~) INSTITUTPASTEUR/ELsEVIER Paris 1990
Res. MicrobioL 1990, 141, 971-979
R E C O M B I N A N T C H O L E R A TOXIN B S U B U N I T A N D G E N E F U S I O N P R O T E I N S F O R O R A L VACCINATION
J. Sanchez (l. 2), S. Johansson (l), B. LOwenadler (3), A.M. Svennerholm (l) and J. Holmgren (1) (i) Department of Microbiology and Immunology, University of GOteborg, S-413 46 Gothenburg (Sweden), (2) Institute for Public Health, Center for Research on Infectious Diseases, Apdo. Postal 222 Oficina Central Correos, Cuernavaca 62000 Mexico, and (3) KabiGen AB, Strandsbergatan 49, S-ii2 87 Stockholm
Summary. The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (noncholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNApolymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coil enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 izg/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a nearnative conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa. However, only hybrid.protein presenting the STa peptide with a free carboxy end was able to also react with another available STa mAb. Our results suggest that even minor modifications of a given antigenic zegion may lead to complete epitope hiding and/or to its lack of antibody reactivity. Alternate positioning of such peptides in the carboxy end of the CTB protein was
Correspondence:Dr J. Sanchez,AppartadoPostalNo 222,Oficinade CorreosNol, C.P. 6200,Cuernavaca, Morelos, Mexico.
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found to assist in antibody recognition and is proposed as a means to help exposure of some foreign epitopes by CTB fusion proteins. The results may be of significance for the development of recombinant oral vaccines based on gene fusions to CTB or to the closely related B subunit of LT. KEY-WORDS:Cholera, Toxin, B subunit; Oral vaccination, Gene fusion.
Introduction. Vibrio cholerae of serogroup 01 may cause severe diarrhoeal disease in infected individuals by releasing cholera toxin (CT) which induces active electrolyte and water secretion from the intestinal epithelium. The prototype bacterial enterotoxin CT is a complex protein built from two types of subunits: a single A subunit of molecular weight (MW) 28,000 and five B subunits, each with a MW of 11,600. The B subunits are aggregated in a ring by tight non-covalent bonds; the A subunit is linked to and probably partially inserted in the B pentamer ring through weaker non-covalent interactions. The two types of subunit have different roles in the intoxication process: the B subunits are responsible for cell binding and the A subunit for the direct toxic activity (Holmgren, 1981). At the genetic level in V. cholerae 01 bacteria, CT is encoded by chromosomal genes for the A and B subunits. These genes have been cloned from several strains, and their nucleotide sequences have been determined (Mekalanos et al., 1983; Kaper et al., 1984; Gennaro and Greenaway, 1983; Sanchez and Holmgren, 1989). The A and B subunit genes of CT are arranged in a single transcriptional unit with the A cistron ( c t x A ) preceding the B cistron (ctxB) (Mekalanos et al., 1983). The synthesis of CT is positively regulated by a gene, t o x R , that increases c t x expression manifold (V.L. Miller and J.J. Mekalanos, 1984). Vaccination against cholera by parenteral injection has yielded only modest shortterm protection (usually less than 50 °7o protection for less than 6 months). Given the strictly mucosal surface location of the cholera infection, interest has therefore turned to development of oral vaccines that stimulate mucosal intestinal immunity more efficiently. Special attention has been drawn to CTB pentamers as a component (together with killed vibrios) of such oral cholera vaccines (Holmgren et al., 1977). CTB is an effective oral immunizing agent which recently, in a large field trial, has been shown to afford protection against both cholera and diarrhoea caused by enterotoxigenic E. coil (ETEC) producing heat-labile toxin (LT) (Clemens et aL, 1986; 1988). To facilitate large-scale preparation of CTB for vaccine production, we have developed overexpression systems for CTB where the gene is under the control of strong foreign (non-cholera) promoeters. Previously, we have reported on expression of C T B gene by an inducible t a c P promoter (Sanchez and Holmgren, 1989) which also expresses high levels of a recombinant fusion protein (Sanchez et al., 1988). We here report on expression of C T B by a constitutive t a c P and by the T 7 RNApolymerase promoter (Tabor and Richardson, 1985).
aa Abs CT CTA CTB
= = = = =
ctx ctxA ctxB
= = =
ELISA = ETEC = HIV =
amino acid. absorbance. cholera toxin. CT A subunit. CT B subunit. cholera toxin gene. ctx A subunit cistron. ctx B subunit cistron. enzyme-linkedimmunosorbentassay. enterotc::igenicEscherichia coll. human immunodeficiencyvirus.
1PTG = LT = LTA = LTB = mAb = MW = SDS-PAGE= STa
isopropylthiogalactoside. heat-labileenterotoxin of E. coll. A subunit of LT. B subunit of LT. monoclonalantibody. molecularweight. sodium dodecyl sulphatepolyacrylamidegel electrophoresis. = methanol-soluble heat-stable enterotoxin of E. coli.
