ES2453391A1 - Recombinant protein ofhaemonchus contortus and use thereof for the production of a vaccine against haemonchosis - Google Patents

Abstract

Recombinant protein from Haemonchus contortus and its application for the production of a vaccine against haemonchosis. A recombinant protein (rHc23) of H. contortus has been obtained after verifying that the native Hc23 protein, with a molecular weight of 23kDa, originating from the soluble extract of adults of the blood-sucking nematode H. contortus, has shown immunoprotective capacity in cattle 5-month-old young sheep against the most important trichostrongylidosis (hemoncosis) from an economic and pathological point of view. The Hc23 protein has been characterized, obtaining its amino acid and genomic sequences, allowing, by molecular biology techniques, recombinant DNA, the production of a stable protein, rHc23. Its use in immunogenic compositions that include the combination of inactivated Propionibacterium acnes + E. coli lipopolysaccharides as adjuvant has shown a high degree of protection in animals (>85% protection).

Description

Recombinant Haemonchus contortus protein and its application for the production of a vaccine against hemoncosis.

SECTOR OF THE TECHNIQUE

The present invention falls within the field of Animal Health. More specifically, it refers to the preparation of immunogenic compositions that include the Hc23 protein or the recombinant Haemonchus contortus rHc23 protein for use in the preparation of vaccines against Haemonchus infestations. It also refers to the use of adjuvants to improve the protection granted by immunogenic compositions.

STATE OF THE TECHNIQUE

Ovine gastrointestinal nematodosis, including hemoncosis,
Produced by Haemonchus contortus, they represent one of the most relevant pathologies from an economic point of view worldwide. H. contortus is a nematode that is located in the ruminant abomasum, shows great fertility and both adults and larval stages (L4) feed on blood, causing blood loss, leading to anemia, anorexia, depression, and including death.

This parasitosis can occur - depending on the number of vermes present in the hosts, their age, environmental factors, immune status - as an acute or chronic process, constituting in both cases an important ruminant disease.

According to FAO- (http://www.fao.org.) -Hemoncosis represents 15% of parasitic gastroenteritis in small ruminants and causes the greatest economic losses of all trichostrongilidosis.

The introduction of more intensive exploitation systems, in irrigated meadows, entails an increase in livestock load and very short herbaceous use cycles. These conditions, together with the apparent inability of young individuals (less than 6 months of age) to respond effectively to infestations, represent an increased risk of clinical significance processes.

The control of this parasitosis is essentially based on the use of anthelmintic treatments and, although anthelmintics have been useful for decades, their indiscriminate use has caused the emergence of drug resistance worldwide (Waller PJ. (1997). Anthelmintic resistance, Veterinary Parasitol., 72: 391-412). There is also a growing concern about the possible presence of residues both in food and in the environment.

The cost of anthelmintic treatments (drugs + labor) has experienced a remarkable rise. The costs associated with the development and commercialization of new anthelmintic compounds have caused the number of new drugs to be very small. Under these conditions, immunoprophylaxis may be an alternative for the control of hemoncosis.

H.contorlus infestations, natural or experimental, cause partial protection against reinfestations. The appearance of the protective response is dependent on the age and race of the animals, it is estimated that up to 6 months of age do not achieve this capacity. This determines that animals, particularly lambs, are receptive to the parasite after weaning without the possibility of natural immunoprotection.

Over the years there have been numerous studies aimed at provoking protective immune responses in sheep against H.contorlus infestations, using infestations with viable L3, attenuated L3, antigen extracts, individual antigens. The lower efficacy of helminth vaccinations is related to the antigenic variations associated with the developmental changes they experience throughout their life cycle (various parasitic stages until reaching adulthood); They also perform intraorganic migrations as a mechanism to evade the immune response and sometimes, as is the case at hand, they are located in certain areas of the host that are not especially sensitive (stomach) against external agents, which makes generation difficult of an adequate response; without entering into the genetic diversity that both parasitic and host populations can present.

Attempts to develop a vaccine against hemoncosis have been very numerous using various parasitic components (Smith, WD and Zarlenga DS (2006). Developments and hurdles in generating vaccines for controlling helminth parasites of grazing ruminants. Veterinary Parasitology, 139: 347 -359) from those found in the nematode when it begins its parasitic life (L3) (US5525508; Goud, GN; Zhan, B .; Ghosh, K .; Loukas, A; Hawdon, J .; Dobardzic, A; Deumic , V .; Liu, S .; Dobardzic, R; Zook, R C .; Qun, J .; Liu, YY; Hoffman, L .; ChungDebose, D .; Patel, R; Mendez, S. and Hotez, PJ (2004) Cloning, yeast expression, isolation and vaccine testing of recombinant Ancylostoma secreted protein 1 (ASP-1) and ASP-2 from Ancylostoma ceylanicum Journal of Infectious Diseases 189: 919-929, Goud, GN; Bottazzi, ME; Zhan, B .; Mendez, S .; Deumic, V .; Plieskatt, J .; Liu, S .; Wang, Y .; Well, L .; Fujiwara, R; Samuel, A; Ahn, SY; Solanki, M .; Apsojo, O. A; Wang, J .; Bethony, J. M .; Loukas, A; Roy, M. and Hotez, P. J. (2005). Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials. Vaccine

