Non-pyrogenic bacterial strains and use of the same

Details Non-pyrogenic bacterial strains and use of the same Non-pyrogenic bacterial strains and use of the same

1999-06-24

2003-04-15

Smith, Lynette R. F. (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C424S234100, C424S241100, C424S245100, C424S253100, C424S258100, C424S260100, C424S261100, C424S001210, C424S240100, C424S249100, C435S069300, C435S170000, C435S252300

Reexamination Certificate

active

06548287

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides gram-negative bacterial strains that stably produce substantially-pure non-pyrogenic lipopolysaccharide or lipid A. The present invention also relates to the use of said strains for the preparation of non-pyrogenic DNA and use of the same for introducing endogenous or foreign genes into animal cells or animal tissue. Further, the present invention relates to a use of said strains for the preparation of non-pyrogenic recombinant mammalian, protozoan and viral proteins. Yet a further use of tile present invention relates to a use of said strains for the preparation of non-pyrogenic bacterial proteins and polysaccharides antigens for use as vaccines. Furthermore, the present invention relates to a use of said strains for the preparation of non-pyrogenic live and inactive bacterial vaccines and vaccine vectors.
BACKGROUND OF THE INVENTION
1. The Use of Bacterial Host Strains in Biotechnology
Medical biotechnology now encompasses a broad range of medical technologies that have veterinary and human applications. At the crux of this technology is the use of recombinant DNA, molecular biochemistry and immunochemical techniques, which allow the identification, characterization and manufacture of proteins and polysaccharides. One of the first products produced using these techniques was cloned recombinant human insulina. Since it's initial implementation, biotechnology has enabled the development of a large array of biological products that have therapeutic or vaccinal properties (Crommelin and Schellekens (eds), in:
From clone to clinic
, Kluwer Academic Publishers, Dorddrecht, The Netherlands (1990); The Biotol Team (eds), In:
Biotechnology innovations in health care
. Butterworth-Heinemann Ltd, (1991); Reidenberg (ed), In:
The clinical pharmacology of hiotechnology products
, Elsevier Science Publishers (1991)).
One of the biotechnology “work horses” are the bacterial host strains, which are used to house cloned genes and for the large scale production of the cloned genes or the products of said cloned genes. Examples of these bacterial hosts strains include HB101, DH5, DH5&agr;, DH5&agr;MCR, DH10, DH10B, C600 or LE392 (Grant et al, Proc Natl Acad Sci (USA) 87:4645-4649 (1990); Sambrook et al (eds), In:
Molecular Cloning
, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1993)). These host strains are designed to stably harbor and express clones genes.
A major problem, however, associated with using bacterial host strains is the process of removing bacterial LPS from the final products. The biological properties of LPS have been extensively investigated (Rietschel et al,
FASEB
, 8(2): 217-225 (1994);
Raetz, J Bacteriol
, 175(18):5745-5753 (1993); and Alving,
Immunobiol
, 187:430-446 (1993)). This molecule has powerful pyrogenic activity, so that in humans nanogram quantities of LPS can induce febrile responses, which are mediated by host proinflammatory cytokines IL-1, IL-6, and TNF-&agr; (Mackowiak (ed), In:
Fever: Basic mechanisms and management
. Raven Press, NY (1991); Abernathy and Spink,
J Clin Invest
, 37:219-226 (1958); Greisman et al,
J Clin Invest
, 43:1747-1757 (1964); Rietschel et al, supra; and Raetz, supra (1993)). For this reason, the United States Food and Drug Administration have strict guidelines on the level of LPS that is acceptable in biomedical products (
Good Manufacturing Practices
, In:
US Code of Federal Regulation
210-211; and
Protection of human subjects
, US Code of Federal Regulation 50, Food and Drug Administration, CBER, Rockville Md.). Since all the currently available host strains produce pyrogenic LPS, this activity must be removed to acceptable levels, resulting in additional manufacturing costs.
2. Gene Therapy and Genetic Immunization
The commercial applications of DNA delivery technology to animal cells are extremely broad and includes delivery of vaccine antigens (Fynan et al,
Proc. Natl. Acad. Sci., USA
, 90:11478-11482 (1993); Katsumi et al,
