Adjuvant-Associated Peripheral Blood mRNA Profiles and Kinetics Induced by the Adjuvanted Recombinant Protein Candidate Tuberculosis Vaccine M72/AS01 in Bacillus Calmette-Guérin-Vaccinated Adults

Abstract

Systems biology has the potential to identify gene signatures associated with vaccine immunogenicity and protective efficacy. The main objective of this study was to identify optimal postvaccination time points for evaluating peripheral blood RNA expression profiles in relation to vaccine immunogenicity and potential efficacy in recipients of the candidate tuberculosis vaccine M72/AS01. In this phase II open-label study (NCT01669096; https://clinicaltrials.gov/), healthy Bacillus Calmette-Guérin-primed, HIV-negative adults were administered two doses (30 days apart) of M72/AS01. Twenty subjects completed the study and 18 subjects received two doses. Blood samples were collected pre-dose 1, pre-dose 2, and 1, 7, 10, 14, 17, and 30 days post-dose 2. RNA expression in whole blood (WB) and peripheral blood mononuclear cells (PBMCs) was quantified using microarray technology. Serum interferon-gamma responses and M72-specific CD4+ T cell responses to vaccination, and the observed safety profile were similar to previous trials. Two different approaches were utilized to analyze the RNA expression data. First, a kinetic analysis of RNA expression changes using blood transcription modules revealed early (1 day post-dose 2) activation of several pathways related to innate immune activation, both in WB and PBMC. Second, using a previously identified gene signature as a classifier, optimal postvaccination time points were identified. Since M72/AS01 efficacy remains to be established, a PBMC-derived gene signature associated with the protective efficacy of a similarly adjuvanted candidate malaria vaccine was used as a proxy for this purpose. This approach was based on the assumption that the AS01 adjuvant used in both studies could induce shared innate immune pathways. Subjects were classified as gene signature positive (GS+) or gene signature negative (GS-). Assignments of subjects to GS+ or GS- groups were confirmed by significant differences in RNA expression of the gene signature genes in PBMCs at 14 days post-dose 2 relative to prevaccination and in WB samples at 7, 10, 14, and 17 days post-dose 2 relative to prevaccination. Hence, in comparison with a prevaccination, 7, 10, 14, and 17 days postvaccination appeared to be suitable time points for identifying potentially clinically relevant transcriptome responses to M72/AS01 in WB samples.

Keywords: adjuvant system; innate immunity; interferon; transcriptome; tuberculosis; vaccine.

Figures

Figure 1

Study design overview. (A) The timing of vaccination and sampling procedures. Abbreviations: AEs, adverse events; CMI, cell-mediated immunity (i.e., antigen-specific CD4+ T cell frequencies); PBMC, peripheral blood mononuclear cell; SAEs, serious adverse events; and WB, whole blood. (B) Participant flow and the numbers entered into the immunogenicity per-protocol (PP) cohort and safety (total vaccinated, TV) cohort.

Figure 2

Safety and immunogenicity outcomes observed in vaccinated subjects. (A) Histograms describing the percentage of subjects (N = 20) reporting solicited adverse events (injection site symptoms and general symptoms of all grades or Grade 3 only) after either Dose 1 or Dose 2 during the 7-day (Days 0–6) postvaccination period. Grade 3 represents, for redness and swelling, a diameter >50 mm; for injection site pain, there is considerable pain at rest; for fever, an axillary temperature >39.5°C; and for other symptoms, normal activity is prevented. Gastrointestinal symptom is abbreviated to gastrointestinal. Error bars describe Fisher exact 95% confidence intervals (95% CIs). (B) Individual and median interferon-gamma (IFNG) concentrations of the evaluated immunogenicity cohort subjects (N = 18, Days 0 [Pre], 30 [PI], 37, 40, and 44; N = 17, Days 31 [1 day PII] and 47). The timing of vaccination is indicated below the x-axis by black triangles. (C,D) Antigen (M72)-specific CD4+ T-cell frequencies of the evaluated immunogenicity cohort subjects (N = 13, Day 0 [Pre]; N = 12, Day 60 [PII]). (C) Individual and median percentage frequencies of antigen-specific CD4+ T cells/million CD4+ T cells expressing two or more immune markers, or (D) Box and whisker plots describing the percentage frequencies of antigen-specific CD4+ T cells/million CD4+ T cells expressing defined combinations of immune markers (indicated below the x-axis) among CD40L, IL2, tumor necrosis factor (TNF), IFNG, IL13, and IL17 after short-term in vitro stimulation. The whiskers extend to the lowest (Min) and highest (Max) values; the box extends to the first quartile (Q1) and third quartiles (Q3) in which the median (Med) is marked by a horizontal line. Significance differences in panels (B,C) between postvaccination concentrations/frequencies and prevaccination concentrations/frequencies (Day 0) are indicated by asterisks (**p < 0.01; ***p < 0.001) and were determined by the Wilcoxon signed-rank test.

