U.S. patent application number 14/986895 was filed with the patent office on 2016-10-06 for immunization against clostridium difficile disease.
The applicant listed for this patent is Sanofi Pasteur Biologics, LLC. Invention is credited to Paul J. GIANNASCA, Wende LEI, Thomas P. MONATH, William D. THOMAS, JR., Zhenxi ZHANG.
Application Number | 20160287689 14/986895 |
Document ID | / |
Family ID | 34700454 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160287689 |
Kind Code |
A1 |
THOMAS, JR.; William D. ; et
al. |
October 6, 2016 |
IMMUNIZATION AGAINST CLOSTRIDIUM DIFFICILE DISEASE
Abstract
The invention provides active and passive immunization methods
for preventing and treating Clostridium difficile infection, which
involve percutaneous administration of C. difficile
toxin-neutralizing polyclonal immune globulin, C. difficile
toxoids, or combinations thereof. Also provided by the invention
are C. difficile toxoids, C. difficile toxin-neutralizing
polyclonal immune globulin, and methods of identifying subjects
that produce C. difficile toxin-neutralizing polyclonal immune
globulin.
Inventors: |
THOMAS, JR.; William D.;
(Somerville, MA) ; GIANNASCA; Paul J.; (Westford,
MA) ; ZHANG; Zhenxi; (Waltham, MA) ; LEI;
Wende; (Waltham, MA) ; MONATH; Thomas P.;
(Harvard, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi Pasteur Biologics, LLC |
Cambridge |
MA |
US |
|
|
Family ID: |
34700454 |
Appl. No.: |
14/986895 |
Filed: |
January 4, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13457821 |
Apr 27, 2012 |
9226956 |
|
|
14986895 |
|
|
|
|
11755459 |
May 30, 2007 |
|
|
|
13457821 |
|
|
|
|
11235609 |
Sep 26, 2005 |
|
|
|
11755459 |
|
|
|
|
10737270 |
Dec 16, 2003 |
6969520 |
|
|
11235609 |
|
|
|
|
09815452 |
Mar 22, 2001 |
6680168 |
|
|
10737270 |
|
|
|
|
09176076 |
Oct 20, 1998 |
6214341 |
|
|
09815452 |
|
|
|
|
60062522 |
Oct 20, 1997 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/56911 20130101;
A61P 1/12 20180101; A61K 39/08 20130101; A61K 2039/55505 20130101;
A61K 2039/55511 20130101; C07K 14/33 20130101; A61K 2039/54
20130101; C07K 2317/76 20130101; G01N 2469/20 20130101; A61K
2039/505 20130101; C12N 9/1051 20130101; G01N 2333/33 20130101;
A61P 31/04 20180101; A61P 1/04 20180101; A61K 39/39 20130101; A61K
39/00 20130101; C07K 16/1282 20130101; A61K 2039/521 20130101 |
International
Class: |
A61K 39/08 20060101
A61K039/08; A61K 39/39 20060101 A61K039/39 |
Claims
1. A vaccine composition comprising a clostridial toxoid and an
aluminum adjuvant.
2. The vaccine of claim 1, wherein the clostridial toxoid comprises
Clostridium difficile Toxoid A and Toxoid B.
3. The vaccine composition of claim 1, wherein the aluminum
adjuvant is selected from the group consisting of aluminum
hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
4. The vaccine composition of claim 1, wherein the composition
further comprises 0.012-0.020% formaldehyde.
5. A vaccine composition comprising (i) a toxoid of Clostridium
difficile Toxins A and B, (ii) an aluminum adjuvant, and (iii)
0.012-0.020% formaldehyde.
6. (canceled)
7. A method of preventing or treating symptomatic Clostridium
difficile disease in a human patient, the method comprising
percutaneously administering to the human patient (i) a clostridial
toxin or toxoid, and (ii) an aluminum adjuvant.
8. The method of claim 7, wherein the aluminum adjuvant is selected
from the group consisting of aluminum hydroxide, aluminum
phosphate, and aluminum hydroxy phosphate
9. The method of claim 7, wherein the toxin or toxoid is a
Clostridium difficile toxin or toxoid.
10. The method of claim 9, wherein the Clostridium difficile toxin
or toxoid comprises Clostridium difficile Toxoid A and Toxoid
B.
11. The method of claim 7, wherein the patient has or is at risk of
developing recurrent Clostridium difficile disease.
12. The method of claim 7, wherein the clostridial toxin or toxoid
is intramuscularly, intravenously, or subcutaneously administered
to the human patient.
13. The method of claim 7, wherein the patient does not have, but
is at risk of developing, symptomatic Clostridium difficile
disease.
14. The method of claim 7, wherein the patient has symptomatic
Clostridium difficile disease.
15. The method of claim 7, wherein the administered clostridial
toxin or toxoid and the aluminum adjuvant are present in a single
composition.
16. The method of claim 15, wherein the composition further
comprises 0.012-0.020% formaldehyde.
17-18. (canceled)
Description
[0001] This application is a continuation of and claims priority
from U.S. patent application Ser. No. 11/755,459, filed May 30,
2007, which is a continuation of U.S. Ser. No. 11/235,609, filed
Sep. 26, 2005 (abandoned), which is a continuation of U.S. Ser. No.
10/737,270 filed Dec. 16, 2003 (U.S. Pat. No. 6,969,520), which is
a continuation-in-part of U.S. Ser. No. 09/815,452, filed Mar. 22,
2001 (U.S. Pat. No. 6,680,168), which is a continuation of U.S.
Ser. No. 09/176,076, filed Oct. 20, 1998 (U.S. Pat. No. 6,214,341),
which claims benefit of U.S. Ser. No. 60/062,522, filed Oct. 20,
1997 (abandoned).
BACKGROUND OF THE INVENTION
[0002] This invention relates to methods and compositions for
preventing and treating Clostridium difficile disease.
[0003] 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 referred to as
antibiotic-associated diarrhea and colitis (LaMont, Bacterial
Infections of the Colon, Textbook of Gastroenterology, second
edition, 1897-1903, 1995).
[0004] Three clinical syndromes caused by C. difficile are
recognized, based on the 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 severe than in PMC, the
characteristic endoscopic appearance of the colon in PMC is absent,
and mortality is low. Finally, antibiotic-associated diarrhea (AAD,
which is also known as C. difficile associated diarrhea (CDAD)) is
the mildest syndrome caused by C. difficile, and is characterized
by mild-moderate diarrhea, lacking both large intestinal
inflammation (as characterized by, e.g., abdominal pain and
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.
[0005] 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 recurrent or 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.
[0006] 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 (.about.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 with
one another, 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). Both
toxins are lethal to animals when administered systemically.
SUMMARY OF THE INVENTION
[0007] 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.
[0008] Also included in the invention are methods of preventing C.
difficile disease in human patients. In these methods, a
toxin-neutralizing antibody raised against 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.
