DE3586181T2 - IMPROVED ANTIQUE COMPOSITION. - Google Patents

Description

Technical area

The present invention relates to a method for producing an antigen composition or preparation capable of producing antibodies in vertebrates that bind to fimbrial type 4 antigens of bacterial pathogens and the antigen preparation itself.

The specific example used to illustrate this invention is that of the production of fimbrial antigens effective as a vaccine against B. nodosus, which is the major causative agent of footrot infection in sheep and other ungulates.

State of the art

B. nodosus is an anaerobic bacterium whose field isolates are characterized by the presence of surface filaments called fimbriae (or more commonly "pili") that are about 6 nm in diameter and up to several hundred nm long. In other bacteria, fimbriae have adhesive properties and although the exact function of fimbriae in B. nodosus has not been clearly defined, it seems likely that they are involved in the attachment to and/or colonization of epithelial tissue in the hoof. Fimbriae of B. nodosus exhibit polar localization on the cell and appear to be involved in surface locomotion through a phenomenon known as ciliary motility. Related fimbriae are found in a number of gram-negative species, including the genera Pseudomonas, Neisseria, Moraxella, Acinetobacter, Bacteroides and Eikenella.

In the case of B. nodosus, a large body of evidence has accumulated over the past decade suggesting that fimbriae play a central role in both the infectivity of the bacteria and the immune response of the sheep. Fimbriae are also involved in pathogenesis and immunity in other bacterial pathogens. Vaccination of sheep with either whole cells or purified fimbriae from B. nodosus confers resistance to homologous footrot infections.

There are at least 8 or 9 major serogroups of B. nodosus (comprising a number of subtypes) classified based on the "K" agglutination test.

It has been found that the fimbriae are the antigen involved in the specific agglutination reaction and, accordingly, the range of effective immunity conferred by vaccination with whole cells or fimbrial preparations is limited to the serogroup or serotype in question.

The commercially available vaccines against footrot consist of a mixture of whole cells of different B.nodosus serotypes. However, the high cost of these vaccines, which is about an order of magnitude higher than that of other comparable vaccines (e.g. against the group of clostridial infections), remains a serious obstacle to their widespread use and acceptance by the sheep industry. As a result, current methods of controlling footrot continue to rely heavily on topical application of therapeutic agents that have little or no effect on preventing the disease.

The high cost of producing vaccines against footrot is directly related to the difficulty of Cultivation of B. nodosus with fimbriae. This bacterium has very difficult growth requirements, including complex culture media and the absence of oxygen. Furthermore, since fimbrial expression is an unstable feature of B. nodosus, particularly under the liquid culture conditions required for commercial production, batch-to-batch variation in fimbrial antigen yields can affect the quality and reliability of vaccine preparations, imposing a significant cost burden in the area of quality control testing.

The Applicant's pending Australian patent application No. 34979/84 describes the production of the B. nodosus fimbrial subunit antigen expressed from cloned gene copies in Escherichia coli. The antigen is assembled into the mature fimbriae in these cells.

This invention describes the further development of this process by describing the generation of supramolecular, extracellular fimbrial structures from recombinant bacterial hosts. This brings significant advantages both in terms of the antigenicity of the preparation (for vaccination purposes) and the practicality and economics of antigen production. The invention utilizes a compatible fimbrial assembly system in a suitable host for type 4 fimbriae, such as Pseudomonas aeruginosa. This process is applicable to the generation of fimbriae as vaccine antigens active against a range of bacterial pathogens that produce type 4 fimbriae.

Disclosure of the invention

The present invention provides a method for producing an antigen preparation for use in generating antibodies effective against a bacterial species which naturally produces type 4 fimbriae, comprising culturing genetically engineered aerobic or facultatively anaerobic host cells having type 4 fimbriae which contain the genes encoding the fimbrial subunit structural antigens of at least one strain of the bacterial species and an endogenously compatible system for morphogenetic assembly of type 4 fimbriae to produce mature fimbriae as extracellular structures, and completely or partially harvesting the fimbriae substantially free from the host cells.

In another aspect, the invention provides a genetically engineered aerobic or facultatively anaerobic bacterium having type 4 fimbriae, the bacterium being characterized by containing exogenous genes encoding the fimbrial subunit structural antigens of at least one bacterial strain that naturally produces type 4 fimbriae, such that expression of the exogenous genes causes the production of fimbriae of the at least one strain as extracellular structures.

B. nodosus fimbrial strands consist of a small repeated polypeptide subunit (protein) of about 150 amino acids, of about 17,000 daltons (17 kD) molecular weight, the exact size of which appears to vary in different serotypes of the bacteria. Similar serological and structural variation occurs in the fimbrial subunits of other pathogens with type 4 fimbriae, such as Neisseria gonorrhoeae, Neisseria meningitidis, and Moraxella bovis, presumably as a mechanism to avoid host immune responses. We have found that fimbrial preparations derived from B. nodosus also contain another significant antigen, a protein of about 80,000 daltons (80 kD) molecular weight. Our studies indicate that this protein is physically attached to the fimbrial strand and may represent the basal protein which which anchors fimbriae to the surface of bacterial cells. In B. nodosus, this protein, like the fimbrial subunit, shows both structural and antigenic variation between different strains and is another major polypeptide antigen against which sheep react when infected with B. nodosus or vaccinated with either whole cell or isolated fimbrial preparations.

We have found that using Western transfer matrix analysis, where fimbriae from a panel of different B. nodosus isolates of different serotypes were separated by gel electrophoresis, transferred to paper and treated with a panel of homologous and heterologous antisera, it is the fimbrial strand (i.e., the 17 kD subunit antigen) that is the serogroup and serotype specific antigen and therefore the primary protective antigen.

The applicant's pending Australian patent application No. 34979/84 describes the production of the subunit structure antigen from B. nodosus fimbriae from cloned gene copies expressed in E. coli. In this case, however, the B. nodosus fimbrial subunit is not assembled into mature fimbrial structures on the cell surface, but is instead embedded in the membrane fraction of the cell. This leads to severe disadvantages both in terms of the antigenicity of the material and the difficulty of isolating and purifying it, although even the expression level of the protein can be increased by appropriate manipulation of the cloned gene. The present invention relates to the further development of the vaccine by describing the production of the specific active antigen component in the form of mature fimbrial structures from new genetically engineered bacterial host cells. The strategies and details of these constructions are described below.

The production of the antigen in the form of mature fimbriae has significant immunological and technical advantages in terms of the efficacy of the vaccine, the simplicity of its components and the ease and economy of its manufacture, which are based on the following considerations:

(i) Mature fimbriae appear to elicit a more intense and better (i.e., K-agglutinating) immunological response than the equivalent dose of fimbrial subunit protein. A serological K-agglutination titre of about 5000 is generally considered to be the minimum response corresponding to sufficient protective immunity against infection with a given strain of B. nodosus. This level of response (and up to an order of magnitude higher) is readily achieved by vaccination with mature fimbriae but not with the isolated subunit protein, which generates only low levels of K-agglutinating serum antibodies.

