Title:  Caulobacter LPS immunoadjuvants

United States Patent:  6,368,599

Assignee:  Univ. of British Columbia (Vancouver, CA)

Filed:  February 22, 2000

The present invention provides immunogenic compositions and methods for inducing enhanced immune responses using an antigen by use of an adjuvant comprising a member selected from a Caulobacter (in particular, C. crescentus) LPS or a fragment or derivative thereof.

SUMMARY OF THE INVENTION

The present invention relates to a novel adjuvant or immunization composition comprising a Caulobacter LPS or fragment thereof having adjuvant properties. For example, the fragment may be LPS lipid A, a LPS lipid A fragment, or a derivative thereof, having adjuvant properties. Preferably, such adjuvants are obtained from Caulobacter crescentus.

In a further aspect, the present invention provides a Caulobacter LPS or fragment thereof having adjuvant properties which is less toxic than other gram-negative LPS molecules. Preferably such adjuvant is LPS or lipid A, a lipid A fragment, or a derivative thereof, having adjuvant properties that are less toxic than other gram-negative LPS molecules and are derived from C. crescentus. As a representative example of a lipid A derivative, there may be mentioned the monophosphorylated derivative of the lipid A fragment of the LPS of C. crescentus. Such derivative may be prepared by monophosphorylating the lipid A fragment by procedures known in the art with respect to lipid A fragments from the LPS of other bacteria.

In another aspect, the present invention provides a method of inducing an immunogenic response in a host, in particular, a protective response when the adjuvant composition described in the above embodiments is administered orally, intramuscularly or parenterally concurrently with or proximately to the administering of an antigen. In one aspect, the adjuvant is mixed with the antigen prior to administration.

It is a further object of the invention to provide a vaccine for treating or preventing bacterial infections which utilizes the adjuvant according to the invention as set forth in the above objects of the invention and utilizes as an antigen a complex of a bacterial periplasmic chaperone protein with a bacterial adhesin protein. Preferably, the antigenic adhesin protein is a pilus adhesin protein. In one aspect, the antigenic periplasmic chaperone protein and pilus adhesin protein are from E. coli; for example, a member selected from the complexes PapD/PapG and FimC/FimH.

In a particular aspect, the invention relates to vaccines formulated with the adjuvant according to the invention and from an antigen which is a type 1 pilus-associated adhesin (or from a mannose-binding fragment thereof) or from antigens which are complexes of chaperone proteins and pilus-associated adhesins for the treatment and/or prophylaxis of diseases caused by pathogenic species of gram-negative bacteria, such as Escherichia coli (E. coli). For example, it relates to treatment and/or prophylaxis of urinary tract infections caused by E. coli with vaccines formulated with the adjuvant according to the invention and from an antigen which is selected from at least one of (1) a fragment of the pilus-associated adhesin FimH that retains mannose binding capability (alone or complexed with its chaperone FimC), (2) the pilus-associated adhesin PapG protein complexed with its periplasmic chaperone protein PapD or (3) the full-length pilus-associated adhesin FimH (alone or in a complex with its chaperone protein FimC). This invention also relates generally to the use of the adjuvant according to the invention in combination with heteropolymeric protein complexes to raise antibodies in non-human mammalian species useful, for example, as diagnostic reagents and vaccines.

In a further aspect of the present invention, Caulobacter adjuvants according to the invention are used in concert or combination with antigens to produce a vaccine which is useful as a therapeutic for treatment of a disease.

Caulobacter crescentus is a stalked, gram-negative bacteria and contains an atypical LPS molecule which has been generally described. LPS from such bacteria have been described as being of a rough type (R type LPS) in that it does not contain heterogeneous O antigen attached to the core sugars. The lipid A equivalent region and core oligosaccharide region are distinct from other LPS molecules. Caulobacter crescentus possesses both rough (R type) and smooth (S type) LPS, the smooth LPS being the rough LPS-equivalent with the addition of a homopolymer antigen of perosamine (with about 40 copies of this sugar). The LPS produced by such Caulobacter comprises an inner core region containing three 2-keto-3-deoxyoctulosonate (KDO) molecules, two .alpha.-L-glycero-D-mannoheptose and one .alpha.-D-glycero-D-mannoheptose; an outer core region of .alpha.-D-mannose, .alpha.-D-galactose, and .alpha.-D-glucose (possibly phosphorylated); and a lipid A portion contain 3-OH-dodecanoic acid attached to a backbone having an undetermined structure.

However, prior to the present invention, nothing was known about the immunomodulating properties of Caulobacter LPS.

Caulobacter crescentus strains are grown, and LPS isolated, and purified as described by Ravenscroft et al., Journal of Bacteriology, 7595-7605 (December 1992), which is incorporated herein by reference.