Hum Gene Ther
, 5(111:1335-1339 (1994); Spooner et al,
Int J Oncol
, 6(6):1203-1208 (1995)), immunotherapeutic agents (Shillitoe et al,
Eur J Cancer
30B(3):143-154 (1994); Hengge et al,
Nature Genetics
, 10(2):161-166 (1995); Vile and Hart,
Ann Oncol
, 5(Suppl 4):59-65 (1994); Miller et al,
Ann Surg Oncol
, 1(5):436-450 (1994); Foa,
Baillieres Clin Haematol
, 7(2):421-434 (1994)), and gene therapeutic agents (Darris et al,
Cancer
, 74(Suppl 3):1021-1025 (1994); Magrath,
Ann. Oncol
., 5(Suppl 1):67-70 (1994); Milligan et al,
Ann. NY Acad. Sci
., 716:228-241 (1994); Schreier,
Pharma. Acta Helv
., 68:145-159 (1994); Cech,
Biochem. Soc. Trans
., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Dropulic and Jeang,
Hum Gene Ther
, U5(8):927-939 (1994); Sorscher et al,
Hum Gene Ther
, 5(10):1259-1277 (1994); Woo,
Trends Genet
, 10(4):111-112; Long et al,
FASEB J
., 7:25-30 (1993); Nabel et al,
Hum Gene Ther
5 (9):089-109 (1994); Manthorpe et al,
Hum Gene Ther
, 4(4):419-431 (1993); Mittal et al,
Virus Res
, 28:67-90 (1993); Setoguchi et al,
Am J Respir Cell Mol Biol
10:369-377 (1994); and Rosi et al,
Pharm. Therap
., 50:245-254 (1991)).
In the aforementioned applications prolonged expression of the eukaryotic expression cassette once in the host tissue is highly desirable (Yang et al,
J Virol
, 69(4):2004-2015 (1995); Wicks et al,
Hum Gene Ther
, 6(3):317-323 (1995) and Alton et al,
Nature Genet
, 5(2):135-42 (1993)). Unfortunately, adenoviral vectors have proven to he highly immunogenic and induce a host responses against cells containing these vector (Yang et al, supra). This host response causes a more rapid clearance of the cells carrying adenovirus-delivered eukaryotic expression cassettes. Similarly, induction of inflammation at the site of “naked” DNA introduction or treatment with DNA encapsulated in cationic lipids can be deleterious to the elicitation of prolonged expression of the introduced eukaryotic expression cassette (Wicks et al, supra). A major cause of inflammation after introduction of DNA into the host is LPS, which co-purifies with DNA (Wicks et al, supra). LPS is a notorious biologically active molecule with potent pyrogenic properties (Mackowiak (ed), supra); Rietschel et al, supra; Raetz, surpa (1993); and Alving,
Immunobiol
, 187:430-446 (1993)).
Thus, sophisticated DNA purification procedures have been devised that remove LPS from the DNA prior to introduction into the host (Yang et al, supra; Nabel et al, supra; and Manthorpe et al, supra). These purification procedures involve removal of LPS with ionic detergents such as Triton X-114 or using polymyxin B columns (Yang et al, supra; Nabel et al, supra; and Manthorpe et al, supra). A weakness of this approach is that it adds additional cost to the commercial production of DNA and even after such purification procedures significant quantities of LPS remains associated with the DNA (Yang et al, supra; Nabel et al, supra; and Manthorpe et al, supra). This LPS will enter the host cells that receive the DNA encoding the eukaryotic expression cassette and exert it's biological effects. Higher primates are more sensitive to LPS than laboratory rodents and under certain pathological conditions states of LPS hypersensitivity can be induced (Mackowiak (ed), supra); Abernathy and Spink, supra; and Greisman et al, surpa). Therefore, it is paticularly important to produce DNA preparations that are free of LPS pyrogenic activity for applications in humans.
3. Bacterial Strains in Vaccine Development
Inactivated and live attenuated bacteria are effective as vaccines (Holmgren et al, In:
Vibrio cholerae and cholera
. Wachsmuth et al. (eds), ASM Press Washington D.C., pp 415-424 (1994); Woodrow and Levine (eds), In:
New Generation Vaccines
, Marcel Dekker, New York (1990); and Cryz (ed) in:
Vaccines and immunotherapy
, Pergamon Press New York (1991)) and as vector vaccines for the delivery of passenger antigens from other pathogens to the host immune system (Woodrow and Levine (eds), supra; and Cryz (ed) supra). In the role of vaccine vector

Affiliated with

Also associated with

Rating

Non-pyrogenic bacterial strains and use of the same does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Non-pyrogenic bacterial strains and use of the same, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Non-pyrogenic bacterial strains and use of the same will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3083628