WO1994004187A1

WO1994004187A1 - Method for improving the growth and the quality of carcases of domestic meat-producer animals, vaccination kit et vaccines

Info

Publication number: WO1994004187A1
Application number: PCT/FR1993/000793
Authority: WO
Inventors: Raymond Dufour; Claude Roulet
Original assignee: Rhone Merieux Societe Anonyme
Priority date: 1992-08-20
Filing date: 1993-08-04
Publication date: 1994-03-03
Also published as: FR2694885A1; BE1007316A3; ITMI931762A0; AU4712693A; IT1264553B1; ITMI931762A1; FR2694885B1; IE930617A1

Abstract

Method for improving the growth and the quality of carcases of domestic swines andruminants intended to the production of meat, whereby the proteinic anabolism is substantially increased, particularly that of the muscular tissues, and the lipogenesis is decreased by augmentation of anabolizing actions of the endogenous growth hormone, the method being characterized in that the animal is administered a valence intended to induce an increase of the basal and pulsed secretion of the growth hormone GH and at least another valence selected amongst valences designed to induce such effect or a GH-potentiating immunomodulation .

Description

Method for improving the growth and quality of carcasses of meat-producing domestic animals, vaccination package and vaccines.

The present invention relates to a process for improving the growth and quality of carcasses of domestic pigs and ruminants intended for meat production, by which the protein anabolism, particularly that of muscle tissue, is substantially increased and lipogenesis, by increasing the anabolic actions of endogenous growth hormone. It also relates to a set of vaccinations and vaccines.

Continuous and prolonged administration of homologous growth hormone, extracted from the pituitary glands or produced by the expression of recombinant DNA in an appropriate host, to ruminants and growing pigs intended for meat production, leads to a increase in weight growth, an improvement in the composition of the carcass by an increase in protein mass compared to that of fats and an improvement in food efficiency.

The zootechnical interest of this hormone, in its applications to the increase of the production of meat or milk, is due to its properties of coordination of the tissue, muscular, mammary, adipose metabolisms, which act in support of physiological processes or dominant growths, which define homeorhesis and which implement the direct distribution of nutrients (Bauman DE et al., Federation Proc, 1982, 41, 2538-2544). Additional studies have shown that growth depends on a variety of factors such as the protein composition of the ration, the amount of hormone administered, and possibly specific environmental conditions.

In pigs, the work of Machlin (J. Anim. Sci., 1972, 35, 794-800), Chunq CS et al (J. Anim. Sci., 1985, 60, 118-130), Etherto TD et al . (J. Anim. Sci., 1987, 64, 433-443), Evock CM. et al (J. Anim. Sci., 1988, 66, 1928-1941), Campbell RG et al (J. Anim. Sci. 1989, 67, 177-186), Smith VG and Kasson (J. Anim. Sci. , 1990, 68, 4109-4116) and Grant AL et al. (J. Endocrinol., 1991, 130, 331-338; 1991, 69, 571-577) have shown that porcine growth hormone administered for several weeks only clearly developed its anabolic effects on muscle growth and food efficiency on animals fed a protein-rich diet. In ruminants, the same conclusions were brought by the work of Pell JM et al. (British Journal of Nutrition 1990, 63, 431-445) and Mac Rae JC (J. Endocrinol., 1991, 130, 53-61) on sheep, and those of Eisemann et al. (J. Nutrition 1986, 116, 2504-2515 and J. Nutrition 1989, 67, 105-115) and de Breir BH et al. (J. Endocrinol., 1988, 116, 169-177) for heifers and steers. The latter have shown in particular that weight gain was correlated with the development of hepatic receptors of great affinity for GH, which also depended on the protein content of the ration.

To overcome the difficulties or constraints linked to the exogenous administration of growth hormone, immunological methods attempt to replace this exogenous supply, either by seeking to increase the activity of endogenous GH, by the action of antibodies directed against certain peptide sequences of GH, either by seeking to increase the secretion of endogenous GH by the action of antibodies directed against certain peptide sequences of GRF (Growth Hormone Releasing Factor) or by the immunoneutral isation of somatostatin (SRIF: Somatotropin Release Inhibiting Factor).

The increase in the activity of GH of external origin on the growth of hypophysectomi ses rats or mice lacking GH was obtained by its administration in the form of complexes with certain monoclonal antibodies vis-à-vis certain epitopes of GH by Holder AT et al (J. Endocrinol., 1986, 107, R9-R12), Aston R. et al. (J. Endocrinol., 1986, 110, 381-388), Aston R. et al. (Mol. Immunol. 1987, 24, 143-150), Wallis R. et al. (Biochem. Biophys. Res. Commun., 1987, 149, 187-193), Aston R. et al. (Mol. Immunol., 1989, 25, 435-446), patent application EP-AO 458 072 (American Cyanamid Company), concerning the monoclonal antibody PS 7.6 associated with pST, and by Battes PC et al. (J. Endocrinol., 1992, 132, 369-371). This increase in GH activity is also observed with certain sera prepared using specific sequences of ovine GH by Bomford R. and Aston R. (J. Endocrinol., 1990, 125, 31-38) or porcine GH by Wang BS et al. (J. Endocrinol., 1990, 127, 482-485).

The properties of the antibodies developed against certain peptide sequences of growth hormone can be implemented directly vis-à-vis the endogenous hormone of growing animals by means of active immunization directed against these sequences . These possibilities are mentioned in patent applications EP-AO 137 234 (Wellcome), EP-AO 303 488 (Coopers Animal Health Limited), EP-AO 284 406 (Coopers Animal Health Limited), EP-AO 429 788 (American Cyanamid Company) and WO 89/00166 (Peptide Technology LTD).

A particular application is described by Pell JM and Aston R. (J. Endocrinol., 1991, 131, R1-R4) using the sequence 134-154 of pST used in the form of an immunogenic conjugate in lamb. The increase in weight and carcass growth and the improvement in the protein composition of the carcass, without this resulting in a significant reduction in the amount of fat, are correlated with immunization against this peptide. The metabolic activities due to the immunomodulation of endogenous GH in this study would be different from those provided by the administration of GH and would not cause significant differences in the plasma concentrations of GH, IGF.I (insul ine- 1 ike growth factor I) and insulin.

