1. A novel vaccine construct comprising of lipidated peptide protects against Mycobacterium tuberculosis by bolstering enduring memory T cell response Javed N Agrewala, PhD, FNA CSIR-Institute of Microbial Technology Chandigarh, INDIA javed@imtech.res.in IMTECH Vaccines-Hyd-2.11..15 115:21

2. Theme of the study To develop a unique and novel construct that can function as a vaccine in TB-endemic regions, where BCG has failed

3. Failure of BCG Despite nine decades of BCG vaccination, TB continues to be a major global health challenge. Clinical trials worldwide have proved the inadequacy of the BCG vaccine in preventing the manifestation of pulmonary TB in adults. Ironically, the efficacy of BCG is poorest in TB-endemic areas. Factors such as non-tuberculous or environmental mycobacteria and helminth infestation have been suggested to limit the efficacy of BCG. Hence, in high TB-burden regions, radically novel strategies of vaccination are urgently required. Gowthaman & Agrewala. Trends Mol Medicine 2012 315:21

4. Antigen presentation Recent series of studies have suggested that M. tuberculosis and environmental mycobacteria (EnM) actively inhibit bacterial antigen processing and presentation by MHC class I and class II pathways, thus slowing the emergence of protective adaptive immunity (Wolf et al. 2007; Soualhine et al. 2007; Gehring et al. 2003, Tobian et al, 2003). BCG and EnM specifically blocks the surface export of mature class II molecules on APCs (Soualhine et al. 2007). M. tuberculosis and EnM impairs in vivo antigen processing by DCs (Wolf et al. 2007). IMTECH Gowthaman & Agrewala. Crit Rev Microbiol. 2014 415:21

5. What ever may be the reason for BCG failure!  But the fact remains that more people have been vaccinated with BCG than any other vaccine.  Still tuberculosis continues to kill some 3 million people annually.  2 billion people worldwide are infected with M. tuberculosis. Thus, the protective efficacy of the BCG vaccine remains doubtful! IMTECH 515:21

6. Any vaccine that require minimum antigen processing will work in TB-endemic population 615:21

7. Peptides can overcome the problem of antigen processing. Since they can directly bind to MHC molecules and require minimum antigen processing. Can peptide based vaccine be successful choice in TB-endemic regions? 715:21

8. PEPTIDE VACCINES  Comprise of synthetic peptides that represent immunodominant epitopes  T cells and B cells epitope can be precisely selected  Epitopes are accurately defined; can avoid autoreactive portions in the antigen  Requires no or minimum processing Х Fails to elicit immune response in genetically diverse human population Х Requires adjuvant IMTECH 815:21

9. Th epitope 1: LFAAFPSFAGLRPT FDTRLM Th epitope 2: SEFAYGSFVRTVSLPVGADE -K- I S I S I Pam2Cys -K- I S I S I Pam2Cys -K- I S I S I Pam2Cys Schematic representation of the epitope-based vaccine candidate. The vaccine will contain Th and CTL epitopes. In all cases the T-helper (Th) epitope occupies N-terminal position and is separated from the cytotoxic T cell epitope (CTL) epitope by a single lysine (K) residue. Where lipid is attached, this is done through ε-amino group of the lysine residue such that the self-adjuvant lipid, linked through two serine residues (S), forms a branch between the Th and CTL epitope ThCTL Th CTL Gowthaman & Agrewala. Trends Mol Medicine 2012 915:21

10. Why peptide vaccine may be better than BCG? Can overcome problem of antigen processing due to EM/Mtb/BCG, since no processing is required. No fear of preformed immune response generated due to exposure to BCG/EM/Mtb. Selective epitopes, can avoid autoreactive/suppressive portions in the antigen. IMTECH 1015:21

11. Whether the construct influences the maturation of DC? F91: free peptide L91: lipidated peptide IMTECH 1115:21

12. Untreated F91 L91 355 T Cell Proliferation Unstimulated L91 Number of conjugates Gowthaman & Agrewala. J Infect Dis. 2011 12 15:21 L91 augments IL-12 production by DCs T cells exposed to L91 treated DCs secreted more IFN-γ IFN-γ (pg/ml) Medium Pam2Cys F91 L91

