Crossplatform Tuberculosis Vaccine and Diagnostics Development in Humans, Livestock, and Wildlife Michelle H. Larsen, PhD Assistant Professor, Department of Medicine Albert Einstein College of Medicine, Bronx, NY National Animal Disease Center 14 September 2008 Mycobacteria Research at EINSTEIN Jacqueline Achkar (M&I) Serologic diagnostic for MTB infection John Blanchard (Biochem) TB drug mechanisms and development James Brust (Medicine) Community based MDR-TB/HIV treatment Arturo Casadavell (M&I) Antibody responses to MTB infection John Chan (M&I) Latent MTB infections; Role of B-cells Glenn Fennelly (Peds) HIV/TB vaccine development Neel Gandhi (Medicine) Clinical epidemiology of MDR/XDR-TB Harris Goldstein (M&I) HIV and MTB immolonogy responses Bill Jacobs (M&I) MTB genetics, diagnostics, drugs, vaccines Michelle Larsen (Medicine) TB/HIV vaccines, biomarkers, XDR-TB Max O’Donnell (Medicine) Treatment outcomes for XDR-TB patients Steve Porcelli (M&I) TB immunology responses Sarita Shah (Medicine) Clinical epidemiology of MDR/XDR-TB Human Tuberculosis ~ Global Considerations • Spread by aerosol route • One third of world’s population is infected • 4,000+ deaths every day from TB • 8-10 million new infections every year • Increasing incidence of TB in HIV-endemic areas • Leading attributable cause of death in HIV infected individuals • Worldwide emergence of drug-resistant TB (MDR-TB & XDR-TB) WHO-Declared Global Health Emergency White Plague Consumption King Tutankhamen John Keats Elizabeth Barrett Browning Edgar Allen Poe Frederic Chopin Ralph Waldo Emerson Emily Bronte Robert Louis Stevenson Eleanor Roosevelt George Orwell Vivien Leigh Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) Tuberculosis Vaccines Considerations • Safety Efficacy Cost Diagnostics compatibility Feasibility Environmental persistence Bridge studies to target populations Types of TB Vaccines • Subunit (protein, protein fusions) Subunit (viral vector; DNA) Live attenuated mycobacteria M. bovis derivatives M. tuberculosis deritives M. smegmatis derivatives Human Tuberculosis Natural History Infection Initial containment – 95% Late Progression - 5% Self-Cure – 90% Early Progression - 5% Human Tuberculosis Natural History Infection Initial containment – 95% Late Progression - 5% Self-Cure – 90% Early Progression - 5% Goals of Vaccination • Reduce/interrupt transmission Reduce disease manifestation Prevent secondary infection Sterilization/Clearance Prevent primary infection Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) Vaccination: M. bovis BCG Bacille Calmette Guerin (A Live Attenuated M. bovis and TB Vaccine) A mutant isolated from bovine tubercle bacillus follow 39 3-week passages (1904 -1908) in bile glycerinated medium 1921 – Newborn Boy Vaccinated for TB 1928 – Adopted as TB Vaccine by the League of Nations Over 3 billion doses administered to humans (most widely used human vaccine in the world) Efficacy Studies 0 to 80% Protection Not safe in immunocompromised individuals Inexpensive to produce Calmette and Guerin 36 Point Subtitle Pediatric vaccines.tiff RD1 region.tiff picrender From Brosch et al PNAS 104:5596-5601 (2007) M. tuberculosis and Attenuation of M. bovis Virulent