CA2285480A1 - Screening method for proline-rich proteins - Google Patents

Abstract

A method for screening a pathogenic microorganism for a secreted or cellsurface protein potentially useful as a vaccine antigen involves the step of identifying proline-rich proteins, either as the amino acid level, or preferably at the DNA level by employing PCR. Suitable consensus sequences are described as (PXm)nP or (PPXq)nPP wherein: P is proline; X is any amino acid, m is from 1 to 5; q is 1 or 2; and n is 2 or more.

Description

SCREENING METHOD
FIELD OF THE INVENTION
- This invention relates to the identification of proteins either secreted from or on the cell surface of microorganisms, and of the genes encoding them. These proteins may be useful as ' S antigens for the preparation of vaccines against the microorganisms.
BACKGROUND TO THE INVENTION
The characterisation of proteins produced by pathogenic microorganisms is, of course, a well known and active field of research.
One specific area of interest is the identification of secreted, exported and cell-surface proteins, which might be suitable for the design and development of vaccines, especially subunit vaccines - compositions which comprise one or more antigens which are capable of eliciting an immune response.
It would be useful if such proteins could be easily identified from the vast amount of uncharacterised sequence information now available from the genomes of microorganisms.
The identification of sequence motifs which are characteristic of exported or surface-located proteins in bacteria facilitates rapid screening of genomes for potential virulence factors and vaccine components.
It has now been found that the presence of a proline-rich region in a protein is a good indicator of its cellular location. This information can be used both to screen sequence information in databases and to amplify DNA from microorganisms, even in the absence of sequence information, by designing primers based on degenerate proline-rich sequences.
DESCRIPTION OF THE INVENTION
According to the present invention, there is provided a method for screening a pathogenic microorganism for a secreted or cell-surface protein comprising the step of identifying ' 25 proline-rich proteins. Preferably, the method further comprises the step of selecting the proline-rich proteins which have been identified.
As used herein, the term "proline-rich" protein denotes a protein having substantially more than the mean proline content of the proteins of the particular microorganism undergoing screening.

