JP6612807B2 - C. Isolated polypeptides of difficile toxin A and toxin B proteins and uses thereof - Google Patents

Description

The present invention relates to an isolated polypeptide containing the receptor binding domains of Clostridium difficile toxin A and toxin B, and its use as a vaccine. This isolated polypeptide provides antitoxin immunity against both toxins.

Clostridium difficile is a leading cause of nosocomial antibiotic-related diarrhea,
It has become a major health problem in hospitals, nursing homes and other nursing homes. Hospital costs are estimated at $ 2 billion in Europe and $ 3.2 billion in the United States.

The causative agent is a gram-positive spore-forming anaerobic bacterium that is commonly found throughout the environment but is also present in the intestinal tract of 2-3% of the healthy adult population. C. Difficile-related disease (CDAD)
Is induced by destruction of the normal colonic flora, usually as a result of antibiotic administration. C. in the environment After exposure to difficile spores, the organism may colonize in the intestinal mucosa where production of disease-causing toxins can lead to CDAD. The disease can vary from diarrhea without mild complications to severe pseudomembranous colitis and toxic megacolon.

CDAD is increasingly becoming a problem in the medical environment. In a recent study, 31% of hospitalized patients given antibiotics had C.I. It has been reported that 56% of those patients colonized with difficile subsequently develop CDAD.
In general, C.I. Difficile is responsible for 10-25% of all antibiotic-related diarrhea, 50-75% of antibiotic-related colitis, and 90-100% of antibiotic-related pseudomembranous colitis.
Treatment of CDAD involves cessation of the causative antibiotic followed by treatment with metronidazole or vancomycin. Recurrence after the antibiotic treatment is stopped occurs in about 20% of patients, which is often C.I. It is a result of colony re-formation by C. difficile.

In 2003, C.E. in Quebec, Canada The occurrence of difficile is North A
C.I. known as melican phenotype 1/27 (NAP1). It showed the emergence of a more toxic strain of difficile. NAP1 is associated with higher toxicity, lower outcome, and higher morbidity and mortality compared to previous strains. The emergence of this strain increases the problems that have already arisen in attempts to prevent the development of CDAD.

Fidaxomycin (Dificid®) for the prevention of recurrent disease is the first in a new class of narrow macrocyclic antibiotics (Revil, P .; Se
rradell, N.M. Bolos, J .; (2006). "Tiacumcin B:
macrolide antibiotic treatment of C.I. diff
"icile-associated diarhe"". Drugs of the.
Future 31 (6): 494-497). This is the actinomycete Dactylosporangium aurantiacum
It is a fermentation product obtained from the subspecies hamdenesis. Fidaxomycin is non-systemic, with minimal absorption into the bloodstream, is bactericidal, exhibits selective eradication of pathogenic Clostridium difficile, and constitutes a normal healthy intestinal flora There is very little destruction against multiple types of bacteria. Maintenance of normal physiological status in the colon can reduce the likelihood of recurrence of Clostridium difficile infection (Johnson, St.
uart (2009-06). ”Recurrent Clostridium dif
file effect: a review of risk factor
s, treatments, and outcomes ". Journal of In
effect 58 (6): 403-410). Although the introduction of this new class of antibiotics could improve the treatment of CDAD, there is still a medical need for prophylactic drugs, especially in high-risk patients, such as the elderly and those who are not immune .

CDAD is a C.I. Two exotoxins produced by difficile, toxin A and toxin B (
It is the result of the action of CTA and CTB, respectively. Both of these toxins
A high molecular weight (˜300 kDa) secreted protein with multiple functional domains (V
oth DE and Ballard JD, Clinical Microbiol
ogy Reviews 18: 247-263 (2005)). The N-terminal domains of both toxins contain ADP-glucosyltransferase activity that modifies Rho-like GTPase. This modification causes a loss of actin polymerization and changes in the cytoskeleton, resulting in the destruction of colonic epithelial tight junctions. This results in excessive fluid oozing into the colon and consequently diarrhea. The central domain contains a hydrophobic domain and is presumed to be involved in membrane transport.
The C-terminal domains of both toxins contain multiple regions of homology called repeat units (RU) involved in toxin binding to target cells (Ho et al, PNAS 102: 18373-18).
378 (2005)). Repeat units are classified as short chains (21-30 amino acids) or long chains (-50 amino acids). The repeating units combine to form a cluster,
Usually contains one long chain and 3-5 short repeat units. Full-length toxin A has 39 repeat units (ARU) organized into 8 clusters (Dove et al. Infe
ct. Immun. 58: 480-488 (1990), while full-length toxin B contains 24 repeat units (BRU) organized into 5 clusters (Barroso et al.
al, Nucleic Acids Res. 18: 4004 (1990); Eich
el-Striber et al, Gene 96: 107-113 (1992
)).

In some studies from both animal models and clinical, C.I. The role of antitoxin antibodies in protecting against difficile-related diseases has been shown. Hamsters immunized with formalin-inactivated toxin A and toxin B produce high levels of anti-toxin antibodies and C.I. Protected against lethal burden of difficile bacteria (Giannasca PJ and Warny M,
Vaccine 22: 848-856 (2004)). Furthermore, passive transmission of mouse antitoxin antibodies protected hamsters in a dose-dependent manner. Kyne L et al. (The Lancet
357: 189-193 (2001)) reported that the development of an anti-toxin A antibody response during the initial expression of CDAD correlates with protection against disease recurrence.

The determinant recognized by the protective antitoxin antibody is located in the C-terminal domain containing a repeating unit that functions as a receptor binding domain. First, Lyly et al. (Curren
t Microbiology 21: 29-32 (1990)), the C-terminal domain of toxin A containing 33 repeat units can induce the production of neutralizing antitoxin antibodies. It was revealed that it could protect against difficile infection. In this study, hamsters were given purified recombinant polypeptides multiple times subcutaneously before being challenged by bacteria,
Only partial protection was achieved. Another study (Ryan et al, Infect.
Immun. 65: 2941-49 (1997)) 720 from the C-terminal end of CTA.
An isolated polypeptide containing amino acid residues and a secretion signal of E. coli hemolysin A (expressed in Vibrio cholerae) induces systemic and mucosal immunity protective against small doses of CTA in a rabbit CDAD model It was shown that.

It has also been reported that antibody responses against the C-terminal domains of both toxins A and B are necessary to achieve full protection (Kink and Williams, Infect
. Immun. 66: 2018-25 (1998), U.S. Pat. S. Pat. No. 5,73
6, 139 (1998)). This study revealed that the C-terminal domain of each toxin was most effective in producing toxin neutralizing antibodies. This demonstrated the effectiveness of an orally delivered avian antibody (antitoxin) raised against the C-terminal domain of CTA and CTB in a hamster lethal model. The results also show that antitoxins can be effective in the treatment and management of CDAD in humans. In another study, human antitoxins A and B
Monoclonal antibodies were produced in C. hamsters. Reported to confer protection against difficile-induced death (Babcock et al., Infect. Immun. 74:
6339-6347 (2006)). Protection was observed only with antibodies to the receptor binding domain of either toxin, and improved protection was observed after treatment with both antitoxin A and B antibodies.

On the other hand, Ward et al. (Infect. Immun. 67: 5124-32 (1999))
For the study of adjuvant activity C.I. 14 repeat units from difficile toxin A (1
4 CTA) was considered. The repeat unit was cloned and expressed with an N-terminal polyhistidine tag (14CTA-HIS) or fused to a non-toxic binding domain from tetanus toxin (14CTA-TETC). Both fusion proteins administered intranasally produced anti-toxin A serum antibodies in mice but showed no response at the mucosal surface. Improved systemic and mucosal antitoxin A responses were observed after co-administration with E. coli heat labile toxin (LT) or its mutant LTR72. Based on the data, Ward et al., Non-toxic 14 CTA-TET as a mucosal adjuvant in human vaccines against Clostridium pathogens.
Suggested use of C-fusion.

C. Recent biochemical studies on the repeat unit domain of difficile toxin have focused on the minimum sequence requirements to form a stable ternary structure (Demarest S J et
al. , J .; Mol. Bio. 346: 1197-1206 (2005)). The 11 repeat unit peptide obtained from Toxin A has the correct ternary structure, but 6 from Toxins A and B
And 7 repeat units were found not to have it. A correctly folded 11 repeat unit segment was found to maintain receptor binding properties. The second study examined the functional properties of a toxin A fragment containing 6, 11 or 15 repeat units (Dingle T, Gly).
cobiology 18: 698-706 (2008)). Only 11 and 15 repeat units could competitively inhibit the toxin neutralizing ability of anti-toxin A antibody. All three fragments were found to have hemagglutination activity, but the longer fragment showed higher hemagglutination activity than the shorter one. Data show that toxin receptor binding domain structure and immunogenicity are 1
It is shown to be retained in domain fragments containing more than 1-14 repeats.

Also, Thomas et al. (WO 97/02836, US Pat. No. 5,919,463 (1
999)) are C.I. Difficile toxin A, toxin B and certain fragments thereof (eg, C-terminal domains containing some or all of the repeating units) have been disclosed. They show that intranasal administration of CTA or CTB significantly improves mucosal immune responses to heterologous antigens such as Helicobacter pyloriurease, ovalbumin, or keyhole limpet hemocyanin (KLH) in multiple mouse compartments; It is shown to be related to the protection against load by Helicobacter. Furthermore, the adjuvant activity of the toxin A fusion protein was evaluated and the 794 C-terminal amino acid residue of CTA containing ARU (toxin A repeat unit) is
The resulting polypeptide GS fused to glutathione-S-transferase (GST)
T-ARU was expressed in E. coli. This study demonstrated a marked improvement in immune response with GST-ARU against co-administered antigens in serum and mucosal secretions.

All of these studies are C.I. Difficile toxin A or toxin B, or a fragment thereof,
Or suggest the potential use of non-toxic recombinant proteins, including combinations thereof, for the production of active vaccines against CDAD. Currently, C.I. Although no vaccine against difficile is commercially available, a candidate vaccine consisting of formalin detoxification total toxins A and B is being evaluated in human Phase I and IIa studies. Parenteral immunization with this vaccine has been reported to induce anti-toxin IgG and toxin-neutralizing antibody responses (Kotoff KL et al., Infect. Immun. 69: 988-
995 (2001); Aboudola S et al. , Infect. Immun
. 71: 1608-1610 (2003)).

The literature further describes C.I. The construction of recombinant fusion proteins containing both difficile toxin A and B receptor binding domains has been shown to be an efficient and commercially viable approach for vaccine development, in whole or in fragments thereof . Such an approach is
Varformomeva et al., 700 base pair fragment of toxin A and 1300 of toxin B
Attempted as a fusion protein consisting of two parts of a base pair fragment (Mol. Gen.
etics, Microb. and Virol. 3: 6-10 (2003)). This approach is also described by Belyi and Varforemeva (FEMS Letters 2
25: 325-9 (2003)) and is divided into three parts: C.I. FIG. 4 shows the construction of a recombinant fusion protein consisting of a fragment of C. perfringens enterotoxin Cpe, followed by two C-terminal domains composed of difficile toxin A and toxin B repeat units. The fusion protein was expressed in E. coli, but the product accumulated in the inclusion bodies and was not stable. Furthermore, the yield of pure product achieved in this study (50 μg per 100 ml of medium) was significantly lower.

Wilkins et al. (WO 00/61762, US Pat. No. 6,733,760 (20
04)) is also recombinant C.I. Describes the use of polysaccharide conjugates for the preparation of difficile toxin A and B repeat units (recombinant ARU and recombinant BRU) and vaccines against CDAD. The resulting recombinant ARU protein contains 867 amino acid residues, while the recombinant BRU protein contains a length of 622 amino acids.
Unlike the studies previously mentioned, this study does not support recombinant ARU and BRU in E. coli.
High level expression of soluble protein was demonstrated. Mice vaccinated with recombinant ARU and polysaccharide-conjugated recombinant ARU both have high levels of neutralizing anti-toxin A antibody and C.I. Extremely protected from lethal loading by difficile toxin A. In addition, Wilki
ns et al. suggested the use of a recombinant fusion protein consisting of both ARU and BRU for vaccine preparation.

There is interest in developing vaccines against CDAD. A recombinant fusion protein consisting of ARU and BRU can potentially be useful as a vaccine.

The present invention relates to C.I. New tools and methods are provided for the design, production and use of toxin A and toxin B from difficile. The present invention relates to an isolated polypeptide C-TAB comprising SEQ ID NO: 2 (C-TAB.G5) or a derivative thereof, SEQ ID NO: 4 (C-TAB.G5.1).
I will provide a. C-TAB. G5 or C-TAB. G5.1 contains 19 repeat units of toxin A C-terminal domain fused to 23 repeat units of toxin B C-terminal domain. The present invention also provides C-TAB. G5 or C-TAB. Compositions and formulations comprising G5.1 isolated polypeptides are included. The composition or formulation may contain isolated polypeptides, additional antigens, adjuvants, and / or excipients. Alternatively, the composition or formulation may consist essentially of an isolated polypeptide free of adjuvants or other active ingredients (provided that excipients such as carriers, buffers and / or stabilizers are optionally included). Included). Further, the compositions or formulations of the present invention may be used in combination with other drugs such as antibiotics, particularly in subjects with recurrent CDAD or subjects requiring frequent and / or long-term use of antibiotics. It may be administered.

The present invention also provides a vaccine comprising the isolated polypeptide of the present invention. The vaccine may further comprise an adjuvant, for example an adjuvant obtained from eg alum, ADP-ribosylated exotoxin, or others. Vaccines are based on a single dose schedule, a double dose schedule (
For example administered within 3 to 20 days after the first dose, eg 10 to 15 days)
It may be administered in a three dose schedule (eg, administered about 7 days and about 21 days after the first dose), or more than four dose schedules, preferably two or three dose schedules,
An amount of 20 μg to 200 μg of the polypeptide of the present invention is included.

The present invention provides methods for preventing, treating, or alleviating one or more symptoms of a disease, such as CDAD, by administering an isolated polypeptide of the present invention to a subject in need thereof. C
-TAB. G5 or C-TAB. A G5.1 isolated polypeptide can be administered to a subject by intramuscular or other delivery routes.

In one embodiment, the present invention provides a CDAD, such as a subject having the following profile:
A method for preventing diseases such as CDAD by administering an isolated polypeptide of the present invention or a composition comprising the polypeptide to a subject at risk of: i) a subject having a weaker immune system, For example, elderly subjects (for example, subjects over 65 years old) or subjects under 2 years old, etc .; ii) subjects without immunity, for example subjects with AIDS, etc .; iii) being administered or scheduled to receive immunosuppressive drugs Iv) subjects who are scheduled to be hospitalized or are hospitalized; v) subjects who are undergoing or will receive intensive care (ICU); vi) who are undergoing gastrointestinal surgery; or Subjects who are scheduled to receive; vii) subjects who are receiving or plan to receive long-term care such as nursing homes; viii) frequent,
And / or subjects with comorbidities that require long-term antibiotic use; ix) subjects who have more than one of the above profiles, such as elderly subjects who are scheduled to undergo gastrointestinal surgery; x ) Subjects with inflammatory bowel disease; and / or xi) recurrent CDAD
A subject having, for example, a subject experiencing one or more episodes of CDAD.

In one embodiment, the present invention provides C-TAB. G5 or C-TAB. A method of producing a G5.1 isolated polypeptide is provided. C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide can be obtained using a bacterial expression system, such as an E. coli expression system, using C-TAB. G5 or C
-TAB. It can be generated from a nucleic acid encoding a G5.1 isolated polypeptide.

In one embodiment, the present invention provides C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide is provided, wherein the 19 repeat unit of toxin A is linked to a 23 of toxin B via a linker consisting of at least 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Connected to a repeat unit. By way of example, the linker of the present invention has the sequence RSMH (Arg-Ser-Met-His
) (Amino acids 439 to 442 of SEQ ID NO: 2 or SEQ ID NO: 4).

In another embodiment, the invention provides a variant of the isolated polypeptide comprising at least one mutation (eg, insertion, substitution or deletion), eg, in ARU and / or BRU. The sequence of the variant is 85%, 86%, 87%, 88% relative to SEQ ID NO: 2,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
Or it may have 99% identity.

The present invention also provides C-TA by recombinant DNA manipulation, bacterial fermentation and protein purification.
B. G5 or C-TAB. Methods are provided for generating G5.1 isolated polypeptides or variants thereof. In one embodiment, the present invention provides C-TAB. G5 or C-TAB
. Methods are provided for constructing nucleic acids encoding G5.1 isolated polypeptides. In another embodiment, the present invention uses a bacterial expression system, such as an E. coli expression system, to produce C-TAB. G5
Or C-TAB. A method of producing a G5.1 isolated polypeptide is provided.

The present invention further provides a method for preventing and treating CDAD in a subject such as a human in need thereof. In this method, C-TAB. G5 or C-TAB. G
5.1 is administered to the subject alone or in combination with one or more adjuvants such as alum or others. The subject is C.I. Healthy individuals at risk of exposure to difficile, C.I. I have been treated or recovered from a difficile infection. Human subjects at risk of reinfection with difficile or currently C.I. Infected with difficile, the condition is C.I. It may be a human subject that can be improved by induction of difficile toxin neutralizing antibodies.

The present invention relates to C-TAB. G5 or C-TAB. An immunogenic composition comprising G5.1 is provided. The immunogenic composition may further comprise an adjuvant and / or a pharmaceutically acceptable carrier that enhances the antigen-specific immune response and / or other components in the formulation suitable for application to a subject in need thereof. May be included. The immunogenic composition is useful for intramuscular (IM) delivery, intradermal (
ID) delivery, subcutaneous (SC) delivery, intraperitoneal (IP) delivery, oral delivery, nasal delivery, buccal delivery,
Or it can be delivered by rectal delivery.

In another embodiment of the invention, the immunogenic composition is native C.I. An antibody is produced that binds to difficile toxin and neutralizes its cytotoxic activity, and thus C.I. Provide long-term active protection and / or treatment against difficile-related disease (CDAD).

Thus, the present invention provides C.I. Immunogenic compositions useful for the prevention or treatment of difficile-related diseases are provided.

In another embodiment, the present invention provides C-TAB. G5 or C-TAB. Nucleic acids encoding G5.1 and fragments or variants thereof are provided. The present invention also provides C-TAB. G5
Or C-TAB. An expression vector comprising a nucleic acid encoding G5.1 is provided.

Another embodiment of the present invention is C-TAB. G5 or C-TAB. Specific for G5.1,
Provided are antibodies and fragments thereof, such as neutralized, humanized, monoclonal, chimeric and polyclonal antibodies. The antibody or fragment thereof can recognize toxin A and / or toxin B.

Another embodiment provides a vaccine comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

Another embodiment of the present invention provides a diagnostic kit comprising a nucleic acid, polypeptide and / or antibody of the present invention.

Other embodiments and advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned from the practice of the invention.

C-TAB. It is a figure which shows the nucleic acid which codes G5 isolation polypeptide (sequence number 1). C-TAB. It is a figure which shows the amino acid sequence of G5 isolation polypeptide (sequence number 2). The amino acid linker between toxin A and toxin B is underlined.

C-TAB. FIG. 5 shows a nucleic acid encoding a G5.1 isolated polypeptide (SEQ ID NO: 3). C-TAB. It is a figure which shows the amino acid sequence of G5.1 isolated polypeptide (sequence number 4). The amino acid linker between toxin A and toxin B is underlined.

C-TAB. C-TAB. By increasing dose of G5 and combined delivery with alum adjuvant. It is a table | surface which shows the improvement of antibody production in a G5 vaccinated mouse | mouth. Mice were vaccinated twice by IM injection. Two weeks after the first and second injections, the IgG titers of anti-C-TAB, antitoxin A and antitoxin B antibodies were evaluated by ELISA.

Increased doses of C-TAB with and without alum by two IM injections. FIG. 3 is a graphical representation of anti-C-TAB, antitoxin A, and antitoxin B IgG induction in mice receiving G5.

C-TAB. in the presence or absence of alum. 2 is a table showing antibody titers over a semi-log dose range in mice immunized with G5. IgG titers were assessed by ELISA 2 weeks after the second immunization. The data show that alum greatly enhances antibody production in vaccinated mice.

C-TAB. 2 is a table showing protective effects in mice vaccinated with G5 (with and without alum) and then exposed to lethal doses of toxin A or toxin B. Mice that received two vaccinations (IM) at 2-week intervals were challenged (IP) after 3 weeks. Toxin A and toxin B neutralizing antibody (TNA) were evaluated 2 weeks after the second injection to determine the percentage of animals that survived a lethal challenge. C-TAB. Increasing doses of G5 resulted in increased protection against lethal load with higher TNA production. The presence of alum further increased TNA production and resulted in higher survival at lower doses.

Antibody responses and C-TAB. In vaccinated young (6-7 weeks old) and old (18 months old) mice. It is a table | surface which shows the comparison of the protection effectiveness of G5. Mice receiving two vaccinations (IM) at 2-week intervals were challenged (IP) after 3 weeks. Anti-C-TAB, anti-toxin A and anti-toxin B antibody ELISA IgG titers, TNA production and overall survival were assessed. Young mice showed a higher antibody response even without alum, and both groups showed improved survival when vaccinated in the presence of alum.

It is a graph which shows the comparison of the reaction rate of an anti- C-TAB IgG antibody increase in a vaccinated young and old mouse | mouth. Young mice showed faster rates and earlier IgG production, and both groups showed improved responses when vaccinated in the presence of alum.

C-TAB. 2 is a table showing a comparison of anti-C-TAB, antitoxin A and antitoxin B antibody production in mice immunized with G5.1 or a mixture of toxoids A and B (1: 1). Mice were vaccinated twice by IM injection. Two weeks after the second injection, IgG titers of anti-C-TAB, antitoxin A and antitoxin B antibodies were evaluated by ELISA. Immunization with toxoid induces antibodies against the N-terminal portion of the toxin molecule, while immunization with C-TAB induces antibodies against the C-terminal portion of the toxin molecule.

C-TAB. 2 is a table showing a comparison of protection against TNA production and challenge with toxin A or B in mice immunized with G5.1 or toxoid A and B mixtures. Mice that received two vaccinations (IM) at two week intervals were challenged with a lethal dose of toxin A or toxin B after 3 weeks (IP).

C-TAB with and without alum. 2 is a table showing anti-C-TAB (A), antitoxin A (B), and antitoxin B (C) IgG production in hamsters immunized with G5.1. Hamsters were vaccinated 3 times by IM injection on day 0 and day 14. On days 14, 28 and 35, IgG titers of anti-C-TAB, antitoxin A and antitoxin B antibodies were evaluated by ELISA.

