CZ18920U1 - General-purpose composition for enhancing stamina, prevention, prophylaxis and therapy of Lyme borreliosis for human and veterinary application - Google Patents

Description

Universal preparation for enhancement of defense, prevention, prophylaxis and treatment of Lyme disease for human and veterinary use

Technical field

The technical solution concerns the composition of a universal preparation for enhancing the defense, prevention, prophylaxis and treatment of Lyme disease for human and veterinary use.

BACKGROUND OF THE INVENTION

Lyme disease is a chronic multisystemic infectious disease that is the most common arthropod-borne infectious disease in Europe and the United States. The importance of this disease is evidenced by the frequency of publications in journals dealing with information problems. In this regard, in the last decade, borreliosis has been classified as an acquired immunodeficiency syndrome, as described, for example, in The Biological and Social Phenomenon of Lyme Disease (Barhour AG, Fish D, Science. 1993 Jun 11: 260 (5114): 1610 -6).

The disease is caused by a group of spirochetes collectively referred to as Borrelia burgdorferi sensu lato. This group of microorganisms consists mainly of three closely related subspecies, namely Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia gariníi. While Borrelia burgdorferi sensu stncto is the cause of virtually all Lyme disease on the North American continent, Borrelia gariníi and Borrelia afzelii predominate in Europe.

Serious damage to the mammalian host organism, as well as problems associated with the diagnosis and therapy of Lyme disease, is an important stimulus for the development of effective vaccine preparations. On the other hand, there are also concerns about the development of vaccines and their use especially in humans. The main concern is that the disease is not transmissible from person to person, it is relatively easy to treat with antibiotics and clinical manifestation with serious consequences affects only a part of infected patients. Moreover, the disease is very rarely fatal. There is therefore concern about the possible side effects of a new vaccine that would be very difficult to accept under these circumstances, as described in Experimental immunization against Lyme borreliosis with recombinant Osp proteins: an overview (Sadziene A, Barbour AG., Infecúon. 1996). Mar-Apr; 24 (2): 195-202.

The first veterinary vaccine against borreliosis was developed in the United States in 1990 for dogs. It was licensed in 1992, as is known from the articles of pfa Borrelia burgdorferi bacte30 rin in borreliosis-endemic areas (Left NT, Lissman BA, Fieke CM., J Am Vet Med Assoe. / 993 Jun 1; 202 (11): 1834-8.) Or Immunization against Lyme disease? (Wormser, GP., Ann Intern Med 123, 627-629, 1995).

The vaccine is whole cell, chemically inactivated, administered with a vehicle containing polymeric adjuvants. The vaccine is injected intramuscularly twice every two to three weeks.

A maintenance dose (booster) is recommended after one year. Vaccination of domestic animals, especially dogs, is recommended regardless of whether the animal has been infected or is under disease. The preparation of this vaccine was based on findings from rodent experiments. The protective effect of whole cell vaccine was first described in hamsters.

The development of the whole cell vaccine for veterinary purposes was supported primarily by the simplicity of the right and also by the low cost. However, this vaccine has not been recommended for human use due to the fact that some Borrelia antigens cross-react with human antigens and therefore induction of immunopathological processes cannot be excluded as described in Molecular mtmiery and Lyme borreliosis: human tissue (Aberer E et al., Ann Neuroil 989 Dec; 26 (6): 732-7) or Serology of response to the Bor45 relia burgdorferi flagellin demonstrates an epitope of the neuroblastoma cell line. (Fikrig et al. 1993, Proc Nat Acad Se and U S A. 1993 Jan 1:90 (1): 183-7).

For example, some Borrelia burgdorferi antigens activate sets of specific T lymphocytes that have been involved in the development of arthritis in hamsters, as described in

- I LZ teVZU U1 bivolvement of CD4 + T lymphocytes in the induction of northern desulphurized Lyme arthritis and inhred LSII hamsters (Lim LC et al., Infect Immun. 1995 Dec; 63 (12): 4818-25).

