CA2069106A1 - Chimeric proteins - Google Patents

Abstract

A chimeric peptide is provided which includes at least a portion of the B subunit of cholera toxin and an epitope region of a desired antigen fused to the N-terminal end of the B subunit of cholera toxin. The epitope region includes an antigenic determinant of the desired peptide. According to certain preferred embodiments, such chimeric peptides are used as a vaccine to elicit an immune response in a subject to a desired antigen. Recombinant-DNA
mediated methods for the production of a chimeric peptide, DNA
sequences, vectors and host organisms used in such methods are also provided.

Description

WO91/07979 PCT/US~/~

t - 1 _ 26~06 DescriDtion CHIMERIC PROTEINS

BACKGROUND OF THE INVENTION
Technical Field The muco~al surfaces of the gut and oral cavity are constantly expo~ed to a large number of organisms which include pathogenic bacteria, fungi, viruses, and protozoa Many of these pathogens use the mucosa as the principal portal of entry into the host, whereupon the systemic immune ~y~tem eliminates the agent Other pathogenq do not have to invAde ths mucos~ in order to be pAthogenic, snd ~ystemic immunity i8 ineffective in these cases The mucosal-as~oci_ted lymphoid tissue (MALT) probAbly developed in response to the noed to protect the exterior ~uxface~ of the host The concept of a common mucosal immune system i5 rel_tively new (McGhee And Michalek, 1981) The predominant form of immunity at tho~e ~ite~ is tho relea-e of dimeric ~-cretory IgA (sIgA) E~skoround Art S-cr-tory IgA can pr-v nt adh-renc- of pathog-ns to the muco~, n-utrall~- vlru~ nd lnactivat- toxln~ (Tom~
1984) Inductlon of ~n lmmun- r-~pon-e in the gut confers ~-cr tory immunlty at dl~tant ~lte~ of tho muco~a ~uch a~ the lung~, or~l cavlty, and th- genitourlnary tract Antigen-prl~ d ly~phocyt-~ ar- dl-~ d n~ted from th- gut by the lymph~tlc- to th--- ~lt-- wh-r- they matur- lnto ~unctlonal f~-ctor c~ McGh ~nd Mlch~l-k, 198~) ~
AttemptJ to tlmul_te MALT by oral lmmunlzAtlon have h~d l~lt-d UCC-J-. Thl- h_~ be-n _ttribut d, ln part, to th- lnductlon of tol-ranc-, a stat- of ~p clflc immunologlc unr ~pon-lv-n -- that dev-lop~ upon xpoJur to _n ~ntlgen Th- d-v lopm nt of tol-rance i~ dependent upon the ~ntlgen, do-age, l-ngth of expo-ure, route of lmmunlz~tion, ~nd genotic background of the ho~t (Webb and Winkel~tein, 1984) SU~TITIITF ~F~
,, ., ~ . . .. : . . . ", , .. .. . . . ~ .. . .. .. . . . . .. . .

... .... .. . . ... . ...... . . .
- ~ , - , ;, j; ; ... ,: .. , ,, -, . -~. ~ . . -Wo9l/07979 PCT/US~

3¢
A condition of oral tolerance develops to many antigens pre~ented to MAL~ (challacombe and Tomasi, 1980; Nossal, 1983) Thi~ phenomenon is believed to be a natural mechanism for preventing the immune system from respondinq to the large load of innocuous antigens, from food and normal microbial flora, that it i~ exposed to daily It was previously thought that a sIgA response could occur in the absence of systemic immunity, and this was ob~erved with certain antigens (Suzuki et al, 1986) However, a recent study indicated that keyhole limpet hemoeyanin (RLH), an antigen that cau~es ~ystemic tolerance, also induce~ tolerance in the secretory compartment (Elson and Ealding, 1984b) Other data has shown that the sIgA and IgG respon~es are under coordinate genetic control, and that the Peyer's patches contain both IgG and IgA producing B
cells (Elson and Ealding, 1984a) $hus, most orally administered antigenQ probably induce tolerance in both the seeretory and systemic immune compartments The development of oral tolerance appears to be under the regulatory control of suppressor and contrasuppressor cells (Green et al , 1982;
R~ehm~n et al, 1978) Although most antigen- induee tol-r~ne- in th-~- e~ , th-re ~re x~mple- of oral lmmunlz-tlon eonf-rrlng Jolid immunity to p~thog-n- Jueh as pollov~ru~ (Tom~, 1984) and Vibrlo ehol-r~e ~Lycke and Holmgr-n, 1987) C-rt~n adv-r-e health condltlon- require treatment or would b- ~ld d by a -er-tory immune r- pon-- Att~mpts to m~k- or~l ~ubun~t v~eeln-- h~ve ~ t with limited uee---, how v-r, due m~lnly to the f~lure of m~ny proteln ~ntlgens pr-~-nt d by thl~ rout- to timulat- ~n ad-guate antlbody r-~pon-- (Ch~llaeomk- ~nd Toma-i, 1980; Crabb- et ~1, 1969;
Pi-re- ~nd Gowan, 1975) Cholera toxin (CT) i~ unu~ual b-e~u~- lt i- on- of the few protein~ that llelt a ~trong immun- r--pon-- wh-n glven or~Ily (Pieree and Gowan, 1975) Wh-n eo-f-d, CT ha~ b-en demonstrated to act a~ an ad~uv~nt for oth-r protein~ whieh normally are not immunogenic when given orally (Lycke and Holmgren, 1986; Liang et al, 1988; - -S"~ST~TUTE SHEET
. .. . ~ ' ,, , . . ,.~ . .
~ ;,: . ,. . ,. ., .-- - ... ~, .. ..

, w09l/07979 PCT/US~/~
~ ~ 3 ~ 2Q~91Q~

Nedrud et al, 1987) In fact, Elson and Ealding (1984b) were able to abrogate tolerance to keyhole limpet hemocyanin (KLH) ln mice th~t were fed this antigen mixed with CT This resulted ln the production of both sIgA and serum IgG to the antigen The role of CT in these cases appears to be greater than simply increaqing the immunogenic mass, as most carrier proteins do, but the mechani~m of this action is unknown CT is produced by the enteric pathogen Vibrio cholerae and is composed of two subunits The toxinogenic A subunit i8 responsible for the ADP-ribosylation of eucaryotic adenylate cyclase This modification increases cAMP
activity, resulting in the diarrhea and fluid 1089 observed in patients with cholers ~Betley et al, 1986) The B ~ubunit of cholera toxin (C~8) is composed of five identical subunits of 11 6 kD~l ~oined together in a noncovalent association (Gill, 1976) The CTB pentamer binds to the monosialoganglioslde GNl (Cuatrecasas, 1973), which appears to be the nAtural ligand of the protein This ganglioside is found in abundance on the surface of inte~tinal epithelial cell~ and probably facilitates entry of the A subunit into the coll Although CTB 1- non-toxic, it i~ ~till immunog-nic when glv-n orally ~Ly¢~- and ~olmgr n, 198~) ~hi- haJ ncouraged ~tt-mpt~ to u~- CT~, ln~t-ad of th- holotoxin, a- a carrier for ch mically cr at-d muco-al vaccin-- B~ n and Fl~ch-tti (1988) hav- lnduced prot-ctiv ~munity in mice that wer lntrana-ally immuniz d with an pitop from ~tr ptococcal M prot-in con~ugat-d to C~B Som tudi-- have ugg--t d that CTB may act a~ more than ~ t ~ impl- carrier for antigen- (Mc~-nzi- and Hal-ey, 1984; Tamura t al, 198~) Mor-ovor, lt ~ay b- po--ibl- to incr-a-e th- i~munoqenicity of th- antig-n ~-n further by con~ugatlng lt to C~B In a tudy by McX nzl- and ~al--y (1984), glutaraldehyd- wa- u--d to coval-ntly coupl- C~B to th- oral tol-raq-n hor--radi~h p roxlda-- (HRP) When thi~ con~uQat- wa~ fed to mic-, ~IgA
waJ pre~ent to both antigen~ at levels ~ignificantly greater than that found from f-eding the two proteins a~ a mixture -SUBSTITUTE SHE--.. . ` . .
` . ` - `.. : , ., `~.` - ~ , , ` ` ` `..... ` . . `...... . .. .

W091/07979 PCT/US~/~
2 G ~ 4 ~ ~'`

This in contrast, however, to the results repcrted by others (Lycke and Holmgren, 1986) Liang et al (1988) were unable to demonstrate an ad~uvant effect for CTB when conjugated to Send~1 v~rus There have also been reports of attempts to use gene fusion a~ an alternative to chemical conjugation In such report~, gene fusion vectors have been reported which produce a chimeric protein including a large portion of the glucosyltransferase B enzyme (GtfB) produced by the cariogenic bacterium Stre~tococu~ mutans and CTB (Dertzbaugh and Macrina, 1987) That chimera, however, appeared to change the structure of CTB because the ability of CTB to bind to mucosal monosialoganglioside GMl, was adversely affected Thus, the~e constructs were not suitable for use as a vaccino Dental caries commonly occurs in people who con~ume sucrose as a part of their diet Although the incidence of d-ntal caries has been reduced in the general population, it ~ -i- still a prevalent diseaJe of man For example, over 24 billion dollars W~8 spent in the United States in 1984 to r palr cariou- le~ions (Loesche, 1986) Bett-r oral hyqiene has r duc d th- ~-verity of the di~-as-, but hygione ~lone cannot limlnat- th- dl~-a~- Th- ~tructur- of the tooth ~urf-c- cont-ln~ fi-~ur-- whor- S mutans i- more likely to CCu~ul~t- ~nd cau~- carl--, v-n wlth good hygien-Furth-r~or , proper m chanical debridoment of the teeth is rath-r l~bor-int-n-ive and, consequently, many peopl- do not cl-an th lr t--th ad quat-ly Currently, th- only r-cour-e to th- probl m Or d-ntal cari-~ is to r-~lr ~h- damag- ait-r lt ha~ occ~rr d A mor- attractiv- approach i- to pr-v-nt carlou~ lon- from d-v loping A way in which to achieve thl- 1- to pr-v nt colonization of the tooth Jurface by S mutan-On- m thod for pr-v-nting colonization of the tooth u~face 1~ to vacc~at- again~t S mutan~ Several studies hav- been performed uslng different co~ponent~ of S mutans as a vaccine The early vaccines were composed of whole SUBSTITUTE S~!EE~

,~
;
. .' ` ,` ~ :.,; .. :` .. , .. : .

WO91/07979 PCT/US~/~

,...
2069~Q~
cells, and these were found to reduce colonization and caries formation in some cases (Gregory and Filler, 1987; Michalek et al, 1983; Russell et al, 1980) However, surface antigens of S mutanq have been found to cross-react with human heart tis~ue, snd this has precluded the use of whole cells vaccLnes for safety reasons (Ayakawa et al, 1985; Ayakawa et al, 1988; Hughes et al, 1980) For this reason, recent efforts have been directed towards the development of subunit vsccines using purified components of S mutans At present, two components of S mutans hsve been identified ss the best candidates for a subunit vsccine A
component of the cell wall of S mutans has been independently identified by two group~ and designated either antigen I/II (Ru~ell and Lehner, 1978) or SpaA (~olt et al, 1982) Vaccination with thi~ purified protein has been ~hown to reduce colonization and dental caries in monkeys (Lehner et al, 1980; Lehner et al, 1981), and it doe~ not appear to induce heart cros~-reactive antibodies (Bergmeier and Lehner, 1983) The antibody response elicit was reported to be predominately ~erum IgG, which reaches the oral cavity via l-akage into the gingival crevicular fluid How v-r, lmmunization~ w ro p-r~orm d u~ing ad~uvant- which m~y not be practical for hu~an u~- Furthermor , par-nt-ral lmmunl~tlon wlth ~ntlg-n I/II did not induc- ~ignificant ~gA r-~pon~-~ ln th~ nim~l- Thi- w~- not ~urprising, con~ld-rlng th~t -cretory immunity requlres local tlmulation in ordor to d-v-lop Anoth r group of proteins that h~v be-n ~luated for u~e in ubunit vaccine- are gluco-yltr~n~-r~ Th-Je enzym - c~lyze-th- formatlon of glucanJ ~rom ~ucro--, ~nd the-e polymer- medl~t- the ~dh-r-nce of S mutans to the tooth surfac- Enzyme pr-par~tion~ have been used to immunize rodents both p rorally ~Smlth et al, 1978; Smith et al, 1979) ~nd par-nt-rally (Bahn et al, 1977) Vaccination with th--e protelns ha~ also prev-nted colonization and caries formation in th-se animals The advantage of oral immunization W85 demonstrated by these studies, becsu~e both sIgA and serum SUBSTITUTE SHEET
.. . . . .. .. .. ..

.

WO 91 /07979 PCl /US90/0681 1 ~i - 6 - ~' Q
.

