Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0258—Escherichia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention generally relates to the preparation of proteins.
• the present invention relates to the preparation and purification of large quantities of at least one colonization factor (CF) and methods of using thereof.
• CF colonization factor
• ETEC may be transmitted by food or water contaminated with animal or human feces. ETEC produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT). ETEC infections may cause profuse watery diarrhea, abdominal cramping, fever, nausea, vomiting, chills, loss of appetite, headache, muscle aches, and bloating.
• ST heat-stable toxin
• LT heat-labile toxin
• the current therapy for travelers' diarrhea is to initiate treatment with agents such as bismuth subsalicylate (Pepto-Bismol®), antidiarrheals such as diphenoxylate with atropine (Lo otil®), loperamide HC1 (Immodium®), attapulgite (Kaopectate®) and the like, rehydration therapy, and combinations thereof.
• agents such as bismuth subsalicylate (Pepto-Bismol®), antidiarrheals such as diphenoxylate with atropine (Lo otil®), loperamide HC1 (Immodium®), attapulgite (Kaopectate®) and the like, rehydration therapy, and combinations thereof.
• the majority of the treatments involve the non-specific removal of the offending agents (i.e. toxins) from the intestinal tract. Only in moderate to severe cases of diarrhea where distressing or incapacitating symptoms are reported is antimicrobial therapy recommended. ETEC is frequently resistant
• Fluoroquinolones such as ciprofloxacin have shown some efficacy. Antibiotics are not usually effective at reducing clinical symptoms of the disease and problems associated with antibiotic resistance can occur. Prophylactic use of antibiotics is not recommended. Thus, therapies that specifically remove ETEC from the intestine are needed to provide more effective treatments for ETEC diarrhea.
• ETEC In order to initiate the infectious process of diarrhea, ETEC must adhere to the host intestinal epithelial cells via the binding between bacterial adhesions, colonization factors (CFs) and host receptors. This binding is commonly referred to as adhesion- receptor interaction. See Beachey, E.H. (1981) J. Infect. Dis. 143:325-345; Satterwliite, T. K., et al. (1978) Lancet 2:181-184; and Warner, L. and Y. S. Kim. (1989) "Intestinal Receptors for Microbial Attachment", Eds. M. J. G. Farthing, and G. T.
• CFs interact with receptors on the host epithelial cells allowing for adherence of the ETEC to the mucosa. See Cassels, F.J. and Wolf, M.W. (1995) J. Indust. Microbiol 15:214-2263. CFs include colonization factor antigens (CFAs), coli surface (CS), and putative colonization factors (PCFs).
• CFAs colonization factor antigens
• CS coli surface
• PCFs putative colonization factors
• ETEC strain B7A expressing colonization factor CS6 that enables colonization of the intestine was successful in causing diarrhea in human subjects.
• ETEC strain H10407 expressing CFA/I colonization factor, mediated the development of disease in 9 out of 10 humans challenged with the plate grown bacteria. See Tacket, CO. et al. (1988) N. Engl. J. Med. 318(19):1240-3124.
• ETEC strain E24377A expressing colonization factor antigen II (CFA/II) containing CS1 and CS3, provided similar attack rates as in the previous studies. See Tacket, CO. (1994) Vaccine 12(14): 1270-1274.
• the ETEC challenge strains used were cultured on either TS A or CFA solid agar plates and harvested by scraping the bacteria from the surface.
• the present invention generally relates to methods for expressing colonization factors (CFs).
• the present invention provides a method of obtaining a preparation comprising at least one colonization factor from a bacterial strain which comprises culturing or fermenting the bacterial strain in DME/F-12 broth.
• the expression levels of the colonization factor are increased, induced, enhanced, or modulated as compared with expression levels of the bacterial strain cultured or fermented in a conventional medium, such as Luria broth, CFA agar, or CFA broth.
• the bacterial strain is an enterotoxigenic Escherichia coli strain, preferably H10407, E9034A, or 60R75.
• the colonization factor is a colonization factor antigen, a coli surface protein, or a putative colonization factor, such as those belonging to the CFA/I family, the CS5 family, the Type IV family, or the distinct group of colonization factors.
• the colonization factor is CSl, CS3, or CFA/I.
• the bacterial strain is cultured or fermented at about 25 °C to about 37 °C.
• the colonization factor is obtained in an amount that is greater than amounts obtained when the bacterial strain is cultured or fermented in a conventional medium, such as Luria broth, CFA agar, or CFA broth. In some embodiments, the amount obtained is about 1 to about 14 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 1 to about 7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 1 to about 4 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium.
• a conventional medium such as Luria broth, CFA agar, or CFA broth. In some embodiments, the amount obtained is about 1 to about 14 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 1 to about 7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium
• the amount obtained is about 12.4 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium. In some embodiments, the amount obtained is about 3.7 times greater than amounts obtained when the bacterial strain is cultured or fermented in the conventional medium.
• the colonization factor is not obtained from the bacterial strain when cultured or fermented in Luria broth. In some embodiments, the colonization factor is not obtained from the bacterial strain when cultured or fermented in CFA broth.
• the preparation has a protein purity of the colonization factor of about 70% or more. In some embodiments, the protein purity is about 80% or more. In some embodiments, the protein purity is about 90% or more. In some embodiments, the bacterial strain was frozen prior to cultivating or fermenting.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising the preparation or the colonization factor made by the methods disclosed herein and a carrier, an adjuvant, or both.
• the pharmaceutical composition may be an oral formulation, an mtranasal formulation, or a transcutaneous formulation.
• the pharmaceutical composition is an injectable formulation.
• the present invention provides methods of treating, preventing or inhibiting an enterotoxigenic Escherichia coli infection in a subject which comprises administering at least one preparation or at least one colonization factor made by the methods disclosed herein.
• the enterotoxigenic Escherichia coli infection is traveler's diarrhea.
• Figure 1 shows the growth curves for HI 0407.
• Figure 2 shows the growth curves for 60F75.
• Figure 3 shows the SDS-PAGE from H10407 when grown in DME/F-12, L- broth, and CFA broth.
• Figure 4 shows the Western blot for HI 0407.
• Figure 5 shows the SDS-PAGE from E9034A when grown in DME/F-12, L- broth, and CFA broth.
• Figure 6 shows the Western blot for E9043 A.
• Figure 7 shows the SDS-PAGE from 60R75 when grown in DME/F-12, L- broth, and CFA broth.
