Vol. 59, No. 7
INFECTION AND IMMUNITY, JUlY 1991, p. 2279-2284 0019-9567/91/072279-06$02.00/0 Copyright © 1991, American Society for Microbiology
Characterization and Immunogenicity of EX880, a Salmonella typhi Ty2la-Based Clone Which Produces Vibrio cholerae 0 Antigen S. R. ATTRIDGE,'* C. DEARLOVE,2 L. BEYER,' L. VAN DEN BOSCH,' A. HOWLES,' J. HACKETT,'t R. MORONA,1 J. LABROOY,2 AND D. ROWLEY' Enterovax Ltd., clo Department of Microbiology and Immunology,' and Department of Medicine,2 The University of Adelaide, Adelaide, South Australia 5001, Australia Received 18 January 1991/Accepted 2 April 1991
EX645 is a derivative of Salmonella typhi Ty2la which carries a plasmid specifying production of Vibrio cholerae 0 antigen. When cultured with exogenous galactose to overcome the galE defect of the vector, EX645 also synthesizes S. typhi 0 antigen, and this can result in the masking of the shorter V. cholerae 0 antigen on the bacterial surface. To determine whether the potential for such masking at least partly underlies the inconsistency of anti-V. cholerae responses elicited by EX645, a derivative of this strain has been isolated, characterized, and tested for immunogenicity in human volunteers. EX880 has an rjb defect which prevents synthesis of S. typhi 0 antigen, and consequently V. cholerae 0 antigen is still detectable on the surface of the clone following growth in the presence of galactose. Compared with EX645, EX880 more consistently elicited significant rises in serum bactericidal antibody levels, although individual responses within a cohort still varied widely.
carrying the plasmid does not vary during passage through the human gut (unpublished observations). The apparent unavailability of thymine in vivo was previously suggested by the finding that a thymine auxotroph derived from an attenuated cholera vaccine candidate lacked the colonizing capacity of its parent (14). Experience with the typhoid vaccine S. typhi Ty2la suggests that the bivalence of vaccine candidates such as EX645 will be dependent upon surface expression of S. typhi O antigen (11), which in turn requires growth of the vaccine strain in the presence of exogenous galactose to overcome the galE defect in Ty2la (10, 11). However, when EX210 (2) and EX645 (this study) are grown under such conditions, V. cholerae 0 antigen is no longer detectable on the bacterial surface. In vitro studies have revealed that S. typhi 0-antigen polysaccharide is considerably longer than that of the V. cholerae type; experiments suggest that at the concentration of galactose recommended for the growth of Ty2la, the Ty2la-based clones synthesize primarily S. typhi 0 antigen, with a resultant masking of the shorter V. cholerae polysaccharide (2). A limiting galactose concentration which results in approximately equivalent surface expression of the two 0-antigen types can be defined (2), and this level was adopted for growth of EX645 for the cholera challenge trial. However the availability of galactose cannot be controlled in vivo, and our experience is consistent with there being sufficient galactose in the human gut to allow the synthesis of smooth LPS by a galE strain of S. typhi (12). Indeed, both EX210 and EX645 elicit much stronger immune responses to S. typhi 0 antigen than to V. cholerae 0 antigen (5, 7, 21). It therefore seemed feasible that the in vivo masking of surface V. cholerae 0 antigen might be one factor contributing to the variability of immune responses elicited by EX645. Although one might expect masked antigens to be made available to the immune system following intracellular processing of vaccine organisms by antigen-presenting cells, immunogenicity trials performed with EX210 suggest that this is not necessarily the case. The consistency with which EX210 elicited anti-V. cholerae responses improved when
One approach to the development of an effective cholera vaccine involves the cloning of genes encoding a protective antigen of Vibrio cholerae into the attenuated live oral typhoid vaccine Salmonella typhi Ty2la. The rationale for this approach has been outlined elsewhere (2); the potential advantages are the safety and bivalence of such a vaccine. Previous reports have described the construction, characterization, safety, and immunogenicity of two Ty2la-based clones, EX210 and EX645 (2, 5, 7, 17). These carry plasmids which specify the synthesis of V. cholerae 0 antigen and have a modified lipopolysaccharide (LPS) core structure which allows linkage of this 0 antigen to the LPS core. The protective efficacy of EX645 has been recently evaluated in a cholera challenge trial. Although the protection rate was only 25%, there were encouraging indications of a reduced severity of infection among vaccinees, who suffered significantly less purging and excreted significantly fewer organisms than controls (21). Of the eight vaccinees challenged, the two who resisted infection were the two who showed the greatest increases in serum bactericidal antibodies following vaccination. This confirms the value of this index of immunogenicity as a guide to the protective efficacy of cholera vaccines (16, 19). The challenge trial demonstrated that EX645 has the potential to stimulate protective host immunity but highlighted the need for a greater consistency of immune responses. In considering options for meeting this objective, there seemed little scope for improved plasmid stability. In progressing from EX210 to EX645, the basis for plasmid retention was changed from tetracycline resistance, which is clinically unacceptable and would provide no selection pressure in vivo, to a system in which Ty21a thyA carries a plasmid which is thyA+ (18). The efficiency of this system is demonstrated by the fact that the percentage of organisms * Corresponding author. t Present address: Department of Animal Sciences, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064,
Australia. 2279
2280
ATTRIDGE ET AL.
