United States Patent:  5,955,080

Assignee:  Commonwealth Scientific and Industrial Research Organisation (Campbell, AU)

Filed: June 9, 1997

The present invention provides self-adjuvanting vaccines for use in raising antibodies to peptides without the use of oil or alum adjuvants. Further, the present invention provides methods of therapy using these vaccines and has particular application in chemical castration. In one aspect the vaccine comprises in admixture a peptide conjugated to 1-3 fatty acids and a peptide conjugated to a carrier protein. In preferred forms the peptide is conjugated to the fatty acids via a tromethamine or ethanolamine derivative. The preferred protein carrier is Type 4 fimbriae.

Summary of the Invention

Accordingly, in a first aspect the present invention consists in a vaccine for use in raising an immune response to a peptide, the vaccine comprising in admixture the peptide conjugated to 1 to 3 fatty acids and the peptide conjugated to a carrier protein.

In a preferred embodiment of the first aspect of the present invention the peptide has or includes one of the following amino acid sequences:

SGGWSYGLRPGG; (SEQ ID NO:1)

WSYGLRP; (SEQ ID NO:2)

WSYGWLP; or (SEQ ID NO:3)

WSYGLQP. (SEQ ID NO:4)

In a second aspect the present invention consists in a vaccine for use in chemical sterilisation the vaccine comprising a peptide which has or includes an amino acid sequence selected from the group consisting of:

SGGWSYGLRPGG (SEQ ID NO:1), WSYGLRP (SEQ ID NO:2), WSYGWLP (SEQ ID NO:3) and WSYLLQP (SEQ ID NO:4), the peptide being conjugated either to 1 to 3 fatty acids or to a fimbrial subunit.

In a preferred embodiment of the invention the carrier protein is a fimbrial sub unit protein, ovalbumin, bovine serum albumin, tetanus toxin, or keyhole limpet haemocyanin. It is presently preferred, however, that the carrier protein is a fimbrial protein subunit and that the subunit proteins are assembled into fimbriae. It is most preferred that the fimbriae are Type 4 fimbriae.

The peptide is preferably conjugated to 1 to 3 fatty acids via a tromethamine or an ethanolamine derivative.

It is presently preferred that the peptide is linked to three fatty acids and more preferably that each are the same fatty acid. It is also preferred that the fatty acid has a carbon chain of 3 to 18 carbon atoms and most preferably 16 carbon atoms.

The present inventors have also found that partial denaturation of the peptide/fimbrial protein carrier results in a higher antigenic response. This partial denaturation is preferably obtained by treatment of the peptide/carrier protein at a pH of less than or equal to 4 and preferably at a pH of about 1. Given the enhanced response that this partial denaturation provides in a preferred form of the invention the peptide/fimbrial protein conjugate is subjected to a a partial denaturation prior to admixture with the peptide/fatty acid conjugate.

As will be appreciated by those skilled in the art while any peptide can be used in the vaccine of the first aspect of the present invention the preferred peptides are LHRH derived peptides. Where the peptide is an LHRH peptide, or a derivative, the vaccine may be used for chemical sterilization of animals or for use as an alternative to the present LHRH agonist and antagonist therapy for human sex hormone dependent cancers. Four of the most commonly occurring human sex hormone dependent cancers are prostate, breast, endometrial and ovarian cancer. These conditions may be susceptible to treatment by vaccination with the vaccine of the present invention using the LHRH peptide or derivative. The vaccine of the present invention could also be used as an adjunct to the present LHRH agonist and antagonist therapy for these diseases.

It will be appreciated by those skilled in the art that a number of modifications may be made to the peptides preferably used in the present invention without deleteriously effecting the biological activity of the peptide. This may be achieved by various changes, such as insertions, deletions and substitutions, either conservative or non-conservative in the peptide sequence where such changes do not substantially alter the nature of the immune response raised by the peptide. By conservative substitutions the intended combinations are:

G,A; V,I,L,M; D,E; N,Q; S,T; K,R,H; and F,Y,W.

The vaccine of the first aspect of the present invention will also have applicability in the treatment of diseases such as AIDS, malaria, influenza, zone pellucida peptide epitopes or hepatitis where peptide epitopes have been identified and a vaccine approach is possible.

The vaccine of the first aspect of the present invention comprising an admixture of peptide/fatty acid conjugate with peptide/carrier protein conjugate when used without oil or alum adjuvants induces high level antibody responses in animals. The ability of this vaccine to induce such high level response without requiring oil or alum based adjuvants is of particular benefit. Such findings will allow the use of this vaccine where the use of oil based adjuvants is not permitted and the use of alum based adjuvants is being questioned, e.g., human vaccines or vaccines for companion animals.

The preferred form of preparing the peptide/type 4 fimbrial protein conjugate involves using the method disclosed in Australian patent application No 17049/88. By using this method the desired peptide can be expressed in association with Type 4 fimbriae by genetically engineered strains of Pseudomonas. This method is, however, to some extent, limited in that the peptide epitope size that can be incorporated into the fimbriae is dependent on the degree of modification which can be made before the fimbriae is no longer properly assembled. Investigations have shown that peptides of up to approximately 20 amino acid residues can be produced in association with the fimbriae in Pseudomonas aeruginosa. Accordingly, it is presently preferred that the peptides are able to be expressed in association with fimbriae in Pseudomonas aeruginosa. If, however, the peptide epitope is too large or cannot be expressed in this fimbrial system it is still possible to use the vaccine of the present invention by simply chemically conjugating the peptide with the fimbrial protein.

The response to the vaccine component of the present invention may also be enhanced by the use of T-cell, H₂ receptor antagonists, e.g., cimetidine or carnosine, or other immunomodulators e.g., cytokines or immunostimulatory peptides which may act as immunomodulators to overcome carrier protein unresponsiveness or to further enhance the immune system.

The present inventors also believe that the antibody response in an animal to an antigen may be heightened by the use of fimbrial protein of a Type 4 fimbriate bacteria.

Accordingly, in a third aspect the present invention consists in a method of enhancing the antibody response to an antigen in an animal, the method comprising to the animal an effective amount of the antigen conjugated to the fimbrial protein of a Type 4 fimbriate bacteria.

As is disclosed in application No PCT/AU90/00599 a wide range of fatty acids may be linked to the peptide via a tromethamine or ethanolamine derivative. In addition, 1-3 fatty acids may be linked to the peptide. It should also be noted that the peptide linked to the fatty acids can be of virtually unlimited size.

Claim 1 of 11 Claims

1. A composition effective for raising antibodies to a peptide, the composition comprising a mixture comprising a peptide conjugated to 1 to 3 fatty acids, and the peptide conjugated to a carrier protein.

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