The fusion protein of Antigen 85B and ESAT-6 was studied and it showed protective immune response.

1. Presented by
Esakki Muthu Lakshmi .V
M.Tech (1st year Industrial
Biotechnology)

2. OBJECTIVE
 To evaluate the potential of a subunit vaccine based on a
fusion protein between two immunodominant antigens, Ag85B
and the 6-kDa early secretory antigenic target (ESAT-6)

3. Introduction
 Tuberculosis(T.B)- contagious disease
 Mycobacterium tuberculosis-causative agent
 Gram positive
 Acid fast bacilli
 highly aerobic
 Resistant to dessication
 Infects lungs
 Lipoarabinomannan and mycolic acids
 Divides every 15-20 hrs

4. Types of tuberculosis
 Latent TB disease
 Active TB infection
 Miliary TB infection
 Symptoms
 Cough with bloody sputum
 Fever
 Loss of appetite
 Night sweat
 Chest pain
 Weight loss

5. Pathology
Ghon
complex
Formation (3-
8 weeks after
exposure)
Tubercle
formation (3
months of post
infection)
Miliary
TB
(delayed
upto 2
years)

7. Diagnosis
 Tuberculin Skin Test (TST)
 Chest Radiograph (X-ray)
 Sputum Smear Microscopy (SSM)
 Culture
 Polymerase Chain Reaction (PCR)
 Blood Test (e.g. the T-SPOT.TB test)

8. BCG vaccine
Effective vaccine against TB
Albert Calmette and Camille Guerin
Live attenuated Mycobacterium bovis
Need for subunit vaccine
BCG vaccine
• Revert back to virulent form
• Infection in immunocompromised patient
• Emergence of MDR strain
• False positive in diagnostic test

9. ESAT-6
 Early secreted protein(6KDa)
 Encoded by esxA in RD1 loci
 ESX-Bacterial virulence
 Mtb specific T cell antigen
 Low immunogenicity molecule
 diagnostic tool

10. Antigen 85B
 30 kDa protein
 Part of Ag85 complex
 Mycolyl transferase activity
 Cell wall of Mtb
 Phagosome of Mtb infected macrophages
 T cell and B cell epitopes
 Elicit protective immune response

11. Expression of recombinant ESAT-6
Amplification of ESAT-6 gene from H37Rv by PCR
Restriction of PCR product by BglII and NcoI
Cloning of ESAT-6 into BglII and NcoI sites of pMCT6 containing
His tag sequence
Expression of ESAT-6 in E.coli XL1 blue
Purification of recombinant protein by metal ion affinity
chromatography containing Ni2+ ions
Analysis of ESAT-6 by SDS PAGE
Primers used:
Forward- 5’GAAGATCTATGACAGAGCAGCAGTGG (BglII site)
Reverse- 5’ CCGCCATGGTAAACACGAGAAAGGGCG (NcoI site)

12. Expression of recombinant Ag85B
Amplification of Ag85B from H37Rv by PCR
Cloning of PCR product into BglII and BamHII site of pMCT16
Expression of His tagged protein in E.coli XL1 blue
Purification of expressed protein on talon column followed by protein
anion exchange chromatography
Analysis of purified protein by SDS PAGE
Primers used: BglII
Ag85B-F1 - GGCAACCGCGAGATCTTTCTCCCGGCCGGGC
Ag85B-R2 –CCTTCGGTGGATCCCGTCAG
BamHI

13. Expression of fusion protein
PCR Amplification of ESAT-6 and Ag 85B using corresponding
primers
Fusion of Ag 85B-ESAT-6 and ESAT-6-Ag85B molecule at HindIII
site using F1-R1 and F2-R2 primer set accordingly
Cloning of fusion molecule into BglII/BamH1 pMCT6 in frame
with eight His residues
Expression of fusion protein, Ag85B and ESAT-6 in E.coli XL1 blue
Purification by ion exchange chromatography and analysis by SDS
PAGE

14. Primers
Ag85B-F1primer- GGCAACCGCGAGATCTTTCTCCCGGCCGGGC
BglII
Ag85B-R1primer- GGCAAGCTTGCCGGCGCCTAACGAACT
HindIII
ESAT-6-F1primer GGCGCCGGCAAGCTTGCCATGACAGAGCAGCAGTGG
HindIII
ESAT6-R1primer- CGAACTCGCCGGATCCCGTGTTTCGC
BamHI
Ag85B -F2primer
GTTCGCAAAGCTTTTCTCCCGGCCGGGGCTGCCGGTCGAGTACC
HindIII
Ag85B -R2primer CCTTCGGTGGATCCCGTCAG
BamHI
ESAT-6 -F2primer GGACCCAGATCTATGACAGAGCAGCAGTGG
BglII
ESAT-6- R2primer
CGGCAGCCCCGGCCGGGAGAAAAGCTTTGCGAACATCCCAGTGACG
HindIII

15. SDS PAGE analysis
kDa
Molecular
standard ESAT-6 Ag 85B Ag85B –
ESAT-6
ESAT-6-
Ag85B

16. Vaccine preparation and immunization
procedure
Emulsification of vaccine doses from 0.01-50 µg with 250 µg
of dimethyl dioctadecylammonium bromide
Addition of 25 µg monophosphoryl lipid A (MPL)
Subcutaneous injection of C57BL/6J mice with 0.2 mL of
subunit vaccine with 2 weeks interval and BCG vaccine
An adjuvant alone as a negative control and naive mice as
control

17. IFN-γ releasing assay
After five weeks of first immunisation, isolation of PBMC on a
density gradient
Culturing of cells in 96 well microtitre plate containing 2 × 105
cells in a volume of 200 µL of RPMI 1640 medium supplemented
with 5× -10-5 M 2-mercaptoethanol, 1 mM glutamine, penicillin-
strepotmycine and 5% (vol/vol) fetal calf serum
Incubation of cells with Ag85B, ESAT-6 and fusion protein (5, 2.5
and 1.3 µg/mL)
After 72h of incubation, harvesting of culture supernatant.
Determination of IFN- γ by enzyme-linked immunosorbent assay

18. Results

19. Experimental infections and bacterial
enumeration in organs.
After first immunization, injection of 100CFU of Mtb per lung through
aerosol route or i.v injection of 5*104 CFU of Mtb H37Rv suspended
in PBS in a volume of 0.2 ml.
Sacrification of mice after 2 weeks (i.v route) and 6 weeks (aerosol
route)
Removal and homogenization of lung and spleen in sterile saline and
plating onto Middlebrook 7H11 agar with 2 mg of 2-thiophene-
carboxylic acid hydrazide per ml
Incubation for 2 weeks at 37ºC and counting of colonies

20. RESULT

21. Conclusion
Induces long term memory of immunity
Fusion protein and BCG vaccine-same efficacy
Superior protective efficacy
Multiple epitopes for B cell and T cell
Diagnostic tool
More defined product

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