MX2013001626A - Method for the manufacture of a recombinant vaccine against shipping fever using recombinant proteins of mannheimia haemolytica bonded to cellular bodies. - Google Patents

Abstract

Method for preparing a vaccine for preventing the bovine, ovine and caprine respiratory disease, especially shipping fever. The vaccine uses fragments of the outer membrane protein PIpE and leukotoxin LktA of Mannheimia haemolytica expressed in a bacterial vector and bonded to cellular bodies for generating a protective immune response of the cellular and humoral type when it is administered to mammals susceptible to the infection with M. haemolytica.

Description

METHOD OF ELABORATION OF A RECOMBINANT VACCINE AGAINST THE EMBARKMENT FEVER USING PROTEINS RECOMBIN BEFORE MANNHEIMIA HAEMOLYTICA UNITED TO CELLULAR BODIES.

DESCRIPTION TECHNICAL FIELD The present invention relates generally to the prevention of respiratory disease in goats and sheep, particularly of the so-called "shipping fever". The present invention also relates to a novel preparation for generating a cellular immune response in an animal. It provides a method for attaching purified proteins to cell bodies. Describes a way to introduce fragments of the genes of the LktA and PlpE proteins of Mannheimia haemolytica in Escherichia coli to express them. It refers to a way to induce the expression of these genes at levels that facilitate the purification of said proteins. Describe a very simple way to purify proteins. It also relates to a way to bind the purified proteins to bacterial cell bodies. The present invention also provides a formulation for developing a protective immune response against infection by M. haemolytica.

BACKGROUND Respiratory infections are one of the main causes of disease and death in intensive beef cattle holdings. The cost of the disease in Mexico is not calculated but for example in the United States it is estimated that it costs annually about 1000 million dollars (Miles DG: Overview of the North American beef cattle industry and the incidence of bovine respiratory disease (BRD).

Anim Health Res Rev 2009, 10: 101-103). Many factors, related to the transport of the animals to the feedlot, contribute to the occurrence of this disease, for this reason it is given the name of Embarque Fever. Commonly, the disease begins with a viral infection that in severe cases is complicated by the proliferation of three species of bacteria, M. haemolytica (formerly Pasteurella haemolytica), Pasteurella mullocida, and in lesser frequency Histophil s somnii (formerly Haemophilus somnus), which they produce pneumonia and in many cases, the death of affected animals. The first to be associated with shipping fever and the most frequently isolated case of pneumonia is M. haemolytica (Cárter, Can J. Comp.Med. 1956. XX, 10) mainly serotype 1 (S1) although it is also has observed serotype 6 (Al-Ghamdi GM et al., JVet Diagn Invest., 2000. 12 (6): 576-578). The prevention and control of boarding fever is oriented to three lines: vaccination against viruses causing respiratory diseases and against M. haemolytica; prophylactic treatment with antibiotics at the arrival of cattle to the feedlot (also called metaphylaxis); and antibiotic therapy for sick animals. The extensive use of antibiotics to control shipping fever increases the possibility of residues of antibiotics in meat and the development of drug-resistant bacteria in livestock, including bacteria with potential impact on human health such as pathogenic Salmonella and Escherichia coli. For this reason it is very important to have effective vaccines to prevent the disease, however, despite the fact that for several decades there have been vaccines developed for this purpose, the disease continues to be a major problem for health. bovine, so the development of effective vaccines to prevent infections by these bacteria is a very active research topic.

Specifically for M. haemolytica attempts to develop an effective vaccine are numerous and in recent years have been oriented towards formulas based on recombinant DNA technology highlighting the following: U.S. No. 5,055,400 issued to Lo et al., (October 8, 1991) discloses the genes and proteins for leukotoxin. The U.S. patents No. 5,476,657 (Potter, December 19, 1995) and U.S. No. 5,871,750 (Potter, February 16, 1991) disclosed vaccines using leukotoxin or truncated forms of leukotoxin. Patents US 5,708,155 (Potter et al., January 13, 1998) and US No. 6,797,272 (Potter et al., September 28, 2004) describe vaccines formed by leukotoxin chimeras with antigens such as somatostatin ( SRIF), the gonadotropin-releasing hormone (GnRH) or the rotavirus viral protein 4 (VP4). And recently, the cloning of cattle in which the molecular receptor of leukocytes has been modified to make them resistant to leukotoxin has been proposed (US Patent 2011/0296545 Al, Srikumaran et al, December 1, 2011).

In addition to leukotoxin, it has been proposed that outer membrane proteins (OMPs) may be important to obtain protection against infection. Of these there is a 45 kDa that has been correlated with resistance against experimental challenge. In 1998, Pandher et al. Reported the cloning, sequencing and characterization of the gene and designated it PlpE (Pandher K, Confer A W, Murphy G L. Infect Immun 1998; 66: 5613-5619). The use of the complete PlpE protein or in fragments and alone or in combination with the LktA protein was disclosed in US patent 7,794,734. The procedure for obtaining the proteins described in this patent consisted of harvesting of the bacteria, a lysis by sonication, a centrifugation to remove the remains of the bacteria, a binding step to affinity columns of Nickel "Pro-Bond" that binds recombinant proteins labeled with a tag of 6 histidines and an elution step with a low pH buffer or containing imidazole.

Despite all these developments and patents mentioned, there are two reasons why no recombinant vaccine has been commercialized to prevent this disease to date.

