U.S. patent application number 14/316662 was filed with the patent office on 2014-12-25 for methods for conjugation of oligosaccharides or polysaccharides to protein carriers through oxime linkages via 3-deoxy-d-manno-octulsonic acid.
This patent application is currently assigned to The U.S.A , as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is National Research Council of Canada, The U.S.A., as represented by the Secretary, Department of Health and Human Services, The U.S.A., as represented by the Secretary, Department of Health and Human Services. Invention is credited to Gil Ben-Menachem, Ariel Ginzberg, Joanna Kubler-Kielb, Teresa Langergard, Vince Pozsgay, Rachel Schneerson, Evguenii Vinogradov.
Application Number | 20140378669 14/316662 |
Document ID | / |
Family ID | 38828681 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140378669 |
Kind Code |
A1 |
Kubler-Kielb; Joanna ; et
al. |
December 25, 2014 |
METHODS FOR CONJUGATION OF OLIGOSACCHARIDES OR POLYSACCHARIDES TO
PROTEIN CARRIERS THROUGH OXIME LINKAGES VIA
3-DEOXY-D-MANNO-OCTULSONIC ACID
Abstract
Methods for preparing an oligosaccharide--protein carrier
immunogenic conjugate or a polysaccharide--protein carrier
immunogenic conjugate. The methods include obtaining an
oligosaccharide or polysaccharide having a KDO moiety located at
the terminal reducing end of the oligosaccharide or polysaccharide
that includes a carbonyl functional group; and reacting the
carbonyl functional group of the KDO moiety with an aminooxylated
protein carrier molecule resulting in a conjugate that includes a
covalent oxime bond between the oligosaccharide and the protein
carrier or the polysaccharide and the protein carrier.
Inventors: |
Kubler-Kielb; Joanna;
(Bethesda, MD) ; Pozsgay; Vince; (Washington,
DC) ; Langergard; Teresa; (Kullavik, SE) ;
Ben-Menachem; Gil; (Rockville, MD) ; Schneerson;
Rachel; (Bethesda, MD) ; Ginzberg; Ariel;
(Jerusalem, IL) ; Vinogradov; Evguenii; (Ottawa,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The U.S.A., as represented by the Secretary, Department of Health
and Human Services
National Research Council of Canada |
Bethesda
Ottawa |
MD |
US
CA |
|
|
Assignee: |
The U.S.A , as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
National Research Council of Canada
Ottawa
|
Family ID: |
38828681 |
Appl. No.: |
14/316662 |
Filed: |
June 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12309428 |
Jan 13, 2010 |
8795680 |
|
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PCT/US07/16373 |
Jul 18, 2007 |
|
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14316662 |
|
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60832448 |
Jul 21, 2006 |
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Current U.S.
Class: |
530/395 |
Current CPC
Class: |
A61K 39/107 20130101;
A61K 39/102 20130101; A61K 47/646 20170801; A61P 31/04 20180101;
A61K 39/099 20130101; A61K 2039/6031 20130101; A61K 39/0283
20130101 |
Class at
Publication: |
530/395 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 39/02 20060101 A61K039/02; A61K 39/112 20060101
A61K039/112; A61K 39/102 20060101 A61K039/102 |
Claims
1. A method for preparing an oligosaccharide--protein carrier
immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate, comprising: obtaining an oligosaccharide or
polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid
moiety located at the terminal reducing end of the oligosaccharide
or polysaccharide that includes a carbonyl functional group; and
reacting the carbonyl functional group of the anhydro
3-deoxy-D-manno-octulsonic acid moiety with an aminooxylated
protein carrier molecule resulting in an oligosaccharide--protein
carrier immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate that includes a covalent oxime bond between
the oligosaccharide and the protein carrier or the polysaccharide
and the protein carrier.
2. The method of claim 1, wherein the oligosaccharide or
polysaccharide is obtained from Haemophilus ducreyi, Bordetella
bronchiseptica, Bordetella parapertussis, Bordetella pertussis,
Vibrio cholere, Shigella sp., Haemophilus influenza, or a mixture
thereof.
3. The method of claim 1, wherein the oligosaccharide or
polysaccharide is obtained from at least one bacteria containing a
lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid
moiety.
4. The method of claim 1, wherein obtaining the oligosaccharide or
polysaccharide comprises: isolating a lipopolysaccharide from at
least one type of bacteria, wherein the lipopolysaccharide includes
a Lipid A domain and at least one polysaccharide or oligosaccharide
domain, the Lipid A domain and the polysaccharide or
oligosaccharide domain being joined together by
3-deoxy-D-manno-octulsonic acid; and cleaving the Lipid A from the
polysaccharide or oligosaccharide domain such that the
3-deoxy-D-manno-octulsonic acid remains linked to the
polysaccharide or oligosaccharide domain.
5. The method of claim 4, wherein cleaving the Lipid A from the
polysaccharide or oligosaccharide domain comprises acid hydrolyzing
the lipopolysaccharide under conditions sufficient for severing a
glycosidic bond between the 3-deoxy-D-manno-octulsonic acid and the
Lipid A domain.
6. The method of claim 4, wherein cleaving the lipid A from the
polysaccharide or oligosaccharide domain comprises treating the
lipopolysaccharide with acetic acid.
7. The method of claim 1, wherein the carbonyl functional group is
a ketone.
8. The method of claim 1, wherein the mol ratio of the carbonyl
functional group on the oligosaccharide or polysaccharide:aminooxy
on the protein carrier ranges from about 0.3:1 to about 1:3.
9. The method of claim 1 wherein the aminooxylated protein carrier
is prepared by treating a protein with at least one agent that
introduces at least one aminooxy functional group onto the
protein.
10. The method of claim 9 wherein the aminooxy-introducing agent is
selected from aminoooxy-alkyl-thiol and aminoooxy-aryl-thiol.
11. The method of claim 9, further comprising treating the protein
with a treatment agent that introduces at least one thiol-reactive
group onto the protein prior to treating the protein with the
aminooxy-introducing agent.
12. The method of claim 11, wherein the thiol-reactive group is a
haloacetamido moiety.
13. The method of claim 1, wherein the oligosaccharide or
polysaccharide is obtained from Bordetella bronchiseptica,
Bordetella parapertussis, or Bordetella pertussis.
14. The method of claim 1, wherein the oligosaccharide or
polysaccharide is obtained from at least one bacteria containing a
lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid moiety
phosphorylated at position C4 on the 3-deoxy-D-manno-octulsonic
acid moiety.
15. The method of claim 1, wherein the oligosaccharide or
polysaccharide is obtained from Shigella flexneri.
16. A method for preparing an oligosaccharide--protein carrier
immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate, comprising: obtaining an oligosaccharide or
polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid
moiety located at the terminal reducing end of the oligosaccharide
or polysaccharide; reacting the anhydro 3-deoxy-D-manno-octulsonic
acid moiety of the oligosaccharide or polysaccharide with a
heterobifunctional compound that includes at least one aminooxy
group; and then reacting the oligosaccharide or polysaccharide with
a protein carrier resulting in an oligosaccharide--protein carrier
immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate that includes a covalent oxime bond between
the oligosaccharide and the protein carrier or the polysaccharide
and the protein carrier.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/309,428, filed on Jan. 13, 2010, which is the U.S.
National Stage of International Application No. PCT/US2007/016373,
filed Jul. 18, 2007, which was published in English under PCT
Article 21(2), which in turn claims the benefit of U.S. Provisional
Application No. 60/832,448, filed Jul. 21, 2006, which applications
are incorporated herein in their entirety.
FIELD
[0002] Disclosed herein are conjugates and methods for making
conjugates from oligosaccharide or polysaccharide antigens.
BACKGROUND
[0003] There are numerous human and animal diseases or infections
that can be caused by Gram-negative bacteria such as, for example,
Bordetella spp. and Haemophilus ducreyi.
[0004] Vaccination has proven effective for preventing infection of
humans and animals by Bordetella spp. Killed whole cell and subunit
vaccines have been used to immunize parenterally to protect humans
against pertussis caused by Bordetella pertussis, a highly
contagious, severe respiratory infection especially of young
children. B. parapertussis causes a milder and less frequent form
of the disease, but its incidence and importance is garnering
increasing attention. No vaccine is known to prevent it.
Vaccination against B. pertussis does not protect against B.
parapertussis. Parapertussis infection followed by pertussis in the
same individuals has been described in literature. B. pertussis is
confined to human, while B. parapertussis is confined to human and
sheep. B. bronchiseptica causes respiratory infections in a variety
of hosts: kennel cough in dogs, atrophic rhinitis in piglets,
bronchopneumonia in rabbits and guinea pigs. Rarely, it infects
humans, but young children, animal handlers and increasingly
immuno-compromised individuals are susceptible. Unlike most
bacterial respiratory pathogens, B. bronchiseptica efficiently
colonizes the ciliated epithelium of the respiratory tract of the
host and may establish chronic infections. A cellular veterinary
vaccine is available but it is of limited efficacy.
[0005] It has been shown that protection to the infections caused
by gram-negative bacteria can be conferred by serum
anti-lipopolysaccharide (LPS) IgG. Cohen et al., "Double-blind
vaccine-controlled randomised efficacy trial of an investigational
Shigella sonnei conjugate vaccine in young adults," Lancet
349(9046):155-159, 1997. The LPSes of all three bordetellae share
several similar features, though none of them is identical in
structure. B. pertussis produce rough-type LPS comprising a Lipid A
domain and branched dodecasaccharide chain, carrying unusual sugars
and free amino and carboxylic groups. On the basis of SDS-PAGE
migration, it is divided into Band B-Lipid A and a branched
nanosaccharide, that if further substituted by a trisaccharide unit
is termed Band A. Almost identical core structure was reported for
B. bronchiseptica LPS. On the contrary, B. parapertussis core
region has a simplified heptasaccharide structure; it does not
contain Band A trisaccharide and Band B lacks one heptose and one
N-acetylgalactosamine substituents. Only B. bronchiseptica and B.
parapertussis synthesize O-specific polysaccharides (O-SP) and
initially it was reported that they carry identical structure of
linear polymers of 1,4-linked
2,3-diacetamido-2,3-dideoxy-.alpha.-galactouronic acid (Di Fabio J
L et al., FEMS Microbiol. Lett. 97:275-282, 1992). However later,
serological differences between B. bronchiseptica strains were
observed and ascribed to the structural variations of the
non-reducing end-groups of LPS O-chains (Vinogradov E. et al., Eur.
J. Biochem. 276:7230-7236, 2000). As it was reported for Vibrio
cholerae O1 serotype Ogawa and Inaba, the non-reducing end-groups
play a significant role as major epitopes in serological reactions
(Wang J., J. Biol. Chem. 273:2777-2783, 1998). Similar observation
was made in case of Salmonella O40 and O43 serotypes.
[0006] Chancroid is a sexually transmitted genital ulcer disease
(GUD) caused by the bacterium Haemophilus ducreyi. Chancroid
presents with characteristic and persistent genital ulcers on the
external genitals, associated with regional lymphadenopathy in 50%
of cases. The disease is common in many developing countries, and
is considered a significant risk factor together with other genital
GUD, e.g. herpes simplex virus 2 (HSV-2) for heterosexual HIV
transmission in geographic areas where both diseases are
prominent.
[0007] A number of putative virulence factors of H. ducreyi have
been described which may play a role in pathogenicity of this
organism. Two of these factors are toxins: a hemolytic toxin and
cytolethal distending toxin. The outer membrane proteins, DsrA and
DltA, have been shown to promote resistance to killing by normal
human serum. The hemoglobin receptor HgbA and the Cu, Zn-superoxide
dismutase both seem to play a role in iron acquisition for H.
ducreyi. Filamentous hemagglutinin like protein is involved in
inhibition of phagocytosis. Heat-shock proteins (HSP) of H. ducreyi
situated on the surface of the bacteria are responsible for
protection of these bacteria against changes in the environment and
enhance H. ducreyi adhesion to mammalian cells. Additionally, a
number of proteins have been shown to play a role in adherence.