(ii) The expression of mature fimbriae on the surface of the recombinant cell allows the simple and convenient separation of the fimbrial antigens from the host bacterium on an industrial scale. This has several advantages:

(a) the production of the antigen for use in an effective vaccine formulation does not require expensive or complicated extraction or purification procedures,

b) the host cells can be removed from the vaccine preparation. This avoids potential problems associated with the use of recombinant organisms in vivo due to biosafety/biohazard considerations and/or the presence of toxic or other undesirable components in the host bacterium,

c) the removal of host cells also eliminates the possibility of antigenic competition between cellular components and the fimbrial antigens,

(d) the use of vaccines based on isolated fimbriae should reduce the occurrence of a necrotic reaction at the vaccination site. This is a common problem with currently available B. nodosus whole cell vaccines, especially when coupled with the use of strong immunological adjuvant conditions such as oil emulsion and/or aluminium sulphate adsorption.

(e) the use of isolated fimbriae in vaccine formulations should facilitate standardisation of ingredients and quality control, in particular in the context of a multivalent vaccine,

iii) the expression of mature fimbriae in a recombinant host allows the possibility of incorporating more than one type of antigen of a structural subunit into the actual strands produced. This would produce a simpler vaccine with broad activity against more than one type of antigen, either with respect to the different serogroups of B. nodosus or as a mixed vaccine effective against type 4 fimbrial antigens from more than one pathogenic species.

The biosynthesis and assembly of fimbrial strands, ie, morphogenetic expression, appears to require the involvement of several genes in addition to the one encoding the structural subunit itself. Studies of the plasmid- and chromosomally encoded fimbrial systems of type 1 or type 2 of uropathogenic and enterotoxigenic strains of E. coli, for example, have shown that at least 5 to 7 genes are involved in the process; one encodes the fimbrial subunit, another one encodes a basal protein. and the rest for other polypeptides whose exact functions are not yet well understood but which appear to act as assembly factors in the construction and assembly of the fimbrial strand. These genes are normally clustered in the genome and occupy up to 5 to 10 kilobases (kb) of DNA sequence information.

The gene encoding the 17 kD subunit structure of B. nodosus serotype A1 fimbriae is located on a 5.5 kb HindIII segment of B. nodosus DNA cloned into an E. coli plasmid vector as described in our pending Australian patent application No. 34979/84. The gene is expressed in E. coli from a promoter within the cloned sequence. This gene is representative of those found in other serotypes of B. nodosus as well as in other bacteria with type 4 fimbriae. In this context, we have also cloned, using the same general strategies, the genes encoding the structural subunits of Pseudomonas aeruginosa strain PAK fimbriae and those of a number of different serotypes of B. nodosus, although at least in the latter cases there appears to be some degree of restriction site polymorphism and sequence divergence between the different serotypes. For example, the gene encoding the structural subunit of B. nodosus serogroup C fimbriae is located on a cloned (HindIII) fragment of about 4.5 kb, which itself contains two additional internal HindIII sites, at least one of which appears to be within the functional gene. The serogroup F fimbrial subunit gene is located on a cloned HindIII fragment of about 3 kb. In no case, however, have we observed the formation of mature fimbrial structures on the surfaces of recombinant E. coli host cells expressing these cloned genes. These clones also do not appear to express the basal protein antigen. The likely reason for the lack of assembly of mature fimbriae in the recombinant cells is that the cloned DNA segments do not contain all the information required for proper morphogenetic expression, ie some or all of the genes encoding the basal protein and assembly factors and/or associated promoters. On the other hand, it is possible that some or all of these other genes are not properly expressed or function in the E. coli host cell environment.

The problem of obtaining morphogenetic expression of fimbriae in recombinant host cells can be addressed by attempting to clone and express the other required genes. The initial strategy here involves extending the cloned sequences flanking the structural subunit gene, assuming that the genes are indeed clustered as in E. coli, by chromosomal walking using a series of overlapping clones generated by digestion with different restriction endonucleases (with recognition sequences of 6 bases) or by partial digestion with a restriction enzyme such as Sau3A (which has a recognition sequence of 4 bases). It is possible that low copy number vectors are required to avoid deleterious gene dosage effects associated with the expression of membrane proteins, and we have evidence to suggest that such genes are located in the vicinity of the B. nodosus fimbrial subunit genes, which has caused problems in cloning these sequences. However, once obtained, an extensive region around the subunit gene can be reconstructed and tested in a number of bacterial hosts for the ability to produce mature fimbrial structures, using standard techniques for fimbriae isolation, immunological and electrophoretic analyses, and electron microscopic Examination of whole cells and isolated fractions. An alternative strategy is to use transposon mutagenesis to generate a series of fim⊃min; mutants and to clone those genes that are affected using the transposon as a genetic or hybridization marker. This strategy does not require that the genes be clustered in the bacterial genome. The genes involved can (in any case) be reconstructed into a functional system containing the fimbrial subunit with appropriate molecular genetic manipulation as required to maximize and stabilize fimbrial biosynthesis and to provide the correct balance of expression of each of the genes involved.

The problem of morphogenetic expression of fimbriae can also be approached by genetic or molecular genetic complementation of the fimbrial subunit gene with a compatible assembly system derived wholly or in part from other bacteria. Although this method involves gene isolation and/or modification, as discussed above, it has the advantage of providing possibly the simplest solution to the problem. Attempts to complement missing functions by subcloning the B. nodosus fimbrial subunit gene into plasmid vectors containing E. coli fimbrial assembly genes have so far been unsuccessful, as have similar experiments carried out with the N. gonorrhoeae fimbrial subunit gene, while Klebsiella pneumoniae fimbriae have been expressed from cloned DNA segments inserted into both E. coli and Salmonella typhimurium. These results suggest that there is some fundamental incompatibility between fimbriae of type 4 and those systems found in Enterobacteriaceae with fimbriae, comprising the genera Klebsiella, Escherichia and Salmonella. There are indeed considerable differences between the structural subunits of type 4 fimbriae and those found in E. coli, particularly with regard to the location of hydrophobic regions, which occur at the N-terminus in the former and at the C-terminus in the latter. On the other hand, there are great similarities between the fimbrial subunits of various bacteria with type 4 fimbriae, including Bacteroides nodosus, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella nonliquifaciens and Moraxella bovis (see Table 1), which are the only species for which detailed data are currently available. Sequence analyses have shown that all fimbrial subunits of these bacteria share the distinguishing feature of possessing the unusual amino acid methylphenylalanine (Me-Phe) as the first residue in the mature protein. These fimbrial subunits also share a very high degree of amino acid sequence homology within the first 20 to 25% of the protein, to the extent that where changes have occurred, they generally involve conservative amino acid substitutions. The total length of these proteins varies in the range of about 140 to 160 amino acids. Strong homology is observed within the first 32 residues, with further zones of homology extending to about residue 50. Furthermore, the subunits also appear to share a similar and unusual 6 to 7 amino acid long, positively charged N-terminal leader sequence in the primary translation product, which is cleaved from the protein at a stage prior to incorporation into the mature fimbrial strand. Interspecies variation among these fimbrial subunits occurs primarily in the two carboxy-terminal thirds of the protein. Intraspecific variation (ie between different serotypes) also occurs in this region and includes not only amino acid substitutions but also small insertions and deletions in the reading frame. Table 1 Comparison of the N-terminal amino acid sequences of the fimbrial subunits of B. nodosus (1), P. aeruginosa (2), N. gonorrhoeae (3), N. meningitidis (4), M. nonliquefaciens (5), and M. bovis (6).