Peptide sequences of the growth hormone causing the formation of potent antibodies isalising the anabolic activity of GH are described in the abovementioned patent applications: application EP-AO 137 234 describes the 7 kDa fragment of the part C-terminal of human growth hormone, application EP-AO 284 406 relates to the fragment 35-53 of growth hormone of different species, application WO 89/00166 relates to passive and active immunization against -vis peptide fragments of the sequence 112-159 of GH of different species, in particular ovine, bovine and porcine, application EP-AO 429 788 relates to the passive and active immunization of the fragments 98-110, 110-118 and 155-163 of porcine growth hormone and equivalent antigenic peptides. These fragments are compared to peptides 35-52, 36-44, 46-53 and 35-43 which are derived from those described in application EP-A-O 284 406.

EP-A-O 284 406 relates to the fragment

35-53 of porcine growth hormone (pGH), ovine or bovine (oGH / bGH), peptides bGH 26-43, bGH 35-43, bGH 37-48, bGH 39-46, bGH 43-54, bGH 43-61 and their human, avian, porcine and fish analogues. Their conjugation to different peptides, to T-cell epitopes and to the somatostat ine is considered from a theoretical perspective. These conjugates are proposed for active immunization intended in particular for the increase of somatogenesis and lactogenesis. Application WO 89/00166, for the same species, relates to the sequence 112-159 of GH, more particularly the sequences 130-150 of bovine GH, 134-154 and 120-140 of porcine GH.

The increase in endogenous secretion of GH by the increase in GRF activity by certain anti-GRF antibodies was the subject of patent application WO 90/15073 (Coopers Animal Health Limited) which proposes the use antibodies directed against peptide sequences located in the region 28-44 of GRF and in particular the peptide sequences 31-44 and 35-44 for improving the growth of lactation or of the composition of the carcass, than these antibodies are of passive or active origin. This same application theoretically proposes the conjugation of these sequences with all or part of somatostatin or growth hormone.

The active immunoneutralization of somatostatin aimed at increasing growth and the consumption index and improving the characteristics of the carcass has been described in its applications to domestic species.

In cattle, active immunization of cattle is reported by Trout WE and Schanbacher BD (J. Endocrinol., 1990, 125, 123-129) and Lawrence ME et al. (J. Anim. Sci., 1987, 215, Suppl. 1 abstract 135) in dairy heifers by Petitclerc G. et al. (J. Anim. Sci., 1988, 66, Suppl. 1389) and in the calf by Vicini JL and al. (Dom. Anim. Endocrinol. 1988, 5, 35). The effects of the anti isosmatostatin immunisatich are not consistent: the increase in the basal secretion of GH is only observed by Petitclerc et al. (1988 cited) and the increase in growth on dairy heifers is only observed by Lawrence et al. on the bouvi lions (1987 cited). Trout WE and Schanbacher (1990 cited) do not observe any effect on GH, on the increase in weight and on the composition of the carcass.

In sheep, work on somatostatin immunization has also led to discordant results. The increase in GH secretion, measured by the basal secretion and pulsed is observed on lambs receiving active immunization from the age of 5 months (Varnes MA et al., Endocrinology 1980, 106, 1027-1032) ; on the other hand, the weight gain of these animals is lower than that of the controls.

These results are not confirmed on lambs subjected to active immunization against isomatostat ine at the same age (4-5 months) and placed in rich or non-rich feeding conditions (Bass JJ et al., J. Endocrinol., 1987, 112, 27-31). Under these conditions, no increase in GH is observed and a slight increase in the growth rate is measured at the limit of significance in animals receiving the richest ration.

These two trials showed the ineffectiveness of active immunization against older somatostat in older lambs on growth and the significant role of food on growth rate. All the work which is then reported was carried out on younger lambs, aged 3 to 4 weeks, subjected to active immunization against somatostatin. They show an increase in the growth rate without modification of the carcass composition and of the basal or pulsed secretion of GH. This is how Spencer GSG et al. (Anim. Prod. 1981, 32, 376) observe an increase in growth of 76%; the following tests confirmed an increase in the rate of growth of the immunized lambs of the order of 15 to 20% (Spencer GSG et al., Livest. Prod. Sci, 1983, 10, 25-37 and 469-477; Chaplin RK et al., Can. J. Anim. Sci., 1984, 64, Suppl. 1, 312-313). However, this increase effect would be less significant in fast-developing breeds and would not be observed in these breeds on females (Spencer GSG et al., Livest. Prod. Sci. 1985, 13, 43-52). This reservation on the lower efficacy of immunization on rapidly developing breeds is invalidated in a more recent trial (Laarveld B. et al., Can. J. Anim. Sci., 1986, 66, 77-83).

In pigs, the first active immunization tests against somatostat ine were carried out by Spencer GSG, 1984 (cited by Spencer GSG, Reprod. Nutr. Develop., 1987, 27 (23), 581-589). For their part, Dubreuil P. et al. (Endocrinology 1989, 125, 1378-1384) subjected pigs to active anti-somatostat immunization to study the secretion of GH before and after external administration of GRF on animals fed or subjected to fasting. They do not describe in this report the effects on growth and carcass composition. They put in evidence, in the immune system, the elevation of the basal secretion of GH and the increase in the amplitude of the response of GH to close injections of GRF. This study shows that the active immunization against i somatostatin lifts a constant inhibition which would be due to somatostatin on the level of basal secretion and increases the amplitude of the peaks of spontaneous or provoked secretion by endogenous or exogenous GRF. It also leads to show that the interval separating two pulsed secretions is due to a refractory state which is partly under the control of SRIF. Active immunization against i somatostat ine suppressing the part of this state due to somatostatin would reveal another state of refractory to the closer pulsed stimulations which would be due to a desensitization of the GH secretory cells to the action of GRF. An identical observation was made on the rat subjected to a passive anti-SRIF immunization (Van Arsenigevic D, et al. Endocrinology, 1987, 121, 1487).

