-i(BORRELIA BURGDORFERI) BACTERIN

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DESCRIPTION

Borrelia burgdorferi bacteria.

Lyme disease was first described by Steere et al. in 1977, who reported an arthritis epidemic in Lyme, Old Lyme, and East Haddam, Connecticut. In 1982, Dr. Willy Burgdorfer discovered a new spirochete in the midgut of the Ixodes dammini ticks (see Burgdorfer, WA et al., (1982) Science 216: 1317-1319). This spirochete was later shown to generate an immune response in rabbits infected equivalent to that in humans with Lyme disease, and it is now known to be the etiological agent of the disease. Later the spirochete was named Borrelia burgdorferi.

Although Ixodes ticks are the most frequent vectors of Borrelia burgdorferi, spirochetes have also been found in deer flies, horse flies, and mosquitoes. Spirochetes enter the host animal when it is bitten by the vector. Spirochetes of B. burgdorferi reside in various tissues of the host animal and can lead to prolonged infection.

Borrelia burgdorferi has recently been shown to possess a unique type of extrachromosomal DNA, linear plasmids, with lengths from 0.5 to 50 kb (Bergstrom, S. et al., (1991) Scand. J. Infect. Dis. - Suppl. 77: 102107). These plasmids contain the genes encoding the two major outer surface proteins (Osp) expressed by B. burgdorferi, OspA and Osp B (Bergstrom et al., 1991). These proteins are believed to be the major antigens involved in immunity against B. burgdorferi infection. Schwan and Simpson (Schwan, TG and Simpson, WJ, (1991) Scand. J. Infect. Dis. - Suppl. 77: 94-101) describe the heterogeneity of Osp A and B proteins in different isolates of B. burgdorferi , as well as in the same isolate cultured at different temperatures in vivo, and studies at different stages during the infection of mice.

The early stage of Lyme disease is characterized by a spreading skin lesion, chronic erythema migrans (Asbrink, E. and Hovmark, A., (1988) Ann. NY Acad. Sci. 539: 4-15; Halperin , JJ, (1991) Scand. J. Infect. Dis. - Suppl. 77: 74-80). Affected individuals frequently contract arthritis. However, only a small percentage of these individuals are chronic arthritis (Steere, AC, (1991) Scand. J. Infect. Dis. - Suppl. 77: 51-54). Borrelia burgdorferi can also infect the myocardium. Cardiac involvement in Lyme disease has been described to be predominantly transient. However, animal studies have shown that the skeletal and cardiac muscles are commonly affected, and that the spirochetes appear to be located within the muscle fibers. This observation indicates that spirochetes are capable of long-term survival in the host, thereby causing chronic cardiac complications (Stanek, G. et al. (1991) Scand J. Infect. Dis. Suppl. 77: 85-87 ).

B. burgdorferi also infects the nervous system in a high percentage of cases, leading to a wide range of acute, chronic and progressive disorders of the central and peripheral nervous system (Reik, L. et al. (1991) Ann. NY Acad Sci. 539: 1-3). Published reports indicate that European populations affected by the disease present very striking clinical manifestations of neurological disorders, while North American patients present milder forms of nervous system involvement (Halperin, 1991; Halperin, JJ et al., (1988) Ann. NY Acad. Sci. 539: 24-34). However, Halperin (1991) has reported that it seems increasingly clear that, at least in terms of nervous system involvement, both populations present the same range of neurological manifestations.

The diagnosis of Lyme disease depends on a combination of recognition of the presented clinical features and also the likelihood of exposure in endemically infected areas. However, the development of arthritis is often attributed to other causes, and is not linked to a spirochete infection. Neurological symptoms that may result from infection with B. burgdorferi can mimic a number of different neurological disorders (Reik et al., 1988). Added to these diagnostic complications is the difficulty of detecting spirochetes in affected tissues.

Borrelia infection is treated with antibiotics, eg, tetracycline, penicillin, amoxicillin, doxycillin, erythromycin, and phenoxymethylpenicillin (Neu, H., (1988) Ann. NY Acad. Sci., 539: 314-316; Skoldenberg, B. et al. (1988) Ann. NY Acad. Sci. 539: 317-323; Weber, K. et al. (1988) Ann. NY Acad. Sci. 539: 325-345; Luft., BJ et al. ( 1988) Ann. NY Acad. Sci. 539: 352-361). Ceftriaxone and compounds of the same chemical type have also been useful as chemotherapeutic agents (Neu, 1988). However, the establishment of protocols for effective chemical treatment of Lyme disease has been hampered by the lack of data to determine an appropriate time period for treatment. Furthermore, studies on the efficacy of drugs in human patients are lacking. This therapeutic scenario is further complicated by the need to achieve and maintain sufficiently high concentrations of antibiotics in the tissues to combat chronic Borrelia infection (Neu, 1988; Skoldenberg et al., 1988).

Given the difficulties in the detection and treatment of Lyme disease, as well as the impracticality of controlling the spread of spirochete vectors, the need for a vaccine to immunize susceptible animals and humans against Borrelia infection has been recognized. burgdorferi. Vaccines have been studied in hamster models (Johnson, RC et al. (1988) Ann. NY Acad. Sci, 539: 258-263) and in rats (Barthold, SW et al. (1988) Ann. NY Acad. Sci . 539: 264-273). A whole cell bacterin from Borrelia has been developed

ES 2 265 643 T3 burgdorferi for use in domestic animals (US Patent No. 4,721,617). Vaccines based on the Osp A and / or B proteins of B. burgdorferi have also been developed. However, these whole cell and subunit vaccines contain, or are derived from, a single isolate of B. burgdorferi.

There are also reports in the literature that have identified the existence of several distinct seroprotective groups in North American and European isolates of B. burgdorferi itself and Borrelia garinii and Borrelia afzelli through in vitro studies. Such reports include Lovrich, SD et al, (1993) Infection and Immunity 61 (10): 4367-4374, and Lovrich, SD et al, (1994), The Journal of Infectious Diseases 170: 115-121. These results have been interpreted by some to indicate that combinations of different isolated strains, their antigens, or their protective immunogenic proteins will be necessary to provide a general vaccine. In relation to the results described in these reports, in addition to other reports, Figrig E. et al. ((1995) J. Exp. Medicine 181: 215-221) noted that evaluations of the efficacy of Lyme disease vaccines should use the natural mode of transmission and not rely on classification systems or trials that do not depend on the vector for transmission of infection, and have, against said reports, successful immunizations with OspA or OspB against heterogeneous B. burgdorferi infection. Other literature documents that corroborate Fikrig's ideas are, for example, EP-A-0 465 204 which describes a vaccine that contains a quantity of the main B. burgdorferi antigens from a single isolated strain that is capable of to protect a susceptible mammal from infection by various strains. See also, Telford et al. J. Exp. Medicine, 1993,178 (2): 755-8, which describe protection against B. burgdorferi of variable antigenic characteristics conferred using an active immunization protocol with recombinant proteins formed by the fusion of glutathione transferase and proteins A or B of the outer membrane surface (OspA or OspB) and natural infection methodology.

In contrast, the bacterin of the present invention contains, or is derived from, at least two isolated strains, without cross-protection, of B. burgdorferi. Accordingly, the bacterin of the present invention will provide immune protection against two different types of isolates of B. burgdorferi, whereas the previously described bacterins provide protection against only one type of isolate. Therefore, the bacterin provided by the present invention will be more useful for vaccinating animals against Lyme disease.

The present invention provides a bacterin comprising in each dose an amount, effective for immunization, of at least two isolated strains, without cross-protection, of inactivated Borrelia burgdorferi, an adjuvant in an amount effective to enhance the immunogenicity of Borrelia. inactivated burgdorferi and a suitable excipient. Non-cross-protective isolates of Borrelia burgdorferi can be selected from the Wisconsin, Chicago, and European seroprotective groups of Borrelia burgdorferi isolates. At present, strains isolated from the Wisconsin and Chicago groups are preferred. However, isolated strains belonging to other seroprotective groups can also be used. The bacterin may further comprise a third isolated, non-cross-protected strain of inactivated Borrelia burgdorferi.

The present invention also provides a bacterin comprising in each dose an effective amount of at least one antigenic subunit from at least one of two isolates, without cross-protection, of Borrelia burgdorferi, an adjuvant in an amount effective to enhance the immunogenicity of the antigenic subunit or subunits or the isolated strain, and a suitable excipient.

The present invention also provides a bacterin comprising in each dose an immunization-effective amount of an antigenic subunit from a first isolated strain of Borrelia burgdorferi, an immunization-effective amount of an antigenic subunit from a second isolated strain, without cross protection, of Borrelia burgdorferi, an adjuvant in an amount effective to enhance the immunogenicity of the antigenic subunits and a suitable excipient. The bacterin may further comprise an immunization-effective amount of an antigenic subunit from a third isolated, non-cross-protected strain of Borrelia burgdorferi.

The present invention also provides a bacterin comprising in each dose an immunization-effective amount of at least two non-cross-protected isolates of inactivated Borrelia burgdorferi and one or more antigenic subunits of the non-cross-protected isolates, an adjuvant in an amount effective to enhance the immunogenicity of the inactivated Borrelia burgdorferi and antigenic subunits and a suitable excipient.

The present invention further provides the use of a bacterin comprising an amount, effective for immunization, of at least two isolated strains, without cross-protection, of inactivated Borrelia burgdorferi, an adjuvant in an amount effective to enhance immunogenicity. of the inactivated Borrelia burgdorferi and a suitable excipient for the preparation of a pharmaceutical composition for the immunization of animals against infection by Borrelia burgdorferi.

The present invention also provides the use of a bacterin comprising an effective amount of at least one antigenic subunit from at least two isolated, non-cross-protected strains of inactivated Borrelia burgdorferi, an adjuvant in an amount effective to enhance the ability immunogenic of antigenic subunits, and a suitable excipient for the preparation of a pharmaceutical composition for the immunization of animals against infection by Borrelia burgdorferi.

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The present invention also provides the use of a bacterin comprising an immunization-effective amount of at least two non-cross-protected isolates of inactivated Borrelia burgdorferi and one or more antigenic subunits of the non-cross-protected isolates, an adjuvant in an amount effective to enhance the immunogenicity of the inactivated Borrelia burgdorferi and the antigenic subunits, and a suitable excipient for the preparation of a pharmaceutical composition for the immunization of animals against infection by Borrelia burgdorferi.

The animal can be a mammal, including, but not limited to, humans and dogs. The present invention contemplates a pharmaceutical composition of such a nature that an additional dose of the vaccine can be applied to the animal in a suitable time interval after the administration of the previous dose.

Figure 1 shows a growth curve of a viable B. burgdorferi culture. The vertical axis indicates the number of cells (x 10⁷) determined by counting in a Petroff-Hauser chamber.

Figure 2 shows dextrose consumption and lactate production in B. burgdorferi cultures. The black circles denote the lactate production, and the empty circles the dextrose consumption in the culture. Values ​​are expressed in grams per liter of culture medium.

Figure 3 shows the values ​​obtained with the plethysmograph showing the displacement of mercury by the legs of control and vaccinated hamsters. The hamsters were inoculated with normal hamster serum (NHS) or with serum from hamsters that had been inoculated with the indicated isolate (297 or 35211) of B. burgdorferi. The vertical axis shows the displacement of mercury in mm Hg. The horizontal axis indicates the number of days after infection in which the mercury shift was measured. Empty circles = initial values ​​corresponding to hamsters not inoculated and not subjected to exposure; black circles = hamsters inoculated with antisera from hamsters inoculated with a live 297 isolate of B. burgdorferi and then exposed to strain 297; black squares = hamsters inoculated with anti-297 antisera and exposed to isolate 35211; empty squares = hamsters inoculated with normal hamster serum and challenged with isolate 35211; and black triangles = hamsters inoculated with normal hamster serum and challenged with strain 297.

Figure 4 shows a test of immunogenicity: Borreliacidal antibodies against strain S-1-10 measured with the borreliacidal activity assay after vaccination with a bivalent B. burgdorferi bacterin and subsequent exposure to B. burgdorferi infected ticks. .

Figure 5 shows a test of immunogenicity: Borreliacidal antibodies against strain C-1-11 measured with the borreliacidal activity assay after vaccination with a bivalent B. burgdorferi bacterin and subsequent exposure to B. burgdorferi infected ticks. .

Figure 6 shows a test of immunogenicity: Antibodies detected against the S-1-10 antigen measured by ELISA after vaccination with a bivalent B. burgdorferi bacterin and subsequent exposure to B. burgdorferi infected ticks.

Figure 7 shows a test of immunogenicity: Antibodies detected against the C-1-11 antigen measured by ELISA after vaccination with a bivalent B. burgdorferi bacterin and subsequent exposure to B. burgdorferi infected ticks.

Figures 8A and 8B show immunoblotting of vaccinated serum extracted at the first vaccination (PV), seven months after the second vaccination prior to tick exposure (PC), and eight weeks after tick exposure (PsC) against the isolated strains S-1-10 (Figure 8A) and C-1-11 (Figure 8B).

Figures 9A and 9B show immunoblotting of vaccinated serum collected at the first vaccination (PV), seven months after the second vaccination prior to tick exposure (PC), and eight weeks after tick challenge (PsC) against the isolated strains S-1-10 (Figure 9A) and C-1-11 (Figure 9B).

Figures 10A and 10B show immunoblotting of vaccinated serum collected at the first vaccination (PV), seven months after the second vaccination prior to tick challenge (PC), and eight weeks after tick challenge (PsC) against the isolated strains S-1-10 (Figure 10A) and C-1-11 (Figure 10B).

Figures 11A and 11B show immunoblots of vaccinated serum collected at the first vaccination (PV), seven months after the second vaccination prior to tick exposure (PC), and eight weeks after tick challenge (PsC) against the isolated strains S-1-10 (Figure 11A) and C-1-11 (Figure 11B).

Figures 12A and 12B show immunoblotting of unvaccinated serum collected seven months after the second vaccination prior to tick challenge (PC) against isolated strains S-1-10 (Figure 12A) and C-1-11 (Figure 12B).

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Figures 13A and 13B show immunoblots of unvaccinated serum collected at eight weeks post-tick challenge (PsC) against isolates S-1-10 (Figure 13A) and C-1-11 (Figure 13B).

Figure 14 shows a summary of the percentage of dogs lame after exposure to ticks, measured over seven months.

Figures 15A and 15B show immunoblots (strain C-1-11) of serum from dogs vaccinated with bacterin formulated at full dose (5 x 10⁸ cells of each isolated strain per dose). Serum was collected before vaccination (PV), before tick challenge (PC), and twelve weeks after challenge.

Figures 16A and 16B show immunoblots (strain S-1-10) of serum from dogs vaccinated with bacterin formulated at full dose (5 x 10⁸ cells of each isolated strain per dose). Serum was collected before vaccination (PV), before tick challenge (PC), and twelve weeks after challenge.

Figures 17A and 17B show immunoblots (strain C-1-11) of serum from dogs vaccinated with bacterin formulated at full dose (5 x 10⁷ cells of each isolated strain per dose). Serum was collected before vaccination (PV), before tick challenge (PC), and twelve weeks after challenge.

Figures 18A and 18B show immunoblots (strain C-1-11) of serum drawn from unvaccinated controls at the time of tick challenge (Figure 18A) and twelve weeks after tick challenge (Figure 18B).

Figures 19A and 19B show immunoblots (strain S-1-10) of serum drawn from unvaccinated controls at the time of tick challenge (Figure 19A) and twelve weeks after tick challenge (Figure 19B).

Figure 20 shows a summary of the percentage of dogs lame after exposure to ticks, measured over twelve months.

The present invention provides a bacterin comprising in each dose an amount, effective for immunization, of at least two isolated strains, without cross-protection, of inactivated Borrelia burgdorferi, an adjuvant in an amount effective to enhance the immunogenicity of Borrelia. inactivated burgdorferi and a suitable excipient.

Typically, the effective amount for immunization of the isolated non-cross-reactive strains of inactivated Borrelia burgdorferi is an amount from about 1 x 10⁴ microorganisms up to about 1 x 10¹⁰ microorganisms of each isolated strain. It is desirable that the amount, effective for immunization, of the inactivated Borrelia burgdorferi isolates be an amount from about 1 x 10⁴ microorganisms up to about 1 x 10⁹ microorganisms of each isolated strain. It is more desirable that the amount, effective for immunization, of the isolated strains of inactivated Borrelia burgdorferi be an amount from about 1 x 10⁴microorganisms up to about 1 x 10⁸ microorganisms of each isolated strain. In a preferred embodiment the amount, effective for immunization, of the inactivated Borrelia burgdorferi isolates is an amount of about 10⁷ microorganisms of each isolated strain. In a particularly preferred embodiment the amount, effective for immunization, of the isolated strains of inactivated Borrelia burgdorferi is approximately 5 x 10⁷ microorganisms of each isolated strain. In another particularly preferred embodiment the amount, effective for immunization, of the isolated strains of inactivated Borrelia burgdorferi is approximately 5 x 10⁸ microorganisms of each isolated strain.

For the purposes of the present invention, an "inactivated" Borrelia burgdorferi isolate is an isolate that is capable of eliciting an immune response in an animal, but is incapable of infecting the animal. Isolated Borrelia burgdorferi strains can be inactivated using an agent selected from the group consisting of binary ethyleneimine, formaldehyde, ^ -propriolactone, or heat. In the presently preferred embodiment of the present invention, the isolated Borrelia burgdorferi strains are inactivated using binary ethyleneimine.

Said at least two isolated strains, without cross protection, of inactivated Borrelia burgdorferi can be selected from the seroprotective groups A, B or C of isolates of Borrelia burgdorferi. As used in the present invention, the term "seroprotective group A of isolates of B. burgdorferi" defines a seroprotective group of isolated strains of B. burgdorferi including strain S-1-10 Wisconsin, and isolates 297 and B31; the expression "seroprotective group B of isolates of B. burgdorferi" defines a seroprotective group of isolates of B. burgdorferi that includes strain C-1-11 Chicago; and the expression "seroprotective group C of isolates of B. burgdorferi" defines a seroprotective group of isolates of B. burgdorferi that includes the European isolates. Example 10 describes in vitro cross-protection studies of nine isolates of B. burgdorferi indicating that six belong to seroprotective group A, one belongs to seroprotective group B, and two belong to seroprotective group C. However, use is contemplated. of isolated strains without cross protection from other seroprotective groups in the bacterin of the present invention. The results set forth in Example 12 (see below) demonstrate that there are at least two Borrelia burgdorferi seroprotective groups in addition to seroprotective groups A, B and C.

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Cross protection can be determined by inoculating an animal, eg, a hamster, with a live isolate of B. burgdorferi. The antiserum from said animal is then administered to cultures of isolates of B. burgdorferi. Antibodies, along with complement, should cause cell death in cultures of the same isolate used to inoculate the animal. If the antibodies recognize an antigenic determinant in a second isolate, they will cause cell death in a culture of that isolate. Therefore, the first and second isolates of B. burgdorferi are said to be cross-protective. Cross protection can also be assessed by inoculating an animal with an isolated strain of B. burgdorferi, isolating the antiserum from the animal and inoculating another animal of the same species with said antiserum. This animal is subsequently exposed to the same or to a second isolate of B. burgdorferi. The antibodies should confer passive immunity to the recipient animal against challenge to the same isolate. If the antibodies also confer passive immunity against a second isolate, the isolates are cross-protected.

Isolated strains that are cross-protective express outer surface proteins (Osp) that share antigenic determinants recognized by the same antibodies. Isolated strains showing cross protection are said to belong to the same seroprotective group. Accordingly, the term "seroprotective group" is a group of isolates of B. burgdorferi that share common outer surface protective proteins. Antibodies that recognize these proteins confer passive immunity against challenge to any isolate belonging to the same seroprotective group.

Antibodies directed against one member of a pair of isolates without cross protection will not confer passive immunity against the other member of the pair. Consequently, isolated strains without cross protection belong to different seroprotective groups. Vaccination with an inactivated B. burgdorferi isolate will not confer passive immunity to an animal against challenge to isolates without cross protection. The bacterin of the present invention, which comprises at least two isolates without cross-protection, will be effective in producing borreliacidal antibodies against members of two seroprotective groups. Consequently, the bacterin provides a broader spectrum of immune protection than is achieved with currently available vaccines, which do not use multiple isolated, non-cross-protected strains of B. burgdorferi.

In one embodiment of the present invention, non-cross-protected isolates of Borrelia burgdorferi are selected from seroprotective groups A and B of isolates of Borrelia burgdorferi. The isolate strain of Borrelia burgdorferi from seroprotective group A can be strain S-1-10 Wisconsin, or strain 297 or B31. In the presently preferred embodiment of the present invention, the isolated Borrelia burgdorferi strain from seroprotective group A is the S-1-10 Wisconsin strain. Preferably, the Borrelia burgdorferi isolate from seroprotective group B is the C-1-11 Chicago strain. However, the present invention can also be practiced with other isolates of inactivated B. burgdorferi, which will belong to the class of the seroprotective group B.

For the purposes of the present invention, an adjuvant is a composition of a material capable of enhancing the immunogenicity of antigens when the adjuvant is administered as part of a bacterin. Helpers useful in the bacterin of the present invention can be selected from the group consisting of aluminum hydroxide, Carbopol, lipopolysaccharide (LPS), and derivatives thereof. The term "lipopolysaccharide", as used herein, refers to a group of polysaccharides known to those of skill in the art due to their presence in the cell wall of bacteria, and which are useful with helpers. Certain examples of lipopolysaccharides and derivatives thereof, which demonstrate an adjuvant effect, have been used in vaccines. The use of lipopolysaccharides or derivatives thereof in connection with the bacterin of the present invention is contemplated. In the presently preferred embodiment of the present invention the adjuvant is aluminum hydroxide. However, any other adjuvant capable of enhancing the immunogenicity of the inactivated Borrelia burgdorferi can be used in the bacterin of the present invention.

As used herein, the term an "effective amount" of an adjuvant is any amount of the adjuvant effective to enhance the immunogenicity of antigens on the bacterin. Typically, the effective amount of the adjuvant is an amount from about 1.0% by volume of a dose of the bacterin to about 15% by volume. It is desirable that the effective amount of the aluminum hydroxide builder is an amount from about 5% by volume to about 10% by volume. Most preferably, the effective amount of aluminum hydroxide builder is 7.5% by volume.

The term "Excipients", as the term is used herein, refers to standard diluents for a bacterin, which are well known to those of skill in the art. In the presently preferred embodiment of the present invention the excipient comprises an aqueous buffer, eg, physiological saline, and preservatives, eg, nystatin and gentamicin.

The bacterin of the present invention may further comprise an immunization-effective amount of a third, isolated, non-cross-protected strain of inactivated Borrelia burgdorferi.

The present invention provides the use of a bacterin of the present invention for the preparation of a pharmaceutical composition for immunizing an animal against Borrelia burgdorferi infection whereby a dose of the bacterin of the present invention is administered to the animal. It is well known to those skilled in the art that Borrelia burgdorferi is the causative agent of Lyme disease and that the infection occurs as a result of the bite of bacteria-carrying ticks in homeothermic host animals.

