Multi-mutant diphtheria toxin vaccines

Details Multi-mutant diphtheria toxin vaccines Multi-mutant diphtheria toxin vaccines

1999-02-16

2002-09-24

Smith, Lynette R. F. (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S350000, C424S183100, C424S184100, C424S185100, C424S203100, C424S236100, C424S245100, C424S239100, C424S136100, C424S143100, C424S238100, C424S178100, C424S150100, C435S069100, C435S069700, C435S029000

Reexamination Certificate

active

06455673

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to vaccines that protect against diphtheria toxin.
BACKGROUND OF THE INVENTION
Diphtheria toxin (DT) is a protein exotoxin produced by the bacterium
Corynebacteria diphtheria.
The DT molecule is produced as a single polypeptide that is readily spliced to form two subunits linked by a disulfide bond, Fragment A (N-terminal ~f21K) and Fragment B (C-terminal ~37K), as a result of cleavage at residue 190, 192, or 193 (Moskaug, et al.,
Biol Chem
264:15709-15713, 1989; Collier et al.,
Biol Chem,
246:1496-1503, 1971). Fragment A is the catalytically active portion of DT. It is an NAD-dependent ADP-ribosyltransferase which specifically targets a protein synthesis factor termed elongation factor 2 (EF-2), thereby inactivating EF2 and shutting down protein synthesis in the cell. Fragment A consists of the diphtheria toxin C domain. Fragment A is linked to the diphtheria toxin Fragment B by a polypeptide loop. Fragment B of DT possesses a receptor-binding domain (the R domain) which recognizes and binds the toxin molecule to a particular receptor structure found on the surfaces of many types of mammalian cells. Once DT is bound to the cell via this receptor structure, the receptor/DT complex is taken up by the cell via receptor-mediated endocytosis. A second functional region on Fragment B (the T domain) acts to translocate DT across the membrane of the endocytic vesicle, releasing catalytically active Fragment A into the cytosol of the cell. A single molecule of Fragment A is sufficient to inactivate the protein synthesis machinery in a given cell.
Immunity to a bacterial toxin such as DT may be acquired naturally during the course of an infection, or artificially by injection of a detoxified form of the toxin (i.e., a toxoid) (Germanier, ed.,
Bacterial Vaccines,
Academic Press, Orlando, Fla., 1984). Toxoids have traditionally been prepared by chemical modification of native toxins (e.g., with formalin or formaldehyde (Lingood et al.,
Brit. J. Exp. Path.
44:177, 1963)), rendering them nontoxic while retaining an antigenicity that protects the vaccinated animal against subsequent challenges by the natural toxin: an example of a chemically-inactivated DT is that described by Michel and Dirkx (
Biochem. Biophys. Acta
491:286-295, 1977), in which Trp-153 of Fragment A is the modified residue.
Another method for producing toxoids is by the use of genetic techniques. Collier et al., U.S. Pat. No. 4,709,017 (herein incorporated by reference) disclosed a genetically engineered diphtheria toxin mutant that bears a deletion mutation at Glu-148 of diphtheria toxin. Glu-148 was originally identified as an active-site residue by photoaffinity labelling (Carroll et al.,
Proc. Natl. Acad. Sci. USA
81:3307, 1984; Carroll et al.
Proc. Natl. Acad. Sci. USA
82:7237, 1985; Carroll et al.,
J. Biol. Chem.
262:8707, 1987). Substitution of Asp, Gln or Ser at this site diminishes enzymatic and cytotoxic activities by 2-3 orders of magnitude, showing that the spatial location and chemical nature of the Glu-148 side-chain greatly affects these activities (Carroll et al.,
J. Biol. Chem.
262:8707, 1987; Tweten et al.,
J. Biol. Chem.
260:10392, 1985; Douglas et al.,
J. Bacteriol.
169:4967, 1987). Similarly, Greenfield et al., U.S. Pat. No. 4,950,740 (herein incorporated by reference) disclosed genetically engineered mutant forms of DT in which the Glu-148 residue is deleted or replaced with Asn, and the Ala-158 residue is replaced with Gly. The DNA sequence and corresponding amino acid sequence of wild-type diphtheria toxin DNA are set forth in
FIG. 1
(SEQ ID NOs:1 and 2, respectively).
SUMMARY OF THE INVENTION