13. L91 Enhances Activation and Maturation of DCs IA d P91 L91 CD74 CD80 CD40 CD86 Medium LPS L91 P91 1315:21

14. Whether the peptides works across MHC barriers? 1415:21

15. Immunization L21, P21, L91, P91 Booster 7-14d In vitro challenge with peptides T cell response Sacrificed 240 days IMTECH [BALB/c, C57BL/6, C3He, FVB] Free peptides were emulsified in IFA. 1515:21

16. IMTECH Peptide works across MHC barriers BALB/c T cell proliferation C3HeFVB C57BL/6 Peptides (nmoles) L21 L91 P91 P21 1615:21

17. Control 4.1% Pam2Cys 4.2% F91 16% L91 52% 136 4 CD44 4 Control Pam2Cys F91 L91 A CD69 Control Pam2Cys F91 L91 CD69 + CD44 + CD4 T cells (Fold Change) B L91 induces proliferation and activation of CD4 T cells 17 15:21 T Cell Proliferation

18. A B C D IL-5 (pg/ml) IFN-γ (pg/ml) IL-4 (O.D) IL-10 (O.D) Control Pam2Cys F91 L91 n.s Control Pam2Cys F91 L91 Immunization with L91 elicits mainly secretion of IFN-γ L91 F91Medium Pam2Cys CD4 IFN-γ 8 8 10 25 A B Control Pam2Cys F91 L91 IFN-γ + CD4 T cells (Fold Change) 11 15 36 Control Pam2Cys F91 L91 IFN-γ TNF-α 18 15:21

19. 1.4 2.3 2.6 9.9 1.4 3.1 1.5 3.4 2.0 1.5 2.4 2.8 L91 immunization evokes IL-17 secretion Immunization L91 BCG Saline Medium L91 Pam2Cys F91 IL-17 CD4

20. 11 3 Control F91 L91 12 4 34 6 CD62L CD44 Central Memory Effector Memory B Untreated F91 L91 Fold induction 93130315 A B CD127 Control F91 L91 CD127 (Fold induction) L91 immunization engenders T cell memory 20 15:21

21. Experimental design for protection studies in mice. Different groups of BALB/c mice were immunized with L91 or controls (F91, LH, BCG and placebo). Peptides were given at a dose of 20 nmol (primary immunization) and 10 nmol (booster). BCG (10 6 CFU/animal) was given as control. Mice were rested for 75 days. Animals were challenged with a low dose aerosol of M. tb H37Rv (~100 CFU/mouse) 75 days post immunization. Thirty days after infection, animals were sacrificed and studied for immune response and pathology. L91 immunization engenders better protection than BCG in mice. Mtb load in lungs was enumerated by CFU plating. Results are depicted as bar graphs with mean ± SD (log 10 value). Data shown are from 3 independent experiments. '*' indicates p<0.05, '**' p<0.01, '***' p<0.001. L91 Vaccination Protects Mice Against Mtb 21 15:21

22. Untreated Pam2Cys L91 19 30 20 27 24 36 CD62L CD44 Untreated Pam2Cys F91 L91 274263488525 CD69 Untreated Pam2Cys L91 35 19 CD127 L91 evokes enduring memory CD4 T cell response in Mtb infected mice Central: CD44 hi /CD6L hi Effector: CD44 hi /CD62L lo 22 15:21

23. Placebo BCG F91 L91 24222632 CD4 TNF-α IFN-γ 5 8 6 15 Placebo BCG F91 L91 CD4 FoxP3 5964 Placebo BCG F91 L91 L91 immunization induces better lung immunity than BCG 1424 1175 2334 1056 Placebo BCG F91 L91 PD-1 23 15:21

24. L91 immunization engenders better protection than BCG in mice. Mtb load in lungs was enumerated by CFU plating. Results are depicted as bar graphs with mean ± SD (log 10 value). Data shown are from 3 independent experiments. '*' indicates p<0.05, '**' p<0.01, '***' p<0.001. Log 10 CFU Placebo BCG L91 F91 LH L91 immunization engenders better protection than BCG in mice 2415:21