Rabbits are protected against infection when the immune serum prepared by immunizing the animals with the immunogenic proteins of the borreliosis strain is applied prior to experimental challenge. Administration of immune serum after infection does not prevent the development of erythema migrans and visceral manifestations of infection. Similar results were also found in mice and hamsters and described in Serum-mediated resolution of Lyme arthritis in mice ”(Barthold SW et al, Lab. Hivest.996, 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 Expenmenial infection of the hamster with

Borrelia Burgdorferi (Johnson RC et al., Ann Acad Sci. 1988; 539: 258-63).

These experimental findings indicated the possibility of developing a safe subunit vaccine. The main candidate antigens for such a vaccine have been outer surface proteins (Osp), referred to as OspA, B, CaD. In particular, the OspA protein has been studied intensively. The protective effect of this subunit vaccine has been confirmed experimentally in mice, hamsters and rabbits and described in the articles of protection against the Lyme disease agent by recombinant OspA (Firking E et al., Science 1990 Oct 26; 250 (4980): 553-6). ) or Long-term protection of mice from Lyme disease by OspA vaccination (Firking E et al., Infect immun. 1992 Mar; 60 (3): 773-7). Laboratory tests have confirmed that application of this protein in the form of purified, lipidated recombinant protein induces protective immunity upon parenteral administration and protects against infection by borreliemia administered not only by injection, but also by means of a tick vector. It has also been confirmed that infectious ticks sucking on such immunized animals lose their incectivity as described in Safety and immunogenicity of 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 tieksfeeding on OspA25 immunized mice (Firking E et al. Proc Natl Acad Sci CSA. 1992 Jun i5; 89 (12): 5418-21).

In the absence of a lipid unit, induction of antibody formation requires the adjuvant effect of Freund's complete adjuvant, or other immune adjuvant, as described in the role of lipid in the immunogenicity of Borrelia burgdorferi OspA (Erdile 1F et al., Infect Immun. 1993 Jan; 6I (1)). 81-90). This suggests a dual possible vaccine effect:

(1) spirochetes are inactivated already in the tick organism to prevent transmission of infection, (2) spirochetes may be inactivated shortly after entry into the host organism prior to antigenic variation and down-regulation of OspA antigen expression.

Two different constructs and vaccine formulas were prepared to verify the protective effect of OspA:

Native and pure recombinant OspA, described in Recombinant outer surface protein and from

Borrelia burgdorferi induces antibodics protective against spirochetal infection in mice (Simon MM et al., J. Infect Dis. 1991 Jul; 164 (1): 123-32)

2. OspA incorporated into the BCG genome (Calmette, Guerin). OspA was expressed as a membrane-associated lipoprotein, as known from Protective immunity elicited by rBCG vac40 cines (Stover CK et al., Dev Biol Stand. 1994; 82: 163-70). In this form, it has been tested for intrapentoneal administration and also for intranasal administration 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). In this form of administration, specific IgG and IgA antibodies have been induced for a long time, as shown in the article Protective immunity elicited by recombinant hacille 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.

At present, the United States is the only producer of such vaccines for human use. The vaccine has been approved by the Food and Drug Administration (FDA) 21 December 1998 and is described in a study of Vaccination against Lyme disease with recombinant Borrelia burgdorferi outersurface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group (AC Steere et al., N

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EnglJ Med. 1998 Jul 23; 339 (4): 209-15. It is a LYMErix product from SmithKline Beecham. According to the third phase of clinical trials, the vaccine provides 80-90% protection, as demonstrated in A vaccine consisting of a recombinant Borrelia burgdorferi outersurface protein A to prevent Lyme disease. Recombinant Outer-Surface Protein A Lyme Disease Vaccine Study Consortium (Sigal LH et al., N Engl J Med. / 998 Jul 23; 339 (4): 216-22. Erratum in: N Engl J Med 1998 Aug 20; 339 ( 8): 571)