[0008] The lipooligosaccharide (LOS) produced by H. ducreyi is a
putative virulence facto, as well. Previous studies have shown that
LOS plays a role in adherence of bacteria to keratinocytes and
human foreskin fibroblasts and also contribute to the development
of lesions in animal models. Structural studies have been performed
on the LOS from a number of H. ducreyi strains, e.g. 35000, ITMA
2665, 3147, 5535, CCUG 7470, 4438 and others. These studies have
shown that the predominant form of the core oligosaccharide of the
LOS is composed of 10 saccharides with a lactosamine or
sialyllactosamine at the non-reducing end and is expressed by
majority of strains.
[0009] H. ducreyi enters the skin or mucosa through wounds and
attaches to extracellular matrix and to cells. This stimulates an
inflammatory response with the development of pro-inflammatory
cytokines and assembly of phagocytic cells; granulocytes and
macrophages, at the infection site. H. ducreyi may be found both
intra and extracellularly. The inflammatory process may clear the
organism partially but may also cause tissue destruction and
chronic infection with granuloma formation as observed in rabbit
model of infection.
[0010] The mediators of immunity to chancroid are not known. Data
from patients and infected volunteers indicate that this local
infection does not confer immunity against subsequent re-infection
and do not induce an antibody response. The results from these
experiments indicate that the cytokine pattern and the type of
cells involved in the early immune response to H. ducreyi, may have
features of a Th1 response, including a poor or no antibody
response. The in vitro studies of interactions of H. ducreyi with
human monocyte-derived-dendritic cells and with macrophages
confirmed an initial Th1 response. Studies in a rabbit model showed
that both antibodies and cellular immunity contributed to reducing
the number of bacteria in the lesions, thus contributing to
protection. Data from a swine model indicated that antibodies
alone, at levels achieved only after more than 3 injections of live
bacteria, are sufficient for protection. Antibodies to different
bacterial cell components were detected in the late stage of
disease in sera from patients with chancroid, but antibodies
neutralizing CDT have been detected only in about 28% of chancroid
cases. It has also been noted that antibodies specific to the LOS
of this organism enhance opsonophagocytic killing of H. ducreyi in
vitro, but such antibodies are not elicited in sufficient amounts
after repeated dermal injections of bacteria to animals. Low level
of induced LOS antibodies may be due to the fact that the LOS
structure resembles terminal saccharides of paraglobise, a major
antigen on human erythrocytes and muscles. Since re-infection with
H. ducreyi can occur, the immunity, including the amount and
specificity of antibodies elicited by this local infection, is
likely not sufficient for protection.
[0011] The covalent binding of oligosaccharide to carrier proteins
by random activation of the saccharide using CDAP and ADH as the
linker, resulted in conjugates that induced higher levels of IgG
anti LOS than repeated injections of the whole cell (Lundquist A,
Ahlman k T. Lagergard, "Preparation and immunological properties of
Haemophilus ducreyi lipooligosaccharide--protein conjugates," ASM
Meeting, abstract e-044, New Orleans, 2004). A vaccine to prevent
chancroid would reduce/prevent the disease burden and have the
added benefit of reducing HIV incidence.
[0012] Shigellae are Gram-negative bacteria, pathogens to humans
only, that can cause endemic and epidemic dysentery worldwide,
especially in the developing countries. The symptoms usually start
with watery diarrhea that later develops into dysentery,
characterized by high fever, blood and mucus in the stool, and
cramps. Shigella flexneri causes dysentery mostly in developing
countries with more fatalities then any other Shigella species. The
disease can be prevented by vaccination using the polysaccharide
part of the LPS as an immunogen.
SUMMARY
[0013] Disclosed herein are methods for preparing an
oligosaccharide--protein carrier immunogenic conjugate or a
polysaccharide--protein carrier immunogenic conjugate. The methods
include:
[0014] obtaining an oligosaccharide or polysaccharide having an
anhydro 3-deoxy-D-manno-octulsonic acid moiety located at the
terminal reducing end of the oligosaccharide or polysaccharide that
includes a carbonyl functional group; and
[0015] reacting the carbonyl functional group of the anhydro
3-deoxy-D-manno-octulsonic acid moiety with an aminooxylated
protein carrier molecule resulting in an oligosaccharide--protein
carrier immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate that includes a covalent oxime bond between
the oligosaccharide and the protein carrier or the polysaccharide
and the protein carrier.
[0016] Also described herein are immunogenic conjugates comprising
the structure of:
Pr--Sp--O--N.dbd.C(COOH)-anh-KDO-OS
[0017] wherein Pr is a carrier protein, Sp is an optional spacer
moiety, anh-KDO is an anhydro moiety from
3-deoxy-D-manno-octulsonic acid, and OS is an oligosaccharide or
polysaccharide residue from the cleavage of Lipid A from a
lipopolysaccharide.
[0018] Further disclosed are methods of eliciting an immune
response in a subject, comprising administering to the subject the
above-described conjugates, thereby eliciting an immune response in
the subject.
[0019] Another embodiment for preparing an oligosaccharide--protein
carrier immunogenic conjugate or polysaccharide--protein carrier
immunogenic conjugate includes obtaining an oligosaccharide or
polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid
moiety located at the terminal reducing end of the oligosaccharide
or polysaccharide. The anhydro 3-deoxy-D-manno-octulsonic acid
moiety of the oligosaccharide or polysaccharide is reacted with a
heterobifunctional compound that includes at least one aminooxy
group. Subsequently, the resulting functionalized oligosaccharide
or polysaccharide is reacted with a protein carrier to produce an
oligosaccharide--protein carrier immunogenic conjugate or
polysaccharide--protein carrier immunogenic conjugate that includes
a covalent oxime bond between the oligosaccharide and the protein
carrier or the polysaccharide and the protein carrier.
[0020] The foregoing and other features and advantages will become
more apparent from the following detailed description, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a conjugation reaction scheme that depicts (a) the
synthesis of aminooxylated protein, (b) the synthesis of an
oligosaccharide or polysaccharide that includes a carbonyl
functional group (i.e, ketone), and (c) the conjugation of the
aminooxylated protein with the carbonyl-functional oligosaccharide
or polysaccharide. Pr is a carrier protein, LPS is
lipopolysaccharide, LOS is a lipooligosaccharide, O-chain is an
O-antigen oligosaccharide or polysaccharide chain, Core is a core
oligosaccharide or polysaccharide chain, KDO4P is
3-deoxy-D-manno-octulsonic acid moiety phosphorylated at position
C4, and anhydro-KDO is described below.
[0022] FIG. 2 is a LPS structure of Bordetella parapertussis and
Bordetella bronchiseptica. A novel pentasaccharide
(-4-.beta.-ManNAc3ANcAN-4-.beta.-GlcNAc3NAcAN-4-.alpha.-GalNAc-4-.beta.-M-
anNAc3NAcA-3-.beta.-FucNAc4NMe-) present between the O-SP and the
core was identified. In addition, besides the reported structure
the O-SP of B. bronchiseptica and B. parapertussis being a
homopolymer of 1,4-linked
2,3-diacetamido-2,3-dideoxy-.alpha.-galacturonic acid, it was found
that both O-SP contain amidated uronic acids, the number of which
varied between strains (Preston et al., J. Biol. Chem., 2006 (in
press)). Certain fragments
(-6-.beta.-GlcNAc-4-.beta.-ManNAc3NAcA-3-.beta.-FucNAc4NMe-) are
not present or partially present (residues with *) in B.
parapertussis. Two types of O-SP end groups (Vinogradov et al.,
Eur. J. Biochem. 267:7230-7237, 2000) (A) were found in B.
bronchiseptica and only one, Ala-type, in B. parapertussis.
[0023] FIG. 3 is a MALDI-TOF spectrum of BSA-ONH.sub.2/BbRb50
(conjugate #2 in Table 2 below).
[0024] FIG. 4 is a MALDI-TOF spectrum of BSA-ONH.sub.2/OS(H.
ducreyi) (conjugate #1 in Table 4 below).
[0025] FIG. 5 is a MALDI-TOF spectrum of TT-ONH.sub.2/OS(H.
ducreyi) (conjugate #2 in Table 4 below).
[0026] FIG. 6 is an ESI-MS spectrum of B. pertussis OS used for
conjugation in Example 3.
[0027] FIG. 7 is an ESI-MS spectrum for B. bronchiseptica OS-core
used for conjugation in Example 3.
[0028] FIG. 8 is an SDS-PAGE gel result showing an increase in
molecular size of BSA-ONH.sub.2/S. flexnerii 2a O-SP conjugate
(line 3) over BSA-ONH.sub.2 (line 2). Line 1 is a marker. 10%
NUPAGE MES gel was used in this experiment. The highest marker line
corresponds to 188 kDa.
DETAILED DESCRIPTION
I. Abbreviations
[0029] ADH: adipic acid dihydrazide [0030] AT: anthrax toxin [0031]
ATR: anthrax toxin receptor [0032] EDAC:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl [0033] EF: edema
factor [0034] GLC-MS: gas-liquid chromatography-mass spectrometry
[0035] kDa: kilodaltons [0036] LC-MS: liquid chromatography-mass
spectrometry [0037] LeTx: lethal toxin [0038] LF: lethal factor
[0039] LOS: lipooligosaccharide [0040] LPS: lipopolysaccharide
[0041] MALDI-TOF: matrix-assisted laser desorption ionization
time-of-flight [0042] OS: oligosaccharide [0043] .mu.g: microgram
[0044] .mu.l: microliter [0045] PA: protective antigen [0046] PBS:
phosphate buffered saline [0047] SBAP: succinimidyl
3-(bromoacetamido) propionate [0048] SFB:
succinimidylformylbenzoate [0049] SPDP: N-hydroxysuccinimide ester
of 3-(2-pyridyl dithio)-propionic acid [0050] SLV:
succinimidyllevulinate [0051] TT: tetanus toxoid
[0052] The saccharide units disclosed herein are abbreviated as
below following conventional oligosaccharide/polysaccharide
nomenclature: [0053] anhKDO: anhydro KDO [0054] Fuc: fucose [0055]
Gal: galactose [0056] Glc: glucose, [0057] GlcNAc:
N-acetylglucosamine [0058] GalNAc: N-acetylgalactosamine [0059]
Hep: glycero-D-manno-heptopyranoside (heptose) [0060] Hex: hexose
[0061] Man: mannose [0062] NeuNAc: N-acetylneuramic acid
II. Terms
[0063] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN
0471186341); and other similar references.
[0064] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. Also, as used
herein, the term "comprises" means "includes." Hence "comprising A
or B" means including A, B, or A and B. It is further to be
understood that all nucleotide sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides or other compounds are approximate, and are
provided for description. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present disclosure, suitable methods and materials
are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0065] In order to facilitate review of the various examples of
this disclosure, the following explanations of specific terms are
provided:
[0066] Adjuvant: A substance that non-specifically enhances the
immune response to an antigen. Development of vaccine adjuvants for
use in humans is reviewed in Singh et al. (Nat. Biotechnol.
17:1075-1081, 1999), which discloses that, at the time of its
publication, aluminum salts, such as aluminum hydroxide (Amphogel,
Wyeth Laboratories, Madison, N.J.), and the MF59 microemulsion are
the only vaccine adjuvants approved for human use. An aluminum
hydrogel (available from Brentg Biosector, Copenhagen, Denmark, is
another common adjuvant).
[0067] In one embodiment, an adjuvant includes a DNA motif that
stimulates immune activation, for example the innate immune
response or the adaptive immune response by T-cells, B-cells,
monocytes, dendritic cells, and natural killer cells. Specific,
non-limiting examples of a DNA motif that stimulates immune
activation include CpG oligodeoxynucleotides, as described in U.S.
Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116;
6,339,068; 6,406,705; and 6,429,199.
