Microbial Pathogenesis 1990 ; 8 : 177-188
Surface co-expression of Vibrio cholerae and Salmonella typhi 0-antigens on Ty2l a clone EX210 Stephen R . Attridge, Denise Daniels, Judy K . Morona and Renato Morona Enterovax Limited, Cl- Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia 5001 (Received October 6, 1989 ; accepted in revised form December 8, 1989)
Attridge, S . R . (Enterovax Limited, C/- Dept of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia 5001), D . Daniels, J . K . Morona and R . Morona . Surface co-expression of Vibrio cholerae and Salmonella typhi 0-antigens on Ty21 a clone EX21 0 . Microbial Pathogenesis 1990 ; 8 : 177-188 . In an attempt to construct a bivalent, live, oral cholera-typhoid vaccine, genes specifying the biosynthesis of Vibrio cholerae 0-antigen have been transferred into a modified version of the attenuated, oral typhoid vaccine strain Salmonella typhi Ty21 a . The present report investigates the production of V. cholerae and S . typhi 0-antigens by one such clone, EX21 0 . When cultured without galactose supplementation EX210 produces surface 0-antigen of V. cholerae type, as detected by haemagglutination-inhibition and bactericidal assays, and by immuno-electron microscopy . However, the protective efficacy of Ty21 a depends upon growth in the presence of exogenous galactose and under these conditions only S . typhi 0-antigen is detectable on the surface of EX210 . Subsequent experiments revealed that the proportion of polysaccharide of S. typhi type is dependent upon the level of galactose supplementation, and identified a limiting sugar concentration which results in surface co-expression of both O-antigens . Visualization of the two polysaccharides on silver-stained polyacrylamide gels indicates that S . typhi 0-antigen subunits are polymerized into longer sidechains, suggesting that at higher galactose concentrations their predominance results in a masking of the shorter V . cholerae 0-antigen . Key words : Vibrio cholerae ; Salmonella typhi Ty2l a ; 0-antigen ; bivalent vaccine .
Introduction Killed parenteral cholera vaccines have consistently failed to provide enduring protection in the field .' 2 Evidently the serum antibodies generated by parenteral immunization are of insufficient concentration and/or inappropriate isotype to contribute significantly to enteric defence via transudation from the circulation . In contrast, natural or induced clinical infection affords strong and enduring resistance to disease ."' It is now generally accepted that to be effective a cholera vaccine will have to be orally administered, to stimulate production of IgA antibodies by the local immune system of the gut . Since Vibrio cho/erae is a non-invasive enteropathogen, the location and isotype of such antibodies make them ideally suited for host defence . Attempts to develop a safe and effective oral cholera vaccine have led to two new vaccine candidates . The more advanced of these is a non-viable formulation, comprising large numbers of inactivated V. cholerae and the isolated B subunit of the cholera toxin .' The design of this vaccine is based upon experimental data which show that 0882-4010/90/030177+12 $03 .00/0
0 1990 Academic Press Limited
178
S . R . Attridge et al.
antibacterial and antitoxic immune responses can act synergistically .' A recent field trial has shown that the combined bacterial-toxoid vaccine confers significant protection for at least 3 years .' A second approach is based upon the premise that the type of vaccine most likely to mimic the potent immunizing effects of natural infection would be a live oral vaccine with the potential to at least transiently colonize the gut . Attempts to attenuate a pathogenic V. cho/erae strain by deletion of the genes encoding cholera toxin have culminated in a vaccine candidate which is well-tolerated, immunogenic and protective in human volunteers .' The relative merits of these two vaccines have been recently discussed .` The most recent approach to cholera vaccine development is aimed at cloning protective antigens of V. cholerae into the live attenuated typhoid vaccine Ty21 a, 8 to produce a bivalent vaccine capable of conferring resistance to both cholera and typhoid . The demonstrated safety of Ty21 a 8 means that such a vaccine might combine the advantages of the two cholera vaccine candidates discussed above ; that is, it offers the safety of an inactivated vaccine with the superior immunogenicity of a viable one . At the commencement of this work there was only one V. cholerae antigen, the lipopolysaccharide (LPS), which had been implicated as a protective antigen . Direct evidence for this had been derived exclusively from experimental cholera models, 6 ' although several lines of circumstantial evidence suggested that antibodies to LPS could protect against clinical disease . Volunteer studies had shown that resistance to cholera challenge is dependent upon pre-existing antibacterial rather than antitoxic immunity .", " Epidemiological studies had identified an inverse correlation between the level of (naturally acquired) serum bactericidal antibodies and the incidence of disease, 12 and LPS is by far the most efficient substrate for such antibodies ." Finally, the occasional finding of serotype-restricted protection in field trials with parenteral vaccines' implicates LPS as a protective antigen, since this is the only bacterial component known to vary between the serotypes of V. cholerae . Construction of a bivalent Ty21 a-based cholera vaccine began with the cloning of the genes required for 0-antigen synthesis in V. cholerae . 14 A recent report describes the introduction of these genes into a modified Ty21 a strain to produce clone EX21 0 (R . Morona, unpublished results) . We now describe studies which analyze the expression of V. cholerae and Salmonella typhi 0-antigens on the surface of EX21 0 .
