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The Human Humoral Immune Response to Salmonella typhi Ty2la Bruce D. Forrest, Justin T. LaBrooy, Les Beyer, Christine E. Dearlove, and David J. C. Shearman
University of Adelaide Department of Medicine, Royal Adelaide Hospital, Adelaide, and Enterovax Limited, Salisbury, South Australia
The development of effective oral vaccines against bacterial infections requires careful analysis of the local and systemic immune response these vaccines generate [1]. This evaluation relies heavily on measuring the immune response at an optimal point. The orally administered typhoid vaccine Salmonella typhi Ty21a represents the most effective oral vaccine developed against an enteric bacterial disease to date. However, this vaccine has been shown to provide variable degrees of protection against typhoid fever when administered to different populations in different formulations and dose schedules [2-9]. This work was initiated because of our earlier studies in which we examined the humoral immune response of candidate oral hybrid typhoid/cholera vaccines based on S. typhi Ty21a [10, 11]; we suspected that we had not sampled serum or jejunal fluid at the optimal time point. Therefore, we were obliged to investigate the time course of the human humoral immune response to S. typhi Ty21a more carefully. In these studies we examined in detail the humoral immune response in the first few weeks after ingestion of S. typhi 1)r21a and defined the optimal time points for sampling serum and intestinal fluid after oral vaccination. We also studied different dose regimens and formulations of this particular vaccine
Received 29 January 1990; revised 25 May 1990. Financial support: Enterovax Limited, Salisbury, Australia. Written and informed consent was obtained from volunteers before entry into the study. Use of human subjects was in accordance with the ethical standards of the Human Ethics Committee of the RoyalAdelaide Hospital, the Committee on the Ethics of Human Experimentation of the University of Adelaide, and the Helsinki Declaration of 1975. Reprints or correspondence: Dr. B. D. Forrest, Communicable Disease Surveillance Centre, Public Health Laboratory Service, 61 Colindale Avenue, London NW9 5DF, United Kingdom. The Journal of Infectious Diseases 1991;163:336-345 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6302-0020$01.00
for their effect on its immunogenicity and relevance to more effective production and oral delivery of attenuated bacterial vaccines.
Subjects and Methods Subjects. Sixty healthy adults (17 women and 43 men, 18-40 years old) agreed to participate in this study. None had previous exposure to typhoid fever, and only 1 (in group D) had been vaccinated againsttyphoid (with parenterally administered heat-inactivated typhoid vaccine) in the past 7 years. None had any history or current symptoms of gastrointestinal tract disease. Subjects were allocated randomly to 10 study groups (A-K). The vaccine organism, route, dose, and schedule used in the various groups are shown in table 1. Oral vaccination schedules required that vaccine be administered to subjects after an 8-h fast and 5 min after oral administration of 50 ml of a 2 % sodium hydrogen carbonate solution. This pretreatment was done where relevant, as gastric acid has an adverse effect on the viability of live, orally administered enteric organisms [12] as well as being able to alter the immunogenicity of inactivated oral vaccine preparations [13]. The vaccine dose was ingested after suspension in 40 ml of 0.9% saline and followed with 100 ml of distilled water. Vaccine preparations. S. typhi Ty21a is an attenuated Vi antigen-negative mutant of the pathogenic strain S. typhi Ty2 [2, 3]. Its safetyhas been confirmed in previous studies. The formalin-killed vaccine doses for group E were supplied by Dr. 1. Hackett (Department of Microbiology and Immunology, University of Adelaide;Australia). The enteric-coated capsules (Typh-Vax [Oral]) used for group H were manufactured by the Swiss Serum and Vaccine Institute (Berne, Switzerland) with the vaccine doses being obtained from the Australian distributor (Commonwealth Serum Laboratories, Parkville, Australia). Careful attention was paid to maintaining the cold chain with this preparation. The monovalent heat-killed typhoid vaccine doses used in group J were also obtained from the Commonwealth Serum Laboratories. All other vaccine doses were supplied by Enterovax Limited, Salisbury, South Australia.
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The short-term kinetics and the effects of different dose regimens and formulations on the humoral immune response induced in human subjects by the live attenuated typhoid vaccine Salmonella typhi Ty21a were examined. Antibody responses in jejunal fluid and serum and by specific antibody production in vitro by peripheral blood lymphocytes to S. typhi lipopolysaccharide were determined. A short vaccination schedule of three doses of 1011 live organisms over 5 days induced significantly greater intestinal 19A antityphoid antibody responses than did two comparable doses 21 days apart. The humoral immune response was dose dependent with 1010 and 1011 live organisms stimulating greater intestinal immune responses than did 1011 killed organisms. No responses were evident with either 109 viable organisms or with an enteric-coated preparation. In the continued development and assessment of oral typhoid vaccines, the effects of different doses and formulations and the timing of sampling on the humoral immune response should be considered.
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Table 1. Salmonella typhi Ty21a vaccine groups, doses, and formulations. Organism description, formulation
Group (n) A (7) B (6) C (5) D (6) E (4) F (7) G (7) H (6) J (7)
K (5)
S, S, S, S, S, S, S, S, S, R,
lyophilized, reconstituted'[ lyophilized, reconstituted'[ lyophilized, reconstitutedf live, freshly harvested formalin-killed lyophilized, reconstituted'[ lyophilized, reconstituted'[ enteric-coated capsule heat-killed, phenol-preserved live, freshly harvested
No. of organisms Total 5.2 5.2 5.2 1.8 1.3 5.2 5.2 5 5.0 1.8
X 1011
x
1011
X 1011
x
1011
X 1011 X 1010 X 10 9
x x x
1010 10 8 1011
Viable (% total) 1.7 1.7 1.7 1.4
°
1.7 1.7 1.1
°
1.3
X 1011 X 1011 X 1011 X 1011
(33) (33) (33) (78)
X 10 9 X 10 9
(33) (33) (2.2)
X 1011
(72)
X 1010
Dose schedule*
Time of intestinal intubations t
0, 2, 5 0, 2, 4 0,21 0, 2, 5 0, 2, 4 0, 2, 5 0,2,5 0,2,4§ 0, 14§ 0,2,4
15, 29, 43 7, 14, 21 21,42 15 14 15 15 14 26 14
*
A single batch of fermenter-grown lyophilized smooth S. typhi Ty21a was used for vaccine doses in this study. The total number of organisms as assessed by direct microscopy in the batch was 5.2 x 1011, with 1.7 x 1011 (33 %) shown to be viable by colony counts. However, it appears that whenever viable counts of S. typhi Ty21a are made directly from fermentation broths or agar plates, the count represents 65% of the total (microscope) count. Considering this observation, the percentage of initially viable cells surviving lyophilization is f\J50%. This discrepancy may be partly accounted for by the probable existence of bacterial cells that are viable but cannot be cultured (unpublished data). The vaccine derived from rough S. typhi Ty21a (group K) was prepared by growing the organisms in the absence of exogenous galactose; thus, the organisms were unable to assemble the a-antigen polysaccharide side chains of the lipopolysaccharide (LPS). This rough strain failed to agglutinate on slides with anti-9, 12 a-antigen typing sera, whereas smooth strains did. Samplecollection. Blood for serum samples and peripheral blood lymphocytes (PBL) was collected before and every 3-4 days after vaccination until the completion of the study. Intestinal fluid samples were obtained from the upper jejunum as previously described [14] when possible on day 14 or 15 after the start of the vaccination course (except for group C) as detailed in table 1. PBL were obtained by Ficoll-Paque (Pharmacia, Uppsala, Sweden) [15] centrifugation of heparinized venous blood and processed as previously detailed [14]. Quantifying specificantibody. Class-specific antityphoid LPS antibodies in serum and secretions were quantified using a previously described ELISA [14]. Because the vaccine strain S. typhi Ty21a does not elaborate the capsular polysaccharide Vi antigen, measurement of anti-Vi antigen antibodies was not possible. Briefly, 96-well polyvinyl microtiter ELISA plates (Costar 2595; Data Packaging, Cambridge, MA) were coated with S. typhi Ty2 LPS (L6386; Sigma Chemical, St. Louis) and blocked with 0.05% (wt/vol) bovine serum albumin in PBS. Subsequently, appropriate dilutions of serum and intestinal fluid obtained from individual vaccinated subjects were added to duplicate wells on the plates and
