U.S. patent application number 14/563920 was filed with the patent office on 2015-04-02 for use of saccharides cross-reactive with bacillus anthracis spore glycoprotein as a vaccine against anthrax.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is National Research Council of Canada, The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Haijing Hu, Joanna Kubler-Kielb, Stephen H. Leppla, John B. Robbins, Rachel Schneerson, Evguenii Vinogradov.
Application Number | 20150093412 14/563920 |
Document ID | / |
Family ID | 40578048 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093412 |
Kind Code |
A1 |
Kubler-Kielb; Joanna ; et
al. |
April 2, 2015 |
USE OF SACCHARIDES CROSS-REACTIVE WITH BACILLUS ANTHRACIS SPORE
GLYCOPROTEIN AS A VACCINE AGAINST ANTHRAX
Abstract
Provided are immunogenic compositions and methods for eliciting
an immune response against B. anthracis and other bacteria that
contain 3-methyl-3-hydroxybutyrate- or
3-hydroxybutyrate-substituted saccharides. Conjugates of
3-methyl-3-hydroxybutyrate- or 3-hydroxybutyrate-substituted
saccharides elicit an effective immune response against B.
anthracis spores in mammalian hosts to which the conjugates are
administered.
Inventors: |
Kubler-Kielb; Joanna;
(Bethesda, MD) ; Vinogradov; Evguenii; (Ottawa,
CA) ; Schneerson; Rachel; (Bethesda, MD) ; Hu;
Haijing; (Montgomery Village, MD) ; Leppla; Stephen
H.; (Bethesda, MD) ; Robbins; John B.; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv
National Research Council of Canada |
Bethesda
Ottawa |
MD |
US
CA |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
CA
National Research Council of Canada
Ottawa
CA
|
Family ID: |
40578048 |
Appl. No.: |
14/563920 |
Filed: |
December 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13909992 |
Jun 4, 2013 |
8926986 |
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14563920 |
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12918281 |
Aug 18, 2010 |
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PCT/US2009/000995 |
Feb 17, 2009 |
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13909992 |
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61066509 |
Feb 19, 2008 |
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Current U.S.
Class: |
424/197.11 ;
424/234.1; 424/246.1; 424/260.1 |
Current CPC
Class: |
A61K 2039/6081 20130101;
A61K 47/643 20170801; A61K 39/0208 20130101; A61K 39/00 20130101;
A61K 2039/521 20130101; A61P 31/04 20180101; A61K 39/104 20130101;
A61K 47/646 20170801; C07H 3/06 20130101; A61K 39/07 20130101; C07H
5/04 20130101 |
Class at
Publication: |
424/197.11 ;
424/234.1; 424/246.1; 424/260.1 |
International
Class: |
A61K 39/07 20060101
A61K039/07; A61K 47/48 20060101 A61K047/48; A61K 39/104 20060101
A61K039/104; C07H 5/04 20060101 C07H005/04; A61K 39/02 20060101
A61K039/02 |
Claims
1. A method of inhibiting a Bacillus infection in a subject, the
method comprising administering to the subject a therapeutically
effective amount of a pharmaceutical composition comprising at
least one immunogenic agent that is cross-reactive with B.
anthracis, wherein the immunogenic agent is selected from: (a) at
least one compound comprising: ##STR00023## (b) an isolated B.
anthracis antigenic component from Shewanella or P. syringae; (c)
killed whole cells of Shewanella or P. syringae; or (d) any
combination or mixture of (a)-(c).
2. The method of claim 1, wherein the Bacillus is B. anthracis.
3. The method of claim 1, wherein the immunogenic agent is killed
whole cells of Shewanella or P. syringae, and the killed whole
cells are administered to a non-human animal that has been, or may
have been, exposed to B. anthracis.
4. The method of claim 1, wherein the subject has been, or may have
been, exposed to B. anthracis.
5. The method of claim 1, wherein the compound is covalently linked
to a polymeric carrier.
6. A method of inhibiting a Bacillus infection in a subject, the
method comprising administering to the subject a therapeutically
effective amount of an immunogenic conjugate, the conjugate
comprising at least one moiety selected from ##STR00024##
covalently linked to a carrier, wherein the conjugate elicits an
immune response in a subject.
7. A method for stimulating an immune response in a subject against
B. anthracis, comprising selecting a subject in need of stimulating
an immune response against B. anthracis; and administering to the
subject at least one immunogenic agent selected from: (a) at least
one compound comprising: ##STR00025## (b) an isolated B. anthracis
antigenic component from Shewanella or P. syringae; (c) killed
whole cells of Shewanella or P. syringae; or (d) any combination or
mixture of (a)-(c).
8. The method of claim 7, wherein the immunogenic agent is the
compound of (a), and the compound of (a) is covalently linked to a
polymeric carrier.
9. The method of claim 8, wherein the polymeric carrier is a
protein.
10. The method of claim 7, wherein the immunogenic agent is an
isolated B. anthracis antigenic component from P. syringae, and the
component is isolated fimbriae from P. syringae.
11. The method of claim 7 wherein the immunogenic agent is killed
whole cells of Shewanella spp. MR-4 or killed whole cells of P.
syringae.
Description
[0001] This is a divisional application of U.S. application Ser.
No. 13/909,992, filed on Jun. 4, 2013, which is a divisional
application of U.S. application Ser. No. 12/918,281, filed on Aug.
18, 2010, which is the U.S. National Stage of International
Application No. PCT/US2009/000995, filed Feb. 17, 2009, published
in English under PCT Article 21(2) which claims the benefit of U.S.
Provisional Application No. 61/066,509, filed Feb. 19, 2008, which
are incorporated herein by reference in their entirety.
FIELD
[0002] The disclosure relates to identifying compounds useful for
generating vaccines against anthrax. Methods of generating and
using the compounds, along with the vaccines developed from the
compounds are also disclosed.
BACKGROUND
[0003] Anthrax is a potentially lethal human infection. The
causative organism is a Gram-positive rod-shaped bacterium,
Bacillus anthracis, which exists in a vegetative or a spore form.
Spores are the infecting agent; infection is initiated by entry of
spores into a mammalian host. Entry can be by intradermal
inoculation, ingestion, or inhalation. The most lethal form of
anthrax in humans is pulmonary infection caused by inhalation of B.
anthracis spores.
[0004] Like all Bacillus species, B. anthracis bacteria form spores
when subjected to adverse conditions. Mature spores are dormant and
highly resistant to heat, dryness, and aggressive chemical
conditions. They can survive in soil for decades. Upon entry into a
suitable host, the spores germinate and multiply rapidly. The
bacteria then release the anthrax toxins toxic to the host.
[0005] Most Bacillus spores consist of a central genome-containing
core surrounded by two protective layers: the cortex and the coat.
The outer layer of most Bacillus spores is the spore coat comprised
of different proteins. Mature spores of Bacillus species such as B.
anthracis contain an additional loose-fitting layer called an
exosporium. The exosporium is the outermost layer for B. anthracis
and interacts with the environment/host. The exosporium is the
primary permeability barrier of the spore and contains spore
surface antigens.
[0006] Analysis of the exosporium identified several protein
including a glycoprotein called BclA (Bacillus collagen-like
protein of anthracis). BclA is a structural component and contains
multiple collagen-like Xaa-Yaa-Gly repeats. BclA is an
immuno-dominant protein on the B. anthracis spore surface because
most of the antibodies raised against spores react with this
protein.
[0007] An unusual tetrasaccharide is attached to the BclA protein,
likely through a GalNAc linkage. This tetrasaccharide consists of
three rhamnose monosaccharides linked to a sugar residue called
anthrose
[2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-.beta.-D-glucose-
]. Anthrose was reported to be unique to B. anthracis spores,
however the anthrose biosynthesis genes were recently identified
also in other bacilli and it was demonstrated that anthrose
expression is not restricted to B. anthracis.
SUMMARY
[0008] In one aspect, different components of Shewanella spp. MR-4
or Pseudomonas syringae are used to develop anthrax vaccines. For
example, capsular polysaccharides from Shewanella spp. MR-4 (and
similar saccharides as described below) are used to generate an
immune response in a subject and to develop anthrax vaccines. In
another example, compounds from the flagella of Pseudomonas
syringae are used to generate an immune response in a subject and
to develop anthrax vaccines.
[0009] In one embodiment, there is disclosed a pharmaceutical
composition comprising at least one immunogenic agent that is
cross-reactive with B. anthracis, wherein the immunogenic agent is
selected from:
[0010] (a) at least one compound comprising:
##STR00001##
[0011] (b) an isolated B. anthracis antigenic component from
Shewanella or P. syringae;
[0012] (c) killed whole cells of Shewanella or P. syringae; or
[0013] (d) any combination or mixture of (a)-(c).
[0014] In another embodiment, there is disclosed a pharmaceutical
composition comprising at least one compound that is cross-reactive
with B. anthracis, wherein the compound comprises
##STR00002##
[0015] and at least one pharmaceutically acceptable additive.