Results Effect of galactose on surface 0-antigen expression Although EX210 was known to be agglutinable by rabbit anti-V. cholerae serum (R . Morona, unpublished results), no information was available relating to the relative production of Inaba 0-antigen by EX210 and its V. cholerae parent strain 596B . In addition, it was of interest to investigate the effect of exogenous galactose on surface expression of both V. cholerae and S . typhi 0-antigens by the clone . The Ty21 a vector strain has a galE defect, and previous studies have shown that its capacity to synthesize smooth polysaccharide, and its protective efficacy, is dependent upon growth in medium supplemented with galactose ."" Initial experiments therefore compared the surface expression of 0-antigen of both V . cholerae and S . typhi types by clone EX210 and the parental strains, 569B and Ty21 a . The three strains were cultured in the presence or absence of 0 .001 % (w/v) galactose, 15 and the resulting (formalin-killed) bacterial suspensions titrated for their capacities to inhibit the agglutination of LPS-sensitized erythrocytes . Results of these haemagglutination-inhibition assays (HIAs) are shown in Table 1, and indicate that the two 0-antigen detection systems are specific, with limits of detection generally
0-antigens of Ty21 a clone EX21 0
179
Table l
Surface 0-antigen expression by clone EX21 0 and parental strains V. cholerae 569B and S . typhi Ty21 a, following growth in the presence or absence of 0 .001 % (w/v) galactose 0-antigen type Inhibitor strain
Growth +gala
V . cho/erae
S. typhi
+ + + +
8 8 0 0 7 0 9 0
0 0 0 9 0 9 0 10
569B Ty2la EX210 569B LPS Ty21a LPS
Bacteria were grown in the presence (+) or absence (-) of exogenous galactose (0 .001% w/v), inactivated by formalin treatment, and resuspended in saline . Bacterial suspensions (dry weights - 2 mg/ml) were tested for surface expression of the two 0-antigen types by HIA (see Materials and methods) . LPS preparations obtained from the parental strains were included as control inhibitors after standardization to 100 pg/ml on the basis of dry weight determinations . Figures show number of wells of inhibition in HIA .
0 .2 pg/ml . When grown in the absence of galactose, EX210 produces significant levels of V. cho/erae 0-antigen . The data in Table 1 confirm that 0-antigen production by Ty2l a (though not 569B) is dependent upon the addition of galactose to the growth medium . Not surprisingly, EX21 0 also produces S . typhi 0-antigen only in the presence of galactose . Under these conditions however, 0-antigen of V. cho/erae type is no longer detectable on the clone (Table 1 ) . The effect of exogenous galactose upon 0-antigen expression by EX210 was also examined by determining the sensitivities of the bacteria to complement-dependent lysis mediated by antibodies specific for V. cholerae or S. typhi LPS . This approach confirmed that growth in the absence of galactose results in primarily V. cholerae 0antigen on the surface of EX210 (Table 2) . In contrast, only S . typhi 0-antigen is detectable on the clone following growth in the presence of galactose (Table 2) . The two EX210 populations showed different sensitivities to lysis effected by antibodies directed against components of the outer membrane (Table 2) . One explanation for Table 2 Detection of 0-antigen on surface of clone EX21 0 by complement-dependent antibody- mediated bacteriolysis Antiserum specificity Indicator strain
V. cholerae 569B S . typhi Ty2Vi EX931 Clone EX210 Clone EX21 0
+gala +
569B-LPS 58 700
Surface co-expression of Vibrio cholerae and Salmonella typhi 0-antigens on Ty2l a clone EX210 Stephen R . Attridge, Denise Daniels, Judy K . Morona and Renato Morona Enterovax Limited, Cl- Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia 5001 (Received October 6, 1989 ; accepted in revised form December 8, 1989)
Attridge, S . R . (Enterovax Limited, C/- Dept of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia 5001), D . Daniels, J . K . Morona and R . Morona . Surface co-expression of Vibrio cholerae and Salmonella typhi 0-antigens on Ty21 a clone EX21 0 . Microbial Pathogenesis 1990 ; 8 : 177-188 . In an attempt to construct a bivalent, live, oral cholera-typhoid vaccine, genes specifying the biosynthesis of Vibrio cholerae 0-antigen have been transferred into a modified version of the attenuated, oral typhoid vaccine strain Salmonella typhi Ty21 a . The present report investigates the production of V. cholerae and S . typhi 0-antigens by one such clone, EX21 0 . When cultured without galactose supplementation EX210 produces surface 0-antigen of V. cholerae type, as detected by haemagglutination-inhibition and bactericidal