WO2012106251A2 - Pertussis vaccine - Google Patents

Abstract

An immunogenic composition, comprising: (a) an immunogenic conjugate comprising the structure of: Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS; (b) diphtheria toxoid; and (c) tetanus toxoid.

Description

PERTUSSIS VACCINE

CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 61/438,190, filed January 31, 2011, all of which is incorporated herein by referenced in its entirety.

BACKGROUND The genus Bordetella comprises Gram-negative bacteria pathogenic for mammals and birds. The most common Bordetella species are the respiratory pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. B. pertussis is an exclusively human pathogen causing paroxysmal coughing in infants (whooping cough) and persistent respiratory infections in adults. Killed whole-cell pertussis vaccines were used to immunize infants and children worldwide for many years but are too reactogenic for adults. Acellular pertussis vaccines containing pertussis toxoid and other proteins such as filamentous hemagglutinin, pertactin and fimbriae have recently largely replaced whole -cell vaccines in most developed countries.

Despite a high rate of vaccination, a resurgence of pertussis in the U.S. and Europe has initiated calls for an improved vaccine. Yet, there is no agreement among experts or regulatory agencies about how to improve the current acellular vaccines. There is, however, agreement that pertussis toxoid is an essential component of the acellular vaccines; a postimmunization level of >100 EU (ELISA units) to pertussis toxin (PT) is likely a protective level, and low levels of anti-PT are related to susceptibility to infection with this pathogen. The confusion is mainly due to the nature of pertussis vaccines' complex mode of action. The primary action of pertussis vaccines is serum IgG anti-toxin immunity that blocks the inactivating action of PT on phagocytic cells thus allowing them to opsonize the B. pertussis, i.e. the antibodies elicited by acellular or whole cell vaccines do not directly kill the pathogen. There is also the "herd" immunity effect of pertussis vaccines that reduces the coughing thus resulting in decreased transmission of B. pertussis in the susceptible population. Similar to the effect induced by widespread immunization with diphtheria toxoid, this indirect effect of antitoxin accounts for the incomplete immunity of both vaccines on an individual basis (approximately 80%). SUMMARY Disclosed herein is an immunogenic composition, comprising:

(a) an immunogenic conjugate comprising the structure of:

Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS;

(b) diphtheria toxoid; and

(c) tetanus toxoid.

Also disclosed herein is an immunogenic composition, comprising:

(a) an immunogenic conjugate comprising the structure of: Pr - Sr - O - N = Kdo - OS (II) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide having a structure of: a-GlcN

17

a-GlcA-2-a-Hep4P

13

a-GlcNAc-4-P-ManNAc3NAcA-3-P-FucNAc4NMe-6-a-GlcN^-P-Glc-4-a-Hep-

14 16

a-Hep a-GalNA

(b) diphtheria toxoid; and (c) tetanus toxoid.

A further embodiment disclosed herein is an immunogenic conjugate comprising the structure of:

PT - Sr - O - N = Kdo - OS (III) in which PT represents a pertussis toxoid carrier protein, Sr is an optional spacer moiety, Kdo is 3 -deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS,

wherein the conjugate can induce an anti-toxin response and a bacteridal response against Bordetella pertussis. Additionally disclosed is a method for inducing a bactericidal response against

Bordetella pertussis in a subject, comprising:

administering to the subject a Bordetella pertussis bactericidal-effective amount of an immunogenic conjugate comprising the structure of: Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3- deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS.

A further embodiment disclose herein relates to a method for preparing an immunogenic conjugate, comprising:

obtaining from a Bordetella bronchiseptica mutant an oligosaccharide having a structure of: a-GlcN

17

a-GlcA-2-a-Hep4P 13

a-GlcNAc-4-P-ManNAc3NAcA-3-P-FucNAc4NMe-6-a-GlcN^-P-Glc-4-a-Hep-5-Kdo4P-2-6-Lipid A

14 16

a-Hep a-GalNA cleaving the Lipid A from the oligosaccharide such that the Kdo moiety remains linked to the oligosaccharide; and

reacting the Kdo moiety with an aminooxylated protein carrier molecule resulting in an immunogenic conjugate that can induce in a subject a bactericidal response against

Bordetella pertussis.

Also disclosed is method for making Bordetella pertussis-bactericidal antisera, comprising:

administering to a subject an immunogenic conjugate comprising the structure of:

Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or

polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS, in an amount sufficient to elicit bactericidal antibodies against Bordetella pertussis; and

collecting the bactericidal antibodies against Bordetella pertussis to make the

Bordetella pertussis -bactericidal antisera.

The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Structure of B. pertussis LPS and B. bronchiseptica RB50 core-Lipid A fraction

(Band A). Star indicates differences in methylation and phosphorylation between the strains as explained below. FIG. 2: BioGel P-10 gel filtration of B. bronchiseptica RBA2b LPS hydrolyzate showing separation of oligosaccharide with different number of Band A non-reducing end trisaccharide repeats; SDS-PAGE of B. bronchiseptica RBA2b LPS; and ESI-MS spectra of separated fractions (each oligosaccharide gives several peaks due to partial presence of methyl group on FucNAc4N (mass of 14 amu) and/or phosphate (mass of 80 amu). Fl - 4 and 5 repeats, F2 - 3 repeats, F2 - 2 repeats; F4 - 1 repeat; each repeat has a mass of 661 Da. FIG. 3: SDS-PAGE (A) and immunoblotting (B, C, D): B - transferred LPSs were reacted with anti-β. pertussis Tohama I serum, one Band A trisaccharide repeat); C - transferred LPSs were reacted with serum raised to BSA/RBA2b-Fl (4 repeats of Band A trisaccharide; similar picture was obtain with sera raised to BSA/RBA2b-F2 and BSA/RBA2b-F3 conjugates with 3 and 2 repeats of Band A trisaccharide); D - transferred LPSs were reacted with serum raised to BSA/ RBA2b-F4 (1 repeat of Band A trisaccharide). 1 - B. pertussis Tohama I LPS, 2 - B. bronchiseptica RBwbmA LPS, 3 - B. bronchiseptica RBA2b LPS; 4 - B. bronchiseptica RBB la LPS.

FIG. 4: Schemes of conjugation of B. pertussis and B. bronchiseptica core OS.

FIG. 5: Incorporation of 3-(0-thiopropyl)hydroxylamine into B. pertussis oligosaccharide (high yield of the reaction between anhydro-Kdo and aminooxy group of the linker).

FIG. 6: Double immunodiffusion of BSA/ B. bronchiseptica RBA2b OS conjugates containing different number of Band A trisaccharide reapts with anti-β. pertussis and anti- BSA sera. A line on identity between the conjugates and the two antisera is shown.

FIG. 7: A LPS structure of Bordetella parapertussis and Bordetella bronchiseptica. A novel pentasaccharide (-4- -ManNAc3ANcAN-4- -GlcNAc3NAcAN-4-a-GalNAc-4- - ManNAc3NAcA-3- -FucNAc4NMe-) present between the O-SP and the core was identified. In addition, besides the reported structure the O-SP of B. bronchiseptica and B. parapertussis being a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-a- galacturonic acid, it was found that both O-SP contain amidated uronic acids, the number of which varied between strains (Preston et al., . Biol. Chem. , 2006 (in press)). Certain fragments (-6- -GlcNAc-4- -ManNAc3NAcA-3- -FucNAc4NMe-) are not present or partially present (residues with *) in B. parapertussis. Two types of O-SP end groups (Vinogradov et al., Eur. J. Biochem. 267:7230-7237, 2000) (A) were found in B.

bronchiseptica and only one, Ala-type, in B. parapertussis. DETAILED DESCRIPTION

/. Abbreviations

AT: anthrax toxin

EF: edema factor

GLC-MS: gas-liquid chromatography-mass spectrometry

kDa: kilodaltons

LC-MS: liquid chromatography-mass spectrometry

LF: lethal factor

LOS: lipooligosaccharide

LPS: lipopolysaccharide

MALDI-TOF: matrix-assisted laser desorption ionization time-of -flight OS: oligosaccharide

