Vaccine for protection against Leptospira serovar Australis

Abstract

The present invention relates to providing protective immunity against Leptospira serovar Australis in dogs using a vaccine comprising non-Australis Leptospira serotypes.

Description

Technical Field

[0001] The present invention generally relates to immunogenic Leptospira compositions capable of inducing a cross-protective immune response in animals, particularly in dogs. The present invention further relates to a method of providing a cross-protective immune response against Leptospira Australis to an animal, particularly a dog.

Background Art

[0002] Leptospirosis is a worldwide zoonotic infection caused by Gram-negative spirochetes belonging to the genus Leptospira. Leptospirosis is common in humans, dogs, horses, cattle, and wildlife. Dogs are very susceptible to infection and, after infection, may develop symptoms such as high fever, jaundice, hemorrhagic diathesis, and abortion, and may die within a few days. In addition, dogs may develop chronic symptoms such as liver, kidney, and gastrointestinal symptoms. Pet dogs live in close relationship with humans and livestock and can be used as an indicator species for environmental risks to humans.

[0003] Studies on the seroprevalence have suggested that the dominant and most widespread serogroups in dogs are Canicola, Icterohaemorrhagiae, Australis, and Grippotyphosa. In addition to these serogroups, Pomona is relevant in the United States and Hbdomadis is relevant in Japan. Various vaccines against leptospirosis in dogs have been approved in Europe, and the oldest vaccine is a bivalent vaccine containing serotype Canicola and either serotype Icterohaemorrhagiae or Copenhageni. More recently, tetravalent and trivalent vaccines have been introduced to the European market, which also included serogroups Grippotyphosa and Australis, or only the serotype of Grippotyphosa.

[0004] Reviews in the literature, such as the "Leptospirosis Fact Sheet" (WHO, Regional Office for South-East Asia, 2009), have shown that although animals and humans can be immunized to some extent, the protection is mostly serotype-specific. The lack of cross-protection is not surprising considering the significant genetic / genomic differences between serotypes, for example, the significant genetic / genomic differences between the genetic mechanisms at the lipopolysaccharide biosynthesis (rfb) locus (Pena-Moctezuma, A. et al, 2001 FEMS Immunology and Medical Microbiology 31(2001)73-81).

[0005] Thus, in dogs, there is little evidence of "cross - protection" between serotypes. Cross - protection or heterologous protection, as used herein, refers to providing protection against Leptospira serotypes by administering an effective amount of different serotypes, for example, an effective amount of non - Australis serotypes of the same genus, such as serotype Portland - vere, Dadas, Copenhageni or Bratislava, to provide protection against the Australis serotype.

[0006] European Patent No. 2874653 discloses a method of providing protection against Leptospira interrogans serotype Copenhageni using a polyvalent vaccine comprising non - Copenhageni serotypes Icterohaemorrhagiae, Canicola, Grippotyphosa and Pomona.

[0007] This was the first disclosure regarding a Leptospira vaccine providing protection in dogs against serotypes not present in the vaccine. This patent was published in January 2014, and since then, no reports in dogs regarding other cross - protections of Leptospira serotypes have been made.

[0008] The paper by Bouvet et al. (Veterinary Immunology and Immunopathology 219(2020)109985) is a scientific publication regarding the same findings and experiments described in European Patent No. 2874653. It has been confirmed that bacterin Leptospira vaccines only provide protection against the same serotype. It is also disclosed that there is very limited evidence of protection against other serotypes within the same serogroup and only in the rodent model. Evidence of the effectiveness of other Leptospira serotypes providing cross - protection in dogs is not shown in the prior art.

[0009] Therefore, there remains a need for protection of animals, particularly dogs, against Leptospira serovars. Since leptospirosis is also a potential zoonotic problem for humans, it would be beneficial to have protection against additional Leptospira serovars, preferably by vaccines already in use. Thus, it would be beneficial to protect animals, particularly dogs, against heterologous Leptospira serovars. Until the present disclosure, no method was known for providing protection against Leptospira serovar Australis using non-Australian serovars.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0012] The object of the present invention is to provide a method for providing protective immunity against a first leptospira serotype, the method comprising the step of administering (one or more) further leptospira serotypes that are different serotypes from the first leptospira serotype. When the (one or more) further leptospira serotypes are a mixture of a plurality of leptospira serotypes (for example, a mixed / polyvalent vaccine), the (one or more) further leptospira serotypes shall not contain a leptospira serotype of the same serotype as the first leptospira serotype for which protective immunity is sought.