[0016] In another embodiment, there is disclosed an antibody that
is immuno-reactive to a compound comprising
##STR00003##
wherein the antibody is also immuno-reactive to B. anthracis
spores.
[0017] According to a further embodiment, there is disclosed a
vaccine comprising at least one immunogenic agent that is
cross-reactive with B. anthracis, wherein the immunogenic agent is
selected from:
[0018] (a) at least one compound comprising:
##STR00004##
[0019] (b) an isolated B. anthracis antigenic component from
Shewanella or P. syringae;
[0020] (c) killed whole cells of Shewanella or P. syringae; or
[0021] (d) any combination or mixture of (a)-(c).
[0022] Another disclosed embodiment is an immunogenic conjugate
comprising at least one moiety selected from
##STR00005##
covalently linked to a carrier, wherein the conjugate elicits an
immune response in a subject.
[0023] There are also disclosed methods for inhibiting a Bacillus
infection in a subject, or stimulating an immune response in a
subject against B. anthracis, that comprise administering the
pharmaceutical compositions or immunogenic conjugates disclosed
herein.
[0024] Another aspect relates to a compound of general formula
##STR00006##
in which [0025] R.sub.1 is selected from the group consisting of
--H, --OH, --CH.sub.3, C.sub.2 to C.sub.6 alkyl, and C.sub.2 to
C.sub.6 alkoxy; [0026] R.sub.2 is selected from the group
consisting of --H, --OH, --CH.sub.3, C.sub.2 to C.sub.6 alkyl, and
C.sub.2 to C.sub.6 alkoxy; [0027] R.sub.3 is selected from the
group consisting of --H, --OH, --CH.sub.3, C.sub.2 to C.sub.6
alkyl, and C.sub.2 to C.sub.6 alkoxy; [0028] R.sub.4 is selected
from the group consisting of --H, --OH, --CH.sub.3, C.sub.2 to
C.sub.6 alkyl, and C.sub.2 to C.sub.6 alkoxy; [0029] R.sub.5 is
selected from the group consisting of --H, --OH, --CH.sub.3,
C.sub.2 to C.sub.6 alkyl, and C.sub.2 to C.sub.6 alkoxy; and [0030]
R.sub.6 is selected from the group consisting of --H, --OH,
--CH.sub.3, C.sub.2 to C.sub.6 alkyl, C.sub.2 to C.sub.6 alkoxy,
and (--OR.sub.7).sub.n, wherein R.sub.7 is a sugar molecule and n
is 0 to 20. The sugars can be straight-chain, furanose, or
pyranose; D- or L-; .alpha.- or .beta.-linked; and linked from any
position on the ring of one sugar to any position on the ring of
another sugar. The compound of general formula is not any of:
##STR00007##
[0031] Another aspect relates to a pharmaceutical composition
comprising the compound of general formula
##STR00008##
and a pharmaceutically acceptable carrier, where the compound of
general formula is not:
##STR00009##
[0032] In another aspect the compound of general formula
##STR00010##
is covalently attached to a polypeptide, where the compound of
general formula is not any of:
##STR00011##
[0033] In another aspect the compound of general formula
##STR00012##
is attached to an immunogenic conjugate.
[0034] Another aspect relates to antibodies raised against the
compound of general formula
##STR00013##
[0035] In another aspect, the compound of general formula
##STR00014##
is used to generate an immune response in a subject. Preferably an
immune response is generated against B. anthracis spores.
[0036] In another aspect, the compound of general formula
##STR00015##
is used to test the selectivity of B. anthracis antibodies.
[0037] Another aspect relates to the use of capsular
polysaccharides from Shewanella and compounds from the flagella of
Pseudomonas syringae as substitutes for BclA tetrasaccharide and
glycoprotein in the development of anthrax vaccines. Alternatively,
the capsular polysaccharide is part of a glycoprotein, such as a
Shewanella MR-4 or P. syringae glycoprotein, or the polysaccharide
is conjugated to another carrier protein such as bovine serum
albumin.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 shows immunodiffusion of two Shewanella spp. MR-4
capsular polysaccharides against serum to B. anthracis spore
spores.
[0039] FIG. 2A to 2D show immunofluorescent staining of B.
anthracis spores treated with four antibodies.
[0040] FIG. 3A to 3B show immunofluorescent staining of B.
anthracis spores and P. syringae cells treated with anti-spore
serum.
DETAILED DESCRIPTION
Abbreviations
[0041] BclA is Bacillus collagen-like protein of anthracis.
[0042] ELISA is enzyme-linked immuno-absorbent assay.
[0043] HMB is 3-hydroxy-3-methylbutyrate.
[0044] HB is 3-hydroxy-butyrate.
TERMS AND DEFINITIONS
[0045] An "adjuvant" is a substance that helps and enhances the
pharmacological effect of a drug or increases the ability of an
antigen to stimulate the immune system. Adjuvants can be used to
improve the immune response to vaccine antigens for several
different purposes, including: (1) increasing the immunogenicity of
weak antigens; (2) enhancing the speed and duration of the immune
response; (3) modulating antibody avidity, specificity, isotype or
subclass distribution; (4) stimulating cell mediated immunity; (5)
promoting the induction of mucosal immunity; (6) enhancing immune
responses in immunologically immature or senescent individuals; (7)
decreasing the dose of antigen in the vaccine to reduce costs or
(8) helping to overcome antigen competition in combination
vaccines. Examples of adjuvants include Freund's complete adjuvant,
Freund's incomplete adjuvant, saponin, aluminum hydroxide (for
example, Amphogel, WYETH Laboratories, Madison, N.J.), MF59,
MTP-PE, QA-21, ISA51, B30-MDP, LA-15-PH, MPL (3-O-deacylated
monophosphoryl lipid A; Corixa, Hamilton Ind.), 3D-MPL, oil
emulsions, lipopolysaccharides, polymers, liposomes, cytokines,
IL-12 (Genetics Institute, Cambridge Mass.), ISCOMs, or other
available adjuvants or adjuvant combinations. A general discussion
of vehicles and adjuvants for oral immunization can be found in
Vaccine Design, The Subunit and Adjuvant Approach, Powell and
Newman (Eds.), Plenum Press, New York (1995).
[0046] "Administration of" and "administering a" compound should be
understood to mean providing a compound, a prodrug of a compound, a
conjugate of the compound, or a pharmaceutical composition that
includes the compound as described herein. Administration may be
for either "prophylactic" or "therapeutic" purpose. When provided
prophylactically, compounds or compositions are provided in advance
of any symptom. The prophylactic administration of the compound or
composition serves to prevent or ameliorate any subsequent
infection. When provided therapeutically, the compound or
composition is provided at (or shortly after) the onset of a
symptom of infection. The compound or composition may, thus, be
provided prior to the anticipated exposure to B. anthracis so as to
attenuate the anticipated severity, duration or extent of an
infection and disease symptoms, after exposure or suspected
exposure to these bacteria, or after the actual initiation of an
infection. For all therapeutic, prophylactic and diagnostic uses,
the polysaccharide-carrier conjugates, as well as antibodies and
other necessary reagents and appropriate devices and accessories
may be provided in kit form so as to be readily available and
easily used.
[0047] Administration includes self-administration by a subject or
administration to a subject by another person. Administered to a
subject may be by a variety of mucosal administration modes,
including by oral, rectal, intranasal, intrapulmonary, or
transdermal delivery, or by topical delivery to other surfaces.
Non-mucosal administration routes include intramuscular,
subcutaneous, intravenous, intra-atrial, intra-articular,
intra-peritoneal, or parenteral routes. Administration may be ex
vivo by direct exposure to cells, tissues or organs originating
from a subject.
[0048] Also included are kits, packages and multi-container units
containing the pharmaceutical compositions, active ingredients,
and/or means for administering the same for use in the prevention
and treatment of anthrax and other bacterial diseases and other
conditions in mammalian subjects. Kits for diagnostic use are also
included. These kits may include a container or formulation that
contains one or more of the polysaccharide, polysaccharide
conjugate and/or other active agent described. In one example, the
polysaccharide conjugate and/or other active agent described is
formulated in a pharmaceutical preparation for delivery to a
subject. The conjugates and/or other biologically active agent
is/are 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 (for example, anthrax)
and/or in what manner the pharmaceutical agent packaged therewith
can be used.
[0049] "Antibody" refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules.
Exemplary antibody molecules are intact immunoglobulin molecules,
substantially intact immunoglobulin molecules, and portions of an
immunoglobulin molecule, including those portions known in the art
as Fab, Fab', F(ab').sub.2 and F(v), as well as chimeric antibody
molecules.
[0050] In one aspect an antibody is characterized as comprising
antibody molecules that immunoreact with B. anthracis spores.
Antibodies are typically produced by immunizing a mammal with an
immunogen or vaccine containing a B. anthracis saccharide-protein
carrier conjugate to induce, in the mammal, antibody molecules
having immunospecificity for the saccharide moiety. Antibody
molecules having immunospecificity for the protein moiety will also
be produced. The antibody molecules may be collected from the
mammal and, optionally, isolated and purified by methods known in
the art.
