Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever

Immunology Letters 93 (2004) 115–122

Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever Rosa Mar´ıa Salazar-González a , Carmen Maldonado-Bernal a , Nora Elena Ram´ırez-Cruz a , Nora Rios-Sarabia a , Jorge Beltrán-Nava a , Jorge Castañón-González b , Noem´ı Castillo-Torres c , José A. Palma-Aguirre d , Manuel Carrera-Camargo a , Constantino López-Mac´ıas a , Armando Isibasi a,∗ a

Unidad de Investigación Médica en Inmunoqu´ımica, 1er piso Hospital de Especialidades, Centro Médico National Siglo XXI, Instituto Mexicano del Seguro Social (IMSS), P.O. Box A-047, C.P. 06703 México D.F., México b Intensive Care Unit, Specialties Hospital, National Medical Centre S. XXI, Mexican Institute for Social Security (IMSS), Mexico City, Mexico c Clinical Laboratory, Specialties Hospital, National Medical Centre S. XXI, Mexican Institute for Social Security (IMSS), Mexico City, Mexico d Medical Research Unit on Pharmacology, Specialties Hospital, National Medical Centre S. XXI, Mexican Institute for Social Security (IMSS), Mexico City, Mexico Received 5 December 2003; accepted 13 January 2004 Available online 19 February 2004

Abstract Typhoid fever remains a serious public health problem. We have developed a vaccine from Salmonella enterica serovar typhi (S. typhi) outer-membrane proteins (OMPs) known as porins. A single subcutaneous dose of 10 ␮g of porins induced a five-fold (P = 0.05) seroconversion index consisting of IgM and IgG at 7 and 15 days after vaccination as well as the production of IgG1 and IgG2 isotypes. The porins-based vaccine induced a two-fold increase (P = 0.05) in bactericidal titres in volunteers, whom also developed a T-cell response characterized by the production of interferon-␥ (INF-␥). Side effects after vaccination were mild and transient. The data showed that our S. typhi porins-based candidate vaccine is safe and immunogenic in healthy humans. © 2003 Elsevier B.V. All rights reserved. Keywords: Typhoid fever; Porins; Vaccine

1. Introduction Typhoid fever remains a serious public health problem in many developing countries. There are an estimated 33 million cases each year world-wide, resulting in 500,000 deaths [1]. This infectious disease also represents a risk for travellers visiting endemic areas [2]. The majority of cases occur in Southeast Asia, where resistance to antibiotics is increasingly prevalent [3–5]. School children are the population with the highest incidence of typhoid fever [6–9], and could represent a captive population for a vaccination program. Several typhoid vaccines are currently available [1]. The oral live attenuated galE mutant Ty21a vaccine is effec∗

Corresponding author. Tel.: +5255-56276915; fax: +5255-57610952. E-mail address: [email protected] (A. Isibasi).

0165-2478/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.01.010

tive in endemic areas, but it is not licensed for use in children under 6-year-old, and it requires three to four doses [10–12]. The polysaccharide vaccine, Vi, is licensed for children over 2-year-old. One injection of Vi provides similar protection to the Ty21a vaccine, but its effect lasts for only 2–3 years. Thus, the major disadvantage of this vaccine is the lack of induction of long-lasting immunity [13–15]. A new typhoid vaccine, based on Vi polysaccharide conjugated to non-toxic recombinant Pseudomonas aeruginosa exotoxin A (Vi-rEPA), has been shown to be safe and protective even in 2–5-year-old children, and is currently being tested in phase-3 clinical trials [16,17]. However, has been reported the emergence of Salmonella enterica serovar typhi Vi antigen-negative strains in an epidemic typhoid fever cases in Calcutta [18] which could direct to the need of new vaccines.

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To design a new typhoid vaccine that induces long-lasting immunity, we first tested the safety and the capacity of S. typhi porins to elicit an immune response in humans. Porins are exposed trimeric outer-membrane proteins (OMPs) from Gram-negative bacteria that function as relatively non-specific channels [19,20]. We have previously demonstrated that the immunization of mice with 30 ␮g of porins protected 90% of the animals from a challenge with 500 lethal doses (LD500 ) of mucin-resuspended S. typhi [21,22]. Acute and convalescent typhoid fever patients, as well as volunteers vaccinated with oral Ty21a vaccine produce high titres of porin-specific antibodies [23,24]. Moreover, they showed porins-specific cellular immune responses [25]. These data indicate that porins are important targets for both cellular and humoral immune responses during S. typhi infection. The study presented here shows that our vaccine candidate prepared from S. typhi porins met the quality and safety requirements for human use and induced a strong specific antibody response, with bactericidal activity, as well as a cell-mediated immune response. Moreover, the porins-based vaccine did not induce significant adverse effects in vaccinated human volunteers.