Although the vaccine is already in distribution, it is under permanent control with a minimum of knowledge of the long-term use of the preparation. Vaccine is injected three times over a 12 month period (0, 1, 12) to maximize immune response. Vaccine efficacy is described, for example, by Wahlberg, who reports that after two doses of recombinant OspA protein bound to aluminum hydroxide in phosphate buffer, the vaccine efficacy was 50% (1 year), after three vaccine doses (20 months) the efficacy was 79%. Antibody titer decreased relatively quickly and was at the level of the first year of vaccination (50% efficiency) after two years. Details of the “booster” effect are not available. To date, it is not permitted to administer the vaccine to children under 15 years of age and to people suffering from autoimmune disease, especially arthritis. In a group of 5,765 vaccinated individuals, two individuals with the HLA-DR4 phenotype developed arthritis as published in the article "Guarded Endorsement for Lyme Disease Vaccine" (Marwick C, JAM A. 1998 Jun 24; 279 (24): 1937-8).

A commercially available vaccine is prepared from the OspA protein of Borrelia burgdorferi sensu stricto. With regard to it regarding to it. that this bacterium is the dominant infectious agent of Lyme disease on the North American continent, Vakein can be relatively effective for the US population. In Europe, however, three pathogenic subspecies, Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, are the main cause of this disease. All these strains have antigenic variability described on the European continent. Therefore, in Europe, a vaccine prepared only from Borrelia burgdorferi sensu stricto is unusable for human and veterinary use. New trends in vaccine technology have also been used in the development of an effective form of vaccine, where, for example, effective vaccine DNA is published in the articles Protective immunization with plasmid DNA containing the outer surface lipoprotein A of Borrelia burgdorferi is independent of the eukaryotic promoter ”(Simon MM et al ., Eur J Immunol. 1996 Dec; 26 (12): 283I-40) or "DNA vaccines expressing and fusion product of outer surface proteins

A and C from Borrelia burgdorferi inducing protective anti-prophylactic agents suitable for prophylaxis but not for the resolution of Lyme disease ”(Waltich R. et al., Infect Immun. 2001 Apr; 69 (4): 2130-6).

Another possible starting point is to prepare a vaccine from the OspC protein, which is expressed on the surface of microorganisms in the host's body and according to literature reports, the humoral immunity against this protein is protective, as described in Protective immunization with plasmid

DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is an independent of the eukaryotic promoter ”(Simon MM et al., Eur. J, Immunol. 26, 283U2840, 1996).

OspC is the major membrane antigen expressed in the early phase of infection. In Borrelia burgdorferi sensu stricto, OspC is highly variable. Epidemiological analysis and sequencing of OspC genes in individual isolates and complementation with GeneBank revealed 21 allelic groups labeled AU for this antigen as described in Four dones of Borrelia burgdorferi sensu stricto invasive infection in humans ”(Seinost G et al., Infect Immun., 67, 3518-3524 (1999).

Outer surface antigen A (OspA) is the major surface antigen that is expressed when Borrelia burgdorferi is resident in a tick. When the tick begins to suck blood in mammals, the synthesis of this antigen is repressed and conversely the synthesis of the OspC antigen is induced. Thus, OspC becomes the major antigen of the outer surface membrane in the early phase of infection, as described, for example, in Induction of the outer surface protein of Borrelia burgdorferi during tiek feeding ”(Schwan TG et al., Proc. Natl. Acad. Sci. USA 92, 2909-2913 (1995). Although OspC has been shown to have limited surface exposure, it is a potent immunogen. Immunization

OspC is protective against Borrelia infection. However, protection is linked to a particular OspC allele, which controls the synthesis of a particular protein, and infection with another type of Borrelia leads to disease in these immunized individuals. This, of course, limits the use of this antigen obtained from only one genomospecies of borrelia to produce a universal vaccine.

Thus, the question of successful vaccination against Lyme Boretiosis remains an open question. Moreover, in Europe it is complicated by the existence of three different genomospecies (Borrelia burgdorferi sensu stricto,

Borrelia cherelia and Borrelia garinii) and Lyme disease, in addition to humans, in various species of domestic and farm animals. Canine borreliosis (fLittman MP, Vet Clin Smáli Anim 33, 2003, 827-862) and Eguine Abortion Associated with Borrelia Parker i-B. turicatae Tick-Borne Relapsing Fever Spirochete Group (Walker RL et al., Journal in Clinical Microbiology, 2002, 40, 4, 1558-1562).

