U.S. patent application number 10/643314 was filed with the patent office on 2004-03-18 for antigenic conjugates of conserved lipopolysaccharides of gram negative bacteria.
This patent application is currently assigned to Wyeth Holdings Corporation. Invention is credited to Apicella, Michael A., Arumugham, Rasappa G., Fortuna-Nevin, Maria, Gibson, Bradford W..
Application Number | 20040052804 10/643314 |
Document ID | / |
Family ID | 29405716 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040052804 |
Kind Code |
A1 |
Arumugham, Rasappa G. ; et
al. |
March 18, 2004 |
Antigenic conjugates of conserved lipopolysaccharides of gram
negative bacteria
Abstract
Antigenic conjugates are provided which comprise a carrier
protein covalently bonded to the conserved portion of a
lipopolysaccharide of a gram negative bacteria, wherein said
conserved portion of the lipopolysaccharide comprises the inner
core and lipid A portions of said lipopolysaccharide, said
conjugate eliciting a cross reactive immune response against
heterologous strains of said gram negative bacteria.
Inventors: |
Arumugham, Rasappa G.;
(Chapel Hill, NC) ; Fortuna-Nevin, Maria;
(Webster, NY) ; Apicella, Michael A.; (Solon,
IA) ; Gibson, Bradford W.; (Berkeley, CA) |
Correspondence
Address: |
WYETH
PATENT LAW GROUP
FIVE GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth Holdings Corporation
Madison
NJ
|
Family ID: |
29405716 |
Appl. No.: |
10/643314 |
Filed: |
August 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10643314 |
Aug 19, 2003 |
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09264747 |
Mar 9, 1999 |
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6645503 |
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60088364 |
Mar 10, 1998 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 2039/6037 20130101; A61K 39/385 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 039/00; A61K
039/38 |
Claims
What is claimed is:
1. An antigenic conjugate comprising a carrier protein covalently
bonded to the conserved portion of a lipopolysaccharide of a gram
negative bacteria, wherein said conserved portion of the
lipopolysaccaride comprises the inner core and lipid A portions of
said lipopolysaccaride, said conjugate eliciting a cross reactive
immune response against heterologous strains of said gram negative
bacteria.
2. An antigenic conjugate as in claim 1, wherein said conjugate
elicits a cross reactive immune response against heterologous
genera of gram negative bacteria.
3. An antigenic conjugate as in claim 1, wherein said
lipopolysaccharide is de-O-acylated.
4. An antigenic conjugate as in claim 1, wherein said carrier
protein is selected from the group consisting of tetanus toxin or
toxoid, diptheria toxin or toxoid, mutant of diptheria toxin
CRM.sub.197, pseudomonas exotoxin A, cholera toxin or toxoid, Group
A streptococcal toxins, pneumolysin of Streptococcus pneumoniae,
filamentous haemagglutinin (FHA), FHA fragments of Bordetella
pertussis; pili or pilins of Neisseria gonorrhoeae, pili or pilins
of Neisseria meningitidis; outer membrane proteins of Neisseria
meningitidis, outer membrane proteins of Neisseria gonorrhoeae; C5A
peptidase of Streptococcus and surface protein of Moraxella
catarrhalis.
5. An antigenic conjugate as in claim 1, wherein said carrier
protein is linked to said conserved portion of the
lipopolysaccharide with a compound selected from the group
consisting of Sulfosuccinimidyl-6-(3-[2--
pyridyldithio]propionamido)-hexanoate (Sulfo-LC-SPDP);
succinimidyl-6-(3-[2-pyridyldithio]propionamido)-hexanoate
(LC-SPDP); Traut's reagent (2-iminothiolane); N-succinimyl-S-acetyl
thioacetate (SATA); N-Succinimidyl-3-(2-pyridyl dithio)propionate
(SPDP), succinimidyl acetyl thiopiopionate (SATP),
succinimidyl-4-(N-maleimido methyl)cyclohexane-1-carboxylate
(SMCC), maleimido benzoyl-N-hydroxy succinimide ester (MBS),
N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB), bromoacetic acid-N-hydroxy
succinimide (BANS) ester, 1-ethyl-3-(3-dimethylamino propyl)
carbodiimide (EDAC), adipic acid dihydrazide (ADH), cystamine and
dithiobis(succinimidyl propionate) (DTSSP).
6. An antigenic conjugate as in claim 1 wherein said gram negative
bacteria is selected from the group consisting of Neisseria
meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae,
non-typeable Haemophilus influenzae, Haemophilus ducreyi,
Helicobacter pylori, Escherichia coli, Chlamydia, Salmonella,
Salmonella typhimurium, Salmonella minnesota, Proteus mirabilis,
Pseudomonas aeruginosa, Moraxella catarrhalis, Bordetella
pertussis, Shigella, Klebsiella, and Vibrio cholerae.
7. An antigenic conjugate as in claim 6, wherein said gram negative
bacterium is Neisseria meningitidis.
8. An antigenic conjugate comprising the carrier protein diptheria
toxin CRM.sub.197 covalently bonded to the conserved portion of a
lipopolysaccharide of Neisseria meningitidis with long chain
N-succinimidyl-3-(2-pyridyldithio)-propionate, and bromoacetic
acid-N-hydroxysuccinimide ester, wherein said conserved portion of
the lipopolysaccharide comprises the inner core and lipid A
portions of said lipopolysaccharide, said conjugate eliciting a
cross reactive immune response against heterologous strains within
the genus Neisseria meningitidis.
9. An antigenic conjugate as in claim 8, wherein said conjugate
elicits a cross reactive immune response against heterologous
genera of gram negative bacteria.
10. A vaccine formulation comprising an effective amount of the
antigenic conjugate of claim 1.
11. A vaccine formulation comprising an effective amount of the
antigenic conjugate of claim 2.
12. A vaccine formulation comprising an effective amount of the
antigenic conjugate of claim 8.
13. A vaccine formulation comprising an effective amount of the
antigenic conjugate of claim 9.
14. A method of immunizing an individual to prevent disease caused
by a gram negative pathogen, comprising vaccinating the individual
with a prophylactically effective amount of vaccine formulation
comprising an antigenic conjugate comprising a carrier protein
covalently bonded to the conserved portion of a lipopolysaccharide
of a gram negative bacteria, wherein said conserved portion of the
lipopolysaccharide comprises the inner core and lipid A portions of
said lipopolysaccharide, said conjugate eliciting a cross reactive
immune response against heterologous strains of said gram negative
bacteria.