25. 1 0 immunization L91 (20nmol) First dose of drug (INH 25mg/kg orally) Aerosol challenge (100 CFU) 4 wk2 wk CFUs: lungs, spleen 2 0 immunization L91 (10nmol) Second dose of drug (INH 25mg/kg orally) 12 wk A novel therapeutic strategy to reinforce the potency of drugs to kill Mycobacterium tuberculosis by concurrently bolstering host immunity by promiscuous peptide conjugated to TLR-2 agonist

26. Lungs (log 10 CFU/g) *** ** *** ns * * * * * * * ns INH+RFP in drinking water INH orally with two dose Lungs (log 10 CFU/g)

27. Lungs IFN- γ (ng/ml) ** *** Spleen IFN- γ (ng/ml) *** Lungs IL-17 (ng/ml) *** ** *** Spleen IL-17 (ng/ml) ***

28. 2815:21 ** * * Spleen IFN-γ + TNF-α + CD4 T cells (%) ** * Lungs IFN-γ + TNF-α + CD4 T cells (%) ** Spleen IFN-γ + IL-17 + CD4 T cells (%) * * * * Lungs IFN-γ + IL-17 + CD4 T cells (%)

29. IFNγ + CD4 T Cells (%) *** ns 2915:21 PBMCs of TB patients on drug therapy on in vitro stimulation with L91 induces secretion of IFN-γ IL-17A + CD4 T Cells (%) ns ***

30. CD8 IFN-γ CD4 IFN-γ IFN-γ + CD8 T Cells (%) IFN-γ + CD4 T Cells (%) *** ** * * * Medium F4.8 Pam2Cys L4.8 2.8±0.13.3±0.3 2.8±0.1 10.6±1.6 3.9±0.16.6±0.3 9.0±0.2 14±0.1 3015:21

31. IMTECH L91 expands central memory pool of CD4 T cells of TB patients 3.6±1.54 n= 14 4.4±2.64 n= 13 13±3.2 n= 14 3±2.3 n= 3 CD45RA CD45RO Untreated 16 kDa F91 L91 3115:21

32. Experimental design for the protection studies in Guinea pigs. Different groups of Duncan-Hartley Guinea pigs were immunized with L91 or controls (F91, LH, BCG and placebo). Peptides were given at a dose of 100 nmol (primary immunization) and 50 nmol (booster). BCG (10 6 CFU/ animal) was given as a control. Animals were rested for 75 days and challenged with a low dose aerosol of M. tb H37Rv (~30 CFU/animal), 75 days post immunization. Thirty days after infection, animals were sacrificed and studied for bacterial burden and pathology. L91 Protects Guinea Pigs from Mtb better than BCG 0.0178 0.0004 0.0316 0.0042 CFU Load of Lung 3215:21

33. Placebo L91 BCG F91 LH L91 immunized Guinea pigs exhibit constrained pathology upon M. tb challenge in Guinea pigs. The protection studies were performed as described in Fig. 3.4. Representative photomicrographs of formalin fixed H & E stained histopathology lung sections of Guinea pigs (left and center panel). Arrows indicate regions of granulomas or necrosis. Right panel shows photographs of gross pathology in lungs of immunized and infected animals. Data are representative of 2 comparable experiments with a minimum of 5 animals per group. L91 immunized exhibit constrained pathology upon Mtb challenge in Guinea pigs 3315:21

34. TLR2 MHC II MHC I CM MHC II MHC I CM CD8 CD4 TLR2 Peptide1 Pam2Cys Peptide2 Linker Pathogen IL-6, IL-12, IFN- γ IFN-Y, TNF α, IL-17, IL-2 Cytokine Receptor CM: Costimulatory molecule Gowthaman & Agrewala. Trends Mol Medicine 2012

35. CONCLUSION Lipidated peptide vaccine may be a future prophylactic strategy to eradicate TB from TB-endemic zones 3515:21

36. IMTECH, CSIR and DBT for Financial Support Juraj Ivanyi, TB & RI Unit, Hammersmith Hospital, London David Jackson, University of Melbourne, Australia Pushpa Gupta and UD Gupta, JALMA, India AK Janmeja, Government Medical College and Hospital, India 3615:21