[0009] The invention also provides methods of preventing or
treating symptomatic C. difficile infection 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. Patients treated by these methods can
have or be at risk of developing, for example, recurrent C.
difficile associated diarrhea (CDAD). 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.
[0010] All of the prophylactic and therapeutic methods described
above can, in conjunction with percutaneous administration (i.e.,
before, during, or after such administration), involve mucosal
administration, such as oral or rectal administration.
[0011] 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.degree. C. or less
(e.g., 15.degree. C. or less, or 5.degree. 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.
[0012] 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.
[0013] Also included in the invention are methods of identifying a
human producing a C. difficile immune globulin. These methods
involve obtaining a blood sample from a human vaccinated with a C.
difficile toxoid; determining the level of antibodies to C.
difficile Toxins A and B in the blood sample by an enzyme-linked
immunosorbent assay (ELISA); and determining the level of in vitro
cytotoxicity neutralization activity against C. difficile Toxins A
and B in the blood sample. Detection of increased levels of
antibodies to C. difficile Toxins A and B in the blood sample, and
detection of in vitro cytotoxicity neutralization activity against
C. difficile Toxins A and B in the blood sample, indicate
identification of a human producing a C. difficile immune globulin.
In addition to humans that have been vaccinated with a C. difficile
toxoid, this method can be carried out with unvaccinated humans to
identify good candidates for vaccination.
[0014] The term "C. difficile immune globulin" is used herein to
describe polyclonal hyperimmune serum raised in subjects (e.g.,
human volunteers) vaccinated with C. difficile toxoids. The immune
globulin contains antibodies that neutralize cytotoxicity and in
vivo effects of Toxin A and Toxin B.
[0015] The term "C. difficile toxoid" is used to describe a C.
difficile toxin (Toxin A or Toxin B) or a mixture of C. difficile
toxins that have been partially or completely inactivated by, for
example, chemical (e.g., formaldehyde) treatment. A toxin is said
to be "inactivated" if it has less toxicity (e.g., 100%, 99%, 95%,
90%, 80%, 75%, 60%, 50%, 25%, or 10% less toxicity) than untreated
toxin, as measured, for example, by an in vitro cytotoxicity assay
or by animal toxicity. Other chemical means for inactivating toxins
can be used including, for example, peroxide or glutaraldehyde
treatment. Toxoids can also be generated by genetic changes that
result in toxin inactivation.
[0016] The invention provides several advantages. For example,
treatment using the methods of the invention specifically results
in inactivation of C. difficile bacterial toxins, without affecting
normal intestinal flora. Both C. difficile Toxin A and Toxin B are
involved in human disease, and the immunotherapy methods of the
invention can be used to target both of these molecules. Recovery
using immunotherapy is more rapid than antimicrobial therapy, which
targets vegetative bacteria, rather than directing toxin
neutralization. The specific neutralization of toxin activity has
the advantage of specifically and rapidly inactivating the cause of
tissue damage. In addition, a single dose of C. difficile immune
globulin, administered percutaneously (e.g., intramuscularly,
intravenously, or intraperitoneally), can be used in the methods of
the invention, rather than the repeating dosing required for oral
administration (Lyerly et al., Infect. Immun. 59:2215-2218, 1991).
Further, the overall dose of C. difficile immune globulin
administered percutaneously is lower than the dose required in oral
methods, due to the longer half life of injected antibodies,
compared to orally administered antibodies (hours vs. weeks or
months). Specific antibody therapy also permits continuation of
treatment of underlying conditions with antibiotics, which may
otherwise have to be withdrawn to permit reconstitution of the
intestinal flora and recovery from C. difficile infection. Also,
treatment using the methods of the invention prevents the emergence
of antibiotic-resistant bacteria. In particular, C. difficile
disease has been traditionally treated with vancomycin and
metronidizole, and use of vancomycin has led to the emergence of
vancomycin-resistant enterococcus. Similar problems may be arising
from metronidizole treatment. In addition, as is described further
below, the methods of the invention have been shown to be effective
in patients with recurrent disease (e.g., recurrent C. difficile
associated diarrhea (CDAD)), which otherwise is difficult to manage
and requires prolonged therapy with metronidazole or vancomycin.
Further, C. difficile is cultured in the methods of the invention
in medium that lacks complex animal products, such as nervous
system products, e.g., the animal products in Brain Heart Infusion
medium. Media containing such complex animal products have been
found to contain the bovine spongiform encephalopathy (BSE) prion.
Thus, in not using such medium, the invention provides safety
against infection by such agents.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a chromatogram tracing of the elution profile of a
C. difficile ammonium sulfate precipitate from an Sephacryl S-300
column.
[0019] FIG. 2 is a graph showing the inactivation kinetics of C.
difficile toxin lot 144.
[0020] FIG. 3 is a schematic representation of a schedule for
active immunization of hamsters with C. difficile toxoid vaccine
for protection from challenge after immunization.
[0021] FIG. 4 is a graph showing that hamsters immunized
intramuscularly with toxoid vaccine are protected from death and
diarrhea after C. difficile challenge.
[0022] FIG. 5 is a schematic representation of a schedule for
passive immunization of hamsters with C. difficile
toxin-neutralizing antibodies.
[0023] FIG. 6 is a graph showing that hamsters treated
intraperitoneally with toxin-neutralizing antibodies are protected
from death and diarrhea after C. difficile challenge.
[0024] FIG. 7 is a schematic representation of a schedule for
passive immunization of hamsters with diarrhea using C. difficile
toxin-neutralizing antibodies.
[0025] FIG. 8 is a graph showing that death and diarrhea are
prevented in hamsters treated with C. difficile toxin-neutralizing
antibodies.
[0026] FIG. 9 is a schematic representation of experiments
addressing the safety and immunogenicity of C. difficile toxoid
vaccine in Rhesus monkeys.
[0027] FIG. 10 is a graph showing the mean toxin-neutralizing
antibody titers in Rhesus monkeys immunized with C. difficile
toxoid vaccine.
[0028] FIG. 11 is a set of graphs showing serum IgG anti-toxin A
(upper panel) and serum IgG anti-toxin B (lower panel) antibody
levels in 3 subjects with recurrent C. difficile antibiotic
associated diarrhea. The subjects received intramuscular
inoculations of a C. difficile toxoid vaccine on days 0, 7, 28, and
56. The highest dilution of serum that neutralized the cytotoxicity
of purified C. difficile toxin A (upper panel) or toxin B (lower
panel) is shown in parentheses for any serum sample that had
detectable neutralizing activity.