Figure 3

The evaluation of RNA expression changes relative to baseline (Day 0) at the level of blood transcription modules (BTMs, see Materials and Methods for definition) (31). A heatmap description of significant enrichment, with coloration indicating the directionality (upregulation or downregulation) of the majority of genes (coloration described in legend), in peripheral blood mononuclear cell (PBMC)-derived (left panel; Days 31 and 44), and whole-blood-derived (right panel; Days 30, 31, 37, 40, 44, and 47) RNA expression data from all study subjects evaluated. BTM titles and reference codes are described to the right of the heatmaps.

Figure 4

The assignment to gene signature-positive (GS+) and gene signature-negative (GS−) groups using RNA expression data from study subjects. (A) Reference gene signatures in RTS,S malaria vaccine efficacy trial (41) with respect to mean RNA expression intensities (levels) for the Cluster-A probe sets (left graph) and Cluster-B probe sets (right graph). Cluster A included 25 probe sets, and Cluster B included 40 probe sets (see Table S1 in Supplementary Material). The graphs show that the mean RNA expression levels differed, 14 days after vaccination, between vaccine recipients who were subsequently protected (P) or not-protected (NP) against malaria sporozoite challenge. (B) Voting system simulation for assignment of subjects to the GS+ and GS− groups based on differences in RNA expression intensities with baseline (ΔRNA; higher [+] or lower [−]). Correspondence (both >0 or both <0) or no correspondence (one >0 and other <0) with a given GS+ reference ΔRNA of the probe set was scored +1 or −1, respectively. If the overall score for all probe sets from both clusters was above 0 then the subject was assigned to the GS+ group, and if the overall score was 0 or below then the subject was assigned to the GS− group. (C) Individual and mean overall RNA expression intensities relative to baseline (Day 0) for Cluster-A probe sets (left graphs) and Cluster-B probe sets (right graphs) for the evaluated peripheral blood mononuclear cell (PBMC) samples (upper graphs) and whole-blood samples (lower graphs) from study subjects assigned to either the GS+ group or the GS− group. Group assignment was based on PBMC RNA expression data at Day 44 (highlighted by gray rectangular outlines). Note that means are only shown for Days 0, 31, and 44. The timing of vaccination is indicated below the x-axes of the lower graphs by black triangles.

Figure 5

Individual and mean overall RNA expression intensities relative to baseline (Day 0) for Cluster-A probe sets (left graphs) and Cluster-B probe sets (right graphs) for the evaluated whole-blood (WB) samples from study subjects assigned to either the gene signature-positive (GS+) group or the gene signature-negative (GS−) group. Group assignment was based on WB RNA expression data at Days 37, 40, 44, and 47 (highlighted by gray rectangular outlines). Note that means are only shown for Days 0, 31, and the day from which group assignment was determined. The timing of vaccination is indicated below the x-axes of the lower graphs by black triangles.

Figure 6

Individual and median serum interferon-gamma (IFNG) concentrations (left graphs) and individual and median frequencies of antigen (M72)-specific CD4+ T-cells/million CD4+ T cells (right graphs), in accordance with group assignment based on RNA expression data from peripheral blood mononuclear cell (PBMC) at Day 44 or whole blood (WB) at Days 37, 40, 44, and 47. The serum IFNG concentrations at Day 31 and the antigen (M72)-specific CD4+ T-cell frequencies at Day 60 are highlighted by gray rectangular outlines. The timing of vaccination is indicated below the x-axis of the lowest IFNG graph by black triangles.