• Figure 8 shows the Western blot for 60R75.
• Figure 9A and Figure 9B show CS 1 expressing ETEC grown on CFA agar.
• Figure 10A and Figure 10B show CSl expressing ETEC grown on CFA broth.
• Figure 11A and Figure 1 IB show CSl expressing ETEC grown on L broth.
• Figure 12A and Figure 12B show CSl expressing ETEC grown on DME/F-12 broth.
• Figure 13 A and Figure 13B show CFA/I expressing ETEC grown on CFA agar.
• Figure 14A and Figure 14B show CFA/I expressing ETEC grown on CFA broth.
• Figure 15A and Figure 15B show CFA/I expressing ETEC grown on DME/F-12 broth.
• Figure 16 is an SDS-PAGE of CS3 from a 300L fermentation. 16% tris-Tricine
• SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 10 and 20 ⁇ g loaded (lanes 2 and 3, respectively) of CS3, Lot 0963 from 300 L fermentation.
• the molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1.
• Figure 17 is an SDS-PAGE of CFA/I from a 10L fermentation. 16% tris-
• Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 30 ⁇ g loaded (lane 2) of CFA/I from 10 L fermentation.
• the molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1.
• Figure 18 is an SDS-PAGE of CFA/I from a 300L fermentation. 16% tris-
• Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 20 and 10 ⁇ g loaded (lanes 1 and 2, respectively) of CFA/I, Lot 1096 from 300 L fermentation.
• a CFA/I standard is included for comparison (20 ⁇ g, lane 4).
• the molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 3.
• the present invention generally provides methods for increasing, inducing, or enhancing the expression levels of at least one colonization factor (CF), such as CSl and CFA/I, as compared to prior art methods.
• CF colonization factor
• the methods for increasing, inducing, or enhancing the expression levels of CFs as provided herein may be used in order to obtain at least one CF in high purities and large amounts that are suitable for approval from governmental regulatory agencies for administration to humans.
• F-12 medium a serum-free medium that was used to support the clonal growth of Chinese hamster ovary and lung cells. See Ham, R. (1965) PNAS USA 53:288-293, which is herein incorporated by reference. Later, it was demonstrated that F-12 also supported the growth of differentiated cells from rat, rabbit and chicken embryos. More importantly, chemical analysis of the medium revealed selenium contamination that proved essential in the absence of serum.
• DME Dulbecco's Modified Eagle's medium
• BME Eagle's medium
• DME and F-12 media were combined to create DME/F-12.
• This combination essentially increased the concentrations of components already present in F-12 by a factor of 2. See Ham R. and McKeehan, W. (1979) in METHODS IN ENZYMOLOGY Jacoby W. B. and Pastan, I. H., eds. vol. 58; and Barnes, D. and Sato, G. (1980) J. Anal. Biochem. 102:255-270, which are herein incorporated by reference.
• DME Dulbecco's Modified Eagle's Medium
• DME/F-12 Dulbecco's Modified Eagle's/Ham's Nutrient Mixture
• DME/F-12 (Sigma- Aldrich, St. Louis, MO) was used to grow enterotoxigenic Escherichia coli (ETEC) strains. When grown in DME/F-12 CFs are expressed at levels per bacterium that are higher than that of the methods known in the art such as those using CFA agar, CFA broth, and L broth.
• the present invention provides methods for increasing, inducing, or enhancing the expression levels of at least one colonization factor (CF) which comprise culturing a bacterial strain in DME/F-12.
• CF colonization factor
• the ETEC strains exhibit prodigious increases in expression levels of CFA/I, CSl, and CS3 when cultured in DME/F-12, which is contrary to the accepted dogma in the art.
• the method disclosed herein was used for CFA/I vaccine production in the BioProcess Production Facility (BPF) at Walter Reed Army Institute of Research and resulted in the highest level of production of any CF to date.
• yields from the method of the present invention far surpasses yields of recombinant CS6 produced according to the methods disclosed in U.S. Patent No. 5,698,416 and U.S. Patent Application No. 20040005662, which are herein incorporated by reference.
• the CFs made by the method of the present invention may be incorporated into various pharmaceutical formulations and delivered to a subject via numerous routes. See e.g. International Application Publication No. WO 02/064162, and U.S. Patent No. 6,309,669, which are herein incorporated by reference.
• the method of the present invention reduces chances for contamination during the production process and reduces the large number of assays required for quality control when small aliquots are pooled to make a final product.
• Example 1 provides methods of making host cells comprising a DNA insert that encodes a CF of interest.
• CFAs colonization factor antigens
• CS coli surface
• PCFs putative colonization factors
• Frozen bacteria can be quickly and easily thawed and reconstituted anytime for use in animal or human challenge studies.
• preparing plate grown strains for challenge studies requires considerable amounts of time and energy, particularly on the day of the challenge.
• Personnel conducting the challenges often find bacterial preparation and manipulation difficult and distracting from the many other demands at challenge which may introduce further variability.
• DME/F-12 may be commercially obtained and prepared according to the manufacturer's instructions or made according to methods known in the art. As provided herein DME/F-12 powder (Sigma- Aldrich, St. Louis, MO) plus NaHCO 3 was dissolved in WFI water and sterile filtered.
• the supernatant may be decanted and the pellet resuspended in tryptic soy broth (TSB)/20% glycerol.
• TSA tryptic soy broth
• the levels of gelatin and glycerol may be adjusted depending on the viability of the thawed bacteria according to methods known in the art.
• a fraction of the bacteria may be enumerated by plating on TSA and the remaining portion may be aliquoted in 1-ml amounts and then frozen to - 80 °C.
• Bacteria previously frozen may be thawed at room temperature and diluted in sodium bicarbonate and CeraVacx® (Cera Products, Jessup, MD). The levels of both may be adjusted to optimize ETEC viability after thawing according to methods known in the art. Thawed bacteria may be re-enumerated using OD600 values and then used in animal challenge studies according to methods known in the art. Enumeration by optical density may be confirmed by serially diluting and plating thawed bacteria on TSA plates.
• ETEC strains cultured in DME/F-12 are as (or more) virulent and as capable of causing disease as plate grown ETEC strains.
• harvested, but not frozen, strains are diluted at several concentrations between 5 . x 10 7 to 5 x 10 9 CFU and given intragastrically to seven-day old preweaned cotton rats according to protocols previously described. See U.S. Provisional Serial Number 60/280,736 and PCT/US02/08234, which are herein incorporated by reference.
