Universal vaccine for treating and preventing lyme disease applicable in human and veterinary science, and method for preparing it

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns a universal vaccine for treating and preventing Lyme disease to be applied in veterinary science, based on a whole-cell bacterial vaccine or bacterial lysates or purified preparations, containing the three most pathogenic genospecies Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, each of which simultaneously contains both immunogenic protective proteins of the outer membranes OspA and OspC.

EFFECT: invention provides developing the protective immunity against a natural tick-borne infection of the three most pathogenic genospecies Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii.

9 cl, 1 ex, 12 tbl

The technical FIELD

The invention relates to compositions of a universal vaccine for the treatment and prevention of Lyme disease for use in human and veterinary medicine and the method of its production.

The current LEVEL of TECHNOLOGY

Lyme disease is a chronic infectious disease characterized by multiple lesions of the organ systems. This is the most common, portable arthropod disease in Europe and the United States. The significance of this disease is shown in a number of publications in journals devoted to infectious diseases. From this point of view, in a number of diseases over the last decade, Lyme disease becomes immediately for acquired immunodeficiency syndrome, as described, for example, in"The biological and social phenomenon of Lyme disease" (Barbour AG, Fish D, Science. 1993 Jun 11;260(5114):1610-6).

The disease is caused by a group of spirochaetes with the titleBorrelia burgdorferi sensu lato. This group of microorganisms, mainly includes three closely related subspecies, i.e. theBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garinii. WhileBorrelia burgdorferi sensu strictois the cause of almost all cases of Lyme Disease in the North American continent, in Europe is dominated byBorrelia gariniiandBorrelia afzeli.

Serious damage of the host body of a mammal, as well as problems associated with diag what asticou and treatment of Lyme disease are significant incentives for the development of effective vaccines. On the other hand, there are doubts regarding the development of vaccines and their use, especially in humans. The main reasons are the lack of contagiousness of the disease, that it is fairly easy to treat with antibiotics, and clinical manifestations with serious consequences occur only in some infected patients. Moreover, this disease is rarely lethal outcome. Thus, the fear associated with possible side effects of a new vaccine, which, in these conditions, would be hardly permitted for use, is not a surprise, as described in article"Experimental immunization against Lyme borreliosis with recombinant Osp proteins: an overview" (Sadziene A, Barbour AG., Infecrion. 1996 Mar-Apr;24(2): 195-202).

The first veterinary vaccine against Lyme borreliosis was designed for dogs in the United States in 1990. The license was obtained in 1992, in accordance with articles"Performance of a Borrelia burgdorferi bacterin in borreliosis-endemic areas" (Levy SA, Lissman BA, Ficke CM., J Am Vet Med Assoc. 1993 Jun 1;202(11):1834-8) and "Immunization against Lyme Disease?" (Wormser, GP., Ann Intern Med 123,627-629,1995).

Whole cell, chemically inactivated vaccine is applied using the media containing the substance is a polymer adjuvant. The vaccine is applied intramuscularly, twice with an interval of two or three weeks. On the maintenance dose (booster) is recommended after one year. Vaccination of domestic animals, especially dogs, are recommended regardless of whether the animal is infected or illness on the stage of progression. Receiving this vaccine is based on the knowledge obtained in experiments on rodents. The protective effect of whole cell vaccine first described in hamsters.

The development of a whole cell vaccine for veterinary purposes, mainly contributed a simple method of preparation and low cost. However, this vaccine was not recommended for use in humans due to the fact that some of the antigens of Borrelia burgdorferi react with the antigens of man, and thus, we cannot exclude the stimulation of immunopathological processes, as described in"Molecular mimicry and Lyme borreliosis: a shared antigenic determinant between Borrelia burgdorferi and human tissue" (Aberer E, et al., Ann Neurol. 1989 Dec; 26(6)732-7) or Serologic response to the Borrelia burgdorferi flagellin demonstrates an an epitope common to a neuroblastoma cell line. (Fikrig et al. 1993, Proc Nat Acad Sci USA. 1993 Jan 1;90(1): 183-7).

