U.S. patent number 4,411,888 [Application Number 06/403,751] was granted by the patent office on 1983-10-25 for composition of a novel immunogen for protection against diarrheal disease caused by enterotoxigenic escherichia coli.
This patent grant is currently assigned to The University of Rochester. Invention is credited to John D. Clements, Richard E. Engert, Frederick A. Klipstein.
United States Patent |
4,411,888 |
Klipstein , et al. |
October 25, 1983 |
Composition of a novel immunogen for protection against diarrheal
disease caused by enterotoxigenic Escherichia coli
Abstract
There is disclosed a novel immunogen which can be used for
immunological protection against acute diarrheal disease caused by
enterotoxigenic strains of Escherichia coli. The novel immunogen is
provided by cross-linking the E. coli heat-stable enterotoxin with
the E. coli heat-labile enterotoxin (either the complete holotoxin
or just the B subunit of this toxin) by a conjugation process in
the presence of a conjugating agent, typically the water soluble
carbodiimide, 1-ethyl-3-(3-diamethylaminopropyl) carbodiimide. This
results in a unique new molecule in that the toxic properties of
each individual toxin are greatly reduced, the heat-labile toxin
(or its B subunit) retains its antigenicity, and the heat-stable
toxin acquires immunogenicity as a function of the reaction.
Immunization with the cross-linked immunogen provides immunological
protection in mammals against water secretion induced by strains of
E. coli which produce the heat-labile or heat-stable enterotoxins,
either singly or together.
Inventors: |
Klipstein; Frederick A.
(Rochester, NY), Engert; Richard E. (Webster, NY),
Clements; John D. (Pittsford, NY) |
Assignee: |
The University of Rochester
(Rochester, NY)
|
Family
ID: |
26957960 |
Appl.
No.: |
06/403,751 |
Filed: |
June 3, 1982 |
PCT
Filed: |
June 03, 1982 |
PCT No.: |
PCT/US82/00763 |
371
Date: |
June 03, 1982 |
102(e)
Date: |
June 03, 1982 |
PCT
Pub. No.: |
WO83/00018 |
PCT
Pub. Date: |
January 06, 1983 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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276409 |
Jun 22, 1981 |
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Current U.S.
Class: |
424/194.1;
424/197.11; 424/236.1; 424/241.1; 424/257.1; 424/261.1 |
Current CPC
Class: |
A61K
39/107 (20130101); A61K 39/0258 (20130101); Y02A
50/474 (20180101); A61K 2039/6037 (20130101); Y02A
50/30 (20180101) |
Current International
Class: |
A61K
39/106 (20060101); A61K 39/108 (20060101); A61K
039/108 (); A61K 039/106 () |
Field of
Search: |
;424/88,92 |
References Cited
[Referenced By]
U.S. Patent Documents
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|
4220584 |
September 1980 |
Limyuco et al. |
4314993 |
February 1982 |
Weynendaele |
|
Other References
Deneke, R. et al., Infect. Immun., vol. 32, pp. 1254-1260, 1981.
.
Gill, D. M. et al., Infect. Immun., vol. 33, pp. 677-682, 1981.
.
Holmgren, J., Nature, vol. 292, pp. 413-417, 1981. .
Clements, J. D., et al., Infect. Immun., vol. 22, pp. 709-713,
1981. .
Pierce, N. F., Infect. Immun., vol. 18, pp. 338-341, 1977. .
Klipstein, F. A., et al., Infect. Immun., vol. 31, pp. 144-150,
1981. .
Klipstein, F. A., et al., Infect. Immun., vol. 32, pp. 1100-1104,
1981. .
Frantz, J. C., et al., Infect. Immun., vol. 33, pp. 193-198, 1981.
.
Giannella, R. A., et al., Infect. Immun., vol. 33, pp. 186-192,
1981. .
Klipstein, F. A., et al., Infect. Immun., vol. 34, pp. 637-639,
1981. .
Klipstein, F. A., et al., Infect. Immun., vol. 23, pp. 592-599,
1979. .
Clements, J. D., et al., Infect. Immun., vol. 24, pp. 760-769,
1979. .
Staples, S. J., et al., J. Biol. Chem., vol. 255, pp. 4716-4721,
1980. .
Chan, S., et al., J. Biol. Chem., vol. 256, pp. 7744-7746. .
Klipstein et al., J. Infect. Dis., vol. 147, No. 2, pp. 318-325,
1983. .
Erlanger, B., Pharmacological Reviews, vol. 25, pp. 271-280, 1973.
.
Peters et al., Ann. Rev. Biochem., vol. 46, pp. 523-551,
1977..
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Primary Examiner: Hazel; Blondel
Attorney, Agent or Firm: LuKacher; Martin
Parent Case Text
BACKGROUND
This application is a continuation in part of application Ser. No.
06/276,409, filed June 22, 1981, now abandoned.
Claims
We claim:
1. A composition for immunization of mammals against diarrheal
disease comprising the product provided by the process which
comprises reacting heat-stable (ST) enterotoxin of Escherichia coli
with a reactant selected from the group consisting of heat-labile
(LT) enterotoxin of Escherichia coli, the B subunit of said heat
labile (LT) enterotoxin, cholera toxin (CT) holotoxin of Vibrio
cholerae, and the B subunit of said cholera toxin (CT) holotoxin at
a molar ratio of ST to reactant of at least 1 to 1 in the presence
of a conjugating reagent.
2. The composition of claim 1 in combination with a suitable
adjuvant to provide an effective, nontoxic immunogen for use in
immunization to protect against diarrheal disease caused by strains
of homologous and heterologous somatic serotypes of Escherichia
coli which produce the heat-labile or heat-stable enterotoxins,
either singly or together.
3. The composition of claim 1 wherein the molar ratio of ST to
reactant in the reaction mixture is at least 5 to 1.
4. The composition of claim 1 wherein the conjugating reagent is a
water-soluble carbodiimide or other effective conjugation
reagent.
5. The composition of claim 4 wherein the carbodiimide is
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
6. The composition of claim 1 wherein the reaction is allowed to
proceed for a period of time in excess of 1 hour.