15. A method as in claim 14, wherein the vaccine formulation is
administered to said individual by a route of administration
selected from the group consisting of intradermal, intramuscular,
intraperitioneal, intravenous, vaginal, subcutaneous, ocular,
intranasal, and oral administration.
16. A method as in claim 14, wherein said vaccine formulation
further comprises a physiological carrier and an adjuvant.
17. A method for preventing bacterial sepsis in a mammal in need
thereof, comprising administering an effective amount of a
formulation comprising an antigenic conjugate comprising a carrier
protein covalently bonded to the conserved portion of a
lipopolysaccharide of a gram negative bacteria, wherein said
conserved portion of the lipopolysaccharide comprises the inner
core and lipid A portions of said lipopolysaccharide, said
conjugate eliciting a cross reactive immune response against
heterologous strains of said gram negative bacterial organisms.
18. A method for preventing bacterial sepsis in a mammal in need
thereof, comprising administering an effective amount of a
formulation comprising an antigenic conjugate comprising a carrier
protein covalently bonded to the conserved portion of a
lipopolysaccharide of a gram negative bacteria, wherein said
conserved portion of the lipopolysaccharide comprises the inner
core and lipid A portions of said lipopolysaccharide, said
conjugate eliciting a cross reactive immune response against
heterologous genera of gram negative bacterial organisms.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/088/364 which was converted from U.S. patent
application Ser. No. 09/037,529, filed Mar. 10, 1998, pursuant to a
petition filed under 37 C.F.R. 1.53(c)(2) filed May 6, 1998.
FIELD OF THE INVENTION
[0002] This invention relates to antigenic conjugates of the
conserved portion of the lipopolysaccharides of certain gram
negative bacteria and to vaccines containing such antigenic
conjugates. The conjugates elicit antibodies which exhibit cross
reactivity against heterologous strains of gram negative bacteria
and the vaccines containing such conjugates induce antibodies which
are functional and protective against such gram negative bacterial
organisms.
BACKGROUND OF THE INVENTION
[0003] Lipopolysaccharides (LPS) are major surface antigens
localized abundantly on the surface of gram negative bacteria. LPS
molecules are comprised of: (1) a lipid A portion which consists of
a glucosamine disaccharide substituted with phosphates,
phosphoethanolamine groups and long chain fatty acids in ester and
amide linkages; (2) an inner core portion attached to the lipid A
portion by an eight carbon sugar, ketodeoxyoctonoate (KDO), which
may be substituted by 1 to 2 additional KDO molecules and by up to
3 heptose moieties; (3) an outer core portion comprising hexoses
such as glucose, galactose, N-acetylglucosamine and
N-acetylgalactosamine; and (4) an O-specific chain comprising
repeating oligo-saccharide units which vary widely among bacterial
strains. Polymerization of these repeating units to structures in
excess of 60,000 daltons is not uncommon. The LPS molecules can
vary extensively at the structural and antigenic level among
bacterial strains, although the structure of the inner core is
largely conserved among bacterial species. A typical structure of
the lipid-A inner core of Salmonella typhimurium LPS is illustrated
in FIG. 1. The immune response responsible for the evolution of
naturally protective antibodies is considered to arise by natural
immunization to this region of the LPS.
[0004] In non-enteric pathogens, the LPS structure lacks repeating
O-antigen units. Moreover, the complete genetic machinery for
assembly of the O-antigen repeating unit appears to be absent in
such pathogens. This has led to the designation of these LPS
structures as lipooligosaccharides (LOS). There are similarities
between LPS and LOS structures in such pathogens. For instance, all
of the LPS and LOS structures link the lipid A regions to the cores
through the KDO junction. The number of KDO residues can vary from
one (e.g., Haemophilus influenzae and Haemophilus ducreyi) to two
(e.g., Neisseria meningitidis and Neisseria gonorrhoeae). Recent
studies indicate that the branched oliogsaccharides are synthesized
separately from the core region and the assembly of the entire LOS
structure is completed on the outer side of the cytoplasmic
membrane (see Preston, et al., "The Lipooligosaccharides of
Pathogenic Gram-Negative Bacteria", Critical Reviews In
Microbiology, 22:139-180 (1996)).
[0005] Accordingly, there is a single core region in such LOS
structures without a distinct inner and outer core region. The core
structure of these pathogens can vary from species to species and
may comprise KDO in the complete absence of heptose (e.g.,
Moraxella catarrhalis); KDO in the presence of a di-heptose
structure (e.g., Neisseria meningitidis and Neisseria gonorrhoeae);
or KDO in the presence of a triheptose structure (e.g., Haemophilus
influenzae and Haemophilus ducreyi). Examples of core structures
from Haemophilus and Neisseria are shown in FIG. 2. The
oligosaccharide units can extend from each of the heptoses and/or
they can be substituted by phosphoethanolamine groups. Typical
examples of completed LOS structures of Haemophilus influenzae
strain 2019 (see Phillips et al., "Structural Characterization of
the Cell Surface Lipooligosaccharides from a Non-Typable Strain of
Haemophilus Influenzae," Biochemistry, 31:4515-4526 (1992)) and
Neisseria gonorrhoeae strain 1291 (see John et al., "The Structural
Bases for Pyocin Resistance in Neisseria gonorrhoeae
lipooligosaccharides," J. Biol. Chem., 266:1903-1911 (1991)) are
shown in FIG. 3.
[0006] The use of LPS in the development of vaccines is known in
the art. It has long been recognized that a specific antibody
response directed against the LPS of a particular bacterial
pathogen can contribute to protection against that specific strain.
It is further known that saccharide structures (e.g., the
saccharide portions of LPS) can be conjugated to a carrier protein,
so that a vaccine composition containing such a conjugate will
elicit the desired T-dependent response. An example of this is the
successful glycoconjugate vaccines against bacteria having
type-specific capsular saccharides see, Vaccine Design: The Subunit
and Adjuvant Approach, Powell, M. F., and Newman, M. J., 673-694
(1995). This category of immune response is the basis for the
effectiveness in human infants of a new generation of
saccharide-protein conjugate vaccines as discussed in Vaccine
Design: The Subunit and Adjuvant Approach, Powell, M. F., and
Newman, M. J., 695-718 (1995).
[0007] However, since the LPS of heterologous strains of such
pathogens demonstrate extensive variation of the outer core
saccharide and/or O-specific chain, efforts to generate an antibody
response to a number of heterologous strains or heterologous genera
of bacteria utilizing a vaccine containing a single LPS have to
date been unsuccessful.