incubated at 37°C for 16 h. After washing, alkaline phosphatase-conjugated goat anti-human 19A, IgG, or IgM antisera (KPL; Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added and plates were incubated at 37°C for 4 h. After washing, 0.100 ml of a 1 mg/ml solution of the substrate p-nitrophenylphosphate (phosphatase substrate 104-105, Sigma) in a 10% diethanolamine buffer was added to all wells and, after further incubation at 37°C for 2 h, optical density (00) was measured using a Titertek ELISA reader at 405 nm. In vitro specific antityphoid antibody production by isolated peripheral blood lymphocytes was assayed as above, except that duplicate wells of 1 x 106 PBLlwell were assayed for each sample obtained for a specific time point; cells were incubated in a 5 % C02 incubator and substrate incubation time was 4 h at 37°C and not 2 h [14]. In each assay, serum from a convalescent typhoid patient with a known high antibody titer directed against S. typhi LPS was used as a positive control; serum from an unexposed individual with a known low antibody titer against S. typhi LPS was used as a negative control. Serum and intestinal specific antibody responses are presented as the reciprocal of the final titration that gave an 00 of 0.15 ELISA absorbance units/0.100 ml (the volume added to each well) and are expressed as units of antibody. This absorbance was chosen because it represented the upper limit of the 95 % confidence interval (CI) above background levels. The intestinal fluid specific antibody units were adjusted for total class-specific immunoglobulin content and were expressed as units of specific antibody per milligram of total class-specific immunoglobulin. A single radioimmunodiffusion method [16] was used to determine the total class-specific immunoglobulin content of intestinal fluid. The standard curve for the determination of total 19A in intestinal fluid was constructed using secretory 19A in the form of human colostrum of predetermined 19A and secretory 19A content [17]. Expressionofresultsand statistics. When assaying serum samples for specific antibody to compare doses and formulations of S. typhi Ty21a, the prevaccination sample and only the postvaccination sample nearest day 14 (days 14 or 15, most commonly) were
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NOTE. Smooth (S) = grown in presence of exogenous galactose, rough (R) = grown in absence of exogenous galactose. Venous samples for serum and peripheral blood lymphocytes (except E) obtained from all subjects at prevaccination, then every 3-4 days from start of vaccination for 21 or 42-43 (A and C) days. * Days from start of vaccination regimen. t Prevaccination intestinal intubation in week before vaccination. Values are time of postvaccination intestinal intubations in days after vaccination. All doses from same batch. § Subjects did not ingest sodium hydrogen carbonate solution before vaccination.
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used. The fold rises quoted and compared were the ratio of the day 14 to day 0 titers. All serially obtained postvaccination serum samples were assayed for kinetics of the antibody response. Sera and the adjusted intestinal antibody responses were transformed using natural logarithms [In(x)] before statistical analysis. Differences between the means of the pre- and postvaccination antibody titers within anyone group were determined using Student's t test. Comparisons of the fold rises in specific antibody titers and the PBL responses where available, between groups, were analyzed using the two-tailed Wilcoxon rank sum test for independent samples. Intragroup analysis of the significance of PBL responses was determined using the nonparametric signed rank test for paired data.
JIO 1991;163 (February)
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Intestinal antibody response. After vaccination with the three-vaccine-dose schedule, a significant increase in jejunalspecific antityphoid antibody level was evident as early as day 7 and peaked at days 14 or 15 (group A, P < .001; group B, P = .014; figure 1). There was no notable difference between day 14 or 15 and the day 21 levels (P = .99). Despite a significant decline in specific IgA antibody from day 15 to day 43 (P < .001), the day 43 level still remained above the prevaccination baseline (P = .041) (group A, figure 1). We have previously shown that a consistent specific intestinal antibody response can be demonstrated only in the IgA class [14]. In this study the same situation prevailed, with only specific IgG and IgM responses determined in exceptional responders; therefore, those results are not presented. The pattern of response to two doses of S. typhi Ty2la administered 21 days apart (group C) differed from that of three doses at 2-day intervals (groups A and B) most strikingly in terms of its consistency in individuals (figure 2C); some subjects responded to a single dose of S. typhi Ty2Ia similarly to those who had three doses close together, while other subjects showed little or no response. These results are also reflected in figure 1, where there is no statistically significant difference seen between the pre- and postvaccination intestinal IgA antibody levels in group C (P = .12), although the mean postvaccination IgA antibody response of group C is not significantly different from that of group B (P = .85) and where the range of the CI of the postvaccination IgA antibody levels in group C is much greater than in groups A and B. Similarly, the specific intestinal IgA response to the second single dose on day 21 was also erratic (figure 2C) with only one of five subjects showing clear evidence of benefit from the second dose. The day 42 mean specific antibody levels of this group were also not statistically different from those at day 0 (P = .16) or day 21 (P = .60). Serum antibody response. The serum-specific IgA antibody response paralleled that of the intestine (figure 3), with a similar significant increase in specific antibody levels oc-
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curring as early as day 7 (P = .003), peaking in the day 10-15 period, followed by a decline in specific antibody level to day 43. As with the intestinal response, the day 43 serum specific antibody level remained elevatedabove the prevaccination level despite this sharp decline (P = .023). The specific antityphoid serum IgG response followed a similar pattern (figure 3), with a significant rise evident as early as day 7 and peaking around day 15 (P = .020). However, unlike the serum IgA response, the IgG level did not decline appreciably from day 15 to day 43 (P = .13) and remained significantly elevated above baseline at that time (P = .014). The baseline serum IgG level was the one parameter that was not comparable between groups, but despite the slightly elevated group A baseline level, the pattern of response between the groups was the same.
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Humoral Immunity to S. typhi Ty2la
1991;163 (February)
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Effect of Vaccine Formulation and Dose on the Humoral Immune Response to S. typhi Ty21a Intestinaland PBLspecific antibodyresponse. (1) Vaccine formulations. Mean baseline prevaccination intestinal an-
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Serum antibody responses. As can be deduced by comparing the individual intestinal specific IgA responses in figure 2 and the individual serum specific IgA responses in figure 4, this study confirmed previously reported observations that the magnitude of specific antibody responses in serum samples obtained at a single time point correlate poorly with the magnitude of local intestinal specific antibody responses [14]. It is broadly apparent, however, that subjects with the greatest intestinal antibody responses were more likely to have the largest serum IgA responses. In addition, we observed that for anyone subject group, a change in the geometric mean fold rise of anyone particular antibody class after vaccination was reflected by a similar response in the other classes. The collated results for each subject group are presented in table 3.