23: 4754-4764; Bethony, J. M .; Loukas, A; Smout, M. J .; Mendez, S .; Wang, Y .; Bottazzi, M. E .; Zhan, B., Williamson, A L .; Lustigman, S .; Correa-Oliveira, R; Xiao, S. H. and Hotez, P. J. (2005). Antibodies against a secreted protein from hookworm larvae reduce the intensity of infection in humans and vaccinated laboratory animals. FASEB Journal19: 1743-1755; Méndez et al., 2005) to components of the adult phase of the parasite, proteases derived from its intestine, including aspartyl-, cysteinyl- and metalloproteases (Knox, DP and Smith, WD (2001). Vaccination against gastrointestinal nematode parasites of ruminants using gut-expressed antigens. Veterinary Parasitology,

100:

21-32; Loukas, A; Bethony, J. M .; Williamson, A L .; Goud, G. N .; Mendez, S .; Zhan, B .; Hawdon, J. M .; Bottazzi, M. E .; Brindley, P. J. and Hotez, P. J. (2004). Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes the fecundity and growth of worms. Journal of Infectious Diseases 189: 1952-1961; Loukas, A; Bethony,

J.

M .; Mendez, S .; Fujiwara, R T .; Goud, G. N .; Ranjit, N .; Zhan, B .; Jones, K .; Bottazzi, M. E. and Hotez, P. J. (2005). Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs. PLoS Medicine 2: e295; Williamson, A L .; Lecchi, P .; Turk,

B.

AND.; Choe, Y .; Hotez, P. J .; McKerrow, J. H .; Cantley, L. C .; Sajid, M. and Loukas, A (2004). A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms. Journal of Biological Chemistry 279:

3560-3567). The use of irradiated larvae is capable of inducing protective immunity in adult animals although it does not protect young animals. The use of purified larval extracts (70-83 kOa of unsheathed L3-Newton, SE and Munn, EA (1999). The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus. Parasitology Today, 15: 116-122) has shown some protection, dependent on the adjuvant used, superior with aluminum hydroxide over QuilA. Partial protection has been achieved in older animals with L4 excretion and secretion antigens and parasite adults (Hc15 / 4-Schallig, HDFH; Van Leeuwen, MAW and Cornelissen, AWCA (1997). Protective immunity induced by vaccination with two Haemonchus contortus excretory secretory proteins in sheep Parasite Immunology, 19: 447-453; Bakker, N .; Vervelde, L .; Kanobana, K .; Knox. OP; Cornelissen, AWCA; de Vries, E. and Yatsuda, AP (2004 Vaccination against the nematode Haemonchus contortus with a thiol-binding fraction from the excretory / secretory products (ES). Vaccine, 22: 618-628). Immunizations with low molecular weight fractions from the soluble adult extract of the parasite caused reductions of less than 60% (Domínguez-Toraño, IA; Cuquerella, M .; Gómez-Muñoz, MT; Méndez, S .; Fernández-Pérez, FJ and Alunda, JM (2000). Vaccination of manchego lambs against Haemonchus contortus with a somatic fraction (p26 / 23) of adult parasites. Parasite Immunology, 22: 131-138) in lambs less than 5 months old. Lower values have been achieved with 3 low molecular weight fractions of the helminth (Alunda J.M .; Angulo-Cubillán, F. and Cuquerella,

M. (2003). Immunization against ovine haemonchosis with three low molecular weight somatic antigens of adult Haemonchus contortus. Journal of Veterinary Medicine, B 50: 70-74). The use of H11 glycoprotein (110kDa) from the intestinal microvilli of L4, preadult and adults of

H. contortus apparently reduced egg elimination by 90% and adult helminth load by 75% (Munn, E.A. (1997). Rational design of nematode vaccines: hidden antigens. International Journal for Parasitology,

27: 359-366). The results were superior using Quil A as an adjuvant (Newton, S.E. and Munn, E.A. (1999). The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus. Parasitology Today, 15: 116-122). Antigens from the intestinal surface of the nematode (galactose-containing glycoprotein complex, H-gal-GP) have been used, this complex also has other components such as aspartin-cysteinyl-metallo-proteases, thrombospondin-like proteins, cystatin and galactin, so it is not clear which of them is responsible for its protective activity. The use as an adjuvant of QuilA intensified its protective role (Knox, OP; Redmond, OL; Newlands, GF; Skuce, PJ; Pettit, O. and Smith, WO (2003). The nature and prospects for gut membrane proteins as vaccine candidates for Haemonchus contortus and other ruminant trichostrongyloids. International Journal for Parasitology, 33: 1129-1137). Vaccines with cysteine proteases have been used to alter the blood supply of the parasite, a fibrinogen degradation complex (35-55 kOa) with considerable reductions (93% egg reduction, 87% adults) (Soisvenue, RJ; Stiff, MI; Tonkinson, LV; Cox, GN and Hageman, R (1992). Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep. American Journal of Veterinary Research, 53: 1263-1265) and binding proteins to thiols (TSSP) similar to cathepsin S that have conferred high protection (Newton, SE and Munn, EA (1999). The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus. Parasitology Today, 15: 116-122). Lately, the use of glycoconjugates in vaccination tests has been proposed (van Stijn, CMW; van den Broek, M .; Vervelde, L .; Alvarez, RA .; Cummings, RO .; Tefsen, S .; Van Die, 1. (2010) Vaccination-induced IgG response to Gala1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection Int. J. Parasitol 40 (2010) 215222).