[0051] Human or humanized monoclonal antibodies are preferred,
including those made by phage display technology, by hybridomas, or
by mice with human immune systems. The antibody molecules may be
polyclonal or monoclonal. Monoclonal antibodies may be prepared
from murine hybridomas according to the classic method of Kohler et
al. [Nature 256, 495-497, (1975)], or a derivative method thereof.
Briefly, a mouse is repetitively inoculated with a few micrograms
of the selected immunogen (for example, a polysaccharide conjugate)
over a period of a few weeks. The mouse is then sacrificed, and the
antibody-producing cells of the spleen isolated. The spleen cells
are fused by means of polyethylene glycol with mouse myeloma cells,
and the excess unfused cells destroyed by growth of the system on
selective media comprising aminopterin (HAT media). The
successfully fused cells are diluted and aliquots of the dilution
placed in wells of a microtiter plate where growth of the culture
is continued. Antibody-producing clones are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures, such as the enzyme-linked immuno-absorbent assay
(ELISA) or a derivative method thereof. Selected positive clones
can be expanded and their monoclonal antibody product harvested for
use. Detailed procedures for monoclonal antibody production are
described in Harlow and Lane, Using Antibodies: A Laboratory
Manual, CSHL, New York (1999). Polyclonal antiserum containing
antibodies can be prepared by immunizing suitable animals with an
immunogen comprising a polysaccharide conjugate.
[0052] Effective antibody production (whether monoclonal or
polyclonal) is affected by many factors related both to the antigen
and the host species. For example, small molecules tend to be less
immunogenic than others and may require the use of carriers and
adjuvants. Also, host animals vary in response to site of
inoculations and dose, with either inadequate or excessive doses of
antigen resulting in low titer antisera. Small doses (ng level) of
antigen administered at multiple intradermal sites appear to be
most reliable. An effective immunization protocol for rabbits can
be found in Vaitukaitis et al. [J. Clin. Endocrinol. Metab. 33,
988-991 (1971)].
[0053] Antibodies may be contained in blood plasma, serum,
hybridoma supernatants and the like. Antibody-containing serum will
be capable of killing, in the presence of complement, 50% of B.
anthracis bacilli or spores at a serum dilution of 1300:1 or more,
typically will do so at a dilution of 32,000:1 or more, and most
typically will be capable of killing 50% of B. anthracis bacilli or
spores at a dilution of 64,000:1 or more.
[0054] Alternatively, the antibodies are isolated to the extent
desired by well known techniques such as, for example, ion
chromatography or affinity chromatography. The antibodies may be
purified so as to obtain specific classes or subclasses of antibody
such as IgM, IgG, IgA, IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4
and the like. Antibodies of the IgG class are preferred for
purposes of passive protection.
[0055] "Antibody fragments" can be used in place of whole
antibodies and can be readily expressed in prokaryotic host cells.
Methods of making and using immunologically effective portions of
monoclonal antibodies, also referred to as antibody fragments, are
well known. Conditions by which a polypeptide/binding agent complex
can form are known in the art.
[0056] The antibodies have a number of diagnostic and therapeutic
uses. They are useful in prevention and treatment of infections and
diseases caused by B. anthracis. They can also be used as in vitro
diagnostic tools or assays to test for the presence of B. anthracis
including in biological samples, in soil samples, in air, in water,
in weapon components, in containers that may have been used to
store weapons or weapon components, and carriers (e.g., human
beings, animals, etc.) who may have been exposed to, or may be
transporting B. anthracis.
[0057] Assays include, but are not limited to, agglutination
assays, radioimmuno assays, enzyme-linked immunosorbent assays,
fluorescence assays, Western blots and the like. In one such assay,
for example, a sample containing B. anthracis is contacted with a
first antibody describes, and a labeled second antibody is used to
detect the presence of B. anthracis to which the first antibodies
have bound. Such assays may be, for example, of direct format
(where a labeled first antibody is reactive with the antigen), an
indirect format (where a labeled second antibody is reactive with
the first antibody), a competitive format (such as the addition of
a labeled antigen), or a sandwich format (where both labeled and
unlabelled antibodies are used), as well as other formats described
in the art.
[0058] In providing antibodies to a recipient mammal, preferably a
human, the dosage of administered antibodies will vary depending
upon such factors as the mammal's age, weight, height, sex, general
medical condition, previous medical history and the like. In
general, it is desirable to provide the recipient with a dosage of
antibodies which is in the range of from about 1 mg/kg to about 10
mg/kg body weight of the mammal, although a lower or higher dose
may be administered. The antibodies are intended to be provided to
the recipient subject in an amount sufficient to prevent, or lessen
or attenuate the severity, extent or duration of the infection by
B. anthracis. Antibodies which specifically immunoreact with HMB-
and HB-linked saccharides are intended to be provided to the
recipient subject in an amount sufficient to prevent, or lessen or
attenuate the severity, extent or duration of the infection by B.
anthracis.
[0059] 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, or 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, Chapter 28 (1995).
[0060] An "antigen" is a compound, composition, or substance that
can stimulate an immune response, such as the production of
antibodies or a T-cell response in an animal, including
compositions that are injected or absorbed into an animal. An
antigen reacts with the products of specific humoral or cellular
immunity, including those induced by heterologous immunogens. The
term "antigen" includes all related antigenic epitopes.
[0061] An "animal" is a living multi-cellular vertebrate organism,
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.
[0062] "Binding agents" can be used to purify and detect
polysaccharides, as well as for detection and diagnosis of B.
anthracis and anthrax. Examples of the binding agents are a
polyclonal or monoclonal antibody (including humanized monoclonal
antibody), and fragments thereof, that bind to any of the
polysaccharides or polysaccharides conjugates disclosed. Binding
agents may be bound to a substrate (for example, beads, tubes,
slides, plates, nitrocellulose sheets, and the like) or conjugated
with a detectable moiety, or both bound and conjugated. The
detectable moieties can include, but are not limited to, an
immunofluorescent moiety (for example, fluorescein, rhodamine), a
radioactive moiety (examples include .sup.3H, .sup.32P, .sup.35S,
and .sup.125I), an enzyme moiety (examples include horseradish
peroxidase and alkaline phosphatase), a colloidal gold moiety, and
a biotin moiety. Such conjugation techniques are standard in the
art
[0063] A "booster" refers to an increased immune response to an
immunogenic composition upon subsequent exposure of the mammalian
host to the same immunogenic composition. Booster injections can be
given at regular intervals, and antiserum harvested when the
antibody titer thereof, as determined semi-quantitatively, for
example, by double immunodiffusion in agar against known
concentrations of the antigen, begins to fall.
[0064] A "conjugate" is inclusive of any construct that includes an
immunogenic compound coupled to a pharmaceutically acceptable
carrier. Thus, "conjugate" is not limited to a conjugate of an
immunogenic compound covalently bound to a protein carrier (which
specific type of conjugate is often referred to in the art as a
"conjugate vaccine").
[0065] "Cross-reactive" refers to the ability of an antibody to
react with similar antigenic sites on different proteins.
Cross-reactivity also comprises the ability of an antibody to react
with or bind an antigen that did not stimulate its production,
i.e., the reaction between an antigen and an antibody that was
generated against a different but similar antigen.
[0066] A "derivative" is a biologically active molecule derived
from a base molecular structure.
[0067] Immune response: A response of a cell of the immune system,
such as a B cell, T cell, or monocyte, to a stimulus. In one
example, the response is specific for a particular antigen (an
"antigen-specific response"). In another example, an immune
response is a T cell response, such as a CD4.sup.+ response or a
CD8.sup.+ response. In yet another example, the response is a B
cell response, and results in the production of specific
antibodies.
[0068] The terms "immunoreact" and "immunoreactivity" refer to
specific binding between an antigen or antigenic
determinant-containing molecule and a molecule having an antibody
combining site, such as a whole antibody molecule or a portion
thereof.
[0069] An "immunogenic composition" or "immunogenic agent" is any
composition or agent that elicits an immune response in a mammalian
host when the immunogenic composition or agent is injected or
otherwise introduced. The immune response may be humoral, cellular,
or both.
[0070] Inhibiting or Treating a Disease: Inhibiting refers to
arresting the development of a disease or condition, for example,
in a subject who is at risk for a disease such as anthrax.
"Treating" or "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.
[0071] An "isolated" biological component is a component that has
been substantially separated or purified away from other biological
components in the cell of the organism in which the component
naturally occurs, i.e., other chromosomal and extra-chromosomal DNA
and RNA, proteins, lipids, and organelles. "Isolated" does not
require absolute purity. For example, the desired isolated
biological component may represent at least 50%, particularly at
least about 75%, more particularly at least about 90%, and most
particularly at least about 98%, of the total content of the
preparation. Isolated biological components as described herein can
be isolated by many methods such as salt fractionation, phenol
extraction, precipitation with organic solvents (for example,
hexadecyltrimethylammonium bromide or ethanol), affinity
chromatography, ion-exchange chromatography, hydrophobic
chromatography, high performance liquid chromatography, gel
filtration, iso-electric focusing, physical separation (e.g.,
centrifugation or stirring), and the like.
