MXPA98005424A - Three synthetic peptides to be used in the vaccination and diagnosis of cisticercosis by taenia sol - Google Patents

Abstract

The present invention relates to the design of a vaccine against porcine cysticercosis, a model of murine cysticercosis caused by Taenia crassiceps was used, which was adequate to identify protective antigens. Of twelve antigenic fractions of Taenia crassiceps cysticercus, 3 induced the highest levels of protection in mice and pigs. Protective antigens included in these fractions were identified in a Taenia crasiceps cysticercus gene library using specific antibodies against the 3 protective antigenic fractions. Four of the recombinant antigens induced protection in the mouse and in the pig. Based on the DNA sequence of 3 of them, 3 sequences that code for three antigenic peptides were identified. They were synthesized and their protective capacity was confirmed in the murine model and its immunogenicity in the pig. Two of the identified peptides are recognized by infected individuals and constitute a tool of interest for the diagnosis of cysticerci

Description

THREE SYNTHETIC PEPTIDES TO BE USED IN THE VACCINATION AND DIAGNOSIS OF CISTICERCOSIS BY TAENIA SOLIUM BACKGROUND Cysticercosis caused in humans and pigs by the larval stage of Taenia solium is one of the main causes of neurological disease in humans (Sotelo, J., Del Brutto OH and Román G. 1996. Cysticercosis., In: Current Clinical Topics in Infectious Diseases, Remington, JS and Swartz MN (Eds) Blackwell Sci, Boston Ma.) And causes significant economic losses in pig farming (Aluja, A. Frequency of porcine cysticercosis in Mexico, 1982. In: Flisser, A., Willms, K., Laclette, JP, Larralde, C, Ridaura, C, Beltrán, F. (Ed.) Cysticercosis: present stage of knowledge and perspectives., NY, Ac. Press, 53-62.). In Mexico and other countries of Latin America, Asia and Africa it represents a serious socioeconomic problem, especially in areas where scarce health resources favor the life cycle of this parasite. The most recent seroprevalence studies indicate that this parasite is widely distributed throughout the Mexican territory, with a higher prevalence in the Bajío. The life cycle of the Taenia solium includes a larval phase (cysticercus) that affects both the man (definitive host) and the pig (intermediate host) and is acquired by the intake of eggs present in food contaminated with human feces. On the other hand, when the man consumes undercooked and infected pork, the cysticerci can develop to the adult state of the parasite, tapeworm or solitary, which produces hundreds of thousands of eggs capable of transforming into cysticerci when they are ingested by man. or the pig, thus completing the life cycle of the parasite. The high frequency of this parasitosis, its consequences on human health and the economic repercussion of this disease justifies the attempts to prevent it. Regarding the alternatives for the prevention of this disease, it has been considered that the indispensable requirement of the pig as intermediate host in its life cycle, makes it possible to interfere in its transmission, increasing the resistance of the pig to cysticercosis. Thus, an alternative is to reduce the susceptibility of pigs to this parasitosis through vaccination as a strategy to interrupt the cycle of transmission of the parasitosis and reduce human disease. There are other alternatives for the control of this parasitosis, among which are: a) The improvement of living conditions, environmental sanitation and educational campaigns for the population, which seem feasible only in the long term. b) The treatment of teniasics in massive form. This measure has been applied in some regions and its epidemiological effect as a single measure is debatable. Thus, an increase in the incidence of porcine cysticercosis has been observed after the massive application of treatment, possibly due to the expulsion of tapeworms associated with inadequate stool processing. Vaccination seems a realistic measure because it does not imply modifying the way of rustic breeding that exposes pigs to a high risk of contracting the disease. In addition, it is not a measure that implies the confiscation of infected pigs and would allow the rustic producer to access a better price for the sale of their pigs, which could favor its application. Finally, considering that the pig is consumed mostly between 8-14 months of age, the vaccine would only have to protect them during this period. The feasibility of controlling this parasite through vaccination seems likely considering that other similar parasitoses such as sheep cysticercosis by Taenia ovis can be controlled through vaccination (Johnson, KS, Harrison, GBL, Lightowlers, MW, O? Oy, KL, Cougle, WG, Dempster, RP, Lawrence, SB, Vinton, JG, Heath, DO and Rickard, MD, 1989. Vaccination against ovine cysticercosis using a recombinant recombinant., 338: 585-587.). The total antigens of Taenia solium cysticerci have also been used as a vaccine against porcine cysticercosis (Molinari, JL, Meza, R., Suarez, B., Palacios, S., Tato, P. 1983. Taenia solium: Immunity in hogs To the cysticercus, Exp. Parasitic 55: 340-357 Molinari, JL, Soto, R., Tato, P., Rodriguez, D., Retana, A., Sepulveda, L., Palet, A. 1993. Immunization against cysticercosis in an endemic area in Mexico: a fíeld and laboratory study, Am. J. Trop. Med. Hyg. 49: 502-512.). Although this vaccine has been evaluated experimentally and has been used in pigs subjected to the natural challenge, the evaluation in this last population has been carried out only through the inspection of the pig's tongue as a diagnostic form. This aspect is a limitation of importance considering that this diagnostic procedure is very low sensitivity. Additionally, there is information that in the total set of cysticerc antigens there are antigens that can facilitate parasitism (Valdez, F., Hernández, M., Govezensky, T., Fragoso, G. and Sciutto, E. 1994. Imrnunization against Taenia crassiceps cysticercosis Identification of the most promising antigens in the induction of protective immunity J. Parasitol 80: 931-936.; Sciutto, E., Aluja, A., Fragoso, G., Rodarte, LF, Hernandez, M., Villalobos, N., Padilla, A., Keilbach, N, Baca, M., Govezensky, T., Diaz, S. and Larralde, C. 1995. Immunization of pigs against Taenia solium cysticercosis. Factors related to effective protection. Vet. Parasitol.60: 53-67.), Which implies a certain risk in the use of total antigens. The three peptides presented in this invention can be used as a synthetic vaccine for the prevention of porcine cysticercosis; its simple and complete composition characterized is of special interest since it implies a vaccine of reduced costs that can be produced in a laboratory of chemical synthesis in a controlled and systematic way. It is important to stress the importance of using the three peptides jointly, which implies inducing an immune response against different components of the parasite and increasing the probability of success of the vaccine. Reducing the incidence of swine cysticercosis would, in addition to reducing the losses from confiscation of infected meat, reduce the costs involved in the human disease. In this sense it is worth remembering that 10% of the budget of specialized institutions in neurology is devoted to the diagnosis and treatment of neurocysticercosis, which is currently the main cause of consultation in these institutions. The development of diagnostic tests for neurocysticercosis is another aspect of great interest in cysticercosis. Currently, the diagnosis of neurocysticercosis is based on clinical studies and very expensive imaging tests such as computed axial tomography and nuclear magnetic resonance. Serological studies are also carried out in search of anti-cysticercus antibodies or parasite antigens. Serological tests are used as a complementary data for the diagnosis of neurocysticercosis because they do not have the sensitivity and specificity necessary to constitute a definitive diagnostic element. Serological tests, currently available, based on the detection of antibodies, present different sensitivity and specificity depending fundamentally on the source of antigen used. The most widely used procedures are based on the use of total antigens, those included in the vesicular fluid, wall or antigens of the cyclocercus scoter of Taenia solium (Espinoza, B. et al., 1982. In: Flisser, A., Willms, K ., Laclette, JP, Larralde, C., Ridaura, C, Beltrán, F. (Ed.) Cysticercosis: present stage of knowledge and perspectives., NY, Ac. Press, 163-170, Larralde, C. Laclette, JP, Owen, Ch. S., Madrazo, I., Sandoval, M., Bojalil, R., Sciutto, E., Contreras, L., Arzate, J. Diaz, ML, Govezensky, T., Montoya, RM and Goodsaid, R. 1986. Reliable serology of Taenia solium cysticercosis with antigens from cyst vesicular fluid: ELISA and Haemagglutination test, Am. J. Trop.Med. Hyg 35 (5): 965-73, Larralde, C, Montoya, RM, Sciutto, E., Diaz, ML, Govezensky, T., and Coltorti, 1989. Deciphering Western blots of tapeworm antigens (Taenia solium, Echinoccocus granulosus and Taenia crassiceps) reacting with sera from neurocysticercosis and hydatid design ase patients. Am. J. Trop. Med. Hyg 40 (3): 284-292; Rosas, N., et al. 1986. Arch. Neurol. 43: 353-356., Nascimento, E., et al. 1987. J. Clin. Microbiol. 25: 1181-1185.). Also, heterologous antigens from the murine cysticercus of Taenia crassiceps have been used (Larralde, C, Sotelo, J., Montoya, RM, et al., 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid: antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium, Arch. Pathol, Lab. Med. 114: 926-928.). Tests based on the use of total antigens or recovered from different compartments of the cysticercus, often present low specificity, since containing many antigens are more likely to share them with other pathogens (Ramos-Kuri, M., Montoya, R.M., Padilla, A., Govezensky, T., Diaz, M.L., Sciutto, E., Sotelo, J. and Larralde, C. 1992. Immunodiagnosis of Neurocisticercosis. Arch. Neurol. 48: 633-36.). In the search to increase the specificity of the antibody detection assays, different isolated antigens have been evaluated. Among them, the B antigen stands out, although in principle it seemed of interest, the results obtained with the use of this antigen were not satisfactory (Espinoza, B. et al., 1986.