Despite partial successes with the components of the helminth in its native form, antigens that have been cloned and expressed have not induced protection against hemoncosis (Newton, SE and Meeusen, ENT (2003). Progress and new technologies for developing vaccines against gastrointestinal nematode parasites of sheep. Parasite Immunology., 25: 283296; Knox, OP (2010). Parasite Vaccines: Recent Progress in, and Problems Associated with their Oevelopment. Open Infect. Dis. J., 4: 63-73 ). The use of recombinant Hc 15/24 only caused partial protection (close to 50%) in animals older than 9 months (Vervelde, L .; Van Leewen, MAW; Kruidenier, M .; Kooyman, FNJ; Huntley, JF; Van Die, 1. and Cornelissen,

A.W.C.A. (2002). Protection studies with recombinant excretory / secretory proteins of Haemonchus contortus. Parasite Immunology, 24: 189-201). The recombinant proteins H11, H-gal-GP and the TSSP were unable to confer protection against hemoncosis, regardless of the adjuvants used. Immunization with a 21.4 kOa defective recombinant protein (p26 / 23) with Freund's complete and incomplete adjuvant did not induce any protection in lambs less than 6 months of age against a challenge with H. contortus although there were very high antibody levels (García Coiradas, L .; Angulo-Cubillán, F .; Valladares, B .; Martínez, E .; de la Fuente, C .; Alunda, JM .; and Cuquerella, M. (2010). Immunization against lamb Haemonchosis with to Recombinant Somatic Antigen of Haemonchus contortus (rHcp26 / 23). Veterinary Medicine International. pp. 1-8).

Given the low efficacy of the recombinant proteins used so far against H. contortus infestations, the economic importance of hemoncosis, the anthelmintic resistance detected against all pharmacological groups of anthelmintics, and the practical impossibility of immunizations with native proteins of the helminth, there is a need to find proteins, native and especially recombinant, as well as formulations that are effective against the parasite cited.

EXPLANATION OF THE INVENTION

One aspect of this invention relates to an adult Haemonchus contortus protein, Hc23, characterized by SEa ID NO: 2, to the recombinant rHc23 protein and to polypeptides with at least 80% identity with SEa ID NO: 2 which maintain the antigenic properties of Hc23. Any of these proteins or polypeptides can be included in an immunogenic composition that allows the production of a vaccine against

H. contortus. The use of the vaccine thus produced achieves protections greater than 85% of the parasitic load and fecal elimination of eggs from the helminth.

The invention also relates to the gene encoding the Hc23 protein of

H. contortus, characterized by SEa ID NO: 1 and sequences with a percentage of identity with SEa ID NO: 1 of at least 70%.

In this specification, the percentage of amino acid sequence identity is understood as the percentage of coincidences of the same amino acids between the two aligned sequences, along the entire length of both sequences. In addition, polypeptides with at least 80% identity with SEQ ID NO: 2 referred to herein are those that maintain the antigenic characteristics of the Hc23 protein. Likewise, the percentage of nucleotide sequence identity is understood as the percentage of coincidences of the same nucleotides between two aligned sequences, along the entire length of both sequences.

Another aspect of the invention relates to the sequence of the Hc23 gene, or to sequences with at least 70% identity with said sequences, cloned into a vector that is also susceptible to be included in prokaryotic or eukaryotic host cells in which are expressed nucleotide molecules.

The invention also relates to an immunogenic composition comprising: the Hc23 protein, the rHc23 recombinant protein or poJypeptides with at least 80% identity with the Hc23 protein; the nucleotide molecule of the Hc23 gene, or sequences with at least 70% identity with said sequence; vectors containing any of these nucleotide sequences or prokaryotic or eukaryotic vector-bearing cells that include the nucleotide molecule of the Hc23 gene, or sequences with at least 70% identity with said nucleotide sequence.

