Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants

Definitions

• 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.
• 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" (Barbo ⁇ r 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 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.
• Spirochaetas may be inactivated shortly after they enter the host organism, just before the antigenic variation and the reduced expression of the OspA antigen.
• 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).
• 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).
• 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.
• 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.
• 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.
• 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.
• 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 is a principal surface antigen that is acteded 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).
• the OspC has a limited surface exposure, it is a potent immunogene.
• the OspC immunization is protective against borrelia infection.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 - 38 0 C 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.
• 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.
• 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.
• the ampoule with the culture is taken out of liquid nitrogen and defrozen at about 30 0 C.
• 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.
• 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 - 35 0 C 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.
• 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 0 C 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.
• 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 0 C for 48 hours.
• OspA 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.
• 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 37 0 C at the ratio of 1 + 9 (1 part of culture + 9 parts of medium) and the incubation takes place at 36 - 38 0 C for 6 to 18 days.
• the culture reproduction is analogical, but the difference is that the cultivation takes place at 36 - 38 0 C 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).
• 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.
• ticks Ixodes ricinus
• 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.
• a mineral or oil immunity adjuvant or with immunity complexes (ISCOM), liposomes and other natural or synthetic immunity adjuvants.
• ISCOM immunity complexes
• 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 ⁇ .

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