C-TAB with or without alum. FIG. 5 is a graphical representation of the increase in anti-C-TAB IgG antibody in hamsters immunized with G5.1.

C-TAB with or without alum. 2 is a table showing a comparison of TNA and protection in hamsters immunized with G5.1. Two weeks after the third vaccination, the hamster received a lethal dose of toxin A or toxin B by IP injection.

A lethal dose of C.I. C-TAB. After intragastric administration of difficile spores. It is a graph which shows the survival rate of the hamster vaccinated with G5.1. Survival data was plotted as a Kaplan-Meier survival fit curve and statistical analysis was performed using log rank analysis. At all spore doses (10 ² , 10 ³ and 10 ⁴ ), 100% survival of hamsters within the vaccinated group was observed, with significantly improved survival compared to the placebo group.

C-TAB. in the presence or absence of alum. 2 is a table showing anti-C-TAB, antitoxin A, and antitoxin B antibody production in cynomolgus monkeys immunized with G5.1. Two groups of monkeys (3 in each group, 4-6 years old) have 200 μg C-TAB. With or without 250 μg alum. Received G5.1. Blood samples were taken on days 0, 14, 28 and 42 of the study. An ELISA method was used to assess anti-C-TAB, antitoxin A and antitoxin B IgG titers.

C-TAB delivered over a dose range of 1-30 μg in PBS or histidine buffer. G5 and C-TAB. It is a graph which shows the comparison of the immunogenicity of G5.1. Mice received two vaccinations (IM) at 2-week intervals. Two weeks after the second injection, IgG titers of anti-C-TAB, antitoxin A and antitoxin B antibodies were evaluated by ELISA. C-TAB. Delivered in PBS or histidine buffer. C-TAB delivered in G5 and histidine buffer. All three antibody titers were not significantly different from G5.1 (T-test analysis).

C-TAB in mice. It is a table | surface which shows the comparison of the immunogenicity of G5, C-TABNCTB, and C-TADCTB. Mice were vaccinated twice with each recombinant protein at 2 week intervals by IM injection. All immunizations were performed in the absence of alum adjuvant. Two weeks after the second injection, IgG titers of anti-C-TAB, antitoxin A and antitoxin B antibodies were evaluated by ELISA. All three fusion proteins are highly immunogenic.

2 is a table showing protection against loading with natural toxin B in mice. Mice were immunized as shown with respect to FIG. 17 and loaded with a lethal dose of native toxin B by IP injection 3 weeks later.

C-TAB. in the absence or presence of alum. 2 is a table showing a comparison of TNA and protection in hamsters vaccinated with G5.1 or C-TADCTB. Two weeks after the third vaccination, the hamster received a lethal dose of toxin A or toxin B by IP injection.

C-TAB. 2 is a table showing protection against TNA production and toxin A or toxin B loading in mice immunized with G5.1. Comparison of TNA production and protection between groups of mice vaccinated three times by IM injection on days 0, 3 and 14 or 0, 7 and 21 or 0, 14, and 28. Three weeks after the last injection, mice were challenged with a lethal dose of toxin A or toxin B (panel A is tabular and panel B is graphical).

10 μg of C-TAB. In mice immunized with a single dose of G5.1 and 12.5 μg alum (in 100 μl). It is a graph which shows the protection (survival rate) with respect to the load by a difficile toxin A (55 ng / mouse). The loading was performed 21 days, 35 days or 49 days after immunization.

Overview The present invention relates to an isolated polypeptide C-TAB. G5 (SEQ ID NO: 2) or a derivative thereof, C-TAB. Including use of G5.1 (SEQ ID NO: 4), C.I. Immunogenic compositions for inducing protective and / or therapeutic immune responses against difficile toxins A and B are provided.

The present invention also provides C-TAB. G5 or C-TAB. Methods of producing G5.1 isolated polypeptides and methods of preparing compositions (eg, vaccines) useful for the prevention and / or treatment of CDAD in mammals are provided. The following description provides further details and examples of the construction, expression, and purification of recombinant isolated polypeptides, inducing specific immune responses, and their use as antigens to assess protection in a subject. The subject may be an animal or a human.

C-TAB. For use in the methods and compositions of the invention. G5 or C-TAB.
G5.1 isolated polypeptides can be prepared using any of a number of standard methods. For example, C-TAB. G5 or C-TAB. G5.1 may be generated using standard recombinant DNA techniques, and a suitable host cell is transformed with an appropriate expression vector containing a portion of a toxin-encoding nucleic acid fragment (eg, Dove et al., Infe
ct. Immun. 58: 480-8 (1990), and Barroso et al.
. , Nucleic Acids Research 18: 4004 (1990)). Any of a wide variety of expression systems can be used to produce a recombinant polypeptide. C-TAB. G5 or C-TAB. G5.1 is a prokaryotic host cell (eg, a bacterium such as E. coli or Neisseria gonorrhoeae) or a eukaryotic host cell (eg, a yeast cell, a mammalian cell (eg, COS1, NIH3T3, or JEG3 cell), or an insect cell (eg, Mushroom (SF9) cells)). Such cells are available, for example, from the American Cultured Cell Line Conservation Organization (ATCC). The method of transformation and transfection and the choice of expression vector will depend on the host system selected. Methods for transformation and transfection are described, for example, in Ausubel et al. , ISBN:
047132938X. In addition, C-TAB. G5 or C-TAB
. G5.1, particularly short chain fragments, can be synthesized by chemical synthesis, for example, Solid Phase Pe.
ptide Synthesis, 1984, 2nd ed. , Stewart and
Young, Eds. , Pierce Chemical Co. , Rockford
Ill. Or can be generated by standard in vitro translation methods.

C-TAB. G5 or C-TAB. In addition to the G5.1 sequence, the present invention provides variants thereof that are functionally active and immunogenic. The mutant is C-TAB. G5 or C-TAB
. It may have the same level of immunogenicity as G5.1. A variant may have amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 2 or SEQ ID NO: 4. C-TAB. G5 or C-TAB. The gene encoding G5.1 or a variant thereof can be made using standard methods (see below, see also eg Ausubel et al.,
See also the above reference).

C-TAB. G5 or C-TAB. In addition to the G5.1 sequence, the present invention provides C-TAB. Further derivatives of G5 are provided. As an example, a fusion protein, C-
TABNCTB (encoded by SEQ ID NO: 18, SEQ ID NO: 17) is C-TAB. G
Similar to 5, CTA 19 repeat units (amino acids 2272 to 2710), CTB 23 repeat units (amino acids 1850 to 2366), and an additional 10 repeats of CTB fused to the C-terminus of CTB (amino acids 1834 to 2057) )including. A further variant, C-TA
The DCTB fusion protein (encoded by SEQ ID NO: 20, SEQ ID NO: 19) is C-T
AB. G5 (19 repeats of CTA and 23 repeats of CTB), and C-TAB. Contains an additional 24 repeat units of CTB (amino acids 1834-2366) fused to the C-terminus of G5. Moreover, a variant is C-TAB. Additional copies of G5 or parts thereof may be included. For example, C-TADCTB is C-TAB. It may contain the double part of the repeating unit of CTB present in G5.

The present invention relates to C-TAB. G5 or C-TAB. Introducing a nucleic acid encoding G5.1 into a bacterial host cell; and C-TAB. G5 or C-TAB. High-level expression C-TAB. In bacterial systems such as E. coli, including expressing G5.1. G5 or C-TAB
. A method for G5.1 is provided.

Furthermore, the C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide is
It may be covalently linked or cross-linked to the adjuvant (eg, Cryz et al., Va
ccine 13: 67-71 (1994); Liang et al. , J .; Immu
theory 141: 1495-501 (1988) and Czerkinsky e
t al. , Infect. Immun. 57: 1072-77 (1989)).

The present invention protects against CDAD and provides treatment for it, C-TAB. G5 or C-TAB. A vaccine comprising a G5.1 isolated polypeptide is provided. The vaccines of the present invention contain novel antibodies and can be delivered by intramuscular (IM), intradermal (ID), subcutaneous (SC), oral, nasal, buccal, or rectal routes. Vaccines can provide immune protection or induce antibodies for passive immunization.

C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide is CDAD
A vaccine is provided to immunize against. C-TAB of the present invention. G5 or C-TA
B. The G5.1 isolated polypeptide or variant thereof may be C.I. Combination vaccine candidates that aim to expand the scope of protection against difficile-related diseases, such as CDAD, to a level that has never been known or published. C. This concept of a single vaccine providing protection from or reducing the severity of difficile-related diseases is unique in global public health management, particularly in reducing the severity of epidemics (eg, nursing homes, cruise ships) Showing progress.

As used herein, “toxin A protein” or “toxin B protein”
Is the main cause of CDAD. It refers to the toxic protein of difficile. Toxin A and toxin B contain multiple repeating units responsible for immunogenicity in the C-terminal binding domain.

As used herein, “wild type” or “natural” refers to a full-length protein comprising a nucleic acid or amino acid sequence as found endogenously in a host cell.

As used herein, “Clostridial difficile-related disease”, “
The terms "Clostridial difficile-related disease", "Clostridial difficile-related disease", "Clostridial difficile toxin-mediated disease", "Clostridial difficile infection" and "CDAD" are directly or indirectly Refers to diseases caused by infection.

An “antigen” refers to a substance that induces a specific immune response when presented to an immune cell of an organism. For example, an antigen can be a nucleic acid, protein, polypeptide, peptide, glycoprotein, carbohydrate,
It may be a complex of lipid, glycolipid, lipoprotein, fusion protein, phospholipid, or combinations thereof. The antigen may be a B cell receptor (ie, an antibody on the B cell membrane) or T
It may comprise a single immunogenic epitope or multiple immunogenic epitopes recognized by cellular receptors. Antigens may be provided as virus-like particles (VLPs) or pathogens or whole microorganisms such as bacteria or virions. The antigen may be an inactive or live attenuated virus. Antigens may be obtained from extracts or lysates from whole cells or membranes only, or antigens may be produced by chemical synthesis or recombinant means. The antigen can be administered alone or with an adjuvant. A single antigen molecule may have both antigen and adjuvant properties.

“Adjuvant” means any substance used to specifically or non-specifically enhance an antigen-specific immune response, possibly by activation of antigen-presenting cells. Examples of adjuvants are oil emulsions (eg complete or incomplete Freund's adjuvant), Mon
Tanide incomplete Seppic adjuvants such as ISA, oil-in-water emulsion adjuvants such as Ribi adjuvant system, syntax adjuvant formulations containing muramyl dipeptide, aluminum salt adjuvant (ALUM), polycationic polymers, especially polycationic peptides, especially polycations Arginine or at least two LysL
peptides containing the euLys motif, in particular KLKLLLLLKLK, immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine dinucleotide (CpG) within a defined base context (eg as described in WO 96/02555) or inosine and Cytidine-based ODN (for example described in WO 01/93903) or deoxynucleic acid containing deoxyinosine and / or deoxyuridine residues (W
O01 / 93905 and WO02 / 095027), in particular Oligo (dIdC
₁₃ (described in WO01 / 93903 and WO01 / 93905), neuroactive compounds,
In particular human growth hormone (described in WO01 / 24822), or combinations thereof, chemokines (eg, defensin 1 or 2, RANTES, MIP1-α, MIP
-2, interleukin-8, or cytokines (eg, interleukin-1β,
-2, -6, -10 or -12; interferon-γ; tumor necrosis factor-α; or granulocyte monocyte colony stimulating factor) (discussed in Noria and Rubin, 1994), muramyl dipeptide variants ( For example, mulabtide, threonyl-M
DP or muramyl tripeptide), synthetic mutants of MDP, heat shock proteins or mutants, mutants of forest-type tropical Leishmania LeIF (Skeiky et al.,
1995, J. Org. Exp. Med. 181: 1527-1537), a non-toxic variant of bacterial ADP-ribosylating exotoxin (bARE) containing a variant at the trypsin cleavage site (Dick)
enson and Elements, (1995) Infection and I
munity 63 (5): 1617-1623) and / or active ADP-ribosylation (Duce et al., 1997) or chemical detoxification bARE (toxoid), QS21, Quill A, N-acetylmuramil-L-alanyl A composition comprising -D-isoglutamyl-L-alanine-2- [1,2-dipalmitoyl-s-glycero-3- (hydroxyphosphoryloxy)] ethylamide (MTP-PE) and metabolizable oils and emulsifiers including. The adjuvant may be administered with the antigen by the same route as the antigen route or by a route different from the antigen route, or may be administered alone. A single adjuvant molecule may have both adjuvant and antigenic properties.

“Effective amount” means, for an antigen, an amount of a therapeutic agent sufficient to induce or enhance an antigen-specific immune response, or for a drug, to treat or diagnose a condition. Such induction of an immune response may be a treatment such as immunoprotection, desensitization, immunosuppression, modulation of autoimmune disease, enhanced immunological surveillance of cancer, or therapeutic vaccination against established infectious diseases. Can provide. Treatment includes healing, amelioration, or prevention.

“Nucleic acid” means a single deoxyribonucleobase or ribonucleic acid, or a sequence thereof linked by a phosphodiester bond.

“Therapeutic agent” means any molecule that can be used in the treatment of a disease, reduction of symptoms of the disease, prevention of the disease, or diagnosis of the disease. For example, the therapeutic agent can be an antigen or a drug.

“Subject” means an animal. The subject may be any animal including any vertebrate. The subject may be a domestic animal, a laboratory animal (including but not limited to rodents such as rats, hamsters, gerbils, or mice) or pet animals. In one embodiment, the animal may be a mammal. Examples of mammals are humans, primates, marsupials,
Includes dogs, monkeys, rodents, cats, apes, whales, dolphins, cows, pigs, and horses. The subject may be in need of treatment for the disease or may be in need of prophylactic treatment.

As used herein, the term “antibody” means an immunoglobulin molecule or fragment of an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies are well known to those skilled in the field of immunology. As used herein, the term “antibody” refers not only to full-length antibody molecules, but also to fragments of antibody molecules that retain antigen binding ability. Such fragments are also well known in the art and usually in vitro
Used both ro and in vivo. Specifically, as used herein, the term “antibody” refers not only to full-length immunoglobulin molecules, but also to the well-known active fragment F.
It also refers to antigen-binding active fragments such as (ab ′) 2, Fab, Fv, and Fd.

As used herein, a “variant” may include a protein and / or polypeptide and / or peptide that is different from a wild-type polypeptide, leaving one or more residues functionally similar. Conservatively substituted with a group, which also exhibits substantially the same functional properties of the wild type polypeptide. Examples of conservative substitutions include substitution of one nonpolar (hydrophobic) residue with another residue (eg, isoleucine, valine, leucine or methionine) of one polar (hydrophilic) residue Substitution with another residue (eg between arginine and lysine, between glutamine and asparagine, between glycine and serine) another residue of one basic residue (eg lysine, arginine or histidine) Or substitution of one acidic residue with another residue (eg, aspartic acid or glutamic acid). Variants may include any polypeptide having a ternary structure substantially identical to a polypeptide of the invention that also exhibits functional properties of the polypeptides described herein. The variant may be a wild type polypeptide mutation.

As used herein, “treatment” can include any type of interference used in an attempt to modify the natural history of an individual or cell. Treatment can include, but is not limited to, administration of a pharmaceutical composition, for example, alone or in combination with other therapies generally known in the art. “Treatment” may be performed prophylactically or after initiation of a pathological event.

As used herein, a “pharmaceutically acceptable carrier”, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. The material may be included. Pharmaceutically acceptable carriers and / or excipients can include buffers, stabilizers, diluents, preservatives, and solubilizers. In general, the nature of the carrier or excipient will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable solutions that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (eg, in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the administered pharmaceutical composition contains trace amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents such as sodium acetate or sorbitan monolaurate. You may contain.

As used herein, “fusion” may refer to nucleic acids and polypeptides comprising sequences that are not found in natural association with each other in the order or context arranged according to the present invention. A fusion nucleic acid or polypeptide does not necessarily include the entire natural sequence of the nucleic acid or polypeptide. The fusion proteins are linked to each other by normal peptide bonds.
Has more than one segment. A fusion nucleic acid has two or more segments joined together by normal phosphodiester bonds.
Isolated polypeptide

The present invention relates to C.I. 19 repeat units of difficile toxin A and C.I. 2 of difficile toxin B
The C-TAB. Of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, comprising 3 repeat units. G5 or C-TAB. A G5.1 isolated polypeptide is provided. C-TAB. G5 homologues such as C-TAB. G5.1 is C-TAB.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids only. It may be different from G5. C-TAB. The G5.1 polypeptide is
C-TAB. Contains the same C-terminal domain of toxin B as G5, but C.I. Difficile 630
The corresponding C-TAB obtained from the strain. C. a homolog of G5 polypeptide. It is a fusion protein that does not contain the C-terminal domain of toxin A obtained from the difficile VPI-10463 strain, and differs by two amino acids at positions 155-156. C-TAB. Described in SEQ ID NO: 3. The G5.1 coding sequence was codon optimized for improved expression in E. coli host cells. C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide is C.I. Can be effective in neutralizing the toxic effects of difficile toxin A and toxin B.

Toxin A and Toxin B are C.I. TrdA of difficile strain 630 (SEQ ID NO: 5)
And the trdB (SEQ ID NO: 7) gene. Structurally, C.I. The difficile toxin includes an ADP-glycosyltransferase domain, a cysteine protease domain, a hydrophobic region, and a receptor binding region. The C-terminal domain contains highly repetitive units (RU) (also known as combinatorial repeat oligopeptides (CROPS)). The RU may be a long or short oligopeptide and repeats consensus Y
It can contain 20 to 50 amino acids with a YF motif. RUs are grouped as clusters. As an example, the strain 630 (SEQ ID NO: 6) encoded by toxin A, wild type trdA gene (SEQ ID NO: 5) has 39 RUs. The 39 RUs are grouped into 8 clusters. Strain 630 (SEQ ID NO: 8) encoded by toxin B, wild-type trdB gene (SEQ ID NO: 7) contains 24 RUs grouped into 5 clusters. Tables 1 and 2 below show the C. elegans encoded by trdA and trdB genes. The respective amino acid positions of RU in difficile toxin A and toxin B are shown.

Table 1: Toxin A repeat unit (ARU)

Table 2: Toxin B repeat unit (BRU)

Therefore, C-TAB. G5 and C-TAB. Each of the G5.1 isolated polypeptides is C.I. 19 RU from the C-terminal domain of difficile toxin A, and C.I. Contains 23 RU from the C-terminal domain of difficile toxin B. C-TAB. G5 or C-TAB.
G5.1 comprises toxin A amino acids 2272-2710 of SEQ ID NO: 6 fused to toxin B amino acids 1850-2366 of SEQ ID NO: 8. C-TAB. G5 or C-TAB. G5.1
The isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

In addition, C-TAB. G5 or C-TAB. Each RU in the G5.1 isolated polypeptide
Is C.I. It may be from a variant of difficile toxin A or toxin B. C-TA
These RUs in the B isolated polypeptide are also C.I. Difficile toxin A or toxin B
Or a combination of naturally occurring or mutants.

C-TAB. G5 or C-TAB. The RU in the G5.1 isolated polypeptide includes a long chain RU and a short chain RU, and the long chain RU and short chain RU are arranged as a cluster. C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide is C.I. 4 clusters of 1 long RU followed by 3 to 5 short RUs of difficile toxin A, and C
. It contains 5 clusters of 1 long RU followed by 3 to 5 short RUs of difficile toxin B.

Short and long RUs contain conserved motifs. Short repeat units are 15, 16
, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acids. Each short repeat unit may contain a conserved tyrosine motif such as YYF, FYF, YFF, FYI, or HYF. The short repeat unit may further comprise an aspartate / histidine residue before the tyrosine motif if the subsequent repeat unit is a long repeat unit. The long chain repeat unit may comprise 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. Each long chain repeat unit is FEYF, FKYF, or YK
It may contain a tyrosine repeat motif such as YF.

In the present invention, C-TAB. G5 or C-TAB. The toxin A and toxin B portions of the G5.1 isolated polypeptide may be fused directly to each other. Toxin A and Toxin B moieties are
It may be separated by a linker region. The linker region is 1, 2, 3, 4, 5, 6,
It may contain 7, 8, 9, 10, 11 to 15, 20 to 30, 40, 45, or 50 amino acids. C-TAB. G5 or C-TAB. The linker region is such that each toxin repeat unit in the G5.1 isolated polypeptide is positioned to optimally expose the potential epitope and retain its immunogenicity in its expressed and folded form. One skilled in the art will recognize that it may be adapted to alter the positioning of the toxin A and toxin B moieties. RUs and clusters in a C-TAB isolated polypeptide may also be separated by a linker. In one embodiment, the linker comprises the peptide RSMH (SEQ ID NO: 2 or 439-442 of SEQ ID NO: 4).

The C-TAB isolated polypeptide of the present invention comprises SEQ ID NO: 2 or SEQ ID NO: 4 and at least 8
5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9
It may have 5%, 96%, 97%, 98%, or 99% sequence identity or sequence similarity. As is known in the art, “similarity” between two polypeptides or polynucleotides refers to the amino acid or nucleotide sequence of one polynucleotide or polypeptide and its conserved nucleotides or amino acid substitutions. Determined by comparison with the sequence of a second polynucleotide or polypeptide. Also, “identity”, which means the degree of sequence relatedness between such sequences, determined by the identity of the match between the two strands of two polypeptides or two polynucleotide sequences, Known in the technical field. Both identity and similarity can be easily calculated (Comp
utational Molecular Biology, Lesk, A .; M.M. , Ed
. , Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Proj
ects, Smith, D.E. W. , Ed. , Academic Press, New Y
ork, 1993; Computer Analysis of Sequence D
ata, Part I, Griffin, A .; M.M. , And Griffin, H .; G.
, Eds. , Humana Press, New Jersey, 1994;
nce Analysis in Molecular Biology, von He
inje, G .; , Academic Press, 1987; and Sequence.
Analysis Primer, Gribskov, M .; and Devereux,
J. et al. , Eds. , M Stockton Press, New York, 1991).
There are several ways to measure identity and similarity between two polynucleotide or polypeptide sequences, but the terms “identity” and “similarity” are well known to those skilled in the art (Sequence Analysis). in Molecular Biolog
y, von Heinje, G. et al. , Academic Press, 1987;
ence Analysis Primer, Gribskov, M .; and Dev
reux, J. et al. , Eds. , M Stockton Press, New York, 1
991; and Carillo, H .; , And Lipman, D.M. , SIAM J. et al. A
published Math. 48: 1073 (1988). A commonly used method for determining identity or similarity between two sequences is Guide to Huge C.
omputers, Martin J .; Bishop, ed. , Academic Pr
ess, San Diego, 1994 and Carillo, H .; , And Lipm
an, D.C. , SIAM J. et al. Applied Math. 48: 1073 (1988), including but not limited to.