Means for Solving the Problems

[0013] In one embodiment and / or an embodiment of the present invention, the method comprises the step of providing protective immunity against Leptospira serotype Australis and administering an immunologically effective amount of a non-Australis Leptospira serotype to an animal in need thereof.

[0014] In another embodiment, the method provides protective immunity against Leptospira serotype Australis by administering a mixed / polyvalent leptospira vaccine. In a particular embodiment, the polyvalent leptospira vaccine comprises Leptospira serotypes Portland-vere, Dadas, Copenhageni, and Bratislava. Nobivac L4 (MSD Animal Health) is such a polyvalent vaccine.

[0015] It was unexpected and surprising to those skilled in the art with current state-of-the-art knowledge in the field of leptospirosis that a vaccine not containing Leptospira serotype Australis could induce protective immunity against Leptospira serotype Australis in dogs. In particular, it was surprising that a vaccine such as Nobivac L4, which includes Leptospira serotypes Portland-vere, Dadas, Copenhageni, and Bratislava, provided immunity against Leptospira serotype Australis.

[0016] Examples show that vaccines such as Nobivac L4, which include Leptospira serotypes Portland-vere, Dadas, Copenhageni, and Bratislava, provided protective immunity against Leptospira serotype Australis.

BRIEF DESCRIPTION OF THE INVENTION

[0017] The present invention includes a method for preventing or reducing infection by a specific serotype of Leptospira by administering a vaccine having one or more Leptospira of different serotypes. The present invention relates to a vaccine composition containing Leptospira serotypes for use in providing protective immunity against Leptospira serotype Australis to dogs. The vaccine does not include Leptospira serotype Australis.

[0018] In one embodiment and / or an embodiment thereof of the present invention, the present invention provides a method for inducing a protective immune response against Leptospira serotype Australis in an animal, the method comprising administering to the animal an effective amount of a non-Australis Leptospira serotype. "Non-Australis Leptospira serotype" means a Leptospira serotype different from serotype Australis. It may be derived from the same or a different serogroup as serotype Australis (serogroup Australis).

[0019] In one embodiment and / or an embodiment thereof of the present invention, the non-Australis Leptospira serotype belongs to serogroup Australis. In a particular embodiment, the non-Australis Leptospira serotype is Bratislava.

[0020] In one embodiment and / or an embodiment thereof of the present invention, the non-Australis Leptospira serotype is delivered as part of a multivalent / mixed vaccine. In a particular embodiment, the non-Australis Leptospira serotype is a serotype selected from the group consisting of Leptospira Portland-vere, Dadas, Copenhageni, and Bratislava, preferably serotype Bratislava, and one or more serotypes selected from Portland-vere, Dadas, and Copenhageni.

[0021] In another embodiment and / or implementation of the present invention, the vaccine comprises Leptospira serotype Portland-vere. In another embodiment, the vaccine comprises Leptospira serotype Dadas. In another embodiment, the vaccine comprises Leptospira serotype Copenhageni. In another embodiment, the vaccine comprises Leptospira serotype Bratislava. In yet another embodiment, the vaccine comprises Leptospira serotype Portland-vere, Dadas, Copenhageni and Bratislava.

[0022] Suitably, the Leptospira Portland-vere strain is Ca-12-000. Suitably, the Leptospira Dadas strain is Gr-01-005. Suitably, the Leptospira Copenhageni strain is Ic-02-001. Suitably, the Leptospira Bratislava strain is As-05-073.

[0023] Suitably, the vaccine provides protective immunity against Leptospira serotype Australis, Grippotyphosa and Icterohaemorrhagiae to dogs.

[0024] As used herein, the term vaccine refers to a pharmaceutical composition comprising at least one immunologically active ingredient that induces an immunological response in an animal and a pharmaceutically acceptable carrier. The vaccine may also be referred to herein as an immunogenic composition. The vaccine may additionally contain further components typical of pharmaceutical compositions. The immunogenic composition and the vaccine are used interchangeably herein.

[0025] Typically, "immune response" includes, but is not limited to, one or more of the following effects. That is, it includes the effect of producing or activating antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma delta T cells specifically directed against one or more antigens contained in the composition or vaccine of interest. Appropriately, the target exhibits either a therapeutic or prophylactic immune response such that resistance to new infections is enhanced and / or the clinical severity of the disease is reduced. Such protection is evidenced by a decrease or absence of clinical signs normally exhibited by an infected host, a shortening of the recovery time, and / or a reduction in the duration, or a decrease in the bacterial titer in the tissues or body fluids or excretions of the infected host.

[0026] The "clinical signs" or "clinical symptoms or clinical reactions" of leptospirosis are, for example, anorexia, delayed or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (meaning dehydration), pallor or jaundice of the mucous membranes (conjunctiva or oral mucosa), and rounding of the back.