Legend: The first 48 residues of the mature fimbrial subunit are shown, numbered from the N-terminal methylphenylalanine (MeF) residue, as well as the leader sequence present in the primary translation product (where this has been determined). The arrow (T) indicates the cleavage site, although in a subset of molecules the cleavage appears to occur on the carboxy-terminal side of the MeF residue (--> ). Dashes indicate the absence of a residue at that point. Residues are listed using the 1-letter code for amino acids as proposed by the IUPAC-IUB Commission on Biochemical Nomenclature.

At N. gonorrhoeae, it has been suggested that the conserved amino-terminal portion of the fimbrial subunit is involved in subunit-subunit interactions within the fimbrial strand, and this may well be the case. However, we reasoned that the high conservation of this sequence across a number of different bacterial species and genera possessing type 4 fimbriae has occurred because this amino-terminal region contains important topogenic signals for fimbrial morphogenesis involving the interaction of the structural subunit with the assembly system, factors, and mechanism. If this is so, the fimbrial subunits containing these signals should function interchangeably in the type 4 (MePhe) group. This is the crux of the invention which we have now demonstrated by the morphogenetic expression and production of the B. nodosus fimbrial type from a cloned subunit gene inserted into the fimbriate P. aeruginosa and the use of this material as an effective vaccine against footrot in sheep. P. aeruginosa was chosen (among available bacteria with type 4 fimbriae) as the (prototype) receiving host on the basis that this species is a genetically well-characterized aerobe which is easily cultured on relatively simple media and for which plasmid shuttle vector systems are available for cloning and gene transfer.

Type 4 fimbriae are defined here as those fimbriae that (i) are commonly found in bacterial species exhibiting the phenomenon known as ciliation, although this is not always present (see below); (ii) exhibit a polar or polar-like localization at the surface of the cell; and (iii) possess a highly conserved sequence in the amino-terminal region of the structural subunit, i.e. (a) a similar short (6 to 7 amino acids long) leader sequence in the primary translation product, followed by (b) a hydrophobic region showing (more than 75%) homology with the following 32 amino acid consensus sequence, which begins with the (modified) phenylalanine residue that normally occurs at position 1 of the mature protein:

(see Figure 1).

The parentheses indicate that any of the residues within can be present in that position. These pairs are conservative in the sense that each of the amino acids in the pair has a similar structure and properties, e.g. isoleucine/valine (I/V), isoleucine/leucine (I/L), glutamine/asparagine (Q/N), arginine/lysine (R/K), and to a lesser extent alanine/serine (A/S).

It should be noted that although ciliation is commonly exhibited by strains containing type 4 fimbriae, this is not always or necessarily the case. For example, some (mutant) strains of P. aeruginosa have been found to retain polar type 4 fimbriae, although they have apparently lost the ability to exhibit ciliation. Fimbriae of type 4 are found in B. nodosus, P. aeruginosa, N. gonorrhoeae, N. meningitidis, M. nonliquefaciens and M. bovis and probably also in Acinetobacter calcoaceticus, Bacteroides ureolyticus, Eikenella corrodens, Moraxella kingae, Moraxella lacunata, Moraxella osloensis, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas maltophilia, Pseudomonas pseudoalcaligenes, Pseudomonas putrefaciens, Pseudomonas solanacearum, Pseudomonas stutzeri and Pseudomonas testosteroni. Fimbriae Type 4 may also be found in some Gram-positive species, such as Streptococcus sanguis.

The activity of the antigen preparation can be represented as the ability to generate antibodies or other immune response in vivo against the species of bacteria in question and/or the ability to bind antibodies against the bacteria. Thus, the antigen preparation can be useful either as a vaccine or as a diagnostic test for the bacteria or their antibodies.

The source of the fimbrial subunit gene can be any species or strain possessing type 4 fimbriae. The host organism can contain the fimbrial subunit gene from a number of different bacteria having type 4 fimbriae. In preferred embodiments of the invention, the host organism contains fimbrial subunit genes for a number of serotypes of a single species having type 4 fimbriae.

The host cell may be any compatible species or strain that expresses type 4 fimbriae and is preferably an aerobe or a facultative anaerobe that can be transformed with and maintain a suitable plasmid vector encoding at least the fimbrial subunit gene. The host cell may contain the exogenous genes either in a plasmid or by insertion into its chromosome. The plasmid may be either exogenous or endogenous.

In preferred embodiments of the invention, the genes for morphogenetic assembly of the fimbriae are endogenous to the host cell, which itself is a species with type 4 fimbriae. In other embodiments of the invention, however, the genes may be exogenous, from the same species as the subunit genes or from a different species with type 4 fimbriae.

In a preferred embodiment, a promoter is used to increase the level of gene expression, which may be any promoter active in the host species, with or without accompanying regulatory sequences. Preferred promoters include the PL and PR promoters of bacteriophage lambda, as well as other strong natural or synthetic promoters, such as the tac hybrid promoter. The regulation of the promoter may be chemical or physical, as is well known in the art. In an alternative embodiment of the invention, the molecular structure of the subunit gene may be altered, particularly to make its leader sequence more homologous to that of the host cell, in order to improve morphogenetic expression in a particular host.

When used as a vaccine, the antigen preparations are preferably administered with a common adjuvant and carrier system.

Short description of the drawings

The following example is described with reference to the attached drawings, in which:

Figure 1 shows the origin and construction of pJSM202. A full description is given in the text. The cross-hatched sequences in pBA121 show those originally cloned from B. nodosus VCS 1001, with the inner dark segment indicating the Dra1 fragment containing the fimbrial subunit gene (see Figure 2 for details). The hatched sequences in pPL-Lambda show the BamH1 segment (originally derived from bacteriophage lambda) containing the PL promoter and into which the Dra1 fragment of pBA121 was cloned (at the Hpa1 site). The exact orientation of this construction in PJSM202 has not been determined (i.e., this segment may be flipped). The thin line in plasmids pBA121, pPL-Lambda and pJSM129 represents sequences derived from pBR322. The thin line in pJSM202 represents sequences derived from pKT240. Various restriction endonuclease sites are indicated accordingly. Numbers refer to the distance in kilobases (kb) around each plasmid. Arrows indicate the direction of transcription and the approximate boundaries of the gene in question. Abbreviations are: fim, fimbrial subunit gene; amp, ampicillin (or carbenicillin) resistance; tet, tetracycline resistance (not functional in pBA121); kan, kanamycin resistance; ori, origin of replication; mob, mobilization functions.

Figure 2 shows the nucleotide sequence of the gene encoding the fimbrial subunit of B. nodosus VCS 1001 and flanking DNA. The deduced amino acid sequence of the fimbrial subunit is shown using the standard one-letter code as proposed by the IUPAC-IUB Commission on Biochemical Nomenclature. The positions of the Dra1 restriction endonuclease sites flanking the gene (TTTAAA) and the PvuII (CAGCTG) and Pst1 (CTGCAG) sites located within the gene are indicated by the dashes.