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Recently, CTB has also attracted great interest as an antigen carrier for development of other, mainly oral vaccines (Holmgren et al., 1989). Construction of"foreign antigen"-carrying CTB proteins by genetic manipulations on CTB (or analogous construction of hybrid proteins based on the closely related B subunit of the heat-labile enterotoxin of E. coil (LTB)) has the advantage that the extra epitope is bound in a defined form and at a defined place in the molecule. This is likely to result in hybrids retaining many of the native properties of the fused proteins, including binding of specific antibodies. We have described the gene fusion of a 10-amino-acid (aa) peptide analogue of the E. coli heat-stable enterotoxin (STa) to the amino end of CTB. Once joined to the B subunit, this decapeptide was efficiently recognized by a neutraiizing monoclonal antibody directed against native STa, and in further support of its proper folding, the hybrid protein also exhibited excretion by V. cholerae host cells, pentamerization and binding to a GMI ganglioside much like the native CTB protein (Sanchez et al., 1988). Likewise, genetic fusion of a glucosyl transferase epitope from Streptococcus mutans to the amino end of CTB (Derztbangh and Macrina, 1990), and fusions of hepatitis B epitopes to the carboxy terminus of LTB (Sch/kiel and Will, 1989) have been reported to result in immunogenic hybrid proteins able to give rise to anti-epitope antibodies. As further extensions of our previous efforts to develop recombinant overexpression systems for CTB associated with the potential for prod,Jction of hybrid proteins, we here report on the genetic fusion of various STa-related peptides at either the amino or carboxy ends of CTB, with analyses of the foreign epitope accessibility as monitored by reaction with specific antibodies and by in vivo immunogenicity studies. Materials and methods. Gene modifications at the C T B 3' and amino ends. -- To facilitate expression of the CTB gene by 7"7 and the inducible tacP promoters and to allow productive gene fusions at the amino end of CTB, we introduced synthetic oligodeoxynucleotides which made possible replacement of the natural CTB leader peptide by that in the closely related protein E. col; LTB protein (Sanchez and Holmgren, 1989). Inclusion of these synthetic oligodeoxynucleotides incorporated single SacI and Xmal sites at the junction between the leader peptide and mature CTB. Gene modifications at the C T B carboxy end. - - To be able to fuse synthetic oligonucleotides at the carboxy end of CTB, we synthesized oligodeoxynucleotides encoding the last 24 aa of the CTB gene carboxy-end and introduced single SacI, XmaI and SpeI sites. In addition, the CTB carboxy end gene was made to be followed, after the stop codon, by a unique HindIII site in order to facilitate further subcloning into other expression vectors. Cloning of the synthetic CTB gene carboxy end was undertaken in plasmid pUCI8. Vector-directed expression of CTB. -- Controlled expression of the CTB gene by the constitutive tocP promoter was achieved by subcloning an EcoRI-HindllI DNA fragment into plasmid vector pKK223-3 (Pharmacia, Uppsala, Sweden). Expression by the 77 RNA-polymerase promoter was obtained by first subcloning the CTB gene as an EcoRI-HindllI fragment into plasmid pT7-5 (encoding resistance to ampicillin) followed by coresidence in the same strain with the compatible plasmid pGP1-2 (encoding resistance to kanamycin) (Tabor and Richardson, 1987). tacP.directed expression of CTB fusion proteins. - - For expression of CTB gene fusion proteins under the inducible taeP, a derivative of plasmid pMMB66 (Ffirste et al., 1986; Sandkvist et al., 1987; Sanchez and Holmgren, 1989) was used. Expression under the constitutive tacP promoter was achieved by subcloning the hybrid gene into pKK223-3. Gene fusions at the CTB gene amino end. - - For gene fusions at either the amino or carboxy end of CTB, double-stranded oligonucleotides with single-stranded Sa¢I
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and X m a I compatible ends were used. Typically, fusion of the inserted oligonucleotide was carried out so as not to reconstitute at least one of the restriction sites used for cloning. Thus digestion of the ligation mixture with the restriction enzyme for that site resulted in linearization of plasmid molecules which had not incorporated the added oligonucleotides, which reduced their aptitude to transform. Gene fusions at the CTB gene earboxy end. - - For insertion of oligonucleotides at the C T B 3' end SacI-XmaI compatible double-stranded oligodeoxynucleotides were first inserted at these unique sites in the pUC18 derivative containing a synthetic 3' portion analogue of the C T B gene (see above). After fusion, the now extended carboxy end portion of the gene was joined as a TaqI.HindIII fragment to the rest of the C T B gene which was excised as an EcoRI-TaqI fragment. The compound EcoRI-TaqI-TaqI-HindIII construct was then cloned in pKK223-3 for expression under the tacP promoter. The oligonucleotides inserted were those encoding a 10-aa non-toxic STa analogue (Sanchez et al., 1988) and those encoding the complete 19-aa STa protein. Some of the STa-peptides fused at the CTB carboxy end carried a short three-amino-acids extension at the carboxy end (table II). To remove those extra amino acids, short oligonucleotides were introduced between the Sphl and SpeI sites to cause translation to terminate immediately after the last Tyr in native STa (table II). Expression of recombinant CTB and fusion proteins in V. cholerae. - - The V. cholerae host JS1569 (Sanchez and Holmgren, 1989) or isolated V. cholerae rifampicin-resistant derivatives were used for expression of recombinant CTB or CTBderived fusion proteins. The pKK223-3 derivatives or both of the plasmids of the T7 system were mobilized into V. cholerae by the aid of plasmid pRK2013 (Ditta et al., 1980) while the pMMB66 derivatives were mobilized from a strain holding RP4 in the chromosome (Simon et al., 1983). Selection of V. cholerae harbouring the desired plasmids was done by plating on rifampicin plus the selective antibiotic.
Enzyme-linked immunoassays for CTB and fusion proteins. - - GM I-ELISA for detection of the CTB antigen or the attached STa epitopes in the fusion proteins was carried out using specific monoclonal antibodies (mAb) or antisera essentially as described (Sanchez et al., 1988). Two mAb, 1:3 and 27:3, were used to detect "neutralizing" STa epitopes in the fusion proteins: both of these STa-specific mAb have been found to completely neutralize the infant-mouse toxicity of STa, yet they seem to recognize slightly different epitopes in the STa molecule (A.-M. Svennerholm et al., to be published) (see also "Results and Discussion"). SDS polyacrylamide gel eleetrophoresis (SDS.PAGE) and Western Blot Analyses. - - Protein samples were examined by SDS-PAGE followed by protein staining and also subjected to Western blot testing on nitrocellulose paper using standard procedures. Protein samples were prepared by affinity-adsorption to GMI-Spherosil as reported (Tayot et al., 1981 ; Sanchez et al., 1988) or by hexametaphosphate precipitation (Mekalanos et al., 1978). The electrophoretic separations were performed with or without prior boiling of the proteins in the SDS sample buffer to allow evaluation of monomers and oligomers, respectively (Hardy et al., 1988). Rabbit immunizations with CTB fusion proteins and antibody determinations. - - For immunizations, the STa-related CTB fusion proteins in bacterial culture supernatants were first isolated either by hexametaphosphate salting-out or by GMI affinity purification (Tayot et al., 1981) and dialysed against PBS. The approximate specific protein concentrations of samples used for immunizations were determined by ELISA using CTB as a standard. Male rabbits weighing 2-3 kg were immunized with 50-250 t~g of CTB equivalents of protein in each immunization. In total, 3-5 infections were given every 3-4 weeks, the first three with the antigen suspended in complete Freund adjuvant and subsequent ones given in incomplete adjuvant. Sera prepared from bleedings taken before the start of the immunizations and c.a. 2 weeks after the third or fourth vaccination were assayed for specific antibodies against CTB and STa by ELISA methods (Sanchez et al., 1988; Svennerholm et al., to be
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TABLE I. - - Expression of CTB by different promoters in V. eholerae. Strain
Promoter
JS1569 JS1569 (pJS162) JBK70 (pJS162) JBKT0 (pOP1-2), pJS7522) JBK70 (pGP1-2, pJS7525) JBKT0 (pJS752-3) JS1569 (pJS752-3) JS1569 (pJSl8)
ctx tacP (*) tacP (*)
CTB ([zg/ml) 0.6 28 66
7"7 (**)
11
T7 (**) tacP (***) tacP (***) taeP (***)
6.7 81 44 17
(*) Induction by IPTG required. (**) Inductionof 77 RNA polymerasefrom P! was undertaken by shift from 30 to 37°C. (***)Constitutiveexpression.
published). In addition, the ability of the sera to neutralize E. coil STa was assayed in the infant mouse test: serum in a dilution of 1/5 was mixed with an equal volume of purified STa, 60 ng/ml, and 0.1 ml of this mixture (which also contained Coomassie blue stain) was then injected into the stomach of baby mice and assayed for ST "diarrhoegenic" activity as described previously (Svennerholm et al., 1986). Results and Discussion.
Overexpression o f CTB by tacP and T7 promoters.