7. The composition of claim 1 in combination with a suitable
adjuvant to provide an effective immunogen for immunological
protection against diarrheal disease due to strains of
enterotoxigenic Escherichia coli.
8. The composition of claim 7 wherein the adjuvant is Freund's
complete adjuvant or other suitable adjuvants such as alum.
9. The composition of claim 1 wherein the reactant is the
heat-labile (LT) enterotoxin.
10. The composition of claim 1 wherein the reactant is the B
subunit of heat-labile (LT) enterotoxin.
11. The composition of claim 1 wherein the reactant is cholera
toxin (CT) holotoxin.
12. The composition of claim 1 wherein the reactant is the B
subunit of cholera toxin (CT) holotoxin.
13. A method of immunization of mammals against diarrheal disease
due to strains of enterotoxigenic Escherichia coli which comprises
administering an effective amount of the composition of claim
1.
14. The method of claim 13 wherein the composition is administered
parenterally.
15. The method of claim 13 wherein the composition is administered
perorally.
16. The method of claim 13 wherein the reactant is the heat-labile
(LT) enterotoxin or its B subunit.
17. The method of claim 13 wherein the reactant is cholera toxin
(CT) holotoxin or its B subunit.
18. The method of claim 13 wherein the mammal is human.
Description
Acute diarrheal disease due to transient colonization of the small
intestine by enterotoxigenic strains of Escherichia coli (ETEC) is
a major health problem of global scope. These organisms and
rotavirus are the two principal causes of acute diarrhea in young
children, which, according to World Health Organization estimates,
accounts for approximately ten million deaths per annum among
infants, mostly in those living in underdeveloped tropical
countries (See Black, R. E., et al. 1981. Incidence and severity of
rotavirus and Escherichia coli diarrhea in rural Bangladesh. Lancet
1:141-143). Enterotoxigenic E. coli are also the principal cause of
acute diarrhea among persons from temperature zones who travel to
the tropics (turista), a common cause of sporadic episodes of
diarrhea among adults living in temperate and tropical areas, and a
major problem in animal husbandry by virtue of their causing
frequently fatal acute diarrhea among weanling animals, especially
in lambs and piglets.
The mechanisms by which ETEC strains cause diarrhea have been
elucidated. Following peroral ingestion, the bacteria adhere to the
surface of the intestinal mucosa of the proximal small bowel; this
process is enhanced by the presence on the surface of the bacteria
of plasmid-induced specific fimbrial antigens which are host
specific and are referred to as colonization factors in human
strains (See, Evans, D. G., et al. 1978. New surface-associated
heat-labile colonization factor antigen (CFA/II) produced by
enterotoxigenic Escherichia coli of serogroups 06 and 08. Infect.
Immun. 21:638-647). There appear to be multiple, antigenically
dissimilar fimbrial antigens in human ETEC strains, and no specific
fimbrial antigen has been detected in the case of some pathogenic
ETEC strains (See Deuke, R. et al. 1981. Serotypes of attachment
pili of enterotoxigenic Escherichia coli isolated from humans.
Infect. Immunm. 32:1254-1260 and Levine, M. M., et al. 1980.
Hemagglutination and colonization factors in enterotoxigenic and
enteropathogenic Escherichia coil that cause diarrhea. J. Infec.
Dis. 141:733-737). The bacteria proliferate in this location and
elaborate two plasmid-induced enterotoxins, either singly or
together: a heat-labile (LT) toxin and a heat-stable (ST)
toxin.
The holotoxin (complete toxin) of the LT toxin has recently been
isolated in purified form and characterized (See, Clements, J. D.,
et al. 1979. Isolation and characterization of homogeneous
heat-labile enterotoxins with high specific activity from
Escherichia coli cultures. Infect. Immun. 24:760-769). It consists
of five B subunits (MW 12,000 daltons each) and one A subunit (MW
31,500 daltons) as reported by Gill, D. M. et al. 1981. Subunit
number and arrangement of Escherichia coli heat-labile enterotoxin.
Infec. Immun. 33:677-682. The B subunits are responsible for
attaching the toxin to specific GM.sub.1 ganglioside receptors on
the surface of the intestinal mucosa, thereby permitting
penetration of the cell by the A subunit which stimulates
intracellular adenylate cyclase which is responsible for secretion
of fluid and electrolytes ito the intestinal lumen. It is
noteworthy that the E. coli LT toxin functionally, structurally and
immunologically resembles the toxin produced by Vibrio cholerae
(cholera toxin). Both LT and cholera toxin (CT) consist of the same
type and number of subunits which have approximately the same
molecular weight; the subunits serve the same functions, and the LT
and CT have shared and distinct antigenic determinants in both of
their A and B subunits (See Holmgren, J., 1981. Actions of cholera
toxin and the prevention and treatment of cholera. Nature
292:413-417 and Clements, J. D. et al. 1978. Shared and unique
immunological determinants of enterotoxins from Vibrio cholerae and
Escherichia coli. Infec. Immun. 22:709-713). Further, immunization
of rats with LT or cholera toxin provides protection against
challenge with the LT toxin (See, Pierce, N. F. 1977. Protection
against challenge with Escherichia coli heat-labile enterotoxin by
immunization of rats with cholera toxin-toxoid. Infect. Immun.
18:338-341, and Klipstein, F. A. et al. 1981. Protective effect of
immunization of rats with holotoxin or B subunit of Escherichia
coli heat-labile enterotoxin. Infect. Immun. 31:144-150).
E. coli heat-stable toxin (ST) obtained from a human strain has
recently been characterized in purified form, as reported in
Staples, S. J., et al., 1980. Purification and characterization of
heat-stable enterotoxin produced by a strain of E. coli pathogenic
for man. J. Biol. Chem. 255:4716-4721. ST has a molecular weight of
approximately 2,000 daltons. It is uncertain how this toxin
attaches to the surface of the mucosal cell; once located
intracellularly, it causes fluid and electrolyte secretion by means
of stimulating intracellular guanylate cyclase. Approximately 20%
of human ETEC strains produce just LT (LT.sup.+ /ST.sup.-), 60%
produce both LT and LT (LT.sup.+ /ST.sup.+) and 20% elaborate only
ST (LT.sup.- /ST.sup.+); each of these types is capable of causing
acute diarrhea.