The possibility of overcoming these problems by preparing so-called "cocktail vaccines" has been attempted by, for example, Livey et al., and published in OspC vaccine candidate "(Abstract of the Symposium on Pathogenesis and Management of Tick-Borne Diseases. 1998 Sept. 28) -30, Vienna, Austria 1998).

With the current problems with the diagnosis and treatment of Lyme disease and the inability of t5 to effectively control and reduce the spread of borrelia vectors, there is an urgent need for a vaccine capable of effectively immunizing susceptible species of domestic and farm animals and humans against infection

Borrelia burgdorferi sensu lato. Borrelia burgdorferi whole cell vaccines have been developed for use in domestic animals, as described, for example, in US Patents 4721617, US 6316005. Vaccines based on the content of OspA, OspC or other outer immunogenic proteins isolated from cultured borrelia cultures expressed as recombinant proteins in various hosts (E. coli) or synthetically prepared, examples of which are WO 94/25596, WO 96/05313, WO 97/49812, US 6716574, US 2004067517, US 6676942, WO 02/16422, US 5530103, US 6486130, US 6464985, EP 633028).

However, these whole-cell or sub-unit vaccines do not include protection against the entire range of pathogenic borelias of all genomospecies, in particular Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii and possibly others. They are derived from only one genomospecies, Borrelia burgdorferi sensu stricto. Furthermore, the use of these preparations remains at risk of inducing undesirable immunopathological processes.

SUMMARY OF THE INVENTION The object of the present invention is to provide a novel universal composition comprising the major immunogenic proteins OspA and OspC in various combinations of at least one, two, preferably all three of the most known pathogenic genomospecies, Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii. it may be successfully applied without territorial restriction, including orally (orally), which excludes the induction of immunopathological processes in this mode of administration.

The essence of the technical solution

This object is achieved by a technical solution which is a versatile preparation for enhancing the defense, prevention, prophylaxis and treatment of Lyme disease for human and veterinary use, based on whole-cell bacterins or bacterial lysates or bacterial purity agents in liquid form or as lyophilisates of at least one or more. genomospecies of pathogenic Borrelia, wherein each of the Borrelia genomospecies selected preferably from the group of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinti contains at least one immunogenic outer membrane protein protein, either OspA or OspC, or both immunogenic protective proteins OspA and OspC, optionally containing other immunogenic protective proteins of the outer membrane, as well as whole cell bacterin or bacterial lysate or bacterial purity in liquid or lyophilized form in the form of a selected immunomodulatory strain Propionibacterium acnes or another bacterial immunomodulatory strain.

In a preferred embodiment, the preparation comprises bacterial lysates of all three of the most pathogenic genomo50 species of Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, each of which is contained.

It simultaneously co-binds both the immunogenic protective proteins of the outer membrane OspA and OspC, and at the same time contains a bacterial lysate of the bacterial immunomodulatory strain Propionibaeterium acnes.

Another object of the invention is that, optimally, the immunogenic protective outer membrane proteins of the strains Borrelia burgdoreri sensu stncto, Borreha afzelii and Borrelia gari5 mi OspA and OspC are preferably present in the vaccine in a ratio of 8: 1: 1 to 1: 1: 1,

It is also preferred that the lyophilized bacterial lysates of Borrelia burgdosterii sensu stncto, Borrelia afzelii and Borrelia garinii are in a 1: 1: 1 ratio relative to each other, a ratio of a mixture of lyophilized bacterial lysates of Borrelia burgdermeri sensu stncto, Borrelia afzelii and Borrelia acanisium acne strain bacterium It is in a ratio of 9: 1 to 1: 10 and the total dry matter content of the lyophilisates of all strains in a single application dose is 1 to 1000 mg.

