CA2188316A1 - Recombinant pgp3, methods of preparation and use in diagnosis and therapy - Google Patents

Abstract

new recombinant form of the plasmid-encoded protein pgp3 from C.trachomatis, serotype D, was purified by ion exchange column chromatography and shown to be suitable for quantitative immunoassay on clinical samples in an ELISA format.

Description

~ WO 95/28487 21~ ~ 31~ r~" o ; nAnt pgp3, methods of preparation ~na U5e in diaanosis An~l thera~v Fi-l~ of th- Invention The invention relates to a l~c ' inAnt form of plasmid-encoded protein pgp3 from Chlnmydia L, tis, serotype D
and the uses thereof in ; _y, particularly quantitative i --~say, such as enzyme linked 1 -- y 10 (ELISA). The invention also relates to methods of production of pgp3.
~- . ' to tbe Inv~ntion 15 Chlamydias are gram-negative bacteria which are obligate intrac~llul~r parasites of eukaryotic cells. They exhibit a form which is extracellular, infective and metabolically pr~ctiAlly inert, known as the ~ 1 body (EB) and an intrA~ l Ar replicative f orm called the reticular body 20 (R~3).
The reticular bodies, after multiplication by binary fissLon, are transformed into e1~ 1 bodies which are released from the host cell and infect new cells. The 25 masses of reticular and P~ 1 bodies inside an infected cell constitute characteristic ninclusions" visible with an optical mi-;LvsCv~.
Chl~lmydia L~ . ~. tis (C. tr~chomntis or CT) is a bacterial 30 species patht~n;~ to man and i8 the et;~-lo~;cAl agent of venereal ly - anuloma (VLG) which gives rise to various infl~ tULy pathologies of the human genitalia and of trachoma, a chronic disease which affects 500 million people and can cause bl;n~ln~C~. Urethritis and cervicitis induced 35 by C. ~ tis if not treated early can lead to chronic ;nfl; tions such a5 vaginitis, salpingitis and pelvic ;nfl; tion which may result in sterility and extrauterine ~Lc:y~a~ y~ Furthermore, the newborn from infected mothers SU~STITLITE SHEET (RULE 2B~
.... . _ ...... . ..... ..... .. .. ... , ,,,, , , _ _ , _ _ _ , , w095l28487 2~ 1B P~ ' --may contract pulmonary and/or ocular infections during delivery .
There i8 therefore a clear and unfulfilled need for accurate 5 diagnostic assay techniques for detecting C. I.L ' Lis infection as well immunological preparations for use in the prevention of infection and the LL-:~; ' of preexisting infection .
10 C. trachomatis (15,16) harbours a ;VIISeL~2~ 7.5-kb plasmid (pCT) (3), which appears to be under positive selective yL~Sr~uLe during natural infections, since it is found in essentially all strains and isolates. The role that this genetic element may have in C. ~ tis physiology has 15 been object of speculation; however, the search for pCT-associated phenotypes has been h;, -ad by the fact that no genetic transformation procedure for chlamydia has been available, so far.
20 The identification and characterization of plasmid ~I.vvded pLV-Iu~L6 is of particular interest, because pCT could encode chlamydial pa~ht~g~n1~ ity factors, as is the ca6e for plasmids of several other path~ nlc bacteria. Although the origin of replication has been identified (28), and several 25 features of pCT transcriptional regulation are known (20, 21, 5), the majority of other pCT features are only hypotheses deduced from DvNA sequencing data (2, 3, 8, 27).
However, a 28-kDa elev~Lv~l.vIe Lic band has been recently reported to cros6-react on Western blots of chlamydial 30 protein extracts with ant~ho~ raised against a 39 kDa chimeric protein, obtained by gene fusion with pCT open reading frame 3 (ORF3) (4). We have c~n~ ively identified a 28-kDa L of C.trachomati6 elementary bodies (EBs) as the pla6mid ~ oded polypeptide pgp3. ~lso, using an 35 i -,~cay with a purified, recombinant form of this protein, we show that it Lt~LC=_.lLS a novel and potentially important i , - in human chlamydial infections.
SU8STITLITE SHEET (RULE 26) ... . .. _ . _ _ _

2 ~ ~ ~ 3 ~ I/11 5 ~

Initial attempt5 to develop an ELISA test for human sera using the previously de5cribed (4) 39-kDa pgp3 fusion protein, purified by electrophoresis on SDS-acrylamide gels, gave ;nroncictent and unsatisfactory results. One of the 5 problems was the ob~L v~tion that, in some patient sera, antibody reaction with minor contaminants from E. coli was 8~LLU~ L than the reaction with the del-aLuL~d fusion protein .
lo situations like this can be adequately resolved by using the Western blot technique, where signals from different antigens can be separately monitored, but they become a serious problem in ELISA. For example, the resulting 39-kDa product was used to show that pgp3 epitopes can be 15 re~o~n i ced on Western blots by antibodies present in sera from patients with chlamydial infections, but not in control sera from healthy donors.
The object of the present invention is to provide a 20 serological test system more suitable than the hlotting ~P~hn;q--- for obtaining r-:~ u-lucible and quantitative data from large numbers of clini~-Al samples.
We attempted to improve both the quality of the recombinant 25 antigen and purification ~LUce-luLes to produce a reagent suitable for use in ELISA.
The present invention therefore relates to an enzyme-linked caAy based on a new r~ inAnt form of the pgp3 30 protein which can be u5ed for Ac6Pc~in~ the prevalence of pgp3 antibodies in people with C. LL ` tis infections.
r-pgp3 was obtained as stable 28-kDa protein, which L~ inP~, water-soluble in the bacterial cell even when over-~,.u,e:~ed 35 by the pT7-7 system, and t~l UUIgllUUt the whole purification uLoceduL ~. Since mis-rolded r~_ ' inAnt proteins typically undergo proteolytic degradation, or tend to ayyL~Le and precipitate t34), the above properties suggest that r-pgp3 SU9STITUTE SHEET (RULE 26) . , _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ wo 9s/28487 2 8 ~

(which contains ~our cy6teine residues) is correctly folded in a ~u~ ~uLe close to its native form. We believe that r-pgp3 may also retain an ~n~i~6-~;Q ~LLUV~UL~ capable of lptPot;nq ant;hoA;ec directed against conformational 5 epitopes, which could be missed by; -'lot analysis.
Surprisingly, a high proportion of r-pgp3 was found in the periplasm of the transformed E.coli BL21 cells, and the posRihil ity of using peripla8mic extracts simplified its 10 purification.
~ummary Or th~ Inv~ntion According to a f irst aspect 0~ the invention there is 15 provided a recombinant Chlamydia L ~.- t~s pgp3 protein, or a derivative or L~ t thereof.
The r~- ' ;n~nt pgp3 protein may be a r~ in~ln~ form of any o~ the allelic variants of the pgp3 protein having a 20 molecular weight of about 28kd, ~n~ ;ng for example pgp3-D (the allele from C. L~ tis s~Iv~y~e D) or pgp3-L2 (the allele from C.L~ :' Lis L2. pgp3-D is a ~LuLv~y~e of the trachoma variants of C. LL - tis and pgp3-L2 is a ~lvtv-y~e of the ly ~ uloma variants of C.LL '.- Li5.
It has been eS~hl; C~lPd that the conformation of pgp3 i8 ~ L for the retonti~n of essential; 107;~'A1 properties .
30 Preferably the ~ ;n~nt protein of the first aspect of the invention is substantially in a conformation capable of recognition by an~; hoA; PC in human serum. This protein is a new ~ in~nt form of pgp3, not previously described in the literature which has adva~ gevus properties when used 35 in; - -~ay, particularly enzyme-linked i . IcSays.
The protein may be a derivative of pgp3 provided that it adopts the conformation of native pgp3. It may therefore be ,-SU~STITUTE SHEET lRULE 26~
_ _ _ _ .

~wos~/28487 ~ 1 8 8 3 ~ 6 P~
a rLc. t of the complete pgp3 or a derivative thereof modified in a way which does not substantially affect it functional properties, particularly its immunological properties as an antigen.
Preferably the L~ ' inJ-nt protein has its native amino sequence and substantially a native conformation.
According to a second aspect of the invention there is 10 provided an; -';Agn~qtic assay comprising at least one step involving as at least one binding partner, a recombinant protein according to the f irst aspect of the invention, optionally l~hell~cl or coupled to a solid support.
The; ~,l;A7n-,=tic assay may be any form of; -qf~Ay employing as an essential ~-~t a diagnostic antigen.
Preferably however, the i -';AgnsFtic assay is an enzyme-20 linked solid phase; -~A~y (ELISA).
According to a third aspect of the invention there is provided an; --iagnosis kit for performing an assay according to second aspect of the invention, comprising at 25 least one recombinant protein according to the first aspect of the invention.
According to a fourth aspect of the invention there is provided a method f or the production of a recombinant 30 protein according to the first aspect of the invention, comprising culturing a host cell transformed with a vector comprising a r. ; nAnt polynucleotide encoding the l~C ~ ;nAnt protein according to the first aspect of the invention and isolating the L~ ~ ;nAnt protein.
The host cell may be any 5uit~ble host capable of producing the r~ ~ ;nAnt protein. Preferably however, the host cell is a bacterium, suitably E.coli.
r SUBSrITUTE SHEET (RULE 2fi\
_ _ _ _ _ _ _ _ _ _ _ _ _ _ .

Wo 95/28487 2 1 8 8 ~

Most preferably the protein is lLoduced by uv~=Le~,uLession in an E.coli expression system, It often found that expression in E. coli gives rise to u~ aLuLell folding and here non-5 native cu-,f-,L~tion. Such expression reguires carefully controlled purification te~ hniQ~ , often requiring denaturation and renaturation to recover native protein.
We have dis-uv~-d, surprisingly, that, in the case of pgp3, lO the protein i5 pLuduCed in native form by E.coli obviating the need f or such ~roc~lluL ~s which add not only to the production cost, but also gives rise to quality control problems associated with the possibility of inadequate removal of reagent employed in purification, such as strong 15 denaturants.
A particularly suitable expression system is the plasmid expression vector pT7-7 optionally in E.coli strain BL21.
In this system, the expression of ORF3 is under the control 20 of an IPTG-;n~ hle promoter and yields the unmodified pgp3 amino acid s~T~nre.
Preferably, the protein is purified from host cell extracts under non-denaturing conditions.
Preferably the purification comprises a step of ion-GYrhAn~e column chromatography on, f or example, mono-Q prepacked columns (Pharmacia) employing an NaCl elution gradient in p iperaz i ne-HC l buf f er .
Extraction from the host cell may employ any known non-denaturing t~hniq~, such as cell lysis, but preferably employs a periplasmic extract obtained for example using polymixin B.
We have surprisingly dis~uvcL~d that, in E.coli the pgp3 protein is pr~-luced in the periplasm, greatly facilitating purif ication .
SUBSrlTUTE SHEET (RULE 26) WO 95/28487 ~ 1 8 ~ 3 1 ~ p~.,,~ .i~

It is believed from the experimental evidence we now have that pgp3 i5 a virulence factor of chlamydial infections.
5 The fourth aspect of the invention also provides l.c ~inAnt pgp3 protein ~L ~.luced by the method of the invention as broadly defined or as specifically ~ 5~rl in the specific description .
10 According to a fifth aspect of the invention, we provide a vaccine or therapeutic composition comprising a . inAnt protein according to the f irst aspect of the invention and a rhA~-^eutically acceptable carrier.
15 According to a sixth aspect of the invention, we provide a vaccine or therapeutic composition comprising a r~_ inAnt protein according to the f irst aspect of the invention and a pharmaceutical carrier.
20 According to a seventh aspect of the invention, we provide a method of t,æai ~ of the human or animal body comprising administering an effective amount of a vaccine or theLe~ ul.ic composition according to the sixth aspect of the invention to prevent infection by C. LL tis or to 25 treat such an infection.
According to an eighth aspect of the invention, we provide a r~ 'jnAnt protein of claims 1 or 2 for use in the manufacture of a medicament for vaccinating against 30 C.~ tiq infection or treating such an infection.
Def initions ~he practice of the present invention will employ, unless 35 otherwise indicated, conv~ntionAl t~rhniqn~q of ~ lAr biology, microbiology, r- -inAnt DNA, and immunology, which are within the skill of the art. Such te~hniq~ are explained ~ully in the literature. See e.g., Sambrook, et SUBSTITUTE SHEET (RULE 26) _, , _ _ _ _ _ _ _ _ _ Wo 9~/28487 ~ 1 ~ 8 ~ ~ ~ r~."L c lo al., I-IOT RCTTT AR CLONING; A LABORATORY MANUAL, SECOND EDITION
(1989); DNA CLONING, VOLUMES I AND II tD.N Glover ed.
1985); OLIGONUCLEOTIDE :jYI~-n~:xlS (M.J. Gait ed, 1984);
NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. Higgins eds.
5 1984); T~ANRrT2TPTION AND TRl~NCTA~ION (B.D. Hames & S.J.
Higgins eds. 1984); ANIMAL CELL CULTURE (R. I . FL,OI.I~ey ed.
1986); .T5~T'n CELLS AND ENZYMES (IRL Pre6s, 1986); B.
Perbal, A PRACTICAL GUIDE TO MnT.Fr~T.~R CLONING (1984); the series, METHODS IN ~ nGy (~ A~ c Press, Inc. ); GENE
10 TR~NSFER VECTORS FOR MAMMAT TAN CELLS tJ . H . Niller and M . P .
Calos eds. 1987 , Cold Spring Harbor Laboratory~, Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), lrqnuNIJcn~.lCAL METHODS IN CELL AND T~TnT T~CTTT ~R BIOLOGY
15 (Academic Press, London), Scopes, (1987), PROTEIN
PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (springer--Verlag, N.Y.), and T~TANnsOr~Y OF ,~ IM~ -.AT. IM--MUNOLOGY, VOLUMES I-IV (D.M. Weir and C.C. Blackwell eds 1986) .
Standard abbreviation5 for nucleotldes and amino acids are used in this specification. All publications ,patents, and patent applications cited herein are incuL~ol<lted by re~erence .
r 1PS 0~ the protein that can be used in the present invention include polypeptide5 with minor amino acid v2riations from the natural amino acid se~u~ e of the protein; in particular, conservative amino acid r~plr - Ls 30 are contemplated. Conservative replAc - c are those that take place within a family of amino acids that are related in their side chain5. Genetically encoded amino acids are generally divided into four families: (1) acidic =
aspartate, glutamate; (2) ba5ic = lysine, arginine, 35 histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylAlAnin~ methionine, LLy~L~han;
and (4) u--- I.aL~:d polar 5 glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. PhenylAlAn;n-"
SUE~STITUTE SHEET (RULE 26) wO gsl28487 ~2 1 8 ~ 3 1 6 A ., ~ 0 LLyyLL~han~ and tyrosine are sometimes classified jointly as 2romatic amino acids. For example, it is reasonably predictable that an isolated r~rl A~ t. of a leucine with an i cole~lcin~ or valine, an aspartate with a glutamate, a 5 threonine with a serine, or a similar cu.-e_L vc~Live replAI L of an amino acid with a DLLuuLuLt~lly related amino acid will not have a major ef~ect on the biological activity. Polypeptide molecules having substantially the same amino acid sequence as the protein but Fncc~cc;n~ minor 10 amino acid substitutions that do not substantially affect the functional aspects are within the definition of the protein.
A signif icant advantage of producing the protein by 15 recombinant DNA technique6 rather than by isolating and purifying a protein from natural sources is that equivalent quantities of the protein can be I~L ùduced by using less starting material than would be required for isolating the protein from a natural source. Producing the protein by 20 recombinant terhn;~lPc also permits the protein to be isolated in the absence of some molecules normally present in cells. Indeed, protein compositions entirely free of any trace of human protein contaminants can readily be ~Lu-luced because the only human protein ~L u-luced by the r~ ; n~nt 25 nu-. hu.la,- host is the recombinant protein at issue.
Potential viral agents from natural sources and viral - pathng~n;C to humans are also avoided.
The term IIL~ n~nt polynucleotide" as used herein intends 30 a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or ~-n;r-l;~tion: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is 35 linked in nature, or (3) does not occur in nature.
The term "polynucleotide" as used herein ref ers to a polymeric form of nucleotides of any length, either SUBSTITUTE SHEET (RULE 26) WO 95/28487 , 2 1 8 8 3 1 6 . ~ o ~

