Immunoscreening test for determining humoral immune response against isolated peptides was carried out in cattle naturally infested with ticks. Hence, it can been concluded that using immunoreactivity tests for characterization of specificity of clones against biologic target used in various selections allowed for validation of clones for development of new immunogens to be used in a potential vaccine .

Figure 6 and Figure 8 refer to ELISA test, in which immunoreactivity of 370 phage clones diluted in culture medium supernatant and obtained from selection Sl, was determined. Said immunoreactivity of phages was determined against sera of chicken anti-protein of teleogines (biological target) . It was possible to choose 48 clones (A) of immunoreactive phages for DNA sequencing in the region corresponding to the insert (library Ph. D. -12 mer) . Figure 7 illustrates a profile of immunoreactivity of 48 clones, obtained after strategy of selection S3 (library Ph.D.-C7C) . The results evaluated by Elisa index values were significant, although showing differentiated profiles between the tested and control phages (wild-type M13 phage) . In this experiment, ELISA index (EI) for assays was determined by dividing the optical density (OD) by cut off value. Cut off was calculated by adding 2 standard deviations to the mean OD value of wild M13 phage (negative) . EI values above 1 were considered as positive and IE values below 1 were considered as negative. It is important to observe that in this step clones are discriminated in conformity with a provisory number and do not represent the final numeration described in this list. Table 9 shows a compilation of methods used for characterizing clones obtained from studies on antigenicity and immunogenic!ty. Firstly, identity of amino acid sequences selected from DNA sequencing of corresponding inserts (SEQ ID NO) , identification code of these clones in sequencing plates (Plate) , as well as their respective observed frequencies (OF) and relative frequency (Freq., %) were determined. Subsequently, results of tests of Phage- ELISA (PE-(IE)), Dot Blot (D), Dot Blot Inhibition (DC), Western blot (WB) , immunizations in mice (IMU) and immunoscreening in bovine serum (IMT) are sequentially represented.

As shown in Table 9, most of the sequences appeared only once, excepting for some clones that overlaid the others, such as clones SEQ ID NO: 91 (93.3%), SEQ ID

NO:47 (48.5%), SEQ ID NO:75 (40.0%), SEQ ID NO:76 (40.0%),

SEQ ID NO:80 (36.8%), SEQ ID N0:18 (29.7%), SEQ ID N0:l

(18.6%) . This shows that the most frequent peptides have a better affinity, in the selection, with biological target. In order to confirm specificity of said peptides, tests for detecting immunoreactivity against sera of chicken previously immunized with total proteins of larvae and adults of B. microplus, were conducted. In the beginning of this study, specific interactions have been determined by Phage- ELISA (Table 9), Figure 8, Figure 9 and Figure 10. In said test, phages were incubated with positive serum, negative serum and buffer, respectively. ELISA index for assays was calculated by dividing the optical density (OD) by the cut off value. Cut off is then calculated by adding 2 standard deviations to the mean OD of M13 phage (negative control of the phage with no insert) . EI values above 1 were considered as positive and EI values below 1 were considered as negative. The results show that said peptides were specific to the target used in the selection. Most of the results were positive, although differentiated profile between phages was shown, with values being superior to ELISA index (EI) .

TABLE 9

FIA = field immunoassay, EI = ELISA index, OF = observed frequency, WB = western blotting, IMU, mice immunization, D = dot-blotting, CD = Competitive do-blotting, Hr = Hostein resistant, Hs = hosltein sensitive, Nr = nelore resistant, Ns = nelore sensitive, P = Positive, N = Negative, ' PF = Weakly Positive , FF = very weakly positive

By using this methodology, it has been possible to confirm specificity selection as most clones have shown positivity (ELISA Index-EI above 1) and also some identified clones presented higher reactivity index compared to the others, such as, for example, SEQ ID NO: 12, SEQ ID N0:l, SEQ ID NO:7, SEQ ID NO:32, SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:56, SEQ ID NO:48 (Figure 8, Figure 9, and Figure 10) . Figure 8 shows reactivity index of clones selected from selection Sl; Figure 9 shows reactivity index for clones obtained from selection S2; and Figure 10 shows reactivity index of clones obtained from selection S3.

In addition to Phage-ELISA Test, immunoreactivity of phages has also been confirmed by Dot Blot assays. In this test, viral particles were adsorbed on strip-cut nitrocellulose membrane and incubated with polyclonal chicken antibodies specific to B. microplus proteins. The highest reactive clones are shown in Table 9 (column 7) and Figure 11. In Table 9, reactivity signal was alternated in decreasing quantitative values (P = Positive, N = Negative, PF = Very Positive, FF = Very Weakly Positive) . In Figure 11, in Dot Blot test, negative control (NC) is represented by wild-type M13 phage, with no insert, and is located in line 7 and positive control (PC) is represented in line 8; this being the adsorption of total tick proteins on the membrane (Figure 11) . By qualitative analyses, it has been verified that most of the clones exhibited a signal higher than the negative control, especially for selections S2 and S3. These data confirm the high degree of positivity in clones determined by ELISA. Among those clones with higher reactivity are: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:47_f SEQ ID NO.-50, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:70, SEQ ID NO:90.

In a Western Blot experiment, the selected phages were separated by SDS-PAGE and transferred to nitrocellulose membrane in order evaluate whether the purified IgYs could recognize fusioned peptides expressed in PIII and PVIII proteins of phage clones (Figure 12, Figure 13, Figure 14 and Figure 15) . Figure 12 shows Western Blot results for phage clone after selection Sl of this experiment. An arrow indicates the fusioned peptide-containing III protein. Also presented are bands with variable signal intensities, thus indicating an increase in the recognition of peptides selected by tick anti-protein antibodies. Figures 13 and 14 show Western Blotting results for phage clones of selections S2 and S3, respectively. Figure 15 lists Western Blotting results for phage clones obtained after selections S4, S5 and S6. In said analyses, PIII protein and PVIII protein, for the respective libraries, in most clones, were specifically recognized by tick anti-protein chicken antibodies. Said data indicate that PIII and PVIII proteins containing recombinant peptides can be specifically recognized when compared to the wild phage (M13) in a region corresponding to the insert.

In competitive assays, tick proteins in different concentrations prevent chicken IgYs from binding to the phage clones adsorbed on nitrocellulose membrane by Dot Blot assays (Figure 16, Figure 17 and Figure 18) . The results of immunoreactivity inhibition of phages obtained from three strategies of selection (S2 and S3) are disclosed in Table 9. Data show that increasing amounts of tick proteins added to serum interfer with IgY recognition in recognizing phages on said membrane (see Figure 16; clone SEQ ID NO:50) . These data indicate that the selected peptides expressed in phage clones may specifically bind chicken IgY and mimic B. microplus epitopes. By this way, reactivity inhibition can be proved by competitive assays for clones SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:69, SEQ ID NO:70.

Animal immunization was effected in order to evaluate the potential of mimotopes as vaccinal antigens

(Table 9, Figure 19 and 20) . Clone characterization has involved analyses of cross-reactivity of mice immunized with phages obtained from seven selections against B. microplus proteins. Evaluation of mice immune response in order to detect IgG in serum against total B. microplus proteins through immunizations with phage clones was carried out by ELISA tests. Various purified phages were used for immunizing BALB/c female mice via subcutaneous injection. Clones were chosen in accordance with the following criteria: higher frequency; reactivity with ELISA, Dot Blot and Western Blotting; and reduced activity in inhibition assays. Each immunization consisted of 10¹² vp (viral particles) diluted in TBS in the absence of adjuvants. TBS buffer and wild-type M13 phage were used as negative controls and total B. microplus proteins as positive controls. Five animals of each treatment group were immunized. Blood samples were taken from every two weeks and booster in weeks 2, 4, 6, and 8. Subsequently, titer of antibodies reactive against B. microplus proteins was determined by ELISA methods in order to prove the existence of mimeticity between natural epitopes and recombinant peptides isolated from phages. ELISA index (EI) for assays was determined by dividing optical density (OD) by the cut off value. Cut off was calculated by adding 2 standard deviations to the mean OD value of wild-type M13 phage (negative control of phage with no control) . EI values above 1 were considered as positive and below 1 as negative. The results show that peptides were specific to the target used in the selection.

