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Department of Medicine, Unit of Infectious Diseases
Department of Pediatrics, University of Vermont College of Medicine, Burlington
Microscience Limited, Wokingham, Berkshire, United Kingdom
Background.
M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC- ssaV-] ZH9) is a live oral-dose typhoid vaccine candidate. M01ZH09 was rationally modified with 2 independently attenuating mutations, including a novel mutation in Salmonella pathogenicity island (SPI)2. We demonstrate that M01ZH09, in a single oral dose, is well tolerated and prompts broad immune responses, regardless of whether prevaccination with a bicarbonate buffer is given.
Methods.
Thirty-two healthy adult subjects were randomized and given 5 × 109 cfu of M01ZH09, with (presentation 1) or without (presentation 2) prevaccination with a bicarbonate buffer. Immunogenicity data included Salmonella Typhi lipopolysaccharide (LPS)specific immunoglobulin (Ig) A antibody-secreting cells (enzyme-linked immunospot assay), IgG serologic responses to Salmonella Typhi LPS, lymphocyte proliferation, and interferon (IFN) production.
Results.
The vaccine was well tolerated; adverse events after vaccination were mild. No fever or prolonged vaccine shedding occurred. Immunogenicity data demonstrated that 88% and 93% of subjects who received presentation 1 and presentation 2, respectively, had a positive response by ELISPOT assay; 81% of subjects in both groups underwent IgG seroconversion on day 14. Both groups had similar cellular immune responses to presentation 1 and presentation 2; lymphocyte proliferation to Salmonella Typhi flagellin occurred in 63% and 67% of subjects, respectively, and 69% and 73% of subjects, respectively, had an increase in IFN- production.
Conclusion.
The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.
Typhoid fever is a major cause of morbidity, with an estimated global incidence of 21.6 million cases and 216,510 deaths in 2000 [1]. Particularly in urban areas, the global burden of typhoid fever may be underestimated [13]. The prevalence of typhoid strains with resistance to most known antibiotics is increasing globally and has caused large-scale outbreaks of disease [4, 5]. Current trends in urbanization, migration, travel, and trade increase the risk of spread of multiantibiotic-resistant strains from areas where they are hyperendemic. At present, the World Health Organization recommends typhoid vaccination for persons living in or traveling to areas with endemic typhoid, as well as in outbreak situations [6]. Multiantibiotic-resistant strains of Salmonella serovar Typhi offer a compelling argument for the development of next-generation typhoid vaccines and the increased use of typhoid vaccination.
Currently available typhoid vaccines include the 4-dose oral Ty21a (Vivotif) and the injectable, purified Vi polysaccharide (Typhim Vi; Typherix). Concerns about Ty21a include the requirement for multiple doses and the poorly understood mechanism of attenuation [8]. The purified Vi polysaccharide vaccine is an injectable product. It is T cell independent and, thus, cannot stimulate protective T helper cells or be used in young children [9]. A next-generation typhoid vaccine should be safe and easy to administer, ideally in a single oral dose. For live vaccines, the use of rationally attenuated Salmonella Typhi strains with minimal risk of reversion to the wild type (wt) is criticalas is the production of robust humoral, gut, and cellular immune responsesin advance of field trials that demonstrate protective efficacy.
M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC- ssaV-] ZH9) is a live oral-dose vaccine that is formulated as a lyophilized preparation and has well-defined attenuating mutations. The novel strategy of M01ZH09 is the targeted mutation of a structural protein (SsaV) of Salmonella pathogenicity island (SPI)2. SPI-2 is a virulence mechanism of Salmonella species that permits a needlelike structure to inject bacterial effector components into host cells, thus permitting the intracellular bacteria to avoid being killed by oxidative burst [10, 11]. Mutation in the ssaV gene destroys the function of SPI-2, thus preventing the systemic spread of Salmonella Typhi. M01ZH09 also contains an additional aromatic mutation (aroC-), which deprives the live vaccine bacterium of essential nutrients it must obtain from the mammalian host [12].
