Vaccine for protection against Leptospira serotype icterohemorrhagiae

Abstract

The present invention relates to providing protective immunity against Leptospira serovar Icterohaemorrhagiae to dogs using a vaccine comprising a non-Icterohaemorrhagiae Leptospira serotype.

Description

Technical Field

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

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 highly 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 risk to humans.

[0003] Studies on the seroprevalence suggest 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 Hebdomadis is relevant in Japan. Various vaccines against leptospirosis in dogs are approved in Europe. 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 into the European market, which also included serogroups Grippotyphosa and Australis, or only the serotype Grippotyphosa.

[0004] Reviews in the literature, for example in the "Leptospirosis Fact Sheet" (WHO, Regional Office for South-East Asia, 2009), indicate that while animals and humans can be partially immunized, the protection is shown to be 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 is defined herein as providing protection against Leptospira serotypes by administering an effective amount of different serotypes (e.g., providing protection against the Icterohaemorrhagiae serotype by administering an effective amount of a non-Icterohaemorrhagiae serotype of the same genus, such as serotype Portland-vere, Dadas, Copenhageni, or Bratislava).

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

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

[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 therein that bacterin Leptospira vaccines provide protection only 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 in dogs of other Leptospira serotypes providing cross-protection is not shown in the prior art.

[0009] Therefore, there is still a need to protect animals, particularly dogs, against Leptospira serotypes. Since leptospirosis is also a potential zoonotic infection problem for humans, it would be beneficial to have protection against additional Leptospira serotypes, preferably by a vaccine already in use. Thus, it would be beneficial to protect animals, particularly dogs, against heterologous Leptospira serotypes. Until the present disclosure, no method was known for providing protection against the Leptospira serotype Icterohaemorrhagiae using a non-Icterohaemorrhagiae serotype.

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 administering to an animal in need thereof an immunologically effective amount of a non-Icterohaemorrhagiae Leptospira serotype, thereby providing protective immunity against Leptospira serotype Icterohaemorrhagiae.

[0014] In another embodiment, the method provides protective immunity against Leptospira serotype Icterohaemorrhagiae 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 containing no Leptospira serotype Icterohaemorrhagiae could induce protective immunity against Leptospira serotype Icterohaemorrhagiae in dogs. In particular, it was surprising that vaccines such as Nobivac L4, which contain Leptospira serotypes Portland-vere, Dadas, Copenhageni, and Bratislava, provided immunity against Leptospira serotype Icterohaemorrhagiae.

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

Mode for Carrying Out 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 a Leptospira serotype for use in providing protective immunity against Leptospira serotype Icterohaemorrhagiae to dogs. The vaccine does not contain Leptospira serotype Icterohaemorrhagiae.

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

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

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

[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 Icterohaemorrhagiae, Australis and Grippotyphosa 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, "immunological response" includes, but is not limited to, one or more of the following effects. That is, the production or activation of 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 immunological 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 decrease 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 reaction" of leptospirosis are, for example, anorexia, delayed or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (indicating dehydration), pallor or jaundice of the mucosa (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 (indicating dehydration), pallor or jaundice of the mucosa (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 delaying agents, and the like. 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, and the like. Examples of isotonic agents may include, among others, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Examples of stabilizers may include, among others, albumin and alkaline 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, for example 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. Preferably, 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 preferably, the at least two doses are administered at intervals of 1 to 12 weeks, more preferably 2 to 10 weeks, more preferably 2 to 8 weeks, more preferably 3 to 6 weeks, and more preferably 3 to 5 weeks.

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

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

[0037] Suitably, the vaccine contains additional antigens that provide immunity against additional non-Leptospira 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 used as an unvaccinated control group.

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

[0041] Inactivated Leptospira strain: 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 carried out at 6 weeks of age, and the second vaccination was carried out at 10 weeks of age. The leptospira challenge was carried out 3 weeks after the second vaccination. Dogs were challenged with leptospira bacteria cultured from the 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 serogroup-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 tests, Clinical signs, and Postmortem macroscopic and histopathological examinations.

[0045] Serology Blood samples for serology and platelet count investigations 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 serogroup-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. Thereafter, the titer was determined, which was the log2 value of the reciprocal of the maximum dilution indicating that ≧50% of leptospira agglutinated in the serum-antigen mixture. Positive and negative rabbit antisera were used as control sera. The test was considered valid when 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 of ≧2 were considered positive.

[0048] Body temperature Body temperature was measured daily, 3 days before challenge and from the day after 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 challenge (-5 days and twice on day 0), and then daily until the end of the study.

[0050] Re-isolation of challenge organisms from blood, urine, kidney, and liver * Blood: Blood samples used to obtain culture results were collected from all dogs on day -5 before challenge and on days 1, 2, 3, 4, 7, 10, 14, and 21 after challenge. 0.5 ml aliquots directly collected from the syringe were inoculated into 10 ml of EMJH medium (containing 200 μg / ml of 5-fluoro-uracil (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 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 from all dogs by cystocentesis. 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 with 1 - 2 grams of tissue pieces. The aseptically collected tissue pieces were placed in 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 the homogenate of each kidney or liver in EMJH (containing 5 - FU and "negative" rabbit serum) was used for culture.

[0056] The 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 leptospirosis challenge, such as concurrent diarrhea due to opportunistic enteric bacteria or parasites, 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 then a postmortem examination was performed immediately. For all dogs that survived until the planned necropsy date, the same procedure was followed. Macroscopic examination was performed, particularly on 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, results were described and judged regarding specific pathological conditions related to canine leptospirosis.

[0063] Example 1 Defense against Serotype Icterohaemorrhagiae [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

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

[0065] Example 3 Defense against Serotype Australis

Table 11

Table 12

Table 13

Table 14

Table 15

Claims

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

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

3. The vaccine composition according to claim 1 or 2, comprising the Leptospira serotypes Portland-vere, Dadas, Copenhageni, and / or Bratislava.

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

5. The vaccine composition according to claim 1, comprising an additional antigen that provides immunity against additional non-leptospira canine pathogens.

6. A method for providing protective immunity to a dog against Leptospira serotype icterohemorrhagiae, comprising administering an immunologically effective amount of the vaccine composition described in claim 1 to the dog.

7. The method according to claim 6, wherein 0.1 to 2.5 ml of the vaccine composition is administered to a dog.

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 to dogs in at least two doses.

10. The method according to claim 9, wherein the vaccine composition is administered to a dog in at least two doses, with at least five days between doses.

11. The method according to claim 6, wherein the vaccine composition comprises an additional antigen that provides immunity against an additional non-leptospira canine pathogen.

12. The method according to claim 11, wherein the additional antigen is selected from canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpesvirus, rabies, canine coronavirus, and combinations thereof.