2. Materials and methods 2.1. Antigens

sist in a 200 pages document with the vaccine production specifications, quality assurance and quality controls [Manual de aseguramiento de la calidad y procedimientos estándar de operación para la producción y el control de calidad de la vacuna contra la fiebre tifoidea a base de porinas de Salmonella typhi 9,12, Vi:d para su aplicación en humanos (Fase 1). Isibasi, A., Maldonado C., Alpizar S. Unidad de Investigación Médica en Inmunoqu´ımica, Hospital de Especialidades del Centro Médico Nacional Siglo XXI IMSS. México D.F. 2000]. The following quality controls were conduced before the vaccine injection in humans. Visual aspect: compared to reference standards on established conditions. The presence of pyrogens was determined on rabbits (New Zealand) using a 0.025 ␮g/(ml kg) dose of porins on the marginal ear venous of each animal. Sterility: the absence of microorganism growth was verified on the dosed porins. Innocuousness: The toxicity of porins was assessed in mice. Half of a human dose of the vaccine was administered on each mouse in order to examine toxicity symptoms, lost of weight and survival was followed during 7 days. In addition, protein quantification, LPS quantification, Limulus Amoebocyte Lysate Assay for LPS content, Antigenicity determined as protein recognition by specific antibodies using ELISA and Western blot tests, pH determination and SDS quantification were performed on the vaccine. These analyses were conducted at the Assessment Department of Drugs, Biological Products and Diagnostic Reagents, IMSS, Mexico.

2.1.1. Purified porins-based vaccine candidate Porins were purified from S. typhi 9,12, Vi:d. ATCC 9993, using the method described by Nikaido [26]. Briefly, S. typhi was grown in glucose-supplemented minimal A medium and porins were extracted from the bacteria according to the method described previously [21]. Proteins were purified by molecular exclusion chromatography using a Sephacryl S-200 column (Waters FPLC System 650E, Millipore, Bedford, MA, USA). Chromatographically purified proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide (LPS) content was evaluated using a Limulus Amoebocyte Lysate Assay (LAL) (Endosafe® KTA, Charles River Endosafe laboratories, Charleston, SC, USA) [27]. Porins integrity was evaluated by enzyme-linked immunosorbent assay (ELISA) using hyper-immune anti-porins rabbit serum. To prepare the vaccine, porins were purified according to Mexican regulations for biological products for human use [28]. The vaccine was a sterile, pyrogen-free and safe solution, containing 20 ␮g of porins dissolved in 1 ml of diluted Nikaido Buffer (0.8 ␮g/ml SDS content) as vehicle.

2.2. Vaccination protocol

2.1.2. Vaccine quality control Quality Control was performed according to specifications of Mexican Pharmacopoeia for human use [28] and Standard Operational Procedures Manual for Porins Vaccine preparation developed in our laboratory, which con-

2.4. Study design

A phase-I study was approved by the ethics committee of the Specialties Hospital of the National Medical Centre of the Mexican Institute for Social Security (IMSS), Mexico City. The study followed the provisions of the Helsinki Declaration and its amendments; it was also developed in compliance with standard good clinical practice (GCP). 2.3. Volunteers Fifteen healthy male volunteers, aged 18–30 years, from the Mexico City area were selected after a medical examination consisting of a complete clinical history, physical examination, and clinical laboratory tests (haematic biometry, blood chemistry, general urine test, and hepatitis test). Volunteers suffering from any disease, as well as those who had been previously both vaccinated against typhoid or treated with immune modulators were excluded from the study. A written informed consent was obtained from all volunteers before vaccination.

To evaluate the immunogenicity and reactogenicity of the vaccine, a prospective comparative double-blind randomized study was performed.