There is evidence that some antigensBorrelia burgdorferiactivate the pool-specific T lymphocytes involved in the development of arthritis in hamsters, see"Involvement of CD4+ T lymphocytes in the induction of severe destructive Lyme arthritis in inbred LSH hamsters" (Lim LC, et al., Infect Immun. 1995 Dec; 63(12):4818-25).

Rabbits were protected from infection, if before the experimental infection was used by the immune serum obtained by the Les immunization of animals immunogenic protein of a strain of Borrelia burgdorferi. Introduction immune serum after infection did not prevent the development of erythema migrans, visceral types of infection. Similar results were also obtained for mice and hamsters and described in the"Serum-mediated resolution of Lyme arthritis in mice" (Barthold SW et al, Lab. Invest. 1996, Jan;74(1):57-67)or"Protective and arthritis-resolving activity in sera of mice infected with Borrelia burgdorferi" (Barthold SW, et al., Clin Infect Dis 1997 Jul; 25 Suppl 1:S9-17.)or"Experimental infection of the hamster with Borrelia Burgdorferi" (Johnson RC, et al., Ann Acad Sci. 1988;539:258-63).

The obtained experimental data highlight the possibility of developing a safe subunit vaccines. The main antigens candidates such vaccines are outer membrane proteins (Osp), labeled OspA, b, C and D. carefully researched OspA protein. Protective effect of this subunit vaccines was confirmed experimentally in mice, hamsters and rabbits and described in the articles in the"Protection of mice against the Lyme Disease agent by immunizing with recombinant OspA" (Firking E et al., Science 1990 Oct 26; 250(4980):553-6)and"Long-term protection of mice from Lyme Disease by vaccination with OspA" (Firking E et al., Infect Immun. 1992 Mar; 60(3):773-7).Laboratory tests have confirmed that the use of this protein in the form of purified recombinant protein obtained by the connection formation with lipids in parenteral stimulates protective immunity and protects against borreliosis infection made by injection or h is rez tick vector infection. Also, it was confirmed that infected ticks, presswise to immunized animals lost their infectivity as described in"Safety and immunogenicity of a recombinant outer surface protein A Lyme vaccine" (Keller D et al., JAMA. 1994 Jun 8;271(22): 1764-8)or"Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice" (Firking E et al.,Proc Natl Acad Sci USA. 1992 Jun 15;89(12):5418-21).

In the absence of lipid particles, the formation of antibodies by stimulating requires adjuvant effect complete adjuvant-blockers or other immune adjuvant, as described in article"Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA" (Erdile IF et al., Infect Immun. 1993 Jan;61(1):81-90).It has been noted that two of the possible effect of vaccination:

(1) Spirochaetes already inactivated in the body of the tick and, thus, prevents transmission,

(2) Spirochaetes can be rapidly inactivated after exposure to the organism of the owner, directly in front of antigenic variation and low levels of antigen OspA.

To confirm the protective effect of antigen OspA were obtained in two different designs and formulations of the vaccine:

1. Native and purified recombinant OspA described in"Recombinant outer surface protein A from Borrelia burgdorferi antibody dosage protective against spirochetal infection in mice" (Simon MM,et al.,.J Infect Dis. 1991 Jul;164(1):123-32).

2. OspA, incorporated into the genome of BCG (Calmette, Guerin). OspA is expressed externally as a membrane-associated lipoprotein, as should the article "Protective immunity elicited by rBCG viccines" (Stover CK et al., Dev Biol Stand. 1994; 82:163-70).In this form of OspA investigated intraperitoneal and intranasal introduction, as published in"Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi" (Langermann S, et al., Nature. 1994 Dec 8;372(6506):552-5).When used in this form, specific antibodies IgG and IgA were worked out over a long period of time, which is obvious from the article"Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme Disease vaccine" (Stover CK et al., J Exp Med. 1993 Jul 1;178(1):197-209).