[0008] In an effort to develop an LPS-based vaccine against
Neisseria meningitidis, tetanus toxoid has been conjugated with
oligosaccharides isolated from the LPS of a number of Neisseria
meningitidis strains (see, Jennings et al., Infect. Immun.,
43:407-412 (1984)). However, the antibodies elicited by this
conjugate were oligosaccharide specific and exhibited a high degree
of serotype specificity.
[0009] Verheul et al., Infect. Immun., 61:187-196 (1993), disclose
the conjugation of oligosaccharides of meningococcal LPS to either
tetanus toxoid or meningococcal outer membrane protein. In mice,
the tetanus toxoid conjugates induced oligosaccharide specific
antibodies which were not bactericidal against meningococci. The
outer membrane protein--LPS conjugates induced antibodies against
the outer membrane protein, but not against LPS. Verheul et al.,
Infect. Immun., 59:843-851 (1991), also studied the immunogenicity
of the conjugates of oligosaccharides of several Neisseria
meningitidis strains and tetanus toxoid in rabbits. The results
demonstrated that the antibodies elicited are directed only towards
the oligosaccharide portion of the LPS which contain the immunotype
specific epitopes.
[0010] The preparation of oligosaccharides from the LPS of
Neisseria meningitidis laboratory adapted wild strain A1 and the
subsequent conjugation thereof to tetanus toxoid as the carrier
protein was disclosed in Gu et al., Infect. Immun., 61:1873-1880
(1993). The conjugates were immunogenic in mice and rabbits and the
majority of the antibodies were directed against
immunotype-specific LPS epitopes. Also, the conjugate antisera
showed less cross reactivity to different immunotypes of LPS than
the LPS antisera. These studies demonstrate that LPS-derived
oligosaccharide conjugates induce antibodies to the specific
oligosaccharide immunotype. However, there was no evidence that the
conjugate induced significant cross reactive antibodies to a common
core saccharide structure present in the majority of Neisseria
meningitidis strains.
[0011] Bhattachaijee et al., J. Infect. Dis., 173:1157-1163 (1996)
disclosed the mixture of the LPS from Escherichia coli with the
outer membrane protein of Neisseria meningitidis group B, resulting
in the formation of unconjugated, non-covalent complexes thereof.
It was found that these complexes elicited antibodies which cross
reacted with a number of gram negative bacteria. However, no
evidence was provided to indicate that these complexes had
properties different from other preparations of unconjugated
saccharide structures which are known to be incapable of eliciting
a T-dependent antibody response which can be boosted upon
administration of subsequent doses. Moreover, it is known that such
unconjugated saccharides do not elicit an appropriate immune
response in infants.
[0012] Gu et al., Infect. Immun., 64:4047-4053 (1996) disclose the
preparation of conjugates of oligosaccharides from nontypeable
Haemophilus influenzae and tetanus toxoid. However, the antisera
induced in rabbits demonstrated bactericidal activity against only
homologous strains.
[0013] Accordingly, there remains a need for antigenic conjugates
and vaccines containing such conjugates which effectively induce an
immunogenic response, preferably a T-dependent response, to a given
species of gram negative bacteria, as well as which exhibit
effective cross reactivity to heterologous strains or serotypes of
gram negative bacteria within a given genus. Moreover, it would be
advantageous for such conjugates and vaccines to elicit antibodies
which exhibit cross reactivity to heterologous genera of gram
negative bacteria.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to antigenic conjugates
comprising a carrier protein covalently bonded to the conserved LPS
portion of a gram negative bacteria, wherein the conserved portion
of the LPS comprises at least the conserved inner core and the
lipid A portion of the LPS. The conjugate elicits a cross reactive
immune response against heterologous strains of gram negative
bacteria and preferably, against heterologous genera of gram
negative bacteria.
[0015] The present invention is further directed to vaccines
comprising these antigenic conjugates and methods for immunizing
individuals with such vaccines to prevent various diseases caused
by gram negative bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A shows the typical structure of the lipid A-inner
core of Salmonella typhimurium.
[0017] FIG. 1B shows the typical structures of the O-antigen or
repeating polysaccharide of Salmonella typhimurium.
[0018] FIG. 1C shows a structure for the heptaacyl Lipid A of
Salmonella typhimurium.
[0019] FIG. 2A shows the core LOS structure of Haemophilus
species.
[0020] FIG. 2B shows the core LOS structure of Neisseria
species.
[0021] FIG. 3A shows the LOS structure of Haemophilus influenzae
strain 2019.
[0022] FIG. 3B shows the LOS structure of Neisseria gonorrhoeae
strain 1291.
[0023] FIG. 4 shows the Western Blot analysis of the activity of
various conjugates against the anti-sera of various bacteria.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention is directed to antigenic conjugates of
a carrier protein and the conserved LPS of gram negative bacteria
and vaccines containing such conjugates. The present conjugates
utilize the LPS of various gram negative bacteria including, but
not limited to: Neisseria meningitidis, Neisseria gonorrhoeae,
Haemophilus influenzae, non-typeable Haemophilus influenzae,
Haemophilus ducreyi, Helicobacter pylori, Escherichia coli,
Chlamydia, Salmonella, Salmonella typhimurium, Salmonella
minnesota, Proteus mirabilis, Pseudomonas aeruginosa, Moraxella
catarrhalis, Bordetella pertussis, Shigella, Klebsiella, and Vibrio
cholerae.
[0025] The present conjugates and the vaccines containing these
conjugates generate a functional polyclonal antibody response
against the conserved LPS portion contained in the conjugates.
Thus, the vaccines are capable of reacting to a large number of
heterologous strains of pathogens, thereby inducing a cross
reactive and cross-functional antibody response against different
strains of gram negative bacteria. This cross reactive response is
demonstrated both against heterologous strains within a given
genus, as well as against heterologous genera of gram negative
bacteria.
[0026] The present invention is thus directed to antigenic
conjugates which generate an antibody response against the common
conserved portions of the LPS of a gram negative bacteria, i.e.,
portions of the LPS which are common to a number of gram negative
bacteria. As used herein, the term "LPS" is meant to include both
smooth LPS and LOS (otherwise known as "rough LPS").