Characterization by enzyme-linked immunosorbent assay of the humoral immune response in patients with neurocysticercosis and its implication in immunodiagnosis. J. Clin. Microb. 24: 536-541). More recently, a set of antigens purified from the antigens of Taenia solium have begun to be used. These antigens are purified using their ability to associate with lectin-lectins and are used in the immunoblot technique (Tsang CWV, Brand, LA and Boyer, AE 1989. An Enzyme-Linked Immunoelectrotransfer Blot Assay and Glycoprotein Antigens for Diagnosing Human Cysticercosis ( Taenia solium), J. Inf. Disease, 1: 50-59.). Although its use has obtained greater specificity and sensitivity, this procedure is laborious, expensive and difficult to reproduce. The positive result is established based on the visualization in strips of nitrocellulose of a set of antigens whose appearance determines a positive diagnosis. By not offering a numerical variable this aspect allows subjective interpretations of the results. An additional drawback of all serological tests and vaccinal products that use total or purified antigens obtained from different batches of parasites is the limitation in the reproducibility of the results obtained, since each batch of parasites could present antigenic differences that are difficult to control. Tests based on the detection of antigens have shown promising results (Correa D., Sandoval, MA, Harrison, LJS, Parkhouse, M., Planearte, A., Mezas-Lucas, A., and Flisser A. 1989. Human cysticercosis : comparision of enzyme immunoassay capture techniques based on monoclonal and polyclonal antibodies for detection of parasite producís, Trans. Roy. Soc. Trop.Med. Hyg. 83: 814-816), although they have not been extensively evaluated. In the present invention, the use of two peptides (KETcl and KETcl2) as a source of antigen to be used in the diagnosis of neurocysticercosis is proposed. These peptides can be used in ELISA assays, a simple and inexpensive procedure with the advantage that they can be synthesized in a laboratory in a systematic and reproducible way in the quantities required and at a very low cost.