[0027] As used herein, "decrease in the incidence and / or severity of clinical signs" or "decrease in the incidence and / or severity of clinical symptoms" means reducing the number of infected animals in the group, reducing or eliminating the number of animals showing clinical signs of infection, or reducing the severity of any clinical signs present in the animals, as compared to infection with a wild-type pathogen. For example, such clinical signs include body temperature, general health score, anorexia, delayed or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (meaning dehydration), pallor or jaundice of the mucous membranes (conjunctiva or oral mucosa), and rounding of the back. Appropriately, these clinical signs are reduced by at least 10% in animals receiving the vaccine composition of the present invention compared to animals infected when not vaccinated.

[0028] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes all kinds of excipients, solvents, growth media, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, inactivators, antimicrobial agents, antibacterial agents and antifungal agents, isotonic agents, adsorption retardants, etc. Such components include those that are safe and appropriate for use in veterinary medicine. Appropriately, stabilizers for use in the present invention include stabilizers for lyophilization or freeze-drying.

[0029] Examples of "diluents" may include water, physiological saline, dextrose, ethanol, glycerol, etc. Examples of isotonic agents may include, inter alia, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Examples of stabilizers may include, inter alia, albumin and alkali salts of ethylenediaminetetraacetic acid.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0031] Appropriately, the vaccine is administered in a volume of about 0.05 to about 5.0 ml, such as 0.1 to 2.5 ml. Appropriately, the vaccine is administered in a volume of 0.2 to 2.0 ml, or 0.25 to 1.5 ml, or 0.3 to 1.2 ml, or 0.4 to 1.0 ml, or 0.5 to 0.9 ml, or 0.6 to 0.8 ml.

[0032] The vaccine can be administered subcutaneously, intramuscularly, intraperitoneally, orally, intranasally, intravitreally, and / or rectally. Appropriately, the vaccine is administered subcutaneously, intramuscularly, orally, intranasally, intravitreally, and / or rectally. Appropriately, the vaccine is administered subcutaneously, intramuscularly, orally, and / or intranasally. Appropriately, the vaccine is administered subcutaneously or intramuscularly.

[0033] Preferably, the vaccine is administered as a single dose. Appropriately, the vaccine is administered in at least two doses. The at least two doses are administered at intervals of 2 to 100 days, preferably 5 to 60 days, more preferably 7 to 50 days, more preferably 10 to 40 days, more preferably 14 to 30 days, and more preferably 15 to 25 days. Preferably, the at least two doses are administered at intervals of 5 to 40 days, preferably 6 to 35 days, preferably 7 to 32 days, preferably 8 to 30 days, preferably 9 to 28 days, preferably 10 to 25 days, preferably 11 to 22 days, preferably 12 to 20 days, preferably 13 to 18 days, and preferably 14 to 16 days. Also, appropriately, the at least two doses are administered at intervals of 1 to 12 weeks, more appropriately 2 to 10 weeks, more appropriately 2 to 8 weeks, more appropriately 3 to 6 weeks, and more appropriately 3 to 5 weeks.

[0034] Preferably, the single dose is updated by annual revaccination. Appropriately, after the single dose, revaccination is performed every 10 to 15 months. Appropriately, the revaccination is every 11 to 14 months, and appropriately, the revaccination is every 12 to 13 months.

[0035] Preferably, the at least two doses are updated by annual revaccination with a single dose. Appropriately, after the last dose of the at least two doses, revaccination is performed every 10 to 15 months. Appropriately, the revaccination is every 11 to 14 months, and appropriately, the revaccination is every 12 to 13 months.

[0036] Preferably, the at least two doses are updated by annual revaccination with at least two doses. Appropriately, after the last dose of the at least two doses, revaccination is performed every 10 to 15 months. Appropriately, the revaccination is every 11 to 14 months, and appropriately, the revaccination is every 12 to 13 months.

[0037] Suitably, the vaccine contains additional antigens that provide immunity against additional non - leptospiral canine pathogens. Suitably, the additional antigens are selected from the group consisting of canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpes virus, rabies, canine coronavirus, Bordetella, and combinations thereof.

[0038] Next, the present invention will be further described by the following non - limiting examples.

[0039] [Examples] Overview A total of 29 healthy puppies with undetectable levels of serum antibodies against the serogroups Canicola, Icterohaemorrhagiae, Grippotyphosa, and Australis were used. Three groups of 7 puppies each were vaccinated. Groups 1 and 2 each received an L4 - LV vaccine with different batches (one dose is 0.5 ml). Group 3 received batch L4 (one dose is 1 ml). The amount of antigen is the same for L4 and L4 - LV, and the only difference is the volume of the dose. Group 4 was the non - vaccinated control group.