The application of passive anti-somatostatin immunization is the subject of US-A-4,599,229 (International Ores and Chemical).

The mechanisms of action of growth hormone or somatotropin are still poorly defined and can involve either a direct action of GH on the metabolism of tissue proteins, or an action dependent on insulin-1 ike growth factor 1 (IGF.1). The latter would involve the endocrine and / or autocrine-paracrine activities of IGF.1 to account for the presence of GH receptors, particularly on the liver and muscle. The endocrine activity of IGF.1 has been the subject of numerous demonstrations. Administration of IGF.1 to hypophysectomized rats stimulates their growth (Schoenle E. et al., Nature 1982, 296, 252-253). GH would increase the secretion of IGF. 1 Hepatigue which would act on the anabolism of proteins in muscle tissue. This mode of action is the conclusion of the study by Grant AL et al. (J. Endocrinol., 1991, 130, 331-338) who observe, after administration of GH to pigs for 1 month, an increase in the plasma concentration of IGF.1 associated with an increase in IGF mRNA. 1 of liver tissue and a decrease in IGF mRNA. 1 of muscle tissue. More indirectly, Chung S. et al. (Endocrinology 1986, 119, 780-786) have shown in pigs receiving a GH supply for 35 days, a positive correlation between the increase in the binding of hepatic receptors to GH, the serum concentration of IGF.1 and weight gain. Pell JM et al. (British Journal of Nutrition 1990, 63, 431-445) also envisage an endocrine action of IGF.1. They also observe in the lamb receiving a contribution of GH a positive correlation between the increase in the capacity of the binding sites of high affinity of GH of the hepatic tissue, the plasma concentration of IGF.1 and insulin. and the growth gains observed, but do not rule out a peripheral action of GH on muscle tissue. Mac Rae JC et al. (J. Endocrinol., 1991, 130, 53-61) also describe the simultaneous increase in IGF.1, insulin and glucose during the continuous administration of GH: they observe that the basic levels and the increases IGF.1 are higher with a high protein diet. Although the correlation between protein anabolism and increased IGF.1 is not significant in animals receiving more protein and disappears in animals receiving a low protein diet, the authors do not rule out hypothesis of the endocrine activity of IGF.1 and envisage a desensitization or a "down-regulat ion" of its receptors in case of insufficient protein intake.

Although the previous observations show in most cases the endocrine role of IGF.1, the property of IGF.1 to reproduce the activity of GH has been questioned by weight and longitudinal growth studies showing that GH is approximately 20 times more active than IGF.1 on a molar basis (cited by Behringer R. et al., Endocrinology, 1990, 127, 1033-1040). The separation of the actions of GH and IGF.1 is difficult to achieve and can only be envisaged on small laboratory animals [transgenic mice (Tg) expressing only IGF.1 and devoid of GH and Tg mice overexpressing GH from Behringer R. et al. (Endocrinology 1990, 127, 1033-1040) or mice lacking GH receiving IGF.1 and / or GH, from Pell JM and Bâtes PE (Endocrinology 1992, 130, 1942-1950)]. The first model showed that the expression of IGF.1 in the absence of GH stimulates weight and longitudinal growth normally and that, moreover, GH and IGF.1 appear to act respectively on the growth of the liver and the brain. The second mouse model lacking GH (Pell JM) made it possible to study the respective roles of GH and IGF.1 on protein metabolism liver, muscle and heart tissue. IGF.1 and GH have specific effects on tissue protein metabolism in this model. GH has greater anabolic activity than IGF.1 on the protein metabolism of muscle and cardiac tissues and acts alone and directly on that of liver tissue. These results, associated with the observation of IGF tissue synthesis, show that the anabolic activity of GH cannot be limited to the endocrine activity of IGF alone.

Immunomodulation of GH by active immunization in growing lamb by the pST sequence 134-154 by Pell JM and Aston R. (J. Endocrinol., 1991, 131, R1-R4) seems to confirm the observation previous one made by Pell JM on the mouse. Immunization against this peptide causes effects comparable to those of an exogenous supply of GH but is not accompanied by any modification of the plasma IGF level. 1. The effects of this immunization imply a direct action of GH on muscle tissue and this action seems to depend on the interaction of certain regions of the hormone with different receptor subtypes.

The work described above has shown that active immunizations, neutralizing for somatostatin, potentiating by modulating the activities of the hormone releasing growth hormone (GHRH or GRF) or those of growth hormone (GH ) using the particular peptide sequences of these two hormones, could have a zootechnical interest. These same studies have however shown that uncertainties remain as to the level intervention and the real effectiveness of these methods. We have thus seen that the immunomodulation of endogenous GH does not lead to a significant reduction in the quantity of fat. For its part, the active immunoneutralization of somatostatin reveals very contradictory results, particularly in cattle and sheep and more particularly in rapidly growing animals.

The object of the present invention is to provide a process which makes it possible to obtain, in all growing ruminants and pigs, intended for the production of meat, an increase in weight growth, an increased efficiency of the food ration and an improvement in composition. of the carcass, obtained by the increase in protein anabolism, particularly that of muscle tissue, and the decrease in lipogenesis, by the increase in the anabolic actions of endogenous growth hormone (GH).

The Applicant has found that this objective can be achieved by means of multiple active immunization involving at least two distinct valences leading to an increase in the basal and pulsed secretion of the growth hormone GH and preferably in addition to a potent ial immunomodulation of this hormone, these two valences having a synergistic effect making it possible to achieve the desired result even in rapidly growing animals.

The present invention therefore relates to a process for improving the growth and quality of the carcasses of domestic pigs and ruminants intended for meat production, by which anabolism is substantially increased protein, particularly that of muscle tissue, and lipogenesis is reduced, by increasing the anabolic actions of endogenous growth hormone, characterized in that the animal is administered a valence designed to induce an increase in basal and pulsed secretion of GH growth hormone and at least one other valence chosen from valences designed to induce this effect or a potentiating immunomodulation of GH.

Despite the contradictory data of the state of the art regarding active immunization antisomatostat ine (supra), the Applicant has also found that it was preferable to ensure an increase in the basal and pulsed secretion of GH by this technique, by the potential by its association with the potentiating immunomodulation of GH.