This immunogenic composition can be used in the preparation of a vaccine for administration to sheep in order to confer protection against diseases caused by H. contortus. In addition, the immunogenic composition may include adjuvants to improve the immune response. The adjuvants of choice are aluminum hydroxide and the combination of inactivated Propionibacterium acnes + lipopolysaccharides of

E. coli. The vaccine made with the immunogenic composition that includes the combination of inactivated Propionibacterium acnes + E. coli lipopolysaccharides as an adjuvant against hemoncosis in young lambs, with protection values far superior to those achieved so far with other vaccines. Vaccination with the immunogenic composition that aluminum hydroxide as an adjuvant induces partial protection against hemoncosis in lambs.

On the other hand, the invention also relates to the method for preparing an immunogenic composition comprising the formulation of at least one of the following products: the Hc23 protein, characterized by SEQ ID NO: 2, the recombinant rHc23 protein or polypeptides with At least 80% identity with SEQ ID NO: 2; the gene that encodes the Hc23 protein from

H. contortus, characterized by SEQ ID NO: 1, or sequences with a percent identity with SEQ ID NO: 1 of at least 70%; a recombinant vector that includes one of these nucleotide sequences; a transformed prokaryotic cell, or a transfected eukaryotic cell, with a recombinant vector as described herein; an adjuvant which, in turn, can be selected from aluminum hydroxide and inactivated Propionibaeterium aenes

+ E. eoli lipopolysaccharides.

Until now, the use of recombinant vaccines against hemoncosis has not shown the expected efficacy, in fact multiple failures are recorded in the literature.

The immunogenic composition of the invention allows obtaining a high degree of protection in animals, in addition to a considerable decrease in parasitic contamination of the environment, it also shows the advantage of being effective in young animals, without the need to wait a year of life (as the majority of the scientific community maintains) so that animals reach immune maturity and are able to respond to hemoncosis.

The immunogenic composition that includes a recombinant protein or polypeptide avoids the problems associated with the use of poorly characterized vaccines from crude parasite extracts that may contain both immunomodulatory and immunosuppressive components. Likewise, the fact that the protein or polypeptides can be synthetically obtained, which can be easily synthesized by molecular biology techniques, avoids the "socially questionable" need for the use of experimental animals (ruminants) as permanent donors for obtaining the extract or the native protein of the parasite that integrates the vaccine unit!

Figure 1. Fragment of about 600 bp obtained by PCR of the positive halos of Soufhem blof from the cONA library of

H. confortus generated in the Uni-ZAP XR vector.

Figure 2. 23 KOa band resulting from the purification of the Hc23 protein.

Figure 3. Fecal removal of eggs (hpg) from H. confortus during the vaccination test described in example 3.

Figure 4. Adult count in the abomasum of the animals of the vaccination test described in example 3.

Figure 5. Hematocrit value throughout the vaccination test described in example 3. Arrow marked the time of infestation.

Figure 6. Eosinophil count throughout the vaccination test described in example 3. Arrow marked the time of infestation.

MODE OF INVENTION

Example 1. Production of the recombinant Hc23 protein (rHc23).

In order to obtain the complete gene encoding the Hc23 protein, it was based on the somatic antigen of H. confortus p26 / 23 partially cloned and expressed in E. coli (García Coiradas, L .; Angulo-Cubillán, F .; Valladares, 8 .; Martínez, E .; de la Fuente, C .; Alunda, JM .; and Cuquerella, M. (2010). Immunization against lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus confortus (rHcp26 / 23). Veferinary Medicine Infemafional pp. 1-8).

From the positive clones that carried the incomplete p26 / 23 gene, conserved in the PGEM-T plasmid at -80'C, a colony PCR was performed.

Plasmid primers were used:

SP6: 5 'ATTTAGGTGACACTATAGAA 3', characterized by SEQ ID NO: 3

T7: 5 'TAATACGACTCACTATAGGG 3', characterized by SEQ ID NO: 4

This allowed the amplification of a 757 bp fragment. The selected

3 colonies that had shown greater purity in electrophoresis.

The 3 bands (one per colony) were purified by "gel extraction kit" (QIAGEN) and sequenced. The result was consistent with the initial (incomplete) sequence of the gene encoding p26 / 23, which used the primers of the insert F and R:

F: 5 'GCAGGACTGTTCGCACAT 3', characterized by SEQ ID NO: 5

R: 5 'TCAGTCTTTCGCGGACTTG 3', characterized by SEQ ID NO: 6

which allowed the amplification of a 580 bp fragment. Subsequently, 2 colonies were chosen that presented the highest purity in amplification and the two bands (one per colony) were purified from the "gel extraction kit" agarose gel (Qiagen).

Once the sequence was obtained, its labeling was carried out, for this we performed a PCR with these 2 colonies using the same primers and adding «OIG-ONA labeling mix» as a marker.

After the construction of a H.conforlus cAON genomic library using the Lambda Uni ZAP XR vector (Stratagene) we used 10-4 dilutions of the cAON library, which made a total of 1.2 x 104 colony forming units ( cfu) scrutinized; they were contacted with the previously constructed probe at a concentration of 25 I ") g / I-Il, under highly astringent conditions (68 ° C). 8 positive halos were selected.