BACKGROUND OF THE INVENTION 1. Development of an experimental model capable of predicting antigens of interest for the design of a vaccine against porcine cysticercosis Considering the economic and experimental difficulties involved in the experimentation with pigs, it has been considered as a strategy to use murine cysticercosis by Taenia crassiceps (Freemann, RS 1962. Studies on the biology of Taenia crassiceps (Zeder, 1800) Rudolphi, 1810 (cestoda), Can. J. Zool, 40: 969-990.), as an experimental model of cysticercosis (Sciuttp, E., Fragoso, G., Trueba, D., Lemus, D., Montoya, RM, Diaz, ML, Govezensky, T., Lomelí, T., Tapia, R. and Larralde, C. 1990. Cysticercosis vaccine: cross protecting immunity with T. solium antigens against experimental murine T. crassiceps cysticercosis, Paras, Immunol 12: 687-96. This cysticercus has a life cycle similar to that of T. solium, being its possible intermediate hosts an extensive set of rodents and their definitive hosts the canids and felines. Both the cysticercus and the tapeworm of both species have a similar macroscopic structure, with some differences: the cysticerci of T. crassiceps are smaller and also have a unique feature that is their ability to divide by multiple polar budding. This parasite reproduces in the peritoneal cavity of mice rapidly, thus offering an important source of antigens obtained under controlled experimental conditions. Thus, after only a few months of infection, they can be recovered from the peritoneal cavity of each infected mouse to grams of antigens. This fact is combined with the fact that their antigens have extensive cross-reactivity with T. solium cysticercos antigens (Larralde, C, Montoya, R.M., Sciutto, E., Diaz, M.L., Govezensky, T., and Coltorti. 1989. Deciphering western blots of tapeworm antigens (Taenia solium, Echinoccocus granulosus and Taenia crassiceps) reacting with sera from neurocysticercosis and hydatid disease patients. Am. J. Trop. Med. Hyg 40 (3): 284-292.), Encouraged us to use the antigens of murine cysticercus in order to evaluate them in their ability to prevent porcine cysticercosis. 2. Immunization with T. crassiceps or T. solium antigens protects against murine cysticercosis.

In the first studies, the protective capacity associated with the antigens included in the complete extract of larvae of T. crassiceps and T. solium was evaluated in immunizing BALB / cAnN mice, challenged with T. crassiceps cysticercos (Sciutto, E., Fragoso , G., Trueba, D., Lemus, D., Montoya, RM, Diaz, ML, Govezensky, T., Lomelí, T., Tapia, R. and Larralde, C. 1990. Cysticercosis vaccine: cross protecting immunity with T. solium antigens against experimental murine T. crassiceps cysticercosis, Paras, Immunol 12: 687-96. The results obtained indicated that both antigenic extracts of T. crassiceps and T. solium effectively protected the mice against cysticercosis by T. crassiceps (Sciutto, E., Fragoso, G., Trueba, D., Lemus, D., Montoya, RM, Diaz, ML, Govezensky, T., Lomelí, T., Tapia, R. and Larralde, C. 1990. Cysticercosis vaccine: cross protecting immunity with T. solium antigens against experimental murine T. crassiceps cysticercosis. Immunol., 12: 687-96.). This observation allowed us to speculate that T. crassiceps antigens were then able to confer protection against porcine cysticercosis. 3. Immunization with cysticercus antigens of T. crassiceps protects against porcine cysticercosis. Considering the previous findings, it was evaluated experimentally if T. crassiceps antigens were able to protect against porcine cysticercosis (Sciutto, E., Aluja, A., Fragoso, G., Rodarte, LF, Hernández, M. , Villalobos, N., Padilla, A., Keilbach, N, Baca, M., Govezensky, T., Diaz, S. and Larralde, C. 1995. Immunization of pigs against Taenia solium cysticercosis. Vet. Parasitol.60: 53-67.). Vaccination at a dose of 400 ug / kg of weight reduced by 58% the parasitic load of pigs challenged experimentally with T. solium eggs orally. This result showed that antigens of murine cysticercus are able to protect against porcine cysticercosis and validated the predictive capacity of the murine cysticercosis model to evaluate antigens of interest in vaccination. A further important observation is the fact that the effects on parasytosis induced by the use of total antigens as an immunogen critically depend on the dose of antigen used. Thus, the dose of 4 mg / kg of weight of the pig favored the parasitosis, increasing the parasitic load in the immunized animals in more than 50% of what was expected (Sciutto, E., Aluja, A., Fragoso, G., Rodarte , LF, Hernández, M., Villalobos, N., Padilla, A., Keilbach, N, Baca, M., Govezensky, T., Diaz, S. and Larralde, C. 1995. Immunization of pigs against Taenia solium cysticercosis Factors related to effective protection, Vet. Parasitol.60: 53-67.). Considering these findings, we decided to identify the protective antigens in this antigenic set too complex in order to use an immunogen of limited heterogeneity that would allow to induce protection in a controlled manner. 4. Identification of fractions of protective antigens of T. crassiceps cysticercus In order to optimize the vaccine, separate antigenic fractions were obtained based on differences in molecular weight in acrylamide gels (7 and 15%). To obtain these fractions, the published procedure was essentially followed (Valdéz, F., Hernández, M., Govezensky, T., Fragoso, G., and Sciutto, E., 1994. Immunization against Taenia crassiceps cysticercosis: identification of most promising antigens in the induction of protective immunity, J. Parasitol 80: 931-936.), which briefly consists in the recovery of the soluble antigens of the cysticercus of Taenia crassiceps. After washing them extensively with saline, the cysticerci suspended in the smallest volume of saline are centrifuged at 10,000 rpm at 4 ° C. The extracted liquid is collected and its protein concentration is quantified. These antigens are separated in an acrylamide gel and they are identified according to their molecular weight. The gel fragments including 12 antigenic fractions were cut and used to immunize as a vaccine. The fractions included in the gel and appropriately quantified were used for the vaccination of mice with the purpose of evaluating their effect on the growth capacity of the parasite. The electrophoretic procedures allowed us to optimize the separation of 12 different antigenic fractions and evaluate them in their protective capacity using initially the murine cysticercosis. Based on the protective capacity induced by each of them, three antigenic fractions of 56 were selected., 66 and 74 kDa that were able to induce the highest levels of protection and can be obtained in accessible quantities for its use (8% of the total proteins in the preparation). Figure 1 shows the identification of the three fractions identified in a polyacrylamide gel used for their separation. The protection capacity of the three fractions used jointly was confirmed by evaluating them in mice as illustrated in Table I. Subsequently, these fractions were used together to evaluate their ability to protect the pig against cysticercosis by T. solium. The immunization of pigs with these antigenic fractions greatly reduced the parasitic load as observed in Table I, obtaining 0.16 cysticerci on average in the vaccinated animals, unlike 5 in the unvaccinated animals, confirming their protective capacity (Manoutcharian , K., Larralde, C, Aluja, A., Fragoso, G., Rosas, G., Hernández M., Villalobos, N., Rodarte, LP, Govezensky, T., Baca, M. and Sciutto, E. 1995. Advances in the development of a recombinant vaccine against Taenia solium pig cysticercosis, Vaccines 95: 63-68.). This strategy of separation of antigenic fractions allowed us to identify protective antigens in the set of antigens of T. crassiceps cysticerci, although this type of isolation is not adequate for the provision of antigens to be used massively, which is why decided to produce them by recombinant DNA methods. Table I. Protective capacity induced by vaccination with antigenic fractions of 56, 66 and 74 kDa. Mice • Pigs Controls 38. l ± 6.7+ (10) + + Controls 5.0 ± 0.85 + (6) + + Immunized 9.7 * 6.6 (10) * Immunized 0.16 * 0.17 (7) * Number of average parasites Number of animals used -standard deviation * Statistically significant differences (non-parametric Wilcoxon scores test).