[0027] As noted above, the LPS of gram negative bacteria comprise
the inner core portion, the Lipid A portion, the outer core portion
and the O-specific antigen. In order to elicit a response to
heterologous strains or genera of bacteria, the structure of the
inner core and lipid A portions must be conserved and utilized in
the present conjugates. Accordingly, the term "conserved portion"
of LPS as used herein is meant to include at least the glucosamine
disaccharide substituted with phosphates, phosphoethanolamine
groups and long chain fatty acids in ester and amide linkages
(i.e., the lipid A portion); and the KDO function of the inner core
and the heptose substituents, if any. The phosphates,
phosphoethanolamine and pyrophosphoethanolamine groups which may be
contained in the inner core may also be included in the "conserved
portion", although they may not be necessary. The portion of the
pathogen contained in the "conserved portion" is highly conserved
among bacterial strains and thus, broadly cross reactive antibodies
can arise from these structures (see, e.g., Apicella et al., "The
Normal Human Serum Bactericidal Antibody Response to the
Lipooligosaccaride of Neisseria gonorrhoeae", J. Infect. Dis.,
153:520-528 (1986).
[0028] It is further within the scope of the present invention that
additional branched carbohydrates of the inner core of LPS-bearing
strains or oligosaccharides of LOS-bearing strains, may be
conserved as part of the "conserved portion" as defined herein.
This, however, is not required or even desirable in many
instances.
[0029] It has been found by the present inventors that the
generation of a conjugate utilizing such a conserved portion of the
LPS structure elicits a boostable, T-cell dependent IgG response in
the individual being treated and that the resultant antibodies
cross react to the surface of heterologous strains within a
particular bacterial genus, as well as to heterologous genera of
grain negative bacteria. Moreover, the surface reactive antibodies
elicited by the present conjugates have been found to be both
bactericidal (i.e., demonstrating a functional property associated
with immunoprotection) and protective.
[0030] The conserved portions of the LPS utilized in the present
conjugates may be prepared by a number of techniques known to those
skilled in the art. For example, the conserved structure may be
prepared by: (1) the chemical synthesis of whole or part of the
conserved core structure, (2) the selection of wild type strains
that will produce LPS which contains predominantly the conserved
structure, (3) the enzymatic cleavage of non-reducing sugar
residues of LPS synthesized by wild type strains, and (4) the
synthesis of the conserved structure of the LPS of a given
bacterial organism by various mutants and progenies derived from
those mutants such as disclosed in PCT Application Publication No.
WO97/19688 (e.g., the production of the conserved core portion of
Neisseria meningitidis LPS by mutant strains defective in the
biosynthesis of LPS; the production of core-defective rough mutants
of Salmonella and Escherichia coli by their exposure to
bacteriophages; the production of Neisseria gonorrhoeae LPS mutants
by the exposure of wild type strains to pyocin and the progenies
derived from these mutants; the generation of transposan-induced
mutations of specific enzymes involved in the biosynthesis of LPS;
and the site-directed mutations of specific enzymes involved in LPS
biosynthesis and the progenies derived from those mutant
strains).
[0031] The preferred means of preparing the conserved portions of
the LPS for use in the present conjugates is through the synthesis
of the LPS conserved portion via mutant bacterial strains and their
progeny (pathway 4 above). More preferably, the following mutant
strains are utilized to synthesize the LPS conserved portion:
organisms that express only the conserved core saccharides of the
LPS, such as the R.sub.a core (both inner and outer core) of
Salmonella, R.sub.c and R.sub.e of Salmonella and J.sub.5 of
Escherichia coli; organisms that do not add glucose to the core
portion of the LPS; organisms that do not add galactose to the core
portion of the LPS, such as the strain 281.25 mutant from
Haemophilus influenzae type b as described in Biochemistry,
35:5837-5947 (1996); organisms that do not add glucose to the core
portion of the LPS due to mutations in the phosphoglucomutase (PGM)
gene, such as the NMB R6 strain of Neisseria meningitidis and
1291-R6 strain of Neisseria gonorrhoeae discribed in J. Biol.
Chem., 269:11162-11169 (1994); organisms that do not add galactose
to the core portion of the LPS due to mutations in the galactose
epimerase (GalE) gene, such as the mutant strain SS3 of Neisseria
meningitidis described in Infect. Immun., 63:2508-2515 (1995); and
organisms that do not add glucose or galactose to the core portion
of the LPS due to mutations in the corresponding transferase
enzymes, such as mutations in the corresponding transferase enzyme,
such as mutations in the glucosyl or galactosyl transferase genes
described in J. Exp. Med., 180:2181-2190 (1994).
[0032] Studies by Lee et al., Infect. Immun., 63:2508-2515 (1995)
have shown that a mutation in galactose epimerase results in the
expression of LPS which is truncated after the outer core or branch
regions containing glucose. A screening of Tn916-generated mutants
of group B Neisseria meningitidis showed that a stable LPS mutant
strain (designated NMB-R6 strain) expressed a deep core LPS with
the structure: GlcNAc-Hep.sub.2(PEA)-KDO.sub.2-LipidA. Additional
studies have shown that transposon is inserted into the putative
phospho-glucomutase gene (see J. Biol. Chem., 269:11162-11169
(1994)). The cell free extracts of the mutant strain demonstrated
no phosphoglucomutase activity. Similarly, another
transposon-induced mutation eliminates UDP-glucose-4 epimerase
activity.
[0033] LPS is biosynthesized by the initial synthesis of the Lipid
A portion followed by the successive addition of the sugar residues
of the inner core and then the outer core. It is known that the
addition of hexose units, such as glucose and galactose, to the
core (e.g., GlcNAc-Hep.sub.2(PEA)-KDO.sub.2-Lipid A of Neisseria
meningitidis) is catalyzed by a series of enzymes involved in the
biosynthesis. For example, glucose is converted to
glucose-6-phosphate by glucokinase. An enzyme, phospho-glucomutase,
converts glucose-6-phosphate to glucose-1-phosphate, which is then
converted to UDP-glucose by
UTP-glucose-1-phosphate-uridyl-tranferase. UDP-glucose is also
converted to UDP-galactose by UDP-glucose-4-epimerase. Hexose units
from these UDP intermediates are then transferred to the core of
the LPS catalyzed by the transferases. The loss of these transfer
activities or the enzymes responsible for the generation of
UDP-glucose and UDP-galactose result in the preparation of LPS
structures containing only the inner core. The pathway necessary
for making the core LPS would not be affected by the loss of
phospho-glucomutase, UTP-glucose-1-phosphate-uridyl transferase,
UDP-glucose-4 epimerase, or the UDP-glucose or UDP-galactose
transferases. Therefore, any defect in these enzymes will result in
the chain termination of LPS.
[0034] Although the LPS of a given gram negative bacteria in any
form may be utilized to prepare the present conjugates, it is
preferred that the conserved LPS portions of the present invention
be de-O-acylated prior to conjugation. The LPS structure can
advantageously be de-O-acylated by the mild alkaline hydrolysis
thereof with sodium hydroxide as described in, e.g., J. Biol.