Additionally, Caulobacter strains can be grown in known media such as peptone-yeast extract under standard culturing conditions and LPS isolated by known methods. Such methods may include nuclease digestion of the disrupted cells followed by incubation with solutions that cause the Caulobacter cell membranes to dissociate more completely. Samples of LPS can be purified by well-known procedures followed by, or including, electrophoresis and/or chromatography.

For example, the rough LPS of Caulobacter can be extracted by the modified Galanos method (described in Quereshi et al, J. Biol. Chem. 258:12947-12951 (1983), incorporated herein by reference) and examined by Thin Layer Chromatography (TLC) and/or SDS-PAGE to determine purity. The smooth LPS of Caulobacter can be purified by NaCl/EDTA extraction and elution from an SDS-PAGE prep gel, followed by extensive washing on an Amicon 30,000 MW cutoff filter against distilled water to remove impurities. MALDI-TOF analysis can be utilized to show that the Caulobacter LPS has a molecular weight of about 10,400 daltons. Gas chromatography of the hydrolyzed LPS contains a homopolymer of a dideoxyaminohexose, tentatively identified as perosamine.

LPS can be cleaved into Lipid A and polysaccharide using 0.1 N HCl at 100^oC. for 30 minutes, followed by extraction with 2.5 volumes of chloroform/methanol (2:1) per volume of aqueous LPS. The lipid A partitions to the chloroform/methanol (or lower) phase, which is then evaporated to dryness in a stream of dry nitrogen gas. The composition of the samples can be determined by standard analytical procedures, such as gas chromatography and the like. Dephosphorylation of LPS samples can be performed by utilizing HF and then removing the HF. Lipid analysis and NMR structural studies can be conducted by known methods.

Thus, the present invention provides a LPS from Caulobacter, or a fragment or derivative thereof, which enhances an immune response to an antigen, as well as adjuvant or vaccine compositions comprising them. The LPS may be in rough or smooth form. The adjuvant may be comprised of mixtures of the rough and smooth form, or either form alone, or may be comprised of a lipid A fragment or derivative of such fragment, alone or in combination with LPS.

As used herein, the terms "portion," "segment," and "fragment," when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to a polynucleotides, such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.

The present invention further provides a method of inducing an immunogenic response in a host (a human or a non-human animal), in particular, a protective response when the adjuvant composition according to the present invention is administered orally, intramuscularly or parenterally concurrently with or proximately to the administering of an antigen. In one aspect, the adjuvant is mixed with the antigen prior to administration. Alternatively, the adjuvant composition is administered proximately to the time of administering the antigen, but is not mixed with the antigen prior to administration.

The antigen useful in practicing the present invention can be derived from a cell, bacteria, or virus particle, or portion thereof. As defined herein, antigen may be a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or combination thereof, which elicits an immunogenic response in an animal, for example, a mammal, bird, or fish. As defined herein, the immunogenic response can be humoral or cell mediated. In the event the material to which the immunogenic response is to be directed is poorly antigenic, it may be conjugated to a carrier such as albumin or to a hapten, using standard covalent binding techniques, for example, with one of the several commercially available reagent kits.

Examples of antigens include viral proteins such as influenza proteins and hepatitis B proteins; and bacterial proteins and lipopolysaccharides such as gram-negative bacterial cell wall and surface proteins.

The adjuvant can also be covalently conjugated with the antigen in accordance with methods well-known to those skilled in the art, usually by covalent linkage between an amino or carboxyl group on the antigen and one or more side groups on the adjuvant. Although in the preferred embodiment the adjuvant and antigen of the vaccine composition are administered simultaneously, in an alternative embodiment, the adjuvant and antigen are administered separately to the same site or to nearby sites. The adjuvant serves to attract cells of the immune system to the site where they then act upon the antigen.

The immunogenic composition can be administered as a vaccine by any method known to those skilled in the art that elicits an immune response, including parenterally, orally, or by transmembrane or transmucosal administration. Preferably, the vaccine is administered parenterally (intravenously, intramuscularly, subcutaneously, intraperitoneally, etc.), and preferably subcutaneously. Nonlimiting examples of routes of delivery to mucosal surfaces are intranasal (or generally, the nasal associated lymphoid tissue), respiratory, vaginal, and rectal. In the vaccine, the adjuvant is added in an amount effective to increase or potentiate the immune response to an antigen. The immune response can be a protective immune response or an immune response effective for treating a disease or condition.

The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., New Jersey current edition).

In carrying out the procedures of the present invention it is of course to be understood that reference to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein.

Claim 1 of 7 Claims

What is claimed is:

1. A composition for inducing an immunogenic response in an animal comprising an antigen and at least one member selected from the group consisting of Caulobacter LPS and a fragment of Caulobacter LPS wherein said member has adjuvant properties.

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