Identification of recombinant protective antigens against cysticercosis A cDNA library was constructed in the Uni-ZapXR vector using messenger mRNA from T. crassiceps cysticerci. The procedures developed for its construction are described in a previously published work. In this library, thirteen recombinant clones were identified that were selected by immunodetection using specific polyclonal antibodies produced in rabbit: anti-56, anti-74 and anti-66. Of the clones identified, those recognized by sera from infected pigs containing antibodies capable of recognizing antigens of T. solium cysticerci were selected. The presence of these antigens in T. solium cysticerci indicated them as proteins of great interest for the prevention against porcine cysticercosis. The clones were designated KETcl, KETc4, KETcl I, KETc7 and KETcl2 (Manoutcharian, K., Larralde, C, Aluja, A., Fragoso, G., Rosas, G., Hernandez M., Villalobos, N., Rodarte , LP, Govezensky, T., Baca, M. and Sciutto, E. 1995. Advances in the development of a recombinant vaccine against Taenia solium pig cysticercosis, Vaccines 95: 63-68.). From the proteins encoded by these clones, specific antibodies against them were purified by affinity from serum of rabbits hyperimmunized with protective antigenic fractions. These antibodies were used for the detection of the presence of specific antigens in the vesicular fluid of T. crassiceps by Immunoelectro transfer. Antibodies specifically recognized native antigens of 56 and 74-78 kDa. This result implies that the 5 recombinant antigens represent parts of native antigens of these molecular weights. Evaluation of the protective capacity of recombinant antigens by vaccination. In order to evaluate the efficacy of the cysticerco recombinant proteins, in the prevention of cysticercosis, mice of the susceptible strain BALB / cAnN were vaccinated. Mice were vaccinated with complete Freund's adjuvant (ACF) and the recombinant proteins included in a Used E. coli infected with lambda phage including the corresponding insert. As controls were used only animals treated with ACF and with ACF and proteins from the Used of E. coli infected with the same phage without insert. The mice vaccinated with the KETcl, KETc4, KETc7 and KETcl 2 clones showed, as shown in Table II, a high level of resistance against murine cysticercosis (Table II) caused by T. crassiceps, reducing the parasitic load on the less than 5 times than expected (Manoutcharian, K., Larralde, C, Aluja, A., Fragoso, G., Rosas, G., Hernandez M., Villalobos, N., Rodarte, LP, Govezensky, T., Baca , M. and Sciutto, E. 1995. Advances in the development of a recombinant vaccine against Taenia solium pig cysticercosis, Vaccines 95: 63-68.). Meanwhile, clone KETcl l did not modify the expected parasitic load and was discarded for prevention purposes.

The protective capacity of clones KETc4, 7 and 12 was evaluated against porcine cysticercosis in two independent experiments (Table II). In one experiment three pigs were immunized with a single dose of the bacterial Used containing the immunogen constituted by KETc 7 and 12, four pigs with a dose of the KETc 7 and 12 antigens and a second dose 15 days later of the KETcl2 antigen. As controls, four pigs were immunized with the used bacterial without the recombinant antigen. All immunizations were performed in Freund's Complete Adjuvant. Thirty days after the last immunization the pigs were challenged with 10 eggs of T. solium and 80 days after they were slaughtered, quantifying the number of cysticerci in order to determine the efficiency of vaccination. Table II shows the reduction in the number of parasites obtained as well as the effect induced on the viability of recovered cysticerci. In a second experiment the effect of the vaccination obtained with the KETc4 antigen was evaluated by performing the same procedure with the difference that was used as adjuvant saponin instead of Freund's complete adjuvant. Table II. Protective capacity induced by vaccination with the 4 recombinant antigens in mice and pigs. Mice Pigs + + Controls 61.3 ± 29.7 + (7) + + Controls 3.3 ± 1.3 (3) Immunized with: Immunized with KETcl 8.5 ± 17.5 (7) * KETc4 0 (2) ^ KETc4 22.0 ± 15.9 (7) * KETc7 42.5 ± 6.4 (7) * KETcl2 26.3 ± 7.2 (7) * Controls 56 ± 14, + (, 4 •) > + "+ ** (46%) Immunized with: KETc7 43.2 ± 7.8+ (3) + + (79%) ** KETc7 + KETcl2 35.0 ± 14.2 + (4) + + (74%) ** Number of recovered cysticerci after infection ± standard deviation Number of animals included in each experimental group * Statistically significant differences (Wilcoxon nonparametric test scores) ** Percentage of cysticercos macroscopically identified as calcified.

