KR100556275B1 - Cat vaccine containing chlamydiacittaci and its manufacturing method - Google Patents

Abstract

The present invention relates to a cat vaccine containing non-reactive Chlamydia psittaci for preventing and treating Chlamydia disease in cats.

Description

Cat vaccine containing chlamydia cittaci and method for producing the same

Feline chlamydiasis is a common conjunctival and respiratory disease in cats known as feline pneumonia (FPn). This highly infectious disease is characterized by sneezing and coughing and is accompanied by mucosal eye and nose release. Cats of all ages are susceptible and have low mortality, but infected kittens and old cats can be severely debilitated. Feline chlamydiasis is a major problem in pet hospitals, clinics and cat breeds because of its severe infectivity. There are some opinions that persistent genital lineage infection by Chlamydia psittaci is a cause of breeding failure in cat kennels.

Inoculation studies with modified-live compositions of Chlamydia psittaci yielded contradictory results. Cello (Am. J. Vet. Med. Assoc. 158: 932-938, 1971) pointed out that this vaccine did not show any significant protection in cats. Shewen, et al (Can. J. Comp. Vet. Res. 44: 244-251, 1980) pointed out that they are partially protected, but McCercher (Am. J. Vet. Res. 13 (557-561, 1952) and others pointed out that these vaccines were almost completely protected. However, inoculated cats have been proposed to be reversed because some vaccine organisms can be transferred to other cats by inoculating live Chlamydia psittaci .

Studies with inactivated chlamydia vaccines resulted in mixed results and unacceptable local and systemic reactions in inoculated cats. Comparative challenge studies conducted with four inactivated vaccine formulations and commercially-modified live vaccines indicated that the inactivated formulation did not provide substantially any protection against chlamydial infection in cats (see, Shewen). et al, Can. J. Comp. Med. 44: 244-251, 1980). Inactivated Chlamydia psittaci vaccines have been described by Chu et al. (US Pat. No. 5,242,686). However, the vaccine is unpurified and causes an unacceptable local reaction when administered to cats (COMPENDIUM).

The modified-live chlamydia vaccine can be spread to other animals, can be recombined with wild strains of Chlamydia psittaci , can be toxically converted, and that commercially available inactivated chlamydia vaccines are ineffective or Due to the fact that it produces an unacceptable local reaction, there is a need for an effective and non-responsive chlamydia vaccine. It is an object of the present invention to provide an inactivated feline chlamydia vaccine composition having no high antigenic mass and having high immunogenicity. Another object of the present invention is to provide a simple method for preparing a suitable purified Chlamydia psittaci for the preparation of non-reactive and effective vaccines as monovalent preparations or mixtures with other cat antigens. will be.

The present invention is directed to inactivated immunogenically purified cells from chlamydia grown to produce vaccines that are contained in chlamydia vaccines at low levels (low antigen loading or low antigen mass) and are administered to cats and are non-reactive but highly effective. Effective Chlamydia psittaci is disclosed . In such vaccines, immunologically effective, low antigenic masses, purified Chlamydia psittaci, are combined with an effective amount of adjuvant and a pharmaceutically acceptable carrier or diluent therefor. The purification procedure to generate Chlamydia psittaci is a seed for inoculation of a mammalian cell line, such as a Buffalo Green Monkey (BGM) cell line, using a yolk sac membrane washed free of yolk material. And several steps of removing cells and cell fragments from the vaccine preparation before inactivation after cell disruption (cytopathic effect or CPE) by Chlamydia is complete. One key requirement in the present invention include chlamydia City City (Chlamydia psittaci) for the cell lines used to grow and infection by Chlamydia City City (Chlamydia psittaci), and the victim would (Chlamydia should be destroyed by the organism within 7 days 7 At least 80% CPE in one day).

1 is a group summary graph of clinical scores (average scores / day reported further in Table 1; when prepared with vaccine, a large amount of Chlamydia psittaci (50-1000 μg / referred to by Chu et al. ml) as well as trace amounts (less than 50 μg / dose) of Chlamydia psittaci indicating protection)

The present invention contains trace amounts of purified, inactivated, immunogenically effective amounts of Chlamydia psittaci in combination with an effective amount of adjuvant, and a pharmaceutically acceptable carrier or diluent, which can be combined with other cat antigens. It provides a vaccine composition. When administered to cats, the vaccine is non-responsive but effective.