the bacteria were cultured in such a way that vibrio 0 antigen was detectable on the cell surface at the time of immunization (7). To assess the significance of in vivo masking, we selected from EX645 a variant which produces surface V. cholerae 0 antigen irrespective of the concentration of exogenous galactose. This clone, EX880, was characterized in vitro and then tested for immunogenicity in human volunteers.
MATERIALS AND METHODS Bacterial strains. EX645, a rifampin-resistant, modified Ty2la strain carrying a plasmid specifying production of V. cholerae 0 antigen (pEVX22), was constructed as described elsewhere (21). EX880 was derived from EX645 by selection for resistance to phage 9NA, which recognizes 0-antigen subunits of certain salmonellae (23). Briefly, 5 x 108 EX645 bacteria were spread onto a nutrient agar plate which had previously been spread with 1010 PFU of phage 9NA; the plate contained 0.2% (wt/vol) galactose to enable the galE vector to produce S. typhi 0 antigen. After incubation at 37°C for 48 h, several of the resulting colonies were picked and purified by restreaking prior to determination of phage resistance patterns. Strains of V. cholerae (569B) and S. typhi (Ty2Vi-, a Viantigen-negative derivative of Ty2) (12) were included as standards in in vitro assays of surface 0-antigen expression. LPSs from these species were purchased from Sigma Chemical Co. (St. Louis, Mo.). Salmonella typhimurium SU418 was obtained from K. Sanderson, University of Calgary, Canada, and S. typhimurium DG1624 (Hfr, K9) was obtained from P. Reeves, University of Sydney, Sydney, Australia. Growth conditions. During the period of in vitro characterization of EX880, strains were cultured in nutrient broth (Difco) (16 g/liter with 5 g/liter of NaCl) with aeration at 37°C. When batches of EX880 were prepared for assessment of immunogenicity in human volunteers, the organisms were grown in parallel 2-liter fermenters in a defined medium supplemented with amino acids and with controlled pH (7.0) and oxygen tension. Bacteriophages and determination of resistance patterns. Bacteriophages were obtained from P. Reeves (C21, Felix 0, and T3); K. Sanderson (P221 and Ffm); and B. Stocker, Stanford University, Stanford, Calif. (9NA). Overnight bacterial cultures were cross-streaked on nutrient agar plates containing 0.2% (wt/vol) galactose when necessary for 0-antigen production; 5 RI of bacteriophage stock (109 PFU per ml) was applied, and sensitivity was scored after overnight incubation at 37°C. Investigation of 0-antigen production by EX880. The nature of the 0 antigen produced by EX880 was compared with that of its predecessor EX645 and with those of the parental strains of V. cholerae and S. typhi. Three approaches were adopted. (i) SDS-PAGE. Bacterial suspensions were analyzed for 0antigen production by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (17). This was performed as described elsewhere, except that the concentration of polyacrylamide in the gel was 20% and piperazine diacrylamide (Bio-Rad) was used instead of bisacrylamide as a cross-linking agent. (ii) HIA. Bacterial suspensions were inactivated by treatment with formaldehyde, suspended in saline to a concentration of 1010 per ml, and titrated for their capacities to inhibit the antibody-mediated agglutination of LPS-sensitized sheep erythrocytes, as described elsewhere (12). V.
