Steffen Stenger Medical Microbiology and Infection Control University Hospital Ulm Mytb Lab

Steffen Stenger Medical Microbiology and Infection Control University Hospital Ulm MyTB Lab

Mycobacterial Lipids as Vaccine Antigens- What have we achieved- and what not?

proteins surface glycoprotein

perforin Mycolic acid

LA arabinogalactan M

peptidoglycan P

granulysin plasma membrane Presentation of exogenous microbial antigens

conventional T-cells donor unrestricted T-cells (DURTs) CD4+ T-cells CD8+ T-cells

CD3 CD3 CD3

TCR  TCR  TCR  CD4 CD8

15 aa 9 aa lipid or peptide peptide lipoglycan

MHC MHC  m  m CLASS II CLASS I 2 CD1 2

Antigen-presenting cells Glycolipids as Vaccine Antigens: Advantages

T-cell - group 1 CD1-molecules (CD1a,b,c) are non-polymorphic (broad population coverage) glycolipids CD1 - lipids are presented by dendritic cells (efficient T-cell priming)

- adjuvant activity of lipids (mediated by DC-SIGN, TLRs, MINCLE) What have we achieved?

- Identification of mycobacterial (glyco-)lipid antigens (Glyco-)lipids presented by group 1 CD1-molecules

CD4-, CD8- TCR /gd autoreactive CD1b,c Porcelli, Nature 1989

CD4-, CD8- TCR  mycobacterial lipid CD1b Porcelli, Nature 1992

CD4-, CD8- TCR  mycolic acid CD1b Beckman, Nature 1994

CD4-, CD8- TCR  lipoarabinomannan CD1b Sieling, Science 1995

CD8+ TCR  mycobacterial lipid CD1b Stenger, Science 1997 n.d. TCR  sphingolipids (autoreactive) CD1 Shamshiev, Immunity 2000

CD4-CD8- TCR  isoprenoid phosphoglycolipids CD1c Moody, Nature 2000 transfectants TCR  mycobactin (lipopeptides) CD1a Moody, Science 2004

CD8+ TCR  diacylated sulfoglycolipid CD1b Gilleron, J Exp Med, 2004

CD4+ TCR  glycerolmonomycolate CD1c Layre, Chem Biol, 2009 The CD1 Antigen Presentation Pathway

Saposin C

CD1e

Winau, Nat. Immunol., 5: 169: 2004 De la Salle, Science, 310: 1321, 2005 Jullien, Stenger, Modlin, J. Clin. Invest., 99:2071, 1997 What have we achieved?

- Identification of mycobacterial (glyco-)lipid antigens

- Molecular mechanisms of antigen presentation and TCR recognition

structural requirements and CD1 trafficking Sugita, Science, 273: 349, 1996 Sugita, J. Immunol, 159: 2358, 1997 Jackmann, Immunity, 8: 2358: 341, 1998 Winau, Nat. Immunol., 5: 169: 2004 de la Salle, Science, 310: 1321, 2005 Gras, Nat. Comm., 27: 13257, 2016

- Functional characterization of group 1 CD1-restricted T-cells Response of „total lipid“-reactive donors to defined lipids

100

80 (%)*

60 donors

40 reactive

Total LAM GroMM PIM Ac2SGL Mycolic Lipid Acid

* IFN-g release threefold above background Response of „total lipid“-reactive donors to defined lipids

100

80 (%)*

60 donors

40 reactive

Total LAM GroMM PIM Ac2SGL Mycolic Lipid Acid

* IFN-g release threefold above background Antimicrobial activity of lipoarabinomannan-specific PBMC

PBMC CD1+ APC adherent cells LAM GM-CSF/IL4 18 hrs. 3 days IFN-g-PE

cell sorting (IFN-g-capture)

non-adherent cells IL-2 (10IU/ml)

unsorted IFN-g negative IFN-g positive LAM-specific T cells mediate antimicrobial activity

+ 8 ) 6 6 *

CFU (10 CFU 4

no T-cells 48hrs 2 unsorted PBMC IFN-g-negative IFN-g-positive (LAM-specific)

0 hrs 48 hrs

Time after Infection What is the Mechanism of Antibacterial Activity? Granulysin

15kDa 9kDa

granulysin perforin overlay perforin

Stenger et. al., Science, 1997 Stenger et. al., Science, 1998 Stenger et al., J. Immunol, 2000 Ochoa et al. , Nat. Med., 2001 Stenger et al., Science, 2001 granulysin Thoma et al., Science, 2003 Stegelmann et al., J. Immunol, 2008 Delivering three punches to knock out intracellular bacteria

hypothesis

Nami-Mancinelli & Vivier, Cell, 157: 1251, 2014 LAM-specific polycytotoxic T-cells: Co-expression of perforin, granzyme B and granulysin

