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Pradeep K Rai CSIR-IMTECH, Chandigarh

Future peptide vaccine for TB endemic regions: Challenges and solutions. MHC I. CD4 CD8. MHC II. Pam2Cys. TLR2. DC. CM. MHC I. CM. TLR2. TLR2. CM. CM. Pradeep K Rai CSIR-IMTECH, Chandigarh. Epidemiology of Tuberculosis. Previously treated 85%.

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Pradeep K Rai CSIR-IMTECH, Chandigarh

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  1. Future peptide vaccine for TB endemic regions: Challenges and solutions MHC I CD4 CD8 MHC II Pam2Cys TLR2 DC CM MHC I CM TLR2 TLR2 CM CM Pradeep K Rai CSIR-IMTECH, Chandigarh

  2. Epidemiology of Tuberculosis Previously treated 85% Best Prophylactic measure Drugs Total no. of deaths 1.5 million MDR: Multi drug resistant Total no. of new TB cases 9 million New cases 15% Vaccine DOTs Treatment BCG Side effects 480,000 Emergence of drug resistant BCG Failure WHO Global tuberculosis report , 2014

  3. Reasons for BCG failure Lack of enduring immunologic memory (Andersen and Doherty 2005, Narayanan 2006, Orme 2010, Singh et al 2010, 2011) Interference by environmental mycobacteria (Masking and Blocking hypotheses) (Andersen and Doherty 2005, Verma and Grover 2009, Flaherty et al 2006, Young et al 2007, Brant et al 2002) Antigen processing and presentation- Blocking phagosome maturation and export of major histocompatibility complex (MHC) molecules (Soualhine et al 2007, Pancholi et al 1993, Sendide et al 2005, Sun et al 2007, Fulton et al 2004, Sendide et al 2004) Helminth infections (Malhotra et al 1999, Elias et al 2001, 2005, Black et al 2002, Rook et al 2004) Regulatory T cells (Hanekom 2005, Ahrens et al 2009, Rook and Kim 2006, Akkoc et al 2010)

  4. Limitations of DOTs Time consuming (3-12 months), so chances of intermittent therapy increases, resulting in increased risk of drug resistance. Side Effects. No cure for latent infection. Urgent need to develop: A potent construct with two edge sword (Prophylactic and Therapeutic potential)

  5. Future TB vaccine: Epitope based vaccine • Minimal antigen processing • Minimal presence of mycobacterial components (except antigenic portions) • Robust Th1 response (even in a Th2 bias environment) • Enduring T cell memory • No preformed antibodies in the population • Vaccine candidate with both prophylactic and therapeutic • properties ХPoor immunogenicity ХBinds to very few HLA-alleles

  6. Which peptide to choose? Is promiscuous peptide right choice? ++++ indicates high-affinity binding (IC50<10µM); +++ intermediate affinity (10 µM<IC50<100µM); and ++ indicates low affinity (100–1000µM). Agrewala and Wilkinson 1997, 1998, 1999

  7. Can coupling promiscuous peptides to Pam2Cys render them immunogenic? Pam2Cys S-[2,3-is(palmitoyloxy)propyl]cysteine induces effective Th1 immune responses, by evoking DCs to secrete IL-12 (Thoma et al 2000, Jackson et al 2004, Zho et al 2004, Ghosh et al 2006) Dendritic cells copiously express TLR2 so Pam2Cys (TLR2 agonist) can be used as vaccine delivery module (Jackson et al 2004, Zho et al 2004) Pam2Cys has ability to mature and activate DCs (Hertz et al 2001, Jackson et al 2004, Zho et al 2004) Safe for Human use (Zeng et al 2002) (91-110) SEFAYGSFVRTVSLPVGADE -K I S I S I Pam2Cys L91

  8. Mechanism of L91 CD4 CD4 APC Pam2Cys MHC II CM MHC I TLR2 IFNγ , IL-17, TNFα CM CM MΦ CM MHC II CM CM Induction of MHC expression Induction of microbicidal activity NOS2 generation MHC II MHC I CM