M. bovis BCG Attenuate (∆RD1, RDs) Virulent M. bovis BCG Attenuate (∆RD1, RDs) Overexpress antigens rBCG30 Virulent M. bovis BCG Attenuate (∆RD1, RDs) Overexpress antigens rBCG30 Virulent M. tuberculosis mc26020 (∆lysA ∆panCD) Non-replicating mc26030 (∆RD1 ∆panCD) Limited Rep. Attenuate Hypothesis: A Mycobacterium tuberculosis derived vaccine can protect better against TB infection than an M. bovis (BCG) strain. RD1 First Generation TB Vaccine Candidates 393537aa panCD lysA mc26020 lysA panCD (non-replicating) mc26030 RD1 panCD (replicating) Derivatives of M. tuberculosis H37Rv Strain Construction & Characterization • Developmental History • BSE Exposure • Media requirements • Identity (PCR/Southern) • in vitro growth kinetics • Morphology • Survival (SCID) • Growth Kinetics (C57Bl/6) • Pathology (C57Bl/6) Safety (Mice) • Bacterial Burden • Pathology • Post-challenge survival Protection (Mice) • Bacterial Burden • Pathology • Survival Safety & Protection (Guinea Pigs) Freedom from Virulent Mycobacteria (Guinea Pig) Intradermal Skin Test (Guinea Pig) • Lot Release Sterility Purity Potency (CFU) Identity (PCR) GMP Seed Lot • Lot Release Sterility Purity Potency Identity Safety GMP Clinical Lot Modified General Safety Test (Mice) 28-day Toxicity (Guinea Pigs) Repeat Repeat Repeat Safety & Protection (Non-human Primates) Pre-Clinical Development Phase I Studies submain-img-su-1.jpg 00026DB3 Eeyore IV BEAE2E52: gp.jpg 00026DB3 Eeyore IV BEAE2E52: in_c57bl.jpg 00026DB3 Eeyore IV BEAE2E52: Pre-clinical animal models for human TB vaccines submain-img-su-1.jpg 00026DB3 Eeyore IV BEAE2E52: gp.jpg 00026DB3 Eeyore IV BEAE2E52: Holstein Calf in Pasture.jpg 00082A8B Eeyore IV BEAE2E52: in_c57bl.jpg 00026DB3 Eeyore IV BEAE2E52: Pre-clinical animal models for human TB vaccines First Generation Pre-clinical studies mc26020 lysA∆panCD mc26030 RD1 panCD Mice - Safety + + Mice – Efficacy + + Guinea pig – Safety (FVA) + + Guinea pig - Efficacy + +/- Non-human primate – Safety + + Non-human primate -- Efficacy +/- - Bovine (neonates) – Safety ND + Bovine (neonates) – Efficacy ND - Non-human primate (neonates; SIV+) -- Safety + ND Feline (FIV+) -- Safety + ND Conclusions: (1) mc26020 and mc26030 are safe, (2) Efficacy varies First Generation Pre-clinical studies mc26020 lysA∆panCD mc26030 RD1 panCD Mice - Safety + + Mice – Efficacy + + Guinea pig – Safety (FVA) + + Guinea pig - Efficacy + +/- Non-human primate – Safety + + Non-human primate -- Efficacy +/- - Bovine (neonates) – Safety ND + Bovine (neonates) – Efficacy ND - Non-human primate (neonates; SIV+) -- Safety + ND Feline (FIV+) -- Safety + ND Conclusions: (1) mc26020 and mc26030 are safe, (2) Efficacy varies 6030 cattle title.tiff First Generation Pre-clinical studies mc26020 lysA∆panCD mc26030 RD1 panCD Mice - Safety + + Mice – Efficacy + + Guinea pig – Safety (FVA) + + Guinea pig - Efficacy + +/- Non-human primate – Safety + + Non-human primate -- Efficacy +/- - Bovine (neonates) – Safety ND + Bovine (neonates) – Efficacy ND - Non-human primate (neonates; SIV+) -- Safety + ND Feline (FIV+) -- Safety + ND Conclusions: (1) mc26020 and mc26030 are safe, (2) Efficacy varies Virulent M. bovis BCG Attenuate (∆RD1, RDs) Overexpress antigens rBCG30 Virulent M. tuberculosis MTBVAC (∆phoP) mc26020 (∆lysA ∆panCD) Non-replicating mc26030 (∆RD1 ∆panCD) Limited Rep. Attenuate Unmarking; Safety mc26206 (∆leuCD ∆panCD) Non-replicating Safety of mc26206 ∆leuCD ∆panCD in SCID mice 105 CFU for mc26206 104 CFU for BCG IV injection JacobsFig1 MHC I Mycobacteria Immune Evasion JacobsFig1 MHC I Mycobacteria Immune Evasion Apoptosis Apoptosis 2 DIC GFP-bacteria TUNEL Quantitation (FACS: TUNEL+) Human THP-1 cells (macrophage cell line); day 1 after infection; MOI 10:1 Blocking the Infection-Induced Suicide Response: M. bovis BCG Inhibits Apoptosis Pro-apoptotic phenotype of mc26208 (∆leuCD ∆panCD ∆secA2) DAY 3: mc26208 MOI 3 DAY 3: mc26206 MOI 3 DAY 3: mc26206 MOI 10 DAY 3: mc26208 MOI 10 Hinchey et al., 2011 Apoptotic assay in THP1 cells CONFIDENTIAL – Unpublished data Uma Ranganathan, Michelle Larsen, Glenn Fennelly, Bill Jacobs Protective immunity against virulent M. tuberculosis challenge in mice following vaccination with mc25222 (∆secA2 ∆lysA) * Ф Ф Ф * * Ф Ф ** Ф Ф Ф Ф ** vaccination with mc25222 (∆secA2 ∆lysA) * Ф Ф Ф * * Ф Ф ** Ф Ф Ф Ф ** JacobsFig1 MHC I Mycobacteria Immune Evasion 24 h Re-screen by FACS analysis M. tuberculosis H37Rv transposon library M IL-12-GFP UT LPS wildtype mmaA4 mmaA4 identified from a high-throughput screen for M. tuberculosis mutants that induce IL12p40 production Loss of methoxy and keto mycolic acids in mmaA4 mutant changes colonial morphology wild type  mmaA4 wild type  mmaA4.c Dao, D. N., K. Sweeney, T. Hsu, S. S. Gurcha, I. P. Nascimento, D. Roshevsky, G. S. Besra, J. Chan, S. A. Porcelli, and W. R. Jacobs. 2008. Mycolic acid modification by the mmaA4 gene of M. tuberculosis modulates IL-12 production. PLoS Pathog 4:e1000081. mycolic acids The mmaA2 Gene of Mycobacterium tuberculosis Encodes the Distal Cyclopropane Synthase of the   alpha -Mycolic Acid* Michael S. Glickman      alpha Dagger Loss of oxygenated mycolic acid enhances vaccine efficacy by promoting greater CD4 T-cell proliferation H37Rv ∆mmaA4 (-) ctrl (+) ctrl Lymphnode Day 4 Virulent M. bovis BCG Attenuate (∆RD1, RDs) Overexpress antigens rBCG30 Endosome escape (listeriolysin; perfringoslysin) rBCG ∆ureC::Hly Attenuate rBCG(mbtB)30 Limited Replication Virulent M. tuberculosis MTBVAC (∆phoP) mc26020 (∆lysA ∆panCD) Non-replicating ∆leuCD ∆panCD ∆secA2 Non-replicating Pro-apoptotic ∆leuCD ∆panCD ∆mmaA4 Non-replicating IL-12 modulation AERAS-407 mc26030 (∆RD1 ∆panCD) Limited Rep. ∆RD1 ∆panCD ∆secA2 Limited Rep Pro-apoptotic ∆RD1 ∆panCD ∆mmaA4 Limited Rep IL-12 modulation Attenuate Overcome immune evasion M. tuberculosis TB Vaccine Candidates First Generation: mc26020 ∆lysA::gdres ∆panCD::gdres-hyg-gdres * # mc26030 ∆RD1::gfp ∆panCD::gdres-hyg-gdres * mc27000 ∆RD1::gfp ∆panCD::gdres * Second Generation: mc26220 ∆lysA ∆panCD mc26230 ∆RD1::gdres ∆panCD::gdres * # mc26206 ∆leuCD ∆panCD * # Third Generation: mc25222 ∆secA2 ∆lysA::gd res hyg sacB gd res mc26207 ∆leuCD ∆panCD ∆mmaA4::gd res hyg sacB gd res * mc26208 ∆leuCD ∆panCD ∆secA2::gd res hyg sacB gd res * # * Reclassified for BSL2 containment # Additional safety studies in SIV-infected neonatal macaques 36 Point Subtitle Pediatric vaccines.tiff Model species