Study Design and Ethics
This Phase I/II, observer blind, randomised, single center, controlled trial was conducted between April 2008 and April 2009 at the City Health Office I, Santa Rosa City, Laguna, The Philippines (www.clinicaltrials.gov NCT00621322). The study protocol and informed consent forms were reviewed and approved prior to initiation of the study by the National Ethics Committee, Philippine Council for Health Research and Development. Written informed consent was obtained from each subject prior to enrollment. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice.
Eligible subjects were randomized (4:4:4:4:1:1) to receive M72/AS01B (40 μg), M72/AS01E (10 μg), M72/AS01E (20 μg), M72/AS02D (10 μg), M72/Saline (40 μg) or AS01B alone. A randomization list generated with standard statistical analysis software (SAS Institute Inc., Cary, NC, USA) was used to number the vaccines, with a block randomization scheme to ensure a balanced allocation between groups. All groups received 2 injections of study vaccine at a one-month interval (Days 0 and 30), and were followed for up to 6 months after the second vaccination (Day 210).
Vaccination was staggered in two sets and each set was observer-blind (i.e., the subject and site or sponsor staff involved in the evaluation of subjects were blinded, whilst staff involved in vaccine administration were aware of the treatment). Subjects of the first set were randomized to receive study vaccine containing the lowest antigen quantities used in this study (M72/AS01E (10 μg) and M72/AS02D (10 μg)) or AS01B alone. The second set (M72/AS01E (20 μg), M72/AS01B (40 μg) and M72/Saline (40 μg) groups) was vaccinated if no safety signals were observed post vaccination of Set 1 subjects.
Study Participants
Healthy subjects were eligible if they were PPD-positive (PPD induration ≥ 3 mm but ≤ 10 mm 48–72 hours after administration of the PPD skin test; Tuberculin PPD RT23 SSI, Statens Serum Institute), HIV-negative, aged between 18 and 45 years, had clinically normal screening laboratory values, no history of TB disease and no sign of active TB disease on chest X-ray. The selection of a skin test induration between 3 and 10 mm was made using the assumption that the test is positive maybe due to an infection with a non-tuberculous mycobacterium and/or a previous BCG vaccination, and not to an infection by Mtb. Histories of BCG vaccination were not collected. Subjects were excluded if they had a history of any acute or chronic illness, or of medication with the potential to interfere with the safety and immunogenicity evaluations. Pregnancy and lactation were exclusion criteria.
Study Vaccines
The M72 antigen has been described previously [9]. Each lyophilised cake of M72 antigen was reconstituted with the appropriate liquid adjuvant or saline. AS01 and AS02 both contain the immunostimulants QS-21 (Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA, USA) and MPL, either combined with liposomes (AS01) or in an oil-in-water emulsion (AS02) [10]. AS01B contained 50 μg MPL and 50 μg QS-21 per injection, and AS01E and AS02D both contained 25 μg MPL and 25 μg QS-21 per injection. Injected volumes were 0.5 mL for all study vaccines, except for M72/AS01E (20 μg) which was a half of the injected volume of M72/AS01B (40 μg), i.e., 0.25 mL. The vaccines were administered by intramuscular injection with a 23-gauge needle in the deltoid muscle.
Study Objectives
The primary objective was to evaluate the safety and reactogenicity of the M72 candidate vaccines in the targeted PPD-positive population. Secondary objectives were to evaluate cell-mediated and humoral immune responses.
Safety Assessment
Safety was assessed by daily recording of solicited local adverse events (AEs) (pain, redness and swelling) and general AEs (fatigue, fever [axillary temperature ≥37.5°C], gastrointestinal (GI) symptoms, headache, malaise and myalgia) for 7 days after each dose (Days 0–6). Unsolicited AEs occurring within 30 days after each dose and serious AEs (SAEs) until study end were also recorded. Biochemical and haematological parameters (complete blood count, renal and liver function tests) were monitored on the day of each vaccination (Days 0 and 30), on Day 7 after each dose (Days 7 and 37) and 30 days after Dose 2 (Day 60). All solicited local AEs were considered vaccination-related; the relationship of all other AEs to vaccination was determined by the Investigator. AE intensities were scored, and grade 3 (severe) AEs were defined as those preventing normal activity, redness or swelling >50 mm in diameter, or an axillary temperature >39.5°C.
