U.S. patent number 9,173,932 [Application Number 14/293,745] was granted by the patent office on 2015-11-03 for vibrio cholerae o139 conjugate vaccines.
This patent grant is currently assigned to The United States of America as Represented by the Secretary of the Department of Health and Human Services. The grantee listed for this patent is The United States of America as represented by the Secretary, Department of Health and Human Servies, The United States of America as represented by the Secretary, Department of Health and Human Servies. Invention is credited to Zuzana Kossaczka, John B. Robbins, Shousun Chen Szu.
United States Patent |
9,173,932 |
Szu , et al. |
November 3, 2015 |
Vibrio cholerae O139 conjugate vaccines
Abstract
The disclosure pertains to conjugates of the capsular
polysaccharide of Vibrio cholerae O139, or a structurally and/or
immunologically related oligo- or poly-saccharide, and a carrier.
These conjugates are useful as pharmaceutical compositions and/or
vaccines to induce serum antibodies which have bactericidal
(vibriocidal) activity against V. cholerae, in particular V.
cholerae O139, and are useful to prevent, treat and/or reduce the
severity of disease caused by V. cholerae infection, such as
cholera. The present disclosure also relates to diagnostic tests
for V. cholerae infection, and/or cholera caused by V. cholerae
infection, using one or more of the oligo- or
poly-saccharide-carrier conjugates or antibodies described
above.
Inventors: |
Szu; Shousun Chen (Potomac,
MD), Kossaczka; Zuzana (Bethesda, MD), Robbins; John
B. (New York, NY) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as represented by the Secretary,
Department of Health and Human Servies |
Washington |
DC |
US |
|
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Assignee: |
The United States of America as
Represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
21741739 |
Appl.
No.: |
14/293,745 |
Filed: |
June 2, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140287476 A1 |
Sep 25, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11695735 |
Apr 3, 2007 |
8852605 |
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10363618 |
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7527797 |
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PCT/US00/24119 |
Sep 1, 2000 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
39/107 (20130101); A61K 47/646 (20170801) |
Current International
Class: |
A61K
39/02 (20060101); A61K 47/48 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0941738 |
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Sep 1999 |
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EP |
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WO 91/01146 |
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Feb 1991 |
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WO |
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WO 93/13797 |
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Jul 1993 |
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WO |
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WO 95/15178 |
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Jun 1995 |
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WO |
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WO 00/48638 |
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Aug 2000 |
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WO |
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WO 02/20059 |
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Mar 2002 |
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WO |
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WO 02/080965 |
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Oct 2002 |
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WO |
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WO 2008/081014 |
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Jul 2008 |
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WO |
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WO 2009/077854 |
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Jun 2009 |
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WO |
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Other References
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Primary Examiner: Minnifield; Nita M
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This is a divisional of U.S. patent application Ser. No.
11/695,735, filed Apr. 3, 2007, which is a continuation of U.S.
application Ser. No. 10/363,618, filed Mar. 3, 2003, now U.S. Pat.
No. 7,527,797, issued May 5, 2009, which is the U.S. National Stage
of International Application No. PCT/US00/24119 filed on Sep. 1,
2000, all of which are each incorporated herein by reference in
their entirety.
Claims
We claim:
1. A method for preparing a conjugate molecule, comprising: (a)
contacting capsular polysaccharide of Vibrio cholerae O139 with
adipic acid dihydrazide in the presence of a carboxyl activating
reagent; and (b) contacting the product of (a) with a recombinant
diphtheria toxin mutant comprising CRMH21G in the presence of a
carboxyl activating reagent, thereby preparing the conjugate
molecule comprising capsular polysaccharide of Vibrio cholerae
O139, covalently bound with adipic acid dihydrazide to the
recombinant diphtheria toxin mutant CRMH21G which functions as a
carrier protein, in which the toxin is covalently bound to the
polysaccharide by coupling with a dicarboxylic acid dihydrazide
linker and wherein the dicarboxylic acid dihydrazide linker
comprises adipic acid dihydrazide, whereby, the conjugate elicits
serum antibodies vibriocidal to at least Vibrio cholerae O139.
2. The method of claim 1, wherein the conjugate molecule consists
essentially of the capsular polysaccharide of Vibrio cholera O139,
hydroxyl coupled to a adipic acid hydrazide-recombinant diphtheria
toxin mutant CRMH21G carrier protein, wherein the polysaccharide to
protein ratio (weight/weight) is about 0.90 and the conjugate
molecule elicits serum antibodies vibriocidal to Vibrio cholera
O139 and diphtheria.
3. The method of claim 1, wherein the conjugate molecule comprises
the capsular polysaccharide of Vibrio cholerae O139, covalently
bound with an adipic acid dihydrazide linker to recombinant
diphtheria toxin comprising CRMH21G, wherein the conjugate
comprises a polysaccharide to protein ratio (weight/weight) of
about 0.76 and elicits serum antibodies vibriocidal to Vibrio
cholerae O139.
4. A method for preparing a conjugate molecule, comprising: (a)
contacting a recombinant mutated diphtheria toxin comprising
CRMH21G with adipic acid dihydrazide in the presence of a carboxyl
activating reagent; and (b) contacting the product of (a) with
capsular polysaccharide of Vibrio cholerae O139, in the presence of
a carboxyl activating reagent, thereby preparing the conjugate
molecule comprising capsular polysaccharide of Vibrio cholerae
O139, covalently bound to the recombinant mutated diphtheria toxin
comprising CRMH21G which functions as a carrier protein, in which
the toxin is covalently bound to the polysaccharide by coupling
with a dicarboxylic acid dihydrazide linker and wherein the
dicarboxylic acid dihydrazide linker comprises adipic acid
dihydrazide, whereby, the conjugate elicits serum antibodies
vibriocidal to at least Vibrio cholerae O139.
5. The method of claim 4, wherein the conjugate molecule consists
essentially of the capsular polysaccharide of Vibrio cholera O139,
hydroxyl coupled to a adipic acid hydrazide-recombinant diphtheria
toxin mutant CRMH21G carrier protein, wherein the polysaccharide to
protein ratio (weight/weight) is about 0.90 and the conjugate
molecule elicits serum antibodies vibriocidal to Vibrio cholera
O139 and diphtheria.
6. The method of claim 4, wherein the conjugate molecule comprises
the capsular polysaccharide of Vibrio cholerae O139, covalently
bound with an adipic acid dihydrazide linker to recombinant
diphtheria toxin comprising CRMH21G, wherein the conjugate
comprises a polysaccharide to protein ratio (weight/weight) of
about 0.76 and elicits serum antibodies vibriocidal to Vibrio
cholerae O139.
7. A method for preparing a conjugate molecule, comprising: (a)
contacting the capsular polysaccharide of Vibrio cholerae O139 with
adipic acid dihydrazide in the presence of
1-cyano-4-dimethylaminopyridinium tetrafluoroborate; and (b)
contacting the product of (a) with a recombinant mutated diphtheria
toxin comprising CRMH21G in the presence of a carboxyl activating
reagent, thereby preparing the conjugate molecule comprising
capsular polysaccharide of Vibrio cholerae O139, covalently bound
to the recombinant mutated diphtheria toxin comprising CRMH21G
which functions as a carrier protein, in which the toxin is
covalently bound to the polysaccharide by coupling with a
dicarboxylic acid dihydrazide linker and wherein the dicarboxylic
acid dihydrazide linker comprises adipic acid dihydrazide, whereby,
the conjugate elicits serum antibodies vibriocidal to at least
Vibrio cholerae O139.
8. The method of claim 7, wherein the conjugate molecule consists
essentially of the capsular polysaccharide of Vibrio cholera O139,
hydroxyl coupled to a adipic acid hydrazide-recombinant diphtheria
toxin mutant CRMH21G carrier protein, wherein the polysaccharide to
protein ratio (weight/weight) is about 0.90 and the conjugate
molecule elicits serum antibodies vibriocidal to Vibrio cholera
O139 and diphtheria.
9. The method of claim 7, wherein the conjugate molecule comprises
the capsular polysaccharide of Vibrio cholerae O139, covalently
bound with an adipic acid dihydrazide linker to recombinant
diphtheria toxin comprising CRMH21G, wherein the conjugate
comprises a polysaccharide to protein ratio (weight/weight) of
about 0.76 and elicits serum antibodies vibriocidal to Vibrio
cholerae O139.
Description
FIELD
This disclosure relates to compositions and methods for eliciting
an immunogenic response in mammals, including responses which
provide protection against, or reduce the severity of, bacterial
infections. More particularly it relates to conjugates of the
capsular polysaccharide of Vibrio cholerae O139, or a structurally
and/or immunologically related oligo- or poly-saccharide, and a
carrier. These conjugates are useful as pharmaceutical compositions
and/or vaccines to induce serum antibodies which have bactericidal
(vibriocidal) activity against V. cholerae, in particular V.
cholerae O139, and are useful to prevent, treat and/or reduce the
severity of disease caused by V. cholerae infection, such as
cholera.
The present disclosure also relates to diagnostic tests for V.
cholerae infection, and/or cholera caused by V. cholerae infection,
using one or more of the oligo- or poly-saccharide-carrier
conjugates, or antibodies described above.
The present disclosure also relates to methods of making oligo- or
poly-saccharide carrier conjugates using CDAP to activate
carboxylic acids on the carbohydrate. The present disclosure also
relates to methods of separating V. cholerae CPS from contaminating
smaller molecules by means of diafiltration.
Abbreviations used: LPS: lipopolysaccharide; CPS: capsular
polysaccharide; O-SP: O-specific polysaccharide; DT: diphtheria
toxin; HSA: human serum albumin; DCC: dicyclohexyl carbodiimide;
EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; CDAP:
1-cyano-4-dimethylaminopyridinium tetrafluoroborate; ADH: adipic
acid dihydrazide; rDT: recombinant diphtheria toxin mutant
CRMH21G.
BACKGROUND
The most successful of all carbohydrate pharmaceuticals so far have
been the carbohydrate-based antibacterial vaccines [48]. The basis
of using carbohydrates as vaccine components is that the capsular
polysaccharides and the O-specific polysaccharides on the surface
of pathogenic bacteria are both protective antigens and essential
virulence factors. The first saccharide-based vaccines contained
capsular polysaccharides of Pneumococci: in the United States a
14-valent vaccine was licensed in 1978 followed by a 23-valent
vaccine in 1983. Other capsular polysaccharides licensed for human
use include a tetravalent meningococcal vaccine and the Vi
polysaccharide of Salmonella typhi for typhoid fever. The inability
of most polysaccharides to elicit protective levels of
anti-carbohydrate antibodies in infants and adults with weakened
immune systems can be overcome by their covalent attachment to
proteins that confer T-cell dependent properties [49]. This
principle has led to the construction of vaccines against
Haemophilus influenzae b (Hib) [37] and in countries where these
vaccines are routinely used, meningitis and other diseases caused
by Hib have been virtually eliminated [50]. Extension of the
conjugate technology to the O-specific polysaccharides of
Gram-negative bacteria has provided a new generation of
glycoconjugate vaccines that are undergoing various phases of
clinical trials [51].
