WO1992003553A1

WO1992003553A1 - Footrot vaccine

Info

Publication number: WO1992003553A1
Application number: PCT/AU1991/000366
Authority: WO
Inventors: Eric Keith Moses; Robert Trygve Good; Christopher John Langford; Julian Ian Rood
Original assignee: Daratech Pty. Ltd.
Priority date: 1990-08-17
Filing date: 1991-08-15
Publication date: 1992-03-05

Abstract

A veterinary vaccine where the antigen is a genetically engineered recombinant polypeptide having an amino acid sequence which is homologous to secreted outer membrane proteins of D.nodosus.

Description

FOOTROT VACCINE

FIELD OF THE INVENTION

This invention relates to veterinary vaccines and more particularly to recombinant vaccines for the treatment or prevention of footrot in ruminants caused by Dichelobacter nodosus previously referred to as Bacteroides nodosus. The invention also relates to a method of treating ruminants to prevent footrot caused by D.nodosus. In an alternative form the invention provides aids for diagnosing virulent, benign and intermediate strains of footrot.

BACKGROUND OF THE INVENTION

Ruminant footrot is an economically important disease particularly in sheep. The disease is characterised by separation of the hoof from the underlying epidermal tissues. Various treatments have been proposed including foot bathing with formalin and zinc sulphate solutions as well as the use of antibiotics. More recently preventative treatments using vaccines have been proposed.

SUMMARY OF PRIOR ART

Immunity to footrot has been demonstrated using whole cell D.nodosus vaccines as described in Australian Patent Specification No. 51387/85. However, to achieve significant levels of protective immunity these vaccines are reguired to contain strains representative of the several known serogroups of this species. In addition, D.nodosus has fastidious growth requirements, is an obligate anaerobe and the expression of fimbriae is unstable in commercial scale liquid culture. Accordingly, these vaccines are difficult and expensive to produce commercially.

In an attempt to overcome these problems, recombinant sub-unit vaccines based on the serogroup specific fimbrial proteins have been developed as described in Australian Patent Specification No 18030/88. While overcoming some of the problems of culturing D.nodosus for vaccine production, these vaccines must still contain fimbrial proteins from the majority of known serogroups and are therefore still complex and expensive vaccine formulations. This complexity gives rise to problems of antigenic competition between the components of the vaccine resulting in incomplete immunity in animals vaccinated with this formulation.

To overcome these problems it is desirable that there be a defined antigen which provides cross-protective immunity against infection with various strains of D.nodosus.

SUMMARY OF THE INVENTION

Accordingly the present invention provides in one form a veterinary vaccine wherein the antigen is genetically engineered and wherein the antigen is substantially homologous to secreted outer membrane proteins of D.nodosus.

A vaccine containing one or more of these genetically engineered antigens or closely related molecules is able to stimulate cross-protective immunity in sheep to infection with D.nodosus strains from various serogroups.

According to a further aspect of the present invention there is provided a veterinary vaccine comprising protein antigens of D.nodosus which are characterised by being members of a family of closely related molecules which are synthesised as 70-75 kDa proteins which are major components of the outer membrane of D.nodosus.

In another aspect of the present invention there is provided a diagnostic test based on the nucleotide sequences of the genes described herein which encode the family of outer membrane proteins. According to this aspect of the present invention the DNA from any strain of D.nodosus can be rapidly amplified using the polymerase chain reaction (PCR) technique (see R.K. Saiki et al., Science 239, 487-491 (1987).) from samples taken directly from the hoof of ruminants. This amplified DNA can then be specifically fragmented by digestion with any one of a number of restriction endonucleases to generate a discrete pattern of DNA molecules of varying sizes which can be visualised by a variety of standard procedures as shown Figure 14. Each strain of D.nodosus produces a unique pattern (or fingerprint) which is recorded and stored in a computer data base.

The fingerprints of strains or isolates which share the same phenotypic characteristics (such as virulence) can be grouped together. New isolates with the same fingerprints can be immediately assigned to one of the defined phenotypic groups. This technique provides a method for typing virulent, intermediate and benign strains of D.nodosus.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 describes the linear restriction maps of cloned D.nodosus genomic DNA fragments. Arrows indicate the location of the coding region of the OMP genes on these DNA fragments. The size of the cloned DNA fragments is indicated by the scale at the bottom.

