1. 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

2. 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

3. 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!

4. 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.

5. 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.

6. TB vaccine candidates. Clinical trials

7. 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»

8. 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,

9. 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

10. 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;

11. 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): The protective role of antibody responses during Mycobacterium tuberculosis infection.
Abebe F, Bjune G.

12. 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

13. 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!

14. 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
- ± ± ±

15. 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

16. 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

17. 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,
Tel.+7(499)