comparisons for human TB vaccine development Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) MTBC phylogeny.tiff Mycobacterium tuberculosis Complex Phylogeny picrender phoP Auxotrophs ∆RD1 Immune Evasion Antigen overexpression Auxotrophs Endosome escape Improved M. bovis and M. tuberculosis Vaccine Platforms Virulent M. bovis BCG Attenuate Overexpress antigens rBCG30 Endosome escape (listeriolysin; perfringoslysin) rBCG ∆ureC::Hly Attenuate rBCG(mbtB)30 Limited Replication Ravenel ∆RD1 AERAS-407 AFN212297 ∆RD1 Virulent M. bovis BCG Attenuate Overexpress antigens rBCG30 Endosome escape (listeriolysin; perfringoslysin) rBCG ∆ureC::Hly Attenuate rBCG(mbtB)30 Limited Replication Ravenel ∆RD1 AERAS-407 AFN212297 ∆RD1 Safety of ∆RD1 strains in SCID mice Intravenous infection with 2 x 106 CFU Neonatal Bovine Study Day 0 Day 190 • M. bovis ∆RD1 1X106 CFU ID n =7 • BCG-Danish 1 X106 CFU ID n=7 • Saline n=7 Challenge with 1000 CFU M. bovis 95-1315 Day 62 Necropsy ---------------Safety--------------- Study Design • Vaccinate Necropsy ---------------Safety--------------- Study Design • Vaccinate 117, 152, 170, and 189 • Comparative Cervical Test (PPD) 7 days prior to necropsy • Analysis after necropsy: -- examined for gross lesions -- samples collected for culture and histopathology -- lung lobes radiographed -- quantitative bacteriology of tracheobronchcial lymph nodes -- lung and lung-associated lymph nodes weighed and scored for lesions • Evaluation of cellular responses Ray Waters and Mitch Palmer, Tyler Thacker, USDA (Iowa), Mark Estes, UTMB (Galveston) ------------------------------- Challenge------------------------------- Gross pathology following challenge with M. bovis 95-1315 Mediastinal ln Tracheobronchial ln Lung Disease Score * * * * * * BCG and M. bovis Ravenel ∆RD1 have reduced pathology Lung bacterial burden following challenge with M. bovis 95-1315 BCG and M. bovis Ravenel ∆RD1 have reduced bacterial burden Non-vaccinated BCG ∆RD1 cfu / g (log10) * * Ravenel RD1 paper.tiff Vaccine 27 (2009) 1201–1209 Includes whole genome seqeuncing of M. bovis Ravenel Comparision to M. bovis AF2122/97 (sequenced by Hewinson, et al) 115 of the 233 SNPs that resulted in differences in the proteins coded for by annotated genes were analyzed. Of the 115 SNPs characterized, nearly half of the M. bovis Ravenel sequences had the consensus residue for the M. tuberculosis (MTB) complex. M. bovis Ravenel is less diverged from the ancestral strain than M. bovis AF2122/97. Among the 54 SNPs between M. bovis AF2122/97 and M. bovis Ravenel in which Ravenel codes for the MTB complex consensus residue were differences in genes involved in carbohydrate utilization, including aceE, glpK, pflA, pykA, and ugpB. These changes may allow M. bovis Ravenel to grow on glycerinated media in the absence of pyruvate, as is true of M. bovis BCG and M. tuberculosis strains but not of M. bovis AF2122/9 Virulent M. bovis BCG Attenuate Overexpress antigens rBCG30 Endosome escape (listeriolysin; perfringoslysin) rBCG ∆ureC::Hly Attenuate rBCG(mbtB)30 Limited Replication Ravenel ∆RD1 95-1315 ∆RD1 AERAS-407 Overcome immune evasion AF2122297 ∆RD1 Ravenel ∆RD1 ∆panCD 95-1315 ∆RD1 ∆panCD Attenuate Ravenel ∆panCD 95-1315 ∆panCD Ravenel ∆RD1 ∆panCD 95-1315 ∆RD1 ∆panCD Ravenel ∆panCD ∆secA2 95-1315 ∆panCD ∆secA2 ∆leuCD for plasmid selection M. tuberculosis H37Rv M. bovis Ravenel M. bovis 95-1315 M. tuberculosis H37Rv M. bovis Ravenel M. bovis 95-1315 Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) Virulent M. bovis BCG Attenuate Overexpress antigens rBCG30 Endosome escape (listeriolysin; perfringoslysin) rBCG ∆ureC::Hly Attenuate rBCG(mbtB)30 Limited Replication Ravenel ∆RD1 95-1315 ∆RD1 AERAS-407 Overcome immune evasion AF2122297 ∆RD1 Ravenel ∆RD1 ∆panCD 95-1315 ∆RD1 ∆panCD Attenuate Ravenel ∆panCD 95-1315 ∆panCD Ravenel ∆RD1 ∆panCD 95-1315 ∆RD1 ∆panCD Ravenel ∆panCD ∆secA2 95-1315 ∆panCD ∆secA2 ∆leuCD for plasmid selection Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) M. bovis Wildlife and Wildlife/Livestock “Pairs” Deer/Cattle (US) Badgers/Cattle (UK) Possum/Cattle (New Zealand) Buffalo/Lion (South Africa) zimbabwe-simbabwe-map-g.gif PoorCondition P5040090Lion Tuberculosis Vaccine Development 6020 in FIV cats.tiff Considerations • Safety  FIV infected domestic cats Efficacy  N/A (no BSL3 for challenge of felines; disease kinetics unknown Cost  production comprable to $$ for other live mycobacterial strains Diagnostics compatibility  fewer reagents for cat immunology Feasibility  intramuscular delivery (simulate darting) Environmental persistence  non-replicating platform Bridge studies to target populations  are FIV+ domestic cats close enough? First Generation Pre-clinical studies mc26020 lysA∆panCD mc26030 RD1 panCD Mice - Safety + + Mice – Efficacy + + Guinea pig – Safety (FVA) + + Guinea pig - Efficacy + +/- Non-human primate – Safety + + Non-human primate -- Efficacy +/- - Bovine (neonates) – Safety ND + Bovine (neonates) – Efficacy ND - Non-human primate (neonates; SIV+) -- Safety + ND Feline (FIV+) -- Safety + ND Conclusions: (1) mc26020 and mc26030 are safe, (2) Efficacy varies Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) Advantages of Recombinant BCG (Jacobs and Bloom; Nature 1987) Only live vaccine currently given at birth Safe -Over 3 billion doses have been administered Can provide long lasting immunity Mycobacterial Cell Walls are the adjuvant component of Freund’s complete adjuvant. Can be an efficient part of prime and boost Genetically tractable To develop recombinant attenuated M. tuberculosis vectors in order to establish protective immune memory against both TB and HIV at the earliest possible time after birth in rhesus macaque model. Non replicating AMTB pro-apoptotic for better presentation of antigens Uma Ranganathan Glenn Fennelly Kristina Abel Development of live attenuated mycobacterial vaccines for TB/HIV Mother-to-Child-Transmission (MTCT) of HIV Prevalence of HIV-1 in pregnant women Hypothesis An attenuated Mtb vector expressing HIV antigens orally administered to infants shortly after birth can protect against both HIV and Mtb infection. Oral administration possible. effective against breast-milk transmission of HIV Potent adjuvant effect. infant dendritic cell activation Magnitude of BCG-induced immune responses in infants is comparable to those observed in adults. potent induction of anti HIV/TB immunity Strain