WO2009062454A2

WO2009062454A2 - Universal vaccine for the treatment and prophylaxis of lyme disease for human and veterinary use and method of its manufacture

Info

Publication number: WO2009062454A2
Application number: PCT/CZ2008/000130
Authority: WO
Inventors: Vladimir Vrzal; Libor Bittner; Jiri Nepereny; Josef Chumela
Original assignee: Bioveta, A.S.
Priority date: 2007-11-14
Filing date: 2008-10-21
Publication date: 2009-05-22
Also published as: CZ301244B6; CZ2007791A3; UA102081C2; EP2219670A2; RU2472525C2; WO2009062454A3; RU2010123897A

Abstract

The universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use, based on whole-cell bacterins or bacterial lysates or purificates of at least one or more borrelia genomospecies, which essence lies in the fact that each borrelia genomospecies, selected preferably from the group of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, contains as a minimum one immunogenic protective protein of outer membrane, either OspA or OspC, or simultaneously both the immunogenic protective proteins OspA and OspC, or possibly other immunogenic protective proteins of outer membrane.

Description

Universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use and method of its manufacture

Technical Field

The invention refers to the composition of universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use and the method of its manufacture.

Contemporary Prior Art

Lyme disease is a chronic multi-system infectious disease. It is the most common arthropod-born disease in Europe and the United States. The importance of this disease is proved with the number of publications in journals dealing with infections. From this viewpoint, lyme disease ranks behind acquired immunodeficiency syndrome over the last decade, as described for example in "The biological and social phenomenon of Lyme disease" (Barboυr AG, Fish D, Science. 1993 Jun 11;260(5114):1 '610-6).

The disease is caused by a group of spirochaetas, entitled collectively Borrelia burgdorferi sensu lato. This group of microorganisms consists mainly of three closely related subspecies, i.e. Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. While Borrelia burgdorferi sensu stricto causes practically all cases of Lyme disease on the North American continent, Borrelia garinii and Borrelia afzeli prevail in Europe.

The serious damage of mammalian host organism, as well as the problems related to the diagnostics and therapy of Lyme disease are significant stimuli for the development of efficient vaccines. On the other hand, there are reservations against the development of vaccines and their use, especially in humans. The main reasons are that the disease is not contagious, may be treated with antibiotics quite easily and the clinical manifestation with serious consequences affects only a part of infected patients. Moreover, this disease is rarely fatal. Therefore, the fear of possible side effects of a new vaccine, which would be hardly acceptable under these circumstances, is not surprising, as described in the article "Experimental immunization against Lyme borreliosis with recombinant Osp proteins: an overview" (Sadziene A, BarboυrAG., Infection. 1996 Mar-Apr;24(2): 195-202).

The first veterinary vaccine against borreliosis was developed for dogs in the United States in the year 1990. It was licensed in the year 1992 according to the 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).

The whole-cell, chemically inactivated vaccine is applied with a vehicle containing polymer adjuvant substances. The vaccine is applied intramuscularly, twice in the interval of two or three weeks. A maintenance dose (booster) is recommended a year later. The vaccination of domestic animals, especially dogs, is recommended irrespective of whether the animal was infected or the disease is just in progress. The preparation of this vaccine was based on the knowledge acquired in experiments performed in rodents. The protective effect of the whole-cell vaccine was described in hamsters for the first time.

The development of the whole-cell vaccine for veterinary purposes was mainly supported with its simple preparation and low price. However, this vaccine was not recommended for human use owing to the fact that some borrelia antigens cross react with human antigens, and so the induction of immunopathological processes cannot be excluded 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 epitope common to a neuroblastoma cell line". (Fikrig et al. 1993, Proc Nat Acad Sci U S A. 1993 Jan 1;90(1):183-7). The finding that some Borrelia burgdorferi antigens activate sets of specific T lymphocytes which participate in the development of arthritis in hamsters was alerting, see "Involvement of CD4+ T lymphocytes in induction of severe destructive Lyme arthritis in inbred LSH hamsters" (Lim LC et al., Infect Immun. 1995 Dec; 63(12):4818-25).

Rabbits are protected against the infection if the immune serum, prepared by immunization of animals with immunogenic proteins of borrelia strain, is applied before experimental challenge. Administration of the immune serum after infection does not impede the development of erythema migrans and visceral manifestations of infection. Similar results were also detected in mice and hamsters and were described in "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 JuI; 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).