ri hnm~c-l eotides or deoxyr i hnn~cl~otides . This term refers only to the primary ~L---_LuLe of the ~ 1PC111P. Thus, this term includes double- and 8ingle-stranded DNA and RNA. It also includes known types of modifications, for example, 5 labels which are known in the art, methylation, "caps", substitution of one or more of t_e naturally occurring nucleotides with an analog, internucleotide modifications such as, f or example, those with uncharged 1 1 nL-rgn~ (e . g ., methyl rhincrhnn~tes, phor~yhcLLiesters, rho~rhnAm;~iltes, 10 carbamates, etc. ) and with charged linkages (e.g., phosphorothioates, pllV yho~odithioates~ etc.), those cnnt~inin~ pendant moieties, such as, for example proteins (;nn~ in~ for e.g., nllfl~ PC, toxins, i~nt;hnA;Ps~ signal peptides, poly-L-lysine, etc. ), those with intercalators 15 (e.g., acridine, psoralen, etc.), thosecontainingchelators (e.g., metals, rAriin~C~iVe metals, boron, oxidative metals, etc. ), those containing alkylators, those with modified l;nk~Pc (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
A "replicon" is ~ny genetic element, e.g., a plasmid, a ,l1L . ~ ~ ~ a virus, a cosmid, etc . that behaves as an c.uL~ unit of polynucleotide replication within a cell;
i . e ., capable of replication under its own control . This 25 may include selectable markers.
A "vector" is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
"Control seq~ c~" refers to polynucleotide seguences which are necessary to effect the expression of coding seguences to which they are ligated. The nature of such control 5eql~n~Pc differs ~iepPnf; n~J upon the host organism; in 35 yLuh~IyuLes, such control sequences generally include promoter, r;i-- l binding site, and tr~nscription termination seuu~ ce; in eukaryotes, generally, such control ~e.l ~- eC include promoters and transcription termination SUBSTITUTE SHEET ~RULE 26) WO 95/U487 ~1~3 8 31~ /~ D

8~ u- e. The term "control 8~-lu~ is intended to include, at a minimum, all ~ ~ whose ~L.s_..ce is n ~c~ Ary for expression, and may also include additional 3rLs whose presence i8 advantageous, for example, 5 leader 8~ c and fusion partner se~uel.c~s.
"Operably linked" refers to a juxLa~osition wherein the - ts so described are in a relationship permitting them to function in their 1 nt~n~l~d manner. A control 10 se~ e..~e "operably linkedn to a coding sequence is ligated in such a way that expression of the coding sequence is nchieved under conditions compatible with the control sequences .
lS An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region Day ré~L~ ~enL a portion of a coding sequence or a total coding ~ e.
20 A "coding sequence" is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory F~quonr~c. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation 25 stop codon at the 3 ' -term; mlc . A coding seqll~n~e can include, but is not limited to, cDNA, and l~ ' inslnt polynucleotide 5~
"E'CR" refers to the t~l~hniT~ of polymerase chain reaction 30 as described in Saiki, et al-, Nature 324:163 (1986); and Scharf et al., Science (1986) 233:1076-1078; and U.S.
4,683,195; and U.S. 4,683,202. As used herein, x is "heterologouS" with respect to y if x is not naturally associated with y in the identical manner; i.e., x is not 35 nssociated with y in nature or x is not associated with y in the same manner as is found in nature.
"~omology" refers to the degree of similarity between x and SUBSTITUTE SHEET (RULE 26) WO 9s)284~7 ` 2 1 8 ~ 3 1 ~ . ~., 3~ lo y. The UULL~'-L~ A ,~-~e between the s= ~ e from one form to imother can be determined by techniques known in the art.
For example, they can be det~ n~d by a direct comparison of the E~ information of the polynucleotide.
5 Alternatively, homology can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous regions (for example, those which would be used prior to Sl digestion), followed by digestion with single-stranded specific n~lrlel~(s), fol-10 lowed by size determination of the digested rL L,,.
As used herein, the term "polypeptide" refers to a polymerof amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are 15 inrll-rl.od within the definition of polypeptide. This term also does not refer to or exclude post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
Tnr~ within the definition are, for example, 20 polypeptides containing one or more analogs of an amino acid (including, for example, ul"~Lu~al amino acids, etc.), polypeptides with substituted 1 ink~ , as well as other modifications known in the art, both naturally occurring and nc,~. n~LuLc,lly occurring.
A polypeptide or amino acid se~tu~ "derived from" a designated nucleic acid s6~ut~ e refers to a polypeptide having an amino acid seqUenCe identical to that of a polypeptide encoded in the seT~nre, or a portion thereof 30 wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is im-munologically identifiable with a polypeptide encoded in the sequence . This terminology also ~ nrl u~9~Y a polypeptide 35 expressed from a designated nucleic acid sequence.
The protein may be used for producing antibodies, either - -~lon~l or polyclonal, specific to the protein. The SU~3S~l~ESHEE~ U E 26) _ _ _ _ -Wo 95/28487 2 18 8 ~1 ~ r~l ~

methods for producing these antibodies are known in the art.
.
~n- ' in~n1- host cells", "host cells," "cells," "cell 5 cultures, " and other 6uch terms denote, for example, mi.L~,uLy~ isms, insect cells, and 11~n cells, that can be, or have been, used as recipients for L~- ' in~nt' vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood 10 that the progeny of a single parental cell may not n~ CcArily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. r - l~c for l~s~n host cells include Chinese hamster ovary (CHO) 15 and monkey kidney tCOS) cells.
Specifically, as used herein, "cell line, " refers to a population of cell5 capable of continuous or prolonged growth and divi5ion in vitro. Often, cell lines are clonal 20 populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transf er of such clonal populations. Therefore, cell5 derived from the cell line referred to may not be precisely identical to the 25 /1~ LLCI1 cells or culture5, and the cell line referred to includes such variants. The term "cell lines~ also inrl~lec immortalized cells. Preferably, cell lines include nonhybrid cell line5 or hybridoma5 to only two cell types.
As used herein, the term "microorganism" inrl~ c prokaryotic and c uk~Lyutic microbial 5pecies such as bacteria and fungi, the latter incl~ in~ yeast and f 11 L,,us fungi .
"Transformation", a5 used herein, refers to the insertion of an ~ J-~ = polynucleotide into a ho5t cell, iLL ~ye-. Live of the method used for the insertion, for example, direct uptake, trAnC~c~ion, f-mating or ele. LLuy.,L~tion. The SU~STITUTE SHEET (RULE Z6~
_ _ _ _ _ _ _ _ _ , WO 95/28~87 ~ r 8 8 ~1~ 1~,,~ . ~

u~ polynucleotide may be 7r- i nt~ i nDd as non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
5 By "genomic" is meant a collection or library of DNA
molecules which are derived rrom restriction r.., Ls that have been cloned in vector~. ~his may include all or part of the genetic material of an organism.
10 By "cDNA" is meant a compli~ Ly mRNA sequence that hybridizes to a complimentary strand of mR~A.
By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide 5 qu~nce, that the indicated 15 molecule is present in the substantial absence of other biological ma~l -le~ e~ of the same type. The term "purified" as used herein preferably means at least 75% by weight, more preferably at least 859s by weight, more preferably still at least 95~ by weight, and most preferably 20 at least 98~6 by weight, of biological ma~., ~leclll~e of the same type present (but water, buffers, and other small molecules, eepe~iAlly molecules having a molecular weight of less than 1000, can be present).
25 Expression systems Once the appropri~te coding s~qu~n~e is isolated, it can be expressed in a variety of di~ferent expression systems; for example those used with l iAn cells, baculoviruses, 30 bacteria, and yeast.
i. I l i~n systems 1 1 jAn expression systems are known in the art. A
1 i An promoter is any DNA s~ lu~ e capable of binding -li;~n RNA polymerase and initiating the ' ~~ u (3') tran~;cription of a coding ~ ,_ ~e (e.g. ~U ~UL~l gene) into mRNA. A promoter will have a transcription initiating SUBSTiTUTE SHEET (RULE 26) _ _ _ _ _ _ _ _ _ , = . .. . _ _ ~ wo 95/28487 ~ 1 8 ~ ~ 1 6 1 ~1/ s IC

region, which is usually placed proximal to the 5 ' end of the coding ~e~lv- -~P, and a TATA box, usually located 25-30 base pairs (bp) ulJ~LL~am of the transcription initiation site. The TATA box is thought to direct RNA polymerase II
5 to begin RNA 6ynthesis at the correct site. A l; An promoter will also contain an upstream promoter element, usually located within 100 to 200 bp uyD~Lea,u of the TATA
box. An uy~LLea~ promoter element ~ rm;nc~c the rate at which transcription i8 initiated and can act in either 10 orientation tSambrook et al. (1989) "Expression of Cloned Genes in T- ~l;An Cells." In Mole~ Ar Clonin~l A
T.2~ r c,t~v MAnllAl 2nd ed.~.
T' liAn viral genes are often highly ~L.a6sed and have a 15 broad host range; therefore sequence5 ~nCor3in~ 1 iAn viral genes provide particularly useful promoter ~e~lu~
Examples include the SV40 early promoter, mou6e mammary tumor virus LTR promoter, adenovirus major late promoter (Ad ~LP), and herpes 6implex virus promoter. In addition, 20 s~ s derived from non-viral genes, such as the murine metallothQi~ P;n gene, also provide useful promoter sequences. Expression may be either constitutive or regulated ( i n~ ; hl f~ lep~n~l; n~ on the promoter can be induced with glucocorticoid in h - responsive cells.
The p~eDc--~e of an ~nhAnt ~r element (enhancer), in~
with the promoter ~l Ls described above, will usually increase expression levels. An enhancer is a regulatory DNA
sequence that can stimulate transcription up to 1000-fold 30 when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. El~hancelD
are also active when they are placed upstream or c' I~DLL. am from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 35 nucleotides from the promoter tManiatis et al. (1987) science 236:1237; Alberts et al. (1989) Mol~ Ar Bioloqv Of the Cell. 2nd ed. ] . P:nh~n~ r elements derived from viruses may be particularly useful, because they usually have a SUBSTITUTE SHEET (RULE 26) Wo 9s/28487 ~ ~ 8 8 3 ~ 6 r~ .. s ~ lo broader host range. Examples include the SV40 early gene enhancer [Dijkema et al tl985) PMF~rl J. 4:761] and the enhancer/promoters derived from the long ~rm1nA1 repeat (LTR) Or the Rous S~rcoma Virus tGorman et al. (1982b) ~Q5~
5 Natl. Acad. Sci. 79:6777] and rrom human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the ~s~.-ce of an inducer, such as a hormone or metal ion [S~sol,e Corsi and Borelli (1986~ TrPn~c Genet. 2:215;
10 Maniatis et al. (1987) Science 236:1237].
A DNA molecule may be ~--~L~ssed intracP~ rly in l~i~n cells. A promoter se~r~ n-e may be directly linked with the DNA molecule, in which case the first amino acid at the N-15 terminus of the r~ ' i n~nt protein will always be a ~ ;r.ninP, which is encoded by the ATG start codon. If desired, the N-tPrmin~c may be cleaved from the protein by ~,~ ~ incubation with cyanogen bromide.
20 Alternatively, foreign proteins can also be secreted rrom the cell into the growth media by creating chimeric DNA
molecules that encode a fusion protein comprised of a leader sequence r. L that provides rOr secretion Or the roreign protein in 1 i~n cells. Prererably, there are 25 processing sites encoded between the leader fL, L and the roreign gene that can be cleaved either n vlvo or }n vi~ro.
The leader s~ lu~ e r. L usually encodes a signal peptide comprised Or ~y~ hobic amino acids which direct the secretion of the protein from the cell. The adenovirus 30 triparite leader is an example of a leader se~lu-,. e that provides for secretion of a foreign protein in ~ n cells .
Usually, transcription termination and polyadenylation 35 sequ~nrQc reco~ni7ecl by li:~n cells are regulatory regions located 3 ' to the translation stop codon and thus, together with the promoter elements, f lank the coding 5~lpnre. The 3 ' terminus of the mature mRNA is formed by SUBSTITUTE SltEET (RULE 26) _ _ _ _ ~wogs/28487 ~ 316 site-specif ic post-tran8criptional cleavage and polya-denylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end pro~-Ps6in7 of eukaryotic RNA. In Tr~ncrrir~tion ~nA svlicin~ (ed. B.D.
5 Hames and D.M. Glover); Proudfoot (1989) TrPn~lc Biochem.
Sci. 14 :105] . These sec~UenCe8 direct the transcription of an mRNA which ean be tr~nslated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived 10 from SV40 [Sambrook et al (1989) nExpression of cloned genes in cultured li~n cells.~ In MolPr~lAr Cloninq: A
T ~hnratorY r5An~
Some genes may be e~ sed more efficiently when introns 15 (also called intervening s~qUPn~ ~C) are present. Several cDNAs, however, have been Pffi~QiPntly ex~r~ssed from vectors that lack splicing signals (also called 5plice donor and ac.c~cI sites) [see e.g., Gothing and S oolc (1981) Na~l~re 293:620]. Introns are intervening nnnro~l;n7 20 c~ s within a coding sPqnc~nre that contain splice donor and acceptor sites. They are removed by a process called "splicing, " following polyadenylation of the primary tLc-r~s~.LLpt [Nevins (1983) Annu. Rev. Blochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al. (1986) Annu.
25 Rev. BioQhem. 55:1119; Krainer and Maniatis (1988) "RNA
splicing." In Tr~ncrrivtion and st:~licin~ (ed. B.D. Hames and D.M. Glover) ] .
Usually, the above described ~, comprising a 30 promoter, polyadenylation signal, and transcription termination sequence are put together into expression ~ .,I.,,L~,~,..~5. ~nh~nQPr5, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression ~ -u- ~, if desired. Expression 35 constructs are often maintained in a replicon, such as an e.L~. -1IL~ 1 element (e.g., plasmids) capable of stable maintenance in a host, such as 1 i ;~n cells or bacteria.
n replication systems include those derived from SUBSTITUTE SHEET (RULE 26~