[0068] Analog, Derivative or Mimetic: An analog is a molecule that
differs in chemical structure from a parent compound, for example a
homolog (differing by an increment in the chemical structure, such
as a difference in the length of an alkyl chain), a molecular
fragment, a structure that differs by one or more functional
groups, a change in ionization. Structural analogs are often found
using quantitative structure activity relationships (QSAR), with
techniques such as those disclosed in Remington (The Science and
Practice of Pharmacology, 19th Edition (1995), chapter 28). A
derivative is a biologically active molecule derived from the base
structure. A mimetic is a molecule that mimics the activity of
another molecule, such as a biologically active molecule.
Biologically active molecules can include chemical structures that
mimic the biological activities of a compound.
[0069] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects, for
example, humans, non-human primates, dogs, cats, horses, and
cows.
[0070] Antibody: A protein (or protein complex) that includes one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon, and
mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
[0071] The basic immunoglobulin (antibody) structural unit is
generally a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms "variable light chain" (VI) and "variable heavy chain" (VH)
refer, respectively, to these light and heavy chains.
[0072] Antibodies for use in the methods and devices of this
disclosure can be monoclonal or polyclonal. Merely by way of
example, monoclonal antibodies can be prepared from murine
hybridomas according to the classical method of Kohler and Milstein
(Nature 256:495-97, 1975) or derivative methods thereof. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New
York, 1999.
[0073] Antigen: A compound, composition, or substance that may be
specifically bound by the products of specific humoral or cellular
immunity, such as an antibody molecule or T-cell receptor. Antigens
can be any type of biologic molecule including, for example, simple
intermediary metabolites, sugars (e.g., oligosaccharides), lipids,
and hormones as well as macromolecules such as complex
carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids
and proteins. Common categories of antigens include, but are not
limited to, viral antigens, bacterial antigens, fungal antigens,
protozoa and other parasitic antigens, tumor antigens, antigens
involved in autoimmune disease, allergy and graft rejection,
toxins, and other miscellaneous antigens. In one example, an
antigen is a lipopolysaccharide antigen.
[0074] Carrier: An immunogenic molecule to which an antigen such as
an oligosaccharide or polysaccharide can be bound. When bound to a
carrier, the bound molecule may become more immunogenic. Carriers
are chosen to increase the immunogenicity of the bound molecule
and/or to elicit antibodies against the carrier which are
diagnostically, analytically, and/or therapeutically beneficial.
Covalent linking of a molecule to a carrier confers enhanced
immunogenicity and T-cell dependence (Pozsgay et al., PNAS
96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976;
Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers include
polymeric carriers, which can be natural (for example, proteins
from bacteria or viruses), semi-synthetic or synthetic materials
containing one or more functional groups to which a reactant moiety
can be attached.
[0075] Examples of bacterial products for use as carriers include
bacterial toxins, such as B. anthracis PA (including fragments that
contain at least one antigenic epitope and analogs or derivatives
capable of eliciting an immune response), LF and LeTx, and other
bacterial toxins and toxoids, such as tetanus toxin/toxoid,
diphtheria toxin/toxoid, P. aeruginosa exotoxin/toxoid/, pertussis
toxin/toxoid, and C. perfringens exotoxin/toxoid. Viral proteins,
such as hepatitis B surface antigen and core antigen can also be
used as carriers.
[0076] Covalent Bond: An interatomic bond between two atoms,
characterized by the sharing of one or more pairs of electrons by
the atoms. The terms "covalently bound" or "covalently linked"
refer to making two separate molecules into one contiguous
molecule. The terms include reference to joining a hapten or
antigen indirectly to a carrier molecule, with an intervening
linker molecule.
[0077] Epitope: An antigenic determinant. These are particular
chemical groups or contiguous or non-contiguous peptide sequences
or saccharide units on a molecule that are antigenic, that is, that
elicit a specific immune response. An antibody binds a particular
antigenic epitope based on the three dimensional structure of the
antibody and the matching (or cognate) epitope.
[0078] Immune Response: A response of a cell of the immune system,
such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a
stimulus. An immune response can include any cell of the body
involved in a host defense response, for example, an epithelial
cell that secretes interferon or a cytokine. An immune response
includes, but is not limited to, an innate immune response or
inflammation.
[0079] Immunogenic Conjugate or Composition: A term used herein to
mean a composition useful for stimulating or eliciting a specific
immune response (or immunogenic response) in a vertebrate. In some
embodiments, the immunogenic response is protective or provides
protective immunity, in that it enables the vertebrate animal to
better resist infection or disease progression from the organism
against which the immunogenic composition is directed. One specific
example of a type of immunogenic composition is a vaccine.
[0080] Immunogen: A compound, composition, or substance which is
capable, under appropriate conditions, of stimulating the
production of antibodies or a T-cell response in an animal,
including compositions that are injected or absorbed into an
animal.
[0081] Immunologically Effective Dose: An immunologically effective
dose of the oligosaccharide-protein or polysaccharide-protein
conjugates of the disclosure is therapeutically effective and will
prevent, treat, lessen, or attenuate the severity, extent or
duration of a disease or condition, for example, infection by
Bordetella parapertussis or Bordetella bronchiseptica.
[0082] Inhibiting or Treating a Disease: Inhibiting the full
development of a disease or condition, for example, in a subject
who is at risk for a disease such as respiratory tract infections.
"Treatment" refers to a therapeutic intervention that ameliorates a
sign or symptom of a disease or pathological condition after it has
begun to develop. As used herein, the term "ameliorating," with
reference to a disease, pathological condition or symptom, refers
to any observable beneficial effect of the treatment. The
beneficial effect can be evidenced, for example, by a delayed onset
of clinical symptoms of the disease in a susceptible subject, a
reduction in severity of some or all clinical symptoms of the
disease, a slower progression of the disease, a reduction in the
number of relapses of the disease, an improvement in the overall
health or well-being of the subject, or by other parameters well
known in the art that are specific to the particular disease.
[0083] Isolated: An "isolated" biological component (such as a
lipopolysaccharide) has been substantially separated or purified
away from other biological components in the cell of the organism
in which the component naturally occurs, such as other chromosomal
and extra-chromosomal DNA and RNA, proteins, glycolipids and
organelles.
[0084] Lipopolysaccharide (LPS): LPS is an endotoxin that is a
major suprastructure of the outer membrane of Gram-negative
bacteria which contributes greatly to the structural integrity of
the bacteria, and protects them from host immune defenses. LPS
typically contains three components: (a) Lipid A (a hydrophobic
domain that typically consists of a glucosamine disaccharide that
is substituted with phosphate groups and long chain fatty acids in
ester and amide linkages); (b) a core polysaccharide or
oligosaccharide that can include, for example, heptose, glucose,
galactose and N-acetylglucosamine units depending upon the genera
and species of bacteria; and (c) optionally, polysaccharide distal
or side chain(s) (often referred to as the "O antigen" that can
include, for example, mannose, galactose, D-glucose,
N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a
dideoxyhexose depending upon the genera and species of bacteria).
Lipid A and the core polysaccharide or oligosaccharide domains are
joined together by one or more units of 3-deoxy-D-manno-octulsonic
acid ("KDO", also known as ketodeoxyoctonate). A
lipooligosaccharide (LOS) (also known as a "short chain LPS")
commonly refers to an LPS that contains Lipid A plus a core
polysaccharide or oligosaccharide (e.g., in H. ducreyi and B.
pertussis that does not naturally contain any 0 antigen chains). As
used herein, the term LPS can include short chain LPS and LOS.
[0085] Oligosaccharide (OS): As used herein, the term
"oligosaccharide" is not necessarily restricted to a molecule
having a specific number of saccharide units. However, in general,
an oligosaccharide is a carbohydrate that contains from about 3 to
about 10 simple sugars (e.g., monosaccharides) linked together.
O-specific oligosaccharide (O-SP) refers to an O-specific
oligosaccharide chain attached to a core oligosaccharide or
polysaccharide chain. The oligosaccharides or polysaccharides
conjugated to the protein carrier do not include a lipid
component.
[0086] Pharmaceutically Acceptable Carriers: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compounds or molecules, such as one or
more SARS-CoV nucleic acid molecules, proteins or antibodies that
bind these proteins, and additional pharmaceutical agents. The term
"pharmaceutically acceptable carrier" should be distinguished from
"carrier" as described above in connection with a hapten/carrier
conjugate or an antigen/carrier conjugate.
[0087] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0088] Polypeptide: A polymer in which the monomers are amino acid
residues which are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used. The terms "polypeptide" or
"protein" as used herein are intended to encompass any amino acid
sequence and include modified sequences such as glycoproteins. The
term "polypeptide" is specifically intended to cover naturally
occurring proteins, as well as those which are recombinantly or
synthetically produced.
[0089] The term "residue" or "amino acid residue" includes
reference to an amino acid that is incorporated into a protein,
polypeptide, or peptide.
[0090] Protein: A molecule, particularly a polypeptide, comprised
of amino acids.
[0091] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified peptide, protein, conjugate, LPS, or other
active compound is one that is isolated in whole or in part from
proteins, lipids or other contaminants. Generally, substantially
purified peptides, proteins, conjugates, LPSs or other active
compounds for use within the disclosure comprise more than 80% of
all macromolecular species present in a preparation prior to
admixture or formulation of the peptide, protein, conjugate, LPS or
other active compound with a pharmaceutical carrier, excipient,
buffer, absorption enhancing agent, stabilizer, preservative,
adjuvant or other co-ingredient in a complete pharmaceutical
formulation for therapeutic administration. More typically, the
peptide, protein, conjugate, LPS or other active compound is
purified to represent greater than 90%, often greater than 95% of
all macromolecular species present in a purified preparation prior
to admixture with other formulation ingredients. In other cases,
the purified preparation may be essentially homogeneous, wherein
other macromolecular species are not detectable by conventional
techniques.
[0092] Therapeutically Effective Amount: A quantity of a specified
agent sufficient to achieve a desired effect in a subject being
treated with that agent. For example, this may be the amount of an
OS-protein or polysaccharide-protein conjugate useful in increasing
resistance to, preventing, ameliorating, and/or treating infection
and disease caused by Bordetella parapertussis, Bordetella
bronchiseptica, Haemophilus ducreyi, Bordetella pertussis, Vibrio
cholere, or Haemophilus influenza infection in a subject. Ideally,
a therapeutically effective amount of an agent is an amount
sufficient to increase resistance to, prevent, ameliorate, and/or
treat infection and disease caused by Bordetella parapertussis,
Bordetella bronchiseptica, Haemophilus ducreyi, Bordetella
pertussis, Vibrio cholere, or Haemophilus influenza infection in a
subject without causing a substantial cytotoxic effect in the
subject. The effective amount of an agent useful for increasing
resistance to, preventing, ameliorating, and/or treating infection
and disease caused by Bordetella parapertussis, Bordetella
bronchiseptica, Haemophilus ducreyi, Bordetella pertussis, Vibrio
cholere, or Haemophilus influenza infection in a subject will be
dependent on the subject being treated, the severity of the
affliction, and the manner of administration of the therapeutic
composition.
[0093] Toxoid: A nontoxic derivative of a bacterial exotoxin
produced, for example, by formaldehyde or other chemical treatment.
Toxoids are useful in the formulation of immunogenic compositions
because they retain most of the antigenic properties of the toxins
from which they were derived.
[0094] Vaccine: A vaccine is a pharmaceutical composition that
elicits a prophylactic or therapeutic immune response in a subject.
In some cases, the immune response is a protective response.
Typically, a vaccine elicits an antigen-specific immune response to
an antigen of a pathogen, for example, a bacterial or viral
pathogen, or to a cellular constituent correlated with a
pathological condition. A vaccine may include a polynucleotide, a
peptide or polypeptide, a polysaccharide, a virus, a bacteria, a
cell or one or more cellular constituents. In some cases, the
virus, bacteria or cell may be inactivated or attenuated to prevent
or reduce the likelihood of infection, while maintaining the
immunogenicity of the vaccine constituent.