Brief description of the figure Figure 1. The three protective antigenic fractions of 56, 66 and 74 kDa against murine and porcine cysticercosis are indicated with arrows to distinguish them from the set of antigens of Taenia crassiceps separated in an acrylamide gel of 7%.

DESCRIPTION OF THE INVENTION Al. Identification of a protective sequence called GK-1 (SEQ ID No. 1) present in the KETc7 antigen Based on the KETc7- antigen sequence (Manoutcharian, K., Rosas, G., Hernández, M., Fragoso, G., Aluja, A., Villalobos, N., Rodarte, LF, and Sciutto, E. 1996. Cysticercosis: Identification and cloning of protective recombinant antigens, J. Parasitol 82 (2): 250-254.), Which codes for a 100 amino acid polypeptide, a prediction of the regions of this polypeptide of higher antigenicity was made. Based on this theoretical prediction (Gevorkian, G., Manoutcharian, K., Larralde, C, Hernandez, M., Almagro, JC, Viveros, M., Sotelo, J., Garcia, E. and E. Sciutto. Immunodominant synthetic peptides of Taenia crassiceps in murine and human cysticercosis, Immunology Letters, 49, 185-189.), 3 peptides were synthesized that were found to be recognized to a different extent by sera from infected individuals. For the identification of the protective peptide, the three peptides were evaluated using the murine model, finding that the so-called GK-1 induces important levels of protection in this model of cysticercosis. The protective capacity was extensively confirmed using the following experimental conditions: mice of the male BALB / cAnN strain are vaccinated at time zero with the immunogen to be evaluated inoculated subcutaneously, using as adjuvant saponin in a concentration of 100 ug per mouse and a group control is subjected to an equivalent procedure immunizing them with the same adjuvant in saline. Fifteen days later, the mice are challenged intraperitoneally with 10 cysticerci of T. crassiceps ORF variety and 30 days later the amount of cysticerci in the peritoneal cavity of each infected mouse is quantified. The number of parasites obtained in the control mice is compared with that recovered in the vaccinated mice to determine the protective capacity of the antigens used. The induced protection capacity is illustrated in Table III and in the sequence listing the nucleotide and amino acid sequence of this peptide. Table III. Protective effect induced by vaccination with the synthetic peptide GK-1. + + Controls 6.87 ± 2.42 + (0/7) Immunized with: GK-1 0.71 ± 0.88 + (4/7) + + + + Number of average cysticerci recovered in each group ± standard deviation. Number of mice fully protected / total of mice used per group. * Statistically significant differences (Wilcoxon nonparametric test scores). A2. Location of the peptide GK-1 GK1 represents a part of the recombinant antigen KETc7. Analyzing the sequence of KETc7 we find that this belongs to the family of the extensins, proteins rich in prolines and hydroxyprolines that occur abundantly in the cell walls of plant cells and some parasites. Antibodies against it were produced in mice for the characterization of this epitope. For this purpose, the immunization protocols conventionally used to obtain polyclonal antibodies capable of specifically recognizing antigens or peptides were used. The procedure used consisted essentially in immunizing mice with the GK-1 peptide intradermally with a dose of 10 -g of peptide per mouse in 100 g of saponin dissolved in saline. The immunization was repeated 5 times with a space of 15 days between each one. After verifying the induction of antibodies in the mice, they were bled white and sacrificed to obtain antibodies in the sera of the immunized animals, 10 days after the last immunization. Using these antibodies, in the case of Taenia solium it has been found that this antigen is in the spiral channel of the cysticercus and in the external parts of the adult worm and very intensely expressed in the oncosphere phase. Its location in the different stages of the Taenia solium parasite has been determined by two ELISA and immunofluorescence immunological methods whose procedures are detailed in previously published works (Larralde, C. Laclette, JP, Owen, Ch. S., Madrazo, I., Sandoval , 'M., Bojalil, R., Sciutto, E., Contreras, L., Arzate, J. Diaz, ML, Govezensky, T., Montoya, RM and Goodsaid, R. 1986. Reliable serology of Taenia solium cysticercosis with antigens from cyst vesicular fluid: ELISA and Haemagglutination test, Am. J. Trop. Med. Hyg 35 (5): 965-73., Parkhouse, RME, &; Harrison, L.J.S. 1987. Cyst fluid and surface associated glycoprotein antigens of Taenia sp.- metacestodes. Paras Immunol. 9: 263-268.). GK-1 has also been localized in other parasites such as Entamoeba histolytica and Trypanosoma cruzi. Its presence in different parasites explains why this peptide has not been of interest in diagnosis. Meanwhile, considering the protective capacity induced by GK-1 in other parasites is of interest to induce protection against other parasitosis. With the identification of GK-1, monoclonal antibodies can be induced, which have the advantage over polyclonal antibodies of having a monospecific antibody produced by the transformed tumor cells in large quantities. The method for obtaining them consists essentially in immunizing mice with the peptide. When the mouse begins to induce antibodies, they are sacrificed and the spleen cells are recovered. These cells fuse with tumor cells that have the property of dividing. From the cells resulting from the fusion, those which are capable of dividing in a tumor form and which secrete the specific antibodies against the peptide of interest are selected. Bl. Identification of two protective sequences called KETcl (SEQ ID No2) and KETc 12 (SEQ ID No3) corresponding to peptides by the KETcl and KETcl 2 clones sequences, respectively. Based on the sequence of the KETcl and KETcl2 clones, two peptides called KETcl and KETcl2 were synthesized, respectively, whose sequences are illustrated in the sequence list. Both peptides were evaluated in the murine cysticercosis model following the previously described procedure, confirming their protective capacity as shown in Table IV. The protective effect induced by the immunization of both KETcl and KETcl2 is observed both in the reduction of the number of average cysticerci recovered and in the induction of complete protection in some of the immunized animals. Table IV. Protective effect induced by vaccination with the synthetic peptides KETcl and KETcl2. Controls 46.6 ± 5.6+ (0/8) + + Immunized with: KETcl 15.7 ± 14.7+ (2/8) + + KETcl2 19.7 ± 15.6+ (2/7) + + Number of average cysticerci recovered in each group ± standard deviation .