I~G were elicited to glucosyltransferase This vaccination route confers both systemic and secretory antibodies which both can play a role in host defense again~t S mutans However, these studies demonstrated the current problem with oral vaccines to S mutans, specifically that in order to induce and maintain high levels of antibody, the animals had to be immunized repeatedly, over a long period of time In general, these experiments have demonstrated that oral vaccination with soluble antigens of S mutans may be an effective method of preventing colonization and caries ~ -formation However, the vaccines u~ed required repeated booster immunizations in order to maintain protective levels of antibody, due to the unique nature of the secretory immune Jy~tem~ The use of an oral ad~uvant may improve the immune response to these vaccine components The present inventors have surprisingly found that peptide sequences composed of only an epitope when fused to the N-terminal end of CTB elicit an antibody response, giving riJ- to a Ju~talned anti~ody response with antibodles dlrected against the entire protein of interest For example, a lS amlno acld epltope of GtfB fu~ed to CT~ glves rl~- to antlbodl-~ again-t the GtfB protein whlch i8 over 1000 ~mlno acldJ ln compo~ltlon ~9l9~Y~ of tho Inventlon lt 1~ an ob~-ct o~ th- pre-ent lnvention to provide an oral vacoln- capabl- of elicltlng both a Jecretory immune r--p~on-- ~nd a Jy-t mic immune reJpon--, thereby cauJlng a ho~t to produc- JIgA and IgG
Anoth-r ob~-ct of th- pr-J-nt inv-~tfon i~ to provide chim ric p ptid-- not toxic to a ho~t which can be produced by r-comblnant DNA m thods Y-t anoth-r ob~-ct 1~ to provide a univerJal vaccine carrl-r that can b fuJ d to form a chimeric p ptlde with any numb-r of antig-n- or pltopes to elicit a ~-cr-tory immune r--ponJ- to that ~pecific antigen or epitope wh-n the chimeric peptide i~ admini~tered orally to a host SUB~T~TUTF ~FF~
-- : . - . . . - ; ` . . - . . . . . . - . .
~"` ' ,' ,' `' ` ' , . ' .~'., ,' ' ,, ' ' . '. ., '., : ' . . , , . , . - . ~ .
' `' , -. ' '. " - ', ,- ' ' , - " ' - ' " '' -' ', . ' " . ' ' '~' ' ' " ' ' / ' ' ' ' ' : ' '. ' . ~ "
''."~'`` ' - ' '. ; . '.. .' ' ' . - ' ' . '" ". . ' . . ' -: - ' . , . . ~ '' ' - ' ''`"" '' '`' '. '' '- "~ ' .''. '-'. '' ''', ;'' ' ' '' '.~ ' ' ' '- ' . '', ,''' ' ' '. , ' '' W091/07979 PCT/US~
- 7 _ It is still another object to provide the recombinant methods, vectors and host organisms that produce peptides for effective oral vaccine compositions The~e ob~ects and other objects are achieved by provid~ng a chimeric peptide including the B subunit of cholera toxin and an epitope region of a desired antigen fused to the N-terminal end of the B subunit of cholera toxin The epitope region includes an antigenic determinant of the desired peptide Such chimeric peptides can be used for an oral vaccine according to certain preferred embodiments in which the epitope fused to the B subunit of cholera toxin gi~eq rise to an immune response, producing antibodies directed against the peptide of interest Rocomblnant methods for producing a chimeric peptide, and the DNA ~equences, v~ctors and hosts used in such recombinant method~ are also provided According to other advantageous feature~ according to certaln preferred embodiment~ of the present invention, an epitope of the GtfB peptide i8 fused to the CT8 peptide to provide an or~l vaccine for the prevention of dental caries It i- to b- under-tood that both the for going general d-~cription ~nd th- followlng det~iled deJcription ~re x-mplary ~nd xpl~n~tory only ~nd ~r- no~ re~trictlv- of the lnv-ntion, ~- cl~lm d Th- ~ccompanying dr~wing~, which are lncorpor-t d in ~nd con-titut- a part of the pecification, llluJtr~t- varlou- mbodiments of the inv ntion and, together with th- d--crlption, ervo to oxplain th- prlncipl-- of the inv-ntlon B~~ cription of th- D~a'wino-Fig 1 Con~truction of th- ctxB fu-ion vectors Pla-mid pVA1662 which contalns th- ctxB gon- ~sh~ded ~rea) wa- dig--t d wlth ~coRI and NdeI to del-te the 5~ end of the g-n- Th-n ~ynth-tlc llnkers with comp~tible ndJ were ln--rt-d ~nd llg~t d to create the fusion vectors hown Fig 2 Construction of pVA1555 Plasmid pHR7, whlch contains a 1 9 kb insert encoding a truncated portion of the gtfB gene of Streptococcu~ mutans (stippled area), was SUBSTI~UsE~ SH'~T

w091/07979 PCT/US~/~

~9~Q6 - 8 - ~

inserted into pVA1542 as a PstI-EcoRI fragment upstream of the ctxB gene (shaded area) The resulting pla~mid, pVAl555, expres~eq a gtfB ctxB fusion protein of 58 kDal in E coli Flg 3 Construction of pVAl782 Plasmid pVAl542 contained a promoterle~s version of the ctxB gene of Vibrio cholerae (qtippled area) that lacked DNA encoding the first 17 amino acids of the leader ~equence of the protein The synthetic oligonucleotide encoding gtfB l (filled area) was in~erted into pVAl542 as an EcoRI-NdeI fragment to create pVAl599 The resulting ~tfB l ctxB gene fusion could be inserted easily into a number of expression vectors as an EeoRI-BamHI fragment, if desired The seeretion vector pINIII om~A2 contained DNA encoding the leader -qequence of ompA (striped area)~ an outer membrane protein of E coli, under eontrol of the indueible lae promoter Promoter aetivity is inhibited by repre~sor, the product of the laei gene, until induetion by IPTG Insertion of the atf~ l ctxB
gene into this veetor resulted in expression of a ehimeric protein of 14 4 kDal upon induction with IPTG
Fig ~ Linker sequeneeJ of the fusion veetors These oligonueleotid-s were used as a tran-lational ~unetion b-tw -n otfB and ctxB The number abov- eaeh link-r d-slgnat-s th- plasmld v-etor containing ~h- J-quenee ~old-fae-d l-tt-rJ eorr-spond to ~equenees aetually eneodod by the ynth-ele l$nk-rs Important re-trietion endonuelease ~ites ar- shown abov- aeh linker; amino aeid seguenees are loeated b-low ach linker Flg 5 The sequ-nce of the gtfB l p-ptid- The hydrophlllcity plot is shown for a portiohrof ~he glucosyltransf-rase B enzyme of the cariogenic baeterlum ~ oseu~ mutans The region eneod~ng am~no aeid r-sidu-s 345-359, eorresponding to peptide gtf~ l, is hown in bl~ek The gtfB l peptide (qhaded area) wa- inserted b-twn-n the leader sequenee for the Qm~A gene of E eoli and th- B sulDunit gene of eholera toxin (etxB) The eleavage site for omDA is indicated by the arrow The numbers above the amino acid sequence correspond to the residue nu~ber of SUBSTITUTE S~lEEt - . , ~ . . ~ . . .
, . . . ... . .

, ` ` ` `-. . ` ` ` , . ... - - - ,` .`
. ,.. . -.
. ; . . . .

WO 91/079J9 PCI`/US90/06811 ~rr, _ 9 . .
2~910~

the mature, secreted form of the chimeric protsin The corresponding DNA sequence is listed above, along with important restriction sites Bold letters indicate the sequence encoded by the synthetic oligonucleotide used to construct the gene fusion Fig 6 Analysis of CTB and the chimera by circular dichroism Mean residue ellipticity (M R E ) is expressed in deg cm2/mole The minima for the oligomeric form of the chimera (dotted line) was shifted relative to native CTB
(solid line), indicating some differences However, the spectra of the monomer (dashed line) was significantly different from both the oligomer and CTB
Fig 7 Immunogenicity of the chimera Protein s~mples were fr~ctionated by 10~ SDS-PAGE and electrophoretically transferred to nitrocellulose sheets Replicate blots were probed with antiserum to either CT~, GtfB, or chimera Arrows denote immunoreactive protein~ of interest Lane 1, extracellular proteins form S mutans GS-5; V1792 chimera; lane 3, CTB
Fig 8 Rinetics of enzyme inhibition Total glucan ~yntheJis wa~ mea~ur~d over time (in hourJ) The enzyme w~ pr-incubat-d in either P~S (control), normal r~bbit ~-rum ~N R S ), anti-Gtf~ J-rum, or anti-Chim ra serum b-~or- ~ddition to th- r-action mixture All ampling wa~
p-rform-d ln trlpllcat-Flg 9 Dlfferential inhibition of qluc~nsynth~ Prot-in ~mple- preincubat-d in either P~S
(~ontrol)~ anti-Gtf~, or ~nti-chimera Jerum w ro ~-ayed for diff-r-ntial gluc~n ynth--i- The r ~c~lon~ proce d d for 8 h at 17C before m ~-uring activity The ample~ w r-~--ay d ln triplicate for both total gluc~n~ and water-in~olubl- glucan~ Total glucans were precipitated with m-th~nol Wat-r-inJolubl- glucan~ were pr cipitated wlth d-ioni~-d w~t-r The amount of water-Joluble qlucanJ
produc-d w~ d-t-rmined by subtracting in~oluble glucans from total glucans The activity of the samples in each as~ay was SUBSTITUTE SH~:T
.. . . . . . . . .. . .
` . ~

` . . . . - : . - : . ~ -`.-. . . . ...

wog1/07979 PCT/US~

~a~9~
determined relative to the control The activity of the control was normalized to 100~
F~g 10 Inhibition of fructan synthesis Extracellular protein from S mutans GS-5 was preincubated for 1 h at 37C in either PBS (control), normal rabbit serum (N R S ), anti-GtfB serum, or anti-chimera serum The ~amples were incubated in substrate containing 14C-(fru)-sucrose for 8 h at 37C Total fructan synthesis was measured by precipitating the polymer in methanol - -Pig llA The DNA sequence encoding the GtfB l/CTB
chimeric peptide Fig llB The amino acid seguence of the GtfB l/CTB chimeric peptide Best Mode for CarrYino Out the Invention Reference will now be made in detail to the pre~ently preferred embodiments of the invention, which, together with the following examples, ~erve to explain the principleQ of the invention Amino acid abbreviations used herein are custom_ry and are defined, for example in Stryer, Biochemistry, ~econd edition, W H Freem_n and Company (1981) Vaccine- encompa~ed by the pre~ent invention can be u-ed for _ny living org_nl-m, _nd the term ~-ub~ect" is used to d-fln- any hum~n or non-hum4n _nlm~l All r-ferenceJ
clt-d h-r-ln _r- h-r-by lncorporat d by r-~er-nc-Th- pr-~-nt ~nv-ntion ~how th_t, u-lng recomblnant DNA
t-ohnlqu--, pl_-mld- c_n b- conJtructed that c_n be tr_n~-ct-d into ho-ts ~nd chimeric, immunogenically active p ptld-- _r xpr--s d whlch includ- _n opltop~ of the p ptld- o~ lnter-~t ~u- d to the N-termin_l nd of CTB Th-r-l_tiv small slz- of th- pitop- _mlno~cld -qu-nc- ~u-ed to th- CTB r duces the ch_nca that th- structure or function of CTB wlll b- _lt-r d La-ge peptide- fu-ed to CTB are more llk-ly to _dv-r--ly _ffect the b~ndlng of CTB to muco-_l tl~-u- Such _lt-r_tlon~ adverJely _ff-ct the ability of the chlm rlc CTB p ptid- to olicit a ~ecretory immune re~pon~e The lnv ntors have urpris~ngly shown that peptides as ~mall an epitope fu~od to CTB elicits the desired immune response.

SUBSTITUT~ S~ E.

WO9l/07s7s PCT/US~

2~691Q~

As defined in the present invention, an ~pitope should have the following characteristics An epitope is defined as ~n antLgenic determinant or that portion of an antigenic molecule th~t Lnteracts by molecular complementarity with the ~nt~gen combining site of an immunoglobulin molecule An epitope is a discrete portion of an entire antigen molecule which can trigger the immune response As defined in the present invention, the epitoFe need not actually trigger an immune response by itself, but includes the particular region of the antigen which brings about the proper interaction to raiqe antibodies agsinst that antigen when the epitope is fused to CTB Moreover, the epitope, according to the present invention, should have little te no impact on CTB
interaction with mucosAl tissue when the epitope is fu~ed to the CTB ~n order to ensure that the epitope according to the present invention does not interfere with CT~
interaction, the epitope should be no greater than 100 amlno acids long and no greater than 11 kDal in size According to c-rt~in preferred embodiments, the epitope~ according to the pre~ent invention ~re in the range of about 10 to 30 amino ~cids long, or 15-20 ~mino acids long Th-re ~re ~tand~rd procedur-8 that on- of ordinary ~klll in th- ~rt know~ how to mploy to loc~te the epitopes o~ ~n ~ntlg-n mol-cul- Epltop-~ or ~ntlbody binding sites o~ prot-ln~ ha~ b--n correlated with region~ of hydrophlllclty (Hopp and Woods, 1981; Lerner, 1982; and T~mura, 1983) and with high J-gmental mobility region- or r~ndom coll r-glon- (G-y-en t al , 1987; and W--thoff, ~984) ~lgh ~egmnntal mobility iJ charac~ riz d by ~ peptide d-void of a highly organlzed ~-condary Jtructure Do~ain~
wlth th--- prop rti-- would probably be located on the urf~c- of th- prot-in, wher- thoy would be acceJsible to an antibody, and would h~ve a highly m411-able -condary ~tructur-, thu~ allowing them to conform to a pre-exi~ting antlbody ~p-ciflcity Bpitopes possess these predicted characteristics SUBSTIT~Te S~E~
~, . ~ . ............. . .. ..
.. . . .. . , . . . . .. . - . . . .. ... . . .

~ . ... .
- . . ..

W09l/07979 PCTtUS~/~
~ Q~ 12 - ~

By synthesizing an oligonucleotide corresponding to a domain of a protein with these properties, an antibody binding sLte or epitope can be identified Available ~lgorithms, such as those included in a software program known as MICROGENIE, from BECRMAN INSTRUMENTS, Palo Alto, California, may assist in finding epitope regions First, the hydrophilicity of regions of a protein can be determi~sd using the algorithm of Hopp and Woods (1983) Seconda~-structure predictions of this region can also be made us_ the algorithm of Garnier et al (1978) Domains that are identified which have both high segmental mobility and hydrophilicity can then be screened to ensure that the~e regions compri~e an antigenic determinant or epitope By following the methods of the pre~ent invention, such regions can be screened by fractionating proteins into peptide~, isolating the peptides containing the putative epitope of interest, and determining it~ reactivity with antisera to the protein of interest Alternatively, expre-sion vectors could be prepared that produce chimeric peptide , including the rogion~ to be screened fused to CTB, and testing the chimeric p-ptid-~ to determine if ~uch peptide~ rai~e antibodies dLr-ctod aga~n~t th- prot-in of int-r--t Th- ~ollowing 1- a non--xhau-tl~- li-t of cert~in p-ptld-~ whlch could b- u--d for fu-lon to CTB for u~o as an oral vaccln- Th- ll-t d p ptld-- ar- known to elicit irmune r--pon--- that r duce and/or eliminato the severity of the r--p ct~v dl--a--Such p ptld-- could be fused to the CTB a- de-cribed ln the pr---nt appllcatlon to form an oral vaccl~ -Residu-~ 141-158 and 200-213 of viru- coat protein I
(VPI) form th- ~aufbeuren strain of Foot and Mouth Disea~e Vlru-, a dl~-a-- of cattle ~D~Marchi, et al , Scienco 232s63~-641, 1986) ~he con~erved region of the M Protein of group A ~tr~ptococci ~re~ldue~ 216-235, 248-269, 275-284), r--pon-lbl- for phary~ngiti~ ~BeJJen and Pischetti, Infect Immun 56 2666-2672, 1988) The conserved tetrapeptide region (Asn-Ala-Asn-Pro) of the circumsporozoite protein of SUBS~U I E S~
.. ...

` .: , .. : ; ` `
., ~ . . . . ,.. . . , , . `
., .` ~ ~ , . . . .
.. ' .
.. . . .. . . . .