Wo 9s/28487 2 ~ 8 ~ 3 1 6 animal viruses, which require tran~ 3-t;n~ factors to replicate. For example, plasmid5 containing the replication sy6tems of papovaviruses, such as SV40 tGluzman t1981) Ç~LL
23:175] or polyomavirus, replicate to e:~Ll- ly high copy 5 number in the presence o~ the appropriate viral T antigen.
Additional examples of 1~ An replicons include tho6e derived from bovine pArill~ virus and Epstein-Barr virus.
Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in - l~An 10 cells for exrression and in a prV}.aL2 Ut_iC host for cloning and amplification. Examples of such 1 iAn-bacteria shuttle vectors include pMT2 [Kaufman et al- ~1989) ~QL
~'Pl 1. Biol. 9:946 and pHEB0 tShimizu et al. (1986) Mol.
Cell . Biol . 6 :1074 ~ .
The transformation E~Lu~e-lu~ ~ Used depends upon the host to be transformed. Methods for i..~o l ~ ~inrl of heterologous polynucleotides into -1 ~An cell8 are known in the art and include dextran-mediated tran8fection, calcium phosphate 20 precipitation, polybrene mediated transfection, protoplast fusion, ele~ u~.,r~tion, Pnc=~psulAtion of the polynucleotide(s) in ~irnS~ --, anddirectmicroiniectionof the DNA into nuclei.
25 T- 1 i;~n cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHû) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey 30 kidney cells (COS), human hepatncPl 1l~1Ar carcinoma cells (e.g., Hep G2), and a number of other cell lines.
ii. Baculovirus Systems 35 The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector co~ u~ ~ion employs te~hniql~P5 which are known in the art.
StJBSTlTLlTE SHEt~ (RULE 26) ~WO95/28487 2 1 ~ 8 3 16 1~l". Io Generally, the _ ts of the expression syste~ include a transfer vector, usually a bacterial plasmid, which 5 contains both a ~L ~ L Or the baculovirus genome, and a convenient restriction site ror insertion of the heterologous gene or genes to be ~A~L-'SSed; a wild type baculovirus with a sequence ~ o~ Ollc to the baculovirus-specific r. ~ t in the transfer vector (this allows for 10 the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
After inserting the DNA 5~ lu-~ ~ e ~n--o~l i nAJ the protein into 15 the transf er vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to ~ ' inG. The r-A~ ~d , ~ ; nAnt virus is l~ L . 6iDed and 1 ~ i nAnt pla~ues are identified and purified. Materials and methods 20 ror baculovirus/insect cell expression systems are commercially available in kit form from, inter alia.
Invitrogen, San Diego CA ("NaxBac" kit). These t rhniTl~A
are generally known to those skilled in the art and fully described in Summers and Smith, Texas A~ricllltural 25 FYn~riment Station Bulletin No. 155S (1987) (hereinafter "Summers and Smithn).
Prior to inserting the DNA s~ n~ o~l ~ nAj the protein into the baculovirus genome, the above described ~ Ls, 30 comprising a promoter, leader (if desired~, coding sequence of interest, and transcription termination se~ n- e, are usually A~ into an illL- 'iAte transplacement c~ .L.u~ ~ (transfer vector) . This construct may contain a single gene and operably linked regulatory elements;
35 multiple genes, each with its owned set of operably linked regulatory G1 ~; or multiple genes, rejulated by the same set of regulatory ~ L~ . Tnt- - - i Ate trnnsplacement c~ll,LLu~ Ls are often r-in~A1n~d in a SUESTITUTE SHEET (RULE 26) WO 95128487 ~ 3 ~ ~; F~

replicon, such as an e~LL ' I ~ 1 element (e.g., plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for 5 cloning and ampli~ication.
Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Nany other vectors, known to those of 5kill in the art, have also been 10 designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which i--LL-,~u~es a 13amHI cloning site 32 basepairs c~ --OLL~am from the ATT; see Luckow and Summers, Viroloqv (1989~ 17:31.
15 The plasmid usually al80 contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann Rev.
Mit-robiol., ~2:177) and a prokaryotic; icillin-resistance (~) gene and origin of replication for selection and pL ~ y~ltion in E.
Baculovirus transfer vectors usually contain a baculovirus LeI. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the ~ LL~am (5 ' to 3 ' ) transcription of a 25 coding seS~u~l~c~: (e.g. 8~L~_LuL~l gene) into mRNA. A
promoter will have a transcription initiation region which i~ usually placed proximal to the 5 ' end o~ the coding 5G~ . This transcription initiation region usually ~nrlt~ an RNA polymerase binding site and a transcription 30 initiation site. A baculovirus transfer vector may ~lso have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
35 StLU- LULCI1 genes, abundantly transcribed at late times in a viral infection cycle, provide particularly u8eful promoter r 1P~ include se~c~ derived from the gene ~in~ the viral polyhedron protein, Friesen et al., SUBSTITUTE SHEET (~ULE Z6~

~Wo 95l28487 2 1 8 8 ~ 1 ~ P~
t1986) "The Regulation of Baculovirus Gene Expression, " in:
ThF- Nolpn~ r Bioloqv of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene onroAin~ the plO protein, Vlak et al., (1988), J. Gen~
5 Virol. ~ 765.
DNA Pn~ o~lin~ suitable signal seq~l~n~ Pc can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) 10 Gene, 73:409). Alternatively, since the signals for 1 ii~n cell po~.Ll_L,Ii-slational modifications (such as signal peptide cleavage, proteolytic cleavage, and rhncrhnrylation) appear to be recogn; 70d by insect cells, and the signals reguired for fiecretion and nuclear 15 a~ l ~tion also appear to be conserved between the invertebrate cell6 and vertebrate cells, leaders of non-insect origin, such a6 those derived from genes encoding human cl-interferon, Maeda et al., (1985), ~3~_ ~:592;
human gastrin-rPlP~cin~J peptide, Lebacg-Verheyden et al., 20 (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. sci. USA, ~:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human uc~r~brosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide f or secretion in insects .
A l~ in~nt polypeptide or polyprotein may be ~ L~ sse~
intr~rPlll-lArly or, if it ic A~L-3sed with the proper regulatory 8~ l _c, it can be secreted. Good intr~Pll~lAr expression of r.o~.~u,,ed foreign proteins 30 usually requires heterologous genes that ideally have a short leader 6e~ `c containing suitable translation lnitiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen 35 bromide.
Alternatively, ~ i n~nt polyproteins or proteins which are not naturally secreted can be secreted from the insect SUBSTITL~E SHEET (RULE 26~

Wo gs/28487 2 ~ ~ ~ 3 ~

cell by creating chimeric DNA -lo^lllo~t that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in im;ects.
The leader soquon~ e rL L usually encodes a signal 5 peptide comprised of IIYdL ~l-obic amino acids which direct the translocation of the protein into the endoplasmic reticulum .
After insertion of the DNA sequence and/or the gene ort- o~ing 10 the expression product ~L~:VUL~ L of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus -- usually by co-tran6f ection . The promoter and tr~nscription termination sequence of the v..~.L,u~ L will 15 usually comprise a 2-5kb section of the baculovirus genome.
Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith ~; Ju et al. (1987); Smith et al., Mnl. Cell. Biol. (1983) 3:2156; and Luckow and Summers 20 (1989) ) . For example, the insertion can be into a gene such as the polyhedrin gene, by ~ logOIl~ double ~Lv5se~.-L
r~ Ation; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
Miller et al., (1989), Bioessav& 4:91.The DNA reqt~on~-e, when 25 cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3' by polyhedrin-specific u~ e~ and is positioned downstream of the polyhedrin promoter .
30 The newly formed baculovirus expression vector is SIII~CG~ Lly pA~ god into an infectious r~ - ' i nAnt baculovirus. Homologous r-c ` ;nAtion occurs at low .C~etuu~ ;y (between about 1% and about 5%); thus, the majority of the virus ~Lv-luced after c~LL~ rection is still 35 wild-type virus. Therefore, a method is necé,ss~Ly to identify re ~ int~nt viruses. An a~v~ of the expression system is a vi5ual screen allowing r~ ; n:~nt viru~es to be distinguished. The polyhedrin protein, which SIJBSTITLITE SHEET (RULE 26) ~wogsl284~7 ~ ~ 8 8 3 6 r~l,L~ I 1~
i~ ~L~,d ced by the native virus, i8 ~L~luced at very high levels in the nuclei of infected cells at late times after viral infection. A- _ 1 Ated polyhedrin protein forms ocrll~qit~n bodies that A150 contain: ~ -' particles.
5 These o~ n bodies, up to 15 /~m in size, are highly refractile, giving them a bright shiny appearance that is readily vi ~ under the light mi~.L~ ".e. Cells infected with r~ ' inAnt viruses lack occlusion bodies. To distinguish . ~ inAnt virus from wild-type virus, the 10 transfection nu~_L~ L~ is plaaued onto a monolayer of insect cells by techniaues known to those skilled in the art. Namely, the plaaues are 5nL~- .-ed under the light mi~ L~,sc~,~e for the ~L~SO~ (indicative of wild-type virus) or absence ( indicative of recombinant virus) of oc~ n 15 bodies. "Current Protocols in ~icrobiology" Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, ~; Miller et al. (1989).
R~.~ ' ;n~nt baculovirus expression vectors have been 20 developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, ~ ~liil: Aedes aeav~ti, Au~ ",ha r ~l i for~ , Bombvx mQ~;L, Drncrr~hil~ m~ n~qter. SDodo~tera fru~iDerda, and Tricho~lllqia ni (PCT Pub. No. W0 89/046699; Carbonell et 25 al., (1985) J. Virol. 56:153; Wright (1986) ~ ~:718;
Smith et al., (1983) l~ol. Cell. Biol. ~:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol.
;~: 225) -30 Cells and cell culture media are commercially available forboth direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology i5 generally known to those skilled in the art . See e . a . . Summers and Smith supra .
The modif ied insect cell8 may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(5) present in the modified insect SU~SlITUTE SHEET (RULE 26~
_ _ _ _ _ _ _ _ _ _ _ _ wo9s/28487 ~ ~ 8~ ~1 6 P~ 1,.. 3!' host. Where the ecpression product gene is under in~lllrihl~
control, the host may be grown to high density, and expression induced. Alternatively, where expression is confititutive, the product will be rontim1~11cly :x~Lessed 5 into the medium and the nutrient medium must be rr~ntin~ llcly circulated, while removing the product of interest and augmenting ~erlet~ nutrients. The product may be purified by such tPrhniTl~c as chromatography, e.g., HPLC, affinity chromato~Lt-p~y, ion pYrh~ , C~ to~L~1-y, etc.;
lO electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove ,,ub Ll--Lially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so a6 to provide a 15 product which i8 at least substantially free of host debris, e.g., proteins, lipids and polysaccharides.
In order to obtain protein expression, L~ ~ inAnt host cells derived from the transformants are incubated under 20 condition5 which allow expression of the Ia_ ini~nt protein ~nrot4in~ se~ . These conditions will vary, c1-~
upon the host cell 6~1 ~ct~d . However, th~ conditions arereadily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
iii. Bacterial Systems Bacterial expression techniques are known in the art. A
bacterial promoter is any DNA s~quonc~e capable of binding 30 bacterial RNA polymerase and initiating the ~ L.aam (311) transcription of a coding 5e~ (e.g. ~>LLU~LUr~1 gene) into mRNA. A promoter will have a transcription initiation region which 1~ y placed proximal to the 5' end of the coding seqn~nre. This transcription initiation region 35 usually inr~u~s an RNA polymera5e binding site and a transcription initiation site. A bacterial ~, L~r may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA
~UBSTiTUTE SHEET (RULE 26) _ _ ~Wo gs/28487 2 1 8 8 ~ 1 6 ~ ic synthesis begins. The operator permits negative regulated (in~-lcihle) transcription, as a gene repressor protein may - bind the operator and thereby inhibit tL ail8~:L iption of a specific gene. Constitutive expression may occur in the 5 absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding seq~onre, which, if present is usually proximal (5 ' ) to the RNA polymerase binding seyu~,ce. An example Or a gene activator protein is 10 the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E.
coli) [Raibaud et al. (1984) ~nml. Rev. Genet. ~:173].
Regulated expression may therefore be either positive or negative, thereby either c-nh:~nrin~ or reducing 15 LL O.IIS~.;L iption .
Se~uc ..~ es ~nro-l i n~ metabolic pathway enzymes provide particularly useful promoter ~ c. Examples include promoter sequences derived from sugar metabolizing enzymes, 20 such as galactose, lactose (lac) tChang et 311. (1977) Nature ~2~:1056], and maltose. Additional examples include promoter seq~l~nc~e derived froD biosynthetic enzymes such as tryptophan (t~) [Goeddel et al. (1980) Nuc. Acids Res.
~:4057; Yelverton et ~1. (1981) Nucl. Ar~ c Res. 9:731; U.S.
25 Patent No. 4,738,921; EP0 Publ. Nos. 036 776 and 121 775].
The g-laotamase (~1~) promoter system tW~iaF~-nn (1981) "The cloning Or interferon and other mistakes." In Interferon 3 (ed. I. Gresser) ], bacteriophage lambda PL [Shimatake et ~1.
(1981) Nature i~2~:128] and T5 [U.S. Patent No. 4,689,406]
30 promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation se~iu~l,cl:s of one bacterial or 35 bacteriophage promoter may be joined with the operon 5~ = of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U. S . Patent No. 4,551,433]. ~or example, the tac promoter is a hybrid SU9STITUTE SHEET (RULE 26) _ _ . _ _ _ _ _ _ _ _ _ Woss/28487 '~ i ~3831~ J ~
~-lac promoter comprised of both ~ promoter and lac operon 6e~ nr~s that is regulated by the ~ L ~:IJL as50 tAmann et al- (1983) Gene i~ 67; de Boer ~ al. (1983) Proc. Natl. Acad. Sci. 80:21]. Fur~h~ a, a bscterial 5 promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA
polymerase and initiate transcription, A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high 10 levels of expression of some genes in p~ uhdLyv~es~ The bacteriophase T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. r~0l. 8iol. 1~:113; Tabor et a]. (1985) Proc Natl. Acad.
&~ ~,:1074~. In addition, a hybrid promoter can al60 be 15 comprised of a bacteriophage promoter And an E-U~LC~tC~l region (EP0 Publ. No. 267 851).
In addition to a functioning promoter seauence, an efficient ribosome binding site is also useful for the expression of 20 foreign genes in prokaryote5. In E. ~Q~i, the ribosome binding site is called the Shine-Dalgarno (SD) A~ nre and i n~ ec an initiation codon (ATG) and a sequence 3-9 nucleotide6 in length located 3-11 nucleotides u~-.L.e~ of the initiation codon [Shine et ~1. (1975) Nature 254:34].
25 The SD se~Aiuence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD se~AiU~n~ e and the 3 1 and of E. ç5~1i 16S rRNA tsteitz et ~. (1979) "Genetic signals and nucleotide ~e~ c in r~ r PcNA. "
In Bioloaical Reaulation and DeveloDment: Gene E~ression 30 (ed. R. F. Goldberger) ] . To express eukaryotic genes and prokaryotic genes with weak ri~ binding site [~ vok et ~1. tl989) "Expre88ion of cloned genes in Escherichia coli." In ?~ol~lll~r Clnnin~: A TAhnratorY MAmlJ~
35 A DNA molecule may be ~ Le~aed intr~c-AllulArly. A promoter ~ ~ ,ue~ c may be directly linked with the DNA ~ lec~ , in which case the f irst amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start SUBSTITUTE SHEET (RULE 26~

