Title:  Immunogenic conjugates comprising a group B meningococcal porin and an H. influenzae polysaccharide

United States Patent:  6,613,336

Issued:  September 2, 2003

Inventors:  Blake; Milan S. (Fulton, MD); Michon; Francis (Bethesda East, MD); Fusco; Peter C. (Burtonsville, MD); Heron; Iver (Bethesda, MD)

Assignee:  Baxter International Inc. (Deerfield, IL)

Appl. No.:  142441

Filed:  May 10, 2002

Abstract

Disclosed is an H. influenzae type b polysaccharide-meningococcal outer membrane protein conjugate, pharmaceutical compositions thereof, and the use thereof to induce an immune response to H. influenzae in an animal.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an H. influenzae type b (Hib) polysaccharide-substantially pure, refolded meningococcal outer membrane protein (rPorB) conjugate.

The present invention also relates to a method of preparing an Hib polysaccharide-rPorB conjugate, comprising

(a) obtaining an Hib polysaccharide,

(b) oxidizing or selectively hydrolyzing said polysaccharide to generate aldehyde groups;

(c) obtaining a rPorB; and

(d) conjugating the polysaccharide containing aldehyde groups to the rPorB by reductive amination.

The present invention also relate to the conjugates obtained according to the methods of the invention. Optionally, the conjugates of the present invention may be combined with DTaP (diphtheria, tetanus, acellular pertussis vaccine).

The present invention also relates to pharmaceutical compositions comprising the conjugates of the invention, optionally comprising DTaP, and a pharmaceutically acceptable carrier.

The present invention also relates to a method of inducing an immune response in an animal to H. influenzae, comprising administering the conjugates of the invention to the animal in an amount effective to induce said immune response.

The invention relates in part to the surprising discovery that the Hib-rPorB conjugates of the invention induce substantially greater immune responses in animals compared to when tetanus toxoid and the recombinantly produced outer membrane P2 protein from H. influenzae is used as the antigenic protein. Substantially greater immunogenic responses were also obtained compared to the Hib-CRM conjugate which is commercially available from Lederle Laboratories, Division of American Cyanamide Company, Pearl River, N.Y. CRM₁₉₇ is a site mutant, non-toxic variant of diphtheria toxin isolated from cultures of Cornebacterium diphtheriae C7(.beta.197). Seid, R. C. Jr. et al., Glycoconj. J 6: 489-498 (1989).

Furthermore, the conjugate of the present invention is especially useful in compositions also comprising DTaP, as immunologic interactions between the components, as well as epitopic suppression, is observed with conventional carrier proteins such as tetanus toxoid. The conjugates of the present invention overcome this serious limitation in combination vaccine compositions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a vaccine for inducing an immune response in an animal comprising the outer membrane meningococcal group B porin protein linked to Hib polysaccharide, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the vaccine may be administered in an amount effective to elicit an immune response in an animal to H. influenzae. In a preferred embodiment, the animal is a mammal selected from the group consisting of humans, cattle, pigs, sheep, and chickens. In another preferred embodiment, the mammal is a human.

By the term "rPorB" is intended the mature, refolded class 2 or class 3 outer membrane protein from N. meningitidis and fusions thereof comprising amino acids 1 to 20 or 1 to 22 of the T7 gene .phi.10 capsid protein. Methods for high level expression of the mature class 2 and class 3 rPorB and fusions thereof, refolding, and purification are described in (47) and U.S. Pat. No. 5,439,808, the disclosures of which are incorporated herein by reference. The recombinant porin may be expressed at high levels from E. coli according to U.S. Pat. No. 5,439,808, or from yeast according to Ser. No. 08/92,302. In a preferred embodiment, the class 3 rPorB is expressed from the host BL21(DE3).DELTA.ompA which has been transformed with the gene coding for the rPorB, is substantially pure and is refolded according to U.S. Pat. No. 5,439,808.

The H. influenzae capsular polysaccharide can be isolated according to methods well known to those of ordinary skill in the art. See, Schneerson et al., J. Exp. Med. 152:361-376 (1980); Marburg et al. J. Am. Chem. Soc. 108:5282 (1986); Jennings et al., J. Immunol. 127:1011-1018 (1981); and Beuvery et al., Infect. Immunol. 40:39-45 (1983). In a preferred embodiment, the organism is cultured, the culture supernatant is microfiltered, and the filtrate is passed through a 300,000 molecular weight cut off filter. The permeate is then concentrated, for example, with a 100,000 molecular weight cut off filter. This 100,000-300,000 molecular weight material is then oxidized with a mild oxidant such as metaperiodate, the product filtered through a 30,000 molecular weight filter and then concentrated with a 5,000 molecular weight filter, to give a polysaccharide having aldehyde groups that may be used directly for conjugation. The preferred polysaccharide has a molecular weight of about 5,000-50,000. A more preferred polysaccharide has a molecular weight of about 10,000-50,000, however other molecular weight ranges may be employed as desired.

It will be understood by those of skill in the art that the capsular polysaccharide-protein carrier conjugates of the vaccine may be produced by several different methods. The types of covalent bonds which couple a polysaccharide to a protein carrier, and the means of producing them, are well known to those of skill in the art. Details concerning the chemical means by which the two moieties can be linked may be found in U.S. Pat. Nos. 5,623,057, 5,371,197, 5,192,540, 4,902,506 and 4,356,170, the contents of which are herein incorporated by reference in their entirety. For a review, see Contributions to Microbiology and Immunology, vol 10, Conjugate Vaccines, volume editors J. M. Cruse and R. E. Lewis, Jr., 1989, and (29). One such method is the reductive amination process described in Schwartz and Gray (Arch. Biochim. Biophys. 181:542-549 (1977)). This process involves producing the polysaccharide in a form which has reducing end groups, and reacting the capsular polysaccharide and rPorB in the presence of cyanoborohydride ions, or another reducing agent. The reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both.

The vaccine of the present invention comprises the Hib-rPor conjugate, in an amount effective depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the meningococcal group B porin protein, fusion protein or vaccine of the present invention can also be administered by an intraperitoneal, intravenous, or intranasal route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts are expected to fall within the range of 5 to 50 .mu.g per animal, more preferably, about 10 .mu.g per animal.

The vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the conjugate vaccine has suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.

The vaccines of the present invention may further comprise adjuvants which enhance production of H. influenzae-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Pat. No. 4,258,029), the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.

Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administration to a human. The conjugate vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The conjugate vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Pat. No. 4,235,877.

In another preferred embodiment, the conjugate of the invention is combined with other immunogens that are used to vaccinate animals. Thus, the conjugate of the invention may be combined with DTaP or DTaP IPV for administration to the animal. DTaP is a combination vaccine for diphtheria, tetanus, and acellular pertussis, which is available from Amvax, Inc., Beltsville, Md. In a preferred embodiment, the acellular pertussis is in an oxidized form as is available from Amvax, Inc.

In another preferred embodiment, the present invention relates to a method of inducing an immune response in an animal comprising administering to the animal the vaccine of the invention in an amount effective to induce an immune response. Optionally, the vaccine of the invention may be coadministered with effective amounts of other immunogens as mentioned above to generate multiple immune responses in the animal.

Claim 1 of 8 Claims

What is claimed is:

1. A method of inducing an immune response in an animal to H. influenzae, comprising administering an H. influenzae type b (Hib) polysaccharide-substantially pure, refolded meningococcal outer membrane protein (rPorB) conjugate to the animal in an amount effective to induce said immune response.

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