Recombinant CTB was expressed in different V. cholerae hosts by the constitutive tacP promoter in plasmid pJS752-3 or by the T7 RNA-polymerase promoter (plasmid pjS7522 or pJS7525) (table I). The levels achieved in culture supernatants demonstrated that high expression of CTB by these two systems surpassed by far the expression from the natural ctx promoter (table I). The best expression was achieved with the constitutive tacP in pJS752-3. Both El Tor and classical V. cholerae strains were able to overexpress CTB from this promoter. We believe that the preparation of CTB for cholera and enterotoxigenic E. coil vaccine production purposes will benefit from these new strains harbouring the pJS752-3 plasmid. These strains can be grown under simpler conditions (without antibiotics and isopropylthiogalactoside; IPTG) and yet produce similar CTB levels as compared with strains harbouring the inducible tacP (Sanchez and Holmgren, 1989). Moreover, for our additional purposes of achieving gene fusions in association with overexpression, plasmid pjS752-3 was as suitable as the previously employed plasmid pJS162 (Sanchez et al., 1988). All gene fusions described below were therefore undertaken using this new plasmid vector (see "Materials and methods" and below). Gene fusions at the redesigned CTB gene amino end.
Modifications at the CTB gene amino end as described (Sanchez and Holmgren, 1989) provided single Sac! and XmaI sites at the junction between the leader peptide and the mature CTB sequence. Synthetic double-stranded oligodeoxynucleotides were fused to the redesigned CTB amino end in plasmid pJS752-3. After insertion, in-
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TABLE II. - - STa-related peptides fused to the amino or earboxy ends of CTB. Strain
Plasmid
Amino acid sequence of peptides fused
189 248 258 268 278
pJS8 pJS7-11 pJS18-11 pJS7-15 pJSIS-8
NH2-aCAELCCNPACpgyahgT--NH2-aNSSNYCCELCCNPACTGCYpgyahgT-----Nga NSSNYCCEL.CCNPACTGCYpgn-COOH ---Nga NSSNYCCELCCNPACTGCY-COOH ---Nga CAELCCNPACpgn-COOH
Note: all plasinidsexceptpJS8 (Sanchezet al., 1988)derivefromp!asmidvectorpjS752-3(seetable I).
frame fusion proteins were created carrying either an STa-related non-toxic decapeptide or the complete STa (table II). Expression of fusion proteins was first determined in E. coli HB101 by GMI-ELISA using specific mAb. Because positive results in this ELISA depend on binding to GM1 as well as reaction with STa-specific mAb the authenticity of the fusion proteins and proper exposure of the fused epitopes were directly ascertained. Transfer of plasmids to V. cholerae JS1569 usually resulted in much higher levels of expression of the hybrid proteins than those obtained from E. coll, We attribute this to the extracellular location of the fusion proteins produced from V. cholerae host cells. Conceivably, secretion helps elude turnover mechanisms which might downregulate the concentration of the fusion proteins in the E. coli periplasm. SDS-PAGE and Western blot analyses (not shown) confirmed the hyb~:qdnature of the CTB fusion proteins. Not only did protein staining of SDS-PAG~ gels show bands in positions corresponding to those expected for CTB with pepfide extensions of the size indicated by the various gene fusions, but immunostaining also confirmed the reactivity of the proteins with both anti-B subunit (mAb LT39) and anti-STa (mAb 1:3) monoclonal antibodies. Furthermore, results from analysing unboiled as well ~s boiled protein preparations by SDS-PAGE and immunoblotting indicated that, similar to native CTB, all STa-CTB hybrids here described were normally in an oligomeric (pentameric) form (data not shown). In the course of isolating our first fusion proteins with the STa decapeptide at the CTB amino end, lack of reactivity with the STa-specific mAb 27:3 was observed even though the hybrid protein reacted strongly with our primary STa-specific mAb 1:3. Our initial suspicion was that this was due to the fact that this decapeptide did not contain all aa in STa, and therefore we proce,eded to fuse oligodeoxynucleotides encoding the entire STa molecule to the amino end of CTB. This hybrid STaCTB protein encoded by pJS7-11 (table II) was, however, also unable to react with mAb 27:3. This led us to consider the possibility that it was instead the joining through the carboxy end of STa that caused its lack of reactivity with mAb 27:3. We thus redesigned the carboxy end of the CTB gone as described (in "Materials and methods") in order to allow for insertion of SYa in this position. Fusions at the redesigned CTB gene carboxy end.
Fusions at the carboxy end of the CTB gene were carried out by insetting the same oligonucleotides at the carboxy end of the synthetic partial CTB gone that were previously employed to construct the amino end fusions. In a similar fashion to the fusions at the amino end, in-frame gene fusions were obtained by introducing synthetic oligodeoxynucleotides as SacI.XmaI inserts. Because of the duplicity of the S a d and
R E C O M B I N A N T C H O L E R A T O X I N F O R O R A L V A C C I N E 977
E = o
2
e~
(o -~
1
iU
.i
189
248
;
258 268 Gene fusion protein
I
2?8
218
FIG. 1. -- Reactivity of different hybrid proteins with mAb against ~ subunit (open bars) and STa, respectively. Open bars = B subunit; filled bars denote reactivity with anti-STa mAb 1:3, and stippled bars in~i;::ate reactivity with anti-STa (mAb 27:3). For specification of strains producing different gene fusion proteins, see table II; strain 213 refers to a strain producing recombinant CTB without any peptide extension.
XmaI restriction sites, the fusions to the redesigned CTB gene carboxy end were first carried out in the pUC18 derivative (see under Materials and Methods). After fusion, the fragment was then joined to the remainder (the amino portion) of the CTB gene. However, the fusion protc~ins containing STa or its non-toxic decapeptide analogue at the carboxy end of CTB still lacked reactivity with mAb 27:3. A remaining possible explanation for the reactivity with only one of the two anti-ST mAb was that the fusion proteins (table II) had a short, three-aa (ProGlyAsn) extension at their carboxy end. To define whether these extra aa interfered with recognition by mAb 27:3 synthetic oligonucleotides were introduced into pJSlS-I 1 (table II) to insert a ~top codon immediately after the last Tyr in the CTB-STa to give plasmid pJS7-1.~ (table II). Indeed, as shown in figure 1, the fusion protein encoded by this last construct now reacted with mAb 27:3, indicating that removal of the three-aa extension at the carboxy end had prevented proper recognition by this antibody. This could not be due to any abnormal overall folding of the hybrid proteins, since both of these proteins reacted with the anti-B subunit mAb and the anti-STa mAb 1:3 in both ELISA and Western blots in a fashion indistinguishable from all other hybrids tested; they also showed a normal oligomerization pattern, as examined by SDS-PAGE.