' 21g8~6 ~WO ss/2s4s7 1 codon. I~ desired, me~hionin~ at the N-t~rmin~lc may be cleaved from the protein by ~ y~ incubation with cyanogen bromide or by either ~ ~yQ on ~ Yitro incubation with a bacterial - hicn;n~ N-t~min~l peptidase tEPO Publ.
S No. 219 237).
Fusion proteins provide an alternative to direct expression.
Usually, a DNA sequ~nre ~nro~in~ the N-terminal portion of an endogenous bacterial protein, or other stable protein, is 10 fused to the 5 ' end of heterologous coding sequences . Upon expression, this ccll~L-u- L will provide a fusion of the two amino acid seguences. For example, the bacteriophage lambda cell gene can be linked at the S' terminus of a foreign gene and ~ L~e~ in bacteria. The resulting fusion protein 15 preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Natu~ 309:810]. Fusion proteins can also be made with se~u~ es from the lacZ [Jia et 31L-(1987) Gene 60:197], tr~E [Allen et al. (1987) J.
20 Biotechnol. 5:93; Makoff et ~Ll. (1989) J- Gen. Microbiol.
~:11], and Chev tEPO Publ. No. 324 647] genes. The DNA
sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a u~iquitin fusion protein. Such a fu5ion protein is made 25 with the ubiquitin region that preferably retains a site for a proc~ccinq enzyme (e.g. ubiquitin specific proc~ccin~-protease) to cleave the ubiguitin from the foreign protein.
Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/T~rhn~lo~y 7:698].
Alternatively, foreign proteins can al50 be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a 5ignal peptide seuu_.,ce LL _ t that provides for secretion of the foreign protein 35 in bacteria [U.S. Patent No. 4,336,336]. The signal e LL, ~ usually encodes a signal peptide comprised of ll~.l-u~hobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into SUBSTITUTE SI~EET (RULE 26) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Wo gs/28487 ~ 6 r~l,.L3~. Io ~

the growth media (gram-positiVe bacteria) or into the periplasmic spece, located between the inner and outer mem~r~ne of the cell (S~ah. n~ ive bacteria). Preferably there are proc~csin~ 5ites, which can be cleaved either 5 ViVQ or ~Ln vitro encoded between the signal peptide fragment and the f oreign gene .
DNA or co~ suitable signal 8~ c can be derived from genes f or secrcted bacterial proteins, 8uch a8 the E;- ~QLi 10 outer membrane protein gene (s~ ) [Nasui ot al. (1983), in:
EYnOri- A1 ~AniplllAtion of Gene 7~ression; Ghrayeb ~ al.
(1984) E~IBO J. 1:2437] and the E. Coli Alk~l ino phosphata_e signal sequence (PhQA) [Oka et ~1. (1985) Proc. Natl. Acad.
~ ;h $2:7212]. As an additional example, the signal 15 sequence of the alpha-amylase gene from varioug ,RAr; 1 111C
strains can be used to secrete heterologous proteins from ~.
~-lhtil~R [Palva et 3~1- (1982) Proc. Natl. Acad. Sci. USA
79:5582; EPO Publ. No. 244 042,.
20 Usually, transcription termination seUu~ .es recognized by bacteria are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding soT7onre. These ~CDTlonr~c direct the transcription of an mRNA which can be translated into the 25 polypeptide encoded by the DNA- Transcription termination se~uc l,ces frequently include DNA sequences of about 50 nucleotides capable of forming stem loop ;,~Lu- Lu~as that aid in terminating transcription. r 1 ~R include transcription termination 8equence8 derived from genes with 30 strong promoters, such as the ~e gene in ~. coli as well as other biosynthetic genes.
Usually, the above described ~8, comprising a promoter, signal s~ re (if desired), coding 8~T~or7~e of 35 interest, and transcription termination s~lu~ e, are put together into expression con8truct8. Expression c~ ,u..LK
are often maintained in a replicon, such as an extra..l., 1 element (e.g., plasmids) capable of stable SU~STITUTE SHEET (RULE 26) ~Wo 9S/28487 2 1 ~ 8 3 1 6 ~ ~1'~ . .C
maintenance in a host, 8uch as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a r^rl;.^oin may be either a 5 high or low copy number pla5mid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually About 10 to about 150. A host cnnt^;n;n~ a high copy number plasmid will pre~erably contain at least about 10, and more preferably at least 10 about 20 plasmid5. Either a high or low copy number vector may be selected, ~-rPnAin~ upon the effect of the vector and the foreign protein on the host.
Alternatively, the expression constructs can be integrated 15 into the bacterial genome with an integrating vector.
Integrating vectors usually contain at least one s~uu~nce homologous to the bacterial ch., - ~ that allows the vector to integrate. Integrations appear to result from ~ ' in^tions between homologous DNA in the vector and the 20 bactedrial ~ . For example, integrating vectors ~ ;Led with DNA from various 8acillus strains integrate into the Bacillus ,I-L- - - (EPO Publ. No. 127 328).
Integrating vectors may also be comprised of bacteriophage or trAncrncnn se Usually, e..~L- C l~L~ - 1 and integrating expression cc,l...L~ucL~ may contain sol_rlAhl~ markers to allow for the g_l PC~ j nn of bacterial strains that have been transformed.
S-IPrt^h~- markers can be e~L~ssed in the bacterial host 30 and may include genes which render bacteria resistant to drugs such as ampicillin, Chl-,L ' ^- ;~^ol, eLyLI~ in, kanamycin (neomycin), and tetracycline [Davies et ~. (1978) Anml. Rev.~;r-robiOl. 32:469]. SelP~^t:~hl., markers may also include bi-.~.yllLI._LiC genes, such as those in the histidine, 35 LLy~L~,~han, and leucine biosynthetic pathways.
Alternatively, some of the above described t2. can be put together in transformation vectors. Transformation SlJaSTITUTE SHEET (RULE 26) WO95/28487 2 1~ ~ ~16 1 . l/~. D

vectors are usually comprised of a sP~ectAhlR market that i5 either maintained in a replicon or developed into an integrating vector, as described above.
5 Expression ~nd transformation vectors, either extra-eplicons or integrating vectors, have beendeveloped for transformation into many bacteria. For example, expression vectors have been developed for, inter ~, the following bacteria: R~r~ C subtilis tPalva Q~
10 al. tl982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ.
Nos. 036 259 ~nd 063 953; PCT Publ. No. Wo 84/04541], Escherichia coli [Shimatake et 311- (1981) Nature j~:l28;
Amann e~ al. (1985) ~elle 40:183; Studier et ~,. (1986) J, Mol. Biol. 189:113; EPO Publ. Nos. 036 776, 136 829 and 136 15 907], Streptococcus cremoris [Powell et al- (1988) ~;L
V;rOn. M;crobiol. 54:655]; Streptococcu6 lividans tPowell . (1988) i~nrll. Envir-~n. Microbiol. 54:655], S~L~L ~es lividans tU.S. Patent No. 4,745,056].
.

20 Methods of introducing ~ y~ u5 DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaC12 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation.
25 Transformation ~oce-luL~ usually vary with the bacterial species to be L,c~ rcL '. See e.g., tMasson et ilL. (1989) FF~Mq Mi~robiol. Lett. 60:273; Palva et al. (1982) Proç.
Natl. Acad. sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541, Bacillus], tMiller et 30 ~. tl988) Proc. Natl. Acad. Sci. 85:856; Wang et ~1. (1990~
J. BacteriQl. ~:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. sci. 69:2110; Dower et al. (1988) ~lucleic Acids Res. 16:6127; Rushner (1978) "An improved method for tr~nsformation of Escherichia coli with ColE1-derived 35 P1;!~F~;~ In Genetic ~n~7jnPPrinr~ Pro~Pp~7innc of thP
Internati~nll S ~lm on GenetiC ~nr~inPPr;n~ (eds. H.W.
Boyer and S. Nicosia); Mandel ~ al. (1970) J. Mol. Biol.
53:159; Taketo (1988) Bir~h~m- Biophvs, Acta 949:318;
SUBSTITUTE SHEET (RULE 26) .

218~3~ ~ ~
Wo 95l28487 1~ o Escherichia], [Chassy et ~1. (1987) FEMr~ Microbiol, Lett.
44:173 Lact-~h~ c]; [Fiealer et ~,. (1988) Anal. Biochem 170:38, PL ~ --]; [AUgUStin et ~. (1990) FEMS
M;crobiol. Lett. 66:203, Staphyl~corcll~], [Barany ~ ~.
5 (1980) J. Bacteriol. ~:698; Harlander (1987) "Transformation oStrept~lcocc~c lactis by ele- ~-u~uLC~ion~
in: S~_-eu~o.;uc~e.l Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infec. T . 32:1295; Powell et ~. (1988) AD~1~ ~nviron, Mirrobiol. 54:655; Somkuti et ~al-(1987) Proc. 4th ~vr. Conq. Biot~rhn~loqv 1:412, Streptococcus ] .
iv. Yeast Expression 15 Yeast expression 6ystems are also known to one of ordinary skill in the art. A yeast promoter is any DNA s~qu~nre capable of binding yeast RNA polymerase and initiating the ..,,~.~am (3 ' ) transcription of a coding 5~q~ nre (e.g.
structural gene) into mRNA. A promoter will have a 2û .c-ne_.iption initiation region which is usually placed proximal to the 5 ' end of the coding sequence. This transcription initiation region usually ; nrl ll~l~c an RNA
polymerase binding site (the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second 25 domain called an u~ ~ activator seuu~ (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (;n~lllr;hle) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby 3û either ~nh lnr;n~ or reducing transcription.
Yeast is a fermenting organism with an active metabolic pathway, therefore se~u~l~aes ~nroS;nJ ensymes in the metabolic pathway provide particularly useful promoter 35 sequences. Examples include alcohol de~.ydLuy~ 'dse (ADH) (EPû Publ. No. 284 044), enolase, glurol~;nS~ce, gluco;~ G
phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPD~), hexokinase, SUBSTITUTE SHEET (RULE 26) wo 95r28487 ~ ~ 8 pho-~horL ~ kin~ce~ 3 ~ Jlyceratemutase~ andpyruvate kinase (PyK) tEPO Publ. No. 329 203). The yeast ~ gene, encoding acid phosphatase, also provides useful promoter [Myanohara et ~. (1983) Proc. Natl. Acad. Sci.
5 USA 8 0 ~
In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS
eC of one yeast promoter may be joined with the 10 transcription activation region o~ another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory 6eguence linked to the GAP transcription activation region (U.S. Patent Nos.
4,876,197 and 4,880,734). Other examples of hybrid 15 promoters include promoters which consist o~ the regulatory sequences of either the ~, ~1, Ç~Q, OR PE~Q5 genes, - ` in-~d with the transcriptional activation region o~ a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556). Furth. æ, a yeast promoter can include naturally 20 occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, ~;

3~, [Cohen et ~. (lg80) Proc. Natl. Acad. ScL. USA
77:1078; Henikoff et ~1. (1981) Natu~e~ ~:835; Hollenberg 25 ~ ~ 1981) t'-rr. ToPics Mirrobiol. T nnl, 96:119;
Hollenberg ~ ~. (1979) "The Expression of Bacterial Antibiotic Resistance Genes i the Yeast Sac~,l,aL ~ -0~
cerevisiae," in: pl~ c of M"~l~r~l. Envi '~1 Anrl 1 T ' ~~ (eds. K~N> Timmis and A. Puhler);
30 Mercerau-Puigalon et ~1. (1980) Gene 71:163; Panthier et ~1.
(1980) ~ rr. Genet. 2:109;].
A DNA molecule may be ~ æssed intrArelll~lArly in yeast.
A promoter Ce~ e may be directly linked with the DNA
35 molecule, in which case the first amino aoid at the N-terminus of the 1~_ ` inAnt protein will always be ~
methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from SU~STlTUrE SHEET (RULE 26) 2188 ~ L~
~WO 95l28487 P

the protein by ~.n vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative for yeast expression systems, as well as in 1 iAn, baculovirus, and bacterial 5 expression systems. Usually, a DNA ~olu- ~ ~ ollro~lin7 the N-terminal portion of an on~logonrlllC yeast protein, or other stable protein, is fused to the 5 ' end of heterologous coding sequences. Upon expression, this C~ .LI.I~_~ will provide a fusion of the two amino acid solu .l~0C. For 10 eYample, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5 ' terminus of a f oreign gene and eA~Le~f~ed in yeast. The DNA s~u-~ at the junction of the two ~mino acid c~-lu~ ro5 may or may not encode a cleavable site. See e.g., EPO Publ. No. 196 056. Another example is 15 a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific pr protease) to cleave the ubiquitin from the foreign protein.
Through this method, therefore, native foreign protein can 20 be isolated (see, e.g., PCT Publ. No. ~0 88/024066).
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA
~ lec~lPc that encode a fusion protein comprised of a leader 25 5 I~ e f~ that provide for secretion in yeast of the foreign protein. Preferably, there are procoFsin~ sites encoded between the leader r- and the foreign gene that can be cleaved either ~LII v vo or La vitro. The leader so~onre rl _ L usually encodes a signal peptide comprised 30 of hydrophobic amino acids which direct the secretion of the protein from the cell.
DNA onco~;n~ suitable signal sequences can be derived from genes f or secreted yeast proteins, such as the yeast 35 invertase gene (EPO Publ. No. 012 873; JPO Publ. No.
62,0g6,086) and the A-factor gene (U.S. Patent No.