Cholera remains an important public health problem. The long-term
control of cholera depends on good personal hygiene, uncontaminated
water supply and appropriate sewage disposal. However, the
improvement of hygiene is a distant goal for many countries. Thus
the availability of an effective cholera vaccine is important for
the prevention of cholera in these countries. Research on new
cholera vaccines has mainly focused on oral formulations that
stimulate the mucosal secretory immune system. Two oral cholera
vaccines have been experimented with on large scale in humans.
The first vaccine, containing inactivated bacterial cells and the
B-subunit of cholera toxin, was tested in Bangladesh from 1985 to
1989. This vaccine, according to the WHO, may prove useful in the
stable phase of refugee/displaced person crises, especially when
given preventively. The second vaccine is a live attenuated vaccine
containing the genetically manipulated V. cholerae O1 strain CVD
103-HgR. Despite its efficacy in adult volunteers, results of a
large-scale field trial carried-out in Indonesia for 4 years have
shown a surprisingly low protection. Moreover, one of the safety
concerns associated with live cholera vaccine is a possible
horizontal gene transfer and recombination event leading to
reversion to virulence. [52]
More recently, conjugates of V. cholerae O1 lipopolysaccharide with
cholera toxin variants were prepared with an adipic acid
dihydrazide linker. In Phase I studies, these conjugates elicited
vibriocidal antibodies in human volunteers, with IgM levels
comparable to, and IgG levels superior to, the Ig levels elicited
by a cellular vaccine [10, 36, 38, 39]. Conjugation of the V.
cholerae O139 CPS to tetanus toxoid, and inoculation of mice with
the conjugate, has also been described by Morris et al. in U.S.
Pat. No. 5,653,986.
Until 1992, V. cholerae serogroup O1 was recognized as the sole
cause of cholera epidemics, whereas the non-O1 serogroups were
associated with sporadic cases of gastroenteritis and
extra-intestinal infections. In late 1992, the etiological agent of
a massive cholera epidemic was identified as non-O1 V. cholerae
serogroup O139. [53] This was the first reported instance of an
encapsulated strain that caused epidemic cholera. [11]
The surface polysaccharide of V. cholerae O1 is a
lipopolysaccharide (LPS), whereas V. cholerae O139, in contrast,
has a capsular polysaccharide (CPS) composed of a hexasaccharide
repeating unit, containing a trisaccharide backbone and two
branches [3, 4, 8, 11, 16, 17, 19, 26, 28, 30, 31, 35, 38, 42, 43,
45]. The repeating unit contains two negatively charged groups, a
galacturonic acid carboxyl group and a cyclic phosphate diester.
This repeating unit is incorporated into the V. cholerae O139
lipopolysaccharide as well as the capsular polysaccharide, and it
is possible that the V. cholerae O139 CPS is in fact a very high
molecular weight LPS.
Passive immunization of mice with antiserum to the V. cholerae O139
capsular polysaccharide has been shown to protect against variants
of V. cholerae O139, and it has been proposed that conjugates of
the V. cholerae O139 capsular polysaccharide with cholera toxin or
toxoid might be "worthy of further study" [38]. A potential live
oral vaccine, comprising a non-pathogenic deletion mutant of V.
cholerae engineered to express the V. cholerae O139 capsular
polysaccharide and core-linked O-polysaccharide, has been described
[62]. The vaccine elicited anti-CPS antibodies in rabbits, but
neither animal protection studies nor clinical results have been
reported to date.
It has been proposed that a critical level of serum IgG to the
surface polysaccharides of V. cholerae O1 and V. cholerae O139
confers serotype-specific immunity to cholera [3, 7, 17, 24, 25,
28, 29-32, 38, 39, 43, 44]. It has also been proposed that the
level of IgG, rather than the total level of vibriocidal antibodies
may correlate more accurately with protection against cholera,
because (1) synthesis of IgG is predictive of long-lived immunity,
probably reflecting induction of T-helper cells to the
antigen-specific B-cells, and (2) IgG antibodies penetrate into the
extracellular spaces and interior of the small intestine more
effectively than IgM. IgG directed to the O-specific polysaccharide
of V. cholerae O1 or V. cholerae O139 could confer protective
immunity to cholera by inactivating the inoculum on the intestinal
mucosal surface.
Currently, vibriocidal antibody titers induced by vaccines are
regarded as being predictive of therapeutic utility, at least for
vaccines that have passed regulatory review: vibriocidal titer is
the only serologic assay required by the U.S. Food and Drug
Administration for licensure of new cholera vaccine lots. [61]
Previously described conjugates of the V. cholerae O139 CPS have
not demonstrated the induction of adequate levels of IgG antibodies
to provide reliable vaccines; accordingly there still remains a
need for improved conjugates.
SUMMARY
The present disclosure provides conjugates comprising the capsular
polysaccharide of V. cholerae O139 and a carrier. The present
disclosure also provides conjugates comprising oligo- or
poly-saccharides which are structurally related and/or
antigenically similar to the capsular polysaccharide of V. cholerae
O139. Preferably, these oligo- or poly-saccharides of the
disclosure are antigenically similar to the capsular polysaccharide
of V. cholerae O139. These oligo- or poly-saccharide conjugates are
immunogenic and elicit serum antibodies that are bactericidal
against V. cholerae, in particular V. cholerae O139, and are useful
in the prevention, treatment, and reduction in severity of disease
caused by V. cholerae. These oligo- or poly-saccharide conjugates,
and the antibodies which they elicit, are also useful for studying
V. cholerae, in particular V. cholerae O139, in vitro, and for
studying its products in patients.
In another embodiment, the present disclosure provides antibodies
which have vibriocidal activity against V. cholerae, in particular
V. cholerae O139, and which react with, or bind to, the capsular
polysaccharide of V. cholerae O139, wherein the antibodies are
elicited by immunization with a carrier-conjugate comprising the
natural V. cholerae capsular polysaccharide, or a structurally
and/or immunologically related natural, synthetic or semi-synthetic
oligo- or poly-saccharide, preferably a semi-synthetic or synthetic
oligo- or poly-saccharide comprising one or more, preferably four
or more, repeating hexasaccharide units of V. cholerae O139
capsular polysaccharide.
The present disclosure also involves carrier-conjugates which are
useful as pharmaceutical compositions and/or vaccines to prevent,
treat and/or ameliorate diseases, such as cholera, caused by V.
cholerae, in particular V. cholerae O139.
Other embodiments of the present disclosure relate to preparing
antibodies for use in the prevention, treatment or amelioration of
cholera. Antibodies elicited by the carrier conjugates of the
disclosure are useful in providing passive protection to an
individual exposed to V. cholerae, in particular V. cholerae O139,
to prevent, treat, or ameliorate infection and disease caused by
the microorganism.
In yet another embodiment of the present disclosure, diagnostic
tests and/or kits are provided for disease caused by V. cholerae,
in particular V. cholerae O139, using one or more of the
carrier-conjugates, and/or antibodies, of the present
disclosure.
In still other embodiments of the present disclosure, a method for
synthesizing a conjugate vaccine comprising V. cholerae O139
capsular polysaccharide covalently linked to a polypeptide, such as
a diphtheria toxin (DT) derivative which has a lower toxicity than
DT and is suitable for clinical use.
Methods are also provided to conjugate the natural, semi-synthetic,
or synthetic oligo- or poly-saccharides of the disclosure with a
carrier.
Methods are also provided for separating CPS from contaminating
smaller molecules by diafiltration.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. SEPHAROSE.TM. (cross-linked agarose) CL-4B gel filtration
profile of the unfractionated V. cholera O139 capsular
polysaccharide (CPS). Refractive index (RI) response (broken line);
Colitose-containing fractions (solid line); the arrow indicates the
point of separation between the retentate and filtrate by
diafiltration (Amicon YM100) of the unfractionated CPS;
Distribution coefficients (Kd) are depicted above each peak.
FIG. 2. .sup.13C NMR spectrum of V. cholerae O139 capsular
polysaccharide. Legend: .sup.13C NMR spectrum of the CPS (50 mg/ml
D.sub.2O) was measured using Varian XL3000 spectrometer by
averaging 50,000 scans with a 10-s decay between acquisition and 10
.mu.s 90.degree. pulse. Prior to Fourier transformation, a 5-Hz
line broadening was applied and zero-filled to 32,000 datum
points.
FIG. 3. SEPHAROSE.TM. (cross-linked agarose) CL-4B gel filtration
profiles of V. cholerae O139 CPS conjugates with rDT.
Representative chromatograph of the CPS.sub.AH-rDT conjugates (A)
prepared by EDC-mediated synthesis or of CPS-rDT.sub.AH conjugates
(B) prepared by CDAP-mediated synthesis. Legend: Polysaccharide
(.box-solid.) and protein (.largecircle.).
FIG. 4. Double immunodiffusion of V. cholerae O139 conjugates with
murine hyperimmune cholera O139 and equine diphtheria toxin
antisera: (A) representative pattern for CPS.sub.AH-rDT conjugates
and (B) representative pattern for CPS-rDT.sub.AH conjugates.
Legend: 1 murine hyperimmune cholera O139 antiserum, 3 .mu.l: 2
equine diphtheria toxin antiserum, 5 .mu.l; 3 conjugate (PS: 1-4
.mu.g; PR: 1-8 .mu.g).
FIG. 5. Structure of the repeating hexasaccharide unit of the V.
cholerae O139 capsular polysaccharide.
DETAILED DESCRIPTION
In preliminary studies the present inventors found that V. cholerae
CPS does not elicit serum antibodies after three injections in
mice. To improve its immunogenicity, CPS was covalently bound by a
variety of different synthetic methods to chicken serum albumin, a
model protein. The resultant conjugates induced serum anti-CPS IgG
in mice with vibriocidal activity. CPS was then covalently bound by
several methods to the diphtheria toxin mutant CRMH21G.
The recombinant diphtheria toxin mutant CRMH21G was prepared by
replacing histidine 21 with glycine in the A-chain of diphtheria
toxin [15]. This mutant protein has a 1.times.10.sup.-4 lower
toxicity than diphtheria toxin (DT) and is suitable for clinical
use. The two synthetic schemes found most successful with the
chicken albumin, involving
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as
activating agents, were adapted to prepare 4 conjugates of V.
cholerae O139 CPS with the recombinant diphtheria toxin mutant,
CRMH21G. Adipic acid dihydrazide was used as a linker.
When injected subcutaneously into young outbred mice in a
clinically relevant dose and schedule, these conjugates elicited
very high levels of serum CPS antibodies of IgG and IgM classes,
with vibriocidal activity to strains of capsulated V. cholerae
O139. Treatment of these sera with 2-mercaptoethanol (2-ME)
reduced, but did not eliminate, their vibriocidal activity. These
results indicate that the conjugates elicited IgG with vibriocidal
activity. The conjugates also elicited high levels of serum
diphtheria toxin IgG.
Convalescent sera from 20 cholera patients infected with V.
cholerae O139 had vibriocidal titers ranging from 100 to 3200.
Absorption with the CPS reduced vibriocidal titer of all sera to
.ltoreq.50. Treatment with 2-ME reduced the titers of 17 of the 20
to .ltoreq.50. These data show that, similar to infection with V.
cholerae O1, infection with V. cholerae O139 induces vibriocidal
antibodies specific to the surface polysaccharide of this bacterium
(CPS) that are mostly of IgM class.
These results clearly indicate that the conjugates of the
disclosure are capable of inducing anti-V. cholerae CPS antibodies
having desirable properties. Based on these data, clinical trials
of the V. cholerae O139 CPS-rDT conjugates of this disclosure are
planned.
