WO1992018624A1

WO1992018624A1 - Plasmides for the expression of filamentous haemagglutinin (fha) and hosts therefor

Info

Publication number: WO1992018624A1
Application number: PCT/EP1992/000814
Authority: WO
Inventors: Kenneth Timmis; Carlos Guzman; Mark Walker
Original assignee: GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Priority date: 1991-04-09
Filing date: 1992-04-09
Publication date: 1992-10-29
Also published as: EP0579685A1; DE4111531A1; JPH06506587A

Abstract

Plasmides for the highly effective expression of unfused filamentous haemagglutinin from Bordetetella pertussis for use in the manufacture of acellular and oral live vaccines against whooping cough, and hosts for these plasmides.

Description

Plasmids for FHA expression and hosts

Plasmids for the highly effective expression of unfused filamentous hemagglutinin from Bordetella pertussis for the production of acellular and oral live vaccines against whooping cough and hosts for these plasmids

Bordetella pertussis is responsible for the development of whooping cough, a respiratory disease, especially in children, which can lead to serious complications and death if you are highly educated. Whilst whole cell vaccine preparations routinely administered in combination with diphtheria and tetanus toxoids offer good protection against a serious disease, there is growing concern about the side effects of the vaccines, which has led to this. that the number of children vaccinated has decreased and whooping cough increases accordingly.

Bordetella pertussis provides a number of virulent factors, the synthesis of which is positively regulated by the products of the bvg locus (1), which include pertussis toxin, adenylate cyclase, filamentous haemagglutinin (FHA), fimbriae and main proteins of the outer membrane (2) . Some of these determinants are potential antigens for inclusion in a new generation of acellular, non-toxic, non-reactogenic vaccines. One of the most promising components for such a vaccine is FHA, which plays a major role in the accumulation of bacteria and the subsequent colonization of the epithelial respiratory tract during the early stages of the disease. FHA can also promote the super infections that are commonly associated with Disease complicate because other bacteria can use this bridging adhesive (3) and because the macro-response can be impaired as a result of specific interactions that are mediated by FHA (4).

In the production of FHA for acetic vaccines, the fermentation of B. pertussis, a human pathological pathogen (problems of safe production) and delicate microorganism (requires an expensive medium) with slow growth rates (long fermentation times, low yields), and the contamination of FHA preparations with other disease factors are problematic, which can contribute to some neo-effects that have been observed during vaccination. Recombinant hybrid proteins containing FHA sequences (5 and 6) have been formed, although hybrid proteins comprising antigens unrelated to the specific immune response are generally recognized as undesirable for vaccination purposes.

According to an embodiment of the invention, a plasmid for the expression of FHA in Escherichia coli or Saimonella

(in particular Saimonella typhimurium) is provided, which is characterized by. that it is a plasmid for the expression of structural genes and the plasmid

- comprises a DNA region encoding FHA, and further

- One or more Koάons of this DNA area to the

Main codon uses (Maj or Codon Usage) in E. coli are adapted.

represent FHA area, be adjusted. According to a further embodiment of the invention, a plasmid for the expression of FHA in Escherichia coli or Salmonella (in particular Salmonella typhimurium) is provided, which is characterized in that it is a plasmid for the expression of structural genes and the plasmid

- comprises a DNA region encoding FHA, and further

the N-terminal FHA region is represented by the DNA sub-region according to FIG. 2D or the following DNA sub-region:

According to a further embodiment, an expression plasmid (or recombinant plasmid) for expressing FHA in Escherichia coli or Saimonella (in particular Salmonella typhimurium;) is provided, which is characterized in that it is a plasmid for the expression of structural genes and the plasmid

a DNA region encoding FHA, and

- In reading directions in front of this DNA area comprises two cistrons in such a way that the ribosome at the start codon of this DNA area begins (again) with translation.

A plasmid according to the invention for the expression of FHA in Escherichia coli or Saimonella (in particular Saimonella typhimurium) can provide the following features in the reading direction: - promoter,

- Shine-Dalgarno sequence,

- first cistron, starting with a start codon and ending with a stop codon,

- intercistronic area,

- Second cistron, starting with or consisting of a start codon,

DNA region encoding FHA and

- at least one stop codon.

A DNA region that encodes FHA (wild-type FHA) is present in pRMB2. In the present context, DNA region is further understood to mean DNA sequences which likewise encode wild-type FHA, but which differ from the DNA region present in pRMB2.

In the plasmid according to the invention, the DNA region encoding FHA may lack the B. pertussis bvg locus.

The plasmid according to the invention can be constructed using a pEX vector, for example the vector pEX31A.