DETAILED DESCRIPTION
[0029] The invention provides methods and compositions for
preventing and treating C. difficile disease in mammals, such as
humans. The methods include passive and active immunization
approaches, which involve percutaneous (e.g., intramuscular,
intravenous, or intraperitoneal) administration of antibodies
(e.g., toxin-neutralizing polyclonal immune globulin) to C.
difficile toxoids, C. difficile toxoids themselves, or combinations
thereof. For example, the invention includes methods of preventing
and/or treating recurrent C. difficile associated diarrhea (CDAD)
by percutaneous administration (e.g., intramuscular) of a vaccine
including toxoid A and/or toxoid B. The invention also includes C.
difficile toxoids, vaccine compositions containing C. difficile
toxoids, methods of producing C. difficile toxin-neutralizing
polyclonal immune globulin, substantially purified C. difficile
toxin-neutralizing polyclonal immune globulin, and methods of
identifying donors of C. difficile toxin-neutralizing polyclonal
immune globulin. These methods and compositions are described
further, as follows.
[0030] The prophylactic and therapeutic methods of the invention
involve vaccination with C. difficile toxoids, whether in carrying
out the treatment itself or in the production of C. difficile
immune globulin for subsequent use in passive immunization. C.
difficile toxoids can be produced by purification of toxins (Toxin
A, Toxin B, or a combination thereof) from C. difficile cultures,
and inactivation of the toxins by chemical, e.g., formaldehyde (see
below), glutaraldehyde, peroxide, or oxygen, treatment (see, e.g.,
Relyveld et al., Methods in Enzymology 93:24, 1983; Woodrow and
Levine, eds., New Generation Vaccines, Marcel Dekker, Inc., New
York, 1990). Alternatively, wild type or mutant C. difficile toxins
that lack or have reduced toxicity can be produced using
recombinant methods. Methods for making toxoids by genetic methods
are well known in the art (see, e.g., U.S. Pat. Nos. 5,085,862;
5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945). For
example, deletion mutations that remove the amino terminal,
enzymatic region of the toxin can be made. Deletion or point
mutations can also be made in the toxin active site. In addition,
deletion or point mutations can be made that prevent receptor or
carbohydrate binding.
[0031] Vaccine compositions containing C. difficile toxoids can be
prepared for administration by suspension of the toxoids in a
pharmaceutically acceptable diluent (e.g., physiological saline) or
by association of the toxoids with a pharmaceutically acceptable
carrier. The toxoids can be administered in the presence or absence
of an adjuvant, in amounts that can be determined by one skilled in
the art. Adjuvants that can be used in the invention include
aluminum compounds, such as aluminum hydroxide, aluminum phosphate,
and aluminum hydroxy phosphate. The antigen can be precipitated
with, or adsorbed onto, the aluminum compound using standard
methods. As a specific example, alum (e.g., Rehydragel LV.RTM.,
Reheis, Inc., Berkeley Heights, N.J.; up to 2 mg AlOH/dose, e.g.,
about 1.5 mg AlOH/dose) can be used. Additional adjuvants that can
be used include RIBI (ImmunoChem, Hamilton, Mont.), QS21 (Aquila),
Bay (Bayer), and Polyphosphazene (Virus Research Institute,
Cambridge, Mass.; WO 95/2415).
[0032] The vaccine compositions of the invention can be
administered by the percutaneous (e.g., intramuscular, intravenous,
or intraperitoneal) route in amounts and in regimens determined to
be appropriate by those skilled in the art. For example, 100 ng-500
.mu.g, 1-250 .mu.g, 10-100 .mu.g, 25-75 .mu.g, or 50 .mu.g toxoid
can be administered. For the purposes of prophylaxis or therapy,
the vaccine can be administered, for example, 1, 2, 3, or 4 times.
When multiple doses are administered, the doses can be separated
from one another by, for example, one week to a month. For the
purposes of stimulating donors of C. difficile immune globulin, a
higher number of doses can be administered. For example, up to 6
doses can be administered, separated from each other by, e.g., one
week to a month. In another example, four doses of 50 .mu.g each
can be administered intramuscularly over any eight week period.
Such a schedule is described in more detail below in the context of
treating recurrent C. difficile associated diarrhea.
[0033] When vaccination is performed to generate C. difficile
polyclonal immune globulin, e.g., human C. difficile polyclonal
immune globulin, serum samples from the immunized donors are first
monitored for the presence of C. difficile Toxin A and Toxin B by
enzyme-linked immunosorbent assay (ELISA) analysis. Briefly, ELISA
plates are coated with carbonate/bicarbonate, pH 8.5, and 1
.mu.g/ml protein (purified Toxin A or Toxin B), and incubated at
4.degree. C. overnight. The wells are contacted with serum samples
diluted in phosphate-buffered saline (PBS), washed, and contacted
with an anti-human antibody coupled to a detectable label, such as
alkaline phosphatase. Detection of a signal of greater than two
times over background is considered positive. Signal is detected by
optical density measurement at 405 nm.
[0034] Samples that test positive in the ELISA assay are then
tested in a toxin neutralization assay. Briefly, serum samples (100
.mu.l) are serially diluted two-fold in MEM, and are pre-incubated
with an equal volume of Toxin A containing 10 MC.sub.50 for 1 hour
at 37.degree. C. The Toxin A concentration is standardized for
challenge of the cells. For example, ten times the concentration
that affects 50% of the cells is used for challenge. The range used
for Toxin A is 10-100 ng. Toxin A/serum mixtures (100 .mu.l) are
then added to confluent IMR90 cell monolayers (American Type
Culture Collection (ATCC, Rockville, Md.); Torres et al., supra).
The overlaid cells are incubated for 16-18 hours at 37.degree. C.,
and are then scored for cytotoxicity. If at least 50% of the cells
are protected from rounding, the sera is rated "protective." The
potency test for Toxin B is performed by the same procedures
described above for Toxin A, except that the serum samples are
pre-incubated with Toxin B prior to determination of cytotoxicity
in the IMR90 cell assay. The amount of Toxin B that has an effect
on 50% of IMR90 cells is 10-100 pg.
[0035] The screening methods described above can also be used to
identify subjects that have not been vaccinated with C. difficile
toxoids, but have higher than normal serum levels of antibodies
against C. difficile toxins. These subjects are good candidates for
vaccination with the toxoids, for production of C. difficile immune
globulin.
[0036] Once an acceptable donor is identified, immune globulin is
obtained from the donor using standard plasmapheresis methods. The
immune globulin is purified using standard methods, such as Cohn
cold-ethanol fractionation, or standard chromatography methods,
such as sizing column chromatography or antibody affinity
chromatography (e.g., using Protein A). Up to two times per week,
whole blood (500 ml-1 L) is obtained from donors, plasma is
isolated by centrifugation, and cells are returned to the donors.
Preferably, the purified sample contains all or predominantly IgG,
but mixtures containing, e.g., IgG, IgA, and IgM, can also be used
in the invention.