: microgram

μΐ: microliter

PA: protective antigen

PBS: phosphate buffered saline

SBAP: succinimidyl 3-(bromoacetamido) propionate

TT: tetanus toxoid

The saccharide units disclosed herein are abbreviated as below following conventional oligosaccharide/polysaccharide nomenclature: anhKDO: anhydro KDO

Fuc: fucose

Gal: galactose

Glc: glucose,

GlcNAc: N-acetylglucosamine

GalNAc: N-acetylgalactosamine

Hep: glycero-D-manno-heptopyranoside (heptose)

Hex: hexose

Man: mannose

NeuNAc: N-acetylneuramic acid //. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term

"comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides or other compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune response to an antigen. Development of vaccine adjuvants for use in humans is reviewed in Singh et al. (Nat. Biotechnol. 17: 1075-1081, 1999), which discloses that, at the time of its publication, aluminum salts, such as aluminum hydroxide (Amphogel, Wyeth Laboratories, Madison, NJ), and the MF59 microemulsion are the only vaccine adjuvants approved for human use. An aluminum hydrogel (available from Brentg Biosector, Copenhagen, Denmark, is another common adjuvant).

In one embodiment, an adjuvant includes a DNA motif that stimulates immune activation, for example the innate immune response or the adaptive immune response by T- cells, B -cells, monocytes, dendritic cells, and natural killer cells. Specific, non-limiting examples of a DNA motif that stimulates immune activation include CpG

oligodeoxynucleotides, as described in U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199.

Analog, Derivative or Mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington {The Science and Practice of Pharmacology , 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (V_L) and "variable heavy chain" (V_H) refer, respectively, to these light and heavy chains.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in a subject, including compositions that are injected, absorbed or otherwise introduced into a subject. An antigen can bind or combine with the product of an immune response such as an antibody or a surface receptor on a T-cell.

Antigens can be any type of biologic molecule including, for example, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In one example, an antigen is an oligosaccharide antigen derived from B. pertussis or B. bronchiseptica.

Carrier: An immunogenic molecule to which an antigen such as an

oligosaccharide or polysaccharide can be bound. When bound to a carrier, the bound molecule may become more immunogenic. Carriers are chosen to increase the

immunogenicity of the bound molecule and/or to elicit antibodies against the carrier which are therapeutically (e.g., prophylactically) beneficial. Covalent linking of a molecule to a carrier confers enhanced immunogenicity and T-cell dependence (Schneerson, Barrera, Sutton, Robbins, J Exp Med 152:361-376, 1980; Pozsgay et al , PNAS 96:5194-97, 1999; Lee et al. , . Immunol. 116:1711-18, 1976; Dintzis et al , PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached.

Examples of bacterial products for use as carriers include bacterial toxins, such as B. anthracis PA (including fragments that contain at least one antigenic epitope and analogs or derivatives capable of eliciting an immune response), LF and EF, and other bacterial toxins and toxoids, such as tetanus toxin/toxoid, diphtheria toxin/toxoid, P. aeruginosa exotoxin toxoid/, pertussis toxin/toxoid, and C. perfringens exotoxin/toxoid. Viral proteins, such as hepatitis B surface antigen and core antigen can also be used as carriers.

Covalent Bond: An interatomic bond between two atoms, characterized by the sharing of one or more pairs of electrons by the atoms. The terms "covalently bound" or "covalently linked" refer to making two separate molecules into one contiguous molecule. The terms include reference to joining a hapten or antigen indirectly to a carrier molecule, with an intervening linker molecule.

Epitope: An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences or saccharide units on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope based on the three dimensional structure of the antibody and the matching (or cognate) epitope.

Immune Response: A response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. An immune response can include any cell of the body involved in a host defense response. An immune response includes, but is not limited to, an adaptive immune response. In some examples, an immune response is stimulated by administering to a subject a vaccine and/or an immunogenic conjugate. In one embodiment, an immune response is induction of a serum antibody response.

Immunogenic Conjugate or Composition: A term used herein to mean a composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective (e.g., provides protective immunity), in that it enables the vertebrate animal to prevent infection or disease progression from the organism against which the immunogenic composition is directed, for example B. pertussis. One specific example of a type of immunogenic composition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, under appropriate conditions, of stimulating the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. In some examples, an immunogen is an oligosaccharide obtained from B. pertussis or B.

bronchiseptica.

Immunologically Effective Amount: An immunologically effective amount of the oligosaccharide -protein or polysaccharide-protein conjugates of the disclosure will prevent, treat, lessen, or attenuate the severity, extent or duration of a disease or condition, for example, infection by B. pertussis. For example, an immunologically effect amount may be a bactericidal-effective amount that is effective in killing B. pertussis. A bactericidal effective-amount may be the amount of administered conjugate sufficient to elicit a measurable level of bactericidal antibodies in a subject. A measurable level of bactericidal antibodies typically is sufficient to confer protection to the subject against infection by B. pertussis. If there is an insufficient amount of bactericidal antibodies that can be measured by a bactericidal assay as described herein, then the subject may not have a protective antibody level against infection by B. pertussis.

Inhibiting or Treating a Disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as respiratory tract infections, for example B. pertussis infection. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Inhibiting" refers to providing a protective immune response in a subject against B. pertussis infection, which may prevent or ameliorate the infection. 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.

Isolated: An "isolated" biological component (such as a lipopolysaccharide) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, glycolipids and organelles. Isolated does not require absolute purity, and can include protein or peptide molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 100% isolated.

Lipopolysaccharide (LPS): LPS is an endotoxin that is a major suprastructure of the outer membrane of Gram-negative bacteria which contributes greatly to the structural integrity of the bacteria, and protects them from host immune defenses. LPS typically contains three components (domains): (a) Lipid A (a hydrophobic domain that typically consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages); (b) a core polysaccharide or oligosaccharide that can include, for example, heptose, glucose, galactose and N-acetylglucosamine units depending upon the genera and species of bacteria; and (c) optionally, polysaccharide distal or side chain(s) (often referred to as the "O antigen" that can include, for example, mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose depending upon the genera and species of bacteria). Lipid A and the core polysaccharide or oligosaccharide domains are joined together by one or more units of 3- deoxy-D-manno-octulosonic acid ("KDO", also known as ketodeoxyoctonate). A lipooligosaccharide (LOS) (also known as a "short chain LPS") commonly refers to an LPS that contains Lipid A plus a core polysaccharide or oligosaccharide (e.g., in B. pertussis that does not naturally contain any O antigen chains). As used herein, the term LPS can include short chain LPS and LOS.

Oligosaccharide (OS): As used herein, the term "oligosaccharide" is not necessarily restricted to a molecule having a specific number of saccharide units. However, in general, an oligosaccharide is a carbohydrate that contains from about 3 to about 20 linked simple sugars (e.g., monosaccharides) linked. O-specific oligosaccharide (O-SP) refers to an O-specific oligosaccharide chain that is attached to a core oligosaccharide or polysaccharide chain. In certain embodiments, the oligosaccharides or polysaccharides conjugated to the protein carrier do not include a lipid component.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers

(vehicles) useful in this disclosure are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more SARS-CoV nucleic acid molecules, proteins or antibodies that bind these proteins, and additional pharmaceutical agents. The term "pharmaceutically acceptable carrier" should be distinguished from "carrier" as described above in connection with a hapten/carrier conjugate or an

antigen/carrier conjugate.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.