C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide is immunogenic. For example, C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide may have at least 50%, 60%, 70%, 80%, or 90% of the immune activity of the corresponding bacterial toxin A, and C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide has at least 50%, 60%, 70%, 80% of the immune activity of the corresponding bacterial toxin B,
Or you may have 90%. C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide can be used as a vaccine for the treatment, prevention, or alleviation of symptoms of CDAD.

In addition, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide is a C-TAB. G5 or C-TAB. G5.
Includes variants of one isolated polypeptide. Variants may have amino acid insertions, substitutions and / or deletions that have little or no effect on the activity, function or shape of the isolated polypeptide. Examples of such substitutions are substitution of one nonpolar residue with another, 1
Substitution of one polar residue with another residue, substitution of one basic residue with another residue, or substitution of one acidic residue with another residue. C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide variant further comprises amino acid insertions that have a minor effect on the activity, function and / or structure of the polypeptide as compared to the amino acid sequence of the extracellular domain of native toxin A or toxin B. Substitutions and / or deletions may be included. One skilled in the art will recognize that unnatural amino acids may be used. Non-natural amino acids include, for example, beta-alanine (
Beta-Ala), or other omega-amino acids such as 3-aminopropionic acid, 2,
3-diaminopropionic acid (2,3-diaP), 4-aminobutyric acid, etc., alpha-amine isobutyric acid (Aib), sarcosine (Sat), ornithine (Orn), citrulline (Ci)
t), t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle) Cysteic acid (Cya) 2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); beta-2-thienylalanine (Thi); and methionine sulfoxide (MSO) )including.

C-TAB of the present invention. G5 or C-TAB. The nucleotide sequence encoding the G5.1 isolated polypeptide may be codon optimized to improve expression in various host cells. Codon optimization is to improve protein expression in the target host cell.
Refers to altering a nucleotide sequence by replacing one or more codons of a native sequence with a gene of that host cell or more frequently used in the host gene from which the cell is derived. Various species exhibit a particular bias for a particular codon of a particular amino acid. The present invention provides C-TAB. For improved expression in E. coli. A codon optimized nucleotide sequence encoding a G5.1 isolated polypeptide is provided.

C-TAB of the present invention. G5 or C-TAB. A G5.1 isolated polypeptide can be prepared by any known technique. For example, an isolated polypeptide can be expressed by genetic engineering. An example is the translation of recombinant DNA. C-TAB. G5 or C-TAB. G
5.1 Isolated polypeptides can also be prepared synthetically. As an example, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide can be obtained from Merrifield (J. Am Ch
em. Soc. 85: 2149-2154) (incorporated herein by reference) and may be synthesized using solid phase synthesis techniques. Other polypeptide synthesis techniques are described, for example, in Kent et al. (1985) Synthetic Peptides in.
Biology and Medicine, eds. Alitaro et al. ,
Elsevier Science Publishers, 295-358. Can be found in

C-TAB of the present invention. G5 or C-TAB. A G5.1 isolated polypeptide can be isolated or obtained in substantially pure form. Substantially pure means that the protein and / or polypeptide and / or peptide is substantially free of other substances that can be found together in a natural or in vivo system to the extent practical and appropriate for its intended use. Means not included. Specifically, C-TAB. G5 or C-TA
B. The G5.1 isolated polypeptide is sufficiently pure to fully utilize other biological components of the host cell, eg, useful in antibody production, sequencing, or production of pharmaceutical preparations. Not included. By techniques well known in the art, substantially pure polypeptides can be generated in light of the nucleic acid and amino acid sequences disclosed herein. Since a substantially purified isolated polypeptide of the present invention can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the isolated polypeptide can only contain a certain weight percent of the preparation. Can occupy. Nevertheless, an isolated polypeptide is substantially pure in that it is substantially separated from substances that may be relevant in a biological system.

The present invention further provides an isolated C-TAB. G5 or C-TA
B. A G5.1 isolated polypeptide is provided. The additional polypeptide may be a fragment of a larger polypeptide. In one embodiment, C-TAB. G5 or C-TAB
. There are one, two, three, four or more additional polypeptides fused to the G5.1 isolated polypeptide. In some embodiments, the additional polypeptide is C
-TAB. G5 or C-TAB. Fused to the amino terminus of the G5.1 isolated polypeptide. In other embodiments, the additional polypeptide is C-TAB. G5 or C
-TAB. Fused to the carboxyl terminus of the G5.1 isolated polypeptide. In a further embodiment, the additional polypeptide is C-TAB. G5 or C-TAB. G
5.1 Adjacent to isolated polypeptide. In a further embodiment, the additional polypeptide is C-TAB. G5 or C-TAB. Dispersed between the toxin A and toxin B portions of the G5.1 isolated polypeptide.

In some embodiments, the additional polypeptide is C-TAB. G5 or C-
TAB. Helps induce secretion or subcellular localization of G5.1 isolated polypeptide. Such polypeptides are called “signal sequences”. Secretion signals are described, for example, in US Pat. No. 6,291,212 and US Pat. No. 5,547,871, both incorporated herein by reference in their entirety. The secretory signal sequence encodes a secretory peptide. The secretory peptide is C-TAB. G5 or C-TAB. An amino acid sequence that acts to induce secretion of G5.1. Secretory peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not limited to) the amino terminus of newly synthesized proteins. The secretory peptide is C-TAB. G5 or C-
TAB. It may be cleaved from the G5.1 isolated polypeptide. Secretory peptides may contain processing sites that allow cleavage of the signal peptide from the mature protein as it passes through the secretory pathway. The processing site may be encoded within the signal peptide,
Alternatively, it may be added to the signal peptide, for example by in vitro mutagenesis. The secretory signal sequence is C-TAB. G5 or C-TAB. It may be necessary for a complex series of post-translational processing steps to enable secretion of G5.1. The signal sequence may follow immediately after the start codon, C-TAB. G5 or C-TAB. G5.1
The signal peptide is encoded at the amino terminus of The signal sequence may precede the stop codon and C-TAB. G5 or C-TAB. It encodes a signal peptide at the carboxy terminus of G5.1. In most cases, the signal sequence is cleaved by a specific protease called a signal peptidase. Examples of secretory signal sequences are ompA, pel
Including, but not limited to, B and ST pre-pro.

In some embodiments, the additional polypeptide is C-TAB. G5 or C-
TAB. Helps stabilize, structure and / or purify the G5.1 isolated polypeptide. In some embodiments, the additional polypeptide may comprise an epitope. In other embodiments, the additional polypeptide may include an affinity tag. As an example, epitopes and / or C-TAB. G5 or C-TAB. Fusion of a polypeptide comprising an affinity tag to a G5.1 isolated polypeptide can assist in the purification and / or identification of the polypeptide. By way of example, a polypeptide segment can be a His-tag, a myc-tag, an S-
Peptide tag, MBP tag (maltose binding protein), GST tag (glutathione S)
-Transferase), FLAG tag, thioredoxin tag, GFP tag (green fluorescent protein), BCCP (biotin carboxyl carrier protein), calmodulin tag,
It may be a Strep tag, an HSV-epitope tag, a V5-epitope tag, and a CBP tag. The use of such epitopes and affinity tags is known to those skilled in the art.

In further embodiments, the additional polypeptide comprises a C-TAB. Containing a site for cleavage of the polypeptide. G5 or C-TAB. A G5.1 isolated polypeptide can be provided.
As an example, a polypeptide can be cleaved by hydrolysis of peptide bonds. In some embodiments, the cleavage is performed enzymatically. In some embodiments, the cleavage occurs within the cell. In other embodiments, the cleavage occurs by artificial manipulation and / or artificial introduction of a cleavage enzyme. By way of example, the cleavage enzyme may comprise pepsin, trypsin, chymotrypsin, thrombin, and / or factor Xa. Cleavage is performed by C-TAB. G5 or C-TAB. Allows easy isolation of G5.1 isolated polypeptides. Cleavage may further allow separation of the toxin A moiety from the toxin B moiety. Cutting is also
For example, C-TAB. Fused to a polypeptide by cleavage of the epitope used to purify the expressed protein. G5 or C-TAB. Isolation of G5.1 isolated polypeptide from other polypeptides may be possible.

C-TAB. G5 or C-TAB. G5.1 isolated polypeptides may further have additional structural changes that are not shared with the same organic synthetic peptide, such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristylation. Good. These additional structural variations may be further selected or preferred by appropriate selection of the recombinant expression system. On the other hand, the fusion polypeptide may have its sequence extended by the principles and practices of organic synthesis.

The present invention also provides C.I. A polypeptide portion obtained from difficile toxin A and C.I. C-TAB. Containing a polypeptide portion derived from difficile toxin B. G5 or C-TA
B. Nucleic acids encoding G5.1 isolated polypeptides are provided. Nucleic acids may include deoxyribonucleotides or ribonucleotides or single or double stranded forms of polymers thereof. The present invention relates to C-TAB. G5 or C-TAB. Ribonucleic acids encoding G5.1 isolated polypeptides are provided. The present invention also provides for C-T under stringent conditions.
AB. G5 or C-TAB. Nucleic acids that hybridize to nucleic acids encoding G5.1 isolated polypeptides and their complements are provided. Stringent conditions refer to the degree of homology between the probe and the filter-bound nucleic acid, the higher the stringency, the higher the percent homology between the probe and the filter-bound nucleic acid. The temperature for stringent washing can be determined based on the Tm of the nucleic acid (based on G / C content). Stringent conditions can also be influenced by the concentration of salts in a buffer such as standard sodium citrate (SSC). The present invention provides about 85%, 86%, 87%, 88%, 89%, 90%, 9% with SEQ ID NO: 1.
Nucleic acids having 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity or sequence identity are provided.

C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide further comprises a linker region, eg, a linker of less than about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. May be included. The linker may be covalently linked to or between the polypeptide moiety derived from toxin A or a portion thereof and the polypeptide moiety derived from toxin B.

The present invention relates to C-TAB. Degenerates to SEQ ID NO: 1 or SEQ ID NO: 3, respectively. G5 or C
-TAB. Nucleic acids encoding G5.1 isolated polypeptides are provided. The degeneracy of the genetic code allows for diversity in the nucleotide sequence of toxin A protein, toxin B protein and / or subject isolated polypeptide, but still has the same amino acid sequence as the polypeptide encoded by the native DNA sequence. Producing a polypeptide having This procedure, known as “codon optimization” (described in US Pat. No. 5,547,871, which is incorporated herein by reference in its entirety), designs such a modified DNA sequence. Provide a means. Codon-optimized gene design is based on codon usage, closest frequency, R
NA stability, potential secondary structure formation, synthetic pathway, and intended future D of the gene
Various factors should be considered, including NA manipulation. In particular, the codon encoding a given isolated polypeptide is modified with a codon that is most easily recognized by yeast if a yeast expression system is used, or by insect cells if an insect cell expression system is used. Available methods can be used for this purpose. The degeneracy of the genetic code also allows the same amino acid sequence to be encoded and translated in many different ways. For example, leucine, serine and arginine are each encoded by 6 different codons, while valine, proline, threonine, alanine and glycine are each encoded by 4 different codons. However, the frequency of use of such synonymous codons varies between genomes between eukaryotes and prokaryotes. For example, synonymous codon selection patterns among mammals are very similar, but evolutionarily distant organisms such as yeast (eg S. cerevisiae), bacteria (eg E. coli) and insects (eg Drosophila melanogaster) Clearly different genomic codon usage patterns are shown (Grantham, R., et al., Nucl. Acid Res., 8
49-62 (1980); Grantham, R .; , Et al. , Nucl. Aci
d Res. 9, 43-74 (1981); Maroyama, T .; , Et al. ,
Nucl. Acid Res. 14, 151-197 (1986); Aota, S .;
, Et al. , Nucl. Acid Res. 16, 315-402 (1988);
Wada, K .; , Et al. , Nucl. Acid Res. , 19 Supp. , 1
981-1985 (1991); Kurland, C.I. G. , FEBS Lett. , 2
85, 165-169 (1991)). These differences in codon selection patterns appear to contribute to the overall expression level of individual genes by adjusting the peptide extension rate (Kurl
and, C.I. G. , FEBS Lett. , 285, 165-169 (1991); Pe
dersen, S.M. , EMBO J. et al. , 3, 2895-2898 (1984);
ensen, M.M. A. , J .; Mol. Biol. , 207, 365-377 (1989)
Randall, L .; L. , Et al. , Eur. J. et al. Biochem. 107
, 375-379 (1980); Curran, J. et al. F. , And Yarus, M .; ,
J. et al. Mol. Biol. 209, 65-77 (1989); Varenne, S .; , E
t al. , J .; Mol. Biol. , 180, 549-576 (1984), Vare.
nne, S.M. , Et al. , J .; Mol, Biol. , 180, 549-576 (19
84); Garel, J .; -P. , J .; Theor. Biol. , 43, 211-225
(1974); Ikemura, T .; , J .; Mol. Biol. , 146, 1-21 (1
981); Ikemura, T .; , J .; Mol. Biol. , 151, 389-409 (
1981)).

Preferred codon usage for synthetic genes reflects the codon usage of nuclear genes obtained from the extracted (or as closely related as possible) genome of cells / organisms intended to be used for recombinant protein expression Should.

Preferred methods for determining identity are designed to give the greatest match between the two sequences tested. The method of determining identity and similarity is organized in a computer program. A preferred computer program method for determining identity and similarity between two sequences is the GCG program package (Devereux, et al.
al. , Nucl. Acid Res. 12 (1): 387 (1984)), BRAS
TP, BLASTN, FASTA (Atschul, et al., J. Mol. Bio
l. 215: 403 (1990)), but is not limited thereto. The degree of similarity or identity described above is determined as the degree of identity between two sequences and often indicates the derivation of the first sequence from the second sequence. The degree of identity between two nucleic acids is determined by the GA provided in a computer program known in the art, such as a GCG program package.
P can be determined using Needleman and Wunsch J. Mol.
Biol. 48: 443-453 (1970)). For purposes of determining the degree of identity between two nucleic acids for the purposes of the present invention, GAP is used in the following settings: GAP generation penalty of 5.0 and GAP extension penalty of 0.3.

The present invention also provides C-TAB. G5 or C-TAB. Vectors comprising nucleic acids encoding G5.1 isolated polypeptides are provided. The vector may be any of a number of nucleic acids into which the desired sequence may be inserted, either by transfer between different production environments or by restriction and ligation for expression in the host cell. The vector is typically DN
Although composed of A, RNA vectors are also available. Vectors include but are not limited to plasmids and phagemids. A cloning vector is a vector that can replicate in a host cell and further features one or more endonuclease restriction sites, at which the vector may be cleaved in a measurable manner, and The desired DNA sequence may be ligated so that the new recombinant vector retains the ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence may occur many times with the plasmid showing an increase in copy number in the host bacterium, or only once per host before the host regenerates by mitosis. In the case of phage, replication can occur actively during the lysis phase or passively during the lysogen phase.

The vector may further contain a promoter sequence. A promoter may comprise untranslated nucleic acid, usually located upstream of the coding region containing the site for initiating transcription of the nucleic acid. The promoter region may also contain other elements that function as regulators of gene expression. In a further embodiment of the invention, the expression vector contains additional regions to aid in the selection of cells that have incorporated the expression vector. The promoter sequence is
In many cases, it is bounded at the 3 'end by the transcription start site (inclusive) and extends upstream (5' direction) to achieve the highest level necessary to initiate transcription at a detectable level above background. Contains a small number of bases or elements. Within the promoter sequence, the transcription start site, and RN
A protein binding domain responsible for the binding of A polymerase is seen. Eukaryotic promoter is
Although not necessarily, it often contains a “TATA” box and a “CAT” box.

The vector may further contain one or more marker sequences suitable for use in the identification and selection of cells transformed or transfected with the vector. Markers include, for example, genes that encode proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, enzymes whose activity can be detected by standard assays known in the art (eg, β -Genes encoding galactosidase or alkaline phosphatase) and genes that clearly affect the phenotype of the transformed, transfected, cell, host, colony or plaque. Preferred vectors are those capable of self-replication and expression of structural gene products present in operably linked DNA segments.

An expression vector is a vector into which a desired nucleic acid can be inserted by restriction and ligation so that it can be operably linked to regulatory sequences and expressed as an RNA transcript. Expression refers to the transcription and / or translation of an endogenous gene, transgene or coding region within a cell.

A coding sequence and a regulatory sequence are operably linked when the expression or transcription of the coding sequence is covalently linked so as to be under the influence or control of the regulatory sequence. If it is desired that the coding sequence be translated into a functional protein, the induction of the promoter in the 5 ′ regulatory sequence results in transcription of the coding sequence, and the nature of the bond between the two DNA sequences is (
1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) the corresponding R translated into a protein
Two DNA sequences are said to be operably linked if they do not interfere with the ability of the NA transcript. Thus, if the promoter region can effect transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide,
The promoter region is operably linked to the coding region.

C-TAB of the present invention. G5 or C-TAB. A G5.1 isolated polypeptide can be produced by expressing the encoding nucleic acid in a host cell. The nucleic acid can be transformed or transfected into a host cell. Accordingly, some aspects of the present invention provide C-TAB. G
5 or C-TAB. Transformation and / or transfection of a nucleic acid encoding a G5.1 isolated polypeptide. Transformation is the introduction of foreign or heterologous nucleic acid into the prokaryotic cell. Transfection is the introduction of foreign or heterologous nucleic acid into the interior of eukaryotic cells. The transformed or transfected nucleic acid is the chromosomal DNA that makes up the genome of the cell
Integrated (covalently bonded) or not. For example, in prokaryotic cells, the transforming nucleic acid can be maintained on an episomal element such as a plasmid or viral vector. For eukaryotic cells, a stably transfected cell is a cell that is integrated into the chromosome so that the transfected nucleic acid is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones that contain a population of daughter cells containing the transfected nucleic acid.

Higher eukaryotic cell cultures may be used to express the proteins of the invention from invertebrate cells, including vertebrates or insects, and their propagation procedures are known (eg, Kruse et a
l. (1973) Tissue Culture, Academic Press
See).

Also, nucleic acid replication and encoded C-TAB. G5 or C-TAB. Host cells and vectors for expression of the G5.1 isolated polypeptide are also provided. Any prokaryotic or eukaryotic vector or host cell may be used. Many vectors and host cells for such purposes are known in the art. Selecting the appropriate set for the desired application is well within the skill of the art.

The DNA sequence encoding toxin A and toxin B, or a portion thereof, is C.I. Difficile,
And can be cloned from various genomic or cDNA libraries obtained from other known toxin A and toxin B expressing prokaryotes known in the art. Techniques for isolating such DNA sequences using probe-based methods are conventional techniques and are well known to those skilled in the art. Probes for isolating such DNA sequences may be based on published DNA or protein sequences. Alternatively, the polymerase chain reaction disclosed by Mullis et al. (US Pat. No. 4,683,195) and Mullis (US Pat. No. 4,683,202) (incorporated herein by reference) (
PCR) method may be used. Selection of libraries and selection of probes for isolation of such DNA sequences is within the level of skill in the art.

Suitable host cells can be obtained from prokaryotes or eukaryotes. Suitable prokaryotic hosts are those of the genus Pseudomonas such as Pseudomonas aeruginosa, E. coli, Staphylococcus such as S. aureus and S. aureus. Epidermis, Serratia marcescens, Neisseria gonorrhoeae, such as Bacillus subtilis and giant fungi,
Clostridium sporogenes, Enterococcus faecalis, Micrococcus sp. Lutheus and M.M. Includes Roseus and Proteus Bulgaris. Suitable host cells for expressing the polypeptides of the invention in higher eukaryotic cells are yeast,
For example, Saccharomyces (eg S. cerevisiae); 293 (human fetal kidney) (ATC
C CRL-1573); 293F (Invitrogen, Carlsbad CA)
293T and mutant 293T / 17 (293tsA1609neo and mutant AT;
CC CRL-11268) (human fetal kidney transformed with SV40 T antigen);
COS-7 (monkey kidney CVI line transformed with SV40) (ATCC CRL1651
BHK (baby hamster kidney cells) (ATCC CRL10); CHO (Chinese hamster ovary cells); mouse Sertoli cells; CVI (monkey kidney cells) (ATCC)
CCL70); VERO76 (African green monkey kidney cells) (ATCC CRL158)
7); HeLa (human uterine cancer cells) (ATCC CCL2); MDCK (canine kidney cells)
(ATCC CCL34); BRL3A (buffer rotat liver cells) (ATCC C
RL 1442); W138 (human lung cells) (ATCC CCL75); HepG2 (human hepatocytes) (HB8065); and MMT 060652 (mouse mammary tumor) (ATC).
C CCL51).

In another embodiment, the present invention provides C-TAB. G5 or C-TAB. Nucleic acids encoding isolated polypeptides, including G5.1 isolated polypeptides, as well as additional polypeptides are provided. Vectors useful for constructing eukaryotic expression systems for the production of fusion polypeptides include nucleic acids encoding isolated polypeptides operably linked to appropriate transcriptional activation sequences, such as promoters and / or operators. Including. Other exemplary features may include appropriate ribosome binding sites, stop codons, enhancers, terminators, or replicon elements. These additional features can be inserted into the vector at the appropriate site (s) by conventional splicing techniques such as restriction endonuclease digestion and ligation.

In some embodiments, the additional nucleic acid is C-TAB. G5 or C-TAB.
It may be fused to a nucleic acid encoding a G5.1 isolated polypeptide. The fusion nucleic acid is C-TA
B. G5 or C-TAB. A polypeptide that can assist in purification and / or immunogenicity and / or stability without shifting the codon reading frame of the G5.1 isolated polypeptide may be encoded. The fusion nucleic acid is C-TAB. G5 or C-TAB. G5.1
It may encode a secretory sequence that may or may not be cleaved from the isolated polypeptide. The fusion nucleic acid may not significantly extend the expressed polypeptide. The fusion nucleic acid is C-
TAB. G5 or C-TAB. The G5.1 isolated polypeptide may encode less than 60 extra amino acids. In some embodiments, the fusion nucleic acid is C-TAB.
. G5 or C-TAB. Following the nucleic acid encoding the G5.1 isolated polypeptide. In other embodiments, the fusion nucleic acid is C-TAB. G5 or C-TAB. Precedes nucleic acid encoding G5.1 isolated polypeptide. In other embodiments, the fusion nucleic acid is C-TAB. G
5 or C-TAB. It is adjacent to a nucleic acid encoding a G5.1 isolated polypeptide.