4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for SUBSTITUTE SHEET (RU~E 26) , , _ _ , _ . _ _ . _ _ _ _ _ _ .

wo gs/28487 2 1 ~ 0 ~ecretion in yeast (EP0 Publ. No. 060 057J.
A preferred class of secretion leaders are those that employ ~I Ll _ L of the yeast alpha-ractor gene, which contains

5 both a "pre" signal 5~1U - re, and a "pro" region. The types of alpha-factor rL c that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U. S . Patent NOB .
10 4,546,083 and 4,870,008; EP0 Publ. No. 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence oS a f irst yeast, but a pro-region from a second yeast alphafactor. (See e.g., PCT Publ. NO.
15 N0 89/02463 . ) Usually, transcription termination sequences reco~ni7-~d by yeast are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the 20 coding s~ . These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator 8eqn~nre and other yeast-r~r.07ni7~cl termination s~ r- ~, such a6 those coding for glycolytic enzymes.
Usually, the above described ~ - Ls, comprising a promoter, leader (if de5ired), coding se~u~..u~ of interest, and tr~nscription termination sequence, ar~ put together into expression CU~ LLUULS. Expression c..JIDLLuuLs are often 30 maintained in a replicon, such as an extra. liL, -element (e.g., plA~ C) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host 35 for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 tBotstein et ~.
(1979) Gene ~:17-24], pCl/1 tBrake et ~1. (1984) Proc. Natl.
Acad. Sci USA 81:4642-4646], and YRpl7 [St inr~ ` e~ al.
SUBSllTUTE SHEET (R~JLE 26) wo 95128487 ~ 6 P~

(1982) J. Mol. Biol. l:~:lS7]- In addition, a replicon may be either ~ high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 5 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be 5elected"l--p~-n~lin-J upon the effect of the vector ~nd the foreign protein on the host- See e.g-, Brake et 3~., su~ra.
Alternatively, the expres5ion construct5 can be integrated into the yeast genome with an integrating vector.
Integrating vectors Usually contain at least one seuuel~ce homologous to a yeast chl - that allows the vector to 15 integrate, and preferably contain two homologous sequences flAnL~ the expre55ion ~ u..~ . L. Integrations appear to result from recombination5 between homologou5 DNA in the vector and the yeast ~ ~ trr-weaver et ~Ll. (1983) Methn-lq in ~n7Vmo~ 1:228-245]- An integrating vector 20 may be directed to a 5pecific locu5 in yeast by selecting the appropriate ~ o~c sequence for i n~ in the vector. See Orr-Weaver et ~1., qU~ra. One or more expression cu.-~-,u~_L may integrate, po55ibly affecting levels of recombinant protein ~r ud.lc~d [Rine et 3~1- (1983) 25 Proc. Natl. acad. Sci. USA 8Q:6750] . The ~ llr~ 1 sequences included in the vector can occur either as a single segment in the vector, which re5ult5 in the integra-tion of the entire vector, or two -- c h~ lo~o~lc to adjacent segment5 in the ~ and flanking the 30 expres6ion c;u..,LLu~ L in the vector, which can result in the stable integration of only the expression uilDLruuL.
Usually, extra~l., l and integrating expression CVI.~ s may contain selectable marker5 to allow for the 35 selection of yea5t strain5 that have been transformed.
S~l~ct~hl ~ marker5 may include bio~y..Ll. _Lic genes that can be e A~,e4~ed in the yeast host, such as ~, ~, L~, ~1, and ALG7, and the G~18 re5istance gene, which confer SUE~STITUTE SHEET (RULE 26) _ _ _ _ _ _ wo 95l28487 2 1 ~ 8 ~ ~ 6 1 ~ I IL ~-- ~ L ~ ~

resLstance in yeast cell8 to tunicamycin and G418, respectively. In addition, a 8uitable sPlect~ble marker may also provide yeast with the ability to grow in the presence of toxic _ _ ~, 6uch as metal. For example, the 5 presence of S~l allows yeast to grow in the ~L~ ce of copper ions [Butt et ill- (1987) Ni~ robiol. Rev. ~L:351].
Alternatively, 50me of the above described ~ Ls can be put together into transformation vectors. Transformation 10 vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either 15 extra~ l.L~ _ 1 replicons or integrating vectors, have been developed for l.L~Ll,_roL~ tion into many yeasts. For example, expression vectors have been developed for, ~n~ ~L~, the following yeasts:Candida Alhi~AnC tKurtz~ et ~1. (1986) Mol.
r~l1. Biol. 6:142], Candida maltosa [Kunze, et ~. (1985) J.
20 Basic l-~icrobiol. 25:141] . ~n~Pn~ polymorpha [Gleeson, e~
- (1986) ~. Gen. Microbiol. ~:3459; p~ en~ et ~al-(1986) Nol. Gen. Genet. 202:302], Kluyveromyces fragiiis tDaS, et ~ (1984) J- Bacteriol- 158:1165], K1UYV~L~ 5 lactis [De Louvencourt et ~1. (1983) J. Bacteriol. ~:737;
25 Van den Berg et, ~1. (1990) Bio/TP~-hnnloqY 8:135], Plchia guillerimondii [Kunze et, i~l. (1985) J. ~ic Mi~robiol, 25:141], Pichia pastoris [cregg~ et ~1. (1985) Mol. CR11.
Biol. ~:3376; U.S. Patent Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae tHinnen ~, al. (1978) Proc. Natl, 30 Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol, ;L~:163], Schizosac~ es pombe [Beach and Nurse (1981) 300:706], and Yarrowia lipolytica [Davidow, et ~;L.
(1985) rllrr. Genet. ~,Q:380471 Gaillardin, et ~l. (1985) ~lrr. Genet. 10:49]-Nethods of introducing ~ J~ c DNA into yeast hosts arewell-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells SUBSTITUTE SHEET (RULE 26) Woss/28487 ~ t~8~16 ,~ o treated with alkali cations. Transformation ~Lu~eduL-~usually vary with the yeast species to be transformed. See e.g., tKurtz et ~. (1986) Mol. Cell. Biol. 6:142; Kunze et A1. (1985) J. R:~cic r~i~ robiol. 25:141; Candida]; [Gleeson - 5 et al. (1986) J. Gen. ~ robiol. ~:3459; D ~g~n~ et ~.
(1986) Mol. Gen. Genet. ~ :302; H~nc~n~ ]; [Das et ~.
(1984) J. Bacteriol. 158:1165; De LUuv~ uuLL et ~1. (1983) J. Bacteriol. 1~:1165; Van den Berg et ~L;L. (1990) Bio/T~ hnnloqy 8:135; R1UY~L~ ~,es]; [cregg ~ ~1. (1985) 10 Mol. Cell. Biol. 5:3376; Kunze et ~l. (1985) J. RAcic Microbiûl, ~:141; U.S. Patent Nos. 4,837,148 and 4,929,555;
Pichia]; [Hinnen et ial- (1978) Proc. Natl. Acad. Sci. USA
75;1929; Ito et ~1. (1983) J. Bacteriol. ;L~:163 Saccharomyces]; [Beach and Nurse (1981) ~a~ 300:706;
15 S~!hi 7ocaC~h~romyces]; [Davidow et i~l- (1985) Curr. Genet.
lQ:39; Gaillardin et ~1. (1985) t`~lrr. Genet. ~:49;
Yarrowia ] .
Nucleic Acid ~Cc~V8 Polynucleotide probes o~ approximately 8 nucleotides or more can be ~I~parcd which hybridize with the positive strand(s) of the RNA or its ~ 1~ L, as well as to cDNAs. These polynucleotide5 serve as probes for the detection, isolation 25 and/or l;~hl-l;n~ of polynucleotides which contain nucleotide se~ c.c~ and/or as primers for the transcription and/or replication of the targeted 5~lu~ s. Each probe cnn~ ~inc a targeting polynucleotide s~ n~-e, which is comprised of nucleotides which are compl ~ALy to a target nucleotide 30 5~ ~e; the F~ e is of sufficient length and Larily with the 8~uuel~c~ to form a duplex which has sufficient stability for the purpo8e intended. For example, if the purpose is the i801ation, via i ~i l; 7-tion, of an analyte cnnt-:-inin~ a target se~u~ ~.ce, the probes wlll 3s contain a polynucleotide region which is Or sufficient length and compl ~L ily to the targeted ~Lyue~,ce to afford sufficient duplex stability to i - i l i 7e the analyte on a solid surface under the isolation conditions. For SU3STITUTE SHEET (RULE 26) _ _ _ _ Wo 95/28487 2 1 8 ~ 3 1 6 P~,l,..,. '~ .C
ex~lmple, also, if the polynucleotide probes are to serve as primers for the transcription and/or replication of target ~ u~naes, the probes will contain a polynucleotide region of sufficient length and 1~ LArily to the targeted 5 5 ~, ~~ _ to allow for replication. For example, also, if the polynucleotide probes are to be used ac label probes, or are to bind to multimers, the targeting polynucleotide region would be of sufficient length and complementarily to form stable hybrid duplex DLLUUl.ULe5 with the label probes 10 and/or multimers to allow detection of the duplex. The probes may contain a minimum of about 4 contiguous nucleotides which are complementary to the targeted S~oq~lon~o; usually the oligomers will contain a minimum of about 8 continuous nucleotides which are complementary to 15 the targeted se~u~lce, and preferably will contain a minimum of about 14 contiguous nucleotides which are 1 - ~ y to the taLyc:~ed so~ nre.
The probes, however, need not consist only of the sequence 20 which is compl L~LY to the targeted sequence. They may contain additional nucleotide ~;o ~ eC or other moieties.
For example, if the probes are to be used as primers for the amplification Of 5~ r~n via PCP~, they may contain se~u_..ces which, when in duplex, form restriction enzyme 25 sites which facilitate the cloning of the amplified ie.lue..-es. For example, also, if the probes are to be used as "capture probesn in hybridization assays, they will be coupled to a "binding paLLI,_L" as defined above.
Preparation of the probes i5 by means known in the art, 30 ~n,-~.7-7;ng, for example, by methods which include excision, transcription or rho7~7i~1 synthesis.
T ~ ' i acmostic ~ ys 35 Antigens can be used in; -- y:, to detect antibody levels (or CUIIV~LS~:1Y antibodies can be used to detect antigen levels) and correlation can be made with disease.
- -- ya~ based on well defined, i~ -~ ' in~nt antigens can Sl)BSTITUTE SHEET (RULE 26) W095128487 ~ 3~6 be developed to replace the invasive diagnostics methods that are used today. Ant i ho~ c to proteins within biological samples, including for eYample, blood or serum samples, can be detected. Design of the i C~ayS i8 5 subject to a great deal of variation, and a variety of these are known in the art. Protocols for the i -- y may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid ~.U~UL ~5, or may be by i , ~cipitation. Most 10 assays involve the use of labeled antibody or polypeptide;
the labels may be, for example, flu~JL-~sc~lL, r~homi ll-min~cont~ radioactive, or dye molecules. A6says which amplify the signals from the probe are also known;
examples of which are assays which utilize biotin and 15 avidin, and enzyme-labeled and mediated i - - ccayS, such as ELISA assays.
Kits suitable for i -'i~n~ and c~ntAinin~ the appropriate labeled reagents are c-,.,z,~Lu~ Led by packaging 20 the appropriate materials, including the compositions of the invention, in suitable containers, along with the r~ inin~
reagents and materials (for example, suitable buffers, salt solutions, etc. ) required for the conduct of the assay, as well as suitable set of assay instructions.
V~ccines Vaccines may either be prophylactic (to prevent in~ection) or theLtl~uLic (to treat disease after infection).
Such vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers, "
which include any carrier that does not itself induce the production of antibodies harm~ul to the individual receiving 35 the composition. Suitable carriers are typically large, slowly D~ holi~o~ macromolecule8 such as proteins, polysaccharides, polylactic acids, polyglycolic acids, poly-meric amino acids, amino acid copolymers, lipid ay~L-:yc,tes SUBSTITUTE SHEET (RULE 26) _ _ _ _ _ _ _ _ Wo 9~l18487 2 ~ ~ ~ 316 r~l,~3~ ^ ~

