Vaccine compositions

Details Vaccine compositions Vaccine compositions

1996-07-19

2002-12-03

Navarro, Mark (Department: 1645)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

C424S184100, C424S193100, C424S197110, C435S252300, C435S320100, C435S325000, C530S350000, C530S402000, C536S023400

Reexamination Certificate

active

06488926

ABSTRACT:

This invention relates to DNA constructs, replicable expression vectors containing the constructs, bacteria containing the constructs and vaccines containing the bacteria or fusion proteins expressed therefrom. More particularly, the invention relates to novel DNA constructs encoding the C-fragment of tetanus toxin, and to fusion proteins containing tetanus toxin C-fragment.
It is known to prepare DNA constructs encoding two or more heterologous proteins with a view to expressing the proteins in a suitable host as a single fusion protein. However, it has often been found that fusing two proteins together in this way leads to an incorrectly folded chimaeric protein which no longer retains the properties of the individual components. For example, the B-subunits of the
Vibrio cholerae
(CT-B) and
E. coli
(LT-B) enterotoxins are powerful mucosal immunogens but genetic fusions to these subunits can alter the structure and properties of the carriers and hence their immunogenicity (see M. Sandkvist et al. J. Bacteriol. 169, pp4570-6, 1987, Clements et al. 1990 and M. Lipscombe et al. Mol. Microbiol. 5, pp 1385, 1990). Moreover, many heterologous proteins expressed in bacteria are not produced in soluble properly folded or active forms and tend to accumulate as insoluble aggregates (see C. Schein et al. Bio/Technology 6, pp 291-4, 1988 and R. Halenbeck et al. Bio/Technology 7, pp 710-5, 1989.
In our earlier unpublished international patent application PCT/GB93/01617, it is disclosed that by providing a DNA sequence encoding tetanus toxin C-fragment (TetC) linked via a “hinge region” to a second sequence encoding an antigen, the expression of the sequence in bacterial cells is enhanced relative to constructs wherein the C-fragment is absent. For example, the expression level of the full length P28 glutathione S-tranferase protein of
S. mansoni
when expressed as a fusion to TetC from the nirB promoter was greater than when the P28 protein was expressed alone from the nirB promoter. The TetC fusion to the full length P28 protein of
S. mansoni
was soluble and expressed in both
E. coli
and
S. typhimurium
. In addition, the TetC-P28 fusion protein was capable of being affinity purified by a glutathione agarose matrix, suggesting that the P28 had folded correctly to adopt a conformation still capable of binding to its natural substrate. It was previously considered that a hinge region, which typically is a sequence encoding a high proportion of proline and/or glycine amino acids, is essential for promoting the independent folding of both the TetC and the antigenic protein fused thereto. However, it has now been discovered, surprisingly in view of the previous studies on CT-B and LT-B referred to above, that when the hinge region is omitted between the TetC and a second antigen such as P28, the proteins making up the fusion do exhibit correct folding as evidenced by affinity purification on a glutathione agarose matrix.
Accordingly, in a first aspect, the invention provides a DNA construct comprising a DNA sequence encoding a fusion protein of the formula TetC-(Z)
a
-Het, wherein TetC is the C fragment of tetanus toxin, or a protein comprising the epitopes thereof; Het is a heterologous protein; Z is an amino acid, and a is zero or a positive integer, provided that (Z)
a
does not include the sequence Gly-Pro.
Typically (Z)
a
is a chain of 0 to 15 amino acids, for example 0 to 10, preferably less than 6 and more preferably less than 4 amino acids.
In one embodiment (Z)
a
is a chain of two or three amino acids, the DNA sequence for which defines a restriction endonuclease cleavage site.
In another embodiment, a is zero.
Usually the group (Z)
a
will not contain, simultaneously, both glycine and proline, and generally will not contain either glycine or proline at all.
In a further embodiment, (Z)
a
is a chain of amino acids provided that when a is 6 or more, (Z)
a
does not contain glycine or proline.
The group (Z)
a
may be a chain of amino acids substantially devoid of biological activity.
In a second aspect the invention provides a replicable expression vector, for example suitable for use in bacteria, containing a DNA construct as hereinbefore defined.
In another aspect, the invention provides a host (e.g. a bacterium) containing a DNA construct as hereinbefore defined, the DNA construct being present in the host either in the form of a replicable expression vector such as a plasmid, or being present as part of the host chromosome, or both.
In a further aspect, the invention provides a fusion protein of the form TetC-(Z)
a
-Het as hereinbefore defined, preferably in substantially pure form, said fusion protein being expressible by a replicable expression vector as hereinbefore defined.
In a further aspect the invention provides a process for the preparation of a bacterium (preferably an attenuated bacterium) which process comprises transforming a bacterium (e.g. an attenuated bacterium) with a DNA construct as hereinbefore defined.
The invention also provides a vaccine composition comprising an attenuated bacterium, or a fusion protein, as hereinbefore defined, and a pharmaceutically acceptable carrier.
The heterologous protein “Het” may for example be a heterologous antigenic sequence, e.g. an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.
Examples of viral antigenic sequences are sequences derived from a type of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, the CD4 receptor binding site from HIV, for example from HIV-1 or -2., hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV).
Examples of antigens derived from bacteria are those derived from
Bordetella pertussis
(e.g. P69 protein and filamentous haemagglutinin (FHA) antigens),
Vibrio cholerae, Bacillus anthracis
, and
E.coli
antigens such as
E.coli
heat Labile toxin B subunit (LT-B),
E.coli
K88 antigens, and enterotoxigenic
E.coli
antigens. Other examples of antigens include the cell surface antigen CD4
, Schistosoma mansoni
P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms, tapeworms,
Chlamydia trachomatis
, and malaria parasites, eg. parasites of the genus plasmodium or babesia, for example
Plasmodium falciparum
, and peptides encoding immunogenic epitopes from the aforementioned antigens.
Particular antigens include the full length
Schistosoma mansoni
P28, and oligomers (e.g. 2, 4 and 8-mers) of the immunogenic P28 aa 115-131 peptide (which contains both a B and T cell epitope), and human papilloma virus E7 protein, Herpes simplex antigens, foot and mouth disease virus antigens and simian immunodeficiency virus antigens.
The DNA constructs of the present invention may contain a promoter whose activity is induced in response to a change in the surrounding environment. An example of such a promoter sequence is one which has activity which is induced by anaerobic conditions. A particular example of such a promoter sequence is the nirB promoter which has been described, for example in International Patent Application PCT/GB92/00387. The nirB promoter has been isolated from
E.coli
, where it directs expression of an operon which includes the nitrite reductase gene nirB (Jayaraman et al, J. Mol. Biol. 196, 781-788, 1987), and nirD, nirC, cysG (Peakman et al, Eur. J. Biochem. 191, 315323, 1990). It is regulated both by nitrite and by changes in the oxygen tension of the environment, becoming active when deprived of oxygen, (Cole, Biochem, Biophys. Acta. 162, 356-368, 1968). Response to anaerobiosis is mediated through the protein FNR, acting as a transcriptional activator, in a mechanism common to many anaerobic respiratory genes. By deletion and mutational analysis the

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