Chem., 250:1926-1932 (1975) and J. Biol. Chem., 256:7305-7310
(1981) or by mild hydrazine treatment as described in, e.g., Eur.
J. Biochem., 177:483-492 (1988). It has been found that
de-O-acylating the LPS improves its immunogenicity and reduces its
toxicity. Alternatively, the non-toxic LPS portion can be isolated
from non-toxic mutants of pathogenic gram negative bacteria, e.g.,
in the manner described in WO/97/19688.
[0035] In producing the antigenic conjugates of the present
invention, the conserved LPS portion is linked to a carrier protein
via an appropriate linker compound. The use of such linker
compounds is known in the art and discussed, e.g., in Conjugate
Vaccines, Cruise et al., 48-114, Karger Publishing (1989).
[0036] For example, reactive groups on the lipid A or inner core
portions of the conserved LPS structure can be bound to known
heterobifunctional and homobifunctional linking agents. The
heterobifunctional linking agents contain heterologous reactive
ends and produce intermediates with the conserved LPS. The
intermediates are linked at one end to the LPS while the opposing
end contains a reactive group. Homobifunctional linking agents also
contain two reactive groups, however they are identical. In
general, the linking agents connect the conserved LPS portion to
the carrier protein via amino, hydroxyl, or carboxyl groups. The
amino and hydroxyl group linkages are formed with the saccharides
of the lipid A or inner core of the conserved LPS. The carboxyl
group linkages are formed with the KDO of the inner core. The
opposing end of the linking agent preferably contains a sulfhydryl
group for further reaction with the carrier protein.
[0037] Suitable linking agents for use in the present invention
include, e.g.,
Sulfosuccinimidyl-6-(3-[2-pyridyldithio]propionamido)-hexanoate
(Sulfo-LC-SPDP);
succinimidyl-6-(3-[2-pyridyldithio]propionamido)-hexanoa- te
(LC-SPDP); Traut's reagent (2-iminothiolane); N-succinimyl-S-acetyl
thioacetate (SATA); N-Succinimidyl-3-(2-pyridyl dithio)propionate
(SPDP), succinimidyl acetyl thiopropionate (SATP),
succinimidyl-4-(N-maleimido methyl)cyclohexane-1-carboxylate
(SMCC), maleimido benzoyl-N-hydroxy succinimide ester (MBS),
N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB), bromoacetic acid-N-hydroxy
succinimide (BANS) ester, 1-ethyl-3-(3-dimethylamino propyl)
carbodiimide (EDAC), adipic acid dihydrazide (ADH), cystamine and
dithiobis(succinimidyl propionate) (DTSSP).
[0038] The conserved portion of the LPS is allowed to react with
the linking agent in a non-amino containing buffer solution (such
as phosphate buffered saline or sodium biocarbonate) at a neutral
to slightly alkaline pH for a suitable period of time (e.g.,
approximately one hour at room temperature). The intermediate is
then removed from unreacted reagent by any suitable means (e.g.,
gel filtration). The carrier protein is then activated by reaction
with a suitable linking agent selected from the group set forth
above. The conserved LPS intermediate is then reacted with the
activated carrier protein, under suitable conditions, to produce
the present conjugates.
[0039] In the case of linkage via sulfhydryl groups, the
LPS-linking agent intermediate is reacted with a suitable reducing
agent or hydroxylamine to reduce the disulfide bond on the linking
agent to expose free sulfhydryl groups. Suitable reducing agents
include dithiothreitol (DTT) and mercaptoethanol. The
sulfhydryl-exposed intermediate is then reacted with an activated
carrier protein to provide a thioether-linked covalently bound
conjugate.
[0040] The formation of an antigenic conjugate via the sulfhydryl
groups is illustrated in the schematic set forth below: 1
[0041] Alternatively, aldehydes may be exposed on the LPS structure
by the periodate oxidation of vicinyl hydroxyl groups on the
saccharide structures as disclosed in, e.g., Morrison, R. T. and
Boyd, R. N., Organic Chemistry, Allyn and Bacon, Inc., 875-905
(1966), or by the treatment of LPS containing a non-reducing
terminal galactose or N-acetyl galactosamine residue with galactose
oxidase to transform the C-6 hydroxyl group to an aldehyde group as
disclosed, e.g., in Avigaeld et al., J. Biol. Chem., 237:2736,
(1962). The aldehyde-containing LPS can then be attached to a
suitable carrier protein, e.g., by reductive amination.
[0042] In another alternative method, the present conjugates may be
formed by the linkage of the conserved portion of the LPS and the
carrier protein via carboxyl groups on the LPS with the use of a
carbodimide reagent such as
1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDAC).
In this method, it is preferred that the conjugates are prepared by
linking the carboxyl groups of the LPS saccharide to the carboxyl
groups of the carrier protein. In this case, the conserved LPS is
first reacted with a suitable linker compound, such as adipic acid
dihydrazide (ADH) in the presence of EDAC. The carrier protein may
then be reacted with the ADH-LPS intermediate in the presence of an
appropriate linker compound, such as EDAC. A carboxyl-linked
conjugate is obtained.
[0043] The formation of an antigenic conjugate via linkage of the
carboxyl groups is illustrated in the schematic set forth below.
2
[0044] In another alternative embodiment, the present conjugates
may be formed by linking the carboxyl groups of the LPS saccharide
to the amino groups of a carrier protein. In this case, the
conserved LPS is first reacted with cystamine in the presence of
EDAC. The sulfhydryl group from the cystamine-LPS intermediate is
exposed by a reducing agent, such as dithiothritol and finally
reacted with a bromo-acetylated carrier protein.
[0045] Carrier proteins useful in the preparation of the present
antigenic conjugates include bacterial toxins and toxoids (e.g.,
tetanus toxin or toxoid, diptheria toxin or toxoid, non-toxic
mutants of diptheria toxin CRM-.sub.197 (as described, e.g., in
Immunochem., 9:891-906 (1972)), pseudomonas exotoxin A, cholera
toxin or toxoid, Group A streptococcal toxins, pneumolysin of
Streptococcus pneumoniae, etc.); filamentous haemagglutinin (FHA)
or FHA fragment(s) of Bordetella pertussis; pili or pilins of
Neisseria gonorrhoeae; pili or pilins of Neisseria meningitidis;
bacterial outer membrane proteins (e.g., outer membrane protein
complexes of Neisseria meningitidis (e.g., such as class 1 outer
membrane protein and class 3 outer membrane protein of Neisseria
meningitidis)); outer membrane proteins of Neisseria gonorrhoeae;
C5A peptidase of Streptococcus; and ubiquitous surface protein of
Moraxella catarrhalis. The preferred carrier protein is
CRM-.sub.197.