Figure 2 describes one arrangement of the E, B, H and C OMP genes in the D.nodosus genome. Also shown is the location of the promoter sequences (P), sequences encoding the hydrophobic leader peptide (hatched) and direction of transcription of each gene (arrows). The circles indicate some of the regions of genome which invert resulting in alternative arrangements of the OMP genes and to the altered expression of the OMP antigens on the surface of the D.nodosus organism.

Figures 3-7 illustrate the entire nucleotide sequence of the 8.5kB DNA fragment described in Figure 2. The coding regions for the OMP antigens are indicated and are located at the following positions. E (26 to 2146), B

(4071 to 2224), H (4356 to 6491) and C (8397 to 6571).

The hydrophobic leader sequence of the H and E genes is shown underlined. The break points which are generated by the process of DNA inversion are indicated with an asterisk.

Figure 8 illustrates the multiple alignment of the deduced amino acid sequences encoded by the E, H, B and C OMP genes. The alignment includes the full sequence of the B and C genes which are generated by the addition of the sequences at the NH₂-terminal end of the sequences described in Figures 3-7 as a result of the DNA inversion process described in Figure 2. Asterisks indicate positions at which the amino acid sequences are conserved in all four OMP proteins. It is noted that regions of homology flank regions of amino acid sequence which are not homologous. Arrowed lines labelled oligo A and oligo C indicate the location of 18 and 16 nucleotide long sequences respectively which are identical in all four OMP genes from D.nodosus strain A198.

Figure 9 shows SDS-PAGE analysis of the recombinant B, E, H and C OMP proteins expressed in E. coli . ROMP C/B is a hybrid antigen comprised of the first 448 amino acids of OMP C fused to the last 221 amino acids of OMP B. ROMP 3'C is the last 355 amino acids of OMP C expressed as a fusion protein. The DNA sequences encoding the E, B, C/B and H OMP antigens were cloned into the IPTG inducible expression vector PMMB66EH. The sequence encoding OMP 3'C was cloned into pGEX-3X to give a fusion with the 3' end of the gene encoding Schistosoma japonicum glutathione S-transferase. The migration of the M.W. standards are shown at left. Figure 10 shows the results of a footrot vaccine trial designed to test the efficacy of rOMP antigens in inducing protective immunity in sheep against infection with D.nodosus strain A198.

Figure 11 shows the reactivity of antibodies raised against rOMP C/B antigen. Fimbriae and proteases purified from D.nodosus strain A198 and D.nodosus A198 whole cells were solubilized by boiling in SDS and subjected to SDS- PAGE. The gel was transferred to nitrocellulose membrane and blotted with the rOMP C/B antibodies. The antibodies react strongly with a 75-80 kDa protein which co-purifies with the fimbriae (arrowed in lane 1) and 75-80 kDa proteins from whole cells (arrowed in lane 2). The antibodies also react weakly with a 35-40 kDa polypeptide in the protease fraction (arrowed in lane 3). Figure 12 shows the Coomassie blue protein staining profiles and Western blotting profiles of cell envelope proteins from virulent and benign D.nodosus strains within eight serogroups. Cell envelope proteins were subjected to SDS-PAGE on 10-17.5% linear gradient gels and stained with Coomassie blue R250. A duplicate gel was subjected to Western transfer and blotting with rOMP C/B antibodies. The antibodies cross-react with major cell envelope proteins of D.nodosus irrespective of serogroup. Figure 13 shows PCR analysis of OMP genes from various D.nodosus strains. Two oligonucleotide primers oligo A and oligo C that are based on conserved nucleotide sequences found in all four OMP genes from D.nodosus strain A198 (1168-1186, 3193-3210 reverse and complement, 5511-5528, 7525-7542 reverse and complement, 1676-1691 reverse and complement, 2688-2703, 6012-6027 reverse and complement and 7044-7059 in Figures 3-7), were used to amplify, by PCR, intervening DNA sequences from the genomic DNA of 16 different D.nodosus strains representative of 9 serogroups. The observation that DNA fragments could be synthesized from all strains tested indicates that the conserved sequences defined by oligo A and oligo C in strain A198 are also conserved in other strains of D.nodosus.