Accordingly, one object of the disclosure is a vaccine that will
induce antibodies with vibriocidal activity against V. cholerae, in
particular V. cholerae O139. These antibodies may be obtained by
parenteral administration of a vaccine containing natural V.
cholerae CPS, or a structurally and/or immunologically related
natural, synthetic or semi-synthetic oligo- or poly-saccharide,
conjugated to a carrier. The oligo- or poly-saccharide, as a
natural, synthetic, or semi-synthetic product, may be bound to both
a carrier saccharide and a non-toxic non-host protein carrier or
directly to a non-toxic non-host protein carrier to form a
conjugate. The present disclosure also encompasses mixtures of the
oligo- or poly-saccharides and conjugates thereof.
The vaccine compositions of the disclosure will preferably induce
protective levels of anti-V. cholerae O139 antibodies, so as to
render the recipient immune to infection by V. cholerae O139, or
resistant to cholera caused by V. cholerae O139, after one or more
doses of vaccine. The levels of antibodies induced by the vaccine
will preferably result in vibriocidal titers of greater than 800,
more preferably greater than 1600, and most preferably greater than
3200, when measured against V. cholerae O139 SPH1168.
The saccharide-based vaccine is intended for active immunization
for prevention of cholera, but may also be used for preparation of
immune antibodies as a therapy. This CPS-based vaccine is designed
to confer specific preventative immunity to infection with V.
cholerae, in particular V. cholerae O139, and to induce antibodies
specific to V. cholerae O139 CPS for prevention and/or treatment of
cholera.
The conjugates of the disclosure, as well as the antibodies
thereto, will be useful in increasing resistance to, preventing,
ameliorating, and/or treating disease, such as cholera, caused by
V. cholerae, in particular V. cholerae O139, in humans.
Specifically, it is expected that conjugates of V. cholerae O139
CPS will elicit serum antibodies specific to V. cholerae O139 CPS,
which should induce complement-dependent killing of V. cholerae
O139. It is also expected that these serum antibodies specific to
V. cholerae O139 CPS will protect against V. cholerae O139,
infection in mammals, including humans.
A number of primary uses for the compounds of this disclosure are
envisioned, for example in the routine immunization schedule of
infants and children living in areas where cholera is endemic, and
in individuals at risk for cholera, such as travelers to areas
where cholera is endemic. It is also intended for the compounds to
be used for intervention in epidemics caused by V. cholerae O139.
Additionally, it is planned to be used for a multivalent vaccine
for V. cholerae and other enteric pathogens for routine
immunization of infants.
The disclosure may also be used to prepare antibodies with
vibriocidal activity against V. cholerae, in particular V. cholerae
O139, for therapy of cholera. The disclosure may also be used to
provide a diagnostic test for cholera caused by V. cholerae, in
particular V. cholerae O139.
The conjugates of the disclosure are also expected to be capable of
inducing anti-DT antibodies which may prevent, lessen or attenuate
the severity, extent or duration of an infection by Corynebacterium
diptheriae.
DEFINITIONS
"Oligosaccharide" as defined herein is a carbohydrate containing up
to twelve monosaccharide units linked together. A "polysaccharide"
as defined herein is a carbohydrate containing more than twelve
monosaccharide subunits linked together.
As used herein, "natural" refers to a native or naturally occurring
oligo- or poly-saccharide which has been isolated from an organism,
e.g., V. cholerae O139, and "semi-synthetic" refers to a native or
naturally occurring polysaccharide that has been structurally
altered. Such structural alterations are any alterations that
render the modified polysaccharide antigenically similar to the
capsular polysaccharide of V. cholerae, in particular V. cholerae
O139. Preferably, the structural alterations substantially
approximate the structure of an antigenic determinant of the
capsular polysaccharide of V. cholerae O139.
In other words, a modified oligo- or poly-saccharide of this
disclosure is characterized by its ability to immunologically mimic
the capsular poly-saccharide of V. cholerae O139, in particular V.
cholerae O139. Such a modified oligo- or poly-saccharide is useful
herein as a component in an inoculum for producing antibodies that
preferably immunoreact with, or bind to, the capsular
polysaccharide of V. cholerae O139.
As used herein, the term "immunoreact" means specific binding
between an antigenic determinant-containing molecule and a molecule
containing an antibody combining site such as a whole antibody
molecule or a portion thereof.
As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules. Exemplary antibody molecules are intact immunoglobulin
molecules, substantially intact immunoglobulin molecules and
portions of an immunoglobulin molecule, including those portions
known in the art as Fab, Fab', F(ab').sub.2, and F(v), as well as
chimeric antibody molecules.
As used herein, the phrase "immunologically similar to" or
"immunologically mimic" refers to the ability of an oligo- or
poly-saccharide of the disclosure to immunoreact with, or bind to,
an antibody of the present disclosure that recognizes and binds to
a native antigenic determinant on the capsular polysaccharide of V.
cholerae O139.
It should be understood that an oligo- or poly-saccharide of the
disclosure need not be structurally identical to the capsular
polysaccharide of V. cholerae O139 so long as it is able to elicit
antibodies that immunoreact with, or bind to, the capsular
polysaccharide of V. cholerae O139.
An oligo- or poly-saccharide of the disclosure includes any
substituted analog, fragment or chemical derivative (either natural
or synthetic) of the capsular polysaccharide of V. cholerae O139 so
long as the oligo- or poly-saccharide is capable of reacting with
antibodies that immunoreact with the capsular polysaccharide of V.
cholerae O139. Therefore, an oligo- or poly-saccharide can be
subject to various changes that provide for certain advantages in
its use. For example, it has been observed that loss of the
colitose residues from the capsular polysaccharide abolishes
antigenicity, and therefore at least one important antigenic
determinant of V. cholerae O139 CPS comprises or consists of one or
more colitose residues. Synthetic portions of V. cholerae O139
capsular polysaccharide, and analogs thereof, may be prepared by
those skilled in the art of carbohydrate synthesis [see, e.g.,
reference 46].
The terms "substitute", "substituted" and "substitution" include
the use of a chemically derivatized residue in place of a
non-derivatized residue provided that the resulting modified oligo-
or poly-saccharide displays the requisite immunological
activity.
"Chemical derivative" refers to a modified oligo- or
poly-saccharide having one or more residues chemically derivatized
by reaction of a functional side group. For example, one or more
hydroxyl groups of the oligo- or poly-saccharide may be reduced,
oxidized, esterified, or etherified; or one or more acetamido
groups may be hydrolyzed or replaced with other carboxamido or
ureido groups, and suitably disposed pairs of hydroxyl groups may
be converted into cyclic phosphate diesters. Such transformations
are well-known and within the abilities of those skilled in the art
of carbohydrate chemistry. Additional residues may also be added
for the purpose of providing a "linker" by which the modified
oligo- or poly-saccharide of this disclosure can be conveniently
affixed to a label or solid matrix or carrier. Suitable residues
for providing linkers may contain amino, carboxyl, or sulfhydryl
groups, for example. Labels, solid matrices and carriers that can
be used with the oligo- or poly-saccharide of this disclosure are
described hereinbelow.
Polymeric Carriers
Carriers are chosen to increase the immunogenicity of the oligo- or
poly-saccharide and/or to raise antibodies against the carrier
which are medically beneficial. Carriers that fulfill these
criteria are described in the art (see, e.g., references 54-59).
Polymeric carriers can be a natural or a synthetic material
containing one or more primary and/or secondary amino groups, azido
groups, or carboxyl groups. The carrier can be water soluble or
insoluble.
Examples of water soluble peptide carriers include, but are not
limited to, natural or synthetic peptides or proteins from bacteria
or virus, e.g., tetanus toxin/toxoid, diphtheria toxin/toxoid,
Pseudomonas aeruginosa exotoxin/toxoid/protein, pertussis
toxin/toxoid, Clostridium perfringens exotoxins/toxoid, and
hepatitis B surface antigen and core antigen. Mutants of these
peptides, derived for example by amino acid substitution or
deletion, may also be employed as carriers. Toxins, toxoids and
mutants of toxins having reduced toxicity are preferred
carriers.
Polysaccharide carriers include, but are not limited to, capsular
polysaccharides from microorganisms such as the Vi capsular
polysaccharide from S. typhi, which contains carboxyl groups and
which is described in U.S. Pat. No. 5,204,098, incorporated by
reference herein; Pneumococcus group 12 (12F and 12A)
polysaccharides, which contain a terminal galactose: and
Haemophilus influenzae type d polysaccharide, which contains an
amino terminal; as well as plant, fruit, or synthetic oligo- or
polysaccharides which are immunologically similar to such capsular
polysaccharides, such as pectin, D-galacturonan,
oligogalacturonate, or polygalacturonate, which are described in
U.S. Pat. No. 5,738,855, incorporated by reference herein.
Example of water insoluble carriers include, but are not limited
to, aminoalkyl-SEPHAROSE.TM. (cross-linked agarose), e.g.,
aminopropyl or aminohexyl SEPHAROSE.TM. (cross-linked agarose), and
aminopropyl glass and the like. Other carriers may be used when an
amino or carboxyl group is added through covalent linkage with a
linker molecule.
Methods for Attaching Polymeric Carriers
The oligo- or poly-saccharides of the disclosure may be bound to
both a carrier saccharide and a non-toxic non-host protein carrier
or directly to a non-toxic non-host protein carrier to form a
conjugate.
When the oligo- or poly-saccharide of the disclosure is bound to
both a carrier saccharide and a non-toxic non-host protein carrier,
it may be bound first to the carrier saccharide, then the
saccharide-carrier conjugate can be bound to the non-toxic non-host
protein carrier. The complex compound would properly be described
as a semi-synthetic complex molecule with three distinct domains
and origins. This complex compound would first contain an oligo- or
poly-saccharide bound to the carrier polysaccharide and then the
two-domain saccharide bound to a protein. Alternatively, the oligo-
or poly-saccharide of the disclosure may be bound to both a carrier
saccharide and a non-toxic non-host protein carrier
simultaneously.
Methods for binding a polysaccharide to a protein, with or without
a linking molecule, are well known in the art. See for example
reference [60], where 3 different methods for conjugating Shigella
O-SP to tetanus toxoid are exemplified. See also reference [22],
which describes methods for conjugating S. typhi Vi and adipic
hydrazide-derivatized proteins. In U.S. Pat. No. 5,204,098 and U.S.
Pat. No. 5,738,855, it is taught that an oligo- or poly-saccharide
containing at least one carboxyl group, through carbodiimide
condensation, may be thiolated with cystamine, or aminated with
adipic dihydrazide, diaminoesters, ethylenediamine and the like.
Groups which could be introduced by this method, or by other
methods known in the art, include thiols, hydrazides, amines and
carboxylic acids. Both the thiolated and the aminated intermediates
are stable, may be freeze dried, and may be stored at low
temperature. The thiolated intermediate may be reduced and
covalently linked to a polymeric carrier containing a sulfhydryl
group, such as a 2-pyridyldithio group. The aminated intermediate
may be covalently linked to a polymeric carrier containing a
carboxyl group through carbodiimide condensation.