It is also the essence of the technical solution that the preparation is formed in lyophilized or liquid form and is applied in buffered saline or with mineral or oil immune adjuvant, optionally with other natural or synthetic immunomodulators or with other adjuvants, preferably vehicle, with natural nature immunomodulators are selected from the group of bacterial immunomodulators or glucans or mineral elements, and the pH of the preparation ranges from 4 to 9.

The present invention achieves a new effect in that, after administration, including oral administration, vaccinated animals or humans produce specific OspA and OspC antibodies that prevent and assist the transfer of pathogenic borelias from the tick to the vaccinated organism from (OspA antibodies). destruction shortly after the eventual transfer of pathogenic borelias to the vaccinated organism (OspC antibody) and possibly to the formation of further undefined postvaccination of protective antibodies and the stimulation of immune mechanisms. Another benefit is that, in addition to normal adult individuals, the preparation can be administered to domestic animals (preferably dogs and cats) and livestock (preferably horses) from 3 weeks of age to induce active protective immunity against Lyme disease. The preparation may also be co-administered with other preparations, medicaments and vaccines against the viral, bacterial, fungal and other diseases of the dog, cat, horse and other animal species for which the preparation is intended and other preparations, medicaments and vaccines for human use in human use. preparation. Another benefit of the technical solution is that in addition to the usual routes of administration (intramuscularly), subcutaneously (subcutaneously), intradermally (intradermally), needle-free (transcutaneous), skin and skin (cutaneous), the preparation can be applied orally (by mouth) to induce active Protective immunity against Lyme disease The use of the preparation orally (by mouth) avoids the induction of undesirable immunopathological processes.

Examples of technical solution

Example 1

This exemplary embodiment demonstrates the verification of the protective properties of a universal preparation after oral immunization of experimental dogs against challenge with virulent strains of Borrelia burgdorferi sensu lato, but in no way limits the rights relating to this invention.

Preparation of the test specimen and test results:

A) Cultivation of Borrelia strains to express OspA:

When cultivating Borrelia production strains to express OspA, one of the items

- BOD-H + rabbit serum medium

- Medium BOD - II completc and production strains Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu strieto according to the following schematic production process:

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Preparation of culture medium

Production strains

Control of growth and purity

Recovery

Inactivation of individual strains

Growth and purity control Number determination Determination of OspA presence

Inactivation test

Concentration, purification of antigens

Determination of absence of serum

Preparation of lysate

Lyophilization

Note: Each Borrelia strain (Borrelia afzelii, garinii and burgdorferi sensu stricto) is cultured, checked, inactivated, lyophilized and tested separately until all strains are mixed (including strains expressing OspC antigens).

The actual cultivation was carried out in plastic or glass Roux bottles intended for cultivation of Borrelia cultures, when the BOD culture medium was enriched before the cultivation with 6% of sterile rabbit serum suitable for Borrelia cultivation. the complete BOD-H medium containing 6% rabbit serum can be used directly. The parent and working production strains are kept frozen at -196 ° C in liquid nitrogen,

For culture recovery, culture vials are removed from liquid nitrogen and thawed at about 30 ° C. Immediately after dissolving, is inoculated into a test tube with a nutrient medium vytemperovaným a temperature of approximately 28 ^at c = 1 + 9 (1 part of culture + 9 parts of medium). Each strain is cultured separately and incubated at 26-35 ° C for 6-18 days.

i5 In culture propagation, a well-grown, viable culture (changing color of the culture medium from red to yellow) is inoculated into a nutrient medium at aseptic conditions at a ratio of 1 part culture + 9 parts medium. Cultivation takes place for 6 to 10 days at 26 to 35 ° C. Further subpassages of well-grown Borrelia are performed in a ratio of 1 part batch: 10 to 100 parts culture medium. The number of subassemblies depends on the required volume of Borrelia for preparation.

In the production of the culture for preparation, the grown cultures from previous cultures under aseptic conditions are used as inoculum in 1000 ml flasks in a ratio of 1 part batch: 10 to 100 parts of culture medium. Cultivation takes place for 6 to 10 days at 26 to 35 ° C.