M01ZH09 at 5 × 109 cfu has been well tolerated when given to healthy adult subjects, including in recent dose-escalation trials of 3 and 16 subjects (B.D.K., unpublished data) [13]. In the latter trial, reactogenicity symptoms did not significantly differ between vaccinated individuals and control subjects who received buffer without vaccine. In these trials, vaccine was prepared with a bicarbonate buffer, and an additional dose of bicarbonate buffer was given before vaccination, to neutralize gastric acid. The first aim of the present study was to determine whether M01ZH09 is well tolerated when given in a single oral dose in 2 vaccine presentations, 1 with and 1 without prevaccination with gastric acidneutralizing bicarbonate buffer. In addition, we sought to define the immunogenicity of M01ZH09 in terms of humoral, cellular, and mucosal immune responses after vaccination with a single oral dose.
SUBJECTS, MATERIALS, AND METHODS
The vaccine.
M01ZH09 is supplied as a lyophilized formulation and is reconstituted immediately before administration. M01ZH09 is derived from virulent Salmonella Typhi strain Ty2 and contains a defined and independently attenuating mutation in each of 2 genes, aroC and ssaV. The aroC gene encodes chorismate synthase, an enzyme involved in biosynthesis of aromatic compounds, and is attenuating, since aromatic compounds are required for growth of Salmonella Typhi but are not available in mammalian tissues. The ssaV gene is a structural gene that encodes part of the type III secretion system of Salmonella species and is found on SPI-2, as described above. Preclinical data demonstrate that Salmonella Typhimurium with the same aroC and ssaV deletions are more attenuated in immunocompromised interferon (IFN) knockout mice than are Salmonella Typhimurium with single aro deletions and that Salmonella Typhi with the same aroC and ssaV mutations are more attenuated in human macrophages than are Salmonella Typhi with a single aroC deletion [12].
Subjects and protocol.
The open-label, randomized trial was performed at the University of Vermont, approved by the institutional review board, and performed under an institutional new drug application issued by the Food and Drug Administration. Healthy subjects 1850 years old with no history of typhoid vaccination within 10 years were recruited. Subjects were screened by history, examination, screening stool culture, pregnancy test, test of study comprehension, laboratory studies (including antibody testing for HIV and hepatitis B and C viruses), and electrocardiogram (for subjects >40 years old). The subjects received the single-dose vaccine (at 5 × 109 cfu of M01ZH09) in 1 of 2 presentations after having fasted for 2 h. The first group received lyophilized M01ZH09 prepared as in previous trials: subjects received 100 mL of a wt/vol sodium bicarbonate solution 520 min before administration of the vaccine or placebo, which was suspended in 50 mL of a 2% sodium bicarbonate solution. All solutions in the first presentation were prepared with bottled water. The second group received M01ZH09 in a single volume (150 mL) of a modified bicarbonate solution (containing 1.75% wt/vol sodium bicarbonate, 1.1% wt/vol ascorbic acid, and 0.02% wt/vol aspartame) prepared with potable tap water; no prevaccination with a bicarbonate buffer was given. Ascorbic acid was used to neutralize the chlorine in the tap water, which, if present in high enough concentrations, would kill the live vaccine strain. Aspartame was added to improve the palatability of the vaccine solution. Both types of bicarbonate solution had been tested, and they were demonstrated to not decrease the M01ZH09 inoculum size.
The subjects were monitored for 90 min after vaccination and were evaluated 1 day after vaccination and then on days 7, 14, and 28, as well as at 3 and 6 months, after vaccination. Subjects recorded all symptoms, including diarrhea, on a diary card and monitored their temperature twice daily for the first 28 days after vaccination. Diary cards were reviewed at each clinic visit. Stool cultures for Salmonella Typhi and enteric pathogens were checked 2 days before vaccination and on days 0, 7, 14, and 28 after vaccination. Hematological and biochemical parameters, urinalysis with culture (if indicated), and vital signs were evaluated at each visit. Heparinized whole blood for derivation of peripheral blood mononuclear cells (PBMCs) and whole blood for derivation of serum were collected on days 0, 7, 14, and 28 after vaccination. Fever was defined as a temperature >38°C. Diarrhea was defined as 3 unformed stools in a 24-h period. Blood cultures were scheduled to be performed if temperature elevation occurred (defined as a temperature 37.5°C on 2 consecutive readings or a temperature 38.0°C on 1 reading).