These experimental findings outlined the possibility of development of a safe subunit vaccine. Principal candidate antigens of such vaccine were outer surface proteins (Osp) marked as OspA, B, C and D. Especially the protein OspA was studied intensively. The protective effect of this subunit vaccine was confirmed experimentally in mice, hamsters and rabbits and was described in the articles "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 confirmed that the application of this protein in the form of purified, lipidated recombinant protein induces the protective immunity at parenteral administration and protects against borrelia infection administered by injection or through a tick vector. It was also confirmed that infected ticks sucking immunized animals lost their infectiosity, 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 case of absence of a lipid unit, the induced creation of antibodies requires the adjuvant effect of Freund's complete adjuvant or another immunity adjuvant, as described in the paper "Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA" (Erdile IF et al., Infect Immun. 1993 Jan;61(1):81-90). This indicates two possible effects of vaccination:

(1) Spirochaetas are inactivated already in the tick organism, and so the transmission of infection is prevented,

(2) Spirochaetas may be inactivated shortly after they enter the host organism, just before the antigenic variation and the reduced expression of the OspA antigen.

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

1. Native and pure recombinant OspA, described in "Recombinant outer surface protein A from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice" (Simon MM et al.,.J Infect Dis. 1991 Jul;164(1 ):123-32) 2. OspA incorporated in the BCG genome (Calmette, Guerin). The OspA was exprimed as a membrane-associated lipoprotein, as results from the article "Protective immunity elicited by rBCG vaccines" (Stover CK et al., Dev Biol Stand. 1994,82:163-70). In this form, the OspA was tested at intraperitoneal and intranasal administration, as published in the "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). At this form of application, specific IgG and IgA antibodies were induced on a long-term basis, which is clear 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 Ju1 1;178(1):197-209).

At present, the only producer of such vaccine for human use is the United States of America. The vaccine was approved by the Food and Drug Administration (FDA) on 21^st December 1998 and is described in the study "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 JuI 23;339(4):209-15). The product LYMErix is manufactured by SmithKline Beecham. According to the third phase of clinical tests, the vaccine provides 80 - 90% protection, as proved in the paper "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 JuI 23;339(4):216-22. Erratum in: N Engl J Med 1998 Aug 20;339(8):571).

Although the vaccine has already been distributed, it is under permanent control in respect of minimum knowledge of its long-term use. The vaccine is applied by injection three times during a 12-month period (0, 1 , 12) to achieve the maximum immunity response. The vaccine efficacy has been described for example by Wahlberg who declares that the vaccine efficacy was 50% (1 year) after two doses of recombinant OspA protein bound to aluminium hydroxide in phosphate buffer and 79% after three vaccination doses (20 months). The antibody titre decreased quite quickly and two years later it was at the level of the first year of vaccination (50% efficacy). No details of booster effect are available. To date, it has not been permitted to apply the vaccine to children under 15 years of age and to people suffering from autoimmunity diseases, in particular arthritis. In a set of 5765 vaccinated individuals, arthritis developed in two persons with HLA-DR4 phenotype, as published in the article "Guarded endorsement for Lyme disease vaccine" (Marwick C, JAMA. 1998 Jun 24;279(24): 1937-8).

The commercially available vaccine is prepared from the OspA protein from Borrelia burgdorferi sensu stricto. Owing to the fact that this bacterium is a dominant infectious producer of Lyme disease on the North American continent, the vaccine may be relatively efficient to the inhabitants of the USA. In Europe, however, this disease is mainly produced by the following three pathogenic subspecies, i.e. Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Antigenic variability has been described in all these strains on the European continent. Therefore, the vaccine for human and veterinary use prepared only from the strain Borrelia burgdorferi sensu stricto is inapplicable in the European conditions. New trends of vaccination technologies were used to develop an efficient form of vaccine. For example, an efficient DNA vaccine is 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 C from Borrelia burgdorferi induce protective antibodies suitable for prophylaxis but not for resolution of Lyme disease" (Wallich R. et al.,. Infect Immun. 2001 Apr;69(4):2130-6).

Another possibility is to prepare the vaccine from the OspC protein exprimed on the surface of microorganisms in host's body. According to the literature, the humoral immunity against this protein has a protective character, 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).