Figure 3 shows Western transfer analysis of cell pellet and supernatant fractions from P. aeruginosa PAK/2Pfs and E. coli E418 cells containing pJSM202. Recombinant cells were cultured on Luria Broth agar medium with the required antibiotic. P. aeruginosa cells were cultured overnight at 37ºC. E. coli cells were incubated for 8 hours at 30ºC, then shifted to 42ºC for 4 hours to induce synthesis of the fimbrial subunit from the (de-repressed) PL promoter. Cells were harvested in phosphate buffered saline (PBS), subjected to mechanical mixing and the cells pelleted by centrifugation. The supernatant was then removed and the cells resuspended in fresh PBS. resuspended. Equal amounts of cell and supernatant fractions were then electrophoresed on SDS-urea-8 to 15% polyacrylamide gradient gels with a standard Tris-glycine (Laemmli) buffer system. The resulting gel separations were then electrophoretically transferred to nitrocellulose paper, incubated with anti-B. nodosus fimbrial antiserum and then with 125I-labeled protein A under standard conditions. The position of bound antibodies was visualized by autoradiography. Lanes 2, 4, 6 and 8 contain cell pellet material. Lanes 3, 5 and 7 contain supernatant fractions. Lanes 2 and 3, 4 and 5 are from two independent primary clones of P. aeruginosa PAK/2Pfs transformed with pJSM202. Lanes 6 and 7 are from E. coli E418 containing pJSM202. Lane 8 shows P. aeruginosa PAK/2Pfs cells containing pJSM125. Lane 1 contains purified fimbriae from B. nodosus VCS 1001.

Figure 4 shows the results of the purification of fimbriae from P. aeruginosa PKN-A1. Cells were cultured on agar plates as described in the text and harvested in phosphate buffered saline (PBS). Cells were removed by centrifugation and the supernatant was saved (supernatant fraction). Cells were then resuspended in PBS, subjected to mechanical mixing, and again removed by centrifugation to yield a mixed supernatant fraction. The supernatant and mixed supernatant fractions were then divided equally and subjected to either isoelectric precipitation at pH 4.5 or precipitation with 0.1 M MgCl2. Precipitates were recovered by centrifugation, redissolved in PBS, and analyzed by SDS-polyacrylamide gel electrophoresis as described in the legend to Figure 3. The protein bands were visualized by staining with Coomassie blue R250. Lanes 2 and 3 contain fimbriae obtained from the supernatant fraction by isoelectric and MgCl₂ precipitation, respectively. Lanes 4 and 5 contain fimbriae obtained from the pooled supernatant fraction by isoelectric and MgCl2 precipitation, respectively. Lanes 2 to 5 all contain an amount of material equivalent to 3.5% of that obtained from a single Petri dish culture of PKN-A1 cells. Lane 1 contains fimbriae purified from P. aeruginosa PAK/2Pfs (approximately 5 µg). Lane 6 contains fimbriae purified from B. nodosus VCS 1001 (approximately 10 µg).

Figure 5 shows the electrophoretic and immunological properties of fimbriae purified from P. aeruginosa PKN-A1. Fimbriae were purified (by two isoelectric precipitations) from two independent primary clones of P. aeruginosa PAK/2Pfs transformed with pJSM202 (lanes 3 and 4) and compared with fimbriae obtained from the host strain P. aeruginosa PAK/2Pfs (lanes 1 and 5) and from B. nodosus VCS 1001 (lanes 2 and 6). These fimbriae (approximately 2 µg in each case) were separated on SDS-polyacrylamide gels as described in the legend to Figure 3 and stained with Coomassie blue R250 (panel A). Similar (unstained) separations were subjected to Western transfer analysis as described in the legend to Figure 3 using either anti-P. aeruginosa PAK/2Pfs fimbrial antiserum (panel B) or anti-B. nodosus fimbrial antiserum (panel C). Only the lower (fimbrial subunit-containing) part is shown.

Figure 6 shows the electrophoretic and immunological analysis of the antigen samples used in the vaccination experiment. Lane 2 contains whole cells of B. nodosus VCS 1001, lane 3 contains fimbriae purified from B. nodosus VCS 1001, lane 4 contains fimbriae purified from P. aeruginosa PAK/2Pfs, lane 5 contains fimbriae purified from P. aeruginosa PKN-A1, lane 6 contains fimbriae purified from P. aeruginosa PKN-A1 (using MgCl₂) (see text for details). Lanes 1 and 7 contain a mixture of standard protein molecular weight markers. Samples were separated by electrophoresis on SDS-polyacrylamide gels and either stained with Coomassie blue R250 (panel A) or subjected to Western transfer analysis using anti-P. aeruginosa PAK/2Pfs fimbrial antiserum (panel B) or anti-B. nodosus VCS 1001 fimbrial antiserum (panel C) as described in the legends to Figures 3 and 5. In panel A the entire gel is shown, while in panels B and C only the lower part containing the fimbrial subunits is shown.

Best mode for carrying out the invention Example

The specific example used to illustrate this invention involves the morphogenetic expression of B. nodosus fimbriae from a cloned subunit gene inserted into a fimbriate strain of Pseudomonas aeruginosa after appropriate genetic engineering manipulations, and the use of fimbrial preparations derived therefrom as an effective vaccine against footrot infection in sheep caused by the homologous B. nodosus strain.

The B. nodosus fimbrial subunit gene used in this example was from B. nodosus VCS 1001, formerly known as strain 198 (ATCC No. 25549), which is the selected prototype strain of serogroup A (subtype 1). The gene is located on a 5.5 kb HindIII fragment of B. nodosus DNA originally prepared by immunological screening of a cloned HindIII library prepared in E. coli using the positive selection plasmid vector pTR262, as described in the applicant's pending Australian patent application No. 34979/84.

The 5.5 kb HindIII segment was then subcloned into the plasmid vector pBR322 to give pBA121 (Anderson, B.J. et al., J.Bacteriol. 160, 748-754 (1984)). Figure 1 shows the restriction map of this plasmid and the location of the structural gene encoding the fimbrial subunit. The nucleotide sequence of this gene and adjacent DNA has now been determined and is shown in Figure 2.

The P. aeruginosa host strain used in this example is P. aeruginosa strain PAK/2Pfs (ATCC No. 53308), which is a multifimbrial mutant of strain PAK. The vector plasmid used in this example is pKT240 (Bagdasarian, M.M. et al., Gene 26, 273-282 (1983)), which is a broad host range plasmid that can be replicated and propagated in both E. coli and P. aeruginosa and contains genetic markers for penicillin (ampicillin, carbenicillin) and kanamycin resistance as well as a number of unique restriction sites for gene cloning (see Figure 1).