R.M. Salazar-Gonz´alez et al. / Immunology Letters 93 (2004) 115–122

A simple randomization procedure consisting in the use of a code to assign all patients to receive one of two different treatments at the start of the trial, and double-blind character in which neither subjects nor investigators, as well as sponsor or investigator staff involved in the treatment or clinical evaluation of subjects, were aware of each subjects assigned treatment. The study included a group of 11 volunteers, who received subcutaneously a single dose of vaccine consisting of 10 ␮g of porins in 0.5 ml vehicle in the left arm, and a control group of four volunteers, who received 0.5 ml isotonic saline solution (ISS) as placebo. The subjects were monitored at 10 min intervals in the intensive care unit (ICU) for 2 h after vaccination. The reactogenicity of the vaccine was assessed in subjects by requesting a description from them of localized symptoms (pain, irritation, redness, induration, inflammation, or arthralgia) and general symptoms (fever, headache, gastrointestinal discomfort, shivering, sickness, fatigue, or malaise). The volunteers were asked to record these symptoms as follows: 0, absent; 1, mild; 2, moderate (sufficiently uncomfortable to interfere with daily activity); and 3, severe (impeding daily activity). The effects were monitored over the subsequent 6 days. The volunteers were also asked to write down any other symptom not included above. To analyze antibody and cellular immune responses, 50 ml blood samples were drawn at 0, 7, and 14 days after vaccination. 2.5. Immune response analysis Venous blood samples were taken from each volunteer before vaccination, and 7 and 14 days after vaccination. An aliquot was processed to obtain the serum, and the remaining was used to collect peripheral blood mononuclear cells (PBMC). 2.5.1. Serology Specific S. typhi porins antibody titres were determined by ELISA. Flat-bottomed 96-well microtitre plates (Costar® , Cambridge, MA, USA) were coated with 10 ␮g/ml S. typhi porins in carbonate buffer (pH 9.5). Wells were blocked for 2 h at 37 ◦ C using a buffer containing 5% milk in phosphate-buffered saline (PBS). Sera were serially two-fold diluted in 5% PBSmilk. Horseradish peroxidase-conjugated goat anti-human antibodies (anti-IgM, anti-total IgG, anti-IgG1, IgG2, IgG3, and IgG4; Zymed, Cambridge, UK), diluted 1:1000 (for conjugated IgM) or 1:3000 (for conjugated IgG) in 5% milk–PBS, were used. Triplicates of each sample were assessed simultaneously. Porins-specific antibody titres were determined as the sample dilution with an optical density ≥0.2 O.D. units above that of the negative controls.

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2.5.2. Salmonella typhi bactericidal assay Serum bactericidal activity was determined using the method described by Peeters et al. [29]. Briefly, serum samples were complement inactivated at 56 ◦ C for 30 min. Duplicates of complement-depleted serum samples were pre-diluted 1:20 and four log 2 dilutions were performed using sterile PBS. Each diluted sample was incubated for 1 h at 37 ◦ C in the presence of 250 ± 50 colony-forming units (CFU) of S. typhi. Normal guinea-pig serum diluted 1:4 in PBS was added as an external source of complement. Samples containing (i) PBS; (ii) guinea-pig serum; or (iii) bacteria without serum, were used as negative controls. When incubation was completed, samples were plated in Petri dishes containing 0.8% agar and 3% trypticasein soy broth. Plates were incubated for 18 h at 37 ◦ C and CFU counted. The percentage of dead bacteria in each sample was calculated based on the number of CFUs in the negative controls. Bactericidal titres were determined from the sample dilution that produced the death of ≥50% of the negative controls. Samples were tested from the eleven vaccinated volunteers and four controls. 2.5.3. Cellular immune response Porins-induced proliferation assays were performed using PBMC at 0, 7, and 14 days after vaccination. PBMC were isolated using Lymphoprep (Nycomed, Oslo, Norway) and 4×105 cells per well were cultured in 96-well plates (Costar) with 200 ␮l serum-free AIM-V medium (Gibco BRL, Grand Island, NY, USA). For cellular proliferation, 0.01 ␮g/ml porins from S. typhi were added. Concanavalin A, 5 ␮g/ml (Sigma, St. Louis, MO, USA) was used as a positive control. Cells with medium only were used to measure basal proliferation. Culture microplates were incubated for 5 days at 37 ◦ C under 5% CO Triplicate samples were tested for each culture conditions and, 18 h before harvesting, cells were pulsed with 1 ␮Cu [3 H] thymidine (Amersham, Arlington Heights, IL) per well. Incorporated radioactivity was measured using a liquid scintillation ␤-counter (Wallac 1450, Perkin-Elmer, Wellesley, MA, USA). Results are expressed as a specific proliferation index calculated as follows: counts per minute (cpm) antigen proliferation/cpm basal proliferation. Samples were tested from ten vaccinated volunteers and four controls. 2.5.4. Determination of cytokine profiles To assess the phenotype of the cellular immune response induced by porins from S. typhi in immunized volunteers, interferon-␥ (INF-␥) and interleukin-4 (IL-4) cytokines were measured in the supernatants of antigen-stimulated cells using an ELISA kit (BD Pharmingen, Opt-EIA, San Diego, CA, USA). PBMC from the nine vaccinated and four control subjects were purified using Lymphoprep, and 2 × 106 cells per well were grown in 1 ml serum-free AIM-V medium in 24-well plates (Costar). Porins from S. typhi (0.1 ␮g/ml) were added. Con A (5 ␮g/ml) was used as a positive control.