• pre-maturely weaned 7 day old cotton rats may be pre-bled to confirm that naive animals possess less than 1 :10 anti- CFA/I, anti-CS3 and anti-CS6 antibody.
• Immunologic assays may be conducted using ELISA methods known in the art.
• Cotton rats with low antibody titer to colonization factor proteins may be pre-treated with acidified water for several days prior to the ETEC challenge. Prior to the ETEC challenge, cotton rats may be treated with 250 ⁇ l of sodium bicarbonate intragastrically to neutralize stomach acid that could adversely impact the virulence of the ETEC challenge strains.
• a separate group of cotton rats may also be challenged with commensal E. coli strain HS as a negative control.
• the on-set and development of diarrhea may be monitored and scored according to protocols known in the art.
• the production of antibodies against CFs may be monitored in serum and stool samples over time using ELISA and other methods known in the art.
• the cotton rats may be sacrificed and dissected to remove the small intestine.
• the small intestine may be vortex in PBS and plated on to Tryptic soy agar (TSA).
• TSA Tryptic soy agar
• a sterile filter may be applied to the plate as bacteria begin to appear and then incubated for an additional time, e.g. about 2 hours.
• the filter may then be screened using antibodies against CS6 produced by strain B7A, CS3 produced by E23347A and CFA/I and CFA E produced by HI 0407 to confirm the presence of disease causing bacteria.
• ETEC strain as well as strain HS, non-pathogenic control after peroral administration of a rice-based buffer (CeraVacx®) and a histamine-2 (H2)-receptor antagonist to neutralize stomach acidity.
• Fecal excretion of E. coli may be monitored daily after challenge by culturing fecal homogenates on MacConkey agar. The identity of presumptive ETEC or E. coli HS isolates may be confirmed by colony blots, with detection by antisera specific for the challenge strains. Monkeys may be examined twice daily for a period of 10 days for evidence of diarrhea. Blood samples may be collected before and 7, 14 and 21 days after experimental infection.
• immune response patterns may be studied by measuring ETEC- specif ⁇ c IgA and IgG antibodies in serum and stool by ELISA. See Ahren, C. et al. (1998) Infect. Immun. 66(7):3311-3316; Baqar, S. et al. (1995) Vaccine 13(l):22-28; and Jertborn, M. et al. (1986) J. Clin. Microbiol. 24(2):203-209, which are herein incorporated by reference.
• CFs produced according to the present invention may be formulated into pharmaceutical compositions and vaccines.
• a CF of the present invention may be formulated into an oral vaccine and tested in a manner similar to that provided in Example 6, wherein an oral vaccine comprising a CF, CS6, microencapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres was tested.
• PLG poly(DL-lactide-co-glycolide)
• PLG is the same biodegradable material used in resorbable surgical sutures.
• the CS6 used in the test material was produced from genes cloned from ETEC strain E8775. See Wolf, M.K., et al. (1997) FEMS Microbiol Lett. 148(l):35-42, which is herein incorporated by reference.
• the oral vaccine was safe and well tolerated in all 6 formulations. None of the vaccine-related symptoms met the definition of severe. All vaccine formulations elicited an immune response; there was at least one responder in each group. The observed immune response was similar in magnitude to experimental infection using a pathogenic strain of ETEC expressing the CF and transcutaneous administration of the CF.
• the present invention also provides compositions comprising microencapsulated CF and an adjuvant, such as LT, LT R192G, CT, and the like, and methods of treating diseases and disorders associated with ETEC comprising administering a therapeutically effective amount of CF and an adjuvant.
• an adjuvant such as LT, LT R192G, CT, and the like
• a CF made by the method of the present invention may be formulated into a variety of formulations suitable for administration.
• a CF of the present invention may be formulated into a patch or biodegradable/biosorbable microspheres according to methods known in the art.
• at least one CF may be incorporated into a trancutaneous patch or incorporated into a lactide-coglycolide biodegradable microsphere (PLGA) for oral administration.
• PLGA lactide-coglycolide biodegradable microsphere
• a CF made by the method of the present invention may be used as one component of a multivalent vaccine against ETEC infections.
• the CF is CSl, CS6, or CFA/I.
• CFAs colonization factor antigens
• CS coli surface proteins
• PCFs putative colonization factors
• antibody refers to immunoglobulin molecules and immunologically active portions that comprise an antigen binding site which specifically binds an antigen, such as a CF.
• immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which may be generated by treating the antibody with enzymes such as pepsin and papain.
• Polyclonal and monoclonal antibodies against the polypeptides of the present invention may be made by conventional methods known in the art.
• Antibodies of the present invention may be produced by conventional methods known in the art. See e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY.
• ANTIBODIES A LABORATORY MANUAL, Cold Spring Harbor Press, NY; Stites, et al. (1986) BASIC AND CLINICAL IMMUNOLOGY. 4th ed. Lange Medical Publications, Los Altos, CA; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE. 2d ed. Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256:495-497, which are herein incorporated by reference. Therapeutic antibodies may be produced specifically for clinical use in humans by conventional methods known in the art. See Chadd, H.E. and S.M. Chamow (2001) Curr.
• the antigens or antibodies the present invention may be administered, preferably in the form of a pharmaceutical composition, to a subject.
• a subject is mammalian, more preferably, the subject is human.
• Preferred pharmaceutical compositions are those comprising at least one CF made by the method of the present invention or at least one antibody against a given CF, and a pharmaceutically acceptable vehicle.
• the immunogenic composition may be an active immunizing agent, such as a CF of the present invention, or a passive immunizing agent, such as an antibody raised against a given CF of the present invention.
• the immunogenic composition may elicit an immune response that need not be protective or the immunogenic composition may provide passive immunity.
• a vaccine elicits a local or systemic immune response that is protective against subsequent challenge by the immunizing agent such as a given CF.
• an "immunogenic composition” can refer to vaccines as well as antibodies.
• a "protective immune response” may be complete or partial, i.e. a reduction in symptoms as compared with an unvaccinated mammal.
• an "immunogenic amount” is an amount that is sufficient to elicit an immune response in a subject and depends on a variety of factors such as the immunogenicity of the antigen, the manner of administration, the general state of health of the subject, and the like.
• the typical immunogenic amounts of a given CF for initial and boosting immunization for therapeutic or prophylactic administration ranges from about 0.001 mg to about 50 mg per about 65-70 kg body weight of a subject with a preferred range of 0.01 to 10 mg.