The OspC is a principal membrane antigen expressed in the early phase of infection. In case of Borrelia burgdorferi sensu stricto the OspC is highly variable. Twenty-one allelic groups marked A-U were detected for this antigen by epidemiologic analysis and sequencing of genes for the OspC in individual isolates and by completing with GeneBank, as described in "Four 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 a principal surface antigen that is exprimed when Borrelia burgdorferi is resident in a tick. At the moment when the tick starts to suck the mammalian blood, the synthesis of this antigen is reprimed and the synthesis of the OspC antigen is induced on the contrary. This way the OspC becomes a principal antigen of outer surface membrane in the early phase 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 was proved that the OspC has a limited surface exposure, it is a potent immunogene. The OspC immunization is protective against borrelia infection. However, the protection is bound to a particular OspC allele which controls the synthesis of a particular protein. Infection with another type of borrelia produces disease in these immunized individuals. Naturally, this limits the use of this antigen acquired only of one borrelia genomospecies for the preparation of a universal vaccine.

Thus, the question of successful vaccination against Lyme disease remains open. Moreover, in Europe it is complicated with the existence of three different genomospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii) and the occurrence of Lyme disease not only in man, but also in various species of domestic and farm animals. The problems concerning this disease in dogs and cats are described in "Canine borreliosis" (Littman MP, Vet CHn Small Anim 33, 2003, 827-862) and the disease in horses is analyzed in "Equine Abortion Associated with the Borrelia parker i- B. turicatae Tick-Borne Relapsing Fever Spirochete Group" (Walker RL et ai, Journal of Clinical Microbiology, 2002, 40, 4, 1558-1562).

Livey et al. tried to overcome these problems by preparing so-called "coctail vaccines". 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).

With the existing problems to diagnose and treat Lyme disease and owing to the fact that it is impossible to control and reduce efficiently the spread of borrelia vectors, there was an urgent need of a vaccine able to immunize efficiently sensitive species of domestic and farm animals and humans against the infection caused by Borrelia burgdorferi sensu lato. Vaccines based on the whole-cell Borrelia burgdorferi bacterin were developed for domestic animals, as described for example in the patents US 4721617, US 6316005. Likewise, vaccines based on the content of OspA, OspC or other immunogenic outer surface proteins, isolated from cultivated borrelia cultures, exprimed as recombinant proteins in various hosts (E. coli), or prepared synthetically, were developed, as described in the 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 do not include protection against the entire width of pathogenic borrelias of all genomospecies, mainly Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii and others, as the case may be. They are always produced of a single genomospecies - Borrelia burgdorferi sensu stricto.

The task of this invention is to introduce a new universal vaccine containing the principal immunogenic proteins OspA and OspC in various combinations of one, two, or preferably all three best-known pathogenic genomospecies, i.e. Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii, or possibly others, that may be applicable successfully without any territorial limitation.

Subject of Invention

The aforementioned goal is achieved by invention of the universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use, based on whole-cell bacterins or bacterial lysates or purificates of at least one or more borrelia genomospecies, which essence lies in the fact that each borrelia genomospecies, selected preferably from the group of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, contains as a minimum one immunogenic protective protein of outer membrane, either OspA or OspC, or simultaneously both the immunogenic protective proteins OspA and OspC, or possibly other immunogenic protective proteins of outer membrane.

The preferential version of the vaccine includes all the three most pathogenic genomospecies - Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garrinii, each of which contains simultaneously both the immunogenic protective proteins of outer membrane OspA and OspC.

Moreover, the subject of invention is that the immunogenic protective proteins of outer membrane OspA and OspC are included in the vaccine preferably at the ratio of 1 : 1 , that the vaccine is produced in the lyophilized or liquid form or is applied in the buffered physiological solution or with a mineral or oil immunity adjuvant, or possibly with other immunomodulators, and that the pH value of the vaccine ranges between 4 and 9.

Finally, the subject of invention is the method of manufacture of the universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use according to claims 1 to 5, during which each production strain of borrelias is cultivated, inactivated and tested independently before blending, where the culture is proliferated and produced preferably at 26 - 35°C for 6 - 18 days (each step) to express the OspA antigen and at 36 - 38⁰C for 6 - 18 days (each step) to express the OspC antigen.

