This document summarizes research on using a "BCG prime - DNA boost" vaccination strategy for tuberculosis. Key points: - Mice vaccinated with BCG followed by a boost with DNA encoding the M. tuberculosis antigen α-crystallin had significantly reduced lung and spleen bacterial loads compared to BCG alone after airborne infection. - The boosted mice also had less severe lung, liver and spleen pathology and granulomas. - Protection lasted for at least 16 weeks and was associated with an increased proportion of the cytokines IL-12 and decreased IL-10 in the lungs. - The results suggest boosting BCG with α-crystallin DNA enhances and prolongs protection against tuberculosis

1. Anil K. Tyagi
Department of Biochemistry
University of Delhi South Campus
New Delhi, India
Vaccination strategies for Tuberculosis….
Can Probiotics help?
YIMPSF Symposium on “Probiotics-from Bench to Community”
7th
– 8th
March 2015, New Delhi
““Promoting Life”Promoting Life”

2. • The human body carries about
100 trillion microorganisms in
its intestines.
Bacteria in the gut fulfil a host of useful
functions for humans: “Forgotten Organ”
In mouth, around the gum
Nasal cavity
Under the armpits
Stomach
Intestines
Genitourinary
Within joints
Under the toenails,
between the toes
• The four dominant phyla in the
gut microbiota are Firmicutes,
Bacteroidetes, Actinobacteria
and Proteobacteria.
• Gut microbiota, consists of a
complex of microorganism
species that live in our digestive
tracts.

3. Probiotics are derived from traditional
fermented foods, from beneficial
commensals or from the environment.
They act through diverse mechanisms
affecting the composition or function of
the commensal microbiota and by altering
host epithelial and immunological
responses.
Probiotics………are live microorganisms that are
thought to have beneficial effects on the host
Probiotics have been used throughout civilization
but it wasn’t until almost 100 years ago that Elie
Metchnikoff discovered the health benefits of
probiotics. Regarded by many as the father of
probiotics, Metchnikoff attributed the long life of
Bulgarian peasants to their consumption of the
probiotic species, Lactobacillus.

4. Certain probiotic interventions have shown promise in selected clinical
conditions where aberrant microbiota have been reported, such as atopic
dermatitis, necrotising enterocolitis, pouchitis and possibly irritable bowel
syndrome.

5. Mechanisms of Action of Probiotics
Modulation of mucosal immune
response by probiotic bacteria.
Pathogen-associated molecular patterns
(PAMPs) derived from probiotic bacteria are
recognized by pattern recognition receptor
such as TLRs on DCs in the epithelium leading
to downstream signaling and induction of
Th1 cytokines.
Crosstalk between probiotic
bacteria and the intestinal
mucosa.

6. Functions of Probiotics….
Vaccine adjuvant
Increase of the lactose
Tolerance and digestion
PROBIOTICS
Stimulation of the immune
response
Positive influence in the
Intestinal microflora
Reduction of intestinal pH
Improvement of the
Intestinal functioning
Cholestrol reduction
Reduction of ammonia:
Other toxic compounds
Production of the B
vitamins:(Folic acid)
Restoration of the non
intestinal microflora
after antimicrobial
therapy
Treatment and
prevention of acute
diarrhea
As new vaccines

7. • More than 30,000 lives are lost due to tuberculosis globally every week
putting TB in the list of top major killers.
• ~1/3rd
of the world population is infected with M. tuberculosis
asymptomatically.
• In India, more than 5,000 people develop TB everyday and more than 1000
people die of the disease each day.
• ~1/4th
of the world’s total TB patients reside in India.
Goals of TB Research
• Development of new, more efficient and rapid methods for diagnosis
• Development of new vaccine(s) for eradication of tuberculosis
• Development of new anti-tubercular drugs – reducing the period of
treatment, MDR-TB
Tuberculosis – A Global Health Emergency

8. BCG – the most used vaccine
in the history but has problems
• BCG has produced more controversy than protection
against TB.
• Protection by BCG against TB in adults remains a
question?
What do we need ?
We need a vaccine which does what BCG fails to do - for
reasons which we do not understand.
"The new vaccine should protect consistently against
adult tuberculosis"

9. Lack of sensitive and
specific diagnostic tools ?
Nature of protective
immunity ?
Host immune
response ? /
Heterogeneity
Why does BCG fail in
some populations ?
Problems and key
issues
surrounding TB
vaccine
development
Surrogate markers for
vaccine induced protection ?
Role of experimental
animal models ?
New challenge models ?
Seeking answers to ………. ?
 Infants never exposed to TB
 Adults who have been exposed
 Those who are already infected
 HIV infected persons

10. Two pronged approach
On long term basis :
 Nature of protective immunity in TB
 Development of surrogate markers
 Identification of new antigens
 Improve existing animal models
 Host immune response
……in order to develop a rational approach for TB
vaccines.
In the meantime :
Based on the available knowledge and
technology….. continue to develop new TB
vaccines and evaluate them in the existing animal
models.
Channel the promising ones for human clinical
trials.