The most practical approach for the prevention of the widespread
morbidity and mortality caused by diarrheal disease due to
intestinal contamination with ETEC strains of E. coli would be by
means of a program of protective vaccination. Three different types
of E. coli antigen have been shown to be effective as immunogens
which provide protection against challenge with ETEC strains in
experimental models.
(i) Immunization with somatic antigens (usually in the form of the
killed whole bacterim) prevents diarrhea by means of reducing
bacterial growth within the small intestine; this, however, extends
only to homologous somatic serotypes and not to heterologous
serotypes of which 164 antigenically dissimilar somatic serotypes
of E. coli are recognized (See, Gay, C. C., 1971. Problems of
immunization in the control of Escherichia coli infection. Ann.
N.Y. Acad. Sci. 176:336-349).
(ii) Immunization with the specific fimbrial antigen responsible
for adherence and colonization of the bacteria on the surface of
the intestinal mucosa also provides protection, but this does not
extend to ETEC strains possessing antigenically different fimbrial
antigens (See, Morgan, R. L., et al., 1978. Immunization of
suckling pigs against enterotoxigenic Escherichia coli-induced
diarrheal disease by vaccinating dams with purified 987P or
K99pili: Protection correlates with pilus homology of vaccine and
challenge. Infect. Immun. 22:771-777) and multiple antigenically
dissimilar fimbrial antigens have been detected among animal and
human ETEC strains (as cited above).
(iii) Immunization with either the E. coli LT holotoxin or its B
subunit arouses an antitoxin response which provides protection
against active challenge with the toxin itself and viable bacterial
strains which produce just LT (LT.sup.+ /ST.sup.-) as well as LT
and ST toxins (LT.sup.+ /ST.sup.+) (See, Klipstein F. A. et al.,
1979. Protective effect of active immunization with purified
Escherichia coli heat-labile enterotoxin in rats. Infect. Immun.
23:592-599 and Klipstein, F. A. et al. 1981. Protective effect of
immunization of rats with the holotoxin of B subunit of Escherichia
coli heat-labile enterotoxin. Infect. Immun. 31:144-150). LT
produced by different somatic serotypes is antigenically
homogeneous and thus immunization with this toxin provides
protection against all LT-producing strains irrespective of their
somatic serotypes or fimbrial antigens (See, Klipstein, F. A., et
al. 1981. Immunization of rats with heat-labile enterotoxin
provides uniform protection against heterologous serotypes of
enterotoxigenic Escherichia coli Infect. Immun. 32:1100-1104).
Immunization with LT or its B subunit does not, however, provide
protection against ETEC strains which produce just the ST toxin
(LT.sup.- /ST.sup.+) (See, Klipstein, F. A., et al., 1979. Cited
above).
The low molecular weight ST toxin is nonantigenic; recently,
however, procedures have been described for obtaining purified ST
from bovine, porcine and human ETEC strains and several studies
have shown that ST is haptenic. Immunization of rabbits or goats
with purified ST coupled to a large molecular weight carrier, such
as bovine serum albumin or bovine immunoglobulin G, has been shown
to arouse antitoxin to ST, as demonstrated by the ability of the
antiserum to neutralize the activity of ST in the suckling mouse
assay (See, Frantz, J. C., et al., 1981. Immunological properties
of Escherichia coli heat-stable enterotoxins: development of a
radioimmunoassay specific for heat-stable enterotoxins with
suckling mouse activity. Infect. Immun. 33:193-198, and Giannella,
R. A., et al., 1981. Development of a radioimmunoassay for
Escherichia coli heat-stable enterotoxin: comparison with the
suckling mouse bioassay. Infect. Immun. 33:186-192). Further,
immunization of rats with a semipurified preparation of ST coupled
to porcine immunoglobulin has been shown to provide protection
against active challenge with either the ST toxin or viable strains
which produce just this toxin, but not against the LT toxin or
LT-producing viable strains (See, Klipstein, F. A., et al. 1982.
Protection in rats immunized with Escherichia coli heat-stable
enterotoxin. Infec. Immun. 34:637-639).
None of these toxin forms are acceptable for use as a vaccine for
immunization against ETEC strains of E. coli. The LT holotoxin
itself (or cholera toxin) is not a practical immunogen for use in
human immunization for several reasons. Firstly, its immunogenic
form retains toxicity which would result in unacceptable side
reactions (muscle and skin inflammation when given parenterally and
diarrhea when given perorally) if administered to humans. Secondly,
immunization with either the LT holotoxin or its B subunit provides
no protection against those ETEC strains that produce just the ST
toxin (See, Klipstein, F. A., et al. 1979. Protective effect of
active immunization with purified Escherichia coli heat-labile
enterotoxins in rats. Infect. Immun. 23:592-599), which are also a
common cause of acute diarrhea. The ST toxin is not practical since
it is toxic; it is nonantigenic and the large molecular weight
carriers of animal protein needed to render it immunogenic are
unsafe for human use; and immunization with this toxin fails to
provide protection against ETEC strains which produce the LT
toxin.
Thus, what is needed is a vaccine composed of a novel immunogen (a)
that would provide immunological protection against ETEC strains
which produce either LT or ST, (b) in which the ST toxin is
rendered immunogenic, (c) whose toxicity is sufficiently attenuated
so that immunization with this material does not yield adverse side
reactions, and (d) which contains the B subunit (either alone or as
part of the LT holotoxin) that would enhance peroral immunization
by virtue of its property of attaching to specific receptor sites
on the surface of the intestinal mucosa, thus permitting prolonged
exposure of the mucosal cells to the immunogen.
SUMMARY OF THE INVENTION
It is, therefore, an object of this invention to provide a novel
composition that is nontoxic but effective in providing
immunological protection in mammals against acute diarrheal disease
caused by enterotoxigenic strains of E. coli which produce LT or
ST, either singly or together.