INFECT. IMMUN.
cholerae and S. typhi bacteria and LPSs derived from them (12.5 ,ug/ml) were included in each assay to check the specificity and sensitivity of the two 0-antigen-detection systems. The antisera used in these tests were prepared by immunization of rabbits with V. cholerae 569B or S. typhi Ty2Vi-. Hemagglutination inhibition assay (HIA) end points are expressed as the number of wells showing a clear inhibition of agglutination compared with the pattern of agglutinated erythrocytes seen in control wells receiving no inhibitors. (iii) Bactericidal assay. A bactericidal assay was used to determine the sensitivity of EX880 to complement-dependent lysis mediated by antibodies of various specificities and has been described elsewhere (12). The antibodies used in these assays were immunoglobulin G (IgG) fractions isolated from polyclonal rabbit antisera, and guinea pig serum was used as a source of complement (2). The bactericidal titers of the IgG fractions were determined by interpolation from plots of bacterial survival versus antibody dilution and represent the reciprocal dilutions required to kill 50% of the indicator organisms. Immunogenicity of EX880 in human volunteers. (i) Subjects. Healthy normal volunteers gave written informed consent for these studies, which were approved by the Human Ethics Committee of the Royal Adelaide Hospital. Volunteers had no known prior contact with cholera or typhoid and had not been previously vaccinated against these diseases. (ii) Vaccine formulation and administration. Six batches of lyophilized EX880 were prepared, each of which had to meet a variety of quality control criteria, which included plasmid integrity and surface 0-antigen expression. Each vial typically contained ca. 3 x 10" lyophilized bacteria, of which -10% (batches 1 to 4) or -20% (batches 5 and 6) were viable; plasmid retention was >90% (except for batch 5, which had 86% retention). The contents of each vial were reconstituted with 10 ml of saline 10 to 30 min prior to use. Volunteers fasted for 8 h before administration of vaccine. They then drank 50 ml of 2% (wt/vol) NaHCO3 to neutralize gastric acidity, followed 5 min later by -4 x 10'° viable plasmid-containing EX880 (the reconstituted contents of one vial). Three such doses were given on days 0, 2, and 5. (iii) Assessment of immunogenicity. The immunogenicity of EX880 was assessed by determining the change in serum bactericidal antibody titer resulting from vaccination. Serum samples collected on days 0 (immediately prior to dosing) and 14 were titrated for bactericidal activity against V. cholerae 569B, by using an assay described elsewhere (12). From the individual bactericidal responses, the percentages of volunteers showing four- or eightfold increases in titer and the geometric mean (GM) fold increase of the cohort as a whole were calculated. Some samples were also titrated by enzyme-linked immunosorbent assay (ELISA) by using a method previously described (6). The antigen used to sensitize ELISA trays was an outer membrane preparation (OMP) isolated from EX931, which is EX880 lacking the PEVX22 plasmid which encodes V. cholerae 0 antigen. EX931 was cultured under conditions identical to those used for the preparation of the EX880 vaccine. The washed bacteria were ruptured by passage through a French pressure cell (Aminco), and cell envelopes were recovered by differential centrifugation (15). Outer membranes were isolated by extraction with the detergent Triton X-100 (15). (iv) Statistics. Bactericidal titers and responses elicited by EX645 and EX880 were compared for statistical significance
VOL. 59,
1991
IMMUNOGENICITY OF S. TYPHI Ty2la-BASED CLONE EX880
2281
by using chi-square and Student's t tests. The latter was performed by using pooled or separate variances, depending on the significance of Levene's F test. -