• the typical immunogenic amount for initial and boosting immunization for therapeutic or prophylactic administration for a human subject ranges from about 0.05 mg to about 5 mg.
• suitable immunization protocols include initial immunization injections at time 0 and 4 or initial immunization injections at 0, 2, and 4 weeks, which initial immunization injections may be followed by further booster injections at 1/2 or 1 years.
• a "therapeutically effective amount” refers to an amount of an antigen that may be used to treat, prevent, or inhibit an infection caused by an organism expressing the antigen in a subject as compared to a control.
• a “therapeutically effective amount” may be an "immunogenic amount”.
• a “therapeutically effective amount” of a CF refers to an amount of CF that may be used to treat, prevent, or inhibit an ETEC infection in a subject as compared to a control.
• a therapeutically effective amount may be readily determined by conventional methods known in the art. It should be noted that treatment of a subject with a therapeutically effective amount of a CF of the present invention can include a single treatment or, preferably, can include a series of treatments.
• compositions of the present invention may include an adjuvant.
• an adjuvant refers to any substance which, when administered with or before the CF the present invention, aids the CF in its mechanism of action.
• an adjuvant in a vaccine is a substance that aids the immunogenic composition in eliciting an immune response.
• Suitable adjuvants include cholera toxin (CT), heat-labile toxin (LT), incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N- acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipa-lmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which comprise three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2%
• compositions of the present invention may be admimstered to a subject by any suitable route including oral, transdermal, intranasal, inhalation, intramuscular, and intravascular administration. It will be appreciated that the route of administration and pharmaceutical formulation will vary with the condition and age of the subject, the nature of the condition to be treated, the therapeutic effect desired, and the particular CF used. In preferred embodiments, the route of administration is oral, intranasal, parenteral, or transcutaneous.
• a "pharmaceutically acceptable vehicle” or “pharmaceutically acceptable carrier” refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.
• Pharmaceutically acceptable vehicles include those known in the art. See e.g. REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20 th ed. (2000) Lippincott Williams & Wilkins. Baltimore, MD, which is herein incorporated by reference.
• compositions of the present invention may be provided in dosage unit forms.
• dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active agent for the treatment of individuals.
• the pharmaceutical formulations of the invention comprise at least one CF of the present invention and may be prepared in a unit-dosage form appropriate for the desired mode of administration.
• the pharmaceutical formulations of the present invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the condition to be treated, and the chosen CF of the present invention.
• compositions of this invention comprise a therapeutically effective amount of at least one CF of the present invention, and an inert, pharmaceutical or diluent.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical or cosmetic administration.
• the pharmaceutical or cosmetic carrier employed may be either a solid or liquid. Exemplary of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time-delay or time- release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
• time-delay or time- release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
• time-delay or time- release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate and the like.
• Supplementary active compounds can also be incorporated into the formulations.
• Supplementary active compounds include antimalarials, antibacterials, antiprotozoal agents, antifungal agents, and antidiarrheals and other compounds commonly used to treat bacterial, protozoal, and fungal infections, preferably ETEC infections.
• Supplementary active compounds include:
• Antibiotics such as penicillin, cloxacillin, dicloxacillin, mefhicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, quinolone, cinoxacin, nalidixic acid, fluoroquinolone, ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, sulfonamide, trimert
• Antiprotozoal agents such as chloroquine, doxycycline, mefloquine, metronidazole, eplornithine, furazolidone, hydroxychloroquine, iodoquinol, pentamidine, mebendazole, piperazine, halofantrine, primaquine, pyrimethamine sulfadoxine, doxycycline, clindamycin, quinine sulfate, quinidine gluconate, quinine dihydrochloride, hydroxychloroquine sulfate, proguanil, quinine, clindamycin, atovaquone, azithromycin, suramin, melarsoprol, eflomithine, nifurtimox, amphotericin B, sodium stibogluconate, pentamidine isethionate, trimethoprim-sulfame hoxazole, pyrimethamine, sulful
• Antifungal agents such as amphotericin B, fluconazole, itraconazole, ketoconazole, potassium iodide, flucytosine, and the like;
• Antidiarrheal such as diphenoxylate, codeine phosphate, paregoric
• a pharmaceutical formulation of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion), transdermal (topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• a variety of pharmaceutical forms can be employed.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g.
• a liquid carrier is used, the preparation will be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.
• a pharmaceutically acceptable salt of at least one CF is dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3M solution of succinic acid or citric acid.
• an organic or inorganic acid such as 0.3M solution of succinic acid or citric acid.
• the agent may be dissolved in a suitable co-solvent or combinations of co- solvents.
• suitable co-solvents include, but are not limited to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0-60% of the total volume.
• the pharmaceutical formulation may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
• the pharmaceutical formulations of the invention may be manufactured in manners generally known for preparing pharmaceutical compositions, e.g. , using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing.
• Pharmaceutical formulations may be formulated in a conventional manner using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries that facilitate processing of a given CF into preparations which can be used pharmaceutically.
• the CFs of the invention may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the ba ⁇ ier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the CFs of the present invention can be formulated readily by combining with pharmaceutically acceptable carriers known in the art.
• Such carriers enable the CFs of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with at least one CF, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
• PVP polyvinylpyrrolidone
• disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used, which may optionally comprise gum horoi, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compounds and agents.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can comprise the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
• the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
• the formulations may take the form of tablets or lozenges formulated in conventional manner.
• Oral formulations generally include an inert diluent or an edible earner. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, a given CF can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral formulations can also be prepared using a fluid carrier for use as a mouthwash, wherein the CF in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can comprise any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• a CF of the present invention may be conveniently delivered in the form of an aerosol spray presentation from syringe-based fine mist particle generators (e.g. Accuspray, Bectin-Dickinson), pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromefhane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromefhane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated comprising a powder mix of at least one CF and a suitable
• the CF of the present invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may comprise formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• Aqueous injection suspensions may comprise substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also comprise suitable stabilizers or agents which increase the solubility of the CF to allow for the preparation of highly concentrated solutions.
• suspensions of the active agents may be prepared as appropriate oily injection suspensions.
• Suitable Hpophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
• the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid horoidsene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the formulation.