Figure 14 shows DNA fingerprints from various D.nodosus strains. These fingerprints were generated by PCR with oligonucleotide primers oligo A and oligo C followed by digestion with the restriction endonuclease Sau3A and agarose gel electrophoresis. A unique DNA fingerprint was generated from each of the D.nodosus strains tested.

Figure 15 descibes the homology between the predicted amino acid sequence of gene E (Figure 2) and two ttryptic peptides derived from the 35-40kDa fraction of proteins isolated from the supernatant of a D.nodosus strain 198 culture.

The antigen is preferably a polypeptide which includes the whole part or analogues of the amino acid sequences set out in Figures 3-8 or an antigenic fragment thereof prepared by recombinant techniques. By part or analogues of the amino acid sequences we mean molecules which have a high homology with those regions of the sequences which are conserved between the OMP proteins. These are indicated by asterisks in Figures 8. By high homology we mean generally at least 80%. Also in this specification the term "closely related molecules" means molecules which have a high level of homology in the regions where the sequences are conserved between the outer membrane proteins of D.nodosus.

Genes eneoding the proteins in this invention are amplified using PCR and the oligos A and C described in Figure 13 generating. DNA fragments hanging in length from 400-600 nucleotide base pairs. The vaccine preferably also comprises a pharmaceutically acceptable carrier or diluent, and more preferably an adjuvant. Suitable carriers, diluents and adjuvants are known to those skilled in the art.

The invention also provides in an alternative form a method for immunising ruminants against D.nodosus infection. This method comprises administering to the ruminant an effective amount of antigen as defined above or antigenic fragment(s) thereof either alone or in combination. In another preferred embodiment the invention provides an antigen(s) of the present invention or an antigenic fragment(s) thereof in a live recombinant expression and delivery system such as a recombinant virus, bacterium or parasite. The antigenic molecules of the present invention are found in the outer membrane of D.nodosus. The predicted size of the mature proteins, based on the nucleotide sequence data, range from 70,120 to 74,779 Dalton. These proteins have been previously reported as ranging in size from 75,000 - 80,000 Dalton as determined by SDS/PAGE. The difference in molecular weight ranges is attributed to measuring techniques. These molecules and their breakdown products, can also be found in the supernatant of D.nodosus cultures. Fragments of this family of molecules co-purify with the 35-40 kDa fraction of proteases extracted from the culture supernatant by affinity chromatography and size exclusion chromatography. When this 35-40 kDa fraction of proteins was used to immunise rabbits, antibodies to both the 75-80 kDa family of proteins described in this invention and to the 35-40 kDa family of extracellular proteases of D.nodosus were produced. This rabbit antiserum was used to identify a recombinant E . coli clone expressing one of the proteins described in this invention.

Peptide sequence data, determined directly from peptides derived from this 35-40 kDa fraction correlate with the predicted amino acid sequence of the genes described in this invention. Other peptide sequences from this fraction correlate with the predicted amino acid sequence of the novel basic protease sequence described in Australian Patent Application No. 18030/88.

This invention also describes the expression of recombinant OMP proteins (rOMPS) in E. coli and their subsequent use as vaccine antigens.

The results of a trial in sheep are set out in Table 1. Table 1 shows the results of a pen trial experiment designed to test the efficacy of the cloned antigens as a vaccine.

The coding sequences of E, B and H genes were cloned into the IPTG inducible expression vector pMMB66EH in order to express the antigens in E. coli . A hybrid gene encoding 448 amino acids from the NH₂-terminus of the OMP C protein fused to the region encoding 221 amino acids from the COOH terminus of the OMP B protein was also constructed in pMMB66EH. The region encoding 335 amino acids from COOH-terminus of the OMP C gene was cloned into pGEX-3X to express a fusion protein with Schistosona iaponicum glutathione-S-transferase.