[0072] "Label" is a detectable compound or composition that is
conjugated directly or indirectly to another molecule to facilitate
detection of that molecule. Specific, non-limiting examples of
labels include fluorescent tags, enzymatic linkages, and
radioactive isotopes.
[0073] "Lymphocytes" are a type of white blood cell that is
involved in the immune defenses of the body. There are two main
types of lymphocytes: B cells and T cells.
[0074] Pharmaceutically acceptable carriers: A "carrier" is a
physiologically acceptable substance with which the therapeutically
or biologically active compound disclosed herein is associated. The
carrier may facilitate a certain type of administration of the
therapeutically or biologically active compound and/or enhance the
immune response induced by the therapeutically or biologically
active compound. 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 (e.g., 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.
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 the BBGL-II herein disclosed.
[0075] "Pharmaceutically acceptable additive" is inclusive of any
ingredient added or included in a pharmaceutical composition,
including a pharmaceutically acceptable carrier, an adjuvant, or a
therapeutically active agent.
[0076] "Polymeric carriers" are chosen to increase the
immunogenicity of a polysaccharide and/or polysaccharide conjugate
and/or to raise antibodies against the carrier which are medically
beneficial. Carriers that fulfill these criteria have been
described in the art. A polymeric carrier can be a natural or a
synthetic material containing one or more functional groups, for
example primary and/or secondary amino groups, azido groups, or
carboxyl groups. The carrier can be water soluble or insoluble.
[0077] Water soluble peptide carriers are preferred, and include
but are not limited to natural or synthetic polypeptides or
proteins, such as bovine serum albumin, and bacterial or viral
proteins or non-toxic mutants or polypeptide fragments thereof.
Examples include tetanus toxin or toxoid, diphtheria toxin or
toxoid, P. aeruginosa exotoxin or toxoid, recombinant P. aeruginosa
exoprotein A (rEPA), B. anthracis protective antigen, recombinant
B. anthracis protective antigen, pertussis toxin or toxoid,
Clostridium perfringens exotoxin or toxoid, C. welchii exotoxin or
toxoid, mutant non-toxic Shiga holotoxin, Shiga toxins 1 and 2, and
the B subunit of Shiga toxins 1 and 2.
[0078] Examples of water insoluble carriers include, but are not
limited to, aminoalkyl SEPHRAROSE, e.g., aminopropyl or aminohexyl
SEPHAROSE (PHARMACIA Inc., Piscataway, N.J.), aminopropyl glass,
and the like. Other carriers may be used when an amino or carboxyl
group is added, for example through covalent linkage with a linker
molecule. Additional polysaccharide carriers include, but are not
limited to, dextran, capsular polysaccharides from microorganisms
such as the Vi capsular polysaccharide from S. typhi (see, for
example, U.S. Pat. No. 5,204,098, the contents of which are
incorporated in their entirety by reference); Pneumococcus group 12
(12F and 12A) polysaccharides; Haemophilus influenzae type d
polysaccharide; and certain plant, fruit, and synthetic oligo- or
polysaccharides which are immunologically similar to capsular
polysaccharides, such as pectin, D-galacturonan,
oligogalacturonate, or polygalacturonate (for example, as described
in U.S. Pat. No. 5,738,855, the contents of which are incorporated
in their entirety by reference).
[0079] Methods for Attaching Polymeric Carriers: Attaching or
linking polysaccharides to protein carriers confers enhanced
immunogenicity and T-cell dependence. Methods for attaching a
polysaccharide to a protein are well known in the art. For example,
a polysaccharide containing at least one carboxyl group, through
carbodiimide condensation, may be thiolated with cystamine, or
aminated with adipic dihydrazide, diaminoesters, ethylenediamine
and the like. Groups which can be introduced by such known methods
include thiols, hydrazides, amines and carboxylic acids. Thiolated
and aminated intermediates are stable, and may be freeze dried and
stored cold. Thiolated intermediates may be covalently linked to a
polymeric carrier containing a sulfhydryl group, such as a
2-pyridyldithio group. Aminated intermediates may be covalently
linked to a polymeric carrier containing a carboxyl group through
carbodiimide condensation.
[0080] The polysaccharide can be covalently bound to a carrier with
or without a linking molecule. To conjugate without a linker, for
example, a carboxyl-group-containing polysaccharide and an
amino-group-containing carrier are mixed in the presence of a
carboxyl activating agent, such as a carbodiimide, in a choice of
solvent appropriate for both the polysaccharide and the carrier.
The polysaccharide is often conjugated to a carrier using a linking
molecule. A linker or cross-linking agent is preferably a small
linear molecule having a molecular weight of about 500 or less, and
is non-pyrogenic and non-toxic in the final product form.
[0081] To conjugate with a linker or cross-linking agent, either or
both of the polysaccharide and the carrier may be covalently bound
to a linker first. The linkers or cross-linking agents are
homo-bifunctional or hetero-bifunctional molecules, e.g., adipic
dihydrazide, ethylenediamine, cystamine, N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-N-(2-iodoacetyl)-.beta.-alaninate-propionate (SIAP),
succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate
(SMCC), 3,3'-dithiodipropionic acid, and the like. Also among the
class of hetero-bifunctional linkers area w-hydroxy and
.omega.-amino alkanoic acids. Other linkers include amino acids,
including amino acids capable of forming disulfide bonds, but can
also include other molecules such as, for example, polysaccharides
or fragments thereof. Linkers can be chosen so as to elicit their
own immunogenic effect which may be either the same, or different,
than that elicited by the polysaccharides and/or carriers disclosed
herein. For example, such linkers can be bacterial antigens which
elicit the production of antibodies to an infectious bacteria. In
such instances, for example, the linker can be a protein or protein
fragment of an infectious bacterium.
[0082] More specifically, attachment of a polysaccharide to a
protein carrier can be accomplished by methods known to the art.
Attachment may be accomplished by derivatized the protein carrier
with adipic acid dihydrazide (ADH) via carbodiimide activation. The
resulting product is reacted with the polysaccharide using
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC). Other examples
include derivatization with succinimidyl 3-(bromoacetamido)
propionate (SBAP), succinimidylformylbenzoate (SFB), and
succinimidyllevulinate (SLV).
[0083] Regardless of the precise method used to prepare the
conjugate, after the coupling reactions have been carried out the
unbound materials are removed by routine physicochemical methods,
such as for example gel filtration or ion exchange column
chromatography, depending on the materials to be separated. For
example, a polysaccharide-protein conjugated product is dialyzed
after the coupling reaction and purify by gel filtration using a
SEPHAROSE CL-6B column. The final conjugate consists of a
polysaccharide and a carrier bound directly or through a linker.
Another method for purification involves ultra filtration in the
presence of ammonium sulfate, as described in U.S. Pat. No.
6,146,902, the contents of which are incorporated in their entirety
by reference. Alternatively a polysaccharide-carrier conjugate can
be purified away from unreacted starting materials by any number of
standard techniques including, for example, size exclusion
chromatography, density gradient centrifugation, hydrophobic
interaction chromatography, or ammonium sulfate fractionation. The
compositions and purity of the conjugates can be determined by
GLC-MS and MALDI-TOF spectrometry.
[0084] Complex structural characteristics determine optimal
immunogenicity for the conjugates. The lengths and densities of the
conjugate must be sufficient to occupy a cognate antibody combining
site and determine the ability of the conjugate to form both
aggregates with the surface receptor, and to permit interaction of
the carrier fragments with T-cells. Conjugates typically have
saccharide:carrier densities of about 5:1 to about 32:1; about 8:1
to about 22:1; and about 10:1 to about 15:1.
[0085] "Pharmaceutically acceptable complexes" complexes or
coordination compounds formed from metal ions. Such complexes can
include a ligand or chelating agent for bonding with an estrogenic
agent.
[0086] "Pharmaceutically acceptable salts" of the presently
disclosed compounds include those formed from cations such as
sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and
from bases such as ammonia, ethylenediamine, N-methyl-glutamine,
lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine,
chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and
tetramethylammonium hydroxide. These salts may be prepared by
standard procedures, for example by reacting the free acid with a
suitable organic or inorganic base. Any chemical compound recited
in this specification may alternatively be administered as a
pharmaceutically acceptable salt thereof. "Pharmaceutically
acceptable salts" are also inclusive of the free acid, base, and
zwitterionic forms. Descriptions of suitable pharmaceutically
acceptable salts can be found in Handbook of Pharmaceutical Salts,
Properties, Selection and Use, Wiley VCH (2002).