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2.5.5. Statistical analysis A Wilcoxon signed-rank test was used for intra-group comparisons of immune responses. The Mann–Whitney U-test was used to compare immune responses between groups. A value of P < 0.05 was considered to be significant.

3. Results

3.2. Adverse reactions The reactivity induced by the porins-based vaccine is summarized in Table 1. Minimal systemic or local adverse reactions were observed in vaccinated volunteers. The most commonly observed effects were mild and transient. We observed local effects such as pain, redness and induration lasting over 8 h maximum. Only two volunteers had systemic reactions, such as malaise, headache, and fatigue during 1 day. None volunteer developed fever or loss of appetite.

3.1. Porins-based vaccine candidate 3.3. Porins-specific antibody responses The porin homotrimers purified by molecular exclusion chromatography from a crude porin preparation using a Sephacryl S-200 column in a high salt buffer are showed in Fig. 1a. Trimers eluted with an estimated molecular weight of 120 kDa. SDS-PAGE analysis under reducing conditions only showed two types of porin monomers of 36 and 38 kDa (OmpC, OmpF) without other contaminants proteins (Fig. 1b). No LPS content in the porins-based vaccine was detected by LAL assay.

Vaccination induced porins-specific IgM and IgG antibody titres in the immunized volunteers. An increase in the porins-specific seroconversion index (P = 0.05) was observed 7 and 14 days after vaccination for both IgM and IgG antibody titres; a five-fold increase was observed for both immunoglobulins in the vaccinated volunteers (Fig. 2a and b). In contrast, seroconversion was not observed in the control group, although low anti-porin-specific antibody titres were present before and after injection (Fig. 2a and b). The IgG antibody isotypes observed in porins-vaccinated volunteers were mainly IgG1 and IgG2 (Fig. 2c and d). A seroconversion increase in these isotypes was observed at 7 and 14 days after vaccination (P = 0.05), whereas in the control group, no seroconversion was observed (Fig. 2c and d). At day 14, IgG2 showed an antibody titre slightly higher than that of IgG1. This was also observed in pre-immune sera from both experimental groups. No IgG3 or IgG4 antibody isotypes were found at any time point evaluated (Fig. 2c and d). In order to evaluate the specificity of seroconversion induced by porins vaccine, we have determined the anti-Escherichia coli K-12 porins IgG antibody titres in Table 1 Adverse reactions in volunteers vaccinated with porins vaccine

Fig. 1. Isolation of porin trimers from Salmonella enterica serovar typhi. Profile of size exclusion chromatography using a sephacryl S-200 column (a). Fractions that correspond to the major peak were pooled and analysed by SDS-PAGE; (lane 1) molecular weight markers; (lane 2) 5 ␮g of purified porins (b).

Vaccinated (n = 11)

Control (n = 4)

Systemic reactions Fever (≥38 ◦ C) Headache Stomachache Shivering Sickness Fatigue Arthralgia Malaise

0 1 0 0 1 1 0 2

0 0 0 0 0 0 0 0

Local reactions Pain Redness Induration Inflammation Irritation

9 4 6 4 1

0 0 0 0 0

Volunteers received a subcutaneous single dose of 10 ␮g of porins from S. typhi in a total volume of 0.5 ml. Control group received subcutaneously 0.5 ml of SSI.