The new effect of the submitted invention consists in the fact that specific OspA and OspC antibodies are produced in vaccinated animals and humans after application of the vaccine, which prevent the transfer of pathogenic borrelias from the tick to the vaccinated organism (OspA antibodies) and support their destruction shortly after a possible transfer of pathogenic borrelias to the vaccinated organism (OspC antibodies) and, possibly, the creation of other postvaccination protective antibodies, not specified in more details, and the stimulation of immunity mechanisms. Another benefit is that besides common adult individuals the vaccine may be applied to young domestic animals (preferably dogs and cats) and to young farm animals (preferably horses) from 3 weeks of age to induce an active protective immunity against Lyme disease. Likewise, the vaccine may be applied simultaneously with other preparations, medicines and vaccines against viral, bacterial, mycotic and other diseases in dogs, cats, horses and other species of animals for which the vaccine is intended.

Examples of Execution

Example no. 1 : This example of execution demonstrates the verification of protectivity of the universal vaccine after immunization of experimental cats, dogs and horses against the challenge by virulent strains of Borrelia burgdorferi sensu lato, however, it in no case limits the patent rights related to this patent.

Preparation of an experimental sample of the vaccine and test results:

A) Cultivation of borrelia production strains to express the OspA antigen:

During the cultivation of borrelia production strains to express the OspA antigen, one of the following items was used as a starting raw material: - BSK-H medium + rabbit serum - BSK-H medium, complete along with the production strains of Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto according to the following scheme of manufacturing procedure:

Preparation of culture medium

I

Production strains

1

Revival Control of growth and purity

Control of growth and purity

Determination of the number

Determination of OspA presence

Inactivation of individual strains Inactivation test

1

Concentration, purification of antigen Determination of serum absence

Control of sterility, pH determination and adjustment

Control of sterility, pH determination

Note: Each borrelia strain (Borrelia afzelii, garinii and burgdorferi sensu stricto) is cultivated, controlled, inactivated and tested separately until blending of all strains (including the strains with the OspC antigen expression). The proper cultivation took place in plastic or glass Roux flasks intended for the cultivation of borrelia cultures. The BSK culture medium is enriched with 6% sterile rabbit serum suitable for borrelia cultivation before the cultivation itself, or the complete BSK-H medium containing 6% rabbit serum may be used. Master and working production strains are maintained frozen in liquid nitrogen at -196°C.

To revive the culture, the ampoule with the culture is taken out of liquid nitrogen and defrozen at about 30⁰C. Immediately after dissolving the culture is inoculated into a test tube with a nutrient medium heated to about 28°C at the ratio of 1 + 9 (1 part of culture + 9 parts of medium). Each strain is cultivated separately. The incubation takes place at 26 - 35°C for 6 to 18 days.

When reproducing the culture, the well-grown, viable culture (which changes the red colour of culture medium into yellow) is checked under microscope and inoculated, under aseptic conditions, into a nutrient medium at the ratio of 1 part of culture + 9 parts of medium. The cultivation takes place at 26 - 35⁰C for 6 - 10 days. Further subpassages of well-grown borrelias are performed at the ratio of 1 part of charge : 10 to 100 parts of culture medium. The number of further subpassages depends on the required volume of borrelias for the vaccine preparation.

When producing the culture for the vaccine manufacture, the grown cultures of previous cultivations are used under aseptic conditions as an inoculum applied into a broth in 1 000-ml flasks at the ratio of 1 part of charge : 10 to 100 parts of culture medium. The cultivation takes place at 26 - 35 ⁰C for 6 - 10 days.

The number of borrelias is determined in the dark field of microscope by means of the PETROFF HAUSSER COUNTING CHAMBER.

In-process controls of culture growth and purity are made by macroscopic, microscopic and cultivation examination. The macroscopic examination consists in the visual observation whether a suitable culture changes the red colour of culture medium into yellow and if sediment is present in the medium. The microscopic examination in the dark field of microscope monitors whether borrelias are adequately movable with a small quantity of detrite, without any signs of bacterial contamination. In case of cultivation examination, 0.5 ml of the evaluated culture of borrelias are transferred on a pre-dried blood agar and the cultivation takes place at 35 - 37 ⁰C for 48 hours.

When evaluating the test, the presence of any undesirable bacterial growth is observed and the cultures with bacterial contamination are rejected.

The presence of OspA is determined by means of protein electrophoresis on the SDS PAGE after staining with Coomasie Blue or by western blot immunodetection method using the anti-OspA serum.

B) Cultivation of borrelia production strains to express the OspC antigen:

Identical raw materials, production strains and scheme of manufacturing procedure as used to express the OspA antigen described under A) are used to prepare the cultivation.