In some embodiments, the fusion nucleic acid is C-TAB. G5 or C-TAB. G5
. A polypeptide that assists in the purification of one isolated polypeptide may be encoded. In some embodiments, the fusion nucleic acid encodes an epitope and / or an affinity tag. Examples of polypeptides that aid in purification include His-tag, myc-tag, S-peptide tag, MB
Including, but not limited to, P tag, GST tag, FLAG tag, thioredoxin tag, GFP tag, BCCP, calmodulin tag, Strep tag, HSV-epitope tag, V5-epitope tag, and CBP tag. In other embodiments, the fusion nucleic acid is
C-TAB with a site that is induced or susceptible to cleavage. G5 or CT
AB. A G5.1 isolated polypeptide may be encoded. In one embodiment, the fusion nucleic acid may encode a polypeptide that includes a site for enzymatic cleavage. In a further embodiment, the enzymatic cleavage is performed using C-TAB. G5 or C-TAB. G
5.1 Can assist in the isolation of isolated polypeptides, and other fusion polypeptide segments.
As an example, C-TAB. G5 or C-TAB. An intermediate nucleic acid encoding an enzymatic cleavage site located between the nucleic acid encoding the G5.1 isolated polypeptide and the epitope is expressed in the expressed C-TAB. G5 or C-TAB. Subsequent separation of G5.1 isolated polypeptides and epitopes may be possible. Such a site may also be present between the toxin A and toxin B moieties.

The present invention also provides C-TAB. G5 or C-TAB. An expression system is provided that is designed to assist in the expression and delivery of the G5.1 isolated polypeptide. The expression system is C-TAB. G
5 or C-TAB. A host cell transformed or transfected with a nucleic acid encoding a G5.1 isolated polypeptide may be included. The host cell may be prokaryotic. The prokaryote may be E. coli. The host cell may be a eukaryotic cell.

The expression system further includes C-TAB. G5 or C-TAB. Agents may be included to aid in selection of host cells that have been successfully transformed or transfected with nucleic acids encoding G5.1 isolated polypeptides. For example, C-TAB. G5 or C-TAB. The nucleic acid encoding the G5.1 isolated polypeptide further comprises a gene that assists host cells that are resistant to antibiotics,
For example, a gene that resists kanamycin or gentamicin or ampicillin or penicillin may be expressed. Such resistance genes, as known to those skilled in the art,
C-TAB. G5 or C-TAB. Allows selection of host cells that have properly incorporated the nucleic acid encoding the G5.1 isolated polypeptide.

Another aspect of the invention relates to the production of antibodies. Examples of antibodies encompassed by the present invention include C-TA
B. G5 or C-TAB. This includes, but is not limited to, antibodies produced by immunizing a subject with a G5.1 isolated polypeptide. C-TAB. G5 or C-TAB. G
5.1 Antibodies produced by immunization with an isolated polypeptide can specifically bind to toxin A or toxin B, or they can be C-TAB. G5 or C-TAB. G
5.1 Can cross-react with isolated polypeptide. C-TAB of the present invention. G5 or C-TAB. Antibodies produced by the G5.1 isolated polypeptide can be characterized using methods well known in the art.

C-TAB of the present invention. G5 or C-TAB. Antibodies generated by using the G5.1 isolated polypeptide include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab ′, F (ab ′) 2, Fv, Fc, etc.), chimeric antibodies, Bispecific antibodies, heavy chain only antibodies, heteroconjugated antibodies, single chain (ScFv), single domain antibodies, variants thereof, isolated polypeptides comprising antibody portions, humanized antibodies, and glycosylation of antibodies Any other modified structure of an immunoglobulin molecule that includes an antigen recognition site of the required characteristics, including variants, amino acid sequence variants of the antibody, and covalently modified antibodies may be included. Preferred antibodies are obtained from mouse, rat, human, rabbit, dog, pig, dromedary, camel, llama, cat, primate, or any other source (including chimeric, fragment and / or humanized antibodies).

In other embodiments, C-TAB. G5 or C-TAB. The antibody produced by immunization with the G5.1 isolated polypeptide is then humanized by methods known in the art. Humanized antibodies are immunoglobulin molecules that contain minimal sequence derived from non-human immunoglobulin. In yet other embodiments, fully human antibodies are obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. In other embodiments, the antibody is chimeric. A chimeric antibody is an antibody that combines properties from two different antibodies. Methods for preparing chimeric antibodies are known in the art.

In other embodiments, a nucleotide sequence encoding the antibody is obtained and then cloned into a vector for expression or propagation. In another embodiment, the antibody is produced and expressed recombinantly using methods known in the art. As an example, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide can be used as an antigen to isolate recombinant antibodies by these techniques. Antibodies can be made recombinantly using gene sequences in order to express the antibody recombinantly in a host cell. Methods for making antibody variants and recombinant antibodies are known in the art.

In other embodiments, the antibody is isolated or purified, eg, natural toxin A or toxin B, or natural toxin A or toxin B or C. in a biological sample or specimen. For use in the detection of difficile, it is bound to the carrier by conventional methods in the art.
Compositions and formulations

The present invention also provides C-TAB. G5 or C-TAB. Compositions comprising the G5.1 isolated polypeptide are provided. The composition is C-TAB. G5 or C-TAB. It may be a pharmaceutical composition comprising a G5.1 isolated polypeptide and a pharmaceutically acceptable carrier. The compositions used in the methods of the present invention are generally, by way of example and not limitation, an effective amount of C of the present invention.
-TAB. G5 or C-TAB. G5.1 isolated polypeptide (eg, an amount sufficient to induce an immune response), or C-TAB. G5 or C-TAB. Antibodies to the G5.1 isolated polypeptide (eg, alleviate infection, reduce symptoms of infection, and / or
Or an amount of neutralizing antibody sufficient to prevent infection). The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, or stabilizer known in the art (generally Remington, (2005) The Science.
and Practice of Pharmacy, Lippincott, Will
see iams and Wilkins).

C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide is CDAD
Can be used in methods for immunizing or treating humans and / or animals suffering from.
Therefore, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide can be used in pharmaceutical compositions. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers and / or excipients useful in the present invention are conventional and can include buffers, stabilizers, diluents, preservatives, and solubilizers. E. W. Remington's Pharmacy by Martin
utical Sciences, Mack Publishing Co. , East
on, PA, 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides disclosed herein. In general, the nature of the carrier or excipient will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable solutions that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (eg, in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the administered pharmaceutical composition contains trace amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents such as sodium acetate or sorbitan monolaurate. You may contain.

In one embodiment, the pharmaceutical composition may further comprise an immunostimulatory substance such as an adjuvant. Adjuvants can be selected based on the method of administration, mineral oil based adjuvants such as Freund's complete and incomplete adjuvants, Montanide incomplete S
epic adjuvants such as ISA, oil-in-water emulsion adjuvants such as Ri
a bi-adjuvant system, a syntax adjuvant formulation containing muramyl dipeptide,
Aluminum hydroxide or aluminum salt adjuvant (alum), polycationic polymer, especially polycationic peptide, especially polyarginine or at least two Lys
Peptides containing the sLeuLys motif, in particular KLKLLLLLKLK, immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine dinucleotide (CpG) within a defined base context (eg as described in WO 96/02555) or inosine And cytidine-based ODN (eg as described in WO01 / 93903) or deoxynucleic acids containing deoxyinosine and / or deoxyuridine residues (as described in WO01 / 93905 and WO02 / 095027), in particular Oligo (d
IdC) ₁₃ (described in WO 01/93903 and WO 01/93905), neuroactive compounds, in particular human growth hormone (described in WO 01/24822), or combinations thereof. Such combinations include, for example, WO01 / 93905, WO02 /
32451, WO01 / 54720, WO01 / 93903, WO02 / 13857, W
According to 02/095027 and WO 03/047602. Preferably,
The adjuvant is an aluminum hydroxide adjuvant.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations administered. The carrier, excipient or stabilizer may further comprise a buffer. Examples of excipients are carbohydrates (eg monosaccharides and disaccharides), sugars (eg sucrose, mannitol,
And sorbitol), phosphates, citrates, antioxidants (eg, ascorbic acid and methionine), preservatives (eg, phenol, butanol, benzanol; alkyl parabens, catechol, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, resorcinol, cyclo Hexanol, 3-pentanol, benzalkonium chloride, benzethonium chloride, and m-cresol), low molecular weight polypeptides,
Proteins (eg, serum albumin or immunoglobulin), hydrophilic polymer amino acids, chelating agents (eg, EDTA), salt-forming counterions, metal complexes (eg, Zn-protein complexes), and nonionic surfactants (eg, TWEEN ™ ) And polyethylene glycol).

The pharmaceutical compositions of the present invention may further comprise additional agents that act to enhance and / or supplement the desired effect. As an example, C-TAB. Of the present invention being administered as a subunit vaccine. G5 or C-TAB. In order to improve the immunogenicity of the G5.1 isolated polypeptide, the pharmaceutical composition may further comprise an adjuvant.

An example of a pharmaceutical composition may be an immunogenic composition. The present invention relates to C-TAB. G5 or C-TAB. An immunogenic composition comprising a G5.1 isolated polypeptide is provided. The immunogenic composition may further comprise a pharmaceutically acceptable carrier or other carrier and / or excipient in a formulation suitable for injection in a mammal. An immunogenic composition is any composition of material that elicits an immune response in a mammalian host when the immunogenic composition is injected or otherwise introduced. The immune response may be humoral, cellular, or both. A booster effect refers to an increased immune response to the immunogenic composition after subsequent exposure of the mammalian host to the same immunogenic composition. The humoral response results in the production of antibodies by the mammalian host after exposure to the immunogenic composition.

The immunogenic compositions of the present invention elicit an immune response in mammalian hosts including humans and other animals. The immune response may be a cell-dependent response or an antibody-dependent response or both, and further, the response may provide an immune memory or booster effect in a mammalian host. These immunogenic compositions are useful as vaccines. It may provide a protective response by a mammalian subject or host against infection by a strain of difficile.

The present invention further provides C-TAB. G5 or C-TAB. A nucleic acid encoding the G5.1 isolated polypeptide was constructed and C-TAB. G5 or C-TAB. G
5.1 Expressing the isolated polypeptide component and expressing the C-TAB. G5 or C-TAB. G5.1 isolated polypeptide is recovered and C-TAB. G5 or C-TAB.
A method for producing an immunogenic composition by conjugating a G5.1 isolated polypeptide to a second protein component and recovering the complex protein and the polysaccharide component is included.
C-TAB. G5 or C-TAB. Nucleic acids encoding G5.1 isolated polypeptides can be maintained throughout host growth with constant and stable selective pressure. Maintenance of the expression vector can be effected by the incorporation of a gene sequence encoding a selective genotype into the expression vector, whose expression in a microbial host cell results in a selective expression type. A selective genotype sequence may also include a gene that complements a conditional lethal mutation. Other gene sequences such as other drug resistance genes or genes that complement lethal mutations may be incorporated into the expression vector. The microbial host can include gram positive bacteria, gram negative bacteria such as E. coli, yeast, filamentous fungi, mammalian cells, insect cells, or plant cells.

The method of the present invention also provides a level exceeding about 50 mg / liter (culture), about 100 m.
levels above g / liter, levels above about 500 mg / liter, or about 1 g / liter
Levels of C-TAB. G5 or C-TAB. G5.1
A level of expression of the isolated polypeptide is provided. The present invention also provides that the protein can be recovered by any number of methods known to those skilled in the art of protein isolation and recovery, such as ion exchange chromatography followed by ammonium sulfate precipitation.

The present invention further provides that the protein component is conjugated to the second protein component by one of several means known to those skilled in the art, such as an amidation reaction. A method for preparing the composition is included.

The present invention also provides C-TAB. For treating and preventing CDAD. G5 or C-
TAB. A formulation comprising a G5.1 isolated polypeptide is provided. In one embodiment, the formulation is a C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide, an adjuvant, and a pharmaceutically acceptable carrier may be included. In another embodiment, the formulation is a C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide or essentially one or more C-TAB. G5 or C-TAB. Consists of G5.1 isolated polypeptide. The formulation is C-TAB. G5 or C-TAB. A G5.1 isolated polypeptide and an adjuvant may be included. The formulation may further comprise additional antibodies or drugs. Further, the formulation may include one or more drugs and may include one or more drugs in addition to the isolated polypeptide and / or adjuvant.

C-TAB. G5 or C-TAB. Formulations containing the G5.1 isolated polypeptide may be in liquid or dry form. Dry formulations can be easily stored and transported. The dry formulation breaks the cold chain required from the vaccine production site to the point where the vaccination takes place. Alternatively, the dry active ingredient of the formulation may itself be an improvement by providing a solid particulate form that is taken up and processed by antibody-presenting cells. These possible mechanisms are not discussed to limit the scope of the invention or its equivalents, but provide insights into the effects of the invention and are discussed to guide the use of the formulation in immunization and vaccination. Is done.

C-TAB. G5 or C-TAB. Dry formulations of G5.1 isolated polypeptides can be provided in a variety of forms such as fine or granular powders, lyophilized powders, uniform membranes, pellets, and tablets. Formulations include air drying, drying at high temperature, freeze drying, freeze drying or spray drying, coating on a solid substrate or drying after spraying, sprinkling on a solid substrate, slow drying under vacuum following quick freezing, Or a combination thereof may be performed. If different molecules are active ingredients of the formulation, they may be mixed in solution and then dried,
Or it may be mixed only in a dry form.

C-TAB in liquid or solid form, for example dry form. G5 or C-TAB. G5.1
A formulation comprising an isolated polypeptide may be applied with one or more adjuvants at the same or remote sites, or by simultaneous or frequent repeated application. The formulation may include other antigens such that administration of the formulation induces an immune response against multiple antigens. In such cases, other antigens will induce an immune response specific for the different antigens,
It may have a different chemical structure. Prior to administration, at least one antigen and / or adjuvant may be maintained in a dry form. Subsequent release of the liquid from the reservoir or entry of the liquid into the reservoir containing the dry ingredients of the formulation will at least partially dissolve that component.

Solids (eg, nanometer or micrometer sized particles) may also be incorporated into the formulation. Solid forms (eg, nanoparticles or microparticles) are added to an adjuvant, C-TAB., To a substrate that can be opsonized by antigen-presenting cells to aid in the dispersion or solubilization of the active ingredient. G5 or C-TAB. A point of attachment of the G5.1 isolated polypeptide, or both, can be provided, or a combination thereof can be provided. Sustained release of the formulation from a porous solid formed as a sheet, rod, or bead acts as a depot formulation.

At least one component or component of the formulation (ie C-TAB.G5 or C-
TAB. G5.1 isolated polypeptide, adjuvant, or drug) may be provided in dry form prior to administration of the formulation. This formulation may also be used in conjunction with conventional enteral, mucosal, or parenteral immunization techniques.

C-TAB. G5 or C-TAB. Formulations containing the G5.1 isolated polypeptide can be manufactured under aseptic conditions approved by appropriate regulatory authorities for biologics and vaccines (eg, Food and Drug Administration, EMEA). Optionally, components such as desiccants, excipients, stabilizers, humectants, preservatives, or combinations thereof may be included in the formulation, even if immunologically inert. However, they may have other desired properties or characteristics.

Processes for manufacturing pharmaceutical formulations are well known. The components of the formulation can be any combination of a pharmaceutically acceptable carrier or vehicle and optional additives (eg, diluents, binders, excipients, stabilizers, desiccants, preservatives, colorants). May be combined. The use of solid carriers and the addition of excipients to aid solubilization of dry components or immunogenic or adjuvant active stabilizers are preferred embodiments. In general, Ullmann's E
ncyclopedia of Industrial Chemistry, 6 ^th
Ed. (Electronic edition, 2003); Remington's
^{Pharmaceutical Sciences, 22 nd (Gennaro,} 20
05, Mack Publishing); Pharmaceutical Dosag
e ^Forms, 2 nd Ed. (Various editors, 1989-1998, Marcel
Dekker); and Pharmaceutical Dosage Forms a
nd Drug Delivery Systems (Ansel et al., 20
05, Williams & Wilkins).

Appropriate manufacturing standards are known in the pharmaceutical industry, such as government agencies (eg Food and Drug Administration, EM
Regulated by EA). Sterile liquid formulations can be prepared by dissolving the intended components of the formulation in a sufficient amount of a suitable solvent followed by sterilization by filtration to remove contaminating microorganisms. In general, dispersions are prepared by incorporating the various sterilized components of the formulation into a sterile vehicle that contains a basic dispersion medium. For the production of solid forms that are required to be sterile, vacuum drying or freeze drying can be used.

In general, solid dosage forms (eg, powders, granules, pellets, tablets) can be made from at least one active ingredient or component of a formulation.

Suitable tableting procedures are known. A formulation may also be produced by encapsulating a solid form of at least one active ingredient or isolating it from a liquid in a compartment or chamber. Using the size of each dose and the interval between doses to the subject, tablets, capsules,
A suitable size and shape of the compartment or chamber can be determined.

Formulations contain an effective amount of the active ingredient (eg, drugs, antigens and adjuvants) together with a carrier or suitable amount of vehicle to provide a pharmaceutically acceptable composition suitable for administration to humans or animals. ).

The relative amount of active ingredient within the dose, e.g. C-TAB. G5 or C-TAB. The amount of G5.1 isolated polypeptide and the dosing schedule can be appropriately adjusted for effective administration to a subject (eg, an animal or human). This modulation may also depend on the particular disease or condition of the subject and whether treatment or prevention is intended. To simplify administration of the formulation to the subject, each unit dose contains a predetermined amount of active ingredient for a single round of immunization.

There are several cases of instability or degradation of polypeptides, including hydrolysis and alteration. In the case of alteration, the arrangement or three-dimensional structure of the protein is disturbed and the protein spreads out of its normal spherical structure. Rather than refolding to the natural configuration, hydrophobic interactions can cause agglomeration of molecules (ie, aggregation) or refolding to an abnormal configuration. Any of these results can be accompanied by a decrease or loss of immunogenicity or adjuvant activity. Stabilizers may be added to reduce or prevent such problems.

The formulation, or any intermediate in its production, may be pretreated with protective agents (ie, anti-freeze and dry stabilizer) and then subjected to a cooling rate and final temperature that minimizes ice crystal formation. . By appropriate selection of the cryoprotectant and the use of pre-selected drying parameters, almost any formulation can be prepared frozen for a suitable desired end use.

In the following discussion, it should be understood that optional additives such as excipients, stabilizers, desiccants, and preservatives are described by their function. Thus, certain chemicals are excipients,
It can act as a combination of stabilizers, desiccants, and / or preservatives. Such chemicals are not immunologically inactive because they do not directly induce an immune response, but by improving the immune activity of the antigen or adjuvant, for example, modification or drying of the antigen or adjuvant And increase the reaction by reducing alterations during the dissolution cycle.

Stabilizers include cyclodextrins and variants thereof (US Pat. No. 5,730,96).
(See 9). Suitable preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran, and glycerin can also be added to stabilize the final formulation (Howell and Miller, 1983). A stabilizer selected from nonionic surfactants, D-glucose, D-galactose, D-xylose, D-glucuronic acid, salts of D-glucuronic acid, trehalose, dextran, hydroxyethyl starch, and mixtures thereof May be added to the formulation. Addition of an alkali metal salt or magnesium chloride, optionally containing serum albumin, can be performed using C-TAB. G5 or C-TAB.
The G5.1 isolated polypeptide can be stabilized and the stability can be further improved by freeze drying. C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide can also be stabilized by contacting with a saccharide selected from the group consisting of dextran, chondroitin sulfate, starch, glycogen, insulin, dextrin, and arginate. Other sugars that can be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (eg, glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). The polyol can stabilize the polypeptide and is water miscible or water soluble. Suitable polyols are polyhydroxy alcohols, monosaccharides and disaccharides, including mannitol, glycerol, ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and polymers thereof. There may be. Various excipients including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelators, polyvinyl pyrrolidone, hydrolysed gelatin, and ammonium sulfate also
The polypeptide can be stabilized.

As an example, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide formulation is
Depending on the preferred choice of excipients or stabilizers, sucrose, trehalose, poly (lactic acid) (P
LA) and poly (lactide-co-glycolide) (PLGA) microspheres can be stabilized (Sanchez et al., 1999). Since sucrose or trehalose is a non-reducing saccharide and therefore does not cause an aminocarbonyl reaction with a substance having an amino group such as a protein, it can be advantageously used as an additive. Sucrose or trehalose may be combined with other stabilizers such as saccharides.

Furthermore, C-TAB. G5 or C-TAB. Formulations containing G5.1 isolated polypeptides include, for example, anesthetics, analgesics, anti-inflammatory agents, steroids, antibiotics, anti-arthritic agents, anti-appetite drugs,
It may also contain therapeutic agents such as antihistamines and anti-neoplastic agents. Examples of such therapeutic agents include lidocaine and nonsteroidal anti-inflammatory drugs (NSAIDs). In another embodiment, the therapeutic agent is an antigen and an adjuvant. In yet another embodiment,
The formulation containing the antigen and / or adjuvant is separate, but for example anesthetics, analgesics,
It may be applied with other therapeutic agents such as anti-inflammatory agents, steroids, antibiotics, anti-arthritic agents, anti-appetite drugs, antihistamines, and anti-neoplastic agents. In a preferred embodiment, the antibiotic is fidaxomycin, metronidazole or vancomycin.

C-TAB. G5 or C-TAB. Formulations comprising the G5.1 isolated polypeptide can be delivered via various routes of administration, such as intramuscularly.

The polymer may be added to the formulation and acts as an excipient, stabilizer, and / or preservative of the active ingredient and saturates the solution used to dissolve the active ingredient in dry form The concentration of can be reduced. Such a reduction occurs because the polymer reduces the effective volume of the solution by filling the “empty” space. Thus, the amount of antigen / adjuvant can be preserved without reducing the amount of saturated solution. An important thermodynamic consideration is that the active ingredient in the saturated solution is “driven” to a lower concentration region. In solution
The polymer can also stabilize and / or preserve the antigen / adjuvant activity of the solubilized component of the formulation. Such polymers include ethylene or propylene glycol, vinyl pyrrolidone, and 0-cyclodextrin polymers and copolymers.