The oligo- or poly-saccharide can be covalently bound to a carrier
with or without a linking molecule. To conjugate without a linker,
for example, a carboxyl-group-containing oligo- or poly-saccharide
and an amino-group-containing carrier are mixed in the presence of
a carboxyl activating agent, such as for example a carbodiimide, in
a choice of solvent appropriate for both the oligo- or
poly-saccharide and the carrier, as is known in the art [58]. The
oligo- or poly-saccharide is preferably conjugated to a carrier
using a linking molecule. A linker or crosslinking agent, as used
in the present disclosure, is preferably a small linear molecule
having a molecular weight of approximately <500 and is
non-pyrogenic and non-toxic in the final product form (54-59). To
conjugate with a linker or crosslinking agent, either or both of
the oligo- or poly-saccharide and the carrier may be covalently
bound to a linker first. The linkers or cros slinking agents are
homobifunctional or heterobifunctional molecules, e.g., adipic
dihydrazide, ethylene diamine, cystamine, N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-N-(2-iodoacetyl)-.beta.-alaninate-propionate (SLAP),
succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate
(SMCC), 3,3'-dithiodipropionic acid, and the like. Dicarboxylic
acid dihydrazides are preferred. In the examples presented herein,
the linker is adipic acid dihydrazide, attached via hydrazide
linkages to carboxyl groups of the oligosaccharide and the
polypeptide. Similar results would be expected with any two- to
ten-carbon dihydrazide linker. Other amino-containing linkers may
similarly be bound to carboxyl groups of the oligo- or
poly-saccharide or the carrier through carbodiimide condensation.
Carboxylic acid containing linkers may be bound to the amino groups
of the carrier by means of carboxyl activating reagents (e.g.,
carbodiimide condensation) or via N-hydroxysuccinimidyl esters or
other reactive derivatives. The unbound materials are removed by
physico-chemical methods such as gel filtration or ion exchange
column depending on the materials to be separated. The final
conjugate consists of the oligo- or poly-saccharide and the carrier
bound through a linker.
In the present disclosure, attachment of the V. cholerae capsular
polysaccharide to a protein carrier is preferably accomplished by
first coupling a dicarboxylic acid dihydrazide linker to the CPS,
by treatment with a carboxyl activating reagent, such as a
water-soluble carbodiimide (e.g.,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (DEC) or
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide (EDC)),
but preferably through one or more hydroxyl groups, using for
example 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP),
to produce a hydrazide-functionalized polysaccharide. Adipic acid
dihydrazide is a particularly preferred linker, but conjugates
employing other linkers, such as the dihydrazides of succinic,
suberic, and sebacic acids, are contemplated to be within the scope
of the disclosure. The linker-functionalized V. cholerae capsular
polysaccharide (CPS.sub.AH) is then coupled to the carrier protein,
preferably with a water-soluble carbodiimide, most preferably EDC.
In an alternative embodiment, the carrier protein (rDT) is first
coupled to the linker, again using a water-soluble carbodiimide,
preferably EDC, and the linker-functionalized carrier (rDT.sub.AH)
is then coupled to the CPS with a carboxyl activating reagent, or
preferably by hydroxyl coupling using for example CDAP. For
preparation of the conjugates of this disclosure, activation of CPS
for coupling (with linker or with rDT.sub.AH), is preferably
carried out with CDAP, and activation of rDT (for coupling with
linker or with CPS.sub.AH) is most preferably carried out with
EDC.
Dosage for Vaccination
The present inoculum contains an effective, immunogenic amount of
oligo- or poly-saccharide carrier conjugate of this disclosure. The
effective amount of oligo- or poly-saccharide carrier conjugate per
unit dose sufficient to induce an immune response to V. cholerae,
in particular V. cholerae O139, depends, among other things, on the
species of mammal inoculated, the body weight of the mammal and the
chosen inoculation regimen as is well known in the art. Inocula
typically contain oligo- or poly-saccharide carrier conjugates with
concentrations of oligo- or poly-saccharide of about 1 micrograms
to about 100 milligrams per inoculation (dose), preferably about 3
micrograms to about 100 micrograms per dose, most preferably about
5 micrograms to about 50 micrograms, and most preferably about 5
micrograms to about 25 micrograms per dose.
The term "unit dose" as it pertains to the inocula refers to
physically discrete units suitable as unitary dosages for mammals,
each unit containing a predetermined quantity of active material
(oligo- or poly-saccharide conjugate) calculated to produce the
desired immunogenic effect in association with the required
diluent.
Inocula are typically prepared as a solution in a physiologically
tolerable (acceptable) diluent such as water, saline or
phosphate-buffered saline or other physiologically tolerable
diluent to form an aqueous pharmaceutical composition.
The route of inoculation may be intramuscular, subcutaneous and the
like, which results in eliciting antibodies protective against V.
cholerae, in particular V. cholerae O139. The dose is administered
at least once. In order to increase the antibody level, a second or
booster dose may be administered approximately 4 to 6 weeks after
the initial injection. Subsequent doses may be administered as
indicated.
Adjuvants, such as aluminum hydroxide, QS-21, TITERMAX.TM.
(immunoadjuvant) (CytRx Corp., Norcross Ga.), Freund's complete
adjuvant, Freund's incomplete adjuvant, interleukin-2, thymosin,
and the like, may also be included in the compositions.
Antibodies
An antibody of the present disclosure in one embodiment is
characterized as comprising antibody molecules that immunoreact
with the capsular polysaccharide of V. cholerae O139.
An antibody of the present disclosure is typically produced by
immunizing a mammal with an immunogen or vaccine containing a
molecular conjugate of the V. cholerae O139 capsular polysaccharide
(or a structurally and/or immunologically related molecule) in an
amount sufficient to induce, in the mammal, antibody molecules
having immunospecificity for the capsular polysaccharide of V.
cholerae O139. The capsular polysaccharide or related molecule is
preferably conjugated to a carrier. The antibody molecules may be
collected from the mammal and isolated by methods known in the
art.
For administration to humans, human or humanized monoclonal
antibodies are preferred, including those made by phage display
technology or by non-human mammals engineered to produce human
antibodies.
The antibody molecules of the present disclosure may be polyclonal
or monoclonal. Monoclonal antibodies may be produced by methods
known in the art. Portions of immunoglobulin molecules, such as
Fabs, may also be produced by methods known in the art.
The antibody of the present disclosure may be contained in blood
plasma, serum, hybridoma supernatants and the like. Alternatively,
the antibody of the present disclosure is isolated to the extent
desired by well known techniques such as, for example, ion
chromatography or affinity chromatography. The antibodies may be
purified so as to obtain specific classes or subclasses of antibody
such as IgM, IgG, IgA, IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4
and the like. Antibodies of the IgG class are preferred for
purposes of passive protection.
The antibodies of the present disclosure have a number of
diagnostic and therapeutic uses. The antibodies can be used as an
in vitro diagnostic agent to test for the presence of V. cholerae,
in particular V. cholerae O139, in biological samples in standard
immunoassay protocols. Such assays include, but are not limited to,
agglutination assays, radioimmunoassays, enzyme-linked
immunosorbent assays, fluorescence assays, Western blots and the
like. In one such assay, for example, the biological sample is
contacted to antibodies of the present disclosure and a labeled
second antibody is used to detect the presence of V. cholerae, in
particular V. cholerae O139, or the capsular polysaccharide antigen
of V. cholerae, in particular V. cholerae O139, to which the
antibodies are bound.
Such assays may be, for example, of direct format (where the
labeled first antibody is reactive with the antigen), an indirect
format (where a labeled second antibody is reactive with the first
antibody), a competitive format (such as the addition of a labeled
antigen), or a sandwich format (where both labeled and unlabelled
antibody are utilized), as well as other formats described in the
art.
The antibodies of the present disclosure are useful in prevention
and treatment of infections and diseases caused by V. cholerae, in
particular V. cholerae O139.
In providing the antibodies of the present disclosure to a
recipient mammal, preferably a human, the dosage of administered
antibodies will vary depending upon such factors as the mammal's
age, weight, height, sex, general medical condition, previous
medical history and the like.
In general, it is desirable to provide the recipient with a dosage
of antibodies which is in the range of from about 1 mg/kg to about
10 mg/kg body weight of the mammal, although a lower or higher dose
may be administered.
The antibodies of the present disclosure are intended to be
provided to the recipient subject in an amount sufficient to
prevent, lessen or attenuate the severity, extent or duration of
the infection by V. cholerae, in particular V. cholerae O139.
Antibodies which immunoreact with DT may also be provided to a
recipient subject in an amount sufficient to prevent, lessen or
attenuate the severity, extent or duration of an infection by
Corynebacterium diptheriae.
The administration of the agents of the disclosure may be for
either "prophylactic" or "therapeutic" purpose. When provided
prophylactically, the agents are provided in advance of any
symptom. The prophylactic administration of the agent serves to
prevent or ameliorate any subsequent infection. When provided
therapeutically, the agent is provided at (or shortly after) the
onset of a symptom of infection. The agent of the present
disclosure may, thus, be provided either prior to the anticipated
exposure to V. cholerae, in particular V. cholerae O139, (so as to
attenuate the anticipated severity, duration or extent of an
infection and disease symptoms) or after the initiation of the
infection.
For all therapeutic, prophylactic and diagnostic uses, the oligo-
or poly-saccharide of the disclosure, alone or linked to a carrier,
as well as antibodies and other necessary reagents and appropriate
devices and accessories may be provided in kit form so as to be
readily available and easily used.
The following examples illustrate certain embodiments of the
present disclosure, but should not be construed as limiting its
scope in any way. Certain modifications and variations will be
apparent to those skilled in the art from the teachings of the
foregoing disclosure and the following examples, and these are
intended to be encompassed by the spirit and scope of the
disclosure.
EXAMPLES
The examples describe two methods for the synthesis of a conjugate
comprising the capsular polysaccharide of V. cholerae O139, with a
homobifunctional linker unit used for covalent attachment to a
mutant diphtheria toxin as a model carrier protein. These examples
are also described in reference 47.
MATERIALS AND METHODS
Materials.
Chicken serum albumin Fraction V (CSA), rabbit CSA antiserum,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), adipic acid
dihydrazide (ADH), 1-cyano-4-dimethylaminopyridinium
tetrafluoroborate (CDAP), and agarose were from Sigma Chemical Co.,
St Louis, Mo.; SEPHAROSE.TM. (cross-linked agarose) CL-4B and
-SEPHADEX.TM. (cross-linked dextran) G-25 from Pharmacia AB,
Uppsala, Sweden; BSA standard solution, Coomassie blue protein
assay reagent, triethylamine (TEA) from Pierce, Rockford, Ill.;
nickel nitrilotriacetic acid (NiNTA) chelating agarose from Qiagen
Inc., Chatsworth, Calif.; acetonitrile from T. J. Baker, Inc.,
Philipsburg, N.J.; diphtheria toxin (DT) from List Biological
Laboratories, Inc, Campbell, Calif., equine antidiphtheria toxin,
Lederle Laboratories, Pearl River N.Y., Lot 152-5456 R a gift from
CBER, FDA; rabbit (3-4 week) complement from Pel-Freez, Brown Deer,
Wis.; dialysis membranes (molecular weight cut off 6-8,000) from
Spectra-Por, Laguna Hills, Calif.; ultrafiltration membrane YM100
and CENTRIPREP.TM. (cellulose membrane) 30 from Amicon, Inc,
Beverly, Mass.; Limulus amebocyte lysate pyrogen (U.S. License No.