11. Approaches
• Subunit vaccines
• Recombinant BCG vaccines
• Attenuated M.tuberculosis strains
• Atypical mycobacterial strains
• DNA vaccines
• Heterologous Prime Boost Approach
• Immunotherapeutic TB Vaccines

12. Development and Evaluation of Candidate Vaccines
Against Tuberculosis
 Identification and characterization of mycobacterial promoters
 Development of expression vectors
 Recombinant BCG Approach
Antigens 85A, 85B and 85C, 19 kDa lipoprotein antigen, ESAT-6, 38 kDa
PstS homolog, α-crystallin and SOD.
- Evaluation against sub-cutaneous challenge of M.tb
 DNA Vaccines
Superoxide dismutase (SOD), α-crystallin and ESAT-6
- Evaluation against sub-cutaneous challenge of Mtb
 Evaluation of promising candidates using heterologous Prime Boost
Approach
Superoxide dismutase, α-crystallin, ESAT-6, Antigen 85C, 38 kDa
antigen
- Evaluation against aerosol challenge of M.tb
 Immunotherapeutic vaccines and attenuated M. tuberculosis strains

13. Strategies
Replacing BCG Boosting BCG
Recombinant BCG over-
expressing antigen 85C
BCGacr prime - DNAacr
boost regimen (R/D)
Enhancing the protective
efficacy of BCG by boosting
with α– crystallin based
DNA vaccine (B/D)
These three regimens have been approved in principle by
the Tuberculosis Vaccine Clinical Trial Expert Group of
DBT for eventual human clinical trials
Preclinical preparations/studies for this are in progress

14. α-crystallin from Mycobacterium tuberculosis
1. Transcriptome and proteome analysis of Mycobacterium
tuberculosis has revealed that α–crystallin (acr, Rv2031c), a
member of DosR-DosS/DosT dormancy regulon, represents one of
the most abundantly produced proteins during exposure to
hypoxia, nutrient starvation and transition of actively dividing bacilli
to a dormant state.
2. Latently infected individuals (healthy PPD+ and household
contacts) exhibit increased lympho-proliferative and IFN-γ
response to α–crystallin as compared to patients with active TB.
3. These observations signify a crucial role of α–crystallin in the
elicitation of protective immune responses and maintenance of
disease free state in these subjects, thus, making this antigen an
attractive target for the development of new TB vaccines.

15. Effect of ‘BCG prime - DNAacr boost’ (B/D) regimen on the bacillary load and
gross pathology
 B/D regimen significantly reduced pulmonary, hepatic and splenic lesions and provided enhanced
protection in comparison to BCG vaccination
 Vaccination with B/D regimen significantly reduced bacillary load in both lung and spleen by > 37 fold
and > 96 fold respectively, when compared to BCG vaccination
10 weeks post infectionImmunization Boosting
5x105 CFU
of BCG
6 wks 100µg
DNA acr
10 wks
Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
ChallengeImmunization Boosting
5x105 CFU
of BCG
6 wks 100µg
DNA acr
10 wks
Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
Challenge
Gross Histopathology
1
2
3
4
5
*
**
φ
*
Saline BCG B/D
10weekpost-infectionnfection
LungscoreLiverscoreSpleenscoreLungscore
1
2
3
4
5
** ** **
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
5
B
/V
B
/D
B
C
G
S
aline
1
2
3
4
5
*
**
φ
*
Saline BCG B/D
10weekpost-infectionnfection
LungscoreLiverscoreSpleenscoreLungscore
1
2
3
4
5
** ** **
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
5
B
/V
B
/D
B
C
G
S
aline
1
2
3
4
5
*
**
φ
*
Saline BCG B/D
10weekpost-infectionfection
LungscoreLiverscoreSpleenscoreLungscore
1
2
3
4
5
** ** **
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
5
B
/V
B
/D
B
C
G
S
aline
1
2
3
4
5
*
**
φ
*
Saline BCG B/D
10weekpost-infectionfection
LungscoreLiverscoreSpleenscoreLungscore
1
2
3
4
5
** ** **
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
5
B
/V
B
/D
B
C
G
S
aline
Saline
B/D
BCG B/V
SpleenLiverLung
Saline B/DBCG
*, p<.05; **, p<.01 ***, p<.001, when compared to saline group; φ, p<.05; φ φ, p<.01, φ φ φ, p<.001, when compared to BCG
group
0
2
4
6
8
**
**
φφ **
Log10CFU/g
Saline BCG B/D B/V
0
2
4
6
8
**
**
φφ
**
Log10CFU/g
Spleen
Lung
Normal
Sal/BD- 2.31, BCG/BD-1.37
Sal/BD-3.44, BCG/BD – 1.96
***
***
φφφ
φφφ

