Passive immunization against clostridium difficile disease

Details Passive immunization against clostridium difficile disease Passive immunization against clostridium difficile disease

2001-03-22

2004-01-20

Minnifield, Nita (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S326000, C435S340000, C424S130100, C424S150100, C424S164100, C424S167100, C424S234100, C424S236100, C530S389100, C530S389500

Reexamination Certificate

active

06680168

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to methods and compositions for preventing and treating
Clostridium difficile
disease.
Clostridium difficile
, a toxin-producing gram positive bacterium, invades the intestinal tracts of patients whose normal intestinal flora is suppressed due to treatment with broad spectrum antibiotics. The bacterial toxins cause varying degrees of damage to the large intestinal (i.e., colonic) epithelium, and cause a spectrum of illnesses, ranging from mild diarrhea to severe colitis. Because antibiotic treatment induces the onset of
C. difficile
disease, the associated syndromes are named antibiotic-associated diarrhea and colitis (LaMont, Bacterial Infections of the Colon, Textbook of Gastroenterology, second edition, 1897-1903, 1995).
Three clinical syndromes caused by
C. difficile
are recognized, based on severity of the infection. The most severe form is pseudomembranous colitis (PMC), which is characterized by profuse diarrhea, abdominal pain, systemic signs of illness, and a distinctive endoscopic appearance of the colon. The case-fatality rate of PMC may be as high as 10%. Antibiotic-associated colitis (AAC) is also characterized by profuse diarrhea, abdominal pain and tenderness, systemic signs (e.g., fever), and leukocytosis. Intestinal injury in AAC is less than in PMC, the characteristic endoscopic appearance of the colon in PMC is absent, and mortality is low. Finally, antibiotic-associated diarrhea (AAD) is the mildest syndrome caused by
C. difficile
, and is characterized by mild-moderate diarrhea, lacking both large intestinal inflammation (as characterized, e.g., by abdominal pain, tenderness) and systemic signs of infection (e.g., fever). These three distinct syndromes occur in an increasing order of frequency. That is, PMC occurs less frequently than AAC, and AAD is the most frequent clinical presentation of
C. difficile
disease.
The populations affected by
C. difficile
are principally hospitalized, elderly patients and nursing home residents who have received broad spectrum antibiotics. Old age, length of hospital stay, underlying illness, and use of antibiotic therapy are recognized risk factors for
C. difficile
infection (McFarland et al., J. Infect. Dis. 162:678-684, 1990; Bennett, Aging, Immunity, and Infection, 216-229, 1994). A frequent complication of
C. difficile
infection is relapsing disease, which occurs in up to 20% of all subjects who recover from
C. difficile
disease. Relapse may be characterized clinically as AAD, AAC, or PMC. There are no specific risk factors or predisposing factors for relapse, but patients who relapse once are more likely to relapse again.
C. difficile
produces two exotoxins, Toxin A and Toxin B, which mediate the disease process caused by
C. difficile
. Toxin A and Toxin B are large (~300 kDa) extracellular proteins, the active forms of which are believed to be homodimers. The toxins are stably expressed in approximately equivalent amounts from a single chromosomal locus (Mitty et al., The Gastroenterologist 2:61-69, 1994). The toxins have nearly 50% amino acid sequence homology, but are immunologically distinct. The 100 kDa carboxyl-termini of the two toxins contain repetitive oligopeptides, and are involved in carbohydrate receptor binding in vivo. Receptor specificity is believed to mediate tissue and host specificity of toxin action. This region is also more immunogenic than the amino terminus. The amino terminal 200 kDa region contains the enzymatic domain, which is believed to glycosylate the GTP binding proteins Rho, Rac, and Cdc42, thereby preventing their phosphorylation, and leading to a loss of actin polymerization and cytoskeletal integrity (Eichel-Streiber, Trends Micro. 4:375-382, 1996). As a result of the cytoskeletal changes, tight junctions between epithelial cells are lost. The epithelial damage in conjunction with local inflammatory events causes fluid exudation into the gut, manifested as diarrhea (Mitty et al., supra).