The term “immunogenically effective amount” as used herein refers to the ability of Chlamydia psittaci in a vaccine to protect a cat from Chlamydia psittaci infection after inoculation. The term "trace" means that the amount of Chlamydia psittaci added to the vaccine is less than 50 μg / dose. The term "purified" refers to the process of eliminating the presence of egg yolk material in the final vaccine. This process is not limited, but is inoculated with brine-free, crushed, inoculated tissue cultures in yolk sac membranes resuspended in phosphate-buffered saline and destroyed by Chlamydia psittaci. Cell lines are used, which include removing cells and cell fragments before inactivating Chlamydia psittaci .

The invention also cultures Chlamydia psittaci in yolk sac, harvests the yolk sac membrane from the yolk sac and freezes the harvest as the main seed, and makes the main seed or the main seed passage highly sensitive. Cultivate in cell lines and grow on the surface until at least 80% of the cell lines are destroyed by chlamydia citaci, harvest the disrupted susceptible cell line and the chlamydia citacy-containing medium accompanying the cell line and destroy the cell line from chlamydia citasi And a method for preparing a vaccine against chlamydia infection comprising inactivating chlamydia citasy and mixing the inactivated chlamydia citacy with an adjuvant and a physiologically acceptable carrier.

Chlamydia psittaci has a developmental cycle with pronounced reproduction (retirement) and infection (base bodies) forms. When grown in the cell lines most commonly used for the growth of chlamydia (eg, dog kidney cells, McCoy cells and CRFK cells), the organism does not produce a full cytopathic effect on the cell sheet. This makes it difficult to determine the optimum time of harvest in the manufacturing setting. It also creates chlamydia organisms with low immunogenicity, requiring proliferation harvest from cells or high concentration after harvest. In some cases (as described in US Pat. No. 5,242,686) they must be inactivated in the secondary culture. From this procedure a cat is obtained which has a high antigen load (high antigen mass including 50 to 1000 μg / dose) and which is reactive to cats after post vaccination. Infection of cells, such as buffalo green monkey cell lines that are highly susceptible to infection of Chlamydia psittaci organisms, results in complete destruction of the cell sheet when the number of infectious organisms in the culture is very high. These organisms can be easily purified from cells and cell fragments by filtration, centrifugation or ultrafiltration, and trace amounts (<50 [mu] g / dose ) of the purified purified Chlamydia psittaci are used as adjuvants and pharmaceuticals. It is immunogenicly effective when formulated in a vaccine containing an acceptable carrier.

According to the invention, chlamydia isolates, such as Cello strains of Chlamydia psittaci, are first propagated in yolk sacs of embryonated chicken eggs. Chlamydia psittaci isolates were inoculated into embryogenic chick eggs and incubated at 37 ° C. for 5-10 days. Eggs were irradiated every day starting at day 5 to determine viability. On the day of 25% egg death, the egg yolk sac membranes of the surviving eggs are harvested and washed with phosphate buffered saline to remove excess yolk protein material and blended aseptically to resuspend the blended membrane in phosphate buffered saline. I was. One milliliter aliquot of this blended and resuspended seed material was stored frozen at −70 ° C. The same procedure was repeated up to five times in order to produce production seeds useful for inoculating tissue culture cells.

Preparation of the Chlamydia psittaci vaccine antigen is accomplished by first preparing tissue cell culture. Buffalo green monkey cells were grown in Dulbecco's MEM containing high glucose content (DMEM / H), 5% fetal bovine serum, 10 mM HERPES buffer and neomycin. When the cells were confluent, they were infected with Chlamydia psittaci in DMEM / H containing neomycin. Chlamydia psittaci was adsorbed to the cells in low volume medium and DMEM / H medium was added again. Infected cells were incubated at 37 ° C. for 5-7 days. Usually 6 days after infection, 90-100% of the cell sheets were disrupted. At this time, Chlamydia psittaci was harvested by harvesting the vessel by swirling the vessel to remove any remaining cells (capture fluid). Cells and cell pieces were removed from the harvest fluid by 5μ filtration or centrifugation. The purified harvest fluid was then inactivated. Inactivating agents include, but are not limited to, binary ethylenimine, formalin, thimerosal and betapropiolactone. The pH was adjusted to neutral and the purified extract fluid was adjusted to an immunologically effective amount for formulation with an adjuvant by diluting with phosphate buffered saline. The immunogenically effective amount of Chlamydia psittaci was usually less than 50 μg / dose and preferably less than 10 μg / dose. Any supplement may be used. Non-limiting examples of auxiliaries used in the present invention include polymers and copolymers, acrylic acid polymers and copolymers, polyacrylic acids such as Carbopol ^R , surfactants such as hexadecylamine, saponins, Quil A, aluminum hydroxide Side, aluminum phosphate, peptides such as muramyl dipeptides, oil emulsions, immunomodulators such as interleukin and interferon, interferon inducers, ethylene maleic unhydride copolymers such as EMA-31, NEOCRYL A640 and POLYGEN ^R (supplied by the MVP lab). Mixtures of such adjuvants or adjuvant systems may also be used.