Immunogenicity assays.
The IgA enzyme-linked immunospot (ELISPOT or antibody-secreting cell ) assay for reaction to Salmonella Typhi lipopolysaccharide (LPS) was performed on the day of vaccination (day 0) and on day 7 after vaccination, as described elsewhere [14, 15], with modifications. Briefly, whole blood was collected in heparinized tubes (Vacutainer CPT; Becton Dickinson) and centrifuged. PBMCs were immediately removed and used for ELISPOT assays. The remaining PBMCs were frozen on liquid nitrogen until further use. For the ELISPOT assays, PBMCs were plated at 2.5 × 106, 5 × 106, and 1 × 107 cells/mL on nitrocellulose microtiter plates (Millipore). Wells were either coated with Salmonella Typhi LPS (Sigma) in 1× Reggiardo's Buffer or left uncoated, for use as a negative control. PBMCs from study subjects and from control subjects with positive and negative ELISPOT results were incubated overnight at 37°C in 5% CO2. After washing with PBS-Tween, an antihuman IgA alkaline phosphatase conjugate was added, and the mixture was incubated for 1 h. 5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate was added to each well for 30 min in the dark, and the reaction was stopped by washing the microtiter plates. ASC "spots" were manually counted by 2 observers, photographed, and expressed as ASCs per 1 × 106 PBMCs. A result of 4 ASCs/1 × 106 PBMCs was considered to be a positive response.
Serum samples for measurement of Salmonella Typhi LPSspecific IgG were frozen at -20°C until use. Microtiter plates were coated with Salmonella Typhi LPS and incubated overnight at 37°C. Plates were washed with PBS-Tween and blocked. After washing, serum was added, and the plates were incubated for 1 h at 37°C. Primary goat antihuman IgG antibody was added, the plates were incubated, and secondary horseradish peroxidaselabeled rabbit antigoat IgG antibody was added. 3,3,5,5-tetramethylbenzidine substrate was used; the reaction was stopped by the addition of 0.3 mol/L sulfuric acid. The microtiter plates were analyzed at 450 nm. ELISA data were analyzed by use of the end-point titer (EPT). A positive result was indicated by an EPT (means of blanks plus 3 SDs) 4-fold greater than the baseline (day-0) result.
Lymphocyte proliferation assays were performed by stimulation of PBMCs with phytohemagglutinin (PHA) (1 g/mL) (positive control), M01ZH09 (3 × 107 cfu), and Salmonella Typhi flagellin (10 g/mL). Salmonella Typhi flagellin was prepared as described elsewhere [16]. A total of 1 × 105 PBMCs/mL (for PBMCs obtained on days 0 and 14) were incubated in RPMI 1640 medium containing 10% human AB serum, in 96-well round-bottom plates, for 4 days at 37°C in 5% CO2 and then were labeled with [3H]-thymidine for 18 h. Six replicate wells were performed per sample. Measurement of proliferation was performed by use of a Wallac Oy Microbeta workstation and expressed as counts per minute. The adjusted counts per minute was the mean counts per minute for replicated wells minus the mean unstimulated control of the day-0 sample. The mean counts per minute of the unstimulated control of the day-0 sample multiplied by 2 was considered to be the background cutoff. A positive proliferative response to an antigen or mitogen after vaccination was defined as an adjusted count on day 14 after vaccination that was 2 times the mean adjusted count on day 0 for the same antigen or mitogen.
IFN- assays were performed after medium (RPMI with human AB sera) and peripheral blood lymphocytes (1 × 106 cells/mL) had been incubated in 48-well plates with PHA (1 g/mL), inactivated and homogenized M01ZH09 (3 × 107cfu/mL), and Salmonella Typhi flagellin (10 g/mL) for 4 days at 37°C in 5% CO2. Cell supernatants were removed and frozen at 20°C until analysis. IFN- was measured by OptEIA sandwich ELISA (BD Biosciences) including 2 sets of quality control samples at 37.5, 125, and 225 pg/mL.
The IFN- level was calculated from the standard curve, if the plate met criteria for range, accuracy, precision, and drift. A positive IFN- response to vaccination was defined as a day-14 IFN- level that was 30% above the day-0 level.