• Prolonged absorption of the injectable compositions can be brought about by including in the formulation an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating a therapeutically effective amount of at least one CF of the present invention in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the CF of the present invention into a sterile vehicle which comprises a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the CF plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the CF may be formulated into ointments, salves, gels, foams, powders, sprays, aerosols or creams as generally known in the art.
• pharmaceutically acceptable excipients or cosmetically acceptable carriers and additives include solvents, emollients, humectants, preservatives, emulsifiers, and pH agents.
• Suitable solvents include ethanol, acetone, glycols, polyurethanes, and others known in the art.
• Suitable emollients include petrolatum, mineral oil, propylene glycol dicaprylate, lower fatty acid esters, lower alkyl ethers of propylene glycol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, stearic acid, wax, and others known in the art.
• Suitable humectants include glycerin, sorbitol, and others known in the art.
• Suitable emulsifiers include glyceryl monostearate, glyceryl monoleate, stearic acid, polyoxyethylene cetyl ether, polyoxyethylene cetostearyl ether, polyoxyethylene stearyl ether, polyethylene glycol stearate, propylene glycol stearate, and others known in the art.
• Suitable pH agents include hydrochloric acid, phosphoric acid, diethanolamine, triethanolamine, sodium hydroxide, monobasic sodium phosphate, dibasic sodium phosphate, and others known in the art.
• Suitable preservatives include benzyl alcohol, sodium benzoate, parabens, and others known in the art.
• CF of the present invention may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• the CF of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., comprising conventional suppository bases such as cocoa butter or other glycerides.
• CF of the present invention may also be formulated as a depot preparation.
• Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
• the CF of the present invention may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a pharmaceutical carrier for hydrophobic CF is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
• the cosolvent system may be a VPD co-solvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD co-solvent system (VPD:5W) comprises VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic CFs well, and itself produces low toxicity upon systemic administration.
• co-solvent component may be varied considerably without destroying its solubility and toxicity characteristics.
• identity of the co-solvent components may be varied, for example: other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
• CF may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers comprising the therapeutic agent.
• sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the CFs for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
• the pharmaceutical formulations also may comprise suitable solid- or gel-phase carriers or excipients.
• suitable solid- or gel-phase carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene gly cols.
• Some of the CF of the invention may be provided as salts with pharmaceutically compatible counter ions.
• Pharmaceutically compatible salts may be formed with many acids, including hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free-base forms.
• a CF of the present invention is prepared with a carrier that will protect the CF against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a carrier that will protect the CF against rapid elimination from the body
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
• the materials can also be obtained commercially from Alza Corporation, Boeringer-Ingelheim Corp., and Nova Pharmaceuticals, Inc.
• Liposomal suspensions can also be used as pharmaceutically or cosmetically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent Nos. 4,522,811 and 6,309,669, and International Application No. PCT/US91/
• Toxicity and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, one may determine the LD 50 (the dose lethal to 50% of the population), and the ED 50 (the dose therapeutically effective in 50% of the population) by conventional methods in the art. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 50 . CFs which exhibit large therapeutic indices are preferred. While compounds or agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds or agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage of the compositions of the present invention lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from assays known in the art.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 5 o (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined by conventional assays.
• IC 5 o i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
• levels in plasma may be measured, for example, by high performance liquid chromatography.
• Immune responses to the CFs of the present invention may be determined using conventional methods known in the art. See e.g. G ⁇ erena-Burgueno, F., et al. (2002) Infect. Immun. 70(4): 1874-1880; Reid, R.H., et al. (1993) Vaccine 11:159-167; Yu et al (2002) Infect. Immun. 70(3):1056-1068; and Katz et al (2003) Vaccine, 21(5- 6):341-346, which are herein incorporated by reference.
• ETEC enterotoxigenic Escherichia coli
• E8775 tox of serotype 025:H42 which was a gift from Alejandro Cravioto.
• E8775 tox is a derivative of E. coli strain E8775 which was originally isolated from Bangladesh.
• DH5 ⁇ which was purchased from Bethesda Research Laboratories, Inc. (Gaithersburg, MD).
• pUC19 was originally purchased from P-L Biochem.
• the antibiotic resistance gene encodes resistance to kanamycin and was purchased from Pharmacia, Uppsala, Sweden (Kan® Genblock®).
• E. coli HB101 ATCC 33694 and batch 91-1 was purchased from the American Type Culture Collection (Rockville, MD).
• the plasmid containing the CS6 genes, the pUC19 origin of replication, and the gene for kanamycin resistance was transformed into E. coli HB101 by transformation using conventional methods known in the art.
• the transformants were selected by growth on L agar supplemented with 0.004% X-gal and 50 ⁇ g/ml kanamycin sulfate and optionally 50 ⁇ g/ml ampicillin.
• the plasmid comprising the CS6 gene was isolated from the strain and examined by agarose gel electrophoresis.
• E9034A (expresses CS3), and 60R75 (expresses CSl) in three different liquid broths was examined in order to show that the amounts of the CFs obtained and studied herein is due to expression level of the bacterial strains rather than the quantity or amount of bacterial cells grown.
• SDS-PAGE, Western blot, and mass spectroscopy analysis were conducted according to methods known in the art. See Kahlil, et al., (1999) Infect. Immun. 67:4019-4026; Hess, S. et al. (2002) Anal. Biochem. 302(1):123-130; and Kahlil, S., et al. (1999) Infect. Immun., 67 (8): 4019-4026, which are herein incorporated by reference.
• ETEC strains were cultured at 37 °C at 200 rpm in a shaking incubator (Innova 4000, New Brunswick Scientific, New Brunswick, NJ) for about 5 to about 16 hours (about 5 to about 9 hours for late log phase growth, about 16 hours for complete growth curve).
• the bacteria were harvested by centrifugation at 8000 X g for 20 minutes at 4 °C (RC 5C Plus, Sorvall/Dupont, Wilmington, DE).
• the final product purity and yield refer to amount of CFA/I after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis.
• the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 °C for 30 minutes, then centrifuged at 8000 X g for 20 minutes. The given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 °C. The ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times. Table 3 shows the final CF products from 200 ml as follows:
• lane 1 is a marker, Mark 12, (Novex/Invitrogen, Carlsbad, CA)
• lane 2 is 25% PE (centrifugation pellet, protein precipitated at 25% saturation of ammonium sulfate) of 1933D in DME/F-12 with NaHCO 3
• lane 3 is 25% PE of 1933D in L Broth
• lane 4 is 25% PE of 1933D in CFA Broth
• lane 5 is Mark 12
• lane 6 is 25% PE of HI 0407 in DME/F-12 withNaHCOs
• lane 7 is 25% PE of H10407 in L Broth
• lane 8 is 25% PE of HI 0407 in CFA Broth.
