Safety and immunogenicity evaluation of recombinant BCG vaccine against respiratory syncytial virus in a randomized, double-blind, placebo-controlled phase I clinical trial

Abstract

Background: Respiratory syncytial virus (RSV) is responsible for most respiratory tract infections and hospitalizations in infants and represents a significant economic burden for public health. The development of a safe, effective, and affordable vaccine is a priority for the WHO.

Methods: We conducted a double-blinded, escalating-dose phase 1 clinical trial in healthy males aged 18-50 years to evaluate safety, tolerability, and immunogenicity of a recombinant Mycobacterium bovis BCG vaccine expressing the nucleoprotein of RSV (rBCG-N-hRSV). Once inclusion criteria were met, volunteers were enrolled in three cohorts in an open and successive design. Each cohort included six volunteers vaccinated with 5 × 10³, 5 × 10⁴, or 1 × 10⁵ CFU, as well as two volunteers vaccinated with the full dose of the standard BCG vaccine. This clinical trial (clinicaltrials.gov NCT03213405) was conducted in Santiago, Chile.

Findings: The rBCG-N-RSV vaccine was safe, well-tolerated, and no serious adverse events related to the vaccine were recorded. Serum IgG-antibodies directed against Mycobacterium and the N-protein of RSV increased after vaccination, which were capable of neutralizing RSV in vitro. Additionally, all volunteers displayed increased cellular response consisting of IFN-γ and IL-2 production against PPD and the N-protein, starting at day 14 and 30 post-vaccination respectively.

Interpretation: The rBCG-N-hRSV vaccine had a good safety profile and induced specific cellular and humoral responses.

Funding: This work was supported by Millennium Institute on Immunology and Immunotherapy from Chile (P09/016), FONDECYT 1190830, and FONDEF D11E1098.

Keywords: BCG vaccine; Human respiratory syncytial virus; Immunogenicity; Phase I clinical trial; Safety; Transmissibility.

Fig. 1

Clinical study flow diagram and timeline. This phase 1 clinical study was double-blinded and dose-escalated. (A) Each cohort included 6 volunteers vaccinated with escalating doses of rBCG-N-hRSV (Cohort A: 5 × 10³ CFU (blue); Cohort B: 5 × 10⁴ CFU (red); Cohort C: 1 × 10⁵ CFU (green)) and 2 volunteers vaccinated with the standard BCG at full dose (BCG-WT 2 × 10⁵ CFU (purple)). A DSMB evaluated the safety data of each cohort after the first 30 days of follow-up and decided if escalation could continue. (B) A timeline indicating the periods of immunization and end of visits is shown. RSV peaks reported during 2017 and 2018 are also indicated.

Fig. 2

PPD- and N-RSV-specific humoral immune response in rBCG-N-hRSV-immunized volunteers. Anti-PPD and anti-N-RSV IgG levels were measured in the sera of all the volunteers at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 × 10³ (blue), 5 × 10⁴ (red), and 1 × 10⁵ (green) CFU and the full dose of BCG-WT (Purple). IgG levels are expressed as the geometric mean (and geometric standard deviation) of the concentrations of IgG (ng/mL) on a logarithmic scale. The responses against (A) PPD and (B) N-RSV antigens are shown. Statistical differences were evaluated by a two-way ANOVA with a posterior Tukey test. If different letters are shown above a specific time point (with their respective color-bar representing the corresponding group), then statistical differences (p<0.05) were found among those groups. If no letters are indicated above a group, then no statistical differences were found among them. n.s. = no statistical differences were found among any of the groups.

Fig. 3

Evaluation of the neutralizing capacity of antibodies obtained from vaccinated subjects. The neutralizing capacities of the sera obtained from the immunized subjects were tested and are shown as fold change (Normalized to Day 0 for each subject). The sera samples were tested at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 × 10³ (A - Blue), 5 × 10⁴ (B - Red), and 1 × 10⁵ (C - Green) CFU and the full dose of BCG-WT (D - Purple). Neutralization was tested with amounts of IgG ranging from 10 µg to 0.1 µg, which are equivalent to dilutions 1/160 and 1/20.480, respectively. Highest amounts resulted in 100% neutralization, while lowest amounts resulted in 0% neutralization. Therefore, the level of neutralization for 0.75 µg of total antibodies -dilution 1/2.560- is shown. Statistical differences were evaluated by a one-way ANOVA with a posterior Tukey test. n.s.= No statistical differences; *=P<0.05.

Fig. 4

PPD- and N-RSV-specific cellular immune response in the rBCG-N-hRSV-immunized subjects evaluated by ELISPOT. Cellular responses to PPD (A and B) and N-RSV (C and D) antigens in all PBMCs samples were measured at 0, 14, 30, 60, 120, and 180 dpv with rBCG-N-hRSV doses 5 × 10³ (blue), 5 × 10⁴ (red), and 1 × 10⁵ (green) CFU and the full dose of BCG-WT (Purple). The IFN-γ- (A and C) and IL-2- (B and D) secreting cells were detected by ELISPOT and plotted as spot-forming cells (SFC) per million cells. Statistical differences were evaluated by a two-way ANOVA with a posterior Tukey test. If different letters are shown above a specific time point (with their respective color-bar representing the corresponding group), then statistical differences (p<0.05) were found among those groups. If no letters are indicated above a group, then no statistical differences were found among them.