It has been found that the adjuvant described above works at an effective amount of 0.01% to 50% v / v of the total vaccine dose. Preferably, the concentration of the adjuvant is adjusted to 1% to 15%. More preferably, POLYGEN ^R is used at a concentration of 1% to 5% and Carbopol ^R is used at a concentration of 0.1% to 0.5%.

The vaccine composition according to the present invention also comprises a pharmaceutically acceptable carrier or diluent and includes feline non-otitis, feline leukemia, feline calcivirus and feline pancreatopenia, feline immunodeficiency virus, feline infectious peritonitis, Toxoplasma gondii) and additional feline antigens for rabies.

Table 1 Group Summary of Clinical Scores-Average Score / Day

Table 2 shows that multi-component vaccines containing inactivated feline nasal tracheitis, feline calcivirus, feline pancreatic leukemia, feline leukemia virus and feline Chlamydia psittaci prepared according to the present invention are effective.

Table 2 Results of efficacy tests of 5-way cat vaccines containing feline Chlamydia psittaci , feline non-otitis, feline leukemia, feline calcivirus and feline pancreatitis virus

C.M. = cumulative mean score

AV.M.S. = Average Median Score

"T" = statistical calculation (one-tailed, Mann Whitney variant of Wilcoxon's two-sample trial) comparing the score of the control group with the clinical score of the vaccinated (VACCINATES) (9 CFR 115.130 , 113.149, 113.131, 115.150, SAM 310 and SAM 311. "T" of ≥ 31 corresponds to protection.

In a further embodiment of the invention, the vaccine composition may be formulated with a sustained release product, eg, a lactide-glycolide copolymer, slowly releasing microparticles and administered as an implant.

The invention also provides a method for preventing chlamydial infection in a cat comprising administering to the cat an immunogenic effective amount of the vaccine composition described above. The route of administration of the invention is parenteral (subcutaneous, intramuscular, intraperitoneal and intradermal) or oral / nasal. Preferred routes of administration are subcutaneous and intramuscular. Effective therapies for treatment include each administration of the vaccine composition at least about 2 times at intervals of about 2 to about 4 weeks, preferably about 14 to about 30 days.

The following examples are intended to illustrate the invention and do not limit its scope.

Example 1 Monovalent Cat Chlamydia psittaci

Thaw cell stocks of buffalo green monkey cells and add them to growth medium containing DMEM / H + 5% fetal bovine serum, 10 mM HERPES buffer and 30 μg / ml neomycin, 10-15 minutes at 400-600 × g. Centrifuged. Centrifuged cell stocks were suspended in fresh growth medium and placed in roller bottles. The roller bottle was incubated by spinning at 37 ° C. until cells grew and adhered. Subsequent cultures of cells were made until the desired number of roller bottles was obtained for production purposes. Usually the production lot varies from 50 to 500 roller bottles. Production of BGM Cells When the subsequent culture roller bottle is 3-5 days old and at least 95% adhered, the growth medium was removed and Chlamydia psittaci seeds were added with multiple infections (MOI) of 0.01 to 0.1. . The medium used for chlamydia growth was DMEM / H plus 30 μg / ml neomycin. Roller bottles were incubated for 1 hour at 36-38 ° C. for adsorption of chlamydia and at least 200 ml of medium was added to each roller bottle. Incubation at 36-38 ° C. was continued for 5-7 days or until CPE was> 80%. The roller bottle was shaken and harvested and the harvested fluid was transferred to one container to remove any remaining cells from the surface of the roller bottle fluid. The harvested fluid was cleared (purified) by filtration through a 5 micron polypropylene filter to remove foreign cell fragments. The cleared fluid was inactivated with 0.01 M binary ethylenimine (BEI) at 2-8 ° C. for 72 hours. After inactivation, BEI was neutralized with sodium thiosulfate at a final concentration of 0.02 M.