The first is that subunit recombinant vaccines generally produce high levels of antibodies and in some cases this response may be long lasting, but they generate a very poor TH1 cellular response and to obtain a long-lasting protective response it is necessary for a vaccine to generate as much a humoral immune response TH2 as a TH1 cellular response (Amanna IJ, Slifka MK, Contributions of humoral and cellular immunity to vaccine-induced protection in humans, Virology, 411, 206-15). This problem is well known (Patent EP0313569, Henningson et al 1993) and to solve it both adjuvants have been sought that favor a cellular response such as Chitosan, as the fusion of recombinant proteins to other proteins or domains that induce strong immune responses (such as the Shiga-like toxin of Escherichia coli). The covalent binding of recombinant proteins to different types of structures has also been tested; which may be microspheres of different materials or particles-like-to-viruses such as those used in the Gardasyl vaccine used to prevent infection with the Human Papilloma Virus (Le Grand, LR, et al., Recombinant vaccines: development , production and application in Pharmaceutical Biotechnology Drug Discovery and Clinical Applications, 2nd edition, Chapter 17 O. Kayser and H.

Warzechaeds Eds. 2012 Wiley-VCH Verlag & Co). All these strategies have been shown to have some value in increasing the immune response mediated by TH1-type cells in vaccines based on recombinant proteins, but they have been slowed down by technical difficulties to bring them to production level in the veterinary industry of production animals.

The other reason why recombinant vaccines have not been used is that, due to the economic limitations of the industry, bovine vaccines must have low cost. And this is why the current vaccines for M. haemolyüca are crude, usually consist of inactivated cultures containing the dead bacteria, the leukotoxin of M. haemolyüca and proteins detached from the surface. In a review of field studies on commercial vaccines for M. haemolytica, Perino and Hunsaker (1997) found that only in 50% of the trials could some efficacy of the vaccines be established (Bov Practitioner 1997; 31: 59-66) and Larson and Step. (2012) state that there is not enough evidence to support the efficacy of currently commercialized vaccines (Vet Clin North Am Food Anim. Pract. 2012, 28 (1): 97-106). There is therefore a great need to improve the compositions and production methods of vaccines to protect cattle against shipping fever.

BRIEF DESCRIPTION OF THE INVENTION The invention is not limited in its application to the details of the described steps and examples presented. The invention is capable of other incarnations and of being practiced or carried out in various forms. It is to be understood that the phraseology and terminology used in this document is solely for the purpose of describing the invention and not to limit its application In connection with the present invention, fragments of the genes of the outer membrane protein PlpE and the protein A of leukotoxin (LktA) were cloned and expressed in Escherichia coli. The fragments of the proteins were purified and the immunogenicity of both purified proteins and added to commercial vaccines available in Mexico was studied in mice, rabbits and sheep. The fragments were able by themselves to induce a strong immune response, comparable or greater than that observed with commercial vaccines. Used in conjunction with a vaccine containing bacterins for other bacteria of importance in shipping fever proteins showed greater immunogenicity. Subsequently, the protective capacity of the different formulations was evaluated against challenges with Mannheimia pathogenic strains in both mice and sheep and it was found that the recombinant formulation protected more than the previously available vaccines. Subsequently an optimization of the expression of the cloned proteins was carried out and it was coupled to a very simple method of purification by means of heating and cooling cycles, with this a slightly less pure preparation was obtained than the previous one but easier to obtain in large quantities, this new preparation showed the same immunogenic characteristics as the previous one. Finally a new preparation was carried out in which the recombinant proteins purified with the heating and cooling method were covalently bound to the cell bodies of M. haemolytica by incubation with formaldehyde to increase the proportion of these proteins in the cell body and it was found that the protection was still higher than that observed with the purified recombinant proteins or to that observed with the recombinant proteins purified and inoculated at the same time as the cell bodies. The innovative aspect of the present invention is that a very simple preparation of the two proteins, covalently linked to cell bodies in the presence of formaldehyde, showed greater protection than the combination of cell bodies and simultaneously injected proteins, this protection was independent of the degree of purity of proteins. The proteins bound to the cell bodies also showed a strong cellular immune response unlike the purified recombinant proteins alone or inoculated at the same time as the cell bodies. Previous art related to the generation of a cellular immune response includes the fusion of proteins to proteins or epitopes that modify the immune response to direct it towards a TH1 response, but this requires the cloning of the fused genes to the specific epitopes that produce internalization in lymphocytes (Patent EP0532090 A2, Donnelly et al, 1993), or the cloning of the gene encoding the antigen in a vector for expression in a specific bacterium that produces an immune cell response such as Listeria (Patent US20120321662 Al, Portnoy, 2012 ) and in some cases it has been found that the processing of the epitopes can vary depending on the amino acids that are flanking them, so that the fusion can alter the quality of the generated response. Alternatively it has been described that by covalently binding the recombinant proteins to particles of very high molecular weight a cellular immune response can also be achieved, these particles can be generated from the same proteins (Patent EP1221968 A2, O'Hagan et al, 2002 ) or they can be very diverse in nature, be generated separately and then be bound to proteins (Patent EP2496265 Al, Schwamberger, et al, 2012). In the invention described herein, the advantage is that the particle is the same bacteria for which it is desired to generate the immune response and thus it has all the bacterial components but in small quantity and with a high concentration of proteins that we know generate a protective response. So what was obtained is a very specific and powerful response. As for the methods to generate the covalent binding, the most frequently described is the fusion of the gene of the protein of interest, with the epitopes generating the desired response (US Patents No. 5,238,823, Potter et al., 24 August 1993, US Pat. No. 5,273,889, Potter et al., December 28, 1993, US No. 5,594,107, Potter et al., January 14, 1997, US No. 6,096,320, Potter et al. al., August 1, 2000). In the case of particle binding, the design of specific chemical reactions is usually used to control very well the sites where the proteins are going to bind, but at the same time they are very expensive (Patent EP2496265 Al, Schwamberger, et al. al, 2012).