709) from BioWhittaker, Inc., Walkersville, Md.; tryptic soy broth
(TSB) from Difco Inc, Detroit, Mich. (TSB containing 1% agarose was
denoted as TSA). Deionized or pyrogen-free water (PFW) and
pyrogen-free saline (PFS) were used in all experiments.
Bacteria.
V. cholerae O139 MDO-12C [8], a heavily capsulated and opaque
variant selected from the isolate MDO-12 (Madurai, India), was used
for preparation of CPS and murine hyperimmune serum. V. cholerae
O139 SPH1168, a clinical isolate from a That patient (Suanphung
Hospital, Thailand), was used as the target strain in the
vibriocidal assay. Both isolates were stored in 20% glycerol at
-70.degree. C.
Purification of V. cholerae O139 CPS.
V. cholerae O139 MDO-12C was propagated from a single colony on TSA
to 4.times.100 ml and then to 4.times.1 L of TSB for 5 h at
37.degree. C. with shaking at 200 rpm. The 4-L inoculum
(A.sub.560.about.3.0) was transferred to a 300-L fermenter
containing 150 L of TSB, 0.1% dextrose and 0.05 M MgS0.sub.4.
Fermentation was conducted at 30% dissolved oxygen, 35.degree. C.
and pH 7.0 (maintained with NH.sub.4OH). After 16 h, formalin was
added to a final concentration of 2% and stirred slowly for 6 h at
room temperature. The suspension was centrifuged and the
supernatant concentrated to 1.2 L by ultrafiltration and stored at
-20.degree. C.
A 500-mL aliquot of the concentrated supernatant was mixed with 3
volumes of 95% ethanol and stored overnight at 4.degree. C. The
supernatant was decanted and the slurry spun down at
10,500.times.g, 10.degree. C. for 30 min. The pellet (20 g wet
weight) was washed with 80% ethanol, dissolved in 800 ml of 10%
saturated sodium acetate, pH 7.5, and extracted with cold phenol 3
times [9]. The final water phase was dialyzed against H.sub.2O for
3 days at 4-8.degree. C. and freeze-dried. The precipitate was
dissolved in 150 ml of 0.1 M CaCl.sub.2 and ultracentrifuged at
145,000.times.g, 10.degree. C. for 5 h. The supernatant was
recentrifuged as above, dialyzed against H.sub.2O, freeze-dried
(yield 1.6 g) and stored at -20.degree. C.
This material (unfractionated CPS) was dissolved in PFW (100 mg/50
ml) and passed through an Amicon membrane YM100. The retentate was
passed through a 2.5.times.90 cm column of SEPHAROSE.TM.
(cross-linked agarose) CL-4B in PFS. The retentate was eluted from
the column as one peak at Kd 0.4. Colitose-containing fractions
were pooled, dialyzed against PFW and freeze-dried. This material
was denoted as CPS and used to prepare conjugates with rDT. In
earlier experiments the filtration through the Amicon membrane was
omitted (see preparation of CPS-AH conjugates below).
.sup.13C NMR Spectroscopy.
.sup.13C NMR spectrum of the CPS (50 mg/ml D.sub.2O) was measured
using Varian XL3000 spectrometer by averaging 50,000 scans with a
10-s decay between acquisition and 10-.mu.s 90.degree. pulse. Prior
to Fourier transformation, a 5-Hz line broadening was applied and
zero-filled to 32,000 datum points.
Murine Hyperimmune V. cholerae O139 Serum.
V. cholerae O139 culture was prepared by transferring a single
colony from TSA to 50 ml of LB and incubating at 37.degree. C., 200
rpm for 5 h (A.sub.560.about.1.0). The culture was inactivated with
1% formalin. Thirty 6-week-old female Swiss mice (NIH) were
injected as follows: 1) 3 subcutaneous injections of 100 .mu.L 1
day apart; 2) after 9 days, 3 intraperitoneal injections of 150
.mu.L 1 day apart; 3) 9 days later, 3 intravenous injections of 200
.mu.L 1 day apart. Mice were exsanguinated seven days after the
last injection. All sera showed a precipitin line by double
immunodiffusion with CPS: a pool was denoted as murine hyperimmune
V. cholerae O139 serum.
Purification of Recombinant Diphtheria Toxin Mutant CRMH21G
(rDT).
The rDT was constructed by site-directed mutagenesis on the A chain
replacing histidine at position 21 with glycine and expressed in
Escherichia coli BL21 (.lamda.DE3) [15]. To facilitate purification
by NiNTA, a 6-histidine tag was attached to the protein carboxyl
terminal. Fermentation of this recombinant strain was performed as
described [5]. The cell paste was suspended in 0.5 M NaCl, 0.02 M
Tris, 0.005 M imidazole, pH 8.0 and the cytoplasm was released by a
French press. The supernatant was passed through a 2.5.times.10 cm
column NiNTA and washed with 0.02 M Tris buffer containing 0.03 M
imidazole (pH 8.0). rDT was eluted with 0.02 M Tris buffer
containing 0.25 M imidazole, pH 8.0 at 3-8.degree. C. The eluate
was dialyzed exhaustively against 0.02 M Tris, pH 8.0 at
3-8.degree. C. (yield 150 mg rDT/L supernatant). Prior to
derivatization, rDT was dialyzed at 3-8.degree. C. against PBS with
multiple changes of outer fluid followed by dialysis against 0.2 M
NaCl, pH 7.2-7.7 (adjusted with 1 M NaOH). The protein solution was
concentrated to .about.10 mg/ml in an Amicon CENTRIPREP.TM.
(cellulose membrane) 30.
rDT had the same R.sub.f in 10% SDS-PAGE and the same circular
dichroism spectrum as DT. A line of identity was formed between rDT
and DT when reacted against equine anti-DT by double
immunodiffusion.
Adipic Acid Hydrazide (AH) Derivatives.
CPS was treated with ADH in the presence of CDAP [20, 21, 23],
while CSA and rDT were treated with ADH in the presence of EDC [22,
37] as the activating agent.
AH Derivative of CSA (CSA.sub.AH).
The AH derivative of CSA (CSA.sub.AH) was prepared by EDC-mediated
condensation of ADH and CSA [18]. Concentrations of the reactants
in the reaction mixture were 10 mg CSA/mL, 0.2 M ADH, and 0.015 M
EDC. ADH (powder) was added to the CSA, the pH adjusted to 5.5 with
0.1 M MES buffer (pH 5.5), and EDC (powder) was added. The reaction
was carried out at room temperature, pH 5.5 to 5.7 for 1 h. The
mixture was dialyzed overnight at 4.degree. C. against saline and
passed through a 2.5.times.40 cm column of SEPHADEX.TM.
(cross-linked dextran) G-25 in saline. The void volume fractions
were pooled, concentrated by ultrafiltration, stored at 4.degree.
C., and designated as CSA.sub.AH.
AH Derivatives of CPS
CPS.sub.AH.
CDAP activation of CPS was performed as described [21] using a
CDAP:CPS ratio of 1:5 (w/w). 40 mg of CPS (not filtered through on
the Amicon YM100 membrane) was dissolved in 1.5 ml of H.sub.2O; pH
5.0. 80 .mu.L of CDAP in acetonitrile (100 mg/ml) were added with
stifling followed in 30 sec with 80 .mu.L of 0.2 M TEA. After 2
min, the pH was adjusted to 7.8 with 0.1 M HCl. Then 1.5 ml of 0.8
M ADH in 0.5 M NaHCO.sub.3 was added and the pH maintained between
8.0 and 8.4 with 0.1 M HCl for 2 h at room temperature. The
reaction mixture was dialyzed overnight against 6 L of water with 2
changes, and passed through a 2.5.times.40 cm column of
SEPHADEX.TM. (cross-linked dextran) G-25 in PFW. The void volume
fractions were freeze-dried, denoted as CPS.sub.AH, and assayed for
AH.
CPS.sub.AH1.
CPS which had been filtered through the Amicon YM100 membrane was
employed. Concentrations of the reactants in the reaction mixture
were 20 mg/mL of CPS, 0.05M ADH, and 0.05 M EDC. MES buffer (pH
5.5) was added to the CPS in water to adjust the pH to 5.5. EDC and
ADH (both in powder form) were then added. The reaction was carried
out for 2 h at room temperature and pH 5.5-5.6 was maintained with
0.5 M MES-acid. The pH was then brought to 7.0 with 0.1 M sodium
phosphate buffer (pH 8.0), dialyzed overnight against water, and
passed through a 1.5.times.24 cm column of Bio-gel P-10 in water.
The void volume fractions were freeze-dried and designated as
CPS.sub.AH1.
CPS.sub.AH2.
CPS which had been filtered through the Amicon YM100 membrane was
employed. The reaction was performed as described [21], at a
CDAP/CPS ratio of 3:10 (w/w). 1 mL of CPS (30 mg/mL water), pH 5.0,
was mixed with 90 .mu.L of CDAP in acetonitrile (100 mg/mL). After
30 sec, 90 .mu.L of 0.2 M TEA was added. During the next 2 min the
pH dropped from 8.1 to 7.2, and 1 mL of 0.8 M ADH in 0.5 M NaHCO3
was added. The reaction was carried out for 2 h at room temperature
and a pH 8.3-8.6 maintained with 0.1 M NaOH. The mixture was
dialyzed overnight against water and passed through a 2.5.times.40
cm column of SEPHADEX.TM. (cross-linked dextran) G-25 in water. The
void volume fractions were freeze-dried and denoted as
CPS.sub.AH2.
AH Derivative of rDT (rDT.sub.AH).
The reaction mixture contained 10 mg/ml of protein, 0.2 M ADH and
0.011 M EDC. The reaction was carried out for 1 h at room
temperature with the pH maintained at 6.2-6.4 with 0.1 M HCl. The
reaction mixture was dialyzed against 0.2 M NaCl, pH 7.0 (adjusted
with 1 M NaOH), and passed through a 1.5.times.25 cm column of
SEPHADEX.TM. (cross-linked dextran) G-25 in the same solution. Void
volume fractions were pooled, concentrated (Amicon CENTRIPREP.TM.;
cellulose membrane) 30), denoted as rDT.sub.AH, and assayed for
protein and AH.
Conjugates with CSA.
Two sets of conjugates, CPS-CSA.sub.AH and CPS.sub.AH-CSA were
prepared using EDC and CDAP as the activating agents [21, 22].
EDC-Mediated Synthesis of CPS-CSA.sub.AH.
The concentrations of reactants in the reaction mixture were 10
mg/mL of CPS and 10 mg/mL of CSA.sub.AH, and 0.02 M EDC. CPS was
mixed with CSA.sub.AH, and the pH was adjusted to 5.5 with 0.5 M
MES buffer (pH 5.5). The mixture was brought to the final volume
with saline, and EDC was added as powder. The reaction was carried
out at room temperature for 3 h, during which the pH rose from 5.5
to 5.7. The conjugation was accompanied by a formation of
precipitate that became gradually heavier. The mixture was dialyzed
overnight against saline and centrifuged (7,000.times.g, 5 min)
before passing through a 1.5.times.90 cm column of SEPHAROSE.TM.
(cross-linked agarose) CL-2B in saline. Fractions were assayed for
polysaccharide and protein. The fractions 21-29 of Vo peak were
pooled and denoted as EDC:CPS-CSA.sub.AH.
CDAP-Mediated Synthesis of CPS-CSA.sub.AH.