Currently there is only one manufacturer of this vaccine for use in humans in the United States of America. The vaccine was approved by the Department for sanitary supervision of food and drug administration (Food and Drug Administration, FDA) 21 December 1998 and described in the study of"Vaccination against Lyme Disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group" (Steere AC et al., N Engl J Med 1998 Jul 23; 339(4):209-15).Product LYMErix is SmithKline Beecham. According to the third phase of clinical trials, the vaccine provides 80-90% protection, which is confirmed in article"A vaccine, consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme Disease. Recombinant Outer-Surface protein A Lyme Disease Vaccine Study Consortium (Sigal LH, et al., N Engl J Med. 1998 Jul 23; 339(4):216-22. Erratum in: N Engl J Med 1998 Aug 20; 339(8):571).

Although the vaccine has already spread, because of the minimal information about e the long-term use, she is under constant supervision. For maximum immune response, the vaccine is applied injection three times within a 12-month period (0, 1, 12). The efficacy of the vaccine described, for example, Wahlberg, who claimed that the efficacy of the vaccine was 50% (1 year) after two doses of recombinant OspA protein associated with aluminum hydroxide in phosphate buffer, and 79% after three doses of vaccine (20 months). Antibody titer quickly decreased and after two years he reached the level of the first year of vaccination (50% efficiency). Data on the secondary immune response is missing. According to the data does not allow the use of vaccines in children under 15 years of age and in people suffering from autoimmune diseases, in particular arthritis. In the group of 5765 vaccinated people arthritis developed in two people with the phenotype HLA-DR4, as published in article"Guarded endorsement for Lyme Disease vaccine" (Marwick C, JAMA. 1998 Jun 24;279(24):1937-8).

Commercially available vaccine derived from protein OspA fromBorrelia burgdorferi sensu stricto. Due to the fact that this bacterium is the dominant infectious causative agent of Lyme disease on the North American continent, the vaccine may be quite effective in the U.S. population. In Europe, however, this disease is mainly caused by the following three pathogenic subspecies, i.e. theBorrelia burgdorferi sensu stricto,Borrelia afzeliithe Borrelia garinii. On the European continent antigenic variability was described for all data types. Thus, the vaccine for use in humans and in veterinary medicine, is obtained only from the typeBorrelia burgdorferi sensu strictois unsuitable for use in Europe. To develop effective forms of vaccine used new directions vaccine technologies. For example, an effective DNA vaccine published in"Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter" (Simon MM, et al., Eur J Immunol. 1996 Dec;26(12):2831-40)or"DNA vaccines expressing a fusion product of outer surface proteins A and From Borrelia burgdorferi induce protective antibodies suitable for prophylaxis but not for resolution of Lyme Disease" (R. Wallich et al., Infect Immun. 2001 Apr;69(4):2130-6).

Another possibility is to obtain the vaccine from OspC protein expressed on the surface of microorganisms present in the host organism. According to the literature, humoral immunity against this protein is protective in nature, as described in"Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter" (Simon MM, et al., Eur. J. Immunol. 26, 2831-2840, 1996).

OspC is a major membrane antigen expressed at the early stage of infection. In the case ofBorrelia burgdorferi sensu strictothe OspC antigen is highly variable. Twenty-one allelic group, known as A-U, was discovered for this antigen with POM is using epidemiological analysis and sequencing of genes OspC-specific strains and using GeneBank, as described in theFour clones of Borrelia burgdorferi sensu stricto case invasive infection in humans" (Seinost G, et al., Infect. Immun. 67, 3518-3524, 1999).

The outer surface antigen A (OspA) is the major surface antigen, whichBorrelia burgdorferiexpresses outward,when is inside the tick. When the tick starts to suck the blood of a mammal, the synthesis of this antigen is resumed, and the synthesis of OspC antigen on the contrary increases. Thus, OspC becomes the main antigen of the outer surface of the membrane at an early stage of infection, which is described, for example, in"Induction of an outer surface protein of Borrelia burgdorferi during tick feeding" (Schwan TG, et al., Proc. Natl. Acad. Sci. USA 92, 2909-2913. 1995).Although it has been shown that OspC is limited on the surface, it is a powerful immunogen. Immunization with OspC protects against borreliosis infection. However, protection is associated with a specific OspC allele, which controls the synthesis of a specific protein. Infection is another form of Borrelia burgdorferi causes disease in individuals subjected to immunization. This, naturally, limits the use of this antigen obtained from only one of genovia Borrelia burgdorferi, upon receipt of a universal vaccine.