Another object of this invention is to provide a novel vaccine for
the prevention of ETEC induced acute diarrheal disease.
Another object of this invention is to provide a vaccine which
provides protection against ETEC organisms which produce either LT
or ST.
Another object of this invention is to provide a composition
combining ST and LT (or its B subunit) wherein the toxic properties
of the individual toxins are reduced, their respective antigenicity
is retained, and the property of the B subunit to adhere to
specific mucosal receptors is maintained.
In accordance with this invention there is provided the product of
the process of reacting the heat-labile (LT) enterotoxin (either in
the form of the holotoxin or just its B subunit) or cholera toxin
(either in the form of the holotoxin or just its B subunit) and
heat-stable (ST) enterotoxin of Escherichia coli in the presence of
a suitable conjugating reagent. While not being held to any single
theory, it is proposed that the product of the above described
process is a cross-linked molecule via the available carboxyl and
amino groups in each of the LT or CT and ST molecules and,
additionally, involving intrachain linking of the LT or CT
molecule. Accordingly, as employed in this specification and
claims, the product of the process of reacting ST with a reactant
selected from the group consisting of LT or CT holotoxin is
referred to as cross-linked ST-LT or ST-CT and that of reacting ST
to the B subunit as cross-linked ST-B.
The above cross-linked molecules are effective in a vaccination
method wherein the composition is administered together with a
suitable adjuvant for both primary immunization and booster
immunizations in typical vaccination procedures such as are
reported in Klipstein, F. A., et al., 1981. Protective effect of
immunization of rats with the holotoxin or B subunit of Escherichia
coil heat-labile enterotoxin. Infect. Immun. 31:144-150 which is
hereby incorporated by reference. The composition of this invention
is effective in mammals including humans.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphical representation of the data obtained and
methodology where an enzyme-linked immunosorbent assay (ELISA) was
used to compare the antigenicity of different immunogens.
Illustrated by a comparison of the LT antigenicity of a preparation
of cross-linked ST-LT with LT alone.
FIG. 2 is a graphical representation of data obtained in
determining the secretory response to purified LT and ST in
unimmunized rats.
FIG. 3 is a graphical representation of the effect of the ratio of
EDAC to total toxin protein or the degree of ST conjugation from a
molar ratio of semipure ST/LT of 100/1. Reaction time was 18 hours.
Values are the percent ST present in the final conjugate.
FIG. 4 is a graphical representation of the effect of the initial
molar ratio of either pure ST to LT or pure ST to the B subunit on
the percentage of ST present in the final conjugate as determined
using a tracer dose of radioiodinated ST. The EDAC/total protein
ratio was 45/1 and the reaction time was 18 hours.
FIG. 5 is a graphical representation of the effect of the duration
of the conjugation reaction of the degree of conjugation of
semipure ST from an ST/LT molar ratio of 100/1 using an EDAC/total
toxin protein ratio of 10/1. Values for antigenicity were derived
from corrected samples adjusted to reflect 100% of the toxin.
DETAILED DESCRIPTION OF THE INVENTION NH.sub.
The development of a vaccine protocol using the ST and LT or CT (or
their B subunit) toxins required that each be manipulated in such a
way as to reduce its biological activity and yet continue to be
sufficiently immunogenic so as to stimulate an appropriate immune
response. Rather than introduce an irrevelant antigen into this
equation as a carrier, LT and CT (or their B subunit) were chosen
as an immunological vehicle for ST. ST is composed of 10 different
amino acids, a total of 18 amino acid residues, one-third of which
are half-cystine. Each molecule has three amino groups available
for cross-linking to a carrier (two asparagine residues, one of
which is the NH.sub.2 -terminal amino acid) and one carboxyl group
available for cross-linking (one glutamic acid residue). LT is
composed of 806 amino acid residues (assuming a structural formula
A.sub.1 B.sub.5) with one hundred and thirty three available amino
groups (mostly as epsilon amino groups in lysine and arginine) and
one hundred forty nine carboxyl groups (present as aspartic acid
and glutamic acid). The invention has as its concept to cross-link
ST and LT or CT (or their B subunit) via the available carboxyl and
amino groups on the two molecules and, additionally, to intrachain
link the LT or CT molecules as a means of reducing toxicity.
Water soluble carbodiimides have been used extensively as
conjugating reagents in the preparation of conjugated antigens. The
most useful water-soluble carbodiimides are
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and
1-cyclohexyl-3-[2-morpholinyl(4)-ethyl] carbodiimide (MCDI). Any
suitable conjugating reagent is useful in the process of the
invention to produce cross-linked ST-LT, ST-CT or ST-B. The above
mentioned water-soluble carbodiimides are presently preferred.
In the following examples and description of this invention
numerous references are made to prior publications. All of these
publications are hereby incorporated herein by reference so as to
provide a more concise description of this invention.
EXAMPLES
Preparation of the toxins
The LT holotoxin was produced in purified form by the methods
described by Clements, J. D. et al., 1979. Isolation and
characterization of homogeneous heat-labile enterotoxins with high
specific activity from Escherichia coli cultures. Infect. Immun.
24:760-769, from E. coli strain 711 (FlLT), a transformed K-12
derivative bearing LT gene(s) of the Ent plasmid from porcine
strain P307, described by So, M, et al. 1978. Characterization of
an Escherichia coli plasmid encoding for synthesis of heat-labile
toxin: Molecular cloning of the toxin determinant. Infect. Immun.
21:405-411. The homogeneity of this toxin preparation was confirmed
by polyacrylamide gel electrophoresis; its biological activity was
established by demonstrating its ability to activate Y-1 adrenal
cells in tissue culture assay (a standard test for LT activity),
and its antigenic homogeneity was confirmed by the demonstration in
immunodiffusion of a homogeneous, single band reaction with goat
monospecific LT hyperimmune serum. The B subunit was separated from
the LT holotoxin by the chromatographic techniques described by
Clements, J. D., et al., 1980. Properties of homogeneous
heat-labile enterotoxin from Escherichia coliInfect. Immun.
29:91-97.