J. S A N C H E Z E T A L .
978
A [i- 189 (R!761)
B =o
,--m
5
~5
125
62,~ ~1~5 15e25
Serumdilution
I~
2~
N
~
Genefusionprotein
Fla. 2. 1 Immunogenicity of different ST-CTB hybrid proteins. (A) Typical ELISA curves against STa for pre- (open squares) versus post-inununization (filled triangles) sera from a rabbit immunized with one of the gene fusion proteins (from strain 189, see table If). (B) Pre- (open bars) versus post-immunization (filled bars) anti-STa antibody titres against different gene fusion proteins. Titres were determined from ELISA titrations ad modum (A) taking the extrapolated serum dilution giving an absorbance value of 0.4 above background as the titre.
lmmunogenicity of hybrid proteins. Immunizations of rabbits with purified CTB fusion proteins carrying the various STa-related epitopes gave rise to a significant anti-STa antibody response (fig. 2). The maximal antl-STa ELISA titres attained were, however, still much lower than the titres against the CTB carrier protein in the same animals and corresponded to approximately 1-15 I~g/ml of specific anti-ST immunoglobulin (as compared with c.a. 300-1000 iLg/ml anti-CTB immunogiobulin). It is therefore not surprising that tht~e immune sera exhibited only borderline neutralizing activity when tested in a f'mal dilution of 1/10 against an STa challenge dose of 30 ng/ml. Complete neutralization of this challenge dose can be calculated to require at least I ~tg/ml of "perfect" anti-STa antibody (as based on two binding sites per IgG antibody of MW 150,000 for STa of MW 2,200), i.e. >>, 10 izg/ml o f specific anti-STa antibody in undiluted serum, levels which were not quite achieved in these immune sera. Interestingly, the length of the fused epitope and/or its position in the CTB molecule did not affect the immunogenicity in any obvious fashion. If, as discussed above, the STa epitope def'med by mAb 27:3 was hidden in the construct encoded by p3S18-11 but not in that encoded by p3S75-15, the fact that immunization with the latter construct did not give rise to higher titres could be taken to suggest that this epitope is not critical for the overall immunogenicity of STa. This remains speculative, however, until additional immunizations resulting in stronger antisera have been performed.
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References.
CLEMENS,.I.D., SACK,D.A., HARRIS,J.R., CHAKRABORTY,J., KHAN,M.R., STANTON,B.F., KAY,B.A., KHAN,M.U., YUNUS,M.D., ATKINSON,W., SWNNERHOLM,A.M. & HOLMOREN, J. (1986), Field trial of oral cholera vaccines in Bengladesh. Lancet, I, 124-127. CLEMENS, J.D., HARRIS,J.R., SACK, D.A., C~'CRAeORTY,J., AHMEO,F., STANTON,B.F., KHAN,M.U., KAY, B.A., HUDA, N., KHAN, M.R., YUNUS,M., RAO, R., SVENNERHOLM,A.M. & HOLMOREN,J. (1988), Field trial of oral cholera vaccines in Bangladesh: results of one year of follow-up. J. infect. Dis., 158, 60-69. DERTZBAUG~,M.T., PET~asON,D.L. & MACR'NA,F.L. 0990), Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein. Infect. lmmun., 58, 70-79. DITTA,G., STANFIELD,S., CORBIN,D. & HELINSKI,D.R. (1980), Broad-host range DNA cloning system for Gram-negative bacteria: construction of gene bank of Rhizobium meliloti. Proc. nat. Acad. Sci. (Wash.), 77, 7347-7351. FORSTE, J.P., P^NSEOaAU,W., FRANK,R., BLOCKER,H., SCHOLZ,P., B^GD^SARIAN,M. & LA~A, E. (1986), Molecular cloning of the plasmid RP4 primase region in a multihost-range tacP expression vector. Gene, 48, 119-131. GENNARO,M.L., GXEaNAWAY,P.J. & BROADnESr,D.A. (1982), The Expression of biologically active cholera toxin in Escherichia coll. Nucl. Acids Res., 10, 4883-4890. HARDY,S.J.S., HOLMGI~N,J., JOHANSSON,S., S~CHEZ, J. & Hmsr, T.R. (1988), Coordinated assembly of multisubunit proteins: Oligomerization of bacterial enterotoxins in vivo and in vitro. Proc. nat. Acad. ScL (Wash.), 85, 7109-7113. HOLMOREN,J. (1981), Actions of cholera toxin and the prevention and treatment of cholera. Nature (Lond.), 292, 412-417. HOLMGREN, J., SVENNERHOLM,A.M., LONNROTH,I., FALL-PERSSON,M., MARKMAN,B. & LUNDB~CK,H. (1977), Development of improved cholera vaccine based on subunit toxoid. Nature (Lond.), 269, 602-604. HOLMOREN,J., CLEMENS,J., SACK,D., SANCHEZ,J. & SVEENNERHOLM,A.-M. (1989), Development of oral vaccines with special reference to cholera, in "Topics in Pharmaceutical Sciences" (D.D. Breimer & A. Midah). Elsevier Medical Press B.V. (Amsterdam). KAP'~R,J.B., LOCWSCa, N, H., B~.DINI,M.M. & LEVJNE,M.M. 0984), Recombinant nontoxigemc Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.), 308, 655-658. MEV.ALANOS,J.M., COLLIER,J.R. & ROMIG,W. (1978), Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxigenic mutants. In, feet. Immun., 20, 552-558. MILLER,V.L. & MEKALANOS,J.J. (1984), Synthesis of cholera toxin in positively rcgnlated at the transcriptional level by tox R. Proc. nat. Acad. $ci. (Wash.), 81, 3471-3475. SANCHEZ,J. & HOLMO~N,J. (1989), Recombinant system for overexpression of cholera toxin B-subunit as a basis for vaccine development. Proc. nat. Acad. Sci. (Wash.), 86, 481-485. SANCHnZ,J., SVe~a~eRHOLM,A.M. & HOLMGREN,J. (1988), Genetic fusion of a non-toxic heatstable enterotoxin-related decapepfide antigen to cholera toxin B subunit. FEBS Letters, 241, 110-114. SANDXVlST,M., HIasT, T.R. & BAGDASARIAN,M. (1987), Alterations at the carbo~yl terminus change assembly and secretion properties of the B subunit of the Escherichia coli heat-labile enterotoxin. J. Bact., 169, 4570-4576. SCHODEL,F. & WILL,H. (1989), Construction of a plasmid for expression of foreign epitopes as fusion proteins with B subunit of Escherichia coil heat-labile enterotoxin. Infect. lmmun., $7, 1347-1350. SIMON,R., PR1EFER,U. & P0HLER,A. (1983), A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Tech., 1, 784-791. SVeNNERHOLM,A.M., WIKSTROM,M., LINDBLAD,M. & HOLMGREN,J. (1986), Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in diagnostic ST ganglioside GMl-enzyme-linkedimmunosorbent assay. J. clin. Microbiol., 24, 585-590. TAeOR, S. & RICHARDSON,C.C. (1985), A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. nat. Acad. Sci. (Wash.), 82, 1074-1078. TAYOT,J.L., HOLMGREN,J., SVENNERHOLM,L., LINDBLAD,M. & TARDY,M. (1981), Receptorspecific large scale purification of cholera toxin on silica beads derivatized with lysoGMl-gangiioside. Europ. J. Biochem., 113, 249-258.