[0037] The C. difficile immune globulin, prepared as described
above, can be percutaneously (e.g., intramuscularly, intravenously,
or intraperitoneally) administered to patients that have, or are at
risk of developing, C. difficile infection. These patient
populations include, for example, patients that have received broad
spectrum antibiotics, such as hospitalized elderly patients,
nursing home residents, chronically ill patients, cancer patients,
AIDS patients, patients in intensive care units, and patients
receiving dialysis treatment. The C. difficile immune globulin is
administered in amounts ranging from 100 .mu.g/kg-100 mg/kg, or
1-50 mg/kg, for example, about 15 mg/kg, depending on donor titer:
the higher the neutralization titer of the immune globulin, the
lower the amount is that needs to be administered. The immune
globulin can be administered in, e.g., one or two doses. For
example, in the case of therapeutic passive immunization, an
initial dose can be administered for treatment and a second dose
can be administered to prevent relapse.
[0038] The methods and compositions of the invention, as well as
experimental evidence supporting the invention, are described in
further detail, as follows.
Vaccine Production
Overview
[0039] C. difficile Toxin A and Toxin B are produced in anaerobic
cultures of C. difficile grown in culture bottles (10-20 L). Master
and working cell banks of C. difficile were manufactured from a
lyophilized research cell bank prepared at the ATCC from C.
difficile strain ATCC 43255. For vaccine production, toxins are
produced by C. difficile cultures grown in dialysis sacs, suspended
in growth medium. Multiple sac cultures are pooled, and viable C.
difficile and spores are removed by centrifugation, followed by
submicron filtration. The resulting filtrate is concentrated and
diafiltered, the toxins are precipitated at 4.degree. C. with 60%
saturated ammonium sulfate, and pellets are stored frozen. The
ammonium sulfate pellets are re-dissolved in phosphate buffer, and
applied to an S-300 Sephacryl size-exclusion column. The peak
containing Toxin A and Toxin B is collected and concentrated
(50-60% toxin, with a ratio of Toxin A to Toxin B of 2:1). The
toxin preparation is then inactivated for 18 days with 4.25 mg/ml
formaldehyde at 4.degree. C.-6.degree. C. in a solution containing
4.25 mg/ml lysine. After inactivation, the formaldehyde
concentration is reduced by diafiltration to 0.016% for use as a
stabilizer. Final product, at a concentration of 2.5 mg/ml, is
filled into glass vials at a fill volume of 0.6 ml. The current
process yields 15-20 mg/L, or 150-200 doses, of toxoid. Lot release
testing assays of identity, potency, and safety have all been
established on preclinical lots. GMP Master and Production cell
banks have been generated, qualified, and stored in a stable
condition. C. difficile toxoid vaccine preparation is described in
further detail, as follows.
Master and Working Cell Banks
[0040] A research seed was prepared and lyophilized under contract
by the ATCC by their standard methods using an ampule of the type
strain ATCC 43255. Oxoid Reinforced Clostridial Medium (RCM) was
used to grow the seed stock (Oxoid Ltd., Hampshire, England). The
bovine-derived materials in media were obtained in Australia, New
Zealand, Holland, and the USA from healthy animals used for human
consumption. Cultures were stabilized in RCM using 5% dextran and
trehalose as preservatives.
Preparation of C. difficile Master Cell Bank (MCB) and Working Cell
Bank (WCB)
[0041] The MCB of C. difficile was prepared by resuspending and
incubating a lyophilized vial of the research seed stock in RCM
(the same lot used by the ATCC), followed by two expansions in
Tryptone (0.48%)-Yeast Extract (0.24%)-Mannitol (0.1%) (TYM)
medium. Glycerol was added as cryopreservative and 250 aliquots of
.about.1 ml each were snap frozen and stored in liquid nitrogen.
The working cell bank was prepared in a similar fashion using a
vial of the MCB as inoculum.
Cell Bank Testing
[0042] The master and working cell banks were tested for viability,
purity, identity, and toxin expression. Viability was demonstrated
by growth on both solid and liquid medium. Purity was tested by
gram stain and colony morphology under anaerobic culture, and by
the absence of aerobic growth. C. difficile identity was
demonstrated by gas chromatography fatty acid analysis and by
clinical anaerobic identity testing. Toxin expression and identity
were confirmed by culturing the cell banks in dialysis sacs and
testing the culture for expression of both toxins by crossed
immunoelectrophoresis. Toxin A expression and identity were also
confirmed by ELISA. Toxin B expression was confirmed by testing for
cytotoxicity and specific neutralization of cytotoxicity. Toxin
expression was measured in parallel with ATCC 43255 standards and
was shown to be comparable.
Culture and Toxin Expression
[0043] Toxins are produced in anaerobic cultures of C. difficile
grown in dialysis sacs (13-14,000 molecular weight cutoff) and
suspended in a media containing a nitrogen source (e.g., tryptone
in a concentration of 1-100 g/L, 5-20 g/L, or 12 g/L), yeast
extract (1-100 g/L, 15-35 g/L, or 24 g/L), phosphate buffer, a
carbon source (e.g., mannitol (1-50 g/L, e.g., 8 g/L), glucose,
glycerol (1-50 g/L, e.g., 4 g/L), or mannitol+glycerol (e.g., in
the amounts set forth above). Production is initiated by expanding
a vial of the working cell bank in a small static culture and using
aliquots of the culture to inoculate dialysis sacs. After growth at
37.degree. C. for approximately 5 days, material in the sacs is
harvested. The harvested product is centrifuged and filtered (0.5 m
followed by 0.2 .mu.m) to remove vegetative cells and spores. The
filtrate is washed, concentrated, and precipitated with ammonium
sulfate.
Preparation of Culture Units
[0044] A culture unit consists of an 8 L or 16 L spinner flask,
with two sidearm ports, a dialysis sac, and a 1 L or 2 L flask of
phosphate buffer. Up to twenty-five 8 L or 16 L units are
inoculated for each production run. The culture unit is prepared by
dissolving media in a spinner flask, suspending the dialysis sac
between the sidearm ports, capping the ends of the ports, and
attaching a flask of 100 mM phosphate buffer to one port. The
entire unit is autoclaved for media sterilization and creation of
anaerobiasis. After cooling to below 50.degree. C., the phosphate
buffer is pumped into the dialysis sac and the unit is equilibrated
overnight at 37.degree. C., prior to inoculation during which
growth nutrients diffuse into the dialysis sac, establishing
conditions suitable for bacterial growth.
Inoculation and Culture
[0045] A vial of the working cell bank is thawed and used to
inoculate 50 ml of anaerobic TY starter medium (tryptone (0.48%)
and yeast extract (0.24%)). The flask is placed in an anaerobic
chamber at 37.degree. C. for 14-16 hours. Approximately 2 ml of
inoculum in an appropriate volume of diluent is added to each
dialysis sac. The culture units are then returned to the incubator
and left undisturbed for 5 days. Anaerobiasis is maintained after
autoclaving by preventing unnecessary agitation.
