First-in-man open clinical trial of a combined rdESAT-6 and rCFP-10 tuberculosis specific skin test reagent

Abstract

Background: Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity.

Methodology: In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1ratio1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 microg or 0.1 microg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN gamma response just below the Quantiferon(R) cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded.

Conclusion: The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease.

Trial registration: ClinicalTrials.gov NCT00793702.

Conflict of interest statement

Competing Interests: The study group: ABA was previously employed at SSI, but since 1998 has been employed at Rigshospitalet and receives no salary from SSI. ABA and PA are co-inventors of a patent related to ESAT-6 but all rights belong to SSI. WB has no conflicts of interest. The SSI that sponsored the study employs PT, BTC, HA, STH, PA and VOT. This does not alter the authors' adherence to the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Flow diagram of study design.

Participants were screened from −28 to −3 days before inclusion. The injections were given at day 0 and day 42 or on day 0 and day 84. Group A and B received 0.01 µg of the investigational skin test antigen. Group C and D received 0.1 µg. All volunteers completed a final follow-up visit 28 days after the last injection.

Figure 2. In vitro IFN γ responses to selected antigens.

PBMCs from two volunteers were tested for in vitro IFN γ responses to selected M. tuberculosis antigens. A:volunteer from group C, TST negative, QFT-IT positive after 1. injection, but negative after 2. injection and at day 28. B: volunteer from group D, TST and QFT-IT positive after 1. injection and hereafter excluded. PBMCs were stimulated for five days with culture medium only (Nil), TB10.4 peptide mixture (TB10.4), ESAT-6 peptide mixture (ESAT-6), CFP10 peptide mixture (CFP10) or Staphylococcal Enterotoxin B (SEB) as a positive control. Subsequently, the supernatants were tested for IFN γ content by ELISA. Bars indicate the mean concentrations of IFN γ in pg/mL of triplicate wells. Error bars indicate the Standard Error of Mean. For each subject, the means of the peptide- and SEB stimulated wells were compared to the mean of the corresponding un-stimulated (Nil) wells by one-way analysis of variance with Bonferoni post-test correction. ***: P<0.001, *:P<0.05, NS: Not Significant (P>0.05).