The St toxin was produced from E. coli strains 18D (042:H37) and
Texas 452 (078:H12), which are LT.sup.- (ST.sup.+ strains obtained
from human sources, and made in purified form by the method
described by Staples, S. J. et al. 1980. Purification and
characterization of heat-stable enterotoxin produced by a strain of
E. coli pathogenic for man. J. Biol. Chem. 255:4716-4721. This
process involves the following steps: 1 Culture filtrate .fwdarw.2
Amberlite XAD-2chromatography .fwdarw.3 acetone precipitation
.fwdarw.4 first Sephadex G-25 chromatography.fwdarw.5 DEAE
Sephacryl chromatography.fwdarw.6 second Sephadex G-25
chromatography.fwdarw.7 thin layer chromatography. The activity of
the various products obtained from each processing step was
determined by the suckling mouse assay as described by Giannella,
R. A., 1976. Suckling mouse model for detection of heat-stable
Escherichia coli enterotoxin. Characteristics of the model. Infec.
Immun. 14:95-99. Values are given in suckling mouse units which are
defined as that amount of toxin which yields an intestinal/carcass
weight ratio of .gtoreq.0.083. Totally pure ST (obtained by step 7)
contained 250 mouse units per ug; this material was used for
developing specific hyperimmune antisera in rabbits and goats and
for the radioiodination studies. Semipure ST, obtained after step
4, contained 185 mouse units per ug; this material was used for the
conjugation and immunization studies.
Concentrations of the toxin preparations and their conjugates are
expressed in terms of protein content, which was determined by the
method of Lowry (See, Lowry, O. H., et al. 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem.
193:265-275.)Molar equivalents for the toxins were based on
published values of 91,450 daltons for the LT holotoxin, 57,400
daltons for the B subunit in their pentamer form, and 2,000 daltons
for the ST toxin.
Cross-linking
The LT and ST toxins were cross-linked using the carbodiimide
reaction, whose use in general has been reviewed by Bauminger, S.
et al. 1980. The use of carbodiimides in the preparation of
immunizing conjugates, p. 151-159. In S. P. Colowick and N. O.
Kaplan (ed.), Methods in enzymology. Academic Press, New York.
Except where specifically noted, the conjugating material was
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). Cross-linked
ST-LT compositions were also prepared using
1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl] carbodiimide (MCDI) in
order to show that the results of the conjugation procedures were a
functiion of cross-linking by a variety of processes rather than
being specifically related to EDAC. The details of the
cross-linking procedures for each of the different compositions
evaluation, and the variable in the cross-linking process (such as
ratio of the toxins, sequence of conjugations, amount of EDAC used,
pH, temperature and time of the reaction), are described below. In
each instance, following completion of the conjugation reaction,
the conjugates were exhaustively dialyzed against water using a
12,000 molecular weight cutoff bag which retained the conjugate but
not unconjugated ST or the conjugating reagent (i.e., EDAC). The
amount of ST present in the final conjugate was indicated by the
increased amount of protein over that of the LT or B subunit
originally added.
Assay of Toxins and cross-linked LT-ST for toxicity and
antigenicity
Each cross-linked ST-LT composition was tested for the properties
of ST and LT toxicity and antigenicity, and the results were
compared to those obtained by similar tests for the unconjugated
toxins. The results are expressed as either the fold change .DELTA.
or the percentage reduction in the cross-linked compositions as
compared to the unconjugated toxins.
(a) LT Toxicity was assessed by means of testing serial two-fold
dilutions of the test material in tissue culture using Y-1 adrenal
cell tissue culture using the methodology described by Sack, D. A.
et al. 1975. Test for enterotoxigenic Escherichia coli using Y-1
adrenal cells in miniculture. Infect. Immun. 11:334-336. This assay
is a measure of the toxin's ability to stimulate adenylate cyclase
and hence, its ability to induce the intestinal mucosa to secrete
water and electrolytes.
(b) LT Antigenicity was tested by enzyme-linked immunosorbent assay
(ELISA) using published methods (See, Klipstein, F. A. et al. 1981.
Protective effect of immunization of rats with the holotoxin or B
subunit of Escherichia coli heat-labile enterotoxin. Infect. Immun.
31:144-150). Two-fold serial dilutions of the test material was
tested directly against goat hyperimmune serum to purified LT
(titer 1:102,000) using rabbit antiserum to goat conjugated with
alkaline phosphatase and p-nitrophenyl phosphate as the substrate.
The ratio of activity of the conjugate to unconjugated LT was
derived from those dosages which yielded an optical density or OD
of 600, such as is illustrated in FIG. 1 in which case the
antigenicity of the cross-linked LT-ST was reduced 1.4-fold.
(c) ST Toxicity was tested by the suckling mouse assay, as
described in Giannella, R. A. 1976 (cited before). Values reported
are for the reduction in the mouse units of the cross-linked
immunogens as compared to the same amount of unattenuated ST.
(d) ST Antigenicity was evaluated by means of a "double-sandwich"
ELISA in which two-fold serial dilutions of the test material were
placed between two different hyperimmune sera to ST, which were
developed in goats and rabbits to ST; antibody titers of these
antisera to ST toxin were 1:131,000. Values for the conjugates were
compared to ST in a similar fashion as that described for LT
anigenicity.
Rat immunization and challenge
Rats were immunized and challenged using procedures which have been
described in detail by Klipstein, F.A. et al. 1981. Protective
effect of immunization of rats with the holotoxin or B subunit of
Escherichia coli heat-labile enterotoxin. Infect. Immun.
31:144-150. Parenteral immunization was given intraperitoneally
using Freund's complete adjuvant for the primary immunization.
Peroral (PO) booster immunizations were given via an intragastric
tube administered two hours after the peroral administration of
cimetidine (available under the trade name Tagamet from Smith,
Kline & French Co., Philadelphia, Pa.)to ablate gastric
acidity.