The latter is preferably obtained by active immunization directed against one or more fragments of GH so as to increase its activity.

According to the invention, it is also possible to combine the immunizing modulation potentiating the GH or the anti isomatostatic immunization with the immunizing modulation potentializing the GRF which also leads to an increase in the basal and pulsed secretion of the GH, by active immunization. against one or more fragments of GRF so as to increase the activity of this hormone.

Finally, in an interesting embodiment of the invention, these three types of active immunization can be combined.

Active somatostatin immunoneutralization is preferably obtained using somatostat ine 14 and / or at least one of its peptide fragments capable of inducing neutralizing antibodies.

The active potentiating immunomodulation of GRF is preferably obtained using at least one of the peptide sequences described in international patent application WO 90/15073, the sequence 28-44 or any of the sequences partial of this region and in particular the sequences 31-44 and 35-44.

The potent ial isatr ice active immunomodulation of the

GH is preferably obtained using at least one of the peptide sequences described on the one hand for pST

(somatotropin or porcine GH) in patent applications EP-AO 284 406 for the sequence 35-53, WO 89/00166 for the sequence 112-159 or fragments of the sequence 112-159 in particular the sequences 133-159 and 128 -147, EP-AO 429 788 for the sequences 98-110, 110-118 and 155-163, EP-AO 303

488 for the sequences 112-138, 119-131, 130-143, 123-137,

133-146 and in the publication of Aston R. et al. (Molecular Immunology, 1991, 28, 41-50) for the sequences 53-73,

134-154 and 174-191, on the other hand for the bST or oST

(bovine or ovine somatotropin or GH) in patent applications EP-AO 284 406 for the sequences 35-53, WO 89/00166 for the sequence 112-159 or any of the partial sequences of this region (in particular 133- 159 and 128-147),

EP-A-0 303 488 for the sequences 122-138, 119-131, 130-143,

123-137, 133-146, and for the publication of Aston R. et al.

(Molecular Immunology, 1991, 28, 41-50) for the sequences

32-46, 80-100, 95-115, 120-140, 130-150, 167-191.

Of course, the invention also covers the use of fragments of these sequences retaining the activity of the sequence from which they originate and of fragments or sequences antigenically and immunologically equivalent to the fragments and sequences thus defined, which also covers sequences incorporating them.

These peptides can be obtained by chemical or biochemical synthesis or by expression of a recombinant DNA in an appropriate host. They are in all cases associated with a carrier molecule responsible for immunogenicity, in the form of a conjugate in which the sequence is covalently linked to the immunogenic carrier molecule or also in the form of an immunogenic fusion protein obtained directly by expression of recombinant DNA.

The valences according to the invention preferably comprise an adjuvant of immunity and are advantageously in the form of an emulsion.

They are preferably valences, in particular in water-in-oil or oil-in-water emulsion, designed to obtain immunization leading to marked and long-lasting activation of GH after one or two administrations. The method according to the invention preferably comprises an early administration of valences in young animals, in particular in the first weeks following birth, preferably at weaning in piglets and between 3 and 4 weeks for lambs and calves.

A second administration can be carried out later, preferably then at least approximately 4 weeks after the first, at the earliest approximately 3 months and later approximately 1 month before the slaughter of the animal, to obtain an immunization leading to an activation (global activation resulting from the increase in the secretion of GH and possibly from the potentiation of its activity) marked and of long duration of the GH.

The so-called early administration also includes, according to the invention, the single administration of the valences in a form ensuring their delayed, continuous or pulsed release, capable of maintaining a high level of antibodies.

The subject of the invention is also a vaccination assembly containing a valence determining an increase in the basal and pulsed secretion of the hormone GH and at least one valence chosen from valences determining this same effect or a potential immunomodulation which is hormone growth hormone, for simultaneous, separate or spread over time to increase the anabolic actions of GH growth hormone.

The valent potent ial i sant the growth hormone can be a vaccine comprising as active ingredient at least one peptide sequence of GH and able to induce antibodies directed against the corresponding sequence of endogenous GH to potential ial activity of the latter.

The determining valences on the secretion of GH can be a vaccine comprising as active ingredient somatostatin and / or at least one of its fragments capable of inducing neutralizing antibodies, to induce antibodies against isomatostatin, and / or a vaccine comprising at least one GRF peptide sequence for inducing antibodies against the corresponding endogenous GRF sequence to potentiate its action.

Said GH and GRF peptide sequences can be chosen from those mentioned above. The peptide sequences and somatostatin or its fragments are associated with carrier molecules as seen above.

By simultaneous use means the use of a mixture, extemporaneous or not, of the different valences. By separate use is meant the separate administration of unmixed valences within a short period of time. Finally, by use spread over time, is meant the separate administration of the valencies over time or the simultaneous or separate administration in the above sense of the valences but repeatedly.

A preferred vaccination set comprises a mixture of valences, made or to be made extemporaneously, preferably in water-in-oil emulsion or in oil-in-water emulsion, which, after a first administration, generally induces a response. immune system with no significant effect on the activity of growth hormone and which, after a second administration, induces immunization leading to marked and long-lasting activation of GH.

A subject of the invention is also muitivalent vaccines containing a valence designed to induce an increase in the basal and pulsed secretion of growth hormone GH and at least one valence chosen from valences designed to induce this same effect or a potentiating immunomodulation. GH growth hormone. The. valences are especially chosen from: a valence comprising as active ingredient the somatostatin and / or at least one of its fragments capable of inducing neutralizing antibodies associated with an immunogenic carrier molecule; a valency comprising as active principle at least one peptide sequence of GH associated with an immunoqene carrier molecule, capable of inducing antibodies potentiating the activity of GH; a valence comprising as active principle a peptide sequence of GRF associated with an immunogenic carrier molecule, capable of inducing antibodies potentiating the activity of GRF. The vaccines are adjuvanted to obtain an immunization leading to a marked and long-lasting activation of GH after one or more, in particular two, administrations. The adjuvants used are preferably an oil-in-water or water-in-oil emulsion. The peptide sequences can in particular be chosen from those mentioned above.

