Safety and Immunogenicity of a Live Oral Bivalent Typhoid Fever

INFECTION AND IMMUNITY, Apr. 1995, p. 1336–1339 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 63, No. 4

Safety and Immunogenicity of a Live Oral Bivalent Typhoid Fever (Salmonella typhi Ty21a)-Cholera (Vibrio cholerae CVD 103-HgR) Vaccine in Healthy Adults S. J. CRYZ, JR.,1* J. U. QUE,1 M. M. LEVINE,2 G. WIEDERMANN,2

AND

H. KOLLARITSCH3

1

Swiss Serum and Vaccine Institute, CH-3001 Berne, Switzerland ; Center for Vaccine Development, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 212022; and Institute of Specific Prophylaxis and Tropical Medicine, University of Vienna, A-1095 Vienna, Austria3 Received 2 September 1994/Returned for modification 31 October 1994/Accepted 25 January 1995

The safety and immunogenicity of the live oral attenuated vaccine strains Vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a were evaluated alone or in a combined bivalent formulation in four groups composed of 185 healthy European adults. All presentations were well tolerated. The serum anti-S. typhi lipopolysaccharide immunoglobulin G and immunoglobulin A antibody responses were comparable for all groups (66 to 72% seroconversion). The serum vibriocidal antibody seroconversion rate ranged from 78 to 92.5% (P > 0.05) among the groups. However, the peak and geometric mean vibriocidal antibody titers were significantly higher (P < 0.005) in the groups which received the bivalent formulation along with two doses of Ty21a than in the group which received CVD 103-HgR followed by two doses of killed Escherichia coli K-12 placebo. The ingestion of a placebo shortly after CVD 103-HgR may have suppressed the magnitude of the immune response. These findings demonstrate the feasibility of producing multivalent live oral attenuated vaccines. Typhoid fever and cholera are endemic in most tropical and subtropical areas of the world, constituting a threat to both residents and visitors from developed nations (3, 20, 27, 30). The recent emergence of Salmonella typhi and Vibrio cholerae strains resistant to multiple antibiotics has complicated therapy (12, 28). Vaccination will, therefore, undoubtedly play an increased role in the prevention of these enteric diseases in the future. The live oral attenuated S. typhi Ty21a vaccine has seen widespread use over the past decade (6, 17). Recently, a liquid vaccine formulation has been developed and field tested at two sites (15, 25). This formulation was found to be significantly more effective than the enteric coated version in Chile but only slightly more so in Indonesia (15, 25). One distinct advantage of the liquid formulation is that it can be readily administered to children 2 to 6 years old, who often have difficulty swallowing a standard-size gelatin capsule (9, 22). The live oral attenuated V. cholerae CVD 103-HgR cholera vaccine (Inaba biotype, O1 serotype) has recently been licensed in several countries (18, 19). This vaccine is presented as a liquid formulation identical to that of Ty21a. Extensive testing has demonstrated the safety of CVD 103-HgR in adults and young children (13, 24) and its ability to protect against experimental cholera (18, 19, 26). Since both cholera and typhoid show considerable overlap in their geographical incidence, a bivalent vaccine is desirable. Such a vaccine would be more cost-effective than currently used vaccines and would facilitate large-scale immunization programs in developing countries. We, therefore, combined both vaccine strains into a single-dose formulation which was evaluated for safety and immunogenicity in healthy adult volunteers.