[0046] Vaccines containing the antigenic conjugates of the present
invention may advantageously contain various adjuvants which are
known to augment the immune response to the vaccine antigen. It is
believed that such adjuvants increase the antibody response by the
non-specific stimulation of the patient's immune system. The use of
adjuvants is well known in the art and is described, e.g., in
"Vaccine Design: The Subunit and Adjuvant Approach", Powell et al.,
Plenum Press (1995). Examples of adjuvants suitable for use in
vaccines containing the present conjugates include: aluminum
phosphate, aluminum hydroxide, monophosphoryl lipid A, 3-deacylated
monophosphoryl lipid A, QS-21(as disclosed in J. Immunol.,
146:431-437 (1991)), as well as various detergents (e.g.,
Triton.TM.X100, zwittergents and deoxycholate) in combination with
the aluminum compounds. In general, the antibody response to the
present conjugates is substantially increased by the inclusion of
one or more adjuvants in the vaccine.
[0047] Many methods are known to be suitable for the administration
of a vaccine formulation to individuals in need thereof. Suitable
methods of administration include, intradermal, intramuscular,
intraperitoneal, intravenous, intraarterial, vaginal, subcutaneous,
ocular, intranasal, and oral administration.
[0048] The vaccine formulations containing the present antigenic
conjugates may comprise the conjugate in a physiologically
acceptable carrier, such as isotonic solution, saline, phosphate
buffered saline, etc. The vaccine formulation is administered to an
individual in a prophylactically effective amount.
[0049] Due to their cross reactivity to a number of different or
heterologous bacterial species, the antigenic conjugates of the
present invention are effective as components of vaccines to
produce an immunologic reaction in humans to disease caused by
LPS-producing bacterial organisms. Vaccines containing the present
conjugates may be prepared by methods and with materials which are
known to those skilled in the art.
[0050] Antibodies generated by the present conjugates may be used
to examine whether an infection has been caused by an LPS-producing
bacterial organism by testing the blood samples, body fluids or
biopsy samples of the infected individual. Therapeutic and
prophylactic applications include the use of present vaccines as
well as the antibodies obtained therewith. Active immunization with
the antigenic conjugates of the present invention may be useful for
the prevention of bacterial infection or diseases.
[0051] The vaccines of the present invention are also useful for
the prophylaxis of septic shock caused by various gram negative
bacteria, e.g., Salmonella, Escherichia coli, Neisseria,
Haemophilus, Shigella, Klebsiella and Pseudomonas. It has been
discovered that, in certain cases, a significant amount of LPS is
released from the dead cells of the bacteria following therapy with
conventional antibiotics J. Infect. Dis., 157:567-568 (1988). This
can lead to endotoxin-induced complications in these patients.
[0052] One approach of preventing septic shock and its related
complications is the administration to the patient of monoclonal or
polyclonal antibodies to the common core region of the LPS of the
potentially involved bacteria. Such antiserum may prevent the toxic
effects of excessively produced LPS by the bacterial organism. The
potential of LPS antibody therapy in animal models using LPS
produced by mutants of Escherichia coli or Salmonella minnesota
immunogens have been reviewed by Applemelk and Cohen in, "Bacterial
Endotoxic Lypopolysaccharides--Vol. II", Immunopharmacology and
Pathology, CRC Press, (1992).
[0053] The present invention will now be illustrated by the
following specific, non-limiting examples.
EXAMPLES
Selection of Bacteria and Growth Conditions
[0054] A Tn916 induced LPS mutant of Neisseria meningitidis strain
NMB-R6 was constructed according to the procedure set forth in Zhou
et al., J. Biol. Chem. 269:11162-11169 (1994). This strain is a
phenotypically stable mutant which expresses LPS with a molecular
mass of approximately 3.1-3.2 KDa. It has been shown that the
inability of this mutant strain to convert glucose-6-phosphate to
glucose-1-phosphate results in a truncated LPS portion containing
only the conserved core LPS structure of Neisseria meningitidis.
The structure of the LPS produced by this mutant strain has been
identified as GlcNAc-Hep.sub.2phosphoethanolamine-KDO.sub-
.2-LipidA.
[0055] This mutant strain was grown on GC agar plate media for 6
hrs at 35.degree. C. in 5% CO.sub.2. Cells from the solid agar
culture were then grown for 18 hours in a supplemented liquid media
which contained 0.2% yeast extract dialysate. The culture was then
transferred to Fernbach flasks and grown for an additional 18-24
hours. The cells were then heat killed and harvested by
centrifugation for the purification of the LPS structure.
LPS Purification
[0056] LPS was extracted from the NMB-R6 cells by the hot
phenol-water extraction method described in Wu et al., Anal. Chem.,
160:298-289 (1987) and purified via ultracentrifugation. More
specifically, cell pellets were suspended in 3 volumes (3 ml
buffer/gm wet weight) of phosphate buffer (pH 7.1) containing 5 mM
EDTA and 0.02% sodium azide. Lysozyme (available from Sigma
Chemical Co.), at a concentration of 2 mg/ml, was then added to the
suspension. This mixture was then digested overnight at 4.degree.
C. The suspension was brought to 37.degree. C. and further digested
with RNAse and DNAse at a concentration of 100 .mu.g/ml for 3
hours. The digest was then brought to 70.degree. C. and an equal
volume of phenol at 70.degree. C. was added thereto. This mixture
was extracted for a period of 15 minutes, and the suspension was
then cooled to 4.degree. C. and centrifuged at 10,000 g for 30
minutes. The aqueous phase was recovered and the phenol phase was
reextracted with an equal volume of water at 70.degree. C. for 15
minutes. This phase was then centrifuged at 10,000 g for 30 minutes
and the aqueous phase was then separated. Sodium acetate was added
to the combined aqueous supernatants at a concentration of 5 mg/ml.
Two volumes of ice cold acetone were then added to this mixture and
the LPS was allowed to precipitate overnight at 4.degree. C. The
precipitated LPS was separated by centrifugation at 10,000 g for 30
minutes. The recovered LPS was suspended in sterile water and
subjected to three rounds of ultracentrifugation at 105,000 g for
three hours. The final pellet was suspended in a small volume of
sterile water for the subsequent experiments. Typically, 3 mg of
LPS was purified from 1 gm wet weight of the NMB-R6 cells.