The plasmid according to the invention can be characterized by the lambda promoter (PL) and / or by the Shine-Dalgarno sequence of the polymerase of the bacteriophage MS2.

In the plasmid according to the invention, the first cistron

have the sequence or a partial sequence of the polymerase of the bacteriophage MS2 between the start and the stop codon.

The plasmid according to the invention can be characterized by an intercistronic range from 1 to 50, in particular 1 to 10 and for example about 5 codons.

In the plasmid according to the invention, the second cistron can comprise approximately 3 to 10 bases. In the case of the plasmid according to the invention, the at least one stop codon can be from the DNA region. the FHA encodes, follows immediately, or is separated by a spacer with about 5 to 100, in particular about 10 to 50, bases.

In all of these plasmids, the DNA region which encodes FHA can be adapted to the main codon use in E. coli, in particular with regard to one or more codons which represent the N-terminal FHA region, for example according to FIG. 2D or according to the following Sequence:

According to a further embodiment of the invention, a plasmid for expressing FHA in Escherichia coli or Salmoneila (in particular Saimoneila typhimurium) is provided, which is characterized in that it is a plasmid for expressing structural genes which

- the atpE transition start area of Escherichia coli and

- Includes a DNA region that encodes FHA.

This DNA area. the FHA encodes the bvg locus of 3rd pertussis.

The plasmid according to the invention can be characterized in that it has been constructed from pJLA 503.

In the plasmid according to the invention, a linker can be found behind the atpE translation start region of E. coli in the reading direction

be present in which the DNA region is inserted or which is followed by the DNA region which codes the FHA.

It can be the Linicer of Figure 2 3. Also, with the invented piasmid in front of the DNA area, the FHA coded, a linker according to FIG. 2B and a linker according to FIG. 2E is provided behind this DNA region.

The plasmid according to the invention can further be characterized in that in the DNA region. the FHA encodes, the sub-region which encodes the first to a maximum of the fifteenth amino acid at the N-terminal, is modified compared to the wild-type FHA without changing the amino acid sequence of the wild-type.

Again, for all of these plasmids, the DNA region encoding FHA can be linked to the main codon usage in E. coli may be adapted, in particular with regard to one or more codons which represent the N-terminal FHA region, for example according to FIG. 2D or according to the following sequence:

According to the invention, an efficient and direct expression of unfused FHA in Escherichia coli is provided. The biological separation of FHA from other virulent B. pertussis determinants which is achieved thereby offers a solution to the problem that FHA preparations are contaminated with other virulent Bordetella determinants. Expression of FHA in E. coli also solves the difficulties associated with fermenting large amounts of the delicate, slow-growing human pathogen.

Furthermore, the expression of FHA in Saimonella typhimurium aro A is achieved according to the invention. The construction of a live vaccine carrier strain of these Saimonella subspecies that expresses B. pertussis antigens can lead to the provision of oral whooping cough vaccines that stimulate both mucosal and systemic immunity. This opens up new perspectives for the development of both subunit vaccines and total vaccines and for the use of purified antigens in the development of kits for serodiagnostic or epidemiological studies on whooping cough.

According to further embodiments of the invention, E. coli and Saimonella, for example Saimonella typhimurium, are provided as hosts for a plasmid according to the invention.

The invention is explained in more detail below on the basis of experimental data and figures. Show it:

Figure 1 shows the production of FHA in a two-cistron system;

Figure 2 shows the construction of a plasmid for the direct expression of FHA;

FIG. 3 shows the direct expression of FHA in E. coli and in Salmonella typhimurium aro A.

The cosmid pRMB2 (7) contains the genetic information encoding the first 3277 amino acids of the fhaB gene (5, 6). A com The implementation of this gene with the bvg locus, which positively regulates FHA expression in B. pertussis, does not lead to its expression in E. coli. Two different strategies were used to achieve an efficient expression of FHA in E. coli.

Two-cistron system

The pEX31 plasmid cloning series (8) is usually used to achieve gene fusions with the first 98 amino acid residues of the NH2 terminus of the replicase protein of the bacteriophage MS2, the expression of which is controlled by the lambda PL promoter. FHA gene fusions are formed in pCGIT (amino acids 882 to 1670), pCG22 (amino acids 1670 to 3241) and pCG24 (amino acids 2028-3241). These gene fusions are used to represent the region containing the epitope (amino acids 1670 to 2028) recognized by the monoclonal antibody P12H3 (9) used to determine FHA expression. A two-cistron system for FHA production is used which is not fused with the MS2 polymerase in order to take advantage of the efficient Lambda PL promoter and the high ribosomal translation initiation rate of the MS2 polymerase Shine-Dalgarno sequence to use. In clone pCG16, the 8.4 kb Sphl-Sphl DNA fragment, which encodes amino acids 16 to 2853, is cloned in the reading direction behind the TAG stop codon, which is located at the XbaI site of the multiple cloning site of the vector pUC18NotI is present. This stop codon is in the reading frame with the MS2 sequence of pEX31A. The intercistronic region is 15 nucleotides in length; after translational termination at the TAG stop codon, the ribosome transfers directly to the ATG start codon within the SphdI site of the FHA fragment; translation of the second cistron begins. The translation is then terminated at the double stop codon (TGA-TGA) present in the plasmid; see. Figure 1 Direct expression of FHA