Number of mice fully protected / total of mice used per group. * Statistically significant differences (Wilcoxon nonparametric test scores). B2. Localization of the KETcl and KETcl2 peptides In order to characterize the KETcl and KETc 12 epitopes, mice were given specific antibodies against both peptides using the previously described procedure for the production of polyclonal antibodies against the GK-1 peptide. Using these antibodies it was determined that both KETcl and KETc 12 are present in the different phases of Taenia solium, cysticercus, adult worm and egg using fluorescence microscopy in the previously reported conditions (Parkhouse, RME, &Harrison, LJS 1987. Cyst fluid and surface associated glycoprotein antigens of Taenia sp.- metacestodes, Paras, Immunol 9: 263-268. B3 Utility of the KETcl peptides (SEQ ID No2) and KETcl2 (SEQ ID No3) for the diagnosis of cysticercosis. The two peptides are not recognized by sera from non-neurocysticercosous individuals, which allows us to suppose that they are not present in other parasites that are frequent in our environment. Meanwhile, both peptides are recognized by neurocysticercosous individuals and not. they react with sera from individuals with other neurological diseases such as brain tumors, tuberculous meningitis, degenerative disorders, which indicates their interest in the design of tests for the diagnosis of cysticercosis. Table V illustrates the reactivity of the KETcl and KETcl2 peptides used simultaneously and antigens present in the vesicular fluid of Taenia solium with cerebrospinal fluid (CSF) of human neurocysticercoses and with other neurological diseases. As can be seen 7 of 9 CSF reacted with T. solium antigens, while 8 of 9 were detected as positive using the two peptides together. The peptides were evaluated using the previously reported ELISA technique (Larralde, C, Sotelo, J., Montoya, RM, et al., 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid: antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium, Arch. Pathol, Lab. Med. 114: 926-928.) with the specification that they were used together in the amount of 1 Mg of each in each well of the ELISA plate and the antibody attached to the peptide in the plate is detected using a second antibody associated with a tag that allows detecting the initial reaction. In this case it was used for a second antibody associated with an enzyme, alkaline phosphatase, whose activity is revealed using its specific substrate, although other enzymes can be used, as well as radioactivity or fluorochromes emitting light. There are other procedures for the detection of antibodies such as immunoblot or immunopoint that detect antibodies using antigens fixed on solid supports such as nitrocellulose. Considering that the immune response by antibodies or humoral immune response, in mammals, is characterized by having antibodies in different humors, blood, plasma, serum, secretions, saliva, cerebrospinal fluid, its presence can be detected in any of them. Table V. Reactivity of cerebrospinal fluid of neurological patients with antigens and peptides used in the ELISA technique. No neurocysticercoses Neurocysticercoses T. solium KETcl + KETcl2 T. solium KETcl + KETcl2 (OD) 405f 0.149 0.109 0.158 0.083 0.127 0.084 0.23 0.198 * .248 0.086 2.7 * 0.204 * .123 0.065 2.8 * 0.14 * .151 0.081 2.6 * 0.14 *. 178 0.084 2.5 * 1.2 * .117 0.067 2.6 * 0.35 * .152 0.064 2.4 * 0.18 * .116 0.084 2.7 * 0.27 * 0.27A 0.122 ^ t Vesicular fluid of Taenia solium cysticerci obtained according to the previously published procedure (Larralde, C. Laclette , JP, Owen, Ch. S., Madrazo, I., Sandoval, M., Bojalil, R., Sciutto, E., Contreras, L., Arzate, J. Diaz, M.L., Govezensky, T., Montoya, R.M. and Goodsaid, R. 1986. Reliable serology of Taenia solium cysticercosis with antigens from cyst vesicular fluid: ELISA and Haemagglutination test. Am. J. Trop. Med. Hyg 35 (5): 965-73.). The optical densities obtained in the ELISA technique are reported according to the previously published procedure (Larralde, C, Sotelo, L, Montoya, RM, et al., 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid: antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium, Arch. Pathol, Lab. Med. 114: 926-928.) A Average of the corresponding optical densities plus three standard deviations. * The values of optical densities above the average of the optical densities obtained with CSF of non-neurocysticercosis individuals plus three standard deviations reacting with the respective antigens are indicated. Utility of the three peptides of the sequences GK1 (SEQ ID Nol), KETcl (SEQ ID No2) and KETcl2 (SEQ ID No3) for use as a vaccine Considering the interest of using the three peptides (SEQ ID Nol, 2, 3) , protectors in the model of murine cysticercosis, their ability to induce a detectable immune response in adult pigs as well as in newly weaned pigs was evaluated. It was determined that immunization with the three peptides used together in doses from 50 to 250 ug of each peptide per pig, in saline and in the presence of saponin (100 to 500 ug per dose), induces detectable levels of antibodies in the vaccinated pigs. Antibodies were detected in vaccinated pigs using the previously reported ELISA technique (Larralde, C, Sotelo, J., Montoya, RM, et al., 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid: antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium, Arch. Pathol, Lab. Med. 114: 926-928.), with the particularity that the three peptides were used individually as antigens in addition to the total cysticercus extract of Taenia crassiceps at a concentration of 1 ug per well. To produce the three peptides proposed in this invention, a chemical synthesis method was used, which involves reduced costs for its production. However, there are other alternatives for their production individually or jointly, using molecular biology methods. For this, plasmids can be used that are essentially vectors that allow introducing and expressing the desired sequences in bacterial or eukaryotic cells. There is a wide repertoire of vectors to be used that contemplate different elements that favor the expression of the desired sequences. Vectors that can infect bacteria and use them for the synthesis of the desired peptides include a promoter region (sequence with the information for RNA synthesis to begin) and a ribosome binding site and antibiotic resistance genes. The latter allow bacteria growing in the presence of said antibiotics to select those infected by the plasmids to which the sequences of interest have been incorporated. These systems can include DNA fragments that contain a sequence that codes for a protein or peptide that favors the stability and / or subsequent purification of the peptide to be produced. In this case, the desired peptide is obtained as fusion protein and, if required, the additional segment can be subsequently separated using enzymatic methods. Protein expression methods using bacteria are generally the most economical alternative for their production by recombinant DNA methods. However, certain sequences require eukaryotic cells to be expressed efficiently. These systems contain promoter elements that direct the transcription of the desired sequences, polyadenylation signals that increase the stability of the transcripts, elements that allow to regulate and increase the expression of the desired sequence as well as selection elements that allow to facilitate the identification of the cells that carry the desired sequence. Vectors that can infect eukaryotic cells include those that can infect mammalian cells. These vectors can also be used for immunization of the individual to be protected through infection of the individual's own cells in vivo, a procedure that is called DNA immunization and consists of inoculating the individual to be protected with a vector capable of infecting and expressing their information in the cells of the individual. It has been observed that this procedure induces the infected individual to generate a response against the proteins capable of coding the sequences that were incorporated into the vector used. The protective capacity of the peptides GK-1, KETcl and KETc 12 can be maintained with functional variants thereof, considering functional variants to all those peptides that retain the same biological properties as the peptides of the present invention and which consist of partial sequences or changes in amino acids not relevant to the biological functions that are associated with them. Considering • the protective capacity of the three peptides observed in the murine cysticercosis model, • that the three peptides belong to fractions of cysticercus antigens of Taenia crassiceps that have been shown to induce very high levels of protection against cysticercosis by Taenia solium in the pig, that the joint immunization with the three peptides induces specific antibodies against each of them that also recognize the antigens of cysticercus, it is proposed the use of the three peptides as a vaccine for the prevention of porcine cysticercosis by Taenia solium. Proposing the use of three different epitopes increases the probability of inducing protection against a complex parasite in a genetically heterogeneous host population. Additionally, these three peptides can be used to induce protection against infections caused by different cestodes (7aen / a crassiceps, Taenia saginata, Taenia ovis, Taenia psiformis, Taenia solium, Taenia hydatygena) in their corresponding hosts: rodents, lagomorphs, cows , sheep, rabbits, humans. For its application it is proposed to use it in doses of 50 to 250 Mg of each peptide per pig, in saline solution and in the presence of saponin (100 to 500 Mg per dose) inoculating intramuscularly at the base of the ear. For the production of this vaccine in quantities suitable for mass use, the conventional chemical synthesis procedures previously reported were used (Gevorkian, G., Manoutcharian, K., Larralde, C, Hernández, M., Almagro, JC, Viveros, M , Sotelo, J., García, E. and E. Sciutto, 1996. Immunodominant synthetic peptides of Taenia crassiceps in murine and human cysticercosis, Immunology Letters, 49, 185-189.). This procedure was used considering it as the most simple and economical. However, it is feasible for the production of the three peptides to use molecular biology methodologies. For this, the DNA sequences coding for each of the peptides must be incorporated in a plasmid, an organism capable of infecting and reproducing bacteria therein. The infected bacteria express the sequences incorporated into the plasmid and become organisms capable of producing the peptides. In order to increase the protective capacity, different compounds called adjuvants whose function consists of immunopotentiating the response induced by. the immunization of immunogens. Among these compounds, the classically used in experimentation is Freund's complete adjuvant (ACF). However, its use is limited to experimental use because its application results in an exacerbated inflammatory reaction and the formation of a granuloma. Evaluating the adjuvant capacity of the ACF and comparing it with other compounds, it was found that saponin induces similar levels of protection. This adjuvant is easy to apply and does not generate adverse reactions to the body, so its use is proposed in the present invention. It is also feasible that other compounds that have been reported to have adjuvant capacity could induce similar results.