C-TAB suitable for administration. G5 or C-TAB. A single or unit dose of a formulation comprising a G5.1 isolated polypeptide is provided. Unit dose adjuvant and / or C-TA
B. G5 or C-TAB. The amount of G5.1 isolated polypeptide can be anywhere within a wide range of about 0.001 μg to about 10 mg. This range is about 0.1 μg to about 1 mg.
The narrower range is from about 5 μg to about 500 μg. Other suitable ranges are between about 20 μg and about 200 μg, such as about 20 μg, about 75 μg, or about 200 μg.
g and the like. C-TAB. G5 or C-TAB. Preferred doses of G5.1 isolated polypeptide are from about 20 μg or up to 200 μg. C-TAB. G5 or CT
AB. The ratio between the G5.1 isolated polypeptide and the adjuvant is about 1: 1 or about 1: 1.
. 25, although higher ratios may be used (eg, about 1:10 or less),
Alternatively, a lower ratio of C-TAB isolated polypeptide to adjuvant may be used (eg, about 10: 1 or greater).

C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide may be used as an antigen and presented to immune cells, inducing an antigen-specific immune response. this is,
C. It can occur before, during, or after infection by a pathogen such as difficile. If the immunogenicity of the formulation is sufficient to not require adjuvant activity, C-TAB. G5 or CT
AB. Only the G5.1 isolated polypeptide is required and no additional adjuvant may be required. The formulation may include additional antigens such that application of the formulation induces an immune response against multiple antigens (ie is multivalent). Antigen-specific lymphocytes may be involved in the immune response, and when B lymphocytes are involved, the antigen-specific antibody may be part of the immune response. The formulations described above may include desiccants, excipients, humectants, stabilizers, and preservatives known in the art.

C-TAB of the present invention. G5 or C-TAB. Formulations containing the G5.1 isolated polypeptide require treatment such as prevention of disease, protection from the effects of infection, reduction or reduction of symptoms of diseases such as CDAD, or combinations thereof Can be used to treat humans or animals). For example, C-TAB. G5 or C-TAB. G5.
A preparation containing one isolated polypeptide is, for example, a CDAD such as a subject having the following profile:
Can be used to treat subjects at risk of: i) subjects with a weaker immune system, such as elderly subjects (eg, subjects over 65 years old) or subjects under 2 years of age; ii) subjects without immunity E.g. a subject with AIDS, etc .; iii) being administered an immunosuppressant,
Or subject to be administered; iv) subject to be hospitalized or hospitalized; v) subject to or will receive intensive care (ICU); vi)
Subjects undergoing or scheduled to undergo gastrointestinal surgery; vii) subjects receiving or willing to receive long-term care such as nursing homes; viii) frequent and / or long-term antibiotic use Ix) subjects who have more than one of the above profiles, such as elderly subjects who are scheduled to undergo gastrointestinal surgery; x
) A subject with inflammatory bowel disease; and / or xi) a subject with recurrent CDAD, such as a subject experiencing one or more episodes of CDAD.

Treatment may vaccinate a subject against infection by a pathogen or against pathogenic effects such as those caused by toxin secretion. The formulation is used to treat existing diseases,
It can be used therapeutically to prevent the disease protectively, to reduce the severity and / or duration of the disease, to improve the symptoms of the disease, or a combination thereof.

C-TAB. G5 or C-TAB. A formulation comprising a G5.1 isolated polypeptide is orally,
Delivered by various routes of administration including, but not limited to, subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intracardiac, intrathecal, intrathoracic, intraperitoneal, intraventricular, and / or sublingual routes obtain.

The formulation may also include one or more adjuvants or combinations of adjuvants. Usually, adjuvants and formulations are mixed prior to presentation to the antigen, but as an alternative,
It may be presented separately within a short time interval.

Adjuvants include, for example, oil emulsions (eg, complete or incomplete Freund's adjuvant), Montanide incomplete Seppic adjuvants such as ISA, oil-in-water emulsion adjuvants such as Ribi adjuvant system, syntax adjuvant formulations containing muramyl dipeptide, hydroxylation Aluminum or aluminum salt adjuvant (ALUM), polycationic polymers, in particular polycationic peptides, in particular polyarginine or peptides containing at least two LysLeuLys motifs,
In particular KLKLLLLLKLK, an immunostimulatory oligodeoxynucleotide (OD) containing unmethylated cytosine-guanine dinucleotide (CpG) in a defined base context
N) (eg as described in WO 96/02555) or OD based on inosine and cytidine
N (for example described in WO01 / 93903) or deoxynucleic acid containing deoxyinosine and / or deoxyuridine residues (WO01 / 93905 and WO02 / 09)
5027), in particular Oligo (dIdC) ₁₃ (WO 01/93903 and WO
01/93905), neuroactive compounds, in particular human growth hormone (WO01 / 248)
22), or combinations thereof, chemokines (eg, defensin 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8, or cytokines (eg, interleukin-1β, -2, -6, -10 or -12; interferon-γ; tumor necrosis factor-α; or granulocyte monocyte colony stimulating factor) (Nohr)
ia and Rubin, 1994), muramyl dipeptide variants (eg, mulabtide, threonyl-MDP or muramyl tripeptide), M
Synthetic mutants of DP, heat shock proteins or mutants, forest tropical Leishmania Le
A variant of IF (Skeiky et al., 1995), a non-toxic variant of bacterial ADP-ribosylating exotoxin (bARE) containing a variant at the trypsin cleavage site (Dickenso)
n and Elements, 1995) and / or active ADP-ribosylation (
Douce et al. 1997) or chemical detoxification bARE (toxoid), QS
21, Quill A, N-acetylmuramile-L-alanyl-D-isoglutamyl-
A composition comprising L-alanine-2- [1,2-dipalmitoyl-s-glycero-3- (hydroxyphosphoryloxy)] ethylamide (MTP-PE) and a metabolizable oil and emulsifier comprising the oil and The emulsifier is a composition present in the form of an oil-in-water emulsion with oil droplets having a diameter of substantially less than 1 micron (eg, EP0398843).
For example). Also, for other adjuvants useful for immunization, Richa
rds et al. (1995).

Adjuvants are antibodies or cell effectors, specific antibody isotypes (eg,
IgM, IgD, IgA1, IgA2, secretory IgA, IgE, IgG1, IgG2, Ig
G3, and / or IgG4), or certain T cell subsets (eg, CTL,
Th1, Th2 and / or T _DTH ) (eg, Munoz et al., 19
90; Glenn et al. , 1995) can be selected to preferentially navigate.

Unmethylated CpG dinucleotides or motifs are known to activate B cells and macrophages (Stacey et al., 1996). Other forms of DNA may be used as an adjuvant. Bacterial DNA is included in a class of structures that have a pattern that allows the immune system to recognize its pathogenic source and stimulate the innate immune response to produce an adaptive immune response (Medzhitov and Janway, 1997, Curr.
Opin. Immunol. 9 (1): 4-9). These structures are called pathogen-associated molecular patterns (PAMPs) and include lipopolysaccharide, teichoic acid, unmethylated CpG motif, double stranded RNA, and mannin. PAMP mediates an inflammatory response, acts as a costimulator of T cell function, and induces endogenous signals that can control effector function. The ability of PAMP to induce these responses plays a role in its potential as an adjuvant, and its target is APC such as macrophages and dendritic cells. PAMP also induces different costimulatory molecules and regulates different effector functions,
It may be used in conjunction with other adjuvants to guide immune responses, such as Th2 to Th1 responses.

Another aspect of the present invention relates to C-TAB. G5 or C-TAB. It relates to the use of the G5.1 isolated polypeptide as a vaccine. The vaccine or immunogenic composition of the invention may use an effective amount of antigen. Later C.I. An amount of antigen is included that causes the subject to generate a specific sufficient immune response to provide protection of the subject from exposure to difficile. The antigen is C-
TAB. G5 or C-TAB. It may be a G5.1 isolated polypeptide. In one embodiment, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide alone is
Or it is administered in combination with an adjuvant.

Another aspect of the present invention relates to C-TAB. G5 or C-TAB. Includes use of G5.1 isolated polypeptide as a subunit vaccine. Subunit vaccine refers to the use of pathogen fragments as an inoculum. One skilled in the art will recognize that subunit vaccines provide a means of generating antibodies against specific portions or regions of pathogens.

The dosage schedule for vaccine administration and its effectiveness can be determined by methods known in the art. The amount of vaccine and immunization regime may depend on the particular antigen and adjuvant used, the mode and frequency of administration, and the desired effect (eg, protection and / or treatment). In general, the vaccine of the present invention may be administered in an amount ranging between 1 μg and 100 mg, such as between 60 μg and 600 μg. C-TAB. G5 or C-TAB. A single dose of a vaccine comprising a G5.1 isolated polypeptide is about 1 μg to about 1 mg, preferably about 5 μg to about 500 μg, more preferably about 20 μg to about 20
It may be in the range of 0 μg. C-TAB. G5 or C-TAB. The ratio between the G5.1 isolated polypeptide and an adjuvant such as alum may be about 1: 1, such as 1: 1.25, but higher ratios may be used (eg, About 1:10 or less), or lower ratios may be used (eg, about 10: 1 or more). In one embodiment, C-T
AB. G5 or C-TAB. In vaccines containing the G5.1 isolated polypeptide, the adjuvant aluminum hydroxide is used in an amount in the range of about 50 μg / mL to about 200 μg / mL, preferably about 125 μg / mL (final formulation).

C-TAB. G5 or C-TAB. A vaccine comprising a G5.1 isolated polypeptide can be administered orally, intravenously, subcutaneously, intraarterially, intramuscularly, intracardiac, intrathecal, intrathoracic, intraperitoneal, intraventricular, and / or sublingually. .

The immunization plan can be determined by methods known in the art. The administration of the vaccine may be repeated as determined by those skilled in the art as needed. For example, one, two, three, or more booster doses may be administered following the prep dose at weekly, biweekly, or monthly intervals. In one embodiment of the invention, one or two booster doses following the prep dose are administered at intervals of about 7 to about 14 days, such as 7 and 21 days after the first dose. In a preferred embodiment, a therapeutically effective amount of vaccine is administered to a subject at intervals of 2 or 3 times, 14 days +/− 1, 2 or 3 days (every 2 weeks). In one embodiment of the invention, a therapeutically effective amount of the vaccine is administered once.

Yet another aspect relates to a population that can be treated according to the present invention. In one embodiment, the population is C.I. Includes healthy individuals at risk of exposure to difficile, especially those who are hospitalized or just staying in a nursing home, and those in hospitals, nursing homes and other nursing homes. In another embodiment, the population includes previously infected patients who have relapsed after cessation of antibiotic treatment or patients who are not effective with antibiotic treatment.

In one further embodiment of the invention, the population comprises individuals at least 18 years of age or older. In one preferred embodiment, the human subject is between 18 and 65 years old. In another preferred embodiment, the human subject is an elderly person over 65 years old. The latter age group is C
. It is the most vulnerable group to be infected with difficile. In some further embodiments, the human subject is younger than 18 years old.
C-TAB. G5 or C-TAB. Method of using G5.1 isolated polypeptide

The invention also provides methods of using the isolated polypeptide. For example, C-TAB. G
5 or C-TAB. The G5.1 isolated polypeptide is C.I. It can be used to prevent or treat diseases associated with difficile. By way of example, introducing an isolated polypeptide of the present invention into a subject's immune system can induce an immune response that includes the subject producing antibodies to the isolated polypeptide. Such antibodies are C.I. Useful for difficile recognition.

The present invention provides a method of delivering an isolated polypeptide to a subject comprising administering the isolated polypeptide to a subject. An isolated polypeptide can be administered as a liquid or a solid. An isolated polypeptide may further comprise a pharmaceutically acceptable carrier.

The present invention also relates to a method for identifying and isolating an antibody variable domain that recognizes and binds to toxin A and / or toxin B, wherein C-TAB.
G5 or C-TAB. Use of G5.1 isolated polypeptide and C-TAB. G5 or C
-TAB. Purifying and then characterizing the antibody produced in response to the G5.1 isolated polypeptide. The identified epitope can be used for cloning additional antibodies or fragments thereof.

One embodiment of the present invention includes C.I. It relates to the treatment, prevention and detection of difficile.
In some embodiments, the subject, such as an animal, is C.I. Receive treatment and / or prevention and / or detection of difficile. In other embodiments, the animal is a human. For example,
The polypeptides of the present invention can be obtained from C.I. It can be used to raise antibodies against difficile. As a further example, a polypeptide of the invention may be administered to a subject whose C.I. It can be used to determine whether to generate antibodies against difficile. In some embodiments, the polypeptide can be used to isolate an antibody. As an example, the polypeptide can be bound to an affinity matrix.

As a further example, the nucleic acids of the invention can be used to transform and / or transfect cells to recombinantly produce the polypeptides and / or antibodies of the invention. The nucleic acids of the invention can also be obtained, for example, when the subject is C.I. It can be used to determine if you are infected with difficile. As an example, this can be achieved using a method of radiolabel hybridization.

As a further example, the antibody of the present invention may be C.I. Can be used to recognize infection by difficile. As an example, an antibody may recognize natural toxin A and / or toxin B as an antigen. The antibodies of the present invention may also be C.I. Can be used to combat infection by difficile. By way of example, humanized antibodies or antibody fragments or monoclonal antibodies are C.I. The subject's own immune response to difficile infection may be used. As a further example, the antibodies of the invention may be conjugated to cytokines or toxins or enzymes or markers to aid in the treatment and detection of infections.

A further aspect of the invention relates to diagnostic assays. The present invention is used with many assays known in the art. One skilled in the art will recognize the broad research-based use of the polypeptides, nucleic acids and antibodies of the present invention. The polypeptides, antibodies and nucleic acids of the invention can be labeled with, for example, radioactive, chemiluminescent, fluorescent and / or stained molecules. The antibodies, nucleic acids and polypeptides of the invention can be used for DNA assays (eg, Southern blotting), RNA assays (eg, Northern blotting), protein assays (eg, Western blotting), chromatographic analysis (eg, gas, liquid, HPLC, size Exclusion), immunoassays (eg ELISA), and structural assays (eg crystallography and NM)
Useful in assays such as (R spectroscopy). The antibodies, polypeptides and nucleic acids of the invention can further be used as probes. Assays that amplify the signal from the probe are also known to those skilled in the art.
kit

The present invention is, by way of example and not limitation, C-TAB. G5 or C-TAB
. A nucleic acid encoding the G5.1 isolated polypeptide, C-TAB. G5 or C-TAB.
G5.1 isolated polypeptide and / or C-TAB. G5 or C-TAB. G5
. A kit comprising an antibody against one isolated polypeptide is provided. The kit may include one or more containers and instructions for use according to any of the methods of the invention described herein. C-TAB of the present invention. G5 or C-TAB. The G5.1 isolated polypeptide and / or antibody may be C.I. It can be used in a variety of assays, including immunoassays for detecting difficile. In one embodiment, C-TAB. G5 or C-TAB. The G5.1 isolated polypeptide serves to function as an antigen for the purpose of detecting antibodies in a biological sample. The container may be a unit dose, a bulk package (eg, a multiple dose package), or a secondary unit dose. The kit of the invention is in a suitable package. Also contemplated are packages for use in combination with certain devices, such as inhalers, nasal administration devices, or infusion devices. The kit may have a sterile access port. The container may also have a sterile access port. The kit may optionally provide additional components such as buffers and interpretation information.

The kit is C.I. To detect the presence of difficile or C.I. It can be used to detect diseases associated with difficile, such as CDAD. The kit is C.I. It can be used to prevent or treat diseases associated with difficile. The kit of the present invention is a C.I. It can be used to reduce the symptoms of diseases associated with difficile.

Without further explanation, one of ordinary skill in the art will be able to make and utilize the claimed invention using the above description and the following illustrative examples. Accordingly, the following examples specifically point out preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure in any way. All articles, publications, patents and literature referenced throughout this application are hereby incorporated by reference in their entirety.

Example 1: C-TAB. G5 and C-TAB. Preparation of G5.1 isolated polypeptide.
This example shows C.I. for expression in E. coli cells. The preparation of an isolated polypeptide comprising a portion of difficile toxin A (CTA) and B (CTB) is described. The method described below is based on CTA
And can be used to make a variety of isolated polypeptides, including CTB. As an example, an isolated polypeptide comprising a portion of the CTA C-terminal domain and a portion of the CTB C-terminal domain is described.
Example 1.1: C-TAB. G5 and C-TAB. Cloning of G5.1 gene construct.

CTA gene encoding the amino acids 2026 to 2710 of the C-terminal domain (Acce
a portion of ssion number YP-001087137) using the following primers:
. Amplified by PCR from genomic DNA of difficile strain 630 (ATCC BAA-1382).
Forward direction: 5′-caccACTTAGtatgaacttagtactactgaggg-
3 ′ (SEQ ID NO: 9) and reverse direction: 5′-CTCGAGttagccatatatccccgggg-3 ”(SEQ ID NO: 10).
Amplification with the forward primer formed a SpeI site, and amplification with the reverse primer formed an Xhol site.

CTB gene encoding amino acids 1850 to 2366 of the C-terminal domain (Acce
ssion number: YP-00108735) using a part of PC
Amplified by R.
Forward direction: 5′-caccATGCATatgagtttattattagaaaaaca
g-3 ′ (SEQ ID NO: 11) and reverse direction: 5′-ggcCTCGAGctattactactatactactattgagc
−3 ′ (SEQ ID NO: 12).
Amplification with the forward primer formed an Nsil site, and amplification with the reverse primer formed an Xhol site.

The PCR reaction was performed using PCR Super-Mix (Invitrogen). The cycling conditions were 95 ° C. for 2 minutes, 95 ° C. for 45 seconds, 55 ° C. for 50 seconds, 68 ° C. for 8 minutes (30 cycles), and 72 ° C. for 10 minutes. The PCR product was purified with a rapid gene extraction kit (Invitrogen) and the PCR 2.1 TOPO vector (Inv
(Itrogen). Using the ligation mixture, E. coli M
ech-1 cells were transformed by heat shock. Transformants were used as Immedia Amp.
Seeded on Blue (Invitrogen) plates. White colonies were picked and cultured in 15 ml tubes with 4 ml LB medium containing 100 μg / ml ampicillin. The culture was incubated overnight at 37 ° C. and the plasmid was extracted with a rapid plasmid miniprep kit (Invitrogen).

PCR 2.1-CPO gene fragment in TOPO / TA vector was converted to SpeI and Xh
The fragment was cloned into an intermediate vector digested with SpeI and XhoI, also using T4 DNA ligase. The three restriction sites (Bg
A linker containing LII-NsiI-SacI) was inserted into the 3 ′ end of the CTA gene fragment by PCR using the following set of synthetic primers:
Forward direction: 5'-AGATCTATGCATGAGCTCctcgagccccaaaacg
aaaggctcagc-3 ′ (SEQ ID NO: 13)
Reverse direction: 5'-cggtccggggccatatatccccggggggttttac
tcc-3 ′ (SEQ ID NO: 14).

PCR 2.1-CTB gene fragment in TOPO / TB was converted to Nsil and Xhol.
The digested CTB gene fragment was ligated to an intermediate vector containing a CTA gene and a linker, which had been digested with NsiI and XhoI. The CTB gene is inserted into the linker at the 3 ′ position, and the construct sequence 5′-CTA-linker-CTB is inserted.
-3 'was formed. This fusion construct is C-TAB. Called the V1 intermediate vector.

C-TAB. The G5 gene was transferred to C-TAB. Amplified by PCR from the V1 intermediate vector.
Forward direction: 5'-caccCCATGatggtaacagaggagttattagaag
a (SEQ ID NO: 15)
Reverse direction: 5′-CTCGAGctattcactataatcactatattgagctg (
SEQ ID NO: 16).
PCR reaction was performed using PCR Super mix (Invitrogen). The cycle conditions were 95 ° C for 2 minutes, 95 ° C for 45 seconds, 55 ° C for 50 seconds, 68 ° C for 4 minutes (
30 cycles) and 72 minutes at 10 minutes. The PCR product was purified with a rapid gene extraction kit (Invitrogen) and the PCR2.1-TOPO vector (Invitrogen).
gen). Using the ligation mixture, E. coli Mech
-1 cells were transformed by heat shock. Transformants were treated with Immedia Amp Bl
Seeded on ue (Invitrogen) plates. White colonies are picked and 10
Cultured in 15 ml tubes with 4 ml LB medium containing 0 μg / ml ampicillin. The culture was incubated overnight at 37 ° C. and the plasmid was extracted with a rapid plasmid miniprep kit (Invitrogen). PCR 2.1-C-TAB. The G5 fusion gene was digested with NcoI and XhoI restriction enzymes.
These C-TAB fragments were ligated into a pET28 expression vector digested with the same restriction enzymes. This resulting construct encodes the C-terminal domain of toxin A from amino acids 2272 to 2710 fused to the C-terminal domain of toxin B from amino acids 1851 to 2366. pET28 / C-TAB. The G5 construct was transformed into E. coli BL21 (DE3) for expression. C-TAB. Five colonies containing the G5 fusion gene were selected for analysis.

C-TAB. The G5.1 coding sequence was obtained by codon optimization for improved expression in E. coli host cells. Codon usage was adapted based on the codon bias of the E. coli gene. In addition, the GC content is adjusted to extend the mRNA half-life and is very high (
> 80%) or very low (<30%) regions of GC content were avoided. Thus, the optimized gene allows a high and stable expression rate in E. coli. Codon optimized C-TAB. The G5.1 gene was synthesized in situ and the expression vector pET-28b
Subcloned into (+).

DNA sequencing: Plasmid DNA sequences were confirmed using dye terminator cycle sequencing chemistry with d-rhodamine staining. Sequencing data was analyzed using Jellyfish software.
Example 1.2: Recombinant C-TAB in E. coli. G5 or C-TAB. Expression of G5.1 fusion protein

C-TAB. G5 and C-TAB. Expression of the G5.1 gene construct can be performed using standard procedures for expression in E. coli.

