Anil K. TyagiDepartment 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”

• 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.

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.

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.

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.

Functions of Probiotics….

Vaccine adjuvant

Increase of the lactoseTolerance and digestion

PROBIOTICS

Stimulation of the immuneresponse

Positive influence in theIntestinal 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 microfloraafter antimicrobial

therapy

Treatment and prevention of acute

diarrhea

As new vaccines

• 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

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"

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

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.

Approaches

• Subunit vaccines• Recombinant BCG vaccines

• Attenuated M.tuberculosis strains• Atypical mycobacterial strains• DNA vaccines• Heterologous Prime Boost Approach• Immunotherapeutic TB Vaccines

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

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

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.

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 Boosting5x105 CFU of BCG 6 wks 100µg

DNA acr10 wks

Euthanasia6 wks ~ 50 - 100

CFU ofMtb

ChallengeImmunization Boosting5x105 CFU of BCG 6 wks 100µg

DNA acr10 wks

Euthanasia6 wks ~ 50 - 100

CFU ofMtb

Challenge

Gross Histopathology

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*, 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

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Spleen

Lung

NormalSal/BD- 2.31, BCG/BD-1.37

Sal/BD-3.44, BCG/BD – 1.96

***

***

φφφ

φφφ

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.

Gran

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ma %

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Live

rL

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g

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ulo

ma %

B/D Normal

10 weeks post infection

Effect of ‘BCG prime - DNAacr boost’ (B/D) regimen on granulomatous pathological lesions

55

3530

5

35

------- ~5 -------

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 Boosting5x105 CFU of BCG 6 wks 100

DNA acr Euthanasia6 wks ~ 50 - 100

CFU ofMtb

ChallengeImmunization Boosting5x105 CFU of BCG 6 wks 100

DNA acr16 wks

Euthanasia6 wks ~ 50 - 100

CFU ofMtb

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/V0

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Long term influence of ‘BCG prime - DNA boost’ B/D regimen on histopathology

Gran

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Saline

BCG B/D B/V0

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 Boosting5x105 CFU of BCG 6 wks 100

DNA acr Euthanasia6 wks ~ 50 - 100

CFU ofMtb

ChallengeImmunization Boosting5x105 CFU of BCG 6 wks 100

DNA acr16 wks

Euthanasia6 wks ~ 50 - 100

CFU ofMtb

Challenge 16 weeks post infectionμg

IL-12

IL-10

PROTECTION INCREASES

CY

TO

KIN

E P

RO

PO

RT

ION

IN T

HE

LU

NG

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

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

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 TKRCloning of α-crystallin gene

Clonal purification of the recombinant clone to remove the wild type MVA

Excise the GFP gene

Uni

nfec

ted

MVA

.WT

rMV

A.A

cr

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

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.tuberculosischallenge

10-30 bacilli Euthanasia Saline (control)

BCG

B/rMVA.acr 107

B/MVA.WT

Saline

12 wks 6 wks

M.tuberculosischallenge

10-30 bacilli Euthanasia BCG

5X105 CFU (i.d.)

6 wks 6 wks

M.tuberculosischallenge

10-30 bacilli Euthanasia BCG

5X105 CFU (i.d.)

6 wks

rMVA.acr1X107 PFU (i.m.)

6 wks 6 wks

M.tuberculosischallenge

10-30 bacilli Euthanasia BCG

5X105 CFU (i.d.)

6 wks

rMVA.acr1X108 PFU (i.m.)

6 wks 6 wks

M.tuberculosischallenge

10-30 bacilli Euthanasia BCG

5X105 CFU (i.d.)

6 wks

MVA.WT1X108 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

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.

Log10

CFU/lung

LUNG

Log10

CFU/spleen

SPLEEN

(1.63)

(2.52)

(0.89)

(0.85)(2.60)

(3.94)

(1.34)

(1.49)

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.

LUNGSPLEEN

LIVER

SALINE BCG B/rMVA.acr 107

SCORESCORE

SCORE

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

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. VaccaeAnHui Langcom, China

M. indicus praniiDBT, Govt. of India, CadilaPharmaceuticals

LIVE RECOMBINANT BCG

VPM1002(rBCG having urease C deletion and expressing listeriolysin )Max Planck, VMP, TBVI, SII

Prime-boost

Prime

Immunotherapeutic

Vaccination Strategy

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

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.

Probiotics as Vaccine adjuvantsPoor 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.

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

MuraliSujoy

V.M. Katoch

U.D. Gupta

P.R. Narayanan

V.D. Ramanathan