Sequence listing SEQ ID No. 1 (GK-1). Gly Tyr Tyr Tyr Pro Ser Asp Pro Asn Thr Phe Tyr Pro Pro Wing Tyr 5 10 15 Wing Wing Information for the sequence SEQ ID No. 1 I. Characteristics of the sequences A. Length: 18 amino acid residues B.Type: sequence of amino acids II. Type of molecule: peptide VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Status of development: cysticercus VII. Immediate source A. Library: built with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETc7 SEQ ID No. 2 (KETcl) Ala Pro Met Ser Thr Pro Ser Ala Thr Ser Val Arg 5 10 Information for the sequence SEQ ID No.2 I. Characteristics of the sequences A. Length: 12 amino acid residues B. Type: amino acid sequence II. Type of molecule: peptide VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Development status: cysticercus VIL Immediate source A. Library: constructed with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETcl SEQ ID No. 3 (KETcl2) Gly Asn Leu Leu Leu Ser Cys Leu 5 Information for the sequence SEQ ID No.3 I. Characteristics of the sequences A. Length: 8 amino acid residues B.Type: sequence of amino acids II. Type of molecule: peptide VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Development status: cysticercus VIL Immediate source A. Library: constructed with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETcl2 SEQ ID No.4 (GK-1) GGTTATTACT ATCCATCTGA TCCAAATACC TTCTACGCTC CACCCTACAG 5 10 15 20 25 30 35 40 45 50 C Information for the sequence SEQ ID No. 4 I. Characteristics of the sequences A. Length: 51 base pairs B.Type: nucleotide sequence II. Type of molecule: cDNA VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Development status: cysticercus VIL Immediate source A. Library: constructed with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETc7 SEQ ID No.5 (KETcl) GCTCCAATGT CTACACCATC GGCTACATCG GTTCGG 5 10 15 20 25 30 35 .. Information for the sequence SEQ ID No.5 I. Characteristics of the sequence A. Length: 36 base pairs B Type: nucleotide sequence II. Type of molecule: cDNA VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Development status: cysticercus VIL Immediate source A. Library: constructed with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETcl SEQ ID No.6 (KETcl2) GGCAACCTCT TATTGAGTTG TTTG 5 10 15 20 Information for the sequence SEQ ID No.6 I. Characteristics of the sequences A. Length: 24 base pairs B.Type: nucleotide sequence II. Type of molecule: cDNA VI. Original source A. Organism: Taenia crassiceps B. Strain: ORF C. Development status: cysticercus VIL Immediate source A. Library: constructed with cDNA in vector? -ZAP (Uni-ZapXR, Stratagene, Co) B. Clone: KETcl2