• the final product purity and yield refer to amount of CS3 after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis.
• the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 °C for 30 minutes, then centrifuged at 8000 X g for 20 minutes.
• the given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 °C
• the ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times.
• Table 5 shows Hie final CF products from 200 ml as follows:
• lane 1 is See Blue
• lane 2 is CS3
• lane 3 is 25% PE of E9034A in DME/F-12 with NaHCO 3
• lane 4 is 25% PE of E9034 in L Broth
• lane 5 is 25% PE of E9034A in CFA Broth.
• the final product purity and yield refer to amount of CS 1 after harvest, heat extraction, ammonium sulfate precipitation at 25% saturation, centrifugation, resuspension of the precipitate, and dialysis.
• the bacterial pellet was suspended by adding 20 ml of PBS. The bacterial suspension was placed in a preheated water bath at 60 °C for 30 minutes, then centrifuged at 8000 X g for 20 minutes.
• the given CF was purified by adjusting the supernatant to 25% saturation of ammonium sulfate, incubated at room temperature for about 2 to about 6 hours or overnight at 4 °C
• the ammonium sulfate precipitate was harvested at 25,000 X g and then subjected to dialysis against PBS three times.
• Table 7 shows the final CF products from 200 ml as follows:
• lane 1 is See Blue
• lane 2 is CSl
• lane 3 is 25% PE of 60R75 in DME/F-12 with NaHCO 3
• lane 4 is 25% PE of 60R75 in L Broth
• lane 5 is 25% PE of60R75 in CFA Broth.
• Example 3 Agglutination Studies were conducted as follows. Bacteria were recovered from broth at late log phase (about 5 hours) by centrifugation (30 min. 7,000 x g), and the pellet suspended in phosphate buffered saline (PBS) to an optical density of 5.0 at 600 nm. Bacteria from CFA plates were scraped into PBS, and suspended as above. 8 ⁇ l of the bacterial suspension was mixed with the same volume of purified antibody from rabbits before (preimmune) and after (postimmune) immunization, as well as with 8 ⁇ l of PBS.
• PBS phosphate buffered saline
• the degree of agglutination was observed visually and graded based on the time of agglutination as well as the clump size. Score of agglutination: 4, agglutination less than about 10 seconds; 3, agglutination about 10 to about 30 seconds; 2, agglutination about 0.5 to about 1 minute; 1, agglutination about 1 to about 2 minutes; 0, no agglutination within about 2 minutes or more.
• the media used was as follows: DME/F-12 (D8900, Sigma- Aldrich, St. Louis, MO) supplemented with 0.12% sodium bicarbonate, L broth containing 1% trypton, 0.5% yeast extract, and 1% sodium chloride, CFA broth containing 1% casamino acids, 0.15% yeast extract, 0.0005% manganese chloride tetrahydrate, and 0.005% magnesium sulfate heptahydrate, and CFA agar containing 1% casamino acids, 0.15%o yeast extract, 0.0005% manganese chloride tetrahydrate, 0.005%) magnesium sulfate heptahydrate, and 2% granulated agar.
• pre- and post-immune antibody was affinity purified on Hi-Trap Protein-G Sepharose (Amersham, Piscataway, NJ) from pooled sera from rabbits 92319 (CFA/I) and 94859 (CSl).
• strain 1933D expressed CFA/I very well in all broths. Although the expression of CFA/I should be turned off in L broth, it is not, thereby indicating a factor independent of growth media conditions that causes an up-regulation of the expression of CFA/I in 1933D.
• Electron microscopy was conducted using methods known in the art. See Harris, R., et al. (1991) ELECTRON MICROSCOPY IN BIOLOGY “Negative Staining” CRC Press, Boca Raton, FL, pp 328-342; and Cassels, F.J., et al. (1992) Infect. Immun. 60:2174-2181, which are herein incorporated by reference. Cell analyzed were according to Example 2. About 1 to about 2 ml samples from growing cultures were obtained and submitted to an electron microscopy facility without centrifugation (neat- no concentration or dilution) for electronmicroscopy according to methods known in the art.
• Figure 9A and Figure 9B show CSl expressing ETEC grown on CFA agar.
• Figure 10A and Figure 10B show CSl expressing ETEC grown on CFA broth.
• Figure 11 A and Figure 1 IB show CSl expressing ETEC grown on L broth.
• Figure 12A and Figure 12B show CSl expressing ETEC grown on DME/F-12 broth.
• Figure 13A and Figure 13B show CFA/I expressing ETEC grown on CFA agar.
• Figure 14A and Figure 14B show CFA/I expressing ETEC grown on CFA broth.
• Figure 15A and Figure 15B show CFA/I expressing ETEC grown on DME/F-12 broth.
• Table 9 as follows summarizes the electron microscopic analysis of CSl and CFA/I expression.
• the bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria.
• B Heat extraction for recovery of CFA/I; purification by tangential flow filtration and ammonium sulfate precipitation.
• the bacteria suspension was placed in a pre-heated water bath at about 60 °C to about 70 °C for 30 minutes. Then the suspension was centrifuged at 8000 X g for 20 minutes. The supernatant was recovered for tangential flow filtration per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-6A 0.2 micron filter. CFA/I passes through the filter to be collected in a reservoir for ultrafiltration.
• the 0.2 um filter retains remaining cells and large cell debris.
• the reservoir solution containing CFA/I was passed over an Amersham (Piscattaway, NJ) AG/T UFP-300-C-6A filter per the manufacturer's instructions. CFA/I was retained on the filter as spent media passes through the filter to waste.
• Retentate of UFP-300-C-6A was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature overnight at 4 °C. The ammonium sulfate precipitate was harvested at 25,000 X g. Then the 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-300-C-4A filter per manufacturer's instructions, with CFA/I recovered from the retentate.
• the bacteria suspension was placed in a pre-heated water bath at about 60 °C to about 70 °C for 30 minutes. Then the suspension was centrifuged at 8000 X g for 20 minutes. The supernatant was recovered for tangential flow filtration per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-6A 0.2 micron filter. CS3 passes through the filter to be collected in a reservoir for ultrafiltration. The 0.2 um filter retains remaining cells and large cell debris. The reservoir solution containing CS3 was passed over an Amersham (Piscattaway, NJ) AG/T UFP-500-C-6A filter per the manufacturer's instructions.