Thus, the question about successful vaccination against Lyme disease remains open. Moreover, in Europe is complicated by the existence of three different genocidal (Borrelia burgorferi sensu stricto, Borrelia afzeliiandBorrelia garinii) and the emergence of Lyme disease not only in humans but also in different species of domestic animals and livestock. Problems related to this disease in dogs and cats, are described in the"Canine borreliosis" (Littman MP, Vet Clin Small Anim 33, 2003, 827-862),and the incidence of horses is discussed in"Equine Abortion Associated with the Borrelia parker i - B. turicatae Tick-Borne Relapsing Fever Spirochete Group" (Walker RL, et al., Journal of Clinical Microbiology, 2002, 40, 4, 1558-1562).

Livey et al. tried to overcome these problems by obtaining so-called "vaccine-cocktail". Their results are published in"OspC vaccine candidate" (Abstract of the Symposium on the Pathogenesis and Management of Tick-borne Diseases. 1998 Sept. 28-30. Vienna, Austria. 1998).

Together with the existing problems of diagnosis and treatment of Lyme disease, as well as due to the inability to effectively control and reduce the spread of vectors of Borrelia, there is an urgent need for a vaccine capable of effective immunization of susceptible domestic animals and livestock, and humans against infections caused byBorrelia burgdorferi sensu lato. The vaccine, created on the basis of whole cell bacterial vaccines fromBorrelia burgdorferi,were developed for domestic animals, as, for example, described in patent documents US 4721617, US 6316005. Similarly, vaccines containing OspA, OspC or other immunogenic outer surface proteins derived from crops cultivated borreli is, produced as recombinant proteins in various cells of the host (E. coli), or obtained synthetically, have been developed as described in documents WO 094/25596, WO 96/05313, WO 9749812, US 6716574, US 2004067517, US 6676942, WO 0216422, US 5530103, US 6486130, US 6464985, EP 633028).

However, these whole cell or subunit vaccines did not include protection against the full spectrum of pathogenic Borrelia all genocidal mainlyBorrelia burgdorferi sensu stricto,Borrelia gariniiandBorrelia afzeliiand others, depending on the specific case. Vaccines are always made from a single genocide -Borrelia burgdorferi sensu stricto.

The task of this invention is the provision of a new universal vaccine containing the major immunogenic proteins OspA and OspC in various combinations of one, two or preferably all three are widely known pathogenic genocidal, ieBorrelia burgdorferi sensu stricto,Borrelia gariniiandBorrelia afzeliior perhaps the other, which can be successfully used without any territorial restrictions.

The OBJECT of the INVENTION

The above-mentioned objective is achieved by the creation of a universal vaccine for the treatment and prevention of Lyme disease for use in humans and in veterinary medicine, created on the basis of whole cell bacterial vaccines or bacterial lysates or purified preparations from at least one or more Geno is the Idov of Borrelia, the essence of which lies in the fact that each geovid Borrelia burgdorferi, preferably selected from the groupBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniicontains at least one immunogenic protective outer membrane protein, either OspA or OspC or both protective immunogenic protein OspA and OspC, or possibly other protective immunogenic proteins of the outer membrane.

The preferred vaccine includes all three of the most pathogenic of genovia -Borrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garrinii, each of which contains both protective immunogenic outer membrane protein OspA and OspC.

Moreover, the object of the present invention is that the protective immunogenic outer membrane proteins OspA and OspC included in the vaccine preferably in a ratio of 1:1, that the vaccine is produced in dried or liquid form or used in a buffered saline solution or with mineral or oil immunological adjuvant, or perhaps with other immunomodulatory agents, and that the pH of the vaccine is in the range of 4-9.