The invention features diphtheria toxoids having multiple mutations as compared with wild-type diphtheria toxin. Thus, the invention features a polypeptide having a mutant diphtheria toxin C domain, a mutant diphtheria toxin T domain, and a mutant diphtheria toxin R domain, wherein the C domain has a mutation at Glu148, the T domain has a mutation at Glu349, and the R domain has a mutation at Lys 516 and/or Phe530 of wild-type diphtheria toxin. In various embodiments, the polypeptide includes any or all of the following mutations: Glu148Ser, Glu349Lys, Lys516Ala, and/or Phe530Ala.
The invention also features a polypeptide having a mutant diphtheria toxin C domain, a mutant T domain, and a mutant loop connecting the diphtheria toxin C and T domains, wherein the C domain has a mutation at Glu148, the T domain has a mutation at Glu349, and the loop has a mutation at Arg190, Arg192, and/or Arg193 of wild-type diphtheria toxin. In various embodiments, the polypeptide (or a mixture of polypeptides) includes any or all of the following mutations: Glu148Ser, Glu349Lys, Arg190Ser, Arg192Gly and/or Arg193Ser. In addition, all of the polypeptides of the invention bind sensitive cells with less affinity than does wild-type diphtheria toxin, and are capable of forming an immune complex with an antibody that specifically recognizes the R domain of wild-type diphtheria toxin.
These so-called “multi-mutant” diphtheria toxoids of the invention can be used as vaccines to provide immunoprotection against diphtheria toxin and against infection by
Corynebacteria diphtheriae.
One approach to vaccination utilizes live, genetically engineered microorganisms (cells or viruses) expressing mutant toxin genes. The multi-mutant toxoids of the invention, and the DNAs which encode them, carry significantly less risk of reversion than do single residue deletion mutants, and so are good candidates for use in a live, genetically engineered vaccine cell that is capable of proliferating in the vaccinee. As discussed below, acellular vaccines also are within the invention.
The invention also includes vectors (e.g., plasmids, phages and viruses) including DNA sequences encoding the diphtheria toxoid mutants described herein. Expression of a diphtheria toxoid polypeptide of the invention can be under the control of a heterologous promoter, and/or the expressed amino acids can be linked to a signal sequence. A “heterologous promoter” is a promoter region that is not identical to the promoter region found in a naturally occurring diphtheria toxin gene. The promoter region is a segment of DNA
5
′ to the transcription start site of a gene, to which RNA polymerase binds before initiating transcription of the gene. Nucleic acids encoding a diphtheria toxoid of the invention can be prepared as an essentially pure preparation, which is a preparation that is substantially free of other nucleic acid molecules with which a nucleic acid encoding diphtheria toxin is naturally associated in Corynebacterium. A nucleic acid encoding a diphtheria toxoid of the invention can be contained in a cell, or a homogeneous population of cells, preferably a
B. subtilis,
Bacillus Calmette-Guerin (BCG), Salmonella sp.,
Vibrio cholerae, Corynebacterium diphtheriae,
Listeriae, Yersiniae, Streptococci, or
E. coli
cell. The cell is capable of expressing the diphtheria toxoid polypeptide of the invention.
Diphtheria toxoids that are “immunologically cross-reactive” possess at least one antigenic determinant in common with naturally occurring diphtheria toxin, so that they are each bound by at least one antibody with specificity for naturally occurring diphtheria toxin. A diphtheria toxoid of the invention is immunologically cross-reactive with naturally occurring diphtheria toxin and possesses at least one of the mutations described herein.
The invention includes various vaccines that can be used to immunize a mammal (e.g., a human) against progression of the disease diphtheria, and against infection by the bacterium
Corynebacterium diphtheriae.
A vaccine of the invention can include any of the various DNAs encoding a diphtheria toxoid of the invention. Alternatively, a cell or virus expressing a nucleic acid of the invention, e.g., a live vaccine cell, can be used as a vaccine. Examples of suitable cells include
B. subtilis,
BCG, Salmonella sp.,
Vibrio cholerae,
Listeriae, Yersiniae, Stre

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