In contrast, the invention presented here allows the economic production and in large quantities of recombinant vaccines that, up to now, have not been able to be introduced to the veterinary market due to cost reasons.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Anti-LKTA antibodies detected by ELISA in rabbits inoculated with: A, a commercial bacterin containing M. haemolytica; B, 30ug of purified recombinant LKTA, 30ug of purified recombinant PLPE and supplemented with a bacterin not containing Mannheimia; C, 60ug of LKTA, 60ug of purified PLPE supplemented with the bacterin without Mannheimia; D, negative control inoculated with the bacterin without Mannheimia; E, negative control not inoculated.

Figure 2. Anti PLPE antibodies detected by ELISA in rabbits inoculated with: A, a commercial bacterin containing M. haemolytica; B, 30ug of purified recombinant LKTA, 30ug of purified recombinant PLPE and supplemented with a bacterin not containing Mannheimia; C, 60ug of LKTA 60ug of purified PLPE supplemented with the bacterin without Mannheimia; D, negative control inoculated with the bacterin without Mannheimia; E, negative control not inoculated.

Figure 3. Anti-LKTA antibodies detected by ELISA in sheep inoculated with: A, a commercial bacterin containing M. haemolytica; B, 50ug of purified recombinant LKTA, 50ug of purified recombinant PLPE and supplemented with a bacterin not containing Mannheimia; C, lOOug of LKTA, lOOug of purified PLPE supplemented with the bacterin without Mannheimia, D, negative control inoculated with the bacterin without Mannheimia; E, negative control not inoculated.

Figure 4. Anti PLPE antibodies detected by ELISA in sheep inoculated with: A, a commercial bacterin containing M. haemolytica; B, 50ug of purified recombinant LKTA, 50ug of purified recombinant PLPE and supplemented with a bacterin not containing Mannheimia; C, lOOug of LKTA, lOOug of purified PLPE supplemented with the bacterin without Mannheimia; D, negative control inoculated only with the bacterin if Mannheimia; E, negative control not inoculated.

Figure 5. Complete anti Mannheimia haemolytica antibodies detected by ELISA in sheep inoculated with: A, a commercial bacterin containing M. haemolytica; B, 30ug of purified recombinant LKTA, 30ug of purified recombinant PLPE and supplemented with a bacterin not containing Mannheimia; C, 60ug of LKTA, 60ug of PLPE purified supplemented with the bacterin without Mannheimia; D, negative control inoculated with the bacterin without Mannheimia; E, negative control not inoculated.

Figure 6. Polyacrylamide electrophoresis of different preparations of the LKTA protein. Lane 1, molecular weight marker; Lanes 2 to 4 total protein of the bacteria; lanes 5 to 9 purified preparations by heating cooling and sedimentation. Figure 7. Polyacrylamide electrophoresis of different preparations of the PLPE protein. Lane 1, molecular weight marker; Lanes 2 to 4 total protein of the bacteria; lanes 5 to 9 preparations purified by heating, cooling and sedimentation.

Figure 8. Challenge in mice. Percentage of survival observed in groups of 20 animals immunized with different concentrations of the recombinant fragments of the recombinant PlpE and LKTA proteins purified with the heating method cooling sedimentation. The dotted line shows the percentage of protection observed in the group immunized with 10 ug of the purified fragments in Ni-NTA columns.

Figure 9. SDS-PAGE electrophoresis of two experiments of crosslinking of the LKTA protein to bacterial cell bodies. Lanes 1 and 2, sediment proteins after 48 hours of incubation of the recombinant protein with the bacteria in the presence of formaldehyde; lanes 3 and 4, LKTA protein purified by heating cooling sedimentation used in each experiment; lanes 5 and 6 supernatant obtained after 48 hours of incubation with formaldehyde.

Figure 10. Challenge in mice with proteins bound to cell bodies. A) Commercial Bacterin; B) Commercial Bacterin with Leukotoxin; C) Recombinants purified by affinity in Ni-NTA columns; D) Recombinants purified by heating cooling; E) Bacterin enriched with LKTA and PlpE; F) Recombinant proteins cross-linked to cell bodies. The bars represent the standard error of 3 experiments.

Figure 11. Delayed type hypersensitivity in mice. Percentage of thickening observed in the ears of the mice after the intradermal injection of the recombinant proteins in animals immunized with:: A) Proteins purified by column; B) Proteins purified by heat; C) Bacterin plus non-crosslinked proteins; D) Bacterin plus cross-linked proteins; E) PBS. The bars represent the standard error.

DETAILED DESCRIPTION OF THE INVENTION In the presented form of the invention, a high efficiency vaccine was obtained by optimizing the expression of fragments of the PlpE and LktA proteins of M. haemolytica to obtain, with a very simple lysis method, proteins purified to more than 90%. These proteins were added to a Mannheimia culture and the mixture was inactivated with formaldehyde which fulfilled the double function of inactivating the bacteria and cross-linking the purified proteins with the cell bodies. The union of the protein with the bacteria was demonstrated because before the treatment a simple centrifugation allowed to separate the bacteria in the tablet and the proteins remained in the supernatant and after the treatment the proteins remained attached to the cell bodies and their separation was no longer possible. by physical methods. The protective capacity of this preparation of cell bodies bound to PlpE and LktA proteins was demonstrated through challenges in laboratory animals where it was demonstrated that the new preparation was more effective in protecting the animals that all the previous preparations, including the vaccine leader in the current market.