37. 3715:21

38. Fails to elicit immune response in genetically diverse human population! Answer: Promiscuous Peptides? IMTECH 3815:21

39. Promiscuous Peptide Selection We generated 141 over lapping peptides from: 16kDa, 19kDa, 30kDa, 38kDa, 65kDa, CFP-10, ESAT-6 All the individuals generated robust T cell proliferation and predominant secretion of IFN-  by PBMCs of the PPD + healthy individuals against 16 kDa (Acr1) antigen of Mtb. IMTECH 1 20 MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRA ELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADE DDIKATYDKGILTVSVAVSEGKPTEKHIQI RSTN (141 AAs) 3915:21

40. Identification of T-Helper Cell Activating Promiscuous Peptides Buffy coat of 20 PPD + healthy volunteers with no history of TB were selected and tested using promiscuous peptides for: (i) Peptides binding to HLA-class II molecules (ii) T cell proliferation (iii) Secretion of IFN-  Agrewala et al. 1997, 1998, 1999 4015:21

41. Peptides of sequence 21-40 and 91-110 showed permissive binding IMTECH HLA DRB1Binding Affinity p21-40 Binding Affinity p91-110 0101+++++++ 0103++++++ 0301+++ 0401+++++++ 0701++++++ 1101+++++++ 1301+++++ 1501++++ 1601+++++++ The HLA type of donors was performed by low resolution PCR with sequence-specific primers (PCR-SSP). All presenting HLA-DR-homozygous L-BCL were chosen to conform at least to the donor’s major serological, and usually to molecular, type. The affinity of the presenting MHC class II molecule for peptides p21-40 and p91- 110 was determined by a competitive MHC binding assay. ++++ indicates high-affinity binding (IC50<10µM); +++ intermediate affinity (10 µM<IC 50 <100µM); and ++ indicates low affinity (100–1000µM). Agrewala et al. 1997, 1998, 1999 4115:21

42. Peptides of sequence 21-40 (p21-40) and 91-110 (p91-110) showed T cell proliferation in all the tested individuals. p91-110 induces mainly secretion of IFN- . Th1 clones (IFN-  ): 90% Th0 clones (IFN-  +IL-4): 10% p21-40 promotes production of IFN-  and IL-4. Th0 clones (IFN-  + IL-4): 75% Th2 clones (IL-4): 25% p21-40: LFAAFPSFAGLRPTFDTRLM p91-110: SEFAYGSFVRTVSLPVGADE Agrewala et al. 1997, 1998, 1999 IMTECH 4215:21

43. Peptides are weak immunogens! Can conjugation with Pam2Cys increase the immunogenecity of peptides? 4315:21

44. Why Pam2Cys? S-[2,3-is(palmitoyloxy)propyl]cysteine Ligand for TLR2, induces signaling through it. Has self-adjuvanting properties when conjugated with peptides. Induces robust Th1 and Th17 type immune responses. Long-term protective effect. Potent immunogenicity can be explained by its ability to mature and activate dendritic cells (suggesting that vaccines containing this lipid moiety may interact directly with DCs to promote immune responses). Jackson et al. 2004, Zhu et al. 2004, Düesberg et al. 2002, Zeng et al. 2002 IMTECH 4415:21

45. Why TLR2? Incorporating the Pam2Cys into peptide structures effectively triggers the TLR2 and secretion of IL-12 by DCs. TLR2 is copiously present on DCs, monocytes, and lung epithelia. Jackson et al. 2004, Zhu et al. 2004, Düesberg et al. 2002, Zeng et al. 2002 IMTECH 4515:21

46. Why DCs? DCs are decisive for efficient immune response. Only cells, capable of activating naïve T cells. Skews immune response towards Th1 and Th17 cells. Constitutive expression of MHCs and costimulatory molecules. IMTECH 4615:21

47. Advantage of Vaccine Totally synthetic No adjuvant is required Can activate CD4 and CD8 T cells Skews immune response to Th1 and Th17 type Can activate naïve T cells IMTECH 4715:21