Res. MicrobioL 1990, 141, 971-979
R E C O M B I N A N T C H O L E R A TOXIN B S U B U N I T A N D G E N E F U S I O N P R O T E I N S F O R O R A L VACCINATION
J. Sanchez (l. 2), S. Johansson (l), B. LOwenadler (3), A.M. Svennerholm (l) and J. Holmgren (1) (i) Department of Microbiology and Immunology, University of GOteborg, S-413 46 Gothenburg (Sweden), (2) Institute for Public Health, Center for Research on Infectious Diseases, Apdo. Postal 222 Oficina Central Correos, Cuernavaca 62000 Mexico, and (3) KabiGen AB, Strandsbergatan 49, S-ii2 87 Stockholm
Summary. The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (noncholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNApolymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coil enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 izg/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a nearnative conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa. However, only hybrid.protein presenting the STa peptide with a free carboxy end was able to also react with another available STa mAb. Our results suggest that even minor modifications of a given antigenic zegion may lead to complete epitope hiding and/or to its lack of antibody reactivity. Alternate positioning of such peptides in the carboxy end of the CTB protein was
Correspondence:Dr J. Sanchez,AppartadoPostalNo 222,Oficinade CorreosNol, C.P. 6200,Cuernavaca, Morelos, Mexico.
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found to assist in antibody recognition and is proposed as a means to help exposure of some foreign epitopes by CTB fusion proteins. The results may be of significance for the development of recombinant oral vaccines based on gene fusions to CTB or to the closely related B subunit of LT. KEY-WORDS:Cholera, Toxin, B subunit; Oral vaccination, Gene fusion.
Introduction. Vibrio cholerae of serogroup 01 may cause severe diarrhoeal disease in infected individuals by releasing cholera toxin (CT) which induces active electrolyte and water secretion from the intestinal epithelium. The prototype bacterial enterotoxin CT is a complex protein built from two types of subunits: a single A subunit of molecular weight (MW) 28,000 and five B subunits, each with a MW of 11,600. The B subunits are aggregated in a ring by tight non-covalent bonds; the A subunit is linked to and probably partially inserted in the B pentamer ring through weaker non-covalent interactions. The two types of subunit have different roles in the intoxication process: the B subunits are responsible for cell binding and the A subunit for the direct toxic activity (Holmgren, 1981). At the genetic level in V. cholerae 01 bacteria, CT is encoded by chromosomal genes for the A and B subunits. These genes have been cloned from several strains, and their nucleotide sequences have been determined (Mekalanos et al., 1983; Kaper et al., 1984; Gennaro and Greenaway, 1983; Sanchez and Holmgren, 1989). The A and B subunit genes of CT are arranged in a single transcriptional unit with the A cistron ( c t x A ) preceding the B cistron (ctxB) (Mekalanos et al., 1983). The synthesis of CT is positively regulated by a gene, t o x R , that increases c t x expression manifold (V.L. Miller and J.J. Mekalanos, 1984). Vaccination against cholera by parenteral injection has yielded only modest shortterm protection (usually less than 50 °7o protection for less than 6 months). Given the strictly mucosal surface location of the cholera infection, interest has therefore turned to development of oral vaccines that stimulate mucosal intestinal immunity more efficiently. Special attention has been drawn to CTB pentamers as a component (together with killed vibrios) of such oral cholera vaccines (Holmgren et al., 1977). CTB is an effective oral immunizing agent which recently, in a large field trial, has been shown to afford protection against both cholera and diarrhoea caused by enterotoxigenic E. coil (ETEC) producing heat-labile toxin (LT) (Clemens et aL, 1986; 1988). To facilitate large-scale preparation of CTB for vaccine production, we have developed overexpression systems for CTB where the gene is under the control of strong foreign (non-cholera) promoeters. Previously, we have reported on expression of C T B gene by an inducible t a c P promoter (Sanchez and Holmgren, 1989) which also expresses high levels of a recombinant fusion protein (Sanchez et al., 1988). We here report on expression of C T B by a constitutive t a c P and by the T 7 RNApolymerase promoter (Tabor and Richardson, 1985).
aa Abs CT CTA CTB
= = = = =
ctx ctxA ctxB
= = =
ELISA = ETEC = HIV =
amino acid. absorbance. cholera toxin. CT A subunit. CT B subunit. cholera toxin gene. ctx A subunit cistron. ctx B subunit cistron. enzyme-linkedimmunosorbentassay. enterotc::igenicEscherichia coll. human immunodeficiencyvirus.
1PTG = LT = LTA = LTB = mAb = MW = SDS-PAGE= STa
isopropylthiogalactoside. heat-labileenterotoxin of E. coll. A subunit of LT. B subunit of LT. monoclonalantibody. molecularweight. sodium dodecyl sulphatepolyacrylamidegel electrophoresis. = methanol-soluble heat-stable enterotoxin of E. coli.
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Recently, CTB has also attracted great interest as an antigen carrier for development of other, mainly oral vaccines (Holmgren et al., 1989). Construction of"foreign antigen"-carrying CTB proteins by genetic manipulations on CTB (or analogous construction of hybrid proteins based on the closely related B subunit of the heat-labile enterotoxin of E. coil (LTB)) has the advantage that the extra epitope is bound in a defined form and at a defined place in the molecule. This is likely to result in hybrids retaining many of the native properties of the fused proteins, including binding of specific antibodies. We have described the gene fusion of a 10-amino-acid (aa) peptide analogue of the E. coli heat-stable enterotoxin (STa) to the amino end of CTB. Once joined to the B subunit, this decapeptide was efficiently recognized by a neutraiizing monoclonal antibody directed against native STa, and in further support of its proper folding, the hybrid protein also exhibited excretion by V. cholerae host cells, pentamerization and binding to a GMI ganglioside much like the native CTB protein (Sanchez et al., 1988). Likewise, genetic fusion of a glucosyl transferase epitope from Streptococcus mutans to the amino end of CTB (Derztbangh and Macrina, 1990), and fusions of hepatitis B epitopes to the carboxy terminus of LTB (Sch/kiel and Will, 1989) have been reported to result in immunogenic hybrid proteins able to give rise to anti-epitope antibodies. As further extensions of our previous efforts to develop recombinant overexpression systems for CTB associated with the potential for prod,Jction of hybrid proteins, we here report on the genetic fusion of various STa-related peptides at either the amino or carboxy ends of CTB, with analyses of the foreign epitope accessibility as monitored by reaction with specific antibodies and by in vivo immunogenicity studies. Materials and methods. Gene modifications at the C T B 3' and amino ends. -- To facilitate expression of the CTB gene by 7"7 and the inducible tacP promoters and to allow productive gene fusions at the amino end of CTB, we introduced synthetic