The presence of the p26 / 23 gene in the 8 isolated phages was checked by a PCR reaction using primers F and R. The 8 isolated phages generated a 580 bp amplicon (Fig. 1), confirming their presence in the phages, however, it remained to be determined if it was complete.

The fragment was purified, using the QiaEX 11 system (Qiagen) following the manufacturer's instructions. The purified samples were subjected to PCR amplification using phage T3 primers and final R sequence to achieve the complete gene sequence:

T3: 5 'AATTAACCCTCACTAAAGGG 3', characterized by SEQ ID NO: 7

R: 5 'TCAGTCTTTCGCGGACTIG 3', characterized by SEQ ID NO: 6

An amplicon of 612 bp was obtained, which we call Hc23.

Once the complete sequence of the gene encoding Hc23 was achieved, the recombinant rHc23 protein was obtained. The PCR amplified products were linked to pGEM®-T Easy Vector (Promega) to ensure the correct position and allow the purification of a complete copy of the gene corresponding to Hc23. Competent cells were used E. coli XL2-Blue strain. Positive colonies were selected and the expression vector was digested with Ndel and Xhol. The insert was linked to the vector pET-29b (+) (Novagen); as competent cells we use E. coli BL21.

The bacteria were grown overnight at 37 oC, in LB medium with kanamycin. Induction of the recombinant protein was obtained with 0.5 mM isopropyl-1-thio-b-0-galactopyranoside (IPTG) (Roche) for 2 h.

The protein was solubilized in buffer [20 mM Tris-HCI pH 8, 0.5 M NaCI, 5 mM imidazole, 10 mM 3-ME and protease inhibitors (Roche)]. The solubilization was optimal with 0.15% sarcosil.

The recombinant protein (rHc23) was associated with a tail of 6 histidines, which allowed its purification in a nickel-nitrilitriacetic acid (Ni-TNA) column (Qiagen) by elution with 100 and 250 mM imidazole. The fractions obtained were subjected to SOS-PAGE electrophoresis. The purified protein was analyzed by electrophoresis and Westem blotting, detecting 2 bands, one 23 kOa (rHc23) and the other 46 KOa (dimer).

The nucleotide sequence of PCR products and clones of positive bacteria in E. col; XL2-blue and BL 21 were determined in the Genetics section of the Animal Production Department of the Veterinary Faculty of the UCM and in the Genomics Service, General Research Support Services, University of La Laguna, La Laguna, Tenerife .

Example 2. Purification of the native Hc23 protein

The native Hc23 protein could be obtained by means of 2 serial affinity chromatographs. To this end, anti-rHc23 hyperimmune sera were previously obtained by inoculations in rabbit (New Zealand breed, white) with 150 I-Ig of rHc23 (intramuscular) -3 times the first with 1 mL of Freund's complete adjuvant and the second and third with Freund's incomplete adjuvant.

The specificity of these sera was analyzed by electrophoresis and Western blotting, being able to recognize the rHc23 protein. ELlSA showed very high antibody titers.

In a first affinity chromatography we used an agarose column with protein A (Roche), following the manufacturer's recommendations, and hyperimmune serum, which allowed us to obtain purified polyclonal antibodies. To determine the fraction containing the immunoglobulins, the fractions were measured by spectrophotometry at 280 nm and the purified immunoglobulins were dialyzed in PBS. Polyclonal antibodies were concentrated after determination of their concentration, using the Bradford method (Bradford, MM (1973). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principie of protein-dye binding. Ann Biochem. , 72: 248254).

An immunoaffinity chromatography (column with "CNBr activated Sepharose 4 B beads" + purified antibodies) was then performed with soluble extract of adults of H. contorfus obtained by homogenization of adults of the parasite and subsequent centrifugation at 30000xg, for 30 minutes, to get the supernatant. The column was maintained at 4 ° C overnight, allowing the antibodies in the column to attach to the soluble adult extract of the parasite. The flow that passed through the column was collected and the column was washed with equilibration buffer until the spectrophotometer reading at 280 nm was the baseline. The fixed protein was eluted by elution buffer and collected in neutralization buffer. Fractions containing protein (measured at 280 nm) were purified and dialyzed with PBS allowing purification of native Hc23 (Fig. 2).

Example 3. Vaccination tests against sheep hemoncosis with Hc23 and rHc23 proteins

Parasites

For our study we used L-3 of an isolate of H. contortus, kindly provided by Merck, Sharp and Dohme (Madrid, Spain) and maintained in our laboratory for more than 2 decades by serial passes on donor animals. The larvae 3 were obtained by the larval migration technique [Baerman method, Ministry of Agriculture, Fisheries and Food (MAFF (Ministry of Agriculture, Fisheries and Food) (1971). Manual of Veterinary Parasitological Laboratory Techniques. HMSO, London)] .

Animals and Experimental Design

48 lambs of the 4 month old Assaf breed were housed in the facilities of the Department of Animal Health. They were kept for the first 15 days under observation, at the same time they were weighed and blood and fecal samples were taken to determine their parasitological status. One month after their arrival, the animals were randomly separated into 6 lots of 7 lambs, which would be subjected to various treatments and 1 lot of 6 animals that would act as witnesses to the experiment.