The proline-rich protein preferably comprises one or more amino acid motifs having the following general sequences:
- (PX",)"P or (PPX~)"PP
wherein: P is proline; X is any amino acid; m is from 1 to 5 (preferably m is from 1 to 3); g is 1 or 2; and n is 2 or more. (preferably n=6). It will be appreciated that, for each of the n repeats, m and g may be the same or different. It will also be appreciated that the various X
residues may be the same or different. ~ .
A preferred motif, referred to hereafter as the proline-rich region (PRR), is represented by the sequence {PX",)~P. For each of the 6 repeats of PX",, the value of m may be the same or different. The PRR thus represents a stretch of seven non-consecutive proline residues, spaced by at least one but no more than five other amino acids. The probability of finding the PRR by chance in a sequence in which the 20 amino acids are randomly distributed is extremely low (7.8x10-~°).
It will be understood that these sequences represent motifs in which proline residues repeat relatively closely together, either singly (eg. PXPXP or PXXPXXPXXP) or doubly (eg.
PPXPPXPPXPP or PPXXPPXXPPXXPP), or as a mixture of both (eg. PPXXPXXPP). In other words, the motifs are of the general sequence (PSXm)"PS, wherein each s is independently 1 or 2.
The X residues in these general sequences, which may be any amino acid, may the same as each other, but are generally different. The upper limit on n is, of course, dependent upon the protein and the microorganism in question, and may be large (eg. greater than 10) for large proline-rich proteins.
The screening method may be applied to any microorganism, but is preferably applied to pathogenic microorganisms such as a bacterium or parasite. The bacterium may be Gram positive or Gram negative and may be, for example, Helicobacter pylori, Neisseria meningitides, Mycobacterium tuberculosis, Escl~erichia coli, Neisseria gonorrhoeae, Haemophilus irtfluenzae, Bordetella pertussis, Vibe°io cholerae and Bacillus subtilis. The parasite may be a protozoan, such as Leishmania, the malarial parasite Plasmodium, or the Legionella. Other suitable microorganisms include Saccharomyces, Chlamydia, and Borrelia burgdorferi.
The screening method may be conducted at the amino acid level, by identifying proteins of known sequence, but unknown cellular localisation. This can conveniently be carried out by -3_ computer, using the FINDPATTERN program available in the GCG Wisconsin Package (version 8.1 or later), for instance. A suitable database for source information on amino acid sequences of microorganisms is the public database SWISSPROT.
Alternatively, and preferably, the screening method is conducted at the DNA
level, by . 5 identifying and amplifying DNA from a library which contains a consensus sequence encoding a proline-rich amino acid motif.
Where the screening method is conducted at the DNA level, the method of the present invention preferably comprises the step of screening a DNA library of a microorganism for DNA encoding a proline-rich amino acid sequence. This first step is preferably followed by selecting DNA which encodes a proline-rich amino acid sequence.
The library is suitably a plasmid genomic library, prepared using known techniques. Suitable libraries for screening Helicobacter pylori, for instance, are described in Censini et al. ( 1996) PNAS USA 93:14648-14653 and Covacci et al. (1993) PNAS USA 90:5791-5795.
The step of screening the DNA library may comprise any suitable screening technique, but preferably involves the screening and amplification of specific DNA sequences containing a DNA sequence encoding a proline-rich amino acid sequence. Suitably the screening step comprises at least one polymerase chain reaction (PCR) step.
In a preferred method, the screening involves the preparation of a degenerate DNA primer encoding a proline-rich amino acid sequence, using known methods. The proline-rich amino acid sequence may be determined by elucidation from known proteins iii general, or more specifically by determining consensus sequences from known secreted or cell-surface proteins either generally or from a specific microorganism of interest.
The degenerate primer is used to amplify specific sequences containing one of the degenerate primer sequences (or sufficiently similar to hybridise thereto) to produce a population of amplified DNA. The sequences thus obtained may then be sequenced and, using a reverse PCR primer and an internal primer, the DNA encompassing the sequence encoding the proline-rich amino acid sequence can be obtained.
The screening process may further comprise the step of expressing the DNA so obtained in a suitable host organism, such as a prokaryotic or eukaryotic host cell.
According to a further aspect of the invention, there is provided a proline-rich protein identified by the screening method of the invention. Such proteins may be formulated with suitable pharmaceutical excipients, such as carriers, and may include other antigens and/or one or more adjuvants.
In particular, there is provided a protein comprising an amino acid sequence depicted in Figure 2 or Figure 3 <SEQ IDs 2-4>, or a functionally active fragment or derivative thereof. As used herein, the term "functionally active" means retaining a substantial biological activity, preferably antigenicity. This protein was identified as a proline-rich protein in H.pylori.
According to a further aspect of the invention, there is provided nucleic acid (eg. RNA or DNA) encoding a protein of the invention (or their functionally active fragments or derivatives). In particular, there is provided a DNA sequence encoding the protein of Figure 2 or a functionally active fragment or derivative thereof. This DNA sequence preferably comprises <SEQ ID 1> or a fragment thereof (eg. the coding sequence of <SEQ ID
1>) According to a further aspect of the invention, there is provided an immunogenic composition comprising a proline-rich protein of the invention or nucleic acid encoding such a protein, and a method for producing such a composition comprising the step of bringing a proline-rich protein of the invention (or nucleic acid encoding such a protein) into association with a pharmaceutically acceptable excipient.
The immunogenic compositions of the invention preferably also comprise one or more further antigenic H.pylori proteins. These further proteins may be proline-rich proteins themselves, but this is not necessarily so, and may be, for example, VacA, CagA, or NAP.
In particular, there is provided an immunogenic composition comprising either the protein of Figure 2 or nucleic acid encoding the protein of Figure 2, and a method for producing such a composition comprising the step of bringing the protein or nucleic acid into association with a pharmaceutically acceptable excipient.
The immunogenic compositions of the invention are preferably formulated as vaccines. These vaccines may be prophylactic or therapeutic, and preferably comprise one or more adjuvants.
Such vaccines typically comprise antigen or antigens in combination with a "pharmaceutically acceptable excipient" ie. any excipient that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents {"adjuvants"). Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
Preferred adjuvants to enhance efficacy of the composition include, but are not limited to: {1) . 5 aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulphate etc. ; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components), such as for example (a) MF59 (W090/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5%
Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer (b) SAF, containing 10%
Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiT"~ adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); (3) saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA}; (5) cytokines, such as interleukins (eg. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. IFN-y), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
Alum and MF59 are preferred.
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
The immunogenic compositions (eg. the antigen, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying agents, pH
buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg.
nonhuman primate, primate, etc. ), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. The amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally eg.
by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648].
According to a further aspect of the invention, there is provided an antibody which is specific for a protein according to the invention.
According to a further aspect of the invention, there is provided a vector including nucleic acid according to the invention. There is also provided a host organism transformed with a vector according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows schematically the PCR techniques used to identify and prepare a proline-rich H.pylori protein. The upper-panel (A) describes the first PCR experiment using a degenerate _.___ _. _ ~