Discussion In assessing the humoral immune response as part of a more complete assessment of the immunogenicity of any enteric bacterial candidate vaccine, the optimal timing of sampling appears to be important. We examined in detail the human humoral immune response to S. typhi Ty21a. As a result, an accurate pattern of the human immune response was determined, enabling assessment of the immune response to occur at the time of maximal response. We noted that the general pattern of the serum and intestinal responses differed only slightly, irrespective of whether a short course of three doses administered over 5 days or two single doses administered 21 days apart (group C) were used. However, the absolute numbers of responders were lower after the first of the two single doses. A second vaccine dose administered 3 weeks after a primary vaccine dose, except for a brief peak in the serum IgG, did not significantly alter the pattern of the response from that of the three-dose schedule at a similar time point. This observation indicated that there were equal numbers of good responders and low or nonresponders after a single dose of S. typhi Ty2la. One possible explanation is that the regimen of three doses simply provides additional opportunities for subjects who did not respond to the first or subsequent dose to produce a primary response. This appears to increase vaccine efficacy without significantly affecting the overall magnitude of the resulting response in that group. It has been observed that a freshly harvested preparation of S. typhi Ty21a given as 5-8 weekly doses of 3 x 1010-1 X 1011 viable organisms conferred substantial protection against disease to previously unexposed recipients [2]. Various factors preclude this effectiveformulation from widespread use: Freshly harvested doses are totally impractical for use in the field, maintaining quality control in minimizing batch variation and maintaining an effective cold chain are expensive, and multiple doses are necessary for maximal protection. In addition, the cost of commercially producing the large doses of 1011 live S. typhi Ty21a organisms is prohibitive.
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could be obtained had a fourfold or greater specific IgA antibody response). The response that was stimulated was not significantly different from that induced by the oral killed vaccine doses (group E). This latter association was not unexpected because nearly all of the organisms making up the enteric-coated capsules were killed ( rv 5 x 1010, or 97.8% of the total dose), comparable with the number of organisms making up the killed vaccine doses (lOll) used in group E. The 109 live organisms present in these doses did not produce a local antibody response detectable by our assay system (as observed in the responses found in group G). Unfortunately, it was not possible to perform the PBL-ELISA on the PBL of subjects in group E who received three doses of 1011 killed organisms. Of those subjects ingesting three doses of 1011 freshly harvested rough S. typhi Ty21a vaccine doses (group K), a fourfold or greater intestinal antityphoid LPS antibody response was detected in only one of five subjects. While the number of responders achieving a fourfold antibody response was much less than that observed with a similar course of smooth organisms, the geometric mean fold rise of the group (K) was not statistically significantly different from that achieved with any other dose regimen or formulation (figure 2). As expected, parenteral immunization with the commercial killed typhoid vaccine (group J) resulted in only one of six subjects just achieving a fourfold or greater rise in specific intestinal IgA antibody; only two had a very low magnitude PBL IgA response occurring by day 7 after vaccination. (2) Vaccine dose. As discussed, a single primary dose of S. typhi Ty21a failed to generate a convincing response with only two of five subjects having a fourfold or greater rise in specific intestinal antibody (group C), with an overall geometric mean fold rise of only 25 % of that produced by the regimen of three doses of 1011 viable organisms (table 2). This response was slightly better than that induced by three doses of 1010 viable organisms (group F), where there were only two of seven satisfactory responders (figure 2). An intestinal IgA immune response was not measurable with our assay system in any of the subjects receiving three doses of 109 live organisms (group G), irrespective of the presence of 3.5 x 109 killed organisms per dose. This was supported by the absence of an in vitro PBL specific IgA antibody response, indicating that this dose may not be sufficientto stimulate a humoral immune response in immunologically naive individuals. This situation is the presumed fringe region of efficacy for S. typhi Ty21a [17], as doses less than this have been reported not to confer protection in any but exceptional cases [18]. Although the magnitude of the specific IgA antibody release in vitro by the subjects' PBL did vary among groups (figure 5), there was little variation in the absolute numbers of responders between groups, with 100 %of subjects in groups A, C, D, F, and K responding, and with only four of six subjects in group H responding.
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JID 1991;163 (February)
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Table 3. Specific serum antityphoid lipopolysaccharide antibody response. Vaccination group
IgA
IgG
IgM
A 49.4 (18.6-131) 210 (90.8-485) .001
226 (85.1-598) 674 (172-2640) .014
272 (79.3-935) 606 (245-1500) .0Il
p
35.6 (16.7-75.9) 308 (69.3-1370) .003
42.1 (25.9-68.4) 546 (79.8-3740) .031
322 (196-531) 744 (485-1140) .016
Before After P
28.5 (7.95-102) 313 (122-803) .012
71.2 (6.19-818) 283 (152-526) .18
428 (377-486) 1230 (574-2620) .016
Before After
46.3 (18.3-Il7) 291 (33.0-2570) .033
266 (115-614) 2060 (424-10,000) .021
375 (159-882) 929 (205-4220) .Il
p
26.8 (11.2-64.2) 97.3 (23.2-407) .084
103 (42.6-251) 742 (251-2190) .048
207 (60.3-707) 213 (34.3-1320) .90
Before After P
38.3 (16.4-91.7) 198 (48.1-817) .003
146 (62.6-343) 340 (129-899) .042
198 (68.4-575) 401 (123-1310) .031
Before After
38.3 (15.8-93.0) 43.8 (18.6-103) .26
164 (78.1-344) 190 (75.9-477) .23
127 (51.6-315) 129 (53.9-310) .91
78.0 (27.8-219) 220 (78.4-619) .11
102 (29.0-359) 425 (245-737) .036
249 (117-532) 612 (293-1280) .016
Il4 (39.8-329) 304 (121-763) 393 (158-979) .15/.079
209 (64.1-680) 575 (257-1290) 447 (283-708) .070I.Il
692 (372-1290) 2350 (853-6480) 1840 (912-3700) .014/.025
Before After
p B Before After C
p E Before After F
G
p H Before After
p J
Before After (1) After (2)
p* K Before After
p
43.5 (18.3-104) 122 (20.5-731) .15
60.8 (27.0-137) 63.6 (14.8-273) .92
450 (285-711) 482 (346-670) .68
NOTE. Data are geometric mean antibody level (95% confidence interval). Postvaccination serum specific antibody levels represent day 14 or IS samples after the start of vaccination and are expressed as the reciprocal of the dilution giving Ol) = O.IS units by ELISA adjusted for total IgA. * First P value relates to change in specific antibody titer comparing first postvaccination (after [I]; day 14) with prevaccination (before); second P value compares second postvaccination (after [2]; day 26) with prevaccination (before).