In the first example, the genes encoding the PlpE and LktA proteins of M. haemolytica were cloned. These proteins were expressed with the inducer Isopropylthio-galacto-pyranoside (IPTG) and subsequently purified by zinc columns that bind to the histidine tags that were added to the amino terminus of the proteins precisely to facilitate this purification . These proteins were used to formulate vaccines that were applied in sheep to test the protective capacity, finding that this formulation protected the animals more than a conventional vaccine formulated with haemolytica and that is commercially distributed. Subsequently, in example 2 a method was developed to obtain in a simple and easily scalable form a preparation of the recombinant proteins with a purity greater than 90% that was used to generate a protective immune response against infection by M. haemolytica. This was achieved by changing the inductor concentrations, growth state of the culture by adding the inducer of the expression and the time the cells were incubated with the inducer. Associated with this optimization of protein expression, a method for obtaining proteins that avoids the methods of extraction of recommended recombinant proteins such as physical homogenization or by means of enzymes and the subsequent purification by Zinc columns was developed. which bind to the histidine tags normally included in the recombinant proteins and which were those used in Example 1, the new procedure is based on a series of temperature changes that can be made compatible with a procedure similar to pasteurization, with this we are breaking the economic barrier that until now has prevented the use Intensive use of recombinant proteins in the formulation of vaccines for use in production areas.

In the last of the examples we show how proteins can be linked very easily to cell bodies, using an incubation procedure with 0.4% formaldehyde, similar to that used in the inactivation of bacterins. This process allowed generating a more powerful immune response capable of protecting animals against challenges greater than those that could be resisted by animals immunized with current commercial vaccines or even those protected with recombinant vaccines prepared with purified proteins.

The combination of these three developments, the optimization of vaccine expression, simple extraction and purification and the binding of proteins to cell bodies through incubation with formaldehyde, have led us to obtain a product with immunological protection characteristics that they surpass those that can be found with currently available vaccines mainly in the aspect that we have a cellular response that in immunological terms is preferable to the single humoral response that the recombinant vaccines that have been previously described have shown. Additionally, by avoiding a laborious process of purification, this product is easy to produce in any biological plant without making modifications. To put into context the importance of this procedure, one should reflect on the fact that there are to date tens of thousands of patents that discover recombinant vaccines and only a dozen of these vaccines have been produced on a commercial scale. The development presented here can change that panorama and make all those vaccines that have been invented finally reach the market helping to improve the health of both animals and humans who depend on them.

It is important to add that, although the invention presented here shows the use of this combination of methods to produce a vaccine that protects cattle against bovine respiratory disease and pneumonic Pasteurellosis, the same methods can be used to optimize the production of other vaccines recombinants against other diseases for which antigens and / or protective epitopes have been identified and cloned, thus improving the protection provided and allowing the manufacture at low cost of these vaccines.

EXAMPLE 1 Cloning and Immunogenicity of recombinant fragments of PlpE and LktA proteins.

For these studies, the plpE and IktA genes were cloned. The DNA was extracted from cultures of the ATCC strains of M. haemolytica, the Illustra Bacteria Genomicprep Mini Spin kit (GE healthcare) was used. The bacteria were inoculated in 200 mL of LB medium and incubated for 16 h at 250 rpm and at 37 ° C. The kit was used as indicated in the insert. DNA quality was verified by absorbance (A260 / A280) and corroborated on 1% agarose gels stained with Safe DNA gel stain (Invitrogen).

The purified DNA was stored at -20 ° C until use. LKTA is a secreted and highly toxic soluble protein. Attempts to study the immunogenicity of its carboxyl terminal region (en-includes the RTX domain) have not been successful (Lainson et al, 1996), for this reason we decided to produce a fusion protein with an LKTA fragment of amino acid 573 to 845. This polypeptide of 273 amino acids includes five repeating hemolysin-like calcium domains, which are highly conserved between different serotypes. For the construction of PlpE, it was decided to eliminate the first 18 amino acids, which code for a signaling peptide and our construction therefore consisted of only 338 amino acids. The base sequences of the primers used for PCR were the following: for the lktA gene, the positive sense primer was 5'-GAAAAGGCCTGATGGTGC AGCAAGTTCTAC-3 'and the antisense primer 5'-GGCACAAGC TTACGAAATC AGCCTCTCGG-3' which was used to amplify a 846-bp fragment encoding 273 amino acids of LKTA. For the plpE gene, the first positive sense was 5'-AATAGGCCTGCGGAGGAAGCGGTAGC-3 'and the antisense primer was 5' -ATAAGCTTATTTTTTCTCGCTAACCATTA-3 'to amplify a 1014-bp fragment encoding 338 amino acids of PlpE. Primers for the Stul enzyme were introduced into the primers in the positive sense and HindHI primers in both antisense primers in order to achieve directional cloning in the expression vector.

The Platinum PCR SuperMix high fidelity enzyme (Invitrogen) was used for the PCR amplifications. The PCR products were visualized on 1% agarose gels stained with SAFE DNA gel stain (Invitrogen). Association temperature (annealing) was initially established by tests with a temperature gradient. All PCR reactions were performed after a single step of denaturation at 94 ° C for 5 min followed by 30 cycles of denaturation at 94 ° C for 1 min, association at 63 ° C for lktA or 60 ° C for plpE for 1 minute and extension at 72 ° C for 1 min, followed by a final extension of 5 min at 72 ° C.