RESULTS Characterization of EX880. EX880 was derived from EX645 by selection for resistance to phage 9NA (see Materials and Methods). Screening with other phages revealed that EX880 was also P221r, whereas EX645 was P221s; both strains were resistant to Felix-O but sensitive to Ffm, T3, and C21. The alteration in phage resistance pattern is consistent with the phenotype caused by an rfb-like gene mutation (23), which was indicated by the following experiment. S. typhimurium phage ES18 was used to transduce hisG::TnlO, a transposon carrying tetracycline resistance and located near rfb, from S. typhimurium DG1624 into S. typhimurium Hfr strain SU418. The resulting strain, RMA90, acts as an Hfr donor and transfers hisG::TnJO and the rfb region as early markers. RMA90 was then mated with EX931, which is unable to synthesize surface S. typhi 0 antigen even when cultured with galactose to circumvent the galE defect inherited from Ty2la. Tetracycline-resistant, rifampin-resistant exconjugants selected from the mating were tested for production of 0 antigen of the S. typhimurium type, and 11 of 32 were positive. Thus the genetic defect preventing 0-antigen synthesis by EX880 is localized near hisG::TnlO, suggesting an rfb mutation. This conclusion was supported by experiments with two other Hfr donor strains capable of transferring rib as an early marker. In vitro production of 0 antigen by EX880. (i) SDS-PAGE. Ty2la, EX645, and EX880 were cultured in the presence and absence of exogenous galactose, harvested, treated with proteinase K, and analyzed by SDS-PAGE. Silver staining allowed visualization of the 0 antigens produced by these strains (Fig. 1). By comparison with the 0 antigen produced by the V. cholerae and S. typhi standards, it is apparent that both clones produce LPS with the shorter vibrio 0 polysaccharide when grown in the presence or absence of galactose. However, EX880 lacks the capacity of Ty2la and EX645 to synthesize the longer 0-antigen chains of the S. typhi type when cultured with galactose (Fig. 1). (ii) HIA. Surface 0-antigen expression by EX880 and EX645 was compared and quantitated by HIA. The clones were cultured with various levels of galactose supplementation and then tested for their capacities to inhibit the antibody-mediated agglutination of LPS-sensitized erythrocytes. Results with the controls in Table 1 show that the two 0-antigen-detection systems are specific and have limits of detection of 0.2 to 0.4 ,ug/ml. As expected for a galE strain, synthesis of S. typhi 0 antigen by EX645 is dependent on the presence of exogenous galactose (Table 1). When cultured with the concentrations of galactose recommended for Ty2la (10), however, V. cholerae 0 antigen was no longer detectable on the surface of EX645. Under these conditions, this 0 antigen is nevertheless detectable by SDS-PAGE (Fig. 1), suggesting that it is masked by the longer 0 antigen of the S. typhi type (2). V. cholerae 0 antigen is detected on the surfaces of EX645 organisms grown in the absence, or in the presence of a limiting concentration, of galactose (Table 1). In all these respects, EX645 behaves like its predecessor EX210 (2). In contrast, EX880 fails to synthesize 0 antigen of the S. typhi type even in the presence of galactose (Table 1). Presumably as a consequence of the absence of the (potentially masking) S. typhi 0 polysaccharide, V. cholerae 0
*
U-
A
FIG.
1.
BC D
SDS-PAGE
based clones.
EF
of LPSs
Ty2la (lanes
G H
produced by Ty2la and Ty2la-
(lanes C and D), and (lanes B, D, excess (0.1%, wt/vol)
A and B), EX880
(lanes G and H) were cultured in the and H) or absence (lanes A, C, and G) of EX645
presence
galactose and then harvested and subjected to SDS-PAGE with silver staining for visualization of LPS. S. typhi (lane E) and V. cholerae (lane F) standards were included for comparison. Symbols show gel positions of full-length S. typhi (~O) and V. cholerae() sidechains.
antigen is
detectable in
equivalent
amounts on the surfaces
of EX880 bacteria cultured with different ments
galactose supple-
(Table 1).
(iii) Bactericidal assay. That EX880 surface 0 antigen
TABLE
1.
Clone
or
expression
typhi and V. cholerae following growth in the presence of various levels of galactosea
Surface
antigens by
in HIA of S.
