Process for the production of vaccines and their use

Details Process for the production of vaccines and their use Process for the production of vaccines and their use

1995-01-10

2001-01-23

Housel, James (Department: 1641)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S184100, C435S252300, C435S173300

Reexamination Certificate

active

06177083

ABSTRACT:

The invention concerns a process for the production of vaccines and their use.
The main purpose of the immunological system in humans and animals is to resist and avoid pathological damage which arises as a result of degenerate cells, infectious viruses, bacteria, fungi or protozoa. A characteristic of the immunological system is that an increasingly stronger resistance occurs after repeated infections with pathogens. The aim of immunization is to build up the power of resistance of the immunological system against certain pathogens without causing corresponding diseases.
Antibodies and cellular T and B lymphocytes are responsible for the specific resistance to pathogens. An important prerequisite for this is the recognition of foreign structures such as e.g. those which occur on a bacterial cell. Depending on the stimulation of the immunological system a temporary or a lifelong immunity to pathogens can be built up by this process after immunization.
It is important for the effectiveness of vaccines that the immune response occurs to a sufficient extent. For this reason it is advantageous to use substances as immunogens which are to a large extent similar in their composition and in their structure to the pathogen against which it is intended to achieve immunity. Thus attenuated or dead bacteria or viruses, processed partial components of pathogens (membrane proteins of bacteria, structural proteins of viruses) or recombinant live vaccines (viruses or bacteria) are used. A disadvantage of using live bacteria or viruses as immunogens is that it is not possible to completely exclude an undesired pathogenic spread of the germs. This danger can be reduced by killing or fragmenting the bacteria and viruses before use as immunogens or vaccines. However, there is a risk that the antigenic determinants will be changed which can lead to a much smaller immune response.
The object of the present invention is therefore to provide immunogens and vaccines against gram-negative bacteria, which can be pathogenic, which do not have these disadvantages.
This object is achieved by a modified bacterium which is obtainable by transformation of a gram-negative bacterium with the gene of a lytically-active membrane protein from bacteriophages or with a lytically-active toxin release gene or with genes which contain partial sequences thereof coding for lytic proteins, culturing the bacterium, expressing this lytic gene, and isolating the bacterium modified in this way from the culture broth. The bacterium is suitable for use as a vaccine or adjuvant.
In the fermentation, the expression of the lytic gene is preferably delayed during the cell growth. This enables an adequate amount of bacteria to be formed first before lysis of these bacteria takes place. The usually impermeable cell wall complex of the bacteria is made permeable in this process such that the cytoplasmic components of the bacteria are released (Eur. J. Biochem. 180 (1989), 393-398). The morphology of the cells, for example, the rod-form of
E. coli
cells, is preserved. A tunnel structure is merely formed in a localized area of the membrane. The tunnel formation is accompanied by a fusion of the inner and outer membrane at the borders of the tunnel. The modified bacteria formed in this way are hereinafter denoted bacterial ghosts. Bacterial ghosts and their production are described for example in Eur. J. Biochem. 180 (1989) 393-398, Biochimie 72 (1990) 191-200 and J. Bacteriol. 172 (1990) 4109-4114. Their schematic structure is shown in FIG.
1
.
The bacterial ghosts consist of a cytoplasmic (inner) membrane, periplasmic space and outer membrane in which the integrity of the cell wall complex is preserved to a large extent. In the case of bacterial strains which have an additional S-layer coat (paracrystalline protein layer outside the outer membrane) this protein layer is also a component of the bacterial ghosts (Ann. Rev. Microbiol. 37 (1983), 311-339).
All gram-negative bacteria, preferably gram-negative pathogens such as those of the genera Neisseria, Escherichia, Bordetella, Campylobacter, Legionella, Pseudomonas, Shigella, Vibrio, Yersinia, Salmonella, Haemophilus, Brucella, Francisella and Bacterioides are suitable as bacteria (Schaechter, M, H. Medoff, D. Schlesinger, Mechanisms of Microbial Disease. Williams and Wilkins, Baltimore (1989)). Examples of pathogenic
E. coli
strains are: ATCC No. 31618, 23505, 43886, 43892, 35401, 43896, 33985, 31619 and 31617.
The bacterial ghosts are surprisingly well suited as immunogens whereby pronounced cellular and humoral immune responses occur.
A further advantage of the bacterial ghosts according to the present invention is that very many antigenic epitopes of the cell wall complex are presented by the bacterial ghosts. In addition, the lipopolysaccharide present in the bacterial envelope acts as a mitogen and also triggers a signal for cell division. As a result, one achieves an effective stimulation of the B-cell specific production of immunoglobulins.
Lytically-active membrane proteins of bacteriophages are preferably understood as membrane proteins from bacteriophages of the Microviridae class, preferably from icosahedral phages, lytic phages and phages containing ssDNA, which can infect Enterobacteriacae. Examples of these are the phages PhiXl74, S13, G4, G6, G14, PhiA, PhiB, PhiC, and PhiR which can infect
E. coli
C strains. Alpha 3, which can infect
E. coli
C and
E. coli
B strains, is also suitable. The phages K9, St-1, PhiK, PhiXtB and U3, which can infect
E. coli
K12 strains, are also suitable (Sinsheimer R. L. (1968) in: Prog. Nucl. Acid Res. Mol. Biol. (Davidson J. N. & Cohn W. W., eds) Vol.8, Academic Press, New York & London, pp. 115-169; Tessman E. S. & Tessmann I. (1978) in: The single-stranded DNA Phages (Denhardt D. T., Dressler D. & Ray D. S., eds.) Cold Spring Harbor Press, Cold Spring Harbor, pp. 9-29; Hayashi M., Aoyama A., Richardson D. L. & Hayashi M. N. (1987) in: The Bacteriophages, (Calendar R., ed.) Plenum Press, New York, pp. 1-71).
The production of genes, which contain partial sequences of lytic proteins or toxin release genes is preferably carried out according to methods used in genetic engineering via protein engineering, protein design or protein redesign as described for example in D. L. Oxender, C. F. Fox “Protein Engineering” A. R. Liss, Inc. New York, 1987.
In a preferred embodiment, the lytic gene contains the DNA sequence of the E-protein, the N-terminal, membrane-spanning domain of the E-protein, the DNA sequence of the L-protein, the C-terminal, membrane-spanning domain of the L-protein or the DNA sequence of the EL-hybrid protein (sequences cf. EP-A 0 291 021). Partial sequences thereof which act lytically are also suitable. Lytic proteins from the above-mentioned bacteriophages as well as other toxin release genes such as the colicin Lytic gene (Microbiol. Sciences 1 (1984) 168-175 and 203-205) are also preferred as lytically-active membrane proteins.
The invention also provides a process for the production of vaccines which is characterized in that a gram-negative bacterium is transformed with a gene of a lytically-active membrane protein from bacteriophages, with a lytically-active toxin release gene or with genes containing partial sequences thereof which code for lytically-active proteins. The bacterium is cultured, the gene is expressed and subsequently the bacterium modified in this way is isolated from the culture broth. The bacterial ghosts are then preferably purified further from non-lysed bacteria and cell fragments which may be still present, for example by density gradient centrifugation (e.g. with saccharose or ficoll).
The transformation by a vector and the expression of the plasmid-coded genes can be carried out according to processes familiar to one skilled in the art. The transformation is preferably carried out by electroporation or conjugation. Further details on suitable lytic genes and vectors for the transformation, expression and lysis may be found in Witte A. and Lubitz W., Eur. J. Biochem. 180 (1989) 393-398

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