Harvest, Filtration, and Precipitation
[0046] Following incubation, culture units are removed from the
incubator, and the contents of the dialysis sacs are pumped out,
pooled, and tested for culture purity and identity. Viable C.
difficile organisms and spores are removed by centrifugation,
followed by filtration through a 0.5 .mu.m pre-filter and then
through a 0.2 .mu.m sterilizing filter. The filtrate is tested for
Toxin A and Toxin B concentration and sterility, and concentrated
10.times. by ultrafiltration with a 30,000 MW cutoff hollow fiber
cartridge. The filtrate is washed with 25 mM Tris, pH 7.5,
resulting in a reduction in low molecular weight media components.
Filtered, saturated ammonium sulfate solution is added to the
concentrate to give a final solution of 60% saturation. The
solution is incubated at 4.degree. C. for 48 hours or longer, the
toxin-containing precipitate is harvested by centrifugation, and
the supernatant decanted. The ammonium sulfate pellet is stored
frozen at -10.degree. C. or colder until processed further.
Purification and Inactivation
[0047] The pellet is thawed by mixing with 100 mM phosphate buffer,
pH 7.4, at room temperature. Solubilized toxin is clarified by
centrifugation and filtered using a 0.45 .mu.m filter. Clarified
material is then fractionated on a Sephacryl S-300 High Resolution
(Pharmacia Biotechnology) gel filtration column. A typical
chromatographic profile is shown in FIG. 1. The toxin peak is
collected and concentrated to 5.0.+-.0.5 mg/ml. Collection begins
with the ascending limb of the toxin peak and continues to the
inflection point on the descending limb, as determined by visual
inspection of the chromatogram.
[0048] After purification, the toxin solution is inactivated for 18
days at 4-6.degree. C. using 4.25 mg/ml of formaldehyde. The
inactivation is carried out at pH 7.0.+-.0.2 in 100 mM phosphate
buffer containing 4.25 mg/ml lysine hydrochloride. The inactivation
period is set to exceed three times the period needed for complete
elimination of lethality in mice. Thus, by day 6 of inactivation,
intraperitoneal inoculation with 0.5 mg of toxoid produces no
lethality or weight change in mice. This corresponds to a reduction
in the cytotoxicity titer in IMR90 cells of approximately 6
log.sub.10. Following 18 days of inactivation, biological activity
is typically reduced another 2 to 3 orders of magnitude, as judged
by effects on IMR90 cells, for a total extent of inactivation of 8
to 9 log.sub.10. Following 18 days of inactivation, the inactivated
toxin is buffer-exchanged in 50 mM phosphate, 100 mM NaCl, pH 7.4,
reducing the formaldehyde concentration to 0.16.+-.0.04 mg/ml. The
soluble, inactivated toxin at 2.5 mg/ml is sterile filtered and
filled into 2 ml Type I borosilicate glass vials with gray butyl
rubber stoppers.
Studies Supporting Conditions of Inactivation and Formulation
[0049] Extensive studies were conducted to establish optimal
conditions for toxin inactivation with formaldehyde. To monitor
loss of biological activity, these studies utilized the IMR90
tissue culture system, which is a highly sensitive indicator of
biological activity of C. difficile toxin (Torres et al., supra).
Parameters studied included concentration of formaldehyde and
toxin, buffer composition, pH, time, temperature, and effect of
added L-lysine, designed to facilitate full toxoiding (Table
1).
TABLE-US-00001 TABLE 1 Parameters Tested Parameters Range tested pH
6.5; 7.0; 7.4; 8.0 Temperature (.degree. C.) 5; 14; 28; 37 Toxin
concentration (mg/ml) 1; 5 Formaldehyde concentration (mg/ml) 0.5;
1.0; 2.0; 2.5; 4.25; 10; 15; 20 Lysine HCl concentration (mg/ml) 1;
2; 4.25
[0050] In general, C. difficile toxins were very sensitive to
inactivation at 37.degree. C. under all conditions, with
inactivation occurring extremely rapidly (e.g., loss of 7
log.sub.10 of activity in 8 hours). Therefore, to maximize control
and reproducibility of the inactivation, we elected to inactivate
at 4.degree. C. Toxoids inactivated at 4.degree. C. induced higher
antibody titers than toxoids inactivated with formaldehyde at
37.degree. C. Under the specified conditions chosen, complete loss
of detectable in vivo biological activity occurs within 6 days of
inactivation, corresponding to a loss of approximately 5-6
log.sub.10 in vitro. To provide a sufficient margin of safety,
inactivation is continued for an additional 12 days, during which
an additional 2-3 log.sub.10 of cytotoxicity are lost. At the end
of the inactivation period, activity in the cell culture system is
just barely detectable, at the threshold of detectability. Kinetics
for a typical inactivation are shown in FIG. 2.
[0051] Low concentrations of formalin are included in the
formulation of the vaccine to prevent toxoid reversion. Reversion
was detected, despite Lys incorporation into the activation site,
which is known to reduce reversion with other toxins (Relyveld,
Prog. Immunobiol. Stand. 3:258, 1969). The choice of formulation
was based on numerous studies undertaken to determine the stability
of the toxoid, including the possibility of reversion, under
various conditions. In general, the toxoid was stable at 4.degree.
C., with or without low concentrations of residual formalin. In the
absence of residual formalin, partial reversion occurred at higher
temperatures (28-37.degree. C.), with the toxoid regaining
detectable biological activity over days to weeks (Table 2).
TABLE-US-00002 TABLE 2 Partial Reversion of C. difficile Toxoid in
Absence of Formalin Time of Incubation MC.sub.50 (IMR90 cell
culture assay) 37.degree. C. (Days) Lot 133A Lot 135A Lot 144A 0
0.2 mg/ml* 0.2 mg/ml* 0.41 mg/ml 7-8 0.10 mg/ml 0.13 mg/ml 0.2
mg/ml 14 0.11 mg/ml 0.13 mg/ml 0.025 mg/ml 35 0.052 mg/ml 0.064
mg/ml Not determined 63 0.014 mg/ml 0.017 mg/ml Not determined
*Estimated data
[0052] As noted, only partial reversion has been detected, even
after exposure to optimal conditions for reversion (37.degree. C.)
for extended periods (over two months). After this time,
approximately 5 log.sub.10 has been regained of the 8-9 log.sub.10
originally inactivated.
[0053] Reversion was completely prevented at all temperatures by
inclusion of formalin at concentrations of 0.010% or higher (Table
3). Therefore, specifications for the formulated toxoid vaccine
have been set to ensure a formalin concentration of
0.012-0.020%.