Group I was immunized with the native Hc23 protein of H. contortus (1 00l-lg) in Aluminum hydroxide [13 mg / ml (SIGMA A-8222)] (9001-11), 3 times, every 2 weeks and 2 weeks after the last dose he underwent an infestation (challenge) with 15,000 L3 of the parasite. Group 11 followed the same immunization and infestation protocol but using the protein

recombinant rHc23 (similar concentration and infestant dose). Group 111 received a first dose of the native protein (100l-lg) in an adjuvant solution containing 2mg of E. coli lipopolysaccharides and 25mg of Propionibacterium sp (Infervac de Laboratorios Calier, SA) (1 mLl1 Okg live weight) ; 2 days later the same inoculation was repeated and the animals were infested; Finally, one week later they received a booster immunization dose that was repeated the following week. In group IV the same immunization and infestation protocol of group 111 was performed, but using recombinant protein, at the same concentration. Group V was the total witness of immunization and infestation. Group VI was the positive witness of the infestation, the animals were subjected exclusively to the parasitic challenge. Group VII (with 4 and a half months of age) was primoinfested with 10,000 L3 of H. contortus, said infestation was terminated on day 35 post-infestation after an anthelmintic treatment with Valbazen ® 10% Albendazole (Pfizer), 2 weeks later it was taken out the reinfestation (challenge) of the animals.

The parasitic challenge remained in all groups for 45 days, to finally proceed to the sacrifice of the animals.

Parasitological determinations

Weekly coprological analyzes were performed to determine fecal removal of eggs throughout the experiment. However, during the end of the prepatence and until the beginning of the patent, analyzes were carried out every 2 days. For coprological determinations we use the modified McMaster method (MAFF (Ministry of Agriculture, Fisheries and Food) (1971). Manual or, Veterinary Parasitological Laboratory Techniques. HMSO, London).

After the necropsy of the animals, the existing adult population was studied. The abomasums were opened by the lesser curvature, placing the gastric content in 2% agarose gels and incubating it in physiological saline solution at 37 ° C overnight. The total load of adults was determined.

Results

All vaccinated groups showed a statistically significant reduction in fecal removal of eggs, of 57% in G 1, 74% in G 11 and 79% in G 111 and GIV compared to the group that only received the parasitic challenge (GVI). This decrease was significant on days 21, 24, 28, 35 and 42 post-infestation, with values of p <0.001 for all groups vaccinated on days 24 and 28, on day 35 a p <0.05 was observed for the GI and <0.001 for the rest of vaccinated groups; also we found significance on day 42 in the Gil groups (with a value of p <0.05) and in GIII and GIV of p <0.001; In addition, the Gil and GIV groups showed significant reduction (p <0.05) on day 21 post-infestation.

The degree of parasitic contamination of the medium, which was estimated as the sum of eliminated eggs (hpg) throughout the experiment, was lower in all vaccinated groups (GI: 75.250, Gil: 42.365, GIII: 36.830 and GIV:

37,575 hpg), with decreases that ranged from 4.5% to 9% less than that generated by the primary group (GVI) that eliminated a total of 323,942hpg (Fig. 3).

The establishment of the adult population (no. Of adults in abomasum) showed a significant decrease in vaccinated groups, from 69% for the GI to more than 85% for the other 3 groups (Gil, GIII and GIV), with parasitic loads 318 ± 385 (GI), 147 ± 203 (Gil), 127 ± 127 (GIII), 131 ± 190 (GIV) compared to the primary group (GVI 1030 ± 383). (Fig. 4)

A positive correlation was observed between fecal elimination of eggs and parasitic establishment (r = 0.85, p <O, 0001).

The values of the determined blood parameters (hematocrit and eosinophils among others) were related to the time of the infestation and the response capacity of the animals. The vaccinated animals showed lower blood losses, with hematocrit values higher than those determined in the primary group, with significance of p <O, 001 throughout the experiment and p <O, 01 on days 63, 77 and 88 post-vaccination (Fig. 5).

A negative correlation was observed between fecal elimination of eggs and the hematocrit value (r = -0.82, p <O, 0001).

The model of the eosinophilic response detected was related to the immunization protocol used. Groups GI and 11 showed a similar behavior throughout the trial, with an increase in their values from day 28 post-vaccination, with a peak on day 7 post-infestation (mean values of 0.87 X 103 eosinophils! JL and 0 , 93 X 103 eosinophils! JL, for GI and Gil respectively.

On the other hand, groups GIII and IV showed a similar behavior throughout the trial, with an increase in the number of eosinophils from day 7 post-confestation, with a peak on day 21 (1.04 X 103 eosinophils! JL and 0.61 X 103 eosinophils! JL, for GIII and GIV respectively.