  9. L91 confers better protection than BCG in Guinea pig model of tuberculosis Placebo L91 BCG F91 LH

  10. Whether L91 has therapeutic potential?

  11. L91 efficiently decreases bacterial burden in the lungs and spleen in conjunction with anti-TB drugs *** *** *** *** L91 L91 ** CFU count T cell response Mtb infection ** *** ns INH+RIF *** *** ns Lungs (log10 CFU/g) *** Spleen (log10 CFU/g) 0 4 6 8 20 weeks

  12. L91 immunization reduces Mtb induced lung pathology 20X 100X Placebo L91 Drug Drug-L91

  13. L91 has enough therapeutic potential to confine bacterial growth in lungs Further, L91 restricts dissemination of bacteria in spleen

  14. Mechanism of protection?

  15. L91 stimulation elicits innate immunity FB US F91 Pam L91 LPS NF-kB F91 (0.01nmol) Pam (0.1nmol) Pam (1nmol) L91 (0.01nmol) F91 (1nmol) F91 (0.1nmol) Pam (0.01nmol) L91 (0.1nmol) L91 (1nmol) LPS UI US iNOS ACTIN

  16. L91 immunization elicits secretion of protective cytokines against Mtb Single cell suspension Lungs ** In vitro stimulation with L91 *** *** *** ** 72h IL-1β (S.I) IL-17A (S.I) IFNγ (S.I) T cell response ** ns

  17. L91 immunization induces generation of multifunctional CD4 T cells * * ** * IL-17+IFN-γ+ CD4 T cells (%) IFN-γ+ TNF-α + CD4 T cells (%)

  18. L91 immunization generates specific subtype of Th17 cells * *** CXCR3+ CCR6+ IFN-γ+ IL17A+ CD4 T Cells (%)

  19. ns ** * * ** L91 immunization rescues CD4 T cells from exhaustion PD-1 (iMFI)

  20. L91 immunization engender long lasting memory CD4 T cells ** ** ** * CD127+ CD4 T cells (%) CD44hi CD62Lhi CD4 T cells (%)

  21. L91 immunization eradicate bacteria by eliciting strong Th1 and Th17 response L91 immunization generates unique subset of IFN-γ+IL-17+ polyfunctional Th17 cells L91 engenders memory generation

  22. Whether immunotherapy with L91 elicits immune response in TB patients?

  23. Lipidated peptide exhibits T cell proliferation in PBMCs isolated from TB patients and their close contacts 96h PBMCs (TB patients) + L91 + IL-2 Immune response **** T cell proliferation (Stimulation index) n= 53 (TB patients)

  24. L91 stimulation elicits IFN-γ secretion by the CD4 T cells of TB patients Medium Pam2Cys L91 3.6±0.6 5.2±0.8 9.7±1.5 IFNγ CD4 *** ** CD4+IFNγ+ T cells (%) ns

  25. L91 stimulation induces IL-17 secretion by the CD4 T cells of TB patients Medium Pam2Cys L91 4.9±0.4 12.5±1.2 5.7±1.6 *** IL-17 ** CD4 CD4+ IL-17+ T cells (%) ns

  26. L91 stimulation generates polyfunctional Th17 cells Medium Pam2Cys L91 2.8±0.4 (n=14) 3.7±0.6 (n=12) 8.3±1.2 (n=14) IFN-γ IL-17A *** ** IFN-γ +IL-17A+ CD4 T cells (%) ns

  27. L91 expands the pool of memory T cell Medium Pam2Cys L91 7.7±0.8 (n=21) 4±0.4 (n=15) 3.8±0.4 (n=28) CD45RO CD45RA *** ** CD45RA +CD45RO+ CD4 T cells (%) ns

  28. F91 specific antibodies are absent in TB endemic population

  29. Conclusions • Promiscuous lipidated peptide can elicit robust Th1 and Th17 response in human PBMCs isolated from TB patients and their closed contacts • L91 immunization induces unique subset of co-expressing CCR6 and CXCR3 Th17 cells, which secrets both IFN-γ and IL-17 • No anti-peptide antibodies in population so L91 has great potential to be a future vaccine candidate for TB endemic area • Immunotherapy with L91 may reduces risk of generation of drug resistant Mtb

  30. Acknowledgment Dr. Javed N Agrewala Professor David C Jackson Professor Ashok K Janmeja CSIR DBT Thanks

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