A. Genetic constructions and manipulations

In the initial constructs, the 5.5 kb HindIII fragment containing the B. nodosus fimbrial subunit gene was cloned directly into the HindIII site of pKT240 (see Figure 1) to give pJSM125. This plasmid was then transformed into competent P. aeruginosa PAK/2Pfs cells (as described below). Immunological analyses of the resulting transformants showed that the B. nodosus fimbrial subunit was expressed in these cells, but at a relatively low level. Nevertheless, there was clear evidence that at least some of the B. nodosus fimbrial subunits produced in these recombinant cells were assembled into mature fimbriae, since the antigen signal copurified with the fimbriae and persisted in the most highly purified fractions. However, the signal was weak and the majority of the fimbriae consisted of the endogenous Pseudomonas subunit normally produced by the host strain.

The problem of low expression of the B. nodosus fimbrial subunit gene in P. aeruginosa was overcome by placing this gene under transcriptional control of the strong promoter PL from bacteriophage lambda, as shown in Figure 1 and detailed below:

pBA121 was digested with the restriction endonuclease DraI, which cleaves at convenient sites flanking the fimbrial subunit gene, once 30 nucleotides upstream of the initiation codon (ATG) and further 69 nucleotides downstream of the termination codon (TAA), to yield a blunt-ended, 576 base pair gene cassette (see Figure 2). This cassette was purified by gel electrophoresis and ligated into the (blunt-ended) Hpa1 site located downstream of the PL promoter in the plasmid pPL-Lambda (Pharmacia P-L Biochemicals) to yield pJSM129 (Figure 1). All constructions involving the PL promoter were carried out in E. coli strain E418 grown at 30°C. E. coli E418 is a lambda lysogen strain containing the temperature-sensitive C1857 repressor gene. The correct orientation of the DraI insertion with respect to the PL promoter in the plasmid was tested by reference to the internal PvuII and PstI sites within the fimbrial subunit gene and confirmed by the overproduction of the antigen in E. coli after shifting the culture to 42ºC. This construction is apparently lethal in E. coli cells lacking a functional C1 repressor, presumably because the cells are unable to cope with the large amount of B. nodosus fimbrial subunit that is produced and deposited in the membrane.

The B. nodosus fimbrial gene/PL promoter construct was then transferred into the vector pKT240 as follows: pJSM129 was the restriction endonuclease BamH1 to release a fragment of approximately 1.75 kb containing the PL promoter and fimbrial subunit gene (see Figure 1). This fragment was purified by gel electrophoresis and ligated into the corresponding site in pKT240 to yield the plasmid designated pJSM202 (ATCC No. 40203).

The presence of the BamH1 fragment in this plasmid was confirmed by restriction endonuclease analysis, although the orientation of this insertion was not determined. Further characterization showed that E. coli E418 cells containing pJSM202 produced high levels of the B. nodosus fimbrial subunit when grown at 42°C (see Figure 3), and that pJSM202, like pJSM129, does not produce viable transformants in E. coli strains lacking the C1 repressor. (In both cases, however, this ability can be recovered by inserting a cloned DNA segment containing the C1857 gene into the plasmids themselves.)

All constructions, manipulations and analyses described above were performed using standard genetic engineering methods.

B. Morphogenetic expression of B. nodosus fimbriae in Pseudomonas aeruginosa PAK/2Pfs (ATCC No. 53308) containing pJSM202 (ATCC No. 40203) Transformation of P. aeruginosa fimbriate with pJSM202

P. aeruginosa strain PAK/2Pfs was made competent for DNA transformation as follows: Cells were cultured overnight at 43ºC (to stationary phase) in KNO₃ medium (2.5% Oxoid Nutrient Broth No. 2, 0.5% yeast extract and 0.4% KNO₃). A sample of the culture was then diluted 1:25 with fresh KNO₃ medium and incubated with shaking at 37ºC. until the culture reached the early-mid log phase of growth. The culture was then rapidly cooled on ice and the cells were recovered by centrifugation. All subsequent steps were carried out at 4ºC. The cells were washed twice by resuspension in one-fifth volume of 0.1M MgCl₂ and recentrifugation. Then the cells were resuspended in 1/8 volume of 0.15M MgCl₂ and kept on ice for 20-30 minutes. The cells were recovered again by centrifugation and finally resuspended in one-twentieth volume of 0.15M MgCl₂ and kept on ice ready for transformation.

pJSM202 was transformed into competent P. aeruginosa PAK/2Pfs cells as follows: Approximately 200 to 400 ng of purified plasmid DNA (in 5 to 10 µl buffer) was added to 0.2 ml of cell suspension, mixed, and placed on ice for 1 hour. The transformation mixture was then heat shocked at 42°C for 2 minutes, subsequently diluted with an equal volume of KNO3 medium and incubated at 37°C for 1 hour to allow phenotypic expression of antibiotic resistance genes on the plasmid. Samples (0.1 ml) were then plated on nutrient (Luria Broth) plates with carbenicillin (1 mg/ml). (P. aeruginosa PAK/2Pfs is not particularly sensitive to ampicillin or kanamycin). Plates were incubated at 37ºC and visible colonies appeared in approximately 2 days.

Several transformants were selected and all were confirmed to contain pJSM202 by plasmid isolation and restriction analysis. This plasmid is therefore not lethal to P. aeruginosa PAK/2Pfs (or other fimbriate strains tested), despite the fact that we found that the PL promoter is highly active in these cells and that large amounts of the B. nodosus fimbrial subunit are produced. However, the transformants were found to grow relatively slowly and to have a short storage stability. than plate cultures, which required regular subculturing to maintain viability. These transformants also do not appear to be stable under standard freezing conditions (as suspensions in 15% glycerol broth at -70ºC). For this reason, we periodically regenerated fresh batches by retransforming P. aeruginosa PAK/2Pfs with pJSM202. These recombinant transformed cells are (generally) referred to as PKN- A1.

(ii) Isolation and analysis of fimbriae produced by P. aeruginosa PKN-A1 (PAK/2Pfs + pJSM202)

An examination of P. aeruginosa PKN-A1 cells has revealed that not only are large amounts of the B. nodosus fimbrial subunit produced in these cells, but that the fimbrial strands assembled by these cells are in fact composed entirely or almost entirely of this protein, with little or no P. aeruginosa-type subunits present.

The morphogenetic expression of B. nodosus fimbriae in PKN-A1 has been demonstrated by fimbrial isolation and purification in conjunction with electrophoretic, immunological and electron micrographic analyses alone or in combination, as described below. Although B. nodosus-type fimbriae are physically very similar to those naturally produced by the P. aeruginosa host strain, they can be distinguished using specific antisera. Furthermore, the fimbrial subunits from the donor strain B. nodosus VCS 1001 and the host strain P. aeruginosa PAK/2Pfs have slightly different molecular weights and thus can also be distinguished by their electrophoretic mobilities in SDS-polyacrylamide gels (see Figures 4, 5 and 6); the apparent molecular weight of the B. nodosus VSC1001 subunit is approximately 17,000, while that of the P. aeruginosa PAK/2Pfs subunit is about 16,000, the actual molecular weights (calculated from the sequence) are 16,218 (151 amino acids) and 15,082 (145 amino acids), respectively.