assays, and by immuno-electron microscopy . However, the protective efficacy of Ty21 a depends upon growth in the presence of exogenous galactose and under these conditions only S . typhi 0-antigen is detectable on the surface of EX210 . Subsequent experiments revealed that the proportion of polysaccharide of S. typhi type is dependent upon the level of galactose supplementation, and identified a limiting sugar concentration which results in surface co-expression of both O-antigens . Visualization of the two polysaccharides on silver-stained polyacrylamide gels indicates that S . typhi 0-antigen subunits are polymerized into longer sidechains, suggesting that at higher galactose concentrations their predominance results in a masking of the shorter V . cholerae 0-antigen . Key words : Vibrio cholerae ; Salmonella typhi Ty2l a ; 0-antigen ; bivalent vaccine .
Introduction Killed parenteral cholera vaccines have consistently failed to provide enduring protection in the field .' 2 Evidently the serum antibodies generated by parenteral immunization are of insufficient concentration and/or inappropriate isotype to contribute significantly to enteric defence via transudation from the circulation . In contrast, natural or induced clinical infection affords strong and enduring resistance to disease ."' It is now generally accepted that to be effective a cholera vaccine will have to be orally administered, to stimulate production of IgA antibodies by the local immune system of the gut . Since Vibrio cho/erae is a non-invasive enteropathogen, the location and isotype of such antibodies make them ideally suited for host defence . Attempts to develop a safe and effective oral cholera vaccine have led to two new vaccine candidates . The more advanced of these is a non-viable formulation, comprising large numbers of inactivated V. cholerae and the isolated B subunit of the cholera toxin .' The design of this vaccine is based upon experimental data which show that 0882-4010/90/030177+12 $03 .00/0
0 1990 Academic Press Limited
178
S . R . Attridge et al.
antibacterial and antitoxic immune responses can act synergistically .' A recent field trial has shown that the combined bacterial-toxoid vaccine confers significant protection for at least 3 years .' A second approach is based upon the premise that the type of vaccine most likely to mimic the potent immunizing effects of natural infection would be a live oral vaccine with the potential to at least transiently colonize the gut . Attempts to attenuate a pathogenic V. cho/erae strain by deletion of the genes encoding cholera toxin have culminated in a vaccine candidate which is well-tolerated, immunogenic and protective in human volunteers .' The relative merits of these two vaccines have been recently discussed .` The most recent approach to cholera vaccine development is aimed at cloning protective antigens of V. cholerae into the live attenuated typhoid vaccine Ty21 a, 8 to produce a bivalent vaccine capable of conferring resistance to both cholera and typhoid . The demonstrated safety of Ty21 a 8 means that such a vaccine might combine the advantages of the two cholera vaccine candidates discussed above ; that is, it offers the safety of an inactivated vaccine with the superior immunogenicity of a viable one . At the commencement of this work there was only one V. cholerae antigen, the lipopolysaccharide (LPS), which had been implicated as a protective antigen . Direct evidence for this had been derived exclusively from experimental cholera models, 6 ' although several lines of circumstantial evidence suggested that antibodies to LPS could protect against clinical disease . Volunteer studies had shown that resistance to cholera challenge is dependent upon pre-existing antibacterial rather than antitoxic immunity .", " Epidemiological studies had identified an inverse correlation between the level of (naturally acquired) serum bactericidal antibodies and the incidence of disease, 12 and LPS is by far the most efficient substrate for such antibodies ." Finally, the occasional finding of serotype-restricted protection in field trials with parenteral vaccines' implicates LPS as a protective antigen, since this is the only bacterial component known to vary between the serotypes of V. cholerae . Construction of a bivalent Ty21 a-based cholera vaccine began with the cloning of the genes required for 0-antigen synthesis in V. cholerae . 14 A recent report describes the introduction of these genes into a modified Ty21 a strain to produce clone EX21 0 (R . Morona, unpublished results) . We now describe studies which analyze the expression of V. cholerae and Salmonella typhi 0-antigens on the surface of EX21 0 .