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119 vaccinated

15

vaccinated control

10

5

IgG titers-log2(x20)

IgM titers -log2(x20)

15

0

7

0

5

0

14

days after vaccination vaccinated

7.5

5.0

2.5

vaccinated

10.0

IgG2 titers -log2(x10)

control

14

7 days after vaccination

(b)

10.0

IgG1 titers -log2(x10)

10

0

(a)

0.0

control 7.5

5.0

2.5

0.0

0

(c)

control

7

14

days after vaccination

0

(d)

7

14

days after vaccination

Fig. 2. Porins from S. typhi induced specific humoral immune response. Antibodies titres were determined by ELISA at days 0, 7 and 14 after vaccination, anti-porins IgM; (a) IgG; (b) IgG1; (c) and; IgG2 (d) antibody titres are shown. Each line represents a median of 11 vaccinated volunteers and 4 control subjects, respectively.

vaccinated volunteers using the ELISA protocol described for anti-S. typhi porins antibody titres determination (data not shown). Basal anti-E. coli porins titres were observed in control and in vaccinated volunteers, titres did not increased after vaccination with porins vaccine, therefore no seroconversion was observed. In addition, sera from a vaccinated volunteer obtained 15 days after S. typhi porins immunization did not recognized E. coli porins in a western blot analysis (data not shown). Taken together these results showed that porins vaccine did not induce seroconversion of anti-E. coli porins cross-reactive antibodies therefore, seroconversion observed after S. typhi porins vaccine injection is specific.

counted in all controls and 50% of these value was taken to determined the bactericidal antibody titre. 3.5. Salmonella typhi porins-induced specific T-cell response PBMC from the volunteers were pulsed with porins from S. typhi and a specific proliferation response was observed. Maximum proliferation index was measured in PBMC from the porins-vaccinated group 7 days after immunization. However, at day 14, proliferation index decreased to values 2 U above the proliferation index for pre-immune Table 2 Bactericidal antibodies induced by porins from Salmonella typhi

3.4. Serum bactericidal activity Basal bactericidal antibody titres were found in both the vaccinated and control groups. However, a two-fold increase (P = 0.05) was observed in sera from porins-vaccinated volunteers 7 days after vaccination, and this bactericidal activity remained at day 15 after immunization. In contrast, no change in bactericidal activity was observed in the control group (Table 2). As negative controls for the bactericidal assay (i) PBS; (ii) guinea-pig serum; or (iii) bacteria without serum were used. An average of 250 ± 40 CFU were

Bactericidal titers

Pre Post-7 Post-15

Vaccinated

Control

2 (2–4) 4 (4–4) 4 (3–4)

3 (2–4) 1 (0–4) 1.5 (0–3)

Pre: pre-immunization. Post-7 and post-15: 7 and 15 days after immunization. Results are representative of 11 vaccinated volunteers and 4 control volunteers. Duplicates determinations were performed for each sample. Bactericidal titres are reported as reciprocal serum dilutions showing ≥50% killing. Data represent median with maximum and minimum values.

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22.5

vaccinated

proliferation index

17.5

cont rol

12.5 12 10 8 6 4 2 0

0 (a)

7 days after vaccination

14

1500

vaccinated

IFN- (pg/ml)

control 1000

500

0

0 (b)

7 days after vaccination

14

Fig. 3. Porins from S. typhi induced specific cellular immune response. Proliferation of peripheral blood mononuclear cells from volunteers before vaccination, 7 and 14 days after vaccination was tested with 0.01 ␮g/ml porins. Stimulation index was calculated: cpm stimulation antigen cells per cpm medium cells. Each line represents the median of 10 vaccinated volunteers and 4 control group subjects, respectively (a). Production of INF-␥ by peripheral blood mononuclear cells. Cells were stimulated with 0.1 ␮g/ml of porins. The amount of produced cytokine in the culture supernatants was measured at 0, 7 and 14 days after vaccination by ELISA. The figure shows the median value from nine vaccinated volunteers and four control subjects Triplicates determinations were performed for each sample (b).

cells (Fig. 3a). In contrast, the control group did not showed an increased on the proliferation index at any time tested, it remained always under the values of the vaccinated group. However, the pronounced proliferation variations among the volunteers created non-statistically significant differences. Cells stimulated with 5 ␮g/ml Con A were used as positive controls. The proliferation values obtained were: 64,290 (6006–78,950) cpm in cells from the immunized group and 59,800 (5520–150,500) cpm from the control group. 3.6. Production of INF-γ and IL-4 by porins-stimulated mononuclear cells PBMC from either the vaccinated or control subjects were pulsed in vitro with 0.1 ␮g/ml S. typhi porins and