The E. coli were grown in bulk liquid cultures and harvested by centrifugation. The E. coli were lysed with the detergent SDS and the proteins solubilized by boiling prior to fractionation by SDS-PAGE. Gel slices containing the recombinant B, C and E proteins were extracted with SDS and the solubilized proteins were then precipitated by methanol or acetone. The precipitate

(vaccine antigen) was harvested by centrifugation and redissolved in phosphate buffered saline. A total of 200 μg of the B, E 3'C and C/B recombinant OMP antigens were mixed together with Alhydrogel and emulsified in Freunds incomplete adjuvant.

Thirty sheep were immunised and bled at 54, 29 and 0 days prior to challenge. Sheep were injected subcutaneously behind the ear with 1 ml of a formulation containing saline, Alhydrogel and Freund's incomplete adjuvant in the ratio 1:1:2. A positive control group of ten sheep were injected with recombinant fimbriae in saline, provided by Arthur Webster Pty. Ltd. Sydney. A negative control group of ten sheep were injected with saline alone. The test group of ten sheep were injected with a mixture comprised of 50μg each of rOMPS B, E, 3'C and C/B in saline. On day 0 all sheep were challenged with D.nodosus strain A198 (the homologous strain with respect to the fimbrial and OMP antigens). Feet were scored for footrot lesions 13, 21, 27, 34, 41 and 48 days after challenge. The results show that the recombinant OMP vaccine induced a significant protective effect in sheep up to 34 days after homologous challenge with D.nodosus. On day 0 every foot of each sheep was subjected to experimental challenge with a large dose of D.nodosus strain A198. The progression of disease on each foot was measured using the standard 0-4 scoring method on days 13, 21, 27, 34, 41 and 48 days after challenge. The results (Figure 10 and Table 1) indicate a favourable control of footrot infection up to 34 days after challenge.

Antibodies raised in sheep in response to immunisation with rOMP C/B reacted specifically with a 75-80 kDa protein of D.nodosus and its natural breakdown products (Figure 11).

These antibodies also react with proteins of molecular weight 75-80 kDa which co-purify with D.nodosus fimbriae which have been physically sheared from the surface of the bacterium (Figure 11).

These antibodies also cross-react with 75-80 kDa proteins found in fifteen strains representative of 8 of the 9 serogroups (A, B, C, D, E, F, G and H; serogroup I was not tested) of D.nodosus (Figure 12). Immunofluorescence of whole unfixed D.nodosus cells using this antiserum shows that the mature protein described in this invention is probably localised on the outer membrane of the bacterium (data not shown).

These and other data are consistent with the characterisation of the antigens described in this invention as members of a family of outer membrane proteins of D.nodosus. In particular, these data are consistent with the characterisation of these cloned molecules as the family of outer membrane proteins previously characterised by others as the OMC protein 1, the basal protein and the basement protein.

This group of outer membrane proteins has also been implicated in the partial cross-protection seen following immunisation with D.nodosus vaccine preparations from various serogroups. Since modifications within the spirit and scope of the invention may be readily effected by persons skilled in the art, it is to be understood that the invention is not limited to the particular embodiment described, by way of example, hereinabove.

Claims

1. A veterinary vaccine wherein the antigen is a recombinant protein which is substantially homologous to secreted outer membrane proteins of D.nodosus.

2. A veterinary vaccine wherein the antigen is a recombinant polypeptide, analogue or fragment which is highly homologous to any one of the amino acid sequences set out in Figures 3-8.

3. A veterinary vaccine as defined in Claim 2 wherein the recombinant polypeptide has molecular weight 70-75 KDa.

4. A veterinary vaccine as defined in Claims 1 or 2 wherein the antigen is a recombinanat protein having groups of amino acid sequences, of length 8-20 amino acids, wherein the groups have a homology of at least 80% with the conserved regions of molecules defined in Figures 3-8.

5. A method for immunising ruminants against D.nodosus infection by administering an effective amount of a vaccine as defined in any one of claims 1 to 4.

6. A method of diagnosing virulent, intermediate and benign strains of D.nodosus by amplifying DNA from any strain of D.nodosus, fragmenting the amplified

DNA by digestion with a restriction endonuclease to produce a fingerprint of DNA molecules, and matching the information to known phenotype groups.