The expression vector pJLA503 (10), which comprises the heat and warmer gulated tandem promoters PR and PL and the highly efficient E. coli atpE translation start region, is modified to provide additional restriction sites to subclone the FHA gene and the Positioning of the stop codons in the reading frame to enable translation to be terminated. This new expression plasmid is called pJLACG1. The first 15 amino acids of the fhaB gene are modified by left-directed mutagenesis in order to break down the expected RNA secondary structure around the ribosome binding site, but to secure the amino acid sequence of the product to be expressed; see. Fig. 2.

The expression plasmids pCG18 (amino acids 16 to 2853), pCG32 (amino acids 1 to 2853) and pCG26 (amino acids 1 to 3277) are transformed into E. coli CAG629 (from Dr. C. Gross), E. coli EC538 (from Dr J. McCarthy) and E. coli EC876 (from Dr. R. Brownlie) and in S. typhimurium aro A SL3261 (11). After induction at 42 ° C, the entire cell extracts are subjected to polyacrylamide gel electrophoresis; the presence of recombinant FHA is checked according to the Western blot test; see. Fig. 3. Maximum expression of FHA in E. coli is achieved with EC538 pCG26. This construct also expresses high levels of FHA in S. typhimurium aro A. The plasmid pCG26 expresses an unfused recombinant protein with an approximate molecular weight of 220 kD. This protein covers the coding sequences of FHA that are required for the expression of all epitopes that are recognized in the immune response against wild-type FHA. Experimental data in connection with Figure 1:

FHA production in a two-cistron system.

(A) The area encoding the epitope recognized by the P12H3 monoclonal antibody (from Dr. C. Parker) is shown by the black bar. Shaded bars correspond to the fhaB gene sequence which is encoded by pCG22, pCG24 and pCG17 in the form of fusion proteins or in the two-cistron system (pCG16). The plus sign and the minus sign represent the presence or absence of a reaction to the proteins which are produced by clones with the monoclonal antibody P12H3 or a polyclonal anti-FHA antiserum in Western blot tests.

(B) SDS-PAGE of whole cell extracts stained with Coomasie-Blue. Lane 1: molecular weight standards; Lanes 2 to 6: E. coli 537 (pCI 857 ^τs ) containing pCG24, pCG22, pCG17, pCGlδ or pEX31A; Arrows indicate the main fusion proteins.

(C) Western blot analysis using the P12H3 monoclonal antibody. Lane 1: molecular weight marker;

Strip 2: purified FHA from B. pertussis (Tohama strain); Lanes 3 to 7: E. coli 537 (pCI857 ^τs ) containing pEX31A (strip 3), pCG22 (strips 4 and 5), pCG16 (strips 5 and 6). For strips 4 and 6, 30 min. long, while strips 3, 5 and 7 induced for 2 hours.

(D) Western blot analyzes using polyclonal

Antiserum against FHA. Lane 1: molecular weight standards; Strip 2: purified FHA from B. pertussis; Lanes 3 through 11: E. coli 537 (pCI857 ^τs ) containing pCG24 (strips 3 and 7), pCG22 (strips 4 and 8), pCG17 (strips 5 and 9), pCG16 (strips 6 and 10), pEX31A (Strip 11). Strips 3 to 6 were 30 min. long while the strips were induced for 7 to 11 for 2 hours. Molecular weight standards (sizes in kD) are indicated by arrows.

(E) Graphical representation of the two-cistron system in the clone

pCG16.

Experimental data in connection with Figure 2: Construction of plasmids for the direct expression of FHA.

(A) Expression plasmid pJLACG1, which contains the lambda promoters PR and PL in tandem arrangement (black arrows), the atpE translation initiation area (black lines), the FD transcription terminator, the β-lactamase gene and the needles / Sall Multiple Cloning Site (MCS).

(B) Sphl / Sall linker, which was used to modify the multiple cloning site of pJLA503 to construct pJLACG1 and subcloned various DNA fragments for cloning FHA in the expression plasmid

enable.