WO91/0797~ PCT/US~/~

2 ~ Q ~

Plasmodium falciParum, the causative agent of ~alaria (Que, et al , Infect Immun 56 2645-2649, 1988) Subunits of gonadotropin for u~e as an anti-fertility vaccine ~Alam, et al , Vaccine 7 129-131, 1989) Synthetic peptide encoding entire preS2 region of hepatitis B virus surface protein (Emini, et al , J Med Virol 28 7-12, 1989) The M genome segment of Rift Valley Fever virus (Schmaljohn et al , Virol 170 184-192) The pilin subunit of HaemoDhilus influenza (Brinton, et al , Pediatrc Infec Dis 8 (Suppl) S51-61 Fimbrial subunit of enterotoxic colibacillosis in neonatal piglets (Greenwood, et al , Vaccine 6 389-92, 1988) Active site residues of Pseudomonas aeruainosa exotoxin A (Lukac, et ~1 , Infect Immun 56 3095-3098, 1988) Peptides corresponding to the cleavage region of VP3 protein of rot~virus which induce neutralizing antlbodies (Stredkert et ~1 , J Virol 62 4265-4269, 1988) Another nonexh~ustive list below includes further antigens which include epitopes which c~n be fused to CTB to produce oral v~ccines Although epitopes of the following llJt h~ve not been characterizod, one of ordinary ~kill in the art hould be able to determine thoJe regions uslng the m thods d--crlb d h-r-in Those methods include, but are not llmlt-d to, th- u-- of ~lqorlthms to locat~ ar-as which are hydrophlllc and whlch hav- h~gh -gm ntal mobility, preparing chlm-rlc prot-lnJ wlth pla-mid- accordlng to the present lnv-ntlon and s~cr-enlng tho-e chlmeric protelns~ for their ablllty to llclt an immune respon-e Altern~tively, peptide fr~m nt~ ld-ntlfi-d by the algorlthm m thod can be te-t-d for cro-~-r actlon wlth antlJ-ra to the pfot-in of lntereJt to ~cr-en for approprlat- epltopeJ for use ln the pr-sent chim rlc p-ptlde, oral vaccines Dlphth-ria toxln Qubunits A (toxin subunlt) and/or B
(bindlng subunlt) from Cor~nebacterium diohtheriae Gr-enfi-ld t al , Proc , Natl Acad Sci 80s6853-6857 ~1983) P-rtus~is toxin su~units A (toxln subunit) and/or B
~blnding Qubunit) from Bordetella ~ertussis, the causative agent of whooping cough Black et al , Science 240 656-659 SUBSTITUTE SHEE~

. i . . .. ` ' , . ~. ` ` , , `, .`
- . ` ` . `- . . .
-.. . . ..

.. . ,` . . . . - , . . ~ .- -- ` ., `

wo91/o797s PCT/US~/~

2P~

(1988) spaA, a surface protein antigen from Stre~tococcus mutans, the causative agent of dental caries gtfC, the glucosyltransferase C enzyme produced by StreDtococcus mutans, the CAu~ative agent of dental caries ftf, the fructosyltransferase enzyme produced by Streetococcus mutans, the c~usative agent of dental caries Fimbrial ~ubunit (an adherence protein) of Bacteroides ain~ivalis, a bacterium associated with periodontitis Type I fimbrial subunit (an adherence protein) of StreDtOCOCCUs sanauis, a bacterium involved in early stages of dental plaque formation Tetanus toxin of Clostridium tetani, the causative agent of tetanus Invasin protein of Yersina ~seudotuberculosis The hemagglutinin (HA) antigen of influenza virus, the causative agent of flu, T~mura et al , Vaccine 6s409-413 (1988) The pilin ~ubunit of ~ac~eroides nodosus, the causative agent of foot rot of sheep Surface antigens of human papilloma virus, the causative agent of genital warts Envelope proteins of herpes simplex virus Tumor-associated antigens in melanoma~ and c~rcinomas The serum and pilin opacity proteins of ~serrip ~onorrh~eae, the cau~ative agent of gonorrhoa Surf~ce prot-lns~ of Gi~ida ~mkl~a, a c~u~ative ag-nt of dlarrhe~ ln man Dongue 1 vlru~ tructural prot-ina, Bray t al , V~rol 63 2853-28S6 (1988) F protein o~ rlnd-rp ~t v~ru~, Barr-tt et al , Vlrol 170~184-192 ~1989) P28 ~ntlq-n o~ Sch~L~a~ aQ~, Wolowazuk et al , J lm~unol, 142~1342-1350 (1989) Avlan influenza virus h~m4gglutlnln antlgen Merozolte antlg-n of Eimoria ~ L~nLLLDl~ th- cau~atlv- agent of coccldlo-1~ ln chlcken~, Xim t al , Infect Immun 57s2434-40 (r9~9) ^
Th- utlllty of the fu-ion v-ctorJ ~or producing chlmeric peptlde~ ha- been demonstrated by the conJtruction accordln~ to th- pr---nt lnvention of the V1555 ch~meric prot-ln contalnlng CIa and a portlon of the GTP protein from S mutanJ Cons~truction of CTB fusion vectors, uJing ~ynth-tic ollgonucl-otlde llnkers, has been ~hown here to be a practical method of producing chimeric proteins for use as vaccine~ The use of synthetic linkers provides a convenient SUBSTITUTE SHEET
`` ` . . - ` . ` . . . .` : -- . ` ,` ; .
. .. . . . . `. ,. ' . .
., ` . ` ;
` ,, ; ' ` - - ;
`. . . . ., . `
. .. . .. . .
` . . , .. . .. ... . .
.. . ` ` . .. ` . .. . . ;
.: : - ` .. - .: ~ , . .
.~

wosl/07979 PCT/US~/~ll ~ - 15 - 2069106 method of inserting a spacer of any iength, rigidity, and composition between the two domains of the chimera The following examples ~how the use of vectors for the con~truction of gene fusions using DNA sequences from S
mutans However, the vectors may be used to make fusions to other DNA as well Once the DNA sequence encoding the antigen or epitope i8 known, an oligonucleotide specifying the immunologically important domains could be synthesized and inserted into the vectors The use of these peptide sequences would increase the specificity of the immune response, and impose minimal conformational changes on CT~ which could affect its immunogenicity An ~deal fus~on vector would contain an inducible promoter and ribosomal binding site, followed by a polylinker cloning site, and then the ctxB gene This construct would allow almost any DNA sequence to be inserted into it and expres~ a CTB fusion protein Although the V1555 chLmeric protein demonstrated the f-asibility of constructing gene fusions with CTB, there were -veral problem~ which affected the u--fulne-s of thi-prot-ln FirJt, lmmunoblotting an~lysl- r-vealed th-pr-~-nc- of 2 ma~or ~p-cl-- of 58 and 72 kDal, in-t-ad of the 58 kD~l prot-ln pr-dlct-d by the nucl-otld- equ-nce data 8ufflcl-nt DMA 1- locat-d upJtream of th- start codon for gtfB to ncod- ~ prot-in of that size There may have been addltlonal tran-l~tional start sites within th- DNA oncoding th- truncat-d gtfB g-ne that were recogniz d by E coll Thl~ would hav- r--ult-d ln th- tr~nJlatlo'n o~ a larg-r proteln har~nq th- sume gu-nce as the S8 kDal chi~era The fact that th- 72 kDal protein crosJ-reacted to both antl~-ra would ~upport thiJ hypotheJiJ, Jince it Jhare~ the ~m pltop-J a~ the 58 kDal protein Furthermore, cell ly~at-- of V1619 a1JO contain-d additional ~mmunor-active polyp-ptlde~ which were larger than the 46 kDal protein predlcted from the nucleotide sequence data (Fig 5) V1619 is an E coli strain that contains pHK7 and expresse~ the SU~STITUTE SHEET

. . , , ',, ', ` ` , ' : ~

w09l/07979 PCT/US~

truncated qtfB gene used in the fu~ion Secon~ the leader ~equence of atf~ wa~ not recognized in E coli which resulted in the protein being trapped in the cytoplasm This made extraction of the protein difficult and contributed additional contaminants which had to be removed Third becau~e the protein could not be exported from the cytoplasm it was degraded by intracellular proteases The resulting degradation products were co-eluted on the GMl affinity column and produced a heterogeneous mixture of proteins which could not be easily resolved from one another Fourth the addition of the 46 kDal GtfB moiety to CTB appeared to affect the ~tructure of the protein based on the reduced affinity of the VlSSS chimera for ÇMl This was not surprising, since the GtfB moiety is over 3 times the ~ize of CTB Genetic fusion of such a large protein to CTB appeared to interfere with it~ function which in turn could affect the putative ad~uvant properties of CTB For the~e reason~
lt wa~ concluded that the Vl555 chimera was not an ideal mod-l y-tem for vaccine ~tudies To overcome the-e drawback-, the pre~ent inventors con~truct-d th- Vl782 chim ra The pre--nt inventor~
b-ll-v-d th~t fu~on of ~m~ller peptid-~ th~n those expre~ed from th- VlSSS con~truct to CTB would prob~bly minimize the ~f-ct th-y h~d on the structure and functlon of the protein Furth- r-, a putative antibody binding ~lte of the GtfB
nzym- h~d b--n ld-ntified by the pr-~-nt inv ntor~ ~8 a r-~ult o~ th- d-gr~d~tion of the VlSSS chim ra (~-- Example II~D)) Nowev-r, th- pr-ci~e location of thi~ ~it- was unknown and wa~ too l~rge to ~ynthe~ize a rcorre~ponding ollgonucleotide to the entire degradation product According to the method~ de-crib-d h-r in, including th- u-- of th- Nopp and Wood~ algorithm and the Garnier ~lgorithm, reglon- of the protein being hydrophilic and havlng hlgh ~egm ntal mobility were located It wa~ a~sumed that the epitope posses~ed these predicted characteristics By ~ynthesizing an oligonucleotide corre~ponding to a domain of GtfB with the~e properties an epitope (antibody ~l~CTlT~lT' S~EE--.. . . , ~ . - ... , ~ .
.. ~, -. , . :

, . .. .
, . ... .
- . ,. .
..

w091/07979 PCT/US~/~

- 17 _ 2Q~
binding site) was identified Genetic fusion of this peptide to CTB re~ulted in expression of a chimeric protein which was recognized extremely well by antiserum to GtfB This tschnique may be helpful for identifying antigenic domains of other large peptide fragmen~s that cannot be located by other means The secretion vector system used to make V1782 chimera provided a convenient method of constructing and expressing small peptides fused to CTB It improved an earlier system by using the inducible lac promoter and a signal peptide sequence to secrete the fusion protein into the periplasm of coli This sy~tem is simil~r to one which has been u~ed to overproduce C~B for production of cholera vaccine (Sanchez ~nd Holmgren, 1989) Placement of the chimera under control of an inducible promoter such ~8 18C perm~tted overproduction of the protein upon induction with IPTG By fusing the chim-ra to the peptide le~der ~eguence of ~Ea, the protein could be tran~ported to the periplasm where it was e~ier to i~ol~te ~nd purify A~ verified by the N-termin~l ~oquence d~t~, the Qme~ d-r w~ prop-rly recognized in E~ coli, von though lt w~- ~u~-d to a for-ign prot-in The ~ecretion of ~or-lgn prot-in- u-lng th- Qmo~ der h~ been previou~ly d--crlb-d ~Ghr~y b t ~1, 1984) B-c~u~e of the w~y in which th- Q~a l-~der w~ fu~-d to the chimer~, two addition~l ~mlno ~cld- w r- l-ft at th- N-t-rminu~ upon proce~-ing by E
coli Although th~ ddition~l re~idueJ could be r-mov d by ollogonucl-otld--dlr-cted mutagon-~ Ghr~yeb ot ~1, 1984), thoy dld not ~pp-~r to lnterfer- wlth th- ~ntlgenlclty or Jtructur- of the chimor~, ~nd wer- thu~ l~ft intact Oth-r v-ctor~ th~t c~n be u~ d to expre-~ the chimeric p-ptld- fu~ion~ once they ~re made ln pVAlS42, pVA1543 or pVAlS44, wlth or wlthout the omDA v-ctor, includ- pRR223-3 ~J BroJlu~, Gen- 27 151-160, 1984~, ~nd p~T translation v-ctor~ The~e ex~mple~ are in no way limiting ~nd ~ny numb-r of vector~ could be used for expres~ing the chimeric peptide SUBST'~TE SHEET

, . . , . . , . . . - . . . . . . .- .

w09l/07979 PCT/US90t~

20~9~
The present invention shows that peptides can be fused to CTB with minimal effect on the structure of the protein Furthermore, chimeras can be constructed that retain the antigenicity of each moiety, which is important in the use of these proteins as subunit vaccines The ability to oligomerize and to bind to GMl shows the liklihood that this protein will be selected out of the intestinal milieu and will induce an immune response in the Peyer's patches The V1782 chimera retains these properties, as well as much of the native structure of CTB
To summarize, a complete system for constructing, expre~sing, and purifying CTB chimera~ by genetic fuqion is provided Using this sy~tem, an antigenic segment of the GtfB enzyme of S mutans was identified and genetically fused to the N-terminus of CTB The structure of thL~ chimeric protein was characterized exten~ively, and demonstrated that 15 amino acids can be added to the N-terminu~ of CTB with minlmal effect on the structure or biological activity of the proteln Antiserum raised to the chlmerlc protein recognized the n~tlve GtfB nzyme ~ well a~ C~B Furthermore, it WaJ
demon-trated 1~ vlt~Q that th- anti~-rum wa- able to inhibit gluco-yltran~f-ra~- activity of S mutan~ Thi~ is the first c~-- ln whlch ~ntl-p ptid- antl~-rum ha- b -n demon-trated to lnhlblt nzym- ~ctlvlty ln ~ mutan-, and ugg--t- that this m~y b- ~ u--ful approach for a Jubunlt vacc~ne again~t dental c~rl--Although th- pr---nt examples ar- dlrect d to the fu-lon of a port~on of GtfB to CTB, th- CTB v ctor~ d--crlbed hereln can b- u~-d to fu~- any numbor of '-~lt~ble pltop-r-glon- to CTB to produc- effective or~l vaccine~ ~oreover, th- particular 15 amino ac~d ~equence of GtfB lncluded in the chim rlc peptide expre~-d by V1782 i~ in no way a limiting x~mpl- Extondlng the length of elther end of that 15 amino ~cld -qu-nco or ~h~ftlng the span of that sequence on the GtfB soquence ~re al~o contempl~ted For a vaccinat~on ~IlR~T~ .lTE SHEEt , . . - . ` . . .
.
. . ~ . .
., ` . , : -- , ' -, . , .;: . ; . ..
- " . . . , :. . .~ . ` . . . :
, .. . .. ... .. ; `