Stool and blood cultures.
Stool samples were cultured directly on desoxycholate agar (DCA) plates and in Selenite broth, both of which were supplemented with aromatic compounds (DCA-Aro and Selenite-Aro, respectively), at the prevaccination stage and at all follow-up visits. After 24 h of incubation, an aliquot of the inoculated Selenite-Aro broth was cultured on DCA-Aro plates. All lactose nonfermenting colonies were further inoculated into triple-sugar iron slants and onto brain-heart infusion (BHI-Aro) plates. Slants demonstrating an alkaline-over-acid pattern without gas or iron (characteristic of the M01ZH09 strain) were further evaluated by screening for agglutination in specific antiserum. Presumptive isolates were first screened by slide agglutination in Salmonella-specific antiserum (serogroups AI plus Vi; Difco). Colonies agglutinating in this antiserum were then screened for agglutination in 09- and Vi-specific sera (Difco). Stool isolates with positive agglutination patterns in polyvalent antisera plus either Vi or O9 were considered to be positive for the M01ZH09 strain. Presence of salmonellae in blood cultures was determined by use of the BacT/Alert system (bioMérieux). Pilot studies performed before the start of the clinical trial indicated that the vaccine strain could grow in this blood culture system (data not shown).
Statistical analysis.
Data were analyzed on the intent-to-treat and per-protocol populations. Statistical analyses were performed by use of the SAS system (version 8; SAS Institute). The primary immunogenicity end point was considered to be the proportion of subjects who had an immunogenic response (either a positive ELISPOT assay on day 7 or a positive LPS-specific IgG response by ELISA on day 28) after receiving a single dose of M01ZH09 vaccine. The primary end point, the proportion of subjects who had an immune response against Salmonella Typhi LPS, was compared with an assumed response of 50% by use of Fisher's exact test (2 sided at the 5% level). Each presentation of M01ZH09 oral typhoid vaccine was compared separately.
The number and percentage of subjects with an immune response was presented along with the 2-sided 95% confidence interval for each presentation of MICRO-TY oral typhoid vaccine. Differences in adverse events that occurred in >10% of the study population were analyzed between groups by use of a 2-tailed Fisher's exact test.
RESULTS
A total of 67 adult subjects were screened for the trial; 32 subjects were enrolled and received 5 × 109 cfu of M01ZH09. Each M01ZH09 vaccine presentation was given to 16 subjects. Subjects for both groups were similar in age, sex, and body mass index. Demographic characteristics are outlined in table 1. All subjects had a prevaccination stool culture that was negative for enteric pathogens but had normal enteric flora present.
In both groups, the vaccine was well tolerated. One subject who received presentation 2 was lost from the study for reasons unrelated to the vaccine; no subjects withdrew from the study or had a serious adverse event related to vaccination. In the absence of fever, no blood or urine cultures were obtained. Fecal shedding of the vaccine was not detected 7 days after vaccination. Antibiotics were not required for treatment of any subject.
Reactogenicity and adverse events recorded by subjects were mild and not statistically significant between groups (table 2). No subject experienced fever. Headache, loose stools, and abdominal cramping were the most common symptoms. Of symptoms possibly or probably related to vaccination, headache occurred in 3 subjects who received presentation 1 and in 4 subjects who received presentation 2. Four subjects who received presentation 1 and 2 subjects who received presentation 2 had loose stools on 1 day, 112 days after vaccination. All events were mild ("no interference with daily activities"), except that 1 subject had moderate ("moderate interference with daily activities") loose stools, which lasted for 1 day (day 1 after vaccination). Two subjects who received presentation 1 had mild episodes of diarrhea on days 0 and 3, respectively, but no subject who received presentation 2 reported diarrhea. One subject reported an episode of moderate diarrhea with abdominal cramping (5 days after vaccination) that abated spontaneously. Although no subject who received presentation 2 reported diarrhea, 1 episode of mild abdominal cramping (day 0) was reported.