[0095] As described above, disclosed herein are methods for
conjugating oligosaccharides or polysaccharides having a
3-deoxy-D-manno-octulsonic acid moiety located at the terminal
reducing end of the oligosaccharides or polysaccharides. According
to the methods, binding the OS by KDO at the reducing end of the OS
means that all of the conserved OS structure remains intact or
unmodified (e.g., none of the saccharide residues are oxidized)
which provides more potential sites for interaction leading to
higher immunogenicity. The conjugates disclosed herein preserve the
external non-reducing end of the OS, are recognized by antisera,
and induce immune responses in mice.
[0096] The oligosaccharide may be obtained from gram-negative
bacteria such as Haemophilus ducreyi, Bordetella bronchiseptica,
Bordetella parapertussis, Bordetella pertussis, Vibrio cholere, or
Haemophilus influenza or any other Haemophilus spp. These bacteria
typically contain a lipopolysaccharide (LPS) with a
3-deoxy-D-manno-octulsonic acid moiety phosphorylated at position
C4 on the 3-deoxy-D-manno-octulsonic acid moiety.
[0097] Also disclosed herein are novel techniques for binding S.
flexnerii O-SP to protein carrier via KDO. This technique can be
applied to other Shigellae like S. dysenteriae and S. sonnei, as
well as to other enterobacteriacea and other gram-negative bacteria
having KDO molecule between Lipid A and oligo/polysaccharide chain
of their LPS.
[0098] The target oligosaccharides or polysaccharides for
conjugation typically are those that carry epitopes in their
structure. Examples of such oligosaccharides or polysaccharides are
described below in more detail in examples 1 and 2. The
oligosaccharides or polysaccharides that are conjugated include a
general structure of:
O-chain(if present)-core OS-anhydro-KDO
[0099] The anhydro-KDO moiety is the moiety that results after acid
hydrolysis treatment of the isolated LOS or LPS as described in
more detail below and it has a structure represented by
(anhydro-KDO could also be referred to as 4, 8(7)-anhydro
derivative of KDO):
anhydro-Kdo
##STR00001##
[0100] The oligosaccharide or polysaccharide typically is derived
from LPS present in the bacteria identified above. The LPS
initially is isolated from the other constituents of the bacteria
cell structure. Illustrative LPS-isolation techniques are
described, for example, in Westphal et al., Meth. Carbohydr. Chem.
5:83-89, 1965, which is incorporated herein by reference in its
entirety, and typically involve isolation or purification via a
phenol-water extraction. Other LPS-isolation techniques include
enzyme digestion and alcohol precipitation, chromatography by gel
filtration and ion-exchange.
[0101] The isolated LPS then is subjected to mild acid hydrolysis
to cleave the Lipid A from the polysaccharide or oligosaccharide
domain such that the 3-deoxy-D-manno-octulsonic acid remains linked
to the polysaccharide or oligosaccharide domain. Such techniques
are described, for example, in Auzanneau, J. Chem. Soc. Perkin
Trans. 1:509-516, 1991 and Rybka et al., J. Microbiol. Methods
64(2):171-184, 2006, both of which are incorporated herein by
reference. Illustrative hydrolysis conditions include treating the
LPS with acetic acid for 1-3 hours at about 100.degree. C., or
hydrolyzing LPS in a mixture of acetic acid and sodium acetate
(e.g., treating 50 mg LPS with a mixture of 73.5 ml of 0.2 M acetic
acid and 26.5 ml of 0.2 M sodium acetate for 5 hours at 100.degree.
C. in 5 ml volume). The acid hydrolysis transforms the KDO
structure in the isolated LPS to an anhydro-KDO structure.
[0102] Conjugation of the oligosaccharide or polysaccharide to the
carrier protein is accomplished via formation of an oxime linkage
between a carbonyl functional group present in the KDO moiety and
an aminooxy functional group present on the carrier protein. The
oxime linkage reaction is a chemoselective ligation since it
involves the aqueous covalent coupling of unprotected, highly
functionalized biomolecules that contain at least a pair of
functional groups that react together exclusively, within a
biological environment. Oxime linkages can be formed in an aqueous
reaction environment, and are stable, from pH 5 to pH 7. Other
advantageous features of forming oxime linkages include a
relatively short reaction time, a good yield, and formation at
ambient temperature. These conditions avoid denaturation of the
carrier protein.
[0103] The reactive carbonyl functional group present in the KDO
moiety can be an aldehyde or a ketone remaining after acid
hydrolysis cleavage of the Lipid A from the LPS. The carrier
protein is functionalized with an aminooxy group. The synthetic
scheme for forming the oxime linkage is shown below:
Pr-Sp-O--NH.sub.2+HOOC--C(O)-anh-KDO-OS.fwdarw.Pr-Sp-O--N.dbd.C(COOH)-an-
h-KDO-OS
[0104] wherein Pr is a carrier protein, Sp is an optional spacer
moiety, anh-KDO is anhydro-KDO, and OS is an oligosaccharide or
polysaccharide residue from the cleavage of Lipid A from LPS.
Condensation between the carbonyl and aminooxy groups leads to a
stable oxime linkage between the OS and carrier protein. The spacer
moiety may have any structure that is present in the linker
reagents as described below. Alternatively, the
HOOC--C(O)-anh-KDO-OS structure could be reacted initially with an
aminooxy reagent, and the resulting aminooxy-functionalized
reactant could be reacted with the protein.
[0105] The oxime conjugation reaction is performed at pH 5 to about
pH 7 at ambient temperature conditions in an aqueous environment.
The reaction time typically ranges from about 8 to about 24 hours.
However, less than 100% conjugation completion can be achieved in
less than 8 hours, and the 8-24 hour reaction time assumes near
100% conjugation completion.
[0106] The carrier protein (or anh-KDO-OS) can be functionalized to
include at least one reactive aminooxy moiety by various techniques
as described, for example, in Kielb et al., J. Org. Chem.
70:6987-6990, 2005 and U.S. Patent Application Publication No.
2005/0169941, both of which are incorporated herein by reference.
Functionalization of the carrier protein can result in the
inclusion of an optional spacer moiety as noted above. In
illustrative examples, a carrier protein (or anh-KDO-OS) may be
reacted with a linker reagent to incorporate the spacer moiety and
the aminooxy functional moiety. The linker reagent typically is a
heterobifunctional compound that includes at least one aminooxy
group and a second functional group that is reactive with the
carrier protein. Suitable linker reagents include aminooxy-thiol
compounds. Illustrative aminooxy-thiol linker reagents include
aminoooxy-alkyl-thiols such as (thiolalkyl)hydroxylamines (e.g.,
O-(3-thiolpropyl)hydroxylamine) and aminooxy-aryl-thiols. In the
case of aminooxy-thiol linker reagents, the carrier protein may be
treated to introduce thiol-reactive groups. For example, the
carrier protein may be treated with a treatment agent that
introduces thiol-reactive haloacetamido or thiol-reactive maleimido
moieties onto the carrier protein. The haloacetamido-containing
protein or maleimido-containing protein is reacted with the
aminooxy-thiol reagent to form the aminooxylated carrier protein
via the formation of stable thioether linkages.
[0107] The amount of oligosaccharide or polysaccharide reacted with
the amount of protein may vary depending upon the specific LPS from
which the OS is derived and the carrier protein. However, the
respective amounts should be sufficient to introduce about 5-20
chains of OS(PS) onto the protein. In certain examples, the mol
ratio of carbonyl groups on OS(PS) to aminooxy groups on the
protein may range from about 0.3:1 to about 1:3, more particularly
1:1 to about 1:2, and more preferably about 1:1. The resulting
number of oligosaccharide chains bound to a single protein carrier
molecule may vary depending upon the specific LPS and the carrier
protein, but in general, about 5 to about 20, more preferably about
10, OS chains can be bound to each protein carrier molecule. The
yield based on the amount of protein ranges from about 70 to about
90% in protein derivatization step and about 70 to about 90% after
the conjugation with the OS.
[0108] Specific, non-limiting examples of water soluble protein
carriers include, but are not limited to, natural, semi-synthetic
or synthetic polypeptides or proteins from bacteria or viruses. In
one embodiment, bacterial products for use as carriers include
bacterial wall proteins and other products (for example,
streptococcal or staphylococcal cell walls), and soluble antigens
of bacteria. In another embodiment, bacterial products for use as
carriers include bacterial toxins. Bacterial toxins include
bacterial products that mediate toxic effects, inflammatory
responses, stress, shock, chronic sequelae, or mortality in a
susceptible host. Specific, non-limiting examples of bacterial
toxins include, but are not limited to: B. anthracis PA (for
example, as encoded by bases 143779 to 146073 of GenBank Accession
No. NC 007322, herein incorporated by reference), including
variants that share at least 90%, at least 95%, or at least 98%
amino acid sequence homology to PA, fragments that contain at least
one antigenic epitope, and analogs or derivatives capable of
eliciting an immune response; B. anthracis LF (for example, as
encoded by the complement of bases 149357 to 151786 of GenBank
Accession No. NC 007322); bacterial toxins and toxoids, such as
tetanus toxin/toxoid (for example, as described in U.S. Pat. Nos.
5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, as
described in U.S. Pat. Nos. 4,709,017 and 6,696,065); P. aeruginosa
exotoxin/toxoid/ (for example, as described in U.S. Pat. Nos.
4,428,931, 4,488,991 and 5,602,095); pertussis toxin/toxoid (for
example, as described in U.S. Pat. Nos. 4,997,915, 6,399,076 and
6,696,065); and C. perfringens exotoxin/toxoid (for example, as
described in U.S. Pat. Nos. 5,817,317 and 6,403,094). Viral
proteins, such as hepatitis B surface antigen (for example, as
described in U.S. Pat. Nos. 5,151,023 and 6,013,264) and core
antigen (for example, as described in U.S. Pat. Nos. 4,547,367 and
4,547,368) can also be used as carriers, as well as proteins from
higher organisms such as keyhole limpet hemocyanin, horseshoe crab
hemocyanin, edestin, mammalian serum albumins, and mammalian
immunoglobulins.
[0109] Following conjugation of the oligosaccharide or
polysaccharide to the carrier protein, the conjugate can be
purified by a variety of techniques well known to one of skill in
the art. One goal of the purification step is to remove the unbound
oligosaccharide or polysaccharide from the conjugation reaction
product composition. One method for purification, involving
ultrafiltration in the presence of ammonium sulfate, is described
in U.S. Pat. No. 6,146,902. Alternatively, the conjugates can be
purified away from unreacted oligosaccharide/polysaccharide and
carrier by any number of standard techniques including, for
example, size exclusion chromatography, density gradient
centrifugation, hydrophobic interaction chromatography, or ammonium
sulfate fractionation. See, for example, Anderson et al., J.
Immunol. 137:1181-1186, 1986 and Jennings & Lugowski, J.
Immunol. 127:1011-1018, 1981. The compositions and purity of the
conjugates can be determined by GLC-MS and MALDI-TOF
spectrometry.
[0110] The conjugates disclosed herein may be included in
pharmaceutical compositions (including therapeutic and prophylactic
formulations), typically combined together with one or more
pharmaceutically acceptable vehicles and, optionally, other
therapeutic ingredients (for example, antibiotics or
anti-inflammatories).
[0111] Such pharmaceutical compositions can be administered to
subjects by a variety of mucosal administration modes, including by
oral, rectal, intranasal, intrapulmonary, or transdermal delivery,
or by topical delivery to other surfaces. Optionally, the conjugate
can be administered by non-mucosal routes, including by
intramuscular, subcutaneous, intravenous, intra-atrial,
intra-articular, intraperitoneal, or parenteral routes. In other
alternative embodiments, the conjugate can be administered ex vivo
by direct exposure to cells, tissues or organs originating from a
subject.
[0112] To formulate the pharmaceutical compositions, the conjugate
can be combined with various pharmaceutically acceptable additives,
as well as a base or vehicle for dispersion of the conjugate.