Purified and inactivated Chlamydia psittaci were diluted to three different antigen levels (three different antigen masses). The highest antigen mass tested was 100 μl / dose, equivalent to approximately 100 μg / dose, and the intermediate antigen mass tested was 50 μl / dose, equivalent to approximately 50 μg / dose, while the lowest antigen mass tested was 25 μl / dose or approximately 25 μg / dose. All antigen masses were diluted with phosphate buffered saline in an amount appropriate for each formulation to be 0.95 ml. To prepare the final vaccine, 0.05 ml volume of POLYGEN ^R was added to each formulation as an adjuvant. Groups of five seronegative cats were administered 1 milliliter dose of these vaccines at three week intervals. The first dose was administered on day 0 and the second dose was administered on day 21. Groups of five seronegative cats matched in age and environment were not inoculated and kept as unvaccinated controls. All cats were inoculated intranasally and in the eye with toxic Chlamydia psittaci 2-8 weeks after the second dose was administered. All cats received clinical signs of Chlamydia psittaci including body temperature, ocular discharge, conjunctivitis, nasal exudate, sneezing, coughing, dyspnea, anorexia, dehydration and depression for 28 days. Were tracked daily. A weighted scoring system was used to analyze the data. The more severe the signs of disease, the higher the numerical values. All data are statistically analyzed at four day intervals and provided in Table 1. These data are shown graphically in FIG. 1.

The results shown in Table 1 and FIG. 1 clearly show that Chlamydia psittaci is effective at less than 25 μg antigen mass per dose when prepared according to the present invention. Since this low antigen mass vaccine provides equivalent protection to high antigen mass vaccines, it is predicted that when prepared according to the method of the invention, low levels of 25 μg per dose are also protective.

Example 2

Inactivated cat Chlamydia psittaci prepared according to Example 1 of the present invention was mixed with four other inactivated cat antigens to prepare a multicomponent vaccine. POLYGEN ^R- assisted Chlamydia psittaci (10 ^6.0 TCID ₅₀ / ml) Feline leukemia, POLYGEN ^R- assisted feline rhinitis at antigen dose of 1.25 ELISA relative potency units supplemented with POLYGEN ^R per 35 microliters per dose 10 ^8.0 TCID ₅₀ per dose of POLYGEN ^R- assisted cat Calicivirus 10 ^7.8 TCID ₅₀ per dose of POLYGEN ^R- assisted pancreatic cytopenia virus and 10 ^5.9 TCID ₅₀ per dose. This 5-way method vaccine was tested for efficacy in the vaccination / inoculation trial conducted in cats. After the start of the experiment, all cats were bled for serum and complete blood samples. Serum was screened for antibodies to feline rhinitis, feline calcivirus, and feline pancreatitis virus titers. All cats started with titers less than 1: 2 for viruses. Cats used in the FeLV protocol were negative for the p27 antigen. Five groups of cats 13-15 weeks old (n = 74) were inoculated and further stimulated with 1.0 ml of 5-way vaccine three weeks later. Approximately half of the cats were subcutaneously inoculated and half were intramuscularly inoculated.

Eight inoculated (four subcutaneous and four intramuscular) cats and one control cat were evaluated for serologic responses after cat pancreatic induction and for further stimulation. A serological response of 1: 8 is considered to be protective for this component. The results after inoculation are listed in Table 2.

Twelve five-way inoculated (six subcutaneous and six intramuscular) cats and four non-inoculated control cats were inoculated with the toxic feline rhinitis virus. Throat swabs and blood samples were taken at each inoculation. All cats were followed for signs of disease after inoculation. Clinical disease signs and temperatures were recorded and analyzed with the median score shown in Table 2. Efficiency is evidenced by a statistically significant difference in clinical score between the inoculated and the control group.