CPS was activated with CDAP and bound to CSA.sub.AH at a CDAP/CPS
ratio of 3:10 (w/w). 10 mg of CPS in water (100 mg/mL) was mixed
with 30 .mu.L of CDAP in acetonitrile (100 mg/mL). The mixture (pH
5.2) was stirred for 30 sec, and 30 .mu.L of 0.2 M TEA was added.
After 2 min, 0.1 M NaOH was added to bring the pH from 7.0 to 8.2.
CSA.sub.AH (10 mg) was added, and the volume adjusted with saline
to 2 mL. The reaction was carried out for 3 h at room temperature,
and a pH of 8.0 to 8.3 was maintained with 0.1 M NaOH. The mixture
was passed through a 1.5.times.90 cm column of SEPHAROSE.TM.
(cross-linked agarose) CL-2B in saline. Fractions were assayed for
polysaccharide and protein. Fractions 30 to 46 were pooled and
denoted as CDAP:CPS-CSA.sub.AH.
EDC-Mediated Synthesis of CPS.sub.AH1-CSA and CPS.sub.AH2-CSA.
CPS.sub.AH1-CSA.
Concentrations of the reactants in the reaction mixture were 10
mg/mL of CPS.sub.AH1, 10 mg/mL of CSA, and 0.02 M EDC. CPS.sub.AH1
was mixed with CSA, and the pH was adjusted to 5.5 with 0.5 M MES
buffer (pH 5.5). EDC was added as powder, and the mixture was
brought to the final volume with saline. The reaction was carried
out at room temperature for 3 h during which the pH rose from 5.5
to 5.6. The reaction mixture was passed through a 1.5.times.90 cm
column of SEPHAROSE.TM. (cross-linked agarose) CL-2B in saline.
Fractions were assayed for polysaccharide and protein. Fractions 36
to 52 were pooled and denoted as EDC:CPS.sub.AH1-CSA.
CPS.sub.AH2-CSA.
Concentrations of the reactants in the reaction mixture were 5
mg/mL of CPS.sub.AH2, 5 mg/mL of CSA, and 0.05 M EDC. The procedure
was performed as described above. Fractions were assayed for
polysaccharide and protein. Fractions 36 to 52 were pooled and
denoted as EDC:CPS.sub.AH2-CSA.
Conjugates with rDT.
Two schemes were used to prepare conjugates with rDT: 1)
EDC-mediated conjugation of the CPS.sub.AH with rDT, and 2)
CDAP-mediated conjugation of the CPS with rDT.sub.AH.
EDC-Mediated Conjugation of CPS.sub.AH with rDT.
Each reaction mixture contained 8 mg/ml of CPS.sub.AH and of rDT,
and EDC of 0.05 M (for I:CPS.sub.AH-rDT) or 0.02 M (for
II:CPS.sub.AH-rDT).
CPS.sub.AH was dissolved in 0.2 M NaCl and the pH adjusted to 6.2
with 0.1 M NaOH. rDT was added and the volume adjusted with 0.2 M
NaCl. After stirring for 1 min, EDC was added. The reaction was
carried out for 3 h at room temperature and the pH maintained at
6.2-6.4 with 0.1 M HCl. The mixture was dialyzed overnight at
3-8.degree. C. against 0.2 M NaCl, 0.005 M sodium phosphate, pH
7.5, and passed through a 1.5.times.90 cm column of SEPHAROSE.TM.
(cross-linked agarose) CL-4B in the same buffer. Fractions were
assayed for polysaccharide and protein. The void volume fractions
were pooled and denoted as I:CPS.sub.AH-rDT and
II:CPS.sub.AH-rDT.
CDAP-Mediated Conjugation of CPS with rDT.sub.AH.
Each reaction mixture contained 8 mg/ml of CPS and of rDT.sub.AH:
the CDAP/CPS was 4:5 (for I:CPS-rDT.sub.AH) or 1:5 (for
II:CPS-rDT.sub.AH).
CDAP (100 mg/ml acetonitrile) was added to CPS in 0.2 M NaCl (pH
5.2) and mixed for 30 sec. An equal volume of 0.2 M TEA to that of
CDAP was added. After 2 min, the pH dropped from 8.5 to 7.2 and
rDT.sub.AH was added. The pH was raised from 7.2 to 8.3 with 0.1 M
NaOH. The reaction was carried out for 2 h at room temperature
during which the pH was stable. The mixture was dialyzed overnight
against 0.2 M NaCl, 0.005 M sodium phosphate buffer, pH 7.5, and
passed through a 1.5.times.90 cm column of SEPHAROSE.TM.
(cross-linked agarose) CL-4B in the same buffer. Fractions were
assayed for polysaccharide and protein and void volume fractions
pooled and denoted as I:CPS-rDT.sub.AH and II:CPS-rDT.sub.AH.
Chemical Assays.
Polysaccharide was assayed by measuring 3,6-dideoxyhexose
(colitose) with the CPS as the standard [18]. Protein was measured
by Coomassie blue assay with BSA as the standard [2]. Hydrazide
content of CPS.sub.AH and rDT.sub.AH was measured by the TNBS
method using ADH as the standard [14]. The degree of derivatization
was expressed in % of AH, and the mol/mol ratio of AH to
polysaccharide or to protein.
Limulus amebocyte Lysate Test.
CPS was assayed for endotoxin by limulus amebocyte lysate test. The
FDA Reference Standard Endotoxin (Lot EC-5) was used as a reference
for the assay. The test conforms with the FDA guideline [41].
Immunodiffusion.
Double immunodiffusion of the conjugates was performed in 1%
agarose gel in 0.15 M NaCl with murine hyperimmune cholera O139
serum and equine diphtheria toxin antiserum.
Immunization of Mice.
Six-week-old female Swiss albino mice (10 per group) were injected
subcutaneously 3 times at 2-week intervals with 100 .mu.L of
immunogen containing 2.5 .mu.g of the CPS alone or as the
conjugate. A control group received 1 injection of 100 .mu.L of
saline. Mice were exsanguinated 7 days after each injection and
sera stored at -20.degree. C.
ELISA.
Flat-bottom 96-well microtiter plates (NUNC-IMMUNO.TM. (coated
polystyrene; Denmark) were coated with CPS (20 .mu.g/ml PBS) and
kept overnight at room temperature. After washing with 0.15 M NaCl,
0.1% Brij and 3 mM sodium azide, plates were blocked with 1% BSA in
PBS for 2 h at room temperature. The plates were washed and 2-fold
serial dilutions of sera in 1% BSA, 0.1% Brij, PBS added. Reference
serum was assayed in triplicates and samples in duplicates. Plates
were incubated overnight at room temperature, washed, and the
alkaline phosphatase-labeled goat antibody specific to mouse IgG or
for IgM was added. After 4 h at room temperature, the plates were
washed, and the 4-nitrophenyl phosphate substrate (1 mg/ml in 1 M
Tris-HCl, 3 mM MgCl.sub.2, pH 9.8) was added. A.sub.405 was
measured by a MRX Dynatech reader.
Anti-CPS IgG was measured in all murine sera; anti-CPS IgM was
measured only in 11 representative sera from mice injected 3 times
with II:CPS.sub.AH-rDT or I:CPS-rDT.sub.AH. Murine hyperimmune V.
cholerae O139 serum was used as the reference for both anti-CPS IgG
and IgM. This serum was arbitrarily assigned a value of 1000 ELISA
units/ml (EU) for IgG and 100 EU for IgM upon the observation that
1/20,000 dilution of anti-IgG and 1/100 dilution of anti-IgM gave
approximately the same A.sub.405.
An analogous ELISA procedure was used to measure anti-DT IgG:
plates were coated with DT (5 .mu.g/ml) and a mouse serum with high
titer of anti-DT IgG, arbitrarily assigned a value of 1000 EU,
served as the reference.
ELISA results were computed with an ELISA Data Processing Program
provided by the Biostatistics and Information Management Branch,
CDC based upon four parameters logistic-log function using Taylor
Series Linearization Algorithm [34]. Anti-CPS IgG and anti-DT IgG
levels are expressed as geometric means.
Statistics.
Comparisons of the geometric means were performed with the
two-sided t test or Wilcoxon analysis.
Vibriocidal Assay.
Eleven representative sera from mice injected three times with
II:CPS.sub.AH-rDT or I:CPS-rDT.sub.AH, and twenty convalescent sera
from cholera patients infected with V. cholerae O139 (Samutskakorn
Hospital, Thailand) [13] were assayed for vibriocidal activity
before and following treatment with 0.1 M 2-ME for 30 min at
37.degree. C. [10, 27]. The patient sera were also tested for
vibriocidal activity after absorption with CPS in vibriocidal
antibody inhibition assay (VAI)[6].
Bacteria were prepared by transferring a single colony from TSA
into 10 ml of TSB and incubating for 2 to 3 h at 37.degree. C. with
shaking at 180 rpm. 100 .mu.L of this inoculum was transferred to
10 ml of TSB and incubated with shaking (180 rpm) at 37.degree. C.
until culture reached A.sub.560 of 0.2-0.24 (3.0-4.0.times.10.sup.7
cells/ml). The bacterial suspension was diluted 10.sup.5-fold in
Dulbecco's buffer.
Vibriocidal assay was performed in sterile non-pyrogenic 24-well
cell culture plates (Costar, Corning, N.Y.) by mixing equal volumes
of serum, bacteria and complement. The tested serum was 2-fold
serially diluted in Dulbecco's buffer (for VAI in 100 mg CPS/ml
Dulbecco's buffer), so that each well contained 100 .mu.L. 100
.mu.L aliquots of the bacteria and of complement were added into
each well. Plates were incubated for 1 h at 37.degree. C. with
shaking. Two 100 .mu.L aliquots from each well were transferred
into empty wells and 1 ml of TSA (46-48.degree. C.) added to all 3
wells. Plates were incubated overnight at 37.degree. C. and the
colonies counted. The vibriocidal titer was defined as the
reciprocal of the highest serum dilution showing .gtoreq.60%
reduction in number of colonies compared to the control (complement
only) [25].
RESULTS
V. cholerae O139 CPS.
The V. cholerae O139 CPS isolated from culture supernatant
(Material and Methods) showed three peaks at Kds of 0.4, 0.71 and
0.91 on SEPHAROSE.TM. (cross-linked agarose) CL-4B with yields of
70%, 29% and 1%, respectively (FIG. 1). Colitose, a component of
the CPS-repeating unit, was detected only in the peak at Kd 0.4.
Fast separation of this peak-material from the lower molecular
weight materials (Kds 0.71 and 0.91) was accomplished by
diafiltration of the unfractionated CPS through an Amicon membrane
YM100. To confirm its purity, the retentate was passed through
SEPHAROSE.TM. (cross-linked agarose) CL-4B and showed only a peak
of Kd 0.4. .sup.13C-NMR spectrum of the retentate (equivalent to
the peak-material of Kd 0.4) [FIG. 2] was identical to a published
.sup.13C NMR spectrum of V. cholerae O139 CPS [19, 35]. The
filtrate spectrum, in contrast, lacked chemical shifts for
colitose, quinovosamine, GluNAc and D-galacturonic acid. The
retentate gave strong reaction with the murine V. cholerae O139
hyperimmune serum by Western blot and double immunodiffusion. The
retentate, denoted as the CPS, showed only <0.5 endotoxin
units/.mu.g as measured by limulus amebocyte lysate test. Fractions
not containing colitose were not antigenic.