Likewise, the proper cultivation runs in the same process with the only difference that when reviving the culture, the ampoule with the dissolved culture is inoculated into a test tube with a broth heated to about 37⁰C at the ratio of 1 + 9 (1 part of culture + 9 parts of medium) and the incubation takes place at 36 - 38 ⁰C for 6 to 18 days. Similarly, the culture reproduction is analogical, but the difference is that the cultivation takes place at 36 - 38 ⁰C for 6 - 10 days. The culture production for the vaccine manufacture, the determination of the number of borrelias, the in- process controls, the control of culture growth and purity and the determination of OspC presence are performed identically with the description under A).

C) Proper preparation of the final appearance of the vaccine:

The concentrated, purified, stabilized and inactivated bacterial strains of Borrelia burgdorferi senzu stricto, Borrelia afzelii and Borrelia garinii with the proved expression of the outer surface protein OspA and the concentrated, purified, stabilized and inactivated bacterial strains of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii with the proved expression of the outer surface protein OspC are blended together so that the OspA and OspC of individual Borrelia genomospecies have an identical ratio. Then the Borrelia component (90 % of the volume) is mixed with aluminium hydroxide gel (10 % of the volume). After a perfect homogenization, the pH is controlled and adjusted to a value between 7.5 and 8.0. The vaccine is filled into storage bottles and a sterility test is performed. Then the vaccine is filled into final dispatch containers, packed and transferred to the final control.

D) Test results a) Efficacy test in cats:

Six cats were vaccinated and revaccinated with an experimental sample of the vaccine and 90 days later they were challenged with ticks (Ixodes ricinus) naturally infected with genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Three non-vaccinated cats served as controls.

The cats were vaccinated subcutaneously with 1 ml of the vaccine and 3 weeks later they were revaccinated identically. Blood was taken before vaccination and revaccination. Further blood taking was performed 1 month after revaccination. Three months after revaccination the cats were challenged with ticks (Ixodes ricinus) collected naturally, who were infected naturally with all the three most pathogenic genomospecies - Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. The level of ticks' infection was verified before starting the challenge experiment.

After infection, all experimental animals (vaccinated and non-vaccinated control cats) were evaluated clinically every day and their state of health was recorded, including measurement of their body temperature. After infection, the blood of all experimental animals was taken probatorily on day 14, 28, 42 and 60. The blood was also taken on the day of challenge, immediately before the infection with ticks. On day 7, 28 and 60 after challenge, skin biopsies were performed on the sites where the ticks were sucked in order to reisolate borrelias from these samples. On day 60 after challenge all the experimental animals were sacrificed and a pathologo- anatomic dissection was carried out and samples were taken from the muscle, articular synovia, lymp-nodes, skin and kidneys in order to reisolate borrelias from these predilection sites.

The acquired results are presented in synoptical tables no. 1 , 2, 3 and 4. It follows unambiguously from the achieved results that the vaccine against borreliosis prepared from the production strains of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, expriming the surface proteins OspA and OspC, is harmless to cats and produces in them the protective immunity against the natural tick-born infection with all the three most pathogenic genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, and possibly other known genomospecies of borrelias due to the principle of cross immunity.

Table no. 1 - Scheme of challenge trial - cats

Explanations:

V - vaccination B - skin biopsy

KO - non-vaccinated control K - blood taking

+ - action performed P - pathologo-anatomic dissection

- - action not performed

Table no. 2 - Clinical evaluation after challenge

Explanations:

+ - clinical symptoms

- - without clinical symptoms

Table no. 3 - Finding of borrelias in skin bioptates after challenge

Explanations:

+ - positive capture

- - negative capture

Table no. 4 - Finding of borrelias in organs

Explanations:

+ - positive capture

- - negative capture

PA - pathologo-anatomic dissection

b) Efficacy test in dogs:

Six dogs were vaccinated and revaccinated with an experimental sample of the vaccine and 90 days later they were challenged with ticks (Ixodes ricinus) naturally infected with genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Three non-vaccinated dogs served as controls. The vaccination, revaccination, blood taking, clinical evaluation, probatory sampling and other acts were performed identically to the tests in cats.