TABLE-US-00003 TABLE 3 Prevention of Reversion by Low
Concentrations of Formalin Lot 133B Time of MC.sub.50 (mg/ml, IMR90
cell culture assay) Incu- Formal- bation No Formaldehyde
Formaldehyde dehyde (Days) formaldehyde 0.05 mg/ml 0.10 mg/ml 0.15
mg/ml 0 0.33 0.20 0.11 0.11 4 0.00028 0.025 0.09 0.11 7 0.00028 --
-- -- 14 0.000095 0.00028 0.12 0.053 28 0.00029 0.00029 0.12 0.12
56 0.00029 0.00029 0.12 0.12
Characterization of C. difficile Toxin (Prior to Inactivation)
[0054] Studies were undertaken to characterize the partially
purified toxin preparation following size-exclusion chromatography,
prior to formaldehyde treatment. Toxin A and Toxin B are not well
separated in Tris-Glycine reducing SDS-PAGE. However, total toxin
(Toxin A and Toxin B) can be estimated by densitometric scanning of
Coomassie stained, Tris-Glycine reducing SDS-PAGE gels. Total toxin
accounts for 50-60% of total protein. Immunoblots of these reducing
gels show a major anti-Toxin A reactive band and a major and
several minor anti-Toxin B reactive bands.
[0055] We have undertaken identification of the major impurities in
the vaccine. SDS-PAGE gels were overloaded with purified bulk toxin
and the proteins were separated under reducing SDS-PAGE conditions.
The gel was cut just below the 244 kDa pre-stained marker to cutoff
the toxin band. The proteins below the toxin band were then
transferred to a PVDF membrane and subjected to amino acid
sequencing for homology comparison to sequence databases. From
N-terminal sequencing, 18-25 cycles, we have identified the
.about.35 kDa impurity as C. difficile 3-hydroxy butryl CoA
dehydrogenase, the 45-47 kDa impurity as C. difficile glutamate
dehydrogenase, and the 60-70 kDa protein as a homologue of groEL or
the bacterial hsp60 family of proteins (.about.70% homology).
[0056] Good separation of Toxin A and Toxin B proteins is achieved
using native PAGE gels, as confirmed by western blotting with
anti-Toxin A and anti-Toxin B antibodies. As with the reducing
gels, a number of lower molecular weight anti-Toxin B reactive
bands are observed.
[0057] Toxin A can also be separated from Toxin B by ion exchange
HPLC using a DEAE-5PW column. The Toxin A/Toxin B ratio is
approximately 2.2, as measured by ELISA, and approximately 1.9, as
measured by ion-exchange chromatography.
Alum-Adsorbed Toxoid Vaccine
[0058] We prepare alum for toxoid adsorption from commercially
available sterile Rehydragel LV.RTM., which contains 20 mg/ml
aluminum oxide (Reheis, Inc., Berkeley Heights, N.J.). This
material is first diluted to 3 mg/ml aluminum oxide with 50 mM
phosphate buffer at pH 7.4, 100 mM NaCl, 100 mg/ml formaldehyde.
The diluted alum is filled aseptically into sterile, pyrogen-free
10 ml capacity glass vials with gray butyl rubber stoppers under
class 100 conditions.
Identification of Candidates for Vaccination to Generate C.
difficile Immune Globulin Donors
[0059] As is discussed above, C. difficile immune globulin donors
can be generated by percutaneous administration of C. difficile
toxoid vaccine. Preferred candidates for vaccination are subjects
that already have C. difficile toxoid neutralizing antibodies.
These donors have been exposed to toxin and would require fewer
booster doses to reach useful titers. We have tested 9 commercial
lots of intravenous immune globulin, and have found them to contain
very low levels of neutralizing antibodies to C. difficile Toxin A
and Toxin B. The titers of antitoxin in these preparations is
<1:50 to both toxins, the titer being higher to Toxin B than to
Toxin A. We also conducted a survey of 100 professional plasma
donors from a center in Nevada. The results indicate that 2% and
13% of these individuals had antitoxin A and antitoxin B
neutralizing antibodies, respectively, but at very low titers.
These data show that selection of plasma from unstimulated,
seropositive plasma donors for the purpose of preparing a
hyperimmune human antitoxin to treat C. difficile would not be
effective, and that it is necessary to stimulate donors by
immunization with toxoid vaccine to produce a therapeutic human
immune globulin.
TABLE-US-00004 TABLE 4 Antitoxin Antibody Levels in Plasma Donors
(n = 100) Antigen Assay* Positive (%) Mean** Toxin A ELISA (>0.2
OD) 15 0.33 .+-. 0.14 Neutralization 2 1:7.5 Toxin B ELISA (>0.2
OD) 40 0.53 .+-. 0.37 Neutralization 11 1:35
Preclinical Evaluation of Active and Passive Immunization
Methods
Active Immunization of Mice
[0060] Groups of 8 female Swiss Webster mice were immunized
intraperitoneally (IP) with 2 doses of alum-adsorbed toxoid vaccine
one week apart. Toxoid was adsorbed to alum to mimic the human
formulation (ratio of 0.144 mg protein per mg of aluminum). Doses
were administered over a range of tenfold dilutions of adsorbed
vaccine. Animals were dosed with four different toxoid lots for
comparison of immunogenicity: one research lot (Lot 27-33) and
three vaccine lots (Lots 133, 135, and 144) manufactured according
to the method for production of the clinical product. One week
after the second immunization, sera were tested for total antibody
by ELISA, and for antibodies to Toxin A and Toxin B by cytotoxicity
neutralization. In the cytotoxicity neutralization assay, toxins
(10.times.MC.sub.50) are incubated with twofold antibody dilutions
for 1 hour at 37.degree. C., and then inoculated onto monolayer
cultures of IMR90 cells. Neutralization titer is expressed as the
highest dilution of antibody that protects 50% of the cells from
rounding.
[0061] ELISA data show that anti-toxin immunity develops in a
dose-dependent manner. Toxin A appears slightly more immunogenic
than Toxin B when the magnitude of response at a particular
dilution of toxoid is compared. The toxoid also elicited
neutralizing antibody responses. The dose of toxoid required to
elicit neutralizing antibodies that protect cells from rounding is
higher for Toxin B than for Toxin A, also demonstrating the higher
immunogenicity of Toxin A in mice.
[0062] To determine whether toxoid vaccine protects mice against
the lethal effects of Toxins A and B, groups of mice were immunized
intraperitoneally with two weekly doses of vaccine. They were then
challenged with five LD.sub.50 of Toxin A (100 ng, IP) or Toxin B
(200 ng, IV). Animals were monitored for illness and death for 14
days. Unimmunized animals died within the first 24 hours after
challenge.
[0063] Results showed that mice were protected from Toxin A at a
dose of adsorbed toxoid that contained >50 ng of protein and
mice were protected from Toxin B at a dose of >5 mg. As in the
immunogenicity experiment described above, Toxin A was protective
at a dose 10-100 fold lower than that required to protect animals
from Toxin B challenge.
[0064] The effect of alum on the immunogenicity of the toxoid was
tested in mice. Groups of ten animals were immunized
intraperitoneally with 3 weekly doses of soluble toxoid or toxoid
adsorbed to alum. Alum adsorptions were performed immediately prior
to dosing by mixing 0.144 mg toxoid protein per mg aluminum.