Screening colonies for expression of recombinant C-TAB fusion protein: For screening, colonies were picked and 4 ml L containing 50 μg / ml kanamycin.
Grow in 15 ml Falcon tubes with B medium. The tube was incubated overnight at 37 ° C. with mixing at 250 rpm. After the initial growth phase, 1 ml of culture from each tube was transferred to a 24-well tissue culture plate and expression was induced with 1 mM isopropyl-β-D-1-thiogalacto-planoside (IPTG) at 30 ° C. for 3 hours. 1 at 12,000 g in a microcentrifuge
Cell pellets were collected by centrifugation for minutes. Cell pellet lysates were prepared and the soluble fraction was analyzed by SDS-PAGE and Western blot analysis for expression of C-TAB fusion protein. Positive clones were selected for further evaluation.

C-TAB. Batch fermentation for G5 expression: Seed cultures were grown in 500 ml shake flasks containing 150 ml Super Broth medium each supplemented with 30 μg / ml kanamycin. The culture is 275 rp until OD ₆₀₀ reaches 2-2.5
grown at 28 ° C. for 12 hours with continuous stirring at m. Using a shake flask, 10L
A fermentor containing the Super Broth was inoculated. The culture was grown at 37 ° C. for about 4.5 hours until OD ₆₀₀ = 3.5-4. 0.1 mM for induction of product expression
Of IPTG was added and growth was continued at 25 ° C. for an additional 4 hours. The cells were then collected by centrifugation and the cell paste was stored frozen at -70 ° C. The typical product specific expression rate achieved by this fermentation process was about 200 mg / ml.

C-TAB. Fed-batch fermentation for G5.1 preparation: 500 μl aliquot of seed bank glycerol stock (stored at −75 ° C.), using 30 μg / ml in 1 L shake flask.
100 ml of preculture medium supplemented with ml kanamycin was inoculated. The preculture was OD ₆₀
Incubate at 37 ° C. for about 7 hours under constant agitation at about 150 rpm until ₀ = 1.0-2.0. 7 L batch fermentation medium was inoculated using 25 mL preculture in a standard industrial 15 L fermentor equipped with a process control system capable of performing fed-batch fermentation. A 7 L batch culture step was performed at 37 ° C. for 12 hours until glucose was depleted (O
D ₆₀₀ = 12-15). Next, a specific growth rate constant μ = 0.
25 / time 37 ° C. for 6 hours _(OD 600 = 40 to 50), was started glucose feed stage (biomass production). One hour prior to switching to a constant feeding stage and induction with a final concentration of 1 mM IPTG (product production), the temperature was reduced to 30 ° C. to reduce the risk of inclusion body formation. The product development phase with constant feeding at 30 ° C. is continued for another 5 hours (OD _60
0 = about 100), a total fermentation process time of 23 hours and a final culture volume of about 8.2 L was obtained. About 1.2 kg of wet cell biomass was recovered by centrifugation and stored at -70 ° C or lower. Typical product-specific expression rates achieved by such fed-batch fermentations are up to 1
. It was 3 g / L.
Example 1.3: Recombinant C-TAB. G5 or C-TAB. Purification of G5.1 fusion protein.

C-TAB. Purification of G5 analysis sample: Thaw frozen cell paste to 10m at pH 5.6
Resuspended in M citrate / NaOH buffer and the cell slurry was passed twice through a homogenizer (GEA Niro Soavi homogenizer) at 550 bar. Suspension twice, 1 minute at 13500 rpm for 30 minutes, and 2nd time in ultracentrifuge at 18000 rpm
And centrifuged for 1 hour. The supernatants were pooled and the pH was adjusted to 5.6 with 50 mM citrate buffer (pH 3). The clarified cell lysate was added to 10 mM citric acid / NaOH at pH 5.6.
Passed through a SP rapid flow column with buffer. Protein is 0 in 20 mM NaPi.
Elute with a linear gradient of sodium chloride increasing from mM to 500 mM. C-TAB. G5
Fractions containing were pooled. The conductivity was adjusted down to 5 mS / cm with distilled H ₂ O. Tris was added to a final concentration of 25 mM. The pooled fractions were passed through a DEAE rapid flow column. The protein was eluted with a linear gradient of sodium chloride increasing from 50 mM to 500 mM in 25 mM Tris. Again, C-TAB. Pool fractions containing G5,
1.5M Na citrate (pH 7.5) was added to a final concentration of 0.4M. C-TAB.
The V1 pool was loaded onto a phenol sepharose HP column equilibrated with 25 mM Tris, 0.4 M Na citrate (pH 7.5). C-TAB. G5 fusion protein
Elute with decreasing salt concentration in a linear gradient using mM Tris, pH 7.5. All columns were monitored by an AKTA Prime chromatography system. Purified C
-TAB fusion protein was buffer exchanged into PBS using a 50K membrane.

C-TAB. Purification of G5.1 bulk preparation: Biomass was stored at −80 ° C. until processing. 450 g of frozen cell paste (corresponding to 2.90 L fermentor) was added to 4 volumes of lysis buffer (
20 mM Hepes, pH 7.5, about 0.6 mS / cm) (eg 450 g paste + 1800 mL buffer) and thus thawed under mechanical agitation for about 1 hour ± 0.5 hours. The optional remaining mass is removed using Ultraturrax (eg 8000
(5 minutes at rpm) can be resuspended. Cell lysis is performed by Niro Soavi Pan
Perform in a da high homogenizer (640 ± 25 bar, 3 cycles). The lysate is cooled to below 10 ° C. using a heat exchanger and maintained at this temperature until centrifugation. 14 crude cell lysate
Batch centrifugation step (Beckm) for 30 minutes at 4 ° C. at 000 rpm (30000 g)
anna Avanti JLA 60.25). The supernatant is collected and pooled.
In order to reduce the risk of clogging the filtration step, the semi-liquid part of the pellet is also discarded. The pooled supernatant is then filtered through Supercap PDH4 100/5 inch deep filter capsules (Pall) (250 cm ² effective filtration area). The remaining lysate in the filter housing is washed away with lysis buffer. After purification, 1M Tris stock solution (pH
7.5) a constant amount of 25 mM. Add to lysate to final concentration of. The buffer composition of the final lysate is 20 mM Hepes, 25 mM Tris (pH 7.5, conductivity about 6 mS / cm)
It is. The lysate may still be slightly turbid after filtration, but this does not affect subsequent capture steps. The capture step is performed at room temperature on a DEAE Sepharose FF (GE Healthcare) in an XK50 / 30 column (GE Healthcare) having a diameter of 50 mm, packed bed height 20 cm and packed bed volume of about 400 mL. The input density is about 0.8 to 1.2 g biomass / mL gel. The process is performed by the Akta Explorer system (GE Healthcare)
Monitor at 280 nm. Equilibration is about 5 CV of 25 mM Tris at 100 cm / hr.
Use 0 mM Hepes, 25 mM NaCl (pH 7.5, conductivity about 5 mS / cm) until pH, conductivity and 280 nm absorbance are stable. The lysate is loaded onto the column at 75 cm / hr and the passing through is discarded. 280n when all filtered lysate is charged
Flow is resumed with about 5 CV equilibration buffer until m absorbance is stabilized. 5CV 25m
Impurities are removed from the column during wash step 2 with M Tris, 175 mM NaCl (pH 7.5, conductivity 19 mS / cm). 3 CV of 25 mM Tris, 375 mM Na
C-TAB protein is eluted from the column by stepwise elution with Cl (pH 7.5, conductivity 36 mS / cm). When the absorbance at 280 nm starts to increase (usually after 1 CV), CT
Begin collection of the AB-containing fraction and continue for about 0.5 CV to 1.0 CV. Pooled fractions containing C-TAB can be stored overnight at 2-8 ° C. The intermediate purification step consists of XK5 having dimensions of 50 mm diameter, packed bed height 20 cm, packed bed volume about 400 mL.
In the 0/30 column (GE Healthcare), SP-Sepharose
Perform at room temperature with FF (GE Healthcare). Maximum input density is about 4-5mg
C-TAB / mL gel. The process is based on the Akta Explorer system (G
E Healthcare) and monitoring at 280 nm. Unless the equilibration, washing and linear gradient elution steps are hindered by excessive back pressure (> 4 bar)
Perform at a maximum flow rate of m / hour (65 mL / min). Equilibrate with about 5-10 CV buffer G at 200 cm / hr until pH, conductivity and 280 nm absorbance are stable. Prior to loading, the DEAE pool needs to be adjusted to allow binding of C-TAB onto the SP-FF resin. S until the final conductivity is 3.5 mS / cm (pH 5.5 ± 0.1) or less.
The DEAE pool is diluted 25-fold with P-FF equilibration buffer (10 mM citrate, 2 mM EDTA (pH 5.5 ± 0.1, conductivity about 2 mS / cm)). If necessary, add additional MilliQ water to achieve the desired conductivity. Note that low conductivity is extremely important to allow C-TAB binding on SP-FF. 1 sample
Put it into the column at 50 cm / hour and discard the passing material. After sample loading, flow is resumed at 200 cm / hr with about 5 CV equilibration buffer until the 280 nm absorbance is stabilized. 0%
From equilibration buffer, 30% over 10 CV 20 mM sodium phosphate, 500 mM
Elution is performed with a linear gradient of 100 cm / hr to NaCl (pH 7.0). Fractions are collected and pooled by UV 280 nm absorbance. The pool starts at 15% of the peak maximum and ends at 15% of the peak maximum. The pool is immediately adjusted to 400 mM citric acid (final pH 7, about 49 mS / cm) using a 1.5 M citric acid stock solution (pH 8.0). The regulated SPFF pool should have a pH of 7 and about 49 mS / cm;
Store overnight at 2-8 ° C.

The polishing chromatography step was performed using an XK50 / 30 column (GE Healthcar) with dimensions of 50 mm diameter, packed bed height of 15 cm and packed bed volume of about 300 mL.
e), Phenyl-Sepharose HP (GE Healthcar
e)) at room temperature. The input density is about 4-5 mg C-TAB / mL gel. The process is performed with an Akta Explorer system (GE Healthcare) and monitored at 280 nm. Equilibration, dosing, washing and elution steps are subject to excessive back pressure (>
4 bar) unless otherwise hindered by a maximum flow rate of 100 cm / hr (33 mL / min). In such a case, it is necessary to reduce the flow rate. Equilibration is about 5-10 CV of 25 mM Tris, 400 mM sodium citrate (pH 7.5, 46 mS) at 100 cm / hr.
/ Cm) until pH, conductivity and 280 nm absorbance are stable. 1 sample
Put into the column at 00 cm / hour and discard the flow-through. After sample loading, flow is resumed at about 100 cm / hr with about 5 CV equilibration buffer until the 280 nm absorbance is stabilized. 1
From 00% equilibration buffer / 0% 5 mM Tris (pH 7.5, 0.5 mS / cm) to 20 C
Up to 100% 5 mM Tris (pH 7.5, 0.5 mS / cm) exceeding V, 100 cm
Elution is performed with a linear gradient per hour. Fractions are collected and pooled by UV 280 nm absorbance. The pool starts at about 10-15% of the peak maximum and ends at about 20% of the peak maximum. The conditioned pool is stored overnight at 2-8 ° C. The final C-TAB drug substance protein solution was prepared by 30 kDa cutoff tangential flow filtration (TFF, Pell) operated at room temperature.
icon 2 membrane, Millipore). Protein solution is permeate p
Formulation buffer (20 mM histidine, 75 mM N until H equals 6.5 ± 0.2.
aCl, 5% sucrose, 0.025% Tween® 80 (pH 6.5))
The membrane is separated.

Following the UV measurement at 280 nm, the final protein concentration is adjusted to 2 mg / mL using 1.566 as the specific extinction coefficient at 280 nm for C-TAB (protein concentration 1 mg / mL, 1 cm cuvette).

SDS-PAGE and Western blot analysis: whole cell lysate and purified C-TAB
. G5 or C-TAB. The G5.1 fusion protein was resuspended in Nu-Page sample buffer containing beta-mercaptoethanol and boiled for 10 minutes. Sample (25 μl)
Was loaded onto a 3-8% Tris-acetate gel. After electrophoresis (150 V for 1 hour), proteins were visualized by simply staining the gel with blue staining or used for Western blot analysis.

C-TAB. G5 or C-TAB. G5.1 specific expression was determined by Western blot analysis using toxin specific antibodies. The protein was transferred to a PVDF membrane at 23 V for 60 minutes using 1 × transfer buffer in 10% methanol. The membrane was blocked with 0.5% casein in phosphate buffered saline (PBS) for 1 hour at room temperature. Transfer membranes were incubated for 2 hours at room temperature with monoclonal antibody against toxin B (GenWay; clone B426M) or with guinea pig polyclonal antibody against proprietary toxin A (List Biological Labs). Washed membranes were incubated with horseradish peroxidase conjugated anti-guinea pig IgG or anti-mouse IgG. The blot was washed and AEC substrate was added. The blot was incubated for 5-10 minutes with gentle mixing. The blot was rinsed with water to stop color development.

RBC hemagglutination: The cell binding domain of toxin A but not toxin B is
) Can be agglomerated. The agglutination process is such that toxin A is rabbit RB
Results of binding to glycan sequences found in blood antibodies to C. Sample (C-T
AB. G5 and native toxin A) are diluted to 100 μg / ml in PBS. In a V-bottom microtiter plate, prepare a 2-fold serial dilution twice across the plate,
Starting with / ml, leave 50 μl of dilution in each well. 50 microliters 0.7
A 5% rabbit RBC / PBS suspension is added to each well of the microtiter plate and the plate is incubated for 1 hour at room temperature. Failure of RBC pellet formation at the bottom of the plate indicates hemagglutination. The hemagglutination titer of the sample is expressed by the concentration of protein present in the well with the highest sample dilution at which no RBC pellet is observed.
Example 2: Recombinant C-TAB in the presence and absence of alum in mice. G
Dose titer measurement of 5 fusion proteins.

This study is a C-TAB efficacy assay with C-TAB with and without alum adjuvant. It was to determine the feasibility of in vivo dose titration of G5. The alum used was Alydragel (alum hydroxide, Brent)
ag). C57BL / 6 female mice (Charles R) between 8 and 9 weeks of age
ever Labs. ) Was used for immunization. All animals received a first immunization on day 0 by intramuscular (IM) injection (50 μl) into the right thigh muscle. The second immunization was performed on day 14 by IM injection into the left thigh muscle. A total of 72 mice were divided into 12 groups vaccinated as follows.
• Group 1: PBS only • Group 2: 100 (154) ng C-TAB. G5
Group 3: 300 (462) ng C-TAB. G5
Group 4: 1,000 (1,540) ng C-TAB. G5
Group 5: 3,000 (4,620) ng C-TAB. G5
Group 6: 10,000 (15,400) ng C-TAB. G5
Group 7: PBS containing 50 μg alum
Group 8: 10.0 (15.4) ng C-TAB. With 50 μg alum OH. G5
Group 9: 30.0 (46.2) ng C-TAB with 50 μg alum OH. G5
Group 10: 100 (154) ng C-TAB with 50 μg alum OH. G5
Group 11: 300 (462) ng C-TAB. With 50 μg alum OH. G5
Group 12: 1,000 (1,540) ng C-TAB with 50 μg alum OH
. G5

In this study, first, the standard protocol Quick Start (trademark) Bradford
Protein concentration was measured according to Protein Assay (Bio-Rad). Later, following the procedure described in Example 1.3, the protein concentration (shown in parentheses) was again measured by UV measurement at 280 nm. In follow-up studies, protein concentration was measured by the UV method.

Blood samples were collected from all animals 2 weeks after the first immunization (study day 14) and 2 weeks after the second immunization (study day 28). Serum was stored at −20 ° C. until analysis.

Serum IgG ELISA: C-TAB. G5 or C-TAB. G5.1 (C-TA
Serum antibodies raised against Toxin A and Toxin B or its toxoid were evaluated in an enzyme-linked immunosorbent assay (ELISA). Briefly, 1.0 μg / ml
Toxin A, toxin B or C-TAB. A stock solution of G5 isolated polypeptide was prepared in PBS and 100 μl was added to each well of a 96 well plate. After overnight incubation at 4 ° C., the plates were washed and blocked with 0.5% casein blocking buffer. The plate was washed again and a serial 2-fold dilution of test serum was added to the plate. After a second overnight incubation at 4 ° C., the plates were washed and peroxidase conjugated anti-mouse IgG (H + L)
Incubated with. After 2 hours incubation at room temperature, the plates were washed again and peroxidase substrate (2,2′-azinobis (3-ethylbenzthiazoline-6-
Sulfonate) was added and color was allowed to develop for 2 hours at room temperature. The reaction was stopped by adding 50 μl of 2% SDS to the wells. The plate was read on an ELISA plate reader with absorbance at 405 nm. Serum antibody titers are reported as the geometric mean of ELISA units, which is the serum dilution that yields an OD 405 nm readout of 1.0. As a negative control, a pooled sample of preimmunized sera obtained from animals prebleeded prior to the first immunization was used to evaluate antibody responses.

C-TAB. Animals given G5 show a dose-dependent increase in antibody titer and alum
Adjuvants have lower C-TAB. G5 doses allowed for significantly improved antibody titers. FIG. 3 shows the titers of anti-C-TAB, antitoxin A and antitoxin B IgG. FIG.
A graphical comparison of antibody titers in the presence or absence of alum is shown.
Example 3: C-TAB in mice. Immunogenicity and protective effect of G5

This test is a C.I. C-TAB. In vaccinated mice subjected to lethal challenge with difficile toxin A or toxin B. It was for evaluating the immunogenicity and protective effect of G5. 6-7 week old female C57BL / 6 mice (Charles River Labs.)
Was used in this study. All animals received an intramuscular (IM) injection (50 μm) into the right thigh muscle on day 0.
Received the first vaccination according to l). A second vaccination was performed on day 14 by IM injection into the left thigh muscle. 116 mice were divided into vaccinated groups as follows.
• Group 1: PBS only • Group 2: 3 μg C-TAB. G5
Group 3: 10 μg C-TAB. G5
Group 4: 30 μg C-TAB. G5
Group 5: 3 μg C-TAB. G5 + 50μg alum OH
Group 6: 10 μg C-TAB. G5 + 50μg alum OH
Group 7: 30 μg C-TAB. G5 + 50μg alum OH
• Group 8: PBS only • Group 9: 3 μg C-TAB. G5
Group 10: 10 μg C-TAB. G5
Group 11: 30 μg C-TAB. G5
Group 12: 3 μg C-TAB. G5 + 50μg alum OH
Group 13: 10 μg C-TAB. G5 + 50μg alum OH
Group 14: 30 μg C-TAB. G5 + 50μg alum OH

Two weeks after the second immunization (study day 28), blood samples were collected from all animals.
Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were then measured by ELISA and reported as ELISA units (EU).

FIG. 5 shows C in mice evaluated 2 weeks after the second immunization (study day 28).
Serum antibody titers against TAB, toxin A and toxin B are shown. This test was conducted using C-TAB. G
It was shown that the 5 fusion protein is highly immunogenic in mice and can induce strong antibody responses against both toxin A and toxin B without the addition of adjuvant. CT
AB. G5 immunogenicity can be greatly enhanced (over 1 log) by combined delivery with hydroxylated alum. C-TAB with or without alum. Animals given G5 showed a 2-fold increase in antibody response over a semi-log dose range.

In addition to antibody titer assessment, C-TAB. Antibodies generated by immunization with G5 are i.
The ability to neutralize native toxins A and B was evaluated in an n vitro toxin neutralization assay (TNA).

Toxin neutralizing antibody assay (TNA). For in vitro analysis, 125 μl of toxin A (5 ng / ml) or toxin B (1 ng / ml) was incubated with a 125 μl serial dilution of antisera obtained from immunized mice. After 1 hour incubation at 37 ° C., the toxin: serum mixture was added to microtiter wells containing Vero cells (monkey kidney cells) and the microtiter plates were incubated for 18 hours. Incubation of toxin A or B and Vero cells resulted in changes in cell morphology and loss of cell adhesion as measured by neutral red staining of toxin-treated cells after removal of non-adherent cells.
Serum toxin neutralization titers are reported as serum dilutions resulting in a 50% reduction in toxin activity.

The results of the TNA assay are shown in FIG. The data is C-TAB. It shows that antibodies produced after immunization with G5 alone can neutralize the toxic activity of natural toxin A, but not toxin B. C-TAB. When G5 was delivered in combination with alum, the TNA titer was enhanced with an approximately 6-fold increase in anti-toxin A TNA and a slightly lower titer in anti-toxin B TNA. This data is based on C-TAB. It has been shown that G5 isolated polypeptides not only retain antibody-recognizing antigenic epitopes present in natural toxins, but also contain important antigenic epitopes required for the generation of functional toxin neutralizing antibodies. Thus, C-TAB. G5 is C.I. It is effective in neutralizing the toxic effects of difficile toxin A and toxin B and is therefore useful for vaccination.

In addition to assessing antibody responses, C-TAB. Protects mice from lethal loading with natural toxins. G
Five immunization capabilities were determined. Three weeks after the second vaccination (study day 35), animals in the vaccinated and non-vaccinated groups (N = 8) received 25 ng of toxin A or 50 n
A lethal dose of g toxin B was received by intraperitoneal (IP) administration. The survival rate of the mice was then monitored over 9 days and the results are shown in FIG. This experiment was performed using C-TAB. It has been demonstrated that immunization of mice with G5 can confer 100% protection against lethal loading with native toxin A and 50% protection against toxin B loading. C-TAB. Combined delivery of G5 with Alum provides a protective immunity against toxin B of 10
Improved to 0% protection. This data is based on C-TAB. It has been shown that G5 vaccination induces an immune response sufficient to protect mice from the toxic effects of both toxin A and toxin B in a lethal load model.
Example 4: C-TAB. In young and old mice. Evaluation of the immunogenicity and protective effect of G5.