In conclusion, the object of the present invention is a production method of a universal vaccine for the treatment and prevention of Lyme disease in humans and veterinary use according to claims 1-5, within which each is th production strain of Borrelia burgdorferi, cultivate, inactivate and independently checked prior to the preparation of the mixture, where culture proliferate and reproduced preferably at 26-35°C for 6-18 day (each phase) for the expression of OspA antigen and at 36-38°C for 6-18 day (each phase) for the expression of OspC antigen.

New effect of the presented invention is that specific antibodies to OspA and OspC are produced in the body subjected to vaccination of animals and humans after administration of the vaccine, which prevents migration of pathogenic Borrelia from ticks subjected to vaccination, the body (antibodies to OspA) and ensure the death of Borrelia soon after possible transfer of pathogenic Borrelia subjected to vaccination, the body (antibodies to OspC) and possibly in the formation of other protective post-vaccination antibody, it is not defined in detail, and in the stimulation of immune mechanisms. Another useful effect is that besides the conventional use in adults, the vaccine can be used in young animals (preferably, dogs and cats) and young cattle (preferably, horses) at the age of 3 weeks for the induction of active protective immunity against Lyme disease. Likewise, the vaccine can be used in conjunction with other drugs, medicines and vaccines against viral, b is sterelny, fungal and other diseases in dogs, cats, horses and other animal species for which the vaccine is.

EXAMPLES of IMPLEMENTATION

Example 1:

This example demonstrates proof of protectively universal vaccine after immunization of experimental cats, dogs and horses against infections caused by virulent strains ofBorrelia burgdorferi sensu latohowever , in none of the cases does not limit patent rights related to this patent application.

Obtaining the experimental sample of the vaccine and the results of the experiment:

A) Culturing the production of Borrelia strains expressing OspA antigen:

During the cultivation, production Borrelia strains expressing OspA antigen, as initial components at the initial stage used the following tools:

environment BSK-H + rabbit serum

environment BSK-H complete

with production by strains ofBorrelia afzelii,Borrelia gariniiandBorrelia burgdorferi sensu strictoaccording to the following scheme of production technology:

In fact, the cultivation is carried out in a plastic or glass mattresses Ru intended for the cultivation of cultures of Borrelia. Culture medium BSK enrich 6% sterile rabbit serum, suitable for cultivation of Borrelia, before the actual cultivation, or you can use the full environment BSK-H containing 6% rabbit serum. Source and production work is haunted strains of support in liquid nitrogen at -196°C.

To restore the culture vial culture removed from liquid nitrogen and thawed at approximately 30°C. Immediately after thawing culture subcultured in a test tube with a nutrient medium, heated to approximately 28°C in the ratio of 1+9 (part 1 culture + 9 parts of the environment). Each strain cultured separately. Incubation is carried out at 26-35°C for 6 to 18 days.

When the reproduction of culture, for good growing, viable culture (when the red color of the medium changed to yellow) is observed in the microscope and in sterile conditions subcultured in nutrient medium at a ratio of 1 part culture + 9 parts of the environment. The cultivation is carried out at 26-35°C for 6-10 days. Further passages of well-growing Borrelia carried out in a ratio of 1 part culture: from 10 to 100 parts of the culture medium. A number of further passages depends on the number of Borrelia required to receive the vaccine.

Upon receipt of culture for the production of a vaccine, adult culture after the previous kultivirovanii in sterile conditions are used as seed culture used in the broth in bottles with a volume of 1000 ml at a ratio of 1 part culture: from 10 to 100 parts of the culture medium. The cultivation is carried out at 26-35°C for 6-10 days.

The number of Borrelia define dark-field microscope using EIT is each counting chamber Petrov-Hausser.

During the process, controlling the growth of culture and purity of conduct macroscopically, microscopically and monitoring of cultivation. Macroscopic observation is visually observing whether the changes associated culture red culture medium to yellow, and is present in the sediment environment. Microscopic observation of dark fields in the microscope exercise control over a sufficient mobility of Borrelia and the presence of small amounts of detritus, without any signs of bacterial contamination. When monitoring cultivation, 0.5 ml estimate of culture of Borrelia transferred onto pre-dried blood agar and carry out the cultivation at 35-37°C for 48 hours.

