Problems and prospects of development of the subunit TB vaccine Gamaleya Research Insitute Russian Ministry of Health Problems and prospects of development of the subunit TB vaccine Vladimir G. Lunin lunin1955@gmail.com

TB may be one of the most common HIV-related opportunistic infections. Globally, TB is the 2nd leading cause of death from an infectious disease (behind HIV) TB is the leading cause of death in HIV globally Active TB may accelerate HIV replication HIV Infection is a Risk Factor for Activation of Latent Tuberculosis 54% of HIV-infected people suffering from tuberculosis More than 30% of HIV-infected people die from tuberculosis HIV Infection as a Risk Factor for Activation of Latent Tuberculosis Carlos Franco-Paredes, MD // medscape

BCG is the only licensed vaccine against tuberculosis The bacille Calmette–Guérin (BCG) vaccine has existed for 80 years and is one of the most widely used of all current vaccines. Protective effect against meningitis and disseminated TB in children It does not prevent primary infection and, more importantly, Does not prevent reactivation of latent pulmonary infection, the principal source of bacillary spread in the community. BCG vaccination can not be used for HIV-infected patients. The impact of BCG vaccination on transmission of Mtb is therefore limited. We need a new vaccine!

What kind of vaccine against TB we need? Antigens lacking in BCG but present in virulent strains –shift and broaden immune repertoire. ESAT6-CFP10 complex Antigens present in BCG: prime-boost strategy. Both, in the form of either fused or mixed components. Looks advantageous. Ag85A Antigens actively secreted by the infect during infection –vaccination against active disease. Antigens expressed by dormant mycobacteria –vaccination against latent disease.

Adjuvants – not less important than antigens Provide a depot for slow, effective immunization. Trigger receptors of innate immunity requisite for the development of acquired, clonally shaped immune responses. In some instances may serve as a tool for intracellular antigen delivery. Very few are certified for human use. http://www.invivogen.com/review-vaccine-adjuvants

TB vaccine candidates. Clinical trials

Structural scheme of subunit protein-based vaccine against TB Antigens Ag85A & ESAT6-CFP10 Immunodominant tuberculosis antigens, it’s strong stimulate Th1 immune response ESAT-6-CFP-10 Ag85A 20 нм Декстран-связывающийдомен Декстран 500 CpG олигонуклеотид M.tb антигены DEAE-Декстран 500 Dextran non-toxic, biodegradable. The dextran is interact with innate immunity receptors and stimulate Th1 response. It’s creates a depot effect for antigens DEAE-Dextran Immobilize and protect CpG ODN CpG ODN Molecular adjuvant, stimulate Th1 immune response by interact with TLR9 (stimulate secretion INFγ, TNFα) Booster vaccine «GamTBvac»

The Drug Discovery Process INDEFINITE Drug Discovery Preclinical Clinical Trials FDA Review Scale-Up to Mfg. Post-Marketing Surveillance ONE FDA-APPROVED DRUG 0.5 – 2 YEARS 6 – 7 YEARS 3 – 6 YEARS NUMBER OF VOLUNTEERS PHASE 1 PHASE 2 PHASE 3 5 250 ~ 5,000 – 10,000 COMPOUNDS PRE-DISCOVERY 20–100 100–500 1,000–5,000 IND SUBMITTED NDA SUBMITTED now, we are here Sources: Drug Discovery and Development: Understanding the R&D Process, www.innovation.org

Lymphocyte proliferation assay & The IFN-gamma ELISPOT assay LPA result Ag85A & ESAT6-CFP10 induce antigen-specific T cell response Dextran binding domain is immunologically inert Adjuvant is not induce non-specific T cell response ELISPOT result ELISPOT result

CFU numbers in the lungs and spleens of infected mice C57Bl6 mice Retro-orbital injection of H37Rv (8.3х106) log10KOE (mean±s.e.m.) CFU counts are expressed as log10 (mean±s.e.m.) 30 days post-infection in lungs or spleens. Data shown are representative of 12 mice for each group in a total of three independent experiments. Statistical differences between strains in the same phase of infection are indicated in the graphic. *P<0.05 versus day 30-infected C57BL/6;

The protective role of antibody responses during Mycobacterium tuberculosis infection are not understood fully Antibodies recognizing mycobacterial antigens were produced in mice  Log10 Ab Ag85A ESAT6 GamTBvac Adjuvant PBS CFP10 Clin Exp Immunol. 2009 Aug;157(2):235-43. The protective role of antibody responses during Mycobacterium tuberculosis infection. Abebe F, Bjune G.

Protective efficacy of GamTBvac and its components % survival Days post-challenge GamTBvac Adjuvant Control,PBS C57Bl6 mice Retro-orbital injection of H37Rv (8.3х106) Ag85A+Adjuvant ESAT6-CFP10+Adjuvant GamTBvac Control, PBS % survival C57Bl6 mice Aerosol challenge of H37Rv (5х107) Days post-challenge

Protective efficacy of GamTBvac and its components Protective efficacy of GamTBvac and its components. Prime-boost immunization BCG+AG85A BCG+GamTBvac BCG+ESAT6-CFP10 BCG Control, PBS C57Bl6 mice % survival Aerosol challenge of H37Rv (5х105) Days post-challenge Heterological prime-boost vaccination is better!

Necrosis, hyperemia, enlarged CFU numbers in the lungs and spleens Lung Spleen GamTBvac Control, PBS log10KOE (mean±s.e.m.) The guinea pig model of tuberculosis Aerosol challenge of H37Rv (1,25х104) Pathomorphological characteristics of lungs and spleen after challenge Immunization Organs Lung Spleen BCG-1 ++++ BCG-1+ 2xGamTBvac 2xGamTBvac +++-  small site of necrosis ++±± enlarged spleen Adjuvant Control, PBS     Necrosis, hyperemia, enlarged - ± ± ±

Safety of the candidate vaccine «GamTBvac» Our animals model: We was investigated: Candidate vaccine «GamTBvac» Adjuvant and it’s components Recombinant proteins (antigens) BALBc mice and had good results: Local tolerance Genotoxicity and carcinogenicity studies Safety pharmacology Pharmacokinetic studies Acute toxicity Chronic toxicity guinea pigs GamTBvac is safety! Rabbits

The next generation of protein-based subunit vaccines against tuberculosis of Gamaleya Research Institute (licensed, manufacture) BCG GamTBvac (Phase 1 clinical trials) Tb vaccines against LTBI (pre-clinical test) 2014 Development of new adjuvant Since 1925

Thank you for your attention! Laboratory bioactive nanostructures In collaboration: Central Institute of Tuberculosis Research Center for Microbiology and Biotechnology Thank you for your attention! Biological testing laboratory, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry Artem P. Tkachuk, Ph.D. Gamaleya Research Institute, E-mail: artem.p.tkachuk@gmail.com Tel.+7(499)193-30-01