For the analyses described below, bacterial cells were grown on solid media, the host strain P. aeruginosa PAK/2Pfs on nutrient (Luria Broth) agar, the recombinant P. aeruginosa strain PKN-A1 (with pJSM202) on the same medium with the addition of 1 mg carbenicillin/ml, recombinant E. coli 418 with pJSM202 on the same medium with the addition of 50 µg ampicillin/ml and B. nodosus cells on "Hoof" agar (as described by Mattick et al (1984), J. Bacteriol. 160, 740-747). Cultures were harvested by scraping into phosphate buffered saline.

The morphogenetic expression of B. nodosus-type fimbriae in P. aeruginosa PKN-A1 cells was initially indicated by the presence of significant amounts of this antigen in the extracellular environment. In many fimbriae-containing bacteria, including B. nodosus and P. aeruginosa, a portion of the fimbriae appear to be secreted into the medium (or resuspension buffer) and remain there after removal of the cells by centrifugation. Additional fimbriae can be removed by mechanical mixing of the cell suspension (see Figure 4) or by heating the culture (at about 60°C) for a short period of time. This is illustrated in Figure 3, which shows Western transfer (immunoblot) analysis of the cell pellet and the resulting supernatant fractions from recombinant cells. These results demonstrate, first, that the B. nodosus fimbrial subunit is very efficiently transcribed from the PL promoter in P. aeruginosa cells with pJSM202 at a level at least two orders of magnitude higher than in the same host with pJSM225, where the gene is transcribed from an associated promoter within the originally cloned segment of B. nodosus DNA. Second, one finds a large proportion of the fimbrial subunit antigen in the supernatant fraction obtained from P. aeruginosa PKN-A1. This is in contrast to the situation in E. coli E418 cells with pJSM202, where the antigen is entirely localized in the cell (pellet) fraction (the traces found in the supernatant apparently derive from cell fragments that have not been removed by centrifugation). A significant amount of the B. nodosus fimbrial subunit is also found on the cells in the case of PKN-A1, but it is not known to what extent this represents fimbriae not removed from the cell or subunits enriched within the cell that have not (yet) been assembled. Third, the size of the fimbrial subunit found in E. coli is indeed slightly larger than that found in P. aeruginosa or in B. nodosus itself. This appears to be due to the inability of E. coli to cleave the short signal peptide from the primary translation product (see Table 1), which is presumably part of the normal process of morphogenetic expression.

The assembly of B. nodosus fimbrial subunits into mature fimbriae in P. aeruginosa PKN-A1 cells was also demonstrated by the presence of this antigen in purified fimbriae isolated under different conditions. Fimbriae can be recovered from cell-free supernatants by either of the independent methods of isoelectric precipitation at pH 4.5 or precipitation induced by the addition of MgCl2. These are standard procedures applicable to the isolation of fimbriae from both P. aeruginosa and B. nodosus. Using both methods, good yields of purified fimbriae (approximately 1 to 1.5 mg per standard (85 mm) Petri dish culture) were obtained from the recombinant PKN-A1 cells, which was confirmed by electron microscopy of negatively stained samples. These fimbriae could also be further purified by isopycnic banding in CsCl gradients. Electrophoretic analysis (Figure 4) showed that the fimbriae isolated from PKN-A1 cells are composed primarily of a structural subunit that gel-co-migrates with that obtained from B. nodosus. Somewhat unexpectedly, there is little or no evidence for the fimbrial subunit characteristic of the host strain PAK/2Pfs (see Figure 4, also below).

These results were confirmed by Western transfer analysis of fimbriae produced by PKN-A1 cells compared with those obtained from B. nodosus strain VCS1001 and P. aeruginosa PAK/2Pfs (Figure 5). This experiment clearly shows that the fimbriae produced by PKN-A1 not only consist of a structural subunit that has the same electrophoretic mobility as that found in B. nodosus itself (Figure 5A), but also that this protein is recognized by antibodies directed against B. nodosus fimbriae (Figure 5C). The reciprocal Western using anti-P. aeruginosa fimbriae antiserum confirms that the fimbriae produced by these recombinant cells contain little or no structural subunit naturally expressed by the host (Figure 5B). Control experiments have shown that the presence of the vector plasmid pKT240 itself does not affect the composition of the fimbriae in P. aeruginose.

The generation of B. nodosus-type fimbriae in PKN-A1 cells was also independently confirmed by immunogold labeling and electron microscopy. Fimbriae were prepared from B. nodosus VCS1001, P. aeruginosa strain PAK/2Pfs and P. aeruginosa PKN-A1, placed on slides and incubated with either anti-P. aeruginosa PAK/2Pfs fimbrial antiserum or anti-B. nodosus VCS1001 fimbrial antiserum. After washing, samples were incubated with gold-labeled protein A, washed again, then negatively stained and visualized by electron microscopy. Antibody binding was indicated by the presence of electron-dense, spherical gold particles and by the clumping of the fimbrial strands. As expected, B. nodosus VCS1001 fimbriae reacted strongly with the homologous antiserum but not with antiserum raised against P. aeruginosa fimbriae. Conversely, P. aeruginosa PAK/2Pfs was also found to react strongly with the homologous antiserum but not with antiserum raised against B. nodosus fimbriae. The pattern is completely reversed in the recombinant P. aeruginosa cells with pJSM202, which now display a profile similar to that observed in B. nodosus. Fimbriae raised by PKN-A1 are not recognized by antibodies directed against P. aeruginosa fimbriae but react strongly with those against B. nodosus fimbriae. These results can be summarized in the following table, where + means antibody detection and - means absence of antibody detection. Strain B.nodosus P.aeruginosa Antiserum VCS1001 (host) PAK/2Pfs (host) PKN-A1 (recombinant) Anti-B.nodosus VCS1001 fimbriae Anti-P.aeruginosa PAK/2Pfs fimbriae

Several other criteria were also used to confirm the production of B. nodosus fimbriae by P. aeruginosa cells expressing pJSM202. These included demonstration that antisera raised against the recombinant fimbriae agglutinated B. nodosus (VCS1001) cells (see the vaccination experiment below) and, conversely, that antisera raised against B. nodosus (VCS1001) agglutinated the recombinant P. aeruginosa PKN-A1 cells. These agglutination reactions are (B.nodosus) serotype specific. Control experiments showed that anti-B. nodosus antisera did not agglutinate the host P. aeruginosa PAK/2Pfs (with or without the vector plasmid pKT240) and that antisera raised against normal P. aeruginosa fimbriae did not agglutinate B. nodosus. Similar results were obtained in standard antibody-antigen solid phase binding assays such as ELISA assays.

These results demonstrate that P. aeruginosa PKN-A1 cells containing the recombinant plasmid pJSM202 produce mature fimbriae structures that are physically, structurally and antigenically indistinguishable from those produced by the B. nodosus strain from which the fimbrial subunit gene was originally derived. As we will demonstrate below, these recombinantly produced fimbriae are also equally effective in inducing protective immunity against footrot.

Similar results were obtained using other fimbriate P. aeruginosa strains as host cells for the recombinant plasmid pJSM202. These include P. aeruginosa PAK (ATCC 25102), the parent strain of PAK/2Pfs, as well as P. aeruginosa PA01 (ATCC 25247), and the derived multifimbrial mutant DB-2, which normally expresses a structurally and serologically different form of the fimbrial subunit, such as that found in PAK strains.