The term "residue" or "amino acid residue" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

Protein: A molecule, particularly a polypeptide, comprised of amino acids.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, conjugate, LPS, or other active compound is one that is isolated in whole or in part from proteins, lipids or other contaminants. Generally, substantially purified peptides, proteins, conjugates, LPSs or other active compounds for use within the disclosure comprise more than 80% of all

macromolecular species present in a preparation prior to admixture or formulation of the peptide, protein, conjugate, LPS or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co- ingredient in a complete pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein, conjugate, LPS or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.

Therapeutically Effective Amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of an OS-protein or polysaccharide -protein conjugate, or an immunogenic composition containing such a conjugate, useful in increasing resistance to, preventing, ameliorating, and/or treating infection and disease caused by Bordetella pertussis in a subject. Ideally, a therapeutically effective amount of an agent is an amount sufficient to increase resistance to, prevent, ameliorate, and/or treat infection and disease caused by Bordetella pertussis without causing a substantial cytotoxic effect in the subject. The effective amount of an agent or composition useful for increasing resistance to, preventing, ameliorating, and/or treating infection and disease caused by Bordetella pertussis infection in a subject will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. Toxoid: A nontoxic derivative of a bacterial exotoxin produced, for example, by formaldehyde or other chemical treatment or by genetic detoxification such as a recombinant toxoid. Toxoids are useful in the formulation of immunogenic compositions because they retain most of the antigenic properties of the toxins from which they were derived.

Vaccine: A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example, a bacterial or viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide, a peptide or polypeptide, a polysaccharide, a virus, a bacteria, a cell or one or more cellular constituents. In some cases, the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent.

///. Overview

In certain embodiments, B. pertussis or B. bronchiseptica OS are bound to an amino- oxylated carrier protein via their terminal Kdo. The two conjugates induced similar levels of serum anti-β. pertussis LPS IgG in mice. Using several B. bronchiseptica deficient in O- specific polysaccharide expression, fractions of core OS with 1 to 4 repeats of a terminal trisaccharide having at the non-reducing end a GlcNAc or GalNAc (see FIG. 2) were isolated, and then bound to BSA (a carrier protein) at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8 to 17 OS per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis and the titers correlated with antibody levels measured by ELISA (r= 0.74). Such conjugates are easy to standardize and, combined with a pertussis toxoid, might induce anti-bacterial and anti-toxin immunity. Thus, disclosed herein are immunogenic compositions that include both a bactericide component and an anti-toxin component. The bactericide component can directly kill the B. pertussis microorganism and the anti-toxin component can inhibit toxins produced by the B. pertussis microorganism.

As explained below in more detail, the pertussis toxoid may be a component of a multi-component physical mixture composition that also includes the conjugate.

Alternatively, the pertussis toxin may be chemically bound with the OS in a conjugate wherein the pertussis toxoid is the carrier protein. Such immunogenic physical mixtures or conjugates may be particularly useful in vaccine compositions for increasing resistance to B. pertussis infection in young children (e.g., less than 4 yrs. old, more particularly less than 2 yrs. old).

IV. Conjugates

The conjugates for use in the immunogenic compositions described herein are oligosaccharides or polysaccharides having a 3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducing end of the oligosaccharides or polysaccharides. According to the methods disclosed herein, binding the OS by KDO at the reducing end of the OS means that all of the conserved OS structure remains intact or unmodified (e.g., none of the saccharide residues are oxidized) which may provide more potential sites for interaction leading to higher immunogenicity. Certain embodiments of the conjugates disclosed herein preserve the external non-reducing end of the OS, are recognized by antisera, and induce immune responses in mice. Methods for conjugating B pertussis core OS or B.

bronchiseptica core OS to an amino-oxylated carrier protein via the terminal Kdo of the core OS are disclosed in WO 2008/013735, which is incorporated herein by reference.

The oligosaccharide may be obtained from Bordetella bronchiseptica or Bordetella pertussis. These bacteria contain a lipopolysaccharide (LPS) with a 3-deoxy-D-manno- octulsonic acid moiety phosphorylated at position C4 on the 3-deoxy-D-manno-octulsonic acid moiety. B. pertussis LPS is comprised of Lipid A and a branched dodecasaccharide, composed of unusual sugars with free amino and carboxylic acid groups but with no O- specific polysaccharide (O-SP) (see FIG 1). SDS-PAGE analysis of B. pertussis LPS reveals two bands, Band A and Band B. Band B is comprised of Lipid A and a branched nonasaccharide core, while Band A consists of Band B further substituted by a trisaccharide. B. bronchiseptica LPS was reported to have an identical core structure to that of B. pertussis but it is further substituted by an O-SP, composed of a linear polymer of 1,4-linked 2,3- diacetamido-2,3-dideoxy-0C-L-galacturonic acid (GalNAc3NAcA), connected to the core by a five sugar linker (see FIG. 7).

B pertussis core OS and B. bronchiseptica core OS both have a low molecular weight (approximately 2,300 Da), and accordingly need covalent binding to a carrier (e.g., a protein) to be immunogenic. B. bronchiseptica is preferred because it is easier to grow than B. pertussis. In one embodiment, the conjugate is a conjugate of the Band A of B.

bronchiseptica RBwbmA LPS to a clinically useful carrier protein.

The target oligosaccharides or polysaccharides for conjugation typically are those that carry epitopes in their structure. Examples of such oligosaccharides or polysaccharides are described below in more detail in examples 1 and 2. The oligosaccharides or polysaccharides that are conjugated include a general structure of:

O-chain (if present) - core OS - Kdo The Kdo moiety is the moiety that results after acid hydrolysis treatment of the isolated LOS or LPS. In some examples the acid hydrolysis treatment results in the formation of anhydro-Kdo as described in more detail below and it has a structure represented by (anhydro-KDO could also be referred to as 4, 8(7)-anhydro derivative of KDO):

anhydro-Kdo

The oligosaccharide or polysaccharide is typically derived from LPS present in the bacteria identified above. The LPS initially is isolated from the other constituents of the bacteria cell structure. Illustrative LPS-isolation techniques are described, for example, in Westphal et al., Meth. Carbohydr. Chem. 5:83-89, 1965, which is incorporated herein by reference in its entirety, and typically involve isolation or purification via a phenol-water extraction. Other LPS-isolation techniques include enzyme digestion and alcohol precipitation, chromatography by gel filtration and ion-exchange.

The isolated LPS then is subjected to mild acid hydrolysis to cleave the Lipid A from the polysaccharide or oligosaccharide domain such that the 3-deoxy-D-manno- octulsonic acid remains linked to the polysaccharide or oligosaccharide domain. Such techniques are described, for example, in Auzanneau, . Chem .Soc. Perkin Trans. 1 :509- 516, 1991 and Rybka et al., . Microbiol. Methods 64(2): 171-184, 2006, both of which are incorporated herein by reference. Illustrative hydrolysis conditions include treating the LPS with acetic acid for 1-3 hours at about 100°C, or hydrolyzing LPS in a mixture of acetic acid and sodium acetate (e.g., treating 50 mg LPS with a mixture of 73.5 ml of 0.2 M acetic acid and 26.5 ml of 0.2 M sodium acetate for 5 hours at 100°C in 5 ml volume). In some examples, the acid hydrolysis transforms the Kdo structure in the isolated LPS to an anhydro-Kdo structure.

Conjugation of the oligosaccharide or polysaccharide to the carrier protein is accomplished via formation of an oxime linkage between a carbonyl functional group present in the Kdo moiety and an aminooxy functional group present on the carrier protein. The oxime linkage reaction is a chemoselective ligation since it involves the aqueous covalent coupling of unprotected, highly functionalized biomolecules that contain at least a pair of functional groups that react together exclusively, within a biological environment. Oxime linkages can be formed in an aqueous reaction environment, and are stable, from pH 5 to pH 7. Other advantageous features of forming oxime linkages include a relatively short reaction time, a good yield, and formation at ambient temperature. These conditions avoid denaturation of the carrier protein.