The acquired results are presented in synoptical tables no. 5, 6, 7 and 8. It follows unambiguously from the achieved results that the vaccine against borreliosis prepared from the production strains of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, expriming the surface proteins OspA and OspC, is harmless to dogs and produces in them the protective immunity against the natural tick-born infection with all the three most pathogenic genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, and possibly other known genomospecies of borrelias due to the principle of cross immunity.

Table no. 5 - Scheme of challenge trial - dogs

Explanations:

V - vaccination B - skin biopsy

KO - non-vaccinated control K - blood taking

+ - action performed P - pathologo-anatomic dissection

- - action not performed

Table no. 6 - Clinical evaluation after challenge

Explanations:

+ - clinical symptoms

- - without clinical symptoms

Table no. 7 - Finding of borrelias in skin bioptates after challenge

Explanations:

+ - positive capture

- - negative capture

Table no. 8 - Finding of borrelias in organs

Explanations:

+ - positive capture

- - negative capture

PA - pathologo-anatomic dissection

c) Efficacy test in horses:

Six horses were vaccinated and revaccinated with an experimental sample of the vaccine and 90 days later they were challenged with ticks (Ixodes ricinus) naturally infected with genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. Three non-vaccinated horses served as controls.

The vaccination, revaccination, blood taking, clinical evaluation, probatory sampling and other acts were performed identically to the tests in cats and dogs. The acquired results are presented in synoptical tables no. 9, 10, 11 and 12. It follows unambiguously from the achieved results that the vaccine against borreliosis prepared from the production strains of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, expriming the surface proteins OspA and OspC, is harmless to horses and produces in them the protective immunity against the natural tick-born infection with all the three most pathogenic genomospecies of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, and possibly other known genomospecies of borrelias due to the principle of cross immunity.

Table no. 9 - Scheme of challenge trial - horses

Explanations:

V - vaccination

KO - non-vaccinated control B - skin biopsy

+ - action performed K - blood taking

- - action not performed P- pathologo-anatomic dissection

Table no. 10 - Clinical evaluation after challenge

Explanations:

+ - clinical symptoms

- - without clinical symptoms

Table no. 11 - Finding of borrelias in skin bioptates after challenge

Explanations:

+ - positive capture

- - negative capture

Table no. 12 - Finding of borrelias in organs

Explanations: + - positive capture - - negative capture PA - pathologo-anatomic dissection

The described examples of the vaccine preparation are not the only possible executions according to the invention, but the whole-cell bacterins or bacterial lysates or purificates containing immunogenic protective proteins may be applied, without any effect on the subject of invention, in the buffered physiological solution or with a mineral or oil immunity adjuvant, or with immunity complexes (ISCOM), liposomes and other natural or synthetic immunity adjuvants.

Industrial utility

The universal vaccine for the treatment and prophylaxis of Lyme disease may be used in the veterinary and human medicine and may be applied successfully intramuscularly, subcutaneously, intradermal^ or transcutaneous^.

Claims

PAT E N T C LA I M S

1. The universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use, based on whole-cell bacterins or bacterial lysates or purificates of at least one or more borrelia genomospecies, characterized in that each borrelia genomospecies, selected preferably from the group of Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, contains as a minimum one immunogenic protective protein of outer membrane, either OspA or OspC, or simultaneously both the immunogenic protective proteins OspA and OspC, or possibly other immunogenic protective proteins of outer membrane.

2. The universal vaccine as per Claim 1, characterized in that the vaccine comprises all the three most pathogenic genomospecies Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garrinii, each of which contains simultaneously both the immunogenic protective proteins of outer membrane, OspA and OspC.

3. The universal vaccine as per Claim 1 or 2, characterized in that the immunogenic protective proteins of outer membrane, OspA and OspC, are included in the vaccine preferably at the ratio of 1 : 1.

4. The universal vaccine as per Claims 1 to 3, characterized in that it is produced in the lyophilized or liquid form or is applied in the buffered physiological solution or with a mineral or oil immunity adjuvant, or possibly, with other immunomodulators.

5. The universal vaccine as per Claims 1 to 4, characterized in that its pH value ranges between 4 - 9.

6. The method of manufacture of the universal vaccine for the treatment and prophylaxis of Lyme disease for human and veterinary use as per Claims 1 to 5, characterized in that each production strain of borrelias is cultivated, inactivated and tested independently before blending, where the culture is proliferated and produced preferably at 26 - 35°C for 6 - 18 days (each step) to express the OspA antigen and at 36 - 38°C for 6 - 18 days (each step) to express the OspC antigen.