To clone the PCR products, the fragments of aga-rose gels were purified by GFX ™ PCR DNA and Gel Band purification Kit (GE Healthcare) and cloned directly into the TOPO pCR 2.1 vector (Invitrogen). The ToplO bacteria were transformed by the CaC12 method and the positive clones were selected by means of the b-galactosidase reaction. Plasmid DNA was obtained using the PlasmidPrep Mini Spin Kit (GE Healthcare). For each insert, at least three clones were sequenced to verify the reported sequence for both genes (GenBank M20730 for LKTA and GenBank AF059036 for PlpE). Only clones that adjusted 100% to the reported sequence were used to subclone and express the fusion protein. Sequences were obtained in an ABI Prism 310 capillary sequencer (AppliedBiosystem). The obtained sequences were compiled with the Chromasv2.31 and software packages of DNASTAR Inc. and compared with databases using the Blast program.

The vector pQE-30 Xa (Qiagen) was used for the expression of the proteins. This introduces a six-histidine tag and a cut-off site for factor Xa. The plasmid DNA with the plpE and IktA PCR inserts previously cloned was digested and ligated to the Stul and Hindffl sites located at the ends of the IktA and plpE sequences. Therefore, two constructs were generated that could express fusion proteins, designated as DLKTA-pQE30Xa and DPlpE-pQE30Xa that theoretically should express products of 30.9 and 38.4 kDa, respectively. The constructs were transformed into E. coli MI 5 (Invitrogen) for the expression of the proteins.

For the expression of fusion proteins, we use isopropyl b-D-l-thiogalactopyranoside (IPTG). One mL of bacterial preculture during the night was used to inoculate 50 ml of medium LB and allowed to grow for 2 h. Then, 1 mM of IPTG was added and it was allowed to grow for three more hours. Finally, total crude extracts were obtained and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To purify the fusion proteins, the QIAexpress Assay System (Qiagen) system was used. It is based on the affinity of the histidine tag, included in the cloned protein, for a nickel-nitrile-acetic resin (Ni-NTA). The protein binds to the column with the resin and then acidic or basic buffers are used to emulate the fusion protein in the column. Total proteins were quantified by the Lowry method (Lowry et al., 1951). A small aliquot of the extract (5 ug of total protein) was examined by SDS-PAGE to confirm the presence of the corresponding proteins.

Obtaining antibodies.

To obtain antibodies, the purified proteins (in the elution buffer 8 M urea, 0.01 M Tris-HCl 0.1 M NaH2P04) were mixed (1/8 v / v) with 0.25% Al (OH) 3 in phosphate saline ( PBS), pH 7.2, as adjuvant. Once prepared, the immunogen was inoculated subcutaneously in New Zealand white rabbits of around 2,000 g. Two rabbits were inoculated subcutaneously with 90ug of each fusion protein on days 0, 14 and 21. Serum was obtained on day 0 to be used as a preimmune control, and after each inoculation on day 14 and 21. Finally, it was obtained total serum of the rabbits by bleeding to white on day 28.

ELISA 96-well polystyrene microplates were covered with complete cells of M haemolytica in lysis buffer or with 100 ng / well of each protein (rLktA or rPlpE) diluted in carbonate buffer (0.1 M sodium bicarbonate, sodium carbonate 0.1 M, pH 9.6). The plates were incubated from 16 to 18 hrs at 4 ° C. The supernatant was removed and washed four times in the buffer (1.25 M NaCl, 250 mM Tris-HCl pH 7.9, 1% Tween-20) blocked with 2% skim milk in 0.1% Tween-PBS and incubated at 37 ° C. ° C for 1 h. Then they were washed and the samples were applied. Serum dilutions were 1: 800 for the cells, rLKTA and rPlpE and were incubated for 1 h at 37 ° C. The microplates were washed four times, anti-rabbit IgG antibody conjugated with HRP (1: 1000, Millipore, Bedford, MA) was added and incubated at 37 ° C for 1 h. O-Phenylenediamine (OPD, Sigma) was added as a substrate for the peroxidase and incubated 5 min at room temperature. They were then read at 415 nm and the results were expressed as absorbance. To determine IgG anti-rL TA and rPlpE in sheep, the same steps were performed. Serum dilutions were also 1: 800 the difference was that rabbit anti-sheep IgG conjugated with HRP (H + L) at a dilution of 1: 1000 (Kirkegaard and Perry Laboratories, Inc) was used for secondary antibody.

Vaccination and production of antibodies in rabbits.

Twenty-five white New Zealand white rabbits of around 2,000 g in weight were divided into five groups. The vaccines in a volume of 500uL were inoculated subcutaneously twice on days 0 and 14. The groups were formed as follows: Group A (positive control) was inoculated with a commercial bacterin containing Mannheimia haemolytica serotype Al; Group B was vaccinated with the recombinant preparation composed of 30 ug of recombinant LKTA (rLktA), 30 ug of recombinant PlpE (rPlpE) supplemented with Al (OH) 3. In group C, the rabbits were inoculated with a preparation composed of 60 ug of rLKTA, 60ug of rPlpE and Al (OH) 3; while the vaccine used in group D (negative control) was a bacterium for Clostridia; and for group E, only PBS and adjuvant were used. For antibody determinations, serum samples were obtained on days 0, 14, 21, 28, 35 and 42. Serum samples were analyzed for anti rLktA, anti rPlpE by enzyme-linked immunosorbent assays (ELISA). The total amount of protein was 2 ug per assay. The results obtained for the rLKTA protein can be seen in figure 1. It can be seen that rabbits inoculated with the two recombinant vaccine preparations (groups B and C) produced antibodies from day 14 whereas rabbits inoculated with the bacterin containing Mannheimia (group A) presented antibodies until day 21. The animals inoculated with the bacterin for Clostridia (group D) and those that only received PBS and aluminum hydroxide (group E) did not present antibodies against the protein at any time of the experiment. In Figure 2, we can see the antibodies that were produced against the PlpE protein. As in Figure 1, the first two groups showed antibodies from day 14 while the animals vaccinated with the commercial bacterin began to show antibodies until day 21.