The statistical analysis determined significant differences on days 49, 63, 77 and 88 between the vaccinated groups (with elevation in the number of eosinophils) and the primary infosted group VI, with values from p <0.05 to p <O, 001 ( Fig. 6).

The GVII group subjected to an infestation and parasitic challenge behaved during primoinfestation in the same way as group VI (primary only) with counts of the elimination of raised eggs. After the challenge, the animals showed a reduction in egg counts and parasite load compared to the GVI group (statistically significant); however, no significant differences were observed with the vaccinated groups.

Although this primary and reinfrested group after the challenge showed good protection, it must be considered that the previous infestation (essential to achieve that degree of protection) caused a high elimination of eggs from the parasite, with the consequent contamination of the environment and a initial decline in physical conditions due to blood loss.

INDUSTRIAL APPLICATION

The veterinary sector and products intended for animal health face the need to increase the quality of the products used in order to meet the technification of the sector and higher demands by consumers. The development of efficient, pure, cheap, safe and environmentally friendly products represents a qualitative leap of great importance for the future of the veterinary sector.

In addition, the development of products obtained by biotechnology such as those of the invention may represent the consolidation of prevention, as an alternative to treatment, for diseases as widespread in cattle as trichostrongilidosis, allowing us to think about significant improvements in the safety and competitiveness of Animal production

The incorporation of the recombinant rHc23 protein, or derivatives thereof, into immunogenic compositions for vaccine production is capable of significantly reducing (up to> 80%) the parasitic load and elimination of eggs in the H. contortus medium, in dependence on the adjuvant employed.

FREE TEXT OF THE LIST OF SEQUENCES

The free text used in the sequence list corresponds in Spanish to the following characteristics:

SEQ ID NO: 3. Primer SP6 of plasmid PGEM-T;

SEQ ID NO: 4. Primer T7 of the plasmid PGEM-T; SEQ ID NO: 7. Primer T3 of the lambda plasmid ZAP CMV XR.

Claims (13)

one.

Isolated nucleic acid molecule, characterized by SEa ID NO: 1 or with at least 70% identity with SEa ID NO: 1, which contains the nucleotide sequence encoding the H. contortus Hc23 protein, characterized by SEa ID NO: 2, or a polypeptide with at least 80% identity with SEa ID NO: 2 that maintains the antigenic properties of Hc23.

2.

Purified amino acid molecule comprising the Hc23 sequence of

H. contortus, characterized by SEa ID NO: 2, or a sequence with at least 80% identity with SEa ID NO: 2 that maintains the antigenic characteristics of Hc23.

3.

Recombinant vector including a sequence comprised in claim 1.

Four.

Prokaryotic host cell transformed with the recombinant vectors of claim 3.

5.

Eukaryotic host cell transfected with the recombinant vectors of claim 3.

6.

Immunogenic composition including: the Hc23 protein, the rHc23 recombinant protein or polypeptides with at least 80% identity with the Hc23 protein; the nucleotide molecule of the Hc23 gene, or sequences with at least 70% identity with said sequence; vectors containing any of these nucleotide sequences; vector-bearing prokaryotic cells that include the nucleotide molecule of the Hc23 gene, or sequences with at least 70% identity with said nucleotide sequence; or eukaryotic vector-bearing cells that include the nucleotide molecule of the Hc23 gene, or sequences with at least 70% identity with said nucleotide sequence.

7.

Immunogenic composition according to claim 6 which includes an adjuvant.

8.

Immunogenic composition according to claim 7 wherein the adjuvant is aluminum hydroxide.

9.

Immunogenic composition according to claim 7 wherein the adjuvant is a combination of inactivated Propionibacterium acnes + E. coli lipopolysaccharides.

10.

Use of the composition of any of claims 6-9 in the preparation of a vaccine against hemoncosis in ruminants.

eleven.

Use according to claim 10 in which the ruminants are sheep.

12.

Use according to claim 11 wherein the sheep are between 4 and 12 months old.

Fig. 1

600 bp

300 bp

100 bp

Fig. 2

KDa

94

67

43

30

20.1

14.4

 Fig. 3 hpg 15000 -f-GI

+

Gil

+

GIII

+

IVG

-B-GVI 16 18 21 24 28 35 42 Days post-confession Fig. 4 2500 .. ~ GI '5 1500 ~ "you C'G 00 Gil or z g GIII 1000 rrm GIV ~ GV 500 ~ GVI or GI Gil GIJI GIV GV GVI Fig. 5 o 14 28 42 49 63 77 88 Post vaccination days Fig. 6 1.5

-

~ M "" "