Although having differentiated profile between sera, most of the results showed reactivity, with the values being superior to ELISA index (EI) . Among the groups tested, the serum corresponding to groups immunized with phages SEQ ID- NO:32, SEQ ID NO:26, SEQ ID NO:75, SEQ ID NO:25, and SEQ ID NO: 15 presented the highest index of reactivity.

Immunogenicity and specificity effects on the immune response developed by chemically synthesized peptides have been evaluated by Dot Blot experiment (Figure 21) . Serum (SEQ ID NO: 12) of animal groups immunized at days 0, 30 and 60 with the synthetic peptide corresponding to SEQ ID NO: 16 was incubated with the own membrane-adsorbed peptide, phage clone SEQ ID NO: 18 and wild-type M13 phage. It was noted that the animal group, which was immunized with synthetic peptide, was capable of generating an intermediate degree of antibodies (IgG) response against the own peptide adsorbed on the membrane and also intensively reacted against the recombinant peptide expressed in the viral particle. Wild-type M13 phage was used as vector negative control. Zero day serum (DO) was used as preimmune control. Theses results reassure the immunogenic capacity of peptides synthesized from selections of phage-expressed libraries. Moreover, said phages may be recognized by animal sera reactive to other mimetic structures other than that of the own phage used in sensitizing animals, thereby confirming the potential of immunity cross-reactivity. In view of this aspect, a hypothesis is made in order to detect the potential of a recombinant peptide to be recognized by sera of naturally infested animals. In accordance with this hypothesis, identification of peptides with cross-reactivity to tick antigens by survey of serologic recognition, in Dot Blot tests (figure 22), of said animals submitted to natural exposure in different situations, may occur. Therefore, a group of bovines containing eight Holstein breed animals and eight Nelore breed animals were naturally infested with tick larvae, under controlled conditions. After a determined time for reaching significant infestation, serum of four animals was collected and prepared - two sera of Holstein breed and two sera of Nelore breed. For each breed, an animal having a high tick count and another having a low tick count were selected. As negative control, wild-type M13 phage representing the control vector was used; and as positive control the total B. microplus proteins were used.

The results show differentiated profiles in recognition of phage clones by serum IgG for four treatments (figure 22) : clone SEQ ID NO:2 was exclusively recognized by Nelore animal serum having low tick count. Clones SEQ ID NO: 48; SEQ ID NO: 51; and SEQ ID NO: 93 were predominantly recognized by Holstein animal serum. Clones SEQ ID NO: 32, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:67, SEQ ID NO:83, SEQ ID NO: 92, and SEQ ID NO: 94 were recognized by serum of four animals. Clones SEQ ID N0:12, SEQ ID N0:15, SEQ ID N0:7β, SEQ ID NO: 75, and SEQ ID NO: 80 reacted more intensively to either high tick count Holstein and Nelore sera or low tick count Nelore sera. Clones SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:67, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:92 and SEQ ID NO:80 reacted more intensively to high tick count Holstein animal sera. Clones SEQ ID NO: 12 and SEQ ID NO: 15 reacted more intensively to high tick Nelore animal sera.

These results support the hypothesis that clones have cross-reactivity and mimic structures of proteins which are constituent parts of specific organs of a parasite that could contact a host in natural infestations and trigger a humoral response, thus rendering the so-called exposed antigens. On the other hand, clones that have not presented reactivity could constitute occult antigens due to the lack of recognition of said clones by sera from tick naturally infested animals. Further occurrence of reactivity between breeds and also between animals having higher tick count has been observed. This can be connected with the degree of exposure of a host to a parasite and because sera of these animals present higher reactivity to mimotopes selected in this work.

Example 5 : Reactivity of anti-sera produced by immunization of peptides obtained from selection S2 in mice and bovines . This Example refers to additional confirmation on immunogenicity of peptides obtained from selection S2 by utilizing animal-immunizing assays.

Twelve days after the first immunization, some mice have not yet responded to the immunization: 1 animal of clone SEQ ID NO: 19, 1 animal of clone SEQ ID NO: 32, 1 animal of clone SEQ ID NO: 37, 1 animal of wild-type M13 phage, and 1 animal of PBS control (Table 10) . Table 11 shows levels of antibody level in mice sera against peptide mimotopes fusioned to phage, wild-type M13 phage, phage pool and PBS, calculated by ELISA test, twelve or nineteen days after the first immunization.

Animals immunized with clone SEQ ID NO: 26 were the ones which response gave the highest EI, thereby being statistically different from the other peptides, wild-type M13 phage, and PBS. The response of mice immunized with clone SEQ ID NO: 18 was statistically different from SEQ ID NO: 26, wild-type M13 phage and PBS, and presented the second highest EI. Nineteen days after the first immunization, and seven days after the second immunization, 1 animal of clone SEQ ID NO: 21 and 1 animal of PBS have not yet responded. The most reactive clones were SEQ ID NO: 22, which was statistically different from all the others, except for SEQ ID NO: 41, which was statistically different from all the others, except for SEQ ID NO:22, SEQ ID NO:18, and pool, the pool that was statistically different from SEQ ID NO: 22, wild-type M13 phage and PBS, wild-type M13 phage that was statistically different from SEQ ID NO:22, SEQ ID N0:41 and pool; and PBS was statistically different from SEQ ID NO: 18, SEQ ID NO:22, SEQ ID N0:41 and pool.

Between days 12 and 19 after immunization, a statistical difference occurred in relation to peptides: SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:41 and pool. Increase in EI was observed in SEQ ID NO:22, SEQ ID NO: 41, and pool. For peptides SEQ ID N0:21 and SEQ ID NO:26, a decrease in EI occurred. Table 11 supports the cross-reactivity by the reactivity of mice serum antibodies generated by immunization with peptides fusioned to the phage, wild-type phage, phage pool, PBS, which showed the reactivity of this serum against total tick larva proteins. This Experiment was performed in accordance with ELISA test, ninety days after the first immunization. Sera from animals immunized with peptide SEQ ID NO: 18, which was statistically different from SEQ ID NO: 41, from Wild-type M13 phage and from PBS, was the one showing the highest reactivity against total tick larva proteins (Table 11) . TABLE 10 Group 12 Days 19 Days

Seq. ID N^≥ 18 3,70 ± 0,557 Ab 3 , 10 + 0 , 173 Abed Seq. ID N^≤ 19 2,03 ± 1,069 AbC 2 , 43 ± 1 , 124 Acde Seq. ID N^ 20 2,60 ± 1,375 Abe 2 , 60 ± 1 , 179 Acde Seq. ID N^≤ 21 1,97 ± 0,851 AbC 1 , 50 ± 0 , 872 Bcde Seq. ID N- 22 1,50 ± 0,265 Bbc 4 , 87 + 0 , 723 Aa Seq. ID N- 26 5, 63 ± 0,764 Aa 2 , 83 + 0 , 473 Bcde Seq. ID N^≤ 32 1,70 ± 1,136 ^Abc 1,87 ± 0,208 ^Acde Seq. ID N² 37 1,47 + 0,513 ^Abc 1,80 ± 0,600 ^Acde Seq. ID N² 41 2,40 ± 1,179 ^Bbc 4,37 ± 0,710 ^Aab Wild M13 1,07 ± 0,153 ^Ac 1,43 ± 0,252 ^Ade Phage Pool 1,63 ± 0,503 ^Bbc 3,27 ± 0,116 ^Abc

PBS 1,07 + 0,351 ^Ac 1,13 + 0,208 ^Ae

Data are mean ± standard deviation.

Different capital letters in the same line represent statistic difference between collection days.

Different lower case letters on the same column represent statistic difference between experimental groups. t-Student test having 5% significance level was used for comparison between collection days.