Vaccination and production of antibodies in sheep.

Ten healthy sheep of around 30 kg, females of a hybrid breed of Pelibuey, Katahdin and Blackbelly were used in groups of two, housed in suitable pens and orally dewormed with Closantil. The animals were divided into five groups and vaccinated in the lateral neck region on days 0 and 14 with 2.5 ml of each vaccine. The groups were as follows: Group A (positive control) was inoculated with a commercial bacterin; Group B recombinant preparation composed of 50 ug rLktA and 50 ug rPlpE; Group C with recombinant preparation composed of 100 ug rLktA, 100 ug of rPlpE; Group D (negative control) with Bacterin for clostridia and Group E (negative control) with PBS and Al (OH) 3.

For antibody determinations, serum samples were obtained on days 0, 14, 21, 28, 35 and 42. Serum samples were analyzed for anti rLKTA, anti rPlpE and against a crude extract of complete bacterial cell proteins from M. haemolytica by enzyme-linked immunosorbent assays (ELISA). In Figure 3 we can see the generation of antibodies against the LKTA protein and in Figure 4 the generation of anti-PlpE antibodies. It can be seen that sheep vaccinated with the recombinant proteins (B and C) generated antibodies, whereas, unlike rabbits, the group vaccinated with the bacterin containing M. haemolytica (A) produced almost no antibodies against LKTA and PlpE. As expected, the negative control groups, groups E and D did not generate antibodies against the recombinant proteins. In figure 5 we see the antibodies against the total proteins of haemolytica in this case the group A was the one that showed the highest levels of antibodies.

Challenge in animals.

Seventy-five BALB / c male mice, weighing 26-32 g, were distributed in the experimental groups. The animals were kept under conditions of light, humidity and controlled temperature. The BALB / c mice were divided into five groups and injected intraperitoneally twice on days 0 and 14 with 250 uL of each preparation. Group 1 (positive control) was vaccinated with commercial vaccine, group B, with a recombinant preparation composed of 10 ug rLKTA + 10 ug rPlpE, Group C, with a recombinant preparation composed of 20 ug rLKTA + 20 ug rPlpE and the control groups negative D and E were inoculated with a commercial vaccine that does not contain Mannheimia and with PBS + Al (OH) 3, respectively. Groups were not included to test both recombinant proteins separately. All mice were challenged on day 28 after the first immunization with approximately 1.6 xlO7 CFU of virulent M. haemolytica intraperitoneally and monitored for survival for 10 days. Necropsies were performed on all the animals that died to check the effect of the bacteria.

Table 1. Challenge in mice of different vaccine preparations.

The protection was analyzed by Fisher's exact test and the results are summarized in table 1. Groups A, B and C were protected in the challenge. Group A (Biobac 11-way) was protected with an efficiency of 93.3% survival; group B, vaccinated with a lower dose of rLKTA + rPlpE recombinant vaccine (10 ug) showed 86.7% of survival, while group C vaccinated with a higher dose (20 ug) of rLKTA + rPlpE was completely protected (100% survival).

Negative controls (groups D and E) were not protected (0% survival) against infection with 1.6x10 7 CFU of M. haemolytica virulent. After the challenge, the mice were monitored for 10 days. Those belonging to groups D and E developed macroscopic hemorrhages, edema and pulmonary congestion. The protection generated by the commercial vaccine (Biovac 11 vias) and the recombinant vaccines were significantly greater (p <0.0001) than that of the negative controls.

EXAMPLE 2 Rapid purification of fragments of recombinant proteins.

Determination of production of recombinant proteins.

To determine the production of the recombinant proteins, the total protein produced by the bacteria was analyzed in 12% Laemli-type Polyacrylamide-Dodecyl Sulfate Sodium (SDS-PAGE) gels in a MiniProtean electrophoresis chamber. using the Page ruler Plus Prestained protein Ladder of the Fermentas brand (# SM1811) as a molecular weight marker. The gels were run at 200mA until the front of the gel advanced to 85% of the total length from separation. The gels were stained with Coomassie Blue with a standard procedure. The gels were photographed and the photographs were analyzed by densitometry with TotalLab Software. Numerical values were obtained both for the total amount of recombinant protein - comparing the area under the peak curve of the recombinant protein with the area under the curve of a bovine serum albumin standard - and for the purity of it calculated as the area under the curve of the recombinant protein divided over the total area of the lane sweep.

Optimization of protein production.

The clones obtained in example 1 were tested under different growth conditions to optimize the production of the recombinant proteins. The conditions analyzed were: inoculate the culture with different densities of bacteria in a range of 0.05 to 0.5 optical density (OD) measured at 600 nm; Different concentrations of the IPTG inductor in a range of 0.5 to 5mM were also studied; and it was also tried to add the inductor in different points of the growth curve studying a range of optical densities of growth between 0.1 to 1.0U; and finally it was also studied to incubate the bacteria different times in the presence of the inducer in a range from 1 to 24 hours. For each of these conditions, the production of the recombinant proteins was studied as described above and both the quantity and the proportion of the obtained recombinant protein were plotted against the variables analyzed.