16. Lung
Saline group : Multifocal coalescing granulomas with extensive necrosis
BCG group : discrete granulomas with or without central necrosis
B/D group : negligible and diffused aggregates of inflammatory cells.
Liver
Saline : Multiple granulomas
BCG group : mild inflammation
B/D group : no evident sign of inflammation.
Granuloma%
Saline BCG
LiverLung
Granuloma%
B/D Normal
10 weeks post infection
Effect of ‘BCG prime - DNAacr boost’ (B/D) regimen on granulomatous
pathological lesions
55
35
30
5
35
------- ~5 -------

17. Long term protective efficacy of ‘BCG prime - DNA boost’ regimen
 Animals vaccinated with B/D regimen showed minimal involvement of lung and spleen and no evidence
of gross lesion in liver, in comparison to BCG immunized animals.
 B/D regimen continued to provide significant protection with > 200 fold and ~47 fold fewer bacilli in lung
and spleen, respectively, when compared to BCG vaccination.
16 weeks post infectionImmunization Boosting
5x105
CFU
of BCG
6 wks 100
DNAacr Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
ChallengeImmunization Boosting
5x105
CFU
of BCG
6 wks 100
DNAacr
16 wks
Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
Challenge
*, p<.05; **, p<.01, ***, p<.001, when compared to saline group; φ, p<.05; φ φ, p<.01, φ φ φ, p<.001 when compared to BCG
group
Spleen
Lung
Saline BCG B/D B/V
0
2
4
6
8
**
φ
Log10CFU/g
0
2
4
6
8
**
φφ
Log10CFU/g
Saline
B/D
BCG B/V
SpleenLiverLung
Saline B/DBCG
Gross pathology
10wee16weekpost-infection
LiverscoreLungscoreLiverscoreSpleenscore
1
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
1
2
3
4
5
*
**
*
1
2
3
4
5
**
** *
10wee16weekpost-infection
LiverscoreLungscoreLiverscoreSpleenscore
1
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
1
2
3
4
5
*
**
*
1
2
3
4
5
**
** *
10we16weekpost-infection
LiverscoreLungscoreLiverscoreSpleenscore
1
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
1
2
3
4
5
*
**
*
1
2
3
4
5
**
** *
10we16weekpost-infection
LiverscoreLungscoreLiverscoreSpleenscore
1
1
2
3
4
5
*
φ
**
1
2
3
4
5
**
** **
1
2
3
4
5
*
**
*
1
2
3
4
5
**
** *
BCG/BD-2.01
BCG/BD – 1.47
***
φφφ
φ
**
μg

18. Long term influence of ‘BCG prime - DNA boost’ B/D regimen on
histopathology
Granuloma%
Saline BCG
LiverLung
Granuloma%
B/D
Saline
BCG
B/D B/V
0
20
40
60
80
100
**
**
**
φφ
Lung
Saline group : Extensive multifocal coalescing granulomas with prominent central coagulative necrosis
BCG group : Scattered areas of inflammation with discrete as well as coalescing granulomas with small
necrotic centres
B/D group : negligible inflammation in lungs.
Liver
Saline : Multiple granulomas, BCG group : mild inflammation, B/D group : no evident sign of inflammation.
Immunization Boosting
5x105
CFU
of BCG
6 wks 100
DNAacr Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
ChallengeImmunization Boosting
5x105
CFU
of BCG
6 wks 100
DNAacr
16 wks
Euthanasia
6 wks ~ 50 - 100
CFU of
Mtb
Challenge 16 weeks post infection
μg