proline primer and the universal primer of the pBluescriptTM vector. The lower panel (B) describes the second PCR experiment, using an internal specific primer and the reverse primer of the vector.
Figure 2 shows the nucleotide <SEQ ID 1> and amino acid <SEQ ID 2> sequences of the proline-rich H.pylori protein. The putative leader peptide and proline-rich consensus sequence are underlined, and "SD" denotes the possible Shine-Dalgarno sequence.
Figure 3 compares the sequences <SEQ IDs 2-4> of the proline-rich proteins from H.pylori type I and type II strains. "----" denotes amino acids not found in proteins from type II strains.
Figure 4 shows Western analysis of H.pylori extracts. The antiserum used was raised against the proline-rich protein of Figure 2.
DETAILED DESCRIPTION OF EMBODIMENTS
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of computer searching, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, 1989);
DNA Cloning, Volumes I and ii (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984);
Transcription and Translation (B.D. Hames & 5.3. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A
Practical Guide to Molecular Cloning ( 1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (ed.
Miller and Calos 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. ( 1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);
Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (ed. Weir and Blackwell 1986).
1 ) Computer analysis of protein sequences It was considered that proline-rich proteins might have specific functions in microorganisms and specifically to be correlated with secreted/exported or cell-surface proteins. On this basis, the FINDPATTERN program (GCG Wisconsin package, version 8.1 ) was used to screen the SWISSPROT database for sequences containing the following sequences:
(PX",)"P or (PPX~)nPP

wherein: P is proline; X is any amino acid; m is from 1 to 3; q is 1 or 2; and n is 2 or more.
These sequences were designed to detect any kind of proline-rich motif in proteins which had already been characterised.
The search was limited at the outset to three microorganism classes: protozoa, Gram positive bacteria, and Gram negative bacteria. The results were as follows, classified by cellular location and/or biological function:
Gram positive bacteria (55 SWISSPROT
entries identified) 27 Membrane-associated proteins 49%

16 Endoglucanases 29%

3 DNA-binding proteins 5%

5 Other 9%

4 Unknown function (hypothetical 7%
proteins) Gram negative bacteria (96 SWISSPROT
entries identified) 63 Membrane-associated proteins 65.5%

14 DNA and RNA-binding proteins 14.5%

2 Endoglucanases 2.5%

9 Other 9%

8 Unknown function 8.5%

Protozoa (53 SWISSPROT
entries identified) 26 Surface antigens 49%

13 Other membrane-located functions24.5%

4 DNA and RNA-binding proteins 7.5%

2 Actin-binding proteins 3.5%

6 Other 1 I

2 Unknown function 3.5%

These results clearly demonstrate that screening for proline-rich motifs produces a highly relevant percentage of membrane-associated proteins. Indeed, amongst the proteins identified in this way are two of the major components of the acellular vaccine developed against Bordetella pertussis, namely filamentous haemagglutinin (FHA) and pertactin [Rappuoli et al.
(1991) TIBTECX 9:232-238].
These results clearly support the general principle that analysis for the presence of proline-rich motifs in an amino acid sequence is useful for the identification of secreted or cell-surface proteins.

In addition, since the invention was initially conceived, additional studies have been carried out which further validate this approach.
Firstly, the 17567 prokaryotic proteins in release 34 of SWISSPROT were searched against S the PRR motif [{PXi_;)~P], using the FINDPATTERN program in version 9.0 of the GCG
Wisconsin package. 1536 motifs were identified in 190 bacterial proteins (ie.
just over 1 % of all proteins analysed). Of these, 98 were from Gram-negative bacteria, and the remaining 92 were from Gram-positive bacteria. On the basis of either known function or predicted cellular location [PSORT algorithm - http://psort.nibb.ac.jp], it was found that 68 of the 98 (69.4%) and 64 of the 92 (69.5%) sequences were surface-located or exported proteins.
Secondly, the published genomes of Escherchia toll, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautothropicum, Methanococcus jannaschii and Mycoplasma pneumoniae were searched for the presence of PRR motifs. Of the 53 proteins identified in this way, 39 (73.6%) were predicted to be membrane-associated or exported, thus confirming 1 S that PRR motifs are preferentially found in proteins localised in the superficial compartments of the bacterial cell. In H. influenzae, all five PRR-containing proteins are predicted to be surface-located or exported.
Number Number Number Source of containingpredicted proteins the PRR to be analysed motif surface-located or secreted SwissProt Gram-negative bacteria- 98 68 Gram-positive bacteria- 92 64 Total 17567 190 132 Genomes E. toll 2438 1 S 12 H. influenzae 1679 5 5 H.pylori 1577 9 7 M jannaschii 1735 3 2 M. thermoautothropicum1871 7 S