Conjugation to Carrier Protein, CRM.sub.197
[0057] The LPS purified in the manner described above, was then
de-O-acylated by the reaction thereof with 45 mM of NaOH at
80.degree. C. for 20 minutes. The de-O-acylated material was then
neutralized with HCl and purified by gel filtration on a Biogel P6
column using 0.1 M NaHCO.sub.3 as an eluant. The de-O-acylated LPS
(referred to hereinafter as "DeA-LPS") was then conjugated to the
carrier protein CRM.sub.197 by linking the amino groups of the
saccharides on the conserved LPS structure to the amino groups of
the carrier protein utilizing the procedure described below.
CRM.sub.197 is a non toxic mutant protein of diphtheria toxin and
has been used as a carrier protein for the commercial production of
glycoconjugate vaccines for human use.
[0058] Long chain
sulfo-N-succinimidyl-3-(2-pyridyldithio)-propionate (sulfo LC-SPDP
available from the Pierce Chemical Company) was used to thiolate
the primary amino group(s) of the DeA-LPS. The sulfo LC-SPDP was
added to 15 mg of the LPS in 0.1 M NaHCO.sub.3 (pH 7.9) at a ratio
of 1:1 (w/w). This mixture was then incubated for an hour at room
temperature. At the end of the reaction, the mixture was purified
on a Biogel P6 column equilibrated in 0.1 M NaHCO.sub.3. The
recovered fractions were assayed for KDO according to the procedure
set forth in Keleti and Lederer, Biochem. Biophys., 74:443-450
(1974) and the fractions containing the KDO were pooled. The
N-pyridyl disulfides present in the SPDP derivatives of the LPS
were reduced with 50-100 mM dithiothreitol (DTT) and gel filtered
on a Biogel P6 column as described above. The thiolated material
containing the KDO positive fractions were again pooled. Thiolation
of the oligosaccharides of the DeA-LPS was monitored in accordance
with the reaction described in Ellman, G. L., Arch. Biochem.
Biophys., 74:443-450 (1958). 0.1 ml of the material was mixed with
0.1 ml of Ellman reagent (i.e., 40 mg of 5,5'-dithiobis(2
nitrobenzoic) acid in 10 ml of pH 8.0 phosphate buffer). After 15
minutes of incubation, the absorbance was 412 nm. Cysteine was used
as the standard sulfhydryl reagent.
[0059] The CRM.sub.197 carrier protein was bromoacetylated
according to the procedure described in Bernatowitz and Matsueda,
Anal. Biochem., 155:95-102 (1986). Bromoacetic acid-N-hydroxy
succinimide ester (available from Sigma Chemical Co.) in 100 mg/ml
of dimethyl formamide was added dropwise to 3 ml of the protein (in
0.1M NaHCO.sub.3) at a ratio of 1:1 (w/w) at 4.degree. C. The
solution was mixed and incubated for 1 hour at room temperature.
The reaction mixture was then gel filtered on a Biogel P6 column as
described above and the void fractions containing the
bromoacetylated protein were pooled. Derivatization of amino groups
on the carrier protein to the bromoacetyl groups was monitored by a
decrease in the amount of free amino groups.
[0060] The bromoacetylated CRM.sub.197 in 0.1 M NaHCO.sub.3 was
then added to the thiolated DeA-LPS at a 1:1.5 ratio of protein to
LPS (w/w) in 0.1M NaHCO.sub.3. The reaction mixture was incubated
overnight at 4.degree. C. The final conjugate (hereinafter referred
to as "DeA-LPS-SPDP-CRM") was purified by gel filtration on a
Biogel P30 (Bio-Rad) column equilibrated in 0.1 M NaHCO.sub.3/1 mM
EDTA, pH7-9.
Immunogenicity Determination
[0061] The immunogenicity of the DeA-LPS-SPDP-CRM conjugate
prepared above was determined in Swiss Webster mice according to
the following procedure. Groups of 6-8 week old female mice, 10 per
group, were immunized subcutaneously with 10 .mu.g LPS, 10 .mu.g
DeA-LPS, 10 .mu.g DeA-LPS-SPDP (i.e., the unconjugated
intermediate) and 10 .mu.g of the DeA-LPS-SPDP-CRM conjugate. 10
.mu.g CRM.sub.197 was also administered to the mice to serve as a
control. Each of these immunogens further contained 20 .mu.g of
QS-21 (available from Aquila) as an adjuvant in a final volume of
0.1 ml containing phosphate buffered saline (PBS), per dose. An
additional group was immunized with 10 .mu.g of LPS without the
QS21 adjuvant. The animals were immunized at weeks 0, 3, and 6 and
blood samples were taken prior to each immunization for antibody
determination. Blood samples were further taken at week 8 for
antibody determination.
[0062] LPS antibody levels were determined by the Enzyme Linked
Immunosorbent Assay (ELISA) procedure against purified LPS from R6
and the other wild type and immunotype specific Neisseria
meningitidis strains identified below. The immunotype specific
strains were obtained from Walter Reed Army Medical Center,
Washington. LPS was purified from these strains by the hot
phenol-water extraction method described above.
[0063] The purified LPS was diluted in endotoxin-free PBS to the
following concentrations: 10 .mu.g/mL for Neisseria meningitidis
strains A1, H44/76, 2996 and Immunotypes L1, L2, L3, L4, L5, L6,
L7, L8, L10, L11 and L12; and 2.5 .mu.g/mL for the R6 strain.
Polystyrene microtiter plates were coated with 100 .mu.L per well
of the diluted LPS-containing mixtures and incubated for 3 hours at
37.degree. C. followed by overnight storage at 4.degree. C. The
unbound LPS was then removed from the plates by suction utilizing
an automatic plate washer. 150 .mu.L per well of PBS/0.1% gelatin
was then added to the plates and the plates were then incubated for
60 minutes at 37.degree. C. Following this incubation, and between
all subsequent steps, the plates were washed with a mixture of PBS
and 0.1% Tween 20 using an automatic plate washer.
[0064] Test mouse sera was serially diluted in a mixture of PBS,
0.05% Tween 20 and 0.1% gelatin. 100 .mu.L per well of the dilution
was added to the plates. The plates were incubated for 60 minutes
at 37.degree. C. Goat anti-mouse IgG alkaline phosphatase (from
Southern Biotechnology), diluted in a mixture of PBS and 0.5% Tween
20, was then added in an amount of 100 .mu.L per well and incubated
for 60 minutes at 37.degree. C. The color was developed using 100
.mu.l of a 1 mg/ml solution of p-nitrophenol phosphate in a
diethanolamine buffer. These materials were allowed to react for 60
minutes at room temperature, after which the reaction was stopped
by the addition of 50 .mu.L per well of 3N NaOH. Absorbance values
were determined using an automated ELISA reader with a 405 nm test
and 690 nm reference filter.