Genotype mc26206 ΔleuCD ΔpanCD mc26208 ΔleuCD ΔpanCD ΔsecA2::hyg mc26434 mc26208 (pJH222) mc26435 mc26208(pJH222:Sivmac239gag) Plasmid Characteristics pJH222 Episomal, kan, alpha ag promoter,19kDa ss pJH222:Sivmac239gag pJH222 with mycobacterial codon optimized SIV gag pVRC4307 CMV promoter, humanized SIV gag, pol and nef Ad5SIVgag expressing SIV gag Strains and Plasmids used in the study mc26434 (pGAG) elicits significant SIV gag - specific CD8+ T cells than the vector only control mc26435 mc26434 mc26435 Prime (wk. 1) Dose/route Boost (wk 4) Dose/route mc26434 107cfu/S.C. mc26434 107cfu/S.C. mc26435 107cfu/S.C. mc26435 107cfu/S.C. mc26435 107cfu/S.C. Ad5 gag 109PFU/i.m pVRC4307 50mg/i.m. mc26435 107cfu/S.C. Prime Wk 0 Boost Wk 4 Sampling Wk 1 Tetramer -spleen Wk 6 Tetramer – spleen - blood, ICS – spleen, lung Wk 3 Tetramer – spleen ICS – spleen Wk 10 Tetramer – spleen - blood, lung ICS – spleen, lung ND ND Ad5 gag 109PFU/i.m IMMUNIZATION SCHEDULE W14 rAMtb-SIV Immunization (ID) Ad5-SIVgag boost (IM) Saline (PO/ID) Saline (IM) * ** * ** Saline controls (n=4) ID (n=6) Oral (n=6) Immunogenicity Study i.m. rAd5-SIVgag (10^8 pfu rAMtb-SIVgag (10^9 cfu) SIV-specific Plasma Antibodies - good induction of SIV-specific plasma IgG and IgA antibodies - oral route might induce better IgA responss SIVgag p27-specific T Cell Responses SIV-specific T cell responses can be detected at week 3, prior to rAd5-SIVgag boost! Mtb-specific CD8+T Cell Response Ag85b PSTS1 IFN-g vs. Granzyme TNF-a vs. IFN-g IFN-g vs. Granzyme TNF-a vs. IFN-g Summary rAMtb-SIVgag vaccine is safe in newborn macaques the vaccine is immunogenic: - activation of mDC - induction of SIV and Mtb-specific CD4+ and CD8+ T cell responses - development of SIV and Mtb-specific antibodies in plasma the oral route appears to be similar effective to the id route for priming a heterologous, systemic boost will enhance immunogenicity a 3-week immunization interval is preferrable Tuberculosis Vaccine Development Human TB Vaccines Cattle TB Vaccines Wildlife TB Vaccines Mycobacterial Vaccine Vectors (diseases other than TB) HIV-Associated MDR & XDR TB in S Africa XDR TB MDR TB Survival in days 60 days 29 days Gandhi et al AJRCCM 2010 HIV co-infection 90% Median CD4: 87 cells/mm HIV co-infection 98% Median CD4: 66 cells/mm Jacobs Lab • Karolin Biermann • Bing Chen • Tsugunda Hsu • Sunhee Lee* • Vasan Sambandamurthy* Porcelli Lab • Joe Hinchey Aeras • Ron Mayner & Lew Barker Duke • Bart Haynes Colorado State • Ian Orme • Angelo Izzo Human Pre-Clinical Vaccine Development FDA • Sheldon Morris & Steve Derrick North Shore Gene Therapy Laboratory • Jim Mason • Kathy Cronin Spring Valley Laboratories Laurie Wiedl Tulane National Primate Research Center • Andrew Lackner & Pyone Aye University of Illinois, Chicago • Zheng Chen University of North Carolina • Miriam Braunstein USDA/UMTB • Ray Waters, Mitch Palmer, & Mark Estes Cattle Vaccine Development AECOM • Tsugunda Hsu • Karolin Biermann • Michelle Larsen National Animal Disease Center • Ray Waters • Mitch Palmer • Tyler Thacker University of Texas Medical Branch • Mark Estes Genomics Institute of the Novartis Research Foundation • Richard Glynne • John Walker • Whitney Barnes