M. pneumoniae 677 14 8 Total 9977 53 39 2) Detailed analysis of PRR-containing proteins The 29 PRR-containing proteins found in the genomes of E.coli, H.pylori, and H.irtfluenzae were analysed in more detail (see page 11 - PRRs are shown underlined).
The average length of the PRRs is approximately 32 amino acids.
The shortest PRR was found in the RhsA transmembrane protein of E. coli ( 14 residues):
1235-PLNPVTNTDPLGLEVFPRPFPLPIPWPKSP-1264 ~ -=
The longest was in the H.pylori Tong homologue Hp1341 (56 residues):

It is apparent that, in addition to the PRRs themselves, the flanking regions are also rich in proline.
The following table shows the bias in the PRRs towards proline residues. The amino acid composition of PRRs is clearly different from the average across the genome:
Amino acids Average PRR
%

P 3.8 28 F Y W (aromatic) 9 2.3 M L I V (hydrophobic) 26.5 11 A T G S (small, non-charged)26 25 D E Q N (negative charge) 20.5 21 K R H {positive charge) 13.3 12.7 Whilst the distributions shown in the final three rows in the table are the same for PRRs as for the genome in general, hydrophobic and aromatic residues are under-represented in PRRs, whereas proline is obviously over-represented. This analysis suggests that PRRs will usually be in hydrophilic regions of a protein.