Desired additives include, but are not limited to, pH control
agents, such as arginine, sodium hydroxide, glycine, hydrochloric
acid, citric acid, and the like. In addition, local anesthetics
(for example, benzyl alcohol), isotonizing agents (for example,
sodium chloride, mannitol, sorbitol), adsorption inhibitors (for
example, Tween 80), solubility enhancing agents (for example,
cyclodextrins and derivatives thereof), stabilizers (for example,
serum albumin), and reducing agents (for example, glutathione) can
be included. Adjuvants, such as aluminum hydroxide (for example,
Amphogel, Wyeth Laboratories, Madison, N.J.), Freund's adjuvant,
MPL.TM. (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,
Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many
other suitable adjuvants well known in the art, can be included in
the compositions. When the composition is a liquid, the tonicity of
the formulation, as measured with reference to the tonicity of 0.9%
(w/v) physiological saline solution taken as unity, is typically
adjusted to a value at which no substantial, irreversible tissue
damage will be induced at the site of administration. Generally,
the tonicity of the solution is adjusted to a value of about 0.3 to
about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about
1.7.
[0113] The conjugate can be dispersed in a base or vehicle, which
can include a hydrophilic compound having a capacity to disperse
the conjugate, and any desired additives. The base can be selected
from a wide range of suitable compounds, including but not limited
to, copolymers of polycarboxylic acids or salts thereof, carboxylic
anhydrides (for example, maleic anhydride) with other monomers (for
example, methyl(meth)acrylate, acrylic acid and the like),
hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives, such as
hydroxymethylcellulose, hydroxypropylcellulose and the like, and
natural polymers, such as chitosan, collagen, sodium alginate,
gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often,
a biodegradable polymer is selected as a base or vehicle, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters and the like can be
employed as vehicles. Hydrophilic polymers and other vehicles can
be used alone or in combination, and enhanced structural integrity
can be imparted to the vehicle by partial crystallization, ionic
bonding, cross-linking and the like. The vehicle can be provided in
a variety of forms, including fluid or viscous solutions, gels,
pastes, powders, microspheres and films for direct application to a
mucosal surface.
[0114] The conjugate can be combined with the base or vehicle
according to a variety of methods, and release of the conjugate can
be by diffusion, disintegration of the vehicle, or associated
formation of water channels. In some circumstances, the conjugate
is dispersed in microcapsules (microspheres) or nanocapsules
(nanospheres) prepared from a suitable polymer, for example,
isobutyl 2-cyanoacrylate (see, for example, Michael et al., J.
Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible
dispersing medium, which yields sustained delivery and biological
activity over a protracted time.
[0115] The compositions of the disclosure can alternatively contain
as pharmaceutically acceptable vehicles substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate, and
triethanolamine oleate. For solid compositions, conventional
nontoxic pharmaceutically acceptable vehicles can be used which
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like.
[0116] Pharmaceutical compositions for administering the conjugate
can also be formulated as a solution, microemulsion, or other
ordered structure suitable for high concentration of active
ingredients. The vehicle can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), and suitable mixtures thereof. Proper fluidity for solutions
can be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of a desired particle size in the case
of dispersible formulations, and by the use of surfactants. In many
cases, it will be desirable to include isotonic agents, for
example, sugars, polyalcohols, such as mannitol and sorbitol, or
sodium chloride in the composition. Prolonged absorption of the
conjugate can be brought about by including in the composition an
agent which delays absorption, for example, monostearate salts and
gelatin.
[0117] In certain embodiments, the conjugate can be administered in
a time release formulation, for example in a composition which
includes a slow release polymer. These compositions can be prepared
with vehicles that will protect against rapid release, for example
a controlled release vehicle such as a polymer, microencapsulated
delivery system or bioadhesive gel. Prolonged delivery in various
compositions of the disclosure can be brought about by including in
the composition agents that delay absorption, for example, aluminum
monostearate hydrogels and gelatin. When controlled release
formulations are desired, controlled release binders suitable for
use in accordance with the disclosure include any biocompatible
controlled release material which is inert to the active agent and
which is capable of incorporating the conjugate and/or other
biologically active agent. Numerous such materials are known in the
art. Useful controlled-release binders are materials that are
metabolized slowly under physiological conditions following their
delivery (for example, at a mucosal surface, or in the presence of
bodily fluids). Appropriate binders include, but are not limited
to, biocompatible polymers and copolymers well known in the art for
use in sustained release formulations. Such biocompatible compounds
are non-toxic and inert to surrounding tissues, and do not trigger
significant adverse side effects, such as nasal irritation, immune
response, inflammation, or the like. They are metabolized into
metabolic products that are also biocompatible and easily
eliminated from the body.
[0118] Exemplary polymeric materials for use in the present
disclosure include, but are not limited to, polymeric matrices
derived from copolymeric and homopolymeric polyesters having
hydrolyzable ester linkages. A number of these are known in the art
to be biodegradable and to lead to degradation products having no
or low toxicity. Exemplary polymers include polyglycolic acids and
polylactic acids, poly(DL-lactic acid-co-glycolic acid),
poly(D-lactic acid-co-glycolic acid), and poly(L-lactic
acid-co-glycolic acid). Other useful biodegradable or bioerodable
polymers include, but are not limited to, such polymers as
poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic
acid), poly(epsilon.-aprolactone-CO-glycolic acid),
poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),
hydrogels, such as poly(hydroxyethyl methacrylate), polyamides,
poly(amino acids) (for example, L-leucine, glutamic acid,
L-aspartic acid and the like), poly(ester urea),
poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,
polyorthoesters, polycarbonate, polymaleamides, polysaccharides,
and copolymers thereof. Many methods for preparing such
formulations are well known to those skilled in the art (see, for
example, Sustained and Controlled Release Drug Delivery Systems, J.
R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other
useful formulations include controlled-release microcapsules (U.S.
Pat. Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid
copolymers useful in making microcapsules and other formulations
(U.S. Pat. Nos. 4,677,191 and 4,728,721) and sustained-release
compositions for water-soluble peptides (U.S. Pat. No.
4,675,189).
[0119] The pharmaceutical compositions of the disclosure typically
are sterile and stable under conditions of manufacture, storage and
use. Sterile solutions can be prepared by incorporating the
conjugate in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated herein, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the conjugate and/or other biologically
active agent into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated herein. In the case of sterile powders, methods of
preparation include vacuum drying and freeze-drying which yields a
powder of the conjugate plus any additional desired ingredient from
a previously sterile-filtered solution thereof. The prevention of
the action of microorganisms can be accomplished by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
[0120] In accordance with the various treatment methods of the
disclosure, the conjugate can be delivered to a subject in a manner
consistent with conventional methodologies associated with
management of the disorder for which treatment or prevention is
sought. In accordance with the disclosure herein, a
prophylactically or therapeutically effective amount of the
conjugate and/or other biologically active agent is administered to
a subject in need of such treatment for a time and under conditions
sufficient to prevent, inhibit, and/or ameliorate a selected
disease or condition or one or more symptom(s) thereof.
[0121] Typical subjects intended for treatment with the
compositions and methods of the present disclosure include humans,
as well as non-human primates and other animals. To identify
subjects for prophylaxis or treatment according to the methods of
the disclosure, accepted screening methods are employed to
determine risk factors associated with a targeted or suspected
disease of condition (for example, coughing disease) as discussed
herein, or to determine the status of an existing disease or
condition in a subject. These screening methods include, for
example, conventional work-ups to determine environmental,
familial, occupational, and other such risk factors that may be
associated with the targeted or suspected disease or condition, as
well as diagnostic methods, such as various ELISA and other
immunoassay methods, which are available and well known in the art
to detect and/or characterize disease-associated markers. These and
other routine methods allow the clinician to select patients in
need of therapy using the methods and pharmaceutical compositions
of the disclosure. In accordance with these methods and principles,
a conjugate and/or other biologically active agent can be
administered according to the teachings herein as an independent
prophylaxis or treatment program, or as a follow-up, adjunct or
coordinate treatment regimen to other treatments, including
surgery, vaccination, immunotherapy, hormone treatment, cell,
tissue, or organ transplants, and the like.
[0122] The conjugates can be used in coordinate vaccination
protocols or combinatorial formulations. In certain embodiments,
novel combinatorial immunogenic compositions and coordinate
immunization protocols employ separate immunogens or formulations,
each directed toward eliciting an anti-LPS or an anti-LOS immune
response. Separate immunogens that elicit the anti-LPS or anti-LOS
immune response can be combined in a polyvalent immunogenic
composition administered to a subject in a single immunization
step, or they can be administered separately (in monovalent
immunogenic compositions) in a coordinate immunization protocol.
For example, a combinatorial or a polyvalent immunogenic
composition could include (i) an oligosaccharide or polysaccharide
obtained from Bordetella bronchiseptica or Bordetella pertussis as
a first component and (ii) oligosaccharide or polysaccharide
obtained from Bordetella parapertussis as a second component.
[0123] The administration of the conjugate of the disclosure can be
for either prophylactic or therapeutic purpose. When provided
prophylactically, the conjugate is provided in advance of any
symptom. The prophylactic administration of the conjugate serves to
prevent or ameliorate any subsequent infection. When provided
therapeutically, the conjugate is provided at (or shortly after)
the onset of a symptom of disease or infection. The conjugate of
the disclosure can thus be provided prior to the anticipated
exposure to Haemophilus ducreyi, Bordetella bronchiseptica,
Bordetella parapertussis, Bordetella pertussis, Vibrio cholere,
Shigella sp. or Haemophilus influenza, so as to attenuate the
anticipated severity, duration or extent of an infection and/or
associated disease symptoms, after exposure or suspected exposure
to the bacteria, or after the actual initiation of an
infection.
[0124] For prophylactic and therapeutic purposes, the conjugate can
be administered to the subject in a single bolus delivery, via
continuous delivery (for example, continuous transdermal, mucosal
or intravenous delivery) over an extended time period, or in a
repeated administration protocol (for example, by an hourly, daily
or weekly, repeated administration protocol). The therapeutically
effective dosage of the conjugate can be provided as repeated doses
within a prolonged prophylaxis or treatment regimen, that will
yield clinically significant results to alleviate one or more
symptoms or detectable conditions associated with a targeted
disease or condition as set forth herein. Determination of
effective dosages in this context is typically based on animal
model studies followed up by human clinical trials and is guided by
administration protocols that significantly reduce the occurrence
or severity of targeted disease symptoms or conditions in the
subject. Suitable models in this regard include, for example,
murine, rat, porcine, feline, non-human primate, and other accepted
animal model subjects known in the art. Alternatively, effective
dosages can be determined using in vitro models (for example,
immunologic and histopathologic assays). Using such models, only
ordinary calculations and adjustments are required to determine an
appropriate concentration and dose to administer a therapeutically
effective amount of the conjugate (for example, amounts that are
effective to elicit a desired immune response or alleviate one or
more symptoms of a targeted disease). In alternative embodiments,
an effective amount or effective dose of the conjugate may simply
inhibit or enhance one or more selected biological activities
correlated with a disease or condition, as set forth herein, for
either therapeutic or diagnostic purposes.
[0125] The actual dosage of the conjugate will vary according to
factors such as the disease indication and particular status of the
subject (for example, the subject's age, size, fitness, extent of
symptoms, susceptibility factors, and the like), time and route of
administration, other drugs or treatments being administered
concurrently, as well as the specific pharmacology of the conjugate
for eliciting the desired activity or biological response in the
subject. Dosage regimens can be adjusted to provide an optimum
prophylactic or therapeutic response. A therapeutically effective
amount is also one in which any toxic or detrimental side effects
of the conjugate and/or other biologically active agent is
outweighed in clinical terms by therapeutically beneficial effects.
A non-limiting range for a therapeutically effective amount of a
conjugate and/or other biologically active agent within the methods
and formulations of the disclosure is about 0.01 mg/kg body weight
to about 10 mg/kg body weight, such as about 0.05 mg/kg to about 5
mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body
weight.
[0126] Upon administration of a conjugate of the disclosure (for
example, via injection, aerosol, oral, topical or other route), the
immune system of the subject typically responds to the immunogenic
composition by producing antibodies specific for LPS, LOS and/or an
antigenic epitope presented by the conjugate. Such a response
signifies that an immunologically effective dose of the conjugate
was delivered. An immunologically effective dosage can be achieved
by single or multiple administrations (including, for example,
multiple administrations per day), daily, or weekly
administrations. For each particular subject, specific dosage
regimens can be evaluated and adjusted over time according to the
individual need and professional judgment of the person
administering or supervising the administration of the conjugate.