For comparison between experimental groups, variance analysis was used and when a significant difference occurred, the Student Newman-Keuls multiple comparison test with 5% significance level (P<0,05) was used.

__^ TABLE 11 Group 19 Days Ag. Total 19 Days

Seq. ID N≤ 18 3, 10 ± 0, 173 ^Abcd 3,00 ± 1, 609 ^Aa

Seq. ID N≤ 19 2,43 ± 1,124 ^Acde 2,20 ± 0,700 ^Aabc

Seq. ID N≤ 20 2, 60 ± 1, 179 ^Acde 2, 60 + 0, 436 ^Aab

Seq. ID N^≤ 21 1,50 ± 0, 872 ^Acde 1, 57 + 0, 493 ^Aabc

Seq. ID N≤ 22 4, 87 ± 0,723 ^Aa 2, 13 ± 0,322 ^Babc

Seq. ID N^ 26 2,83 + 0, 473 ^Acde 1,37 ± 0,252 ^Aabc

Seq. ID N≤ 32 1, 87 ± 0,208 ^Acde 1, 87 ± 0,306 ^Aabc

Seq. ID N≤ 37 1,80 ± 0, 600 ^Acde 2,40 ± 0,436 ^Aabc

Seq. ID N^≤ 41 4,37 ± 0,710 ^Aab 0, 83 ± 0,153 ^Bc Wild Ml 3 1 , 43 + o, 252 Ade

1 , 17 + o, 153 Abe

Pool 3 , 27 + o, 116 Abe

1 , 57 + o, 404 Babe

PBS Ae

1 , 13 + o, 208 1 , 00 + 0 , 200 Abe

Data are mean ± standard deviation.

Different capital letters in the same line represent statistic difference between collection days.

Different lower case letters on the same column represent statistic difference between experimental groups. t-Student test having 5% significance level was used for comparison between collection day 19 of peptides with total antigen.

For comparison between experimental groups, variance analysis was used and when a significant difference occurred, the Student Newman-Keuls multiple comparison test with 5% significance level (P<0,05) was used.

Twelve days after the first immunization (Table

12), sera from two immunized bovines immunized with pool showed reactivity with peptides SEQ ID NO: 19, SEQ ID NO:20,

SEQ ID NO:21, SEQ ID NO: 22, and one of the bovines has not responded to SEQ ID NO: 37, wild M13 and PBS. The most reactive clones were the following: SEQ ID NO: 41, statistically different from SEQ ID NO: 19 and SEQ ID NO: 21; SEQ ID NO:26 and SEQ ID NO:18, which had not statistically differed from the other clones, Wild-type M13 phage, pool and PBS (Table 12) . Table 12 shows antibody levels, as calculated by ELISA test, in serum of bovines immunized with peptide pool against each one of the peptides fusioned to the phage, phage pool, PBS, and of bovines immunized with wild-type phage against the own wild-type M13 phage, at days 12 and 19 after the first immunization.

The most reactive bovine clones were the following: SEQ ID NO:18, SEQ ID NO:26 and SEQ ID N0.-41. These first three clones did not show statistically difference between them but were different concerning the others, excepting for SEQ ID NO: 41. Between days 12 and 19 after immunization, it is observed a statistical difference only for peptide SEQ ID NO: 21, where an increase in EI occurred.

TABLE 12

Group 12 Days 19 Days

Seq. ID Aa

N≤ 18 1,60 ± 0,424 Aab 2,15 ± 0,071

Seq. ID N≤ 19 0, 65 + 0,354 Ab 1,00 + 0,141 Ae

Seq. ID N≤ 20 0,80 ± 0,283 Aab 1,15 ± 0,071 Acde

Seq. ID N≤ 21 0,70 + 0,000 Bb 1,45 ± 0,071 Abed

Seq. ID N≤ 22 1,00 + 0,000 Aab 1,50 ± 0,283 Abed

Seq. ID N≥ 26 1,70 ± 0,141 Aab 2,10 + 0,141 Aa

Seq. ID Aab

N≤ 32 1,50 + 0,141 1,55 + 0,071 Abe

Seq. ID N≤ 37 1,35 ± 0,495 Aab 1,30 ± 0,141 Acde

Seq. ID N≤ 41 1,85 + 0,212 Aa 1,85 ± 0,071 Aab

Wild M13 1,00 + 0,424 Aab 1,30 + 0,141 Acde

Phage Pool 1,20 + 0,283 Aab 1,50 + 0,141 Abed

PBS 1,05 + 0,212 Aab 1,05 + 0,071 Ade

Data are mean ± standard deviation. Different capital letters in the same line represent statistic difference between collection days .

Different lower case letters on the same column represent statistic difference between experimental groups. t-Student test having 5% significance level was used for comparison between collection days.

For comparison between experimental groups, variance analysis used and when a significant difference occurred, the Student Newman-Keuls multiple comparison test with 5% significance level (P<0,05) was used. ELISA index was calculated such as for the mice.

Evaluation of similarity of the selected peptides SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:22 and SEQ ID N0:41 with tick B. microplus proteins deposited in GenBank

(http : //www. ncbi .nlm. gov) has shown that, among the four aligned target proteins, calreticulin was the one that obtained the highest number of alignments, including 3 of the 4 peptides cited.

Bovines challenged twelve days after the first inoculation have not presented, 24 days after the first infestation, ticks having between 4.5 and 8.0. The amount of larvae that are able to be fixed on a susceptible animal and thus to complete the cycle is known to be 20% (Willadsen et al., 1977 cited by Verissimo, 1990) .

Oviposition test (Figure 23) has shown changes in morphologic aspects of teleogines in the treatment group (A) compared to control group (B) . Teleogines presented a blackened color suggesting hemorrhagic damages. Visual Reduction in the mass of eggs after egg laying was observed in the treatment group compared to the control group.

In conclusion, this Example shows that: Immunorespon.se is obtained in mice and bovines against the selected peptides; Infested bovines have not presented any amount of tick between 4.5 and 8.0 mm-size for counting; Peptides that conferred immunoresponse with the highest titer machted with calreticulin protein.

From the Examples given above, one may conclude that mimotopes obtained for total B. microplus proteins through selections using Phage Display process presented promising results to develop a vaccine. After three rounds of selection, excepting for experiment S4 that occurred in four selections, clones were characterized by validation. CFU number as obtained after each selection cycle, provided for enrichment, thereby indicating that the process was effective. It was possible to identify 107 distinct sequences amongst 286 viable sequences obtained from DNA sequencing. Deduced amino acid sequences were analyzed by using different bioinformatics methodologies, whereby important motifs have been produced. Characterization of mimotopes specificity could be done by immunoreactivity tests. Immunization with mimotopes in mice confirmed the cross-reaction against tick proteins. By immunoscreening test, cross-reactivity and identification of differentiated reactivity profiles between strains in recognizing phage clones by IgG of serum from naturally infested animals were confirmed. Furthermore, after challenge in bovines, the significant degree of damages in tissues of teleogines after parasitism in animal inoculated with recombinant phages was determined. Further, released females containing between 4.5 and 8.0 mm were nonexistent. These results represent an advance towards production of new immunogens for preparing a new vaccine.

The present invention shows the existence of a damage factor generated against the parasites as evidenced by the obtained results. These results validate clones as important candidates for vaccinal B. microplus antigen epitopes and show the great importance of the present invention in the field of vaccines . The presence of a molecular tool that directs the development of a polyimmunogenic vaccine is based on the use of a huge number of data based on peptide sequences. In this work, a hundred seven different sequences representing different bovine tick epitopes were produced. Said sequences represent a bank of epitopes selected by immunointeraction with antibodies that recognize total proteins in the stage of larvae and teleogines of B. Microplus ready to be tested in different formulations.