In the evaluation test, the presence of any visible unwanted bacterial growth entails umbraculifera cultures with bacterial contamination.

The presence of OspA was determined by electrophoresis of proteins in SDS page in the presence of LTOs (SDS) after staining blue Kumasi or immunological method Western blotting using antisera against OspA.

B) Culturing the production of strains of Borrelia burgdorferi for the expression of OspC antigen:

For the implementation of cultivation used the same source components, production strains and the J. of manufacturing technology, as for the generation of antigen OspA described in (A).

Similarly, the actual cultivation proceeds in the same way with the only difference that when restoring a culture vials thawed culture subcultured in a test tube with liquid medium heated to approximately 37°C in a ratio of 1+9 (part 1 culture + 9 parts of the environment) and carry out incubation at 36-38°C for 6 to 18 days. Similarly, the reproduction of culture carried out in a similar way, but with the difference that the cultivation is carried out at 36-38°C for 6-10 days. Culture for vaccine production, the determination of the number of Borrelia, the monitoring process, the monitoring of the growth and purity of the culture and the presence of OspC carried out in the same manner as described in (A).

C) the Proper way to obtain the final form of the vaccine:

Concentrated, purified, stable and inactivated bacterial strains ofBorrelia burgdorferi senzu stricto,Borrelia afzeliiandBorrelia gariniithat are proved to develop outer membrane protein OspA, and concentrated, purified, stable and inactivated bacterial strains ofBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniithat are proved to develop outer membrane protein OspC, mixed together in such a way that OspA and OspC individual genovia Borrelia burgdorferi were present in Odin the new ratio. Next component of Borrelia (90% of the volume) was mixed with the gel-like aluminum hydroxide (10% volume). After thorough homogenization checked pH and brought up to a value between 7,5 and 8,0. Vials for storage of filled vaccine and conducted the test for sterility. Next, the vials to send filled vaccine, Packed and sent for final review.

D) the Results of research

a) checking the efficiency in cats:

Six cats were subjected to vaccination and revaccination with the experimental sample of the vaccine and after 90 days they were infected ticks (Ixodes ricinus)naturally infected genocideBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garinii. As control was used three cats, not subjected to vaccination.

Cats were vaccinated subcutaneously with 1 ml of the vaccine and after 3 weeks the same way were re-vaccinated. Blood was taken before vaccination and re-vaccination. Then blood was taken 1 month after revaccination. Three months after revaccination cats infected collected naturally ticks (Ixodes ricinus), which were naturally infected with all three of the most pathogenic genocide -Borrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garinii. The degree of infection of ticks tested before experimental infected who I am.

After infection, every day, all experimental animals (subjected and not subjected to vaccination, employees control cats) have conducted clinical examination and recorded their health status, including measurement of body temperature. After infection, the blood of all the experimental animals were taken to test for 14, 28, 42 and 60 days. Blood was also taken on the day of infection, immediately after becoming infected ticks. At 7, 28 and 60 days after infection, samples were taken of the skin biopsy in those places where it was sucking ticks in order to re-allocate Borrelia burgdorferi from these samples. At 60 days after infection, all the experimental animals were killed and held the anatomic dissection and as samples were taken muscle, joint fluids, lymph node, skin, and kidneys in order to re-allocate Borrelia burgdorferi from these predisposing sites.

The results are presented in summary tables 1, 2, 3 and 4. From the obtained results clearly imply that the vaccine against Lyme borreliosis, obtained from the production of strains ofBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniiexpressing surface proteins OspA and OspC, is harmless to cats and promotes the development they have protective immunity against natural tick-borne infection is the most pathogenic of all three genocidalBorrelia burgdorferi ensu stricto ,Borrelia afzeliiandBorrelia gariniiand, possibly because of cross-immunity, other known genocidal of Borrelia.

b) checking the efficiency in dogs:

Six dogs were subjected to vaccination and revaccination with the experimental sample of the vaccine and after 90 days they were infected ticks (Ixodes ricinus)naturally infected genocideBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garinii. As control was used three dogs, not subjected to vaccination. Vaccination, a booster vaccination, blood sampling, clinical assessment, collecting samples for research and other manipulations were performed in the same way as in the study for cats.