In some embodiments, the antibody response of a subject
administered the compositions of the disclosure will be determined
in the context of evaluating effective dosages/immunization
protocols. In most instances it will be sufficient to assess the
antibody titer in serum or plasma obtained from the subject.
Decisions as to whether to administer booster inoculations and/or
to change the amount of the composition administered to the
individual can be at least partially based on the antibody titer
level. The antibody titer level can be based on, for example, an
immunobinding assay which measures the concentration of antibodies
in the serum which bind to a specific antigen, for example, LPS
and/or LOS.
[0127] Dosage can be varied by the attending clinician to maintain
a desired concentration at a target site (for example, the lungs or
systemic circulation). Higher or lower concentrations can be
selected based on the mode of delivery, for example,
trans-epidermal, rectal, oral, pulmonary, or intranasal delivery
versus intravenous or subcutaneous delivery. Dosage can also be
adjusted based on the release rate of the administered formulation,
for example, of an intrapulmonary spray versus powder, sustained
release oral versus injected particulate or transdermal delivery
formulations, and so forth. To achieve the same serum concentration
level, for example, slow-release particles with a release rate of 5
nanomolar (under standard conditions) would be administered at
about twice the dosage of particles with a release rate of 10
nanomolar. The methods of using conjugates, and the related
compositions and methods of the disclosure, are useful in
increasing resistance to, preventing, ameliorating, and/or treating
infection and disease caused by Bordetella, H. ducreyi, Vibrio
cholere, Shigella sp. or Haemophilus influenza in animal hosts, and
other, in vitro applications. These immunogenic compositions can be
used for active immunization for prevention of infection, and for
preparation of immune antibodies. The immunogenic compositions are
composed of non-toxic components, suitable for infants, children of
all ages, and adults.
[0128] The methods of the disclosure are broadly effective for
treatment and prevention of bacterial disease and associated
inflammatory, autoimmune, toxic (including shock), and chronic
and/or lethal sequelae associated with bacterial infection.
Therapeutic compositions and methods of the disclosure for
prevention or treatment of toxic or lethal effects of bacterial
infection are applicable to a wide spectrum of infectious agents.
Non-lethal toxicities that will be ameliorated by these methods and
compositions can include fatigue syndromes, inflammatory/autoimmune
syndromes, hypoadrenal syndromes, weakness, cognitive symptoms and
memory loss, mood symptoms, neurological and pain syndromes and
endocrine symptoms. Any significant reduction or preventive effect
of the conjugate with respect to the foregoing disease condition(s)
or symptom(s) administered constitutes a desirable, effective
property of the subject composition/method of the disclosure.
[0129] The instant disclosure also includes kits, packages and
multi-container units containing the herein described
pharmaceutical compositions, active ingredients, and/or means for
administering the same for use in the prevention and treatment of
bacterial diseases and other conditions in mammalian subjects. Kits
for diagnostic use are also provided. In one embodiment, these kits
include a container or formulation that contains one or more of the
conjugates described herein. In one example, this component is
formulated in a pharmaceutical preparation for delivery to a
subject. The conjugate is optionally contained in a bulk dispensing
container or unit or multi-unit dosage form. Optional dispensing
means can be provided, for example a pulmonary or intranasal spray
applicator. Packaging materials optionally include a label or
instruction indicating for what treatment purposes and/or in what
manner the pharmaceutical agent packaged therewith can be used.
[0130] The subject matter of the present disclosure is further
illustrated by the following non-limiting Examples.
Example 1
Bordetella Conjugates
[0131] Bacteria and Cultivation.
[0132] The following strains were obtained from ATCC: B.
bronchiseptica ATCC 10580, Rb50 (ATCC BAA-588), and B.
parapertussis ATCC 1589; B. bronchiseptica 15374, 3145 and B.
parapertussis 12822 were obtained from Dr. M. Perry (NRC Canada).
Bacteria were grown on Bordet-Genguo (BG) agar plates and then
transferred to Stainer-Scholte (S--S) media (Stanier D W, Scholte M
J. A simple chemically defined medium for the production of phase I
Bordetella pertussis. J Clin Pathol. 25:732-733, 1970). Bacteria
were harvested, killed by boiling for 1 hour and frozen for LPS
extraction.
[0133] Oligosaccharides.
[0134] LPS was isolated by phenol-water extraction and purified by
enzyme treatment and ultracentrifugation as described in Westphal
O., Jann K., Meth. Carbohydr. Chem. 5:83-89, 1965, which is
incorporated herein by reference in its entirety. To isolate
O-specific oligosaccharide (O-SP), LPS (100 mg) was treated with 1%
acetic acid (10 ml) for 60 minutes at 100.degree. C.,
ultracentrifuged and the carbohydrate-containing supernatant was
fractionated on a BioGel P-4 column (1.0.times.100 cm) in
pyridine/acetic acid/water buffer (4/8/988 ml) monitored with a
Knauer differential refractometer. 28 mg of O-SP was eluted in void
volume and used for conjugation. Alternatively, LPS was deaminated
in the following way: 100 mg of LPS was dissolved in the mixture: 6
ml 30% acetic acid, 6 ml 5% sodium nitrite, 6 ml water. Reaction
was carried out in room temperature, 6 hours, on the magnetic
stirrer followed by ultracentrifugation. The supernatant was
lyophilized and purified on BioGel P-4 column using conditions as
above. 23 mg of O-SPdeam was eluted in void volume and used for
conjugation.
[0135] Analytic.
[0136] Protein concentration was measured by the method of Lowry
(O. H. Lowry et al., J. Biol. Chem. 193:265, 1951). SDS-PAGE used
14% gels according to the manufacturer's instructions. Double
immunodiffusion was performed in 1.0% agarose gel in PBS.
Spectroscopy.
[0137] MALDI-TOF mass spectra of the derivatized carrier proteins
and the conjugates were obtained with an OmniFlex MALDI-TOF
instrument (Bruker Daltonics) operated in the linear mode. Samples
for analysis were desalted and 1 .mu.l was mixed with 20 .mu.l of
sinapic acid matrix made in 30% CH.sub.3CN and 0.1% trifluoroacetic
acid. Next, 1 .mu.l of mixture was dried on the sample stage and
placed in the mass spectrometer.
[0138] Methods.
[0139] NMR spectra were recorded at 30.degree. C. in D.sub.2O on a
Varian UNITY INOVA 500, 600, or 800 instrument, using acetone as
reference for proton (2.225 ppm) and carbon (31.5 ppm) spectra.
Varian standard programs COSY, NOESY (mixing time of 400 ms), TOCSY
(spinlock time 120 ms), HSQC, and gHMBC (long-range transfer delay
100 ms) were used with digital resolution in F2 dimension <2
Hz/pt. ESI-MS and NMR spectroscopy was used to confirm the
structure of bordatellae LPS structure.
[0140] Molecular mass obtained from MALDI like 135 kDa is a mass of
conjugate, from which is subtracted a mass of aminooxylated
protein-like aminooxylated-BSA is 73 kDa; the difference is a mass
of oligo(poly) saccharide introduced on protein.
[0141] Conjugation.
[0142] (1) BSA-ONH.sub.2/O-SP.
[0143] An aminooxylated bovine serum albumin (BSA) was prepared via
a two-step procedure as described in Kielb et al., J. Org. Chem.
70:6987-6990, 2005, which is incorporated herein by reference in
its entirety. First, the protein was treated with succinimidyl
3-(bromoacetamido)propionate (SBAP) to introduce thiol-reactive
bromoacetamido moieties. Next, it was coupled with
O-(3-thiolpropyl)hydroxylamine, a heterobifunctional linker, to
form the aminooxylated protein through stable thioether linkages
(BSA-ONH.sub.2). For conjugation with O-SP, BSA-ONH.sub.2 (5 mg)
was reacted with 10 mg of O-SP in 1.5 ml Buffer A (PBS, 0.1%
glycerol, 0.005 M EDTA, pH 7.4), at pH 5.7, for 15 hours. Next, it
was purified by Sephadex G100 gel filtration in 0.2 M NaCl as
eluant and the void volume fraction characterized by protein and
sugar assays, immunodiffusion, SDS-PAGE and MALDI-TOF spectroscopy.
Three conjugates were obtained this way and named as
BSA-ONH.sub.2/Bb10580 (#1), BSA-ONH.sub.2/BbRb50 (#2) and
BSA-ONH.sub.2/Bp15898 (#3).
[0144] (2) BSA-ONH.sub.2/O-SPdeam BSA was derivatized to
BSA-ONH.sub.2 as above and 5 mg was reacted with 10 mg of O-SPdeam
using the same condition as above. Next, it was purified by
Sephadex G100 gel filtration and assayed as above. The products
were named BSA-ONH.sub.2/Bb10580d. (#4), BSA-ONH.sub.2/BbRb50d.
(#5) and BSA-ONH.sub.2/Bp15898d. (#6).
[0145] Immunization.
[0146] 5 to 6-weeks-old female NIH Swiss Webster mice were
immunized s.c. 3 times at 2 weeks intervals with 2.5 .mu.g O-SP as
a conjugate in 0.1 ml PBS and groups of 10 exsanguinated 7 days
after the second or third injections. Controls received PBS.
Hyperimmune mice sera against B. bronchiseptica strains 10580 and
Rb50, and against B. parapertussis strain 15898 were induced by
multiple intraperitroneal immunization of mice with heat killed
whole bacterial cells.
[0147] Antibodies.
[0148] Serum IgG antibodies were measured by ELISA. Nunc Maxisorb
plates were coated with B. bronchiseptica 10580 LPS, Rb50LPS or B.
parapertussis 15898 LPS at 5 .mu.g/ml in PBS containing 1%
trichloroacetic acid as described in Hardy et al., "Enhanced ELISA
sensitivity using TCA for efficient coating of biologically active
lipopolysaccharides or lipid A to the solid phase," J Immunol
Methods 176(1):111-6, 1994. Concentration and the composition of
buffer for coating antigen were determined by checkerboard
titration. Plates were blocked with 1% BSA in PBS for hyperimmune
sera or 1% HSA in PBS for conjugate-induced sera for 1 hour at room
temperature. A MRX Dynatech reader was used. Antibody levels were
calculated relative to hyperimmune standard serum diluted 1:20,000
for B. bronchiseptica 10580; 1:15,000 for B. bronchiseptica Rb50
and 1:10,000 for B. parapertussis 15898 and assigned a value of
1000 ELISA units (EU). Results were computed with an ELISA data
processing program provided by the Biostatistics and Information
Management Branch, CDC.
[0149] Inhibition ELISA was done by incubating hyperimmune mice
sera, diluted to the concentration that gave an A.sub.405
absorption of 1.0, with 10 or 50 .mu.g of inhibitor per well, for 1
hour at 37.degree. C. and overnight at 4.degree. C. The assay was
then continued as above. Sera with and without inhibitor, at the
same dilution, were compared. Percent inhibition was defined as
(1-A.sub.405 adsorbed serum/A.sub.405 non-adsorbed
serum).times.100%.
[0150] The results are shown below in Table 1.
TABLE-US-00001 TABLE 1 Inhibition ELISA. Plates were coated with B.
bronchiseptica 10580 LPS, Rb50 or B. parapertussis 15989,
respectively at 5 .mu.g/ml and reacted anti-B. bronchiseptica 10580
hyperimmune mice serum diluted 1:40000, Rb50 1:20000 and anti-B.
parapertussis 15989 1:20000. Inhibitors were used at 50 .mu.g/well
O-SP Amidation end- of second % of inhibition of hyperimmune whole
cell sera Inhibitor group O-SP sugar Anti-Bb10580 Anti-BbRb50
Anti-Bp15989 Bb 10580 O-SP Ala - 92 2 50 Bb 110H O-SP Ala - 96 3 45
Bb Rb50, O-SP Lac - 0 93 1 Bb 512 O-SP Lac - 1 95 3 Bp 15898 O-SP
Ala + 42 5 97 Bp 15311 O-SP Ala + 50 3 98 H. ducrei O-SP na Na 0 0
0
[0151] Results.