Insertion of pJSM202 into P. aeruginosa fimbriate effectively resulted in complete substitution of the B. nodosus fimbrial subunit for that normally produced by the cells. The fact that the fimbriae produced by these recombinant cells contain little or no endogenous P. aeruginosa structural subunits suggests that the high expression level of the B. nodosus subunit either prevented entry of the endogenous subunit into the assembly pathway or resulted in feedback inhibition (presumably via common signals in the conserved region) of synthesis of the endogenous subunit at either the transcriptional or translational stage. In any case, the provision of a strong promoter to increase expression of the cloned B. nodosus fimbrial subunit gene in the P. aeruginosa host cell is clearly an important feature of the invention. However, the precise nature of the promoter used does not appear to be critical. We have obtained similar results using alternative constructs, with the fimbrial subunit gene under the transcriptional control of the strong PR promoter of bacteriophage lambda. Expression of the fimbrial subunit gene from either the PL or PR promoter can be regulated by inserting a cloned copy of the C1857 repressor gene into the plasmid, thereby reducing the selective pressure against the plasmid during normal propagation of transformed cells. It is also possible to consider the use of other strong promoters, such as tac, which can be induced by chemical signals rather than by temperature change.

The results presented here support our prediction that type 4 fimbrial subunits from different bacterial species share common topogenic signals for fimbrial morphogenesis and can be exchanged in compatible hosts. This is certainly the case for B. nodosus and P. aeruginosa and should be equally applicable to other type 4 species that share the conserved hydrophobic amino-terminal region of the fimbrial subunit. Since variation in the carboxy-terminal region of this protein does not seem to disturb the process of fimbrial assembly, it is also possible that the co-cloning of two or more genes encoding related type 4 fimbrial subunits in the same compatible host cell results in the formation of mixed or hydride fimbriae (consisting of a random arrangement of the introduced subunits), which can then provide a multivalent antigen preparation active against more than one serotype or possibly even against more than one species.

There are various other modifications of the basic system that may be considered in special circumstances. For example, in some cases it may be possible to eliminate the expression of the fimbrial subunit produced by the host cell. This can be achieved by a number of methods including traditional mutant selection, transposon mutagenesis, or in vitro modification of the gene followed by recombination insertion in vivo. The system can also be improved by modifying (the expression of) genes involved in the fimbrial assembly process. Changes can also be made within the conserved amino-terminal sequence of the fimbrial subunit itself to improve the efficiency of fimbrial assembly in a heterologous type 4 species. This can be done, for example, by gene fusions or site-directed mutagenesis in vitro using synthetic oligonucleotides to alter specific amino acid codons so that the consensus sequence is brought into close agreement with that normally active in and recognized by the preferred host.

It will be apparent to those skilled in the art that numerous other variations and modifications can be made to the invention described above without departing from the spirit or scope of the invention as broadly described.

C. Vaccination trial

The efficacy of fimbriae produced by recombinant P. aeruginosa PKN-A1 cells in inducing protective immunity against footrot infection in sheep was tested in a vaccination trial. The vaccination trial was conducted as follows: A total of 73 normal sheep were randomly divided into 7 groups, each containing either 10 or 11 individuals per group. Two groups were designated as negative controls - one received no treatment (group 1) and the other was vaccinated with isolated fimbriae from the P. aeruginosa host strain PAK/2Pfs (group 4). There were also two positive control groups vaccinated with either whole cells (group 2) or isolated fimbriae (group 3) from B. nodosus VCS1001. Two experimental groups (groups 5 and 6) were vaccinated with different amounts of fimbriae isolated from the recombinant P. aeruginosa strain PKN-A1 (containing pJSM202). A third experimental group (group 7) was also vaccinated with fimbriae from PKN-A1, in this case isolated by an alternative procedure (see below). Group Vaccine antigen Dose Total cells B. nodosus VCS1001 Isolated fimbriae B.nodosus VCS1001 Isolated fimbriae PAK/2Pfs Isolated fimbriae PKN-A1 Isolated fimbriae PKN-A1 (MgCl₂)

Cells were cultured on agar plates and harvested by scraping into sterile phosphate buffered saline (PBS). To isolate fimbriae, these cell suspensions were subjected to mechanical mixing and then cells were removed by centrifugation. Fimbriae were recovered from the cell-free supernatant by isoelectric precipitation with 0.1M Na acetate (pH 4.5) or, in the case of group 7, by precipitation with 0.1M MgCl2. Fimbriae were then redissolved in sterile PBS and purified by another round of precipitation using the method originally used. The content and purity of all antigen preparations used in the vaccination trial was then determined by electrophoretic separation and Western transfer analysis using anti-B. nodosus VCS1001 and anti-P. aeruginosa PAK/2Pfs antisera (Table 7).

Vaccines were prepared using a standard alum/oil adjuvant system: an appropriate amount of antigen was resuspended in sterile PBS to which one seventh volume of 10% potassium alum (AlK(SO4)2) was added and the pH was adjusted to 6.2. One hundredth volume of Tween 80 was added and the mixture emulsified with an equal volume of Freund's incomplete adjuvant. Each individual dose was in a volume of 2 ml and contained the amount of antigen specified in the table above. Each animal received 2 doses - a primary vaccination followed by a booster shot 28 days later. Four days after the booster, animals were prepared for challenge by standing in pens under humid conditions (on wet mats). The challenge was artificially administered three days later (i.e., one week after booster) by applying cultured cells of B. nodosus VCS1001 to each foot. Animals from all groups were penned together for the duration of the experiment. Blood samples were taken during the experiment to determine the agglutination titer against B. nodosus VCS1001 in serum. The presence and severity of infection was assessed 21 days after challenge by examining each individual foot for lesions. Data are summarized in Table 2. Table 2 Group Vaccine Affected sheep Severe (underfoot) lesions (% feet) Agglutination titre + 2 weeks + 3 weeks B.nodosus cells (5 x 10⁹) B. nodosus fimbriae 250 µg P. aeruginosa fimbriae 250 µg PKN-A1 fimbriae 250 µg PKN-A1 fimbriae 500 µg PKN-A1 fimbriae MgCl⁹ 250 µg

Key to Table 2: Affected animals are defined as having at least one foot with a lesion score of 3 or 4, or at least 2 feet with a lesion score of 2 three weeks after challenge. The lesion score was scored on a standard scale of 2 to 4 as follows: 2 visible interdigital dermatitis, 3 underrunning lesions of the soft horn of the heel, and 4 underrunning lesions of the hard horn of the hoof. (A lesion score of 1 is normally used for feet showing symptoms of mild interdigital dermatitis. This may be due to other causes and was therefore not included in the score). Severe lesions are those that underrun the hoof, i.e. a score of either 3 or 4. G.M.F.S. is the mean foot score of the group and was calculated as the sum of the lesion scores for all feet of all animals in the group divided by the number of feet in the group. The agglutination titer was determined as the reciprocal of the maximum dilution at which visible K agglutination of B. nodosus (VCS1001) cells was observed. Two-fold serial dilutions were performed. Data are shown for samples collected 2 weeks and 3 weeks post-challenge (i.e., 3 and 4 weeks post-boost). Data are expressed as the geometric mean for the group (rounded to the nearest hundred). n.d. = not determined.