Within Set 1, all safety and reactogenicity data collected for 6 days following vaccination were reviewed in a blinded manner by the Investigator group and a sponsor Safety Review Team to see if pre-defined holding/suspension criteria were met, prior to vaccination of Set 2. Vaccination would be put on hold pending unblinded safety review by an independent sponsor Vaccine Safety Monitoring Board if more than 8 subjects were withdrawn for severe or unexpected AEs judged to be related to vaccination 1 week after the last subject was vaccinated, in the event of a death or life-threatening SAE judged to be related to vaccination, or if a subject experienced anaphylactic shock following vaccination.
Immunological Assessment Time-Points
Blood samples were collected prior to each vaccination (Days 0 and 30) and 1 and 6 month(s) after the second vaccination (Days 60 and 210, respectively). Laboratory assessments were conducted in a blinded manner.
Humoral Responses
M72-specific IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA) based on a previously described method [9] with an assay cut-off of 2.8 ELISA units (EU)/ml (i.e., the lower limit of quantitation).
T-Cell Responses Assessed by Intracellular Cytokine Staining (ICS) and Flow Cytometry
M72 or PPD-specific CD4+ and CD8+ T cells expressing the immune markers IFN-γ and/or IL-2 and/or TNF-α and/or CD40L were detected by ICS upon short-term in vitro stimulation, based on a previously described methodology [18]. We used an adaptation of the method described by Maecker et al. [19, 20], in which PBMCs were stimulated ex vivo by incubation with antigen in the presence of costimulatory antibodies to CD28 and CD49d, and of Brefeldin A to inhibit cytokine secretion and allow intracellular accumulation. PBMCs were then stained using fluorochrome-conjugated antibodies before enumeration by flow cytometry.
To measure the M72-specific T-cell responses, PBMC were stimulated with a pool of 15-mer peptides (Eurogentech s.a., Seraing, Belgium; final concentration 1.25 μg/ml of each peptide) overlapping by 11 amino acids and spanning the entire sequence of the M72 antigen. The peptides were shown to have >80 % purity by high-performance liquid chromatography. Lyophilized peptides were reconstituted in phosphate buffered saline (PBS)/dimethylsulfoxide (<0.1 % final concentration). For assessment of PPD-specific T-cell responses, PBMC were stimulated with PPD (10 μg/ml; Staten Serum Institut, Denmark).
For the M72/AS01E (10 μg) vaccine group, an additional ICS assay was done. Th1/Th2 cytokine profiles (at Days 0 and 60 only) were characterized by measuring frequencies of CD4+ T cells expressing IFN-γ and/or IL-2 and/or TNF-α and/or CD40L and/or IL-13, following stimulation of PBMC with pools of overlapping 15-mer peptides of either 10-kDa culture filtrate protein (CFP-10), early secreted antigenic target (ESAT-6) (both 1.25 μg/ml; Eurogentec s.a., Seraing, Belgium), or M72 (1.25 μg/ml), or with PPD (10 μg/ml).
Cell Stimulation and Staining
Purified PBMC were thawed, washed twice in culture medium (RPMI 1640, Cambrex, East Rutherford, NJ, USA), supplemented with 10 % heat-inactivated fetal calf serum (FCS; PAA Laboratories GMbH, Austria), 100 IU/ml penicillin, 100 μg/ml streptomycin sulfate, 2 mM L-glutamine, MEM nonessential amino acids, 100 mM sodium pyruvate, 50 mM 2-mercapto-ethanol (all Life Technologies, Belgium), examined for viability and counted (Trucount, BD Biosciences, San Jose, CA USA), washed again, and resuspended to 2 × 107 cells/ml in culture medium. PBMC (1 × 106 cells per well) were incubated in 96-well microtiter plates with unconjugated and azide-free costimulatory anti-human CD28 and CD49d antibodies (1/250 dilution each) and stimulated for 20 h at 37 °C with the appropriate antigen. Brefeldin A (BD Pharmingen, San Diego, CA, USA; final concentration 1 μg/ml) was added for the last 18 h of culture. Positive (Staphylococcal enterotoxin B (SEB), 1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) and negative controls (unstimulated; no antigen) were included in each assay. PMA/ionomycin-stimulated CD4+ T cells were used as a positive control for IL-13 staining.