v v c c ~ c~ m °
m 3 a E ~ ~a°. '° d ~ a .c .c u~ ca ~, E E c a v ' aa~ c Ua<".U ~C a'~C O~~_cd U N d ~ N ~ ~ a.
'D C U O E O .~ ~C LZ ~ c p E 'O w ai N O ~ . f0 °
47 N a~ ~ O ~ ~, tin f0 O ~ O m N ~ d C ~ ~ .a - ~ m c c ~ ~ .d .C U is WU V1 O E a E ~ E ' -c o _c .52 c O O ~ ~ ~ fn O N O L'~ N O 1p C ~Ø. ~_ r0.. E ,~ ~ ~ v a. .o .a ~ a~ -o c ~ ~ N v. E C ~ C c, '~? ~ ~ o "~ °~ ~ m O c c ,a ~ N O fc0 ~ ~ .Q ~ ~ 'O ~ Q .~ .C ~ .a E O V m ~ c ~ Q
U ~ ~ W .~ i= O ~ C C ~ ~ r~r ~ C ~~ ~ ~ V ~~ O V O
~ O U U ~ ~ .O lp ~ U O '~ V (~U V ~ ~ T ~ ~ y ~N
o E a ~ ~ Q
cV ~ :o c1 a, 'v o- :n _c ~ :g ~. °
d E c~ E ~ W' E I~ ~ d d (~ E D V ~ ' ~ ~ N ~ X y,~ c. c c '° a a _ Q d a ° ~ ~. ~ _~ c U
X X ~ lfJ CNO p ~ O ~ Iv N N Lf~ tn C~ U~ U Q Q LL .~ O ~ tf~ OO N I~ T c!' tC'~ O N
_a C c O N ~ f6 c ~ N c0 f~- 1~ ~ M p O O O _ M_ N_ O M
O.~.C C O O O_OO_O_ _ a x = = x x ~ x x x x x x x x x N
O ~ M
N
a v ~o a _~
M ,~
o, ~ a ~ cue. .~ 2 0 > °~ cn o a i a ~ ~ Q z 4 cn w cn a °' N H W yJ w~", Y O
O
~ LLJ Y >, a b~ Vj M ~ ~ D .O >
Q Q
a 2 N Y ~~ UJ
Q ui a cn cC p~ a~
N .b Y OD
4. o ~ a a a C~ ~ z ~_ w °o C'J Q ~ Y W t4 0. ~ Y
.a .s a z ~ ~ "' > a '~ ~ >
.~.. .~ ~ cn ~ n. c.~ a u.r ~ Y y >> a cu'in~a °' ~ Y ~ ~ m z a ~ u~.~ ~ cY. ~ ~ c~ ~ ~ Y
z = ~ > Y a a '~ a ~ Y u°'.~ ~
a z a a a a ~ o a o '~-~ Y a d f3, ~ z ~-. ~ ~ ~ ~c o a ~v Y a°, w W.~u ~ ~ Y o a ~ d a zQ ~ ~ w '!' t- '- ,Yl, cQn ~ ~ a Y a w Q Q ~ ~ c° a a E w > a ~ a oW~ ~ ~~ Y> c°Y az ~ a YaM
~ a I- a~ Y
°~ c~r~aa ~~ ~ ~ a aC~'l o a wa ~ ~~,a -c'~na ~ o a a~ ~ a Y°~-a a H a Fa-. a a a a ~ ~ a a ~i o a M a a z ,M
> c~ w ~ ~ a a ~ a a n o a ~ ~ c~ Y
w a a ~ a a ~ '~ a z o Y a a a g a i a z " a ~ ~ a > ~ a o a ~ a ~ ~ a w a a a a a uQ.. J d c~ a N ~ a c, a 4~ ~- ~ a ~ a ,'~; a z ~ ~ o a a M ~ ~ ~
z > m ,.,., o a a ~ uW~ ~-- r. i- n. .r~ Y a -, cn o, n. ~ c~ ~- a cn a a a a a > ~ ~ ~ a a c~ a d ~ ~ a a d o a ac a a o. a c9 ~ ~ a > ~ a o Q ~ ~ ~ a mu c3 z c~ C~ a, a a z a ,r j ~ w a ~ OJ., 0 w ~ a Y d > Y ~ ~ ~ a a I- I- Y a p~ Y ~ ~ J Y -i m ~ c~ ,.~. > a > a a .,; ~ m uW-a a a ~ Q ~ a a a a ° a ~ a ~.a: ~ Z z z z ° a a a z > Y
Y g ,-- a o. i- a a E- Y z a ~ ~ a a a n. a ° W - a ~ c~ ~, n. E- Cy-- f- ~, n~ Y z x n. a a a, a z w ~ c~ Q ,~, a a u~ ~~ cn " ,l, a c~ a a ,.fl a a a a a ~.u a <t >
w a a a ° > "' a -~ a Y a a ~ a d a a °- c~ c~ ~ a a s-Q- a °- '.'.' z z a > > Y m > a o > Y Q a a a m z ~ z z o a ~ _ ,~ u.r Y mu u.i a ~ ~ ~. ~ fn > Q D ~ C'1 ti N a ~- H" ~-- E., f- 'L x U' Y a a Y
E-- > a ~ cn o a o- a m cn a c m a ~ n. a ~ a y- z Y a z E- a c~na~a~g~°aa~a~~ ~ ua.~z~zz': aa~YwwacaCmi o ~aa~o°~~~aQ~~aa.n~..~aa~aa~°. ~aa~
a Q~aaazc~E->oQC.~ma>,~~ >c~aC3C~ q"',aQx > ~ g ~ ~ J J a o ~ ~- n. a, ~ o z c~ z z ~, w ~ cn z °- Y E- i- u.
a z a a E- C3 ~ u., ~ u.W- z ~- f" .~; Y C~J a u. Q w c~ CJ a ~ o > z a a a Q cVn ~ ~.u ~ '' w Cl ~ a a ~ w z a o. u.~ a Q Y
,1, o D c~ C! a Q m a '~ a ~ ~ > ~:; o a z ~ z z .4 Y Y ~ ~ f.- u' n. Q U
a a a a a a a a a- a a a, a a~ m-- um- ~- c a cW-- Y ~ u.~ > x cn .n a s , ii ~ a a ~ a a a a a a. Y J J
L~ O a~O CO COD N N O ~ M CAD O N M M ~ I- (00 M ~ N N ~ ~ O ~ ~ ~ M <t' p r' M r~ r., f" Cfl N 'ct' I~ O O

3) Analysis of Hpylori ~enome library In order to extend the principles demonstrated above, the microorganism H.pylori was chosen to demonstrate the utility of the screening methods. The experiments were aimed at the identification and characterisation of surface-exposed proline-rich proteins common to type I
$ and type II strains ofH.pylori to use as candidate antigens in a new vaccine against H.pylori.
PCR was used to identify and amplify DNA sequences encoding proline-rich proteins. from a genomic library of H.pylori type I strain CCUG 17874 (Culture Collection of the University of Gotheborg). The library comprised HindIII-digested genomic DNA in pBluescript(SK+) [Censini et al. & Covacci et al., supra].
The library was screened for six different proline-rich pentapeptide sequences. According to a codon preference table compiled for H.pylori using the GCG Wisconsin package, these six sequences were translated into the following degenerate 15mer primers:
P A P A P