An advantageous vaccine according to the invention, requiring a single administration, can be prepared according to any known technique in a form ensuring delayed, continuous or pulsed release of valences and thus maintaining a high level of antibodies.

The emulsions according to the invention preferably consist of a mixture of highly purified mineral oils and non-ionic surfactants.

The invention will now be described in more detail by the presentation of methods for preparing vaccines and of embodiments of the method according to the invention.

NEED FOR AN IMMUNOGENIC CARRIER MOLECULE

The different peptides indicated for the implementation Work of the invention, whether obtained by chemical or biochemical synthesis or by expression of a recombinant DNA in an appropriate host, cannot by themselves trigger an immune response and lead to the appearance of antibodies. To be immunoqene, they must be coupled to carrier molecules which are generally proteins (albumin, ovalbumin, tetanus toxoid, alpha-globulin, thyroglobulin, hemocyanin for example) by covalent bonds or be expressed in an appropriate host from recombinant DNA in the form of an immunogenic fusion protein, for example a protein from the periplasm (MalE) or the outer membrane (TraTp) of E. coli or any other protein with similar properties.

The conjugation of a peptide on a protein (or a glycoprotein) involves the creation of a covalent bond between the peptide and the protein. This bond is made on the reactive groups of the amino acid residues of the carrier protein (aspartic acid, glutamic acid, histidine, tyrosine, cysteine and especially lysine) or on oligosaccharides after periodic oxidation (for glycoproteins). For the peptide, the binding can also take place on the amino acid residues, on the NH ₂ and COOH ends (when they are free); an additional amino acid can be added such as tyrosine (which also allows labeling with radioactive iodine) and especially cysteine which can react quantitatively with certain groups.

Many conjugation agents have been proposed. They can be classified into 3 main classes: homobifunctional agents, heterobifunctional agents and condensing agents.

Homobi functional agents essentially consist of an aliphatic and / or aromatic hydrocarbon chain, having at its ends two identical groups capable of reacting with good yield with the amino acid residues carried by the peptide and the carrier molecule . Many groups have been proposed: methyl bisimidates, N-hydroxysuccinimide diesters, bis isothiocyanates, dialdehydes, all of which react on the amine functions, bisdiazotized benzidine, which copulate on the residues of tyros i ne, bi smal eimides, which is' addi t on thiols. The most widely used homobi functional agent is glutaraldehyde. The homobi functional agents lead, in addition to the peptide-carrier protein bond sought, to dimerization of the peptide and of the carrier protein and, if the protein or the peptide has several reactive groups, to crosslinking of the conjugate rendering its Difficult reproduc ibility and characterizat ion.

To overcome these drawbacks, some authors have proposed the use of heterobi functional agents. These consist of a hydrocarbon chain having at its ends two different reactive groups. The coupling is done in 3 stages:

(1): addition of the heterobi functional agent on the carrier molecule (or the peptide), by only one of the two reactive groups,

(2): (for certain agents only): release of the other reactive group, (3): addition of the peptide (or of the carrier molecule) which reacts with the group released previously.

Practically, in most heterobi functional aqents, one of the reactive groups is an ester of N-hydroxysuccinimide (or N-hydroxysul fosuccinimide, more soluble in water) which reacts initially in an aqueous medium on amine residues to give an amide bond with the coupling agent. The other group is generally either a group capable of releasing thiols [S-acetγl thioglycolate from SATA, hydrolyzable by hydroxyamine or dilute bases, (pyr idy 1 -2) di thio from SPDP, reduced by di thiothreitol ], or on the contrary a group capable of reacting with thiols (maleimide of MBS, SMPB, SMCC or GMBS), (2-pyridyl) di thio of SPDP, iodoacetyl of SIAB. In the category of heterobifunctional agents, one can add iminothiolane.

In most cases, it is possible to simply measure the quantity of fixed reagent, and to determine the average level of substitution of the conjugate.

Finally, in the condensation reagents, there is no ligand between the carrier molecule and the peptide. To this category belong the carbodi imides and the periodic oxidation of ol igosaccharides in sugars.

The carbodi imides allow the formation of an amide bond between the amino and carboxylic acid groups carried by the two molecules, peptide and carrier protein. The reaction is carried out by activating the carboxylic acid function (O-acyl isourea) on which the amino groups react. It has been proposed to catalyze the reaction with N-hydroxysuccinimide, the reactive species is alόrs an ester of N-hydroxysuccinimide, whose half-life is longer than that of isourea O-acyl. Although the reaction is widely used for the preparation of conjugates with in particular N-ethyl-N '- (3-dimethylamino-propyl) carbodiimide (EDCI), with or without N-hydroxysuccinimide, this method suffers from the same drawbacks as those using homobifunctional agents: possible dimerization of the peptide, crosslinking of the carrier protein, complex final mixture. In addition, it has been shown that the carbodiimide can add up to give a stable and more antigenic N-acyl urea group. As for the coupling on glycoproteins by oligosaccharides, it is mainly used for the preparation of conjugates with immunoglobulins and certain enzymes, such as peroxidase. After blocking the amino groups, the glycoprotein is oxidized by the periodic acid, then purified. Then the other protein reacts with its amino functions on the aldehyde groups and finally the conjugate formed is reduced by the borohydride. Recently, it has been proposed to perform coupling on aldehydes originating from the periodic oxidation of sugars by hydrazines or semicarbazides carried by the other molecule. The vaccines of the invention intended for domestic ruminants and pigs consist in particular of two or three immunogens associated with immunity adjuvants, forming the valences of the vaccines. These valences can be constituted as follows: - mixing of the respective conjugates or fusion proteins with somatostatin and a GH peptide sequence,

- mixing of the respective conjugates or fusion proteins with somatostatin and a GRF sequence, - mixing of the respective conjugates or fusion proteins with a GRF sequence and a GH sequence, - mixing of the conjugates or proteins respective fusion to somatostatin, a GRF sequence and a GH sequence.