Claims (31)

1. Having described the invention as above we claim our property, contained in the following: CLAIMS 1. A peptide or a functional variant thereof, capable of inducing protection against infection by cestodes in mammals characterized because it has the amino acid sequence of the SEQ ID No. 1
2. 2. A peptide or a functional variant thereof, capable of inducing protection against infection by cestodes in mammals characterized in that it has the amino acid sequence of SEQ ID No. 2
3. 3. A peptide or a functional variant thereof , capable of inducing protection against infection by cestodes in mammals characterized in that it has the amino acid sequence of SEQ ID No. 3
4. 4. A nucleotide sequence characterized in that it encodes the peptide of claim 1.
5. 5. A characterized nucleotide sequence because it codes for the peptide of claim 2.
6. 6. A nucleotide sequence characterized by e encodes for the peptide of the vindication 3.
7. 7. The nucleotide sequence in accordance with the claims nos. 4 to 6 characterized because the sequence is DNA.
8. 8. The nucleotide sequence according to claim 4, characterized in that it has the nucleotide sequence of SEQ ID No4.
9. 9. The nucleotide sequence according to claim 5, characterized in that it has the nucleotide sequence of SEQ ID No5.
10. 10. The nucleotide sequence according to claim 6, characterized in that it has the nucleotide sequence of SEQ ID No6.
11. 11. A vector comprising the nucleotide sequence according to claim 4, 5, 6 or 7.
12. 12. A vector comprising the nucleotide sequence according to claim 8, 9 or 10.
13. 13. The vector according to claim 11 or 12 characterized in that it is a plasmid.
14. 14. A host cell transformed with a vector as defined in claim 11 or 12.
15. 15. The host cell according to claim 14, characterized in that it is a bacterium.
16. 16. A process for the preparation of a peptide that is defined in any of claims 1 to 3, characterized in that the process comprises: a) the coupling of the amino acids with dicyclohexylcarbodimide using the solid phase synthesis method, b) the release of the peptide of the solid phase using trichloromethane sulfonic acid, c) elimination of salts for obtaining the pure peptide using gels of Sephadex, d) lyophilization of the peptide for its preservation, e) purity control by high pressure chromatography.
17. 17. A process for the preparation of a peptide that is defined in any of claims 1 to 3, characterized in that the process comprises: a) culturing in a culture medium under the appropriate conditions the transformed cell of claim 14 or 15, b ) recovering the protein produced by said cell from the culture medium, c) isolating the protein from the culture medium,
18. 18. An antibody capable of recognizing the peptide of claim no.1.
19. 19. An antibody capable of recognizing the peptide of the claim. no 2.
20. 20. An antibody capable of recognizing the peptide of claim no 3.
21. 21. The antibody in accordance with the claims nos. 18 to 20, characterized in that it is monoclonal.
22. 22. A hybridoma capable of producing the antibodies of claim 21.
23. 23. A vaccine composition for the prevention of cysticercosis comprising a peptide according to claim 1, 2 or 3, or mixtures thereof and a pharmaceutically acceptable carrier. .
24. 24. The vaccine composition of claim 23, characterized in that it also contains an adjuvant.
25. 25. The vaccine composition of claim 24, characterized in that the adjuvant is saponin.
26. 26. A diagnostic method for cysticercosis and taeniasis in mammals characterized in that it comprises: a) obtaining a biological sample from the mammal in question b) contacting said sample with the peptide of claim 2 or 3 or mixtures thereof and c) detect the presence of specific antibodies in the sample.
27. 27. The method of claim 14 characterized in that the biological sample is selected from the group comprising the cerebrospinal fluid, blood, plasma, saliva and serum.
28. 28. A diagnostic kit for the detection of cysticercosis and taeniasis in biological samples of mammals comprising: a) A peptide according to claim 2 or 3 mixtures thereof. b) Suitable means for the interaction between the peptide of part a) and the sample and c) An agent for detecting the interaction generated in part b).
29. 29. The diagnostic kit of claim 26, characterized in that the detection agent is selected from the group consisting of labeled antibodies, labeled G protein and labeled protein A.
30. 30. The diagnostic kit of claim 27, characterized in that the tag is selected from the group consisting of peroxidase, alkaline phosphatase, fluorochromes and radioactive isotopes.
31. 31. The use of a peptide as defined in claim 1 to 3 or mixtures thereof for the manufacture of a vaccine for the prevention of cysticercosis in mammals.