• CS3 was retained on the filter as spent media passes through the filter to waste.
• Retentate of UFP-500-C-6A was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature overnight at 4 °C.
• the ammonium sulfate precipitate was harvested at 25,000 X g.
• the 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-500-C-4A filter per manufacturer's instructions, with CS3 recovered from the retentate.
• the contents of the three flasks of culture were aseptically transferred to inoculate a fermentor containing 300 L DME/F-12 and 30 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increased from a low level (about 5% to about 40%) to about 15%) above the lowest level.
• the cells were harvested by continuous flow centrifugation (Sharpies AS-26SP) (12,000 rpm at a flow rate of about 1.5 to about 2.0 L per minute). Then the bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria.
• the bacterial suspension was aseptically pumped into a 30 L New Brunswick fermentor and heated at about 60 °C to about 70 °C for 30 minutes.
• the cells were harvested by continuous flow centrifugation (Sharpies AS-26SP) (12,000 rpm at a flow rate of about 0.75 to about 1.5 L per minute).
• the supernatant was recovered for tangential flow filtration and conducted per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-2-E-65 0.2 micron filter.
• CFA/I passes through the filter to be collected in a reservoir for ultrafiltration.
• the 0.2 ⁇ m filter retains remaining cells and large cell debris.
• the reservoir solution containing CFA/I was passed over an Amersham (Piscattaway, NJ) AG/T UFP-300-C-55 filter per the manufacturer's instructions. CFA/I was retained on the filter as spent media passes tlirough the filter to waste. Retentate of UFP-300-C-55 was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature for 2 hours followed by about 18 to about 72 hours at 4 °C The ammonium sulfate precipitate was harvested at 25,000 X g (50 minutes). The 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-300-C-4A or UFP-300-C-5A filter per the manufacturer's instructions, with CFA/I recovered from the retentate.
• the contents of the three flasks of culture were aseptically transferred to inoculate a fermentor containing 300 L DME/F-12 and 30 ml antifoam (New Brunswick Scientific, New Brunswick, NJ) and fermented for about 6 to about 8 hours or until dissolved oxygen rapidly increased from a low level (about 5% to about 40%) to about 15% above the lowest level.
• the cells were harvested by continuous flow centrifugation (Sharpies AS-26SP) (12,000 rpm at a flow rate of about 1.5 to about 2.0 L per minute). Then the bacterial pellet was suspended in PBS at about 3 to about 4 ml/g wet weight of bacteria.
• the bacterial suspension was aseptically pumped into a 30 L New Brunswick fermentor and heated at about 60 °C to about 70 °C for 30 minutes.
• the cells were harvested by continuous flow centrifugation (Sharpies AS-26SP) (12,000 rpm at a flow rate of about 0.75 to about 1.5 L per minute).
• the supernatant was recovered for tangential flow filtration and conducted per the manufacturer's instructions using an Amersham (Piscattaway, NJ) AG/T CFP-l-E-65 0.1 micron filter.
• CS3 passes through the filter to be collected in a reservoir for ultrafiltration.
• the 0.1 ⁇ m filter retains remaining cells and large cell debris.
• the reservoir solution containing CS3 was passed over an Amersham (Piscattaway, NJ) AG/T UFP-500-C-55 filter per the manufacturer's instructions.
• CS3 was retained on the filter as spent media passes through the filter to waste.
• Retentate of UFP-500-C-55 was adjusted to 25% saturation of ammonium sulfate and incubated at room temperature for 2 hours followed by about 18 to about 72 hours at 4 °C.
• the ammonium sulfate precipitate was harvested at 25,000 X g (50 minutes).
• the 25% precipitate was dialfiltered against PBS using an Amersham AG/T UFP-500-C-5A filter per the manufacturer's instructions, with CS3 recovered from the retentate.
• Table 10 summarizes the harvest parameters, yield and purity of CFA/I and CS3 in DME/F-12 broth as compared with those of a CS6 expressing strain according to Example 1.
• Figure 16 is an SDS-PAGE of CS3 from a 300L fermentation. 16% tris-Tricine
• SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 10 and 20 ⁇ g loaded (lanes 2 and 3, respectively) of CS3, Lot 0963 from 300 L fermentation, strain E9034A grown in DME-F-12.
• the molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 1.
• Figure 17 is an SDS-PAGE of CFA/I from a 10L fermentation. 16% tris-
• Tricine SDS-PAGE gel (Invitrogen, Carlsbad, CA) with 20 and 10 ⁇ g (lanes 1 and 2, respectively) of CFA/I, Lot 1096 from 300 L fermentation, strain 1933D grown in DME/F-12. A CFA/I standard is included for comparison, Lot 0633 (20 ⁇ g, lane 4).
• the molecular weight standards (Mark 12, Invitrogen, Carlsbad, CA) are in lane 3.
• Example 6 Oral Formulation Comprising Microencapsulated CS6 Vaccine preparations comprising a CF, such as CS6, have been tested in subjects. See e.g. Yu et al. (2002) Infect. Immun. 70(3):1056-1068 (transcutaneous mice); G ⁇ erena-Burgueno, F., et al (2002) Infect.
• Immunity 70(4): 1874-1880 transcutaneous human
• de Lorimier et al (2003) Vaccine 21(19-20):2548-2555, (intranasally mice); and Katz et al (2003) Vaccine, 21(5-6):341-346 (oral microencapsulated human); and Bryd and Cassels (2003) Vaccine 21:1884-1893, (intranasally and intragastrically mice), which are herein incorporated by reference.
• the formulations tested included 1 and 5 mg doses of CS6, either encapsulated in biodegradable polymer poly (D,L)-lactide-co-glycolide, or as free protein, administered orally in a solution of either normal saline or a rice-based buffer.
• Three doses of CS6 were given at 2 week intervals. Blood was collected immediately before and 7 days after each dose. All formulations were well tolerated.
• CS6 and CS6 microencapsulated in PLG microspheres were produced under current good manufacturing practices (cGMP) at the WRAIR Pilot Bioproduction Facility, Silver Spring, MD.
• the bacterial strain used for the production of CS6 was constructed from E. coli (HB101) containing a recombinant plasmid carrying the four genes necessary for CS6 expression. See Wolf, M.K., et al. (1997) FEMS Microbiol Lett. 148(l):35-42, which is herein incorporated by reference.