(such as oil droplets or li -~), and inactive virus par-ticles. Such carriers are well known to those of ordinary skill in the art~ Additionally, these carriers may function as ~ - imulating agents (nad~uvantsn). Furthermore, the 5 antigen may be c~i,juyc.Led to a bacterial toxoid, such as a toxoid from diphtheria, tetanu8, cholera, H. mvlori etc.
p~tho9ens .
Preferred adjuvants to enhance effectiveness of the compo-10 sition include, but are not limited to~ min1lm salts (alum), such as aluminum hydroxide, ~lllmimlm phosphate, Alllmin--m sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific ~imulating agents such as muramyl peptides (see 15 below) or bacterial cell wall c --nts), such as for example (a) MF59 (PCT Publ. No. Wo 90/14837), containing 5~6 Sgualene, 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 20 microfluidizer such a5 ~odel llOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, cnr~ nin~ 10%
Sgualane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle 2s size ~ 1 ~inn, and (c) RibiTR adjuvant system (RAS), (Ribi T nnll~-m~ Hamilton, MT) containing 2% Sql~ nP, 0.2% Tween 80, and one or more bacterial cell wall ~ _ ents from the group consisting of - ,'-,'r ylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably 30 MPL + CWS (DetoxT ); (3) saponin adjuvants, such as StimulonTR (Ca~ridge Ris~ci~nr~, Worcester, MA~ may be used or particles generated therefrom such as ISCOMs ( i - Limulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, 35 such as interleukins (IL-l, IL-2, etc.), ma.;Lu~ colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act a~ imulating agents to enhance the ef~ectivenes5 of the composition.
SU~STITLITE SHEET (RULE 26) ~883~ 6 ~ woss~8487 r : 41 Alum and MF59 are preferred.
As mentioned above, muramyl peptides include, but are not limitedto,N-acetyl-muramyl-L-t~ yl-D-isoglutamine (thr-5 MDP),N-acetyl - ~yl-L-alanyl-D-isoglutamine(nor-MDP), N-acetylmuramyl-L-alanyl-D-~ tAm;nyl-L-alanine-2-(1'-2~-dipalmitoyl-sn-glycero-3 huydL~Ay~ hcly 1OAY) -ethylamine (NTP-PE), etc.
The ; j ;c compositions (e.g., the antigen, phArr^^eutically acceptable carrier, and adjuvant) typically will contain Ail--~nt~, such as water, saline, glycerol, ethanol, etc. Additionally, allY;liAry substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Typically, the ; , ;c compositions are ~L~paLed as injectables, either as liquid solutions or sll~pDn~;nn~;
solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be pL~pal.d.
The preparation also may be emulsified or onrArs~ ted in 1;,-~ for ~nhAn~ed adjuvant effect, as A;F---F-ed above under pharmaceutically acceptable carriers.
T , ; ~ compositions used as vaccines comprise an ; -lc7ic~lly effective amount of the antigenic polypeptides, as well as any other of the ab~.~ r tioned ~ , 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 dep~n~;n~ upon the health and physical condition of the individual to be treated, the ~ ;c group of individual to be treated (e.g., nnnhllr~n primate, primate, etc.), the capacity of the individualls immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor~5 A~- L of the medical situation, and other rel-~UBSTITUTE SHEET (RULE 26) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .

Wo gs/28487 ~ 6 ~1 C

evant factors. It is ~Yrect~d that the amount will fall ina relatively broad range that can be determined through routine trials.
5 The i -, ; c compositions are conventionally administered parenterally, e.g., by in~ection, either subcutaneously or illl,L -_ lArly. Additional formulation~ suitable for other modes of administration include oral and p~ y formulations, suppositOries, and trAn~ l applications.
10 Dosage treatment may be a single dose 8l h~AIlle or a multiple dose sch~ llle. The vaccine may be administered in conjunc-tion with other 1 ~:yulatory agents.
15 Briof D~cr~pt~on o~ th~ Dr~w~ ng~
Figure lA shows part of a silver-stained qel showing chlamydial EB proteins with M, between 25,000 and 40,000 and pI values between 4 and 5 (non-linear pH gradient). EBs. The 20 arrow shows the spot which was eluted for N-terminal amino acid sequencing.
Figure lB shows part of an 1 -- lot Or the map region shown on the left, developed with a pgp3-specific rabbit 25 serum.
Figure 2 shows ~c~ Blue stained gels after SDS-PAGE
following expression in l~.coli BL21tDE3), and purification of r-pgp3. L~n- A: size markers. Lan-- B, Ct pellet tB) and 30 ~u~L..dt~nt tc) fractions of pgp3-expressing E.coli cells after Polymyxin B treatment and centrifugation. L~nu- D, lS:
pellet tD) and supernatant tE) samples of per~rl~ ic fractions tas in lane C), after dialysis against piperazine-}icl buffer pH 5.4, and centrifugation. L~n~ F:pooled peak-35 fractions after ion-~ hAn~e ~IILI t.UYLCI~hY.
Figure 3 shows j -~1 nt analysis of human sera with puriried r-pgp3. Typical positive and negative results (A
SUBSTITUTE SHEET (RLILE 2lj) ~wog5/28487 2 ~L 8 g 3 ~

and C) are shown. Immunoblots with crude ~3.coli extracts are also shown (B and D): these gave variable patterns o~
reactivity to ~.coli antigens, served ~or patient identification, and as a positive control for negative 5 samples. Blot- A n~ B: serum of a patient affected by salpingitis and NIF-positive ~or C. L~ . The ELISA
L~ .se of this serum is 5hown in Fig. 4C (curve with int~ te r~ se). Blot- C ~nl~ D: serum from a healthy blood donor, MIF-negative for C.LL ' tis. This serum was 10 used as a negative ELISA control in the present study.
Figure 4(i) is a graph showing the reaction between plastic bound ,. inAnt pgp3 protein of the present invention and various human sera. Sera C, A and 3 were from women with 15 ~:Al~;n~;tis Serum 8 was from a male with isolation-positive chlamydial urethitis and serum 13 was from a healthy blood donor .
Figure 4 (ii) shows typical pOsitive and negative pgp3-ELISA
20 results. Semilogarithmic plots of OD readings (each point is the average value of duplicate samples) against 2-fold dilutions o~ the sera, starting from 1/100. A: 10 healthy blood donor sera which gave negative MIF results with purified chlamydial E8s o~ all three species, and did not 25 react with r-pgp3 on immunoblots. The upper curve (dotted ~quares) was given by a positive control serum (immunoblot-positive, MIF-positive) assayed on the same microtiter plate. B: 10 sera from patients with C. rn i~ infection (MIF >512). The upper curve (open squares) is the positive 30 control. These results are comparable to those obtained with healthy subjects in panel A. C: positive pgp3-ELISA results obtained with 10 of 46 salpingitis ~era DYAm;nD~; positive and negative control 8era (top and bottom curves, respectively) are ;nrl~ D~. D: 5 negative tcurves with OD ~
35 0.25), and 2 positive pgp3-ELISA results (solid ~ and triangles) obtained with 7 O~ 40 male urethritis sera .-YAmi nD~l . A positive (dotted squares) and negative (open triangles) control are also shown.
SUBSTITUTE SHEET (RULE 26 wo 95t28487 2 1 8 ~ 3 ~ 6 Figure 5 shows the prevalence of anti-pgp3 IgGs in human sera nnd comparison with anti-E8 sUrface IgGs (~IF) prevalence. ~op p~n~l2 results obtained with a group of 40 5 male urethritis (NGU) sera . ItiA_l- p~n~l s results obtained with 46 ~alrinqitis sera. For convenience, the more general term PID tfor pelvic infla~Dmatory disease~ has been used in the table. Bottol~ pan-ls summary of all results, including those from the 10 C.~n~ pogitive sera, 50 healthy 10 blood donor sera and re8ults obtained with a group of 24 sera from women with various symptoms of genital tract infection or sec~ .y sterility.
~-t~ilo0 D~l~cription Or r~hOA;~
The examples presented herein are provided as a further guide to the practitioner of ordinary skill in the art and are not to be construed as limiting the invention in any way.
Production of ~-- ;nJ~nt s~q~3 in E.coli.
ORF3 DNA (pCT segment from bp 4054 to bp 5013, according to ref . 3 ) was r~h1 ~ i nPd by a polymerase chain reaction (PCR) 25 (22) using 10 ng of plasmid pUC8-pCTD (3), as a template, and 20 pmoles each of the following primers:
5 '--GGG~... L~t~G-,AAA~ C L ~ ' L ~ - 3 ', and 5 '--C(`C'c tg~g~ ~ At'-TTAc. . ~ ,--3 Tag DNA polymerase and the GeneAmp kit (Perkin-Elmer) were used according to manufacturer's r~ tions. Primers were designed with NdeI and PstI re8triction ~ites (low case characters in the above ~lu ~ c) at their 5' ends, in 35 order ~o orient the amplified DNA fragment into the .~ r1inq sites of expression plasmid vector pT7-7 (29, 30, 35). The PCR product was purified using Centricon cartridges, digested with NdeI/PstI endonucleases SUBSTITUTE SHEET (RULE 26) Wo ss/28487 2 1 ~ 8 3 I ~ P~l/5~ o (Boehringer) and ligated into pT7-7. The ligase reaction mix was used to transform E.coli strain DH5 t7) which was plated on LB agar t23) containing 100 ~g/ml ampicillin. rOlnn;pc were s~ L~ d for the ~es~-,. e of the ORF3 insertion by 5 hybridization with ORF3-specific synthetic ol i gnntl~-leotides, end labelled with 32p t23). DNA from positive colonies were then used to transform E.coli BL21tDE3) competent cells t29, 30) which were ~1ected first on 5 iC-;llin-LB agar plates t23) and then by their cArAhil;ty of expressing the 10 r~ ' inlnt protein.
Bacteria from positive cnlnnipc were grown overnight in 10 ml of LB medium containing 100 ,ug/ml _mpicillin. Overnight cultures were diluted (1:200) with fresh LB medium without 15 ampicillin and grown at 37 C until O.D.500= 0.6. Since expression in the pT7-7/BL21tDE3) system is under the control of an IPTG-in~oihle promoter, ORF3 expression was achieved by adding 0 . 4m'!l IPTG to the medium and further incubating for 2 . 5 hours with vigorous shaking. Bacterial 20 cells, collected by centrifugation were r- I~Qn~ed in 1/20 of the initial volume of 25% Sucrose, 50mM Tris-Hcl, pH 8, containing 1 mg/ml of polymyxin B sulfate tSigma Ch~mir5~18) t18) and incubated at room temperature for 2 hr.
After centrifugation tlO min in an E5u~5lldùLL centrifuge) 25 most o~ r-pgp3 was found in the :~u5y~5LIlaL~nL tperirlAC~ic fraction). The integrity of the bacterial cells was checked by ensuring that the cytor,s~F-nic beta-galactoeidAce activity tl4) remained all in the pellet.
30 r-pgp3 was detected by polyacrylamide gel ele~ LLv5~huL~sis, using 12.5% polyacrylamide, 1% SDS gels tSDS-PAGE) according to Laemmli tl2). Gels were stained with Cc cc;~ brilliant blue t0 . 05% w/v) in 10% tv/v) acetic acid, 30~ tv/v) methanol. T -hlot analysis t31) was performed with a 35 pgp3-specific rabbit serum, as described t3). One expression competent 13.coli clone was eventually 5~1e~-ted for further work. The 1~ ;ns~nt pT7-7 cu--:,LLu~:t h~scLuult5d by thls clone was checked by 5equencing, Using the dideoxy SUBSTITUTE SHEET (RULE 26~
. . _ _ ... . . .. .. . . _ _ _ _ _ Wo ssn8487 21 8 ~ ~1 6 r~.,~.. o terminators t~chni~ (25) and the S~5rlnAC~ kit tUS
BiorhPmir~lq) the entire ORF3 DNA insert, which was identical to the s~-lu~ originally described for ORF3.
5 Pl~rif ication of r~ ' i nAnt ~cro3 Crude perirl~iC fractions were ~bl Ain~d, as described above, from 40 ml or 200 ml bacterial cultures,and dialyzed against 30mM piperazine-HCl, pH 5.4. This cau~ed extensive 10 protein precipitation, but left r-pgp3 in solution. Further purification was obtained by ion-exchange chromatography on Dono-Q prepacked columns (Pharmacia) in the same piperazine-HCl buffer. Selective elution was oht~in~d with a NaCl concentration gradient O to lN. FPLC equipment (Pharmacia) 15 was used. In a typical run 4 mg o~ total protein were loaded and lml fractions were collected, and analyzed by SDS-PAGE
followed by Coomassie blue staining and i -' lot analysis, as above described. Peak fractions containing purified pgp3 were pooled and dialyzed against sterile PBS ( lOmM-sodium 20 phosphate pH 7.4, 15mN NaCl). pgp3 purity was estimated as >90% by total protein determination (Biorad Protein Assay) and PAG~ together with protein standards (increasing amounts of titrated bovine serum albumin solution) followed by ~ --ie 81ue staining and photod~nci ~Ly r - . ~5 25 using an Ultroscan XL Densitometer (LKB).
Two d;r i onAl elec-J vi,hv.,stic AnAlysis of FP~ ~ro~ nq Large scale ~.~aLations of C. Lr ' tls L2/343/Bu 30 elementary bodies (EB) were obtained from Vero cells cultures in rolling bottles, according to described methods (1). EBs were purified by two cycles of density gradient centri~ugation (1) and stored at -20 C for s-lhsQ~ nt electrophoretic analysis.
TWO-~ nAl gel ele.~.v~hur~8i8 wa8 performed using the -' ilin~/ polyacrylamide system, essentially as described by Hochstrasser et al. (1988), and Hughes et al. (1992).
SUBSTITUTE SHEET (RULE 26~