the supernatants were tested for INF-␥ and IL-4 production. The vaccinated volunteers showed a peak of INF-␥ response at day 7 after immunization further increasing at day 14. In contrast, only one control subject produced INF-␥ at day 14 (Fig. 3b). No IL-4 production (above 3 pg/ml) was detected in the supernatants of porins-stimulated PBMCs from either the vaccinated or control subjects. Supernatants from Con A-stimulated PBMCs were used as positive controls. For the vaccinated group, the median INF-␥ concentration was 1682 (746–2462) pg/ml and the median IL-4 concentration was 25.6 (0–73.7) pg/ml, whereas PBMC from the control group produced 1910 (1742–2081) pg/ml of INF-␥ and 44.09 (0–83.7) pg/ml of IL-4. The concentrations of INF-␥ were always higher than the concentrations of IL-4 in the samples tested.

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4. Discussion The current research evaluated the immunogenicity and biosafety in human volunteers of a typhoid fever vaccine candidate based on porins obtained S. typhi. The results show that the vaccine formula is safe and clinically well tolerated. No serious adverse reactions were observed after vaccination; side effects were mostly related to local pain. The local and general reactions observed in volunteers were mild. As it has been shown for porins from other bacteria [30,31], S. typhi porins were immunogenic in humans. Our results show that the porins vaccine induces cellular and humoral immune responses. As expected, the study group showed low basal levels of cellular and humoral immune response, probably caused by previous contact with non-pathogenic Salmonella species common in Mexico City [32]. The induction of a specific antibody response after porins vaccination was shown by the increase in seroconversion observed for both IgM and IgG porins-specific antibodies. The porins-specific antibody isotypes analyzed showed that IgG response is characterized mainly by the presence of IgG1 and IgG2, which indicates that porins antibody response requires the involvement of CD4+ Tcells. Porins have not always been found to induce bactericidal antibodies [33]. However, porins from Neisseria and Haemophilus generate bactericidal antibodies in humans and animals [34–36]. Bactericidal antibodies are important in protecting against Salmonella infection [37,38]. Therefore, we evaluated the induction of Salmonella bactericidal antibodies induced by porins in volunteers. We found low titres of bactericidal antibodies before vaccination and a significant increase in bactericidal activity was detected in the sera of vaccinated volunteers after immunization, indicating that porins induced the generation of anti-Salmonella bactericidal antibodies (Table 2). Vaccinated volunteers developed porins-specific T-cell responses. In most cases, the volunteers showed an increase in T-cell responses 7 days after vaccination. However, this was not statistically significant because of the great variability in the results obtained within the study group. In contrast, the placebo group did not show increase on proliferation responses, suggesting that the T-cell response observed in the vaccinated group is porins-specific. These results are in agreement with previous findings using Neisseria porins [39]. There was no further increase in T-cell proliferation at day 14 after vaccination, but rather a decrease in the response, although the values recorded were above the values obtained for pre-immune cells. The kinetics of T-cell responses observed in the porins-vaccinated volunteers are similar to those observed in humans vaccinated with the oral typhoid vaccine Ty21a [40]. Therefore, our data demonstrate that the S. typhi porins vaccine can induce T-cell responses in humans.

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INF-␥ and IL-4 cytokines induced by the porins-based vaccine were measured in the supernatants of in vitro porins-stimulated mononuclear cells from the volunteers. We found that S. typhi porins induced INF-␥ production. In summary, we conclude that subcutaneous injection of 10 ␮g of porins was safe and induced antibodies with bactericidal activity in healthy humans. In addition, porins vaccine induced a TH1-type T-cell response. Further clinical experiments are planned that include evaluation of booster injections and the optimal vaccine dose.

Acknowledgements We wish to thank all the staff at the Intensive Care Unit, Clinical Laboratory, and the ethics committee of the Specialties Hospital of the National Centre of the Mexican Institute for Social Security (IMSS, Mexico). We thank the IMSS Biological Quality Control Department for the help received to evaluate the quality of porins vaccine. We thank Dr. José Moreno for providing us with the liquid scintillation beta counter. We thank all of the volunteers who participated in this work. We thank Dr. Edmundo Calva and Dr. José Luis Puente from Instituto de Biotecnolog´ıa de la Universidad Nacional Autónoma de México for providing us with E. coli K-12 strain. This work was supported by grants F243-MP207 and 0757P-M 9506 from Consejo Nacional de Ciencia y Tecnolog´ıa (CONACYT), Mexico City.

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