Borrelia counts are performed in a dark field microscope using PETROFF HAUSSER

COUNTING CHAMBER.

Interoperative growth and purity controls of the culture are performed during the process by macroscopic examination, microscopic examination and culture examination. The macroscopic examination consists in visual observation when a suitable culture changes the culture medium color from red to yellow and sediment is present in the medium. Microscopic dark field microscopic examination of the microscope monitors whether Borrelia are reasonably movable with a small amount of detritus, with no signs of bacterial contamination. For the culture examination, the test Borrelia culture is transferred in an amount of 0.5 ml onto pre-dried blood agar and cultivated at 35-37 ° C for 48 hours.

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The assessment of the test should be carried out for any bacterial growth that is undesirable and discard cultures with bacterial contamination.

Determination of OspA is performed by protein electrophoresis on SDS PAGE after Coomassie Blue staining or by Western Blot immunodetection using anti OspA serum.

B) Cultivation of Borrelia strains to express OspC antigen:

In preparing the culture, the same raw materials and the same production strains are used and the same schematic manufacturing procedure as for the expression of the OspA antigen described under A) is used.

The cultivation itself is carried out in the same process, except that during culture recovery, the culture ampoule is inoculated into a test tube with a nutrient medium at a temperature of about 37 ° C in a ratio of 1 + 9 (1 part culture + 9 parts medium). it is run at 36-38 ° C for 6-18 days. Also, the culture propagation proceeds analogously except that the cultivation takes place for 6 to 10 days at 36 to 38 ° C. Identical to the description in (A), culture of the vaccine is made, determination of the number of borelias, in-process controls, growth and purity control of the culture, and determination of the presence of OspC,

(C) Cultivation of Propionibacterium acnes:

This process takes several steps:

(a) Cultivation

The strain is kept in a lyophilized state. The ampoule lyophilisate is rehydrated, plated on Petri dishes with Reinforced Clostridial Agar (VL agar). This is followed by cultivation in an anaerobic medium at a temperature of 35-39 ° C for 48 hours.

b) Extraction

Extraction is carried out at 2-8 ° C for 22-26 hours.

c) Antigen concentration and purification

Concentration and purification is by ultrafiltration through a 300 kDa porous cassette.

d) Inactivation

The purified retentate is placed in a water bath heated to 56-62 ° C, followed by inactivation for 60 minutes. After removal from the bath, the bottles were placed at 2-8 ° C the next day. This is followed by reheating to 56 to 62 ° C. This inactivation is repeated 3 times. e) Lyophilization

After freeze-drying, the contents of each bottle are squeezed and homogenized. Samples are taken to determine residual moisture and the lyophilisate is stored in an airtight container in a dry and dark place at 2-8 ° C.

D) Preparation of the final form:

All lyophilisates of individual production vaccine strains of Borrelia burgdorferi sensu stricto lysatum cryodesiccatum, Borrelia garinii lysatum cryodesiccatum, Borrelia afzelii lysatum cryodesiccatum and Propionibacterium acnes lysatum cryodesiccatum, are mixed so that the proportion of individual lyophilisates of lysaticum stratum lysaticum cryodesiccatum, Borrelia afzelii lysatum cryodesiccatum was equivalent (1: 1: 1), and the ratio of the mixture of lyophilisates of bacterial lysates of Borrelia burg40 dorferi sensu stricto cryodesiccatum, Borrelia garinii lysatum cryodesiccatum, Borrelia afzelii lysatum cryodesiccatum libacteria lyophilisate of bacterial lysatium cryodesiccatum to lyophilisate of bacterial lysatum the dry matter volume of all 4 strains in the final product was 5 mg per capsule (i.e. one application rate). All lyophilisates of bacterial lysates are mixed with excipients (vehicle) by adding 200 mg of vehicle to 5 mg of active ingredients and a single dose per capsule having a total weight of 205 mg.