It is herein hypothesized that said peptides may represent different epitopes having different degrees of importance for generating immune response in bovines. By this way, some of these peptides have been subjected to. mice and bovine immunization assays in order to measure immunogenicity, an essential characteristic of vaccines, although the absolute majority showed antigenic reactivity to B. microplus protein recognizers through immunochemical tests. Hence, object of the present invention certainly will be improved or modified without departing from the concept and scope of the invention, which is limited to the contents of the accompanied claims.

The present invention that is directed to the development of immunogens for controlling bovine tick has the following advantages :

I)A vaccine with potential for controlling ticks, based on the expression in vectors or chemical synthesis;

2) Highly purity levels because its production does not involve complicated purification processes; 3) Complete antigen characterization.

4) Safety due to the absence of contaminants;

5) Medium scale reproducibility;

6) High stability in view of the expression system or chemical synthesis, rendering it more suitable for storage; and

7 ) Low production costs on industrial scale due to the fact that phage exhibits unlimited reproduction and simplified purification.

Social and commercial value of the present invention is of great importance since the knowledge of how to effectively control these parasites is fundamental for formulation and application of animal sanitation; for suitable use of already known methods; and for development of new control tools. On the other hand, the lack or inaccuracy of this knowledge severely compromises the effectiveness of strategies of controlling these parasites, increases waste of public resources; increases costs of farming and cattle raising business; and exposes rural populations to reduction of income due to low productivity.

In this scenario, the knowledge of an effective control in the form of vaccines represents a substantial advance for consumers. Because said form is residue-free, it will improve food quality and favor safety food matters and environmental issues.

Examples will be better understood by the detailed description of the adopted experimental procedures given herein below. Peptide mimotopes were obtained by using biological selection methodology through Phage Display filamentous phage libraries. Said technique refers to a selection manner in which a peptide or protein library having randomized sequences is expressed in the outside of a viral particle, while coding genetic material for each residue is found in viral genome. Hence, it is possible to have a correlation between each variant protein sequence and its respective DNA sequence, with which fast characterization based on binding affinity for a target molecule can be effected.

Carrying out of immunoaffinity biological selection firstly requires the production of B. microplus anti-protein polyclonal antibodies for selecting peptides expressed in Phage Display libraries. In this process, biologic targets were chicken polyclonal IgY obtained by immunizations with total protein extract from B. microplus larvae stages and teleogines .

Inoculum to be used in said immunizations are obtained by extracting total proteins from B. microplus larvae, macerating in nitrogen liquid, and then adding ethyl ether for degreasing the proteins for 18 hours. Further extraction buffer (4OmM HEPES pH 7.4, 10 mM EDTA, 2mM EGTA, ImM DTT, ImM Benzamidine, 0.5mM PMSF) was added to the macerate and centrifuged for 40 minutes at 4.000 g. The supernatant was collected and extensively dialysed in saline and quantified. The protein profile was measured by SDP-PAGE

(10%) . For immunization, White-Leghor three-week-old chickens were used. Immunization, scheme was described by Barbas et al . , 2001, with some modifications. The first dose containing 200 μg and the three subsequent doses containing total B. microplus proteins were administered by intramuscular route at 14 days interval, for each dose. Said first dose contained complete Freund' s adjuvant (Sigma Chemical Co., USA) and said subsequent doses contained incomplete Freund' s adjuvant (Sigma Chemical Co., USA) . Immunized animals were bled from the third dose and measured for production of antibodies, using ELISA method.

For Immunoenzymatic Assay (ELISA) , high affinity microtiter plates (NUNC) were sensitized with 10 μg total tick proteins, and then diluted with carbonate/bicarbonate buffer, overnight, at 4°C. Next, said plate was blocked with 5% PBST, for addition of chicken serum, before and after immunization, and incubated for 60 minutes at 37⁰C. After this period of time, said plates were washed, followed by addition of secondary antibodies (chicken anti-IgY rabbit IgG) diluted in 5% blotto, for 60 minutes. The Immunoenzymatic conjugate [rabbit anti-IgG goat IgG coupled to peroxidase (Sigma Chemical Co., USA)] was diluted in 5% Blotto and incubated at 37°C for 60 minutes. Specific binding was resolved by addition of enzyme substrate containing o-phenylenediarαino (OPD) (Sigma Chemical Co., USA) . After 15 minutes, the reaction was finalized with sulfuric acid, and reading was made at 492 nm in a microplate reader (Flow Titertek Multiskan Plus - USA) . After a satisfactory titer is observed, blood and also eggs of said chickens inoculated with antigens were collected for the preparation of polyclonal immunoglobulins .

IgY specific for adults (teleogines) tick proteins were obtained from eggs and then firstly concentrated by using method of dilution in water and subsequently precipitated with ammonium sulfate. Further DEAE-Sephacel chromatography (buffer 0.02M tris-HCl pH 4.5) was performed and IgY-containing fractions were lyophilized, and stored at 4°C. Purification of IgY from chicken serum was carried out through 5 mL HiTrap IgY Purification HP Column (Amersham Biosciences) . Peristaltic pump was filled with elution buffer (2OmM Na₂HPO₄ pH 7.5) and then connected to the column. Said column was washed with 5 volumes (25.0 mL) of each buffer: binding (2OmM Na₂HPO₄, 0.5M K₂SO₄, pH 7.5), elution and cleaning (2OmM Na₂HPO₄, 30% v/v isopropanol, pH 7.5) . Next, said column was balanced with 5 volumes of binding buffer and then sample (6.0 mL serum) was injected. Said column was washed with 10 volumes (50.0 itiL) of the same binding buffer. IgY' s were then diluted with 10 column volumes of elution buffer and 1.0 inL samples were collected and further quantified in a spectrophotometer at 280 ran. Said column was regenerated with 8 volumes (40.0 mL) of cleaning buffer, and rebalanced with 5 volumes of binding buffer. As said samples were filtered through the purification columns, optical readings (OD₂80nm) from the aliquots were taken. For dialysis of the obtained samples, a

Fisherbrand^® cellulose membrane (regenerated cellulose- DYALYSIS tubing) was used. The purified samples were arranged in dialysis tubes (tubes 1 to 8), concentrated in sucrose for 1 hour at 4°C, and then dialysed in PBS (Saline phosphate buffer solution: 137mM NaCl, 2.7mM KCl, 12mM Na₂HPO₄, 1.2mM KH₂PO₄, pH 7.4) for 12 hours at 4⁰C. The samples were quantified in spectrophotometer at 280 nm.

The quality of the purified IgY' s samples were determined by SDS-PAGE (16%) . The sample buffer contained 1% SDS, 1% Tris pH 6.8, 0.02% bromophenol blue, and 20% glycerol, with or without reducing agents. Electrophoresis run was performed at room temperature with upper buffer 0. IM Tris, 0.1M Tricyne, and 1% SDS and with lower buffer 0.2M tris-HCl, pH 8.9, until the indicators leave, at 100V. After this run, polyacrylamide gel was poured into a solution of Coomassie blue R-250 as protein dye.

Western Blotting analysis for the total proteins, electrophoresis (16% SDS-PAGE) and gel electrophoresis of proteins in nitrocellulose membrane (Sigma Chemical Co., USA) were carried out in accordance with techniques described by Sambrook et al . (Cold Spring Harbor, pl8. 60- 18.75 (1989)), with some modifications. Said nitrocellulose membrane was blocked by 5% blotto and incubated with purified antibodies (IgY) diluted with 5% blotto overnight at 4⁰C. Rabbit anti-IgY antibodies (Sigma Chemical Co., USA) diluted in 5% blotto were incubated at room temperature for two hours. Finally, rabbit anti-IgG goat antibodies marked with alkaline phosphatase (Sigma Chemical Co., USA) were incubated for 2 hours at room temperature and specific reactivity was determined by addition of NBT/BCIP substrate (Sigma Chemical Co., USA) .

By this way, the next step was the carrying out of selection of recombinant peptides through combinatory libraries constructed from phages. Filamentous phages (M13, fl, fd, among others) pertain to the bacteriophage family Inoviridae and contain single-stranded DNA (ssDNA) as genetic material. For the preparation of B. microplus mimotopes, six distinct libraries constructed from said filamentous phages comprising three different vectors (M13, fUSE and f88) were used.