(C) and (D)

Original coding region (C) and modified coding region (D) of the Ndel / Sphl linker. The nucleotide sequence of the N-terminal FHA region is shown in the one-letter notation. The Ndel restriction site (CATATG) and the Sphl restriction site (GCATGC) are specified. Base pair changes, which correspond to differences between the original nucleotide sequence and the synthetic olionucleotide linker Ndel / SphI, are indicated by small arrows, the large arrows continuing part of the atpE translation initiation region which extends from Expression plasmid pJLA503 is encoded. Furthermore, the RNA stability +/- 50 bases from the ATG start codon is indicated.

(E) Sphl / EcoRI linker for pCG32.

Experimental data in connection with FIG. 3: Direct expression of FHA in E. coli and Saimonella typhimurium aro A.

(A) Graphical representation of the clones pCG26, pCG18 and pCG32 for the expression of FHA. The presence or absence of the Ndel / Sphl linker is indicated by black bars. Shaded bars correspond to the fhaB gene sequence encoded by pCG26, pCG18 and pCG32. The level of FHA expression, which was determined by Western blot analysis with the monoclonal antibody P12H3, ranges from low level (+/-) to intensive level {+++).

(B) Whole cell extracts from clones subjected to Western blot analysis. Lane 1: molecular weight standards; Strip 2: Purified FHA; Strips 3: B. pertussis (Stam Tohama !; Strips 4 to 7: E. coli CAG629 containing pJLACG1, pCG18, pCG32 and pCG26; Strips 8 to 10: E. coli 876 containing pCG18, pCG32 and pCG26 ; Strips 11 to 13, E. coli EC538 containing pCGIS, pCG32 and pCG26; strips 14 to 17: Saimonella typhimurium aro A SL3261 containing pJLACG1, pCGIS, pCG26 and PCG32. Living material etc. literature or source

E. coli 537 (pCI857 ^TS )

CAG 629

EC 538

EC 876

S. typhimurium aro A SL 3261 11

B. pertussis (Tohama strain)

MS2

Lambda PL

P CG 16 DSM 6 439

p CG 17 DSM 6 440

P CG 18 DSM 6 441

p CG 22 DSM 6 442

p CG 24 DSM 6 435

p CG 26 DSM 6 436

p CG 32 DSM 6 437

p EX 31 8

p EX 31A

p JLA 503 10; Medac (Hamburg) p JLA CG 1 DSM 6 438

p UC 18 Not I

p RMB 2 DSM 6 443

P 12 H3 9

MS2 polymerase

REFERENCES

1) Knapp S., and JJ. Mekalanos. 1988. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J.Bacteriol. 170: 5059-5066.

2) Robinson A., and L.A.E. Ashworth. 1988. A cellular and defined component vaccines against pertussis, in A.C. Wardlaw, and R. Parton (eds), Pathogenesis and Immunity in Pertussis. John Wiley and Sons, Chichester. pp 399-417.

3) Tuomanen E. 1986. Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis. Infect.Immun. 54: 905-908.

4) Relman D., E. Tuomanen, S.Falkow, D.T. Golenbock, K. Saukkonen, S.D.Wright. 1990. Recognition of a bacterial adhesin by an integrin: macrophage CR3 (Mß2, CD11b / CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 61: 1375-1382.

5) Domenighini M., D.Relman, C.Capiau, S.Falkow, A.Prugnola, V.Scarlato, R.Rappuoli. 1990. Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor. Mol. Microbiol. 4: 787-800.

6) Relman DA, M.Domenighini, E. Tuomanen, R.Rappuoli, S.Falkow. 1989. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc.Natl.Acad.Sci.USA. 86: 2637-2641. 7) Brownlie RM, JG Coote, R.Parton, JESchultz, A.Rogel, E.Hanski. 1988. Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis. Microb.Path. 4: 335-344.

8) Strebel K., E. Beck, K. Strohmaier, H. Schaller. 1986. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J.ViroI. 57: 983-991.

9) Frank D.W., C.D. Parker. 1984. Isolation and characterization of monocional antibodies to Bordetella pertussis. J.Biol.Stand. 12: 353-365.

10) Schauder B., H. Blöcker, R. Frank, and J.E.G. McCarthy. 1987. Inducible expression vectors incorporating the E.coli atpE translational initiation region .Gene. 52: 279-283.

11) Hoiseth S.K., and B.A.D. Stocker. 1981. Aromatic-dependent Saimonella typhimurium are non-virulent and effective as live vaccines. Nature. 291: 238-239.