The immunologic assays demonstrated strong responses, without differences between vaccine presentations (table 3). Salmonella Typhi LPSspecific IgA ELISPOT responses, a measure of mucosal immune system priming, were demonstrated in 14 (88%) of 16 subjects who received presentation 1 and in 14 (93%) of 15 subjects who received presentation 2, with mean ELISPOT responses of 180 and 167 ASCs/1 × 106 PBMCs, respectively. By the ELISPOT assay performed before vaccination, no ELISPOT responses were observed for any subject.
Serum IgG responses to LPS (a 4-fold greater EPT) were observed on day 14 for 13 (81%) of 16 and 11 (73%) of 15 subjects who received presentation 1 and presentation 2, respectively. Geometric mean titers (GMTs) on day 14 were 3490 and 1600, respectively. By day 28 after vaccination, 13 (81%) of 16 subjects who received presentation 1 had anti-LPS responses (GMT, 2167), but responses were slightly lower in subjects who received presentation 2 (8 [53%] of 15 subjects; GMT, 1292).
An IFN- response to Salmonella Typhi flagellin on day 14 was detected in 71% of subjects. Results were equivalent between presentations. A total of 69% of subjects who received presentation 1 had an IFN- response, compared with 73% of subjects who received presentation 2 (table 4). Mean IFN- levels on day 14 were 6099 and 4296 pg/mL (presentation 1 and presentation 2, respectively) in response to flagellin stimulation. IFN- responses to the killed, phenol-extracted vaccine strain were somewhat lower, at 56% and 60%, respectively.
T cell proliferative responses to Salmonella Typhi flagellin were detected on day 14 in 63% and 60% of subjects who received presentation 1 and presentation 2, respectively (figure 1). No other association between immunologic results and age, sex, body mass index, or vaccine presentation was noted.
DISCUSSION
We have demonstrated that the M01ZH09 live typhoid vaccine is well tolerated in a single oral dose and can be easily given, without prevaccination with a bicarbonate buffer, after reconstitution of a lyophilized formulation. Our data demonstrate that M01ZH09 evokes humoral, mucosal, and cellular immune responses, regardless of presentation. M01ZH09 is a promising next-generation typhoid vaccine candidate and may also be a useful vector system for presentation of heterologous antigens.
M01ZH09 was constructed with a rational 2-mutation attenuation strategy, to reduce the chances of reversion to virulence/wt [12, 13]. M01ZH09 has a targeted mutation in a structural protein (SsaV) of SPI-2 that destroys both SPI-2 function and the ability of Salmonella Typhi to spread systemically by evading host killing via the macrophage NADPH phagocyte oxidase system. The ssaV- mutation is additive to an aromatic mutation (aroC-), which deprives the live vaccine bacterium of essential nutrients obtained from the mammalian host [11, 12].
In the present randomized trial, M01ZH09 was well tolerated in all subjects. Concerns related to other candidate typhoid vaccinessuch as fever or prolonged fecal shedding of the vaccine strainwere not demonstrated here. Adverse effects were generally mild and did not differ between groups. Headache was the most common symptom, occurring in 19% and 25% of subjects who received presentation 1 and presentation 2, respectively, during the 28 days after vaccination. Signs of systemic illness, such as chills, musculoskeletal pain, or fever, were not noted. Although symptoms were mild and infrequent, presentation 1 appeared to be associated with slightly more gastrointestinal symptoms, such as loose stools (4 vs. 2 subjects), mild abdominal cramping (3 subjects vs. 1 subject), and mild diarrhea (2 vs. 0 subjects). The addition of prevaccination with a bicarbonate buffer may have contributed to additional gastrointestinal symptoms early after vaccination; however, no buffer-only control group was available for comparison of symptoms. Protection against typhoid fever by vaccination is thought to require stimulation of multiple arms of the immune system [17]. M01ZH09 prompted broad humoral, cellular, and mucosal immune responses without significant differences between presentation groups. Fourteen days after vaccination, serologic responses (IgG to Salmonella Typhi LPS), indicated by a 4-fold increase in EPT, were observed for 81% and 73% of subjects who received presentation 1 and presentation 2, respectively. Responses decreased to 81% and 53%, respectively, at 28 days after vaccination. These data meet or exceed IgG LPS ("O" antigen) seroconversion rates found for other licensed and candidate typhoid vaccines given in a single dose, including Ty21a (18% seroconversion), Vi polysaccharide (26%83% seroconversion), and CVD 908-htr-A (46%49% seroconversion) [18, 19].