CD3 granzyme B perforin granulysin

Monocytotoxic (M-CTL)

Dicytotoxic (D-CTL)

Polycytotoxic (P-CTL) Frequency of LAM-reactive polycytotoxic T cells in susceptible and protected individuals

PBMC Susceptible IFN-g + IFN-g +/granulysin+

0.9% 60% 4% 23%

SSC

SSC SSC + perforin 61% 12% CD1+ APC IFN-g granulysingranulysin granzyme B

A. Protected + IFN-g + IFN-g +/granulysin+ 0.7% 87% 7% 67%

LAM

SSC

SSC perforin

12% 14% P IFN-g granulysin granzyme B The frequency of LAM-specific polycytotoxic T cells is associated with protection against tuberculosis

Protected donors Susceptible donors (n=38) (n=51)

granulysin + + + + granzyme B + - + - perforin + - - +

Busch et al., Am J Resp Crit Care Med, 149: 345, 2017

Balin et al., al., et Balin PresortCD3 72.5 Polycytotoxic Sci in the Immunol, 3, eaat7668, eaat7668, 3, Immunol, 2018 8.9 CD3 + CD8 6.3 7.1 + NKG2c CD8 CD3 46.4 T + pos CD8 - cells are highly enriched highlyare cells and + NKG2A 11.8 26.5

7.3 NKG2c (activating) NKG2a + C NKG2a(inhibitory) - CD3 CD3 neg + polycytotoxic 3.5 CD8 + T CD8 2.7 - + cells subset 4 + NKG2C 89.7 + A - M P D N - - - - CTL CTL CTL CTL Polycytotoxic T cells have stronger activity against Mtb as compared to other CD8+ subsets 80 80

c 60

60 a

o 40 r 0.7348

m 2

t 40 i 20 r = 0.73

% 0 0 20 40 60 80 100 20 % P-CTL

80 antimicrobial activity(%) antimicrobial

< 3 c 60

a i 2 b r = 0.77 NKG2A- NKG2A+ NKG2A- o 40 r 0.7709

NKG2C- NKG2C- NKG2C+ m

t i 20

% frequency of polycytotoxic T-cells 0 0 20 40 60 80 100 % N-CTL Balin et al., Sci Immunol, 3, eaat7668, 2018 Lipoarabinomannan specific T cells - are detectable in 2/3 of subjects responding to mycobacterial lipids

- produce Th1-cytokines (IFN-g, TNF-)

- kill intracellular Mycobacterium tuberculosis

- include a polycytotoxic subset that is associated with protection in humans and has profound antimicrobial activity against Mtb

Polycytotoxic T cells What have we achieved?

- Identification of mycobacterial (glyco-)lipid antigens

- Molecular mechanisms of antigen presentation and TCR recognition

structural requirements and CD1 trafficking Sugita, Science, 273: 349, 1996 Sugita, J. Immunol, 159: 2358, 1997 Jackmann, Immunity, 8: 2358: 341, 1998 Winau, Nat. Immunol., 5: 169: 2004 De la Salle, Science, 310: 1321, 2005 Gras, Nat. Comm., 27: 13257, 2016

- Functional characterization of group 1 CD1-restricted T-cells

- Detection and quantification of glycolipid-specific T-cells by CD1 tetramers Kasmer, J Exp Med, 208: 1741, 2011; Ly, J Exp Med, 210: 729, 2013; Kasmar, J Immunol, 19: 4499, 2013; Layton, J Immunol Meth, 458: 44, 2018 Hydrophobic antigens

Activation of effective adaptive immune response

TLR Cytotoxic T-cell CD1 Optimize the deliveryActivation and immunogenicity of LAM

Release of Perforin , Activation of innate Granulysin and Granzyme B immune response

Tuberculosis Gilleron M. et al.: J Exp Med 2004 Bastian M. et al.: J Immunol 2008 Liu P. et al: Science 2006 Bruns H. et al.: J Clin Invest 2009 Freeze fracture and TEM of LIPLAM Nanoparticle Tracking Analysis of LIPLAM

size approx. 280 nm

n = 2 Liposomes promote LAM-specific T cell responses

LIPLAM 800 ***

640 SSC

0.9% /ml)