Claims

P A T E N T A N S P R Ü C H E

1. Plasmid for the expression of FHA in tscnencma coli or Saimonella (in particular Saimonella typnimurium). characterized in that it is a plasmid for the expression of structural genes and the pl asmi d

- Includes a DNA region that encodes FHA. and further

- One or more codons of this DNA area are adapted to the Hauot codon use (major codon usage) in E. coli.

2. Plasmid according to claim 1, characterized in that on or mener codons, which represent the N-terminal FHA region, are adapted.

3. Plasmid for the expression of FHA in Escherichia coli or Salmonella (in particular Salmonelle tyommurium). characterized in that it is a plasmid for the expression of structural genes and the plasmid

- Contains a DNA area, aer FHA eroded, and further

- The N-termal FHA area may represent the DNA sub-area according to FIG. 2D or the following DNA sub-area

4. Plasmid for the expression of FHA in Esch ric hia coli or Salmonella (in particular Sal mone l l a typhi muri um), characterized in that it is a plasmid for the expression of structural genes and the plasmid

- a DNA region that encodes FHA, as well as

- In the laser direction in front of this DNA area comprises two cistrons in such a way that the ribosome at the start codon of this DNA area begins (again) with translation.

5. Plasmid for the expression of FHA in Esch ric hia coli or Salmonella (in particular Saimoneila typhimurium; with the following features in the reading direction:

- promoter,

- Shine-Dalgarno sequence,

- First cistron, starting with a start codon and ending with a stop codon.

- intercistronic area,

- Second cistron. Starting with or ending with a stop codon.

DNA areas encoding FHA and

- at least one stop codon.

6. Plasmid according to claim 4 or 5, characterized in that the DNA area. the FHA encodes the Dvg locus from B. pertussis.

7. Plasmid according to one of the narrowing Ansorϋcne, constructible using a pEX vector, for example the veκtors pEX31A.

3. Plasmid according to one of the foregoing Ansorücne, characterized by the Lampda promoter (PL).

3. Plasmid according to one of the preceding claims. characterized by the Shine-Dalgarno seduence of the polymerase of bacteriobnagen MS2.

10. Plasmid according to one of the preceding claims, characterized in that the first cistron between the start and the stop codon has the seduence or a partial sedence of the polymerase of the bacteriophage MS2.

11. Plasmid according to one of the preceding claims.

characterized by an interciströmischen range from 1 to 50, in particular 1 to 10 and for example about 5 codons.

12. Plasmid to one of the previous assays. dadurcn characterized in that the second cistron comprises about 3 to 10 bases.

13. Plasmid according to one of the preceding claims, characterized in that the at least one stop codon immediately follows the DNA region which encodes FHA or is separated by a spacer with about 5 to 100, in particular about 10 to 50, bases.

14. A plasmid according to one of the foregoing Ansorücne, additionally characterized by the features according to claim 1, 2 or 3.

15. Escherichia coli, comprising a plasmid according to one of Ansorücne 1 to 14

15. Salmonella, for example Saimonella typhimurium. comprising the plasmid according to any one of claims 1 to 14.

17. Plasmid for the expression of FHA in Escherichia coli or Salmonella (especially Salmonella tyonimurium;, characterized in that it is a plasmid for the expression of

Structural genes. the

- the atpE translation start areas of Escnerichia coli and

- comprises a DNA region encoding FHA.

18. Plasmid according to claim 17, characterized in that the DNA region encoding FHA lacks the Dvg locus of B. pertussis.

19. Plasmid according to claim 7 or 8, characterized in that it has been constructed from pJLA 503.

20. Plasmid according to one of claims 7 to 9, characterized in that a linker is provided behind the atpE translation start region of E. coli, into which the DNA region is inserted or followed by the DNA region, the FHA encodes.

21. Plasmid according to Ansoruch 20, characterized by the linker of FIG. 2 3.

22. A plasmid according to claim 20, characterized in that a linker according to FIG. 2 B is provided in front of the DNA region encoding FHA and a linker according to FIG. 2 E is provided behind the DNA region.

23. Plasmid according to one of the Ansorücne 17 to 22. dadurcn characterized in that in the DNA-3eraιch. the FHA encoded. the minor sub-area, which encodes the N-tarminal the first bis maxi ma l the fifteenth amino acid, is modified compared to the FHA wild type without changing the amino acid consistency of the wild type.

24. Plasmid according to claim 23, additionally marked by the features according to claim 1, 2 or 3.

25. E. coli. comprising a plasmid according to one of claims 17 to 24.

25. Saimoneila, for example Salmonella typhimurium, comprising a plasmid according to one of claims 17 to 24.