wos1/o7s~s PCT/US~

2~9~ 06 regimen, it is also contemplated to administer a combination of different chimeric peptide~ which include different port1onq of an antigen ~SAMPL~S
I. MAT~RIALS AND METHODS
A Enzymes and Reagents Restriction endonucleases and T4 ligase (400 unit/ul) were purcha~ed from New ~ngland Biolabs (severly~ MA) Enzymatic reactions were performed according to the manufacturer~s directions A 5~ end-labeling kit was purch~ed from Bethesd~ Re~earch Laboratories (Gaithersburg, MD) DNA polymerase I (Klenow fragment) and dNTP mix were purcha~ed from Internation~l Biotechnology, Inc (New Haven, CT) lgamma-32P]dA~P (4286 Ci/ mmol) and l4C-sucro~e wa~
obtained from New England Nuclear (Boston, MA) Isopropyl-beta-D-galacto~ide (IPTG), CTB, and GMlganglio3ide were purch~sed from Sigma Chemical Co ~St Loui~, MO) DEAE-dextr~n, Tl0 dextran, and Sephadex G-l00 were purchased from Ph~rm4ci~ (Pi~cataway, NJ) Sphoro~il XOC-005 was purchasod from Sup-lco (Bellefonte, P~) Go~t anti-CTB was purchased ~rom C~lbioch-m (~Joll~, CA) Pho-ph~t~-e-l~beled ~-cond ant~ bodi-~ ~nd BCIP/NBT ub-tr~t- w r- purch~-d from XlrX~ rd ~nd ~-rry L~bor~tori-- (G~lth-r~burg, ND) Pool d, norm l r~bblt -r~ w~- purch~--d from Flow ~bor~torl-- (McCl-~n, VA) The BCA ~-~ay (Pierce Chemical Co ~ Rockford, IB) w~- u~ed for the gu~ntitation of tot~l prot-in A ilv r ~t~ln klt w~ purch~ d from Bio-~ad (Rlch~ond, CA) ~nd u- d ~- recommend d by th- m~nufacturer B Bac~teria and Plau~id~
B~cterl~l ~tr~ln- and pl~-mid~ u- d in thi~ p~por are ll-t d ln T~bl- II Pl~-mid pJBR30, cont~inlng ~
promoterl--- ver-ion of the ~txB geno from the El Tor train of Vibrio chol-r~-, wa- obt~in d from J B K~per, Univor~ity o~ M~ryl~nd School of Medicine, Baltimore, MD (Rapor et al, 198~) Pl~mid pHR7 w~s provided by H R Xuramit~u, Northwe~tern Univer~ity, Chicago, IL This plasmid encoded the ~mino terminal one-third of the atfB gene of S mutans SIJBSTITUTE SHEEJ
.` , . . .... . . . ... . . . .. . . .. .. . . . .

`, ' . . ' ' , . `: . ' :. .' ' ' ': : : : ' ' ' ', ' : ' : ' ' ,' ' ' ; '-, : ' , .
.. . ! . " . . ' ' ' '.. , . . . , ' ;: :, , .~ ', ' . ' ' ' ' ' . ' " '''''' ' ' ' :''.' ~ " . ' ' ,' ' ' " ~ ;" '''' '- ,' ', '' " ', ' " ' ' ' wo91/07s79 PCT/US~tl GS-5 (Aoki et al, 1987) Plasmid DNA was prepared by SDS-high ~alt lysis (Guerry et al, 1973) followed by dye-buoyant den~ity equilibrium centrifugation (Welch et al, 1979) E
li HBlOl w~s transformed with plasmid DNA by the CaC12-heat ~hock method (Morrison, 1977) The cells were grown at 37C
in M-9 media (Miller, 1972) supplemented with 10 mg/ml ca~amino acids (Difco), 20 g/ml leucine and proline, and 2mg/
ml thiamine V1555 was grow~ to an O D 660 = O 6, washed twice in phosphate-buffered saline (PBS, pH 7 3), and then ly~ed in a French pressure cell (SLN Aminco; Urbana, IL) The ly~ate was clarified by centrifugation before u~e For fermenter ~cale growth of V1782, cel}s were grown to an O D
660 - O 6 before the addition of 1 mM IPTG to induce the lac promoter Culture~ were ~llowed to grow for an additional 2 h at 37C before harvesting the contents of the periplasm by o~motic shock (Neu and Heppel, 1965) Cells were washed twlce at 25C in buffered ~aline (30 m~ NaCl, 10 mM HEPES, pH
7 3), then resuspended for 10 min in 0 1 volume of hypertonic Jolutlon (15% ucro8e, 50 mN EDTA, 50 mM Tri-, p~ 8 0) The c-ll~ w re pelleted and su~pended in 1 0 volume of cold d-loniz d w~t-r for 10 mln Th- ~hock fluld wa~ cl~rifi-d of ¢-11~ by c-ntrlfug~t~on, and th-n wa- ad~u-t d to pH 6 8 u~lng 0 1 volum of ~ lOX conc-ntr~t- of wa-h buff-r ~10 mN
N-PO~, 200 mM N~Cl, 0 02~ NaN3, pH 6 8) C ~n~y ~ ~r-p~r tlon ~ xtr~¢-llul~r prot-ln~ of S mutans w re obtained ~
follow~ 8tr~1n GS-5 ~Bratthall ~-rotyp- c) wa~ grown in 3 1 Todd-H ~ltt broth, und-r an~-roblc cond~t~on-, to ~n O D
C60~0 6 Th- c-ll- w r- r-mov-d by c-nt~lug~tlon, and ammonium ~ulfat- w~- ~dd d to 60~ atur~t~on The pr-ciplt~te cont~$nlng xtrac-llular prot-ln~ wa~ collected by c-ntrlfugatlon, r-~u~p nded in 300 ml PBS ~pH 7 3), ~nd th-n dlalyz d 3 tlm - ag~ln~t PBS Approxlm~t-ly 30 ml of thl~ ~t-rlal wa~ allquot d and ~tored at -70C ThiJ
mat-rlal wa~ u--d for all enzyme a~ay~. The r~m~ining ~ample wa~ dlluted in PBS + 6 M ure~ and sub~ected tO
dlaflltration through a YM-100 ultrafiltrAtion membrane SUBSTITUTE SHEET
... ~.. . . .. . .
.. , .... ~ - , .
`
.... ... ~ `` .. - . ` .

.. .. ;. . .. , ... - . ` . ; .. . .

w091/07979 PCT/US~/~lt ~$qI.Q~

(Amicon; Danvers, MA) The clarified sample was concentrated by ultrafiltration to 20 ml and then dialyzed in P~S (pH 7 3) ln order to remove the urea The samples were aliquoted and stored at -70C This preparation was used as a control for immunoblotting analyse~
D Antisera Rabbit anti erum to the glucosyltransferase s enzyme of S mutans GS-5 was obtained from H K Kuramitsu, Northwestern University, Chicago, IL Antiserum to the purified, monomeric form of the V1782 chimeric protein was raised in female New Zealand White rabbits One mg of protein was emulsified in complete Freund~s ad~uvant and in~ected subcutaneously into the hind quarters of the animal Three weeks later, the rabbit was boosted with the protein emulsifed in incomplete Preund~s ad~uvant Then, one week later, the rabbit was bled The serum was collected, aliquoted, and stored at -20C
E Oligonucleotides Oligonucleotides and their complementary strands were synthesized on an Applied ~iosystems 380A synthesizer The link-r- w r- i-olat d by thin-layer chromatography on X1-~-lg-1 60 F2S4 silica g-l pl~tes ~M rck; Darmstadt, W
G-rm4ny) in 1-propanol, ~mmonium hydroxide, and w~tor (~5 3S 10) Th- DNA band~ w r- vi-ualized by a hand-held W
light ~d th-n crap d off the plat- The DNA wa~ extracted by wa-hlng th- ~ilica gol three tim - with water, and then con¢-ntrat-d by vaporating th aqueou- pha-- to dryn---Th- larg-r oligonucl-otid-- w-r- s-parated by using a 20%
polyacrylamid- g-l containing 8 M ur-a ~G~l f~agm~nt-containing the DNA war minced and extraet-d 3 time~ with an qual volum of xtraction buffer (0 5 M ammonium acetate, 1 mM EDTA, pH 8 0) The oligonucl-otid-~ w r- purified u~ing a N n-orb affinity column (Dupont, Wilm~ngton, DE) bofor- use Th- ~orward r-action of T4 kinaJ- WaJ u-od to pho~phorylate th- 5~ nds of the oligonucleotides in order to improve the effieiency of the ligation reaction (MAniatis, et al , 1982 Unincorporated dATP was removed by purifying the SUBSTITUTE SHEET

, .. . j., . ...... , . ` ,; . . ... ... ` .. . : : -.. ` . ....
.. ,. . .. ;: . - , .~........ . `
.... .. ... ~,. .. . .

W091/07979 PCT/US~

oligonucleotides with a Nensorb column One pmol of purified vector was mixed with 21 pmol of each complementary linker in annealing buffer (20 mM Tris pH 7 5, 10 mM MgC12, 50 mM NaCl, 1 mM dLthiothre~tol) The ~ample was heated to 2, 90C for 10 min snd then incubated overnight at 42C to reanneal the DNA Un$ncorpor~ted oligonucleotides that could interfere with the ligation were removed by washing the sample in annealing buffer u~ing a Centricon 30 ultrafiltration unit (Amicon; Danvers, MA) The retentate was suspended in ligation buffer with T4 ligase (400 units) for 6 h at 25C
P Construction of the Fusion Vectors Plasmid pJBK30, containing the promoterless ctxB gene, was prepared for insertion of the linker~ by deleting two reJtrictlon sites that could interfere with their placement The NdeI site, located near the origin of replicstion of the plasmid, and the EcoRI site located at the 3~ end of the ctxB
insert w~re both deleted from the plasmid (Fig 1) Plasmid pJBK30 was partially digested by restriction enzyme and the lin-arized DNA was isolated by electro-lution from agarose g-ls ~Maniatis et al, 1982) The 5~ ends were filled-in u~ing DNA polymera-- I (Xlenow fragment) and theJe blunt end~
w r- llgat-d tog-th-r u-ing appropriat- r-action conditions ~Manlati~ t al, 19~2) Th- pla~mid containlng the two d-l-t d r-~trlctlon ~lt-~ wa- designat d pVA1662 Plasmid pVA16C2 wa~ dlg-~t d wlth ~I and E~QRI and th largor of th- two r-~ultlng fragment- wa~ i~olat d by lectro lution from n agaro~- g l Th- linker~ w r in--rt~d into thi-frag~ nt a~ d--crib d ln Example I~E) ~al-o -- Flg 4) The pla~mid wa~ tran~orm d into ~ QQL~ HU~r, and clon-~ th~t w r t-tracycllne (Tc)- and umplclllln (Ap)-r~ tant were ~-l-ct d and cr n d for in~ertlon of th- llnker by r-~triction mapplng Pla-mid~ containing ach llnk-r were i~olat d and d-~ignat d pVA1542, pVA1543, and pVA1544 These v ctor~ contaln d a truncated verJion of th- ctxB g-n in SUBSTltUlTE SHEET
. .. . .. . . . . . .

: ' ' . ~' ` , ': , . ` ' . ' .' 2Q~lQ~
which the 5~ end of the gene was deleted to remove the ribo~omsl binding site and DNA encoding the leader sequence of the protein G Gene Fusions V1555 was constructed as shown in Figure 2 Plasmid pHK7 contained a 1 9 kb PstI-EcoRI fragment from S mutans GS-5, Bratthall serotype c (Hamada and Slade, 1980), that encoded a truncated portion of atfB The PstI-EcoRI fragment from pHR7 was directionally cloned into each fusion vector E coli transformants that were Tc-resistant and Ap--~ensitive were screened for expression of a protein that was recognized by antiserum to C~B A clone wa~ selected, containing a pla~mid derived from pVA1542, and de~ignated V1555 ~ he strategy used to construct V1782 is shown in Figure 3 The oligonucleotide~ were in~erted into the fusion vector pVA1542 at the 5~ end of ctxB as previously deJcribed in part I(E) Proper insertion of the oligonucleotides into pVA1542 was monitored by reJtriction enzyme digestion of plaJmid DNA
prepar-d from minilysates (Macrina et al, 1982) In order to xpr-~ the tran~lational fu~ion protein, the chimeric ctxB
q-n- ~ tr~n~f-rr d to th- ~-cr-t~on v-ctor pINIII Qm~A2 8cr nlnq Pl~mld DNA w~ obt~in d from c~ by ~ r~pid mlnlly~l~ proc-dur- (~acrlna t al, 1982) The DNA wa~
ch-ck-d ~or pla~mid cont-nt by agaro~e gel electrophore~s ~nd r--triction enzyme analy~is For protein analy~i~, a whol--c-ll ly-~t- of th- clone- wa~ propar-d by a modl~ic~tion o~ th- DNA minily~i~ procodur- CellJ
containing the ecretion voctor wer- ~cro~'n-d for expr ~-ion o~ a chim ric prot-in by fir~t cultlvatlng th m on M-9 m dia cont~lnlng 1 mM IPTG After dige~tlon in lysozymo, th- cells w r- ~u~p nd-d ln 0 5 ml of a hypotonic ~olut~on (10 mM ~ri~, 30 mM N~Cl, pH 7 3) and then ~ub~ected to a rapid fr -zo-thaw In-olubl- mat-rlal wa~ removed by centrifugat~on, and the ly~ate~ were assayed for expre~sion of the chlmeric protein by an enzyme-linked immunosorbent a~say tELISA) that SVBSTITUTE SHEET

.... . . .... . . ... . .... . . .... . .. . .
, , , . .. ; , .. ~ ., .. .. ~. ~ . . . . . .