This study was performed on C-TAB. This was to compare the immune response to G5. Female C57BL / 6 mice (Charles River Labs.), 6-7 weeks old and 18 months old, respectively, were used in this study. All animals received the first vaccination on day 0 by intramuscular (IM) injection (50 μl) into the right thigh muscle.
A second vaccination was performed on day 14 by IM injection into the left thigh muscle. 192 mice were divided into vaccinated groups as follows.
-Group 1: PBS to young mice
-Group 2: PBS to old mice
-Group 3: 10 [mu] g C-TAB. G5
-Group 4: 30 [mu] g C-TAB to young mice. G5
-Group 5: 10 [mu] g C-TAB. G5
-Group 6: 30 [mu] g C-TAB. G5
Group 7: 10 μg C-TAB to young mice. G5 + 50μg alum OH
-Group 8: 30 μg C-TAB to young mice. G5 + 50μg alum OH
-Group 9: 10 [mu] g C-TAB. G5 + 50μg alum OH
-Group 10: 30 μg C-TAB to aged mice. G5 + 50μg alum OH
-Group 11: PBS to young mice
-Group 12: PBS to old mice
-Group 13: 10 [mu] g C-TAB. G5
-Group 14: 30 [mu] g C-TAB. G5
-Group 15: 10 [mu] g C-TAB to aged mice. G5
-Group 16: 30 μg C-TAB 5 ^th to old mice
Group 17: 10 μg C-TAB to young mice. G5 + 50μg alum OH
-Group 18: 30 μg C-TAB to young mice. G5 + 50 μg alum OH
-Group 19: 10 [mu] g C-TAB. G5 + 50μg alum OH
-Group 20: 30 [mu] g C-TAB. G5 + 50μg alum OH

Three weeks after the second vaccination (Study Day 35), animals in the vaccinated and non-vaccinated groups (N = 6) were intraperitoneally (IP) with 25 ng toxin A or 50 ng toxin B.
) It was lethal by injection. Mice survival was then monitored over 9 days.

Blood samples were collected from all animals 2 weeks after the first immunization (study day 14) and 2 weeks after the second immunization (study day 28). Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were then measured by ELISA and reported as ELISA units (EU). Toxin A and toxin B neutralizing antibody (TNA
) Was determined using Vero cells treated with cytotoxic amounts of recombinant toxin A and toxin B.

C-TAB. Juvenile animals given the G5 vaccine showed significantly higher levels of all tested antibodies compared to older animals. C-TAB. In the presence of alum hydroxide. Particularly high antibody titers were obtained in young mice vaccinated with G5 (FIG. 7). Toxin B
A particularly significant improvement in TNA titer was achieved. At the same time, there was no significant difference between young and old mice in their ability to withstand toxin A and toxin B loading. However, both groups showed improved protection when vaccinated in the presence of alum.
FIG. 7 shows C-TAB. In young versus old mice. A comparison of G5 immunogenicity and protective effect is shown. FIG. 8 shows the kinetics of increasing anti-C-TAB antibody in young and old mice.
Example 5: C-TAB. Comparison of immunogenicity and protective effect of G5.1 and toxoids A and B.

This test was conducted using C-TAB. It was to compare the immunogenicity and protective effect of G5.1 vs. toxoid A / B. The toxoid A / B used was equivalent (1: 1) toxoid A
(Lot # 100932) and Toxoid B (Lot # 1001333). Toxoid was prepared by formalin fixation and provided by TechLab. 6-7 week old female C57BL / 6 mice (Charles River Labs.)
Was used in this study. All animals received an intramuscular (IM) injection (50 μm) into the right thigh muscle on day 0.
Received the first vaccination according to l). A second vaccination was performed on day 14 by IM injection into the left thigh muscle. 180 mice were divided into vaccinated groups as follows.
-Group 1: PBS only-Group 2: 10 [mu] g C-TAB. G5.1
-Group 3: 30 [mu] g C-TAB. G5.1
-Group 4: 10 [mu] g C-TAB. G5.1 + 50 μg alum OH
-Group 5: 10 [mu] g C-TAB. G5.1 + 50 μg alum OH
Group 6: 30 μg toxoid A / B
Group 7: 10 μg toxoid A / B
Group 8: 30 μg toxoid A / B + 50 μg alum OH
Group 9: 30 μg toxoid A / B + 50 μg alum OH
-Group 10: PBS
-Group 11: 10 [mu] g C-TAB. G5.1
-Group 12: 30 [mu] g C-TAB. G5.1
-Group 13: 10 [mu] g C-TAB. G5.1 + 50 μg alum OH
-Group 14: 30 [mu] g C-TAB. G5.1 + 50 μg alum OH
Group 15: 10 μg toxoid A / B
Group 16: 30 μg toxoid A / B
Group 17: 10 μg toxoid A / B + 50 μg alum OH
Group 18: 30 μg toxoid A / B + 50 μg alum OH

Three weeks after the second vaccination (Study Day 35), animals in the vaccinated and non-vaccinated groups (N = 6) were intraperitoneally (IP
) It was lethal by injection. Mice survival was then monitored over 9 days.

Blood samples were collected from all animals 2 weeks after the first immunization (study day 14) and 2 weeks after the second immunization (study day 28). Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were then measured by ELISA and reported as ELISA units (EU). Toxin A and toxin B neutralizing antibody (TNA
) Was determined using Vero cells treated with cytotoxic amounts of recombinant toxin A and toxin B.

This study demonstrated C-TAB. In mice after two vaccinations. Demonstrate the immunogenicity and protective effect of G5.1 and toxoid A / B. C-TAB. Animals given G5.1 showed lower but significant anti-C-TAB antibody titers compared to animals given toxoid A / B. Also, the combined delivery of alum greatly enhanced all tested antibody responses. As a result, C-TAB. The levels of anti-C-TAB and anti-toxin A antibodies achieved in animals immunized with G5.1 or toxoid A / B are similar. The mouse was C-TAB. When immunized with G5.1, slightly lower antibody titers were observed for anti-toxin B antibody compared to mice immunized with toxoid A / B. Of note, C-TAB. At the C-terminal portion of the toxin molecule. Unlike the antibody generated against the G5.1 recognition epitope, the antibody induced by toxoid immunization is the N of the toxin molecule.
Specific for the terminal portion, which was read in an antitoxin ELISA. Therefore,
C-TAB. Antitoxin A and antitoxin B antibodies produced in mice immunized with G5.1 and toxoid A / B are antibodies of different specificities and therefore cannot be directly compared. However, the data is C-TAB. It shows that the antibody response to G5.1 immunization is remarkably high as in the case of immunization with toxoid. In addition, the toxin tolerance test is a C-TAB. It was demonstrated that the ability of G5.1 immunization is comparable to the protective effects of toxoids A and B. FIG. 9 shows C-TAB. G5.
A comparison of the immunogenicity of 1 and toxoid A / B is shown. FIG. 10 shows C-TAB.comparison with mice immunized with toxoid A / B. Shown are toxin neutralization and protection data for mice immunized with G5.1.
Example 5.1: C-TAB in different immunization regimes. Comparison of antibody titer and protective effect of G5.1.

This study demonstrated that C-TA in mice receiving three doses of vaccine with different immunization regimes.
B. This was to compare the immunogenicity and protective effect of G5.1. 6-7 week old female C57BL / 6 mice (Charles River Labs.) Were used for this study. 135 mice were divided into 14 groups. All animals in group Nos. 2-13 were given 3 by intramuscular (IM) injection (50 μl) into the right or left thigh muscle on the following days:
Vaccinated. Mice in groups 1 and 8 were not vaccinated and served as negative controls. Immunization was performed as follows.

Blood samples were collected from all animals on Trials 0, 3, 7, 14, 21, 28, 35 and 42 days. Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were measured by ELISA and reported as ELISA units (EU). On test day 42, toxin A and toxin B neutralizing antibody (TNA) were determined using Vero cells treated with cytotoxic amounts of recombinant toxin A and toxin B.

Three weeks after the last vaccination (study day 49), animals in the vaccinated and non-vaccinated groups (N = 8) were intraperitoneally injected with 28 ng toxin A or 50 ng toxin B (IP
) It was lethal by injection. Mice survival was then monitored over 9 days.

This study is a 0/14/28 immunization plan, although all antibody titers measured 2 weeks after the third vaccination or on days 35 and 42 of the trial are comparable in all immunization plans Demonstrates the best immune response. Comparing antibody titers measured 2 weeks after the second vaccination, the 0/14/28 immunization plan was better than the 0/7/21 immunization plan, and 0/3/14 Much better than the immunization plan. This study confirmed that the antitoxin A / B antibody titer was significantly improved when the antigen was injected in combination with aluminum hydroxide (data not shown). Studies have also shown that even when two vaccine doses with alum are performed at two week intervals, high antibody levels comparable to those obtained after the three vaccine doses can be elicited.

The level of toxin A / B neutralizing antibodies is much higher in the 0/7/21 and 0/14/28 immunization regimes than in the 0/3/14 immunization regimes.

Complete protection against toxin A loading does not require alum in the 0/7/21 and 0/14/28 immunization regimes, but does require in 0/3/14. alum
The 0/14/28 immunization regime with the highest level of protection against toxin B load (8
7.5%), while the 0/7/21 immunization regime provides 37.5% protection,
/ 3/14 indicates 28.6% protection. The results of this test are shown in FIG.
Example 6: Recombinant C-TAB in hamsters. Evaluation of immunogenicity and protective effect of G5.1 fusion protein.

This study is based on the recombinant fusion protein C-TAB. Administered in different animal models with or without adjuvant. It was for further evaluation of the immunogenicity of G5.1.

Female hamsters (Harlan) weighing 80-90 g over 7 weeks of age were used in this study. All animals received a first vaccination on day 0 by bolus (50 μl) intramuscular (IM) injection into the right thigh muscle. Second vaccination is IM on left thigh muscle on day 14
The third vaccination was performed by IM injection on day 28. Hamsters were divided into groups (N = 6) and vaccinated as follows.
• Group 1: formulation buffer only • Group 2: 10 μg C-TAB. G5.1
Group 3: 10 μg C-TAB. G5.1 + 100 μg alum OH
Group 4: 30 μg C-TAB. G5.1
Group 5: 30 μg C-TAB. G5.1 + 100 μg alum OH
Group 6: 100 μg C-TAB. G5.1
Group 7: 100 μg C-TAB. G5.1 + 100 μg alum OH
Group 10: formulation buffer only. Group 11.10 μg C-TAB. G5.1
Group 12.10 μg C-TAB. G5.1 + 100 μg alum OH
Group 13.30 μg C-TAB. G5.1
Group 14.30 μg C-TAB. G5.1 + 100 μg alum OH
Group 15. 100 μg C-TAB. G5.1
Group 16. 100 μg C-TAB. G5.1 + 100 μg alum OH

Two weeks after the third vaccination (study day 42), animals in the vaccinated and non-vaccinated groups (N = 6) were intraperitoneally injected with 75 ng toxin A or 125 ng toxin B (
IP) under fatal load by injection. On day 44, 12 additional hamsters were used for dose titration of toxin A or toxin B load. Hamster survival was monitored over the next 8 days.

Two weeks after the first immunization (study day 14), two weeks after the second immunization (study day 28), and two weeks after the third immunization (study day 35), Blood samples were collected from all animals. Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were then measured by ELISA and reported as ELISA units (EU). Toxin A and toxin B neutralizing antibodies (TNA) were determined using Vero cells treated with cytotoxic amounts of recombinant toxin A and toxin B.

In this test, hamsters were treated with C-TAB. It was demonstrated to respond positively to G5.1 vaccination. C-TAB. Animals given G5.1 show a dose-dependent increase in all tested antibody titers, whereas alum adjuvant is C-TA
B. The antibody titer was greatly improved at all doses of G5. Maximum antibody titers were observed 2 weeks after the second dose (Study Day 28). FIG. 11 (AC) shows antibody titers for each group of immunized hamsters. FIG. 12 shows C-TAB. In the presence or absence of hydroxyl alum. The kinetics of increasing anti-C-TAB antibody in hamsters immunized with G5 are shown.

The results of the TNA assay are shown in FIG. These results are similar to those obtained for mice, and C-TAB. Antibodies raised against the G5.1 fusion protein are C.I. It has been shown to be effective in neutralizing the toxic effects of difficile toxin A and toxin B.

FIG. 13 shows C-TAB. After lethal toxin loading. Protection data in hamsters immunized with G5.1 are shown. C-TAB in the absence of adjuvant. High protection was also achieved with vaccination with G5.1. By adding alum to the vaccine, the level of protection was improved to 100%.
Example 7: C.I in clindamycin-treated hamsters. C-TAB for difficile spore loading. Protective effect of G5.1 fusion protein.

After antibiotic treatment, C.I. Difficile can colonize the gastrointestinal tract and, if it produces toxins, can cause antibiotic-related diarrhea. Human C.I. Difficile-related disease (CDAD) is typically modeled in hamsters using clindamycin within days of inoculation with a toxin-producing strain to make animals susceptible to colonization, diarrhea and death. C-TAB. To assess G5.1 vaccine efficacy, vaccinated and non-vaccinated hamsters were treated with clindamycin and C.I. Difficile strain 630 was loaded.
100 [mu] g C-TAB. G5.1 was mixed with 125 μg hydroxyl alum adjuvant. Female adult hamsters weighing approximately 100 g are intramuscular (IM) on days 0, 14 and 28
Three vaccinations were received by injection. The placebo was PBS. Forty-eight hamsters were divided into groups of 8 to be vaccinated as follows.
Group 1: PBS only +10 ² spore load Group 2: C-TAB. G5.1 + 10 ² spore load Group 3: PBS only +10 ³ spore load Group 4: C-TAB. G5.1 + 10 ² spore load group 5: PBS only + 10 ⁴ spore load group 6: C-TAB. G5.1 + 10 ⁴ spore load

On day 42, all animals in all groups received an oral dose of 10 mg clindamycin phosphate / kg body weight. On day 43, all animals in all groups received C.I. Difficile strain 63
Zero washed spores were dosed by oral gavage. Three levels of spore loading were used (approximately 1
0 ² , 10 ³ and 10 ⁴ ). Observation was continued until day 54 without treatment. At the end of the study, all surviving animals had no disease for more than 5 days.

On days 0, 14, 28, 42 and 54 (study end), blood samples were taken to obtain sera for serological tests. On days 1 and 42, feces were collected either directly from the hamster's anus or from the bed as needed.

The results are shown in FIG. 14, which shows a survival curve after spore loading in hamsters. Survival data was plotted as a Kaplan-Meier survival fit curve and statistical analysis was performed using log rank analysis. One hamster in the vaccinated group at all spore doses
A 00% survival rate was observed, and the survival rate was significantly improved compared to the placebo group: p = 0.0245 for 10 ² spores, p = 0.006 for 10 ³ spores, p <0.00 for 10 ⁴ spores. 0001.
Example 8: C-TAB in monkeys. Immunogenicity and protective effect of G5.1.

This study was performed using C-TAB. To evaluate the immunogenicity and protection of G5.1. 6 of age between 4 and 6 years and weight between 2 and 4 kg
One female cynomolgus monkey was used for the study. Two groups of 3 monkeys were prepared and the first group (Group 1) was given 200 μg C-TAB. G5.1 and the second group (Group 2) was given 200 μg C-TAB. G5.1 and 250 μg alum were given. As an alum adjuvant, Rehydragel (Reheis, Lot # 534401, 2 mg in PBS
Diluted to / ml) was used. Animals were shaved (if necessary) prior to blood collection or immunization.

The first (study day 0) and third (study day 28) immunizations were given to the left arm (deltoid muscle), and the second immunization (study day 14) was given to the right arm (deltoid muscle). For group 1, 0.5 ml 1 × P
200 [mu] g C-TAB in BS. Only G5.1 was given by IM injection and group 2 was given a 0.
200 μg C-TAB in 5 ml 1 × PBS. G5.1 and 250 μg alum
Was given by IM injection.

At established time points (Trials 0, 14, 28 and 42 days)
3 mL whole blood was collected in a serum separator tube. Serum samples were frozen at approximately -20 ° C. Then EL
ISA method was used to assess anti-C-TAB, antitoxin A and antitoxin B IgG titers.
Antibody titers were expressed in ELISA units (EU).

FIG. 15 shows increasing doses of C-TAB. G5.1 has shown an increase in antibody production that recognizes all three proteins, while the presence of alum has greatly improved antibody levels. Maximum antibody titers were observed in the two vaccinations on day 42. These data include recombinant C-TAB. For vaccination for subjects in need thereof. G5 or C-TAB. The feasibility of using the G5.1 fusion protein is clearly shown.
Example 9: C-TAB. G5 and C-TAB. Comparison of immunogenicity of G5.1.

This test was conducted using C-TAB. G5 and C-TAB. The immunogenicity of G5.1 and CT
It was to compare the effect of two different buffers supplied with AB. 8 to 9
C57BL / 6 female mice (Charles River Labs.) Between the ages were used for immunization. All animals received a first immunization on day 0 by intramuscular (IM) injection (50 μl) into the right thigh muscle. The second immunization was performed on day 14 by IM injection into the left thigh muscle. A total of 72 mice were divided into 12 groups vaccinated as follows.
Group 1: C-TAB. In 1 μg PBS. G5
Group 2: C-TAB in 3 μg PBS. G5
Group 3: C-TAB in 10 μg PBS. G5
Group 4: C-TAB in 30 μg PBS. G5
Group 5: C-TAB. In 1 μg histidine buffer. G5
Group 6: C-TAB in 3 μg histidine buffer. G5
Group 7: C-TAB. In 10 μg histidine buffer. G5
Group 8: C-TAB. In 30 μg histidine buffer. G5
Group 9: C-TAB. In 1 μg histidine buffer. G5.1
Group 10: C-TAB. 3 in 3 μg histidine buffer. G5.1
Group 11: C-TAB. In 10 μg histidine buffer. G5.1
Group 12: C-TAB. In 30 μg histidine buffer. G5.1

Two weeks after the second immunization (study day 28), blood samples were collected from all animals.
Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, toxin A and toxin B were measured by ELISA and reported as ELISA units.

FIG. 16 shows that all antibody titers (anti-C-TAB, antitoxin A and antitoxin B) were not significantly different over the volume range of 1-30 μg in the three vaccine formulations (T-test analysis). More obvious). C-TAB. In histidine buffer compared to PBS. A slightly higher antibody production was achieved in the G5 formulation. C-TAB. G5 and C-TAB. No significant difference was observed between immunizations with the G5.1 histidine formulation. Therefore, this test was conducted using C-TAB. G5 and C-TAB. The equivalent immunogenicity of the G5.1 construct was demonstrated.
Example 10: Preparation and evaluation of alternative C-TABNCTB and C-TADCTB fusion proteins.

In this example, C.I. The preparation of two other fusion proteins comprising one part of the CTA C-terminal domain from C. difficile VPI-10463 and two parts of the C-terminal domain of CTB is described. C-TABNCTB fusion protein (SEQ ID NO: 18)
Is C-TAB. Similar to G5, 19 repeat units of CTA (amino acids 2272 to 2710)
, 23 repeat units of CTB (amino acids 1850-2366), and an additional 10 repeats of CTB fused to the C-terminus of CTB (amino acids 1834-2057). CT
The ADCTB fusion protein (SEQ ID NO: 20) is C-TAB. G5 sequence (19 repeats of CTA and 23 repeats of CTB), and C-TAB. Contains an additional 24 repeat units of CTB (amino acids 1834-2366) fused to the C-terminus of G5. Therefore, C-TADC
TB contains the double part of the repeating unit of CTB. C-TABNCTB and C-TADCT
Cloning of the B gene construct was performed in the same manner as described in Example 1.1. The recombinant fusion protein was expressed in E. coli cells and purified using standard procedures as described in Example 1.2. Isolated polypeptides were evaluated in immunogenicity and protection studies in animals.
Example 10.1 C-TAB. G5, C-TABNCTB and C-TA
Comparison of immunogenicity and protective effect of DCTB.

This study was performed in mice vaccinated with 5 antigen doses over a 2 log range.
C-TAB. It was to compare the immunogenicity and protective effect of G5, C-TABNCTB and C-TADCTB. 6-7 week old female C57BL / 6 mice (Charle
s River Labs. ) Was used in this study. All animals received 2 vaccinations, the first vaccination was intramuscular (IM) injection (50 μl) into the right thigh muscle on day 0
Made by. A second vaccination was performed on day 14 by IM injection into the left thigh muscle. All immunizations were performed in the absence of alum. 2 weeks after the second immunization (Study 28
On day 1, blood samples were collected. Serum was stored at −20 ° C. until analysis. Serum antibody titers against C-TAB, Toxin A and Toxin B were measured by ELISA and E shown in FIG.
Reported as LISA units (EU).

This study consists of alternative fusion proteins C-TADCTB and C-TABNCTB, and C-TAB. It has been demonstrated that G5 is highly immunogenic and can induce a strong antibody response against both toxin A and toxin B without the addition of an adjuvant.

In addition to assessing antibody response, C-TADC protects mice from lethal loading of natural toxin B
The ability of TB and C-TABNCTB immunization was determined. Three weeks after the second vaccination (Study Day 35), animals in the vaccinated and non-vaccinated groups (N = 6)
A lethal dose of 0 ng toxin B was received by intraperitoneal (IP) administration. The survival rate of the mice was then monitored over 9 days, and the results are shown in FIG. This experiment shows that immunization of mice with 33 μg C-TADCTB in the absence of alum can confer 100% protection against lethal burden with natural toxin B, while the same dose of C-TAB. . G5 and C-TABNCTB were demonstrated to induce only partial protection. This data
C-TAB. Similar to G5, two other fusion proteins C-TADCTB and C-TA
It has been shown that BNCTB can be protective against lethal loads from natural toxins.
Example 10.2: C-TAB in hamsters. Comparison of immunogenicity and protective effect of G5.1 and C-TADCTB.

This study was to further evaluate the immunogenicity of the alternative fusion protein C-TADCTB administered with or without alum adjuvant in different animal models.

The test was designed as described in Example 6: female hamster, 100 μg.
Vaccinated 3 times by IM injection in the presence or absence of alum hydroxide alum (Trials 0, 14 and 28). Two weeks after the third vaccination (Study Day 42), all animals were lethally challenged by intraperitoneal (IP) injection of 75 ng toxin A or 125 ng toxin B. Blood samples were collected on Trials 14, 28 and 35 and serum antibody titers against C-TAB, Toxin A and Toxin B were determined by ELISA. Toxin A and toxin B neutralizing antibody (TNA) were measured in day 35 serum. Hamster survival was monitored and reported as% of protection.

