Study design and participants
This phase 2, randomised, observer-blind study was conducted in eight centres in three countries (Canada, Germany and the UK) between March 2018 and September 2020 (ClinicalTrials.gov identifier: NCT03443427). The observer-blind study, which lasted 18 months (vaccination phase), was followed by a 6-month open follow-up period (Fig. 2). A study summary is available at www.gsk-studyregister.com (study identifier, 207759).
Adults aged 40–80 years with a smoking history of at least 10 pack-years were administered four intramuscular injections: three injections of the AS01E-adjuvanted NTHi-Mcat vaccine containing 10 µg of each NTHi antigen (PD and PE-PilA) and 3.3 µg of the Mcat antigen (UspA2) and one injection of placebo (saline solution). The vaccine antigens were described previously [15, 16]. AS01E is an adjuvant system containing 3-O-desacyl-4′-monophosphoryl lipid A (MPL), Quillaja saponaria Molina, fraction 21 (QS-21; licensed by GSK from Antigenics LLC, a wholly owned subsidiary of Agenus Inc., a Delaware USA corporation) and liposome (25 μg MPL and 25 µg QS-21) . All study inclusion and exclusion criteria are provided in the online supplementary methods.
Participants were randomly assigned (1:1 ratio) to one of two study groups. One group received three doses of NTHi-Mcat vaccine at Days 1, 61 and 181, and a placebo injection at Day 361 (0–2–6 months schedule; 0–2–6 group) and the second group received two doses of NTHi-Mcat vaccine at Days 1 and 61, a placebo injection at Day 181, and a third NTHi-Mcat vaccine dose at Day 361 (0–2–12 months schedule; 0–2–12 group) (Fig. 2). Randomisation of supplies was performed at GSK using MATerial Excellence (MATEX), a program developed by GSK for use with Statistical Analysis Systems software (SAS; Cary, NC, USA). Participants were allocated to a study group at each site via a central randomisation system. Due to differences in the appearance of the study vaccine and the placebo formulation for the third and fourth injections, and because the vaccine was prepared and reconstituted on site, the study was conducted in an observer-blind manner, i.e. formulation recipients and those responsible for the evaluation of any study endpoint were unaware of whether a third vaccine dose or placebo was administered, and each formulation was prepared and administered by authorised medical personnel who did not participate in any of the study’s clinical evaluations.
The primary objective of the study was to assess the safety and reactogenicity of the investigational NTHi-Mcat vaccine administered in a 0–2–6 or 0–2–12 months schedule and secondary objectives were to evaluate the vaccine’s long-term safety and its humoral and cellular immunogenicity. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. The protocols and associated documents were reviewed and approved by the ethics committee of each participating centre. All participants provided written informed consent before study entry.
Safety and reactogenicity
Solicited local (pain, redness and swelling at the injection site) and general (fatigue, gastrointestinal symptoms, headache, myalgia, chills and fever) adverse events (AEs) were recorded for 7 days after each injection and unsolicited AEs for 30 days after each injection on diary cards. In addition, all participants were contacted by telephone 7 days after each injection and any safety concerns during the previous 7 days were recorded in the electronic case report form. AE intensity was graded on a 0–3 scale. Grade 3 (severe) intensity was defined as redness or swelling of diameter > 100 mm, temperature ≥ 39.0 °C and, for all other AEs, prevention of normal activities. Data on potential immune-mediated diseases (pIMDs) , serious AEs (SAEs) and AEs leading to withdrawal were collected throughout the 24-month study.
Humoral and cellular immunogenicity
Blood samples were collected for the assessment of humoral immunogenicity before the first, third and fourth injections (Days 1, 181 and 361), 30 days after the second, third and fourth injections (Days 91, 211 and 391) and 18 and 24 months after the first injection (Days 541 and 721) (Fig. 2). Immunoglobulin G antibody levels to each vaccine antigen were measured by enzyme-linked immunosorbent assay (ELISA), developed and qualified by GSK. Sera were stored at − 20 °C or below until assayed. Standardised procedures and in-house-made reference serum were used for each assay. The assay cut-off (lower limit of quantification) was 153 ELISA units (EU)/mL, 16 EU/mL, 8 EU/mL and 28 EU/mL for anti-PD, anti-PE, anti-PilA and anti-UspA2, respectively.
This humoral immune response evaluation was complemented with an investigation of cell-mediated immunity (CMI), specifically, antigen-specific T cells. The CMI subset was selected from sites able to process blood samples according to GSK procedures for peripheral blood mononuclear cell preparation. Blood samples for CMI analysis were taken before the first, third and fourth injections and 30 days after the second, third and fourth injections (Fig. 2) in a subset of participants (around 20 per group). CMI responses (antigen-specific CD4+ and CD8+ T cells) were measured by flow cytometry using intracellular cytokine staining on peripheral blood mononuclear cells, following an adaptation of previously described methods . The gating strategy for measuring antigen-specific T cells is described in the online supplementary methods (Additional file 1: Fig. S1). No CD8+ T cell response was observed. Numbers of antigen-specific CD4+ T cells expressing, upon stimulation, at least two different markers among CD40 ligand (CD40L), interleukin (IL)-2, tumour necrosis factor (TNF)-α, interferon (IFN)-ɣ, IL-13 and IL-17 were calculated.
A sample size of 100 participants in each group allowed a two-sided 95% confidence interval (CI) with width equal to 0.165, calculated using the Clopper–Pearson CI formula , assuming that 20% of participants would experience at least one grade 3 symptom following a third vaccine dose. With this sample size, the probability of observing at least one serious or severe event with incidence of 1.5% was 95%.
The safety analysis was performed on the total vaccinated cohort, including all vaccinated participants. The incidence of AEs per study group was calculated with exact 95% CIs on all safety data after each injection up to 18 months after the first vaccine dose and on all safety data derived from the 6-month follow-up period ending 24 months after the first vaccine dose.
The immunogenicity analysis was performed on the per-protocol immunogenicity cohort, including eligible adults who received the study vaccine as specified in the protocol and complied with study procedures and for whom post-vaccination immunogenicity results were available for at least one antigen. Antibody geometric mean concentrations (GMCs) were determined with 95% CIs. Post-dose 1 GMCs were calculated using an ANCOVA model including pre-dose 1 antibody concentration as covariate and treatment, country, age category and smoking status as factors, while pre-dose 1 GMCs were calculated via an ANOVA including treatment, country, age category and smoking status as factors. Geometric mean ratios (GMRs) with 95% CIs were calculated to describe the change in antibody concentration at a specific time point with respect to the antibody concentration at an earlier time point (‘baselines’, i.e. pre-dose 1, pre-dose 3 and pre-dose 4). GMRs were calculated using an ANOVA on the log10 ratio between antibody concentrations at specific time points and previous log10 antibody concentrations at an earlier time point, with treatment, country, age category and smoking status as factors. Descriptive statistics were used to summarise the frequency, by study group, of specific CD4+ T cells expressing at least two cytokine markers.
The primary endpoint was related to safety and planned to be descriptive only (i.e. no treatment comparison with regards to safety was planned). No alpha adjustment for multiple testing of the secondary endpoints (immunogenicity) was performed; CIs and differences between schedules should be interpreted with caution.
The statistical analyses were performed using SAS within the Life Science Analytics Framework system version 9.4.
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