[0087] "Pharmaceutical compositions" include therapeutic and
prophylactic formulations of a polysaccharide conjugate and/or a
polysaccharide-based immunogen typically combined together with one
or more pharmaceutically acceptable vehicles and, optionally, other
therapeutic ingredients (for example, antibiotics, or
anti-inflammatories).
[0088] To formulate a pharmaceutical composition, a polysaccharide
and/or polysaccharide conjugate can be combined with various
pharmaceutically acceptable additives, as well as a base or vehicle
for dispersion of the polysaccharide and/or the polysaccharide
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. One or more adjuvants may be
included.
[0089] 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.
[0090] 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.
[0091] A polysaccharide and/or polysaccharide conjugate can be
dispersed in a base or vehicle, which can include a hydrophilic
compound having a capacity to disperse the polysaccharide and/or
polysaccharide 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.
[0092] The polysaccharide and/or polysaccharide conjugate can be
combined with the base or vehicle according to a variety of
methods, and release of the polysaccharide and/or polysaccharide
conjugate can occur by diffusion, disintegration of the vehicle, or
associated formation of water channels. In some circumstances, the
polysaccharide and/or polysaccharide conjugate is dispersed in
microcapsules (microspheres) or nanocapsules (nanospheres) prepared
from a suitable polymer, for example, isobutyl 2-cyanoacrylate, and
dispersed in a biocompatible dispersing medium, which yields
sustained delivery and biological activity over a protracted
time.
[0093] Pharmaceutical compositions for administering a
polysaccharide and/or polysaccharide 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 polysaccharides and/or
polysaccharide conjugate can be brought about by including in the
composition an agent which delays absorption, for example,
monostearate salts and gelatin.
[0094] In certain aspects, the polysaccharide and/or polysaccharide
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 a controlled release formulation is desired,
controlled release binders suitable for use include any
biocompatible controlled release material which is inert to the
active agent and which is capable of incorporating the
polysaccharides and/or polysaccharide conjugate. Numerous such
materials are known. 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.
[0095] 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 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-cyanoacrylate),
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,
lactic acid-glycolic acid copolymers useful in making microcapsules
and other formulations and sustained-release compositions for
water-soluble peptides.
[0096] 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
polysaccharides and/or polysaccharide 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
a polysaccharide and/or polysaccharide conjugate 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 polysaccharide and/or
polysaccharide 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.
[0097] The term "purified" does not require absolute purity;
rather, it is intended as a relative term. Thus, for example, a
purified peptide preparation is one in which the peptide or protein
is more enriched than the peptide or protein is in its natural
environment within a cell. For example, a compound preparation is
purified such that the desired polysaccharide protein conjugate
represents at least 50%, more particularly at least about 90%, and
most particularly at least about 98%, of the total content of the
preparation.
[0098] "Subject" includes humans as well as non-human primates and
other animals that are susceptible to an infection by B.
anthracis.
[0099] "T Cell" is a white blood cell critical to the immune
response. T cells include, but are not limited to, CD4.sup.+ T
cells and CD8.sup.+ T cells. A CD4.sup.+ T lymphocyte is an immune
cell that carries a marker on its surface known as "cluster of
differentiation 4" (CD4). These cells, also known as helper T
cells, help orchestrate the immune response, including antibody
responses as well as killer T cell responses. CD8.sup.+ T cells
carry the "cluster of differentiation 8" (CD8) marker. A CD8 T
cells may be a cytotoxic T lymphocyte or a suppressor T cell.
[0100] "Therapeutically active agent" is a compound or composition
that causes induction of an immune response, as measured by
clinical response. Examples include increase in a population of
immune cells, production of antibody that specifically binds, or
measurable resistance to infection with B. anthracis.
Therapeutically active agents can also include organic or other
chemical compounds that mimic the effects of BBGL-II.
[0101] The term "unit dose" as it pertains to the inocula refers to
physically discrete units suitable as unitary dosages for mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired immunogenic effect in association
with the required diluent. Inocula are typically prepared as
solutions in physiologically tolerable (acceptable) diluents such
as water, saline, phosphate-buffered saline, or the like, to form
an aqueous pharmaceutical composition. Adjuvants, such as aluminum
hydroxide, may also be included in the compositions. The route of
inoculation may be intramuscular, subcutaneous or the like, which
results in eliciting antibodies protective against B. anthracis. In
order to increase the antibody level, a second, or booster, dose
may be administered approximately 4 to 6 weeks after the initial
injection. Subsequent doses may be administered as indicated
herein, or as desired by the practitioner.
[0102] "Vaccine" is an antigen that elicits an immune response that
results in a decrease in anthrax burden of a least about 30% in a
subject in relation to a non-vaccinated (e.g., adjuvant alone)
control subject. Preferably, the level of the decrease is about
50%, and most preferably, about 60 to about 70% or greater.
[0103] "Vaccination" refers to eliciting an immune response in a
subject by administration of a vaccine. The quantity to be
administered depends upon factors such as the age, weight and
physical condition of the subject considered for vaccination. The
quantity also depends upon the capacity of the subject's immune
system to synthesize antibodies, and the degree of protection
desired. Effective dosages can be readily established by one of
ordinary skill in the art through routine trials establishing dose
response curves.
[0104] Dosage for Vaccination: An inoculum contains an effective,
immunogenic amount of a polysaccharide and/or
polysaccharide-carrier conjugate. The effective amount of a
polysaccharide and/or polysaccharide-carrier conjugate per unit
dose sufficient to induce an immune response to B. anthracis spores
depends, among other things, on the species of mammal inoculated,
the body weight of the mammal, and the chosen inoculation regimen,
as is well known in the art. Inocula typically contain a
polysaccharide and/or polysaccharide-carrier conjugates with
concentrations of polysaccharide from about 1 micrograms to about
10 milligrams per inoculation (dose), about 3 micrograms to about
100 micrograms per dose, and most about 5 micrograms to 50
micrograms per dose.
[0105] The descriptions are provided solely to aid the reader, and
should not be construed to have a scope less than that understood
by a person of ordinary skill in the art or as limiting the scope
of the appended claims. Unless otherwise explained, all technical
and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
disclosure belongs. 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. It is further to be understood
that all base sizes or amino acid sizes, and all molecular weight
or molecular mass values, given for 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 this disclosure, suitable methods and
materials are described below. The term "comprises" means
"includes". In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. All chemical
compounds disclosed herein include both the (+) and (-)
stereoisomers, either the (+) or (-) stereoisomer, and/or any
tautomers thereof, unless the context indicates otherwise or the
description states otherwise.
[0106] One significant unknown during a deliberate or accidental
release of B. anthracis is which part of B. anthracis has entered
the host. While B. anthracis spores are the infectious agent,
ingestion of the anthrax toxin also causes anthrax. With respect to
the B. anthracis spores, they can be mutated to form virulent
(e.g., the Ames strain) or non-virulent varieties (e.g., the Sterne
strain). Ideally therefore, the development of anthrax vaccines
would occur over a broad front, i.e., use different spore proteins
to generate a wide variety of vaccines. Vaccines that recognize
different immuno-dominant spore proteins are most useful because if
one does not work, another can be administered. The BclA
glycoprotein is merely one of many possible immuno-dominant spore
proteins that can be used to develop anthrax vaccines.
[0107] The BclA tetrasaccharide has the structure
2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-.beta.-D-glucose--
(1.fwdarw.3)-.alpha.-L-rhamnopyranosyl-(1.fwdarw.3)-.alpha.-L-rhamnopyrano-
syl-(1.fwdarw.2)-L-rhamnopyranose, as shown next:
##STR00016##
which can also be represented by
##STR00017##
[0108] The anthrose portion is thought to be the immunodominant
part of the BclA tetrasaccharide. Mehta et al., Chem. Eur. J. 12,
9136-9149 (2006), synthesized the BclA tetrasaccharide, along with
a series of analogs, and determined that the 3-methyl-butyryl chain
of the anthrose sugar is an important antigenic component of the
tetrasaccharide. In other words, the 3-methyl group appears to be
the most important while the 3-hydroxyl group appears not to be
important. However, Wang et al., Proteomics 7, 180-184 (2007),
determined that the anthrose monosaccharide is marginally reactive
while the anthrose-containing tetrasaccharide is highly
reactive.
[0109] From the current scientific data, it appears that: (1) the
BclA tetrasaccharide is unique to B. anthracis; (2) the
3-methyl-butyryl chain of the anthrose sugar is the important
antigenic component of the tetrasaccharide; and (3) the remaining
trisaccharide portion of BclA is also important. In other words,
according to the current scientific data, any anthrax vaccines
developed using BclA as the antigen require both anthrose and the
three rhamnose saccharides. Furthermore, because the BclA
tetrasaccharide is unique to B. anthracis, development of these
vaccines must use anthrax spores or portions thereof.