Four libraries comprising f88 vectors (f88 15 mer, f88 cys 3, f88 cys 6) and fUSE vector (fUSE 6 mer) kindly furnished by Smith were used; the peptides being from libraries f88 15 mer, f88 cys 3, f88 cys 6 expressed in PVIII and peptides from the library fUSE 6 mer expressed in pill. F88 15 and fUSE vectors express linear peptides and f88 cys vectors express cyclic peptides.

Other two libraries were purchased from New

England Biolabs . Said libraries are composed of random peptides followed by a short spacing sequence Gly-Gly-Gly fusioned to the minor capsid protein N-terminal region

(Protein III) of filamentous M13 bacteriophages. Ph. D.12 mer library and Ph. D.7 cys library present 12 and 7 random peptides, respectively. Ph. D.7 cys library contains cyclic peptides, while Ph. D.12 mer is linearized by delimiting cysteins in the peptide ends. These libraries consisted of approximately 2.8 x 10¹¹ independent clones, which represent 1.9 x 10^s possible combinations contained in amino acid residues. All five copies of the phage capsid III protein comprise random peptide amino terminals (Noren & Noren, METHODS, V. 23, pp. 169-178, 2001) .

Seven distinct strategies were used for the biological selection in accordance with the following classification:

Strategy 1: Chicken polyclonal antibodies were subjected to purification by affinity with the immunogen in conformity with techniques described by Sambrook et al . (1989), with some modifications. Tick protein extract was adsorbed on a nitrocellulose membrane (Ge Healthcare) overnight at 4⁰C and subsequently blocked with 5% milk powder for one hour at room temperature. Said membrane containing the proteins was incubated with partially purified IgY fraction overnight at 4⁰C and subsequently washed with PBST (once) and more three times with PBS, monitored, in a spectrophotometer reader, after washing is complete, to investigate release of proteins from the membrane. Antibodies were eluted with 10OmM glycine pH 2.8, and immediately after elution, they were neutralized with 10OmM Tris pH 8.0 and concentration of the eluate was evaluated by spectrophotometry (OD 280) . By this way, in order to enhance the specificity of the peptide selection, said polyclonal IgY' s used in the selection were subjected to affinity purification against the total parasite proteins by eluting the binding fraction. By this method, only specific tick IgY' s were subjected to selection against the random peptide library.

In a well of a 96-well microtiter plate (Maxisorp - Nunc) 150 UI of polyclonal total anti-protein IgY of teleogines purified by affinity with immunogen (100 μg/mL in 0.1M NaHCO₃, pH 8.6) and incubated overnight at 4°C in a container containing a humidified paper tissue. Said plate was blocked with 250 ul blocking buffer (0.1M NaHCO₃, pH 8.6, 5 mg/mL BSA) for 2 hours at 4°C. Said plate was then washed (six times) with TBST (TBS containing 0.1% Tween 20 v/v) , and 4 x 10^1Q phages from the Ph.D.12mer library (New England Biolabs) diluted in 100 ul TBST were added to the plate holes and maintained under stirring for 1 hour at room temperature. Non-binding phages were removed by washing said plate holes, 10 times, with TBST (0.5% Tween-20) in the first round and, in the subsequent rounds, with TBST (0.5% Tween-20) . Binding phages were eluted with 100 ulelution buffer (0.2M glycine-HCl, pH 2.2, containing 1 mg/mL BSA) for 10 minutes at room temperature and immediately neutralized with 15 ul neutralizing buffer (IM Tris-HCl, pH 8.0) . Aliquots of the eluted phages were used for titer determination. The remaining phage-containing eluate was used for re-amplification, in the next round, by infection with E. coli ER 2738. Threes rounds of enrichment of phages containing the binding peptides were conducted.

Strategy 2: In this process, the same library and the same selection process as described above were used, except for the alteration in the biological target by adsorption on ELISA plates of anti-total proteins of B. microplus larva tick polyclonal IgY' s, purified by affinity with immunogen.

Strategy 3: This process was similar to the former one excepting for the use Ph. D.7 cys library (New England Biolabs) . The selection was carried out in the same form as that of strategy 1, excepting for competitive elution, and the polyclonal antibodies, as selection biological target, which were not purified by affinity to immunogen. Strategy 4: A linear f88 15mer library was used.

Microtiter plates (NUNC MAxisorp) were sensitized with streptovidin for 1 hour at 37⁰C and then blocked by using blotto at 37⁰C for 1 hour. Anti-B. microplus larva IgY' s and also preimmune IgY' s as negative control were conjugated with biotin. After washing the plate three times with TBST, IgY' s purified from pre- and post-immune serum of animals immunized with tick larva proteins were added into holes for 2 hours at room temperature, with stirring. After six washings with TBST, pre-adsorption of the library against preimmune IgY' s was performed for 1 hour at 37°C. The supernatant was collected and subsequently incubated with IgY' s obtained from post-immune serum (biological target) . Four selections for enrichment of the phage-subpopulation were effected.

Strategy 5: Selection was carried out in accordance with strategy 3, except for the use of Cys 3 library. For this library, the entry titer was 2xlO¹² TU at the first entry and 1 x 10¹² TU at the remaining entries of a new selection cycle .

Strategy 6: Conformational f88 cys 6 library was used. In this case, anti-IgY rabbit antibodies were adsorbed on holes of a microtiter plate (NUNC MAXISORP) for 1 hour at 37⁰C and subsequently blocked with TBSTB for 1 hour at 37⁰C. After washed with TBST, the animal serum of animals pre- and post-immune to tick larva proteins was added to separate holes for incubation for 2 hours at room temperature, with stirring. After washing (6 times) with TBST, pre-adsorption of the library against preimmune IgY' s was carried out for 1 hour at 37°C. The supernatant was collected and further incubated with IgY' s obtained from post-immune serum

(biological target) . Three selections for enrichment of phage subpopulation were conducted. Strategy 7 : Methodology similar to that used in

Strategy 6 except that the library used was a fUSE βmer.

For Ph.D. libraries (New England Biolabs), during and after immunoaffinity selection, characterization of the selected clones were effected through α-complementary system. A phage vector used contains lacZ gene thus allowing for reactive clones to be selected by coloring colonies (blue positive or white negative) . For titrations, a mixture of phages and bacteria in agarose on the surface is used. Said mixture is plated on a LB medium comprising X-gal (40 μg/mL) and IPTG (0.5 mM) . In the course of the steps, a small amount of eluate (10.0 μL) was titrated and the titrant was used for re- amplification, performed in 20.0 mL of tetracycline- comprising E. coli (ER2537) culture in the initial growth phase (OD₆oo ≤0.3) . Said culture was incubated in a shaker for 4-5 hours, at 37°C, before the step of precipitating phages and subsequent titration. Said culture was transferred to an Oakridge tube

(Hitachi, 50 mL) and centrifuged (10 minutes, 10.000 rpm) .

Residual cells were discarded and the supernatant transferred to a clean tube and then centrifuged. Eighty percent of supernatant were pipetted into a sterilized tube and 20% by volume of PEG/NaCl (20% w/v Polyethylene glycol- 8000, 2.5M NaCl, water), and the mixture was kept standing for 12 hours at 4⁰C.

In the next day, the precipitate was centrifuged for 15 minutes at 10,000 rpm, at 4°C. The supernatant was discarded and the tube was briefly centrifuged so that residual supernatant could be removed. This precipitate was resuspended in 1.0 mL TBS, transferred to a microtube and centrifuged for 5 minutes, a 10,000 rpm, at 4°c for precipitation of cell residues. The supernatant was transferred to a new microtube for addition of PEG/NaCl (1/6 volume) . The resulting mixture was incubated on ice for 1 hour and then centrifuged (10 minutes, 14,000 rpm, 4⁰C) . The supernatant was discarded, and centrifugation was repeated for removal of residual supernatant. The precipitate was resuspended in 200.0 μL TBS thus rendering the amplified eluate that was ready for titration.