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Animals have been shown to synthesize antibodies in response to inoculation with Borrelia burgdorferi, and therefore vaccination with inactivated Borrelia burgdorferi, or a subunit thereof, is a feasible strategy for the prevention of Lyme disease. Given the difficulty of correct diagnosis of Lyme disease, the uncertainty of treatment protocols, and the impossibility of controlling disease vectors, a preventive strategy is necessary to control the disease. Previously used B. burgdorferi vaccines contained only one isolated strain of the bacterium. Therefore, said vaccines are, at best, capable of immunizing only against exposure to isolated strains belonging to the same seroprotective group as the isolated strain used for vaccination. The bacterin supplied by the present invention can provide immunity against isolated strains belonging to at least two seroprotective groups, thus offering more extensive protection than currently available vaccines.

Typically, the animal to which the vaccine of the present invention is administered is a mammal. In a preferred embodiment, the mammal is human. In another preferred embodiment of the present invention, the mammal is a dog. In the practice of the present invention, the person skilled in the art will be able to determine, for each animal to be inoculated, the appropriate age at which the animal is immuncompetent, that is, it has an immune system that functions properly. . Accordingly, it would be appropriate to administer the immunization at the time the animal is immuncompetent and, preferably, prior to challenge with Borrelia burgdorferi. For example, when the inoculated animal is a dog, the dog is at least about twelve weeks of age, preferably from about six weeks to about sixteen weeks of age, at the time the bacterin is first administered.

The present invention also contemplates that the bacterin of the present invention is to be administered to the animal in an additional vaccine dose at a suitable time interval after the administration of the previous dose. Thus, the present invention contemplates that multiple doses of the inactivated Borrelia burgdorferi bacterin are to be administered to an animal. Administration of more than one dose of a bacterin is intended to enhance the immune response of the vaccinated animal against bacterial antigens, compared to the situation where a single dose is administered. The administration of each dose should be carried out separately by a suitable time interval to allow the immune system of the animal to respond more effectively to multiple doses of the bacterin than in the case in which a single dose is administered. A "suitable" time interval between the administration of multiple doses of a bacterin to an animal is, therefore, any time interval between the administration of doses of a vaccine sufficient to elicit the generation of an immune response in the animal greater than that obtained when a single dose is administered. Typically, the suitable time interval between administration of doses of the bacterin of the present invention is from about two weeks to about five weeks, preferably about three weeks. The present invention further contemplates administering an additional dose of the bacterin to the animal approximately one year after the first administration, and at annual intervals thereafter. Such administration, known as a "booster" dose, is standard practice in the vaccination technique. The inactivated Borrelia burgdorferi bacterin can be administered via any art-accepted route for vaccine administration. The currently preferred route of administration of the bacterin is intramuscular injection.

The present invention also provides a bacterin comprising in each dose an immunization-effective amount of one or more antigenic subunits from a first isolated strain of Borrelia burgdorferi, an immunization-effective amount of one or more antigenic subunits. from a second isolated strain, without cross protection, of Borrelia burgdorferi, an adjuvant in an amount effective to enhance the immunogenicity of the antigenic subunits, and a suitable excipient. It is well known in the art that Borrelia burgdorferi antigenic determinants are called "outer surface proteins" (Osp) and are encoded by plasmids contained in bacteria. Currently known B. burgdorferi Osp proteins are Osp A, Osp B, Osp C, Osp D and Osp E proteins. Of these, the Osp A and Osp B proteins are the most studied and characterized. The bacterin of the present invention contemplates the use of an Osp A protein or a combination of the Osp A protein and the Osp B protein of at least two isolated strains, without cross protection, of B. burgdorferi. The bacterin also contemplates the use of other Osp proteins, either alone or in combination with Osp A and Osp B proteins, which are known to generate a borreliacidal antibody response similar to that found in the case of Osp A protein and the combination of Osp A / Osp B proteins. For a review of the preparation of vaccines against Lyme disease using antigenic subunits and polypeptides derived from the outer surface proteins of B. burgdorferi, see PCT International Application No. PCT / US94 / 08529, published February 9, 1995 as International Publication No. WO 95/04145, the contents of which are incorporated herein by reference.

As explained hereinabove, cross-protective isolates share antigenic determinants that are capable of being recognized by antibodies, which bind to them. Thus, Osp proteins from cross-protective isolates share antigenic determinants and are subject to recognition by the same antibodies. Isolated cross-protective strains are said to belong to the same seroprotective group. Vaccination with a member of a seroprotective group will generate immunity against exposure to other isolates classified in the same seroprotective group. Antibodies directed against one member of a pair of isolates without cross protection will not confer passive immunity against the other member of the pair. The isolated strains without cross protection belong to different seroprotective groups. Vaccination with a member of a pair of isolates without cross protection will not confer cross immunity against challenge.

ES 2 265 643 T3 to the other member of the pair. Thus, vaccination with an effective amount of an Osp A protein or with a combination of Osp A protein and Osp B protein from an isolate will confer passive immunity against challenge to the same isolate, as well as against the other isolates with protection. crusade.

Typically, the effective amount for immunization of each of the antigenic subunits derived from non-cross-protective isolates of Borrelia burgdorferi is an amount from about one microgram to about 1000 micrograms.

The bacterin of the present invention may further comprise an immunization-effective amount of one or more antigenic subunits derived from a third, non-cross-protective isolate of Borrelia burgdorferi. However, the bacterin is not limited to containing one, two or three isolates without cross-protection, but may additionally contain four or more such isolates.

The present invention provides the use of a bacterin comprising three or more isolates without cross-protection of B. burgdorferi for the preparation of a pharmaceutical composition for immunizing an animal against disease caused by B. burgdorferi.

The present invention provides a bacterin comprising in each dose an amount, effective for immunization, of at least two isolated strains, without cross protection, of inactivated Borrelia burgdorferi and one or more antigenic subunits of the isolated strains without cross protection, a adjuvant in an amount effective to enhance the immunogenicity of the inactivated Borrelia burgdorferi and antigenic subunits, and a suitable excipient. Also provided is the use of said bacterin for the preparation of a pharmaceutical composition for immunizing an animal against the disease caused by B. burgdorferi, with which a dose of said bacterin is to be administered to the animal.

The present invention further provides the use of a bacterin comprising an amount, effective for immunization, of at least two isolated strains, without cross-protection, of inactivated Borrelia burgdorferi, an adjuvant in an amount effective to enhance immunogenicity. of the inactivated Borrelia burgdorferi and a suitable excipient for the preparation of a pharmaceutical composition for the immunization of animals against infection by Borrelia burgdorferi.

In one embodiment of the use described above, the immunization-effective amount of the non-cross-reactive isolates of inactivated Borrelia burgdorferi is an amount from about 10⁴ microorganisms up to about 10¹⁰ microorganisms of each isolated strain. In a preferred embodiment the effective amount for immunization of the isolated non-cross-reactive strains of inactivated Borrelia burgdorferi is an amount from about 10⁴ microorganisms up to about 10⁹ microorganisms of each isolated strain. In another preferred embodiment the effective amount for immunization of the isolated non-cross-reactive strains of inactivated Borrelia burgdorferi is an amount from about 10⁴ microorganisms up to about 10⁸ microorganisms of each isolated strain. In a preferred embodiment the effective amount for immunization of the isolated non-cross-reactive strains of inactivated Borrelia burgdorferi is approximately 10⁷ microorganisms of each isolated strain. In a particularly preferred embodiment the amount, effective for immunization, of the isolated, non-cross-reactive strains of inactivated Borrelia burgdorferi is approximately 5 x 10⁷ microorganisms of each isolated strain. In another particularly preferred embodiment the amount, effective for immunization, of the isolated non-cross-reactive strains of inactivated Borrelia burgdorferi is approximately 5 x 10⁸ microorganisms of each isolated strain.

The invention also provides the use of a bacterin comprising an immunization-effective amount of at least two non-cross-protected isolates of inactivated Borrelia burgdorferi and one or more antigenic subunits of the non-cross-protected strains isolated, a adjuvant in an amount effective to enhance the immunogenicity of the inactivated Borrelia burgdorferi and the antigenic subunits, and a suitable excipient for the preparation of a pharmaceutical composition for the immunization of animals against infection by Borrelia burgdorferi.

In a preferred embodiment of the use described above, the immunization-effective amount of the antigenic subunits derived from each of the isolates without cross-protection of Borrelia burgdorferi is an amount from about ten micrograms to about 10,000 micrograms.

The invention is further illustrated in the experimental section below. The experimental section and Examples mentioned herein are presented to facilitate understanding of the invention. This section is not intended to limit in any way, nor should it be construed as such, the invention set forth in the claims that follow.

ES 2 265 643 T3

Example 1

Borrelia burgdorferi bacterin preparation

Necessary equipment:

Sterile: 20 ml tubes, 250 ml bottles and 10 liter vessels; 200, 500 or 3,000 liter fermenter; serological pipettes; magnets; tanks for mixing and storage; and centrifuge.

Non sterile: motors for magnetic stirring; pipette material; peristaltic pumps; tubes and tube clips; Petroff / Hauser counting chamber; dark field microscope; gowns, gloves, caps, masks, shoe covers, and face masks; micropipettes and micropipette tips; whites with saline solution.

Microorganisms used: Two isolates of Borrelia burgdorferi are used for the preparation of the product: isolates C-1-11 and S-1-10 of B. burgdorferi obtained from Dr. Steven M. Callister, Gundersen Medical Foundation, La Crosse , WI. The bacterin will contain adjusted counts from each Borrelia strain culture to meet standard potency requirements.

Identity of each microorganism and frequency of identification methods: Identification is carried out on the basis of morphological and serological characteristics. Serological identification of the original stock cultures for sowing will be carried out with specific antisera, using an indirect immunofluorescence test.

Virulence, purity, maintenance and type of cultures: Virulence of cultures is not an essential criterion of antigenic capacity. Strain purity is determined by careful examination of the culture using dark field microscopy, Gram stain, serological identification with specific antisera, and testing according to 9 CFR Part 113.27. using 0.5 ml to 1.0 ml samples.

The cultures are maintained in Barbour-Stoenner-Kelly (BSK) medium, which is prepared as described below (see Example 5). Subcultures are prepared in BSK medium, and the number of subcultures is limited to 10.

Composition of crops for sowing and for production: Crops for sowing and for production are grown in the medium of BSK. Seed stock cultures are grown in BSK medium and frozen after adding glycerol (final concentration 10-14%). Frozen stock cultures are kept at -70 ° C, or below.

Nature, size and shape of containers used for culture growth: Frozen stock cultures are grown in 20 ml screw cap tubes containing ten milliliters of medium. The 20 ml culture tubes will be used to inoculate 250 ml flasks or flasks containing 200 ml of medium. The 250 ml flasks or flasks will be used to inoculate 10 liter vessels containing six to eight liters of medium. The 10 liter vessels can be used to inoculate 40 liter vessels containing 25 to 35 liters of medium, a 200 liter fermenter containing 90 to 175 liters of medium, a 500 liter fermenter containing 175 to 375 liters of medium, or a 3000 liter fermenter containing 800 to 2500 liters of medium. The 200 liter fermenter can be used to inoculate a 3,000 liter fermenter containing 300 to 2300 liters of medium.

Seed culture storage conditions: Stock cultures are kept in BSK medium as frozen cultures at -70 ° C or below.

Methods for the preparation of suspensions for seeding or inoculation: Four one-milliliter vials of the desired strain of B. burgdorferi are thawed and 9.0 ml of Barbour-Stoner-Kelly (BSK) medium are added to each (see more go ahead). The mother and seed cultures are propagated for 10 to 96 hours, controlling the growth rate, at 32 ° C ± 1 ° C before their transfer to BSK medium. Seed cultures are systematically checked by microscopy for purity before transfer to production medium. The cell growth of the sample tubes was measured by counting in a Petroff / Hauser chamber at 12 hours and at each subsequent hour, until their propagation by passage. Cultures of B. burgdorferi are propagated by passage at a concentration greater than or equal to 1 x 10⁸ cells per milliliter. When the cell concentration is greater than 1 x 10⁸ cells / ml Gram staining is performed in each culture tube and two acceptable culture tubes are mixed to form a 250 ml inoculum. Four 250 ml flasks containing 196.0 ml of BSK medium are inoculated with four milliliters of the inoculum described above and then incubated at 32 ° C stationary for 48 to 60 hours. Cell growth is determined by counting in a Petroff / Hauser chamber at 24 hours and then every six hours, until propagation by passage. Passage propagation is carried out at concentrations greater than or equal to 1 x 10⁸ cells per milliliter. When the cell concentration reaches this level, Gram staining is performed in each culture flask, and two acceptable flasks are mixed for inoculation in a ten liter vessel. Each of the three ten liter jars, containing 5880 ml of BSK medium, is inoculated with 120 ml of the culture described above. The flasks are then incubated at 32 ° C for 36 to 48 hours, slowly shaking with a magnetic bar. The cell growth of a vessel is determined by counting in a Petroff / Hauser chamber at 10 hours, and then every six hours, until its propagation by passage, which is carried out at a concentration greater than or equal to 1 x 10⁸ cells per milliliter. When the concentration of cells in the cultures reaches this level, the staining is carried out.

ES 2 265 643 T3 of Gram in each culture vessel, and a vessel containing six liters is used to inoculate a 200 liter fermenter containing 144 liters of BSK medium. The fermenters are incubated at 32 ° C for approximately 96 hours, shaking slowly (55 rpm), maintaining the pH at 7.2 ± 0.2. At 12, 24, 48 and 72 hours, and then every four hours, cell growth is determined by counting in a Petroff / Hauser chamber and Gram's staining is monitored. The culture becomes inactivated when it reaches the late logarithmic phase of growth (approximately 5-7 x 10⁸ cells per milliliter).

Figure 1 shows a growth curve of a Borrelia burgdorferi culture. As can be seen in the figure, exponential growth of the culture began between about 24 and about 48 hours, and continued until about 96 hours, at which point growth stabilized.

Figure 2 shows the dextrose consumption and lactate production of a Borrelia burgdorferi culture. As can be seen in the figure, lactate production increased constantly in the culture, indicating the importance of pH control in the culture. In addition, it can be seen in the figure that the consumption of dextrose increases with time, which indicates that the bacteria are using it as an energy source, so it is necessary to add more to maintain the viability or growth of the culture.

Inoculation technique of medium for sowing and for production: The medium for sowing is inoculated by direct transfer from the frozen stock culture. The medium for production is inoculated by direct transfer from the culture for sowing. The inoculation of the culture does not use more than 2% of the inoculum.

Incubation Time Period, Conditions, and Temperature Level: After inoculation, vessels are incubated at 32 ° C ± 1 ° C for 10 to 96, and production fermenters are incubated at 32 ° C ± 1 ° C for 24 to 120 hours. Sterile NaOH solution is added as necessary to adjust the pH to 7.2 ± 0.2.

Nature and extent of growth: Daily throughout incubation, inoculated production cultures are examined microscopically for quantification in a Petroff / Hauser chamber. At the end of the incubation period, the cultures are monitored microscopically for contamination using Gram stain and / or dark field scanning.

Minimum and maximum times for the collection of cells: The minimum period of incubation until the collection of cells is 24 hours. The maximum incubation period until cell harvest is 120 hours.

Cell harvesting technique: At the end of the incubation period, each production culture is monitored by microscopy for purity by Gram stain and / or dark field scanning, and samples are removed for total cell counts. Culture vessels are stored at 2 ° C to 7 ° C.

Specifications of acceptable material for cell collection: Acceptable production media contain pure Borrelia burgdorferi cultures, verified by Gram stain and / or dark field microscopy, and have a Borrelia cell count of not less than 1 x 10⁸ microorganisms per milliliter. The concentration is determined using a Petroff / Hauser counting chamber (or equivalent).

Disposal of discarded materials: All materials that have not been used in the preparation of the bacterin are disposed of in accordance with Circular No. 800.56 of the Veterinary Services.

Example 2

Preparation of the product

Inactivation method: The production culture growth is inactivated with binary ethyleneimine (BEI). The inactivation is carried out at 32 ° C ± 2 ° C. The appropriate amount of BEI is prepared as a 0.344 M (7.05%) solution. A 7.05% BEI solution is prepared by dissolving 70.48 grams of 2-bromoethylamine hydrochloride (molecular weight 204.89) and 8.00 grams of NaOH (MW: 40.00) in one liter of deionized water. The dissolved solution is incubated at 37 ° C for two hours and then added to the culture at a rate of 30 ml per liter with continuous shaking (slow shaking). The inactivation is carried out for not less than 24 hours at a pH of 7.3 ± 0.2 with agitation (slow agitation). After the inactivation is complete, the BEI is neutralized by adding sterile sodium thiosulfate. The appropriate amount of sterile sodium thiosulfate solution is prepared as a 3.015 M (47.7%) solution by dissolving 477.0 grams of sodium thiosulfate in one liter of deionized water. This solution is filtered through a 0.2 micron filter before use, and is added to the inactivated culture at a rate of 10.6 ml per liter with continuous stirring (slow stirring). The neutralization reaction is allowed to proceed for not less than 6 hours at 32 ° C with continuous stirring (slow stirring) at a pH of 7.3 ± 0.2. Gentamicin sulfate and nystatin are added to the culture at final concentrations of 30.0 micrograms per milliliter, and 30.0 units per milliliter, respectively. The microorganisms from the production culture are then grouped in sterile holding tanks for further processing.

Inactivation Testing: A bulk culture inactivation test is conducted in accordance with 9 CFR Part 113.100. A sample of the inactivated culture is drawn and a tube containing 29 ml of BSK medium is inoculated with 1.0 ml of the sample. At the same time, a sample of the same microorganism at a concentration of

ES 2 265 643 T3 cells per milliliter, which is known to be viable as a positive growth control, is diluted 1:30 in freshly prepared BSK medium using a 29.0 ml BSK medium blank and diluted in medium BSK from 10 'to 10 ⁴ using 9.0 ml BSK media blanks. The tubes are incubated for 14 days at 32 ± 2 ° C and then examined for growth. Lack of Borrelia growth in the tube containing the inactivated specimen indicates that the test is successful. The tubes inoculated with the live microorganism must have an ID₅₀ <100 and> 1 according to the Reed-Muench method.

Composition, proportions, preservatives and helpers:

1. Aluminum hydroxide adjuvant: Add aluminum hydroxide [Al (OH)₃] Rehydragel® HPA obtained from Reheis Chemical Company (Berkley Heights, NJ) to suspension of bacteria at a final concentration of not less than 1.5 mg (0.15%) of aluminum oxide (A1203) per dose.

2. Saline solution: Sterile physiological saline solution (0.85% NaCl, pH 7.2 ± 0.2) can be added as a diluent.

3. Preservatives: Gentamicin sulfate and nystatin are added to the product at a final concentration not exceeding 30.0 micrograms per milliliter, and 30.0 units per milliliter, respectively.

Four. pH: The pH of the completed product is adjusted to 7.3 ± 0.1 with sterile NaOH or HCl.

Concentration method and magnitude: The material from the Borrelia harvest is concentrated by centrifugation and resuspension in sterile saline containing gentamicin sulfate and nystatin to final concentrations of 30.0 micrograms / ml and 30.0 units / ml, respectively. . Borrelia harvest material can be concentrated up to 1/20 of the original volume.

Procedures for antigen normalization: Bacterin is prepared using a Petroff / Hauser chamber count determined after concentration and resuspension of the pellet material. The Borrelia concentration in each culture is determined as described above. A mixture of cultures with high and low concentrations can be used to adjust the final concentrations.

Preparation of a series:

1. Preparation of units to form a series: Sufficient bulk storage containers of individual mixtures of each Borrelia isolate are removed from the refrigerator kept at 4 ° C to 7 ° C. If necessary, sterile physiological saline (0.85% NaCl) is used as diluent. The diluent is autoclaved at 121 ° C ± 2 ° C for 30 minutes, or when the probe in the liquid reaches 119 ° C for no less than 20 minutes.

2. Example of preparing a series:

<td>Ingredients</td><td>Microorganisms per ml in Concentration</td><td>Finished product Liters</td><td>Final accounts by 1.0 ml dose no less than</td>

<td>B. burgdorferi Strain C-1-11</td><td>8.0 x 10⁹</td><td> 18,750</td><td>5.0 x 10⁸</td>

<td>B. burgdorferi Strain S-1-10</td><td>8.0 x 10⁹</td><td> 18,750</td><td>5.0 x 10⁸</td>

<td>Aluminum oxide (Al₂OR₃ at 2%)</td><td></td><td> 22,500</td><td></td>

<td>Gentamicin Sulfate (20.0 mg per ml)</td><td></td><td> 0,394</td><td></td>

<td>Nystatin (25,000 units per ml)</td><td></td><td> 0,315</td><td></td>

<td>Physiological saline solution</td><td></td><td> 239,291</td><td></td>

<td>Total volume</td><td></td><td> 300,000</td><td>liters</td>

The pH is adjusted to 7.3 ± 0.1 with sterile NaOH or HCl. The average size of the series is 300 liters. The maximum size of the series is 900 liters. Suspensions of inactivated Borrelia are mixed in a sterilized stainless steel tank for preparation. After mixing, the entire series is stored in a sterilized stainless steel preparation tank or divided into smaller containers and stored at 2 ° C to 7 ° C until filling, or the product can be introduced immediately in vials and store at 2 ° C to 7 ° C.

3. Filling volume and type of vial used: The bacterin is introduced into vials at 1.20 ml ± 0.05 ml for each dose vial. The vials to be used are two two-milliliter glass vials.

Method and technique for filling and sealing the final container: Sterile vials are filled by means of a sterile mechanical or manual procedure from the batch with continuous shaking. The filled vials are covered with sterile stoppers, mechanically or manually, and sealed with aluminum caps.

ES 2 265 643 T3

Amount of antigenic material per dose in the final container: each dose contains amounts of microorganisms not less than the following:

Isolated strain C-1-11 of B. burgdorferi: 5 x 10⁸ microorganisms / ml.

Isolated strain S-1-10 of B. burgdorferi: 5 x 10⁸ microorganisms / ml.

Example 3

Bacterin tests

Purity: Each series or sub-series is tested for the presence of bacteria and fungi in accordance with 9 CFR Part 113.26.

Safety: Safety testing is conducted on bulk or final containers in accordance with 9 CFR Parts 113.38 and 113.40, which provides for a safety test on dogs using a 2X (2cc) dose administered. by intramuscular injection.

Potency: Potency tests are performed on bulk or final prepared containers. Potency tests are performed using an antigen capture ELISA test. Successful runs must have a relative potency&gt; 1.0 determined by version 3.0 of the relative potency calculation software of the US Department of Agriculture Veterinary Biological Reagents Program.

Example 4

Stages after preparation

Shape and size of the final packages in which the product is to be distributed: The bacterin is marketed in a 2.0 ml, single-dose vial (one dose: 1.0 ml). Labeling of the final container is completed.

Collection, Storage, and Shipping of Representative Samples: Representative samples are drawn from each series or subseries for APHIS-USDA pursuant to 9 CFR Part 113.3 using a company authorized sample collector.

Product expiration date: The expiration date shown on the product will be 24 months from the start of satisfactory potency tests pursuant to 9 CFR Part 114.13. The date will be confirmed pursuant to 9 CFR Part 114.13.

Use, dosage and route of administration for each animal species: The bacterin is recommended for the vaccination of healthy dogs against the disease caused by B. burgdorferi. Each dose contains 1.0 ml (1.0 cc). Healthy dogs are vaccinated at 12 weeks of age, or greater, with two doses, separated by an interval of two to three weeks. Puppies less than 6 weeks old should be re-vaccinated every 2 to 3 weeks until they reach an age of at least 12 weeks. Annual revaccination with one dose is recommended. The route of administration is intramuscular injection.