By virtue of their inhibition of cytoskeleton structure, both toxins cause the rounding of cells in tissue culture at very low concentrations. The dose that causes morphologic change in 50% of cells (MC
50
) for Toxin A on IMR90 cells is 0.4 ng/ml and for Toxin B is 3.5 pg/ml (Torres et al., Infect. & Immun. 63:4619-4727, 1995). Toxin A is an enterotoxin that causes fluid accumulation in ligated animal intestinal loops. Although Toxin B does not induce fluid secretion in animal intestinal loops, both it and Toxin A elicit inflammatory changes in vivo and in vitro (Mitty et al., supra). Both toxins are lethal to animals when administered systemically.
SUMMARY OF THE INVENTION
The invention provides methods of treating
Clostridium difficile
disease in human patients. These methods involve percutaneously (e.g., intramuscularly, intravenously, or intraperitoneally) administering to a patient human
C. difficile
polyclonal immune globulin that neutralizes both Toxin A and Toxin B (hereinafter “immune globulin”) (e.g., 0.01-100 mg/kg body weight). These methods can also include percutaneous administration of a clostridial toxin or toxoid to a patient, to stimulate an anti-
C. difficile
immune response in the patient. When administered as treatment in affected individuals, the injected immune globulin will also prevent relapse.
Also included in the invention are methods of preventing
C. difficile
disease in human patients. In these methods, a toxin-neutralizing antibody to a
C. difficile
toxin or toxoid (e.g., a
C. difficile
polyclonal immune globulin (e.g., 0.01-100 mg/kg body weight)) is percutaneously (e.g., intramuscularly, intravenously, or intraperitoneally) administered to a human subject at risk of becoming infected with
C. difficile
. The
C. difficile
immune globulin used in these methods can be produced, e.g., in a human. These methods can also include percutaneous administration of a clostridial toxin or toxoid containing Toxin A and Toxin B epitopes to the patient.
The invention also provides methods of preventing or treating intestinal clostridial disease in human patients, which involve percutaneously administering a clostridial (e.g.,
C. difficile
) toxin or toxoid to a patient, in the presence or absence of an adjuvant, such as alum. An additional method included in the invention involves administering
C. difficile
immune globulin, as described above, to rapidly treat or protect a patient, while simultaneously administering toxoid for long-term, active protection by means of stimulation of the patient's immune system.
All of the prophylactic and therapeutic methods described above can, in conjunction with percutaneous administration, involve mucosal administration, such as oral or rectal administration.
Also included in the invention are methods of producing
C. difficile
toxoid. These methods involve providing
C. difficile
bacteria; culturing the bacteria in media containing suitable animal products (e.g., casein products) to generate a culture; co-purifying clostridial Toxin A and clostridial Toxin B from the culture to generate a mixture of co-purified Toxin A and Toxin B; and inactivating the co-purified Toxin A and Toxin B by incubation in formaldehyde at a temperature of about 25° C. or less (e.g., 15° C. or less, or 5° C. or less) to generate the clostridial toxoid. The co-purified Toxin A and Toxin B can be present in the mixture at a ratio in the range of 0.1:1 to 10:1, for example, 2:1. The invention also includes a
C. difficile
toxoid produced by this method, and a vaccine composition containing this toxoid and 0.012-0.020% formaldehyde. Optionally, this composition can contain an adjuvant, such as alum.
The invention also provides methods of producing human, toxin-neutralizing
C. difficile
immune globulin. In these methods,
C. difficile
toxin or toxoid containing, e.g., Toxin A and/or Toxin B, is administered to a human, and
C. difficile
immune globulin is isolated from the human.
C. difficile
immune globulin produced using these methods is also included in the invention.
Als

Affiliated with

Also associated with

Rating

Passive immunization against clostridium difficile disease does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Passive immunization against clostridium difficile disease, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Passive immunization against clostridium difficile disease will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3210303