Reamplified phages from the first selection cycle were used in a second cycle and thus subsequently, totalizing three cycles, wherein from the second cycle, stringency of washing buffer was increased from 0.1% to 0.5% Tween 20, in all washings.

All titrations used 1.0 μL phages diluted in 9.0 μL of LB culture medium. Dilutions (10^"1 to 10^~4 for non- amplified eluate and 10^~8 to 10^~u for amplified phages) were incubated with 200 μL E. coli (ER2738) in initial growth phase for 5 minutes and plated on LB medium containing IPTG (0.5 mM) and X-gal (40 μg/mL) , together with 3.0 mL Agarose Top (10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 1 g MgCl₂.6H₂O/liter) .

After incubation in a muffle overnight at 37°C, blue colonies were counted to render entry and exit titers for all selection cycles (number of blue colonies x dilution factor) .

The blue-colored colonies, demonstrating break at X-GIa substrate and expression of phage β-galactosidase gene of phages by ER2738 bacteria, were separately reamplified in Deepwell plates for storing the selected clones. For this, each isolated colony obtained from the 4^th cycle (non- amplified) was discarded in a Deepwell well comprising 1.0 mL of a culture medium containing E. coli in initial growth phase. This plate was incubated for 12 hours at 37⁰C, with stirring. Deepwell plate was centrifuged for 60 minutes at 3,700 rpm for removal of supernatant from the culture. Its contents were transferred to another Deepwell plate, followed by addition of 1/6 volume of PEG/NaCl (20% polyethylene glycol-8000, 2.5M NaCl) and incubation thereof for 12 hours at 4°C. Said plate was centrifuged for 1 hour at 3,700 rpm, the supernatant discarded and the precipitate suspended in 200.0 μL PBS.

Amplification of phages from the libraries expressed in Fd-tet vectors of each bopanning selection cycle started by adding 5 mL culture of E. coli K91 in the exponential growth phase. After incubation for 10 minutes at 37°C, tetracycline resistance started by addition of 95 mL LB medium comprising tetracycline in a concentration of 0.2 μg for 30 minutes at 37⁰C, with stirring, at 250 rpm. Tetracycline was added to a final concentration of 15 μg/mL. The propagation of infected bacterial cells was realized at 37°C overnight with stirring at 250 rpm. The suspension of bacterial cells was two-folded concentrated (10.000 rpm and 12 rpm) . The resulting phages were purified from the supernatant by precipitation in PEG8000/NaCl (1/6 volume) overnight at 4⁰C. In a second precipitation, the pellet diluted in TBS and PEG8000/NaCl (1/6 volume) was incubated on ice for 2 hours and then centrifuged (10 minutes, 4°C) . The supernatant was discarded, and centrifugation was carried out again for removal of residual supernatant. The pellet was re-suspended in 200.0 μL TBS to render an amplified eluate ready for titration.

Concentration of phages (viral particles per mL) was determined by UV absorbance at a wavelength between 240 and 320 nm and the highest peak of absorption at 269 nm. Viral concentration calculation was based on the following formula: viral particles per mL =(A269 x 6xlO^lβ/number of nucleotides in the phage genome) . The final phage stock was stored at 4°C. The final eluate was titrated to provide for separate colonies so that said colonies are substantially individualized in order to be propagated in a independent form for subsequent analysis.

To obtain phage DNA, 10.0 μL of purified phages in Deepwell plate were added to 1.0 mL ER2738 initial growth phase culture in 15 mL Falcon tubes, where they remain under strong stirring for 20 hours at 37°C. After growth, said cultures were transferred to microtubes for settling of bacteria, which were centrifuged for 10 minutes at 4,000 rpm. Five hundred microliters of supernatant were transferred to another tube and 200.0 μL PEG-NaCl were added thereto, and said tube was then incubated at room temperature for 10 minutes. After centrifuging for 10 minutes at 14.000, the supernatant was discarded, and the pellet was resuspended with 100.0 μL iodide buffer (1OmM Tris-HCl, ImM EDTA, 4M NaI), and then 250.0 μL absolute ethanol were added thereto followed by incubating said tube for 10 minutes at 14,000 rpm. The supernatant is then discarded followed by washing the pellet with 500.0 μL 70% ethanol and again centrifuging for 10 minutes, at 14,000 rpm. The supernatant was discarded and the pellet suspended in 20.0 μL of sterile ultra-pure water.

Sequencing was carried out by using Dye Terminator Kit (Amersham Biosciences) and a MegaBace 1000 Automatic Sequencer (Amersham Biosciences) . The following primers were used in the sequencing reaction: M13 5'-

_HOCCCTCATAGTTAGCGTAACG-3 '-Amersham Biosciences), f88-4/15- mer (5' -_HOAGAAGTCCGAAGACGATA-3' -Invitrogen) and fUSE5/6-mer

(5'-_HoGGAGTATGTCTTTTAAGT-3'-Invitrogen) that amplifies the region of random peptide-coding amino acids in recombinant phages.

Analysis of DNA sequences from automatic sequencer has been processed in a software of the own equipment (Sequence Analyser, BASE CALLER, Cimarron 3.12, Phred 15) . Immediately after this pre-analysis, vector sequences were withdrawn and only those inserts with perfect residues were translated.

DNA sequences produced by sequencing were analyzed by on-line available bioinformatics programs:

• Translation of amino acid sequences obtained in sequencing occurred through DNA2PRO7 program

(http://relic.bio.anl. gov/dna2pro7. aspx) ;

• Peptide population was characterized for amino acid frequency using AAFREGS program (http : //relic .bio . anl . gov/aafreqs . aspx) .

• Protein motifs of peptides were produced by M0TIF2 (http: //relic .bio . anl .gov/motif2.aspx) . This program does not allow for substitutions of conservative amino acids, but identical joints. A motif is regarded as conservative for aligned peptides when they are long enough for generating a partial secondary structure.

• FASTAcom program (http: //relic .bio . anl . gov/fastaconsen. aspx) seeks for homologies among consensus sequences, wherein they can be continuous, discontinuous, and have multiple proteins known from Boophilus microplus obtained from "GenBank".

• Search for similarity among peptide sequences was performed by ClustalW program through multiple alignments.

• Search for similarity, between Boophilus microplus peptides and proteins, was carried out by research in public data bank, deposited in "GenBank", by BLAST (Basic Local Alignment Search Tool- http: //www. ncbi.nl, .nih.gov/BLAST/) .

• the produced protein motifs were analyzed for homologies with tick proteins deposited in "GenBnak" by BLAST program -Basic Local Alignment Search Tool (http : //www . ncbi . nlm. nih . gov/BLAST) ; • Search for protein patterns and taxonomic lineage referring to protein families and domains common in species, genus or taxons to which peptides pertain was made by using PROSCAN program (PROSITE SCAN) . This program investigates all peptides against PROSITE, a data bank of protein families and domains, which consists of sites, patterns and profiles biologically significant that help to reliably identify a known protein family, in case it exists (http: //npasa-pbil .ibcp.fr/cgi- bin/npsa automat .pl?page=npsa prosite.html) .

In order to identify and validate clones obtained by biological selection, immunoreactivity and immunogenicity studies have been carried out in in vitro and in vivo experiments. Firstly, clones were tested for determination of antigenicity by ELISA, Dot Blot, Dot Blot inhibition and Western Blot. Immunogenicity profile was investigated by immunization assays in mice and bovines, in addition to studies for determining immune recognition in naturally infested animals (immunoscreening) . Animals were also immunized and challenged for evaluating possible effects concerning protection of a host from parasite.

