Whole blood RNA signatures in tuberculosis patients receiving H56:IC31 vaccine as adjunctive therapy

Introduction Therapeutic vaccination in tuberculosis (TB) represents a Host Directed Therapy strategy which enhances immune responses in order to improve clinical outcomes and shorten TB treatment. Previously, we have shown that the subunit H56:IC31 vaccine induced both humoral and cellular immune responses when administered to TB patients adjunctive to standard TB treatment (TBCOX2 study, NCT02503839). Here we present the longitudinal whole blood gene expression patterns in H56:IC31 vaccinated TB patients compared to controls receiving standard TB treatment only. Methods The H56:IC31 group (N=11) and Control group (N=7) underwent first-line TB treatment for 182 days. The H56:IC31 group received 5 micrograms of the H56:IC31 vaccine (Statens Serum Institut; SSI, Valneva Austria GmbH) intramuscularly at day 84 and day 140. Total RNA was extracted from whole blood samples collected in PAXgene tubes on days 0, 84, 98, 140, 154, 182 and 238. The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR (Biomark HD system, Standard BioTools). Results The targeted gene expression profiling unveiled the upregulation of modules such as interferon (IFN) signalling genes, pattern recognition receptors and small nucleotide guanosine triphosphate (GTP)-ases in the vaccinated group compared to controls two weeks after administration of the first H56:IC31 vaccine. Additionally, the longitudinal analysis of the Adolescent Cohort Study-Correlation of Risk (ACS-COR) signature showed a progressive downregulation in both study arms towards the end of TB treatment, in congruence with reported treatment responses and clinical improvements. Still, two months after the end of TB treatment, vaccinated patients, and especially those developing both cellular and humoral vaccine responses, showed a lower expression of the ACS-COR genes compared to controls. Discussion Our data report gene expression patterns following H56:IC31 vaccination which might be interpreted as a lower risk of relapse in therapeutically vaccinated patients. Further studies are needed to conclude if these gene expression patterns could be used as prognostic biosignatures for therapeutic TB vaccine responses.


Figure 1 .
Figure 1.-Post-vaccination gene expression differences in A) H56:IC31 group compared to Controls; and, B) vaccine Responders compared to Controls.Figure 2.-Principal Component Analysis of the expression of the 183 pre-selected genes in H56:IC31 vaccinated patients at all time points.Figure 3.-Heatmap and clustering analysis of the H56:IC31 group based on the expression of preselected genes.Figure 4.-Evolution of the ACS-COR signature within the H56:IC31 group and Control group.

Figure 2 .
Figure 1.-Post-vaccination gene expression differences in A) H56:IC31 group compared to Controls; and, B) vaccine Responders compared to Controls.Figure 2.-Principal Component Analysis of the expression of the 183 pre-selected genes in H56:IC31 vaccinated patients at all time points.Figure 3.-Heatmap and clustering analysis of the H56:IC31 group based on the expression of preselected genes.Figure 4.-Evolution of the ACS-COR signature within the H56:IC31 group and Control group.

Figure 4 .-
Figure 1.-Post-vaccination gene expression differences in A) H56:IC31 group compared to Controls; and, B) vaccine Responders compared to Controls.Figure 2.-Principal Component Analysis of the expression of the 183 pre-selected genes in H56:IC31 vaccinated patients at all time points.Figure 3.-Heatmap and clustering analysis of the H56:IC31 group based on the expression of preselected genes.Figure 4.-Evolution of the ACS-COR signature within the H56:IC31 group and Control group.
Figure 2.-Principal Component Analysis of the expression of 183 pre-selected genes in H56:IC31 vaccinated patients at days 84, 98, 140, 154, 182, and 238.We use PCA to project gene expression datasets of H56:IC31 vaccinated TB patients onto the first two components.Three H56:IC31 subgroups of patients are analysed: Non-Responders (NR) in purple, Responders (R) in orange and Partial Responders (PR) in green.The contribution of the first two principal components is given in brackets.

Table 3 .-H56:IC31 vaccine mediated responses.
Table shows the individual Cytokine+ CD4+ T cell responses and anti-H56 IgG titers in the H56:IC31 and Control groups at days 84, 98, 140 and 154 and the ratio of responses at day 98 vs day 84, and day 154 vs day 84.CD4+ T cell responses are represented in Spots Forming Units (SFU) per 300,000 cells.Humoral responses are defined by log transformed anti-H56 IgG serum levels (EU/ml).Patients who showed a ≥ 2-fold increase in CD4+ T cell responses are highlighted in red.Patients who showed a 2-fold increase in humoral responses are highlighted in green.Responders (R); Partial Responders (PR), and Non-Responders (NR) are specified in the table.