09l/07979 PCT/US~
~ 24 -used GM1 ganglioside-coated microtiter wells as the solid pha~e snd antiserum to CTB ac the primary antibody (Sack et ~1, 1980) I Purification A GMl ganglioside affinity column was prepared and u~ed to purify the CTB chimeras according to the method of Tayot et al (1981) Lyso-GM, was prepared from 50 mg of GMl and then covalently coupled to 15 g of DEAE-dextan coated Spherosil The samples were recirculated through the column for 24 h at 4C and at a linear flow rate of 1 5 ml/min The column wa~ wsshed with 10 volumes of wash buffer (10 mM
NaP04, 200 mM NaCl, pH 6 8) and then the chimera wa~ released u~ing elution buffer (50 mM citrate, 200 mM NaCl, pH 3 0) The eluate wa8 ad~u~ted to pH 7 3 and then concentrated by diafiltration in PBS (10 mM NaP04 140 mM NaCl, p~ 7 3) using a YM10 ultrafiltration membrane (Amicon; DAnver~, MA) The retentate was quantitated for total protein a8 well a~ for reactivity in the E~ISA Native CTB was used a~ the r-f-rence standard for the E~ISA A 1 6 x 200 cm Sephadex G-100 column was prepared and uJed to separate monomeric and oligom ric fraction- of tho V1782 chimera A 5 mg ~ample wa~
load-d onto th- column and elut-d ln PBS (pH 7 3) at a lineAr ~low rat- o~ 0 13 ml/mln Fractlon- w r- coll-ct-d in 2 ml volum 8 and Analyzed for tot~l prot-ln cont-nt and immunoreactivity 3 ~rot i~ AD~1Y~1-~ h- N-t-rminal qu nc- of th- monom rlc prot-in (40 g) wa- d-t-rmln d u-lng an Appli d Bio~y-tcm~ Mod-l 47OA
S-qu-nc-r, wlth ln-lln- PIH-~mino acld analyJ1-Tho proc dur of Gro~ and Witkop (1962) wa~ uJed tocl-av prot-ln by treatment with cyanog-n bromide The prot-ln wa~ dl~-olved in 200 1 of 70~ formic acid and d-~-r~ted by fluJhing with n~trog-n A 100-fold molar exce~
of cyanog n bromid- ovor methionine thon wa~ added ~he r-action wa~ allowad to proceed for 13 hour~ in the dark at SUSSTITUTE SHEET

..
: :, ` . . - , . . . - , ~ - .
- - .. , . . . . ... - , .. ... . ` ~ -, . . ,. : ., .. . - ~ - , ~. ,. .- , -wosl/07s7s PCTtUS~/~
~ - 2s - 2Q~

room temperature in a tightly sealed tube The reaction was terminated by removal of the reagents in a Savant Speedvac Concentrator The peptides produced by cyanogen bromide cleavage were sep~rated by high performance liquid chromatography on a C-18 reverse phase column (Varian), using a linear gradient of 0 1~ trifluoroacetic acid in water and 0 1~ trifluoroacetic acid in 50% acetonitrile A flow rate of 1 ml/min and a total time of 60 min was used The effluent was monitored at 215 nm Peaks were collected, and analyzed by amino acid analysis Samples of protein or peptides were hydrolyzed in sealed, evacuated tubes with constant boiling HCl for 24 hr at 110C Amino acid analy~es were performed with a Durrum MBF amino acid analyzer u~ing o-phthaldehyde as the detection reagent In some cases, the sample~ were alkylated with lodoacetamide prior to hydrolysis Each sample was reacted with iodoacetamide (1 mole sample/10 moles iodoacetamide) in 50 mM Tris (pH 8 0J for 1 h at 25C
The monomer, oligomer, and CTB were analyzed by circular dichroi~m Sample~ were extenslvely dlalyzed agaln-t PBS (pH 7 3) uJing a C-ntricon 30 (Amicon) Th-~ltrat- wa~ ~v d for u~- a- the buffer blank A 1 5 ml campl- ~0 15 mg/ml) of ach prot-ln wa- load-d $nto a cell and ~n~ly~-d on a J~-co J-500C p-etropolarLm ter An~lytloal polyacrylamlde gel electrophoreJiJ wa~
p-rform d with a BloRad ~lab gel apparatu~ wlth a 15S
polyacrylamld- g-l A 4% tacking g-l waa uJod Th- gel and buff-r formul~tionJ w re tho~e of O'Farrerl (~975), ln ~ome ca~-- modlfl-d to include 8 Mur-a ln th- g-l and Jample buffer unoblottLng Analy-~J
Proteln ~a~ples were 8eparatod by SDS-PAGE and then lectrophoretic~lly tran-ferred to nitrocelluloJe Jh-et8 (Towbin t al, 1979) Nonspeciflc binding of antibody wa~
pr~vented by blocking the sheets for 1 h at 25C with a 5%
solution of chicken serum in-tris-buffered saline ~TBS; 20 mM

. . ` ' - ~
. . : . `.` ` . . . ~ . . ; ` . . . . . ` . .` .. ` .. ~ ' . .. .-. . .. . ` `. . .. . . . -2~ Q~3 Tris, 500 mM NaCl, pH 7 5) The sheets were incubated for 6 h in primary anti4era diluted 1 1000 in TBS with 5% chicken serum The sheets were washed 3 times for 10 min in TBS with 0 054 Tween 20 (TTBS), and then incubated for 2 h in enzyme-labeled second antibody After washing again 3 times in TTBS, the qheets were developed with substrate L Enzyme Assay~
Glucosyltransfera~e activity was determined by mea~uring the amount of 14C-gluco~e converted into glucan polymer from specifically labeled sucro~e Fructosyltransferase activity was measured by a similar me~n~, exeept t~t ~ucro~e labeled with 14C in the fruetose moiety wa~ uoed a~ the ~ubstrate The reaetion mixture was ineub~ted at 37C and conJl~ted of 10~1 enzyme, 5~1 T10 dextran (5 mg/ml), 5~1 labeled ~ubstr~te (261 mCi/mmol, 20 Ci/ml), and 80~1 ~ub~trate buffer (lO mM
imidazole, 10 mM suerose, 0 02~ sodium azide, pH 6 5) A
100~1 sample of the reaetion mixture was pipeted onto 2 4 cm glaJJ fiber filter~Whatman GFA; Maid~tone, England) All ~ampl-J were performed in triplieate Total polymer wa~
eolleeted onto th- filter~ by preeipitation ln mothanol The filt-r- wer- wa~h d 6 tim - wlth methanol u-ing a vaeumn m ni~old apparatu- ~Milllpor-; ~edSord, MA) Water-ln-oluble polym r wa~ eoll-et d by wa~hin~ the ~ilt-r- 4 tim ~ wlth d-lonl~-d wat-r ~nd th-n 2 tlm ~ wlth m thanol The filters w r ~ir-drl-d, plaeed lnto vials with 5 ml ~eintillation eoektall (Am r-ham OCS; Arlington Height~ ), and thon count d ~or 2 mln ln a ~elntlllation eount-r (B ekm~n ~S-1800) Th- m an eount~ por minut- ~CP~) o~ aeh ~ampl-w r- ~ubtraet d ~rom a baekground eontrol eontalnlng no nzyme For inhibition as-ay~, enzym wa- pre-ineub~ted wlth an gual volum- of serum for 1 h at 37C prior to addition to th- r-aetion mixture Enzyme wa~ pre-ineubated wlth an equal volum of PBS for u-e a- a positive eontrol Sera w~-dilut-d in PBS, whon neeesJary SUBSTlTUTe SHE_ .
... .. .. . .... ~ - .- - ....... ..

. . . ~ , . - . . . - .

W091/07979 PCT/US~/~

- 27 _ 2~1Q~
M An~al studie~
The protocol to be used is approved by the Virginia Commonwealth Vniversity Institutional Animal Care and Use Commlttee M~le C57B1/6 mice can be obtained from Charles River (Wilmington, MA) The mice are used at 6-8 weeks of age, and should be fasted overnight before feeding the protein The chimera is administered using an intragastric feeding needle (G Tiemann & Sons, Long Island, NY) The GMl-purified chimera is diluted in 0 2 M Na2HC03 and is delivered in a volume of O S ml Control animals receive saline solution For comparison, the antigen is injected intraperitoneally into some animals After the antigen is diluted in MAALOX, it was in~ected in a volume of 0 1 ml Mice ~re immunized on day O and 14 On day 2i, serum and lnt-stinal secretions are collected and assayed to assay for ~pecific antibody Microtiter wells are coated with either CTB or synthetic peptide composed of the same 15 amino acid s~equence a8 found in the chimera The assay for detecting peptide-~pecific antibody in intestinal washings hould be ~en-itlve onough Juch that 0 01 ng/ml of peptide-specific antlbody m~y be detected A protocol for t-slting the effectivene-s~ of the chim-rlc poptlde v~ccln-- ~or prevontlng dental c~rio~, ~ccordlng to th- pro~-nt lnv-ntion, i- ~et forth in Mori~aki e ~ nf-ctlon and Immunlty, 40s577-591 ~M~y 1983) II RESULTS
A Fu-~on Vectors~
A -t of clonlng vector~ h~ve boen developed for con~tructlng ~nd xpr -Jing ctxB gene fu~ions~ ~n E~ s~LLi Th- pl~-mld pJ~R30 w~- u- d ~SI the b~ of con~truction of th- fu-lon v-ctor~ b-c~u~e lt contaln-d a promot-rl--s ctxB
g-n- ~nd dld not xpro~- this protein in ~ coli By making CT~ th- C-t-rmlnal end of the chimera, ~n ELISA could be used to det-ct ~ucc-J~ful fu~ion, becau-e the gene would only be xpr--~ed if it wero tran~lationally in-frame with soquences loc~t-d upstre~m The region located between the EcoRI and ites of ctx8 encoded a putative hydrophobic leader Su~sTlTuT~
~ ,, . . . , . .. . . . . .... . - . . - .

. - , . - , .. . . .-. : . ... , . -,: .. :

.; . . :

~: . - . .. ,~ , - .. :. , ., . -. . . . -09l/07979 PCT/US~
~ 6 28 -which was not present in the mature protein (Lockman and Kaper, 1983) By eliminating thi~ region from the fusion vectors, the chance of abberant cleavage or folding of the chimeric protein was minimized The linkers were designed to be compatible with any DNA that contained an EcoRI site within its open reading frame (Fig 4) ~he 3~ end of the linkers encoded a sequence compatible with an NdeI site located within the open reading frame of ctxB Only one reading frame in each linker allowed translation of ctxB to occur Although translation relative to the EcoRI site occurred in a different reading frame, the length of the linkers wa~ ad~u~ted ~o that the last codon terminated at the ~unction of the NdeI site Sequencing data has shown that this ~unction separat-s codons translated within ctxB
(Lockman and Raper, 1983) A unique restriction site wa~
encoded into each linker to serve a~ a marker These were very helpful for determining which linkor was insert-d in the v-ctor ~Fig 4) The linker~ contained codons tha~ have been tr~nJlated by other g-ne~ oxpresJed in E coli Small, polar n-utral amino acids were ncoded wh-nev-r po-Jible in ordor to mlnlmize conformatlonal chang-~ ln th- prot-ln- DNA
ncodlng an a-partyl-prolyl umino acld -qu-nc- wa~ includod ln ach llnk-r to allow po-t-tran~latlonal cl-a~ag- of tho chlm-ra, lf d--lr d Thl~ ~qu-nce 1- ~ound rarely in prot-ln-, and contaln- a poptide bond ~u-ceptible to acid hydroly-1- (Landon, 1977) a. Con-truct~on of V15SS
A 1 9 kb fragm nt, encodlng the gluco-yltran-~-ra~- 8 g-no (at~B) of S mutan~ GS-5 (Bratthall~rotype c), wa-u--d to construct a fu-ion with ctx~ Thl~ fragment, contain d ln pHR7 ~Fig 2), con~titutively expre~ed a non-n~ymatically active 48 XDal truncat d polyp ptlde thatcro---r act d with antl~erum to Gt~B Th- im~unoblotting analy-ls of th~ V15S5 chimera peptide was performed by purlfylng th- p-ptlde ~y G~l ganglioside affinity chromatography A clarified whole cell ly~ate from E coli strain V1555 was loaded onto the column (ly~ate) ~ound SUBSTlTlJTE S~E--l' 2Q~91~

material was eluted from the column by decrea~ing the pH
stepwise (p~ 4, pH 3) The column was cleaned by washing it with 0 1 M NaOH Duplicate blots were probed either with anti~erum to glucosyltransferase B (anti-GTF) or to the B
subunit of cholera toxin (anti-CTB) Purified CTB and a lysate of V161~ were used as positive controls for the antisera E coli strain V1619 contains the plasmid pHK7 which expresses a 46 kDal truncated GTF protein The fragment was inserted into all three fusion vectors, but immunopositive clones were isolated from only one, pVA1542, indicating that the reading frame of ~tfB was compatible to the linker contained in this plasmid This result was verified by the nucleotide sequence data for ~tfB (Shiroza et al, 1987) One of the clone~ was selected and designated V1555 Plasmid DNA from this clone was mapped by restriction enzyme analy~is to verify construction of the gene fusion (Fig 2) The results demonstrated that the fu~ion vectors could be successfully used for constructing CT~ chimeras C Purificatlon of the V1555 rh~ra Analysis of V1555 revealed that the chimeric protein WaJ trapp-d in th cytopla~m of ~ coli The leader ~equence for GtfB wa~ not r-cogniz-d by th- hoJt, ev-n though this prot-in i~ norm~lly -cr-t-d by S mutanJ This phenomenon h~ b--n ob~-rr d with other extracellular ~troptococcal prot-ln- that ar- xpre-Jed in E coli (Aoki et al, 1986) In ord-r to lib rate tho protein from the hoJt, the cell~ had to b- ly- d in a French preJsuro cell Onco the c-lls were ly~-d, th- prot-ln wa~ ready to bo purifi-d by afflnlty chromatography CTB has a hlgh a~finity for GMl ganglloslde ~Slllerud t al, 1980) Th- u-- of a GMl ganglio~de affinity column h~ b--n hown pr viou~ly to be a rapid and fficient method o~ purifying chol-ra toxin ~Tayot et al, 1981) In order to optimi~- binding of the V1555 chimera to the G~l affin~ty column, the lysate wa~ recirculated through the column overnight at 4C The ma~ority of the activity was eluted at p~ 4, which was higher than that required to elute native SUBSTITUTE Sl-!EEt os1/o~s7s PCT/US~/~

- 30 - ~ ~

cholera toxin from the column (Tayot et al, 1981) Immunoblottlng analysis of the column eluate revealed the pre~ence of two novel proteins of 58 kDal and 72 kDal thst cro~s-reacted with anti~era to either GtfB or CTB Based on the publi~hed DNA sequences of both genes, the chimera was predicted to be 58 kDal in size The presence of the 72 kDal protein currently cannot be accounted for, but it does cross-react with antisera to both GtfB and CTB Passage of the V1555 lysate through the affinity column was effective for removing many of the contaminating proteins from E coli Cleaning the column with 0 1 M NaOH only eluted a small ~môunt of ~dditional immunoreactive material, indicating that elution of the chimera wa~ complete Smaller immunopo~itive polypeptlde~ were observed on the Western blot, particularly to GtfB, and these were postulated to be proteolytic degradation products Proteolysi~ of foreign protein~ in E
coli ha~ been documented (Goff et al, 1984), and such fragm nts would co-elute with the chimera a~ long as they r-talned the GMl binding site of CTB The addition of protease lnhibitors to the lysate did not alter the pattern ob--rv d on SDS-PAGE and, th-refore, app-are~ to occur in the ho-t pr~or to ly~i- Th- abillty to elute the chimera from th- GMl column at a hlgh-r pH than n~tive chol-ra toxin ~ugg-~t d th~t th- conformatlon of the CTB moiety wa~ altered by th- g n- fu~lon Xowever, the e~tent of the change wa~
not d-t-rmln d D~ ction of ~17a2 An ~nt~g nlc ~-gm nt of the glucooyltran~f-ra~- B
n~ym of ~ mY~a~ wa- id-ntified from ~unoblotting ~n~ly~ of th- V1555 chimeric protein One of th- peptide ~r g~ nt~ cro~-r-~ct d wlth antl~-ra to elther GtfB or CTB, lndlcatlng that it contained antigenlc ~-gment~ from both prot-in- The mol-cular weight of this fragment wa~ ~lightly larg-r th~n that for CTB Ther-fore, a~ ng that tr~n-l~tlon of th- V15S5 chimera terminated at the ~top codon for CTB, the fragment contained the entire length of the CTB
protein and a ~mall portion of the GtfB protein which was SUBSTITUTE SHE~t - , , .. . . - .