C G C G C

A C A C A

P T P T P

CCT ACT CCT ACT CCT

C G C G C

A C A C A

P E,K,Q P E,K,Q P

C A C A C

A C A C A

P T N T P

C G C G C

A C C A

P P Q P P

$ CCT CCT CAA CCT CCT

C C C C

A A A A

P I~ P I P

CCT TTA CCT ATT CCT

C C G C C C

A C A A

_ _ L_ _. ~___ _. __ ____ _~_ _ In six separate reactions, 100ng plasmid DNA and SOpmol each of degenerate primer and the upstream universal pBluescript T3 primer was used for PCR amplification (Figure 1 A).
In some of the reactions, more than one amplification product was obtained, indicating the presence of more than one proline-rich sequence. Each product obtained from this first PCR
a 5 reaction was completely sequenced, giving a sequence with the T3 primer sequence at one end and the proline-primer sequence at the other. From within this, a sequence was used to design a specific internal primer. --This internal primer was used for a second PCR reaction, this time coupled to the downstream T7 primer from pBluescript (Figure 1B). This amplifies a region with the T7 primer sequence at one end, and the internal primer sequence at the other, and which spans the proline-rich sequence.
The two sequences obtained in the two PCR reactions can be combined to give the complete DNA sequence between the pBluescript primers. This sequence was searched for open reading frames (ORFs). Where an ORF was not complete within a single plasmid, the terminal sequence was used to screen further clones.
4) Further analysis of a specific H.p lori proline-rich~rotein One of the several proline-rich ORFs identified in this way was singled out for further analysis. This ORF encodes a 223 amino acid protein (25.SkDa) with the sequence PASAP
near the C-terminus (Figure 2 <SEQ ID 2>). The 15 residue N-terminus signal peptide suggests that the protein is a secreted or cell-surface protein, although no canonical promoter region was located. Secondary structure prediction analysis results in a continuous a-helical structure, except for the proline-rich motif, which appears to be folded as a loop. No hydrophobic segments (suggesting transmembrane regions) were detected.
This protein can be formulated as an immunogenic composition and can, for instance, be used to produce antibodies.
. Although no significant homologies were detected when this sequence was first identified, subsequent sequencing of the H.pylori genome has identified this protein as ORF257 [Tomb et al. (1997) Nature 388:539-547]. No function has been described for this protein, although it has homology to conserved secreted proteins.

Using the TBLASTN algorithm, weak homology (27-31 % identity) can be seen with ORF6 of the.flaA locus of B.subtilis, ORF6 of the flagellar operon of Agrobacterium tumefaciens, and ORFB found in a similar locus of Rhizobium meliliti (GenBanlc L76929).
The fact that the PCR reaction identified a protein containing a PASAP motif, whereas the degenerate oligonucleotide primer was designed for the PAPAP motif indicates that, depending upon the stringency used for amplification, additional proline-rich proteins can be selected beyond those expected.