This study demonstrated that the fusion protein C-TADCTB can induce an anti-toxin antibody response in hamsters, similar to mice. The alum adjuvant significantly improved all tested antibody titers. The results of the TNA assay shown in FIG.
Antibodies raised against TB are C.I. It has been shown to be effective in neutralizing the toxic effects of difficile toxin A and toxin B. FIG. 19 also shows C-TAB. G5.1 or C
-Shows a comparison of protection data for hamsters immunized with TADCTB. High protection was achieved by vaccination with both recombinant fusion proteins.
Example 11: C-TAB. An open-label phase I study evaluating the safety, immunogenicity and dose response of pharmaceutical compositions containing G5.1

Three different doses: 20 μg with Al (OH) ₃ (alum), each with Al (OH
) 75 μg and 200 μg with or without ₃ , 0, 7 by intramuscular (IM) injection
And C-TAB. Administered in 3 vaccinations on day 21. A pharmaceutical composition comprising a recombinant fusion protein consisting of G5.1, cleaved Clostridium difficile (C. difficile) toxin A and toxin B.
Test purpose Main purpose:
-C-TAB. 6 months after the third vaccination. To investigate the safety and tolerability of pharmaceutical compositions containing G5.1.
Secondary purpose:
• 0, 7, 14 after the first vaccination to obtain the first indication of optimal dose and formulation
, 21, 28, 113, 201, vaccine antigen C-TAB. For three different doses and two formulations. G5.1 and C.I. To investigate the immune response measured against difficile natural toxins A and B.
・ C. C-TAB to neutralize difficile toxins A and B in vitro. G5.1
To investigate the ability of vaccine-derived IgG antibodies.
Exam design

好ましい態様：
好ましいポリペプチドおよびその使用：
１．配列番号２に記載のアミノ酸配列に対して少なくとも８５％、より好ましくは少な
くとも９０％、さらにより好ましくは少なくとも９５％、最も好ましくは９９％の配列同
一性を有するアミノ酸配列を含む単離ポリペプチド。

２．配列番号４に記載のアミノ酸配列に対して少なくとも８５％、より好ましくは少な
くとも９０％、さらにより好ましくは少なくとも９５％、最も好ましくは９９％の配列同
一性を有するアミノ酸配列を含む単離ポリペプチド。

３．クロストリジウム・ディフィシルの毒素ＡのＣ末端ドメインから得られる１９反復
単位、およびクロストリジウム・ディフィシルの毒素ＢのＣ末端ドメインから得られる２
３反復単位を含む、態様１または２に記載の単離ポリペプチド。

４．配列番号２に記載のアミノ酸配列を有する、態様１に記載の単離ポリペプチド。

５．配列番号４に記載のアミノ酸配列を有する、態様１に記載の単離ポリペプチド。

６．配列番号４に記載のアミノ酸配列に対して少なくとも８５％、より好ましくは少な
くとも９０％、さらにより好ましくは少なくとも９５％、最も好ましくは９９％の配列同
一性を有するアミノ酸配列を含むポリペプチド。

７．前記単離ポリペプチドをワクチン接種されたハムスターは、全ての芽胞用量（１０
^２、１０^３および１０^４）において、致死用量のＣ．ディフィシル芽胞の胃内投与に対し
生存する、態様６に記載のポリペプチド。

８．クロストリジウム・ディフィシルの毒素ＡのＣ末端ドメインから得られる１９反復
単位を含む、態様６または７に記載のポリペプチド。

９．クロストリジウム・ディフィシルの毒素ＢのＣ末端ドメインから得られる２３、３
３または４７反復単位を含む、態様６から８のいずれか１つに記載のポリペプチド。

１０．配列番号２、配列番号４、配列番号１８、配列番号２０および配列番号２、配列
番号４、配列番号１８または配列番号２０のいずれかと９５％、９６％、９７％、９８％
、９９％同一であるポリペプチドからなる群から選択される、態様６から９のいずれか１
つに記載のポリペプチド。

１１．単離されている、態様６から１０のいずれか１つに記載のポリペプチド。

１２．医薬品における使用のための、態様６から１１のいずれか１つに記載のポリペプ
チド。

１３．ＣＤＡＤの予防および治療のための、態様６から１１のいずれか１つに記載のポ
リペプチド。

１４．ＣＤＡＤのリスクを有する対象におけるＣＤＡＤの予防のための、態様６から１
１のいずれか１つに記載のポリペプチド。

１５．ＣＤＡＤのリスクを有する対象におけるＣＤＡＤの予防のための、態様６から１
１のいずれか１つに記載のポリペプチドであって、ＣＤＡＤのリスクを有する前記対象は
、ｉ）６５歳を超える対象、もしくは２歳未満の対象；ｉｉ）ＡＩＤＳを有する対象；ｉ
ｉｉ）免疫抑制薬を投与されている、もしくは投与される予定がある対象；ｉｖ）入院の
予定がある対象、もしくは入院している対象；ｖ）集中治療を受けている、もしくは受け
る予定がある対象；ｖｉ）消化管手術を受けている、もしくは受ける予定がある対象；ｖ
ｉｉ）養護施設等の長期介護を受けている、もしくは受ける予定がある対象；ｖｉｉｉ）
頻繁な、および／もしくは長期的な抗生物質の使用を必要とする共存症を有する対象；ま
たはｉｘ）再発性ＣＤＡＤを有する対象である、ポリペプチド。

１６．医薬品における使用のための医薬の製造のための、態様６から１１のいずれか１
つに記載のポリペプチドの使用。

１７．ＣＤＡＤの予防および治療のための医薬の製造のための、態様６から１１のいず
れか１つに記載のポリペプチドの使用。

１８．ＣＤＡＤのリスクを有する対象におけるＣＤＡＤの予防のための医薬の製造のた
めの、態様６から１１のいずれか１つに記載のポリペプチドの使用。

１９．ＣＤＡＤのリスクを有する対象におけるＣＤＡＤの予防のための医薬の製造のた
めの、態様６から１１のいずれか１つに記載のポリペプチドの使用であって、ＣＤＡＤの
リスクを有する前記対象は、ｉ）６５歳を超える対象、もしくは２歳未満の対象；ｉｉ）
ＡＩＤＳを有する対象；ｉｉｉ）免疫抑制薬を投与されている、もしくは投与される予定
がある対象；ｉｖ）入院の予定がある対象、もしくは入院している対象；ｖ）集中治療を
受けている、もしくは受ける予定がある対象；ｖｉ）消化管手術を受けている、もしくは
受ける予定がある対象；ｖｉｉ）養護施設等の長期介護を受けている、もしくは受ける予
定がある対象；ｖｉｉｉ）頻繁な、および／もしくは長期的な抗生物質の使用を必要とす
る共存症を有する対象；またはｉｘ）再発性ＣＤＡＤを有する対象である、使用。

２０．態様１から１１のいずれか１つに記載のポリペプチドを含む、対象におけるＣ．
ディフィシル感染を検出するための診断キット。

好ましい核酸：
１ａ．態様１から１１のいずれか１つに記載のポリペプチドのいずれかをコードするヌ
クレオチド配列を含む核酸。

２ａ．態様１から１１のいずれか１つのポリペプチドをコードするヌクレオチド配列か
ら本質的になる、態様１ａに記載の核酸。

３ａ．配列番号１、配列番号３、配列番号１７および配列番号１９の群から選択される
ヌクレオチド配列を含む、態様１ａまたは２ａに記載の核酸。

４ａ．配列番号１、配列番号３、配列番号１７および配列番号１９の群から選択される
ヌクレオチド配列から本質的になる、態様１ａまたは２ａに記載の核酸。

好ましい薬学的組成物：
１ｃ．態様１から１１のいずれか１つに記載のポリペプチドまたは態様１ａから４ａの
いずれか１つに記載の核酸、および薬学的に許容される担体または賦形剤を含む、薬学的
組成物。

２ｃ．Ｃ．ディフィシル毒素ＡおよびＢの両方を中和する抗体を惹起する、態様１ｃに
記載の薬学的組成物。

３ｃ．Ｃ．ディフィシル毒素ＡおよびＢに対する、対象における防御免疫反応を惹起す
る、態様１ｃまたは態様２ｃに記載の薬学的組成物。

４ｃ．アジュバントをさらに含む、態様１ｃから３ｃのいずれか１つに記載の薬学的組
成物。

５ｃ．アジュバントが、ａｌｕｍを含む、態様４ｃに記載の薬学的組成物。

６ｃ．追加的な抗原または薬物をさらに含む、態様１ｃから５ｃのいずれか１つに記載
の薬学的組成物。

好ましい抗体：１ｄ．態様１から１１のいずれか１つに記載のポリペプチドに対する抗体
であるが、Ｃ．ディフィシル毒素Ａ（配列番号６）およびＢ（配列番号８）のいずれかま
たは両方を認識しない抗体。

好ましい方法
１ｅ．態様１から１０のいずれか１つに記載のポリペプチドを生成するための方法であ
って、宿主細胞内に、ポリペプチドをコードする核酸を導入することと、ポリペプチドの
発現を可能とする条件下で宿主細胞を培養することと、ポリペプチドを単離することとを
含む方法。

２ｅ．宿主細胞は、大腸菌である、態様１ｅに記載の方法。

３ｅ．対象におけるＣ．ディフィシル関連疾患（ＣＤＡＤ）を治療および／または予防
する方法であって、それを必要とする対象に、態様１から１１のいずれか１つに記載の単
離ポリペプチドを投与することを含む方法。

４ｅ．対象においてＣ．ディフィシルの毒素ＡおよびＢの両方に対する特異的免疫反応
を誘導する方法であって、態様１から１１のいずれか１つに記載のポリペプチドを対象に
、または態様１ｃから６ｃのいずれか１つに記載の薬学的組成物を投与することを含む方
法。

５ｅ．対象におけるＣ．ディフィシル感染により引き起こされる原発性疾患を予防する
方法であって、態様１から１１のいずれか１つに記載のポリペプチドを対象に、または態
様１ｃから６ｃのいずれか１つに記載の薬学的組成物を投与することを含む方法。

６ｅ．Ｃ．ディフィシル関連疾患（ＣＤＡＤ）のリスクを有する対象における Ｃ．デ
ィフィシル感染により引き起こされる原発性疾患を予防する方法であって、ＣＤＡＤのリ
スクを有する前記対象は、ｉ）６５歳を超える対象、もしくは２歳未満の対象；ｉｉ）Ａ
ＩＤＳを有する対象；ｉｉｉ）免疫抑制薬を投与されている、もしくは投与される予定が
ある対象；ｉｖ）入院の予定がある対象、もしくは入院している対象；ｖ）集中治療を受
けている、もしくは受ける予定がある対象；ｖｉ）消化管手術を受けている、もしくは受
ける予定がある対象；ｖｉｉ）養護施設等の長期介護を受けている、もしくは受ける予定
がある対象；ｖｉｉｉ）頻繁な、および／もしくは長期的な抗生物質の使用を必要とする
共存症を有する対象；またはｉｘ）再発性ＣＤＡＤを有する対象であり、前記方法は、態
様１から１１のいずれか１つに記載のポリペプチドを前記対象に、または態様１ｃから６
ｃのいずれか１つの薬学的組成物を投与することを含む方法。 ７ｅ．ポリペプチドまた
は薬学的組成物は、対象に、筋肉内、皮内、皮下、経口、経鼻、または経直腸投与、好ま
しくは筋肉内投与される、態様１ｅから６ｅのいずれか１つに記載の方法。

８ｅ．ポリペプチドまたは薬学的組成物は、短い間隔（１週間毎または２週間毎）に少
なくとも２回以内の投薬で対象に投与される、態様１ｅから７ｅのいずれか１つに記載の
方法。

９ｅ．生体試料中のＣ．ディフィシルを検出する方法であって、生体試料を態様１から
１１のいずれか１つに記載のポリペプチドと接触させることと、生体試料に対するポリペ
プチドの結合を検出することとを含み、ポリペプチドの結合は、生体試料中のＣ．ディフ
ィシルの存在を示す方法。 This study is an open-label, partially randomized dose-escalation phase 1 study consisting of Part A in healthy adults 18 years old and younger than 65 years, and Part B in healthy elderly people 65 years of age and older. Groups are C.I. It is the most vulnerable group to be infected with difficile. Part A consists of 20 μg C-TAB. G5.1 vaccine, and 75 μg and 200 μg C-TAB. With or without adjuvant, respectively. To test the safety of G5.1 vaccine and the dose response to it, it will be performed on the 0, 7 and 21 day vaccination schedules in 5 treatment groups of 12 healthy adult subjects. Safety and immunogenicity were analyzed after all adult subjects in Part A received a third vaccination, and all safety data were collected before Data Safety Monoto before subject participation from Part B.
It is reviewed by ring Board (DSMB). If treatment groups that are not safe or ineffective (ie, doses that do not induce a significant IgG response) are identified during the interim analysis, these treatment groups will be discontinued and not brought into Part B .
Part B of the study explores dose confirmation in the elderly population. Therefore, Part B is performed in 5 treatment groups of 20 elderly healthy subjects per group. 0, 7 and 21
Apply day vaccination schedule. This study design allows for comparison of dose response in both adults and the elderly. The latter age group is C.I. In a prophylactic vaccination procedure that is a major target population for difficile vaccines and is age-based or age-risk based Two target indicators in the developmental path of difficile vaccine, namely recurrent C. cerevisiae. Prevention of difficile diarrhea and primary C.I. Indicates the most vulnerable population for prevention of difficile infection. However, older subjects may be less responsive to vaccination than younger adults and therefore require dose confirmation in the older target population from early developmental stages. By interim analysis after all adults from Part A have been vaccinated, subjects within the elderly group will be treated with potentially unsafe or ineffective doses (eg, the lowest dose) and / or formulations (eg, non- To reduce the risk of exposure to the adjuvant formulation) doses / formulations that are not safe or do not induce significant IgG responses in adults can be discontinued.
C-TAB. The G5.1 vaccine was generated by standard methods, 20 mM L-histidine, 75 mM NaCl, 5% sucrose, 0.025% Tween® 8
C-TAB in 0 (pH 6.5). It is an aqueous solution of G5.1.
Array:

Preferred embodiment:
Preferred polypeptides and uses thereof:
1. An isolated polypeptide comprising an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95%, most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

2. An isolated polypeptide comprising an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95%, most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

3. 19 repeat units obtained from the C-terminal domain of Clostridium difficile toxin A and 2 obtained from the C-terminal domain of Clostridium difficile toxin B
The isolated polypeptide of embodiment 1 or 2, comprising 3 repeat units.

4). 2. The isolated polypeptide according to aspect 1, having the amino acid sequence set forth in SEQ ID NO: 2.

5. The isolated polypeptide according to aspect 1, having the amino acid sequence set forth in SEQ ID NO: 4.

6). A polypeptide comprising an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95%, most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

7). Hamsters vaccinated with the isolated polypeptide will produce all spore doses (10
² , 10 ³ and 10 ⁴ ), lethal doses of C.I. The polypeptide of embodiment 6, wherein the polypeptide survives intragastric administration of difficile spores.

8). The polypeptide of embodiment 6 or 7, comprising 19 repeat units derived from the C-terminal domain of Clostridium difficile toxin A.

9. 23,3 derived from the C-terminal domain of Clostridium difficile toxin B
A polypeptide according to any one of aspects 6 to 8, comprising 3 or 47 repeat units.

10. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 18 or SEQ ID NO: 20 and 95%, 96%, 97%, 98%
Any one of aspects 6 to 9, selected from the group consisting of polypeptides that are 99% identical
The polypeptide according to 1.

11. 11. A polypeptide according to any one of aspects 6 to 10, which is isolated.

12 12. A polypeptide according to any one of aspects 6 to 11 for use in medicine.

13. 12. A polypeptide according to any one of aspects 6 to 11 for the prevention and treatment of CDAD.

14 Embodiments 6 to 1 for prevention of CDAD in a subject at risk for CDAD
The polypeptide according to any one of 1.

15. Embodiments 6 to 1 for prevention of CDAD in a subject at risk for CDAD
The polypeptide of any one of 1 wherein the subject at risk for CDAD is i) a subject over 65 years old, or a subject under 2 years old; ii) a subject with AIDS; i
ii) subjects who are or will be receiving immunosuppressants; iv) subjects who are scheduled to be hospitalized or who are hospitalized; v) are undergoing or will receive intensive care Subjects; vi) subjects undergoing or scheduled to undergo gastrointestinal surgery; v
ii) Subjects receiving or willing to receive long-term care such as nursing homes; viii)
A polypeptide having a comorbidity that requires frequent and / or long-term antibiotic use; or ix) a subject with recurrent CDAD.

16. Any one of embodiments 6 to 11 for the manufacture of a medicament for use in medicine.
Use of the polypeptide according to 1.

17. Use of a polypeptide according to any one of aspects 6 to 11 for the manufacture of a medicament for the prevention and treatment of CDAD.

18. Use of a polypeptide according to any one of aspects 6 to 11 for the manufacture of a medicament for the prevention of CDAD in a subject at risk for CDAD.

19. Use of a polypeptide according to any one of aspects 6 to 11 for the manufacture of a medicament for the prevention of CDAD in a subject at risk of CDAD, wherein said subject at risk of CDAD comprises i ) Subjects over 65 years old or subjects under 2 years old; ii)
Subjects with AIDS; iii) subjects who are or will be receiving immunosuppressive drugs; iv) subjects who are scheduled to be hospitalized or who are hospitalized; v) are undergoing intensive care, Or subjects who are scheduled to receive; vi) subjects who are undergoing or will undergo gastrointestinal surgery; vii) subjects who are receiving or willing to receive long-term care such as nursing homes; viii) frequent and / Or subjects with comorbidities that require long-term antibiotic use; or ix) uses that are subjects with recurrent CDAD.

20. C. in a subject comprising a polypeptide according to any one of aspects 1-11.
Diagnostic kit for detecting difficile infection.

Preferred nucleic acids:
1a. A nucleic acid comprising a nucleotide sequence encoding any of the polypeptides according to any one of aspects 1 to 11.

2a. The nucleic acid according to aspect 1a, consisting essentially of a nucleotide sequence encoding a polypeptide of any one of aspects 1-11.

3a. The nucleic acid according to embodiment 1a or 2a, comprising a nucleotide sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 and SEQ ID NO: 19.

4a. The nucleic acid according to embodiment 1a or 2a, consisting essentially of a nucleotide sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 and SEQ ID NO: 19.

Preferred pharmaceutical compositions:
1c. A pharmaceutical composition comprising a polypeptide according to any one of aspects 1 to 11 or a nucleic acid according to any one of aspects 1a to 4a and a pharmaceutically acceptable carrier or excipient.

2c. C. The pharmaceutical composition according to embodiment 1c, which raises antibodies that neutralize both difficile toxins A and B.

3c. C. The pharmaceutical composition according to aspect 1c or aspect 2c, which elicits a protective immune response in a subject against difficile toxins A and B.

4c. The pharmaceutical composition according to any one of aspects 1c to 3c, further comprising an adjuvant.

5c. The pharmaceutical composition according to aspect 4c, wherein the adjuvant comprises alum.

6c. The pharmaceutical composition according to any one of aspects 1c to 5c, further comprising an additional antigen or drug.

Preferred antibodies: 1d. An antibody against the polypeptide according to any one of aspects 1 to 11, wherein C.I. An antibody that does not recognize either or both of difficile toxin A (SEQ ID NO: 6) and B (SEQ ID NO: 8).

Preferred method 1e. A method for producing the polypeptide according to any one of aspects 1 to 10, comprising introducing a nucleic acid encoding the polypeptide into a host cell and allowing the expression of the polypeptide Culturing the host cell under and isolating the polypeptide.

2e. The method of embodiment 1e, wherein the host cell is E. coli.

3e. C. in the subject. A method of treating and / or preventing difficile-related disease (CDAD), comprising administering an isolated polypeptide according to any one of aspects 1 to 11 to a subject in need thereof.

4e. In subjects, C.I. A method of inducing a specific immune response against both difficile toxins A and B, wherein the method is directed to a polypeptide according to any one of aspects 1 to 11 or to any one of aspects 1c to 6c. Administering a pharmaceutical composition as described.

5e. C. in the subject. A method for preventing a primary disease caused by a difficile infection, which is directed to the polypeptide according to any one of aspects 1 to 11 or the pharmaceutical composition according to any one of aspects 1c to 6c. Administering a product.

6e. C. C. in subjects at risk for difficile-related disease (CDAD). A method of preventing a primary disease caused by a difficile infection, wherein said subject at risk for CDAD is i) a subject older than 65 years or a subject younger than 2 years; ii) A
Subjects with IDS; iii) subjects who are or will be receiving immunosuppressants; iv) subjects who are scheduled to be hospitalized or who are hospitalized; v) who are undergoing intensive care, Or subjects who are scheduled to receive; vi) subjects who are undergoing or will undergo gastrointestinal surgery; vii) subjects who are receiving or will receive long-term care such as nursing homes; viii) frequent and Or / or a subject with a comorbidity requiring long-term antibiotic use; or ix) a subject with recurrent CDAD, wherein the method comprises the polypeptide according to any one of aspects 1-11. To the subject, or aspects 1c to 6
administering a pharmaceutical composition of any one of c. 7e. The polypeptide or pharmaceutical composition is administered to a subject intramuscularly, intradermally, subcutaneously, orally, nasally, or rectally, preferably intramuscularly, according to any one of aspects 1e to 6e. Method.

8e. The method according to any one of aspects 1e to 7e, wherein the polypeptide or pharmaceutical composition is administered to the subject in a short interval (every week or every two weeks) in a dose of at least two times.

9e. C. in a biological sample A method for detecting difficile comprising contacting a biological sample with a polypeptide according to any one of aspects 1 to 11 and detecting binding of the polypeptide to the biological sample, Binding is determined by C.I. A method of indicating the presence of difficile.

Claims (10)

An isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, or
A polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 , said polypeptide capable of inducing an immune response against Clostridium difficile toxins A and B Method to generate.   Introducing a nucleic acid encoding said polypeptide into a host cell, culturing said host cell under conditions permitting expression of said polypeptide, and isolating said polypeptide. Item 1. The method according to Item 1. 3. The method of claim 2 , wherein the nucleic acid encoding the polypeptide has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. 4. The method according to claim 2 or 3 , wherein the nucleic acid encoding the polypeptide is inserted into an expression vector.   5. The method of claim 4, wherein the expression vector is transformed into a suitable host cell.   6. The method of claim 5, wherein the host cell is a prokaryotic, eukaryotic or insect host cell.   7. The method of claim 6, wherein the host cell is E. coli.   5. The method of claim 4, wherein the expression vector further comprises a nucleic acid encoding a selection factor such as an antibiotic resistance gene. Level of expression of the polypeptide in a host cell, 50 mg / l (culture) higher than A process according to any one of claims 1 to 8. A method for producing an immunogenic or vaccine composition for inducing an immune response against Clostridium difficile toxins A and B, comprising:
An isolated polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, or
A polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and capable of inducing an immune response against Clostridium difficile toxins A and B. Said method.

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