[0110] Separation and purification of the BclA tetrasaccharide from
the thousands of proteins in the B. anthracis spore is an extensive
process and the overall yield is low. Moreover, working with
anthrax spores is difficult and can be dangerous. Alternatively,
the BclA tetrasaccharide can be synthesized. However, synthetic
preparation of the BclA tetrasaccharide is expensive and the yields
are also low. Therefore, compounds similar to the BclA
tetrasaccharide is useful. Also useful is a source of compounds
similar to the BclA tetrasaccharide, a source that is easy to grow
and from which compounds similar to the BclA tetrasaccharide can be
harvested.
[0111] Shewanella is a Gram-negative bacterium that lives in
aquatic and sub-surface environments. Shewanella spp. strain MR-4
is a metabolically versatile bacterium that can use a diversity of
organic compounds and metals to obtain energy needed for growth and
survival. Shewanella is related to Escherichia; therefore, tools
and techniques developed for Escherichia work with Shewanella.
Shewanella also has the ability to tolerate oxygen, which is a
useful ability that makes it easier to work with in the laboratory.
Vinogradov et al., Carbohydrate Res. 340, 1750-1753 (2005),
analyzed the capsular polysaccharides of Shewanella spp. MR-4. They
determined that the capsular polysaccharides had a regular
structure with a repeating unit of five monosaccharides:
4-amino-4,6-dideoxy-.alpha.-glucopyranose;
.alpha.-glucopyranosyluronic acid; .beta.-mannopyranose;
N-acetyl-.beta.-glucosamine; and .beta.-glucopyranose. The
structure of the repeating unit is shown next. The number of the
repeating units is variable and can depend upon the growing
conditions. In general, based on the gel filtration profile, the
molecular size of the capsular polysaccharide is between about 20
to about 500 kDa, and thus it contains about 20 to about 500
repeating units.
[0112] The repeating unit also includes either
3-hydroxy-3-methylbutyrate (HMB) or 3-hydroxy-butyrate (HB). HMB
and HB are commonly found in most organisms, including bacteria.
For example, HMB is a metabolite of leucine and a precursor of
cholesterol. While there are a number of known bacterial
polysaccharides containing HB, bacterial polysaccharides containing
HMB are much rarer. In the repeating capsular polysaccharide of
Shewanella spp. MR-4, HMB or HB is found linked to the
4-amino-group of the terminal .alpha.-glucopyranose saccharide.
Whether the capsular polysaccharide contained HMB or HB depends on
the medium on which Shewanella spp. MR-4 was grown.
[0113] Structurally, the capsular polysaccharides of Shewanella
spp. MR-4 are substantially different from the BclA tetrasaccharide
of B. anthracis. While it is true that both contain HMB (or the
related HB), the sugar moieties are very different. This is not
surprising because Shewanella and B. anthracis are significantly
different organisms. For example, one significant difference is
that B. anthracis is infectious while Shewanella is benign. It is
likely therefore that the function of the surface polysaccharides
in B. anthracis is different from those in Shewanella.
##STR00018##
HMB-Substituted Capsular Polysaccharide from Shewanella Spp.
MR-4
##STR00019##
[0114] HB-Substituted Capsular Polysaccharide from Shewanella Spp.
MR-4
##STR00020##
[0115] BclA Tetrasaccharide from B. anthracis
[0116] The molecular size of the HMB-substituted capsular
polysaccharide or the HB-substituted capsular polysaccharide can
range from 5 to 5000, preferably 20 to 500 kDa, and the number of
repeating units can range from 5 to 5000, preferably 20 to 500.
[0117] The immunogenicity of saccharides, alone or as protein
conjugates, is related to several variables: 1) species and the age
of the recipient; 2) molecular weight of the saccharide; 3) density
of the saccharide on the protein; 4) configuration of the conjugate
(single vs. multiple point attachment); and 5) the immunologic
properties of the protein.
[0118] In other words, one of ordinary skill in the art would not
expect that HMB-substituted capsular polysaccharide from Shewanella
would be a suitable antigen for the development of vaccines against
B. anthracis. As noted previously, both the entire anthrose
monosaccharide (i.e., not just the HMB group, which is a common
compound and found in other bacterial polysaccharides) and the
three rhamnose saccharides are important for a fully-reactive B.
anthracis antigen. Therefore, one of ordinary skill in the art
would not expect that antibodies raised against the HMB-substituted
capsular polysaccharide from Shewanella would be reactive towards
B. anthracis spores. One of ordinary skill in the art would also
not expect that antibodies raised against the HB-substituted
capsular polysaccharide from Shewanella would be reactive towards
B. anthracis spores. The HB-substituted capsular polysaccharide
from Shewanella lacks both the 3-methyl group and the three
rhamnose saccharides--both of which have been shown to be
important.
[0119] Furthermore, because the 3-methyl group is known to be
important, one of ordinary skill in the art would also not expect
that other HB-capsular polysaccharides would be suitable antigens
for anthrax vaccines. Indeed, thousands of HB-linked
polysaccharides are known and none of them have been shown to be
effective antigens for anthrax vaccines.
[0120] Pseudomonas syringae is a Gram-negative, flagellated,
bacterium. It is a plant pathogen that can infect a wide variety of
species. The bacterial flagella are primarily composed of
flagellin. The glycosylation pattern of the flagellin is
species-dependent. The glycosylated flagellin of P. syringae pv.
tabacci comprises HB and two rhamnose monosaccharides
(2-O-methyl-4-(3-hydroxy-butanamido)-4,6-dideoxy-.beta.-D-glucose-(1.fwda-
rw.3)-.alpha.-L-rhamnopyranosyl-(1.fwdarw.2)-L-rhamnopyranosyl-1-O-serine)
as shown next:
##STR00021##
P. syringae Flagellin Glycan
[0121] The P. syringae flagellin glycan differs from the B.
anthracis BclA tetrasaccharide in several ways. One, the B.
anthracis BclA tetrasaccharide contains HMB while the P. syringae
flagellin glycan contains HB. Two, the B. anthracis BclA
tetrasaccharide contains three rhamnose monosaccharides while the
P. syringae flagellin glycan contains two rhamnose monosaccharides.
Three, the linkage between the first and second rhamnose
monosaccharides in the B. anthracis BclA tetrasaccharide is
1.fwdarw.3, while in the P. syringae flagellin glycan it is
1.fwdarw.2. Four, the P. syringae flagellin glycan contains an
O-linked serine, while the B. anthracis BclA tetrasaccharide is
thought to link to the protein through a GalNAc moiety. Therefore,
one of ordinary skill in the art would not expect that antibodies
raised against the P. syringae flagellin glycan would be reactive
towards B. anthracis spores.
[0122] The saccharides described herein may be isolated from
Shewanella MR-4 or P. syringae or they may be synthesized.
[0123] In certain embodiments, the saccharides described above can
be coupled to a carrier to form an immunogenic conjugate. For
example, the HMB-substituted capsular polysaccharide from
Shewanella MR-4, the HB-substituted capsular polysaccharide from
Shewanella MR-4, and/or the P. syringae flagellin glycan can be
covalently linked to a polymeric carrier to form an immunogenic
conjugate. The carrier can be covalently linked to the saccharide
structure via a carboxyl group or an amino group present on at
least one of the monosaccharide units via conjugation methods as
described above.
[0124] In certain embodiments, the saccharide(s) described above
can be administered to a subject without conjugation to a carrier.
For example, the pharmaceutical composition may include at least
one of the saccharides and at least one pharmaceutically acceptable
additive. In another example, the saccharide(s) can be administered
without any other additive.
[0125] In another embodiment, killed whole cells of Shewanella MR-4
or P. syringae that includes the saccharides may be administered to
the subject, particularly to non-human animals. Generally, the
first step in making such a killed whole cell vaccine is to isolate
or create an organism, or part of one, that is unable to cause the
disease, but that still retains the antigens responsible for
inducing the host's immune response. This can be done in many ways.
One way is to kill the organism using heat or formalin; vaccines
produced in this way are also called "inactivated" or "killed"
vaccines. The killed whole cells Shewanella MR-4 or P. syringae are
particularly useful for administration to non-human animal subjects
such as cows that contact anthrax.
[0126] According to another embodiment, only a B. anthracis
antigenic component of Shewanella MR-4 or P. syringae flagellin,
for example the isolated capsule of Shewanella MR-4, or the
isolated fimbriae of P. syringae, is administered to the subject
(these types of vaccines are also known as "acellular vaccines").
Acellular vaccines exhibit some similarities to killed vaccines:
neither killed nor acellular vaccines generally induce the
strongest immune responses and may therefore require a "booster"
every few years to insure their continued effectiveness.
[0127] The mechanics of immune response are unpredictable.
Compounds that appear similar physically or chemically elicit
widely varied immune responses. Furthermore, antibodies raised
against one antigenic compound often do not recognize physically-
or chemically-similar antigenic compounds. It is therefore
unexpected that antibodies raised against HMB-substituted capsular
polysaccharide from Shewanella, HB-substituted capsular
polysaccharide from Shewanella, and the P. syringae flagellin
glycan would recognize and react with spores of B. anthracis. It is
also unexpected that antibodies raised against B. anthracis spores
would recognize and react with the HMB-substituted capsular
polysaccharide from Shewanella, the HB-substituted capsular
polysaccharide from Shewanella, or the P. syringae flagellin
glycan.