Wo 95/28487 ~ 3 1 ~ o Approximately 45 I-g (analytical run) or lmg (yL-=yaLc~ive run) of total EB protein were u6ed ~or each run. EB6 were pollPted by low 6peed centrifugation and I~D~ 1"d in 8M
urea, 4% CHAPS ( 3-t (3 cholamidopropyl)dimethyl~ m]_1_ 5 prop~n~-c~ onate), 40mM Tris base, 65mM dithioerythritol (DTE) and trace amount6 of ~L~ nl Blue. The first d1 ~inn was carried out on 1 -~11n~ strips providing ~-non-linear pH gradient (IPG 6trips, PhArr~ ) ranging from pH value 3 to 10. Voltage was linearly increased from 300 10 to 3500 V during the fir6t three hours, then stabilized at 5000 V for 22 hours (total Volt.hour product 110 kVh ).
After elecLLvyhuL~sis, IPG strips were equilibrated for 12 min against 6M urea, 30% glycerol, 2% SDS, 0.05M Tris.HCl, pH 6.8, 2% DTE, and ~-~hcequ~ntly for 5 min in the same 15 urea/SDS/Tris buffer solution but substituting the 2% DTE
with 2.5~ io~l~acet.~ . The second di----inrl was carried out on 9-16% polyacrylamide linear gradient gels (18 cm x 20 cm x 1.5 mm), at 40 mA/gel ~U~ID~ IL current, for approxiDately 5 h until the dye front reached the bottom of 20 the gel. Analytical gels were stained with ;ACA1 silver nitrate, as described (9, 17).
The pH gradient was monitored with carbamylated creatine kinase (CPK standard, B.D.H. ) ~nd corrected, at the non-25 linear acidic end, according to internal EB proteinreference spots, which were identified by i -' lotting with - --lrn~l antibodies speci~ic for known chlamydial proteins (a gift from G. Christiansen and S. gir~ n~?) 30 N-terminAl DeCTuencinn o~ the native 28 kDa ~~hl~mvdial antigen For N-terminal primary D~L~LuLe determination, protein spots were electroeluted ~rom the gels onto polyvinylidene 35 difluoride ~ ' c.nes (BioRad PVDF membranes 20 x 20 cm, 0.2 micron pore size) ) according to Matsudaira (1987) . Blots were stained with 0.1% (w/v1 Coomassie Brilliant Blue R250 in 50~ aqueous methanol for 5 minutes, and destained in 40%
SUBSTITUTE SHEET (RULE 26) w0 95/28487 ~, ~ 8 8 ~ ~ ~ r~
methanol, 10% acetic acid. Membrane5 were dried at 37 C
(24) and stored at -20 C for further analysis. The membrane area containing the main pgp3 protein spot, was exciced from five identical blots, and the pooled material waEI submitted 5 to Edman deyradation using an automatic Protein/Peptide ~oq on~ or (mod 470A; Applied Biosystem Inc. ) connortod on-line with a phenylthiohydantoin-amino acid analyzer model 120A and a control/Data Module model 900A (Applied Biosystems Inc . ) .

~n7yme inl~o~7 i - cc~y The purif ied pgp3 antigen was used to set up an enzyme linked { be:l~L assay (ELISA) test. 200 ng of protein 15 were adsorbed onto pla6tic wells of Maxisorp microtiter plates (NUNC) in 100 ~1 of coating buffer (PBS pH 8.0, 0.005% Tween 20, 0.02% NaN3), first for 2 hours at 37 C and then overnight at 4 C. After washing with coating buffer, the wells were saturated with 200 ~1 of 2 . 7%
20 Polyvinylpyrrolidone for 2 hours and washed again. 100 ~11 of serum samples, diluted in coating buffer as re~auired, were added to individual wells and incubated for 2 hours at 37 C. After repeated w~ Rh i nlJ_, bound serum-Ant i h~r7 i Pc were detected by incubation with A 1 kA 1 i no phosphatase 1 Aho7 1 ~d 2~ anti-rabbit or anti-human IgG antibody ~Cappel), diiuted in coating buffer 1:5000, at 37 C for 2 hours. For colorimetric detection of enzyme activity ELISA Substrate and Buffer (Sclavo Diagnostics srl) were used as ~
by the manufacturer. Optical density readings were 30 performed, usually after 1 hr of color dev~ 7: ~, with a Multiscan MCC densitometer (Titertek).
~ rr~i ~'luoresron.-O
35 Twofold dilutions of sera were titrated by single-antigen micro-immunofluuL~ce~l~ e (MIF) (33) using ~.UVLV5.. gradient purified EBs of C.L~ ' ~ 'c L2/434/Bu and s ~u~y~ D
strain Go/86, C.psittaci 6BC and A22, and C.p~ ii~o IOL-SU~STITUIE SHEET (RULE 2~) WO 95/28487 2~ 1 ~ 8 3 ~ 6 P~

207. Fluorescein conjugated rabbit anti-human IgGs (Dako~
were used for detection with a W mi~ L~sc-~e (Zeiss) . Sera for which prl~ih~e cross reaction between C.LL ~. tis and C.~ i~o antiho~l;e~ was s-~cpect~od, were .e 2~f~C~
5 using a commercially available kit which di~criminates anti-C. ~L ' tis from anti-C.p -;AO ~ 5C (T ~- , PBS Orgenics, Israel). In this case, titers were deduced from a single dilution (1:200) readings, according to manufacturer 1~ '-tions.
Cl inica] 6ammles Sera from patients who underwent laparoscopical examination for suspected salpingitis were collected at the Centre 15 Hospitalo-Universitaire, university of Picardie, Amiens, France. other sera were obtained from women attending the Laboratoire Départemental de la Somme, Amiens, because of ~y L of genital tract infection. A third group of sera was selected from the collection of the Rioh~nqu~o de 20 Picardie, Amiens; these included sera from healthy s~Lo.,c~ltive subjects, the group of C., iA~ positive sera, and sera from volunteers who accepted to submit to periodic rl inirAl examinations at the Centre de Prevention et d'examen de santé d'Amiens. All the above sera were 25 aliquoted in 200 microliter resin straws, and stored at -80 C, at the Biobanque de Picardie. Healthy blood donor, and male urethritis sera from patients attending the STD clinic of St Orsola Hospital, Bologna were rollected by the Institute of Microbiology, Bologna University, Bologna, 30 Italy. For the male urethritis patients, oYrll~ ion of oc -1 infection, and C. LL ' Lis isolation from urethral swabs (positive in 50% of cases), were performed as previously described (19).
35 Identification of ~tive p~3 Elementary bodies of C. L. tis L2/434/BU were grown in Vero cell cultures, and purif ied by two cycles of density Sl~aSTlTUTE SHEET (~ULE 26) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ . _ Wo 9Sl2848~1 2 ~ o gradient centrifugation. Aliquots of this ~L~aL~Lion t45 l~g or 1 mg of total protein for analytical or preparative runs, respectively) were dissolved in urea/CWStTris/DTE solution and charge-fractionated by elecLL~ oLesis on IPG 1 ~ilin~
5 strips. After reaching ir-~el~ctric ~r~llihrium, the IPG
strips were equilibrated with a new buffer, treated with buffered 2.5% io~ aretAmi~ and loaded on 18x20 cm polyacrylamide gels ~or protein separation according to ~ize. Two gels were prepared under i~ ntieAl conditions lO (initial loading 0.05 mg of total EB protein): the first was silver stained, and the second one was electroblotted onto a cellulose nitrate membrane and probed with a pgp3-specific rabbit serum. The silver stained gel showed a pattern of >500 distinct spots in the 10-150 kDa, and pI=3.5-9 ranges.
15 T -hl r~t analysis of the EB protein map showed that only two spots were reco~ni 70CI by the anti-pgp3 serum: a major one with coordinates CULL~ ;n~ to N,=28,000 and pI=4.6, and a minor one of similar M" but shifted towards the acidic side of the map (Fig. lB) . This minor spot was not 20 further investigated, however, the ~Lèse-.ce, beside a major protein species, of one or more satellite spots ("charge trains") with same molecular weight, decreasing intensity, and increasing negative charge is a typical pattern which is often observed in two-dimensional eleeLLu~hol~Lic maps.
25 These patterns are usually given by ~Lu~Le6sive deamidation of Asn or Gln residues, generating the CULL~ 1;n~
negatively charged acidic residues. By matching the -~ lot with the silver-stained gel, the i èactive 28-kDa protein was identified on the map (Fig. L~) .
In order to purify thi8 protein for further analysis, 5 mg of total EB protein were separated by two-~ nA l electrophoresis on five identical gels (1 mg total protein/gel) and electroblotted onto polyvinylidene 35 membranes. These were then lightly stained with C - i~
Blue and, the major 28-kDa i ~active protein spot on each blot was located by pattern comparison with the silver stained protein map and carefully excisêd. Protein from the SUBSTITUTE Sl iEET (RU~E 26) wo 95/28487 ~ ~ 8 ~ o six spots was pooled for amino acid 6~lu~ ~ e analysis. The f irst 10 N-terminal re6idues of this protein could be clearly identii'ied in the following 5i~qu~nre Gly-Asn-Ser-Gly-Phe-Leu-Leu-Tyr-Asn. This 8equence is identical to the 5 one previously predicted from ORF3 (2, 3), apart rrom an initial Met residue, ~ 1rihl~ from the translation of the first ATG codon of ORF3 but not present in the protein purified from EBs.
~Ynression of ORF3 in E.coli and murirication of recombin~nt ~q~3 ORF3 DNA was cloned in the plasmid vector pT7-7 and e..i~Lessed in E.coli BL21 cells under the control of a T7 15 bacteriophage promoter which can be indirectly activated by the addition of IPTG to the medium. Extracts from a ~:slec~P~
E.coli BL21 clone were shown, by PAGE analysis, to produce large amounts of l~ 'inAnt pgp3 (r-pgp3). It uas also observed that a large proportion of r-pgp3 was present in 20 the periplasmic fraction which was ~ht:~in~ either by controlled treatment of the bacteria with polymyxin B
(ref.18; Fig.2: B and C) or, by osmotic shock (ref 6; data not shown) . Since per;rlA~~ic- extracts had a reduced protein Yity, supernatants obtained by centrifugation of 25 polymyxin-treated BL21 cell8 (Fig. 2C) were Used as starting material for further r-pgp3 purification. Preliminary tests showed ti~iat ion-~YrhAn~Jc ~ IL~ LC~i411~ on mono-Q columns ( Pharmac ia ) cou ld be an ef f ective puri f ication i4. ~c~ 1u, e .
Before loading on mono-Q columns, bacterial extracts were 30 dialyzed against piperazine-HCl buffer, pH 5.4. During dialysis several protein species formed a precipitate, which was removed by centrifugation, while r-pgp3 r~ in~d in solution (Fig.2: D and E). Chromatography of the dialyzed e,.L,c.~;Ls was performed in the 8ame piperazine-HCl pH 5.4 35 buffer and elution with a NaCl c~ ceil~Lcition gradient. Most r-pgp3 was collected in a major peak with a purity ~90~c, as judged by PAGE, C -^cie Blue staining and photodensitometry (Fig. 2F) . Some minor bands in the 80-90 SUBSTITUTE SHEET (RULE 26) . . , , _ _ _ Wo 95/28487 2 1 ~ 6 ~1 IU

kDa region co-purif ied with r-pgp3 . Since Western blot and ELISA data 6howed that these rnnt~m;n~nts did not generate any appreciable ba~}~yL~lull~ in the assay of human sera, no further purification was attempted.
S~ecific detection of Anti--l~t7p3 Ant;hn~i~c bv ET~T~A
Purified r-pgp3 was initially tested for its ability to bind to N~NC microtiter plate wells. Simple incubation in PBS-10 0.05g6 Tween wa8 found to be satisractory. Rabbit sera,raised either against the 39-kDa fusion protein, or the 28-kDa r-pgp3 here described, were initially used for setting up the assay. Variable amounts (500 ng to 50 ng) of puri~ied antigen in each well were tested against the anti-pgp3 15 rabbit sera and the amount of 200 ng/well was eventually chosen as a ~.La~ l assay condition. In order to test if normal human 6erum ~ntC could interfere with the detection of anti-pgp3 antibodies, pgp3-ELISA was tested with cel~ted groups of human sera which were previously zO analyzed for the pI~c..ce/ab8ence of anti-chlamydia antibodies (IgGs) by ~IF, and for anti-pgp3 IgGs by -~lot analysis with purified r-pgp3 preparations.
EYneri- ' 1 In a first experiment, a panel of 21 human sera was used to test the ELISA peLrUL~anCe with r-lin;C~l 8amples. All sera gave negative MIF l. .uu-.-e to C.psittaci and C.,~n- - iA~.
When tested for C.tL '. tis antibodies, using purified L2-30 seL. Ly~e EBs, 15 8era were 8cored a8 llIF negative, and 6positive, with titers comprised between 1:32 and 1:256.
These sera were also te8ted for their ability to react with the purified pgp3 ~L~:l,aLation on We8tern blots: all the MIF
negative sera gave negative re8ult8, whereas the MIF
35 po~itive sera reacted, to variou8 extent8, with the 28-kDa band only.
Two-fold serial ~ t;~nc of the sera were tested, in SUBSTITuTE SH~ET (RU~E Z6~

'2l88~
~0 95128487 1 ~ r~ o d~ at~ ples, w~th th~ p51p3-ELISA OD r~'{n~ w~re taken afe~r 30 ~in, 1 hr ~nd ~ter O/N ~tor~ge ln the cold (-20 C) All 15 n~g~tiv ~era gavQ con~lGt-ntly low OD
L~ r~ (below 0 1~, vh r-a~ th- 6 po-itlv- ~ra gav 5 ~l{~t~ y hlgher OD r~ ich ~roportlonally ~
Ylth th- ~-rum _ JC L~ion ln th- a~ Th~ tOD VL
dilution] ourv-s o~ the po~itlv- ~-r- co f~r teQt~d correlat~d with th- IllF titr~
10 The reault~ or reacting v~rlow hu~n -ra ~t t~ilot~nq ~rom 200 to 6800 rOld with the plastic-~ound ~_ oi~-nt pgp3 prot in ar~ p~ i C~ l l y $n Flgur~
OD~ ~ro aver~g~ v~lU-8 ^ht- i n~d rro~ dupllcate ~ampl~s 5 C. ~ ) - ~ ~ MIF tltr-~ Or th s~r~ Itop to bottom) w~r ~:256; 1 128; 1 1285 ~ 132; z~tro SQr~l C, A nd 3 w~r~ rrOm wom~n wlth ~alpinq~tl~; th- erum 8 w~ ~ro~ a mal- with i~ol~t~o r ItiV chl~mydl~l ur~thrltl~ m 13 wa~ rro a h~althy blood donor .-r ~ t 2 , Ir a ~cond xp~rl~nt, ~ pan-l Or 10 hullun ~-r~ giving a NIF posltiv ~., ~~ to C ~ t~ (Cq!-llIF po~itiv 1 25 wlt~ tlt-r~ ~64, nd SO h ~lthy blood donor, cr-~ar n gativ-~ra ~ lr~ All CT-lltE po~itiv~ -r~ r-act d on -~lot~ Ylth th r~ 3 b n~ only, vh r~ 11 the Cq~-~IF n~qativ er~ w~rn al-o; -~ t negativ~ for pgp3 Th ~ vor-- th~n ~ 1 by E~SA ag~ln~ t ~-WP3 bound 30 to NUNC microtit-r pl~ Two--fold ~crlal dilution- Or ach ~ru~ VQre t~t-ll, in tr~p~ n~pl-- OD ,~ w~r-t-k n ~rt~r 1 hr o~ colour ~ I2` and -~lot n~g~t$v- r~ ga~u ~~ t Iy low OD ~ g~
(below O l, ~e- Flg 4~ii)A~, wh~lr ~- th~ CF nd i -~lot-3S po-~tive ~ra g~v v~ri-bl- but con-i~t-ntly high r OD
readLslg- ~hich proportionally ~ ~ - ~ ~ith th~ ~-rum d l~ in th~ ~laDpl~ C ~ lng th- hlqh pr-val-nc~ Or antibody ag~ Ç.~ Lea in the llt61~L~, w~
RECTIFIED SHEET(RULE 91) ISAfEP