ADDITIVES

The adjuvanting substances of immunity, or adjuvants, are of very varied chemical nature and have a mode of action still little known. The adjuvants most widely used in animal vaccine preparations can be classified into three groups: aluminum gels, saponins and emulsions.

a) Aluminum gels

I I these are additives based on aluminum, hydroxide or phosphate.

Aluminum hydroxides are either prepared in situ, by adding alum to the antigen solution, or separately, before being added to the antigen. These apparently amorphous gels, however, contain boehmite microcrystals (AlOOH) (Shirodkar S. et al., Pharm. Res., 1990, 7, 1282-1288).

b) Saponins

Saponins are substances extracted from plants belonging to various families (Caryophyllaceae, Rosaceae, Araliaceae ...); their essential property is to lower the surface tension and many of them are hemolytic. At the chemical level, saponins are very complex mixtures of heterosides of triterpenes or sterols, chemically very similar. The bark of Panama wood has provided under the name Quil A a purified fraction, but which is still a complex mixture of tri terpene heteroids some of which are adjuvants and whose complete chemical structure is known only for a very small number of them.

The saponin Quil A forms with certain proteins (generally amphiphilic) complexes called isσoms (immunost imulat ing complex) which are very powerful adjuvants.

c) Emulsions

The emulsions are macroscopically homogeneous dispersions of a liquid in another immiscible liquid. One of the liquids being generally water, the other oil or an oil miscible substance, we will speak of water-in-oil emulsion (W / O) and oil emulsion -in-water (O / W) depending on whether the dispersed phase is water or oil respectively. The emulsion is made stable by a third component, which is the surfactant.

The oils used until now are mineral oils (hydrocarbons) obtained by refining petroleum. Their exact chemical composition is not precisely known. These oils are characterized by various parameters (viscosity, density, refractive index) that i are linked to the average molar mass and to the type of hydrocarbon: isoparaffinic or cycloparaffic (naphthenic); linear hydrocarbons and aromatics having been eliminated during refining. Currently hydrocarbons obtained by "oligomerization" of alkenes, chemically much better defined than mineral oils, are available.

Vegetable oils, which are biodegradable, are possible, but have not yet led to the industrial production of emulsion vaccines.

Surfactants are substances having a hydrophilic part, and a lipophilic part. These substances, alone or mixed together, are put at the water-oil interface and, depending on the relative importance of the hydrophilic and lipophilic parts (hydrophi le-1 ipophi le or HLB balance of the Anglo-Saxon authors), lead to oil emulsions -in-water or water-in-oil, whose properties are different, both physically (viscosity, behavior towards containers ...) and biologically

(adjuvant power, tolerance).

An important point, in particular for the emulsions intended to be injected, is the chemical nature of the surfactants, in particular hydrophilic groups. In general, for reasons of safety (absence of irritation in particular) the hydrophilic groups will preferably be nonionic.

An interesting property of lipophilic surfactants with very low HLB such as Span ^® 85 (sorbitan trioleate) or Pluronic ^® L121 (ethylene oxide-co-propylene oxide) is that they are themselves adjuvants (Woodward LF, Lab. Anim. Sci. 1989, 39, 222-225). This is to be linked to the adjuvant power of vegetable oils (weaker, however, than that of hydrocarbons) which can be considered as very low HLB surfactants.

It should be added that the emulsions may contain bacterial bodies used either as immunogens or as an adjuvant. The use of killed mycobacteria, in the complete Freund's adjuvant in particular, is possible in the laboratory, but is impossible for farm animals. The search for the adjuvant substance present in mycobacteria and the smallest chemical structure which has this property has led to the isolation, and the characterization of muramy ldipept ide (MDP), then to its synthesis.

PREPARATION OF SOMATOSTATIN CONJUGATES

The conjugate with somatostat ine can be constituted from the naturally occurring hormone, somatostat ine 14 (or somatostat i ne 15-28), or fragments thereof capable of inducing neutralizing antibodies. It is obtained by one or other of the conjugation methods described above.

The somatostat ine is a tetradecapept ide. Its formula is as follows:

For example, conjugates can be produced by a coupling between a polymer (Lys-Ala) and the somatostat ine by carbodiimide in the presence of N-hydroxysulfosuccinimide, by a coupling between the tetanus toxoid and the somatostat ine by glutaraldehyde, by a coupling between thyroglobul i ne and the somatostat ine by carbodiimide or by the coupling between alpha-globulin ine and the somatostat ine by glutaraldehyde.

EXAMPLES OF ANTISOMATOSTATIN VACCINE PREPARATION:

1) The conjugate is obtained by adding 50 mg of somatostat ine and 200 mg of human alpha globulin dissolved in 40 ml of 0.1 M ammonium acetate, 26 ml of 20 mM glutaraldehyde solution (0 , 2%) over a period of 1 hour. The mixture is left overnight at ambient temperature, then is dialyzed several times at 4 ° C. against 0.15 M NaCl buffer 0.01 M phosphate, pH 7.5.

The conjugate, which forms an ocher suspension, is concentrated by ultrafiltration on an Amicon ultrafiltrating membrane with a cutoff threshold of 10 kDa, until a final volume of 25 ml is obtained.

The antigen is emulsified using an industrially used formulation, into which enter highly purified mineral oil and nonionic surfactants, one lipophilic, the other hydrophilic; the final product is an oil-in-water emulsion.

2) The conjugate is obtained by adding, to 12 mg of thyroglobulin and 4 mg of somatostatin dissolved in 2 ml of water, 2.3 mg of N-ethyl-N '- (3-diethylamino propyl) carbodiimide and 0, 83 mg of N-hydroxysul fosuccinimide dissolved in water. The pH is adjusted around 6-7. The mixture is left overnight at room temperature. The solution is then centrifuged, desalted by σhromatographi e on Sephadex G-50 in 0.05 M NH ₄ HCO ₃ buffer, then lyophi 1 ised.

The antigens are taken up in 2.5 ml of 150 mM NaCl buffer - 10 mM phosphate pH 7.5, then are emulsified in an oily phase used industrially to obtain a water-in-oil emulsion. This oily phase contains highly purified mineral oil and non-ionic, lipophilic and hydrophilic surfactants.