The reactive carbonyl functional group present in the Kdo moiety can be an aldehyde or a ketone remaining after acid hydrolysis cleavage of the Lipid A from the LPS. The carrier protein is functionalized with an aminooxy group. The synthetic scheme for forming the oxime linkage is shown below: Pr - Sp - O - NH₂ + Kdo - OS → Pr - Sp - O - N = Kdo - OS wherein Pr is a carrier protein, Sp is an optional spacer moiety, Kdo is -deoxy-D- mano octulosonic acid or a derivative thereof such as anhydro-Kdo, and OS is an oligosaccharide or polysaccharide residue from the cleavage of Lipid A from LPS.

Condensation between the carbonyl and aminooxy groups leads to a stable oxime linkage between the OS and carrier protein. The spacer moiety may have any structure that is present in the linker reagents as described below. Alternatively, the -Kdo - OS structure could be reacted initially with an aminooxy reagent, and the resulting aminooxy- functionalized reactant could be reacted with the protein. The oxime conjugation reaction is performed at pH 5 to about pH 7 at ambient temperature conditions in an aqueous environment. The reaction time typically ranges from about 8 to about 24 hours. However, less than 100% conjugation completion can be achieved in less than 8 hours, and the 8-24 hour reaction time assumes near 100% conjugation completion.

The carrier protein (or -Kdo - OS) can be functionalized to include at least one reactive aminooxy moiety by various techniques as described, for example, in Kielb et al., . Org. Chem.70:6987-6990, 2005 and U.S. Patent Application Publication No. 2005/0169941, both of which are incorporated herein by reference. Functionalization of the carrier protein can result in the inclusion of an optional spacer moiety as noted above. In illustrative examples, a carrier protein (or -Kdo - OS) may be reacted with a linker reagent to incorporate the spacer moiety and the aminooxy functional moiety. The linker reagent typically is a heterobifunctional compound that includes at least one aminooxy group and a second functional group that is reactive with the carrier protein. Suitable linker reagents include aminooxy-thiol compounds. Illustrative aminooxy-thiol linker reagents include aminoooxy-alkyl-thiols such as (thiolalkyl)hydroxylamines (e.g., 0-( - thiolpropyl)hydroxylamine) and aminooxy-aryl-thiols. In the case of aminooxy-thiol linker reagents, the carrier protein may be treated to introduce thiol-reactive groups. For example, the carrier protein may be treated with a treatment agent that introduces thiol-reactive haloacetamido or thiol-reactive maleimido moieties onto the carrier protein. The haloacetamido-containing protein or maleimido-containing protein is reacted with the aminooxy-thiol reagent to form the aminooxylated carrier protein via the formation of stable thioether linkages.

The amount of oligosaccharide or polysaccharide reacted with the amount of protein may vary depending upon the specific OS and on the carrier protein. However, the respective amounts should be sufficient to introduce about 5-20 chains of OS onto the protein. In certain examples, the mol ratio of carbonyl groups on OS to aminooxy groups on the protein may range from about 0.3:1 to about 1 :3, more particularly 1 : 1 to about 1 :2, and more preferably about 1 : 1. The resulting number of oligosaccharide chains bound to a single protein carrier molecule may vary depending upon the specific LPS and the carrier protein, but in general, about 5 to about 20, more particularly 8-17, and most particularly 10-15, OS chains can be bound to each protein carrier molecule. The yield based on the amount of protein ranges from about 70 to about 90% in protein derivatization step and about 70 to about 90% after the conjugation with the OS. Specific, non-limiting examples of water soluble protein carriers include, but are not limited to, natural, semi-synthetic or synthetic polypeptides or proteins from bacteria or viruses. In one embodiment, bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls), and soluble antigens of bacteria. In another embodiment, bacterial products for use as carriers include bacterial toxins. Bacterial toxins include bacterial products that mediate toxic effects, inflammatory responses, stress, shock, chronic sequelae, or mortality in a susceptible host. Specific, non-limiting examples of bacterial toxins include, but are not limited to: B. anthracis PA (for example, as encoded by bases 143779 to 146073 of GenBank Accession No. NC 007322, herein incorporated by reference), including variants that share at least 90%, at least 95%, or at least 98% amino acid sequence homology to PA, fragments that contain at least one antigenic epitope, and analogs or derivatives capable of eliciting an immune response; B. anthracis LF (for example, as encoded by the complement of bases 149357 to 151786 of GenBank Accession No. NC 007322); bacterial toxins and toxoids, such as tetanus toxin/toxoid (for example, as described in U.S. Patent Nos.

5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, as described in U.S. Patent Nos. 4,709,017 and 6,696,065); P. aeruginosa exotoxin/toxoid/ (for example, as described in U.S. Patent Nos. 4,428,931, 4,488,991 and 5,602,095); pertussis toxin/toxoid (for example, as described in U.S. Patent Nos. 4,997,915, 6,399,076 and 6,696,065); and C. perfringens exotoxin/toxoid (for example, as described in U.S. Patent Nos. 5,817,317 and 6,403,094). Viral proteins, such as hepatitis B surface antigen (for example, as described in U.S. Patent Nos. 5,151,023 and 6,013,264) and core antigen (for example, as described in U.S. Patent Nos. 4,547,367 and 4,547,368) can also be used as carriers, as well as proteins from higher organisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, and mammalian immunoglobulins. Particularly useful carriers include pertussis toxoid, filamentous haemagglutonin (FHA), pertactin and fimbrial agglutinogen.

In certain embodiments, the carrier protein conjugated to OS is a pertussis toxoid. B. pertussis, which is the etiological agent of pertussis, produces during the virulent stage (stage I) a series of toxic components among which the pertussis toxin (PT) represents not only the principal pathogenic agent of the disease but also the major immunogen. PT, which has the structure of a hexamer consisting of five different subunits (SI, S2, S3, S4, and S5, in the ratio of 1 :1 : 1 :2: 1) is capable in fact of inducing in humans antibody levels sufficient to impart a protection against pertussis. Illustrative pertussis toxoids (also referred to herein as pertussis toxins) may be detoxified pertussis toxins which are chemically detoxified or genetically detoxified. Such toxins are described, for example, in U.S. Patent No. 6,696,065.

In a further emdodiment, the pertussis toxoid may be a toxoid used, or proposed for use, in an acellular vaccine. Such toxoids may be toxic proteins produced and secreted by virulent B. pertussis, detoxified with a variety of chemicals reagents such as formaldehyde (Sato et al. (1983), Infect. Immun., 41, 313-320), glutaraldehyde (Quentin-Millet et al.

(1988) J. Bio. Stand., 16, 99-108), tetranitromethane (Siber et al. 1988; Windberry et al., 1988, International Workshop of Bordetella pertussis, Hamilton, Mo.),

trinitrobenzensulfonic acid (Fisch et al., 1984, Infect. Immun, 44, 1-16), or hydrogen peroxide (Sekura et al., (1983), Infect. Immun., 113:806-813). Genetically detoxified PT includes PT mutants or recombinant PT as described, for example, in U.S. Patents Nos.

6,168,928; 7,666,436; and 5,358,868, all of which are incorporated by reference herein.

Following conjugation of the oligosaccharide or polysaccharide to the carrier protein, the conjugate can be purified by a variety of techniques well known to one of skill in the art. One goal of the purification step is to remove the unbound oligosaccharide or polysaccharide from the conjugation reaction product composition. One method for purification, involving ultrafiltration in the presence of ammonium sulfate, is described in U.S. Patent No. 6,146,902. Alternatively, the conjugates can be purified away from unreacted oligosaccharide/polysaccharide and carrier by any number of standard techniques including, for example, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation. See, for example, Schneerson, Barrera, Sutton , Robbins, J Exp Med 152:361-376, 1980; Anderson et al., . Immunol. 137: 1181-1186, 1986 and Jennings & Lugowski, . Immunol. 127: 1011- 1018, 1981. The compositions and purity of the conjugates can be determined by GLC-MS and MALDI-TOF spectrometry.