oligodeoxynucleotides which made possible replacement of the natural CTB leader peptide by that in the closely related protein E. col; LTB protein (Sanchez and Holmgren, 1989). Inclusion of these synthetic oligodeoxynucleotides incorporated single SacI and Xmal sites at the junction between the leader peptide and mature CTB. Gene modifications at the C T B carboxy end. - - To be able to fuse synthetic oligonucleotides at the carboxy end of CTB, we synthesized oligodeoxynucleotides encoding the last 24 aa of the CTB gene carboxy-end and introduced single SacI, XmaI and SpeI sites. In addition, the CTB carboxy end gene was made to be followed, after the stop codon, by a unique HindIII site in order to facilitate further subcloning into other expression vectors. Cloning of the synthetic CTB gene carboxy end was undertaken in plasmid pUCI8. Vector-directed expression of CTB. -- Controlled expression of the CTB gene by the constitutive tocP promoter was achieved by subcloning an EcoRI-HindllI DNA fragment into plasmid vector pKK223-3 (Pharmacia, Uppsala, Sweden). Expression by the 77 RNA-polymerase promoter was obtained by first subcloning the CTB gene as an EcoRI-HindllI fragment into plasmid pT7-5 (encoding resistance to ampicillin) followed by coresidence in the same strain with the compatible plasmid pGP1-2 (encoding resistance to kanamycin) (Tabor and Richardson, 1987). tacP.directed expression of CTB fusion proteins. - - For expression of CTB gene fusion proteins under the inducible taeP, a derivative of plasmid pMMB66 (Ffirste et al., 1986; Sandkvist et al., 1987; Sanchez and Holmgren, 1989) was used. Expression under the constitutive tacP promoter was achieved by subcloning the hybrid gene into pKK223-3. Gene fusions at the CTB gene amino end. - - For gene fusions at either the amino or carboxy end of CTB, double-stranded oligonucleotides with single-stranded Sa¢I
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and X m a I compatible ends were used. Typically, fusion of the inserted oligonucleotide was carried out so as not to reconstitute at least one of the restriction sites used for cloning. Thus digestion of the ligation mixture with the restriction enzyme for that site resulted in linearization of plasmid molecules which had not incorporated the added oligonucleotides, which reduced their aptitude to transform. Gene fusions at the CTB gene earboxy end. - - For insertion of oligonucleotides at the C T B 3' end SacI-XmaI compatible double-stranded oligodeoxynucleotides were first inserted at these unique sites in the pUC18 derivative containing a synthetic 3' portion analogue of the C T B gene (see above). After fusion, the now extended carboxy end portion of the gene was joined as a TaqI.HindIII fragment to the rest of the C T B gene which was excised as an EcoRI-TaqI fragment. The compound EcoRI-TaqI-TaqI-HindIII construct was then cloned in pKK223-3 for expression under the tacP promoter. The oligonucleotides inserted were those encoding a 10-aa non-toxic STa analogue (Sanchez et al., 1988) and those encoding the complete 19-aa STa protein. Some of the STa-peptides fused at the CTB carboxy end carried a short three-amino-acids extension at the carboxy end (table II). To remove those extra amino acids, short oligonucleotides were introduced between the Sphl and SpeI sites to cause translation to terminate immediately after the last Tyr in native STa (table II). Expression of recombinant CTB and fusion proteins in V. cholerae. - - The V. cholerae host JS1569 (Sanchez and Holmgren, 1989) or isolated V. cholerae rifampicin-resistant derivatives were used for expression of recombinant CTB or CTBderived fusion proteins. The pKK223-3 derivatives or both of the plasmids of the T7 system were mobilized into V. cholerae by the aid of plasmid pRK2013 (Ditta et al., 1980) while the pMMB66 derivatives were mobilized from a strain holding RP4 in the chromosome (Simon et al., 1983). Selection of V. cholerae harbouring the desired plasmids was done by plating on rifampicin plus the selective antibiotic.
Enzyme-linked immunoassays for CTB and fusion proteins. - - GM I-ELISA for detection of the CTB antigen or the attached STa epitopes in the fusion proteins was carried out using specific monoclonal antibodies (mAb) or antisera essentially as described (Sanchez et al., 1988). Two mAb, 1:3 and 27:3, were used to detect "neutralizing" STa epitopes in the fusion proteins: both of these STa-specific mAb have been found to completely neutralize the infant-mouse toxicity of STa, yet they seem to recognize slightly different epitopes in the STa molecule (A.-M. Svennerholm et al., to be published) (see also "Results and Discussion"). SDS polyacrylamide gel eleetrophoresis (SDS.PAGE) and Western Blot Analyses. - - Protein samples were examined by SDS-PAGE followed by protein staining and also subjected to Western blot testing on nitrocellulose paper using standard procedures. Protein samples were prepared by affinity-adsorption to GMI-Spherosil as reported (Tayot et al., 1981 ; Sanchez et al., 1988) or by hexametaphosphate precipitation (Mekalanos et al., 1978). The electrophoretic separations were performed with or without prior boiling of the proteins in the SDS sample buffer to allow evaluation of monomers and oligomers, respectively (Hardy et al., 1988). Rabbit immunizations with CTB fusion proteins and antibody determinations. - - For immunizations, the STa-related CTB fusion proteins in bacterial culture supernatants were first isolated either by hexametaphosphate salting-out or by GMI affinity purification (Tayot et al., 1981) and dialysed against PBS. The approximate specific protein concentrations of samples used for immunizations were determined by ELISA using CTB as a standard. Male rabbits weighing 2-3 kg were immunized with 50-250 t~g of CTB equivalents of protein in each immunization. In total, 3-5 infections were given every 3-4 weeks, the first three with the antigen suspended in complete Freund adjuvant and subsequent ones given in incomplete adjuvant. Sera prepared from bleedings taken before the start of the immunizations and c.a. 2 weeks after the third or fourth vaccination were assayed for specific antibodies against CTB and STa by ELISA methods (Sanchez et al., 1988; Svennerholm et al., to be
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TABLE I. - - Expression of CTB by different promoters in V. eholerae. Strain
Promoter
JS1569 JS1569 (pJS162) JBK70 (pJS162) JBKT0 (pOP1-2), pJS7522) JBK70 (pGP1-2, pJS7525) JBKT0 (pJS752-3) JS1569 (pJS752-3) JS1569 (pJSl8)
ctx tacP (*) tacP (*)
CTB ([zg/ml) 0.6 28 66
7"7 (**)
11
T7 (**) tacP (***) tacP (***) taeP (***)
6.7 81 44 17
(*) Induction by IPTG required. (**) Inductionof 77 RNA polymerasefrom P! was undertaken by shift from 30 to 37°C. (***)Constitutiveexpression.
published). In addition, the ability of the sera to neutralize E. coil STa was assayed in the infant mouse test: serum in a dilution of 1/5 was mixed with an equal volume of purified STa, 60 ng/ml, and 0.1 ml of this mixture (which also contained Coomassie blue stain) was then injected into the stomach of baby mice and assayed for ST "diarrhoegenic" activity as described previously (Svennerholm et al., 1986). Results and Discussion.
Overexpression o f CTB by tacP and T7 promoters.