The results are presented in summary tables No. 5, 6, 7 and 8. From the obtained results clearly imply that the vaccine against Lyme borreliosis, obtained from the production of strains ofBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniiexpressing surface proteins OspA and OspC, is harmless to dogs and promotes the development they have protective immunity against natural kresevo the infection of all three of the most pathogenic genocidal Borrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniiand, possibly because of cross-immunity, other known genocidal of Borrelia.

c) checking the efficiency in horses:

Six horses were subjected to vaccination and revaccination with the experimental sample of the vaccine and after 90 days they were infected ticks (Ixodes ricinus)naturally infected genocideBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia garinii. As control was used three horses, not subjected to vaccination.

Vaccination, a booster vaccination, blood sampling, clinical assessment, collecting samples for research and other manipulations were performed as in the study for cats and dogs.

The results are presented in summary tables No. 9, 10, 11 and 12. From the obtained results clearly imply that the vaccine against Lyme borreliosis, obtained from the production of strains ofBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorrelia gariniiexpressing surface proteins OspA and OspC, is harmless to horses and promotes the development they have protective immunity against natural tick-borne infection is the most pathogenic of all three genocidalBorrelia burgdorferi sensu stricto,Borrelia afzeliiandBorelia garinii and, possibly because of cross-immunity, other known genocidal of Borrelia.

Examples of receiving the vaccine are not the only way of implementing the invention, but whole cell bacterial vaccines or bacterial lysates or purified compounds containing protective immunogenic proteins can be used without any impact on the subject according to the invention in physiological buffer solutions or with mineral or oil immunological adjuvant or with the help of immunological complexes (ISCOM), liposomes and other natural or synthetic immunological adjuvants.

INDUSTRIAL APPLICABILITY

A universal vaccine for the treatment and prevention of Lyme disease can be used as a drug in humans and in veterinary medicine and can be successfully applied intramuscularly, subcutaneously, intradermally or transdermally.

1. Universal VA is the CIN for the treatment and prevention of Lyme disease for veterinary use, on the basis of whole cell bacterial vaccines or bacterial lysates or purified preparations, including three of the most pathogenic of genovia Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garrinii, each of which contains both protective immunogenic outer membrane protein OspA and OspC.

2. Universal vaccine according to claim 1, characterized in that the protective immunogenic outer membrane proteins, OspA and OspC included in the vaccine preferably in a ratio of 1:1.

3. Universal vaccine according to claim 1, characterized in that it is received in dried or liquid form, or used in the buffer saline solution, or with a mineral or oil immunological adjuvant or perhaps with other immunomodulatory agents.

4. Universal vaccine according to claim 2, characterized in that it is received in dried or liquid form, or used in the buffer saline solution, or with a mineral or oil immunological adjuvant or perhaps with other immunomodulatory agents.

5. Universal vaccine according to claim 1, characterized in that the pH of the vaccine is in the range from 4 to 9.

6. Universal vaccine according to claim 2, characterized in that the pH of the vaccine is in the range from 4 to 9.

7. Universal vaccine according to claim 3, characterized in that the pH of the vaccine will is designed in the range from 4 to 9.

8. Universal vaccine according to claim 4, characterized in that the pH of the vaccine is in the range from 4 to 9.

9. Method for the production of a universal vaccine for the treatment and prevention of Lyme disease for veterinary use according to claims 1 to 8, characterized in that each production strain of Borrelia burgdorferi cultivated, inactivate and independently checked prior to the preparation of the mixture, where culture proliferate and reproduced preferably at 26-35°C for 6-18 day (each phase) for the expression of OspA antigen and at 36-38°C for 6-18 day (each phase) for the expression of OspC antigen.