EX645 and EX880
control
% (wt/vol) Galactose
Inhibition (no. of wells) with the following type of 0 antigen
V. cholerae
S. typhi
EX645
0 0.0001 0.001 0.1
7 7
INFECTION AND IMMUNITY, JUlY 1991, p. 2279-2284 0019-9567/91/072279-06$02.00/0 Copyright © 1991, American Society for Microbiology
Characterization and Immunogenicity of EX880, a Salmonella typhi Ty2la-Based Clone Which Produces Vibrio cholerae 0 Antigen S. R. ATTRIDGE,'* C. DEARLOVE,2 L. BEYER,' L. VAN DEN BOSCH,' A. HOWLES,' J. HACKETT,'t R. MORONA,1 J. LABROOY,2 AND D. ROWLEY' Enterovax Ltd., clo Department of Microbiology and Immunology,' and Department of Medicine,2 The University of Adelaide, Adelaide, South Australia 5001, Australia Received 18 January 1991/Accepted 2 April 1991
EX645 is a derivative of Salmonella typhi Ty2la which carries a plasmid specifying production of Vibrio cholerae 0 antigen. When cultured with exogenous galactose to overcome the galE defect of the vector, EX645 also synthesizes S. typhi 0 antigen, and this can result in the masking of the shorter V. cholerae 0 antigen on the bacterial surface. To determine whether the potential for such masking at least partly underlies the inconsistency of anti-V. cholerae responses elicited by EX645, a derivative of this strain has been isolated, characterized, and tested for immunogenicity in human volunteers. EX880 has an rjb defect which prevents synthesis of S. typhi 0 antigen, and consequently V. cholerae 0 antigen is still detectable on the surface of the clone following growth in the presence of galactose. Compared with EX645, EX880 more consistently elicited significant rises in serum bactericidal antibody levels, although individual responses within a cohort still varied widely.
carrying the plasmid does not vary during passage through the human gut (unpublished observations). The apparent unavailability of thymine in vivo was previously suggested by the finding that a thymine auxotroph derived from an attenuated cholera vaccine candidate lacked the colonizing capacity of its parent (14). Experience with the typhoid vaccine S. typhi Ty2la suggests that the bivalence of vaccine candidates such as EX645 will be dependent upon surface expression of S. typhi O antigen (11), which in turn requires growth of the vaccine strain in the presence of exogenous galactose to overcome the galE defect in Ty2la (10, 11). However, when EX210 (2) and EX645 (this study) are grown under such conditions, V. cholerae 0 antigen is no longer detectable on the bacterial surface. In vitro studies have revealed that S. typhi 0-antigen polysaccharide is considerably longer than that of the V. cholerae type; experiments suggest that at the concentration of galactose recommended for the growth of Ty2la, the Ty2la-based clones synthesize primarily S. typhi 0 antigen, with a resultant masking of the shorter V. cholerae polysaccharide (2). A limiting galactose concentration which results in approximately equivalent surface expression of the two 0-antigen types can be defined (2), and this level was adopted for growth of EX645 for the cholera challenge trial. However the availability of galactose cannot be controlled in vivo, and our experience is consistent with there being sufficient galactose in the human gut to allow the synthesis of smooth LPS by a galE strain of S. typhi (12). Indeed, both EX210 and EX645 elicit much stronger immune responses to S. typhi 0 antigen than to V. cholerae 0 antigen (5, 7, 21). It therefore seemed feasible that the in vivo masking of surface V. cholerae 0 antigen might be one factor contributing to the variability of immune responses elicited by EX645. Although one might expect masked antigens to be made available to the immune system following intracellular processing of vaccine organisms by antigen-presenting cells, immunogenicity trials performed with EX210 suggest that this is not necessarily the case. The consistency with which EX210 elicited anti-V. cholerae responses improved when
One approach to the development of an effective cholera vaccine involves the cloning of genes encoding a protective antigen of Vibrio cholerae into the attenuated live oral typhoid vaccine Salmonella typhi Ty2la. The rationale for this approach has been outlined elsewhere (2); the potential advantages are the safety and bivalence of such a vaccine. Previous reports have described the construction, characterization, safety, and immunogenicity of two Ty2la-based clones, EX210 and EX645 (2, 5, 7, 17). These carry plasmids which specify the synthesis of V. cholerae 0 antigen and have a modified lipopolysaccharide (LPS) core structure which allows linkage of this 0 antigen to the LPS core. The protective efficacy of EX645 has been recently evaluated in a cholera challenge trial. Although the protection rate was only 25%, there were encouraging indications of a reduced severity of infection among vaccinees, who suffered significantly less purging and excreted significantly fewer organisms than controls (21). Of the eight vaccinees challenged, the two who resisted infection were the two who showed the greatest increases in serum bactericidal antibodies following vaccination. This confirms the value of this index of immunogenicity as a guide to the protective efficacy of cholera vaccines (16, 19). The challenge trial demonstrated that EX645 has the potential to stimulate protective host immunity but highlighted the need for a greater consistency of immune responses. In considering options for meeting this objective, there seemed little scope for improved plasmid stability. In progressing from EX210 to EX645, the basis for plasmid retention was changed from tetracycline resistance, which is clinically unacceptable and would provide no selection pressure in vivo, to a system in which Ty21a thyA carries a plasmid which is thyA+ (18). The efficiency of this system is demonstrated by the fact that the percentage of organisms * Corresponding author. t Present address: Department of Animal Sciences, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064,
Australia. 2279
2280
ATTRIDGE ET AL.