AH Derivatives of CSA.
CSA.sub.AH contained .about.9 moles of AH per mole CSA. CSA.sub.AH
formed a line of identity with CSA when reacted with rabbit
anti-CSA serum by double-immunodiffusion.
AH Derivatives of CPS(CPS.sub.AH, CPS.sub.AH1, CPS.sub.AH2) and rDT
(rDT.sub.AH) [Table 1].
CPS.sub.AH1, prepared by EDC-mediated reaction, contained 0.08
moles of hydrazide per mole of CPS-repeating unit. CPS.sub.AH2,
prepared by the CDAP method, contained 0.12 moles of hydrazide per
mole of CPS repeating unit. CPS.sub.AH contained 3.4% of AH, which
represents .about.1 AH per 5 CPS-repeating units. All three AH
derivatives formed a line of identity with CPS when reacted with
murine hyperimmune V. cholerae O139 serum by double
immunodiffusion.
rDT.sub.AH contained 7.2 moles of AH per mole of protein and formed
a line of identity with rDT when reacted with equine DT antiserum
by double immunodiffusion.
TABLE-US-00001 TABLE 1 Adipic acid hydrazide derivatives (AH) of
Vibrio cholerae O139 capsular polysaccharide (CPS) and of
recombinant diphtheria toxin mutant (rDT) Activating Derivative
agent AH content % mol/mol* CPS.sub.AH CDAP 3.44 0.21 rDT.sub.AH
EDC 1.90 7.12 *CPS-repeating unit (M.sub.r 1053), rDT (M.sub.r
~67,000)
Conjugates. (FIG. 3, Table 2).
EDC-Mediated Synthesis of CPS.sub.AH-CSA Conjugates
(EDC:CPS-CSA.sub.AH).
Gel filtration profile of this conjugate, prepared by EDC-mediated
binding of CPS to CSA.sub.AH, showed 2 peaks (Vo and Kd 0.52) that
contained both polysaccharide (PS) and protein (PR). The Vo
material consisted mostly of PR(PS/PR 0.15). The majority of PS was
detected in the second peak (Kd 0.52). Formation of this conjugate
was accompanied by the development of a protein precipitate that
accounted for about 30% of total PR. Only Vo material was included
in the final pool of the conjugate, and the yield (by the recovery
of PS) was 2.7%.
CDAP-Mediated Synthesis of CPS.sub.AH-CSA Conjugates
(CDAP:CPS-CSA.sub.AH).
This conjugate was prepared from the same components as
EDC:CPS-CSA.sub.AH but using CDAP as the activating agent. Gel
filtration of this conjugate showed that PS was present in
fractions 30-65, similar to the elution range of CPS alone (34-60).
PR was detected within Fr 30-70, that is 20 fractions before the
elution range of CSA alone. Only fractions 30-47 of the PS and PR
overlapping region were included in the final pool of the
conjugate. The PS/PR ratio of the conjugate was 2.6, and the yield
based on the recovery of PS was 51%.
EDC-Mediated Synthesis of CPS.sub.AH-CSA Conjugates
(EDC:CPS.sub.AH1-CSA and EDC:CPS.sub.AH2-CSA).
Both conjugates were prepared by EDC-mediated conjugation of the AH
derivative of CPS with CSA, but the w/w ratio of EDC/PR was 5-fold
higher in the synthesis of EDC:CPS.sub.AH2-CSA. It should be also
noted that CPS.sub.AH1 and CPS.sub.AH2 had a different content of
hydrazide and were prepared by different derivatization reactions.
Gel filtration of the conjugates showed that PS and PR peaks
overlapped in the range of 36-52 (EDC:CPS.sub.AH1-CSA) and 36-60
(EDC:CPS.sub.AH2-CSA). There was more PR eluted at Kd 0.78
(identical to Kd of CSA alone) in the first conjugate. Only
fractions 36-52 were included in the final pools of each conjugate.
The PS/PR (w/w) ratio and yield, by the recovery of PS, were higher
for EDC:CPS.sub.AH1-CSA than for EDC:CPS.sub.AH2-CSA.
EDC-Mediated Synthesis of CPS.sub.AH-rDT Conjugates.
Identical reaction conditions, except for the concentration of EDC,
were used to prepare I:CPS.sub.AH-rDT (0.05 M EDC) and
II:CPS.sub.AH-rDT (0.02 M EDC). Gel filtration of either conjugate
on SEPHAROSE.TM. (cross-linked agarose) CL-4B yielded 3 peaks at
Vo, Kd 0.4 and Kd 0.76 (FIG. 3A). Two peaks, at Vo and Kd 0.4,
consisted of both polysaccharide and protein, while the peak at Kd
0.76 contained only protein. Since the Kds of the free CPS and rDT
on SEPHAROSE.TM. (cross-linked agarose) CL-4B are 0.4 and 0.76,
respectively, the presence of unreacted CPS and/or rDT within the
range of Kd 0.4-0.76 could not be excluded. Accordingly, only the
void volume fractions were pooled and denoted as I:CPS.sub.AH-rDT
and II:CPS.sub.AH-rDT.
I:CPS.sub.AH-rDT had a lower polysaccharide/protein ratio (w/w)
than II:CPS.sub.AH-rDT (0.46<0.76). The yields of both
conjugates were about 20% based upon the recovery of
polysaccharide. Double immunodiffusion of either conjugate against
murine V. cholerae O139 and equine DT toxin hyperimmune sera showed
a single precipitin line (FIG. 4A).
CDAP-Mediated Synthesis of CPS-rDT.sub.AH Conjugates.
I:CPS-rDT.sub.AH and II:CPS-rDT.sub.AH were prepared under the same
conditions except for the w/w ratio of CDAP/CPS that was 4:5 and
1:5, respectively. Gel filtration of either conjugate on
SEPHAROSE.TM. (cross-linked agarose) CL-4B showed 2 peaks (Vo and
Kd 0.4) both containing polysaccharide and protein (FIG. 3B). The
void volume fractions were pooled and denoted as conjugates
I:CPS-rDT.sub.AH and II:CPS-rDT.sub.AH: their
polysaccharide/protein ratios were 0.99 and 0.90, respectively. The
yield of I:CPS-rDT.sub.AH was 45%. The yield of II:CPS-rDT.sub.AH
could not be determined because of an accidental loss of some
material. Both conjugates formed a single precipitin line when
reacted with murine V. cholerae O139 and equine DT hyperimmune sera
by double immunodiffusion (FIG. 4B).
The structural differences between CPS-rDT.sub.AH and
CPS.sub.AH-rDT are unknown, but differing points of attachment and
differing levels of crosslinking seem likely.
TABLE-US-00002 TABLE 2 Composition of V. cholerae O139 capsular
polysaccharide (CPS) conjugates with recombinant diphtheria toxin
mutant (rDT). Conjugation PS/PR Conjugate Method (w/w) Yield*
I:CPS.sub.AH-rDT EDC (0.05M) 0.46 28.0% II:CPS.sub.AH-rDT EDC
(0.02M) 0.78 20.1% I:CPS-rDT.sub.AH CDAP:CPS (4:5) 0.90 45.0%
II:CPS-rDT.sub.AH CDAP:CPS (1:5) 0.99 ** *Yield determined by the
amount of polysaccharide in conjugate ** Accidental loss of some
material prevented accurate determination
Serum Antibody Responses Elicited by Conjugates (Table 3).
Anti-CPS IgG:
All conjugates elicited a significant rise after the second
injection (P<0.006). Among the rDT conjugates, only
II:CPS.sub.AH-rDT, I:CPS-rDT.sub.AH and II:CPS-rDT.sub.AH elicited
a booster after third injection (P<0.003).
Anti-DT IgG:
All rDT conjugates elicited significant rises after the 2nd and 3rd
injections. After the third injection, II:CPS.sub.AH-rDT elicited
the highest and statistically significantly different level
compared to other conjugates (P<0.0007).
Anti-CPS IgG:
CPS alone did not elicit an antibody response compared to saline
(0.22 vs. 0.19, NS).
None of the rDT conjugates elicited a statistically significant
antibody response after the first dose. All four conjugates
elicited significant rises of anti-CPS IgG after the second dose
(P<0.006). However, only II:CPS.sub.AH-rDT, I:CPS-rDT.sub.AH and
II:CPS-rDT.sub.AH, elicited a booster response following the third
dose compared to the second one (P<0.003). There were no
significant differences between the post-third levels elicited by
these three conjugates (10.3 vs. 11.5 vs. 4.21 NS): all were
significantly higher than those elicited by I:CPS.sub.AH-rDT (10.3,
11.5, 4.21 vs. 0.43, P<0.0001).
Anti-Diphtheria Toxin IgG.
All conjugates elicited significant rises of anti-DT IgG after the
second and third injections compared to the first injection
(P<0.0001). II:CPS.sub.AH-rDT induced the highest level of
anti-DT IgG of all conjugates, however, only the post-third
injection level was statistically significantly higher (1050 vs.
245, 255, 279, P<0.0007).
TABLE-US-00003 TABLE 3 Serum IgG response specific to CPS and DT
elicited in mice (n = 10/group) by V. cholerae O139 CPS conjugates
with rDT ELISA units/ml (25-75 centiles) Conjugate Dose anti-CPS
IgG anti-DT IgG I:CPS.sub.AH-rDT 1 0.13 (0.11-0.17) 0.05
(0.03-0.06) 2 0.35 (0.24-0.5) 52.8 (33.8-87.4) 3 0.43 (0.28-0.58)
254. (121-376) II:CPS.sub.AH-rDT 1 0.23 (0.16-0.28) 0.60 (0.2-2.6)
2 1.04 (0.35-0.68) 186. (155-233) 3 10.3 (1.67-12.2) 1050.
(715-1210) I:CPS-rDT.sub.AH 1 0.11 (0.11-0.14) 0.70 (0.37-1.3) 2
0.89 (0.39-1.14) 81.1 (52.3-128) 3 11.5 (4.99-29.7) 255. (128-548)
II:CPS-rDT.sub.AH 1 0.22 (0.18-0.27) 0.80 (0.34-2.8) 2 0.49
(0.35-0.68) 146. (90-214) 3 4.21 (1.67-12.2) 279. (175-388) CPS
alone after 3 injections did not elicit anti-CPS IgG as compared to
saline (0.21 EU vs 0.19 EU, NS).
Vibriocidal Activity of Murine Sera (Table 4).
Representative sera from mice injected 3 times with CPS conjugates
were tested for vibriocidal activity. The CPS-rDT conjugate induced
titers ranged from 1600-6400: II:CPS.sub.AH-rDT induced slightly
higher titers (3200-6400) than I:CPS-rDT.sub.AH (1600-3200). These
two groups of sera showed a similar range of anti-CPS IgG levels,
while the anti-CPS IgM levels were slightly higher in mice injected
with II:CPS.sub.AH-rDT than with I:CPS-rDT.sub.AH. Similar
vibriocidal results were demonstrated with the heavily capsulated
V. cholerae O139 MDO12C variant (the strain which was used for
purification of the CPS) and other clinical isolates as the target
strains.
Following treatment with 2-ME, the vibriocidal titers of most sera
declined about 4-fold, however, all retained significant levels of
vibriocidal activity.