Purification of proteins.

Cells were harvested by centrifugation at 3,000xG for 5 minutes. They were resuspended in PBS and subjected to a process of incubation cycles at high and low temperature. The high temperature was handled in a range between 85-100 ° C and the low temperature was handled between 4 and -12 ° C. The times that were handled for each cycle were between 1 and 30 minutes at each temperature. Subsequently, it was clarified by centrifugation at 3000xG 5 minutes. The supernatant was analyzed by SDS-PAGE and the gel was studied by densitometry as described above.

Immunogenicity of proteins.

To obtain antibodies, the purified proteins (in PBS) were mixed (1/8 v / v) with 0.25% Al (OH) 3 also in PBS, pH 7.2, as an adjuvant. Once prepared, the Immunogen was inoculated subcutaneously in New Zealand white rabbits of around 2,000 g. Two rabbits were inoculated subcutaneously with 90ug of each fusion protein on days 0, 14 and 21. Serum was obtained on day 0 to be used as a preimmune control, and after each inoculation on day 14 and 21. Finally, it was obtained total serum of the rabbits by bleeding to white on day 28.

Challenge in animals.

One hundred and forty BALBc male mice between 21 and 23 gr were divided into 7 groups of 20 animals. Six groups were inoculated with 15, 7.5, 3.7, 1.87, 0.93 and O.Oug of each of the recombinant proteins in a volume of 250uL and using aluminum hydroxide as adjuvant, the remaining group was inoculated with lOug of the recombinant proteins purified by affinity; no tests were performed on each protein separately. Two inoculations were carried out on days 0 and 14 of the experiment and the animals were challenged with 5xl07cfu of a fresh culture of M. haemolytica in BHI medium (25xLD50) on day 21, this number of bacteria corresponds to 30x the LD50 observed in our tests and 3x the dose used in the previous experiments.

Results Under the conditions of our laboratory, we found that the highest concentrations of recombinant proteins LktA and PlpE of M. haemolytica were obtained when we inoculated LB medium ampicillin with an overnight preculture at an optical density of 0.1 OD and incubated at 37 ° C for 3 hours reaching an optical density of 0.3; at that time the EPTG inducer was added at a concentration of 0.3mM and the cells were left under agitation at 37 ° C in the presence of the inducer for 16 hours. Subsequently the cells were harvested and incubated at 95 ° C for 10 minutes, then they were passed at 0 ° C and they were incubated another 10 minutes. The samples were pelleted after 10 minutes of incubation and the supernatant was transferred to 2 ml vials and stored at -20 ° C for later analysis by electrophoresis.

Figure 6 shows the results of electrophoresis obtained for the LKTA protein. Lanes 2 to 4 show us the total proteins obtained from the bacteria and lanes 5 to 9 show us the proteins purified according to the procedure described here. The densitometric analysis indicated that the purity of the proteins obtained by this method was in a range of 85 to 93%.

Similar results were obtained with the PlpE protein as can be seen in figure 7, where we can see in lanes 2 to 4 the total proteins obtained from the bacteria and in lanes 5 to 9 the results obtained in different preparations made according to the procedure described in this example.

The immunogenicity studies of the proteins obtained by this method showed results similar to those obtained with the proteins purified by a ique 1-nitriloacetic column of example 1.

Finally the results of the challenge in mice made with the recombinant proteins shown in Figure 8 showed that the maximum protection was achieved with 7.5ug of each protein and that increasing the amount of proteins inoculated above this amount did not increase the level of protection . In the same figure we can see that the preparation of the recombinant proteins described in this example generated a protective response equal to that obtained with the purified proteins in the affinity columns. EXAMPLE 3 Crosslinking of proteins to cell bodies. 250mL of a F. escoe culture of M. haemolytica was taken in BHI medium obtained from a reactor grown under standard conditions to obtain the bacterin. To these were added 150 mg of each of the purified proteins and 1 ml of 35% formaldehyde. The mixture was incubated at 25 ° C for 48 hours. After this period sterility tests were carried out inoculating different means to guarantee the total inactivation of Mannheimia culture. To verify that the proteins were bound to the cell bodies, the cells were separated by centrifugation and samples of the supernatant were analyzed by SDS-PAGE.

Hypersensitivity of delayed type.

Mice weighing between 20 and 23 grams were taken and immunized intraperitoneally on days 0 and 14 with 250 ul of the different immunogenic preparations to be evaluated. One week after the second immunization, the mice were anesthetized with ether and 10 ul of PBS containing 7.5ug of each of the proteins were intradermally injected into one of the ear pavilions. In the opposite auricular pavilion, only PBS is injected. After 24 hours, the mice were again anesthetized and measurements were taken from both pavilions with a vernier or thickener. The measure obtained from the injected pavilion was divided between the measurement obtained from the pavilion injected with PBS to obtain a percentage value of the fatness attributable to the hypersensitivity reaction.

Challenge in animals.

One hundred and forty BALBc mice between 21 and 23 g were divided into groups of 20 animals that were inoculated intraperitoneally with 250uL of different preparations. Two inoculations were carried out on days 0 and 14 of the experiment and the Animals were challenged on day 21 with 5xl07ufc of a fresh culture of M. haemolytica in BHI medium, corresponding to 30x the LD50 observed in our animals with the Mannheimia strain used.