PEPTIDE SEQUENCES OF GH AND GRF

The peptide sequences of the GRF and GH hormones indicated in the patent applications or in the aforementioned articles, can be used at a rate of one per type of hormone for the preparation of conjugates or fusion proteins.

The peptide sequences of GH which can be used are in particular the following:

- bovine or ovine hormone GH 35-53

NH ₂ -Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn-Thr-Gln-Val-AlG-Phe-Cys-COOH

- of the porcine hormone GH 35-53

Ala-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn-Ala-Gln-Ala-Ala-Phe-Cys - of the porcine hormone GH

98-110 Thr-Asn-Ser-Leu-Val-Phe-Gly-Thr-Ser-Asp-Arg-Val-Tyr

110-118 Tyr-G 1 u-Lys-Leu-Lys-Asp-Leu-G lu-Glu

155-163 Leu-Leu-Lys-Asn-Tyr-Gl γ-Leu-Leu-Ser - sequences 122-138, 119-131, 130-143, 123-137 and 133-146 (bovine, ovine hormones, swine). bovine 122-138:

Ala-Leu-Met-Arg-Glu-Leu-Glu-Asp-Gly-Thr-Pro-Arg-Ala-Gly-Gln-Ile-Leu Ovine 122-138:

same sequence as bovine GH, except in 130 where Gly is replaced by Val

Pork 122-138:

same sequence as bovine GH, except in 131 where Thr is replaced by Ser

Bovine 130-143:

Gly-Thr-Pro-Arg-Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp

Sheep 130-143:

130 Gly is replaced by Val Porcine 130-143:

Gly-Ser-Pro-Arg-Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp Bovine, ovine or porcine 133-146:

Arg-Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp-Lys-Phe-Asp

Fragments of the sequence 112-159;

in part i cu l i er o l i gopep t i of regions 133-159 and 128-147 comprising from 6 to 7 peptides at least and 20 at most.

Some have been described by Aston et al. Cited above.

- porcine hormone

53-73

Cys-Phe-Ser-Glu-Thr-Ile-Pro-Ala-Pro-Thr-Gly-Lys-Asp-Glu-Ala-Gln-Gln-Arg-Ser-Asp-Val

134-154

Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp-Lys-Phe-Asp-Thr-Asn-Leu-Arg-Ser-Asp-Asp-Ala

174-191

Thr-Tyr-Leu-Arg-Val-Met-Lys-Cys-Arg-Arg-Phe-Val-Glu-Ser-Ser-

Cys-Ala-Phe

- bovine or ovine hormone

32-46

Phe-Glu-Arg-Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln

80- 100

Leu-Leu-Leu-Ile-Gln-Ser-Trp-Leu-Gly-Pro-Leu-Gln-Phe-Leu-Ser- Arg-Val-Phe-Thr-Asn-Ser 95-115

Arg-Val-Phe-Thr-Asn-Ser-Leu-Val-Phe-Gly-Thr-Ser-Asp-Arg-Val¬

Tyr-Glu-Lys-Leu-Lys-Asp

120-140

Ile-Gln-Ala-Leu-Met-Arg-Glu-Leu-Glu-Asp-Gly-Ser-Pro-Arg-AlaGly-Gln-Ile-Leu-Lys-Gln

130-150

Gly-Thr-Pro-Arg-Ala-Gly-Gln-Ile-Leu-Lys-Gln-Thr-Tyr-Asp-Lys¬

Phe-Asp-Thr-Asn-Met-Arg 167-191

Lys-Asp-Leu-His-Lys-Thr-Glu-Thr-Tyr-Leu-Arg-Val-Met-Lys-CysArg-Arg-Phe-Gly-Glu-Ala-Ser-Cys-Ala-Phe

Preparation of a conjugate with a GH sequence: example of conjugation with the GH fraction (134-154).

In an opaque bottle are dissolved successively

42 mg of GH peptide (134-154) and 60 mg of pure hemocyanin from Megathura crenulata (KHL). Then 0.8 ml of 68.5 mM glutaraldehyde solution is added very slowly, over a total period of 5 hours, at a rate of 40 μl every 15 minutes. The solution is then dialyzed at 4ºC against an isotonic buffer (0.15 M NaCl - 0.01 phosphate - pH 7.5) then is stored at - 20 ° C until used.

The GRF peptide sequences which can be used are in particular the following:

The sequences 28-44 porcine, bovine (or ovine or caprine) and more particularly the sequences 31-44 or 35-44

28-44 pig

Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val- Arg-Leu 28-44 cattle, sheep, goats

Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu

and the sequences 31-44 and 35-44 of the GRFs of the different species or peptides which are antigenically and immunologically equivalent.

Preparation of a conjugate with a GRF sequence: example of conjugation with the GRF fraction (28-44).

50 mg of GRF peptide (28-44) and 36 mg of lyophilized hemocyanin from Megathura crenulata (KLH) are successively dissolved in an opaque bottle containing 11.5 ml of 0.1 M NaHCO ₃ . Then 475 μl of 68.5 mM glutaraldehyde are added over a total period of 5 hours (i.e. 50 μl every 30 minutes). Then the solution is immediately dialyzed at + 4ºC against an isotonic buffer (0.15 M NaCl 0.01 M phosphate - pH 7.5).

Preparation of plurivalent vaccines

Taking into account the recipient animal, a vaccine is prepared comprising, in a water-in-oil or oil-in-water emulsion, an immunogenic conjugate of one of the above GRF sequences and / or GH and an immunogenic conjugate of somatostatin 14. The first administration of the vaccine induces an immune response with no significant effect on the activity of the growth hormone. The second administration of the vaccine is necessary so that the immunization and its effects on the potential GH immunizing immunity become marked and prolonged. The emulsions are stable and made from a mixture of highly purified mineral oils and non-ionic surfactants.

The vaccination schedule is as follows:

- pig: first vaccine at weaning of piglets;

- cattle, sheep: first vaccine administered to calves and lambs between 3 and 4 weeks old;

- for all, second vaccine at least 4 weeks after the first, at the earliest 3 months and at the latest 1 month before slaughter.