Example 5

Barbour-Stoenner-Kelly (BSK) medium preparation

Composition: The medium is used to grow Borrelia cultures for use in the preparation of bacterins. After washing with detergent, all glassware is thoroughly rinsed with deionized water and then autoclaved. All ingredients are added slowly in the order mentioned in the list with slow and continuous stirring. The base medium is prepared as follows:

Ingredients: Quantity

<td> 1)</td><td>Deionized water</td><td>500.00 ml</td>

<td> 2)</td><td>HEPES</td><td>4.60 g</td>

<td> 3)</td><td>Neopeptone</td><td>3.83 g</td>

<td> 4)</td><td>Sodium citrate</td><td>0.54 g</td>

<td> 5)</td><td>Glucose</td><td>3.83 g</td>

<td> 6)</td><td>Sodium bicarbonate</td><td>1.69 g</td>

<td> 7)</td><td>TC Yeastolate</td><td>1.92 g</td>

<td> 8)</td><td>Pyruvic acid, sodium salt</td><td>0.61 g</td>

<td> 9)</td><td>N-acetyl-glucosamine</td><td>0.31 g</td>

ES 2 265 643 T3

The solution is slowly stirred to dissolve all components. 76.70 ml of 10 X CMRL 1066 without glutamine are added to the mixture, with continuous and slow stirring.

Preparation of the complete medium: For 10.0 ml tube or 200.0 ml bottle cultures, the following ingredients are slowly added to the base medium, with continuous stirring:

Ingredients: Quantity / liter

1) Sterile rabbit serum, heat inactivated 49.00 ml (The sterile rabbit serum is heated for 30 minutes at 58.5 ° C ± 0.5 ° C)

2) Bovine serum albumin 38.34 g

3) Adjust the pH to 7.3 ± 0.1 with 2N NaOH.

A sufficient quantity (qs) is added to 800 ml with deionized water at room temperature. The mixture is sterilized using a 0.2 micron filter with low protein binding.

4) Sterile gelatin 200.00 ml

Sterile gelatin is prepared by adding 10.74 g of gelatin to 200.0 ml of deionized water. The suspension is autoclaved for 30 minutes at 121 2 ° C or when a probe in the liquid reaches 119 ° C for not less than 20 minutes. The solution is cooled to at least 45 ° C ± 2 ° C prior to the addition of the base medium.

For 10 liter jar cultures or 100 liter or larger fermenters, the following ingredients are added slowly with continuous agitation to the base medium:

Ingredients: Quantity / liter

1) Sterile rabbit serum, heat inactivated 49.00 ml (The sterile rabbit serum is heated for 30 minutes at 58.5 ° C ± 0.5 ° C)

2) Bovine serum albumin 38.34 g

The pH is adjusted to 7.3 ± 0.1 with 2N NaOH. Sufficient amount of deionized water is added to make up one liter. The mixture is sterilized using a 0.2 micron filter with low protein binding.

Ingredient information:

1. Bovine serum albumin (BSA): BSA powder is purchased from a commercial supplier. Powdered BSA used in production may be tested under 9 CFR Parts 113.50 and 113.53 and authorized for use. In the absence of such tests, powdered BSA can be gamma irradiated at no less than 2.7 megarads.

2. Rabbit serum: Rabbit serum is purchased from a commercial supplier. Rabbit serum used in production may be tested under 9 CFR Parts 113.50 and 113.53 and authorized for use. Rabbit serum can be gamma-irradiated to not less than 2.7 megarads according to the tests provided in 9 CFR Parts 113.50 and 113.53.

All chemicals were USP grade, or equivalent.

Testing: The medium is considered sterile, therefore it is not tested.

Conservation and use: The sterile medium is stored at 4 ° C to 30 ° C until use. The medium can be used immediately after preparation or for a period not exceeding six months. The medium is used to grow Borrelia cultures for use in bacterin production.

Example 6

Hamster vaccination - serological and exposure studies

Hamsters vaccinated with Borrelia burgdorferi bacterin, inactivated with binary ethyleneimine (BEI), heat, or formalin (FORM.) Were bled and the animals were challenged for two to three weeks, as indicated, after vaccination. only. The vaccinated animals were challenged with a mixture of three isolates of Borrelia burgdorferi. Non-vaccinated animals were used as controls that had not been inoculated with the Borrelia burgdorferi bacterin, but were challenged with the individual isolate, as indicated by the name of the isolate, or with a mixture of three strains. isolated, as indicated by Tri.

ES 2 265 643 T3

TABLE 1

Exposure two weeks after vaccination

<td colspan="4">Vaccinated</td>

<td>Bacterin</td><td>ELISA</td><td>AB</td><td>Percentage of infection</td>

<td>EIB</td><td> 0,258 ± 0,070</td><td> 56 ± 23</td><td> 0 (0/3)</td>

<td>HOT</td><td> 0,157 ± 0,068</td><td> 43 ± 30</td><td> 0 (0/5)</td>

<td>FORM.</td><td> 0,139 ± 0,132</td><td> 35 ± 32</td><td> 25 (1/4)</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td>Exposure to</td><td>ELISA</td><td>AB</td><td>Percentage of infection</td>

<td>isolated strain</td><td></td><td></td><td></td>

<td>P / Bi</td><td> 0,003 ± 0,008</td><td> 0±0</td><td> 0 (0/3)</td>

<td>Wis.</td><td> 0,000 ± 0,000</td><td>NT</td><td> 100 (4/4)</td>

<td>Chi.</td><td> 0,000 ± 0,000</td><td>NT</td><td> 100 (3/3)</td>

<td>Tri.</td><td>NT</td><td>NT</td><td> 80 (4/5)</td>

Exposure three weeks after vaccination

<td colspan="4">Vaccinated</td>

<td>Bacterin</td><td>ELISA</td><td>AB</td><td>Percentage of infection</td>

<td>EIB</td><td> 0,218 ± 0,018</td><td> 34 ± 25</td><td> 50 (2/4)</td>

<td>HOT</td><td> 0,259 ± 0,059</td><td> 29 ± 19</td><td> 75 (3/4)</td>

<td>FORM.</td><td> 0,384 ± 0,202</td><td> 40 ± 18</td><td> 100 (4/4)</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td>Exposure to</td><td>ELISA</td><td>AB</td><td>Percentage of infection</td>

<td>isolated strain</td><td></td><td></td><td></td>

<td>P / Bi</td><td> 0,000 ± 0,000</td><td> 4±2</td><td> 0 (0/4)</td>

<td>Wis.</td><td> 0,000 ± 0,000</td><td>NT</td><td> 50 (2/4)</td>

<td>Chi.</td><td> 0,000 ± 0,000</td><td>NT</td><td> 50 (2/4)</td>

<td>Tri.</td><td>NT</td><td>NT</td><td> 67 (2/3)</td>

AB = Borreliacidal activity assay

The results presented in Table 1 demonstrate that there is no significant difference (P = 0.05) in the serological response with respect to the B. burgdorferi cell inactivation method at 2 or 3 weeks after vaccination. Serological responses decreased or increased slightly from 2 to 3 weeks after vaccination; however, protection (relative to isolates C-1-11 and S-1-10) decreased in each vaccinated group at 3 weeks post-vaccination. The isolated strain P / Bi is not virulent, and it was found not to infect the tissues of the vaccinated or control animals.

Example 7

ELISA studies in unvaccinated dogs and in dogs vaccinated intramuscularly

The ability of the bacterin to stimulate an immune response in dogs was tested. Dogs were either vaccinated with the bacterin provided herein (V), or were not vaccinated (NV), and their antibody levels against S-1-10 and C-1-11 coat antigens were determined by the ELISA technique at three weeks after the first vaccination and at two weeks after a second subsequent vaccination, as indicated.

ES 2 265 643 T3

TABLE 2

Coating antigen = S-1-10

Prevac Group.

V 0.013 ± 0.020

NV 0.012 ± 0.016

Prevac Group.

V 0.033 ± 0.015

NV 0.023 ± 0.019

Three weeks after vaccination

0,672 ± 0,128 0,027 ± 0,011

Coating antigen = C-1-11

Three weeks after vaccination

0,511 ± 0,122 0,017 ± 0,013

Two weeks after vaccination

1,369 ± 0,167

0,014 ± 0,015

Two weeks after vaccination

1,550 ± 0,148

0,037 ± 0,016

The data demonstrate that inoculation with the bacterin of the present invention results in the production of antibodies against both isolates of B. burgdorferi: S-1-10 from Wisconsin and C-1-11 from Chicago. Vaccinated dogs had a higher serum level of antibodies against S-1-10 and C1-11 coat antigens than unvaccinated animals. These levels of antibody production increased after a second dose of the bacterin was administered to the dogs. Accordingly, these results demonstrate that the bacterin of the present invention can stimulate the production of antibodies against Borrelia in animals that will be useful against subsequent exposures to the bacteria.

Example 8

Borreliacidal Activity Assay Using Dog Serum

Dogs were vaccinated with the Borrelia burgdorferi bacterin. Serum from these dogs was isolated and mixed with a growing culture of B. burgdorferi strain S-1-10. The percentage of dead Borrelia cells in the cultures that had been grown in the presence and in the absence of this immunized dog serum is indicated in Table 4 (see below).

TABLE 3

Borreliacidal Activity Assay - Dog Serum Percentage of Borrelia Cells Killed

<td></td><td>Prevac.</td><td>Three weeks after 1.^to vaccination</td><td>Two weeks after the 2.^to vaccination</td>

<td>No vac.</td><td> 0±0</td><td> 0,5 ± 1,7</td><td> 1,5 ± 2,7</td>

<td>Vac. IM</td><td> 0±0</td><td> 59,5 ± 6,3</td><td> 87,9 ± 10,3</td>

<td>Vac. Subc.</td><td> 0±0</td><td> 63,7 ± 20,6</td><td> 86,1 ± 12,0</td>

Non-vaccinated: serum from unvaccinated dogs; Vac. IM: dogs vaccinated intramuscularly; Vac. Subc .: dogs vaccinated subcutaneously; Prevac .: serum from dogs before vaccination.

As can be seen in Table 3, serum extracted from dogs prior to their vaccination with the bacterin (prevac.) And from unvaccinated animals (Non-vac.) Was unable to trigger cell death in a growing culture of Borrelia. In contrast, the immunized dog serum was able to trigger cell death. The serum isolated from dogs vaccinated intramuscularly (Vac. IM) with the bacterin was able to cause a similar percentage of cell death to that of the serum isolated from dogs vaccinated by the subcutaneous route (Vac. Subc.). Serum isolated from dogs that had received a second dose of the bacterin caused a higher rate of cell death compared to serum from dogs vaccinated only once.

Example 9

Isolated strain 297 of Borrelia burgdorferi

Serum from dogs before and after inoculation of Borrelia burgdorferi isolate N-40 was tested for borreliacidal antibodies directed against strain 297 cells using the borreliacidal activity assay.

ES 2 265 643 T3

TABLE 4

Serum dilution

<td>Serum</td><td> 1:20</td><td> 1:40</td><td> 1:80</td><td> 1:160</td><td> 1:320</td><td> 1:640</td>

<td>Preinoc. alive</td><td> 140</td><td> 158</td><td> 173</td><td> 133</td><td>NC</td><td>NC</td>

<td>Preinoc. dead</td><td> 6</td><td> 7</td><td> 13</td><td> 7</td><td>NC</td><td>NC</td>

<td>5 weeks after infection, alive</td><td> 1</td><td> 0</td><td> 2</td><td> 15</td><td> 100</td><td> 102</td>

<td>5 weeks after infection, dead</td><td> 51</td><td> 39</td><td> 57</td><td> 33</td><td> 23</td><td> 7</td>

NC: No count.

The data presented in Table 4 demonstrate that dogs inoculated with the isolate N-40 of B. burgdorferi produce borreliacidal antibodies against a different isolate (i.e., strain 297) but from the same seroprotective group to which N-40 belongs. , which is not present in non-inoculated dogs, free of B. burgdorferi. Isolated strain N-40 of Borrelia burgdorferi

Serum from dogs before and after inoculation of the Borrelia burgdorferi isolate N-40 was analyzed for borreliacidal antibodies directed against the cells of the N-40 strain using the borreliacidal activity assay.

TABLE 5

Serum dilution

<td>Serum</td><td> 1:20</td><td> 1:40</td><td> 1:80</td><td> 1:160</td><td> 1:320</td><td> 1:640</td>

<td>Preinoc. alive</td><td> 154</td><td> 226</td><td> 204</td><td> 219</td><td> 280</td><td> 135</td>

<td>Preinoc. dead</td><td> 0</td><td> 9</td><td> 6</td><td> 5</td><td> 5</td><td> 3</td>

<td>5 weeks after infection, alive</td><td> 0</td><td> 0</td><td> 3</td><td> 1</td><td> 138</td><td> 99</td>

<td>5 weeks after infection, dead</td><td> 54</td><td> 65</td><td> 72</td><td> 48</td><td> 3</td><td> 5</td>

The data presented in Table 5 demonstrate that dogs inoculated with B. burgdorferi isolate N-40 produce borreliacidal antibodies against the inoculated isolate which is absent in uninoculated dogs, free of B. burgdorferi.

Example 10

In vitro cross-protection studies

Table 7 (see below) presents the natural hosts of various isolates of Borrelia burgdorferi used in in vitro cross-protection studies, the origin of the isolates from the host animals, and the geographic locations where the strains were isolated. .

TABLE 7

Borrelia burgdorferi isolates

<td>Name</td><td>Host</td><td>Source</td><td>Location</td>

<td> 297</td><td>Humans</td><td>CSF</td><td>Eastern US</td>

<td>B31</td><td>I. dammini</td><td> ---</td><td>Eastern US</td>

<td>S-1-10</td><td>P. leucopus</td><td>kidney</td><td>Wisconsin</td>

<td>IPT</td><td>I. pacificus</td><td> ---</td><td>West Coast of the USA</td>

<td>MMTI</td><td>I. dammini</td><td> ---</td><td>Minnesota</td>

<td> 35211</td><td>I. ricinus</td><td> ---</td><td>Swiss</td>

<td>Chicago (C-1-11)</td><td>M. pennsylvanicus</td><td>kidney</td><td>Illinois</td>

<td>PBi</td><td>Humans</td><td>CSF</td><td>Germany</td>

<td>G25</td><td>I. ricinus</td><td> ---</td><td>Sweden</td>

CSF: cerebrospinal fluid.

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Hamsters were inoculated with a live form of one of the isolates indicated in the first column of Table 8. Antiserum was extracted from these hamsters and added to cultures of the isolated strains of B. burgdorferi indicated in the corresponding row of the Table. 8. The degree of recognition of the bacteria in culture by the antibody, and hence the degree of cell death caused, was measured as follows. Serum from hamsters infected with live, individual isolates of Borrelia burgdorferi was extracted, diluted in BSK medium, heat inactivated, and 100 µl was added to each reaction tube. Ten microliters of complement was also added. The density of the culture was then adjusted to a concentration of 10⁵ cells per milliliter, and then 100 µl of the culture was added to each reaction tube. The resulting reaction tubes with antibody / B. burgdorferi were incubated at 32 ° C for two hours, and then 800 µl of fresh BSK medium was added. The reaction tubes were then incubated at 32 ° C for four days, and then viability was assessed as a percentage of intact cells, determined with the Coulter cytometer. The results of the in vitro cross-protection studies are presented below in Table 8.

TABLE 8

In vitro cross-protection studies

<td>Antisera</td><td> 297</td><td>B31</td><td>S-1-10</td>

<td>BSK</td><td> 100,0 ± 3,0</td><td> 100,0 ± 5,8</td><td> 100,0 ± 3,5</td>

<td>NHS</td><td> 100,0 ± 3,8</td><td> 107,9 ± 7,9</td><td> 99,5 ± 3,5</td>

<td> 297</td><td> 0,6 ± 0,2</td><td> 0,9 ± 0,0</td><td> 1,0 ± 0,2</td>

<td>B31</td><td> 0,5 ± 0,1</td><td> 0,9 ± 0,2</td><td> 0,9 ± 0,1</td>

<td>S-1-10</td><td> 0,3 ± 0,1</td><td> 0,7 ± 0,1</td><td> 0,8 ± 0,1</td>

<td> 35211</td><td> 0,3 ± 0,1</td><td> 0,7 ± 0,1</td><td> 1,0 ± 0,1</td>

<td>MMTI</td><td> 2,4 ± 1,6</td><td> 0,5 ± 0,0</td><td> 1,0 ± 0,0</td>

<td>IPT</td><td> 0,4 ± 0,1</td><td> 3,3 ± 0,1</td><td> 0,9 ± 0,0</td>

<td>Chicago</td><td> 32,6 ± 1,7</td><td> 55,7 ± 3,8</td><td> 91,2 ± 7,3</td>

<td>PBi</td><td> 93,5 ± 2,8</td><td> 97,5 ± 10,9</td><td> 103,5 ± 5,2</td>

<td>G25</td><td> 103,5 ± 5,3</td><td> 113,5 ± 8,0</td><td> 97,1 ± 8,9</td>

<td>Antisera</td><td> 35211</td><td>MMTI</td><td>IPT</td>

<td>BSK</td><td> 100,0 ± 7,7</td><td> 100,0 ± 12,4</td><td> 100,0 ± 9,0</td>

<td>NHS</td><td> 99,5 ± 3,5</td><td> 103,9 ± 4,1</td><td> 108,9 ± 1,4</td>

<td> 297</td><td> 1,0 ± 0,2</td><td> 2,5 ± 0,4</td><td> 0,3 ± 0,0</td>

<td>B31</td><td> 0,9 ± 0,1</td><td> 11,0 ± 1,0</td><td> 0,4 ± 0,0</td>

<td>S-1-10</td><td> 0,8 ± 0,1</td><td> 0,7 ± 0,3</td><td> 0,3 ± 0,1</td>

<td> 35211</td><td> 1,0 ± 0,1</td><td> 1,7 ± 0,3</td><td> 0,3 ± 0,0</td>

<td>MMTI</td><td> 1,0 ± 0,0</td><td> 0,9 ± 0,2</td><td> 0,2 ± 0,0</td>

<td>IPT</td><td> 0,9 ± 0,0</td><td> 1,9 ± 0,2</td><td> 0,2 ± 0,0</td>

<td>Chicago</td><td> 91,2 ± 7,3</td><td> 84,7 ± 5,4</td><td> 100,5 ± 2,8</td>

<td>PBi</td><td> 103,5 ± 5,2</td><td> 104,2 ± 13,4</td><td> 96,2 ± 3,6</td>

<td>G25</td><td> 97,1 ± 8,9</td><td> 106,4 ± 13,2</td><td> 106,3 ± 9,6</td>

<td>Antisera</td><td>Chicago</td><td>PBi</td><td>G25</td>

<td>BSK</td><td> 100,0 ± 3,5</td><td> 100,0 ± 8,0</td><td> 100,0 ± 7,2</td>

<td>NHS</td><td> 111,2 ± 14,5</td><td> 105,7 ± 7,8</td><td> 104,4 ± 5,8</td>

<td> 297</td><td> 99,2 ± 3,6</td><td> 113,4 ± 3,0</td><td> 91,5 ± 2,9</td>

<td>B31</td><td> 114,2 ± 6,7</td><td> 108,9 ± 8,9</td><td> 96,4 ± 8,9</td>

<td>S-1-10</td><td> 99,5 ± 7,3</td><td> 85,1 ± 6,6</td><td> 90,7 ± 6,4</td>

<td> 35211</td><td> 107,3 ± 11,4</td><td> 102,1 ± 3,8</td><td> 92,2 ± 3,8</td>

<td>MMTI</td><td> 102,5 ± 8,5</td><td> 108,7 ± 9,1</td><td> 96,5 ± 6,0</td>

<td>IPT</td><td> 109,5 ± 8,5</td><td> 119,5 ± 8,0</td><td> 97,1 ± 6,8</td>

<td>Chicago</td><td> 0,8 ± 0,1</td><td> 93,6 ± 3,6</td><td> 105,2 ± 8,2</td>

<td>PBi</td><td> 114,8 ± 11,4</td><td> 0,2 ± 0,0</td><td> 0,3 ± 0,0</td>

<td>G25</td><td> 115,7 ± 0,4</td><td> 0,2 ± 0,0</td><td> 0,3 ± 0,0</td>

BSK: Barbour-Stoenner-Kelly medium; NHS: normal hamster serum, that is, serum that does not contain antibodies against Borrelia.

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Viability values ​​in Table 8 are expressed as a percentage of cell viability in culture, compared to 100% viability of "BSK" controls. Control BSK cultures are cultures that are contacted with Barbour-Stoenner-Kelly medium rather than antiserum from hamsters inoculated with an isolated strain of B. burgdorferi. Therefore, these cultures should not undergo cell death due to antibody reactions. Consequently, the viability of these cultures is set at 100%, compared to that of cultures to which antiserum is added. The low viability of a culture compared to control values ​​indicates that cell death has occurred in the culture, due to antibody reactions with cell surface determinants.

The data in Table 8 demonstrate that cultures of a strain contacted with antiserum from a hamster inoculated with the same isolated strain show low viability; that is, the antibodies synthesized against the inoculated isolate recognize the isolate in culture and, together with complement, cause cell death. For example, a culture of strain 297 of B. burgdorferi contacted with BSK or NHS medium, that is, serum that does not contain antibodies against Borrelia, showed 100% viability. However, a culture of strain 297 put in contact with antiserum extracted from a hamster inoculated with strain 297 showed a viability of 0.6%. In addition, a culture of the Chicago strain of B. burgdorferi contacted with the NHS had a viability of 111%, while a culture of the Chicago strain contacted with an antiserum against the Chicago strain had a viability of 0.8%.

The data in Table 8 also demonstrate that antiserum from hamsters inoculated with an isolate caused cell death in the cultures of some other isolates. As discussed above, the viability of cultures that had been contacted with BSK medium is set at 100%. The data demonstrate that the viability of cultures that had been contacted with normal hamster serum (NHS) is also approximately 100%, as expected. However, the cultures of the first six isolated strains of B. burgdorferi mentioned in the list, that is, strains 297, B31, S-1-10, 35211, MMTI and IPT, when put in contact with antiserum obtained from Hamsters inoculated with any of the same six isolates all presented significantly lower viability values ​​than controls. These data demonstrate that the viability of the cultures was low when antiserum against an isolate was added to the cultures of the same isolate. The viability of the cultures was low when the cultures were contacted with antisera directed against other isolates. For example, antisera against strain 297 were added to cultures of strains 297, B31, S-1-10, 35211, MMTI, and IPT. The viability of the cultures was 0.6%, 0.9%, 1.0%, 1.0%, 2.5%, and 0.3%, respectively, indicating that the antibodies had recognized and reacted with the determinants of said isolated strains.

The data in Table 8 therefore demonstrate that isolates 297, B31, S-1-10, 35211, MMTI and IPT are cross-protected. That is, as explained above, antibodies against one isolate recognize and react with the surface determinants of other isolates. These isolated strains are therefore members of the same seroprotective group. This group is known as the seroprotective group A of Borrelia burgdorferi isolates.