Animals received 10 .mu.g toxoid alone or 10 .mu.g toxoid adsorbed
to alum. Anti-toxin immune responses were measured by ELISA and
cytotoxicity neutralization in serum samples. Total antibody titers
determined by ELISA were comparable for soluble toxoid and alum
adsorbed toxoid. Neutralizing antibody titers against both toxins
were higher in groups that received alum adsorbed toxoid.
[0065] Mice are very sensitive to parenterally administered
purified Toxin A and Toxin B, and thus can be used to monitor
toxoid inactivation. The LD.sub.50 of purified Toxin A and Toxin B
tested individually are approximately 50 ng. The partially purified
toxin preparation, prior to inactivation, has an LD.sub.50 of less
than 20 ng total protein, which corresponds to approximately 4-8 ng
of each toxin, suggesting that the toxins may act synergistically
when administered together.
[0066] Following inactivation with formalin, no toxicity is
observed in mice when animals receive the largest dose of
inactivated toxoid that has been administered, containing 1.25 mg
total protein, corresponding to about 500 mg of Toxin A toxoid and
250 .mu.g of Toxin B toxoid. These data show a minimum reduction in
lethality of over 6.25.times.10.sup.4 fold. The actual extent of
inactivation is at least 8 orders of magnitude, as determined by a
more sensitive tissue culture assay. The mouse safety assay is also
used to define the duration of inactivation. Toxoid (0.5 mg) is
typically fully tolerated at Days 5-6. Inactivation is stopped
after three times the length of inactivation required to show no
lethality in mice after a 0.5 mg intraperitoneal challenge.
[0067] Since vaccination protects the mouse from the biologic
effects of Toxin A and Toxin B, the mouse model has been adapted to
serve as the principal potency assay for the manufactured toxoid
vaccine. In this assay, mice are immunized and then bled to recover
serum, which is tested for toxin neutralization activity in vitro
in the IMR90 tissue culture system.
[0068] To utilize this assay, we first determined that protection
in mice correlates with in vitro neutralization activity, as
measured in the IMR90 system. Four lots of toxoid vaccine were used
to vaccinate mice intraperitoneally with two weekly doses. Toxoid
was adsorbed to alum as described above and tested over a range of
tenfold doses. Animals were bled 7 days after the second dose of
vaccine and sera from individual mice was tested for its ability to
neutralize the effects of Toxins A and B on IMR90 cells. Animals
were allowed to recover for 4 days, and then challenged with lethal
doses of either Toxin A or Toxin B. The correlation of neutralizing
antibody titer and survival from challenge with both toxins was
highly significant (p<0.0001 by the Wilcoxon Rank Sums
Test).
Active Immunization of Hamsters
[0069] The hamster provides an excellent model of C. difficile
infection, as this species is highly sensitive to the actions of
Toxin A and Toxin B. After a single dose of clindamycin in the
presence of C. difficile, hamsters die within 2-3 days with
fulminant diarrhea and hemorrhagic cecitis. Hamsters immunized with
the toxoid vaccine intramuscularly are protected from death and
diarrhea when subsequently challenged with C. difficile. This route
of immunization induces serum antibodies (IgG), but does not induce
detectable mucosal antibodies, indicating that high titers of IgG
can protect from the intestinal disease caused by C. difficile.
[0070] To quantitate the level of protection from death and
diarrhea, groups of hamsters were vaccinated intramuscularly on
days 0, 14, and 21 with approximately 100 .mu.g of toxoid (A and B)
in solution (n=15) or placebo (n=10). Two weeks after the final
dose, hamsters were challenged intragastrically (IG) with 1.0 mg
clindamycin followed by 1.times.10.sup.5 C. difficile (ATCC strain
43255). Animals were monitored for weight loss, diarrhea, and
survival for 14 days after challenge (FIG. 3). Hamsters immunized
with toxoid vaccine were protected from death (p.gtoreq.0.0001) and
diarrhea (p=0.0057), compared to sham immunized controls (FIG. 4).
When sera from each individual animal were analyzed,
toxin-neutralizing antibody levels were present and correlated with
protection.
Passive Immunization in Hamsters and Mice
[0071] Experiments in two animal models confirm the therapeutic
efficacy of passive immunization. Treatment with toxin-neutralizing
antibody preparations protects mice from lethal challenge.
Utilizing the clindamycin challenge model described above, hamsters
given toxin-neutralizing antibodies were shown to be protected from
death and diarrhea. Hamsters developing diarrhea can be cured by
parenteral administration of toxin-neutralizing antibodies.
Protective therapeutic activity of passively administered antibody
is dose-dependent and correlates with passive serum neutralizing
antibody levels achieved in animals treated with the antibody
containing preparation.
Passive Protection of Mice from Lethal Challenge
[0072] To test the protective capacity of toxin-neutralizing
antibodies, mice were given hyperimmune mouse ascites containing
antibodies to both Toxin A and Toxin B by the intraperitoneal (IP)
route and then challenged with Toxin A or Toxin B. The mice
received a single dose of pooled ascites at doses of 100, 10, or 1
.mu.l, and were bled daily to determine the level of neutralizing
antibodies passively obtained in serum. Animals were then
challenged with Toxin A (5.times.LD.sub.50 IP) or Toxin B
(5.times.LD.sub.50 IV) and monitored for 7 days. Direct
administration of Toxin A and Toxin B leads to death in mice. The
100 .mu.l dose of ascites protected 60% of animals against Toxin A
and 80% of animals against Toxin B lethality. The mice were
protected from Toxin A challenge for up to 23 days after antibody
administration. Antibodies had to be administered within 30 minutes
of toxin injection to obtain this level of protection from
lethality. Laboratory measurements indicated all surviving animals
had reciprocal serum neutralization titers of .gtoreq.200 as a
result of the infusion with ascites. The half-life of
toxin-neutralizing antibodies was estimated to be 2 weeks, under
the conditions of the study.
Passive Protection of Hamsters Following Clindamycin Challenge
[0073] Groups of female hamsters were given a single dose of
hyperimmune mouse ascitic fluid IP. Graded doses were administered
(6, 2, 0.6, or 0.2 ml ascites per animal). Ascites from non-immune
mice served as a negative control. Animals were bled in order to
measure passive serum antibody levels the day following receipt of
the ascites and challenged with clindamycin 2 days later. Animals
were monitored for 3 weeks after challenge for survival, diarrhea,
and weight loss (FIG. 5). Protection against death was achieved in
the three highest dose groups, and protection against both death
and diarrhea was seen in animals receiving the highest dose of
antibodies (FIG. 6). Levels of neutralizing antibodies correlated
with protection from death and diarrhea. Fully protected animals
had reciprocal serum neutralizing antibody of .about.800 with the
least effective titer being 200. The half-life of neutralizing
antibodies in hamster serum in this study was estimated to be 14
days.