The mode of administration of these formulations is preferably transcutaneous, in particular using a needle-free injection apparatus by pressure jet, in particular according to patent application FR-A-2 652 257.

The effectiveness of active immunization is measured by the immune responses and the effects on weight growth and the water, protein and fat composition of muscle tissue. The antibody titers are determined by radioimmunoassay up to the time of slaughter and relate to the anti isomatostatic antibodies and anti-peptide sequences of the GRF and GH hormones.

Claims

1. Process for improving the growth and quality of the carcasses of domestic pigs and ruminants intended for meat production, by which the protein anabolism, particularly that of muscle tissue, is appreciably increased and the lipogenesis is reduced, by l increase in the anabolic actions of endogenous growth hormone, characterized in that the animal is administered a valence designed to induce an increase in the basal and pulsed secretion of growth hormone GH and at least one other valence chosen from valences designed to induce this effect or a potential GH-immunizing immunomodulation.

2. Method according to claim 1, characterized in that the increase in the basal and pulsed secretion of GH is obtained by active immunization antisomatostatin and / or by potent ial immunomodulation of the growth hormone releasing hormone (GRF).

3. Method according to claim 1 or 2, characterized in that the potential immunomodulating ion GH and / or GRF is obtained by active immunization directed against one or more peptide fragments of the corresponding hormone so as to increase l activity.

4. Method according to claim 2 or 3, characterized in that the valence designed to induce a basal and pulsed increase in GH comprises somatostatin 14 and / or at least one of its fragments peptides, associated with an immunogenic carrier molecule.

5. Method according to claim 3, characterized in that the valence designed to induce a potentiating immunomodulation of GH comprises at least one of the following GH sequences:

porcine GH sequences 35-53, 112-159, 133-159, 128-147, 98-110, 110-118, 155-163, 122-138, 119-131, 130-143, 123-137, 133 -146, 53-73, 134-154, 174-191;

bovine or ovine GH sequences 35-53, 112-159, 133-159, 128-147, 122-138, 119-131, 130-143, 123-137, 133-146, 32-46, 80-100 , 95-115, 120-140, 130-150, 167-191; - fragments of these sequences conserving the activity of the sequence from which they originate and fragments or sequences antigenically and immunologically equivalent, the fragments or sequences being associated with an immunogenic carrier molecule.

6. Method according to claim 3 or 5, characterized in that the active immunization directed against GRF is obtained by a valence comprising at least one of the sequences 28-44, 31-44, 35-44 or fragments of these sequences retaining the activity of the sequence from which they originate and antigenically and immunologically equivalent fragments or sequences, the fragments or sequences being associated with an immunogenic carrier molecule.

7. Method according to any one of claims 1 to 6, characterized in that the valences comprise an adjuvant of immunity.

8. Method according to any one of claims 1 to 7, characterized in that the valences are designed to obtain an immunization leading to marked and long-lasting activation of GH after one or two administrations.

9. Method according to claim 8, characterized in that the sonic valences: in water-in-oil emulsion or in oil-in-water.

10. Method according to any one of the claims 1 to 9, characterized in that it comprises an early administration of valences in young animals, especially in the first weeks after birth.

11. Method according to claim 10, characterized in that this early administration takes place at weaning in piglets and between 3 and 4 weeks in lambs and calves.

12. Method according to claim 10 or 11, characterized in that the valences are in a form ensuring them a delayed, continuous or pulsed release.

13. Method according to any one of claims 8 to 11, characterized in that a second administration is carried out approximately 4 weeks after the early administration, at the earliest approximately 3 months and at the latest approximately 1 month before the slaughter of the animal.

14. Vaccination kit containing a valence determining an increase in the basal and pulsed secretion of growth hormone GH and at least one other valence determining this same effect or a potent ial immunodulation ionizing growth hormone GH, for simultaneous, separate or spread over time to increase the anabolic actions of growth hormone GH.

15. Vaccination assembly according to claim 14, characterized in that it comprises a valence constituted by a vaccine comprising as active principle at least one peptide sequence of GH and capable of inducing antibodies directed against the corresponding sequence of the Endogenous GH to potentiate the activity of the latter.

16. Vaccination assembly according to claim 14 or 15, characterized in that it comprises a valency constituted by a vaccine comprising as active ingredient somatostatin to induce antibodies against isomatostat ine.

17. Vaccination assembly according to any one of claims 14 to 16, characterized in that it comprises a valence constituted by a vaccine comprising as active principle at least one peptide sequence of GRF for inducing antibodies against the sequence correspondent of endogenous GRF to potentiate its action.

18. Vaccination assembly according to claim 14, characterized in that it comprises a mixture, made or to be made extemporaneously, valences according to claims 14 to 16 intended to induce an immunization leading to marked and long-lasting activation of GH after one or two administrations.

19. Multivalent vaccine containing a valence designed to induce a basal and pulsed increase in GH growth hormone and at least one other valence selected from valences designed to induce this effect or a potentiating immunomodulation of GH growth hormone.

20. Vaccine according to claim 19, characterized in that the valences are chosen from valences comprising as active principle somatostatin and / or at least one of its peptide fragments, associated with an immunogenic carrier molecule, at least one peptide sequence of GH associated with a molecule immunogenic carrier, capable of inducing potential antibodies that are GH activity, and at least one peptide sequence of GRF associated with an immunogenic carrier molecule, capable of inducing antibodies potentiating GRF activity; the vaccine being adjuvanted to induce immunization leading to marked and long-lasting activation of GH after one or more administrations.

21. Vaccine according to claim 19 or 20, characterized in that it is in a form ensuring delayed, continuous or pulsed release of valences.

22. Vaccination or vaccine assembly according to claim 15 or 20, characterized in that the peptide sequence or sequences of GH are chosen from the sequences or fragments defined in claim 5.

23. A vaccination assembly according to claim 17, characterized in that the GRF peptide sequence or sequences are chosen from the sequences or fragments defined in claim 6.

24. Vaccine according to claim 20, characterized in that the GRF peptide sequence or sequences are chosen from the sequences or fragments defined in claim 6.