the bacteria were cultured in such a way that vibrio 0 antigen was detectable on the cell surface at the time of immunization (7). To assess the significance of in vivo masking, we selected from EX645 a variant which produces surface V. cholerae 0 antigen irrespective of the concentration of exogenous galactose. This clone, EX880, was characterized in vitro and then tested for immunogenicity in human volunteers.
MATERIALS AND METHODS Bacterial strains. EX645, a rifampin-resistant, modified Ty2la strain carrying a plasmid specifying production of V. cholerae 0 antigen (pEVX22), was constructed as described elsewhere (21). EX880 was derived from EX645 by selection for resistance to phage 9NA, which recognizes 0-antigen subunits of certain salmonellae (23). Briefly, 5 x 108 EX645 bacteria were spread onto a nutrient agar plate which had previously been spread with 1010 PFU of phage 9NA; the plate contained 0.2% (wt/vol) galactose to enable the galE vector to produce S. typhi 0 antigen. After incubation at 37°C for 48 h, several of the resulting colonies were picked and purified by restreaking prior to determination of phage resistance patterns. Strains of V. cholerae (569B) and S. typhi (Ty2Vi-, a Viantigen-negative derivative of Ty2) (12) were included as standards in in vitro assays of surface 0-antigen expression. LPSs from these species were purchased from Sigma Chemical Co. (St. Louis, Mo.). Salmonella typhimurium SU418 was obtained from K. Sanderson, University of Calgary, Canada, and S. typhimurium DG1624 (Hfr, K9) was obtained from P. Reeves, University of Sydney, Sydney, Australia. Growth conditions. During the period of in vitro characterization of EX880, strains were cultured in nutrient broth (Difco) (16 g/liter with 5 g/liter of NaCl) with aeration at 37°C. When batches of EX880 were prepared for assessment of immunogenicity in human volunteers, the organisms were grown in parallel 2-liter fermenters in a defined medium supplemented with amino acids and with controlled pH (7.0) and oxygen tension. Bacteriophages and determination of resistance patterns. Bacteriophages were obtained from P. Reeves (C21, Felix 0, and T3); K. Sanderson (P221 and Ffm); and B. Stocker, Stanford University, Stanford, Calif. (9NA). Overnight bacterial cultures were cross-streaked on nutrient agar plates containing 0.2% (wt/vol) galactose when necessary for 0-antigen production; 5 RI of bacteriophage stock (109 PFU per ml) was applied, and sensitivity was scored after overnight incubation at 37°C. Investigation of 0-antigen production by EX880. The nature of the 0 antigen produced by EX880 was compared with that of its predecessor EX645 and with those of the parental strains of V. cholerae and S. typhi. Three approaches were adopted. (i) SDS-PAGE. Bacterial suspensions were analyzed for 0antigen production by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (17). This was performed as described elsewhere, except that the concentration of polyacrylamide in the gel was 20% and piperazine diacrylamide (Bio-Rad) was used instead of bisacrylamide as a cross-linking agent. (ii) HIA. Bacterial suspensions were inactivated by treatment with formaldehyde, suspended in saline to a concentration of 1010 per ml, and titrated for their capacities to inhibit the antibody-mediated agglutination of LPS-sensitized sheep erythrocytes, as described elsewhere (12). V.