Hamster antisera inoculated with the Chicago, PBi and G25 strains did not cause cell death in the cultures of these isolates and therefore belong to one or more different seroprotective groups. Antisera against the Chicago strain did not cause a significant degree of cell death in the cultures of any of the other isolates tested. Therefore, the Chicago strain (also called C-1-11) is the only strain tested that belongs to the seroprotective group B.

The antiserum against the PBi strain produced cell death in both the PBi and G25 cultures, as was also the case with the antiserum against the G25 strain. Therefore, PBi and G25 are isolates that are cross-protective and are classified in the same seroprotective group, the "European" group of Borrelia burgdorferi isolates.

Thus, the results of the cross-protection studies presented in Table 8 indicate that the isolates of Borrelia burgdorferi can be classified into at least three different seroprotective groups.

Example 11

Hamster vaccination - serological and exposure study

The hamsters were inoculated with live cells of Borrelia burgdorferi strain 297. Serum from these hamsters, or normal hamsters, ie not inoculated, or normal hamster serum (NHS) was then administered to other hamsters. These hamsters were then challenged with strain 297 or 35211 (Switzerland) as indicated in Figure 3. The ability of the antisera to transfer passive immunity to recipient hamsters against infection by B. burgdorferi, on successive days after infection, as indicated.

Figure 3 presents the results of the serological and exposure study. Protection against infection by

B. burgdorferi was evaluated from the values ​​obtained with the plethysmograph of animals subjected to exposure, in comparison with the initial values ​​for the control, that is, non-inoculated animals. The plethysmograph measures the amount of mercury displaced by a hamster's legs. As explained above, one of the symptoms caused by B. burgdorferi infection is arthritis and swelling. In this way, infected hamsters

ES 2 265 643 T3 with Borrelia should have swollen legs, which displace greater amounts of mercury than the legs of normal hamsters. Hamsters protected against B. burgdorferi infection should not have swollen legs. Consequently, an increase in the paw size of an infected hamster, ie an increase in the amount of displaced mercury, indicates that the antisera administered had not protected the hamster against exposure to B. burgdorferi.

The results presented in Figure 3 indicate that normal hamster serum (NHS), as expected, did not protect hamsters against infection by isolate B. burgdorferi strain 297 (the value obtained with the hamster plethysmograph inoculated with NHS / exposed to 297 increased to a maximum of approximately 1.0 mm Hg eight days after exposure, compared to a baseline value of approximately 0.35). Nor did normal hamster serum protect hamsters against infection by B. burgdorferi isolate 35211 (the value obtained with the hamster plethysmograph inoculated with NHS / exposed to 35211 increased to a maximum of approximately 0.85 mm Hg nine days after infection, compared to baseline).

In contrast, Figure 3 indicates that antiserum against strain 297 protected hamsters against subsequent challenge to isolate 35211. The values ​​obtained with the 297 / exposed hamster plethysmograph were approximately the same as the values initial between two and fourteen days after exposure. These results confirm in vitro studies, which show that Borrelia burgdorferi isolates can be cross-protected, that is, antibodies against one strain can confer passive immunity against the other isolate. The results indicate that isolated strains 297 and 35211 show cross protection, and therefore belong to the same seroprotective group.

Example 12

Summary of Borrelia burgdorferi isolates

Forty-three individual isolates of B. burgdorferi from the United States and Europe were examined. Exponential growing cultures of the individual strains were incubated with antisera against Borrelia burgdorferi isolate 297 belonging to seroprotective group A, isolate PBi from seroprotective group C and a strain C-1-11 from seroprotective group B.

TABLE 9

<td rowspan="2">"TO" anti-297</td><td rowspan="2">Antiserum "C" anti-PBi</td><td rowspan="2">"B" anti-Chicago</td><td colspan="3">Isolated strains</td>

<td>USA</td><td>European</td><td>Total</td>

<td>S</td><td>R</td><td>P</td><td> 15</td><td> 1</td><td> 16</td>

<td>R</td><td>S</td><td>R</td><td> 0</td><td> 13</td><td> 13</td>

<td>R</td><td>R</td><td>S</td><td> 1</td><td> 0</td><td> 1</td>

<td>S</td><td>R</td><td>S</td><td> 3</td><td> 3</td><td> 6</td>

<td>S</td><td>S</td><td>S</td><td> 1</td><td> 0</td><td> 1</td>

<td>R</td><td>R</td><td>R</td><td> 0</td><td> 6</td><td> 6</td>

S: Sensitive to lethal effect; A: Resistant to lethal effect; PE: Partial lethal effect.

The sensitivity (S) to the lethal effect caused by the anti-297 antiserum, but not by the anti-PBi or anti-Chicago antisera, indicates that an isolated strain of Borrelia burgdorferi is classified in the same seroprotective group, that is, seroprotective group A , like strain 297. Resistance (R) to the lethal effect caused by anti-297 antiserum indicates that a strain should be classified in a different seroprotective group. Table 9 indicates that 15 of the 16 isolates classified in seroprotective group A were isolated in the US Table 9 also indicates that 13 of the 13 isolates sensitive to the lethal effect only in the case of anti-PBi antiserum, and therefore classified in seroprotective group C, they were isolated in Europe. Table 9 also shows that 6 of the 43 isolates were resistant to the lethal effect caused by anti-297, anti-PBi and anti-Chicago antisera, that is, the antibodies did not recognize the cell surface determinants expressed by them. strains. Therefore, these six isolates did not show cross protection with any of the other isolates tested. Therefore, the data demonstrate that there is at least one other seroprotective group of isolates of B. burgdorferi, in addition to seroprotective groups A, B or C.

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Example 13

Natural exposure to ticks using a bivalent vaccine composition

Introduction

Lyme disease, caused by the spirochete Borrelia burgdorferi (Bb) and transmitted by Ixodes ticks, is a multisystem disease that can affect the skin, nervous system, heart and joints of humans and unprotected dogs. (Anderson, JF, et al. (1988) J. Clin. Microbiol. 26: 2209-2212; Appel, et al. (1993) J. Inf. Dis. 167: 651-664; Lane, RS et al. ( 1989) J. Clin. Microbiol. 27: 2344-2349; LeFebvre, RB et al. (1990) J. Clin. Microbiol. 28: 700-707; Levy, SA et al. (1992) Canine Practice 17: 5-14; Nelson, JA, et al. (1991) J. Clin. Microbiol. 29: 1732-1734; Piesman, J. et al. (1990) Am. J. Trop. Med. Hyg. 42: 352-357; Steere, A.

C., et al. (1983) N. Engl. J. Med. 308: 733-740; Steere, AC, et al. (1977) Ann. Intern Med. 86: 685-698). Although antibiotic treatment can be effective, it must be given early in the disease. Antibiotics given during the late stages of the disease may not be effective in completely eliminating the organism from the host. Hamsters or mice vaccinated with experimental bacterins containing inactivated whole cells or antigen subunits generate an immune response that will protect them from artificial or natural routes of infection. (Fikrig, E., et al. (1990) Science 250: 553-556; Fikrig, E., et al. (1992) Proc. Natl. Acad. Sci. 89: 5418-5421; Johnson, RC et al. (1986) Immun. 54: 897-898; Johnson, RC et al. (1986) Zbl. Bakt. Hyg. A. 263: 45-48; Kochi, SK et al. (1988) Infect. and Immun. 56: 314-321; Schmitz, JL et al. (1991) Infect. and Immun. 59: 3815-3818). More recently, a commercialized vaccine has been shown to be effective against exposure in the laboratory and in the field. (Hsien-Jue, C., LG Chavez, Jr., BM Blumer, RW Sebring, TL Wasmoen, and WM Acree. (1992) JAVMA 201: 403-411). Although vaccines provide protection, it is important that immunity (borreliacidal antibodies) be present prior to infection, as infected animals can remain infected despite the presence of circulating borreliacidal antibodies (Callister, SM et al. (1991) J Clin. Microbiol. 29: 1773-1776; Johnson, RC et al. (1986) Immun. 54: 897-898; Johnson, RC et al. (1986) Zbl. Bakt. Hyg. A. 263: 45-48 ; Kochi, SK et al. (1988) Infect. And Immun. 56: 314-321; Schmitz, JL et al. (1991) Infect. and Immun. 59: 3815-3818).

Table 10 presents a list of Bb seroprotective groups that have been characterized in passive protection studies in hamsters and in vitro tests (Borreliacidal Activity Assay) that can differentiate between seroprotective groups on the basis of antibody response after infection (Lovrich, SD et al. (1993) Infect. and Immun. [In press]). Antibodies raised against a strain isolated from one seroprotective group may not provide protection in dogs, or other species, against exposure to a strain isolated from a different seropositive group. Therefore, it is necessary to incorporate isolated strains representing different seroprotective groups to ensure a vaccine of wide efficacy.

Ixodes scapularis ticks infected with Bb can infect mice or dogs when they attach to animals and are allowed to feed on them. (Appel, Max JG et al. (1993) J. Inf. Dis. 167: 651-664; Fikrig, E. et al. (1992) Proc. Natl. Acad. Sci. 89: 5418-5421; Rowhrig, JT et al. (1992) Journ. Immunol. 149: 3648-3653). The immune response to Bb of syringe inoculated dogs is different from that of tick transmission (Rowhrig, JT et al. (1992) Journ. Immunol. 149: 3648-3653). For this reason, we have evaluated the immunogenicity of a bivalent vaccine against natural exposure to ticks.

I. Materials and methods

A. Animals

Four (4) B. burgdorferi (Bb) -free bloodhounds, over 21 weeks of age, from Solvay Animal Health, Inc., Charles City, IA., Were used in a tick challenge model test. Thirty (30) Bb-free bloodhounds, 5-16 weeks old, from Harlan Sprague Dawley, Indianapolis, Indiana, were used for immunogenicity testing (19 vaccinated and 9 unvaccinated). Dogs were found to be free of Bb by serological tests and blood culture in Barbour-Stoenner-Kelly (BSK) medium.

B. Preparation of the bacterin

The bacterin was prepared as described above. The bacterin was prepared with two isolates, C-111 and S-1- 10, which had been propagated by passage 8 times (propagation level by passage: X + 8) from the original stock culture used for the seeding.

C. Vaccination

Nineteen dogs were vaccinated according to the following instructions:

1. The vaccine was allowed to warm to room temperature.

2. The vial was lightly shaken before removing its contents with aseptic technique.

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3. One dose (1.0 ml) was applied intramuscularly to the lower part of the muscle. The initial dose was administered at 5 to 16 weeks of age. The second dose was administered three weeks after the initial vaccination.

The dogs were visually inspected for abnormal reactions to vaccination.

D. Exposure

Male and female Ixodes scapularis ticks were collected from two areas of Wisconsin where Lyme disease is endemic (rural areas near Hixton and Ettrick, WI.). The midguts of 50 and 48 male I. scapularis ticks from Hixton and Ettrick, WI., Respectively, were examined by a Bb-specific fluorescent antibody (FA) assay. These tick pools were pooled and used for both challenge model testing and immunogenicity testing.

1. Detection of B. burgdorferi by immunofluorescence using a monoclonal antibody.

a) Sample preparation:

i. Ticks - The head was detached using a scalpel. The midgut of the tick was removed and smeared on a microscope slide using an applicator stick, and then the smear was thoroughly dried.

ii. Culture - The cultured spirochetes were concentrated by centrifugation (13,000 xg, room temperature) and washed in 0.01 M PBS pH 7.2. Fifteen microliters of the washed suspension were placed on a microscope slide, and dried thoroughly.

b) Dried slides were fixed in acetone for 10 minutes and dried.

c) Mouse monoclonal antibody H5332 (initially obtained from Alan Barbour, University of Texas, San Antonio) was diluted 1:40 using PBS and applied to the slide. The slides were incubated for 30 minutes at 37 ° C in a humid chamber.

d) After incubation, the slides were flushed with PBS and immersed for 5 to 10 minutes in a Coplan cuvette containing PBS.

e) The slides were then reacted with a FITC-labeled antibody conjugate against mouse IgG, diluted 1: 400 with PBS. The slides were incubated for 30 minutes at 37 ° C in a humid chamber.

f) After incubation, the slides were rinsed with a jet of PBS and immersed for 5 to 10 minutes in a Coplan cuvette containing PBS, and the slides were blotted dry.

g) Buffered glycerol and a coverslip were placed on the slides and observed by fluorescence microscopy.

E. Exposure methods

1. Exposure model test. To determine the method of attachment and infection, 4 female ticks and 1 or 2 male ticks were placed in a shaved area located on each side of the thorax of each of 4 dogs. The females were placed in the dogs to feed, while the males are present to encourage full feeding of the females (Appel, Max JG et al. (1993) J. Inf. Dis. 167: 651-664). Ticks were allowed to feed for 1 week; they were then collected, if possible, and examined for the presence of Bb. Skin biopsies were taken from the left side of the fixation site one week after fixation, and from the right side two weeks after fixation.

2. Immunogenicity test. Four female ticks and one male tick were placed in a shaved area located on the right side of each vaccinated and unvaccinated animal one week after the second vaccination. Ticks were allowed to feed for 9 days, then collected, if possible, and examined for the presence of Bb. Fourteen to fifteen days after tick attachment, skin biopsies were taken from the tick attachment site and from a site located approximately 15 cm from the attachment site.

F. Skin biopsies

The sites where skin biopsies were to be collected were shaved, washed with Solvahex ™ solution for surgical scrub, and rinsed thoroughly with sterile water, to remove traces of disinfectant. Skin biopsies

ES 2 265 643 T3 were collected by making elliptical incisions through the dermal and subcutaneous layers of the skin. Each skin biopsy was subdivided into sections, which were placed in 9.0 ml of fresh BSK medium. The samples were homogenized and 1.0 ml was placed in fresh BSK medium containing agarose, with or without rifampin (40 micrograms per milliliter). The cultures were incubated at 32 ° C for 6 weeks and periodically examined for the presence of spirochetes. In the case of the cultures that were positive in the spirochete test, it was confirmed that the germ was Bb by FA. After 6 weeks of incubation, the negative cultures were propagated by passage in fresh BSK medium by inoculating 1.0 ml of the negative culture into 9.0 ml of fresh BSK medium containing agarose and rifampin (40 micrograms per milliliter). . These cultures were incubated at 32 ° C for 6 weeks and examined as described above.

G. Blood (Exposure model and immunogenicity tests)

Blood was drawn into tubes containing sodium citrate at the time of tick attachment and weekly after tick attachment. For each sample 3.0 ml of blood from each dog was placed in 27.0 ml of fresh BSK medium. Blood cultures were further diluted 1: 100 and 1: 1000 using 9.0 ml of BSK blanks. The cultures were incubated at 32 ° C for at least 3 weeks and examined as described above. In the case of the cultures that were positive in the spirochete test, the germ was confirmed to be Bb by FA.

H. Serological tests

1. Exposure model test. Blood was drawn at the time of tick attachment and weekly after tick attachment. Borreliacidal activity assay and ELISA tests were performed on serum obtained from collected blood samples.

2. Immunogenicity test. Blood was drawn at the time of the first and second vaccinations, at the time of tick attachment, and weekly after tick attachment. Serum was obtained and AB tests were performed on samples collected at weeks 0, 4, 9 and 10. ELISA tests were performed on samples collected at weeks 0, 3, 4, 5, 6, 8, 9 Y

10.

I. Borreliacidal Activity Assay Procedure

a) A 72-hour culture of B. burgdorferi (Bb) strain C-1-11 or S-1-10 was quantified in a Petroff / Hauser chamber and diluted to 1 x 10⁵ cells per milliliter with fresh BSK medium.

b) The serum to be tested was diluted 1:20 in fresh BSK medium, filtered through a 0.2 micron filter and heat inactivated at 56 ° C for 45 minutes.

c) To carry out the test, 100 µl of the diluted culture and 100 µl of the heat-inactivated, diluted and filtered serum were added in a 1.5 ml tube with a screw cap. Fifteen (15) microliters of guinea pig complement was added to the same tube. The contents were mixed and incubated at 32 ° C for 2 hours.

d) After the 2 hour incubation period, 800 µl of fresh BSK medium was added to the tube. The tube was then incubated at 32 ° C for 5 to 7 days.

e) After the incubation period of 5 to 7 days, the growth of Bb was determined by withdrawing a sample of 300 to 600 µl from the reaction tube and placing it in a vial containing 10.0 ml of 2.0% saline. . The Bb count in the diluted sample was performed using a Coulter cytometer (Model ZBi). The percentage of dead cells was determined using the following equation:

Percent. Dead cells = 100 Mean cell count in test serum

Mean cell count in negative serum

X100

J. ELISA procedure

1. Whole cell antigen preparation:

to)

a) Isolated strains C-1-11 or S-1-10 of Borrelia burgdorferi were grown in BarbourStoenner-Kelly (BSK) medium to the late log phase (approximately 2 x 10⁸ cells per milliliter) and were inactivated with binary ethyleneimine.

b)

b) Cells were washed with sterile saline (0.85% NaCl, pH 7.10 ± 0.2) three times.

c) Total protein content was determined using a commercial kit.

c)

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2. Test protocol:

a) Immulon-3 plates were coated with C-1-11 or S-1-10 whole cell antigen (0.3 micrograms whole cell antigen in 100 microliters carbonate coating buffer, per well) . The plates were incubated at 4 ° C for 15 to 17 hours in a humid chamber.

b) The contents of each plate were removed (whole cell antigen solution) and 5% (w / v) powdered milk solution was added to each well (400 microliters per well). The plates were incubated at 37 ° C for 60 minutes in a humid chamber.

c) The contents of each plate were removed (5% powdered milk solution) and the plates were allowed to dry for 30 minutes. (Plates were either stored at 4 ° C at that time, or used immediately).

d) Serum samples were diluted 1: 200 in 0.01 M PBS pH 7.2, 0.05% Tween-20, and analyzed in triplicate by adding 50 microliters of the diluted serum to each of two wells. Positive and negative control serum samples were also tested in duplicate on each plate. The plates were incubated at 37 ° C for 60 minutes.

e) The contents of each plate were removed (serum sample dilutions) and the plates were washed three times with a 0.9% NaCl, 0.05% Tween-20 solution.

f) Peroxidase-labeled goat antibody conjugate against canine IgG (heavy and light chains) diluted 1: 1500 in 0.01 M PBS pH 7.2, 0.05% Tween-20 ( 50 microliters per well). The plates were incubated at 37 ° C for 60 minutes in a humid chamber.

g) The contents of each plate were removed (conjugate solution) and the plates were washed as in step 6 above.

h) The O-phenylenediamine substrate (30.0 mg) was dissolved in a solution of 0.051 M dibasic sodium phosphate, 0.0224 M citric acid, 0.012% hydrogen peroxide (74.83 ml) and added to each well (100 microliters per well). The substrate was allowed to react for 8 to 10 minutes.

i) The substrate reaction was stopped with 50 microliters per well of 2N sulfuric acid. The optical density of each well was measured at 490 nm. The mean optical density values ​​of the test wells were normalized against the optical density values ​​of the positive control (i.e .: the mean optical density values ​​of the test wells of a tested serum sample were divided by the mean values ​​of the optical density of the positive control).

K. Clinical signs

After challenge, all dogs were observed for clinical signs such as fever, lethargy, lameness, and loss of appetite.

L. Statistical analysis

Results of skin biopsy cultures were compared using Fisher's exact test.

II. Results

A. Vaccination

There were no abnormal reactions to vaccination.

B. Ticks

The samples analyzed by FA from male ticks collected at Hixton and Ettrick were found to have infection levels of 52% and 54%, respectively.

C. Exposure model test

The ticks were fixed in a period of between 1 and 2 hours. After 2 days, the ticks swelled up. Three days later, most of the dogs had shed their attached ticks, however, three swollen ticks were collected from one of the four dogs (# 450). Bb cells were cultured from blood drawn from the hemocele of 1 of the 3 ticks, and the midgut of the same tick was positive in the FA test for Bb. Skin biopsies taken from the attachment site of this dog one week after attachment were not positive for Bb. In fact, one week after tick fixation, spirochetes were recovered from skin biopsies only in the case of one dog (Table 11a). As opposed,

ES 2 265 643 T3 two weeks after tick fixation, spirochetes were recovered from skin biopsies performed on the opposite side of all 4 dogs (Table 11a). Dogs had spirochetes in their blood 2 and 9 weeks after tick attachment (Table 11b). Borreliacidal antibodies were detectable and increased at 10 and 11 weeks after tick attachment (Table 12a). Antibodies against both C-1-11 and S-1-10 antigens were detected, determined by ELISA after tick fixation (Table 12b).

TABLE 11a

Exposure model test: B. burgdorferi cells cultured from skin biopsy samples taken at the tick attachment site one and two weeks after tick attachment Skin biopsy cultures

Dog # One week after fixation Two weeks after fixation

<td> 450</td><td> 0/5^to</td><td> 3/3</td>

<td> 400</td><td> 4/5</td><td> 2/4</td>

<td> 402</td><td> 0/5</td><td> 4/4</td>

<td>R</td><td>_Cb</td><td> 2/2</td>

^to Positive samples by number of samples analyzed.

^b Contaminated.

TABLE 11b

Exposure model test: B. burgdorferi cells cultured from blood taken from dogs after tick fixation Blood cultures

Weeks after fixation

<td>Dog No.^°</td><td> 1</td><td> 2</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td> 13</td><td> 14</td>

<td> 450-</td><td>_-</td><td> +</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td>

<td> 400-</td><td> -</td><td> +</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 402-</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>R-</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

+ (Growth)

- (No growth)

TABLE 12a

Exposure model test: Borreliacidal activity assay values ​​(means ± sd of the percentage of dead cells in four dogs) against C-1-11 and S-1-10 after tick fixation

Borreliacidal activity assay Mean percentage of dead cells

Weeks after fixation^to

<td>Isolated strain</td><td> 0</td><td> 1</td><td> 2</td><td> 9</td><td> 10</td><td> 11</td>

<td>C-1-11</td><td> 6±4</td><td> 6±4</td><td> 6±6</td><td> 2±3</td><td> 14 ± 10^b</td><td> 21 ± 9^b</td>

<td>S-1-10</td><td> 2±2</td><td> 5±5</td><td> 1±1</td><td> 7 ± 13^b</td><td> 13 ± 17</td><td>20 ± 18b</td>

^to AB tests for weeks 12-16 are ongoing.

^b Standard deviations were high due to negative AB responses in some dogs.

ES 2 265 643 T3

TABLE 12b

Exposure model test: Antibodies detected against S-1-10 and C-1-11 antigens measured by ELISA after tick fixation

ELISA

Mean optical density values ​​Weeks after fixation

Isolated strain: C-1-11

<td></td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td> 13</td><td> 14</td><td> 15</td><td> 16</td>

<td>Average ±</td><td> 0,408</td><td> 0,392</td><td> 0,446</td><td> 0,429</td><td> 0,464</td><td> 0,447</td><td> 0,490</td><td> 0,455</td>

<td>DT^to</td><td> 0,233</td><td> 0,251</td><td> 0,261</td><td> 0,262</td><td> 0,258</td><td> 0,262</td><td> 0,306</td><td> 0,266</td>

<td>Isolated strain:</td><td>S-1-10</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average ±</td><td> 0,499</td><td> 0,374</td><td> 0,385</td><td> 0,406</td><td> 0,414</td><td> 0,601</td><td> 0,559</td><td> 0,573</td>

<td>DT^to</td><td> 0,251</td><td> 0,256</td><td> 0,226</td><td> 0,259</td><td> 0,263</td><td> 0,365</td><td> 0,373</td><td> 0,326</td>

^to Standard deviations were high because one dog had uniformly low ELISA responses against both C-1-11 and S-1-10 antigens.