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E) Test results:

Efficacy test on dogs:

Oral administration of the preparation to 6 dogs was performed with the test specimen and 90 days after the last applied dose of challenge with naturally infected ticks (Ixodes ricinus) by Borrelia genomospecies, Borrelia burgdorteri sensu stricto, Borrelia afzelii and Borrelia garinii. including infection of 3 dogs that were not given to control dogs. The preparation was provided with capsules orally (orally) for 10 days, followed by a 20-day rest period, a 10-day rest period, a 20-day rest period and a 10-day rest period. days.

Blood collection was performed prior to application of the preparation. Three months after application of the preparation, challenge was carried out with ticks (ixodes ricinus) from natural collection, which was naturally infected with all three most pathogenic genomospecies - Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. The degree of tick infection was verified prior to the challenge experiment.

and? After infection, all test dogs were clinically evaluated daily and their health status, including body temperature measurements, was recorded. After infection, probate blood samples were taken from all test animals on day 14, day 28, day 42, and day 60. Blood was also collected on the day of challenge just prior to infection by ticks. On the 7th, 28th and 60th days after challenge, skin biopsies were performed at the site of tick tick to re-isolate Borrelia from these samples. The 60th day after challenge was

2o all experimental animals were sacrificed and a pathological anatomical autopsy was performed with sampling of muscle, articular synovia, lymph nodes, skin and kidneys to re-isolate the borrelia from these predilection sites.

The results are presented in tables 1, 2, 3 and 4. The results clearly show that the preparation against borreliosis prepared from production strains

Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii expressing OspA and OspC surface proteins and the immunomodulatory strain Propionibactenum aenes are harmless to dogs and, after oral administration, develop active protective immunity to natural infection via infected ticks of the three most stratospheric senses , Borrelia afzelii and Borrelia garinii and, due to the principle of cross-immunitis, possibly against other known Borrelia genomospecies.

Table 1 - Scheme of challenge trial

Pesč. Immunization 1 Čelenž 1 Collection of biological material after challenge / days Note 7! ¹⁴ 28 35 42, 49 56 60 IV + + ; b TO BK - TO - - BKP Clinic - daily 2V: + + (B) TO BK - TO - •, BKP Clinic - Daily '3V + + (B) TO ! BK TO - ^: - BKP Clinic - daily 4 V + -and- (B) : κ BK - TO , - BKP Clinic - daily i 5V j + 1 B TO í BK - TO | - [BKP Clinic - daily 1 , 6V ’ 1 + 1 T 1 ^B i TO i ^{BK -} ) TO 1 - BKP Clinic - daily 1 '7KO - + B i TO i BK TO - Γ BKP Clinic - daily 8KO - + (B) TO 'BK - TO - - I BKP Clinic - daily 9KO ^{1 B} i TO I BK; - TO 1 1 - í BKP Clinic - daily!

Explanatory notes:

V - application of the preparation

KO - no application (control) + - action performed

- action not taken

B - skin biopsy

K - blood collection

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P - pathological anatomical autopsy. Table 2 - Clinical trial after challenge

Dog c. Clinical changes after challenge - days 56 60 Note 7 '14 21 '28 35 42 49 IV ’- - - - - 1 - ! - Clinic - monitored daily '2 May - - - - Khmka - monitored daily. 3V '- - 1 - '- - i - - Clinic - monitored daily 4V ~ 1 Clinic - monitored daily '5V' -  - , _ _ - '-. - - Clinic - monitored daily ¹ 6V - - _____ - - Clinic - monitored daily 7KO '- - T '+ ; Clinic - monitored daily, SKO í _ : - - + Clinic - monitored daily 9KO I - - _ 1 1 ⁺ Clinic - monitored daily

Explanatory notes:

+ - clinical signs

- without clinical signs.

Table 3 - Finding Borrelia in Skin biopsy after challenge

Dog. The term of skin biopsy collection - days. Note i ~ 7 28 í 60 1_

IV ~ j ”~

2V___ ¹ ·

V! · ¹ · ¹

ÓV__ _u -___ 7KO i ____ 8KO I + | Í 9KO '+ ~ T +

Explanatory notes:

+ - - positive capture

- negative capture.