INFECT. IMMUN.
cholerae and S. typhi bacteria and LPSs derived from them (12.5 ,ug/ml) were included in each assay to check the specificity and sensitivity of the two 0-antigen-detection systems. The antisera used in these tests were prepared by immunization of rabbits with V. cholerae 569B or S. typhi Ty2Vi-. Hemagglutination inhibition assay (HIA) end points are expressed as the number of wells showing a clear inhibition of agglutination compared with the pattern of agglutinated erythrocytes seen in control wells receiving no inhibitors. (iii) Bactericidal assay. A bactericidal assay was used to determine the sensitivity of EX880 to complement-dependent lysis mediated by antibodies of various specificities and has been described elsewhere (12). The antibodies used in these assays were immunoglobulin G (IgG) fractions isolated from polyclonal rabbit antisera, and guinea pig serum was used as a source of complement (2). The bactericidal titers of the IgG fractions were determined by interpolation from plots of bacterial survival versus antibody dilution and represent the reciprocal dilutions required to kill 50% of the indicator organisms. Immunogenicity of EX880 in human volunteers. (i) Subjects. Healthy normal volunteers gave written informed consent for these studies, which were approved by the Human Ethics Committee of the Royal Adelaide Hospital. Volunteers had no known prior contact with cholera or typhoid and had not been previously vaccinated against these diseases. (ii) Vaccine formulation and administration. Six batches of lyophilized EX880 were prepared, each of which had to meet a variety of quality control criteria, which included plasmid integrity and surface 0-antigen expression. Each vial typically contained ca. 3 x 10" lyophilized bacteria, of which -10% (batches 1 to 4) or -20% (batches 5 and 6) were viable; plasmid retention was >90% (except for batch 5, which had 86% retention). The contents of each vial were reconstituted with 10 ml of saline 10 to 30 min prior to use. Volunteers fasted for 8 h before administration of vaccine. They then drank 50 ml of 2% (wt/vol) NaHCO3 to neutralize gastric acidity, followed 5 min later by -4 x 10'° viable plasmid-containing EX880 (the reconstituted contents of one vial). Three such doses were given on days 0, 2, and 5. (iii) Assessment of immunogenicity. The immunogenicity of EX880 was assessed by determining the change in serum bactericidal antibody titer resulting from vaccination. Serum samples collected on days 0 (immediately prior to dosing) and 14 were titrated for bactericidal activity against V. cholerae 569B, by using an assay described elsewhere (12). From the individual bactericidal responses, the percentages of volunteers showing four- or eightfold increases in titer and the geometric mean (GM) fold increase of the cohort as a whole were calculated. Some samples were also titrated by enzyme-linked immunosorbent assay (ELISA) by using a method previously described (6). The antigen used to sensitize ELISA trays was an outer membrane preparation (OMP) isolated from EX931, which is EX880 lacking the PEVX22 plasmid which encodes V. cholerae 0 antigen. EX931 was cultured under conditions identical to those used for the preparation of the EX880 vaccine. The washed bacteria were ruptured by passage through a French pressure cell (Aminco), and cell envelopes were recovered by differential centrifugation (15). Outer membranes were isolated by extraction with the detergent Triton X-100 (15). (iv) Statistics. Bactericidal titers and responses elicited by EX645 and EX880 were compared for statistical significance
VOL. 59,
1991
IMMUNOGENICITY OF S. TYPHI Ty2la-BASED CLONE EX880
2281
by using chi-square and Student's t tests. The latter was performed by using pooled or separate variances, depending on the significance of Levene's F test. -