D. Test of immunogenicity

One to four ticks were collected from all but 6 vaccinated dogs (the dogs had released or detached the bandages in the later stages of feeding, thus losing the ticks that had attached and fed). Some of the recovered ticks were damaged or dead. Bb-positive ticks were recovered from 3 of 19 vaccinated and 3 of 9 controls (Table 13). The existence of Bb-positive ticks did not necessarily correlate with the observed infection.

Four of nine (44%) unvaccinated dogs had positive skin biopsies (Table 4). All skin biopsies performed on vaccinated dogs were negative for Bb, indicating a significant reduction (P = 0.0016, Fisher's exact test) in infection in the vaccinated group. In all cultures in which spirochetes grew, these were identified as Bb by FA. All cultures propagated by blind passage remained negative. None of the skin biopsies performed approximately 15 cm from the tick attachment zone in the control groups or with vaccinated animals was positive for Bb (Table 13).

(Table goes to next page)

ES 2 265 643 T3

TABLE 13

Immunogenicity test: B. burgdorferi cells identified by FA in ticks recovered from dogs and cultured from skin biopsies performed at the tick attachment site and at another remote site approximately two weeks after tick attachment

No. of ticks pos. by AF for Vaccinated n. of analyzed

Fixation site No. of biop. pos. by

n. of analyzed

Remote site No. of biop. pos. by

n. of analyzed

<td>KCP</td><td>NT^to</td><td> 0/4</td><td> 0/2</td>

<td>PIP</td><td> 1/2</td><td> 0/4</td><td> 0/2</td>

<td>KHP</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>PGP</td><td> 0/3</td><td> 0/4</td><td> 0/2</td>

<td>OGO</td><td>NT</td><td> 0/4</td><td> 0/2</td>

<td>JAO</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>HXO</td><td> 0/4</td><td> 0/4</td><td> 0/2</td>

<td>KIP</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>PDP</td><td>NT</td><td> 0/4</td><td> 0/2</td>

<td>PBP</td><td> 0/2</td><td> 0/4</td><td> 0/2</td>

<td>JIO</td><td>NT</td><td> 0/4</td><td> 0/2</td>

<td>LUP</td><td> 0/3</td><td> 0/4</td><td> 0/2</td>

<td>PZP</td><td>NT</td><td> 0/4</td><td> 0/2</td>

<td>PQP</td><td> 0/3</td><td> 0/4</td><td> 0/2</td>

<td>HZO</td><td> 1/3</td><td> 0/4</td><td> 0/2</td>

<td>OEP</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>IXP</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>OWO</td><td>NT</td><td> 0/4</td><td> 0/2</td>

<td>RJP</td><td> 1/3</td><td> 0/4</td><td> 0/2</td>

<td>Dogs pos. /n.° of exposed</td><td></td><td> 0/19</td><td> 0/19</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td>OIP</td><td> 2/4</td><td> 3/3</td><td> 0/2</td>

<td>LLO</td><td> 1/4</td><td> 0/4</td><td> 0/2</td>

<td>QDP</td><td> 0/2</td><td> 0/4</td><td> 0/2</td>

<td>JXP</td><td> 0/2</td><td> 3/4</td><td> 0/2</td>

<td>OPP</td><td> 0/2</td><td> 0/4</td><td> 0/2</td>

<td>JBO</td><td> 0/1</td><td> 1/4</td><td> 0/2</td>

<td>JCO</td><td> 0/1</td><td> 0/4</td><td> 0/2</td>

<td>IOP</td><td> 2/2</td><td> 0/4</td><td> 0/2</td>

<td>KYO</td><td> 0/2</td><td> 2/4</td><td> 0/2</td>

<td>Dogs pos. /n.° of exposed</td><td></td><td> 4/9</td><td> 0/9</td>

^to NT (Not analyzed. Ticks were damaged or viable ticks were not recovered).

Table 14 presents the results of the blood cultures. One of nine unvaccinated animals had positive blood cultures at 1:10 and 1: 100 dilutions approximately one week after tick attachment (ie, during feeding). The rest of the blood cultures were negative, except in week 6 after the fixation of the tick. At that time, 9 of 19 vaccinated animals and 6 of 9 control animals had positive blood cultures at the 1: 100 and 1: 1000 dilutions (determined by growth and by FA), but not at the initial 1:10 dilution. As this was a questionable result, the blood samples were cultured again, and the blood and the 1:10 dilution were analyzed by FA. There was no spirochete growth in the recultured blood and no spirochetes were detected in the blood or at the 1:10 dilution by FA.

ES 2 265 643 T3

TABLE 14

Immunogenicity test: B. burgdorferi cells cultured from blood taken from dogs before and after tick fixation Blood cultures

Weeks after fixation

<td>Vaccinated</td><td> 0</td><td> 1</td><td> 2</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>KCP</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td>

<td>PIP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>KHP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>PGP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>OGO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>JAO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>HXO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>KIP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>PDP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>PBP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>JIO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>LUP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>PZP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>PQP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>HZO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>OEP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>IXP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>OWO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>RJP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>Dogs pos. by no. of analyzed</td><td></td><td></td><td></td><td></td><td></td><td> 9/19</td><td></td><td></td><td></td><td></td>

<td>Not vaccinated</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>OIP</td><td>_-</td><td> +</td><td>_-</td><td>_-</td><td>_-</td><td> +</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td>

<td>LLO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>QDP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>JXP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>OPP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>JBO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>JCO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> +</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>IOP</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>KYO</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>Dogs pos. by no. of analyzed</td><td></td><td> 1/9</td><td></td><td></td><td></td><td> 6/9</td><td></td><td></td><td></td><td></td>

^to Blood collected at the time of attachment of the ticks to the dogs. + (Growth)

- (No growth)

Figures 4 and 5 illustrate borreliacidal antibody responses against isolates S-1-10 and C-1-11 after vaccination and tick attachment. Serum samples from vaccinated animals showed a strong antibody response against S-1-10 and C-1-11 one week after the second vaccination. Five weeks after tick attachment, borreliacidal antibody responses against S-1-10 and C-1-11 increased slightly in

ES 2 265 643 T3 the group with unvaccinated animals. However, 6 weeks after tick attachment, a more intense borreliacidal antibody response was observed only against the S-1-10 strain in the unvaccinated group.

The serum samples analyzed by ELISA also showed a potent antibody response against the S-1-10 and C-1-11 antigens one week after the second vaccination (Figures 3 and 4). Afterward, the antibody response slowly decreased. Higher antibody responses were observed in unvaccinated animals at weeks 8, 9 and 10 (4, 5 and 6 weeks after tick attachment - Figures 6 and 7).

None of the dogs exhibited adverse reactions to vaccination, either immediately, or during observation periods after vaccinations and tick fixation.

To date, none of the dogs have exhibited the clinical signs associated with Lyme disease.

III. Discussion

All dogs in the challenge model study were infected, as determined by skin biopsy cultures (4 of 4 positive dogs), blood cultures (4 of 4 positive dogs), and serological tests (all dogs except dog No. 450, presented seroconversion, determined by AB or ELISA). Dogs are currently under observation for clinical signs. The ticks used to infect these dogs were also used to infect the immunogenicity test dogs.

Ticks infected 4 of 9 unvaccinated animals and none of 19 vaccinated animals, indicating a significant reduction (P = 0.0016, Fisher's exact test) in infection and correlating with the presence of borreliacidal antibody. There was a significant reduction (P = 0.0422, Fisher's exact test) in infection in the vaccinated group when infection from skin biopsies and the presence of spirochetes in blood were jointly assessed.

The animals vaccinated for the immunogenicity test exhibited potent borreliacidal antibody responses against both strains isolated from the vaccine at the time of tick attachment. Following tick attachment, AB-measured antibody responses decreased in the vaccinated group for both isolates. In contrast, borreliacidal antibodies against S-1-10 increased in the unvaccinated group. This increase reflected the infections observed in this group. An increase in the unvaccinated group directed against both C-1-11 and S-1-10 antigens was determined by ELISA. These observations corroborate those cited above (Lovrich, SD et al. (1993) Infect. And Immun. [In press]) in the sense that the borreliacidal activity assay distinguishes between isolates of different seroprotective groups, while the ELISAs are unable to make such a distinction. The data indicate that Borrelia cells carried by ticks can probably be classified in the same seroprotective group to which S-1-10 belongs. However, the mean AB responses increased against both isolates in the post-tick challenge challenge model test. It is possible that all the ticks were infected with some of the isolates from one of the seropositive groups, with both groups, or perhaps with other seroprotective groups.

Bacteria grown in a medium rich in proteins and incorporated in multi-component bacterins are capable of producing adverse reactions (edema, swelling, inflammation, lethargy, hypersensitivity) caused by the interaction between the animal, the vaccine and the environment. In the present study, no abnormal reactions were observed in any of the vaccinated animals after each vaccination with a bacterin containing 2 isolated strains of Bb.

IV. Conclution

The ticks used in these studies were able to infect dogs with B. burgdorferi.

The vaccine generated high levels of borreliacidal antibodies, was effective against natural exposure to ticks, and was safe.

Example 14

Immunogenicity test and study of the duration of immunity.

I. Materials and methods

A. Animals

Bloodhounds from the colony located at Solvay Animal Health, Inc., Charles City, Iowa were used in this study. Dogs were 12-24 weeks of age at the time of vaccination (Table 15) and were seronegative (&lt;1: 20) to B. burgdorferi as determined by whole cell ELISA.

ES 2 265 643 T3

TABLE 15

Dogs used in the study

Dog # Sex Date of birth Age (weeks) in the first vac.

Vaccinated

<td> 558</td><td>H</td><td> 5-10-92</td><td> 18</td>

<td> 560</td><td>H</td><td> 5-06-92</td><td> 20</td>

<td> 562</td><td>H</td><td> 5-11-92</td><td> 20</td>

<td> 564</td><td>H</td><td> 5-11-92</td><td> 20</td>

<td> 566</td><td>H</td><td> 5-11-92</td><td> 20</td>

<td> 568</td><td>H</td><td> 5-11-92</td><td> 20</td>

<td> 570</td><td>H</td><td> 5-25-92</td><td> 18</td>

<td> 574</td><td>H</td><td> 5-25-92</td><td> 18</td>

<td> 580</td><td>H</td><td> 5-26-92</td><td> 16</td>

<td> 582</td><td>H</td><td> 5-26-92</td><td> 16</td>

<td> 584</td><td>H</td><td> 6-19-92</td><td> 12</td>

<td> 586</td><td>H</td><td> 6-19-92</td><td> 12</td>

<td> 609</td><td>M</td><td> 4-14-92</td><td> 24</td>

<td> 615</td><td>M</td><td> 4-14-92</td><td> 24</td>

<td> 617</td><td>M</td><td> 4-22-92</td><td> 20</td>

<td> 627</td><td>M</td><td> 4-22-92</td><td> 22</td>

<td> 639</td><td>M</td><td> 5-06-92</td><td> 18</td>

<td> 641</td><td>M</td><td> 5-06-92</td><td> 18</td>

<td> 643</td><td>M</td><td> 5-06-92</td><td> 18</td>

<td> 647</td><td>M</td><td> 5-11-92</td><td> 18</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td> 554</td><td>H</td><td> 4-22-92</td><td> 22</td>

<td> 556</td><td>H</td><td> 5-10-92</td><td> 20</td>

<td> 598</td><td>H</td><td> 8-12-92</td><td> 4</td>

<td> 608</td><td>H</td><td> 8-12-92</td><td> 4</td>

<td> 610</td><td>H</td><td> 8-17-92</td><td> 4</td>

<td> 612</td><td>H</td><td> 8-17-92</td><td> 4</td>

<td> 651</td><td>M</td><td> 5-25-92</td><td> 18</td>

<td> 655</td><td>M</td><td> 5-26-92</td><td> 18</td>

<td> 661</td><td>M</td><td> 7-10-92</td><td> 11</td>

<td> 663</td><td>M</td><td> 7-14-92</td><td> 11</td>

<td> 665</td><td>M</td><td> 7-14-92</td><td> 11</td>

<td> 669</td><td>M</td><td> 8-12-92</td><td> 4</td>

<td> 671</td><td>M</td><td> 8-12-92</td><td> 4</td>

<td> 673</td><td>M</td><td> 8-12-92</td><td> 4</td>

<td> 677</td><td>M</td><td> 8-12-92</td><td> 4</td>

B. Preparation of the bacterin

Bacterin was prepared with B. burgdorferi serotypes S-1-10 and C-1-11 after eight propagation passages from the original stock culture for seed, as described above. The bacterin was formulated to contain 5 x 10⁸ cells of each serotype for each one-milliliter dose, and aluminum hydroxide (Rehydragel HPA, Reheis Chemical Co., Berkeley Hts., New Jersey) was used as adjuvant at a concentration of 1.5 mg of aluminum oxide for each dose. of a milliliter. The vaccine was stored at 4 ° C until use.

C. Vaccination

Twenty dogs were vaccinated intramuscularly in the lower thigh area using two 1.0 ml doses separated by an interval of three weeks. The first dose was administered at 12-24 weeks of age. The second

ES 2 265 643 T3 dose was administered three weeks after the first dose. The dogs were observed for abnormal reactions; Temperatures were recorded and injection sites were palpated daily for one week after each vaccination. A group of fifteen unvaccinated dogs served as controls.

D. Serological tests

Blood was collected before and after vaccination, and at intervals after challenge. Serum was tested for antibodies to B. burgdorferi using a borreliacidal antibody assay, whole cell ELISA and OspA ELISA, as well as immunoblotting.

E. Whole cell ELISA

The antibody response against surface antigens of B. burgdorferi was determined using a modification of a whole cell ELISA test used at the Regional Animal Health Laboratory, Baron, WI. The late log phase cultures of both strains C-1-11 and S-1-10 were inactivated with binary ethyleneimine (BEI). After neutralization of BEI with sodium thiosulfate, cells were washed by centrifugation three times with sterile saline. The total protein content of the inactivated microorganisms was determined using the bicinchoninic acid (BCA) protein assay (Pierce Co., Rockford, IL). Wells of Immunol-3 plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with whole cell antigens at a level of 0.3 pg in 100 µl of sodium carbonate coating buffer. The plates were incubated in a humid chamber at 4 ° C for 15 to 17 hours. After incubation, the contents of the plate were discarded and the wells were filled with PBS containing 5% skimmed milk powder (NFDM) and incubated in a humid chamber for 60 minutes at 37 ° C. The wells were emptied and 50 µl of test serum diluted in PBS containing 0.05% Tween20 (PBSTW) was added to duplicate wells, and incubated in a humid chamber for 60 minutes at 37 ° C. Positive and negative dog control serum was included in each plate. Plates were washed three times with saline containing 0.05% Tween-20, and 50 µl aliquots per well of peroxidase-labeled goat antibodies to canine IgG (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg , MD) and diluted 1: 1500 in PBS-TW. The plates were incubated in a humid chamber for 60 minutes at 37 ° C and washed three times with PBSTW. The substrate was prepared by dissolving 30.0 mg of O-phenylenediamine in a solution of 0.051 M dibasic sodium phosphate, 0.024 M citric acid, 0.012% hydrogen peroxide, and 100 µl aliquots were added to each well. The reaction was stopped with 50 µl per well of 2N sulfuric acid, and the optical density of each well was determined at 490 nm in an ELISA reader. The titer was defined as the reciprocal of the last dilution that gave an optical density of 30% of the maximum optical density.

E. Osp A ELISA

Wells of a 96-well microtiter plate were coated with 50 ng of recombinant OspA and incubated overnight at 4 ° C. After coating the wells were treated with 5% NFDM in PBS for 30 minutes at 37 ° C. The wells were washed three times with PBS-TW and 50 µl of two-fold dilutions of dog serum were added to the wells. The plates were incubated at 37 ° C for one hour. The wells were washed with PBS-TW and 50 µl of HRP-labeled goat antibodies against canine IgG (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD) were added to the wells. After incubating for one hour at 37 ° C, bound antibodies were detected by adding the ABTS substrate (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD). The optical density of each well was determined at 405 nm in an ELISA reader. The titer was defined as the reciprocal of the last dilution that gave an optical density of 30% of the maximum optical density.

F. Immunoelectroblot

B. burgdorferi cultures in the mid to late log phase were harvested by centrifugation at 15,000 xg, 4 ° C, 30 minutes, and washed three times by centrifugation with sterile saline. A suspension of approximately 1 x 10⁸ Cells were boiled in electrophoresis sample buffer for nine minutes and electrophoresed on a 10% SDS-polyacrylamide gel (Laemmli, EK (1970) Nature 227: 680-685). Proteins were electroblotted onto a PVDF Immobilon ™ membrane (Millipore Corp., Bedford, MA) using a modification of the procedure described by Towbin (Towbin, H. et al. (1979) Proc. Natl. Acad. Sci. 76: 4350 -4354). The PVDF membrane was incubated for 90 minutes at 22 ° C in 20 mM Tris, 150 mM NaCl, pH 7.2 (TBS) with 5% NFDM. The strips were incubated with dog serum or monoclonal antibody against OspA diluted 1:75 in the blocking buffer for 60 minutes at 22 ° C. The strips were then washed twice in TBS containing 0.2% Triton X-100, and once in TBS. Bound antibody was detected by adding antibodies, labeled with horseradish peroxidase, against canine IgG or against murine IgG (Kirkegaard &amp; Perry Laboratories Inc., Gaithersburg, MD). Protein bands were visualized with a membrane peroxidase TMB substrate system (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD).

G. Detection of borreliacidal antibodies by flow cytometry

The borreliacidal activity assay was carried out using a modification of the procedures described above (Lim, LC et al., (1993) Clin. Diag. Lab. Immunol. (In press; Sachsenmeirer, KF et al. (1992) J Clin. Microbiol. 30: 1457-1461) Briefly, 72-hour mid-log cultures of B. burgdorferi isolates C-1-11 and S-1-10 in Barbour-Stoenner-Kelly medium (BSK) were quantified in a

ES 2 265 643 T3 chamber of Petroff-Hauser and diluted to 1 X 10⁶ cells / ml of BSK medium. Aliquots of 100 µl of diluted, heat-inactivated serum were mixed with 100 µl of each B. burgdorferi suspension, and 10 µl of guinea pig serum complement (210 CH units) were added.₅₀; GIBCO Laboratories, Grand Island, NY). The suspension was mixed gently and incubated at 32 ° C for 16 to 24 hours. After incubation of the reaction tubes, 100 μl of the reaction mixture was diluted with phosphate buffered saline (PBS) containing 5.4 X 10 acridine orange.^-9 M. Detection of borreliacidal activity was carried out using a modification of the procedures described above (Callister, SM et al., (1992) J. Infec. Dis. 167: 158-164). Cell death caused by borreliacidal antibodies causes bulges to appear on the cell walls of B. burgdorferi, thereby absorbing higher concentrations of acridine orange into cells and damaged cell walls. Consequently, dead B. burgdorferi organisms fluoresce much more intensely than normal live spirochetes. An increase in fluorescence intensity of&gt; 16% compared to microorganisms exposed to normal dog serum was considered a positive test for borreliacidal activity. The end point titer was expressed as the reciprocal of the last dilution at which a&gt; 16% increase in fluorescence intensity was observed. Dogs with a titer of &lt;1:20 were considered negative.

H. Exposure of dogs to ticks infected with B. burgdorferi

A total of 631 male and 732 female I. scapularis ticks were collected from an area near Ettrick, Wisconsin, where Lyme disease is endemic. To determine the overall rate of infection of ticks by B. burgdorferi, the midguts of 50 male I. scapularis ticks were examined by an immunofluorescence (FA) test using the monoclonal antibody against Osp A provided by Dr. AG. Barbour. At seven months after the second vaccination, the dogs were challenged with 10 female and 6 male ticks. Female ticks are the only adult ticks that carry the disease. Male ticks are necessary for proper feeding of female ticks. For each dog, five female and three male ticks were randomly selected from the pool of ticks, and placed in two small Petri dishes that were fixed to a shaved area of ​​the anterior left thoracic area of ​​each dog. The ticks were allowed to feed for a week. During this time the ticks were observed at two-day intervals. One week after fixation, the ticks were recovered and the midguts were examined for the presence of B. burgdorferi by FA using the monoclonal antibody against B. burgdorferi.

I. Observation of dogs for clinical signs of Lyme disease

The dogs were observed daily after challenge for clinical signs associated with Lyme disease. The symptom used as the main indicator of the clinical course of Lyme disease was lameness. Lameness was defined as a reluctance to bear weight on the affected limb, with or without swelling and increased temperature in the affected joint, stiff legs, and migration of lameness from one joint to another or from one limb to another. The dogs were also observed for lethargy and fever.

J. Isolation of B. burgdorferi from dogs after challenge

An attempt was made to isolate B. burgdorferi from the skin, blood, joints and organs of dogs. Skin biopsies were performed from anesthetized dogs at eighteen to nineteen days after tick fixation and at the time of autopsy. Skin biopsies were performed at the tick attachment site and, in some dogs, skin biopsies were performed at sites remote from the tick bite site. The remote sites were located ventral to the tick bite site, posterior to the tick bite site, and on the right side of the anterior dorsal thoracic area on the opposite side of the dog. The sites to be performed skin biopsies were shaved, washed with Solvahex ™ solution for surgical scrub, and rinsed thoroughly with sterile water to remove traces of disinfectant. An elliptical incision was made through the dermal and subcutaneous layers of the skin. Approximately one gram of skin was placed in nine milliliters of BSK medium containing 0.15% agarose and 40 pg rifampicin / ml. The biopsy sample was homogenized and two additional 10-fold dilutions of homogenate were prepared in nine milliliters of BSK medium blanks. The cultures were incubated at 32 ° C for six weeks and then examined by microscopy at weekly intervals for the growth of spirochetes. In the case of the cultures showing spirochete growth, it was confirmed that the germ was Bb by FA, ​​using a monoclonal antibody against B. burgdorferi. After six weeks of incubation the negative cultures were subcultured in fresh BSK medium by inoculating one milliliter of the negative culture into nine milliliters of fresh BSK medium. These cultures were incubated at 32 ° C for another six weeks and examined as described above. Cultures that were negative for spirochete growth were discarded.

The whole heart, spleen, kidneys and bladder were homogenized separately in 50 ml of BSK containing agarose and rifampin using a Stomacher apparatus (Seward Medical, London, England). A 50 ml sample was poured and diluted 10^-1 and 10 ² in the middle of BSK. The 50 ml sample and dilutions were incubated for six weeks at 32 ° C, observed and confirmed that the germ was B. burgdorferi as described.

A two to three milliliter sample of cerebrospinal fluid was added to nine milliliters of BSK medium and another 1:10 dilution was prepared in the same medium, incubated and observed as described.

ES 2 265 643 T3

Tissue was removed from the elbow, carpal, knee, and tarsal joints. Tissue from each joint was added to nine milliliters of BSK medium containing agarose and rifampin. Another 1:10 dilution was prepared in the same medium and the cultures were incubated and observed as described.