-

O ~ J le

'OR 1.0 .s -t-GI ! w ..... Gil ...... GIII -f .. GIV 0.5 o 14 28 42 49 63 77 88 Post vaccination days <110> Complutense University of Madrid <120> Recombinant Haemonchus contortus protein and its production of a vaccine against hemoncosis <130> 2012_2 <160> 7 <170> BiSSAP 1.0 <210> 1 <211> 612 <212> ONA <213> Haemonchus contortus <220> <221> source <222> 1 .. 612 <223> / mol_type = "ONA" / organism = "Haemonchus contortus" <220> <221> COS <222> 1,609 <223> / transl_table = l
application for / translation = "MRTLVLVAISVAAVSAAGLFAHHPPPECGLPPFVNOLPAOOQAKLKOIWKN WKEGOKCYHEQGLTROLVETLPTEIRRKISKOALLPPPVRKAPEEVQEQFRKIINOKTIPVEEKH KKMNELAQKVLTGONLKEYNEFTAHIEORHKAVAOKAATLSPEAKAAYOKIAKLEKEKHOIIASL NEQAQEELFQVFKLRHSKSAKO " <400> 1 atgcgaacac tggtattagt ggcaatcagc gttgcggctg tcagtgctgc aggactgttc 60 gcacatcatc ctcctcctga atgtgggctc ccaccattcg tcaacgatct tccagctgat 120 agctgaaaga gaccaagcaa tatttggaag aactggaagg aaggcgataa gtgctatcat 180 gagcaaggcc tcaccagaga tctggtggaa acactcccaa ctgaaattcg tagaaaaatc 240 agcaaggatg cacttctgcc accgcctgtg aggaaggcgc cagaagaagt ccaggaacag 300 ttccgtaaaa tcatcaacga caagacgatc ccagttgaag aaaagcacaa gaagatgaat 360 gaactagcac agaaggttct cactggtgac aatctcaagg agtataacga attcactgcg 420 catattgagg atcgccataa ggcggttgcg gacaaggcag ccacactttc accagaagct 480 aaggcggcat acgacaagat cgccaaattg gaaaaggaga aacacgatat tattgcatcg 540 cttaacgagc aggctcaaga agaactattc caggtcttaagagagagagagagagagagaga <210> 2 <211> 203 <212> PRT <213> Haemonchus contortus <220> <221> SOURCE <222> 1.203 <223> / mol_txpe = "rotein" / note = Echoes .1 .. 609 from SEQ ID NO 1 "/ organism =" Haemonchus contortus " <400> 2 Met Arg Thr Leu val Leu val Ala rle ser val Ala Ala val ser Ala 1 5 10 15 Ala Gly Leu Phe Ala His Hi s Pro Pro pro Glu cys Gly Leu pro pro 20 25 30 phe val Asn ASp Leu pro Wing ASp ASp Gln Wing Lys Leu Lys ASp rle 35 40 45 Trp Lys Asn Trp Lys Glu Gly ASp Lys cys Tyr His Glu Gln Gly Leu 50 55 60 Thr Arg ASp Leu val Glu Thr Leu Pro Thr Glu rle Arg Arg Lys rle 65 70 75 80 Ser Lys ASp Ala Leu Leu Pro pro val Arg Lys Ala Pro Glu Glu 85 90 95 val Gln Glu Gln Phe Arg Lys rl e rle Asn ASp Lys Thr rl e pro val 100 105 110 Glu Glu Lys His Lys Lys Met Asn Glu Leu Wing Gln Lys val Leu Thr 115 120 125 Gly ASp Asn Leu Lys Glu Tyr Asn Glu phe Thr Ala His rle Glu ASp 130 135 140 Arg His Lys Wing val Wing ASp Lys Wing Wing Thr Leu Ser pro Glu Wing 145 150 155 160 Lys Wing Wing Tyr ASp Lys rle Wing Lys Leu Glu Lys Glu Lys His ASp 165 170 175 rle rle Ala Ser Leu Asn Glu Gln Ala Gln Glu Glu Leu Phe Gln val 180 185 190 Phe Lys Leu Arg His Ser Lys Ser Ala Lys ASp 195 200 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <222> 1..20 <223> / mol_tr, pe = "DNA" / note = 'sP6 primer from the PGEM-T plasmid " / organi sm = "Arti fi ci al sequence" <400> 3 atttaggtga cactatagaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <222> 1..20 <223> / mol_tr, pe = "DNA" / note = 'T7 p'rimer from the PGEM-T plasmid " / organism = 'Artificial sequence " <400> 4 taatacgact cactataggg 20 <210> 5 <211> 18 <212> DNA <213> Haemonchus contortus

<220> <221> source <222> 1. .18 <223> / mol_type = "ONA" / organism = "Haemonchus

contortus "

<400> 5 gcaggactgt tcgcacat

18

<210> 6 <211> 19 <212> ONA <213> Haemonchus

contortus

<220> <221> source <222> 1. .19 <223> / mol_type = "ONA" / organism = "Haemonchus contortus"

<400> 6 tcagtctttc gcggacttg

19

<210> 7 <211> 20 <212> ONA <213> Artificial

dry ce

<220> <221> source <222> 1..20 <223> / mol_ty, pe = "ONA" / note = 'T3 p, rimer from / organism =' Artificial

lambda ZAP sequence " CMV XR vector"

<400> 7 aattaaccct cactaaaggg

twenty