[0128] An HMB- or HB-saccharide-based vaccine is intended for
active immunization, for prevention of anthrax infection, and for
preparation of immune antibodies as a therapy, preferably for
established infections. In one aspect, the vaccines are designed to
confer specific preventative immunity against infection with
anthrax, and to induce antibodies specific to B. anthracis spores.
The anthrax vaccine may be composed of non-toxic components
therefore making it suitable for infants, children of all ages, and
adults.
[0129] The conjugates and/or compositions thereof, as well as the
antibodies thereto, will be useful in increasing resistance to,
preventing, ameliorating, and/or treating anthrax infection in
humans, and in reducing or preventing anthrax in humans.
[0130] Also provided is a method for screening compounds against B.
anthracis spores, the method comprising providing a compound
selected from:
##STR00022##
adding an antibody that is immuno-reactive with a B. anthracis
spore; and determining a level of reactivity of the compound with
the antibody.
[0131] Compositions provided include, but are not limited to,
mammalian serum, plasma, and immunoglobulin fractions, which
contain antibodies which are immuno-reactive with anthrax spores,
and which preferably also contain antibodies which are
immuno-reactive with the BclA tetrasaccharide, the BclA
glycoprotein, anthrose, anthrose-linked polysaccharides, HMB-linked
saccharides, and HB-linked saccharides. These compositions, in the
presence of complement, have bacteriostatic or bactericidal
activity against B. anthracis. These antibodies and antibody
compositions are useful to prevent, treat, or ameliorate infection
and disease caused by B. anthracis. Antibodies in isolated form are
also included.
[0132] High titer anti-anthrose sera, or antibodies isolated
therefrom, could be used for therapeutic treatment for patients
with B. anthracis infection. Antibodies elicited by the conjugates
described may be used for the treatment of established anthrax
infections, and are also useful in providing passive protection to
an individual exposed to B. anthracis spores. For example, the sera
could be administered to a subject to induce passive immunization
against B. anthracis.
[0133] Also included are diagnostic tests and/or kits for anthrax
infection and/or colonization, using the conjugates and/or
antibodies, or compositions thereof.
[0134] Routine immunization schedule of infants and children, and
in individuals at risk for anthrax infection is included. Use for
intervention in epidemics and terrorist attacks caused by B.
anthracis is also included. Conjugates may be used as components of
multivalent vaccines for B. anthracis and other pathogens that
contain HMB- or HB-linked saccharides. Conjugates may be used in
diagnostic test for anthrax infection and/or colonization.
[0135] In accordance with various treatment methods, a
polysaccharide and/or polysaccharide 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. 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, anthrax) 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 polysaccharide
and/or polysaccharide conjugate 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.
[0136] For prophylactic and therapeutic purposes, the
polysaccharide and/or polysaccharide 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 polysaccharide and/or polysaccharide
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.
Alternatively, effective dosages can be determined using ice 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 polysaccharide
and/or polysaccharide conjugate (for example, amounts that are
effective to elicit a desired immune response or alleviate one or
more symptoms of a targeted disease). Alternatively, an effective
amount or effective dose of the polysaccharide and/or
polysaccharide 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.
[0137] The actual dosage of the polysaccharide and/or
polysaccharide 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
polysaccharide and/or polysaccharide 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
polysaccharide and/or polysaccharide conjugate is outweighed in
clinical terms by therapeutically beneficial effects. A
non-limiting range for a therapeutically effective amount of a
polysaccharide and/or polysaccharide conjugate 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. Antibodies will typically be administered in a dosage
ranging from about 1 mg/kg body weight to about 10 mg/kg body
weight of the subject, although a lower or higher dose can be
administered.
[0138] Upon administration of a polysaccharide and/or
polysaccharide conjugate, 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 the polysaccharide and/or polysaccharide
conjugate. Such a response signifies that an immunologically
effective dose of the polysaccharide and/or polysaccharide
conjugate or related immunogenic composition 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 polysaccharides and/or polysaccharide
conjugates. In some aspects, 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
immuno-binding assay which measures the concentration of antibodies
in the serum which bind to a specific antigen, for example,
polysaccharide and/or polysaccharide conjugate. The ability to
neutralize in vitro and in vivo biological effects of the B.
anthracis can also be assessed to determine the effectiveness of
the treatment.
[0139] 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.
[0140] The methods of using polysaccharides and/or polysaccharide
conjugates, and the related compositions and methods, are useful in
increasing resistance to, preventing, ameliorating, and/or treating
infection and disease caused by bacilli in animal hosts, and other,
in vitro applications. The methods and compositions are useful in
increasing resistance to, preventing, ameliorating, and/or treating
infection and disease caused by B. anthracis infection in animals
and humans. These immunogenic compositions can be used for active
immunization for prevention of B. anthracis infection, and for
preparation of immune antibodies. Immunogenic compositions and
methods are typically designed to confer specific immunity against
infection with B. anthracis, and to induce antibodies specific to
B. anthracis polysaccharides and/or polysaccharide conjugates. The
immunogenic compositions are composed of non-toxic components,
suitable for infants, children of all ages, and adults.
[0141] The methods 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. One or more symptoms
or associated effects of exposure to and/or infection with anthrax
may be prevented or treated by administration to a mammalian
subject at risk of acquiring anthrax, or presenting with one or
more anthrax symptom(s), of an effective amount of a polysaccharide
and/or polysaccharide conjugate. 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
polysaccharide and/or polysaccharide 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.
[0142] The compositions and methods are particularly useful for
treatment and prevention of infection and toxic/morbidity effects
of exposure to anthrax and/or other disease- or illness-causing
bacilli. Also included are diagnostic compositions and methods to
identify individuals at risk for exposure, infection, toxic
effects, or long term deleterious effects of exposure to pathogenic
bacteria, for example B. anthracis. The methods and compositions
are useful for identification of environmental agents, including B.
anthracis and other bacilli expressing a polysaccharide and/or
polysaccharide conjugate, including food-borne pathogenic bacilli.
Certain individuals exposed to small amounts of bacterial products,
such as those derived from B. anthracis, presenting certain genetic
or physiological backgrounds, are predisposed to development of
chronic syndromes, including fatigue syndromes,
inflammatory/autoimmune syndromes, hypoadrenal syndromes, weakness,
cognitive symptoms and memory loss, mood symptoms, neurological and
pain syndromes and endocrine symptoms. In this context, the methods
and compositions of the disclosure are employed to detect, and
alternatively to treat and/or ameliorate, such ubiquitous
environmental exposures and associated symptoms. For example,
antibodies provide for screening for polysaccharides and/or
polysaccharide conjugates in mammalian subjects or food products at
risk of contact/infection with a Bacillus that expresses a
polysaccharide and/or polysaccharide conjugate.
[0143] Also provided are compositions, including but not limited
to, mammalian serum, plasma, and immunoglobulin fractions, which
contain antibodies that are immuno-reactive with a polysaccharide
and/or polysaccharide conjugate of B. anthracis or another Bacillus
species or strain. These antibodies and antibody compositions can
be useful to prevent, treat, and/or ameliorate infection and
disease caused by the microorganism. The disclosure also provides
such antibodies in isolated form. High titer anti-polysaccharide
and/or anti-polysaccharide conjugate sera, antibodies isolated
therefrom, or monoclonal antibodies, can be used for therapeutic
treatment for patients with infection by B. anthracis or another
Bacillus species or strain. Antibodies elicited can be used for the
treatment of established B. anthracis or other Bacillus infections,
and can also be useful in providing passive protection to an
individual exposed to B. anthracis or another Bacillus.
[0144] The following examples are exemplary of the present
processes and incorporate suitable process parameters for use
herein. These parameters may be varied, however, and the following
should not be deemed limiting.
Example 1
Preparation of Shewanella Polysaccharides
[0145] This example describes the preparation of HMB- and
HB-substituted capsular polysaccharides from Shewanella spp.
MR-4.
[0146] Shewanella sp. strain MR-4 was grown in either Triptic Soy
Broth (TSB, DIFCO Laboratories) or in chemically defined medium
(CDM), prepared as described in Vinogradov et al., for 20 h at
25.degree. C. with stifling and aeration; the pH was maintained at
7.2. The capsular polysaccharides were isolated from cell surface
by vigorous shaking and purified. They were ultracentifuged at
35,000 rpm, for 5 h at 5.degree. C. to remove lipo-polysaccharide
contamination. The supernatant was lyophilized and then passed
through a SEPHAROSE CL-6B column (1.times.100 cm) in 0.2 M NaCl.
Two products were obtained depending on which medium was used for
bacterial growth. Shewanella sp. grown on TSB produced
predominantly HMB-substituted capsular polysaccharides while
Shewanella sp. grown on CDM produced predominantly HB-substituted
capsular polysaccharides. The chemical structures of the capsular
polysaccharides were confirmed by NMR experiments. The sugar
concentration was measured by the phenol/H.sub.2SO.sub.4 assay.