19. IL-12
IL-10
PROTECTION
INCREASES
CYTOKINEPROPORTIONINTHELUN
IFN-γ, TNF-α, TGF-β, IL-10, IL-12
PANEL OF CYTOKINES STUDIED
 The degree of protection does not
correlate with the levels of individual
cytokines.
 It is more important to measure the
relative abundance of cytokines in
the cytokine milieu and consider the
dynamic interplay of cytokines which
shows correlation with the
protection.
 Degree of protection increases with
an increased proportion of IL-12 and
decreased proportion of IL-10.
PULMONARY CYTOKINE PROFILE: BY REAL-TIME RT-PCR ANALYSIS

20. B/D vaccination confers enhanced protection against M. tuberculosis challenge in mice. The
figure depicts the bacillary load in lungs and spleen of mice at 4 weeks post-infection.
Vaccine induced protection was associated with increased frequency of PPD and antigen
specific multifunctional CD4 T cells (2+ and 3+) along with higher production of IFN-γ, TNF-
α and IL-2.
Induction of CD4 Th1 cell responses by BCG-DNAacr (B/D) regimen
Frequency of CD4 T cells producing different cytokines (1+
, 2+
and 3+
) along with MFI
and iMFI for these cytokines are compared in spleen among the vaccinated groups at 12
weeks post-immunization
[B]1.69
0.67
1.84
1.35

21. Generation of marker free recombinant MVA expressing α-crystallin gene of M.tuberculosis
GFP gene and direct repeat
sequence were sequentially
cloned in pSC65 to generate
pSC65.GFP. DR
Recombinant MVA foci were
selected on the basis of GFP
fluorescence
rMVA.Acr induced
cytopathic effect
rMVA.Acr showing no
visible fluorescence
pSC65.GFP.DR
TKL MCS Pe/l DR P7.5 GFP TKR
Cloning of α-
crystallin
gene
Clonal purification of
the recombinant clone
to remove the wild type
MVA
Excise the
GFP gene
UninfectedMVA.W
T
rMVA.Acr
16 kDa
Expression analysis of α-
crystallin protein from
rMVA.Acr infected CEF cell
line
66
43
29
20
14
α-crystallin
Select the
marker free
recombinant via
limiting dilution
method

22. Experimental protocol for evaluation of short term protective efficacy of
BCGprime-rMVAacr boost regimen against M.tuberculosis in guinea pigs
Inhalation chamber
12 wks 6 wks
M.tuberculosis
challenge
10-30 bacilli EuthanasiaSaline (control)
BCG
B/rMVA.acr 107
B/MVA.WT
Saline
12 wks 6 wks
M.tuberculosis
challenge
10-30 bacilli Euthanasia
BCG
5X105 CFU (i.d.)
6 wks 6 wks
M.tuberculosis
challenge
10-30 bacilli Euthanasia
BCG
5X105 CFU (i.d.)
6 wks
rMVA.acr
1X107 PFU (i.m.)
6 wks 6 wks
M.tuberculosis
challenge
10-30 bacilli Euthanasia
BCG
5X105 CFU (i.d.)
6 wks
rMVA.acr
1X108 PFU (i.m.)
6 wks 6 wks
M.tuberculosis
challenge
10-30 bacilli Euthanasia
BCG
5X105 CFU (i.d.)
6 wks
MVA.WT
1X108 PFU (i.m.)
B/rMVA.acr 108
Parameters for evaluation of
protective efficacy
 Bacteriological evaluation:
Reduction in lung and spleen
bacillary load
 Pathological assessment
Gross pathology
Histopathology

23. BCGprime-rMVA.acr boost regimen provides superior protection than
BCG vaccination alone against M.tuberculosis challenge
Boosting BCG vaccinated animals with rMVA.acr provides superior protection than
BCG vaccination alone in guinea pigs with 9 fold and 34 fold fewer bacilli in lungs
and spleen, respectively.
Log10CFU/lung
LUNG
Log10CFU/spleen
SPLEEN
(1.63)
(2.52)
(0.89)
(0.85)
(2.60)
(3.94)
(1.34)
(1.49)

24. Boosting BCG vaccinated animals with rMVA.acr reduces the
pathological damage to the organs of guinea pigs
•Sham immunized animals
exhibited maximal damage
with numerous necrotic
tubercles.
•Animals with BCG
vaccination and those
boosted with rMVA.acr
exhibited minimal damage
with very few small tubercles
and normal size.
LUNGSPLEENLIVER
SALINE BCG B/rMVA.acr 107
SCORESCORESCORE

25. Histopathological changes in the lungs and liver of vaccinated animals
following M. tuberculosis challenge
While sham immunized animals exhibited 70% and 20% granuloma in lung and liver
respectively, there counterparts from both groups of vaccinated animals exhibited less
than 20% granuloma in lungs and negligible damage in liver.
Boosting BCG vaccinated animals with rMVA.acr reduces the
histopathological damage to the organs of guinea pigs
SALINE BCG B/rMVA.acr 107
LUNGLIVER