Immunogenic Compositions The conjugates disclosed herein may be included in pharmaceutical compositions

(including therapeutic and prophylactic formulations), typically combined together with one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antibiotics or anti-inflammatories). In one particular embodiment, the conjugates disclosed herein are included in a multi-component immunogenic composition that includes a diphtheria toxoid component and a tetanus toxoid component. The multi-component mixture in the form of a physical mixture or combination. The diphtheria toxoid may be chemically or genetically detoxified as known in the art. The tetanus toxoid may be chemically or genetically detoxified as known in the art. Combination diphtheria/ tetanus/ pertussis (DTP) vaccines are known. The conjugate disclosed herein may be employed as the pertussis component in DTP vaccines. In one specific embodiment, the immunogenic composition includes:

(a) an immunogenic conjugate having the structure of: PT - Sr - O - N = Kdo - OS in which PT represents a pertussis toxoid carrier protein;

(b) diphtheria toxoid; and

(c) tetanus toxoid.

In another specific embodiment, the immunogenic composition includes:

(a) an immunogenic conjugate having the structure of: Pr - Sr - O - N = Kdo - OS in which Pr represents a carrier protein (other than pertussis toxoid);

(b) diphtheria toxoid;

(c) tetanus toxoid; and

(d) pertussis toxoid.

In the latter embodiment, the conjugate may confer bactericidal protection against B.

pertussis and the pertussis toxoid (which is not chemically conjugated to the OS) may confer anti-toxin protection against B. pertussis.

Such pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the conjugate can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, or parenteral routes. In other alternative embodiments, the conjugate can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the conjugate can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the conjugate. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The conjugate can be combined with the base or vehicle according to a variety of methods, and release of the conjugate can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the conjugate is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al., . Pharmacy Pharmacol. 43: 1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

In many cases, it will be desirable to include isotonic agents, for example, sugars, poly alcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the conjugate can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the conjugate can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the conjugate and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be

biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include poly gly colic acids and polylactic acids, poly (DL-lac tic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.- aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly( amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2- hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the conjugate and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze -drying which yields a powder of the conjugate plus any additional desired ingredient from a previously sterile -filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, the conjugate can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the conjugate and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof.

Typical subjects intended for treatment with the compositions and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease of condition (for example, coughing disease) as discussed herein, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods, which are available and well known in the art to detect and/or characterize disease-associated markers. These and other routine methods allow the clinician to select patients in need of therapy using the methods and pharmaceutical compositions of the disclosure. In accordance with these methods and principles, a conjugate and/or other biologically active agent can be administered according to the teachings herein as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments, including surgery, vaccination, immunotherapy, hormone treatment, cell, tissue, or organ transplants, and the like. The conjugates can be used in coordinate vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-LPS or an anti-LOS immune response. Separate immunogens that elicit the anti-LPS or anti-LOS immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol. For example, a combinatorial or a polyvalent immunogenic composition could include (i) an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica or Bordetella pertussis as a first component and (ii) oligosaccharide or polysaccharide obtained from Bordetella parapertussis as a second component.

The administration of the conjugate of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the conjugate is provided in advance of any symptom. The prophylactic administration of the conjugate serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the conjugate is provided at (or shortly after) the onset of a symptom of disease or infection. The conjugate of the disclosure can thus be provided prior to the anticipated exposure to Bordetella pertussis so as to attenuate the anticipated severity, duration or extent of an infection and/or associated disease symptoms, after exposure or suspected exposure to the bacteria, or after the actual initiation of an infection.

For prophylactic and therapeutic purposes, the conjugate can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the conjugate can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the conjugate (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the conjugate may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the conjugate will vary according to factors such as the particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the conjugate for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the conjugate and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. For example, the conjugate may be administered at 10 to 25 μg per dose.

Upon administration of a conjugate of the disclosure (for example, via injection, aerosol, oral, topical or other route), the immune system of the subject typically responds to the immunogenic composition by producing antibodies specific for LPS, LOS and/or an antigenic epitope presented by the conjugate. Such a response signifies that an

immunologically effective dose of the conjugate was delivered. An immunologically effective dosage can be achieved by single or multiple administrations (including, for example, multiple administrations per day), daily, or weekly administrations. For each particular subject, specific dosage regimens can be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the conjugate. In some embodiments, the antibody response of a subject administered the compositions of the disclosure will be determined in the context of evaluating effective dosages/immunization protocols. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and/or to change the amount of the composition administered to the individual can be at least partially based on the antibody titer level. The antibody titer level can be based on, for example, an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, for example, LPS and/or LOS.

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth. To achieve the same serum concentration level, for example, slow-release particles with a release rate of 5 nanomolar (under standard conditions) would be administered at about twice the dosage of particles with a release rate of 10 nanomolar.

The methods of using conjugates, and the related compositions and methods of the disclosure, are useful in increasing resistance to, preventing, ameliorating, and/or treating infection and disease caused by Bordetella in animal hosts, and other, in vitro applications. These immunogenic compositions can be used for active immunization for prevention of infection, and for preparation of immune antibodies. The immunogenic compositions are composed of non-toxic components, suitable for infants, children of all ages, and adults.

The methods of the disclosure are broadly effective for treatment and prevention of bacterial disease and associated inflammatory, autoimmune, toxic (including shock), and chronic and/or lethal sequelae associated with bacterial infection. Therapeutic compositions and methods of the disclosure for prevention or treatment of toxic or lethal effects of bacterial infection are applicable to a wide spectrum of infectious agents. Non-lethal toxicities that will be ameliorated by these methods and compositions can include fatigue syndromes, inflammatory/autoimmune syndromes, hypoadrenal syndromes, weakness, cognitive symptoms and memory loss, mood symptoms, neurological and pain syndromes and endocrine symptoms. Any significant reduction or preventive effect of the conjugate with respect to the foregoing disease condition(s) or symptom(s) administered constitutes a desirable, effective property of the subject composition/method of the disclosure.

The subject matter of the present disclosure is further illustrated by the following non-limiting Examples.

Examples

Conjugates of B. pertussis and B. bronchiseptica OS isolated from native and genetically modified strains with multiples of Band A terminal trisaccharide and were prepared and their chemical, serological and immunological properties as a potential addition to pertussis vaccine were studied. Materials and methods

Bacteria and cultivation.

B. pertussis Tohama I (ATCC BAA-589) was from the Culture Collection of

Goteborg (CCUG) and the non-toxin producing mutant of B. pertussis, Tax 113 strain was provided by Jerry Keith (NIH, NICHD). B. bronchiseptica RB50 (ATCC BAA-588) was obtained from ATCC (Manassas, VA). Genetically modified strains: RBwbmA is a mutant of B. bronchiseptica RB50 in which a large chromosomal Xbal fragment encompassing wbmA-wbmZ is replaced with a kanamycin-resistance cassette; RBA2b and RBBla are B. bronchiseptica RB50 mutants in which wbmA (RBA2b), or wbmB (RBB la) coding sequences are disrupted by insertion of a tetracycline resistance cassette. B. pertussis and B. bronchiseptica strains were cultivated on Bordet-Gengou (BG) agar plates. B.

bronchiseptica was then transferred to Stainer-Scholte (S-S) liquid medium containing 50 mM MgS0₄. After 16 to 24 hours of cultivation at 37 °C with shaking in baffled flasks, bacteria were harvested by centrifugation, killed by boiling for 1 h and stored at -20 °C for LPS extraction.

LPS and oligosaccharide preparation.