K. Blood cultures

Heparinized blood samples were collected for isolation of B. burgdorferi at the time of challenge, at weekly intervals after challenge, and at the time of autopsy. Each dog's blood was diluted 10^-1, 10 ² and 10 ³ in the middle of BSK. The cultures were incubated at 32 ° C for six weeks and examined weekly for growth of B. burgdorferi as described.

L. Statistical analysis

Significant differences were compared by Pearson's chi-square analysis.

II. Results

A. Vaccination

The dogs were vaccinated with two doses of vaccine separated by an interval of three weeks. After the first vaccination, 14 out of 20 dogs developed a mild fever of 0.83 ° C (1.5 ° F) above baseline temperatures (Table 16). Temperatures were maintained in most of these dogs for only three days. Slightly elevated temperatures were recorded in dogs 566 and 568 for seven days after vaccination. A swollen vaccination site was observed in dog 560, which was maintained for 4 days and then disappeared. After the second vaccination, 13 of 20 dogs developed a slight fever of 0.83 ° C (1.5 ° F) above the initial temperature, which was maintained for three to four days (Table 17). Dog 615 had elevated temperatures that lasted for eight days. The dogs did not show any other post-vaccination reactions, and no hypersensitivity or anaphylaxis reactions were observed.

(Table goes to next page)

ES 2 265 643 T3

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<td></td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td>

<td></td><td></td><td></td><td></td><td></td><td> ▼“</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>v—</td><td></td><td></td><td>Y·</td><td></td>

<td></td><td></td><td>CO</td><td>CO</td><td>in</td><td>You</td><td>M</td><td>CO</td><td>M</td><td>CN</td><td></td><td></td><td>CN</td><td>CN</td><td>CN</td><td>ID</td><td>r-</td><td>CD</td><td>or</td><td> 05</td><td> 05</td><td>or</td>

<td></td><td></td><td>CO</td><td>CN</td><td>co</td><td>CN</td><td>γ—</td><td>CN</td><td>CN</td><td>CN</td><td>"or</td><td>co</td><td>CO</td><td>CO</td><td>CO</td><td>CO</td><td>CN</td><td>CO</td><td>co</td><td>Y-</td><td>CN</td><td>CO</td>

<td></td><td></td><td>OR</td><td>OR</td><td>or</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>OR</td><td>or</td><td>or</td><td>OR</td><td>OR</td><td>OR</td><td>OR</td><td>or</td><td>OR</td><td>or</td><td>or</td><td>OR</td><td>or</td>

<td></td><td></td><td></td><td>Y-</td><td></td><td></td><td></td><td> ▼“</td><td></td><td> ▼—</td><td></td><td></td><td></td><td>Y"</td><td></td><td></td><td></td><td></td><td></td><td> ▼“</td><td></td><td></td>

<td></td><td></td><td>in</td><td>t-.</td><td>co</td><td>CN</td><td>CN</td><td>Yt</td><td>M</td><td>CN</td><td>CN</td><td></td><td> 05</td><td> 05</td><td>or</td><td>Tf</td><td>CN</td><td>co</td><td> 00</td><td>or</td><td> 00</td><td>F-</td>

<td></td><td>or</td><td>γ—</td><td>Y-</td><td>co</td><td>Y-</td><td>Y-</td><td>CN</td><td>CN</td><td>Y"</td><td>CN</td><td>CN</td><td>CM</td><td>Y-</td><td>co</td><td>CN</td><td>Y-</td><td>CN</td><td>Y-</td><td>CN</td><td>CN</td><td>CN</td>

<td></td><td></td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>OR</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>or</td><td>or</td><td>or</td><td>OR</td><td>or</td><td>OR</td><td>or</td><td>OR</td><td>OR</td><td>OR</td>

<td></td><td></td><td></td><td>Y"</td><td>Y ™</td><td></td><td></td><td></td><td></td><td></td><td>Y"</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Y-</td>

<td></td><td>or</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>fc</td><td>Yt</td><td>co</td><td>ID</td><td></td><td> 05</td><td> 00</td><td>or</td><td>CN</td><td>Yt</td><td>CD</td><td> 00</td><td>or</td><td>or</td><td>CN</td><td>Yfr</td><td> 05</td><td>b-</td><td>Y-</td><td>co</td><td> 1^</td>

<td></td><td>Φ</td><td>r--</td><td> 00</td><td>Y-</td><td>CN</td><td>co</td><td>ID</td><td>CD</td><td>CD</td><td>CD</td><td>CD</td><td>CD</td><td>I--</td><td>oo</td><td> 00</td><td> 00</td><td>OR</td><td></td><td></td><td>Yt</td><td>Yt</td>

<td></td><td> 0.</td><td>ID</td><td>ID</td><td>co</td><td>CD</td><td>CD</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>ID</td><td>CD</td><td>CD</td><td>co</td><td>co</td><td>co</td>

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<img file="ES2265643T3_D0001.tif" />

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B. Antibody response against whole cell antigens of B. burgdorferi

An ELISA test using whole B. burgdorferi cells was used to demonstrate the serological response of vaccinated dogs exposed to B. burgdorferi surface antigens. Before vaccination all dogs were seronegative. At three weeks after the second vaccination the geometric mean of the titer (GMT) was 1,236 against the S-1-10 strain and 816 against the C-1-11 strain (Table 18). Six months later, at the time of challenge, antibody titers in vaccinated animals had decreased to 343 and 234 for S-1-10 and C1-11, respectively. Antibody titers against both strains remained essentially unchanged in vaccinated animals after challenge to infected ticks. Unvaccinated control dogs were seronegative throughout the pre-challenge period. After challenge, all control dogs were seropositive for both S-1-10 and C-1-11. The MGTs in the control dogs were 1167 and 1404 against S-1-10 and C-1-11, respectively, and were four to five times higher than the titers of the vaccinated dogs after challenge. However, the antibody titers against both strains in dog # 669 were 8 to 16 times lower than the MGT of control dogs.

(Table goes to next page)

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<img file="ES2265643T3_D0003.tif" />

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C. Immunoelectroblotting

Sera from vaccinated dogs and non-vaccinated control dogs were tested before and after challenge for reactivity of the antibody with B. burgdorferi specific antigens. All dogs were seronegative before vaccination. Seven months after the second vaccination, the serum of vaccinated dogs contained antibodies that reacted mainly against the Osp A protein (31 kilodaltons) and against a 34 kilodalton protein that corresponded to the molecular weight described for Osp B (Figures 1 to 4 ). The antibodies showed almost the same degree of reaction to these proteins from the S-1-10 and C-1-11 strains. However, the differences in the staining pattern and the relative mobility of the proteins indicated the existence of clear differences between the proteins of the two strains. Antibodies against other B. burgdorferi proteins such as flagellin (41 kilodaltons) and against proteins of higher molecular weight were detected in the serum of some vaccinated animals. After exposure to ticks there were hardly any changes in the immunoblot profile of the vaccinated dogs. Antibodies from vaccinated dogs reacted primarily against Osp A and Osp B. The immunoblot antibody profile obtained in unvaccinated control dogs after exposure to ticks was different from the immunoblot profile of vaccinated dogs. Unvaccinated controls were seronegative before challenge (Figure 5). After challenge, unvaccinated animals produced antibodies against the 41 kilodalton flagellin protein and against a 39 kilodalton protein, as well as against the higher molecular weight B. burgdorferi proteins (Figure 6). Antibodies against Osp A and Osp B were hardly detected in the serum of control dogs after exposure to ticks. Studies have shown that unvaccinated animals produce very low amounts of antibodies against OspA and OspB after exposure to ticks infected with B. burgdorferi (Appel, et al. (1993); Burgdorfer, et al. (1982); Roelerig, et al. (1992); Schaible UE et al., (1993) Immunol. Let. 36: 219-226).

D. OspA ELISA

Recombinant Osp A was used in an ELISA test to determine the antibody response against one of the main protective antigens in dogs after vaccination with the bacterin. The vaccinated dogs generated antibody titers against OspA in the range of 320 to 2560 after the second vaccination (Table 19). The MGT against OspA was 640 after vaccination and 279 before challenge. Antibodies against OspA were not detected in unvaccinated control dogs at eight weeks post-tick challenge.

TABLE 19

Antibody response in ELISA against Osp A

ELISA antibody titer against recombinant Osp A^to

<td>Dog No.</td><td>Before vaccination</td><td>After vaccination^b</td><td>Before the exhibition</td>

<td>Vaccinated</td><td></td><td></td><td></td>

<td> 574</td><td>NEG^c</td><td> 640</td><td> 160</td>

<td> 586</td><td>NEG</td><td> 320</td><td> 160</td>

<td> 615</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 627</td><td>NEG</td><td> 2560</td><td> 320</td>

<td> 639</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 558</td><td>NEG</td><td> 320</td><td> 160</td>

<td> 560</td><td>NEG</td><td> 1280</td><td> 320</td>

<td> 562</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 564</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 566</td><td>NEG</td><td> 1280</td><td> 640</td>

<td> 568</td><td>NEG</td><td> 320</td><td> 320</td>

<td> 570</td><td>NEG</td><td> 1280</td><td> 320</td>

<td> 580</td><td>NEG</td><td> 320</td><td> 320</td>

<td> 582</td><td>NEG</td><td> 320</td><td> 160</td>

<td> 584</td><td>NEG</td><td> 640</td><td> 80</td>

<td> 609</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 617</td><td>NEG</td><td> 320</td><td> 320</td>

<td> 641</td><td>NEG</td><td> 640</td><td> 320</td>

<td> 643</td><td>NEG</td><td> 1280</td><td> 640</td>

<td> 647</td><td>NEG</td><td> 640</td><td> 320</td>

<td>GMT^d</td><td>NEG</td><td> 640</td><td> 279</td>

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TABLE 19 (continued)

ELISA antibody titer against recombinant Osp A^to

<td>Dog No.</td><td>Before vaccination</td><td>After vaccination^b</td><td>Before the exhibition</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td> 598</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 655</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 663</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 669</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 677</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 610</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 612</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 651</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 661</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 671</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 673</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 554</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 556</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 608</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 665</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td>MGT</td><td>NEG</td><td>NEG</td><td>NEG</td>

^to Antibodies against recombinant Osp A protein bound to wells of the plate. ^b Three weeks after the second vaccination. ^c Negative - corresponds to a title &lt;1:20. ^d Geometric mean of the title.

E. Borreliacidal antibody response

The functional activity of the antibodies generated against B. burgdorferi as a consequence of vaccination with the bacterin was measured with the borreliacidal antibody test. The presence of borreliacidal antibodies was demonstrated by inhibition of the growth of B. burgdorferi due to antibody-mediated lysis of the microorganism. The vaccinated dogs generated high titers of borreliacidal antibodies against S-1-10 and C-1-11 (Table 20). The MGT of borreliacidal activity was 520 against strain S-1-10 and 1,076 against strain C-1-11. Similar to the case of the antibody response against whole cells and against OspA, the antibody titers Borreliacides decreased from the second vaccination to the time of challenge. At the time of challenge, seven months after the second vaccination, borreliacidal antibody titers of 113 and 279 were detected for S-1-10 and C-1-11, respectively, in the vaccinated animals. Vaccinated dogs also generated borreliacidal antibodies against a different strain of B. burgdorferi in the same seropositive group as S-1-10 (Table 21). Borreliacidal antibodies were not detected in any of the unvaccinated control dogs before or after challenge (data not shown). The absence of borreliacidal antibodies in control dogs after challenge is similar to the results that showed the lack of detection of antibodies against OspA by OspA ELISA and immunoblotting in control dogs after challenge.

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<img file="ES2265643T3_D0005.tif" />

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TABLE 21

Borreliacidal antibody response against homologous and heterologous isolates of B. burgdorferi

Before the exhibition

<td>Dog No.</td><td>C-1-11</td><td>S-1-10</td><td> 297^to</td>

<td>Vaccinated</td><td></td><td></td><td></td>

<td> 574</td><td> 320</td><td> 20</td><td> 16</td>

<td> 586</td><td> 80</td><td> 160</td><td> 32</td>

<td> 615</td><td> 320</td><td> 80</td><td> 16</td>

<td> 627</td><td> 320</td><td> 160</td><td> 64</td>

<td> 639</td><td> 320</td><td> 320</td><td> 64</td>

<td> 558</td><td> 320</td><td> 80</td><td> 32</td>

<td> 560</td><td> 160</td><td> 160</td><td> 32</td>

<td> 562</td><td> 320</td><td> 160</td><td> 64</td>

<td> 564</td><td> 320</td><td> 160</td><td> 64</td>

<td> 566</td><td> 320</td><td> 80</td><td> 64</td>

<td> 568</td><td> 640</td><td> 160</td><td> 128</td>

<td> 570</td><td> 320</td><td> 160</td><td> 32</td>

<td> 580</td><td> 320</td><td> 160</td><td> 128</td>

<td> 582</td><td> 320</td><td> 80</td><td> 16</td>

<td> 584</td><td> 80</td><td> 80</td><td> 16</td>

<td> 609</td><td> 160</td><td> 320</td><td> 128</td>

<td> 617</td><td> 320</td><td> 160</td><td> 128</td>

<td> 641</td><td> 320</td><td> 40</td><td> 32</td>

<td> 643</td><td> 640</td><td> 320</td><td> 256</td>

<td> 647</td><td> 320</td><td> 20</td><td> 16</td>

<td>GMT^b</td><td> 279</td><td> 113</td><td> 66</td>

<td>Not vaccinated</td><td></td><td></td><td></td>

<td> 598</td><td>NEG^c</td><td>NEG</td><td>NEG</td>

<td> 655</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 663</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 669</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 677</td><td>NEG</td><td>NDd</td><td>NEG</td>

<td> 610</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 612</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 651</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 661</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 671</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 673</td><td>NEG</td><td>ND</td><td>NEG</td>

<td> 554</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 556</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 608</td><td>NEG</td><td>NEG</td><td>NEG</td>

<td> 665</td><td>NEG</td><td>NEG</td><td>NEG</td>

^to Isolated strain of human cerebrospinal fluid from Connecticut.

^b Geometric mean of the title.

^c Negative - corresponds to a titer &lt;1:20 for the isolates C-1-11 and S-1-10, and &lt;1:16 for the isolate 297.

^d Unrealized.

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F. Exposure of dogs to ticks infected with B. burgdorferi

A total of 1,363 I. scapularis ticks (732 females and 631 males) were collected in the vicinity of the Ettrick, WI area. The infection rate for B. burgdorferi in ticks was found to be 44%, determined by FA analysis of the midgut of male ticks. This infection rate is similar to the 47% infection rate described by Lacombe et al. (Lacombe, E, et al., (1993) J. Infect. Dis. 167: 1236-1238). Calculations were performed to determine the probability that each dog received at least one tick at the time of exposure. The results show that there was a 99.4% probability that the last of the 35 exposed dogs received at least one infected tick (Table 22). At the time of challenge, 10 female and 6 male ticks were placed on each dog and allowed to feed for one week. In general, ticks attached within 24 hours, and most ticks fed fully or almost fully swollen during the one-week exposure period. Ticks that had been shed from the dog remained on the plate. At the end of the one-week tick attachment period, the recovered ticks were analyzed by FA for the presence of B. burgdorferi. Ticks were recovered from 18 to 20 vaccinated animals, and at least one tick infected with B. burgdorferi was recovered from 8 of the 18 (44%) animals. Ticks were recovered from 14 to 15 unvaccinated dogs, and 12 of 14 (86%) were positive for B. burgdorferi. Fikrig has also described (15) that fewer infected ticks are recovered from vaccinated animals than from unvaccinated animals (Fikrig, E. et al. (1992) PNAS 89: 5418-5421).

G. Recovery of B. burgdorferi from skin biopsy samples

Isolation of B. burgdorferi from animal skin has been used as an indicator of B. burgdorferi infection. Therefore, skin biopsies were performed from anesthetized dogs 18 days after exposure to ticks to check for infection with B. burgdorferi-infected ticks. B. burgdorferi was isolated from the skin of 1 of 20 (5%) of the vaccinated dogs, compared to 14 to 15 (93%) of the unvaccinated controls (Table 23).

(Table goes to next page)

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Table 22 (continued)

<img file="ES2265643T3_D0007.tif" />

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In Table 22, the theoretical number of infected ticks placed on dogs was based on the current infection rate. For example, 322/732, or 44%, of the ticks were theoretically infected. Therefore, 40% of 10 ticks analyzed indicates a maximum of 5 infected ticks, in the worst case, with the probability of not having enough infected ticks for some of the last dogs in the study. This number was used to assess the new population infection rates for the next dog.

In calculating the probability of having at least 1 infected tick, 10 probabilities (1 tick, 2 ticks, 3 ticks, 4 ticks, ... and 10 ticks) were added based on the current population of infected ticks, total ticks, and total ticks remaining. As a mathematical resource, a hypergeometric distribution was used to determine the probability of having 1 infected tick out of a sample of 10 taken from a population of 732 ticks with 322 infected ticks. To obtain at least 1 infected tick, the probabilities of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ticks were calculated and summed.

TABLE 23

Characterization of infection in vaccinated and unvaccinated animals after exposure to ticks infected with B. burgdorferi

<td>Dog No.</td><td>Skin biopsy^to</td><td>Date lameness was observed</td><td>Affected legs</td><td>Lethargy and fever</td>

<td>Vaccinated</td><td></td><td></td><td></td><td></td>

<td> 574</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td>

<td> 586</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 615</td><td> -</td><td>Jul.</td><td> 2</td><td> -</td>

<td> 627</td><td> -</td><td>Sept.</td><td> 1</td><td> -</td>

<td> 639</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 558</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 560</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 562</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 564</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 566</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 568</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 570</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 580</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 582</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 584</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 609</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 617</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 641</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 643</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 647</td><td> -</td><td> -</td><td> -</td><td></td>

<td>Not vaccinated</td><td></td><td></td><td></td><td></td>

<td> 598</td><td> +</td><td>Jul.</td><td> 4</td><td>L</td>

<td> 655</td><td> +</td><td>Sept.</td><td> 1</td><td> -</td>

<td> 663</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 669</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 677</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 610</td><td> +</td><td>Jun.</td><td> 3</td><td>L, F</td>

<td> 612</td><td> +</td><td>Oct.</td><td> 1</td><td>F</td>

<td> 651</td><td> +</td><td>Jun.</td><td> 1</td><td>F</td>

<td> 661</td><td> +</td><td>Sept.</td><td> 1</td><td>L, F</td>

<td> 671</td><td> +</td><td>Oct.</td><td> 1</td><td>F</td>

<td> 673</td><td> +</td><td>Sept.</td><td> 1</td><td>F</td>

<td> 554</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 556</td><td> +</td><td> -</td><td> -</td><td> -</td>

<td> 608</td><td> +</td><td>Nov.</td><td> 1</td><td>L, F</td>

<td> 665</td><td> +</td><td>Nov.</td><td> 1</td><td>F</td>

<td colspan="3">^to Skin biopsies performed at tick two bite sites</td><td colspan="2">weeks after tick fixation.</td>

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H. Occurrence of lameness in dogs after exposure

The main clinical sign of Lyme disease in dogs is lameness. Protection of dogs against Lyme disease was assessed as a significant reduction in the number of lame dogs between vaccinated animals and controls. So far, dogs have been observed daily for lameness for seven months after exposure to ticks. The number of dogs exhibiting lameness and associated clinical signs are shown in Table 23. Lameness was observed in two vaccinated dogs. One of the vaccinated animals had one affected limb, while the other vaccinated animal had two affected limbs. Ten unvaccinated controls presented lameness, and the associated signs of lethargy and / or fever appeared in 9 of the 10 dogs. The control dog had three affected limbs. Lameness was more pronounced in unvaccinated dogs, in that the dogs were more reluctant to carry weight on the affected limb. In most of the unvaccinated dogs, the affected joints were swollen and hot to the touch.

The temporal observation of lameness in dogs after exposure is presented in Table 24. Lameness was first observed in dogs approximately two months after exposure to ticks. At that time, one vaccinated dog and two control dogs were lame. At the beginning of September, four months after exposure, lameness had been observed in another vaccinated dog and in four other control dogs. Since it was not known if any of the rest of the dogs would become lame, it was decided to autopsy five vaccinated animals and five unvaccinated animals to determine whether B. burgdorferi could be isolated from the dogs. The recovery of the spirochete from the dogs that had not presented lameness would indicate the survival of the microorganism in the dogs, even if they did not present clinical signs. Two of the five dogs in each group had lame and three dogs in each group had remained normal. Because three dogs had been withdrawn from the population of each test group, the number of dogs in the groups with vaccinated animals and in the control group with unvaccinated animals was reduced to 17 and 12, respectively. At seven months after exposure to ticks, lameness was observed in a total of 2 of 17 (12%) vaccinated animals and 10 of 12 (83%) unvaccinated controls. From this a protection index score of 85.6% is deduced, which represents a significant reduction (p &lt;0.01) in clinical disease between vaccinated animals and controls. A summary of the percentage of dogs that presented lameness after exposure is shown in Figure 14.

An attempt was made to autopsy lame dogs within three to four days of the lameness episode, in order to isolate B. burgdorferi from the skin, joints, and organs of the dogs (Table 25). B. burgdorferi was not isolated from the skin, joints, or organs of either of the two lame vaccinated dogs, but the organism was isolated from the skin and joint of the vaccinated dog without lameness that was initially positive on skin biopsy. In contrast, B. burgdorferi from skin biopsy samples, taken at autopsy from the site of the tick bite or from sites remote from the site of the tick bite, from all unvaccinated control dogs that were lame. The spirochete was also isolated from the joints or organs of all unvaccinated lame control dogs. The unvaccinated control dog that was initially skin biopsy negative remained skin biopsy negative at the time of autopsy, and B. burgdorferi was not recovered from the dog's joints or organs.

(Table goes to next page)

ES 2 265 643 T3

Table 24. Number of dogs with lameness after exposure to ticks.

