rBCG induces strong antigen-specific T cell responses in rhesus macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector

PLoS One. 2008;3(11):e3790. doi: 10.1371/journal.pone.0003790. Epub 2008 Nov 21.


Background: BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals).

Methods and findings: Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha(+) T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha(+) T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity.

Conclusion: AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Animals
  • Antigens, Bacterial / genetics
  • BCG Vaccine / administration & dosage*
  • BCG Vaccine / genetics
  • Bacterial Toxins / genetics
  • Female
  • Genetic Vectors
  • Hemolysin Proteins / genetics
  • Immunity, Cellular
  • Immunization, Secondary
  • Interferon-gamma / biosynthesis
  • Lymphocyte Activation
  • Macaca mulatta
  • T-Lymphocytes / immunology*
  • Tuberculosis Vaccines / genetics
  • Vaccines, Synthetic / administration & dosage
  • Vaccines, Synthetic / genetics


  • Antigens, Bacterial
  • BCG Vaccine
  • Bacterial Toxins
  • Hemolysin Proteins
  • Tuberculosis Vaccines
  • Vaccines, Synthetic
  • Clostridium perfringens theta-toxin
  • Interferon-gamma