LPS was isolated by hot phenol-water extraction and purified by enzyme treatment and ultracentrifugation as described in Westphal O, Jann K (1965) Extraction with phenol-water and further application of the procedure. Methods Carbohydr Chem 5:83-91. LPS was heated in 1 % acetic acid for 90 min at 100 °C, ultracentrifuged at 35,000 rpm for 5 h at 4 ⁰ C and the carbohydrate -containing supernatant was passed through a 1 x 100 cm column of BioGel P-4, or in the case of B. bronchiseptica RBB2a Bio-Gel P-10, in pyridine/acetic acid/water buffer (4/8/988 ml) monitored with a Knauer differential refractometer. Fractions were collected based on the refractometer profile and freeze-dried twice to remove traces of the buffer components.

Conjugation.

Bovine serum albumin (BSA, Sigma, St. Louis, MO) was converted to aminooxylated derivatives in a two step procedure, as similarly described for B. bronchiseptica and B. parapertussis in Kubler-Kielb J, Vinogradov E, Ben-Menachem G, Pozsgay V, Robbins JB, et al. (2008) Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates. Vaccine 26: 3587-3593: (1) BSA was treated with succinimidyl 3-(bromoacetamido)propionate (SBAP, Pierce, Pittsburgh, PA) to introduce thiol-reactive bromoacetamido groups (BSA-Br); (2) BSA-Br was coupled to O- (3-thiopropyl)hydroxylamine, a heterobifunctional linker, to form the aminooxylated protein through stable thioether linkages (BSA-ONH₂) as described in Kubler-Kielb J, Pozsgay V (2005) A new method for conjugation of carbohydrates to proteins using an aminooxy-thiol heterobifunctional linker. Org Chem 70: 6987-6990. For conjugation, BSA-ONH₂ (5 mg) was reacted with OSs isolated from different B. pertussis or B. bronchiseptica stains in the following amounts: 2.5 mg (conjugates #1, 4, 5, 7, 9, 10, 11, 12, 13, see Table 1), 5 mg (conjugates #3, 6, 8) 7.5 mg (conjugate #2,) of OS in 1.5 ml Buffer A (PBS, 0.1% glycerol, 5 mM EDTA), at pH 5.7, for 15 hours. The ratio of BSA to OS was adjusted in order to achieve a range of OS chains incorporated per BSA. Next, the reaction mixture was passed through a 1x100 cm Sephadex G-75 column in 0.2 M NaCl as eluent and the void volume fraction characterized by protein assay, immunodiffusion, SDS-PAGE and MALDI-TOF spectroscopy.

Reactivity of B. pertussis oligosaccharide with 0-(3-thiopropyl)hydroxylamine.

B. pertussis Tax 113 OS (5 mg) of was reacted with 1 mg of 0-(3- thiopropyl)hydroxylamine in 1.5 ml Buffer A (PBS, 0.1% glycerol, 5 mM EDTA), at pH 5.7, for 15 hours. Next, the reaction mixture was passed through a 1 x 100 cm BioGel P-4 column in pyridine/acetic acid/water buffer (4/8/988 ml) monitored with a Knauer differential refractometer, then fractions were collected and freeze-dried. ESI-MS spectra of B. pertussis OS before and after reaction were recorded.

Immunization.

5-6 week-old female NIH Swiss Webster mice were injected subcutaneously 3 times at 2 week intervals with 2.5 μg saccharide as a conjugate in 0.1 ml PBS. Groups of 10 mice were exsanguinated 7 days after the second or third injection. Controls received PBS.

Hyperimmune antiserum against B. pertussis Tohama I was prepared with heat-killed bacteria.

Analytic.

Protein concentration was measured by the method of Lowry JO, Rosenbrough NJ, Farr AL, Randall R (1951) Protein measurement with the Folin phenol reagent. Biol Chem 193:265-275. Double immunodiffusion was performed in 1 % agarose gel in PBS. SDS- PAGE gel and immunoblotting used 16% Tris-Glycine gels and PVDF membranes and were performed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Endotoxin activity was measured by the limulus amebocyte lysate assay as described by the manufacturer (Cambrex, Walkersville, MD).

Spectroscopy.

MALDI-TOF mass spectra of the derivatized carrier proteins and of the conjugates were obtained with an OmniFlex MALDI-TOF instrument (Bruker Daltonics, Bil!erica, MA) operated in the linear mode. Samples for analysis were desalted and 1 μΐ, mixed with 20 μΐ of sinnapinic acid matrix made in 30% CH₃CN and 0.1% trifluoroacetic acid. Next, 1 μΐ of mixture was dried on the sample stage and placed in the mass spectrometer. NMR spectra were recorded at 30°C in D₂0 on a Varian UNITY INOVA 600 instrument using acetone as reference for both proton (2.225 ppm) and carbon (31.5 ppm) spectra. Varian standard programs COSY, NOESY (mixing time of 400ms), TOCSY (spinlock time 120 ms), HSQC, and gHMBC (long-range transfer delay 70 ms) were used.

Antibodies.

Serum IgG and IgM antibodies were measured by ELISA. Nunc Maxisorb plates were coated with B. pertussis Tohama I LPS at 10 μg/ml in PBS; 1% human serum albumin (HSA) in PBS was used in a blocking step. The optimal concentration of the coating antigen was determined by checkerboard titration. A PA MRX Dynatech reader was used. Antibody levels were calculated relative to the hyperimmune standard antiserum diluted 1:5000 for IgG and 1 :3000 for IgM and assigned for each a value of 100 ELISA Units (EU). Results were computed with an ELISA data processing program provided by the Biostatistics and Information Management Branch, CDC.

Bactericidal assay.

The bactericidal activity of sera was tested in vitro as described in Archambault D, Rondeau P, Martin D, Broudeur BR (1991) Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A. Gen

Micro 137:905-911. Briefly, antisera were inactivated at 56°C for 15 min and diluted tenfold in PBS with 0.15 mM CaCl₂, 0.5 mM MgCl₂ and 0.1% BSA (Buffer B). B. pertussis Tohama I was diluted to about 300-500 bacteria per 25 μΐ Buffer B. A pre-colostral calf serum (PCS) was used as a source of complement. Antisera, 45 μΐ, were mixed with 25 μΐ of bacteria and 15 μΐ of undiluted PCS. Finally, 140 μΐ of Buffer B were added (the final volume of reagents was 225 μΐ). Mixtures of bacteria in Buffer B without PCS or without serum were used as controls. After incubation for 60 min at 37 °C, 100 μΐ of each mixture was plated onto Bordet-Gengou agar, incubated for 3-5 days, then colonies were counted. The bactericidal titer of the antiserum was defined as the highest dilution giving killing of 50 % of the inoculum. The lowest positive titer was a dilution of 1 : 10.

Results

Chemical characterization of LPSs.

B. bronchiseptica RB50 LPS has a core OS structure similar to B. pertussis, strains Tohama I and Tax 113, but has an additional component an O-SP. The dodecasaccharide core without O-SP (called commonly a Band A) of B. bronchiseptica RB50 was separated on a BioGel P-4 column: the first fraction eluted in the column's void volume contained Band A substituted with O-SP (-80% by weight). The second fraction contained non- substituted Band A, which was used in these Examples (-20% by weight). ESI-MS and NMR analysis confirmed the published structure (see FIG. l). Mass spectra showed some minor differences between B. pertussis and B. bronchiseptica Band A. In B. pertussis phosphate was not detected and methylation of the N-4 of FucNAc4N was complete (a peak with mass of 2294 amu, Kdo was present only in the anhydro-Kdo form), whereas in B. bronchiseptica RB50 -50% of N-4 of FucNAc4N was methylated and -30% of 0-4 of Hep next to GlcA was phosphorylated (peaks with mass with anhydro-Kdo, P and Me of 2374 amu, without Me of 2360 amu, without P and with Me of 2294 amu, and without both P and Me of 2280 amu, were detected).