This is especially so for fresh doses grown in brain-heart infusion broth, because the medium is expensive and the yield poor ("-'2 X 109 cfu/ml in fermenters). Overnight static cultures of S. typhi Ty21a (grown either in brain-heart infusion broth or in semidefined medium) yield only 5 x 108 cfu/ml; therefore, one dose represents "-'200 ml of medium. Fermentergrown cultures using semidefined medium have better yields (l x 1010 cfu/ml); nevertheless, one dose of 1011 S. typhi Ty21a still requires "-'10 ml of culture medium (unpublished data). The use of lyophilization to reduce dose variation has been
associated with an adverse effect on one experimental live typhoid vaccine's immunogenicity [19]. Here we have observed that after lyophilization S. typhi Ty21a retained "-'50% viability and demonstrated no impairment in its immunogenicity compared with that of an equivalent dose regimen of freshly harvested organisms. The presence of killed organisms is unlikely to have contributed significantly to the immune responses observed after vaccination with the lyophilized doses, since the immune responses after administration of "-'lOll formalin-killed organisms (group E) were not significant. The retention of immunogenicity by S. typhi Ty21a as implied by
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D
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enteric vaccine organism may be useful with respect to its protective efficacy through providing an indication of its ability to successfully colonize the small intestinal lymphoid tissue. We also suggest that previous failures to detect a specific humoral immune response to a candidate enteric vaccine organism may also reflect factors such as inadequate vaccine dosage or inappropriate dose regimen, adverse effectsof dose formulation, suboptimal timing or techniques of sampling, inadequate processing of samples [21-23], or insensitive assay systems. Acknowledgment We thank Richard Pridham, Wendy Ferguson, and Cathy Danz for assistance with the recruitment and management of the volunteers, and Linda Mundy, Pauline Dixon, and Cornelia Fieles for their excellent technical assistance.
References 1. World Health Organization. Intestinal immunity and vaccine development: a WHO memorandum. Bull WORLD Health Organ 1979;57: 719-34. 2. Gilman RH, Hornick RB, Woodward WE, et al. Evaluation of a UDPglucose-s-epimerase-less mutant of S. typhi as a live oral vaccine. J Infect Dis 1977;136:717-23. 3. Wahdan MH, Serie C, Germanier R, et al. A controlled field trial of live oral typhoid vaccine Ty 21a. Bull WORLD Health Organ 1980;58:469-74. 4. Wahdan MH, Serie C, Cerisier Y, Sallam S, Germanier R. A controlled field trial of live S. typhi strain Ty2la oral vaccine against typhoid: three-year results. J Infect Dis 1982;145:292-5. 5. Black RE, Levine MM, Ferreccio C, Clements ML, Germanier R, Chilean Typhoid Committee. Field trials of the efficacy of Ty21a attenuated S. typhi oral vaccine in Santiago, Chile [abstract]. XI International Congress for Tropical Medicine and Malaria, Calgary, Canada, 1984:15. 6. Levine MM, Ferreccio C, Black RE, Germanier R, Chilean Typhoid Committee. Large-scale field trial ofTy2la live oral typhoid vaccine in enteric-coated capsule formulation. Lancet 1987;1:1049-52. 7. Ferreccio C, Levine MM, Rodriguez H, Contreras R, Chilean Typhoid Committee. Comparative efficacy of two, three, or four doses of TY2la live oral typhoid vaccine in enteric-coated capsules: a field trial in an endemic area. J Infect Dis 1989;159:766-9. 8. Levine MM, Ferreccio C, Black RE, Tacket CO, Germanier R, Chilean Typhoid Committee. Progress in vaccines against typhoid fever. Rev Infect Dis 1989;1l(suppl 3):S552-67. 9. WHO Diarrhoeal Diseases Control Programme. Programme for control of diarrhoeal diseases. Interim programme report 1988. Geneva: WHO Report number WHO/CDD/89.31, 1989:35. 10. Forrest BD. The development of a bivalent vaccine against diarrhoeal disease. Southeast Asian J Trop Med Pub Health 1988;19:449-57. 11. Forrest BD, LaBrooy JT, Attridge SR, et al. A candidate live oral typhoid/cholera hybrid vaccine is immunogenic in humans. J Infect Dis 1989;159:145-6. 12. Giannella RA, Broitman SA, Zamcheck N. Influence of gastric acidity on bacterial and parasitic infections. Ann Intern Med 1973 ;78:271-6. 13. Clemens JD, Jertborn M, Sack D, et al. Effect of neutralization of gastric acid on immune responses to an oral B subunit, killed whole-cell cholera vaccine. J Infect Dis 1986;154:175-8.
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the humoral immune responses after lyophilizationmay reflect a retention in protective efficacy. In this study, we found that three sequential doses of 109 live S. typhi Ty21a failed to stimulate a measurable immune response in the intestine of previously unexposed subjects. This confirms other reports of the failure of this dose to effectively stimulate a local antibody response in individuals from nonendemic regions [17, 20]. It is unlikely that this reflects any limitations of the assay for intestinal specific antibody, as specific antibody responses were also not identifiable at this dose using the subjects' PBL. This suggests that the meager response that was observed with the enteric-coated formulation was most likely attributable to the 5 X 1010 killed organisms present rather than to the 1.1 X 109 viable ones. This response was also substantially less than that of three doses of 1011 or 1010 live organisms in the presence of a comparable number of killed vaccine organisms. This study supports previous findings of the inadequacy of single-point determinations of antigen-specific serum antibody irrespective of class [14] for the accurate determination of an intestinal antibody response. Although the sequential determination of a group's response within an antibody class is of most value, it is often the least practicable. Ifa point determination of serum antibody response is the only method available for the evaluation of the immunogenicity of a particular preparation, then the magnitude of the serum IgG response may provide the most consistent and sensitive indicator; significant antibody responses were evident in this class when absent in or not significant in others (e.g., groups Band H; table 3), although the magnitude of the response may not correlate with that of the intestine. This finding has been recently supported in field trials, in which the serum IgG anti-LPS O-antigen antibody responses were observed to correlate roughly with vaccine efficacy [8]. The PBL-ELISA was confirmed as a highly sensitive indicator of the primary intestinal exposure of an individual to an enteric bacterial organism. While the PBL-ELISA correlated closely with the specific intestinal IgA immune responses of a group at doses of 1011 viable vaccine organisms, it was unable to discriminate between responses of groups receiving dose regimens of 1011 or 1010 viable organisms. At dose schedules of 109 viable organisms, this assay was unable to detect an IgA-specific immune response in keeping with the results obtained from the intestinal-fluid assays. The PBL-ELISA, however, continues to remain the most rapid and sensitive measure of an immune response in a specific individual following primary oral vaccination. The intestinal antibody response as determined using our model system appeared to be particularly useful for discriminating between the immunogenicities of various vaccine dosages and formulations. These studies of the humoral immune response to typhoid vaccines suggest that the measurement of this response to an
1ID 1991;163 (February)
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Humoral Immunity to S. typhi Ty21a
14. Forrest BD. The identification of an intestinal immune response using peripheral blood lymphocytes. Lancet 1988;1:81-83. 15. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21(suppl 97):77-89. 16. Mancini G, Carbonara AO, Heremans JE Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965;2:235-7. 17. Bartholomeusz RCA, LaBrooy JT, Johnson M, Shearman DJC, Rowley D. Gut immunity to typhoid: the immune response to a live oral typhoid vaccine Ty2la. J Gastroenterol Hepatol 1986;1:61-7. 18. Hirschel B, Wuthrich R, Somaini B, Steffen R. Inefficacy of the commercial live oral Ty21a vaccine in the prevention of typhoid fever. Eur J Clin Microbiol 1985;4:295-8. 19. Levine MM, DuPont HL, Hornick RB, et al. Attenuated, streptomycin-
20.