Results To search for a greater immunity of the animals, tests were performed in which the recombinant proteins were covalently cross-linked to bacterial cell bodies with the purpose of modifying the immune response, of a humoral response that is the one observed with the purified recombinant proteins. to a cellular response that provides the greatest protection.

To generate covalent bonds between proteins and cells, inter-cross-linking agent was selected for formaldehyde, as it is low cost and naturally included in the chain of production of many bacterins. Other agents such as m-Maleimidobenzoyl-N-hydroxysuccinimide ester gave similar results in laboratory tests but considering their high cost, especially at the concentrations and volumes that would be necessary for the production of a vaccine, no further tests were performed with these compounds .

To verify that the proteins were bound to the cell bodies, the bodies were separated by centrifugation and the proteins were analyzed in the supernatant and in the pellet. In Figure 9 we can see a SDS PAGE gel where samples were loaded from two different experiments of crosslinking with formaldehyde, in lanes 1 and 2 we see the proteins that were found in the sediment, in lanes 3 and 4 the preparations of the purified LKTA protein to identify the position it reaches in the gel and in the lanes 5 and 6 we can see that in the supernatants the LKTA protein is practically not found.

The choice of this method, which is the same as that used for inactivation of the bacterins, facilitates the flow of production because all that is required is to add the fragments of the purified PlpE and LktA proteins according to example 2, to the cells which are used for the preparation of the conventional Mannheimia bacterin.

This preparation was used to make potency tests in mice comparing the new formulation with the following formulations: A) a conventional bacterin that is commercially distributed in Mexico; B) a commercial vaccine containing a bacterin of M haemolytica plus leukotoxin; C) The recombinant proteins purified by Ni-NTA columns according to example 1; D) the recombinant fragments purified according to example 2; E) A bacterin for M. haemolytica to which the purified proteins were added but which was not crosslinked with formaldehyde; and in F) we have the vaccine with the recombinant proteins cross-linked to the bacterial cell bodies of haemolytica.

The results can be seen in Figure 9 and showed that the new preparation protected more than the formulas that are distributed commercially or that the recombinant proteins purified either by the columns of zinc hydroxide or by the technique of heating, cooling and sedimentation.

Hypersensitivity of delayed type.

To detect the cellular immune response to the vaccines, delayed-type hypersensitivity tests were carried out in mice. Fifty male mice between 20 and 22 grams of weight were divided into 5 groups that were immunized with 250 uL of the following preparations: A) The recombinant proteins purified by Ni-NTA columns according to example 1; B) the recombinant fragments purified according to example 2; C) A bacterin for M. haemolyticaa which was added to the purified proteins but was not cross-linked with formaldehyde; D) the vaccine with the recombinant proteins cross-linked to the bacterial cell bodies of M. haemolytica; The group "was inoculated with PBS in both pavilions." The percentage of difference in inflammation observed in the pavilion inoculated with the purified antigens is shown in figure 10. We can observe that mice inoculated with proteins purified by affinity or by method of heating, cooling and sedimentation showed practically no induration, those inoculated with the protein mixed with the bacterin presented moderate indurations, while those inoculated with proteins crosslinked with formaldehyde to the bacterial cell bodies showed inflammation greater than 100%.

REFERENCES • 1. Essential Immunology, Eight Edition Ivan Roitt, Blackwell Scientific publication. • 2. Vaccines, Third edition.S. Plotkein W. Orenstein, W.B. Saunder's company • 3. Vaccines Prospects & perspectives Harminder sigh, rajesh Bhatia, forward publishing company, Delhi • 4. Immunotherapy of cancer. Mary L. Disis, Humana Press, Totowa, N.J., USA. • 5. DNA vaccine. Douglas B. Lowrie, Robert to Whalen, Humana press, Totowa N.J., USA. • 6. Handbook of cancer vaccines. Micheal A. Morse, Timothy M. Clay, H. Kiva Lyerly. Humana press Totowa N.J., USA. • 7. Cellular Microbiology. Bian Henderson, Michael Wilson, John Wiley & sons

Claims (8)

CLAIMS Having described my invention enough, I consider as a novelty and therefore claim as my exclusive property, what is contained in the following clauses:

1. A mmunogenic composition, characterized in that it comprises recombinant fragments of the PlpE and Lkta proteins of M. haemolytica, covalently bound to cell bodies, and a pharmacologically acceptable adjuvant.

2. An immunogenic composition according to claim 1, characterized in that the proteins are linked to bacterial cell bodies.

3. The immunogenic composition according to claim 2, wherein the fragments are overexpressed in a microorganism.

4. An immunogenic composition according to claim 2 wherein the covalent attachment of the proteins is carried out by incubation in the presence of formaldehyde.

5. The immunogenic composition according to claim 2, wherein the proteins have been covalently bound to cell bodies by incubation with formaldehyde.

6. An immunogenic composition as described in claim 1 wherein the adjuvant is aluminum hydroxide.

7. The use of the immunogenic composition according to the preceding claims, for the preparation of a vaccine to prevent infection by M. haemolytica.

8. A method for obtaining an immunogenic composition comprising recombinant fragments of the PlpE and Lkta proteins of M. haemolytica, joined covalently to cell bodies, where the method comprises the steps of: a) purification of proteins, b) obtaining bacterial cultures, c) covalent binding of proteins to bacterial cell bodies. The method according to claim 8 wherein the proteins have been purified by heating and cooling cycles and separation by sedimentation or filtration.