. ~ ,: - - . . . . . . .

- .

. . . ' ' ' ! ' ' ' ' ' ' . ' ~ ' ' W09l/07979 PCT/US~

2~9~ 06 located near the junction of the fusion Once identified, the sequence of the qtfB gene (Shiroza et al, 1987) was used to creste a hydropsthy plot of this antigenic region, based on the algorithm of Hopp and Woods (1983) Secondary structure predictions of this region were al~o made using the algorithm of Garnier et al (1978) Several domains of this region were identified which had both high segmental mobility snd hydrophilicity One such domain, atfB l, is shown in Figure 5 This domsin was relatively hydrophilic and was devoid of a highly organLzed secondary structure A set of synthetic oligonucleotides were ~ynthesized which were compatible with the amino acid sequence of thi~ region The atfB l oligonucleotides were genetically fused to the promoterles~
ctxB gene in pVA1542 (Fig 3) Insertion of atf~ l into pVA1542 did not re~ult in expres~ion of ctxB, due to the lack of a promoter and ribosomal binding ~ite Thi~ waJ provided by the secretion vector pINIII om~A2, which encoded the l-ad-r poptide ~equence of the E coli om~A gene (Ghrayeb et al, 1984) Tran~cription of om~A was controlled by the inducibl- lac promot-r Tran~fer of th- ctxB gone fuJion~
lnto pINlII QE~a2 r--ult-d in xpr-~-ion of chimoric proteinJ
th-t could b d-t-ct-d ln whol--cell-ly-~te~ u-ing ~n E~ISA
a~-ay for CTB Th- -cr-tion v-ctor containing th- ctxB
chim r wa- d--ignat d pVA1782 (Fig 3) ~ coli tr~inJ
cont~lnlng thi- pla-mid w-re able to e~pre~- a chimeric prot-ln that wa~ imwunor-active with anti--rum to C5~ or to ~t~ Immunoblottlng analy-i~ Or the V1782 chim ra w~-p rform d by pr-parin~ ly-at-- from lnd-pendently i-olat-d clon-- from c-ll- gro~n on M-9 agar plat-~ containing 1 mM
IPTG T~- a~pl-~ w re ~eparated by 15~ SDS-PAGE under r duclng condition~ and then electrophoretically trans2erred to nitroc-llulo-- ~h-et~ Proteins that reacted with antl--rum to th- gluco-yltran~ferase 8 enzyme are indicated by arrow~ Th- V1555 lysate contains two truncated verJions of Gtf8, and was used as a control Immunoreactive proteins were detect d in the lysates from V1555 and the clones ~1 IOI!~ITI ~'~ ~1~

, .' ` ; . - ` ' ~ . ,' -~ ` . !

w091/07979 PCT/VS~t~
~9~Q6 - 32 - ~

expressing gtfB l, but not from V1784 which was used as the background control The ~bility to overproduce the chimeric protein is a de~lrable trait for large-scale vaccine production Induction of expression in V1782. Cell lysate~ containing the V1782 chimer~ or V1784, contsining the plasmid vector, were grown in M-9 broth to an O D 660 = 0 6 before induction with 1 mM IP~G Upon induction, the cells were allowed to expre~s for 2 h before harvesting the contents of the peripla~m by osmotic ~hock As controls, parallel cultures of each strain were grown in the absence of IPTG Samples of the shock fluid were ~eparated by 15~ SDS-PAGE and then blotted to nitrocellulo~e The blot~ were reacted with antl~erum to CT~ The addition of IPTG resulted in elevated levels of chimera belng produced However, overexpres~lon of foreign genes in E coli can be deleterious to the ho~t The laci gene, encoding repres~or for the lac promoter, W~8 locat~d on the same pla~mid in order to maximize expres~ion of ropr-JJor The laci gene reduced promoter activity Jlgnlficantly, but it did not completely inhlbit expression of th- protein Although not tlghtly r-gulat d, promoter actlvlty wa~ ~uff$cl-ntly r-pr-J- d, ln th- ab~ence of IPTG, to ~llow nor~al growth o~ th- ho~t O-motic ~hock xp rlm nt~ d~mon~trat d that mo-t oS th- prot-ln wa~ -~
~-or t-d lnto th p rlpla-m of ~ coll wh$ch wac con~$Jtent wlth th ch$m rlo prot-ln b lng fuJ d to th- g~aA l-ad-r No xtr~c-llul~r act$vity wa- d-t-ct d Pr v~ou- work w$th the Vl555 chlm-r~ r -ult d ln th- prot-ln b lng tr pp d ln th-oytopl~-~ of ~1 coll Th$~ made xtractton o~-the prot-ln dl~flcult, but th- u-- of Qm~p allowed -cr tlon of th-prot-ln lnto th p r~pla~m where $t could be xtr~cted easily ~rom th- cell by o-motlc ~hock The ch$m rlc prote$n derlved ~rom Vl782 w~ u~ d for all subJequ nt ~tudle~ ln thi~ paper Purlfic~tion of th Vl782 Chi--r~
Th- chi~ rlc prot-in wa~ purifled by GMl afflnity chro~atography, ba~ed on the ~ffinity of the CT~ moi~ty for GNl; Tho column w~ u~ed to concentrate the chim~ra from the SUBSTITUTE SHE~
. . - - - , `. , . . , . . - ., . . - . .
.. ~ . . . . .~, . .................... . -, .

. . i, . ., ' ., ~ .. ~ -. ., ......... . . . : .

. ; . ., . , ` ~ .. . . .

w091/07979 PCT/US~

2069~ o~

shock fluid by recirculating the fluid through the column Saturstion at 4C was achieved within 24 h of initial loading of the column The chimera was eluted from the ~olumn only upon decre~sing to pH 3 0 The use of a higher pH did not re~ult in elution of the protein Table III lists the purification data from a typical experiment Approximately 23 g of protein could be collected from one elution Silver-staining of a sample from the column, separated by SDS-PAGE, revealed that most contaminating proteins were removed by this method No proteolysis of the protein was observed as wa8 seen in the V1555 chimera A sample of the affinity-purified chimera wa~ loaded onto ~ gel filtration column (Sepha,dex G-100) and eluted in PBS Gel filtration profile of the V1782 chimera The GMl column-purified chimera was fractionated through a 1 6 x 200 cm column cont~ining Sephadex G-100 Samples of the column fractions were reducod and then separated by 15% SDS-PAGE
Prot-in~ were vi~uali~ed by ~ilver-staining. Activity wa~
m ~ur~d by an E~ISA a-~ay, which d-pend~ on the ability of th- chimera to blnd to GMl-coated well- and to be recognized by anti~-rum to C~B Tot~l prot-in waJ m ~ur d u-ing the BCA ~ y (Pi-rc-) ~h- ~ampl- wa- fractionat-d into two m~or Jl~- cl-~ U~ing n~tlv mol-cular w ight ~t~ndard~, th- prot-ln~ w r- -tlm~t d to b 54 ~nd 17 4 kDal (CTB - 33 kD~l) 8umpl-~ ~rom th- column fraction- wer ~eparat~d by SDS-PAG~, which r-veal d th- pr-~ence of two p ci-- (~6 4 ~nd 17 4 XD~l) th~t w r- common to both fraction-I~munoblottlng ~n~lyJl~ d mon~tr~t d th~t th-~- ~p cl-~ w r-lmmunologlc~lly ld-ntlc~l r-gardle-~ o~ ~;th-~ ~ntlbody to C~B or to GtfB w~- u~ d B cau-e the compo-ltlon and ~munor~ctlvlty of th- column fractlon~ w re ldentlc~l, it w~ um d th~t th- fr~ctlon~ contaln d monom ric ~nd ollgom rlc for~J of th- chimera A hlgh molecular w-lght prot-ln wa~ ob--rved ln th oligomerlc fractlon that dld not r-act wlth anti~era in the immunoblot, and wa~ not pre~ent in ~UP~TITIITl: ~:~C~
s ; ;-091/07979 PCT/US~/~

~69~ ~ 34 - ~
the fraction containing monomer Biological ~ctivity, based on ELISA, indicated that most of the reactive material was associated with the oligomer P Analysis of the V1782 Chimera The monomeric form of the V1782 chimera was subjected to 25 cycles of Edman degradation The amino acid sequence w8~ found to be identical to that predicted by the construction of such a fusion protein (Fig 5 According to the qequence data, the leader sequence of the _A protein was absent from the periplasmic form of the protein Fusion of the chimeric protein to the om~A leader did not appear to affeet the ability of E coli to recognize ~nd cleave the sign~l sequenee The data also su~gested the ab~enee of sny ~ddition~l N-terminal seguenees in the sample The ~mino ~eid eomposition of the C-terminal end~ of monomerie and oligomerie forms of the ehimera were also eompared to that of native CTB Protein samples were eleaved by reaetion with eyanogen bromide and the peptide fragments wer- fraetionated by HP~C Elution of the protein was monltored by absorbanee at 215 nm The resultant peptide~
w re th-n an~lyz d for their tot~l ~mino aeid eompo~ition (T~bl- IV) DNA -gu-ne- data for the etxB gone pred~eted th~t cy~nog-n bromid- el-~Y~g- would r-l-~ n ~ Jn dlp ptld- fr~gm nt lf tr~n-l~tlon o~ th- g-n-~ termin~ted prop rly In ~ eoL~ (ToeXman and ~p r, 1983) T~bl- IV
Jho~ th~t p-ptld- A h~d thi~ eompo~ition ThiJ dipeptide wa~ $-ol~t d from ~11 thr-e ~mpl-- The ~mino aeid compo-ltion of p ptid- B w~ found to m~tch th~t exp ct d for r-Jidu-- 61-92 of th- chim r~ Peptid- ~ ri- ~pp~r-ntly the unr--olv d mixtur- of th- r m~ining ey~nog n bromid-p ptid~- For th- ehim r~, peptide~ A ~nd B wor- Golleeted, ~naly~ d, ~nd hown to b- pr-~-nt in e--enti~lly ~ 151 r~tio, r-coverin~ 0 51 nmol-J A/0 62 nmoleJ B. Thi~ W~J ~Jonti~lly id-ntie~l to th- mount of e~eh obt~in-d from th- n~ti~e CTB
(0 96 nmole~ A~l 2 nmole~ B) Therefore, the poJJibility th~t th- J~mple 19 ~ mixture of native CT8 and ehimera wa~
ruled out It ean bo eoneluded that, within the limit~ of SU~STITI~TF ~u~tf .
., ,~ . . ~ . . . ` `, . ` - . , -, `... . ~ . - - . .. .

w09l/07979 PCT/US~

20~9~ 06 measurement, translation of the protein uniformly terminated ~t the ~top codon normally recognized for ctxB
G. SDS-PAGE of the V1782 Chimera An~ly~i~ of the effluent from the gel filtration column by SDS-PAGE i3uggested that there were two forms of the chimera in the monomeric and oligomeric fractions of 17 4 and 16 4 kDal in size Samples of CTB, monomer, and oligomer were reduced and alkylated, in order to block disulfide bond form~tion, and then fractionated by SDS-PAGE. The samples were fractionated by 15% SDS-PAGE and then stained with Coomassie blue Some samples were reduced with 2-mercAptoethanol and then alkylated with iodoacetamide prior to loading them onto the gel Certain qamples were boiled for 5 min in loading buffer containing 2 5%
2-mercaptoeth~nol and 1% SDS Other samplei3 were prepared similarly, except 8 M urea was included in the sample buffer and gel Reduction and alkylation had no effect on the profile of these ~ample~ when fractionated by SDS-PAGE When the-e ~ame samples were fractionated by SD~-PAGE using geli3 contalnlng 8 M urea, only a single species of 14 4 kDal was ob--rv-d Thl- protein wa~ tho s~me sise a~ the one pr-dlctod by the nuclootld- equenco data for the monomeric ~orm of th- chim ra Dl-ulfld- Bond Th- monom rlc and oligomeric form~ of the chimera were analy~-d for th- pre~ence of dl~ulflde bond~ by alkylation of th- prot-in- w$th iodoac-t~mid- The alkylated ~ampleJ were hydrolyz-d and th-n analyzed for thelr amlno acid compo~itlon After alkylation with iodoac~etu~d-, 2 2 mole~
oi carboxym thylcy-t-lne per mole of protoin were found for th- monomer, while none was detected ln the ollgo~er Thls wa~ con~i~t-nt with the monomer having two freo ~ulfhydryl group-, and ~ugge~t-d that the monomeric fraction contained a r duc-d form of the chimera which lacked the proper intramolecular di~ulfide bond SUBSTITUT~: SHEEt .. . . . . .. .

