Vaccine production of the Bacillus anthracis protective antigen

Details Vaccine production of the Bacillus anthracis protective antigen Vaccine production of the Bacillus anthracis protective antigen

1999-02-25

2001-07-31

Stucker, Jeffrey (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Recombinant or stably-transformed bacterium encoding one or...

C424S093460, C424S093462, C424S184100, C424S235100, C424S236100, C435S069300, C435S252300, C435S252310, C435S320100, C435S480000, C435S485000, C530S825000, C536S023700

Reexamination Certificate

active

06267966

ABSTRACT:

The present invention relates to the production of immunogenic proteins such as the protective antigen (PA) of
Bacillus anthracis
using recombinant DNA technology, to expression vectors and hosts used in the production process and to methods of their preparation.
Bacillus anthracis
, the causative agent of anthrax possesses two main virulence factors, a poly-D-glutamic capsule and a tripartite protein toxin. PA, the non-toxic, cell-binding component of the toxin, is the essential component of the currently available human vaccine. The vaccine is usually produced from batch cultures of the Sterne strain of
B. anthracis
, which although avirulent, is still required to be handled as a Class III pathogen. In addition to PA, the vaccine contains small amounts of the anthrax toxin moieties, edema factor and lethal factor, and a range of culture derived proteins. All these factors contribute to the recorded reactogenicity of the vaccine in some individuals. The vaccine is expensive and requires a six month course of four vaccinations. Futhermore, present evidence suggests that this vaccine may not be effective against inhalation challenge with certain strains (M. G. Broster et al., Proceedings of the International Workshop on Anthrax, Apr. 11-13, 1989, Winchester UK. Salisbury med Bull Suppl No 68, (1990) 91-92).
Previous workers have attempted to produce PA in
Escherichia coli
(M. H. Vodkin et al., Cell, (1983)34, 693-697) and
Salmonella typhimurium
(N. M. Coulson et al., Vaccine (1994) 12, 1395-1401) but for reasons which are not known, the level of production of PA was low in these organisms.
B. subtilis
is a harmless bacterium usually found in the environment. The possibility of using a genetically transformed
B. subtilis
to produce just PA, without other, undesirable components of the anthrax toxin, and without the need for rigorous containment has previously been proposed (B. E. Ivins et al., Infection and Immunity (1986), 54, 537-542). In particular, the gene encoding the protective antigen moiety of the tripartite exotoxin of
B. anthracis
was cloned into
B. subtilis
IS53 using the plasmid vector pUB110. Two clones, PA1 and PA2, were obtained, both of which produced more PA in liquid cultures than the Sterne strain of
B.anthracis
with levels of up to 41.9 mg/l being achieved. However, the organism also produced proteolytic enzymes, albeit in low quantities, which degraded the PA and made subsequent purification difficult.
This PA expression system (
B. subtilis
IS53 (pPA102)) has been evaluated (L. W. J. Baillie et al., Lett Appl. Microbiol. (1994) 19, 225-227). The system suffered from a down-regulation of the PA gene in early fermentation and was not proposed for large-scale production of PA antigen.
For production on an industrial scale, for example in vaccine production, it is important to maximise yields of protein for cost reasons. It is also helpful to obtain protein in the form of full length protein as this will be easier to purify than a selection of proteolytic fragments. The applicants have identified a number of expression factors which lead to improved levels of PA production.
Hence the present invention provides a recombinant microorganism which is able to express
Bacillus anthracis
protective antigen or a variant or fragment thereof which is able to generate an immune response in a mammal, said microorganism comprising a sequence which encodes PA or said variant or fragment thereof wherein either (i) a gene of said microorganism which encodes a catabolic repressor protein and/or AbrB is inactivated,and/or (ii) a region of the said PA sequence which can act as a catabolic repressor binding site is inactivated; and/or (iii) a region of the said PA sequence which can act as an AbrB binding site is inactivated.
Variants and fragments of PA must be able to produce an immune response in a mammal to whom they are administered. The immune response is suitably protective against infection by
Bacillus anthracis
although the protective effect may be seen only after repeated applications, as would be determinable by methods known in the art. Variants comprise peptides and proteins which resemble PA in their effect, but have different amino acid sequence. For example, variants may be 60% homologous to PA protein, suitably 80% homologous and more particularly at least 90% homologous. Fragments are suitably peptides which contain at least one antigenic determinant of PA, or variants thereof.
As used herein, the expression “functional equivalent” refers to moities such as nucleotide sequences or proteins, which although different to the reference moieties in certain respects, qualitively fulfill the same biological function.
A suitable microorganism for use as a host organism is a strain of
Bacillus subtilis
. Suitable strains are available from various sources including the Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio, USA from where strains such as IA147 and IA172 may be obtained. Additional strains are described in the literature, for example by Perego et al., Molecular Microbiology, (1988) 2, 689-699 where strains JH642 and JH12575 are described.
Preferably however, the microorganism of the invention comprises a strain which produces little or no proteases, since PA is very susceptable to decomposition by protease. A particularly preferred strain of
Bacillus subtilis
is a protease deficient strain. One such strain is
B.subtilis
WB600. This organism has been engineered to be deficient in six extracellular proteases (Xu-Chu Wu et al., J. Bacteriol. (1991) 173, 4952-4958). This strain is able to produce high yields of PA, for example of up to 40 mg/l which allowed the development of a purification strategy.
Catabolite repression of gene expression involves the trans-acting factors Catabolite control protein A (CcpA) and the phosphocarrier protein Hpr (Saier et al., Microbiology (1996), 142, 217-230). It has been proposed that CcpA binds to a catabolite-responsive element sequence in the control region of catabolite-sensitive operons and prevents transcription when glucose is present (Henkin et al. Molecular Microbiology (1991) 5, 575-584).
AbrB is a transition state regulator which prevents inappropriate gene expression during vegetative growth. Like CcpA, AbrB binds to DNA and prevents gene transcription (Strauch et al., J. of Bacteriology (1995), 177, 6999-7002).
Comparison of the level of PA expression from wildtype and mutant strains revealed that PA is subject to catabolite repression and AbrB regulation. In particular, it was found that PA levels from pPA101-1 are three fold higher in a ccpA mutant than in an otherwise isogenic parent, and eight fold higher in an abrB mutant. Thus, the introduction of mutations affecting catabolite repression and growth phase regulation into strains which are not deficient in these may result in an increase in the yield of PA in this host-vector system.
Screening of the PA control region for potential catabolite repressor binding sites revealed a region with 81% homology which started 37 bases downstream of the translational start point (see
FIG. 3
hereinafter). Screening with the abrB consensus sequence produced three regions which showed between 82-89% homology. The closest match was for a region which included the P2 translational start point and overlapped the ribosome binding site. Thus PA repression may be due to Catabolite control protein A (CcpA) and AbrB binding directly to these target sequences.
Suitably therefore, one or both of these sites are inactivated so as to increase the expression of PA. Inactivation may be effected by for example by mutation of the relevant site. The skilled person would be able to produce these, for example using site directed mutagenesis, and test for the required inactivation using routine techniques.
Preferably however, these activities are inhibited by inactivation of the gene which produce the relevant proteins (e.g. AbrB or CcpA). Either a host strain which is deficient in the genes which produce either or both or these proteins are emp

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