Results Effect of galactose on surface 0-antigen expression Although EX210 was known to be agglutinable by rabbit anti-V. cholerae serum (R . Morona, unpublished results), no information was available relating to the relative production of Inaba 0-antigen by EX210 and its V. cholerae parent strain 596B . In addition, it was of interest to investigate the effect of exogenous galactose on surface expression of both V. cholerae and S . typhi 0-antigens by the clone . The Ty21 a vector strain has a galE defect, and previous studies have shown that its capacity to synthesize smooth polysaccharide, and its protective efficacy, is dependent upon growth in medium supplemented with galactose ."" Initial experiments therefore compared the surface expression of 0-antigen of both V . cholerae and S . typhi types by clone EX210 and the parental strains, 569B and Ty21 a . The three strains were cultured in the presence or absence of 0 .001 % (w/v) galactose, 15 and the resulting (formalin-killed) bacterial suspensions titrated for their capacities to inhibit the agglutination of LPS-sensitized erythrocytes . Results of these haemagglutination-inhibition assays (HIAs) are shown in Table 1, and indicate that the two 0-antigen detection systems are specific, with limits of detection generally
0-antigens of Ty21 a clone EX21 0
179
Table l
Surface 0-antigen expression by clone EX21 0 and parental strains V. cholerae 569B and S . typhi Ty21 a, following growth in the presence or absence of 0 .001 % (w/v) galactose 0-antigen type Inhibitor strain
Growth +gala
V . cho/erae
S. typhi
+ + + +
8 8 0 0 7 0 9 0
0 0 0 9 0 9 0 10
569B Ty2la EX210 569B LPS Ty21a LPS
Bacteria were grown in the presence (+) or absence (-) of exogenous galactose (0 .001% w/v), inactivated by formalin treatment, and resuspended in saline . Bacterial suspensions (dry weights - 2 mg/ml) were tested for surface expression of the two 0-antigen types by HIA (see Materials and methods) . LPS preparations obtained from the parental strains were included as control inhibitors after standardization to 100 pg/ml on the basis of dry weight determinations . Figures show number of wells of inhibition in HIA .
0 .2 pg/ml . When grown in the absence of galactose, EX210 produces significant levels of V. cho/erae 0-antigen . The data in Table 1 confirm that 0-antigen production by Ty2l a (though not 569B) is dependent upon the addition of galactose to the growth medium . Not surprisingly, EX21 0 also produces S . typhi 0-antigen only in the presence of galactose . Under these conditions however, 0-antigen of V. cho/erae type is no longer detectable on the clone (Table 1 ) . The effect of exogenous galactose upon 0-antigen expression by EX210 was also examined by determining the sensitivities of the bacteria to complement-dependent lysis mediated by antibodies specific for V. cholerae or S. typhi LPS . This approach confirmed that growth in the absence of galactose results in primarily V. cholerae 0antigen on the surface of EX210 (Table 2) . In contrast, only S . typhi 0-antigen is detectable on the clone following growth in the presence of galactose (Table 2) . The two EX210 populations showed different sensitivities to lysis effected by antibodies directed against components of the outer membrane (Table 2) . One explanation for Table 2 Detection of 0-antigen on surface of clone EX21 0 by complement-dependent antibody- mediated bacteriolysis Antiserum specificity Indicator strain
V. cholerae 569B S . typhi Ty2Vi EX931 Clone EX210 Clone EX21 0
+gala +
569B-LPS 58 700