W095128487 21883 ~1~ r~ o al30 te-tad by pgp3-ELISA a group o~ l0 ~-ra Sron p~ti~nt~
with re~plratory ~ e vho ~er- Mrr-negative rOr C.L. ' -ti~, but h~t high ~IIF tlt~r~ (>~12) or n~iho~
gain~t C p -t~ ra gav~ p~p3-~rSA L~
~imilar to thos- obt~in~d Yith the hoalthy blood donor control ~Qra (Fig 4(il)~ W~ that cro~s ro~c~lon~
betWeQn pgp3 and nt~ c d~v~lop d ln ~. e to C~ r 1~ ,r ar~ not l~k-ly to occur 10 ~003-E:r TC~ 1 n~ of ~atl~nt - a In ordcr to ov~luato the pr-val-nc~ o~ anti-pgp3 nn~ or~
in individual- vho h~v d~Yolopod, or ~r- d-v~loping an lmmune r~ ~ to C.tL ' ~ 'ic lnrection, Ye studi~d thr~-lS groups Or p~t ianta Vith IIL . ~ ~ ital tract lnf 1~ t 1~
~ t : 46 f-mal- p~tient~l Yith ~ y confirn~d F-1r~itl~; 24 p~ti~nt~ Yith ~rariou~ condltion~ o~t~n a~ocl~tod to C L. ~ inf-ction~ (lower g nlt~l t~ct inf l ~ t~rillty); 40 c~ or ~alo non-20 'J.~. .0~ ur~thritl~ (NGU) All ~e ~ wer~ initially r- ~ ~, by ~ rlenc-d ~t~r~, ~t th~ N-JY~i~.;l1 Or origin by l~IF u~lng puri~led EB~ or C .~r~,ho~tis, and C.~ . Th- 40 NCU C~--J w r~ r~-r~~-~ ' hy ~F art~r th~ pgp3-lSLr6A t-~t~ Th- 70 ~m~l-5 ~er~, on ~ _Lio~ t th~ la~ , w~r~ al-o I wlth 1~ ially avall~bl~, con~ Datory t-~t, which Orrlcl ntly di-cr1"~1- ~ b tY On C ~r_ ' Lfs and C~p ~ ~ ~r~ ~ ~ ~ torrila Rt ~ np ~ r~ult~) 30 t`~n~ ing th F~ ty Or cro~ wit~
~ ;~ t E~ urrac~ q LES~ in dl~er-nt chla~ydial 5p~Ci~9 (1~ ra wh~ch gav a po~itiv~ llIF
r~action wlth C ~ t~ ut al~o h~d qu~l or hlgh-r tlt r8 aqnln~t C-l tr- I~B~ W~r- ~cor-d ~ccordln~ to th~
35 conr1 t~ t----t l ~, th~ --r~ o~ th$-- group which th--T - ' tel~t lndicat~d ~ o~itlv~ ror C l - 1 ~lt~ - but n-gativ~ ror C l~ , ~ r~
in thi~ ~tu~y a- givin~ r~ po~itiv CT-NIF r~ult-FlECTIFIED SHEET (F~ULE 91 ~SNEP

WO 95/28487 2 1 8 8 ~ io SS
All th~ 2~0v~ nera ~er~ ~nalyzed by pgp3-ErISA on duplicat~
~cts Or cix twofold d~ in P~S, ~o~ l 100 to 1 3,200 In g~n~ral th~ ~o-t infor~tlv (l e IYi~l di~r~r~nce betw~-n ampl~ ~nd n-gativ- control~ ~mpl~ t~ t ~ WQr~
S tho~ bctw~n l ~OO nnd 1 ~00 Do~/r~ curv~l~ on ~mi-logarithDliC plot~ v~r- u~-d to va~u~t- individual ~a~pl-~
R~ults w-rc ~ wlth tho~ f~ from po~$tive ~nd neg~tive control ~lera, w~lich w-r~ i LL~ CLI in ach xperl~ental se~uion r - ~ ~ally~ ~r~ wer~ ~cor-d ~ anti-10 pgp3 po~itiv~ ~hen the ELISA l~o~ r ~er~ c~c~ ntly~reat~r th~n tho~e of th~ neg~tiY~ rQf erenc~ ~ru~ ~or ~veral Dlatching di lur~^n Yalu ~ 5~ yi-lding OD Yalu~
consistently low~r th~n two- to Lh~, ~ol ~ th~ n~gati~re control- v~re ~ s neg~tive lS
Typic~l r-5ult- ~r~ ~ovn ln Fig 4~ii)C ~nd 4~ii)D A
~um~ry of CT-HIF and pgp3-Er~SA cor~ giv n in Figur 5 and Tabl~ I

RECTIFIED SHEET (RULE 91 j ISAIEP

Wo 9s/28487 ~ 1 8 ~ 3 ~ 6 ~ ' L_ TAi3LE I
No. ~%) Patient group No of pgp3-ELISA
5 and illness cases positive CT-MIF pO5. ~ STD patients PID 31 25t80.6 %) various 24 20 (83 . 3 %o NGU 13 10 (76 . 9 %) 15 CT-NIF neg., STD patients PID 15 8 (53 . 3 %) NGU 27 3(11.1 S) CT-MIF neg.
20 healthy subjects 50 o(o%) CT-MIF neg.
CPn-MIF positive 10 o (%) 0 2 5 Table Prevalence of pgp3-ELISA positive findings in diverse ~roups of human sera. As in Figure 5, PID re~ers to the s~lrintJitis cases. CPn: Chlamydia E~n~ t~-SUBSTITUTE StlEET (RULE 26) wo gs/28487 ~ 1 8 8 ~ 1 6 P~ o The avsi l::~h; 1 ity of a pgp3-specific ELISA allowed an A~ L of the prevalence of humoral anti-pgp3 ~ P~
in patients with symptoms of infection of the uro-genital tract. A total of 130 human sera were ~Y-s-m~nPd by pgp3-5 ELISA: for all of them ~JL-~Sel~C~ or absence of an humoral ,JOIIS~ to chlamydial surface Ant;7~ was A~ e~ by MIF.
For 81.5% of all sera PY:minPd, MIF and pgp3-ELISA agreed in detecting a positive or negative r~unse to their respective antigens tFigure 5, bottom panel).
Of 110 STD patient sera PY_m; ned, 68 were CT-MIF positive and 42 were CT-MIF negative; of these, 81S in the first group, and 26% in the second group were also pqp3-ELISA
positive. These overall results indicate that most patients 15 which are developing an immune Le~ ,.,GC to C. tr..~ 9ti~
surface antigens make also ant i ho~ against pgp3 ( p <
0 . 0005) -I~ the two group6 of STD patients with a relatively well 20 defined pathological condition (NGU and salpingitis) areevaluated, the results show some interesting variation.
The 40 male NGU cases (positive CT-MIF prevalence 32 . 5%) show an overall anti-pgp3 prevalence of 32.5%, which becomes 76.9% in the CT-NIF positive sub-group. CT-MIF and anti-pgp3 25 ELISA agree in giving a positive or negative response in 85%
of cases (75% for the 20 isolation-positive 6amples, and 95%
for the 20 isolatiu-- n~ya~ive 6amples). The statistical correlation between ~L~ e of anti-pgp3 antibodies and MIF
po6itivity in the NGU group is again significant (p <
30 0.0005) . For these patients cell culture isolation of viable chlamydia from urethral swabs was also performed and it may be interesting to note that Or the 13 ELISA-positive NGU
cases, 11 (84.6S) were also culture positive (p=0.007), whereas o~ the 27 ELISA negative cases 9 (33 . 3%) were 35 culture positive and 18 (66. 6%) were culture negative.
The group of 46 salpingitis cases (positive CT-MIF
prevalence 67.4%) show an anti-pgp3 antibody prevalence of 71.7%, which becomes 80.6% in the CT-MIF positive sub-group.
~ S~
_ _ _ _ _ wo g~l28487 ~ 1 ~ 8 3 1 6 1 ~ s lo For this group of sera the a~L~ --t between CT-MIF and anti-pgp3 ELISA in giving a positive or negative response was lower t69.6% of 5amples) than for the rest of sera, and the ~.~,E~ e of anti-pgp3 Ant;ho~lies did not correlate well 5 with MIF positivity (p=0.115).
Eleven (26%) of the 42 CT-MIF negative STD patients were positive for the pgp3-ELISA test: of these 8 (53.3~) 10 belonged to the salpingiti5 group and 3 (11.1%) to the NGU
group. Such antibody pattern could be given, in some cases, by differences in levels and persistence of anti-EB-surface versus anti-pgp3 antibodies. Alternatively pgp3-ELIsA could detect cross r~A~tir~nC with antibodies generated by 15 infection with microorganisms other than chlamydia or by other pathological conditions, like autoi ity The probability of obtaining positive pgp3-ELISA results due to specific ~L-~ss-ieaC~ nC needs to be evaluated in further studies on larger and well defined populations; however, t_e 20 results obtained with animal preimmune sera (not fihown) and with 60 CT-MIF negative human 5era (healthy, or non-STD
patient groups) indicate that pgp3-ELISA false positive results due to aspecific binding of normal serum are not likely to occur, 5ince none of these sera yielded a 25 positive r~ or De.
In contrast, thirteen (19%) of the 68 STD patient sera which were CT-MIF positive did not show ~ectAhle levels of anti-pgp3 IgGs. This group may comprise patients with a recent 30 first-time infection who have already developed an anti EB-surface reD~.-De but not yet a 5ignificant response to pgp3.
In fact, 2 sera from volunteers who submit to regular controls, and which were surely collected after a recent infection, belong to this group- Another po5sibility is that 35 some patients may not develop any anti-pgp3 Le~ e because of some peculiarity of their immune response to the chlamydial $nfection.
.

SUEST~ E SHEET (~ULE 26) WO 95/28487 2 ~ 8 ~ 3 1 ~
ss The purpose of the serological survey here presented is to show that sera of patients with a C. trachomatis-related disease reco~ni7~ pgp3; however, further epid~iQIogical studies may better asses5 the frequence and significance of 5 such antibody pattern5. Al80, est~h~ i Ch~d animal models of chlamydial infection could be usefully employed to assesE~
the relative timing of appearance of anti-pgp3 and anti-EB
surf ace antibodies .
10 The function and role of pgp3 in the chlamydial cell and life cycle are still unknown; however, since chlamydia induced disea5e is thought to be largely determined by the host im~une r~cprmC~C to the infection, the data here reported show that pgp3 is one of several molecules which 15 are potentially i L~L for the pathogenicity of chlamydial infections of the urogenital tract.
It will be understood that the invention is described above by way of example only and variations are possible within 20 the spirit and scope of the invention.

SL~STITUTE SHEEl (RULE 26) Wo 95~28487 ~ 1 8 8 31 ~

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SIJBSTITUTE SHEET (RULE 26)

Claims (18)

Claims

1. Chlamydia trachomatis pgp3 protein, or a derivative thereof, which has the same immunological properties as native pgp3.

2. A protein derivative according to claim 1, wherein said immnuological properties include antigenicity.

3. Chlamydia trachomatis pgp3 protein, or a derivative or fragment thereof, which possesses one or more of the conformational epitopes present in native pgp3.

4. Protein obtainable by a process comprising:
- transforming a bacterial cell with a vector encoding a protein according to any one of the previous claims;
- culturing the cell under conditions such that said protein is expressed;
- isolating the periplasmic fraction of the resulting cells;
- dialysing said fraction against piperazine-HCl buffer, pH 5.4;
- loading the dialysed solution through an ion-exchange column;
- eluting the column with a NaCl gradient;
- collecting the pgp3-containing fractions.
- dialysing pgp3 back into a physiological buffer solution.

5. An immunodiagnostic assay comprising at least one step involving as at least one binding partner a recombinant protein according to any one of the previous claims, optionally labelled or coupled to a solid support.

6. An immunodiagnostic assay according to claim 5, wherein the assay is ELISA.

7. An immunodiagnostic kit for performing an assay according to claim 5 or 6, comprising at least one protein according to any one of claims 1 to 4.

8. A vector comprising a polynucleotide encoding a protein according to any one of claims 1 to 4.

9. A vector according to claim 8, comprising the ORF3 gene of any Chlamydia trachomatis serotype variant under IPTG-inducible expression control.

10. A host cell transformed with a vector according to claim 8 or 9.

11. A host cell according to claim 10, wherein the host cell is E. coli.

12. A method for the production of a protein according to any one of claims 1 to 4, comprising culturing a host cell according to claim 10 or 11 and isolating the protein.

13. A method according to claim 12, wherein the isolation includes one or more purification steps under non-denaturing conditions.

14. A method according to claim 12 or 13, wherein the isolation includes the step of ion-exchange chromatography.

15. A method according to any one of claims 12-14, wherein the isolation includes the step of preparing a periplasmic extract.

16. A vaccine or therapeutic composition comprising a protein according to any one of claims 1 to 4 and a pharmaceutical carrier.

17. A method of treatment of the human or animal body comprising administering an effective amount of a vaccine or therapeutic comosition accoridng to claim 16 to prevent infection by Chlamydia trachomatis or to treat such an infection.

18. A recombinant protein according to any one of claims 1 to 4 for use in the manufacture of a medicament for vaccinating against Chlamydia trachomatis infection or treating such an infection.