Following incubation, the cells were washed (in PBS containing 1 % FCS) and stained with anti-CD4-PerCP and anti-CD8-APC-Hi7 (all BD Pharmingen). The cells were then washed again, fixed, and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen) according to instructions and stained with anti CD3-AF700, anti-IFN-γ-FITC, anti-IL-2-APC, anti-TNF-α-PE Cy7 and anti-CD40L-PE (all BD Pharmingen). Following washing (Perm/Wash buffer, BD Pharmingen), the cells were analyzed by flow cytometry.
The same protocol and antibodies were used for the characterization of Th1/Th2-expressing T cells, with the exception that the intracellular staining was done with anti-CD3 PO (Caltag-Medsystems Ltd, UK), anti-IFN-γ-AF 700 and anti-IL-2-FITC (both BD Pharmingen) instead of anti CD3-AF700, anti-IFN-γ-FITC and anti-IL-2-APC, respectively, and that anti-IL-13-APC (Biolegend, San Diego, CA, USA) was used.
Flow Cytometry
Cells were acquired on a FACSCanto II flow cytometer or a LSR II flow cytometer (Becton Dickinson) using seven-color panels. Data were analyzed using FlowJo software (TreeStar, San Carlos, CA). Background (unstimulated control) was subtracted from all values. The remaining positive events were regarded as significant. Samples were only included for analysis if viability was ≥80 %.
Statistical Methods
Safety Evaluation
The evaluation of safety was performed on the Total Vaccinated Cohort, which included all subjects with at least one vaccine administration documented.
Descriptive statistics were performed for the percentage of doses followed by at least one solicited AE (local or general) with exact 95 % confidence interval (CI), and for the proportion of subjects reporting an unsolicited AE, classified by the MedDRA-preferred term level, with exact 95 % CI. Similar analyses were conducted for grade 3 AEs and for the AEs considered to be related to vaccination. Any reported SAEs were described. Any biochemistry and/or haematology values outside of the predefined reference ranges were assessed for clinical significance.
Immunogenicity Evaluation
Immunogenicity analysis was performed on the According to Protocol (ATP) cohort, i.e., all subjects meeting all eligibility criteria, complying with protocol defined procedures, with no elimination criteria and for whom data concerning immunogenicity endpoint measures were available.
Subjects were considered to be seropositive if their anti-M72 IgG antibody concentrations were ≥2.8 EU/mL. Seronegative subjects were given an arbitrary value of half the cut-off. Anti-M72 seropositivity rates and geometric mean concentrations (GMCs) were calculated with a 95 % CI as described previously [9].
Descriptive statistics of the CD4+ and CD8+ T-cell frequencies and serum levels of IgG antibodies were performed at each time-point using SAS version 8.2.
In the ICS assays, results were evaluated as the background-subtracted percentages of antigen-specific CD4+ and CD8+ T cells, identified as expressing any or a combination of IFN-γ and/or IL-2 and/or TNF-α and/or CD40L, or expressing any or a combination of these 4 markers and/or IL-13, upon short term in vitro stimulation. Total immune markers-expressing CD4+ and CD8+ T-cell responses were calculated by summation of the frequencies of the phenotypes expressing one, two, three, four, or five of the above immune markers upon in vitro stimulation over the background level.
A comparison of the magnitudes of vaccine-induced M72-specific responses between groups was made by evaluating for each group the proportions of subjects that responded to vaccination. This was performed descriptively by calculating at each post-vaccination time-point and for each subject the fold increases (at least 2-fold, at least 4-fold, at least 6-fold and at least 8-fold) in the frequency of M72-specific CD4+ T cells expressing at least 2 immune markers (among CD40L, IL-2, IFN-γ and TNF-α) over the frequency of these cells at pre-vaccination.
The selection of the candidate vaccine for further evaluation was based on statistical comparisons of M72-specific CD4+ T-cell responses expressing at least two immune markers (among IL-2, TNF-α, IFN-γ and CD40L) between all groups at Day 60. The expression by CD4+ T cells of at least 2 markers (instead of one) has been used as selection criterion to increase the sensitivity of the assay, allowing for a lower cut-off [21–23].
The frequencies of M72-specific CD4+ T cells were compared pair-wise between vaccine groups using a nonparametric ANOVA on the ranks (Kruskal-Wallis test) without correction for multiplicity. A sample size of 40 subjects per group for the adjuvanted vaccines provided a power of 82 % to detect at least one formulation similar to M72/AS01B (40 μg). The Wilcoxon signed rank test was used to compare ICS results at different time-points within a given vaccine group. Each two treatment groups or time-points compared were considered significantly different if p ≤ 0.05.