[0040] L4 - LV and L4 contain antigens of the following four serotypes.

[0041] Inactivated leptospira strains: Serotype Portland - vere (strain Ca - 12 - 000) 3550 - 7100 U* Serotype Copenhageni (strain Ic - 02 - 001) 290 - 1000 U* Serotype Bratislava (strain As - 05 - 073) 500 - 1700 U* Serotype Dadas (Gr-01-005) 650 - 1300 U* *Antigen amount ELISA unit.

[0042] The first vaccination was performed at 6 weeks of age, and the second vaccination was performed at 10 weeks of age. The leptospira challenge was performed 3 weeks after the second vaccination. Dogs were challenged with leptospira bacteria cultured from positive organs or homogenates of positive organs of experimentally infected hamsters.

[0043] To evaluate the efficacy after challenge, the following tests or measurements were performed.

[0044] Microscopic agglutination test (MAT) to determine the serum group-specific antibody titers against serogroups Australis, Canicola, Grippotyphosa, and Icterohaemorrhagiae, Body weight, Body temperature, From blood, urine, kidneys, and liver Culture of challenge organisms, Urine test, Clinical signs, and Postmortem gross and histopathological examinations.

[0045] Serology Blood samples for serology and platelet count were collected from all puppies 5 days before challenge (= 16 days after the second vaccination), and at 3, 7, 14, 21, and 28 days post-challenge (pc).

[0046] Serum was tested by microscopic agglutination test (MAT) to determine the titers of serum group-specific agglutinating serum antibodies against serogroups Australis, Canicola, Grippotyphosa, and Icterohaemorrhagiae.

[0047] Briefly, serially two-fold dilutions of dog sera were incubated with each of the live antigens of the above serum groups. Subsequently, the titer was determined, which was the log2 value of the reciprocal of the maximum dilution indicating that ≧50% of the leptospira had agglutinated in the serum-antigen mixture. Positive and negative rabbit antisera were used as control sera. The test was considered valid if the titer of the negative control serum was ≦1 and the titer of the positive control serum was ≧5 with the homologous antigen. All test samples showing agglutination at a dilution ≧2 were considered positive.

[0048] Body temperature The body temperature was measured daily, 3 days before the challenge and from the day after the challenge until the end of the study.

[0049] Body weight The body weight of each dog was measured on the arrival day (-12 days), before the challenge (-5 days and twice on day 0), and then daily until the end of the study.

[0050] Re-isolation of the challenge organisms from blood, urine, kidneys, and liver *Blood: Blood samples used to obtain culture results were collected from all dogs on day -5 before the challenge and on days 1, 2, 3, 4, 7, 10, 14, and 21 after the challenge. 0.5 ml aliquots directly collected from the syringe were inoculated into 10 ml of EMJH medium (containing 200 μg / ml of 5-fluorouracil (5-FU) and 1% (v:v) rabbit serum negative for antibodies against four related serum groups of leptospira).

[0051] To evaluate the total number of days with positive blood samples per dog, if euthanasia and necropsy were performed earlier than the planned necropsy day (day 28 after the challenge), positive results were used for the uncollected blood samples. This was necessary to avoid an inaccurate comparison with the vaccinated group without uncollected samples due to the absence of euthanized dogs.

[0052] *Urine: Three days before the challenge, and on the 3rd, 7th, 14th, 21st, and 28th days after the challenge, urine samples (at least 2.5 ml) were collected by bladder puncture from all dogs. 1 ml of urine was directly inoculated into 10 ml of the above EMJH medium. The remaining 1.5 ml of urine from each dog was used for rapid urine testing (see below) (Rapid urine testing).

[0053] To evaluate the total number of days with positive urine samples + kidney samples for each dog, if euthanasia and necropsy were performed earlier than the planned necropsy date (28 days after the challenge), positive results were used for the uncollected urine samples. This was necessary to avoid an inaccurate comparison with the vaccinated group that had no uncollected samples because there were no euthanized dogs.

[0054] *Kidney and liver: After euthanasia, one cortex of the kidney and the liver were sampled for culture, taking 1 - 2 grams of tissue fragments. The aseptically collected fragments were placed into 10 ml of the above culture medium.

[0055] The tissue fragments of the kidney and liver were homogenized in 10 ml of EMJH medium. A 100 - fold dilution of each kidney or liver homogenate in EMJH (containing 5 - FU and "negative" rabbit serum) was used for culture.