21.
22.
23.
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dependent S. typhi oral vaccine: potential deleterious effectsof lyophilization. J Infect Dis 1976;133:424-9. Sirianni MC, Turbessi G, Scarpati B, Russo G, Mascellino T, Aiuti E A preliminary report on the immunological responses after-oral vaccine (Ty 21a). Boll 1st Sieroter Milan 1984;63:352-6. LaBrooy JT, Davidson GP, Shearman DJC, Rowley D. The antibody response to bacterial gastroenteritis in serum and secretions. Clin Exp Immunol 1980;41:290-6. Elson CO, Balding W, Lefkowitz 1. A lavagetechnique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J Immunol Methods 1984;67:101-8. Samson RR, McClelland RR, Shearman DJe. Studies on the quantitation of immunoglobulins in human intestinal secretions. Gut 1973; 14:616-26.
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The Human Humoral Immune Response to Salmonella typhi Ty2la Bruce D. Forrest, Justin T. LaBrooy, Les Beyer, Christine E. Dearlove, and David J. C. Shearman
University of Adelaide Department of Medicine, Royal Adelaide Hospital, Adelaide, and Enterovax Limited, Salisbury, South Australia
The development of effective oral vaccines against bacterial infections requires careful analysis of the local and systemic immune response these vaccines generate [1]. This evaluation relies heavily on measuring the immune response at an optimal point. The orally administered typhoid vaccine Salmonella typhi Ty21a represents the most effective oral vaccine developed against an enteric bacterial disease to date. However, this vaccine has been shown to provide variable degrees of protection against typhoid fever when administered to different populations in different formulations and dose schedules [2-9]. This work was initiated because of our earlier studies in which we examined the humoral immune response of candidate oral hybrid typhoid/cholera vaccines based on S. typhi Ty21a [10, 11]; we suspected that we had not sampled serum or jejunal fluid at the optimal time point. Therefore, we were obliged to investigate the time course of the human humoral immune response to S. typhi Ty21a more carefully. In these studies we examined in detail the humoral immune response in the first few weeks after ingestion of S. typhi 1)r21a and defined the optimal time points for sampling serum and intestinal fluid after oral vaccination. We also studied different dose regimens and formulations of this particular vaccine
Received 29 January 1990; revised 25 May 1990. Financial support: Enterovax Limited, Salisbury, Australia. Written and informed consent was obtained from volunteers before entry into the study. Use of human subjects was in accordance with the ethical standards of the Human Ethics Committee of the RoyalAdelaide Hospital, the Committee on the Ethics of Human Experimentation of the University of Adelaide, and the Helsinki Declaration of 1975. Reprints or correspondence: Dr. B. D. Forrest, Communicable Disease Surveillance Centre, Public Health Laboratory Service, 61 Colindale Avenue, London NW9 5DF, United Kingdom. The Journal of Infectious Diseases 1991;163:336-345 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6302-0020$01.00
for their effect on its immunogenicity and relevance to more effective production and oral delivery of attenuated bacterial vaccines.
Subjects and Methods Subjects. Sixty healthy adults (17 women and 43 men, 18-40 years old) agreed to participate in this study. None had previous exposure to typhoid fever, and only 1 (in group D) had been vaccinated againsttyphoid (with parenterally administered heat-inactivated typhoid vaccine) in the past 7 years. None had any history or current symptoms of gastrointestinal tract disease. Subjects were allocated randomly to 10 study groups (A-K). The vaccine organism, route, dose, and schedule used in the various groups are shown in table 1. Oral vaccination schedules required that vaccine be administered to subjects after an 8-h fast and 5 min after oral administration of 50 ml of a 2 % sodium hydrogen carbonate solution. This pretreatment was done where relevant, as gastric acid has an adverse effect on the viability of live, orally administered enteric organisms [12] as well as being able to alter the immunogenicity of inactivated oral vaccine preparations [13]. The vaccine dose was ingested after suspension in 40 ml of 0.9% saline and followed with 100 ml of distilled water. Vaccine preparations. S. typhi Ty21a is an attenuated Vi antigen-negative mutant of the pathogenic strain S. typhi Ty2 [2, 3]. Its safetyhas been confirmed in previous studies. The formalin-killed vaccine doses for group E were supplied by Dr. 1. Hackett (Department of Microbiology and Immunology, University of Adelaide;Australia). The enteric-coated capsules (Typh-Vax [Oral]) used for group H were manufactured by the Swiss Serum and Vaccine Institute (Berne, Switzerland) with the vaccine doses being obtained from the Australian distributor (Commonwealth Serum Laboratories, Parkville, Australia). Careful attention was paid to maintaining the cold chain with this preparation. The monovalent heat-killed typhoid vaccine doses used in group J were also obtained from the Commonwealth Serum Laboratories. All other vaccine doses were supplied by Enterovax Limited, Salisbury, South Australia.
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The short-term kinetics and the effects of different dose regimens and formulations on the humoral immune response induced in human subjects by the live attenuated typhoid vaccine Salmonella typhi Ty21a were examined. Antibody responses in jejunal fluid and serum and by specific antibody production in vitro by peripheral blood lymphocytes to S. typhi lipopolysaccharide were determined. A short vaccination schedule of three doses of 1011 live organisms over 5 days induced significantly greater intestinal 19A antityphoid antibody responses than did two comparable doses 21 days apart. The humoral immune response was dose dependent with 1010 and 1011 live organisms stimulating greater intestinal immune responses than did 1011 killed organisms. No responses were evident with either 109 viable organisms or with an enteric-coated preparation. In the continued development and assessment of oral typhoid vaccines, the effects of different doses and formulations and the timing of sampling on the humoral immune response should be considered.