ELISA tests were performed in order to measure reactivity of recombinant peptides (expressed in selected phages) against antibodies present in sera from chickens immunized with total proteins of cattle tick. A high affinity plate (NUNC) was sensitized with 1 μg/well of anti- tick IgY purified polyclonal antibody diluted with 100.0 μL/well of carbonate buffer (6OmM NaHCO₃, pH 9.6) and incubated for 12 hours, with stirring, at 4°C. After three washings with 0.05% PBS-T (PBS + 0.05% v/v Tween 20), 250 μL blocking buffer (0.05% PBS-T - 5% skimmed milk powder) were added and the plate incubated for 1 hour at room temperature. Further, the plate was washed more β times and 100.0 μL of a phage-containing solution (one clone for each well) were added. After incubation for 1 hour at 37°C, six additional washings were conducted. For detection, a peroxidase-marked anti-M13 anti-body conjugate diluted 1:5,000 (GE healthcare) in blocking solution and incubated for 1 hour at 37°C was used. Next, said plate was washed six additional times and then resolved with 100.0 μL/well of a OPD-containing solution (O-phenylenediamine dihydrochloride - Sigma Chemical), H₂O₂ and citrate buffer (0.1M) . The reaction was quenched by adding 10.0 μL 4M H₂SO₄. Absorbance reading was effected in a Multiscan Plus reader (Thermo Plate) version 2.03, 492 nm filter. Wild-type M13 phage was used as a negative control and also preimmune sera purified polyclonal antibodies, which are non-reactive to total tick proteins .

In Dot Blotting experiment with tick anti-protein chicken polyclonal antibody, a nitrocellulose membrane was sensitized with 2 μL of each spot-selected clone (total of 107 clones) . Said membrane was incubated, washed three times for 5 minutes each with TBST (TBS + 0.1% v/v Tween 20) . After washing is complete, said membrane was blocked for 1 hour at room temperature with 1 mL blocking buffer (0.1% PBS-T - 5% skimmed milk powder) . Hundred microliters of blotto with polyclonal sera in 1/100 dilution in blocking buffer were pipetted over the recounted membrane disposed at individual troughs. Prior to incubation of serum in said membrane, said serum was pre-adsorbed with proteins of ER2738 bacterial cell extract and viral wild-type M13 phage particles. This membrane was then incubated for 2 hours at room temperature with orbital stirring. After this period of time, the membrane was again washed (5 times) with TBST. Next, a chicken anti-IgG secondary antibody conjugated with 1:5.000 alkaline phosphatase (Sigma Chemical) in blocking buffer and incubated for one additional hour at room temperature. The reaction was resolved with NBT/BCIP substrate (nitroblue tetrazolium/5 - bromo-4-chloro-3~ indolyl phosphate - Sigma Chemical) . Wild-type M13 phage was used as a negative control and the total tick antigens were used as positive controls.

Inhibition experiments have been performed in the same way as Dot Blot, except for addition of a step of pre- incubating serum with tick protein prior to membrane incubation. Varying concentrations of total proteins in the order of 0 to 200 μg/trough were used.

In Western Blot experiments, samples referring to clones were treated with DTT and immediately heated in water bath, for 2 minutes at 95°C and applied to electrophoresis gel. Said samples were then electrotransferred to a nitrocellulose membrane (Amersham Pharmacia) and blocked with blocking solution. Total anti-B. microplus protein chicken serum-purified polyclonal IgY' s were used as primary antibody. After washing (5 times) with TBST, 5 minutes each, said membranes were incubated with chicken anti-IgG secondary antibody marked with alkaline phosphatase in a 1/5.000 proportion in blocking buffer, for 1 hour at room temperature. After this period of tmie, said membranes were washed (5 times) with TBST and after addition of NBT/BCIP substrate (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate - Sigma Chemical) resolution occurred. This process was required for assuring that peptides expressed in III proteins of the selected clones exhibit recognition by tick protein specific antibodies.

For amplification of each clone to be tested in immunization and immunoscreening tests, 1 μL tetracycline (20 mg/mL) for each 1 mL of liquid LB medium (Luria Bertane) in Erlenmeyer flasks (final concentration of 20 μg/mL) amounting to 5 mL was used. Into this medium, an isolated bacteria colony, tetracycline-resistant E. coll strain ER2738, was inoculated, which multiplied for 6 hours at 37°C with stirring at 200 rpm. The culture medium was diluted in a 1/100 proportion, i.e. 5 mL of bacteria in 500 mL of LB medium and tetracycline (200 μg/mL) . Said phage-infected bacteria were prepared by inoculation of 50 μL

(approximately 1 x 10^u CFU) of phage in a medium comprising 1/100 diluted bacteria and kept at 37°C for 20 hours. Said culture was transferred to centrifuge tubes and subjected to 10,000 g for 10 minutes, at 4⁰C, twice, in order to eliminate bacteria pellets. Approximately 80% supernatant were transferred to another tube followed by addition of 80 ml PEG (polyethylene glycol 8000 + 2.5M NaCl) in a 1/6 proportion, and incubated overnight at 4⁰C to precipitate phages. After incubation, the preparation was centrifuged at 10,000 g for 15 minutes. The supernatant was discarded and the pellet with phages was again centrifuged for 1 minute. Said pellet with phages was diluted in 10 mL TBS buffer in 15ml Falcon Tubes and then centrifuged at 4.000 g for 40 minutes at 4⁰C. The supernatant was transferred to another microtube, to which 1/6 PEG/NaCl was added and it was then incubated for 60 minutes. After centrifugation at 4,000 g for 40 minutes at 4°C, the supernatant was discarded. Residual supernatant was removed and the pellet diluted in 4 mL TBS. The phages were quantified in viral particles per mL through direct spectrophotometry in a UV spectrophotometer at 269 nm, as described for the former experiments .

For titration of clones, the phage solution to be titrated has undergone 10-fold serial dilution in solid LB medium. For amplification, 10⁷ to 10¹² dilutions were used. To each dilution 200 μL of ER2738 culture in mid-log phase were added. Said mixture was briefly vortexed and incubated for 5 minutes at room temperature. The thus infected cells were transferred to culture tubes containing 3 mL of Agarose Top, at 45°C, rapidly poured and spread on a Petri dish containing a solid LB medium with IPTG/X-gal and tetracycline. For each dilution, a plate was confectioned. Said plates were sealed with parafilm, inverted and incubated at 37⁰C, for 16 hours. After this period of time, blue colonies were counted and plates having approximately 100 colonies were identified as carrying dilution representing the titers. In order to test the ability of peptides to induce immune response, mice and bovines were inoculated. These animals were kept isolated and free from ticks, flies and other parasite infestations. Two mice-immunization experiments were carried out. In the first (Example 3), for immunization with selected clones, six-week female mice having an average weight of 28 g were used. Fifty groups, five animals in each group, were immunized with thirty six different clones, wild-type M13 phage and fd tet phages, three synthetic peptides, three phages representing these peptides and suitable reaction controls. The dosage contained 1 x 10¹² viral particles, in a 100 μL volume. The following clones were tested: SEQ ID NO:1, SEQ ID N0:2, SEQ ID NO: 7, SEQ ID NO:10, SEQ ID NOrIl, SEQ ID NO:12, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:17, SEQ ID N0:18, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID N0:51, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:75_r SEQ ID NO:76_f SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID N0:91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94. No immunization adjuvants were used, except for the synthetic peptides and the corresponding controls, where complete Freund' s adjuvant for the first immunization and incomplete Freund' s adjuvant for the other ones (Sigma Chemical Co., USA), via subcutaneous route, were employed. Animals were immunized every two weeks, totalizing seven immunizations. Blood was collected from orbital vein at days 0, 30, 60 and 90 post- immunization .

Specific antibody levels in mice serum were measured as follows: Each blood sample was aliquoted in Eppendorf-type tubes and stored at -20⁰C. Flat bottom microtiter plates (96 wells: Nunc, Roskilde, Denmark) were sensitized with total proteins diluted in PBS, pH 7.2, in 1 μg/mL dose, overnight, at 4⁰C. After washing, animal sera were added, diluted in blocking solution in 1:100 dilution and incubated for 2 hours at 37°C. Thereafter, plates were washed and incubated with peroxidase-complex anti-mice conjugate, and then diluted in a 1:5,000 dilution blocking solution, for 1 hour, at 37°C. OPD was used for resolving the substrate, after quenching the reaction with sulfuric acid. Absorbance reading was determined at 492 nm in a microplate reader (Flow Titertek Multiskan Plus - USA) .