[0065] The data demonstrating the immunogenicity of the
DeA-LPS-SPDP-CRM conjugate against homologous LPS from the R6
strain at weeks 0, 3, 6, and 8 is shown in Table 1. As can be seen
from this data, the conjugate produced a significant boostable IgG
antibody response.
1 TABLE 1 IgG antibody response to R6 LPS at week* Immunogen 0 3 6
8 LPS <50 365 769 17,633 LPS + QS-21 <50 1,079 24,225 84,491
DeA-LPS + QS-21 ND ND ND 221 DeA-LPS-SPDP-CRM.sub.197 + QS-21
<50 203 6,647 56,062 CRM.sub.197 + QS-21 <50 <50 <50
<50 DeA-LPS: DeO-acylated LPS, unconjugated DeA-LPS-SPDP:
Activated deO-acylated LPS, unconjugated DeA-LPS-SPDP-CRM:
DeO-acylated LPS conjugated to CRM.sub.197 by SPDP ND =Not Done
*The value represents end point dilutions at which the diluted
serum gives a value of an O.D. of 0.1.
[0066] The cross reactive immunogenicity of the conjugate produced
in the present examples to heterologous LPS of various strains of
Neisseria meningitidis was also examined according to the procedure
described above and the data obtained is set forth in Table 2. The
strains examined were: A1, R6, H44/76, 2996, Immunotypes L1, L2,
L3, L4, L5, L6, L7, L8, L10, L11, and L12. As can be seen in Table
2, the LPS-protein conjugate produced a significant antibody
response, particularly in comparison to the unconjugated LPS.
2TABLE 2 Strains DeA-LPS-SPDP DeA-LPS-SPDP-CRM.sub.197 R6 443
33,067 A1 <100 36,923 H44/76 147 24,811 2996 <100 11,467 L1
<100 11,963 L2 350 4500 L3 304 7,936 L4 412 12,820 L5 259 16,251
L6 129 11,600 L7 <100 16,533 L8 232 16,278 L10 141 11,982 L11
<100 3200 L12 142 4,870 DeA-LPS-SPDP: Activated deO-acylated
LPS, unconjugated DeA-LPS-SPDP-CRM: DeO-acylated LPS conjugated to
CRM.sub.197 by SPDP
[0067] The cross reactivity of anti-LPS conjugate antisera (the
antisera against DeA-LPS-SPDP-CRM) was further examined by western
blot analysis against purified LPS from various strains of several
gram negative bacteria. Purified LPS samples of Neisseria
meningitidis, Haemophilis influenzae, Neisseria gonorrhoeae,
Moraxella catarrhalis, and Helicobacter pylori were first digested
with protease and subjected to a standard SDS-PAGE (18%) separation
procedure. The samples were then transferred to nitrocellulose
membrane by standard western blot procedure. The membrane was
blocked with 3% Bovine Serum Albumin (BSA) in a mixture of
PBS/0.05% Tween 20 for 30 min. and reacted with 1:100 dilution of
test mouse sera. The blots were then washed with a mixture of
PBS/0.05% Tween 20 and incubated with goat anti-mouse Ig alkaline
phosphatase diluted in a mixture of PBS/0.05% Tween 20. Following
the washing procedure, the blots were developed using
5-bromo-4-chloro-3-indo- ylphosphate (BCIP)/nitroblue tetrazolium
concentrate (NBT) phosphatase substrate system as described by the
manufacturer (Kirkegaard and Perry Laboratories, Inc., Md.). The
development procedure comprised mixing one part each of the BCIP
and NBT concentrates with ten parts of Tris buffer solution in a
glass container and adding these mixtures to the blots. After color
development, the reaction was stopped by rinsing the blots with
reagent quality water.
[0068] As can be seen in FIG. 4, with the exception of Helicobacter
pylori, the LPS of each of the organisms reacted strongly with the
anti-sera. Although less intense, it appears that there was also a
slight cross reactivity to the LPS from Helicobacter pylori. These
results clearly indicate that the antibodies generated from the LPS
conjugate of Neisseria meningitidis cross reacted with a number of
other gram negative organisms.
[0069] The bactericidal activities of the antisera were further
examined using the R6 strain, the group A strain (A1) and two group
B strains: H44/76 and 2996. Serum samples were diluted in 5 .mu.l
of PCM (PBS containing Ca and Mg) and this dilution was added to
reaction mixtures containing 2-5.times.10.sup.3 Neisseria
meningitidis (10 .mu.l), human serum complement (10 .mu.l) and PCM
(25 .mu.l). This mixture was then incubated for 45 min. at
36.degree. C. in 5% CO.sub.2 The reaction was then terminated by
dilution with 200 .mu.l of PBS. Two aliquots (50 .mu.l) of the
mixture were then plated onto GC agar plates and further incubated
in 5% CO.sub.2 at 36.degree. C. The bactericidal titers (BC50) were
then determined. Bactericidal titers represent the reciprocal of
the dilution of antiserum that kills 50% of the colony forming
Neisseria meningitidis in the assay. The data is set forth below in
Table 3.
[0070] As can be seen in Table 3, the conjugate antisera was able
to kill bacteria expressing different LPS immunotypes. Such
conjugates induced boostable T cell dependent IgG response.
3TABLE 3 BC.sub.50 titers Strain Strain Strain *Strain Immunogen
H44/76 2996 A1 R6 LPS <50 <50 <50 <50 DeA-LPS-SPDP
<50 <50 <50 ND DeA-LPS-SPDP-CRM.sub.197 50 70 350 <50
Normal mouse serum <50 <50 <50 <50 *Positive control
antisera (mouse anti-Al LPS) was used in assay and gave a titer of
100. ND = Not Done DeA-LPS-SPDP: Activated deO-acylated LPS,
unconjugated DeA-LPS-SPDP-CRM: DeO-acylated LPS conjugated to
CRM.sub.197 by SPDP
[0071] Accordingly, it can readily be seen from the data set forth
above that the antigenic conjugates of the present invention
produce a significant immune response to the LPS of a given
bacterial organism. Moreover, this data demonstrates that the
present conjugates induce a cross reactive response to different
strains of the bacterial organism as well as to different species
of bacterial organisms.
[0072] The present invention may be embodied in other specific
forms without departing from the spirit and essential attributes
thereof and accordingly, reference should be made to the appended
claims, rather than to the foregoing specification, as indicating
the scope of the invention.
* * * * *