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Table 1. Salmonella typhi Ty21a vaccine groups, doses, and formulations. Organism description, formulation
Group (n) A (7) B (6) C (5) D (6) E (4) F (7) G (7) H (6) J (7)
K (5)
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lyophilized, reconstituted'[ lyophilized, reconstituted'[ lyophilized, reconstitutedf live, freshly harvested formalin-killed lyophilized, reconstituted'[ lyophilized, reconstituted'[ enteric-coated capsule heat-killed, phenol-preserved live, freshly harvested
No. of organisms Total 5.2 5.2 5.2 1.8 1.3 5.2 5.2 5 5.0 1.8
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x
1011
X 1011
x
1011
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Time of intestinal intubations t
0, 2, 5 0, 2, 4 0,21 0, 2, 5 0, 2, 4 0, 2, 5 0,2,5 0,2,4§ 0, 14§ 0,2,4
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*
A single batch of fermenter-grown lyophilized smooth S. typhi Ty21a was used for vaccine doses in this study. The total number of organisms as assessed by direct microscopy in the batch was 5.2 x 1011, with 1.7 x 1011 (33 %) shown to be viable by colony counts. However, it appears that whenever viable counts of S. typhi Ty21a are made directly from fermentation broths or agar plates, the count represents 65% of the total (microscope) count. Considering this observation, the percentage of initially viable cells surviving lyophilization is f\J50%. This discrepancy may be partly accounted for by the probable existence of bacterial cells that are viable but cannot be cultured (unpublished data). The vaccine derived from rough S. typhi Ty21a (group K) was prepared by growing the organisms in the absence of exogenous galactose; thus, the organisms were unable to assemble the a-antigen polysaccharide side chains of the lipopolysaccharide (LPS). This rough strain failed to agglutinate on slides with anti-9, 12 a-antigen typing sera, whereas smooth strains did. Samplecollection. Blood for serum samples and peripheral blood lymphocytes (PBL) was collected before and every 3-4 days after vaccination until the completion of the study. Intestinal fluid samples were obtained from the upper jejunum as previously described [14] when possible on day 14 or 15 after the start of the vaccination course (except for group C) as detailed in table 1. PBL were obtained by Ficoll-Paque (Pharmacia, Uppsala, Sweden) [15] centrifugation of heparinized venous blood and processed as previously detailed [14]. Quantifying specificantibody. Class-specific antityphoid LPS antibodies in serum and secretions were quantified using a previously described ELISA [14]. Because the vaccine strain S. typhi Ty21a does not elaborate the capsular polysaccharide Vi antigen, measurement of anti-Vi antigen antibodies was not possible. Briefly, 96-well polyvinyl microtiter ELISA plates (Costar 2595; Data Packaging, Cambridge, MA) were coated with S. typhi Ty2 LPS (L6386; Sigma Chemical, St. Louis) and blocked with 0.05% (wt/vol) bovine serum albumin in PBS. Subsequently, appropriate dilutions of serum and intestinal fluid obtained from individual vaccinated subjects were added to duplicate wells on the plates and
incubated at 37°C for 16 h. After washing, alkaline phosphatase-conjugated goat anti-human 19A, IgG, or IgM antisera (KPL; Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added and plates were incubated at 37°C for 4 h. After washing, 0.100 ml of a 1 mg/ml solution of the substrate p-nitrophenylphosphate (phosphatase substrate 104-105, Sigma) in a 10% diethanolamine buffer was added to all wells and, after further incubation at 37°C for 2 h, optical density (00) was measured using a Titertek ELISA reader at 405 nm. In vitro specific antityphoid antibody production by isolated peripheral blood lymphocytes was assayed as above, except that duplicate wells of 1 x 106 PBLlwell were assayed for each sample obtained for a specific time point; cells were incubated in a 5 % C02 incubator and substrate incubation time was 4 h at 37°C and not 2 h [14]. In each assay, serum from a convalescent typhoid patient with a known high antibody titer directed against S. typhi LPS was used as a positive control; serum from an unexposed individual with a known low antibody titer against S. typhi LPS was used as a negative control. Serum and intestinal specific antibody responses are presented as the reciprocal of the final titration that gave an 00 of 0.15 ELISA absorbance units/0.100 ml (the volume added to each well) and are expressed as units of antibody. This absorbance was chosen because it represented the upper limit of the 95 % confidence interval (CI) above background levels. The intestinal fluid specific antibody units were adjusted for total class-specific immunoglobulin content and were expressed as units of specific antibody per milligram of total class-specific immunoglobulin. A single radioimmunodiffusion method [16] was used to determine the total class-specific immunoglobulin content of intestinal fluid. The standard curve for the determination of total 19A in intestinal fluid was constructed using secretory 19A in the form of human colostrum of predetermined 19A and secretory 19A content [17]. Expressionofresultsand statistics. When assaying serum samples for specific antibody to compare doses and formulations of S. typhi Ty21a, the prevaccination sample and only the postvaccination sample nearest day 14 (days 14 or 15, most commonly) were
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NOTE. Smooth (S) = grown in presence of exogenous galactose, rough (R) = grown in absence of exogenous galactose. Venous samples for serum and peripheral blood lymphocytes (except E) obtained from all subjects at prevaccination, then every 3-4 days from start of vaccination for 21 or 42-43 (A and C) days. * Days from start of vaccination regimen. t Prevaccination intestinal intubation in week before vaccination. Values are time of postvaccination intestinal intubations in days after vaccination. All doses from same batch. § Subjects did not ingest sodium hydrogen carbonate solution before vaccination.
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used. The fold rises quoted and compared were the ratio of the day 14 to day 0 titers. All serially obtained postvaccination serum samples were assayed for kinetics of the antibody response. Sera and the adjusted intestinal antibody responses were transformed using natural logarithms [In(x)] before statistical analysis. Differences between the means of the pre- and postvaccination antibody titers within anyone group were determined using Student's t test. Comparisons of the fold rises in specific antibody titers and the PBL responses where available, between groups, were analyzed using the two-tailed Wilcoxon rank sum test for independent samples. Intragroup analysis of the significance of PBL responses was determined using the nonparametric signed rank test for paired data.
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curring as early as day 7 (P = .003), peaking in the day 10-15 period, followed by a decline in specific antibody level to day 43. As with the intestinal response, the day 43 serum specific antibody level remained elevatedabove the prevaccination level despite this sharp decline (P = .023). The specific antityphoid serum IgG response followed a similar pattern (figure 3), with a significant rise evident as early as day 7 and peaking around day 15 (P = .020). However, unlike the serum IgA response, the IgG level did not decline appreciably from day 15 to day 43 (P = .13) and remained significantly elevated above baseline at that time (P = .014). The baseline serum IgG level was the one parameter that was not comparable between groups, but despite the slightly elevated group A baseline level, the pattern of response between the groups was the same.
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Effect of Vaccine Formulation and Dose on the Humoral Immune Response to S. typhi Ty21a Intestinaland PBLspecific antibodyresponse. (1) Vaccine formulations. Mean baseline prevaccination intestinal an-
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Serum antibody responses. As can be deduced by comparing the individual intestinal specific IgA responses in figure 2 and the individual serum specific IgA responses in figure 4, this study confirmed previously reported observations that the magnitude of specific antibody responses in serum samples obtained at a single time point correlate poorly with the magnitude of local intestinal specific antibody responses [14]. It is broadly apparent, however, that subjects with the greatest intestinal antibody responses were more likely to have the largest serum IgA responses. In addition, we observed that for anyone subject group, a change in the geometric mean fold rise of anyone particular antibody class after vaccination was reflected by a similar response in the other classes. The collated results for each subject group are presented in table 3.