WO91/07979 PCT/US~/~
~ Q~ - 36 -I Circular Dichroism Samples of monomer, oligomer, and CT3 were subjected to ~nalysls by circular dichroism The spectra of these samples ~re shown in Figure 6 The results indicated a shift in the minima for the oligomer compsred to that of native CTB This ~ugge~ted that the oligomer has less organized secondary structure than CTB, but this could not be correlated with a detectable difference in its activity On the other hand, the conformation of the monomer was radically different from both CTB and the oligomer, and this could be correlated with a loss of activity J Immunoreac-ivity of the ChimerA
Previous immunoblotJ demon~tr~ted that the Vl782 chimera was antigenic for antisera either to CTB or to GTF
~owevor, it wa~ not clear that this protein was immunogenic or that anti~erum raised to the chimera would recognize the native GTF enzyme iIn order to determine this, replicate blotJ were prepared containing CTB, GTF, and chimera The blotJ were probed with antiserum to either CTB, GTF, or chimera The re~ult~ demonstr~ted that the chimera elicited ~ntibodi-J which r-~cted with CTB and to GTF ~Fig 7) The r-~ctlon of th- ~nti-ch~m-r~ ~ntlbody with GTF WaJ weaker th~n that produc-d by th- ~ntl--rum to GTF, but thi~ WaJ
xp Ct-d ~lnc- th- ~ntl-chlmer~ antlbody l~ ~poclflc for r-l~tlvely tiny r-gion of the lS0 kD~l enzyme ~lbltlon o~ ~n~y e Act~vity Antl~-rum to th- chim r~ w~ evaluat-d for lt~
pot-ntlal to lnhlblt gluco~yltr~n~f-r~ ctlvlty ~ vitro Th- ~p clficity of th~ y w~ v rl~ f~r- p rforming th- lnhibition ~tudi-~ Extr~c-llul~r protein- of ~ mutans GS-5 w re ~ y d for glucoJyltran~fer~- activity by m ~urinq th- incorpor~tion of l4c-(glu)-~ucro-e into glucan polym r Tot~l polym r W~J preclpltat-d in m thanol ~nd coll-ct d on gl~oJ fib~r filtorJ In ordor to detormine if nzym ~ctivity was ~ctually being measured, the a~Jay was verified in two w~ys (A), glucan synthesis was shown to increase linearly over 20 hours of incubation; (~)~ the SUBSTITUTE SHEET

wos1/07s~s PCT/US~

~91 ~6 amount of glucan synthesized was inversely prc?ortional tO
the ~mount of protein used Sampling was performed in triplicate The mean counts per minute for each sample was subtracted from a background control containing PBS It was demonstrated that the assay increased linearly over time and that dilution of the enzyme resulted in a decrease in polymer form~tion The enzyme preparation was diluted 1 2 in either PBS
~control)~ pooled normal rabbit serum (N ~ S ), rabbit anti-GtfB serum, or rabbit anti-chimera serum The samples were then preincubated at 37C for 1 h before performing the assay Total glucan was mea~ured after the reaction proceeded for 18 h at 37C All samples were performed in triplicate Pre-incubation of the enzyme in undiluted antiJerum to either the chimera or GtfB resulted in a reduction in total glucan synthesis, relative to the control Pooled normal rabbit serum (NRS) did not inhibit enzyme activity In order to assess the kinetics of inhibition, enzyme activity wa~ moasured over time The results d-monJtr~ted that the antl~erum to GtfB decreased the r~te of total glucan ynth~ (water ooluble + water-in~oluble), but did not pr-v-nt ~ynth--i- of glucan~ (Flg 8) This ~ugg-~t-d that th- inhlbltion 1- probably due to steric hind-r~nc- rath-r than diJruption of the active ~ite of the nzym Th- antl--ra w r- compared for their lev-l of lnhibitlon by dilutlon Anti--rum to lth-r GtfB or the chim ra was dllut d ln PBS prior to prelncubation wlth tho n~ym Th- ynth--is of total glucans w~ m a~urod after th- r ~ction had proc--d~d for 8 h at 37C Sampling was p r~orm d ln trlpllcat- R ~ultJ indlcat d that the ability o~ th- ~ntl-chim ra anti~-rum to inhlblt actlvlty waJ
appro~ t-ly half that of the anti-GTF antl~-rum Again, thls w~ not ~urprl~lng due to the l~mited ~pecificity of the antls- D to the chimera Water-insoluble glucan~ have been implicated as having a greater role in the accumulation of S
mutans to the tooth Jurface than water-~oluble polymerJ In SUBSTITuTE S~ E~T

: .' ' '', :.. ~ . - ', , ~ ' ' . . . ~ , :
: :
. . . ~ .
. ~ . ,~ , , ,. . -:
~' . ., , , ,~, wo9l/o79~s PCT/US~/~
~9~ ~6 38 -order to determine if the antisera had any effact on the type of glucan polymer formed, water-insoluble assays were performed (FLg 9) The results clearly demonstrated a dr~m~tic effect on the synthesis of water-insoluble glucans W~ter-~oluble glucans comprise most of the total polymer synthesized, and the antiserum to GtfB inhibited this enzyme activity by approximately 50% However, the formation of water-insoluble glucans was inhibited greater than 90% by the antiserum The effect of the antiserum on fructan synthesis was determined as well (Pig 10) The results demonstrated that the antisera were also able to inhibit total fructan synthesis, although much less than for glucans This result was unexpected, but upon comparison of the sequence data for atfB l and fructosyltransferase, the presence of two tripeptides common to both peptides (phe-asp-asp; ala-trp-asn) was revealed The antisera to the chimera may partially recognize these sequences, and thus, interfere with the synthe8is of fructans The data has shown that antiserum to peptides of GTP can inhibit glucan synthesis and suggests that these anti-peptide antibodies may impair the abil~ty of S mutan~ to colonize tho tooth ~urface III DI8C~88ION OF RES~TS
Puriflcation o~ the V1782 ehim ra wa~ gr-atly ~lmpll~l-d by two ~aCtor~ FlrJt, by ~eer-ting th- ehimera lnto th- p-rlpla~m of E coli, the contents of the periplasm could b r~lea~ed from the rest of th- cell and th-n ~-par~t d by c-ntrlfugation This r-mo~ d th- ma~ority of pot-ntlal eontam~nant~ Seeond, th- high affinity of CTB for GNl ganglio-lde wa~ u~ed to purify th ehim ra-by af~lnity chromatography This provid-d a rapid m thod of romoving the ma~ority of the remaining contaminants from the chimera As Tabl~ hows, a large amount of prote~n could be purified by a ~ingle lution from the column, which rend-red this ~y~t-m am nable to vacc~n--scale production of prot-in ~ost of th- high molecular weight proteins ob~erved in the eluate are prob~bly oligomeric forms of the chimerA that were not separated by reducing SDS-PAGE, becau~e they were reactive in TITUTE SHEEl`
.. . . . . . . . .......... .. . ~ . .
.. ` . . `. `:`:`. . . .- -. . . . : . . .

` . . ... . .. .

WO 91tO7979 PCl'/US90/06811 :

_ 39 _ 2~3~ 06 immunoblots The v1555 chimera was previously ~hown to bind to GMl Elution of the protein was effective at pH 4 0, which was higher than the pH required to elute native CT from the ~ffinity column (Tayot et al, 1981) It was postulated that fusion of CTB to a relatively large protein altered its affinity for GMl In the V1782 chimera, fusion of a 15 amino acid ~eguence to CTB did not appear to alter its affinity for GMl, since it could only be eluted from the column at pH 3 0 Elution at this pH did not appear to degrsde the protein Thus, the GMl binding domain of CTB appeared to have been unaltered by fusion of the peptide sequence to CTB The present inventor~ have di~covered that the u~e of large pept$de fragments may affect the conformation of the GM
bindlng domain of CTB
The primary ~tructure of the V1782 chimera was verified by sequence and compo~itional analysis The amino acid composition of the chimera agreed with that predicted by the nucl-otide equonc- data The sequence of the first 25 amino acid r-~idue~ of the mature form of the chimera wa~
d-t-rmln-d, and v-rlfied that the Qm~A l-ader was cleaved at th- it- normally r-cogniz-d by E coli (Ghrayeb et al, 198~) Cyanog-n bro~id- cl--v~g- provld d conv-nient m thod o~ l~ol-tlng th- C-t-rminal dlp-ptid- fragm nt of the chlm r~ and comp rlng lt to th~t of CTB A~ predicted, the C-t-r~ln~l nd of th- chimera wa~ no dilf~r nt from th~t of C~B, thu- conflrmlng th~t tran-lation of th- chim r~ wn-t-rmln-t d t th- top codon norm lly r-cognlz d ~or CTB
G l ~lltr~tlon chrom~tography of tb- G~l -purlfl d ch~m r- ~-p r t d th- prot-in into two fr ction- B --d on SDS-PAGE ~nd immunoblottlng analy-l~, it wa~ concluded that th- chim r~ w~ -par~t d into itJ monom ric and oligom ric ~orm~, ~inc- the am immunoreactive protein~ appeared in -ch ~r~ctlon N-tiv- CTB is compo~ d of five id-ntical ubunit-, arrang-d ln a non-covalent a~sociation (Gill, 1976) Each ~ubunit contains an intramolecular diJulfide bond which is crucial for-CTB to bind to GMl (Ludwig et al, . - ,- - . , ; - :.-- :
. .
: . ' . . . ~ . .; . , , . .: .
.. . . . . ...

w09l/079~9 PCT/US~/~

~,Q~ Q ~

1985) Assay of the monomer by ELISA, which is dependent on the protein binding to GMl-coated microtiter wells, demonstrated almost no activity However, the monomer was ~tLll lmmunoreactive in a We~tern blot, suggesting that it l~cked the di~ulfide bond required for GMl binding This was verified by reacting s~mples of the monomer snd oligomer with lodoacetamide Only the monomer was found to contain reduced forms of cy~teine In order for the monomer to co-elute from the affinity column, it mu~t have been able to bind to GM
~he ~ubunits of CTB ~re reversibly dissociated by acidific~tion (Hardy et al, 1988), and it may be that a fraction of the chimera was di~sociated and reduced during elution from the column The monomer c~n be renatured by gr_dually dlalyzlng lt _w_y from 8 M ur-a under alkallne condition~ Ren_turatlon resulted in the formation of oligomers and re_ctlvlty in ELISA The dl~ulfide bridge of CTB appears to ha~e a dra~tic effect on lt~ conformation and function, since the _bsence of it preclude~ binding of chim r_ to GMl and forming oligomor~ The re~ultJ reported her- wlth the chimer_ are con~l~tent wlth those reported for n_tlv CT8 _nd ~ugg-~t that addition of a peptide to the N-t-rmin~l nd of CT8 ha~ minimal ef~ect on it~ ablllty to bind to it~ llgand or to form ollgom r~ ~
Att-apt~ to ~timat- th- mol-cular w ight of the chlm ~ und-r r ducing condition- by SDS-PAGE r-veal d the pr-~-n¢- of two formJ of the chi r_ ~16 4 _nd 17 4 kDal) ~h-~- ~o~J w-r- both immunor _ctiv _nd ~uch larg-r th_n pr dlct d (14 q kD~l) R ductlon ~nd _lkyl_tion of th-monom r or ollgom r dld not _ff-ct the p~t~-rn-ob--rv d by SDS-PAG~ At flr~t, th--- r ~ult~ wer- thought to d-~on-tr,t- that th- chimer_ con~isted of two diff-r nt prl~ry ~tructure~, with ~dditional ~m- no acldJ at either the N or C-t-rminu- of the larger ~p-cl-J How ver, the reJult from ~d~n d-gr~dation cl-arly ~tabll-h-d that only th-exp ct-d N-t-rminal Je~u-nce w~ obJerved Likewi~e, the C-t-rminu- w~ cl-arly demonstrated to be only that expected from th- proposed construction Therefore, it wa~ concluded ~!II~TITIIT~ CiU~T

... . . - . . . .- , , . . . ,: ~.. - ., W091/07979 PCT/US~/~

2~s9l 06 that the appearance on SDS gel must be due to higher level ~tructur~l effects This was confirmed, since fractionation of the ~ampleq by SDS-PAGE in 8 M urea reduced the pattern observed to a single protein of the expected size Other species of protein were observed in samples of oligomer, but these were postulated to be undenatured, multimeric forms of the chimera Thu~, the chimera appeared to exist in two preferred conformations under reducing SDS-PAGE which were not observed with CTB This suggested that the addition of the peptide to the N-terminal end of CTB has changed the way the protein fold~ in such a way that SDS alone did not completely unfold it under the conditions normally used However, the ~ddition of a strong chaotropic agent such as ure~ was able to completely denature the structure of the protein The ability of the chimera to form oligomer~ and to bind to GNl suggested that the conformational differences ob~erved did not ~iqnificantly affect the physical properties of the CTB moiety However, whether theJe difference~ had a ~lgnlflcant effect on the ad~uvant propertles of CTB ha- yet to b- d-termin d An~ly-~- of th- prot-ln by circular dlchrol-m rev~ d om- dlff-r-nc-~ ln th- conform~tlon of CT~ ~nd the oll~o~ rlc prot-ln How-v-r, th- conformatlon of th- monomer W~J r~dlc~lly dlff-r-nt Th- change ln structure of the ~ono~ r w~- corr ~-t d wlth the lo-~ of G~l-blndlng activity ~nd ~bll~ty to a--oci~t- w~th other zubunit- Th -- re~ults de~on-tr~t d th~t th- addltlon of extr~ a~lno ~cld~ to th-N-t-~mln~l nd of CTB ha- min~m~l ff-ct on th- conformatlon of th- prot-ln or it~ functlon Immunoblotting ~naly~ nd nzym inhlbition ~tudie~
h~v- ~hown th~t th- gtfB l poptid- wa~ immunog-nic, ~nd that ~ntl--rum to th- p ptld- could recognlze th- natlve prot-in ~h- gtf~ l p ptld- wa~ d-rived from the ~equence of gtfB
n~ym , whlch c~t~lyz-~ the formation of primarily water-ln~oluble glucan~ from sucrose It wa~ not known if ~nti~erum to tho gtfB l peptide would inhibit enzyme activlty However, antlsorum to gtfB l not only inhlbited SUBSTITUTE SHEET
.. .. " ~ . .. , .,, .. , , . ... ... . .. ., . " ,, , ". .. ... . . . . .. ... . . . . .. . ..
.. ., i, - - - ~
. ~ , ` . . .

W091/07979 PCT/US~/~

~Q~ 42 _ ~ , the formation of water-insoluble glucans, but also the synthesis of soluble glucans as well Soluble glucans are produced by the product of the atfD gene, which has recently been cloned (Hanada and Kuramitsu, 1989), but not yet sequenced Another enzyme, the product of the atfC gene, cat~lyzes the formation of both soluble and insoluble glucans Comparison of the nucleotide sequence data for atf and atfC demonstrated that the gtfB l peptide was located in regions of both gene products that shared sianificant homology with one another Although soluble glucan ~ynthesis was somewhat reduced, compared to the control, insoluble gluean synthesis was almost completely eliminated The rea~on for this difference is unelear, but it suggests that the anti~erum inhibited the atf~ gene product more than the other gluco~yltransferases ~his m~y be due to minor d~fferenees in the conformation of the peptide between the gene produets, whieh affeet antlbody binding It may indie~te Jignifieant ~truetural differonee- between tho nzym-~ The peptide m~y be more expo-ed on tho surf~ee of GtfB, where it i~ _eee~sible to antibody, eompared to the oth-r on~ym J Alt-rn~tiv~ly, the peptido may play _ great~r rol- ~n forwatlon of th- aetlv- lt- of Gt~B, eompar-d to the oth-r n~ym -, ~n whieh ea-- th- antlbody aff-et~ lt~
~truotur- .
St w~ll b appar-nt to tho-e ~kill d ln tho ~rt that varlou- modlfleation~ and variatlon~ ean b mad- ln the proe----- and produet- of th- pr --nt inv ntlon Thu~, lt i8 lnt-nd-d that th- pr-~-nt lnv-ntion eov-r th- modiflcation-and variAtlon~ of thi~ invention provid d th-y com wlthin th- eop- of th- app nd d elaim~ And th-lr qulval-nt~

SUBSTrrUTE S~

- . . ' ' '.

WO9l/07979 PCT/US~

Table I. Glucosyltransferases of Streptococcus mutans.

Size of Cloned Glucan Reference GeneGene Product Synthesized (kDal) . . _ gtB 150 water-insolublel Aoki et al, 1986 gtfC 140 water-solublel Hanada et al, 1988 water-insolublel gtfD 155 water-~oluble2 Hanada et al, 1989 ~ nzyme does not reguire dextran pr.imer for activity.
2Regulre~ dextr~n primer for enzyme activity.