For mucosal pathogens, the IgA ELISPOT assay demonstrated priming of the mucosal immune response by measuring circulating Salmonella Typhi LPSspecific IgAsecreting plasma cells, which home to the intestinal tract [14, 15, 20]. M01ZH09 prompted markedly positive ELISPOT assays in most subjects (88% and 93% in the group that received presentation 1 and the group that received presentation 2, respectively), with a total ASC geometric median number of 91 spot-forming cells. The ELISPOT assay has been demonstrated to correlate with field trial efficacy [14], and response to M01ZH09 vaccination compares favorably with response to the 3-dose Ty21a vaccine in enteric capsules (90%) and the candidate vaccine CVD-908-htr (100%) [9, 14, 18, 21].
Lymphocyte proliferation and production of IFN- were also measured after vaccination with M01ZH09. Since Salmonella Typhi is an intracellular pathogen, these cellular immune responses are an important component of protective immunity and permit activation and proliferation of effector T cells. Our data demonstrate that vaccination with M01ZH09 prompts the activation and proliferation of Salmonella Typhi antigen-specific lymphocytes in the majority of vaccinated subjects. In the evaluation of all immunogenicity assays together (ELISPOT, IgG serologic response, lymphocyte proliferation, and IFN- response), all subjects mounted 1 immune response to vaccination. Approximately half of the subjects (16/31 [51%]) had immune responses detected by all 4 assays. Fourteen (45%) of the 31 subjects had responses detected by 2 or 3 assays, and only 1 subject had a response detected by only 1 assay (ELISPOT). There was no clear association with the magnitude of the various immune responses, suggesting that an effective host response to vaccination may vary among individuals.
A common dosing practice of live oral enteric vaccines is to neutralize gastric acidity by prevaccination with a bicarbonate buffer (or similar) solution, to minimize killing of the vaccine strain by gastric acid [22]. M01ZH09 vaccine is prepared with a bicarbonate buffer solution, which provides gastric-acid neutralization at the time of vaccination. However, a simplified dosing regimen that allows for vaccine preparation with potable tap water rather than (chlorine-free) bottled water and that does not involve prevaccination with a bicarbonate buffer affords the same safety and immunologic responses as does a 2-step presentation. M01ZH09 prepared for vaccination contains the vaccine strain reconstituted from the lyophilized formulation in a buffer solution that is prepared with clean water (tap water was used in the present trial) and administered immediately.
A simplified or single oral-dose typhoid vaccination would facilitate typhoid vaccination in all populations, including travelers to and residents of areas where Salmonella Typhi is endemic or epidemic. The spread of antibiotic-resistant Salmonella Typhi globally and the recognition of typhoid prevalence in areas of hyperendemicity may encourage increased use of vaccination against typhoid fever [5,25]. M01ZH09 is a promising novel typhoid vaccine candidate and a potential vector for heterologous antigens. Field trials are needed to confirm its safety and protective efficacy.
Acknowledgments
We thank Christopher Huston, W. Kemper Alston, and Lou Polish for helpful reviews of the manuscript and statistical assistance, and we also thank the study subjects for their time and commitment.