RESULTS Characterization of EX880. EX880 was derived from EX645 by selection for resistance to phage 9NA (see Materials and Methods). Screening with other phages revealed that EX880 was also P221r, whereas EX645 was P221s; both strains were resistant to Felix-O but sensitive to Ffm, T3, and C21. The alteration in phage resistance pattern is consistent with the phenotype caused by an rfb-like gene mutation (23), which was indicated by the following experiment. S. typhimurium phage ES18 was used to transduce hisG::TnlO, a transposon carrying tetracycline resistance and located near rfb, from S. typhimurium DG1624 into S. typhimurium Hfr strain SU418. The resulting strain, RMA90, acts as an Hfr donor and transfers hisG::TnJO and the rfb region as early markers. RMA90 was then mated with EX931, which is unable to synthesize surface S. typhi 0 antigen even when cultured with galactose to circumvent the galE defect inherited from Ty2la. Tetracycline-resistant, rifampin-resistant exconjugants selected from the mating were tested for production of 0 antigen of the S. typhimurium type, and 11 of 32 were positive. Thus the genetic defect preventing 0-antigen synthesis by EX880 is localized near hisG::TnlO, suggesting an rfb mutation. This conclusion was supported by experiments with two other Hfr donor strains capable of transferring rib as an early marker. In vitro production of 0 antigen by EX880. (i) SDS-PAGE. Ty2la, EX645, and EX880 were cultured in the presence and absence of exogenous galactose, harvested, treated with proteinase K, and analyzed by SDS-PAGE. Silver staining allowed visualization of the 0 antigens produced by these strains (Fig. 1). By comparison with the 0 antigen produced by the V. cholerae and S. typhi standards, it is apparent that both clones produce LPS with the shorter vibrio 0 polysaccharide when grown in the presence or absence of galactose. However, EX880 lacks the capacity of Ty2la and EX645 to synthesize the longer 0-antigen chains of the S. typhi type when cultured with galactose (Fig. 1). (ii) HIA. Surface 0-antigen expression by EX880 and EX645 was compared and quantitated by HIA. The clones were cultured with various levels of galactose supplementation and then tested for their capacities to inhibit the antibody-mediated agglutination of LPS-sensitized erythrocytes. Results with the controls in Table 1 show that the two 0-antigen-detection systems are specific and have limits of detection of 0.2 to 0.4 ,ug/ml. As expected for a galE strain, synthesis of S. typhi 0 antigen by EX645 is dependent on the presence of exogenous galactose (Table 1). When cultured with the concentrations of galactose recommended for Ty2la (10), however, V. cholerae 0 antigen was no longer detectable on the surface of EX645. Under these conditions, this 0 antigen is nevertheless detectable by SDS-PAGE (Fig. 1), suggesting that it is masked by the longer 0 antigen of the S. typhi type (2). V. cholerae 0 antigen is detected on the surfaces of EX645 organisms grown in the absence, or in the presence of a limiting concentration, of galactose (Table 1). In all these respects, EX645 behaves like its predecessor EX210 (2). In contrast, EX880 fails to synthesize 0 antigen of the S. typhi type even in the presence of galactose (Table 1). Presumably as a consequence of the absence of the (potentially masking) S. typhi 0 polysaccharide, V. cholerae 0
*
U-
A
FIG.
1.
BC D
SDS-PAGE
based clones.
EF
of LPSs
Ty2la (lanes
G H
produced by Ty2la and Ty2la-
(lanes C and D), and (lanes B, D, excess (0.1%, wt/vol)
A and B), EX880
(lanes G and H) were cultured in the and H) or absence (lanes A, C, and G) of EX645
presence
galactose and then harvested and subjected to SDS-PAGE with silver staining for visualization of LPS. S. typhi (lane E) and V. cholerae (lane F) standards were included for comparison. Symbols show gel positions of full-length S. typhi (~O) and V. cholerae() sidechains.
antigen is
detectable in
equivalent
amounts on the surfaces
of EX880 bacteria cultured with different ments
galactose supple-
(Table 1).
(iii) Bactericidal assay. That EX880 surface 0 antigen
TABLE
1.
Clone
or
expression
typhi and V. cholerae following growth in the presence of various levels of galactosea
Surface
antigens by
in HIA of S.
EX645 and EX880
control
% (wt/vol) Galactose
Inhibition (no. of wells) with the following type of 0 antigen
V. cholerae
S. typhi
EX645
0 0.0001 0.001 0.1
7 7