• the major components in the production included: bacterial fermentation, recovery of the CS6 from the fermentation broth by tangential flow filtration, ammonium sulfate precipitation, buffer exchange from PBS into 4% sucrose, microencapsulation by a solvent evaporation procedure using an emulsion of CS6/sucrose and PLG in methylene chloride, homogenization in polyvinal alcohol, removal of methylene chloride by evaporation, lyophilization, and storage at -80 °C.
• Unencapsulated CS6 was produced in an identical manner to the microencapsulated CS6, but the material was stored in PBS at -80 °C
• the vaccine was administered orally in three doses. There were 6 vaccine groups (Table 3). Groups I-III (low dose; 1 mg each dose) were vaccinated on days 0, 14, and 28. Groups IV- VI received their three doses (high dose; 5 mg, 4 mg, and 4 mg, respectively) on days 7, 21, and 35. On the day of vaccination, all subjects were observed 90 minutes prior to and after vaccine administration. The subjects drank from a cup which contained either CS6 or CS6-PLG in 100 ml of either normal saline or buffer (Cera Products LLC, Jessup, MD). Subjects then drank from a second cup containing 50 ml of either normal saline or buffer (to match the solution in the first cup).
• Subjects kept a diary to record any symptoms that occurred for the seven days following each dose.
• the diary collected solicited and unsolicited symptoms and intensities. Symptom presence and intensity was graded: not present; mild (noticeable, but did not interfere with routine activities); moderate (interfered with routine activities); or severe (unable to perform routine activities).
• Fever was defined as a temperature greater than 100.5 °F and documented by Temp-dot (3M, Rochester, MN) disposable thermometers.
• the number of vomiting episodes were recorded as were the number of episodes diarrhea (defined as about 3 or more loose stools over a 24 hour period), and loose stools.
• the subjects returned on the day after vaccination and seven days post- vaccination for clinical assessment and to monitor for any possible side- effects. The diaries were reviewed by the study physicians with the subjects.
• CS6-specific antibody-secreting cell (ASC) immune responses to the vaccine antigen were chosen as a surrogate of intestinal mucosal immune response. See Wenneras, C, et al (1992) Infect Immun. 60:2605-11, which is herein incorporated by reference.
• Whole blood was collected for ASC's weekly from the day of the first vaccination (prior to receiving the vaccine) until 2 weeks after the third vaccination (a total of 7 samples). Blood specimens were collected using EDTA treated tubes (Becton Dickinson Vacutainer Systems, Rutherford, NJ).
• PBMC Peripheral blood mononuclear cells
• the plates After being washed with PBS, the plates were blocked with complete Iscove's medium (GibcoBRL, Grand Island, NY) supplemented with 5% fetal calf serum (GibcoBRL) and 50 ⁇ g/ml gentamycin (GibcoBRL).
• the PBMC were adjusted to 2 x 10 7 viable cells/ml in complete Iscove's medium.
• a final 0.1 ml suspension containing 10 6 PBMC was added to each well, and plates were incubated for 4 hours at 37 °C in 7.5 % CO 2 .
• a positive ASC response was defined as about a 2-fold or more increase over baseline value of the ASC's per 10 6 PBMC, when the number of ACS's was about 0.5 per 10 6 PBMC or more in the baseline sample.
• Bound antibodies were visualized by addition of rabbit anti-human IgA or IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, Westgrove, PA) and , incubated at room temperature for 90 minutes followed by addition of o- phenylenediamine (OPD)-H 2 O 2 (Sigma, St. Louis, MO).
• OPD o- phenylenediamine
• the endpoint titers were assigned as the interpolated dilutions of the samples giving an absorbance value at 450 nm of 0.4 above background. Titers were adjusted in relation to a reference specimen included in each test to compensate for day-to-day variation. Pre- and post-dosing serum samples from the same individual were tested side by side.
• the antibody titer ascribed to each sample represented the geometric mean of duplicate determinations performed on different days. Reciprocal endpoint titers less than 5 were assigned a value of 2.5 for computations. Based on calculations of the methodological error of each ELISA, a response was defined as about a two-fold increase or more in endpoint titer between pre- and post-immunization, with the added criterion that the post- immunization reciprocal titer be about 10 or more. Seroconversion after any dose was defined as a positive response. [165] The Fishers exact test was used to compare proportions. Graphs were constructed to contrast each group (formulation). There were no significant differences in the frequency or the magnitude of the serum antibody or ASC response to CS6 between the 6 groups. Therefore, data were pooled for further presentation.
• the ages of the 29 subjects who participated in this study ranged from 20 to 44 years of age. Nineteen of the subjects were African American, six were Caucasian, one was Hispanic, and three were of other nationalities/ethnicities. Among the 29 subjects who received a study agent, three subjects received one dose, three subjects received two doses, and 23 received all three doses. Only one subject withdrew because the subject was unable to drink the vaccine (lmg CS6 in buffer) due to the taste of the buffer. Five subjects did not receive the full three doses. Specifically, two did not receive the full three doses due to scheduling conflicts, two did not receive the full three doses due to lack of follow-up, and one did not receive the full three doses due to illness. Only the 26 subjects that received 2 or more doses of the vaccine were included in the safety and immune data analysis.
• Serum IgA responses to microencapsulated CS6 was greater than the response to unencapsulated CS6 (1 of 8), and responses to vaccines given with buffer (5 of 18) were greater than responses given in normal saline (1 of 8).
• the formulations tested included CS6 encapsulation in microspheres in 1 mg and 5 mg doses, and a rice-based buffer.
• the microspheres are biodegradable and permit slow and continued release of antigen for increased exposure to the immune system.
• the microspheres were made of biodegradable polymer poly(D,L-lactide-co-glycolide) (PLG).
• PLG biodegradable polymer poly(D,L-lactide-co-glycolide)
• the test vaccine was produced using Good Manufacturing Practices (GMP) at the Walter Reed Army Institute of Research Bioproduction Facility in Silver Spring, Maryland.
• GMP Good Manufacturing Practices
• Three doses of CS6 in PLG microspheres were given in normal saline (NS) or CeraVacx (CV) a rice-based bicarbonate solution to neutralize stomach acid. Human subjects were divided into groups and administered the formulations according to Table 13.
• ASC antibody-secreting cells
• Table 14 summarizes the number of subjects reporting symptoms possibly related to the vaccination.

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