Three mutants deficient in O-SP expression, derived from B. bronchiseptica RB50, were used. RBwbmA, which lacks the complete wbm locus and cannot produce O-PS, produced core OS with a structure almost identical to B. pertussis Band A, differing only, as the parental strain RB50, in the degree of phosphorylation and methylation. Another example of an O-SP deficient mutant is described in more detail in Preston et al, Genetic Basis for Lipopolysaccharide O-Antigen Biosynthesis in Bordetellae, Infection and Immunity, Aug. 1999, p. 3763-3767, which is incorporated herein by reference in its entirety. RBB la and RBA2b produced LPS containing several repeats of the Band A trisaccharide. RBA2b produced oligomers of up to five repeats of Band A trisaccharide, visible by SDS-PAGE and in mass spectra. OS with different numbers of repeats were isolated in separated fractions on a BioGel P-10 column from the acetic acid RBA2b LPS hydrolysate (FIG. 2). ESI-MS spectra confirmed that the OS in each fraction differed by one additional Band A trisaccharide repeat (of mass 661 amu). NMR spectra of the RBA2b structures with repeats of Band A trisaccharide of various lengths were identical to each other except for a new spin system of 0C-GlcNAc linking Band A trisaccharide repeats together appeared, compared to the core fraction of 1 Band A trisacchride only. LPS of RBBla strain also contained repeats of the Band A trisaccharide but in a lesser amount and about 50% of the terminal GlcNAc was replaced by GalNAc.

Serological characterization of LPS.

Western immunoblot analysis showed that anti-β. pertussis Tohama I antiserum binds to the homologous LPS and to the three B. bronchiseptica mutant LPSs, confirming the similarity of these structures (FIG. 3). However, this serum bound only weakly to B. bronchiseptica RBA2b LPS containing several repeats of the Band A trisaccharide. The same was observed for a serum induced by a BSA/RBA2b-F4 conjugate prepared with only 1 repeat of Band A trisaccharide (conjugate #10; the same structure as of B. pertussis Band A). On the other hand, sera induced by BSA/RBA2b-F3, F2, Fl conjugates prepared with 2, 3 or 4 Band A trisaccharide repeats (Conj. #11, #12, #13) bound to LPS with 1 Band A trisaccharide repeat, as well as with several repeats of Band A trisaccharide.

Characterization of conjugates

Schemes for preparing conjugates by derivatization of BSA with 0-( - thiopropyl)hydroxylamine in a two step procedure, followed by binding to a carbonyl group of acetic acid-hydrolyzed LPS to form an oxime linkage are shown in FIG. 4. This procedure yielded high molecular mass conjugates, detected by MALDI mass spectrometry (Table. 1). The reactivity of the aminooxy group of the linker with anhydro-Kdo of the OS was established and ESI-MS spectra showed that all OS molecules incorporated the linker (FIG. 5). The number of OS chains per protein was calculated from the molecular masses of the aminooxylated BSA, the OS and the conjugate. For example, the average molecular mass of the aminooxylated BSA was 72 kDa, the molecular mass of the OS from B.

pertussis Tohama I was 2294 Da and that of the conjugate was 94 kDa (94 kDa - 72 kDa / 2.294 kDa) leading to an estimate of 8 chains per one BSA molecule (Conjugate #1).

All conjugates formed of an identity line between each conjugate and the anti-β. pertussis Tohama I and anti-protein sera by immunodifusion. An example of

immunodiffusion assay between conjugates containing RBA2b OS with different number of Band A trisaccharide repeats and anti-β. pertussis Tohama I and anti-BSA sera is shown on FIG. 6. All conjugates had < 5 EU of endotoxin activity per 1 μg of the conjugate as measured by a LAL assay. Antibodies to B. pertussis LPS and their bactericidal activity.

The conjugates varied in their chain lengths (number of Band A trisaccharide repeats), non-reducing terminal saccharide (GlcNAc or a mixture of GlcNAc/GalNAc) and by the density of OS chains per BSA (8-25 chains), see Table 1 below. All conjugates, injected subcutaneously at 2.5 μg of sugar (saccharide) per mouse, induce significantly higher antibody levels than the controls (Table 1). The antibody response elicited by the conjugates depended on the density of OS chains per protein: for B. pertussis Tax 113 the highest antibody levels were obtained with 17 chains per BSA (conjugate #3 vs #2 and #4, Table 1) and for B. bronchiseptica RBB la 12 chains was significantly better that 20 chains per protein (#8 vs #9, p=0.03, Table 1); (2) conjugate #9 containing a mixture of OS with terminal GlcNAc/GalNAc (about 1 : 1 ratio) induced antibody levels similar to those induced by the conjugates containing terminal GlcNAc only (conjugate #1, #3, #5, #10); (3) conjugates with 2-4 repeats of Band A trisaccharide induced significantly lower antibody levels to B. pertussis LPS than the conjugate with only 1 repeat (conjugates #10 vs #11, #12, #13, p<0.005).

Bactericidal activity and IgM levels were measured from antisera representing various antibody levels (see Table 2 below). The correlation coefficients between the reciprocal bactericidal titers of these antisera and their antibody levels measured by ELISA were for IgG r=0.66; for IgM r=0.73; and for IgG plus IgM r=0.74.

Table 1. Composition and geometric mean (GM) of mouse IgG of anti-β. pertussis Tohama I LPS induced by different conjugates of B. pertussis and B. bronchiseptica core fractions bound to bovine serum albumin (BSA).

*B.p., B. pertussis; B.b., B. bronchiseptica

All conjugates induced statistically higher antibody levels than PBS group; Conjugate #10 vs. #11, #12 and #13 p< 0.005; conjugate #9 vs. #8 p= 0.03.

Mice (10 per group) were injected with 2.5 μg of saccharide as a conjugate per mouse, subcutaneously, 3 times 2 weeks apart and bled one week after last injection.

Table 2. Bactericidal activity, IgG and IgM anti-β. pertussis Tohama I LPS

(ELISA) elicited by different conjugates.

#5 B.b. RB50 core 9 1787C 9.6 1.7 500

#5 B.b. RB50 core 9 1787G 64.9 2.8 750

#6 B.b. RBwbmA 19 2064A 2.4 3.1 20

#7 B.b. RBwbmA 11 2063A 0.06 0.3 <10

#7 B.b. RBwbmA 11 2063C 0.04 0.07 <10

#9 B.b. RBBla 12 2093H 38.4 13.9 750

#9 B.b. RBBla 12 2093J 14.0 1.6 10

#10 B.b. RBA2b, Fl 8 2152B 5.3 0.6 <10

#11 B.b. RBA2b, F2 9 2153D 4.2 0.7 10

#12 B.b. RBA2b, F3 11 2154H 4.1 0.2 10

#13 B.b. RBA2b, F4 9 2155D 39.3 0.1 50

#13 B.b. RBA2b, F4 9 2155F 2.1 3.8 <10

#13 B.b. RBA2b, F4 9 2155H 0.8 0.5 <10

PBS - 2156A <0.01 <0.01 <10

*B.p., B. pertussis; B.b., B. bronchiseptica

Correlation coefficient between reciprocal bactericidal titer and (1) IgG r= 0.66; (2) IgM r= 0.73; (3) IgG plus IgM r= 0.74.

Conclusions

The dodecasaccharide part of detoxified B. pertussis LPS (Band A) was bound to a carrier protein by a one -point attachment at its reducing end. Conjugation was performed between the carbonyl group of anhydro-Kdo released by acetic acid hydrolysis, and an aminooxy group of a bi-functional linker bound to the protein. This method allows the binding of saccharide components of different lengths and at different densities per protein molecule in order to select the most immunogenic construct. The final product is easy to characterize and standardize. When injected subcutaneously in PBS at a fraction of an estimated human dose it induced antibodies with bactericidal activity. This methodology is suitable for clinical use.