Example 2
Preparation of Shewanella Polysaccharide-Protein Conjugates
[0147] This example describes the preparation of protein conjugates
using HMB- and HB-capsular polysaccharides from Shewanella obtained
as described in Example 1.
[0148] To form a protein conjugate of the substituted capsular
polysaccharides, bovine serum albumin (BSA) was first derivatized
with adipic acid dihydrazide (ADH) via carbodiimide activation.
Protein concentration was measured by the method of Lowry [J. Biol.
Chem. 193, 265-275 (1951)]; hydrazide concentration by the TNBS
assay [Schneerson et al., J. Exp. Med. 152, 361-376 (1980)]. The
product (BSA-AH) contained adipic acid hydrazide groups. HMB- or
HB-substituted capsular polysaccharide (10 mg) was dissolved in 1
ml of 0.2 M NaCl and the pH was adjusted to 5.5. 10 mg of BSA-AH
was added in 0.5 ml of 0.2 M NaCl, followed by
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) to obtain a
final concentration of EDC as 0.1 M. The reaction was carried out
under automatic titrator at pH 5.5 for 4 h at room temperature. The
product was dialyzed against 0.2 m NaCl overnight at 4.degree. C.
and purified by gel filtration using SEPHAROSE CL-6B column
(1.times.100 cm) in 0.2 M NaCl. The conjugates were designated
BSA/CPS.sub.TSB (HMB-substituted polysaccharide-protein conjugate)
or BSA/CPS.sub.CSM (HB-substituted polysaccharide-protein
conjugate).
Example 3
Recognition of Polysaccharide-Protein Conjugates by Anti-Anthrax
Antibodies
[0149] This example shows that HMB- and HB-capsular polysaccharides
are recognized by antibodies against whole anthrax spores
(anti-spore) and against synthetic anthrax spore trisaccharide-KLH
conjugate (anti-sacch).
[0150] 5- to 6-week-old female NIH Swiss Webster mice were injected
subcutaneously 3 times at 2 weeks intervals with 2.5 .mu.g of
either the BSA/CPS.sub.TSB conjugate or the BSA/CPS.sub.CSM
conjugate in 0.1 ml phosphate buffered saline (PBS). Controls
received PBS. Groups of 10 mice were exsanguinated 7 days after the
third injections. Hyperimmune sera against Shewanella sp. MR-4 were
prepared with heat-killed whole bacteria.
[0151] Double immunodiffusion were performed in 1% agarose gel in
PBS. Antibody levels to the Shewanella capsular polysaccharides
were measured by ELISA using Nunc chemically modified COVALINK
plates. Plates were coated with either CPS.sub.TSB or CPS.sub.CMP
(5 .mu.g/ml) dissolved in 10 mM 1-methylimidazole buffer (pH 7.0)
and EDC added to a final concentration of 50 mM. The antigens were
applied at 100 .mu.l per well and incubated at 37.degree. C.
overnight. Plates were washed 6 times with 0.1% Brij 35-saline and
blocked with 1% HSA in PBS for 1 h at room temperature. Two-fold
dilutions of anti-spore or anti-sacch sera were made in 1% HSA-0.1%
Brij 35-saline and incubated at 37.degree. C. for 4 h. Plates were
washed, goat anti-mouse IgG conjugated to alkaline phosphatase was
added, and the plates were incubated at 37.degree. C. for 3 h.
4-Nitrophenylphosphate (1 mg/ml in 1 M Tris-HCl buffer, pH 9.8,
containing 0.3 mM MgSO.sub.4) was added, and the absorption at 405
nm (A.sub.405) was read after 30 min in an MR600 microplate reader
(Table 1).
TABLE-US-00001 TABLE 1 Serum dilution that gave A.sub.405 of IgG
titer in the ELISA assay IgG titers* Coating antigen Anti-spore
Anti-saccharide CPS.sub.TSB 1600 12800 CPS.sub.CMP 3200 500
[0152] B. anthracis Ames 35 strain (pXO1+ pXO2-) was grown on
nutrient sporulation agar at 37.degree. C. for 2 days. Spores were
purified by washing the agar plate with deionized water and
incubating the spore suspension at 65.degree. C. for 30 min. After
heat treatment, the spore suspension was washed twice with
deionized water before fluorescent staining. Immunofluorescent
staining was performed as follows. Spores were put on 1%
polylysin-treated cover slip, and blocked in 3% milk in PBS for 30
min. Spores were stained with primary mice antibodies for 30 min.
After three washes in PBS, the coverslips were treated with a
secondary antibody, AF488 conjugate anti-mouse. After staining,
coverslips were mounted to slides and they were examined by a Nikon
fluorescent microscope.
[0153] Both the anti-spore and the anti-sacch precipitated both
CPS.sub.TSB and CPS.sub.CDM in the immunodiffusion assay, as shown
in FIG. 1. In another experiment microtiter plates were coated with
CPS.sub.TSB or CPS.sub.CDM and an ELISA assay was performed. Table
2 shows that anti-sacch serum recognized CPS.sub.TSB better then
CPS.sub.CDM, but the anti-spore sera recognized CPS.sub.CDM better.
The first result was in agreement with structural data since
anti-trisaccharide serum was raised against anthrose, which was
substituted with HMB group, as in CPS.sub.TSB. This group was
almost not present in CPS.sub.CDM.
[0154] The BSA/CPS.sub.TSB conjugate induce higher level of
antibodies to CPS.sub.TSB than to CPS.sub.CDM (54 vs. 43 EU) but
the difference was not statistically significant. Similarly, there
was no statistical difference in antibody levels induced by the
BSA/CPS.sub.TSB conjugate while tested against either of
CPS.sub.TSB or CPS.sub.CDM (31 vs. 32 EU).
TABLE-US-00002 TABLE 2 Composition and levels of mouse IgG of
anti-Shewanella MR-4 CPS induced by conjugates of CPS.sub.TSB and
CPS.sub.CDM bound to BSA. ELISA Units after 3.sup.rd injection
Protein:Sugar Coating antigen Conjugate ratio (wt:wt) CPS.sub.TSB
CPS.sub.CDM BSA/CPS.sub.TSB 2:1 54 43 BSA/CPS.sub.CDM 1:1.4 31 32
Mice (10 per group) were injected with 2.5 .mu.g of saccharide as a
conjugate per mouse, s.c., 3 times, 2 weeks apart and bled one week
after last injection. Antibody levels were calculated relative to
the hyperimmune mouse serum and assigned a value of 100 ELISA Units
(EU).
Example 4
Recognition of P. syringae Bacteria by B. anthracis Spore
Antibodies
[0155] This example shows that serum antibodies raised against B.
anthracis spores recognize the P. syringae bacteria.
[0156] P. syringae bacteria were recognized by anti-B. anthracis
spore serum by fluorescent microscopy, confirming the
cross-reactivity with the spores.
Example 5
Recognition of Shewanella Sp. MR-4 by B. anthracis Spore
Antibodies
[0157] This example shows that serum antibodies raised against B.
anthracis spores recognize the Shewanella sp. MR-4 bacteria.
[0158] Shewanella sp. MR-4 bacteria were recognized by anti-B.
anthracis spore serum by fluorescent microscopy, confirming the
cross-reactivity with the spores.
Example 6
Recognition of B. anthracis Spores by Shewanella Polysaccharide
Antibodies
[0159] This example shows that serum antibodies raised against
CPS.sub.TSB and CPS.sub.CDM recognize B. anthracis spores.
[0160] Hyperimmune serum raised against Shewanella spp. MR-4 as
well as sera induced by conjugates reacted with B. anthracis spores
as tested by fluorescent microscopy (FIG. 2A to 2D). As noted
previously, in CPS.sub.CDM the hydroxybutyrate group is present in
place of hydroxymethylbutyrate, which suggests that the methyl
group is not important for the cross-reactivity. That corroborates
the antigenicity results where the anti-spore sera recognized both
CPS, non-methylated CPS.sub.CDM even with a higher titer (see Table
1). It is also possible that both methylated and non-methylated
sugars are present on the spores.
Example 7
Recognition of P. syringae Bacteria by Shewanella Polysaccharide
Antibodies
[0161] This example shows that serum antibodies raised against
CPS.sub.TSB and CPS.sub.CDM recognize P. syringae bacteria.
[0162] P. syringae pv. tabaci 6605 was grown on King's medium for
24 h at 25.degree. C. Hyperimmune serum against Shewanella sp. MR-4
reacted with P. syringae bacteria in immunodiffusion assay
confirming that the common sugar present on either the capsule or
the flagellum, respectively, is a cross-reactive moiety. FIG. 3A to
3B.
[0163] It will be apparent that the precise details of the methods
or compositions described may be varied or modified without
departing from the spirit of the disclosure. We claim all such
modifications and variations that fall within the scope and spirit
of the claims below.
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