<td></td><td>! Feb.</td><td> 2/17</td><td> 10/12 |</td>

<td></td><td>φ</td><td> 1^</td><td>CM t—</td>

<td></td><td>In</td><td>CM</td><td>or</td>

<td></td><td rowspan="2">or</td><td>h *.</td><td>CM</td>

<td></td><td></td><td>V</td>

<td></td><td>Q</td><td>CM</td><td>or v—</td>

<td></td><td> &gt;</td><td>b-</td><td>CM</td>

<td></td><td>n</td><td></td><td></td>

<td></td><td>z.</td><td>CM</td><td>OR</td>

<td></td><td></td><td>r-</td><td>CM</td>

<td></td><td>or</td><td>Y</td><td>t—</td>

<td></td><td>OR</td><td>CM</td><td>co</td>

<td></td><td>Q</td><td>OR CM</td><td>IT</td>

<td></td><td>Φ</td><td></td><td></td>

<td></td><td>w</td><td>CM</td><td>CO</td>

<td></td><td>ώ</td><td>OR</td><td>IT</td>

<td></td><td>or</td><td>CM</td><td>V</td>

<td></td><td>CT</td><td> —»</td><td></td>

<td></td><td> &lt;</td><td></td><td>CO</td>

<td></td><td>or</td><td>or</td><td>IT</td>

<td></td><td>Zz</td><td>CM</td><td></td>

<td></td><td>Ώ</td><td></td><td> ·«·»».</td>

<td></td><td></td><td></td><td>ου</td>

<td></td><td>or</td><td>OR</td><td>IT</td>

<td></td><td>c</td><td>CM</td><td> ▼—</td>

<td></td><td> □ 0</td><td>OR</td><td>CM</td>

<td></td><td>ayo i</td><td> /20</td><td>tn</td>

<td></td><td> 2</td><td>or</td><td>or</td>

<td></td><td></td><td></td><td>ω</td>

<td></td><td></td><td></td><td>or</td>

<td>OR</td><td></td><td>swims</td><td rowspan="2">Ό CD C 3 OR</td>

<td>Q.</td><td></td><td> □</td>

<td> 3</td><td></td><td>OR</td><td></td>

<td> 1</td><td></td><td>m</td><td>or</td>

<td>or</td><td></td><td> &gt;</td><td>Z</td>

ES 2 265 643 T3

TABLE 25

Recovery of B. burgdorferi from lame dogs

Reisolation of B. burgdorferi at the time of lameness / autopsy

<td>Dog No.</td><td>Biopsy cutaneous^to</td><td>Date of observation of lameness</td><td>Skin</td><td>Joints</td><td>Organs^b</td>

<td>Vaccinated</td><td></td><td></td><td></td><td></td><td></td>

<td> 574</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td><td>_-</td>

<td> 586</td><td> +</td><td> -</td><td> +</td><td> +</td><td> -</td>

<td> 615</td><td> -</td><td>Jul.</td><td> -</td><td> -</td><td> -</td>

<td> 627</td><td> -</td><td>Sept.</td><td> -</td><td> -</td><td> -</td>

<td> 639</td><td> -</td><td> -</td><td> -</td><td> -</td><td></td>

<td> 558</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 560</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 562</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 564</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 566</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 568</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 570</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 580</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 582</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 584</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 609</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 617</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 641</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 643</td><td> -</td><td> -</td><td></td><td></td><td></td>

<td> 647</td><td> -</td><td></td><td></td><td></td><td></td>

<td>Not vaccinated</td><td></td><td></td><td></td><td></td><td></td>

<td> 598</td><td> +</td><td>Jul.</td><td> +</td><td> +</td><td>_-</td>

<td> 655</td><td> +</td><td>Sept.</td><td> +</td><td> +</td><td> -</td>

<td> 663</td><td> +</td><td> -</td><td> +</td><td> +</td><td> -</td>

<td> 669</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 677</td><td> +</td><td> -</td><td> +</td><td> +</td><td> +^B</td>

<td> 610^c</td><td> +</td><td>Jun.</td><td> +</td><td> +</td><td>+ B, H, K</td>

<td> 612</td><td> +</td><td>Oct.</td><td> -/+^d</td><td> +</td><td> -</td>

<td> 651</td><td> +</td><td>Jun.</td><td> +</td><td> +</td><td> +^S</td>

<td> 661</td><td> +</td><td>Sept.</td><td> +</td><td> +</td><td> -</td>

<td> 671</td><td> +</td><td>Oct.</td><td> +/+</td><td> +</td><td> -</td>

<td> 673</td><td> +</td><td>Sept.</td><td> +/+</td><td> +</td><td> -</td>

<td> 554</td><td> +</td><td> -</td><td></td><td></td><td></td>

<td> 556</td><td> +</td><td> -</td><td></td><td></td><td></td>

<td> 608</td><td> +</td><td>Nov.</td><td> -/+</td><td> +</td><td>₊H</td>

<td> 665</td><td> +</td><td>Nov.</td><td> +/+</td><td> +</td><td> -</td>

^to Skin biopsies performed at tick bite sites two weeks after tick attachment.

^b B: Bladder / H: Heart / K: Kidney / S: Spleen ^c B. burgdorferi was also isolated from cerebrospinal fluid.

^d Biopsy performed at the tick bite site / Biopsy performed at a site remote from the tick bite site.

ES 2 265 643 T3

III. Discussion

The humoral immune response has been shown to play a critical role in protecting animals against Lyme disease (Barthold, SW et al. (1993) Infect. Immunol. 61: 4696-4702; Callister, et al. (1991) ); Callister, et al. (1992); Johnson, et al. (1986); Johnson, et al. (1986); Johnson, et al. (1988); Kochi, et al. (1988); Schaible, UE et al. (1990) PNAS 87: 3768-3722; Schmitz, et al. (1991)). In the present study, various serological tests were used to characterize the antibody response in dogs vaccinated with the bacterin. The whole cell ELISA test was used to reveal the antibody response against surface antigens of B. burgdorferi. The generation of antibody titers that persist for seven months after vaccination is a test of the antigenic capacity of the bacterin. The specificity of the antibody response was demonstrated by immunoblotting. At the time of challenge, seven months after vaccination, antibodies in the serum of vaccinated dogs reacted mainly with the surface proteins OspA and OspB. Other investigators have also shown that the predominant antibody response against in vitro cultured B. burgdorferi cells is directed against external surface proteins (Appel, et al. (1993); Gern, L. et al. (1993) J. Infect. Dis. 167: 971-975; Roerhign et al. (1992)). The OspA ELISA test further demonstrated the presence of high antibody titers in the serum of vaccinated dogs and confirmed the ability of the bacterin to elicit an antibody response against a protective spirochete antigen.

The borreliacidal antibody assay was used to demonstrate the functional activity of the antibody against B. burgdorferi in the serum of vaccinated dogs. Antibody-mediated lysis of Borrelia cells is the result of the combination of specific antibodies with complement components and the formation of the membrane attack complex. In effect, growth inhibitor of lysis, mediated by antibodies, of B. burgdorferi can be considered analogous to virus neutralizing antibodies. Studies have shown that protection of laboratory animals against Lyme disease correlates with borreliacidal antibody titers (Jobe, DA et al., (1994) J. Clin. Microbiol. (In press); Lourich, SD et al. . (1991) Infect. Immunol. 59: 2522-2528)). Consequently, the presence of borreliacidal antibodies in vaccinated dogs can be used to demonstrate the immunogenicity of the bacterin. Elevated levels of borreliacidal antibodies were found in all vaccinated dogs, which remained detectable at the time of challenge, seven months after vaccination. Antibodies in vaccinated animals were also borreliacidal for heterologous B. burgdorferi strain 297. Results showing that antibodies from unvaccinated control dogs after challenge did not react with OspA and were not borreliacidal indicate that much of the borreliacidal activity is due to antibodies that react with OspA and other protective surface proteins. Callister et al. Have shown that the borreliacidal activity of antibodies against B. burgdorferi in human serum can be adsorbed with recombinant OspA protein (Callister, SM et al. (1992)). In this way, the dogs vaccinated with the bacterin generated a protective response of antibodies against different strains of the strain present in the vaccine.

In the present study, a natural tick exposure model, similar to that described by Appel et al. (1993)), was used to induce Lyme disease in dogs. The model used represents the natural route of exposure, dose and virulence of B. burgdorferi present in natural conditions on the ground.

The ticks used in the present study were collected from an area where Lyme disease is endemic, and they were found to have a 44% infection rate with B. burgdorferi. This infection rate is similar to that described by Lacombe (Lacombe et al. (1993)), although lower than that described by Appel (Appel et al. (1993). Shih and Spielman have shown that infected ticks transmit B. burgdorferi to mice after feeding on them for only 2 days (Shih C. et al., (1993) J. Clin. Microbiol. 31: 2878-2881). Therefore, the one-week tick feeding period used in the present study would be more than sufficient. In fact, the effectiveness of the exposure was demonstrated in two ways. The whole cell ELISA test detected a serological response against B. burgdorferi in 14 of 15 unvaccinated control dogs after challenge. The results correlated with the results of the skin biopsies in the sense that a dog with little serological response was the same dog whose skin biopsy was negative after exposure and whose skin biopsy was negative at autopsy. Interestingly, fewer B. burgdorferi-infected ticks were recovered from vaccinated dogs than from unvaccinated control dogs. These observations are similar to those made by Fikrig et al. (Frikig et al., PNAS (1992)). These authors conjectured that the reduced number of B. burgdorferi in vaccinated dog ticks was due to increased antibody-mediated clearance of spirochetes.

Lameness is the main manifestation of Lyme borreliosis in dogs, and it was used in the present study as the main criterion of clinical disease. Post-challenge, the temporal development of lameness in dogs was variable over the seven-month period post-challenge. Lameness was observed in two unvaccinated control dogs one month after challenge. However, most lameness episodes did not appear until four to five months after exposure. Appel also described that lameness in dogs did not appear until two to five months after exposure (Appel, et al. (1993)). Ten out of twelve unvaccinated control dogs that were positive on post-challenge skin biopsies developed lameness, and B. burgdorferi was re-isolated from the skin and joints or other organs of all these dogs. The observation that B. burgdorferi remained on the skin of these dogs for up to seven months after exposure and in locations completely remote from the bite site

ES 2 265 643 T3 of the tick highlights the evolutionary development of the parasite to ensure host-to-host transmission. B. burgdorferi was not isolated again from the two vaccinated dogs that presented lameness. The lameness of these two dogs cannot be explained on the basis of the humoral immunity response. Another possible explanation includes the genetic predisposition of dogs, the dose of spirochetes, and the efficiency of transmission from ticks. Although B. burgdorferi has been extensively studied in recent years, the pathogenic mechanisms of the microorganism are not well characterized. The ability of other Borrelia species to be sequestered in immunologically privileged sites of the host and to circumvent its defense mechanisms is well known (Geogitis, K. et al. (1992) J. Infect. Dis. 166: 440-444; Levy et al. (1992), Ramachandra, RN et al. (1992) J. Med. Entomol. 29: 818-826), and this behavior has been conjectured in the case of B. burgdorferi.

The efficacy of the bacterin was assessed by a significant reduction in lameness between vaccinated animals and non-vaccinated dogs exposed to B. burgdorferi-infected ticks. The overall incidence of lameness of 2 out of a total of 17 (12%) vaccinated animals, compared to 10 out of a total of 12 (83%) unvaccinated controls yields a protection index score of 86%. This value represents a significant reduction (p &lt;0.01) in the appearance of the clinical manifestations of Lyme disease between vaccinated animals and controls.

In the present study, the safety of the bacterin was demonstrated. Efficacy and duration of immunity were demonstrated by the ability of the vaccine to reduce the development of clinical disease in vaccinated dogs compared to controls at seven months after exposure to B. burgdorferi-infected ticks.

In comparing the results of Example 13 and Example 14, Example 14 is considered to provide better results due to the use of more ticks, as well as healthier ticks and better skin biopsies in Example 14. As a result, the control dogs were found to have clinical signs of Lyme disease, and consequently, a reduction in the clinical signs of the vaccinated animals is demonstrated compared to the controls when the bacterin of the present invention is used.

Example 15

Reduced dose study

I. Materials and methods

A. Dogs

Bloodhounds from the colony located at Solvay Animal Health, Inc., Charles City, Iowa were used in this study. The mean age of the dogs at the time of vaccination was 13 weeks (Table 26). All dogs were seronegative (titer &lt;20) to B. burgdorferi as determined by whole cell ELISA.

TABLE 26

Sex and age of the dogs used in the study.

Dog # Sex Date of birth Age (weeks) in the first vac.

Full-dose vaccinated

<td> 760</td><td>H</td><td> 5-23-93</td><td> 15</td>

<td> 774</td><td>H</td><td> 6-02-93</td><td> 13</td>

<td> 786</td><td>M</td><td> 6-16-93</td><td> 11</td>

<td> 831</td><td>M</td><td> 5-16-93</td><td> 16</td>

<td> 843</td><td>M</td><td> 5-13-93</td><td> 16</td>

<td> 849</td><td>M</td><td> 5-23-93</td><td> 15</td>

<td> 853</td><td>M</td><td> 5-25-93</td><td> 15</td>

<td> 859</td><td>M</td><td> 6-12-93</td><td> 12</td>

<td> 871</td><td>M</td><td> 6-16-93</td><td> 11</td>

<td> 881</td><td>M</td><td> 6-28-93</td><td> 10</td>

ES 2 265 643 T3

TABLE 26 (continued)

<td>Dog No.</td><td>Sex</td><td>Date of birth</td><td>Age (weeks) in the first vac.</td>

<td>Full-dose vaccinated</td><td></td><td></td><td></td>

<td> 746</td><td>H</td><td> 5-16-93</td><td> 16</td>

<td> 758</td><td>H</td><td> 5-23-93</td><td> 15</td>

<td> 768</td><td>H</td><td> 6-02-93</td><td> 13</td>

<td> 782</td><td>H</td><td> 6-12-93</td><td> 12</td>

<td> 835</td><td>M</td><td> 5-16-93</td><td> 16</td>

<td> 847</td><td>M</td><td> 5-23-93</td><td> 15</td>

<td> 851</td><td>M</td><td> 5-25-93</td><td> 15</td>

<td> 855</td><td>M</td><td> 6-02-93</td><td> 13</td>

<td> 863</td><td>M</td><td> 6-12-93</td><td> 12</td>

<td> 879</td><td>M</td><td> 6-28-93</td><td> 10</td>

<td>Unvaccinated witnesses</td><td></td><td></td><td></td>

<td> 756</td><td>H</td><td> 5-23-93</td><td> 15</td>

<td> 762</td><td>H</td><td> 5-23-93</td><td> 15</td>

<td> 764</td><td>H</td><td> 5-25-93</td><td> 15</td>

<td> 766</td><td>H</td><td> 5-25-93</td><td> 15</td>

<td> 770</td><td>H</td><td> 6-02-93</td><td> 13</td>

<td> 772</td><td>H</td><td> 6-02-93</td><td> 13</td>

<td> 776</td><td>H</td><td> 6-02-93</td><td> 13</td>

<td> 845</td><td>M</td><td> 5-23-93</td><td> 15</td>

<td> 857</td><td>M</td><td> 6-02-93</td><td> 13</td>

<td> 861</td><td>M</td><td> 6-12-93</td><td> 12</td>

<td> 865</td><td>M</td><td> 6-12-93</td><td> 12</td>

<td> 867</td><td>M</td><td> 6-12-93</td><td> 12</td>

<td> 869</td><td>M</td><td> 6-16-93</td><td> 11</td>

<td> 883</td><td>M</td><td> 6-28-93</td><td> 10</td>

B. Preparation of the bacterin

Two bacterins were used in the study. The full dose of bacterin was prepared from B. burgdorferi serotypes S-110 and C-1-11 in the eighth round of passage by passage of the original stock culture for sowing, as described in Examples 1 and 2 previous. This bacterin was formulated to contain 5 x 10⁸cells of each serotype for each dose of one milliliter, and 7.5% (w / v) aluminum hydroxide (Rehydragel HpA, Reheis Chemical Co., Berkeley Hts., New Jersey) was used as adjuvant. Reduced dose bacterin was prepared by diluting the full dose of bacterin 1:10 in adjuvant diluent composed of saline containing 7.5% (w / v) aluminum hydroxide (Rehydragel HPA) and gentamicin and nystatin as preservatives. Both bacterins were stored at 4 ° C until they were used.

C. Vaccination

Ten dogs were vaccinated intramuscularly in the lower thigh area using two 1.0 ml doses separated by an interval of three weeks for each bacterin. After each vaccination the dogs were observed for abnormal reactions, temperatures were recorded, and injection sites were palpated daily for five days. A group of fourteen unvaccinated dogs served as controls.

D. Serological tests

Blood was collected before and after vaccination, and at intervals after challenge. Serum was tested for antibodies to B. burgdorferi using whole cell ELISA, OspA ELISA, borreliacidal activity assay, and immunoblotting.

ES 2 265 643 T3

1. Whole cell ELISA

The antibody response in dogs against B. burgdorferi surface antigens was determined using a modification of a whole cell ELISA test used at the Regional Animal Health Laboratory, Baron, WI. The late log phase cultures of B. burgdorferi strains C-1-11 and S-1-10 were inactivated with binary ethyleneimine (BEI). Wells of Immunol-3 plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with S-1-10 or C-1-11 whole cell antigens at a level of 0.3 pg in buffer sodium carbonate coating. The plates were incubated in a humid chamber at 4 ° C for 15 to 17 hours. The contents of the wells were discarded and unbound reactive sites were blocked by adding phosphate buffered saline (PBS) containing 5% skimmed milk powder (PBS-NFDM), and incubated in a humid chamber for 60 minutes at 37 ° C. The wells were emptied and 50 µl of test serum diluted in PBS containing 0.05% Tween-20 (PBS-TW) was added to duplicate wells, and incubated in a humid chamber for 60 minutes at 37 ° C. Positive and negative dog control serum was included in each plate. Plates were washed three times with saline containing 0.05% Tween-20, and 50 µl aliquots were added per well of peroxidase-labeled goat antibodies to canine IgG (Kirkegaard &amp; Perry Laboratories, Inc. , Gaithersburg, MD) and diluted 1: 1500 in PBS-TW. The plates were incubated in a humid chamber for 60 minutes at 37 ° C and washed three times with PBS-TW. The substrate was prepared by dissolving 30.0 mg of O-phenylenediamine in a solution of 0.051 M dibasic sodium phosphate, 0.024 M citric acid, 0.012% hydrogen peroxide, and 100 µl aliquots were added to each well. The reaction was stopped with 50 µl per well of 2N sulfuric acid, and the optical density of each well was determined at 490 nm in an ELISA reader. The titer was defined as the reciprocal of the last dilution that gave an optical density of 30% of the maximum optical density.

2. OspA ELISA

Wells of a 96-well microtiter plate were coated with 75 ng of recombinant OspA from S-1-10 or C-1-11 and incubated overnight at 4 ° C. After coating the wells were treated with 5% NFDM in PBS for 30 minutes at 37 ° C. The wells were washed three times with PBS-TW and 50 µl of two-fold dilutions of dog serum were added to the wells. After incubation at 37 ° C for one hour, the wells were washed with PBS-TW and 50 μl of HRP-labeled goat antibodies against canine IgG (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD) were added to the wells. ). Bound antibodies were detected by adding the ABTS substrate (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD). The optical density of each well was determined at 405 nm in an ELISA reader. The titer was defined as the reciprocal of the last dilution that gave an optical density of 30% of the maximum optical density.

3. Immunoelectroblotting

Cultures of B. burgdorferi strains S-1-10 and C-1-11 in the mid to late log phase were harvested by centrifugation at 15,000 xg at 4 ° C for 30 minutes. The bacteria were washed three times by centrifugation with sterile saline. Suspensions of approximately 1 x 10 were boiled⁸ cells in electrophoresis sample buffer for nine minutes, and electrophoresed on a 10% SDS-polyacrylamide gel (Laemmli, 1970). The separated proteins were electroblotted onto a PVDF Immobilon ™ membrane (Millipore Corp., Bedford, MA) using a modification of the procedure described by Towbin (Towbin et al. 1979). The PVDF membrane was incubated for 90 minutes at 22 ° C in 20 mM Tris, 150 mM NaCl, pH 7.2 (TBS) with 5% NFDM. The strips were incubated with canine serum or with monoclonal antibody against OspA diluted 1:75 in the blocking buffer for 60 minutes at 22 ° C. The strips were then washed twice in TBS containing 0.2% Triton X-100 and once in TBS. Bound antibody was detected by adding HRP-conjugated goat antibodies to canine IgG or to murine IgG (Kirkegaard &amp; Perry Laboratories Inc., Gaithersburg, MD). Protein bands were visualized with a membrane peroxidase TMB substrate system (Kirkegaard &amp; Perry Laboratories, Inc., Gaithersburg, MD).

F. Detection of borreliacidal antibodies by flow cytometry

The borreliacidal activity assay was carried out using a modification of the procedures described previously (6, 7). Briefly, the 72-hour mid-log phase cultures of B. burgdorferi isolates C-1-11 and S-1-10 in modified Barbour-Stoenner-Kelly (BSK) medium were quantified in a PetroffHauser chamber and diluted to 1 x 10⁶ cells / ml of BSK medium. Aliquots of 100 µl of diluted, heat-inactivated serum were mixed with 100 µl of each B. burgdorferi suspension, and 10 µl of guinea pig serum complement (210 CH units) were added.₅₀; GIBCO Laboratories, Grand Island, NY). The suspension was mixed gently and incubated at 32 ° C for 16 to 24 hours. After incubation of the reaction tubes, 100 µl of the reaction mixture was diluted with PBS containing 5.4 x 10 acridine orange.^-9 M. Detection of borreliacidal activity was carried out using a modification of the procedures described above (Callister, et al. (1994). Cell death caused by borreliacidal antibodies causes the appearance of bumps on the cell walls of B. burgdorferi, thereby adsorbing higher concentrations of acridine orange on damaged cell walls and in. Consequently, killed B. burgdorferi fluoresce much more intensely than normal living spirochetes. An increase in fluorescence intensity of&gt; 16% compared to microorganisms exposed to normal dog serum was considered a positive test for borreliacidal activity. The end point titer was expressed as the reciprocal of the last dilution at which a&gt; 16% increase in fluorescence intensity was observed. Dogs with a titer of &lt;1:20 were defined as seronegative.

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G. Exposure

A total of 300 male and 360 female I. scapularis ticks were collected from an area near Ettrick, Wisconsin, where Lyme disease is endemic. To determine the overall rate of infection of ticks by B. burgdorferi, the midguts of 49 male I. scapularis ticks were examined by an immunofluorescence (FA) test using the monoclonal antibody against Osp A provided by Dr.

AG Barbour. At four weeks after the second vaccination, each dog was challenged with 10 female and 6 male ticks. Female ticks are the only adult ticks that carry the disease. Male ticks are necessary for proper feeding of female ticks. For each dog, five female and three male ticks were randomly selected from the pool of ticks and placed in two small Petri dishes that were fixed to a shaved area of ​​the anterior left thoracic area of ​​each dog. The ticks were allowed to feed for one week and were observed at two-day intervals. One week after fixation, the ticks were recovered and the midguts were examined for the presence of B. burgdorferi by FA using the monoclonal antibody against B. burgdorferi.

H. Observation for clinical signs

The dogs were observed daily after exposure to detect clinical signs associated with Lyme disease. The clinical signs used as main indicators of the clinical course of Lyme disease were lameness, lethargy, and fever. Lameness was defined as a reluctance to bear weight on the affected limb, with or without swelling and increased temperature in the affected joint, stiff legs, and migration of lameness from one joint to another or from one limb to another. The dogs were also observed for lethargy and fever.

I. Isolation of B. burgdorferi

For the isolation of B. burgdorferi, samples were taken from the skin, blood, joints and organs of the dogs. Skin biopsies were performed at the tick attachment site and at sites remote from the tick bite site in anesthetized dogs 21 days after tick attachment and at the time of autopsy. The remote sites were located ventral to the tick bite site, posterior to the tick bite site, and on the right side of the anterior dorsal thoracic area on the opposite side of the dog. The skin was shaved and washed with Solvahex surgical scrub solution, and rinsed thoroughly with sterile water to remove disinfectant traces. An elliptical incision was made through the dermal and subcutaneous layers of the skin. Approximately one gram of skin was placed in nine milliliters of BSK medium containing 0.15% agarose and 40 pg rifampicin / ml. The biopsy sample was homogenized and two additional 10-fold dilutions of homogenate were prepared in nine milliliters of bSk medium blanks. The cultures were incubated at 32 ° C for six weeks and then examined by microscopy at three and six weeks after inoculation, to detect the growth of spirochetes. In the case of the cultures showing spirochete growth, it was confirmed that the germ was B. burgdorferi by FA, ​​using the monoclonal antibody against B. burgdorferi. Cultures that were negative for spirochete growth were discarded. At autopsy, the heart, spleen, kidneys, and bladder were removed and homogenized in 50 ml BSK containing agarose and rifampin using a Stomacher apparatus (Seward Medical, London, England). A 50 ml sample was poured and 10-fold dilutions were made in BSK medium. The cultures were incubated for six weeks at 32 ° C, observed and confirmed that the germ was B. burgdorferi by FA. A two to three milliliter sample of cerebrospinal fluid was added to nine milliliters of BSK medium and another 1:10 dilution was prepared in the same medium, incubated and observed as described. Tissue was removed from the elbow, carpal, knee, and tarsal joints. Tissue from each joint was added to nine milliliters of BSK medium containing agarose and rifampin. Another 1:10 dilution was prepared in the same medium, and the cultures were incubated and observed as described.