5) Presence of the protein in tune II strains The presence of this CCUG 17874 (type I strain) ORF in H.pyloni type II
strains was investigated using the G21 and G50 strains [Censini et al. (1996) supra]. The PCR fragment containing the complete ORF was used as a probe in Southern analysis of HindIII-digested chromosomal DNA from CCUG 17874, G21 and G50 strains under highly stringent conditions, and a similar band was detected for all three strains.
PCR primers were designed against the ORF and these were used to amplify DNA
from the G21 and G50 chromosomes:
5'-CCACCCAGAGAGCGAGAATTT-3' & 5'-CTAATTCATGGACAAAGATC-3' The amplified DNA (925bp) was sequenced, and the results are shown in Figure 3. The ORF
is well conserved between type I and II strains, except for a four amino acid deletion at positions 197-200 and a few other point mutations.
DNA encoding amino acids 19-183 of the ORF was amplified using the following PCR
primers:
S'-TGTGTGGGAATTCGCCAAGAATTGTTGCAATGCTCTGCG-3' 5'TGTGTGAAGCTTTTTTTGCCACAACTCTGTCAAAGC-3' which also introduced EcoRI and HindIII sites (underlined). This fragment was inserted into the pGEX-2T vector [Guan et al. (1991) Anal Biochem 192:262-267]. The truncated protein lacks the signal sequence and the 40 C-terminus residues. The protein was expressed as a GST-fusion in E.coli and was purified using glutathione affinity chromatography. Rabbit antiserum was raised against this recombinant protein, in the form of an immunogenic composition, and was used in Western analysis.
1 _ _ _ _ _T _ Against total protein extracts from H.pylori, a 34kDa protein species was detected (Figure 4A, lane I ). In an isogenic knock-out strain (produced by allelic exchange), the protein was not detected (lane 3). The discrepancy between the theoretical MW (251cDa) and observed MW
(34kDa) can probably be attributed to the high proline-content of the protein, which is known to cause abnormal migration in SDS gels.
The antiserum also reacted with protein extracts from H.pylori strains G27, 60190, 326, 639, 646, 6120 & 686 (all type I strains) and 621, 650, ZU+ & ZU- (all type II
strains), although the apparent MW was 30kDa (Figure 4A, lanes 2, 4, 5, 6, 7). The difference in MW
may be due to the 4 residue deletion described above.
Protein fractions from G27 strain culture supernatant, whole cells, and periplasm (polimixin B) preparations were tested by Western analysis (Figure 4B). The protein was detected in the whole cells (lane 1 ) and periplasmic fractions (lane 3), but not in the culture supernatant (lane 4), suggesting a periplasmic location. The band at lower molecular weight is also found in the isogenic mutant, and appears to be unrelated to the protein of interest.
It will be appreciated that the invention has been illustrated by means of example only, and that modifications to these may be made whilst remaining within the scope and spirit of the invention.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
S (A) NAME: Chiron SpA
(B} STREET: Via Fiorentina 1 (C} CITY: Siena , (E) COUNTRY: ITALY
(F) POSTAL CODE (2IP): 53100 Siena w (ii) TITLE OF INVENTION: Screening Method (iii) NUMBER OF SEQUENCES: 4 IS (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk 720K
(B} COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Ver 2.0/Microsoft Word 97 (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: PCT/IB98/
2S (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 895 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA
3S (xi) SEQUENCE DESCRIPTION FOR SEQ ID NO: 1 SS
(2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 223 amino acids (B} TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii)MOLECULE
TYPE:
protein S (xi)SEQUENCE PTION NO: 2 DESCRI FOR
SEQ
ID

Met ArgLys IleLeu LeuMetGly LeuIleLeu GlnAla LeuPheGly Glu GluAla AlaGln GIuLeuLeu GlnCysSer AlaIle PheGluSer Lys LysAla GluLeu LysAspAsp LeuArgGln LeuSer GluLysGlu Gln SerLeu ArgIle LeuGlnThr GluAsnAla ArgLeu LeuAspGlu Lys ThrAsp LeuLeu AsnGlnLys GluLysGlu ValGlu GluLysLeu Lys AsnLeu AlaAla LysGluGlu AlaPheLys ThrLeu GlnThrGlu ZS Glu LysLys ArgLeu LysAsnLeu IleGluGlu AsnGlu GluIleLeu Arg GluIle LysGln AlaLysAsp SerLysIle GlyGlu ThrTyrSer Lys MetLys AspSer LysSerAla LeuIleLeu GluAsn LeuProThr Gln AsnAla LeuGlu IleLeuMet AlaLeuLys ProGln GluLeuG1y 3S 195 150 155. 160 Lys IleLeu AlaLys MetAspPro LysLysAla AlaAla LeuThrGlu Leu TrpGln LysPro ProLysGlu AsnLysGlu AsnGln LysThrThr Glu ProThr ProAla SexAlaPro ProIleAla ProThr ProProLys Glu ProMet IleLys AspProAsn ThrLysGlu ProAla GlyVal SO (2) INFORMATION FOR SEQ ID N0: 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids (B) TYPE: amino acid SS (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein GO (xi) SEQUENCE DESCRIPTION FOR SEQ ID NO: 3 _lg_ Met ArgLys IleLeuLeu MetGly LeuIleLeu GlnAlaLeu PheSer Glu GluAla AlaGlnGlu LeuLeu GlnCysSer AlaIlePhe GluSer $ 20 25 30 Lys LysAla GluLeuLys AspAsp LeuArgGln LeuSerGlu LysGlu 10Gln SerLeu ArgIleLeu GlnThr GluAsnAla ArgLeuLeu AspGlu Lys ThrAsp LeuLeu.AsnGlnLys GluLysGlu ValGluGlu LysLeu 1$