26. PHASE I PHASE IIa PHASE IIb PHASE III
SUBUNIT
 ID93 + GLA-SE
(Antigens: Fusion of Rv2608,
Rv3619, Rv3620, and Rv1813)
IDRI,Aeras
VIRAL VECTOR
 Ad5Ag85A
(Antigen: Ag85A)
McMaster, CanSino
LIVE ATTENUATED
 MTBVAC
(M.tuberculosisΔphoPΔfadD26)
TBVI, Zaragoza, Biofabri
SUBUNIT
 Hybrid 1 + IC31
(Antigens: Fusion of Ag85B and
ESAT6)
SSI, TBVI, EDCTP, Intercell
 Hybrid 4 + IC31
(Antigens: Fusion of Ag85B and
TB10.4)
SSI, Sanofi-Pasteur, Aeras,
Intercell
 Hybrid 56 + IC31
(Antigens: Fusion of Ag85B,
ESAT6 and Rv2660c)
SSI, Aeras, Intercell
SUBUNIT
 M72 + AS01E
(Antigens: Fusion of Rv1196 and
Rv 0125)
GSK, Aeras
VIRAL VECTOR
 MVA85A/ AERAS-485
(Antigen: Ag85A)
Oxford, Aeras
FRAGMENTED M.tuberculosis
 RUTI
(Detoxified M.tuberculosis in
liposomes)
Archivel Farma, S.L.
WHOLE CELL
 M. Vaccae
AnHui Langcom, China
 M. indicus pranii
DBT, Govt. of India, Cadila
Pharmaceuticals
LIVE RECOMBINANT BCG
 VPM1002
(rBCG having urease C deletion
and expressing listeriolysin )
Max Planck, VMP, TBVI, SII
Prime-boost
Prime
Immunotherapeutic
VaccinationStrategy
WHOLE CELL
 Dar-901
(M.vaccae)
Dartmouth Geisel School of
Medicine
VIRAL VECTOR
 Crucell Ad35/AERAS-402
(Antigens: Fusion of Ag85A, Ag85B
and TB10.4)
Crucell, Aeras
TB vaccine clinical trial pipeline

27. Probiotics against tuberculosis - Nyaditum resae
It is believed that tuberculosis develops because the body reacts to the invading
bacteria with an overly intense inflammation which ends up being harmful to the
body itself.
The probiotic Nyaditum resae
(Mycobacterium manresensis extract), a
product of Manremyc, is administered in
pills as a food supplement that generates
natural tolerance in the body against the
TB infection.
It is able to induce a balanced immune
response that diminish the risk of acquiring
TB and the inflammation generated is
much less.
The final dose consists of a heat killed culture of an environmental mycobacteria
belonging to Mycobacterium fortuitum complex plus excipient. When
administered daily for 14 days, the preclinical assays demonstrated that it is able
to stop the progression towards active TB.

28. Probiotics as Vaccine adjuvants
Poor immunogenicity and limited ability to induce mucosal and cell mediated immunity are problems
often associated with live attenuated, killed, inactivated or subunit vaccines,
Thus, enhancing mucosal immunity offers an effective strategy for preventing pathogen adhesion to
host tissues.
Pathogens such as S. pneumoniae, C. diphtheriae, B. pertusis, H. influenza, enterotoxigenic E.coli and
rotavirus all enter the host via respiratory or gastrointestinal mucosa and therefore, these infections
might be subverted if mucosal immunity is enhanced.
Summary of Probiotic Adjuvant effects to Parenterally administered vaccines.Probiotics have been
shown to have pleiotropic
effects on innate and
adaptive immune
responses including
modulation of DCs, T and B
cells as well as antibody
and cytokine production.
There are numerous
evidences from human
clinical trials wherein the
use of probiotics have
shown promising effects on
vaccine induced immunity.

29. Acknowledgements
:: Financial Resources ::
Department of Biotechnology
Government of India
Vikram
Ramandeep
Tanupriya
Priti
Vibha
Garima
Vineel
Rupangi
Praveen
SeemaAnil Koul
Amit
Akshay
Aparna
Ritika
Prachi
Priyanka
Ruchi
Vivek
Neeraj
Bappaditya
Nisheeth
Deepak
Murali
Sujoy
V.M. Katoch
U.D. Gupta
P.R. Narayanan
V.D. Ramanathan