MATERIALS AND METHODS Vaccine, placebo, and buffer. S. typhi Ty21a and V. cholerae CVD 103-HgR were separately grown to early stationary phase in fermentors. Ty21a cells were harvested by centrifugation, while CVD 103-HgR was collected by tangentialflow filtration (Prostak; Millipore AG, Volketswil, Switzerland). The cell concentrates were each mixed with a cryoprotective medium consisting of sugars and amino acids and lyophilized in bulk under aseptic conditions. The number of CFU per gram of lyophilizate was determined. To produce the bivalent vaccine, each bulk lyophilizate was appropriately diluted with a mixture of lactose, sorbitol, and aspartame. Equal amounts of each lyophilizate were then mixed and were used to fill aluminum foil sachets (2 g per sachet). One dose of monovalent Ty21a vaccine contained 4.5 3 109 CFU, while one dose of CVD 103-HgR vaccine contained 5.8 3 108 CFU. One dose of the bivalent vaccine contained 4.18 3 109 CFU of Ty21a and 4.96 3 108 CFU of CVD 103-HgR. The placebo consisted of 5 3 108 CFU of heat-killed Escherichia coli K-12. The excipients were the same as those used for the vaccines. The placebo was also packed in aluminum foil sachets. Each buffer sachet contained NaHCO3 (2.5 g) and ascorbic acid (1.65 g). All preparations were tested for microbial purity and freedom from toxicity by standard tests. Vaccination. The study protocol was reviewed and approved by the Ethical Committee of the University of Vienna and the Austrian Drug Commission, Federal Ministry of Health, Sports, and Consumer Protection. Written informed consent was obtained from each volunteer. Participants consisted of healthy adults of both sexes, aged 16 to 56 years (mean, 24.1 years), recruited from the student body of the Medical University of Vienna, Vienna, Austria, and their relatives. Exclusion criteria consisted of the following: febrile illness within the past 2 weeks, diarrhea within the preceding 3 weeks, receipt of antibiotics within the past 2 weeks, pregnancy, chronic gastrointestinal disease, chronic systemic inflammatory disease, alcoholism, and receipt of antacids or H2 blockers. Participants (N 5 185) were randomized into four groups. Group 1 (N 5 30) received three doses of Ty21a vaccine on days 1, 3, and 5 (a regimen designated T/T/T). Group 2 (N 5 35) received a single dose of CVD 103-HgR on day 1. To keep the study blind, a placebo consisting of 5 3 108 CFU of killed E. coli K-12 was also administered to group 2 on days 3 and 5 (a regimen designated C/P/P). Group 3 (N 5 60) received the combined Ty21a–CVD 103-HgR vaccine on day 1 and monovalent Ty21a vaccine on days 3 and 5 (the T1C/T/T regimen). Group 4 (N 5 60) received monovalent Ty21a vaccine on days 1 and 3 and the bivalent Ty21a–CVD 103-HgR vaccine on day 5 (the T/T/T1C regimen). The preparations were administered as follows. The participants were instructed not to eat for at least 1 h before and after vaccination. The buffer was dissolved in 100 ml of water, the contents of the vaccine (or placebo) were then added, and the mixture was stirred for 5 to 10 seconds and was then ingested. Venous blood samples were obtained before vaccination on day 1 and on days 10, 14, 21, and 28, and the serum was isolated before being stored at 2208C. Each participant completed an adverse-reaction report form after the ingestion of each dose of vaccine. Reactions monitored included nausea, vomiting, diarrhea, abdominal discomfort, skin rash, fever, and ‘‘other.’’ On day 10 (after

* Corresponding author. Mailing address: Swiss Serum and Vaccine Institute, P.O. Box 2707, CH-3001 Berne, Switzerland. Phone: 41-31980-6345. Fax: 41-31-980-6785. 1336

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TABLE 1. Adverse reactions following vaccinationa No. of reactions/total no. of vaccinations per group (%) Group (vaccine regimen)

1 2 3 4 a b

(T/T/T) (C/P/P) (T1C/T/T) (T/T/T1C)

Nausea

Diarrhea

Abdominal discomfort

Vomiting

Rash

Fever

Otherb

5/90 (5.5) 3/102 (3) 8/171 (4.6) 8/177 (4.5)

18/90 (20) 11/102 (11) 17/171 (9.9) 25/177 (14)

4/90 (4.4) 11/102 (11) 11/171 (6.4) 8/177 (4.5)

0/90 (0) 0/102 (0) 0/171 (0) 0/177 (0)

0/90 (0) 1/102 (1) 1/171 (0.5) 2/177 (1.1)

2/90 (2) 0/102 (0) 0/171 (0) 1/177 (0.5)

1/90 (1) 1/102 (1) 3/171 (1.7) 3/177 (1.6)

Each volunteer was immunized on days 1, 3, and 5. T, S. typhi Ty21a vaccine; C, V. cholerae CVD 103-HgR vaccine; P, placebo (killed E. coli K-12). Includes fever, malaise, gas, and dizziness.

all three doses were administered), the report forms were collected by the attending physician and the volunteers were interviewed about the acceptability of the vaccines. Serological assays. Inaba vibriocidal assays were performed in microtiter plates as described elsewhere (5). Fourfold or greater rises in antibody titer were considered significant. Antibodies to S. typhi lipopolysaccharide (LPS) were determined by means of a previously described enzyme-linked immunosorbent assay (ELISA) (16) with slight modification. Alkaline phosphatase-labeled antihuman immunoglobulin G (IgG) and IgA (Sigma Chemical Co., St. Louis, Mo.) were diluted in phosphate-buffered saline, pH 7.4, containing 1% (vol/vol) fetal calf serum to final dilutions of 1:2,000 for anti-IgG and 1:3,000 for anti-IgA. Test serum (baseline and postimmunization) samples were used in individual reactions against both secondary antibodies. The sera were first tested at a 1:25 dilution. Serum samples from those individuals whose baseline samples yielded an optical density above the linear portion of the curve were retested at a 1:100 dilution. An increase of $0.15 optical density units (.3 standard deviations above background) over the baseline value at the same dilution was considered a significant response. Statistical analyses. Significance between group response rates was determined by chi-square analysis. Significance between geometric means was determined by using a t test.