In the second test of mice inoculations for immunization with the selected clones, six-week female mice having an average weight of 28 g were used. Twelve groups, each having three animals, were immunized with nine different clones (SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID N0:2, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:37, and SEQ ID NO:41), PBS buffer control, wild-type M13 phage, and phage pool (set of these clones in a same dosage) in a dose of 2.5 x 10¹² CFU in a 100 μL volume, together with an incomplete Freund' s adjuvant (Sigma Chemical Co, USA) via intraperitoneal route. These animals were immunized at days 0 and 12. Blood was collected from orbital vein at days 0, 12 and 19, post-immunization .

After these immunizations, specific antibody levels in the mice serum were measured as follows: Each blood sample was aliquoted in Eppendorf-type tubes and stored at -20⁰C. Flat bottom microtiter plates (96 wells: Nunc_r Roskilde, Denmark) were sensitized with the clones or even the total proteins diluted in PBS, pH 7.2, in a dose of 5 x 10¹⁰ CFU/mL, , overnight, at room temperature. After washing, animal sera were added, diluted in 1:100 PBS and incubated for 1 hour at 37⁰C. Thereafter, plates were washed and incubated in anti-mice conjugate, in the case of mice, and anti-animal models made in rabbit in the case of bovines, diluted in 1:2,000 PBS, for 1 hour at 37°C. OPD was used for resolving the substrate, after quenching the reaction with sulfuric acid. Absorbance reading was determined at 492 nm in a microplate reader (Flow Titertek Multiskan Plus - USA) .

Assays of bovine tick infestations: Firstly, an immunoscreening test was performed, where 16 bovines, consisting of 8 Nerole breed and 8 Holstein breed, were naturally infested with 5,000 tick larvae for each animal. After observing a distinct tick count pattern, four animals were selected (2 Holstein and 2 Nelore) , each breed having one animal with high count and another with low count. This was done in an attempt to search for an effect among the animals having higher susceptibility and others having more resistance to natural infestation. After determining a significant statistics of an infestation response pattern, animal serum was collected, processed and submitted to Dot Blot assay in order to evaluate immunoreactivity of bovine IgG' s with clones selected for this test. The following clones were selected: SEQ ID N0:l, SEQ ID N0:2, SEQ ID NO: 7, SEQ ID NO:10, SEQ ID N0:ll, SEQ ID N0:12, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:17, SEQ ID N0:18, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:47, SEQ ID NO:48, SEQ ID N0:50, SEQ ID N0:51, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID N0:91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94. Wild-type M13 phage was used as negative control and total tick proteins were used as positive control. In further experiments for tests concerning potentially vaccinal effects of the obtained peptides, tests of inoculations in bovines together with challenge of ticks in animals vaccinated with phage clones were performed.

To this effect, one month prior to immunization, the animals underwent a series of four baths, every seven days, with a commercial carrapaticide in an attempt to eliminate all ticks from former field infestations.

In those preliminary immunizations, two groups of two male animals being approximately eighteen months old were used for inoculation and challenge tests. These animals were intramuscularly immunized with a pool of more reactive clones (SEQ ID NO: 18, SEQ ID N0:19, SEQ ID NO:20, SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:37, SEQ ID N0:41) in a 2.5 x 10¹² dose (Group 1) and wild-type M13 phage in the same dose (Group 2) in a 1 ml volume with incomplete Freund' s adjuvants (Sigma Chemical

Co, USA) . Said animals were immunized at days 0 and 12. Blood was collected at days 0, 12 and 19, post-immunization.

Twelve days after the first inoculation, these animals were infested with B. microplus (field strain) tick larvae, totalizing two infestations of 5.000 larvae each, at one day interval. These animals were constantly monitored twice a day during the first inoculation days to detect possible cutaneous hypersensitivity reactions to phages and adjuvants .

Clinical observations were weekly made until the 24^th day after the first infection in order to verify any challenge development. After their release, the adult ticks were collected from the barn floor, which was duly prepared for this collection, and identification of ticks referring to each barn was then conducted. Each female was weighed on a precise scale and thereafter prepared for egg laying in a muffle at 27°C, 80% humidity (OBA, 1976, Revista da Faculda.de de Veterinaria da Universidade de Sao Paulo, 13:409-420) . Eggs were weighed and placed in a muffle for 30 days for hatching. Larvae were also weighed on a precision scale (Massard et al . , 1995, Revista Brasileira de Medicina Veterinaria 17:167-173) .

Specific antibody levels in serum were measured in bovines . Each blood sample was aliquoted in Eppendorf-type tubes and stored at -20⁰C. Flat bottom microtiter plates (96 wells: Nunc, Roskilde, Denmark) were sensitized with clones or even total proteins diluted in PBS, pH 7.2, in a 5 x 10¹⁰ CFU/mli dose, overnight, at room temperature. After washing, animal sera were added, diluted in 1:100 PBS and incubated for 1 hour at 37⁰C. Thereafter, plates were washed and incubated with peroxidase-complex anti-mice conjugate, and then diluted in a 1:2,000 dilution blocking solution, for 1 hour, at 37°C. OPD was used for resolving the substrate, after reaction was quenched with sulfuric acid. Absorbance reading was determined at 492 nm in a microplate reader (Flow Titertek Multiskan Plus - USA) .

ELISA index (EI) for mice sera wag calculated by dividing optical density (OD) by the cut off value. Cut off was calculated by adding 2 standard deviations to the mean of the preimmune sera. EI values above 1 were considered as positive and EI values below 1 were considered as negative.

Biological parameters were analyzed after counting and weighing adult females, weighing eggs and larvae, as well as hystopathologic parameters.

Claims

1. Bovine tick antigen mimetic recombinant peptides (mirαotopes) and their reverse sequences, or synthetic sequences, or artificial sequences, or natural sequences characterized in that they comprise the sequences SEQ ID N0:l to SEQ ID NO:110.

2. Bovine tick antigen protein motifs, in accordance with Claim 1, characterized in that they comprise the sequences SEQ ID N0:l to SEQ ID NO: 110, including very short protein motifs.

3. Peptides, their reverse sequences and protein motifs, in accordance with Claim 1 or 2, characterized in that they are used as probes for detecting in vitro the presence of circulating antibodies or other binding molecules against arthropods, preferably bovine ticks.

4. Peptides, their reverse sequences and protein motifs, in accordance with Claim 1, 2 or 3, useful for in vitro immunodiagnosis process as probes for detecting binding molecules, such as circulating antibodies, which react against tick, characterized in that they detect through immunoassays, such as, for example, Immunoenzymatic tests (such as ELISA, Western blot, immunofluorescence, immunohystochemistry, EIA and others), immunoagglutination tests, electrochemical sensors, or any other form of detection directly or indirectly related to fluid samples, such as saliva, urine, blood, preferably blood.

5. Use of peptides, their reverse sequences and protein motifs, as defined in Claim 1 or 2, characterized in that it is in the preparation of a vaccinal composition for stimulating human and animal immune system as a vaccinal therapeutic method against arthropods, mainly tick. β. Vaccinal composition, in accordance with Claim 5, characterized in that it comprises one or more compounds described in the sequences SEQ ID NO: 1 to SEQ ID NO: 110, preferably those related as follows: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID N0:14, SEQ ID N0:15, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:2β, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO: 83, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO:94.

7. Peptides, their reverse sequences and protein motifs, either synthetic or recombinant, comprising DNA, RNA and proteins sequences as defined in Claims 1, 2, 3, 4, 5, or 6, characterized in that they are used in diagnosis methods or vaccinal compositions in any combination or in a number of repetitions used in an isolated form or in conjunction, applied to any platform type or associated with any molecule such as molecule carrier, linker and fusion protein.