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, Nov 2008

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

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rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

et al. (2008) 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 3(11): e3790. doi:10.1371/journal.pone.0003790 rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector Isabelle Magalhaes 0 Donata R. Sizemore 0 Raija K. Ahmed 0 Stefanie Mueller 0 Lena Wehlin 0 Charles 0 Scanga 0 Frank Weichold 0 Giulia Schirru 0 Maria Grazia Pau 0 Jaap Goudsmit 0 Sharon Ku hlmann- Berenzon 0 Mats Sp angberg 0 Jan Andersson 0 Hans Gaines 0 Rigmor Thorstensson 0 Yasir A. W. Skeiky 0 Jerry Sadoff 0 Markus Maeurer 0 Jacques Zimmer, Centre de Recherche Public-Sante, Luxembourg 0 1 Microbiology, Tumor and Cell Biology Center, Karolinska Institutet, Solna, Sweden, 2 The Swedish Institute for Infectious Disease Control , Solna, Sweden, 3 Aeras Global TB Vaccine Foundation, Rockville, Maryland , United States of America , 4 Crucell Holland BV, Leiden , The Netherlands , 5 Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge , Stockholm , Sweden 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 PLoS ONE | www.plosone.org - Funding: This work was supported by the Aeras Foundation, Vetenskapsradet, the So derberg foundation and Karolinska Institutet. IM is supported by a Marie Curie Host Fellowship for Early Stage Researchers Training. The authors state that they have a financial interest. Competing Interests: The authors have declared that no competing interests exist. Bacille Calmette-Guerin (BCG) is a safe live vaccine against M. tuberculosis (Mtb) introduced in 1921 and it is still widely used in newborns. BCG confers protection against disseminated tuberculosis (TB) during childhood, yet it fails to protect against pulmonary disease in adults. An improved BCG, combined with boosts targeting biologically relevant Mtb antigens, represents a reasonable strategy to augment, broaden and prolong immune protection against TB. An orchestrated cellular immune response of CD4+ [1] and CD8+ T cells [2,3,4] is required to effectively contain Mtb infection. Induction and expansion of antigen-specific, long-lived immune responses in CD4+ and CD8+ T cells present therefore worthy goals for vaccine development and for a rational boost strategy design. We combined a BCG prime followed by an adenovirus (Ad)delivered Ag85A, Ag85B [5] and TB10.4 [6] boost. rAd35 was choosen due to the prevalence of Ad5-specific immune responses in Africa [7], where the TB burden is high and novel vaccination strategies are needed. For example, the seroprevalence in subSaharan Africa patients infected with HIV-1 of Ad5 is 90% and 20% for anti-Ad35 reactivity [8]. rAd35 induces low levels of neutralizing antibodies in non-human primates (NHPs) [9], a valuable model for preclinical TB vaccine trials: NHPs are (...truncated)


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Isabelle Magalhaes, Donata R. Sizemore, Raija K. Ahmed, Stefanie Mueller, Lena Wehlin, Charles Scanga, Frank Weichold, Giulia Schirru, Maria Grazia Pau, Jaap Goudsmit, Sharon Kühlmann-Berenzon, Mats Spångberg, Jan Andersson, Hans Gaines, Rigmor Thorstensson, Yasir A. W. Skeiky, Jerry Sadoff, Markus Maeurer. 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, 11, DOI: 10.1371/journal.pone.0003790