480

(

g -

IFN-g IFN 320 LAM

160 SSC

<32 0.1%

LIPLAM LAM LIP

representative result, n=5 IFN-g Kallert et al. Tuberculosis, 2015 Toll like receptor triggering of a vitamin D-mediated human antimicrobial response

Pam3Cys

Pro-VitD

D3H

VitD vitamin D VitD receptor RIP retinoid X cathelicidin receptor (LL-37) VitD

Genes VDR-RE

Liu et al., Science, 311: 1770, 2006 TNF- -release (pg/ml) Kennerknecht Kennerknecht et al.,  1000 1500 500 0 promotes Pam 3 Med Cys H56 Micobiol - cytokine incorporation incorporation Immunol Pam H56 , in press , 3 Cys release into by

IL-12-release (pg/ml) H56/CAF01 human human 10.000 15.000 5000 0 macrophages H56 liposomes Pam H56 3 Cys What have we achieved?

- Identification of mycobacterial (glyco-)lipid antigens

- Molecular mechanisms of antigen presentation and TCR recognition

structural requirements and CD1 trafficking Sugita, Science, 273: 349, 1996 Sugita, J. Immunol, 159: 2358, 1997 Jackmann, Immunity, 8: 2358: 341, 1998 Winau, Nat. Immunol., 5: 169: 2004 De la Salle, Science, 310: 1321, 2005 Gras, Nat. Comm., 27: 13257, 2016

- Functional characterization of group 1 CD1-restricted T-cells

- Formulation of LAM into adjuvant-containing liposomes Hiromatsu, J Immunol, 169: 330, 2002; Larrouy-Maumus, Gilleron, Puzo, Vaccine, 35: 1395, 2017; Kallert, Tuberculosis, 95: 452, 2015 Kennerknecht, Med Micro Immunol, in press ……..and what not

demonstrate protective role of glycolipid-specific T-cells against tuberculosis in vivo

technology: large scale synthesis of glycolipid antigens pathogen: availability of lipid-deficient mycobacterial strains host: lack of a suitable preclinical animal model Limitations of the mouse model for confirming and understanding results derived from human studies

murine APC do not express group 1 CD1 molecules and hence do not present lipid antigens to T lymphocytes

mice do not have a granulysin homologue

murine cathelicidin (CRAMP) does not have a vitamin D response element (Wang, J Immunol, 173: 2909, 2004)

mouse neutrophils are deficient in the expression of defensins (Eisenhauer et al., Infect Immun, 60: 3446, 1992)

mice do not form “human-like“ tuberculous (caseous) granulomas

murine tuberculous granulomas are not hypoxic (Via, Infect Immun, 76: 2333, 2008)

nitric oxide is a key effector molecule for antimicrobial activity in mice, but it`s role in humans is unclear CD1 a,b,c transgenic mice

Mycobacterial lipids and TB infection induce

Mtb-lipid-specific T cell responses Felio, J Exp Med., 206: 2497, 2009

TB infection induces polyfunctional mycolic acid specific T cells that accumulate in lung granulomas and contribute to protection in mice transgenic for the mycolic acid specific T cell receptor Zhao, Elife, doi 10.7554/elife.0825, 2015

T-cell activation is promoted by aerosol delivery of mycolic acid via micellar nanoparticles Shang, Front. Immunol., 9: 2709, 2018 Guinea pigs

- Lipid immunization of guinea pigs induces CD4-negative, cytotoxic T-cells that recognize Mtb-infected cells Hiromatsu, J Immunol, 169: 330, 2002

- Lipid vaccination reduces the bacterial load and pathology in aerosol tuberculosis challenge model Dascher, Int Immunol, 15: 915,2003

- Ac2SGL and PIM vaccination induces protection, albeit less efficiently than BCG Larrouy-Maumus, Vaccine, 35: 1395, 2017) Non human primates Mycobacterial Lipids as Vaccine Antigens- What have we achieved- and what not?

- strong evidence for a protective phenotype of glycolipid-specific CD1-restricted T-cells in vitro

- Lipid vaccination induces T-lymphocytes with a protective phenotype in transgenic mice and guinea pigs

- Lipid vaccination induces protection from TB infection in guinea pigs, which is not as efficient as BCG

Major challenge is the development of a preclinical model to allow for testing of basic parameters: formulation, route of application, dose, adjuvant