[0152] Chemical Characterization of LPSes
[0153] Chemical analysis indicated that strains B. bronchiseptica
10580, 15374 and 5137, as well as strains B. parapertussis 15898
and 12822 belong to the "Ala-type" (terminal non-reducing
residue--2,3,4-triamino-2,3,4-trideoxy-alpha-galactouronamide is
formylated on position 3 and 4 and has N-formyl-L-alanyl or
L-alanyl substituents at N-2), whereas strain B. bronchiseptica
Rb50 belongs to the "Lac-type" (the same terminal residue is
acetylated on position 2, formylated at position 3 and the amino
group at position 4 bears a 2-methoxypropionyl substituent).
[0154] Serum Antibodies.
[0155] Immunogenicity was checked by injection to mice. The average
molecular mass of deaminated O-SP from B. bronchiseptica strain
10580, as assayed by ES-MS, was established as 5108 Da. The number
of O-SP chains bound per one BSA was estimated to be 15 in
conjugate #4. The average mass of O-SP was calculated on the bases
of detailed structural analysis of studies LPSes, as reported
elsewhere, was established as average of 6588 Da for conjugate #1,
2 and 3. The number of O-SP chains bound per one BSA was estimated
to be 10, 10 and 15, respectively. The result are shown below in
Table 2.
TABLE-US-00002 TABLE 2 Composition and serum GM of IgG anti-B.
bronchiseptica and B. parapertussis LPS in mice by conjugates of
O-SP and O-SP.sub.deam bound to bovine serum albumin (BSA). Mice
(10 per group) were immunized with 2.5 .mu.g of polysaccharide as a
conjugate/mouse, injected s.c., 3 times, 2 weeks apart. Mol. Ratio
Mol O- ELISA units after 3rd injection mass.sup.1 protein/ SP/Mol
Coating antigen # Conjugate [kDa] sugar Protein 10580 LPS Rb50 LPS
15898 LPS 1 BSA-ONH.sub.2/Bb10580 135 1:0.9 9 4.9 0.3 0.4 2
BSA-ONH.sub.2/BbRb50 137 1:0.9 9 2.4 132 0.4 3
BSA-ONH.sub.2/Bp15898 165 1:1.4 14 0.3 0.3 12 4
BSA-ONH.sub.2/Bb10580.sub.deam 130 1:1.0 11 55 0.6 4.8 5
BSA-ONH.sub.2/BbRb50.sub.deam 105 1:0.5 8 0.1 3.5 0.3 6
BSA-ONH.sub.2/Bp15898.sub.deam 116 1:0.7 10 0.5 0.1 15.6 .sup.1Mol
mass was assayed by Maldi-tof, Mol mass of BSA-ONH.sub.2 was 74.2
kDa The "Ratio protein/sugar" is the mass ratio of the two
components of the final conjugate. The "Mol O-SP/Mol Protein is the
mole ratio of the two components of the final conjugate.
[0156] A novel pentasaccharide was identified in B. bronchiseptica
and B. parapertussis LPS. B. bronchiseptica O-SP differed in their
non-reducing end-groups: the "ala-type" and the "lac-type." In
contrast, all B. parapertussis strains analyzed belonged to the
"ala-type." No cross reaction between the two types of B.
Bronchiseptica LPS was observed. Inhibition assays showed that the
terminal residues of O-SP are immunodominant. BSA/O-SP conjugates
were specific and induced antibodies only to the homologous type of
O-SP. Accordingly, and based upon epidemiological data, at least
two types of LPS should be included in a vaccine according to a
preferred embodiment.
Example 2
Haemophilus ducreyi Conjugates
[0157] Bacteria and Cultivation.
[0158] Haemophilus ducreyi strains 35000 was obtained from Culture
Collection Goteborg University (CCUG 7470). Bacteria were
cultivated on chocolate agar plates Grand Lux (GLV-3) (Department
of Bacteriology, Sahlgrenska Hospital, Goteborg, Sweden),
containing 5% brain heart infusion (BHI) agar, 1% horse blood, 1.5%
horse serum, 0.06% yeast autolysate, 0.015% IsoVitalex (BBL) and 3
mg/ml vancomycin. The plated bacteria were incubated at 33.degree.
C. for 48 hours in high humidity in an anaerobic jar with
Anaerocult C (Merk, Darmstad, Germany) for generation of an
oxygen-depleted and CO.sub.2 enriched atmosphere. The bacteria were
harvested and frozen at -20.degree. C. for LOS extraction.
[0159] Oligosaccharides.
[0160] LOS was isolated by phenol-water extraction and purified by
enzyme treatment and ultracentrifugation as described in Westphal
et al., Meth. Carbohydr. Chem. 5:83-89, 1965. To isolate
oligosaccharide (OS), LOS (100 mg) was treated with 1% acetic acid
(10 ml) for 60 minutes at 100.degree. C. and the
carbohydrate-containing supernatant was fractionated on a BioGel
P-4 column (1.0.times.100 cm) in 0.05 M pyridine acetate buffer (pH
5.6) and monitored with a Knauer differential refractometer.
[0161] Analytic.
[0162] Protein concentration was measured by the method of Lowry
(Lowry et al., J. Biol. Chem. 193:265, 1951), sugar concentration
by phenol/H.sub.2SO.sub.4 assay (Dubois et al., "Colorimetric
method for determination of sugars and related substances," Anal.
Chem., 28:350-356, 1956), incorporation of benzaldehyde groups by
colorimetric reaction with 2-hydrazinopyride (Solulink protocol),
and hydrazide by TNBS assay as reported (Habeeb A F, "Determination
of free amino groups in proteins by trinitrobenzenesulfonic acid,"
Anal Biochem. 14(3):328-336, 1966).
[0163] Spectroscopy.
[0164] Sugars were analyzed according to Sawardeker et al
(Sawardeker et al., "Quantitative determination of monosaccharides
as their alditol acetates by gas-liquid chromatography," Analyt.
Chem. 37:1602-1604, 1965). A 0.5 mg sample of each polysaccharide
was hydrolyzed in 1 ml of 10 M HCl for 30 minutes at 80.degree. C.,
reduced peracetylated and analyzed by GLC-MS using Hewlett-Packard
apparatus, model HP 6890, with a type HP-5 glass capillary column
(0.32 mm.times.30 m) and temperature programming at 8.degree.
C./minute, from 125-250.degree. C. in the electron ionization (106
eV) mode. Methylation was performed as described in Ciucanu et al.,
"A simple and rapid method for the permethylation of
carbohydrates," Carbohydr. Res. 131:209-217, 1984. Methylated
compounds were hydrolyzed, converted to alditol acetates, and
analyzed by GLC-MS as above. MALDI-TOF mass spectra were obtained
with an OmniFlex MALDI-TOF instrument (Bruker Daltonics) operated
in the linear mode. Samples for analysis were desalted and 1 .mu.l
was mixed with 20 .mu.l of sinnapinic acid matrix made in 30%
CH.sub.3CN and 0.1% trifluoroacetic acid. Next, 1 .mu.l of mixture
was dried on the sample stage and placed in the mass spectrometer.
ESI-MS spectra were recording on the Agilent Series LC/MSD
instrument in the negative ion mode .sup.1H, .sup.13C and .sup.31P
NMR spectra were recorded at 300 MHz using Varian spectrometer.
Solutions of 5-13 mg of analytats in D.sub.2O (99.96 atom % D) were
used, with acetone as an internal reference at 2.225 ppm and 31.0
ppm, for .sup.1H and .sup.13C respectively, or 85% H.sub.3PO.sub.4
containing 10% D.sub.2O as an external reference for .sup.31P at
-0.73 ppm.
[0165] Conjugation.
[0166] Conjugation by Oxime Formation.
[0167] Aminooxylated BSA or Tetanus toxoid (TT) was prepared via a
two step procedure as described in Kielb et al., J. Org. Chem.
70:6987-6990, 2005, which is incorporated herein by reference in
its entirety. First, the protein was treated with succinimidyl
3-(bromoacetamido)propionate (SBAP) to introduce thiol-reactive
bromoacetamido moieties. Next, it was coupled with
O-(3-thiolpropyl)hydroxylamine, a heterobifunctional linker
featuring terminal aminooxy and thiol groups, to form the
aminooxylated protein through stable thioether linkages
(Pr--ONH.sub.2). For conjugation with O-SP, Pr--ONH.sub.2 (10 mg)
was reacted with 10 mg of O-SP in 1.5 ml Buffer A (PBS, 0.1%
glycerol, 0.005 M EDTA, pH 7.4), at pH 5.7, for 15 hours. Next, it
was purified by Sephadex G100 gel filtration in 0.2 M NaCl as
eluant and the void volume fraction characterized by protein and
sugar assays, immunodiffusion, SDS-PAGE and MALDI-TOF spectroscopy.
Two conjugates were obtained this way and named as BSA-ONH.sub.2/OS
(#1) and TT-ONH.sub.2/OS (#2).
[0168] Characterization of H. ducreyi Oligosaccharide Epitpopes in
the Conjugates by Monoclonal Antibodies.
[0169] The maxi-sorp ELISA plates were coated with the conjugates
(1-2) in concentration 2 .mu.g/ml of sugar as a conjugate and 10
.mu.g/ml LOS over night. As a negative control BSA (10 .mu.g/ml)
was used. Plates were blocked with 1% BSA and the medium
(concentrated 5.times.) containing monolonal antibodies (MAHD6 or
MADH7 [v]) was added. Plates were incubated 3 hours, washed and
anti-mouse alkaline phosphatase conjugate was added. After further
incubation, plates were washed and developed. The absorbance at 403
nm was monitored.
[0170] Oxime Formation with Hemiacetal Groups.
[0171] D-Glucose (10 mg), D-maltose (10 mg) maltotriose (25 mg),
D-glucosamine (10 mg) or N-acetyl-D-mannosamine (10 mg) were
reacted with O-(3-thiolpropyl)hydroxylamine (6 mg) in 1 ml D.sub.2O
adjusted to pH 5.5 with 30% solution of NaOD at 37.degree. C. for
15 hours. Progress of reaction was monitored by .sup.1H NMR.
Maltotriose-SH (30 mg) was separated from linker by passing through
BioBel P-2 column in 0.05 M pyridine acetate buffer as above and
freeze-dried. Next 30 mg of maltotriose-SH was reacted with
bromoacetamido-derivatized BSA (15 mg), prepared as above to form
maltotriose-BSA conjugate by thioether linkages. Reaction was done
in buffer A, pH 7.4, 3 hours and solution was purified on Sephadex
G-100 column as above. Extent of conjugation was evaluated by
MALDI-TOF. Molecular mass of bromoacetamido-BSA was 73545 Da, while
maltotriose-BSA conjugate was 81673 Da, indicated incorporation of
16 maltotriose molecules per BSA.
[0172] Immunization.
[0173] 5 to 6-weeks-old female NIH Swiss Webster mice were
immunized sc 3 times at 2 weeks intervals with 2.5 .mu.g OS or PGA
as a conjugate in 0.1 ml PBS and groups of 10 exsanguinated 7 days
after the second or third injections. Controls received PBS.
[0174] Antibodies.
[0175] Serum IgG antibodies were measured by ELISA (Taylor et al.,
Infect. Immun. 61:3678-3687, 1993). Nunc Maxisorb plates were
coated with H. ducreyi LOS, 10 .mu.g/ml PBS (determined by
checkerboard titration). A MRX Dynatech reader was used. The
reference serum to O-SP and BSA was a pool of sera obtained from
mice immunized 3 times with 5 .mu.g of oligosaccharide as a
conjugate BSA-CHO/AH-O-SP (Conj. #3), diluted to 1.2000 in the
first well and assigned a value of 1000 ELISA units (EU). The
reference serum to TT was a pool of sera obtained from mice
immunized 3 times with 5 .mu.g of oligosaccharide as a conjugate
TT-NOS/O-SP (Conj. #2), diluted to 1:5000 in the first well and
assigned a value of 100 ELISA units (EU). Results were computed
with an ELISA data processing program provided by the Biostatistics
and Information Management Branch, CDC.