The results of the vaccination trial clearly demonstrate that the recombinant fimbriae confer the same level of protection as whole B. nodosus cells or fimbriae against a homologous footrot challenge. There is no significant difference between the serum agglutination titres or the level of protective immunity observed in groups vaccinated with either the natural or the recombinant material. Complete immunity against Footrot challenge is rarely observed in such vaccination trials, particularly when a highly virulent strain such as VCS1001 is involved. The response of individual animals to vaccination is quite variable. However, although neither the natural nor the recombinant material conferred full immunity to footrot infection in this trial, there is a large and practically important difference between the vaccinated groups and the negative controls. All animals in the negative control groups (i.e., either untreated or vaccinated with normal P. aeruginosa fimbriae) were affected by severe footrot, with approximately 65% of all feet showing subcutaneous lesions. The mean lesion score for these groups was 2.4. Such lesions are relatively rare in the groups vaccinated with B. nodosus cells or fimbriae or fimbriae produced by recombinant cells, with severe symptoms observed in only 15 to 20% of all feet. The mean lesion score in these groups was only 0.9. This actually means the difference between sheep that are weakened by the disease and those that, despite some exceptions, have only been slightly affected and bothered. This practical difference is important in the field situation.

Claims (32)

1. A method for producing an antigen preparation for use in generating antibodies effective against a bacterial species that naturally produces type 4 fimbriae, comprising culturing genetically engineered aerobic or facultatively anaerobic host cells having type 4 fimbriae containing the genes encoding the fimbrial subunit structural antigens of at least one strain of the bacterial species and an endogenously compatible system for morphogenetic assembly of type 4 fimbriae so that mature fimbriae are produced as extracellular structures, and partially or completely harvesting the fimbriae substantially free from the host cells. 2. A process for producing an antigen preparation according to claim 1, wherein the genes encoding the fimbrial subunit structural antigens are derived from B. nodosus or M. bovis. 3. A process for preparing an antigen preparation according to claim 1, wherein the genes encoding the fimbrial subunit structural antigens are from a species selected from the group consisting of P. aeruginosa, N. gonorrhoea, N. meningitidis and M. nonliquefaciens. 4. A process for producing an antigen preparation according to any one of claims 1 to 3, wherein the genetically manipulated host cells contain genes for a plurality of fimbrial subunit structural antigens, the genes being derived from different species or genera. 5. A process for producing an antigen preparation according to any one of claims 1 to 3, wherein the genetically manipulated host cells contain genes for a plurality of fimbrial subunit structural antigens, the genes being derived from different serotypes of a single bacterial species. 6. A process for the preparation of an antigen preparation according to any one of claims 1 to 5, wherein the mature fimbriae or fragments thereof are from at least one serotype of B. nodosus. 7. A process for producing an antigen preparation according to claim 1, wherein the host cell is a strain of a genetically engineered species of the genus Pseudomonas. 8. A process for producing an antigen preparation according to claim 7, wherein the host cell is a strain of genetically engineered P. aeruginosa. 9. A process for producing an antigen preparation according to claim 8, wherein the genetically engineered P. aeruginosa is P. aeruginosa PAK/2Pfs (ATCC No. 53308) or a derivative thereof. 10. A process for producing an antigen preparation according to any one of claims 1 to 9, wherein the gene encoding the fimbrial subunit structural antigen has been modified to molecularly enhance morphogenetic expression in the host cell. 11. A process for preparing an antigen preparation according to any one of claims 1 to 10, wherein the gene encoding the fimbrial subunit structural antigen is under the transcriptional control of a strong promoter active in the selected host. 12. A process for producing an antigen preparation according to claim 11, wherein the strong promoter is regulatable. 13. A process for the preparation of an antigen preparation according to claim 12, wherein the promoter is the PL or PR promoter derived from bacteriophage lambda. 14. A process for producing an antigen preparation according to any one of claims 1 to 13, wherein the gene encoding the fimbrial subunit structural antigen is contained in a plasmid. 15. A process for the preparation of an antigen preparation according to claim 14, wherein the plasmid is plasmid pJSM202 (ATCC No. 40203). 16. A process for producing an antigen preparation according to any one of claims 1 to 12, wherein the gene encoding the fimbrial subunit structural antigen is inserted into the chromosome of the bacterial host cell. 17. A genetically engineered aerobic or facultatively anaerobic bacterium having type 4 fimbriae, the bacterium being characterized by containing exogenous genes encoding the fimbrial subunit structural antigens of at least one bacterial strain that naturally produces type 4 fimbriae, such that expression of the exogenous genes results in the production of fimbriae of the at least one strain as extracellular structures. 18. A genetically engineered bacterium with fimbriae type 4 according to claim 17, wherein the exogenous genes are from B. nodosus or M. bovis. 19. A genetically engineered type 4 fimbriated bacterium according to claim 17, wherein the exogenous genes are from a species selected from the group consisting of P. aeruginosa, N. gonorrhoea, N. meningitidis and M. nonliquefaciens. 20. A genetically engineered bacterium with fimbriae of type 4 according to any of claims 17 to 19, wherein the exogenous genes originate from different species or genera. 21. A genetically engineered bacterium with type 4 fimbriae according to any one of claims 17 to 20, wherein the exogenous genes encode fimbrial subunit structural antigens from different serotypes of a single bacterial species. 22. A genetically engineered bacterium with type 4 fimbriae according to any one of claims 17 to 21, wherein the exogenous genes are from at least one serotype of B. nodosus. 23. A genetically engineered bacterium with fimbriae type 4 according to any one of claims 17 to 22, wherein the genetically engineered bacterium is from the genus Pseudomonas. 24. A genetically engineered bacterium having type 4 fimbriae according to claim 23, wherein the genetically engineered bacterium is a strain of P. aeruginosa. 25. A genetically engineered bacterium with type 4 fimbriae according to any one of claims 17 to 24, wherein the exogenous genes have been modified to molecularly enhance morphogenetic expression in the genetically engineered cell. 26. A genetically engineered bacterium with type 4 fimbriae according to any one of claims 17 to 25, wherein the exogenous genes are under the transcriptional control of a strong promoter active in the genetically engineered bacterium. 27. A genetically engineered bacterium with type 4 fimbriae according to claim 26, wherein the strong promoter is regulatable. 28. A genetically engineered bacterium with type 4 fimbriae according to claim 27, wherein the promoter is the PL or PR promoter derived from bacteriophage lambda. 29. A genetically engineered bacterium with fimbriae of type 4 according to any of claims 17 to 28, wherein the exogenous genes are contained in a plasmid. 30. A genetically engineered bacterium with fimbriae type 4 according to claim 29, wherein the plasmid is pJSM202 (ATCC No. 40203). 31. A genetically engineered bacterium with fimbriae of type 4 according to any of claims 17 to 28, wherein the exogenous genes are inserted into the chromosome of the genetically engineered bacterium. 32. Pseudomonas aeruginosa PAK/2Pfs (ATCC No. 53308) transformed with plasmid pJSM202 (ATCC No. 40203).