RESULTS A total of 185 healthy adults were randomized into four treatment groups. Completed adverse-reaction report forms were returned by 180 (97.3%) of the volunteers. The reactions associated with immunization are listed in Table 1. The types, frequencies, and degrees of severity of adverse reactions of the groups were comparable. The only statistically significant difference observed was for the incidence of diarrhea between groups 1 and 3 (P 5 0.024). A total of 144 reactions were reported, of which 131 (91%) were classified as mild by the study participants, while the remaining 13 (9%) were described as moderate. No reaction resulted in the curtailing of normal activities. The most frequently reported adverse event was mild diarrhea characterized by one or two loose stools within 24 h. The percentages of subjects who reported no reaction after all three doses were 63, 50, 63, and 63%, respectively, for groups immunized with T/T/T, C/P/P, T1C/T/T, and T/T/T1C. It is interesting that for group 2, whose second and third doses consisted of a placebo, the frequencies and degrees of severity of diarrhea and abdominal discomfort were nearly identical after the ingestion of CVD 103-HgR and the placebo. However, all three cases of nausea reported in this group occurred within 24 h of the volunteer receiving CVD 103-HgR. The serum anti-S. typhi LPS antibody response is shown in Table 2. Vaccination with S. typhi Ty21a engendered both IgA and IgG antibodies. The percentage of participants mounting a significant IgA antibody response was the highest (52%) for the group immunized with only monovalent typhoid vaccine (group 1). However, the response rate was not significantly higher than those of the groups which received the bivalent vaccine formulation (groups 3 and 4) (P . 0.05). The anti-S. typhi IgG LPS response rates were comparable among the three groups receiving Ty21a (60 to 63%). The combined IgA and IgG anti-S. typhi LPS antibody responses were nearly iden-

tical in these three groups (66 to 72%). Two participants (6%), who received the CVD 103-HgR cholera vaccine, presented with a significant IgG and/or IgA antibody response. The serum Inaba vibriocidal antibody response is shown in Table 3. Immunization with a single dose of CVD 103-HgR either alone or in combination with Ty21a resulted in a significant (P , 0.001) rise in vibriocidal antibody titers over baseline values (15.3- to 71-fold rise). Interestingly, the peak geometric mean vibriocidal antibody titer (GMT) was significantly higher (P , 0.005) for the two groups which received the bivalent vaccine (groups 3 and 4) than for the group which was vaccinated with only CVD 103-HgR (group 2). Although the highest peak GMT was seen in group 3, which received the bivalent vaccine on day 1, it was not significantly greater than that for group 4, which received the bivalent vaccine on day 5. Even though the vibriocidal seroconversion rate was also the highest for group 3 (92.5%), it was not significantly greater (P . 0.05) than that of either group 2 (83%) or group 4 (78%). Immunization with Ty21a alone did not stimulate a significant vibriocidal antibody response. The kinetics of the vibriocidal antibody responses are shown in Fig. 1. The peak GMT for all groups which received the CVD 103-HgR vaccine occurred on day 14. The GMTs on days 14, 21, and 28 were significantly higher for groups 3 and 4 than for group 2. There was no significant difference between group 3 and group 4 for these three time points. The amounts of decline in titer from day 14 to day 28 of groups 2, 3, and 4 were comparable. The GMTs on day 28 were 34, 26, and 40% of those seen on day 14 for groups 2, 3, and 4, respectively. DISCUSSION Bacterial enteric infections continue to exact a substantial toll upon individuals residing in developing areas of the world (21). Implementation of sanitary improvements, the most effective control measure, has been hampered by civil unrest and a deteriorating economic environment in many areas. The use of classical parenteral vaccines against typhoid or cholera has had a limited public health impact because of a combination of

TABLE 2. Serum anti-S. typhi IgG and IgA antibody responses Group (vaccine regimen)

1 2 3 4

(T/T/T) (C/P/P) (T1C/T/T) (T/T/T1C)

Response (%)a (95% confidence interval) IgA

IgG

IgA or IgG

15/29 (52) (32–71) 1/29 (3) (0–13) 17/53 (32) (21–49) 25/60 (42) (29–57)

18/29 (62) (43–79) 2/29 (7) (2–18) 32/53 (60) (46–75) 38/60 (63) (49–74)

19/29 (66) 2/29 (7) 38/53 (72) 43/60 (72)

a The response values represent the number of samples with a significant rise (an increase of $0.15 optical density units) at one or more time points as a fraction of the total number of samples.