[0176] Immunology.
[0177] SDS-PAGE and Western-blotting used 14% gels according to the
manufacturer's instructions. Double immunodiffusion was performed
in 1.0% agarose gel in PBS.
[0178] Results
[0179] Characterization of LOS and LOS-Derived
Oligosaccharides.
[0180] Mass spectroscopic and NMR analysis of isolated products
confirmed the structure of the sugar chain of H. ducreyi strain
35000 LOS. The data were in agreement with the published structure
(Melaugh et al., "Structure of the major oligosaccharide from the
lipooligosaccharide of Haemophilus ducreyi strain 35000 and
evidence for additional glycoforms," Biochemistry
33(44):13070-13078, 1994) as represented below:
##STR00002##
[0181] The sialilation of non-reducing end was estimated at about
60% by NMR and GLC-MS analysis. Hydrolysis of LOS with 1% acetic
acid cleaved O-SP from Lipid A on the KDO residue, removing at the
same time all sialic acid residues from non-reducing end. The
observed molecular mass of this product, recorded by ESI-MS in
negative mode was EM-1]=1675.8, which is in agreement with the
structure of Hex3HexNAcHep.sub.4-anhydro-Kdo as the O-SP for H.
ducreyi strain 35000. No phosphate group on KDO was detectable also
by .sup.31P-NMR suggested the beta-elimination of phosphate from
KDO as was reported that results in anhydro-KDO groups at the
reducing end (Auzanneau et al., "Phosphorylated sugars. Part 27.
Synthesis and reactions, in acid medium, of 5-O-substituted methyl
3-deoxy-.alpha.-D-manno-oct-2-ulopyranosic acid 4-phosphates," J.
Chem. Soc. Perkin Transl. 1:509-517, 1991; Vinogradov et al., "The
structure of the carbohydrate backbone of the core-lipid-A region
of the lipopolysaccharide from Vibrio cholerae strain H11
(non-O1)," Eur J Biochem. 218(2):543-554, 1993). A reactive ketone
group was shown to form during beta elimination of a model
compound, 5-O-methyl-KDO-4-phosphate. The ketone group was used for
conjugation of the OS to the protein carrier.
[0182] Characterization of Conjugates.
[0183] Conjugation of O-SP to aminooxylated protein gave a
conjugate containing average 15 chains of oligosaccharide per
protein (#1 and #2). Protein/sugar ratio was analyzed by
colorimetric assays and by increase of molecular mass using
MALDI-TOF spectroscopy. Although not bound by any theory, it is
believed that the conjugate is formed by the reaction of a ketone
group on the terminal KDO molecule with O-alkyl hydroxylamine on
the protein.
[0184] In order to identify the core structure of the H. ducreyi
LOS in the conjugates two monoclonal antibodies were used to
structurally defined epitopes on the H. ducreyi LOS [V]
##STR00003##
[0185] Conjugates #1 and 2 showed similar levels of recognition as
LOS itself (see Table 3 below).
TABLE-US-00003 TABLE 3 Binding (ELISA) of Mabs specific to H.
ducreyi LOS with OS-protein conjugates. Plates were coated with
conjugates and reacted with Mabs. Absorbance at 403 nm Conjugates
Mab MAHD6 MAHD7 BSA-ONH.sub.2/OS 2.73 4.18 TT-ONH.sub.2/OS 1.86
3.95 LOS 1.77 3.6 BSA 0.08 0.08
[0186] Immunology.
[0187] The conjugates were injected into mice at a dose of 2.5 or 5
microgram of OS as a conjugate per mouse and the IgG anti-H.
ducreyi LOS levels were assayed by ELISA. The results are presented
in Table 4 below. Negligible levels of anti-LOS antibodies were
detected in sera. However, when plates were coated with conjugate
#2, high level of antibodies was detected in sera induced by
conjugate #1. This means that the conjugates induce antibodies to
sugar part of this LOS while it is presented on other carrier
protein in ELISA assay. Since carrier proteins are different, the
antibodies seem to be induced against either common sugar part or
the linker moiety. It indicated that epitopes presented on ELISA
plates by coating with LOS is different then by coating with
conjugate.
TABLE-US-00004 TABLE 4 Composition and serum GM of IgG anti-H.
ducreyi LOS in mice by conjugates of O-SP bound to bovine serum
albumin (BSA), and tetanus toxoid (TT) and by lacto-N-neotetraose
and sialyl-lacto-N-neotetraose bound to human serum albumine (HSA).
Mice (10 per group) were immunized with 2.5 .mu.g of
oligosaccharide as a conjugate/mouse and injected s.c., 3 times, 2
weeks apart. Ratio Mol Microgr. Mol. protein/ OS/Mol Pr/OS Anti-
Anti- Ani- # Conjugate mass.sup.1 sugar Protein injected LOS
Protein conjugate 1 BSA-ONH.sub.2/OS 105 kDa 2:1 18 5/2.5 2 656 29
(#2) 2 TT-ONH.sub.2/OS Nd 6:1 15 15/2.5 4 2297 295 (#1)
.sup.1assayed by MALDI-TOF The H. ducreyi OS/protein conjugate had
limited immunogenicity in mice.
Example 3
B. pertussis and B. bronchiseptica Conjugates
[0188] Methods:
[0189] Bacteria and Cultivation.
[0190] B. pertussis ATCC BAA-589 (Tohama I) and B. bronchiseptica
ATCC 10580 were grown on Bordet-Gengou (BG) agar plates and
transferred to Stainer-Scholte (S--S) media. Bacteria were
harvested and killed with 1% formalin.
[0191] Oligosaccharides.
[0192] LPS was isolated by hot phenol-water extraction and purified
by enzyme treatment and ultracentrifugation. To isolate core
oligosaccharide (OS), LPS was treated with 1% acetic acid at 10
mg/ml for 60 min at 100.degree. C., ultracentrifuged and the
carbohydrate-containing supernatant fractionated on a 1.0.times.100
cm column of BioGel P-4 in pyridine/acetic acid/water buffer
(4/8/988 ml) monitored with a Knauer differential
refractometer.
[0193] Conjugation.
[0194] BSA-ONH.sub.2/OS. Bovine serum albumin (BSA, Sigma, St.
Louis, Mo.) was derivatized to aminooxylated derivatives in a two
step procedure as described in Kielb et al., J. Org. Chem.
70:6987-6990, 2005, which is incorporated herein by reference in
its entirety.: (1) BSA was treated with succinimidyl
3-(bromoacetamido)propionate (SBAP, Pierce, Pittsburgh, Pa.) to
introduce thiol-reactive bromoacetamido moieties (BSA-Br); (2)
BSA-Br was coupled with O-(3-thiopropyl)hydroxylamine, a
heterobifunctional linker, to form the aminooxylated protein
through stable thioether linkages (BSA-ONH.sub.2). For conjugation
with OS, BSA-ONH.sub.2 (5 mg) was reacted with 7 mg of OS in 1.5 ml
Buffer A (PBS, 0.1% glycerol, 5 mM EDTA), at pH 5.7, for 15 hours.
Next, it was passed through a 1.times.100 cm Sephadex G-50 column
in 0.2 M NaCl as eluent and the void volume fraction characterized
by protein assay, immunodiffusion, SDS-PAGE and MALDI-TOF
spectroscopy. The obtained conjugates were named BSA-ONH.sub.2/Bp
(#1), BSA-ONH.sub.2/B. b-core (#2)
[0195] Immunization was performed as described above in Example
1.
[0196] Results:
[0197] Oligosaccharides:
[0198] B. pertussis LPS contains only a core region composed of 12
sugars:
##STR00004##
[0199] B. bronchiseptica LPS contains the same core structure as B.
pertussis but it could be further substituted by O-specific chains.
For this study only free core, with no O-SP was used, after
separation on BioGel P-4 column. First fraction eluted from the
column contained core substituted with O-SP and second fraction
contains free core used in this study. ESI-MS (FIGS. 6 and 7) and
NMR analysis confirmed the above structure with small variations in
case of B. bronchiseptica: the methylation of Fuc4NMe is only 50%
(2280 kDa pick), while in B. pertussis is 100% and Hep is
phosphorylated in about 30% (2374 Da pick), while in B. pertussis
Hep is not phosphorylated.
[0200] Conjugates. SDS-PAGE gel and Maldi analysis showed increase
in molecular mass of both conjugates to average 94 kDa comparing to
BSA-ONH.sub.2 71 kDa. Since the mass of OS is 2295 Da, the increase
indicates average incorporation of 100S chains per one BSA molecule
in both cases.
[0201] Both conjugates reacted with anti-B. pertussis hyperimmune
serum and anti-BSA serum with an observed line of identity. Both
conjugates induced serum antibody responses on a similar level as
assayed by ELISA against B. pertussis LOS.
Example 4
S. flexnarii 2a Conjugates
[0202] Methods:
[0203] Bacteria and Cultivation.
[0204] Shigella flexneri type 2a strain 2457T was grown in
ultrafiltered Triptic Soy Broth (Difco Laboratories) with 5 g of
glucose and 5 mM magnesium sulphate per liter, for 20 h at
20.degree. C. with stirring and aeration; the pH was maintained at
.about.7.5 by addition of ammonium hydroxide. The identity of
bacteria was confirmed by culture, Gram staining and agglutination
with typing antisera. LPS was extracted by hot phenol method and
after dialysis recovered from each phase.
[0205] Oligosaccharides.
[0206] The LPSs (20-80 mg) were treated with 1% acetic acid at
100.degree. C. for 1 h, precipitate of lipid A removed by
centrifugation, O-specific chain (O-SP) was separated by gel
chromatography on Sephadex G-50 column.
[0207] Conjugation. BSA-ONH.sub.2/OS.
[0208] Bovine serum albumin (BSA, Sigma, St. Louis, Mo.) was
derivatized to aminooxylated derivatives in a two step procedure as
described in Kielb et al., J. Org. Chem. 70:6987-6990, 2005, which
is incorporated herein by reference in its entirety. (1) BSA was
treated with succinimidyl 3-(bromoacetamido)propionate (SBAP,
Pierce, Pittsburgh, Pa.) to introduce thiol-reactive bromoacetamido
moieties (BSA-Br); (2) BSA-Br was coupled with
O-(3-thiopropyl)hydroxylamine, a heterobifunctional linker, to form
the aminooxylated protein through stable thioether linkages
(BSA-ONH.sub.2). For conjugation with OS, BSA-ONH.sub.2 (1 mg) was
reacted with 3 mg of O-SP in 0.3 ml Buffer A (PBS, 0.1% glycerol, 5
mM EDTA), at pH 5.7, for 15 hours. Next, it was passed through a
1.times.100 cm Sephadex G-50 column in 0.2 M NaCl as eluent and the
void volume fraction characterized by protein assay,
immunodiffusion and SDS-PAGE. The obtained conjugates were named
BSA-ONH.sub.2/Sf-OSP.
[0209] Results:
[0210] O-SP:
[0211] S. flexneii O-SP contains a core region composed of 10
sugars substituted with a repeating unit:
[0212] Core region:
##STR00005##
[0213] PPE-phosphoethanolamine; RU-repeating unit
[0214] Repeating unit (about 5-15 repeats)
##STR00006##
[0215] Conjugates.
[0216] SDS-PAGE gel analysis showed increase in molecular mass of a
conjugate comparing to BSA-ONH.sub.2 to about 250 kDa. The obtained
conjugate reacted with anti-S. flexnerii 2a hyperimmune serum and
anti-BSA serum with an observed line of identity.
[0217] In view of the many possible embodiments to which the
principles of the disclosed conjugates and methods may be applied,
it should be recognized that the illustrated embodiments are only
preferred examples and should not be taken as limiting the scope of
the invention.
* * * * *