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Antigen Discovery and TB vaccines

Enzymes of the sulfate assimilation pathway induced during intracellular growth are novel protective antigens of Mycobacterium tuberculosis. Warwick Britton, Lisa Leotta , Erin Shanahan, Nick West, Tom Leyh , Rachel Pinto, Jamie Triccas . Centenary Institute &

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Antigen Discovery and TB vaccines

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  1. Enzymes of the sulfate assimilation pathway induced during intracellular growth are novel protective antigens of Mycobacterium tuberculosis. Warwick Britton, Lisa Leotta, Erin Shanahan, Nick West, Tom Leyh, Rachel Pinto, Jamie Triccas. Centenary Institute & Deptof Infectious Diseases & Immunology University of Sydney

  2. Antigen Discovery and TB vaccines Screen for M. tb genes expressed in host cells Clone genes, express proteins, test vaccine potential in animal models Tests recognition of novel antigens by TB patients

  3. Sulphur Assimilation Pathway cytoplasm Sulpholipids cysDNC CysDNC subI Sulphate Sulphate PAPS APS cysTWA (ATP sulfurylase/Kinase) (APS reductase) Serine Cell membrane CysE (Serine Acetyl transferase) CysH O-Acetyl-L-serine (Sulfite Reductase) Sulphite Sulphide CysK1 SirA (O-Acetly-L-Serine Proteins Fe-Sulphur clusters Mycothiol Coenzymes Methionine Pinto R, Triccas J et al, JID, 2013 Sulfhydralase/OASS) Cysteine

  4. M. tuberculosis Sulphate Activation Complex (SAC) Sulphate ATP Sulphurylase (CysDN) CysD CysNC The ‘SAC’ CysNC CysD APS PAPS APS kinase (CysC) Cys, Met (proteins) Triccas Lab (USyd) Leyh lab (AECOM, New York) Pinto et al, Microbiology 2004 Pinto et al, J. Bact 2007 Sun et al, JBC, 2005

  5. A B 6 5 cysC cysD cysN CysDNC are expressed as an operon during intracellular growth of M. tuberculosis & in vivo infection APS Kinase ATP sulfurylase GTPase 4 Relative CysDNC expression 3 2 1 C D 0 in vivo in culture Relative CysDNC expression 8 6 Det No Det CFU/mL (x107) 4 2 0 Pinto R, Triccas J et al, JID, 2013 Days

  6. 100 80 60 40 20 0 * * 200 160 120 80 40 0 Ag85B CysDNC Unstimulated Protein CysDNC stimulates T cell response in Mtb-infectedmice and in TB patients A * * Number of INF- producing cells per 105 MLN B C * * * Pinto R, Triccas J et al, JID, 2013 Stimulation Index Ag85B CysDNC CFP Protein

  7. DNA vaccines expressing CysD & CysNC induce protective immunity against Mtb infection A. Lung * 6.5 Log10CFU/Organ 6.0 5.5 5.0 4.5 4.0 cysD +cysNC cysD cysNC BCG B. Spleen pCDNA3 * 5.0 Groups 4.5 4.0 3.5 3.0 2.5 2.0 cysD +cysNC cysD cysNC BCG Pinto R, Triccas J et al, JID, 2013 pCDNA3

  8. Sulphur Assimilation Pathway cytoplasm Sulpholipids cysDNC CysDNC subI Sulphate Sulphate PAPS APS cysTWA (ATP sulfurylase/Kinase) (APS reductase) Serine Cell membrane CysE (Serine Acetyl transferase) CysH O-Acetyl-L-serine (Sulfite Reductase) Sulphite Sulphide CysK1 SirA Proteins Fe-Sulfur clusters Mycothiol Coenzymes Methionine (O-Acetly-L-Serine Pinto R, Triccas J et al, JID, 2013 Sulfhydralase/OASS) Cysteine

  9. A. mRNA in infected Mp CysH, SirA, CysK1 & CysE are expressed in Mtb-infected macrophages & stimulate T cell responses in mice 8 * * B. T cell responses during Mtb infection * 7 200 * 6 160 5 120 Relative expression 4 80 3 40 Number of INF- producing cells per 105 MLN 2 0 1 SirA CysE CysH CysK1 0 unstimulated SirA CysE CysH CysK1 Protein Transcript Pinto R, Triccas J et al, JID, 2013

  10. CysH, SirA, CysK1 & CysE elicit T cell responses in Tuberculosis patients * * * * * Stimulation Index Pinto R, Triccas J et al, JID, 2013 CysE CysK1 CysH SirA CFP Protein

  11. CysD & CysN proteins in MPL/DDA induce protective immunity against Mtb infection * * * 5.5 5.0   4.5 Log10 CFU/Organ B. Spleen A. Lung 4.0 3.5 3.0 * * * 5.1 4.6   4.1 3.6 3.1 2.6 BCG BCG pCDNA3 pCDNA3 cysD+cysN cysD+cysN DNA-SAP mix DNA-SAP mix Groups Pinto R, Triccas J et al, JID, 2013

  12. CysD & CysN proteins in MPL/DDA boost BCG-induced protective immunity against Mtb infection * * 6.5 B. Spleen A. Lung 6.0 * * ** 5.5 6.0 5.0 Log10 CFU/Organ 5.5 4.5 5.0 NS 4.5 4.0 4.0 3.5 3.5 3.0 2.5 BCG/ MPL-DDA BCG/ MPL-DDA unvacc unvacc BCG/ CysDN prot BCG/ CysDN prot Groups Pinto R, Triccas J et al, JID, 2013

  13. Components of SAP as TB vaccines • Enzymes of sulphur assimilation pathway are induced during intracellular growth of Mtb • Proteins of sulphur assimilation pathway are potent T cell antigens during human and murine TB infection • CysD and CysN as DNA and protein vaccines induce protective immunity against Mtbinfection • CysD and CysN as protein vaccines boost BCG-induced protective immunity against Mtbinfection CysD & CysN are strong candidate antigens for TB subunit vaccines to boost BCG

  14. Acknowledgments University of Sydney Infectious Disease & Immunology Jamie Triccas Rachel Pinto Lisa Leotta AECOM, New York Tom Leyh Centenary Institute Mycobacterial Research Warwick Britton Nick West Erin Shanahan Funding: NHMRC, Uni Sydney ARC

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