Twelve 5-way inoculated (six subcutaneous and six intramuscular) cats and four non-inoculated control cats were inoculated with toxic feline calicivirus. Neck sponges and blood samples were taken after inoculation. In addition, cats were followed for body temperature and clinical signs of disease. Clinical signs are recorded, scored, analyzed and provided in Table 2. Efficiency is evidenced by a statistically significant difference in clinical score between inoculation and control.

Twenty 5-way inoculated (10 subcutaneous and 10 intramuscular) cats and 10 non-inoculated control cats were inoculated with the toxic cat Chlamydia psittaci and the disease described in Example 1 The clinical signs of were tracked and scored. Efficacy is demonstrated by the statistically significant difference between the inoculated and the control median score. Summary results of these inoculations are also shown in Table 2.

Twenty-two 5-way inoculated (11 subcutaneous and 11 intramuscular) cats and nine unvaccinated control cats were inoculated with toxic FeLV. This attack requires the cat to be immunosuppressed first and then attacked for two consecutive days. Cats were checked for viremia for 3-12 weeks after inoculation by detecting indirect immunofluorescence (IFA) p27 antigen in blood. Protection is determined using the guidelines described by Shibly et al. (JAVWA 1991, 199: 1402-1406) and is shown in Table 2.

From Table 2 it is proven to be a multi-component vaccine containing the cat when Chlamydia Theta (Chlamydia psittaci) of the present invention effective. Which it would inoculated any vaccine in the different studies carried out on a multi-component vaccine containing the Chlamydia City City (Chlamydia psittaci) vaccine or Chlamydia City City (Chlamydia psittaci) in this study also did not show the local or systemic reactions to the vaccine It should be noted that. Resistance mass Chlamydia psittaci was the main reason for the lack of reactivity.

While the present invention has been described above with respect to the specific embodiments described above, it should be recognized that various changes and modifications apparent to those skilled in the art are possible and are within the scope of the invention as defined in the following claims.

Claims (12)

a) incubating Chlamydia psittaci in yolk sac; b) harvesting the yolk sac membrane from the yolk sac and freezing the harvest as the main seed; c) culturing said main seed or said main seed passage in a highly sensitive cell line to grow on the surface until at least 80% of the cell line is destroyed by chlamydia citaci; d) harvesting the disrupted susceptible cell line and the chlamydia cittash containing medium accompanying the cell line; e) removing the broken cell line from chlamydia cytaci; f) inactivating chlamydia citaci; g) A method of making a vaccine against chlamydia infection comprising mixing an inactivated chlamydia cittaci with an adjuvant and a physiologically acceptable carrier. The method of claim 1, wherein the highly sensitive cell line is buffalo green monkey cells. The method of claim 1, wherein inactivation comprises adding an effective amount of inactivating agent to inactivate Chlamydia psittaci while maintaining immunogenicity. 4. The method of claim 3, wherein said inactivating agent is selected from the group consisting of binary ethylenimine, beta propiolactone, formalin, methirate, thimerosal, glutaraldehyde and detergents. The method of claim 3, wherein the deactivator is a binary ethyleneimine. The method of claim 1, wherein the adjuvant is selected from polymers and copolymers, acrylic acid polymers and copolymers, polyacrylic acids such as Carbopol ^R , surfactants such as hexadecylamine, saponins, Quil A, aluminum hydroxide, aluminum phosphate Peptides such as muramyl dipeptides, oil emulsions, immunomodulators, such as interleukin and interferon, interferon inducers, ethylene maleic unhydride copolymers such as EMA-31, NEOCRYL A640 and POLYGEN ^R and their Method selected from the group consisting of mixtures. The method of claim 1, wherein the adjuvant is POLYGEN ^R. The method of claim 1, wherein the physiologically acceptable carrier is selected from the group consisting of phosphate buffered saline, saline and minimal essential medium. An inactivated immunogenically effective non-reactive Chlamydia psittaci vaccine containing less than 50 μg / dose Chlamydia citaci prepared by the method according to any one of claims 1 to 8. . 10. The inactivated immunogenicly effective non-reactive Chlamydia psittaci vaccine of claim 9, further comprising chlamydia cittaci and another feline antigen. A method for preventing chlamydial infection in a cat comprising administering to the cat an immunogenic effective amount of the vaccine of claim 9. A method for preventing chlamydial infection in a cat comprising administering to the cat an immunogenic effective amount of the vaccine of claim 10.