[0056] Cultures of blood, urine, kidney, and liver homogenates in EMJH were incubated at 29°C. Dark - field microscopy was used to observe for the presence of motile bacteria with a typical leptospira shape at least once a week for at least 8 weeks. After that, negative cultures were discarded. Leptospira in some of the positive cultures (at least one per treatment group) were tested using MAT for serogroup identification by agglutination.

[0057] Clinical signs Between 1 and 30 days after the challenge, the dogs were checked twice daily for clinical signs, paying special attention to the following nonspecific signs related to leptospirosis in dogs: anorexia, delayed or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (indicating dehydration), pallor or icterus of the mucous membranes (conjunctiva or oral mucosa), and arching of the back.

[0058] If the presence of leptospirosis was confirmed by the results of one or more laboratory tests, one or more clinical signs and corresponding clinical score values were assigned to the dogs. In this way, the potential interference of concurrent (mild) clinical signs due to causes unrelated to the leptospira challenge, such as concurrent diarrhea caused by opportunistic bacteria or parasites in the intestinal tract, was significantly reduced.

[0059] To evaluate the total clinical score value for each dog, the following clinical score values were used for the following clinically confirmed signs in the laboratory.

Table 1

[0060] For dogs that were not euthanized earlier than the end date, the total clinical score of the dog was defined as the sum of all individual scores for each day and all days up to the end of the study. For dogs that were euthanized earlier than the planned necropsy date (due to reaching the humane endpoint), a total clinical score value of 150 was used.

[0061] Necropsy, macroscopic examination, and histopathology If there were severe clinical signs after the challenge, the dogs were euthanized after appropriate sedation, and a postmortem examination was performed immediately thereafter. For all dogs that survived until the planned necropsy date, the same procedure was followed. Macroscopic examination was performed, especially for the lungs, liver, kidneys, and spleen. Histological examination was performed on tissue samples (collected from lesions) from the liver, kidneys, spleen, and any organ / tissue suspected of having lesions.

[0062] Tissue samples from the liver, kidney, spleen, and any organ / tissue suspected of having lesions were processed and the sections were stained with hematoxylin and eosin (HE) for histopathological examination according to standard procedures. In addition, Warthin - Starry staining was performed on kidney and liver sections to detect Leptospira in kidney and liver tissues. Based on microscopic examination of all slides of all sampled organs and tissues, the results were described and judged regarding specific pathological conditions related to canine leptospirosis.

[0063] Example 1 Defense against Serovar Australis [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0064] Example 2 Defense against Serovar Grippotyphosa [Table 7] [Table 8] [Table 9] [Table 10]

[0065] Example 3 Defense against Serovar Icterohaemorrhagiae

Table 11

Table 12

Table 13

Table 14

Table 15

Claims

1. A vaccine composition for providing protective immunity to dogs against Leptospira serotype Australis, comprising an immunologically effective amount of at least one non-Australis Leptospira serotype selected from the group consisting of serotype Portland-vere, Dadas, Copenhageni, and Bratislava, and not containing Leptospira serotype Australis.

2. The vaccine composition according to claim 1, comprising at least two Leptospira serotypes selected from the group consisting of serotypes Portland-vere, Dadas, Copenhageni, and Bratislava.

3. The vaccine composition according to claim 1, comprising the serotypes Portland-vere, Dadas, Copenhageni, and Bratislava.

4. The vaccine composition according to claim 1, wherein the serotype Portland-vere of Leptospira is strain Ca-12-000, the serotype Copenhageni of Leptospira is strain Ic-02-001, the serotype Dadas of Leptospira is strain Gr-01-005, and the serotype Bratislava of Leptospira is strain As-05-073.

5. The vaccine composition according to claim 1, which provides dogs with protective immunity against the serotypes Australis, Grippotyphosa, and Icterohaemorrhagiae of the Leptospira serotype.

6. A method for providing protective immunity to a dog against Leptospira serotype Australis, comprising administering to the dog an immunologically effective amount of the vaccine composition according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the vaccine composition is administered in a volume of 0.1 to 2.5 mL.

8. The method according to claim 6, wherein the vaccine composition is administered subcutaneously.

9. The method according to claim 6, wherein the vaccine composition is administered in at least two doses.

10. The method according to claim 9, wherein the at least two doses are administered with an interval of at least five days between them.

11. A vaccine composition according to any one of claims 1 to 5, further comprising at least one antigen that provides immunity against non-leptospira canine pathogens.

12. The vaccine composition according to claim 11, wherein the at least one antigen is derived from a pathogen selected from the group consisting of canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpesvirus, rabies, canine coronavirus, and combinations thereof.