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TABLE 3. Serum Inaba antibody response following immunization with monovalent or bivalent vaccines Group (vaccine regimen)

1 2 3 4

(T/T/T) (C/P/P) (T1C/T/T) (T/T/T1C)

GMT (range) Prevaccination

Peaka

27 (,10–640) 18 (,10–80) 36 (,10–640) 38 (,10–20,480)

42.9 (,10–5,120) 331 (,10–10,240) 2,549 (,10–20,480) 1,389 (,10–20,480)

Fold rise in GMT

Seroconversion rateb (%) (95% confidence interval)

1.6 15.3 71 37

2/29 (7) (1–27) 24/29 (83) (65–94) 49/53 (92.5) (83–99) 47/60 (78) (65–90)

a

Reflects the highest value obtained following immunization. Seroconversion was defined as a greater-than-fourfold rise in titer at any time point over the baseline value. The seroconversion rate represents the number of samples that exhibited seroconversion as a fraction of the total number of samples. b

variable efficacy, a high rate of adverse reactions, and the need for frequent booster doses (10, 14). Live oral attenuated vaccines offer the potential to efficiently evoke long-lasting immunity without attendant adverse reactions (20). Such vaccines are also well suited for large-scale immunization programs by virtue of their ease of administration (14) and avoidance of transmitting adventitious viral agents, a possibility with the use of injectable vaccines. Combined vaccines would offer the distinct advantage of affording protection against multiple enteric pathogens in a given geographical area and simplifying immunization programs. Therefore, in the present study, the live oral attenuated S. typhi Ty21a and the V. cholerae CVD 103-HgR vaccine strains were combined to address issues relating to safety and possible immune interference. Additionally, since only a single dose of CVD 103-HgR is required, versus three doses of Ty21a, the effect of two dosing regimens on the immune response was investigated. Overall, the safety profiles of Ty21a and CVD 103-HgR were comparable whether the vaccines were given alone or in combination. The types, frequencies, and degrees of severity of adverse events associated with immunization were similar for the various groups. Most reactions were mild and transient and did not interfere with normal activities. Therefore, increasing the bioburden by combining the two vaccines did not compromise their safety (19, 22, 24, 25). Interestingly, in all four study groups, the incidence of diarrhea was greater than previously noted (19, 22, 24, 25). This difference may reflect the diligence of the student study population in reporting any potential adverse reaction. A similar increased rate of adverse events

FIG. 1. Kinetics of vibriocidal antibody response following immunization. The bars represent the standard errors of the means. Group 1 (}) volunteers were immunized with T/T/T; group 2 (å) volunteers were immunized with C/P/P; group 3 (F) volunteers were immunized with T1C/T/T; and group 4 (h) volunteers were immunized with T/T/T1C.

with CVD 103-HgR has been reported in an open study involving English travellers (2). Some of the reactions noted may be attributed to the ingestion of the sodium bicarbonate buffer, although it was neutralized with ascorbic acid in an attempt to increase tolerability. Approximately 5% of the volunteers who ingested the buffer alone in a previous study reported mild gastrointestinal upset (data not shown). A rise in serum anti-S. typhi antibodies has been found to correlate with protection against typhoid fever in the field and may serve as a prediction of immunity (14, 23). In Chilean schoolchildren, three doses of Ty21a presented as enteric coated capsules resulted in a 64% seroconversion rate (14). Similarly, three doses of liquid formulation Ty21a engendered a 69% response rate in young Thai children (9). However, the response rate of individuals residing in an area where typhoid is endemic may be influenced by previous natural exposure. Limited data are available on the immune response mounted to Ty21a vaccine among immunologically naive individuals residing in developed areas. When the same ELISA system was used, approximately 55% of North Americans or Swiss who ingested Ty21a presented as enteric coated capsules demonstrated a significant rise in serum anti-LPS antibodies (data not shown). Ambrosch et al. (1), using a different ELISA system, reported an 86% seroconversion rate among Austrian adults who ingested three doses of enteric coated capsules. The present study represents the first time the commercial liquid formulation of Ty21a has been used in such a population. It was encouraging that the immune response rate for healthy adult Europeans was comparable to that previously observed for individuals residing in an area where typhoid is endemic (14). These findings indicate that the priming of the immune system by prior exposure to S. typhi is not required to mount an immunological response. The response rates were essentially the same for Ty21a given alone and in combination with CVD 103-HgR, indicating an absence of immune interference or suppression. Serum vibriocidal antibodies provide the best correlate of protection against cholera (4, 11, 19, 26). The magnitude of the vibriocidal response following immunization of susceptible individuals with either live or non-living vaccines is a rough predictor of vaccine efficacy (4, 11, 19, 26). Immunization with CVD 103-HgR and Ty21a combined engendered the same seroconversion rate as previously reported for CVD 103-HgR alone. The observation that the magnitude of the vibriocidal antibody response was significantly higher for the two groups which received the bivalent vaccine formulation than for the group which received CVD 103-HgR alone was unexpected. However, a similar phenomenon was reported by Wasserman et al. (29) for a crossover study involving CVD 103-HgR. Participants in that study who first received a single dose of killed E. coli K-12 placebo followed by vaccination 1 week later