Immunized rats were challenged with the LT and ST toxins and with
viable organisms of E. coli strain PB 258, which produces just LT
(LT.sup.30 /ST.sup.31), strain H 10407 which produces both LT and
ST (LT.sup.30 /ST.sup.30), and strain Texas 452 which produces just
ST (LT.sup.31 /ST.sup.30). Each test material was given at that
amount which yields maximum secretion in unimmunized animals (as
illustrated in FIG. 2). Each datum point was obtained by testing 3
animals. The results are expressed as the mean .+-. standard error
of the mean (SEM) of the percent reduced secretion in immunized
rats as compared to the value in unimmunized animals similarly
challenged. Reduced secretion of >50% in immunized animals
represents a statistically significant (p<0.001) difference
between the amount of secretion in these rats and in unimmunized
control animals.
OBSERVATIONS
(1) Factors affecting the cross-linking of ST to LT toxin.
(a) Effect of the ratio of conjugating agent (EDAC) to toxins
Initial studies (the data for which were summarized in Table 2 of
the original patent application) indicated that a ratio (by weight)
of EDAC to total toxin protein of 45/1 yielded significant
cross-linking of ST to LT with reduced LT toxicity (>1000-fold)
and ST toxicity (>100-fold) and strong retained antigenicity,
whereas an EDAC/toxin ratio of 0.45/1 did not achieve this.
In order to determine precisely the optimal ratio of EDAC to toxins
necessary to yield the maximum degree of cross-linking, an initial
100/1 molar ratio of ST/LT was exposed to ratios of EDAC to toxin
protein that varied between 2/1 and 200/1. The reaction was run at
a pH of 7.0, at 20.degree. C., with a reaction time of 18 hours.
The results, which are graphically illustrated in FIG. 3, confirm
the fact that maximum cross-linking of ST to LT occurs under these
conditions at an EDAC/toxin ratio of approximately 45/1.
(b) Effect of the ratio of ST conjugated with LT
Under conditions of conjugation with 100 mg EDAC for a 1 hour
exposure time, increasing the initial ST/LT ratio (by weight)
resulted in a greater proportion of ST in the final conjugate. The
data obtained is presented in Table 1 below.
TABLE 1
__________________________________________________________________________
EFFECT OF THE RATIO OF ST CONJUGATED WITH LT. Characteristics of
the Conjugation Process.sup.a cross-linked toxins ST:LT ST + LT +
EDAC Protein .DELTA.LT .DELTA.ST Ratio mg mg mg Total % ST Y1 ELISA
SM ELISA
__________________________________________________________________________
15:1 3 0.2 100 436 77 ND 1.4 ND.sup.b 3.8 10:1 2 0.2 100 307 45 ND
1.6 ND 3.8 5:1 1 0.2 100 250 32 ND 1.4 ND 14.2 1:1 0.1 0.2 100 227
25 ND 2.8 ND 21.9
__________________________________________________________________________
.sup.a All reactions were run for 60 min at pH 7.0. .sup.b ND
signifies not determined.
In order to confirm these observations, pure ST was radioiodinated
with carrier free I.sup.125 by the method of chloramine-T method
described by Hunter, R. 1970. Standardization of the chloramine-T
method of protein iodination. Proc. Soc. Exp. Biol. Med. Molar
ratios of ST/LT or ST/B varying from 1/1 to 200/1 together with a
tracer dose of ST.sup.125 were conjugated at 4.degree. C. for 18
hours in the presence of an EDAC/toxin protein ratio of 45/1.
The results are shown graphically in FIG. 4.
Cross-linked ST-LT
Progressively higher initial ST/LT ratios resulted in a linear
increase in the percentage of the ST added to the initial mixture
that was retained in the conjugate, thus resulting in progressive
increases in the proportion of ST of the final conjugate. Using an
initial ST/LT molar ratio of 100/1, the final conjugate contained
96% ST by moles.
Cross-linked ST-B
Progressively higher initial ST/B ratios also resulted in a linear
increase in the proportion of ST in the conjugate. Using an initial
ST/B molar ratio of 125/1, the final conjugate contained 97% ST by
moles.
(c) Effect of duration of the conjugation process
An 18 hour conjugation reaction using an EDAC/toxin protein ratio
of 45/1 yielded significantly greater reduction in toxicity of the
LT toxin than the 1 hour reaction time. LT and ST antigenicity were
not appreciably reduced by the 18 hour reaction in the conjugation
sequence of LT+ST+EDAC, but antigenicity was moderately reduced in
the other sequences of conjugation. The data obtained is set forth
below in Table 2.
TABLE 2
__________________________________________________________________________
EFFECT OF THE DURATION OF THE CONJUGATION PROCESS Characteristics
of the cross-linked toxins Conjugation process.sup.a Protein
.DELTA.LT .DELTA.ST Sequence Duration Total % ST Y1 ELISA SM ELISA
__________________________________________________________________________
LT + ST + EDAC 60 min. 254 24 4,333 1.6 >100 0 LT + ST + EDAC 18
hr. 463 59 69,333 2.1 >100 1.6 LT + EDAC + ST 60 min. 263 35
1,078 2.5 >100 0 LT + EDAC + ST 18 hr. 363 48 657,894 8.4
>100 8.6 ST + EDAC + LT 60 min. 223 24 267 3.5 >100 3.6 ST +
EDAC + LT 18 hr. 432 56 17,333 6.7 >100 3.2
__________________________________________________________________________
.sup.a All reactions were with an ST:LT ratio of 10:1 run at pH
7.0, at 20.degree. C. for 60 min. and 4.degree. C. for 18 hr.
Additional studies evaluated the effect of varying the conjugation
time using a conjugation mixture added in the sequence of
LT+ST+EDAC with a molar ratio of ST/LT of 100/1 and an EDAC/toxin
protein ratio of 10/1 at pH 7.0 and 4.degree. C. As shown in
summary form in FIG. 5, increasing the conjugation reaction time
between 2 and 192 hours resulted in a progressive increase in the
amount of ST coupled to LT in the absence of significant
attenuation of the antigenicity of either conjugated toxin.
(d) Comparison of the effectiveness of different carbodiimides in
cross-linking LT and ST
Under conditions of conjugation of 18 hours with 100 mg of
carbodiimide, both EDAC and MCDI yielded a cross-linked ST-LT
molecule which had the characteristics of reduced toxicity with
persistent antigenicity. The cross-linked molecule produced with
EDAC had appreciably greater reduced toxicity with persistent
antigenicity. The data obtained is presented in Table 3 below.