Recombinant CTB was expressed in different V. cholerae hosts by the constitutive tacP promoter in plasmid pJS752-3 or by the T7 RNA-polymerase promoter (plasmid pjS7522 or pJS7525) (table I). The levels achieved in culture supernatants demonstrated that high expression of CTB by these two systems surpassed by far the expression from the natural ctx promoter (table I). The best expression was achieved with the constitutive tacP in pJS752-3. Both El Tor and classical V. cholerae strains were able to overexpress CTB from this promoter. We believe that the preparation of CTB for cholera and enterotoxigenic E. coil vaccine production purposes will benefit from these new strains harbouring the pJS752-3 plasmid. These strains can be grown under simpler conditions (without antibiotics and isopropylthiogalactoside; IPTG) and yet produce similar CTB levels as compared with strains harbouring the inducible tacP (Sanchez and Holmgren, 1989). Moreover, for our additional purposes of achieving gene fusions in association with overexpression, plasmid pjS752-3 was as suitable as the previously employed plasmid pJS162 (Sanchez et al., 1988). All gene fusions described below were therefore undertaken using this new plasmid vector (see "Materials and methods" and below). Gene fusions at the redesigned CTB gene amino end.
Modifications at the CTB gene amino end as described (Sanchez and Holmgren, 1989) provided single Sac! and XmaI sites at the junction between the leader peptide and the mature CTB sequence. Synthetic double-stranded oligodeoxynucleotides were fused to the redesigned CTB amino end in plasmid pJS752-3. After insertion, in-
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TABLE II. - - STa-related peptides fused to the amino or earboxy ends of CTB. Strain
Plasmid
Amino acid sequence of peptides fused
189 248 258 268 278
pJS8 pJS7-11 pJS18-11 pJS7-15 pJSIS-8
NH2-aCAELCCNPACpgyahgT--NH2-aNSSNYCCELCCNPACTGCYpgyahgT-----Nga NSSNYCCEL.CCNPACTGCYpgn-COOH ---Nga NSSNYCCELCCNPACTGCY-COOH ---Nga CAELCCNPACpgn-COOH
Note: all plasinidsexceptpJS8 (Sanchezet al., 1988)derivefromp!asmidvectorpjS752-3(seetable I).
frame fusion proteins were created carrying either an STa-related non-toxic decapeptide or the complete STa (table II). Expression of fusion proteins was first determined in E. coli HB101 by GMI-ELISA using specific mAb. Because positive results in this ELISA depend on binding to GM1 as well as reaction with STa-specific mAb the authenticity of the fusion proteins and proper exposure of the fused epitopes were directly ascertained. Transfer of plasmids to V. cholerae JS1569 usually resulted in much higher levels of expression of the hybrid proteins than those obtained from E. coll, We attribute this to the extracellular location of the fusion proteins produced from V. cholerae host cells. Conceivably, secretion helps elude turnover mechanisms which might downregulate the concentration of the fusion proteins in the E. coli periplasm. SDS-PAGE and Western blot analyses (not shown) confirmed the hyb~:qdnature of the CTB fusion proteins. Not only did protein staining of SDS-PAG~ gels show bands in positions corresponding to those expected for CTB with pepfide extensions of the size indicated by the various gene fusions, but immunostaining also confirmed the reactivity of the proteins with both anti-B subunit (mAb LT39) and anti-STa (mAb 1:3) monoclonal antibodies. Furthermore, results from analysing unboiled as well ~s boiled protein preparations by SDS-PAGE and immunoblotting indicated that, similar to native CTB, all STa-CTB hybrids here described were normally in an oligomeric (pentameric) form (data not shown). In the course of isolating our first fusion proteins with the STa decapeptide at the CTB amino end, lack of reactivity with the STa-specific mAb 27:3 was observed even though the hybrid protein reacted strongly with our primary STa-specific mAb 1:3. Our initial suspicion was that this was due to the fact that this decapeptide did not contain all aa in STa, and therefore we proce,eded to fuse oligodeoxynucleotides encoding the entire STa molecule to the amino end of CTB. This hybrid STaCTB protein encoded by pJS7-11 (table II) was, however, also unable to react with mAb 27:3. This led us to consider the possibility that it was instead the joining through the carboxy end of STa that caused its lack of reactivity with mAb 27:3. We thus redesigned the carboxy end of the CTB gone as described (in "Materials and methods") in order to allow for insertion of SYa in this position. Fusions at the redesigned CTB gene carboxy end.
Fusions at the carboxy end of the CTB gene were carried out by insetting the same oligonucleotides at the carboxy end of the synthetic partial CTB gone that were previously employed to construct the amino end fusions. In a similar fashion to the fusions at the amino end, in-frame gene fusions were obtained by introducing synthetic oligodeoxynucleotides as SacI.XmaI inserts. Because of the duplicity of the S a d and
R E C O M B I N A N T C H O L E R A T O X I N F O R O R A L V A C C I N E 977
E = o
2
e~
(o -~
1
iU
.i
189
248
;
258 268 Gene fusion protein
I
2?8
218
FIG. 1. -- Reactivity of different hybrid proteins with mAb against ~ subunit (open bars) and STa, respectively. Open bars = B subunit; filled bars denote reactivity with anti-STa mAb 1:3, and stippled bars in~i;::ate reactivity with anti-STa (mAb 27:3). For specification of strains producing different gene fusion proteins, see table II; strain 213 refers to a strain producing recombinant CTB without any peptide extension.
XmaI restriction sites, the fusions to the redesigned CTB gene carboxy end were first carried out in the pUC18 derivative (see under Materials and Methods). After fusion, the fragment was then joined to the remainder (the amino portion) of the CTB gene. However, the fusion protc~ins containing STa or its non-toxic decapeptide analogue at the carboxy end of CTB still lacked reactivity with mAb 27:3. A remaining possible explanation for the reactivity with only one of the two anti-ST mAb was that the fusion proteins (table II) had a short, three-aa (ProGlyAsn) extension at their carboxy end. To define whether these extra aa interfered with recognition by mAb 27:3 synthetic oligonucleotides were introduced into pJSlS-I 1 (table II) to insert a ~top codon immediately after the last Tyr in the CTB-STa to give plasmid pJS7-1.~ (table II). Indeed, as shown in figure 1, the fusion protein encoded by this last construct now reacted with mAb 27:3, indicating that removal of the three-aa extension at the carboxy end had prevented proper recognition by this antibody. This could not be due to any abnormal overall folding of the hybrid proteins, since both of these proteins reacted with the anti-B subunit mAb and the anti-STa mAb 1:3 in both ELISA and Western blots in a fashion indistinguishable from all other hybrids tested; they also showed a normal oligomerization pattern, as examined by SDS-PAGE.