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with CVD 103-HgR mounted a vibriocidal antibody response more pronounced than that mounted when those antigens were administered in the opposite manner. The authors postulated that the placebo either enhanced the immune response by immunological priming when given before the vaccine or partially abrogated the response when administered subsequent to vaccination. In the present study, the GMT for the groups which received the bivalent vaccine was comparable to that which has been previously reported for North Americans and Europeans (7, 8, 13, 19), while the GMT for those participants who received CVD 103-HgR alone was significantly lower. This suggests that killed E. coli K-12 can interfere with the immune response to CVD 103-HgR. Interestingly, ingestion of two doses of Ty21a vaccine, either before or after administration of CVD 103-HgR, did not exert a similar immune suppressive effect, perhaps because of different mechanisms of interaction with gut-associated lymphoid tissue. Additionally, immunization with CVD 103-HgR did not appear to influence the immune response to Ty21a. Additional studies wherein either killed E. coli or buffer alone will be given after immunization with CVD 103-HgR are planned to pursue these observations. The current data demonstrate the feasibility of producing combined live oral attenuated vaccines against multiple enteric pathogens. The fact that combining the Ty21a and CVD 103HgR vaccines did not adversely affect either their safety or their immunogenicity provides the basis for the continued study of this and other multivalent live oral vaccines. On the basis of these results, it appears that the combined vaccine could be given first, followed by two doses of monovalent Ty21a to allow for a rapid immune response to both vaccines. REFERENCES 1. Ambrosch, F., A. Hirschl, P. Kremsher, M. Kundi, C. Kunz, E. Rappold, and G. Wiederman. 1985. Orale Typhus-Lebenimpfung. Neuere Aspekte. Muench. Med. Wochenschr. 127:775–778. 2. Barret, P., P. Clarke, and S. J. Cryz, Jr. 1993. Oral cholera vaccine well tolerated. Br. Med. J. 307:1425. 3. Centers for Disease Control. 1992. Cholera associated with international travel, 1992. Morbid. Mortal. Weekly Rep. 41:664–667. 4. Clements, J. D., F. van Loon, D. A. Sack, J. Chakraborty, M. R. Rao, F. Ahmed, J. R. Harris, M. R. Khan, M. Yunus, S. Huda, B. A. Kay, A.-M. Svennerholm, and J. Holmgren. 1991. Field trial of oral cholera vaccines in Bangladesh: serum vibriocidal and antitoxic antibodies as markers of the risk of cholera. J. Infect. Dis. 163:1235–1242. 5. Clements, M. L., M. M. Levine, C. R. Young, R. E. Black, Y.-L. Lim, and R. M. Robins-Browne. 1982. Magnitude, kinetics and duration of vibriocidal antibody response in North Americans after ingestion of Vibrio cholerae. J. Infect. Dis. 145:465–473. 6. Cryz, S. J., Jr. 1990. Post-marketing experience with live oral Ty21a. Lancet 341:49–50. 7. Cryz, S. J., Jr., M. M. Levine, J. B. Kaper, E. Fu ¨rer, and B. Althaus. 1990. Randomized, double-blind, placebo-controlled trial to evaluate the safety and immunogenicity of the live oral cholera vaccine strain CVD 103-HgR in Swiss adults. Vaccine 8:577–580. 8. Cryz, S. J., Jr., M. M. Levine, G. A. Losonsky, J. B. Kaper, and B. Althaus. 1992. Safety and immunogenicity of a booster dose of Vibrio cholerae CVD 103-HgR live oral cholera vaccine in Swiss adults. Infect. Immun. 60:3916– 3917. 9. Cryz, S. J., Jr., N. Vanprapaz, U. Thisyakorn, T. Olanratmanee, G. Losonsky, M. M. Levine, and S. Chearskul. 1993. Safety and immunogenicity of

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