Treatment of Diarrhea in Hamsters Using Neutralizing Antibodies
[0074] The hamster model of antibiotic-associated diarrhea is a
useful one for the evaluation of prophylactic strategies against C.
difficile. However, C. difficile disease is very severe in hamsters
with acute cecitis and death occurring rapidly after clindamycin
challenge. The severity of the infection can be reduced by the
administration of a predetermined amount of neutralizing antibodies
against Toxin A and Toxin B designed to protect from death but not
diarrhea. The dose that prevents death but not diarrhea was defined
in dose ranging experiments. During the period when animals had
diarrhea, additional neutralizing antibodies could resolve the
diarrhea. Animals given ascites from non-immune animals continued
to suffer from diarrhea and most eventually died. Treated animals
recovered from diarrhea within 24 hours after treatment, without
relapse. The experimental design and diarrhea outcome are shown in
FIGS. 7 and 8. This experiment shows that toxin-neutralizing
antibodies can be used to treat C. difficile associated diarrhea.
Recovery was rapid.
Immunogenicity of Toxoid Vaccine in Non-Human Primates
[0075] Neutralizing antibodies to both Toxin A and Toxin B were
induced in rhesus monkeys after immunization with our toxoid
vaccine. Groups of 3 animals were given fluid vaccine, with either
the vaccine adsorbed to alum or placebo. The study was designed to
demonstrate the ability of the vaccine to raise high titer
neutralizing antibodies in non-human primates. Placebo controls
were included primarily for safety comparisons. Animals received 5
doses of vaccine (110 .mu.g) in solution, adsorbed to alum, or
placebo. Vaccine was administered on days 0, 8, 29, 65, and 118 in
a 0.5 ml volume by the intramuscular route in the gluteal area.
Immune response and clinical pathology were monitored (FIG. 9). No
adverse pathology or sensitivities were noted after the 5 doses
were given. All immunized animals responded with both binding and
neutralizing antibodies. Several vaccine doses were required to
induce significant neutralizing antibodies; a booster dose at days
65 and 118 raised neutralizing antibody levels further. Alum
adsorbed vaccine induced more rapid and higher responses in some
animals (FIG. 10). The studies showed the feasibility of inducing
levels of neutralizing vaccine-induced antibodies suitable for
processing into immune globulin preparation and documented the
ability of booster doses in primed animals of eliciting high titers
of protective antibodies. This experiment also demonstrated that
hyper-immunization with multiple booster doses of toxoid was safe
in non-human primates.
Clinical Evaluation of Active Immunization Methods
[0076] In the studies described below, a parenteral C. difficile
vaccine containing toxoids A and B was administered to three human
subjects with a history of multiple episodes of recurrent C.
difficile associated diarrhea (CDAD). Subjects received four 50
.mu.g intramuscular inoculations of the vaccine over an 8 week
period. Two subjects showed an increase in their serum IgG
anti-toxin A and anti-toxin-B antibody levels and developed serum
cytotoxin neutralizing activity against both toxins. After
vaccination, all three subjects discontinued treatment with oral
vancomycin without any further recurrence of CDAD. This shows that
use of a C. difficile vaccine is effective in treating subjects at
high risk for CDAD.
[0077] The aims of this study were to examine whether the C.
difficile vaccine would be safe, immunogenic, and prevent relapse
in patients with multiple recurrences of CDAD. An open-label, pilot
study was performed in 3 subjects (one male, aged 51 years, and two
females, aged 71 and 33 years). Each subject had developed CDAD
following antibiotic use and had a documented history of recurrent
CDAD with positive stool tests for C. difficile toxins. Their
diarrhea had improved on treatment with metronidazole or
vancomycin, but in all cases CDAD recurred on at least 3 occasions
within 3 to 14 days of discontinuing antibiotic treatment. The
subjects had also failed to respond to a variety of other
treatments for recurrent CDAD, including oral probiotic therapy,
cholestyramine, rifampicin, and intravenous immunoglobulin. As a
result, at the time of study entry they had required nearly
continuous treatment with metronidazole or vancomycin for periods
of 10, 22, and 9 months respectively.
[0078] The C. difficile toxoid vaccine was produced as described
previously (Kotloff et al., Infect. Immun. 69(2):988-995, 2001).
Briefly, toxins A and B were partially purified from the culture
filtrate of C. difficile strain ATCC 43255 by ammonium sulfate
precipitation. Toxins were further purified using an S300 Sephacryl
size exclusion column and were inactivated with formaldehyde. The
total protein concentration of the vaccine was 0.52 mg/ml, of which
toxins A and B comprised about 44% at a 1.5:1 toxin A to toxin B
ratio. The vaccine was diluted to contain 50 .mu.g protein per 0.4
ml and this was the dose delivered in each innoculation.
[0079] After completing their enrollment evaluation, the subjects
received four intramuscular inoculations of the C. difficile
vaccine in the deltoid region on days 0, 7, 28, and 56. All
subjects continued to take vancomycin orally (at least 125 mg bid)
until day 56 when it was discontinued. Blood samples were obtained
at each visit and on day 70. Serum anti-toxin antibody
concentrations were measured by ELISA and serum toxin neutralizing
activity was determined using a tissue culture cytotoxin assay
described previously (Kyne et al., New England Journal of Medicine
342(6):390-397, 2000; Kyne et al., Lancet 357(9251):189-193, 2001;
Kotloff et al., Infect. Immun. 69(2):988-995, 2001; Kelly et al.,
Antimicrobial Agents & Chemotherapy 40(2):373-379, 1996).
[0080] Vaccination was well tolerated and subjects reported minimal
discomfort at the injection sites. One subject (the 71 year old
woman) developed transient polyarthralgia after the fourth
inoculation. Two months later, polymyalgia rheumatica was diagnosed
and was considered to be possibly related to vaccination. She
received oral corticosteroid therapy with good effect.
[0081] Two subjects showed an increase in their serum IgG
anti-toxin A and anti-toxin-B antibody levels after vaccination
(FIG. 11). Both of these subjects also developed
cytotoxin-neutralizing activity against toxin A and toxin B in
their sera. One subject (the 51 year old man) did not demonstrate
increased serum anti-toxin antibody levels or neutralizing
activity. All three subjects were followed for two months after
they completed vaccination and discontinued vancomycin treatment
and none developed recurrent CDAD.
[0082] This study shows that a C. difficile toxoid vaccine can be
effective in inducing protective immune responses against toxin A
and toxin B in patients with recurrent CDAD. After vaccination all
three subjects who had previously required long-term treatment with
vancomycin were able to discontinue therapy without further
recurrence of CDAD.
[0083] All referenced cited herein are incorporated by reference in
their entirety. Other embodiments of the invention are within the
following claims.
* * * * *