Lys AsnLeu AlaValLys GluGlu AlaPheLys ThrLeuGln ThrGlu Glu LysLys ArgLeuLys AsnLeu IleGluGlu AsnGluGly IleLeu 20 loo l05 llo Arg GluIle LysGlnAla LysAsp SerLysIle GlyGluThr TyrSer 2$Lys MetLys AspSerLys SerAla LeuIleLeu GluAsnLeu ProThr Gln AsnAla LeuGluIle LeuMet AlaLeuLys ProGlnGlu LeuGly Lys IleLeu AlaLysMet AspPro LysLysAla AlaAlaLeu ThrGlu Leu TrpGln LysProPro LysGlu AsnLysGlu SerGlnLys ThrIle 3$ 180 185 190 Pro ProThr ProProIle AlaPro ThrProLeu LysGluPro MetIle 40Lys AspPro AsnThrLys GluPro AlaGlyVal (2) INFORMATION FORSEQ ID
NO:

4$

(i)SEQUENCE CHARACTERISTICS :

(A) LENGTH:219 amino ids ac (B) TYPE: acid amino (C) STRANDEDNESS: ngle si $0 (D) TOPOLOGY:
linear (ii)MOLECULE TYPE:
protein (xi)SEQUENCE DESCRIPTION FOR SEQ NO: 4 ID

S$

Met ArgLys IleLeuLeu IleGly LeuIleLeu GlnAlaLeu PheSer Glu GluAla AlaGlnGlu LeuLeu GlnCysSer AlaIlePhe GiuSer _._T ___ _ __ _. __..__ .__ _ ___ _ _ _ __.__._ _- _.T

Lys Lys AlaGluLeu LysAspAsp LeuArg GlnLeuSer GluLysGlu Gln Ser LeuArgIle LeuGlnThr GluAsn AlaArgLeu LeuAspGlu ~ 50 55 60 Lys Thr AspLeuLeu AsnGlnLys GluLys GluValGlu GluLysLeu Lys Asn LeuAlaAla LysGluGlu AlaPhe LysThrLeu GlnThrGlu Glu Lys LysArgLeu LysAsnLeu IleGlu GluAsnGlu GlyIleLeu Arg Glu IleLysGln AlaLysAsp SerLys IleGlyGlu ThrTyrSer Lys Met LysAspSer LysSerAla LeuIle LeuGluAsn LeuProThr Gln Asn AlaLeuGlu IleLeuMet AlaLeu LysProGln GluLeuGly $ Lys Ile LeuAlaLys MetAspPro LysLys AlaAlaAla LeuThrGlu Leu Trp GlnLysPro ProLysGlu AsnLys GluSerGln LysThrIle Pro Pro ThrProPro IleAlaPro ThrPro LeuLysGlu ProMetIle Lys Asp ProAsnThr ThrGluPro AlaGly Val

Claims (12)

1. A method for screening a pathogenic microorganism for a secreted or cell-surface protein, comprising the step of identifying and selecting proline-rich proteins.

2. A method according to claim 1, wherein the protein comprises one or more amino acid motifs having the following general sequences:

(PX m)n P or (PPX q)n PP

wherein:

P is proline; X is any amino acid; m is from 1 to 5; q is 1 or 2; and n is 2 or more.

3. A method according to claim 2, wherein m is from 1 to 3.

4. A method according to claim 2, wherein n is 6.

5. A method according to any preceding claim, wherein the method comprises the step of screening a DNA library of a microorganism for DNA encoding a proline-rich amino acid sequence.

6. A method according to any preceding claim, comprising the step of screening and amplification of specific DNA sequences containing a DNA sequence encoding a proline-rich amino acid sequence by PCR.

7. A method according to claim 6, wherein the screening involves the preparation of a degenerate DNA primer encoding a proline-rich amino acid sequence.

8. A proline-rich protein identified by a screening method according to any preceding claim.

9. A proline-rich protein according to claim 8, comprising the sequence <SEQ
ID 1>, or a functionally active fragment or derivative thereof.

10. Nucleic acid encoding a proline-rich protein according to claim 8 or claim 9.

11. A vaccine composition comprising a protein according to claim 8 or claim 9, or nucleic acid according to claim 10.

12. A method for producing a vaccine according to claim 11, comprising the step of bringing a proline-rich protein according to claim 8 or claim 9, or nucleic acid according to claim 10, into association with a pharmaceutically acceptable excipient.