Genetically modified strains that produce LPS with several repeats of the terminal trisaccharide were also tested, hypothesizing that longer OS will be immunogenic. OS with 1 to 4 repeats of the trisaccharide were isolated from the B. bronchiseptica RBB2a strain and bound separately to BSA. The repeats were not recognized in Western blotting experiments by hyperimmune antiserum against B. pertussis or by antiserum raised against conjugates prepared with OS containing only 1 trisaccharide repeat. An interpretation of these results is that the epitope of B. pertussis LPS may include not only terminal trisaccharide but also additional sugars from the core, for example the side chain Hep of the terminal pentasaccharide, as observed in STD NMR experiments. This could explain why antibody levels induced by conjugates containing 2-4 repeats were statistically lower than B. pertussis LPS derived conjugates or than the B. bronchiseptica conjugate containing one repeat only by having a modified epitope. On the bases of these experiments a

dodecasaccharide with only one repeat is a preferred vaccine candidate.

Other variables that may influence the immunogenicity of conjugates were the density of OS chains per protein molecule and the non-reducing end molecule. In this study the range of optimal density was wide with an average number of chains between 8 and 17. For the evaluation of the non-reducing end molecule a B. bronchiseptica mutant RBB la was used, which expressed Band A with either terminal GlcNAc or GalNAc in about a 1 : 1 ratio. Conjugates prepared with this OS at the optimal densities, were as immunogenic as conjugates with terminal GlcNAc only.

To summarize, antibody levels induced by most immunogenic conjugates of B. bronchiseptica and B. pertussis OS were similar. The two B. bronchiseptica genetically modified strains RBB la and RBA2b, expressing molecules equivalent to slightly modified B. pertussis LPS, had no advantage over the RBwbmA strain, which produces substantially the same LPS as B. pertussis. The advantage of using B. bronchiseptica RBwbmA instead of B. pertussis is that it can be grown on simple media, in a short time, and with much higher yield.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

What is claimed is:

1. An immunogenic composition, comprising:

(a) an immunogenic conjugate comprising the structure of:

Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS;

(b) diphtheria toxoid; and

(c) tetanus toxoid.

2. An immunogenic composition, comprising:

(a) an immunogenic conjugate comprising the structure of:

Pr - Sr - O - N = Kdo - OS (II) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide having a structure of:

a-GlcN

17

a-GlcA-2-a-Hep4P

13

a-GlcNAc-4-P-ManNAc3NAcA-3-P-FucNAc4NMe-6-a-GlcN^-P-Glc-4-a-Hep-

14 16

a-Hep a-GalNA

;

(b) diphtheria toxoid; and

(c) tetanus toxoid.

3. The composition of claim 1 or 2, further comprising a pertussis anti-toxin component.

4. The composition of claim 1 or 2, wherein the pertussis anti-toxin component is pertussis toxoid.

5. The composition of claim 3 or 4, wherein the composition is a combination vaccine comprising a mixture of the immunogenic conjugate, diphtheria toxoid, tetanus toxoid, and the pertussis anti-toxin component.

6. The composition of any one of claims lor 3 to 5, wherein the OS is obtained from Bordetella bronchiseptica LPS and the OS includes at least: a-GlcN

17

a-GlcA-2-a-Hep4P

13

α-01οΝΑο-4-β-Μ ηΝΑο3ΝΑοΑ-3-β-ΡυοΝΑο4ΝΜε-6-α-01οΝ-4-β-01ο-4-α-Ηερ-

14 16

a-Hep a-GalNA

7. The composition of claim 6, wherein the OS is obtained from Bordetella bronchiseptica RB50 LPS or Bordetella bronchiseptica RBwbmA LPS.

8. The composition of claim 6, wherein the OS is obtained from Bordetella bronchiseptica RBwbmA LPS.

9. The composition of any one of claims 4 to 7, wherein the pertussis toxoid is the carrier protein Pr for the conjugate.

10. The composition of any one of claims 3 to 9, wherein the conjugate is present in a bactericidal-effective amount and the pertussis anti-toxin component is present in an anti-toxin-effective amount.

11. An immunogenic conjugate, comprising the structure of:

PT - Sr - O - N = Kdo - OS (III) in which PT represents a pertussis toxoid carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS,

wherein the conjugate can induce in a subject an anti-toxin response and a bactericidal response against Bordetella pertussis.

12. An immunogenic conjugate, comprising the structure of: PT - Sr - O - N = Kdo - OS (IV) in which PT represents a pertussis toxoid carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D-manno-octulosonic acid or a derivative thereof, and OS includes at least:

a-GlcN

17

a-GlcA-2-a-Hep4P

13

α-01οΝΑο-4-β-Μ ηΝΑο3ΝΑοΑ-3-β-ΡυοΝΑο4ΝΜε-6-α-01οΝ-4-β-01ο-4-α-Ηερ- 14 16

a-Hep a-GalNA wherein the conjugate can induce in a subject an anti-toxin response and a bactericidal response against Bordetella pertussis.

13. The conjugate of claim 11 or 12, wherein the pertussis toxoid is a chemically detoxified pertussis toxin or a genetically detoxified pertussis toxin.

14. The conjugate of claim 11 or 12, wherein the pertussis toxoid is recombinant pertussis toxin.

15. A method for inducing a bactericidal response against Bordetella pertussis in a subject, comprising:

administering to the subject a Bordetella pertussis bactericidal-effective amount of an immunogenic conjugate comprising the structure of:

Pr - Sr - O - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3- deoxy-D-manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS.

16. A method for inducing a bactericidal response against Bordetella pertussis in a subject, comprising:

administering to the subject a Bordetella pertussis bactericidal-effective amount of an immunogenic conjugate comprising the structure of: Pr - Sr - O - N = Kdo - OS (II) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS includes at least: a-GlcN

17

a-GlcA-2-a-Hep4P

13

α-01οΝΑο-4-β-Μ ηΝΑο3ΝΑοΑ-3-β-ΡυοΝΑο4ΝΜε-6-α-01οΝ-4-β-01ο-4-α-Ηερ- α-Hep α-GalNA

17. The method of claim 15 or 16, wherein the bactericidal response includes inducing bactericidal antibodies in the subject.

18. The method of any of claims 15 to 17, further comprising inducing an antitoxin response against Bordetella pertussis by administering a pertussis toxoid to the

subject.

19. A method for preparing an immunogenic conjugate, comprising:

obtaining from a Bordetella bronchiseptica mutant an oligosaccharide having a structure of:

a-GlcN

17

a-GlcA-2-a-Hep4P

13

a-GlcNAc-4-P-ManNAc3NAcA-3-P-FucNAc4NMe-6-a-GlcN^-P-Glc-4-a-Hep-5-Kdo4P-2-6-Lipid A

14 16

a-Hep a-GalNA cleaving the Lipid A from the oligosaccharide such that the Kdo moiety remains linked to the oligosaccharide; and

reacting the Kdo moiety with an aminooxylated protein carrier molecule resulting in an immunogenic conjugate that can induce in a subject a bactericidal response against

Bordetella pertussis.

20. The method of claim 19, wherein the protein carrier is pertussis toxoid.

21. The method of claim 19 or 20, wherein the Bordetella bronchiseptica mutant is Bordetella bronchiseptica RBwbmA LPS.

22. The method of claim 19 or 20, wherein the Bordetella bronchiseptica mutant is deficient in O-SP expression.

23. A method for making Bordetella pertussis -bactericidal antisera, comprising: administering to a subject an immunogenic conjugate comprising the structure of: Pr - Sr - 0 - N = Kdo - OS (I) in which Pr represents carrier protein, Sr is an optional spacer moiety, Kdo is 3-deoxy-D- manno-octulosonic acid or a derivative thereof, and OS is an oligosaccharide or

polysaccharide obtained from Bordetella bronchiseptica LPS, Bordetella pertussis LPS, or Bordetella parapertussis LPS, in an amount sufficient to elicit bactericidal antibodies against Bordetella pertussis; and

collecting the bactericidal antibodies against Bordetella pertussis to make the Bordetella pertussis -bactericidal antisera.