Table 4 - Finding Borrelia in Organs

Dog. , Γ Preserved organs (60th day - autopsy) ¹ Note muscle i skin lymph node articular synovia kidney | PA - neg. 2V i - I Τ ' ¹ ' _ 1 _ | PA - neg. ! 3V 1 1 i 1 PA - neg. 1 | 4 V ^! - - PA - neg. ¹ 5V 1 I PA - neg. ί _; 6V | i -! - PA - neg. '7KO ¹ - -. + + _ i " 1 PA - pos. | 8KO + _t -, 9 - + PA - pos. 9KO | Her PA - pos.

Explanatory notes:

+ - positive capture

- negative capture

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PA - pathological-anatomical autopsy.

The described composition, preparation and use example are not the only possible compositions, embodiments, and uses of the present invention, but without affecting their nature, whole cell bacteria or bacterial lysates or purities may be in liquid form or as lyophilizates containing specific and non-specific immunogenic protective proteins applied in buffered saline or with mineral or oil immune adjuvants, optionally with immune complexes (1SCOM), liposomes and other natural (bacterial immunomodulators, glucans, mineral elements, etc.) or synthetic immune adjutants or other adjuvants, preferably vehicle.

Industrial Applicability

The universal preparation for enhancement of the defenses, prevention, prophylaxis and treatment of Lyme disease can be used in veterinary and human medicine as a food supplement and can be successfully administered intramuscularly, subcutaneously (subcutaneously), intradermally (intradermally) or needle-free (transcutaneously) , by mouth (orally), on the skin and skin (cutaneously), or by any other means that elicits an active protective immune response of the organism to which the preparation is administered.

Claims (7)

PROTECTION REQUIREMENTS Universal preparation for enhancing the defense, prevention, prophylaxis and treatment of Lyme disease for human and veterinary use, based on whole-cell bacterins or bacterial lysates or bacterial purates in liquid form or as lyophilisates of at least 2o one or more genomospecies of pathogenic Borrelia, characterized in that each of the Borrelia genomospecies selected preferably from the group Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii contains at least one immunogenic protective membrane protein of the outer membrane, either OspA or OspC, or both immunogenic protective proteins OspA and OspC, optionally containing other immunogenic protective proteins of the outer membrane, and 25 either a whole cell bacterin or a bacterial lysate or a bacterial purity in liquid or lyophilized form of a selected immunomodulatory strain of Propionibacterium acnes or another bacterial immunomodulatory strain. A multivariate preparation according to claim 1, characterized in that it contains bacterial lysates of all three most pathogenic genomospecies of Borrelia burgdorferi sensu stricto, Borrelia 30 afzelii and Borrelia garinii, each containing both the immunogenic protective proteins of the outer membrane of OspA and OspC, together with a bacterial lysate of the bacterial immunomodulatory strain Propionibacterium acnes. Universal formulation according to claim 1 or 2, characterized in that the immunogenic protective outer membrane proteins of the strains Borrelia burgdorferi sensu stricto, Borrelia afzelii and Preferably, Borrelia garinii OspA and OspC are included in the vaccine in a ratio of 8: 1: 1 to 1: 1: 1. Universal preparation according to claims 1 to 3, characterized in that the lyophilized bacterial lysates of the strains Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii are in a ratio of 1: 1: 1 to one another and a ratio of a mixture of lyophilized bacterial lysates of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii to bacterial lysates of the strain Propionibacterium acnes is in a ratio of 9: 1 to 1: 1 and the total dry matter content of lyophilisates of all strains in a single application is 1 to 1000 mg. The universal preparation according to any one of claims 1 to 4, characterized in that it is formed in a lyophilized or liquid form and is applied in a buffered saline solution. Or with a mineral or oil immune adjuvant, optionally other natural or synthetic immunomodulators or other adjuvants, preferably a vehicle, Universal preparation according to one of claims 1 to 5, characterized in that the immunomodulators of natural character are selected from the group of bacterial immunomodulators. 5 or glucan or mineral elements. Universal formulation according to any one of claims 1 to 6, characterized in that its pH is in the range of 4 to 9.