TABLE 3 ______________________________________ COMPARISON OF THE
EFFECTIVENESS OF DIFFERENT CARBODIIMIDE REAGENTS IN CONJUGATING LT
AND ST Characteristics of the Protein cross-linked toxins
Conjugation.sup.a Total ST .DELTA.LT .DELTA.ST Sequence (mg) (%) Y1
ELISA SM.sup.b ELISA ______________________________________ LT +
463 59 69,333 1.8 >500 1.4 ST + EDAC LT + 932 80 10 2.6 >100
7.4 ST + MCDI ______________________________________ .sup.a All
reactions used an ST:LT ratio of 10:1 with 100 mg of carbodiimide
reagent and were run for 18 hr. at 4.degree. C. .sup.b Tested at
500 ng and 2,500 ng.
(e) Conjugation of ST with cholera toxin
E. coli ST may be conjugated with CT holotoxin (or its B subunit)
by the same conjugation conditions employed for E. coli ST
cross-linking to LT or its B subunit. Conjugation of ST to CT toxin
holotoxin or its B subunit follows the same pattern. As pointed out
above, the CT toxin is structurally and immunologically similar to
the LT toxin. As an example, conjugation of ST to CT using an ST/CT
molar ratio of 100/1 and an EDAC/total conjugate ratio of 10/1 for
18 hours yields a conjugate with 54 percent, by weight, and 98
percent, by moles, of ST. The antigenicity of the ST (50 percent)
and of CT (51 percent) are maintained in the reaction product.
(2) Optimal cross-linked vaccines and their effectiveness
The previously described observations identified optimum
conjugation conditions for cross-linking ST to either LT or its B
subunit as an initial molar ratio of 100/1 of ST/LT or ST/B, an
EDAC/total toxin protein ratio of 10/1 by weight, and a conjugation
reaction time of 96 hours at 4.degree. C.
(a) Cross-linked ST-LT vaccine
The properties of the cross-linked ST-LT immunogen prepared using
these conjugation conditions are presented in Table 4 below.
TABLE 4 ______________________________________ PROPERTIES OF THE
CROSS-LINKED ST-LT VACCINE. Adjusted to 100%.sup.a Per Unit
(ug).sup.b % Toxicity Antigenicity Toxicity Antigenicity Toxin Wgt
% % % % ______________________________________ ST 34 0.15 82 0.06
29 LT 66 0.06 83 0.03 51 ______________________________________
.sup.a Concentrations of the conjugates were adjusted to reflect
100% of each toxin. .sup.b Direct measurements on the uncorrected
vaccine.
Immunization
Rats were immunized with this vaccine in dosages of a single 1000
ug intraperitoneal prime and 2500 ug peroral boosts given twice.
The total peroral immunization contained approximately 1450 ST
antigen units (dosage x antigenicity) and 2550 LT antigen units. As
shown in Table 5, this immunization resulted in significant
protection against both the LT and ST toxins and viable bacteria
which produce these toxins.
TABLE 5 ______________________________________ RESULTS OF CHALLENGE
IN RATS IMMUNIZED WITH THE CROSS-LINKED ST-LT VACCINE. % Reduced
secretion Immuno- after challenge with.sup.a gen LT ST used toxin
LT.sup.+ /ST.sup.- LT.sup.+ /ST.sup.+ toxin LT.sup.- /ST.sup.+
______________________________________ LT alone 92 .+-. 2 69 .+-. 3
54 .+-. 1 0 3 .+-. 2 ST-LT 87 .+-. 7 70 .+-. 5 67 .+-. 3 68 .+-. 2
67 .+-. 2 ______________________________________ .sup.a All values
of >50% represent a significant (p < 0.001) reduction i
secretion.
(d) Cross-linked ST-B vaccine
The properties of the cross-linked ST-B subunit vaccine prepared
using the optimal conjugation conditions listed above under item 2
are shown in Table 6 below.
TABLE 6 ______________________________________ PROPERTIES OF THE
CROSS-LINKED ST-B VACCINE. Adjusted to 100% Per Unit (ug) Toxin %
Toxicity Antigenicity Toxicity Antigenicity
______________________________________ ST 30 0.14 81 0.06 22 B 70
0.00 100.sup.a 0.00 .sup. 43.sup.a
______________________________________ .sup.a Determined by ELISA
using hyperimmune antiserum to the B subunit.
Immunization
Based on the properties shown in Table 6, the cross-linked ST-B
vaccine contained 2,150 B subunit antigen units and 1,100 ST
antigen units in a total peroral dosage of 5,000 ug. Immunization
of rats with this dosage yielded strong protection against
challenge with the viable LT producing strain PB 258 (LT.sup.+
/ST.sup.-) (52.+-.2% reduced secretion) or the viable ST-producing
strain Texas 452 (LT.sup.- /ST.sup.+) (62.+-.1% reduced
secretion).
(3) Use of Synthetic ST in the cros-linked vaccine
The novel immunogen of cross-linked ST-LT or ST-B can be created by
conjugating ST derived by purification of the toxin from growth of
an enterotoxigenic strain of E. coli (biologic ST) as described
herein or by conjugating synthetic ST produced by an amino acid
synthesizer. The sequence of the 18 amino acids which compose
purified biologic ST obtained from growing strain 18D (ad described
in Staples et al. 1980. Purification and Characterization of
heat-stable enterotoxin produced by a strain of E. coli pathogenic
for man. J. Biol. Chem. 255:4716-4721) has been described by Chan
and Giannella, 1981. Amino acid sequence of heat-stable enterotoxin
produced by Escherichia coli pathogenic for man. J. Biol. Chem.
256:7744-7746. A synthetic ST with this sequence was produced by
Dr. Richard Houghten, Scripps Clinic and Research Foundation, La
Jolla, California. This synthetic ST toxin has been shown to be
biologically and immunologically identical to biologic ST in terms
of its ability to evoke fluid secretion in the suckling mouse assay
or ligated rat ileal loops and in the capacity of hyperimmune
rabbit antiserum to biologic ST to neutralize the secretory effect
of either synthetic and biologic ST in the suckling mouse
assay.
