Problems and prospects of development of

the subunit TB vaccine

Vladimir G. Luninlunin1955@gmail.com

Gamaleya Research InsituteRussian Ministry of Health

TB may be one of the most common HIV-related opportunistic infections.

More than 30% of HIV-infected people die from tuberculosis

54% of HIV-infected people suffering from tuberculosis

Globally, TB is the 2nd leading cause of death from an infectious disease (behind HIV)TB is the leading cause of death in HIV globallyActive TB may accelerate HIV replicationHIV Infection is a Risk Factor for Activation of Latent Tuberculosis

HIV Infection as a Risk Factor for Activation of Latent TuberculosisCarlos 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?

ESAT6-CFP10 complex

•Antigens lacking in BCG but present in virulent strains –shift and broaden immune repertoire.

Ag85A

•Antigens present in BCG: prime-boost strategy.

•Both, in the form of either fused or mixed components. Looks advantageous.

•Antigens actively secreted by the infect during infection –vaccination against active disease.•Antigens expressed by dormant mycobacteria –vaccination against latent disease.

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.

Adjuvants – not less important than antigens

http://www.invivogen.com/review-vaccine-adjuvants

TB vaccine candidates. Clinical trials

ESAT-6-CFP-10

Ag85A

20 нм 20

нм

Декстран-связывающийдомен

Декстран 500

CpG олигонуклеотид

20 нм

M.tb антигены

DEAE-Декстран 500

Antigens Ag85A & ESAT6-CFP10Immunodominant tuberculosis antigens, it’s strong stimulate Th1 immune response

Dextrannon-toxic, biodegradable. The dextran is interact with innate immunity receptors and stimulate Th1 response. It’s creates a depot effect for antigens

DEAE-DextranImmobilize and protect CpG ODN

CpG ODNMolecular adjuvant, stimulate Th1

immune response by interact with TLR9 (stimulate secretion INFγ, TNFα)

Booster vaccine «GamTBvac»

Structural scheme of subunit protein-based vaccine against TB

INDEFINITE

Drug Discovery

Preclinical Clinical Trials FDA Review

Scale-Up to Mfg.

Post-MarketingSurveillance

ONE FDA-APPROVED

DRUG

0.5 – 2 YEARS6 – 7 YEARS3 – 6 YEARS

NUMBER OF VOLUNTEERS

PHASE 1

PHASE 2

PHASE 3

5250~ 5,000 – 10,000

COMPOUNDS

PR

E-D

ISC

OV

ER

Y

20–100

100–500 1,000–5,000

IND

SU

BM

ITTED

ND

A S

UB

MIT

TED

Sources: Drug Discovery and Development: Understanding the R&D Process, www.innovation.org

now, we are here

The Drug Discovery Process 

Lymphocyte proliferation assay & The IFN-gamma ELISPOT assay

Ag85A & ESAT6-CFP10 induce antigen-specific T cell responseDextran binding domain is immunologically inertAdjuvant is not induce non-specific T cell response

Ag85A & ESAT6-CFP10 induce antigen-specific T cell responseDextran binding domain is immunologically inertAdjuvant is not induce non-specific T cell response

LPA result

ELISPOT result ELISPOT result

CFU numbers in the lungs and spleens of infected mice

log

10K

OE

(m

ean±

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;

C57Bl6 mice

Retro-orbital injection of H37Rv (8.3х106)

Antibodies recognizing mycobacterial antigens were produced in mice 

The protective role of antibody responses during Mycobacterium tuberculosis infection are not understood fully

CFP10

1

10

100

1000

10000

100000

BCG Аг 85А ESAT-6 CFP-10

Группы иммунизированных мышей

Рец

ип

рокн

ые

знач

ени

я ти

тров

ан

тите

л

BCG BCG+2 вакцинации ГИ 2 вакцинации ГИ адъювант контроль

Log10

Ab

Ag85A ESAT6

GamTBvac

GamTBvac Adjuvant PBS

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

C57Bl6 mice

Retro-orbital injection of H37Rv (8.3х106)%

su

rviv

al

Days post-challenge

GamTBvac

AdjuvantControl,PBS

Ag85A+Adjuvant

ESAT6-CFP10+Adjuvant

GamTBvac

Control, PBS

%

surv

ival

Days post-challenge

C57Bl6 mice

Aerosol challenge of H37Rv (5х107)

Protective efficacy of GamTBvac and its components. Prime-boost immunization

% s

urvi

val

Days post-challenge

BCG+AG85ABCG+GamTBvacBCG+ESAT6-CFP10BCGControl, PBS

C57Bl6 mice

Aerosol challenge of H37Rv (5х105)

Heterological prime-boost vaccination is better!

Lung Spleen

GamTBvacGamTBvac Control, PBS

log

10K

OE

(m

ean±

s.e.m

.)

Aerosol challenge of H37Rv (1,25х104)

Pathomorphological characteristics of lungs and spleen after challenge

CFU numbers in the lungs and spleens

ImmunizationOrgans

Lung SpleenBCG-1 ++++ ++++

BCG-1+ 2xGamTBvac ++++ ++++

2xGamTBvac+++-

small site of necrosis

++±±enlarged spleen

Adjuvant ++++ ++++

Control, PBS

Necrosis, hyperemia, enlarged

- ± ± ±enlarged spleen

The guinea pig model of tuberculosis

Safety of the candidate vaccine «GamTBvac»

Local toleranceGenotoxicity and carcinogenicity studiesSafety pharmacologyPharmacokinetic studiesAcute toxicityChronic toxicity

Our animals model:

BALBc mice

guinea pigs

Rabbits

We was investigated:

and had good results:

Candidate vaccine «GamTBvac»Adjuvant and it’s componentsRecombinant proteins (antigens)

GamTBvac is safety!

The next generation of protein-based subunit vaccines against tuberculosis of Gamaleya Research Institute

Development of new adjuvant

Since 1925

2014

Thank you for your attention!

Artem P. Tkachuk, Ph.D.Gamaleya Research Institute,E-mail: artem.p.tkachuk@gmail.comTel.+7(499)193-30-01

Laboratory bioactive nanostructuresIn collaboration:

Central Institute of Tuberculosis

Research Center for Microbiology and Biotechnology

Biological testing laboratory, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry