Yersinia SPP. Polypeptides and Methods of Use

a technology of yersinia spp. and polypeptides, which is applied in the field of yersinia species, can solve the problems of limited ability to correctly diagnose the causative agent, difficult to determine the incidence of human disease caused by y. enterocolitica in the u.s., and the administration of antibiotic therapy may be too late for effective intervention

Inactive Publication Date: 2011-08-25
EPITOPIX LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The present invention provides a composition including two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 40 kDa, 38 kDa, or 37 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersinia enterocolitica when incubated in media containing an iron chelator and not isolatable when grown in the media without the iron chelator. In some aspects, the composition may include two different 83 kDa polypeptides isolatable from a Y. enterocolitica when incubated in media comprising an iron chelator. The composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729. The composition may further include an isolated polypeptide having a molecular weight of 268 kDa, 92 kDa, 79 kDa, 54 kDa, 45 kDa, 31 kDa, 28 kDa, or a combination thereof, and isolatable from a Y. enterocolitica when grown in the media without the iron chelator.
[0021]The present invention also provides a composition including two isolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 268 kDa, 79 kDa, or 45 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersinia enterocolitica when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. The composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729.
[0022]The present invention further provides a composition including isolated polypeptides having molecular weights of 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, and 28 kDa, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides are isolatable from a Yersinia enterocolitica, and the composition protects a mouse against challenge with Y. enterocolitica ATCC strain 27729. The polypeptides may be isolatable, or in some aspects isolated from Y. enterocolitica is ATCC strain 27729.
[0024]The present invention also provides a composition including two isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, and two isolated polypeptides having molecular weights of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, 20 kDa, or a combination thereof, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa, are isolatable from a Yersinia pestis when incubated in media comprising an iron chelator and not isolatable when grown in the media without the iron chelator. The composition protects a mouse against challenge with Y. pestis strain KIM6+. The composition can further include a pharmaceutically acceptable carrier. The polypeptides may be isolatable, or in some aspects isolated from Y. pestis strain KIM6+.
[0025]The present invention further provides a composition including isolated polypeptides having molecular weights of 254 kDa, 104 kDa, 99 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 60 kDa, 46 kDa, 44 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, and 20 kDa wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The polypeptides are isolatable from a Yersinia pestis, and the composition protects a mouse against challenge with Y. pestis strain KIM6+. The polypeptides may be isolatable, or in some aspects isolated from Y. pestis strain KIM6+.

Problems solved by technology

The incidence of human disease caused by the Y. enterocolitica in the U.S. is difficult to determine, simply because infections associated with this organism are typically self-limiting and insufficient detection techniques have limited the ability to correctly diagnose the causative agent.
These symptoms are often misdiagnosed early, and antibiotic therapy may therefore be administered too late for effective intervention.
Therefore, although antibiotic therapies are available and effective if administered early, the rapid onset of pneumonic plague and the misdiagnosis of septicemic plague are major obstacles in treatment of the disease.
Y. pseudotuberculosis and some serotypes of Y. enterocolitica can also spread to the vascular system and cause fatal cases of septicemia (Bottone, E. J., Clin. Microbiol. Rev., 10, 257-276, (1997), Lenz, T., et al., J Infect Dis, 150, 963, (1984)), although these more invasive infections are typically limited to susceptible individuals.
Although natural infection by Y. pestis is rare in this country, there is fear that the organism will become a bioterrorism agent.
Second, there is a high mortality rate associated with Y. pestis infection if left untreated, and the pneumonic faun of plague is distinguished by a rapid onset of symptoms that may be recognized too late for an effective intervention.
However, there is evidence that KWC immunizations offer little protection against pneumonic plague (Cohen, R. J. and J. L. Stockard, JAMA, 202, 365-366, (1967), Meyer, K. F., Bull World Health Organ, 42, 653-666, (1970)), and an additional drawback to these vaccines is that multiple injections over several months are required for protective immunity.
However, strain EV76 is not fully avirulent, causing death in approximately 1% of vaccinated mice (Russell, P., et al., Vaccine, 13, 1551-1556, (1995)).
In contrast to the extensive search for protective plague vaccines, very little research efforts have been focused on preventing infections by the enteropathogenic Yersinia species.
However, these strains were engineered primarily as live oral vaccine carriers, and no further testing of these strains for prevention of yersiniosis has been reported.
This study demonstrated that hsp60 mRNA was present in various host tissues following immunization, but protection against Y. enterocolitica challenge was limited to the spleen and no protection was observed in the intestinal mucosa.
In mammalian hosts, available iron is extremely limited; intracellular iron is complexed with storage proteins, and extracellular iron is bound by the host proteins transferrin and lactoferrin.