Synthetic ST was conjugated to the B subunit in reaction which
employed and EDAC/total protein ratio of 1.5/1, an ST/B subunit
molar ratio of 50/1, and a reaction time of 18 hr. As shown in
Table 7, the properties of this vaccine are practically identical
to those of the cross-linked biological ST-B vaccine whose
properties are summarized in Table 6.
TABLE 7 ______________________________________ PROPERTIES OF
CROSS-LINKED SYNTHETICST-B Subunit VACCINE Adjusted to 100% Per
Unit (ug) Toxin % Toxicity Antigenicity Toxicity Antigenicity
______________________________________ ST 36 0.13 85 0.06 42 B 64
0.00 .sup. 85.sup.a 0.00 .sup. 59.sup.a
______________________________________ .sup.a Determined by ELISA
using hyperimmune serum to the B subunit.
Immunization of rats with the synthetic ST-B vaccine by the same
techniques and at the same dosages described for the biologic ST-B
vaccine yielded significant protection against both the LT and ST
toxins and viable strains which produce these toxins, as shown in
Table 8 below.
TABLE 8 ______________________________________ RESULTS OF CHALLENGE
IN RATS IMMUNIZED WITH THE CROSS LINKED SYNTHETIC ST-B Subunit
VACCINE % Reduced secretion after challenge with.sup.a Syn- Bio- LT
thetic logic toxin LT.sup.+ /ST.sup.- LT.sup.+ /ST.sup.+ ST ST
LT.sup.- /ST.sup.+ ______________________________________ 94 .+-. 3
61 .+-. 2 68 .+-. 2 70 .+-. 1 97 .+-. 3 76 .+-. 2
______________________________________ .sup.a Values of >50%
represent a significant (p < 0.001) reduction of secretion in
immunized rats.
These observations show that conjugation of ST toxin, derived
either by purification of bacterial growth or produced
synthetically, to either LT or CT toxins (either holotoxin or just
its B subunit) in the presence of a carbodiimide results in a new
molecule,cross-linked ST-LT and ST-CT (or cross-linked ST-B), which
has the properties of reduced toxicity and persistent antigenicity
for each of the component toxins, and acquired immunogenicity for
the ST toxin. EDAC was the most effective conjugating agent but our
observations indicate that cross-linking is not specific for this
reagent. In vitro studies showed that the following variables
influence the cross-linking process: the sequence of conjugation,
the concentration of carbodiimide used, the ratio of ST to LT (or
its B subunit) used, and the reaction time. The optimum
cross-linking reaction yielded a molecule which contained 96% ST by
mols, the toxicities of the cross-linked toxins were reduced to
.ltoreq.0.15% that of the unconjugated toxins while their
antigenicities were maintained at >80% as determined by ELISA
assay. Similar conjugation conditions also yielded equivalent
properties when the ST toxin was cross-linked with the B subunit of
the LT enterotoxin or with CT. The immunogenic potency of these
molecules was documented by the fact that they provided strong
protection in immunized rats against challenge with either the LT
or ST toxins themselves or viable bacteria which produce these
toxins, either singly or together.
The ratio of ST or LT (or its B subunit) in the conjugate can be
varied as can the degree of intramolecular LT holotoxin or B
subunit cross-linking by varying the reaction conditions. The ST-LT
(or ST-B) conjugate, as such, is unique in that the biological
toxicities of both molecules are greatly reduced, the LT (or its B
subunit) retains its antigenicity, and ST has aquired
immunogenicity as a function of the reaction.
In use, the novel immunogen of this invention can be administered
to subjects, animal or human, in a variety of ways. Exemplary
methods include parenteral (subcutaneous) administration given with
a nontoxic adjuvant such as an alum precipitate or peroral
administration given after reduction or ablation of gastric
activity, or in a pharmaceutical form that protects the immunogen
against inactivation by gastric juice (e.g., a protective capsule
or microsphere).
The above results provide evidence that treatment of humans will be
accomplished by administering effective amounts of the novel
cross-linked ST-LT (or ST-B) composition of this invention.
Another aspect of this invention is the discovery that the
heat-labile enterotoxin (either the holotoxin or its B subunit) of
either Escherichia coli or Vibrio cholerae may be employed as a
carrier in place of the usual large molecular weight protein. Thus,
in accordance with this invention there is provided a novel carrier
to which toxins of relatively lower molecular weight can be
cross-linked. The LT or CT portion (or its B subunit) of the novel
cross-linked product is both a carrier and an immunogen. Both
toxins, as indicated above, have greatly reduced toxicity in the
combined form yet both act as immunogens. The presence of the B
subunit (either as the subunit alone or as part of the holotoxin)
enhances the value of this immunogen as a peroral vaccine by virtue
of the property of the B subunit to adhere to specific GM.sub.1
receptors on the surface of the intestinal mucosa thus rendering
close, persistent contact between the immunogen and the mucosa.
Although this property of the B subunit (either alone or as part of
the LT or CT holotoxin) which enhances peroral immunization has
been described as part of the novel cross-linked product of ST-LT
or ST-B (in which instance B serves as both a carrier and an
immunogen), this unique property of the B subunit of either LT or
cholera toxin can be employed when it is used exclusively as a
carrier for immunogens other than ST to enhance their effectiveness
when given as a peroral vaccine.
Although the invention has been described with respect to specific
examples of reagents, adjuvants and conditions, other equivalent
compositions and conditions can be utilized without departing from
the scope of this invention. The invention has been described
largely with respect to specific examples of providing a novel
immunogen by cross-linking the ST toxin to the E. coli LT holotoxin
or its B subunit. The structural, functional and immunological
close similarities between the cholera toxin holotoxin and its B
subunit and the E. coli LT holotoxin and its B subunit make it
evident that a similar novel immunogen with the properties
described herein is created by cross-linking the E. coli ST toxin
to either the cholera toxin holotoxin or its B subunit.
* * * * *