~tl~CTITl1TE SHEE'r .. .. . . .... .. . . . " ,~ .. . . . . .

wogl/o7g79 PCTIUS~/~

~ 44 -Table II Bacterial ~trains and plasmids B~cteri~ or Pl~mLd Genotypel Reference . col~
HB101 1euB6proA2ara-14xyl-5 Boyer and galRmtl-llacYl thi Roulland-Dussoix, supE44recA13rpsL20 1969 V1555 Ap, ~c Present Application V1619 Ap Pre~ent Application V1782 Ap Pre~ent Application V1784 Ap Present Application S ~ut~nJ
GS-5 Present Application ~l ~f d pJBK30 Ap, Te Raper et al, 1984 pHR7 Ap Pro-ent Applie~tion plNlIIQmpA2 Ap Ghr~y~b et al, 1984 pVAlS~2 Ap, TePr---nt Applieation pVA1543 Ap, TePr--ent Applie~tion pVAlS~4 Ap, TePre~ent Applie~tion pVAl~S~ Ap, TePr--ent Applie~tion pVA1599 Ap, Te, r Pr--ent Applieation pVA1662 Ap, TePr--ent Appl$e~t$on pVA1782 ApPre~ent Applieation pVA1784 ApPre~ent Applieation ~Con ~ of ~nt$blotie~ u-ed in all med~ were~ Ap - 50 ~g/ml umpieill$n; Te - 20 ~g/ml tetr~eyeline WO 91/07979 PCI'/VS90/06811 .
7"
_ 45 _ 2~`3~Q~ `

~ ~o ~ ~ ~
o~ ~ o ,~
o o , ~
c: ~ In 8 ~
C~
o ~ ~o u ~ ~ O s t~ O O ~ bO~ E
o-_l ~ u ,~ ~ O
c 8 .. , a~ o :~ o ~ ~ 3 a~
O
.~
~ o U CU~ ~ 8 .~ :~o o 1~ ~ ~ ~D ~D ~ ~ C ~ S ~ ~
.~ . . '~ -~ ~ 0 U ~:
U O ~ ~o ~ ~ 0 C~ ~ E
u~ o~ C 3 ~P~ ~ I~ ~ ~ Q ~
u~ ~ ~ 8 N ~~ ,C U /1 co _I c ~ ~ c ~ c~
U _~ ~ C ~ O
S~ ~ m ~ 0~ ~I J
~ ~ 0~ Z~
~9 ~ ~ o ~ 9~ ~ ~ ~ C u ~ ~
J ~ _1 J.C 0~ Oc 9~ .,C O ~ ~j J @ 3~
a 3 - O O ~ ~ O ~ ~j c ICi g-- NO ~ C $ ~ g ~ ~ UO U ~ ~U W
~ ~ ~ .C gW ~ C
9~ O :~ ~ ~ ~o J3 ~O 8.
n~ o~,~ P ~ P ~ P

... ~ / . . . . ,.. ` .. , . ~ ; - ., i ,., . . , .. ; ,,,: . . . . . .

. . . . .. . . ... ~ .~ . .. . . . . .

WO91/07979 PCr/US90/068 ~ 46 -Table rv Ami no acid compo~ition of the cyanogen bromide peptide~
Number of Residues' Amino _ CTB Chimera AcLd Peptide A Peptide 8Peptide A Peptide B
Asp 0 96 (1) 2 0 (2)1 1 (1) 2 0 (2) Thr 1 0 (1) 1 3 (1) Ser 1 7 (2) 1 4 (2 Glu 5 7 (5) 5 5 (5) Pro N D (1) N D (l) Gly 2 3 (2) 2 2 (2) Ala 1 0 (1) 2 7 (3)1 0 (1) 2 9 (3) V~l 1 8 (2) 1 9 (2) Met o O (1) o O (1) Ile 3 4 (5) 3'3 (5) Leu o O (0) o O (0) Tyr ~ () ' () Phe 1 7 (2) 1 9 (2) Hi- 0 9 ~1) 1 1 (1) Ly~ 2 8 (3) 2 7 (3) Arg 1 1 (1) 1 1 (1) lThe values reported are for 24 h hydrolysates The value of a~partlc acld wa~ a-~igned a~ 1 0 and 2 0 for peptides A and B, r-~p ctively All other v~lues were determined relative to thi~ Valu-- ~n parenthe-e- are tho~e oxpected from the known ~quence of th protein Methionine wa~ converted to homo~-rine by th- r-action with cyanogen bromido, and wa~ not d-t- d n-d b-c~u~- homo--rin- coelute- with glutamlc acid ~rol~n- wa~ not d-t-rmin-d ince it doe~ not react with the o-phth~l~ld-hyd- u--d ~or d-t-ction ell~sTITUTE SHEE~L
` . .` .` ` . , '.' '.` ' - ''. ', ' ' , ' - -' , . ,., . . ;,.,. ... ,,.. . . . ,... .. . ... ;...~ ... .. .
.. ~.. . . ., . , .~. . . .. .. ...... .... .. . ..... .. . ..... . . . . . ... .

W091/07979 PCT/US~/~
~ - 47 - 2~6~Q~
LITERATURE CITED
Aoki, H , Shiroza, T ., Hayakawa, M ., Sato, S , and Kuramitsu, H. (1986) Cloning of a Streptococcus mutans glucosyltrsnsferase gene coding for insoluble glucan ~ynthesi~ Infect Immun 53 587-594.
A~em, K , Cornish-Bowden, A , and Cole, J ( 1986) A
comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes Microbios 47 53-66.
Ayakawa, G , Bleiweis, A., Crowley, P , and Cunningham, M (1988) Heart cro~-reactive antigens of mutans streptococci ~hare epitopes with group A streptococci and myosin J Immunol 140 253-25~.
Ayakawa, G , Siegel, J , Crowley, P , and Bleiweis, A
~1985) Immunochemistry of the Streptococcu~ mutan~ ~HT cell membr~nes detection of determinants cro~-reactive with human heart ti~ue Infect Immun 48 280-286 BAhn, A , Shklair, I , and H~yashi, J (1977) Immunization with dextran~ucra~es, levansucrAses, and glyco-idic hydrolases from or~l streptococci II
Immunlzatlon with glucosyltranJfer~eJ, fructo~yltr~nof-r~ , and glyco~idic hydrol~-s from oral ~tr-ptococci ln monk-y- J D-nt R-- 56 1586-1598 ~ rgm 1-r, ~ ~nd L-hn-r, ~. (1983) L~ck of ~ntibodie~
to hum n h-~rt tl--u- in ~-ra of rh--u~ monk-ys immuniz-d wlth Str ptococcu- mutan- antigens and comp~r~tive ~tudy with r~bbit ~nt$--r~ Inf-ct Immun 40t1075-1082 B~---n, D ~nd Fl-ch-tt$, V ~1988) Influ-nce o~
lntr~n~-~l immunl~tion with ynth-tlc p ptid-- corr -pondlng to con--rv d pltop-- of M proteln on muco-~l~colonlzatlon by ~roup A tr ptococci Infect Immun 56 2666-2672 ~ -tl-y, M , Mlller, V , ~nd Mek~l~nos, J (1986) G n-tic- of bact-rlal entorotoxln~ Ann Rov Nlcrobiol 40~577-605 Boy-r, H and Roulland-Du~oix, D (1969) A
complementation an~lysi~ of the restriction and modification of DNA in E~cherichia coli J Mol Biol 41s459-472.

~O~T~ IT~ ~U~

~., .,. ~ . .
., ~ ., ., - - - ; . ..

wo91/07979 PCT/US~
2069~ 48 _ Carlsson, J (1967) A medium for isolation of Streptococcus mutans Arch Oral Biol 12 1657-1658 Ch~llscombe, S and Tomasi, T (1980) Systemic toler~nce and secretory immunity after oral immunization J
Exp Med 152 1459-1472 Clarke, J tl924) On the bacterial factor in the ~etiology of dental caries Br J Exp Pathol 5 141-147 Crabbe, P , Nssh, D , Bazin, H , Eyssen, H , and Herem~ns, J (1969) Antibodies of the IgA type in intestinal pla~ma cells of germfree mice after oral or parenteral immunization with ferritin J Exp Med 130 723-739 Cuatrecasa~, P (1973) Gangliosides and membrane receptors for cholera toxin Biochem 12s3558-66 Curti~, R (1986) Genetic ~nalysis of Streptococcus mut~n~ virulence ~nd pro~pects for ~n ~ntic~rie~ vacclne J
Dent Re~ 65s1034-1045 Dertzbaugh, M and Macrina, F (1989) Molecular genetic ~pproache~ to the ~tudy of oral microflor~ In My~r~, H
(Ed ) New Technologles in Dental ReJe~rch S Rarger, B~el (ln pre~) El~on, C and E~lding, W (1984a) Generalized ~y~temlc ~nd muco~l immun$ty ln mlco ~fter muco~l stlmul~tion with chol-r~ toxln J Immunol 132s2736-2741 El-on, C ~nd E~ldlng, W (1984b) Cholor~ toxln f-odlng dld not lnduc- or~l tol-r~nc- ln mlce ~nd ~brog~ted or~l tol-r~nc- to ~n unr-latod protoln antigon. J Immunol 133~2892-2897 ~ -nwlck, B ~nd O-burn, B (1986) V~ccln- pot-ntl~l of H~-~ophllu~ pl-uropn-umon~- oligo-~cch~rld -tot~nu~ toxoid con~ug~t-~ Inf-ct Immun 54tS83-586 Fuhrm~n, J ~nd C-br~, J (1981) Sp cl~r f-~ture~ of th- prlm~ng proco~ for ~ ~ecretory IgA ro~pon~o J Exp N d 153~534-544 G~rnl-r, J , O-guthorpe, D , ~nd Rob~on, B (1978) Analy~1~ of the ~ccur~cy and implicatlon~ of ~imple method~
for pr-dlctlng the ~-cond~ry ~tructure of globul~r proteins J Mol Biol120 97-120 . -c~~
, ., ~ . . . . . .. . , - . - . . . ... . . . .

.. . ~ :.; . .:. . - - -wO91/07979 PCT/US~/~
~ _ 49 _ 2~69~0S
Geysen, H., Tainer, J., Rodda, S., Mason, T., Alexander, H., Getzoff, E., and Lerner, R. (1987) Chemistry of antibody binding to a protein. Science 235:1184-1190.
Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Masui, Y., and Inouye, M. (1984) Secretion cloning vectors in Escherichia coli. EM~0 3:2437-2442.
Gill, D. (1976) The arrangement of subunits in cholera toxin. Biochem. 15:1242-1248.
Go, ~08 Altos, CA, pp. 271-287.
Welch, R., Jones, K., and Xacrina, F. (1979) Tr~nsferable lincosamide-macrolide re~iqtance in Bscteroides.
Pla~mid 2:261-268.
Westhof, E., Altschuh, D., Moras, D., Bloomer, A., Mondragon, A., Klug, A., and Van Regenmortel, M. (1984) Correlation between segmental mobility and the location of ~ntigenlc determinant~ in protein~. Nature 311:123-126.

C~ITUlE SHEE~

, : - . ~ "``` .: . ``: , - .

. . ` . :

. -. ~ . . .. . - .
.

Claims (19)

What is claimed is:

1. A chimeric peptide comprising at least a portion of the B subunit of cholera toxin and an epitope region of a desired antigen fused to the N-terminal end of the B subunit of cholera toxin, said epitope region comprising an antigenic determinant of the desired peptide.

2. A chimeric peptide as in claim 1, wherein said epitope region comprises no more than one hundred amino acids.

3. A chimeric peptide as in claim 2, wherein said epitope region comprises about 15 to 20 amino acids.

4. A vaccine for administration to a subject to elicit an immune response in the subject to a desired antigen, comprising a chimeric peptide having at least a portion of the B subunit of cholera toxin and an epitope region of a desired antigen fused to the N-terminal end of the B subunit of cholera toxin, said epitope region comprising an antigenic determinant of the desired peptide, and a pharmaceutically acceptable carrier.

5. A vaccine as in claim 4, wherein said epitope region comprises no more than one hundred amino acids.

6. A vaccine as in claim 5, wherein said epitope region comprises about 15 to 20 amino acids.

7. A vaccine as in claim 4, wherein the B subunit of cholera toxin enhances the immune response of said epitope region when administered orally.

8. A vaccine as in claim 4, wherein said vaccine is administered to help prevent dental caries and said epitope region includes a segment of the glucosyltransferase B
protein no greater than one hundred amino acids long.

9. A vaccine as in claim 8, wherein said epitope comprises the following amino acid sequence: .

10. A substantially pure DNA sequence coding for a chimeric peptide comprising at least a portion of the B
subunit of cholera toxin and an epitope of a desired peptide fused to the N-terminal end of the B subunit of cholera toxin, said epitope region comprising an antigenic determinant of the desired peptide.

11. A substantially pure DNA sequence as in claim 10, wherein said epitope region comprises a 15-20 amino acid sequence of the glucosyltransferase B protein.

12. A vector containing the DNA sequence of claim 10, said vector being capable of expression in a host organism.

13. A vector containing the DNA sequence of claim 11, said vector being capable of expression in a host organism.

14. A vector comprising pVA1782.

15. A vector comprising one of pVA1542, pVA1543 and pVA1544.

16. A substantially pure epitope region of the glucosyltransferase B protein comprising the following amino acid sequence:.

17. A host organism transfected with the vector of claim 10.

18. A method of vaccinating a subject comprising administering orally to the subject a composition in a dosage sufficient to elicit an antibody response thereby raising antibodies in the patient directed against a given peptide, said composition including a chimeric peptide comprising at least a portion of the B subunit of cholera toxin and an epitope region of the given peptide to which an antibody response is desired fused to the N-terminal end of the B
subunit of cholera toxin, said epitope region being an antigenic determinant of the peptide to which an antibody response is desired, and a pharmaceutically acceptable carrier.

19. A recombinant-DNA mediated method for the production of a chimeric peptide comprising:
(a) preparation of at least one portable DNA
sequence encoding at least a portion of a B subunit of cholera toxin and an epitope capable of eliciting an antibody response in a patient, the DNA encoding said epitope being at the 5' end of the DNA portion encoding the B subunit of cholera toxin;
(b) cloning the portable DNA sequence into at least one vector capable of being transferred into and replicated in a host microorganism, said vectors containing elements for the expression of the chimeric peptide encoded by the portable DNA sequences;
(c) transferring the vectors containing the portable DNA sequences into a host microorganism capable of producing at least one chimeric peptide under the direction of the vector;
(d) culturing the host microorganism under conditions appropriate for maintenance of the vectors and synthesis of the chimeric peptide; and (e) harvesting the chimeric peptide.