Discussion In assessing the humoral immune response as part of a more complete assessment of the immunogenicity of any enteric bacterial candidate vaccine, the optimal timing of sampling appears to be important. We examined in detail the human humoral immune response to S. typhi Ty21a. As a result, an accurate pattern of the human immune response was determined, enabling assessment of the immune response to occur at the time of maximal response. We noted that the general pattern of the serum and intestinal responses differed only slightly, irrespective of whether a short course of three doses administered over 5 days or two single doses administered 21 days apart (group C) were used. However, the absolute numbers of responders were lower after the first of the two single doses. A second vaccine dose administered 3 weeks after a primary vaccine dose, except for a brief peak in the serum IgG, did not significantly alter the pattern of the response from that of the three-dose schedule at a similar time point. This observation indicated that there were equal numbers of good responders and low or nonresponders after a single dose of S. typhi Ty2la. One possible explanation is that the regimen of three doses simply provides additional opportunities for subjects who did not respond to the first or subsequent dose to produce a primary response. This appears to increase vaccine efficacy without significantly affecting the overall magnitude of the resulting response in that group. It has been observed that a freshly harvested preparation of S. typhi Ty21a given as 5-8 weekly doses of 3 x 1010-1 X 1011 viable organisms conferred substantial protection against disease to previously unexposed recipients [2]. Various factors preclude this effectiveformulation from widespread use: Freshly harvested doses are totally impractical for use in the field, maintaining quality control in minimizing batch variation and maintaining an effective cold chain are expensive, and multiple doses are necessary for maximal protection. In addition, the cost of commercially producing the large doses of 1011 live S. typhi Ty21a organisms is prohibitive.
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could be obtained had a fourfold or greater specific IgA antibody response). The response that was stimulated was not significantly different from that induced by the oral killed vaccine doses (group E). This latter association was not unexpected because nearly all of the organisms making up the enteric-coated capsules were killed ( rv 5 x 1010, or 97.8% of the total dose), comparable with the number of organisms making up the killed vaccine doses (lOll) used in group E. The 109 live organisms present in these doses did not produce a local antibody response detectable by our assay system (as observed in the responses found in group G). Unfortunately, it was not possible to perform the PBL-ELISA on the PBL of subjects in group E who received three doses of 1011 killed organisms. Of those subjects ingesting three doses of 1011 freshly harvested rough S. typhi Ty21a vaccine doses (group K), a fourfold or greater intestinal antityphoid LPS antibody response was detected in only one of five subjects. While the number of responders achieving a fourfold antibody response was much less than that observed with a similar course of smooth organisms, the geometric mean fold rise of the group (K) was not statistically significantly different from that achieved with any other dose regimen or formulation (figure 2). As expected, parenteral immunization with the commercial killed typhoid vaccine (group J) resulted in only one of six subjects just achieving a fourfold or greater rise in specific intestinal IgA antibody; only two had a very low magnitude PBL IgA response occurring by day 7 after vaccination. (2) Vaccine dose. As discussed, a single primary dose of S. typhi Ty21a failed to generate a convincing response with only two of five subjects having a fourfold or greater rise in specific intestinal antibody (group C), with an overall geometric mean fold rise of only 25 % of that produced by the regimen of three doses of 1011 viable organisms (table 2). This response was slightly better than that induced by three doses of 1010 viable organisms (group F), where there were only two of seven satisfactory responders (figure 2). An intestinal IgA immune response was not measurable with our assay system in any of the subjects receiving three doses of 109 live organisms (group G), irrespective of the presence of 3.5 x 109 killed organisms per dose. This was supported by the absence of an in vitro PBL specific IgA antibody response, indicating that this dose may not be sufficientto stimulate a humoral immune response in immunologically naive individuals. This situation is the presumed fringe region of efficacy for S. typhi Ty21a [17], as doses less than this have been reported not to confer protection in any but exceptional cases [18]. Although the magnitude of the specific IgA antibody release in vitro by the subjects' PBL did vary among groups (figure 5), there was little variation in the absolute numbers of responders between groups, with 100 %of subjects in groups A, C, D, F, and K responding, and with only four of six subjects in group H responding.
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Table 3. Specific serum antityphoid lipopolysaccharide antibody response. Vaccination group
IgA
IgG
IgM
A 49.4 (18.6-131) 210 (90.8-485) .001
226 (85.1-598) 674 (172-2640) .014
272 (79.3-935) 606 (245-1500) .0Il
p
35.6 (16.7-75.9) 308 (69.3-1370) .003
42.1 (25.9-68.4) 546 (79.8-3740) .031
322 (196-531) 744 (485-1140) .016
Before After P
28.5 (7.95-102) 313 (122-803) .012
71.2 (6.19-818) 283 (152-526) .18
428 (377-486) 1230 (574-2620) .016
Before After
46.3 (18.3-Il7) 291 (33.0-2570) .033
266 (115-614) 2060 (424-10,000) .021
375 (159-882) 929 (205-4220) .Il
p
26.8 (11.2-64.2) 97.3 (23.2-407) .084
103 (42.6-251) 742 (251-2190) .048
207 (60.3-707) 213 (34.3-1320) .90
Before After P
38.3 (16.4-91.7) 198 (48.1-817) .003
146 (62.6-343) 340 (129-899) .042
198 (68.4-575) 401 (123-1310) .031
Before After
38.3 (15.8-93.0) 43.8 (18.6-103) .26
164 (78.1-344) 190 (75.9-477) .23
127 (51.6-315) 129 (53.9-310) .91
78.0 (27.8-219) 220 (78.4-619) .11
102 (29.0-359) 425 (245-737) .036
249 (117-532) 612 (293-1280) .016
Il4 (39.8-329) 304 (121-763) 393 (158-979) .15/.079
209 (64.1-680) 575 (257-1290) 447 (283-708) .070I.Il
692 (372-1290) 2350 (853-6480) 1840 (912-3700) .014/.025
Before After
p B Before After C
p E Before After F
G
p H Before After
p J
Before After (1) After (2)
p* K Before After
p
43.5 (18.3-104) 122 (20.5-731) .15
60.8 (27.0-137) 63.6 (14.8-273) .92
450 (285-711) 482 (346-670) .68
NOTE. Data are geometric mean antibody level (95% confidence interval). Postvaccination serum specific antibody levels represent day 14 or IS samples after the start of vaccination and are expressed as the reciprocal of the dilution giving Ol) = O.IS units by ELISA adjusted for total IgA. * First P value relates to change in specific antibody titer comparing first postvaccination (after [I]; day 14) with prevaccination (before); second P value compares second postvaccination (after [2]; day 26) with prevaccination (before).
This is especially so for fresh doses grown in brain-heart infusion broth, because the medium is expensive and the yield poor ("-'2 X 109 cfu/ml in fermenters). Overnight static cultures of S. typhi Ty21a (grown either in brain-heart infusion broth or in semidefined medium) yield only 5 x 108 cfu/ml; therefore, one dose represents "-'200 ml of medium. Fermentergrown cultures using semidefined medium have better yields (l x 1010 cfu/ml); nevertheless, one dose of 1011 S. typhi Ty21a still requires "-'10 ml of culture medium (unpublished data). The use of lyophilization to reduce dose variation has been
associated with an adverse effect on one experimental live typhoid vaccine's immunogenicity [19]. Here we have observed that after lyophilization S. typhi Ty21a retained "-'50% viability and demonstrated no impairment in its immunogenicity compared with that of an equivalent dose regimen of freshly harvested organisms. The presence of killed organisms is unlikely to have contributed significantly to the immune responses observed after vaccination with the lyophilized doses, since the immune responses after administration of "-'lOll formalin-killed organisms (group E) were not significant. The retention of immunogenicity by S. typhi Ty21a as implied by
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Forrest et al.
enteric vaccine organism may be useful with respect to its protective efficacy through providing an indication of its ability to successfully colonize the small intestinal lymphoid tissue. We also suggest that previous failures to detect a specific humoral immune response to a candidate enteric vaccine organism may also reflect factors such as inadequate vaccine dosage or inappropriate dose regimen, adverse effectsof dose formulation, suboptimal timing or techniques of sampling, inadequate processing of samples [21-23], or insensitive assay systems. Acknowledgment We thank Richard Pridham, Wendy Ferguson, and Cathy Danz for assistance with the recruitment and management of the volunteers, and Linda Mundy, Pauline Dixon, and Cornelia Fieles for their excellent technical assistance.