TABLE-US-00004 TABLE 4 Vibriocidal activity of representative sera
from mice injected 3 times with the conjugates of V. cholerae O139
capsular polysaccharide (CPS) and chicken serum albumin (CSA) or
recombinant diphtheria toxin mutant (rDT). vibriocidal titer
anti-CPS (EU) untreated treated Conjugate IgG IgM serum with 2-ME
CDAP:CPS-CSA.sub.AH 18.4 1.68 1000 -- 73.9 1.67 2000 -- 102.3 1.50
1000 -- 325.0 11.53 8000 -- EDC:CPS.sub.AH1-CSA 14.5 2.51 2000 --
58.9 2.38 4000 -- 71.9 0.91 2000 -- 118.1 2.50 2000 --
EDC:CPS.sub.AH2-CSA 18.1 2.38 1000 -- 62.4 2.21 2000 -- 70.3 2.32
4000 -- 103.5 9.92 4000 -- II:CPS.sub.AH-rDT 13.2 5.43 3200 400
17.5 3.64 1600 400 42.2 6.94 3200 800 54.5 9.11 6400 1600 180.8
10.5 >6400 1600 I:CPS.sub.AH-rDT 15.5 4.02 1600 400 18.9 1.45
1600 800 27.2 2.05 1600 200 29.7 2.38 1600 400 36.3 2.54 3200 800
68.5 2.32 3200 800 Serum anti-CPS IgG and IgM levels are expressed
in ELISA units/ml (EU) compared to a murine hyperimmeune cholera
O139 serum arbitrarily assigned 1000 EU for anti-CPS IgG and 100 EU
for anti-CPS IgM. The vibriocidal assay was performed with V.
cholerae O139 isolate SPH1168 as the target strain and 2-fold
serially diluted sera starting from 1:50 dilution. The vibriocidal
titer is defined as the reciprocal of the highest serum dilution
that caused a .gtoreq.60% reduction in the number of bacteria
compared to the complement control. Sera from mice injected with
saline or CPS had vibriocidal titer <50.
Vibriocidal Activity in Convalescent Sera of Cholera Patients
Infected with V. cholerae O139 (Table 5).
Vibriocidal titers of 20 patient sera ranged from 100 to 6400.
After absorption with CPS, titers of all sera declined to
.ltoreq.50 (baseline for the assay).
Treatment with 2-ME reduced the vibriocidal activity to .ltoreq.50
in 17/20 sera. The vibriocidal titer of SK 639-2 remained at the
same level (400) as found in the untreated serum.
TABLE-US-00005 TABLE 5 Serum vibriocidal titers of convalescent
sera from patients infected with V. cholerae O139 measured before
and after absorption with CPS or treatment with 2-mercaptoethanol
(2-ME) vibriocidal titer Patient ID untreated CPS-absorbed
2-ME-treated SK 391-2 3200 <50 <50 SK 395-2 1600 <50
<50 SK 428-2 800 <50 <50 SK 456-2 400 <50 <50 SK
458-2 3200 <50 <50 SK 494-2 100 <50 <50 SK 504-2 400
<50 <50 SK 522-2 1600 <50 <50 SK 577-2 1600 <50
<50 SK 591-2 1600 <50 <50 SK 597-2 800 <50 <50 SK
599-2 3200 <50 <50 SK 622-2 400 <50 50 SK 639-2 400 <50
400 SK 646-2 3200 <50 <50 SK 720-2 1600 <50 <50 SK
741-2 800 <50 <50 SK 749-2 >6400 <50 100 SK 755-2 1600
<50 200 SK 760-2 1600 <50 50 Each vibriocidal assay was
performed with 2-fold serially diluted tested serum starting from a
1:50-dilution and using V. cholerae O139 SPH1168 as the target
strain.
Probably because of its complex structure [19, 35] and relatively
tight folded conformation [11], development of synthetic schemes
for preparation of V. cholerae O139 CPS conjugate vaccine was
difficult and required the use of a readily available protein
carrier (chicken serum albumin, CSA) to optimize the synthetic
methods. Slight modifications of the two most successful synthetic
schemes were then used to prepare conjugates with the medically
useful rDT.
Both synthetic schemes involved adipic acid dihydrazide as the
linker and two different activating agents, CDAP and EDC. CDAP was
used to prepare AH derivative of CPS(CPS.sub.AH), and EDC was used
to prepare CSA.sub.AH and rDT.sub.AH. Conjugation of CPS.sub.AH
with rDT and CSA was mediated by EDC; alternatively conjugates were
prepared by binding rDT.sub.AH and CSA.sub.AH with CDAP-activated
CPS.
Conjugates such as EDC:CPS-CSA.sub.AH and CDAP:CPS-CSA.sub.AH,
although prepared from the same components but using different
activating agents, are structurally different molecules. EDC
activates carboxyls, while CDAP activates hydroxyls for the
reaction with nucleophilic groups [63, 64]. In addition, the
chemistry of both conjugations is complex because the potentially
activated groups (carboxyls or hydroxyls) are present on both CPS
as well as CSA.sub.AH, and they can react with both hydrazides and
amines (s amine group of lysine) on the protein. It should be
pointed out that hydrazides are stronger nucleophiles than amines,
therefore, activated carboxyls or hydroxyls will react
preferentially with hydrazides.
Synthesis of EDC:CPS-CSA.sub.AH is representative of several
conjugation experiments: all were accompanied with precipitation of
protein, and the resultant conjugates were large in molecular size,
had low w/w PS/PR ratios (.ltoreq.0.15), and were poor immunogens.
Together these findings indicate that during EDC-mediated
conjugation of CPS and CSA.sub.AH the protein became
self-cross-linked, and such structural alteration of carrier
protein could explain the low immunogenicity of this conjugate.
Self-cross-linking of protein could be a direct result of the
comparatively higher reactivity of the CSA-carboxyls than the
CPS-carboxyls.
In contrast, no protein precipitation was observed during
EDC-mediated binding of CPS.sub.AH with CSA. In this synthesis the
higher reactivity of protein carboxyls relative to CPS carboxyls
favors the reaction of protein carboxyls with they hydrazides of
CPS.sub.AH, which results in the formation of conjugate. The lower
reactivity of CPS carboxyls reduces the extent of self-crosslinking
of CPS molecules. Both resultant conjugates, EDC:CPS.sub.AH1-CSA
and EDC:CPS.sub.AH2-CSA, had high PS/PR ratios and were
significantly better immunogens than EDC:CPS-CSA.sub.AH.
In contrast to the EDC-mediated coupling of CPS to CSA.sub.AH, the
CDAP-mediated coupling of the same components resulted in the
formation of the highly immunogenic conjugate CDAP:CPS-CSA.sub.AH.
It is also of interest, that although CSA.sub.AH was prepared by an
EDC-mediated derivatization, this exposure to relatively mild
conditions (10 mM EDC for 1 h) had no apparent negative effect on
the carrier protein or on the immunogenicity of the resultant
conjugate.
The resultant conjugates elicited serum anti-CPS IgG after the
second injection and a booster after the third injection when
administered to mice by a clinically relevant method and route.
Similarly to the immunologic properties of the V. cholerae O1
serotype Inaba O-specific polysaccharide conjugates with cholera
toxin [10], the V. cholerae O139 CPS-CSA and CPS-rDT conjugates
elicited high titers of serum vibriocidal antibodies in mice.
Treatment with 2-ME reduced (.about.4-fold) but did not eliminate
the CPS-rDT induced vibriocidal activity, indicating that much of
this activity was mediated by anti-CPS IgG.
I:CPS-rDT.sub.AH elicited the highest level of anti-CPS IgG after
the third injection (11.4 EU) but this was not statistically
different from the levels elicited by II:CPS.sub.AH-rDT (10.3 EU)
or II:CPS-rDT.sub.AH (4.21 EU). On the basis of these data,
I:CPS-rDT.sub.AH and II:CPS.sub.AH-rDT will be clinically
evaluated.
All four conjugates elicited significant rises of anti-DT IgG after
the second and third injections. II:CPS.sub.AH-rDT elicited the
highest post-third injection level of anti-DT IgG that was
significantly different from those of other 3 conjugates
(P<0.0007).
I:CPS.sub.AH-rDT, prepared by synthesis of CPS.sub.AH with rDT at
the higher concentration of EDC (0.05 M), elicited the lowest level
of anti-CPS IgG. The level of anti-DT IgG induced by this conjugate
was comparable to those elicited by both of CPS-rDT.sub.AH
indicating that there was no correlation between the antibody
elicited to the CPS and to the protein carrier.
There is some confusion about the vibriocidal activity of
convalescent sera from patients infected with V. cholerae O139 [3,
17, 25, 28, 40]. Patient sera convalescent from cholera O139 was
found to be uniformly vibriocidal. The data variation among
laboratories may be explained by the different complement dilutions
used for the vibriocidal assays. We found that highly diluted
complement, used in the vibriocidal assay for V. cholerae O1, is
not sufficient to mediate killing of V. cholerae O139 which has a
capsule. We showed that the undiluted baby rabbit serum, as the
source of complement, is a reliable reagent to demonstrate
antibody-initiated lysis of V. cholerae O139.
Similar to the serologic response of humans to the V. cholerae O1
infection [1, 24, 29, 32, 34], our results showed that vibriocidal
activity of sera from patients infected with serotype O139 was
mostly specific to its surface polysaccharide (CPS) and mediated by
IgM. This is also true for parenterally administered killed whole
cell cholera O1 vaccine or orally administered attenuated cholera
O1 strains [7, 27, 44]. In contrast, parenterally administered
polysaccharide-protein conjugate vaccines elicit, in addition to
IgM, high levels of serum anti-polysaccharide IgG (2-ME resistant)
[10, 38]. We proposed that it is IgG that penetrates on to the
intestinal epithelium and initiates complement-mediated lysis of
the bacterial inoculum and that measurement of the
conjugate-induced serum IgG specific to the surface polysaccharides
of both V. cholerae O1 and O139 should provide a reliable method
for standardization of these vaccine candidates [36, 39].
Diafiltration through YM100 allowed a rapid separation of the
low-molecular weight impurities from V. cholerae O139 CPS. When the
material eluted at Kd 0.91 from SEPHAROSE.TM. (cross-linked
agarose) CL-4B, representing only 1% (by weight) of the
unfractionated CPS, was concentrated 100-fold and analyzed by
SDS-PAGE/Western blot with murine hyperimmune cholera O139
antiserum, it showed two fast-moving bands, similar to that
reported for LPS of V. cholerae O139 [4, 45]. Our results indicate
that diafiltration could be adapted for rapid separation of CPS
and/or LPS from other medically useful polysaccharides.
In summary, V. cholerae O139 CPS conjugates with rDT elicited high
levels of serum anti-CPS IgG in mice with vibriocidal activity. The
vibriocidal activity of convalescent sera from patients infected
with V. cholerae O139 was mediated mostly by anti-CPS IgM. To
verify whether a critical level of anti-CPS IgG will confer
immunity to V. cholerae O139, clinical trials of the two most
immunogenic CPS-rDT conjugates are planned.
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Modifications of the above described modes for carrying out the
disclosure that are obvious to those of skill in the fields of
immunology, protein chemistry, medicine, and related fields are
intended to be within the scope of the following claims.
Every reference cited hereinabove is hereby incorporated by
reference in its entirety.
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