Method used

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  • Yersinia SPP. Polypeptides and Methods of Use
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Production and Isolation of Metal Regulated Proteins

[0091]The compositions used in the following examples were prepared using the proteins derived from Y. enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ (obtained from R. D. Perry, University of Kentucky). The two strains were each inoculated from frozen stocks into 25 ml tryptic soy broth (TSB) containing 160 μM 2,2-diprydyl or 300 μM FeCl3, and incubated at 37° C. while shaking at 400 rpm. Following 12 hours of incubation, 5 ml of each culture was transferred into 500 ml of pre-incubated (37° C.) media containing 160 μM 2,2-diprydyl or 300 μM FeCl3 and incubated at 37° C. while stirring at 100 rpm. After 8 hours of incubation, the cultures were centrifuged at 10,000×g for 20 minutes. The bacterial pellets were resuspended in 100 ml of sterile physiological saline and centrifuged at 10,000×g for 10 minutes to remove any contaminating media proteins. The bacterial pellets were then resuspended in 40 ml of Tris-buffered sa...

example 2

Preparation of the Immunizing Compositions Derived from Y. Enterocolitica

[0092]The proteins made from Y. enterocolitica as described in Example 1 were used to prepare a composition for administration to animals. The composition contained polypeptides having molecular weights of 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa. The polypeptides having molecular weights of 83 kDa, 70 kDa, and 66 kDa were expressed only under iron limited conditions, and the expression of polypeptides having molecular weights of 268 kDa, 79 kDa, and 45 kDa was enhanced under iron limited conditions.

[0093]A stock vaccine was prepared from the composition by emulsifying the aqueous protein suspension (500 μg total protein / ml) into the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) using an IKA Ultra Turrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio). The vaccine was administered to mice to give a final dose of 50 μg tota...

example 3

Preparation of Challenge Organism

[0094]When used as a challenge, the Y. enterocolitica ATCC strain 27729 was prepared as follows. Briefly, the isolate from a frozen stock was streaked onto a blood agar plate and incubated at 37° C. for 18 hours. A single colony was subcultured into 50 ml Tryptic Soy Broth (Difco) containing 25 μg / ml 2,2′ dipyridyl. The culture was incubated at 37° C. for 6 hours while rotating at 200 rpm, at which point the culture was centrifuged at 10,000×g for 10 minutes at 4° C. to pellet the bacteria. The bacterial pellet was washed twice by centrifugation in physiological saline at 4° C. The final pellet was resuspended in 25 ml of physiological saline and used for challenge. Just prior to challenge, 1 ml of the above bacterial suspension was serially diluted ten fold to enumerate the number of CFU / mouse dose.

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Abstract

The present invention provides isolated polypeptides isolatable from a Yersinia spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

Description

[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 60 / 646,106, filed Jan. 21, 2005, which is incorporated by reference herein.BACKGROUND[0002]There are three Yersinia species that are pathogenic to humans: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis is the causative agent of plague, while Y. pseudotuberculosis and specific pathogenic serovars of Y. enterocolitica cause gastrointestinal illnesses. Other species of Yersinia, including Y. rohdei, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, and Y. moolaretti, are considered enterocolitica-like opportunist pathogens with the ability to cause diarrheal illness in susceptible individuals (Agbonlahor, J Clin Microbiol, 23, 891-6, (1986), Cafferkey, et al., J Hosp Infect, 24, 109-15, (1993), Loftus, et al., Dig Dis Sci, 47, 2805-10, (2002)). The Yersinia can also infect other animal species causing a range of illnesses. Most wild and domestic species of m...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K7/00A61K38/10C07K14/24A61K38/16A61P31/04
CPCA61K39/00A61K39/025A61K2039/552A61K2039/55505A61K39/0291C07K14/24A61K2039/55566C07K16/1228A61K2300/00A61P1/04A61P1/12A61P31/00A61P31/04Y02A50/30A61K2039/55A61K2039/54A61K2039/575
Inventor EMERY, DARYLL A.STRAUB, DARREN E.WONDERLING, LAURA
Owner EPITOPIX LLC
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