Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-07-21
2004-01-20
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069300, C424S200100, C424S258100, C424S093200, C424S191100
Reexamination Certificate
active
06680182
ABSTRACT:
This invention relates to DNA constructs, replicable expression vectors containing the constructs, attenuated bacteria containing the constructs and vaccines containing the said bacteria.
In recent years, there has emerged a new generation of live oral salmonella vaccines based upon strains of Salmonella which have been attenuated by the introduction of a non-reverting mutation in a gene in the aromatic biosynthetic pathway of the bacterium. Such strains are disclosed, for example, in EP-A-0322237. The aforesaid live oral salmonella vaccines are showing promise as vaccines for salmonellosis in man and animals, and they can also be used effectively as carriers for the delivery of heterologous antigens to the immune system. Combined salmonella vaccines have been used to deliver antigens from viruses, bacteria, and parasites, eliciting secretory, humoral and cell-mediated immune responses to the recombinant antigens. Combined salmonella vaccines show great potential as single dose oral multivaccine delivery systems [C. Hormaeche et al, FEMS Symposium No. 63, Plenum, New York; pp 71-83, 1992].
There are problems to be overcome in the development of combined salmonella vaccines. A major consideration is obtaining a high level of expression of the recombinant antigen in the salmonella vaccine so that it will be sufficient to trigger an immune response. However, unregulated high level expression of foreign antigens can be toxic and affect cell viability [I. Charles and G. Dougan, TIBTECH 8, pp 117-21, 1990], rendering the vaccine ineffective or causing loss of the recombinant DNA. Several possible solutions to this problem have been described, such as expression from plasmids carrying essential genes, “on-off” promoters or incorporation of the foreign genes into the salmonella chromosome.
An alternative approach to overcoming the aforesaid problem would be to use a promoter which is inducible in vivo, and one such promoter is the
E. coli
nitrite reductase promoter nirB which is induced under anaerobiosis and has been used in biotechnology for the production of tetanus toxin fragment C (TetC) of
Clostridium tetani
[M. D. Oxer et al Nucl. Ac. Res., 19, pp 2889-92, 1991]. It has previously been found by the inventors of this application (S. N. Chatfield et al Bio/Technology, Vol. 10, pp 888-92 1992) that an Aro Salmonella harbouring a construct expressing TetC from the nirB promoter (pTETnir15) elicited very high anti-tetanus antibody responses in mice. The article by Chatfield et al was published after the priority date of this application.
However, we have also found that when it was attempted to express the P28 antigen from
Schistosoma mansoni
alone from nirB, the resulting construct was not immunogenic.
Tetanus toxoid has been extensively used as an adjuvant for chemically coupled guest epitopes [D. A. Herrington et al. Nature, 328, pp 257-9 1987]. The potent immunogenicity of TetC in Salmonella suggested to us that it may be possible to exploit this character to promote the immune response of the guest peptides or proteins. However, fusing two proteins together often leads to an incorrectly folded chimaeric protein which no longer retains the properties of the individual components. For example the B subunit 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 carrier and hence their immunogenicity [see M. Sandkvist et al. J. Bacteriol 169, pp 4570-6, 1987, Clements 1990 and M. Lipscombe et al [Mol. Microbiol 5, pp 1385 1990]. Moreover, many heterologous genes 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, 19893.
It is an object of the invention to overcome the aforementioned problems.
We have now found that efficient expression of recombinant antigens, and in particular fusion proteins, can be achieved in bacteria such as salmonellae, by the use of an inducible promoter such as nirB and by incorporating a flexible hinge region between two antigenic components of the fusion protein. The resulting recombinant antigens have been shown to have good immunogenicity. It has also been found, surprisingly, that enhanced expression of a protein can be obtained when a gene coding for the protein is linked to the gene for tetanus toxin C fragment.
Accordingly, in a first aspect, the present invention provides a DNA construct comprising a promoter sequence operably linked to a DNA sequence encoding first and second proteins linked by a hinge region, characterised in that the promoter sequence is one having activity which is induced in response to a change in the surrounding environment.
In another aspect, the invention provides a DNA construct comprising a promoter sequence operably linked to a DNA sequence encoding linked first and second proteins, wherein the first heterologous protein is an antigenic sequence comprising tetanus toxin fragment C or one or more epitopes thereof.
In a further aspect, the invention provides a replicable expression vector, suitable for use in bacteria, containing a DNA construct as hereinbefore defined. In a another aspect, the invention provides a fusion protein, preferably in substantially pure form, the fusion protein comprising linked (e.g. by a hinge region) first and second proteins, the fusion protein being expressed by a replicable expression vector as hereinbefore defined.
In a further aspect the invention provides a process for the preparation of an attenuated bacterium which comprises transforming 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 first and second proteins are preferably heterologous proteins and in particular can be polypeptide immunogens; for example they may be antigenic sequences derived from a virus, bacterium, fungus, yeast or parasite. In particular, it is preferred that the first said protein is an antigenic sequence comprising tetanus toxin fragment C or epitopes thereof.
The second protein is preferably an antigenic determinant of a pathogenic organism. For example, the antigenic determinant may be an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.
Examples of viral antigenic sequences for the first and/or second heterologous proteins are sequences derived from a type of human immuno-deficiency 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 or B 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 a
Chatfield Steven Neville
Dougan Gordon
Hormaeche Carlos Estenio
Khan Mohammed Anjam
Villarreal-Ramos Bernardo
Acambis Research Limited
Portner Ginny Allen
Wolf Greenfield & Sacks P.C.
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