References
1. Crump JA, Mintz ED. The global burden of typhoid fever. Bull World Health Org 2004; 82:34653. First citation in article
2. Lin FY, Vo AH, Phan VB, et al. The epidemiology of typhoid fever in the Dong Thap Province, Mekong Delta region of Vietnam. Am J Trop Med Hyg 2000; 62:6448. First citation in article
3. Brooks WA, Hossain A, Goswami D, et al. High incidence of bacteremic typhoid fever among young children in a urban slum in Dhaka, Bangladesh: implications for prevention. Emerg Infect Dis 2005; 11:3269. First citation in article
4. Mermin JH, Villar R, Carpenter J, et al. A massive epidemic of multidrug-resistant typhoid fever in Tajikistan associated with consumption of municipal water. J Infect Dis 1999; 179:141622. First citation in article
5. Murdoch DA, Banatvla NA, Bone A, Shoismatulloev BI, Ward LR, Threlfall EJ. Epidemic ciprofloxacin-resistant Salmonella typhi in Tajikistan. Lancet 1998; 351:339. First citation in article
6. World Health Organization (WHO). Background document WHO/V&B/03.07: the diagnosis, treatment and prevention of typhoid fever. Geneva: WHO, 2003. First citation in article
7. Hone DM, Attridge SR, Forrest B, et al. A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans. Infect Immun 1988; 56:132633. First citation in article
8. Silva BA, Gonzales C, Mora GC, Cabello F. Genetic characteristics of the Salmonella typhi strain Ty21a vaccine. J Infect Dis 1987; 155:10778. First citation in article
9. Levine MM. Typhoid fever vaccines. In: SA Plotkin, Orenstein WA, eds. Vaccines. Philadephia: Saunders, 2004:105793. First citation in article
10. Deiwick J, Nikolaus T, Shea JE, Gleeson C, Holden DW, Hensel M. Mutations in Salmonella pathogenicity island 2 (SPI 2) genes affecting transcription of SPI 1 genes and resistance to antimicrobial agents. J Bacteriol 1998; 180:477580. First citation in article
11. Vazquez-Torres A, Fang FC. Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect 2001; 3:131320. First citation in article
12. Khan SA, Stratford R, Wu T, et al. Salmonella typhi and S. Typhi murium derivatives harbouring deletions in aromatic biosynthesis and Salmonella pathogenicity island-2 (SPI-2) genes as vaccines and vectors. Vaccine 2003; 21:53848. First citation in article
13. Hindle Z, Chatfield SN, Phillimore J, et al. Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers. Infect Immun 2002; 70:345767. First citation in article
14. Kantale A. Antibody-secreting cells in the evaluation of the immunogenicity of an oral vaccine. Vaccine 1990; 8:3216. First citation in article
15. Baqar S, Nour El Din AA, Scott DA, et al. Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation. Clin Diagn Lab Immunol 1997; 4:3759. First citation in article
16. Doig P, Kinsella N, Guerry P, Trust TJ. Characterization of a post-translational modification of Campylobacter flagellin: identification of a sero-specific glycosyl moiety. Mol Microbiol 1996; 19:37987. First citation in article
17. Sirard J, Niedergang F, Kraehenbuhl J. Live attenuated Salmonella: a paradigm of mucosal vaccines. Immunol Rev 1999; 171:526. First citation in article
18. Levine MM, Ferreccio C, Black RE, et al. Progress in vaccines to prevent typhoid fever. Rev Infect Dis 1989; 11(Suppl 3):S55267. First citation in article
19. Tacket CO, Sztein MB, Wasserman SS, et al. Phase II clinical trial of attenuated Salmonella enterica serovar Typhi oral live vector vaccine CVD 908-htrA in US volunteers. Infect Immun 2000; 68:1196201. First citation in article
20. Kantele A, Arvilommi H, Jokinen I. Specific immunoglobulin-secreting human blood cells after peroral vaccination with Salmonella typhi. J Infect Dis 1986; 153:11269. First citation in article
21. Tacket CO, Ferreccio C, Robbins JB, et al. Safety and characterization of the immune response to two Salmonella typhi Vi capsular polysaccharide vaccine candidates. J Infect Dis 1986; 154:3425. First citation in article
22. Black RE, Levine MM, Young C, et al. Immunogenicity of Ty21a attenuated Salmonella typhi given with sodium bicarbonate or in enteric-coated capsules. Dev Biol Stand 1983; 53:914. First citation in article
23. Tarr PE, Kuppens L, Jones TC, et al. Considerations regarding mass vaccination against typhoid fever as an adjunct to sanitation and public health measures: potential use in an epidemic in Tajikistan. Am J Trop Med Hyg 1999; 61:16370. First citation in article
24. Taylor DN, Levine MM, Kuppens L, Ivanoff B. Why are typhoid vaccines not recommended for epidemic typhoid fever J Infect Dis 1999; 180:208990. First citation in article
25. Wain J, Hoa NTT, Chinh NT, et al. Quinolone-resistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment. Clin Infect Dis 1997; 25:140410. First citation in article