II. Results

A. Vaccination

The dogs were vaccinated with two doses of bacterin separated by an interval of three weeks. No local or systemic reactions were observed in any of the dogs. The temperatures of the dogs in both vaccination groups remained normal after the first and second doses of bacterin, Tables 27 and 28, respectively.

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TABLE 27

Dog temperatures after the first vaccination

<td></td><td colspan="6">Temperatures (° F) in the days after vaccination</td>

<td>Dog</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td colspan="7">Vaccinated - full dose</td>

<td> 746</td><td> 100,4</td><td> 100,6</td><td> 100,2</td><td> 100,2</td><td> 100,3</td><td> 100,3</td>

<td> 758</td><td> 101,4</td><td> 101,6</td><td> 100,6</td><td> 101,1</td><td> 101,7</td><td> 100,2</td>

<td> 768</td><td> 100,4</td><td> 101,1</td><td> 100,9</td><td> 100,8</td><td> 101,2</td><td> 100,4</td>

<td> 782</td><td> 100,8</td><td> 100,5</td><td> 100,7</td><td> 101,1</td><td> 100,2</td><td> 100,6</td>

<td> 835</td><td> 101,2</td><td> 101,1</td><td> 100,6</td><td> 100,9</td><td> 100,9</td><td> 100,6</td>

<td> 847</td><td> 101,8</td><td> 100,5</td><td> 101,6</td><td> 101,1</td><td> 100,1</td><td> 100,5</td>

<td> 851</td><td> 100,8</td><td> 100,9</td><td> 100,8</td><td> 100,6</td><td> 101,2</td><td> 100,2</td>

<td> 855</td><td> 100,8</td><td> 101,4</td><td> 101,7</td><td> 100,6</td><td> 101,3</td><td> 100,9</td>

<td> 863</td><td> 100,8</td><td> 100,1</td><td> 101,2</td><td> 100,8</td><td> 101,1</td><td> 100,5</td>

<td> 879</td><td> 101,1</td><td> 101,6</td><td> 101,9</td><td> 101,2</td><td> 101,2</td><td> 102,2</td>

<td colspan="7">Vaccinated - reduced dose</td>

<td> 760</td><td> 101,3</td><td> 101,4</td><td> 101,1</td><td> 100,1</td><td> 101,1</td><td> 101,1</td>

<td> 774</td><td> 101,2</td><td> 100,9</td><td> 100,7</td><td> 100,1</td><td> 100,6</td><td> 100,4</td>

<td> 786</td><td> 100,9</td><td> 100,2</td><td> 100,9</td><td> 102,1</td><td> 101,1</td><td> 101,0</td>

<td> 831</td><td> 100,7</td><td> 100,7</td><td> 100,1</td><td> 101,1</td><td> 100,5</td><td> 101,9</td>

<td> 843</td><td> 101,6</td><td> 100,6</td><td> 101,2</td><td> 100,4</td><td> 100,7</td><td> 100,0</td>

<td> 849</td><td> 100,7</td><td> 100,7</td><td> 100,4</td><td> 100,3</td><td> 100,2</td><td> 100,4</td>

<td> 853</td><td> 101,5</td><td> 100,3</td><td> 100,4</td><td> 101,6</td><td> 100,6</td><td> 101,2</td>

<td> 859</td><td> 101,9</td><td> 100,5</td><td> 101,4</td><td> 101,3</td><td> 101,1</td><td> 102,0</td>

<td> 871</td><td> 100,5</td><td> 100,1</td><td> 101,1</td><td> 101,6</td><td> 100,7</td><td> 101,1</td>

<td> 881</td><td> 101,1</td><td> 101,7</td><td> 100,8</td><td> 101,1</td><td> 101,4</td><td> 101,7</td>

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TABLE 28

Dog temperatures after the second vaccination

<td></td><td colspan="6">Temperatures (° F) in the days after vaccination</td>

<td>Dog</td><td> 0</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td colspan="7">Vaccinated - full dose</td>

<td> 746</td><td> 102,6</td><td> 102,1</td><td> 103,0</td><td> 102,2</td><td> 102,7</td><td> 103,0</td>

<td> 758</td><td> 102,2</td><td> 102,0</td><td> 102,8</td><td> 101,6</td><td> 102,7</td><td> 102,1</td>

<td> 768</td><td> 101,5</td><td> 101,9</td><td> 102,4</td><td> 101,8</td><td> 102,6</td><td> 101,8</td>

<td> 782</td><td> 101,9</td><td> 101,6</td><td> 102,0</td><td> 102,0</td><td> 102,3</td><td> 102,0</td>

<td> 835</td><td> 102,6</td><td> 101,7</td><td> 103,0</td><td> 102,2</td><td> 102,5</td><td> 102,2</td>

<td> 847</td><td> 102,0</td><td> 102,4</td><td> 101,7</td><td> 101,4</td><td> 101,9</td><td> 101,3</td>

<td> 851</td><td> 102,5</td><td> 102,1</td><td> 102,3</td><td> 101,7</td><td> 102,8</td><td> 102,1</td>

<td> 855</td><td> 102,1</td><td> 102,8</td><td> 102,4</td><td> 101,8</td><td> 102,3</td><td> 102,1</td>

<td> 863</td><td> 101,7</td><td> 102,0</td><td> 101,8</td><td> 101,7</td><td> 102,4</td><td> 101,9</td>

<td> 879</td><td> 102,0</td><td> 102,3</td><td> 101,9</td><td> 101,6</td><td> 101,7</td><td> 101,6</td>

<td colspan="7">Vaccinated - reduced dose</td>

<td> 760</td><td> 101,3</td><td> 101,4</td><td> 101,1</td><td> 100,1</td><td> 101,1</td><td> 101,1</td>

<td> 774</td><td> 101,2</td><td> 100,9</td><td> 100,7</td><td> 100,1</td><td> 100,6</td><td> 100,4</td>

<td> 786</td><td> 100,9</td><td> 100,2</td><td> 100,9</td><td> 102,1</td><td> 101,1</td><td> 101,0</td>

<td> 831</td><td> 100,7</td><td> 100,7</td><td> 100,1</td><td> 101,1</td><td> 100,5</td><td> 101,9</td>

<td> 843</td><td> 101,6</td><td> 100,6</td><td> 101,2</td><td> 100,4</td><td> 100,7</td><td> 100,0</td>

<td> 849</td><td> 100,7</td><td> 100,7</td><td> 100,4</td><td> 100,3</td><td> 100,2</td><td> 100,4</td>

<td> 853</td><td> 101,5</td><td> 100,3</td><td> 100,4</td><td> 101,6</td><td> 100,6</td><td> 101,2</td>

<td> 859</td><td> 101,9</td><td> 100,5</td><td> 101,4</td><td> 101,3</td><td> 101,1</td><td> 102,0</td>

<td> 871</td><td> 100,5</td><td> 100,1</td><td> 101,1</td><td> 101,6</td><td> 100,7</td><td> 101,1</td>

<td> 881</td><td> 101,1</td><td> 101,7</td><td> 100,8</td><td> 101,1</td><td> 101,4</td><td> 101,7</td>

B. Antibody response against whole cell antigens of B. burgdorferi

An ELISA test using whole B. burgdorferi cells was used to demonstrate the serological response of vaccinated dogs exposed to B. burgdorferi surface antigens. Before vaccination all dogs were seronegative. At four weeks after the second vaccination, dogs vaccinated with the full dose of bacterin had an antibody MGT of 788 against C-1-11 and an MGT of 520 against S-1-10 (Table 29). Dogs vaccinated with the reduced dose bacterin had a slightly lower MGT of 485 against C-111 and an identical MGT of 520 against S-1-10. After challenge, the antibody titers against C-1-11 and S-1-10 in both bacterin groups had decreased to almost the same level. At 12 weeks post challenge, the whole cell antibody MGT of the unvaccinated control dogs was higher than the whole cell antibody MGT of the vaccinated dogs. The MGT in the unvaccinated controls was 1502 and 2301 against C-1-11 and S-110, respectively. An unvaccinated control dog remained seronegative 12 weeks after challenge.

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<img file="ES2265643T3_D0008.tif" />

881 NEG NEG 320 320 160 160

MGT NEG NEG 485 520 184 160

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<img file="ES2265643T3_D0009.tif" />

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C. Immunoelectroblotting

Sera from vaccinated and unvaccinated control dogs were tested before and after vaccination and challenge for reactivity of the antibody with B. burgdorferi specific antigens. All dogs were seronegative before vaccination. Antibody responses against B. burgdorferi strains C-1-11 and S-110 in dogs vaccinated with the full dose of bacterin are presented in Figures 1 and 2. The antibody response was mainly directed against the OspA and OspB proteins of C-1-11 and S-1-10. Dogs vaccinated with the full dose of bacterin showed almost the same degree of reaction against the proteins of molecular weights of 31 and 34 kilodaltons of OspA and OspB, respectively, of C-1-11 and S-1-10. The differences in the staining patterns of the OspA proteins of C-1-11 and S-1-10 indicate that there is a clear antigenic difference between the two isolates. In some dogs a slight reactivity against other spirochete proteins was observed. Dogs vaccinated with the bacterin at a reduced dose also generated antibodies against the OspA and OspB proteins of C-1-11 and S-1-10 (Figures 3 and 4). The intensity of the staining of the antibody profile in dogs vaccinated with the reduced dose bacterin indicated that the level of response against OspA was the same as that of dogs that had received the full dose of bacterin. At 12 weeks post challenge, the antibody profile remained essentially unchanged in dogs in the full dose and reduced dose bacterin groups. Reactivity against OspA and OspB remained the main antibody response detected. However, there was little antibody response against other Borrelia proteins after exposure. After challenge, a decrease in the intensity of staining against OspA and OspB was observed in some vaccinated animals, but it occurred in dogs of both bacterin groups. Although non-specific staining was observed in some dogs, all unvaccinated control dogs were seronegative to B. burgdorferi prior to vaccination, and vaccinated control dogs were seronegative to B. burgdorferi prior to vaccination. No reactivity against OspA and OspB was detected in any of the unvaccinated control dogs (Figures 5 and 6). The antibody profile in the post-challenge control dogs differed dramatically from the antibody profile observed in the vaccinated post-challenge dogs. Unvaccinated control dogs generated little response against OspA and OspB proteins after challenge, but generated antibodies against several different antigens of B. burgdorferi.

D. OspA ELISA

A recombinant Osp A ELISA test was used to determine the antibody response against one of the major protective antigens in dogs vaccinated with the full and reduced doses of bacterins. There were no significant differences in the MGT of antibodies against OspA of isolates C-1-11 or S-1-10 of B. burgdorferi between dogs that had received the full dose and the reduced dose of bacterins. Dogs vaccinated with the full dose of bacterin generated an MGT of antibodies against OspA of 452 for C-1-11, compared to a MGT of 368 against the C-1-11 strain in dogs that had received the bacterin at a reduced dose. (Table 30). The MGT of antibodies against OspA in the groups with the full dose and the reduced dose of bacterin against S-1-10 were 299 and 394, respectively. The fact that C-1-11 and S-1-10 OspA antibodies were not detected by ELISA in any of the unvaccinated control dogs after tick challenge correlates with the lack of detection of OspA antibody by immunoblotting.

(Table goes to next page)

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Table 30 (continued)

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E. Borreliacidal antibody response

The functional activity of the antibodies generated against B. burgdorferi as a consequence of vaccination with the bacterin was measured with the borreliacidal antibody test. The presence of borreliacidal antibodies was demonstrated by inhibition of the growth of B. burgdorferi due to antibody-mediated lysis of the microorganism. Dogs vaccinated with full or reduced dose bacterin generated high titers of borreliacidal antibodies against S-1-10 and C-1-11 (Table 31). The MGT of borreliacidal antibodies in dogs that received the full dose of bacterin was 970 against C-1-11, compared to a MGT of 640 against C-1-11 in dogs that received the bacterin at a reduced dose. The MGT of borreliacidal antibodies against S-1-10 was 597 in both groups with bacterin.

(Table goes to next page)

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<img file="ES2265643T3_D0014.tif" />

859 NEG NEG 640 640

871 NEG NEG 640 1280

881 NEG NEG 320 320

MGT NEG NEG 640 597

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<img file="ES2265643T3_D0015.tif" />

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F. Exposure of dogs to ticks infected with B. burgdorferi

A total of 660 I. scapularis ticks (360 female and 300 male) were used to challenge the dogs. The infection rate with B. burgdorferi in ticks was found to be 45%, determined by AF analysis of the midgut of male ticks. This infection rate is similar to the 47% infection rate described in Example 14 above, and, once again, similar to the 47% infection rate described by Lacombe et al. (Lacowbe et al. (1993). In general, ticks attached within 24 hours, and most ticks fed fully or almost fully swollen during the one-week exposure period. Ticks that had been shed from the dog remained on the plate. At the end of the one-week tick attachment period, the recovered ticks were analyzed by FA for the presence of B. burgdorferi. Ticks were recovered from 10 of 10 animals vaccinated with the full dose, and at least one tick infected with B. burgdorferi was recovered from 3 of 10 (30%) animals. In the reduced dose group, ticks were recovered from 10 of 10 vaccinated animals and 40% had at least one tick infected with B. burgdorferi. Ticks were recovered from 14 of 14 unvaccinated dogs, and 12 of 14 (86%) had at least one tick infected with B. burgdorferi. The observation that fewer infected ticks are recovered from vaccinated animals than from unvaccinated animals was previously described in the Investigation Report of the Immunogenicity Test and Duration of Immunity Study, December 28, 1993, and has been published in Fikrig et al. (1992).

G. Isolation of B. burgdorferi from skin biopsy samples

Dog skin has been shown to be a good source for the recovery of B. burgdorferi after exposure to infected ticks (Investigation Report of the Immunogenicity Test and Duration of Immunity Study, 28 December 1993 ). Therefore, isolation of B. burgdorferi from skin biopsies of dogs can be used to check for infection with B. burgdorferi and as an indicator of the disappearance of spirochetes in dogs. At 21 days after tick challenge, dogs were anesthetized and skin biopsies were performed on all vaccinated and unvaccinated dogs. B. burgdorferi was not isolated from the skin biopsies of any of the 10 dogs in the group with the full dose of bacterin. In the reduced dose group, B. burgdorferi was recovered from 1 of 10 dogs (Table 32). In contrast, skin biopsies performed on 12 of 14 (86%) unvaccinated control dogs 21 days after challenge were positive for B. burgdorferi. Interestingly, the only unvaccinated control dog that was seronegative after challenge by whole cell ELISA was one of two control dogs whose skin biopsies were negative.

(Table goes to next page)

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<img file="ES2265643T3_D0016.tif" />

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<img file="ES2265643T3_D0017.tif" />

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H. Appearance of clinical signs in dogs after exposure

The clinical signs of Lyme disease in the dogs used to assess the level of protection conferred by the bacteria were lameness, lethargy, and fever. Eight months after exposure to ticks, one dog (10%) in the full-dose bacterin group was lame and two dogs (20%) in the reduced-dose bacterin group were lame (Table 32). Similar to the immunogenicity / duration study, dogs that had received the full dose or the reduced dose of bacterins did not lame after four months post challenge. Lameness has been observed in 8 of 14 dogs (57%) in the unvaccinated control group, indicating an 83% protection rate for the full-dose bacterin group and a 65% protection rate for the group with the bacterin at a reduced dose. Lameness affecting one leg was observed in vaccinated animals and unvaccinated controls. As described in the immunogenicity / duration study, the occurrence of lameness in dogs with the natural tick exposure model occurred over a prolonged period of time. The time course of the observation of lameness in the dogs in the reduced dose study is presented in Figure 7. Compared to the immunogenicity / duration study, the occurrence of lameness in the unvaccinated control dogs in the reduced dose study occurred for a slightly longer period of time. The dogs were observed for other clinical signs of Lyme disease, including fever and lethargy. The only lame dog in the full-dose bacterin group had no fever&gt; 103.5 ° F (39.7 ° C) and no lethargy was observed in the dog. One of the two lame dogs in the reduced dose group had a fever of 103.7 ° F (39.8 ° C) for one day. All unvaccinated control dogs had temperatures&gt; 103.5 ° F (39.7 ° C) at the time lameness was observed. Lethargy was not observed in any of the lame dogs.

I. Isolation of B. burgdorferi from lame dogs

Dog autopsies were performed within three to four days of the lameness episode and samples of the skin, joints, and organs were cultured to isolate B. burgdorferi. B. burgdorferi was not isolated from vaccinated lame animals in the groups with the full dose and the reduced dose of bacterin (Table 32). In contrast, B. burgdorferi was isolated from the skin and joints of all unvaccinated lame controls and from the spleen of a lame control dog. In all control dogs, the spirochete was recovered from skin biopsy samples taken from locations remote from the tick bite site. In the immunogenicity / duration study, results were reported showing the recovery of B. burgdorferi from unvaccinated lame control dogs, but not from vaccinated lame animals.

III. Discussion

The full dose of bacterin used in the study contained 5 x 10⁸ cells from each of the isolated strains of B. burgdorferi belonging to two different seroprotective groups. The results demonstrate that the vaccinated dogs generated borreliacidal antibodies directed against the main protective antigens of B. burgdorferi. A tick exposure model was established and used to assess the ability of the bacterin to protect dogs against the clinical manifestations of Lyme disease. At seven months after the second vaccination, the dogs were challenged with ticks infected with B. burgdorferi and were observed for another seven months. The bacterin protected the dogs against the clinical signs of Lyme disease, including lameness, fever and lethargy. The observation that B. burgdorferi was not recovered from vaccinated lame animals but was recovered from all lame control dogs indicating an increase in spirochete shedding in vaccinated dogs. The study data were accepted by APHIS (Animal and Plant Health Inspection Service) as a demonstration of the immunogenicity and duration of immunity of the bacterin. The tick exposure model was incorporated into the bacterin efficacy tests at a relatively late stage in the development of the bacterin described above. Therefore, no minimum dose immunization studies were conducted. This Example presents vaccination of dogs with a bacterin containing a 10-fold reduction in the numbers of each isolate of B. burgdorferi. For comparison with the reduced dose bacterin group, more dogs were vaccinated with the bacterin containing the original full dose of B. burgdorferi. Because the duration of immunity had been previously demonstrated, the dogs in the reduced dose antigen study were challenged at 4 weeks after the second vaccination. The tick challenge model was used once again in the reduced dose antigen study because the challenge model is the primary test of the vaccine's efficacy.

As a first step to determine whether the reduction in the numbers of B. burgdorferi cells affected the efficacy of the bacterin, the humoral immunity response of the dogs was evaluated. The borreliacidal antibody titers against whole cells and OspA of the dogs in the reduced dose group were measured. In general, the antibody titers in dogs vaccinated with the full dose of bacterin were slightly higher than the titers in dogs vaccinated with the reduced dose bacterin. However, the antibody titers between the bacterin groups did not differ more than twice, and were not statistically different. The antibody titers in the reduced dose study were comparable to the titers described in the study for immunogenicity and duration (Table 33). The fact that the bacterins used in the reduced-dose study were stored for another year at 4 ° C serves as proof of the stability of the bacterin. Most importantly, the results demonstrated the reproducibility of the experimental conditions and the ability of the reduced dose and the full dose of bacterin to consistently generate similar humoral immunity responses against B. burgdorferi.

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<img file="ES2265643T3_D0018.tif" />

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Isolation of B. burgdorferi from dog tissue samples has been used as an indicator of spirochete infection. Example 14, using the full dose of bacterin, demonstrates that upon challenge to B. burgdorferi infected ticks, the spirochete is recovered from the skin of fewer vaccinated dogs than unvaccinated control dogs. This Example also shows that B. burgdorferi was recovered from skin biopsies of fewer vaccinated dogs than control dogs after exposure to ticks. There were no significant differences between the reduced dose and full dose bacterin groups in the recovery of B. burgdorferi from post-challenge skin biopsies. The difference between vaccinated and control dogs when it comes to isolating B. burgdorferi from tissue samples was also observed at the time of autopsy of lame dogs. B. burgdorferi was not recovered from any tissue from the single lame dog in the full dose bacterin group or from either dog in the reduced dose group. These results are identical to the results of the study of immunogenicity and duration. In contrast, B. burgdorferi from all unvaccinated lame control dogs in both the immunogenicity / duration study and the reduced dose study. Although the mechanisms have not been defined, these results indicate that the full dose and the reduced dose of bacterins had a similar ability to reduce the spirochete load of dogs at the time of infection and / or to generate an immune response that gave lead to increased spirochete shedding.

The main clinical signs of Lyme disease in the dog are lameness and, to a lesser extent, fever and lethargy. The immunogenicity / duration study carried out with the full dose of bacterin in Example 14 showed that lameness and other clinical signs were observed in 12% of vaccinated animals, compared to 83% of non-vaccinated controls. vaccinated. Similar results were observed in this reduced dose study, with lameness reported in 10% of the animals in the full dose group and 20% of the animals in the reduced dose group. In both studies, lameness in unvaccinated control dogs began to manifest at the beginning of two months after challenge and at periodic intervals thereafter. At the end of the immunogenicity / duration study, eight months after challenge, lameness was described in approximately 80% of the unvaccinated control dogs. In the reduced dose study, at eight months after exposure to ticks, lameness had been described in approximately 60% of the unvaccinated controls. The chronic nature of the disease and the delayed onset of clinical signs are not unexpected features when using an exposure model that closely mimics natural field exposure. In Appel et al. (1993) describe that the clinical manifestations of Lyme disease, including lameness, were observed up to five months after exposure of dogs to infected ticks. The reason for this long time interval for the manifestation of clinical disease in the reduced dose study is not known. In the present study with reduced dose, the parameters of the exposure to ticks such as the number of infected ticks / dog and the infection rates of the ticks were similar to the parameters used in the study of the immunogenicity / duration of Example 14 . Although the tick exposure model represents the natural exposure route, dose and virulence of B. burgdorferi found by the dog, it is these same parameters that most directly affect the exposure result.

The data presented in this Example demonstrate that a bacterin formulated with a reduced dose level of antigen is as effective as the full dose of the bacterin in generating a protective antibody response against B. burgdorferi and in reducing spirochete infection thereafter. of exposure to ticks. Both the full dose and the reduced dose of bacterins protected the dogs against the clinical signs of Lyme disease.

References

Anderson, JF, et al. (1988) J. Clin. Microbiol. 26: 2209-2212.

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