References 1. World Health Organization. Intestinal immunity and vaccine development: a WHO memorandum. Bull WORLD Health Organ 1979;57: 719-34. 2. Gilman RH, Hornick RB, Woodward WE, et al. Evaluation of a UDPglucose-s-epimerase-less mutant of S. typhi as a live oral vaccine. J Infect Dis 1977;136:717-23. 3. Wahdan MH, Serie C, Germanier R, et al. A controlled field trial of live oral typhoid vaccine Ty 21a. Bull WORLD Health Organ 1980;58:469-74. 4. Wahdan MH, Serie C, Cerisier Y, Sallam S, Germanier R. A controlled field trial of live S. typhi strain Ty2la oral vaccine against typhoid: three-year results. J Infect Dis 1982;145:292-5. 5. Black RE, Levine MM, Ferreccio C, Clements ML, Germanier R, Chilean Typhoid Committee. Field trials of the efficacy of Ty21a attenuated S. typhi oral vaccine in Santiago, Chile [abstract]. XI International Congress for Tropical Medicine and Malaria, Calgary, Canada, 1984:15. 6. Levine MM, Ferreccio C, Black RE, Germanier R, Chilean Typhoid Committee. Large-scale field trial ofTy2la live oral typhoid vaccine in enteric-coated capsule formulation. Lancet 1987;1:1049-52. 7. Ferreccio C, Levine MM, Rodriguez H, Contreras R, Chilean Typhoid Committee. Comparative efficacy of two, three, or four doses of TY2la live oral typhoid vaccine in enteric-coated capsules: a field trial in an endemic area. J Infect Dis 1989;159:766-9. 8. Levine MM, Ferreccio C, Black RE, Tacket CO, Germanier R, Chilean Typhoid Committee. Progress in vaccines against typhoid fever. Rev Infect Dis 1989;1l(suppl 3):S552-67. 9. WHO Diarrhoeal Diseases Control Programme. Programme for control of diarrhoeal diseases. Interim programme report 1988. Geneva: WHO Report number WHO/CDD/89.31, 1989:35. 10. Forrest BD. The development of a bivalent vaccine against diarrhoeal disease. Southeast Asian J Trop Med Pub Health 1988;19:449-57. 11. Forrest BD, LaBrooy JT, Attridge SR, et al. A candidate live oral typhoid/cholera hybrid vaccine is immunogenic in humans. J Infect Dis 1989;159:145-6. 12. Giannella RA, Broitman SA, Zamcheck N. Influence of gastric acidity on bacterial and parasitic infections. Ann Intern Med 1973 ;78:271-6. 13. Clemens JD, Jertborn M, Sack D, et al. Effect of neutralization of gastric acid on immune responses to an oral B subunit, killed whole-cell cholera vaccine. J Infect Dis 1986;154:175-8.
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the humoral immune responses after lyophilizationmay reflect a retention in protective efficacy. In this study, we found that three sequential doses of 109 live S. typhi Ty21a failed to stimulate a measurable immune response in the intestine of previously unexposed subjects. This confirms other reports of the failure of this dose to effectively stimulate a local antibody response in individuals from nonendemic regions [17, 20]. It is unlikely that this reflects any limitations of the assay for intestinal specific antibody, as specific antibody responses were also not identifiable at this dose using the subjects' PBL. This suggests that the meager response that was observed with the enteric-coated formulation was most likely attributable to the 5 X 1010 killed organisms present rather than to the 1.1 X 109 viable ones. This response was also substantially less than that of three doses of 1011 or 1010 live organisms in the presence of a comparable number of killed vaccine organisms. This study supports previous findings of the inadequacy of single-point determinations of antigen-specific serum antibody irrespective of class [14] for the accurate determination of an intestinal antibody response. Although the sequential determination of a group's response within an antibody class is of most value, it is often the least practicable. Ifa point determination of serum antibody response is the only method available for the evaluation of the immunogenicity of a particular preparation, then the magnitude of the serum IgG response may provide the most consistent and sensitive indicator; significant antibody responses were evident in this class when absent in or not significant in others (e.g., groups Band H; table 3), although the magnitude of the response may not correlate with that of the intestine. This finding has been recently supported in field trials, in which the serum IgG anti-LPS O-antigen antibody responses were observed to correlate roughly with vaccine efficacy [8]. The PBL-ELISA was confirmed as a highly sensitive indicator of the primary intestinal exposure of an individual to an enteric bacterial organism. While the PBL-ELISA correlated closely with the specific intestinal IgA immune responses of a group at doses of 1011 viable vaccine organisms, it was unable to discriminate between responses of groups receiving dose regimens of 1011 or 1010 viable organisms. At dose schedules of 109 viable organisms, this assay was unable to detect an IgA-specific immune response in keeping with the results obtained from the intestinal-fluid assays. The PBL-ELISA, however, continues to remain the most rapid and sensitive measure of an immune response in a specific individual following primary oral vaccination. The intestinal antibody response as determined using our model system appeared to be particularly useful for discriminating between the immunogenicities of various vaccine dosages and formulations. These studies of the humoral immune response to typhoid vaccines suggest that the measurement of this response to an
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14. Forrest BD. The identification of an intestinal immune response using peripheral blood lymphocytes. Lancet 1988;1:81-83. 15. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21(suppl 97):77-89. 16. Mancini G, Carbonara AO, Heremans JE Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965;2:235-7. 17. Bartholomeusz RCA, LaBrooy JT, Johnson M, Shearman DJC, Rowley D. Gut immunity to typhoid: the immune response to a live oral typhoid vaccine Ty2la. J Gastroenterol Hepatol 1986;1:61-7. 18. Hirschel B, Wuthrich R, Somaini B, Steffen R. Inefficacy of the commercial live oral Ty21a vaccine in the prevention of typhoid fever. Eur J Clin Microbiol 1985;4:295-8. 19. Levine MM, DuPont HL, Hornick RB, et al. Attenuated, streptomycin-
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dependent S. typhi oral vaccine: potential deleterious effectsof lyophilization. J Infect Dis 1976;133:424-9. Sirianni MC, Turbessi G, Scarpati B, Russo G, Mascellino T, Aiuti E A preliminary report on the immunological responses after-oral vaccine (Ty 21a). Boll 1st Sieroter Milan 1984;63:352-6. LaBrooy JT, Davidson GP, Shearman DJC, Rowley D. The antibody response to bacterial gastroenteritis in serum and secretions. Clin Exp Immunol 1980;41:290-6. Elson CO, Balding W, Lefkowitz 1. A lavagetechnique allowing repeated measurement of IgA antibody in mouse intestinal secretions. J Immunol Methods 1984;67:101-8. Samson RR, McClelland RR, Shearman DJe. Studies on the quantitation of immunoglobulins in human intestinal secretions. Gut 1973; 14:616-26.
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