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A [i- 189 (R!761)
B =o
,--m
5
~5
125
62,~ ~1~5 15e25
Serumdilution
I~
2~
N
~
Genefusionprotein
Fla. 2. 1 Immunogenicity of different ST-CTB hybrid proteins. (A) Typical ELISA curves against STa for pre- (open squares) versus post-inununization (filled triangles) sera from a rabbit immunized with one of the gene fusion proteins (from strain 189, see table If). (B) Pre- (open bars) versus post-immunization (filled bars) anti-STa antibody titres against different gene fusion proteins. Titres were determined from ELISA titrations ad modum (A) taking the extrapolated serum dilution giving an absorbance value of 0.4 above background as the titre.
lmmunogenicity of hybrid proteins. Immunizations of rabbits with purified CTB fusion proteins carrying the various STa-related epitopes gave rise to a significant anti-STa antibody response (fig. 2). The maximal antl-STa ELISA titres attained were, however, still much lower than the titres against the CTB carrier protein in the same animals and corresponded to approximately 1-15 I~g/ml of specific anti-ST immunoglobulin (as compared with c.a. 300-1000 iLg/ml anti-CTB immunogiobulin). It is therefore not surprising that tht~e immune sera exhibited only borderline neutralizing activity when tested in a f'mal dilution of 1/10 against an STa challenge dose of 30 ng/ml. Complete neutralization of this challenge dose can be calculated to require at least I ~tg/ml of "perfect" anti-STa antibody (as based on two binding sites per IgG antibody of MW 150,000 for STa of MW 2,200), i.e. >>, 10 izg/ml o f specific anti-STa antibody in undiluted serum, levels which were not quite achieved in these immune sera. Interestingly, the length of the fused epitope and/or its position in the CTB molecule did not affect the immunogenicity in any obvious fashion. If, as discussed above, the STa epitope def'med by mAb 27:3 was hidden in the construct encoded by p3S18-11 but not in that encoded by p3S75-15, the fact that immunization with the latter construct did not give rise to higher titres could be taken to suggest that this epitope is not critical for the overall immunogenicity of STa. This remains speculative, however, until additional immunizations resulting in stronger antisera have been performed.
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References.
CLEMENS,.I.D., SACK,D.A., HARRIS,J.R., CHAKRABORTY,J., KHAN,M.R., STANTON,B.F., KAY,B.A., KHAN,M.U., YUNUS,M.D., ATKINSON,W., SWNNERHOLM,A.M. & HOLMOREN, J. (1986), Field trial of oral cholera vaccines in Bengladesh. Lancet, I, 124-127. CLEMENS, J.D., HARRIS,J.R., SACK, D.A., C~'CRAeORTY,J., AHMEO,F., STANTON,B.F., KHAN,M.U., KAY, B.A., HUDA, N., KHAN, M.R., YUNUS,M., RAO, R., SVENNERHOLM,A.M. & HOLMOREN,J. (1988), Field trial of oral cholera vaccines in Bangladesh: results of one year of follow-up. J. infect. Dis., 158, 60-69. DERTZBAUG~,M.T., PET~asON,D.L. & MACR'NA,F.L. 0990), Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein. Infect. lmmun., 58, 70-79. DITTA,G., STANFIELD,S., CORBIN,D. & HELINSKI,D.R. (1980), Broad-host range DNA cloning system for Gram-negative bacteria: construction of gene bank of Rhizobium meliloti. Proc. nat. Acad. Sci. (Wash.), 77, 7347-7351. FORSTE, J.P., P^NSEOaAU,W., FRANK,R., BLOCKER,H., SCHOLZ,P., B^GD^SARIAN,M. & LA~A, E. (1986), Molecular cloning of the plasmid RP4 primase region in a multihost-range tacP expression vector. Gene, 48, 119-131. GENNARO,M.L., GXEaNAWAY,P.J. & BROADnESr,D.A. (1982), The Expression of biologically active cholera toxin in Escherichia coll. Nucl. Acids Res., 10, 4883-4890. HARDY,S.J.S., HOLMGI~N,J., JOHANSSON,S., S~CHEZ, J. & Hmsr, T.R. (1988), Coordinated assembly of multisubunit proteins: Oligomerization of bacterial enterotoxins in vivo and in vitro. Proc. nat. Acad. ScL (Wash.), 85, 7109-7113. HOLMOREN,J. (1981), Actions of cholera toxin and the prevention and treatment of cholera. Nature (Lond.), 292, 412-417. HOLMGREN, J., SVENNERHOLM,A.M., LONNROTH,I., FALL-PERSSON,M., MARKMAN,B. & LUNDB~CK,H. (1977), Development of improved cholera vaccine based on subunit toxoid. Nature (Lond.), 269, 602-604. HOLMOREN,J., CLEMENS,J., SACK,D., SANCHEZ,J. & SVEENNERHOLM,A.-M. (1989), Development of oral vaccines with special reference to cholera, in "Topics in Pharmaceutical Sciences" (D.D. Breimer & A. Midah). Elsevier Medical Press B.V. (Amsterdam). KAP'~R,J.B., LOCWSCa, N, H., B~.DINI,M.M. & LEVJNE,M.M. 0984), Recombinant nontoxigemc Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.), 308, 655-658. MEV.ALANOS,J.M., COLLIER,J.R. & ROMIG,W. (1978), Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxigenic mutants. In, feet. Immun., 20, 552-558. MILLER,V.L. & MEKALANOS,J.J. (1984), Synthesis of cholera toxin in positively rcgnlated at the transcriptional level by tox R. Proc. nat. Acad. $ci. (Wash.), 81, 3471-3475. SANCHEZ,J. & HOLMO~N,J. (1989), Recombinant system for overexpression of cholera toxin B-subunit as a basis for vaccine development. Proc. nat. Acad. Sci. (Wash.), 86, 481-485. SANCHnZ,J., SVe~a~eRHOLM,A.M. & HOLMGREN,J. (1988), Genetic fusion of a non-toxic heatstable enterotoxin-related decapepfide antigen to cholera toxin B subunit. FEBS Letters, 241, 110-114. SANDXVlST,M., HIasT, T.R. & BAGDASARIAN,M. (1987), Alterations at the carbo~yl terminus change assembly and secretion properties of the B subunit of the Escherichia coli heat-labile enterotoxin. J. Bact., 169, 4570-4576. SCHODEL,F. & WILL,H. (1989), Construction of a plasmid for expression of foreign epitopes as fusion proteins with B subunit of Escherichia coil heat-labile enterotoxin. Infect. lmmun., $7, 1347-1350. SIMON,R., PR1EFER,U. & P0HLER,A. (1983), A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Tech., 1, 784-791. SVeNNERHOLM,A.M., WIKSTROM,M., LINDBLAD,M. & HOLMGREN,J. (1986), Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in diagnostic ST ganglioside GMl-enzyme-linkedimmunosorbent assay. J. clin. Microbiol., 24, 585-590. TAeOR, S. & RICHARDSON,C.C. (1985), A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. nat. Acad. Sci. (Wash.), 82, 1074-1078. TAYOT,J.L., HOLMGREN,J., SVENNERHOLM,L., LINDBLAD,M. & TARDY,M. (1981), Receptorspecific large scale purification of cholera toxin on silica beads derivatized with lysoGMl-gangiioside. Europ. J. Biochem., 113, 249-258.
