Recent advances on the complex and multifaceted T memory and effector immunity elicited to S. Typhi in humans

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1 Recent advances on the complex and multifaceted T memory and effector immunity elicited to S. Typhi in humans Marcelo B. Sztein, M.D. Immunology Group Center for Vaccine Development University of Maryland 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Dhaka, Bangladesh March 1, 2013

2 Lack of known immunological correlates of protection in typhoid fever Ab to S. Typhi antigens (e.g., Vi, LPS O) are likely to play an important role in defense against typhoid bacilli when they are extracellular. In contrast, since S. Typhi persists intracellularly, thereby avoiding destruction by Ab and C, CMI is expected to be essential in eliminating S. Typhi from the infected cells. Both adaptive immune mechanisms (CMI & Ab) are expected to provide critical support to innate immunity in the mucosal microenvironment and elsewhere

3 Effector Ab responses to S. Typhi 1. Serum IgG and IgA Abs to S. Typhi antigens LPS O, flagella, Vi, OMPs, hsp, others? (Elisa) Opsonophagocytosis, ADCC, bactericidal (Functional) 2. IgG and IgA ASC to S. Typhi antigens in circulation LPS O, flagella, Vi, OMPs, others? 7-10 days after immunization (longevity in the gut and other tissues unknown) 3. Anti-S. Typhi specific secretory IgA (SIgA) in intestinal lavage fluids and stools of subjects exposed to S. Typhi 4. B memory cells (e.g., LPS, Vi)

4 Immunological correlates of protection in typhoid fever: Ab summary It is not known whether Ab to common S. Typhi antigens, particularly to O, H and Vi, particulalry those with defined functional activities, actually (1) Mediate protection, (2) Act in conjunction with other innate and adaptive responses or (3) Serve as a surrogate for the presence of other more dominant protective immune responses (e.g., CMI) that will eventually lead to the elimination of this intracellular bacteria from the host

5 Immunological correlates of protection in typhoid fever: Hypothesis Both CMI and Ab responses play central roles in protection in typhoid fever and are elicited following immunization with attenuated S. Typhi vaccine candidates

6 Vaccine Development The never ending search for the optimal balance Immunogenicity Reactogenicity What responses?

7 Attenuated S. Typhi vaccine strains Organism Strain Deletions Mutations Metabolic Pathways S. Typhi CVD 908 ΔaroC ΔaroD Biosynthesis of aromatic aa S. Typhi CVD htra aroc ΔaroC arod ΔaroD htra ΔhtrA S. CVD htra Typhi CVD ΔaroC guaba arod ΔaroD htra ( fragc of TT) ΔhtrA, viab Biosynthesis of aromatic aa heat - shock protein Biosynthesis of of aromatic guanine aa heat - shock protein Constitutively expresses Vi Chemical S. Typhi Ty21a Many mutagenesis

8 Key Effector CMI to S. Typhi in orally immunized subjects (1) Effector responses to S. Typhi-infected targets: Cytotoxic T lymphocytes (CTL) activity ( 51 Cr-release assays; granzyme; CD107 staining by flow cytometry) IFNγ production (TNF-α, others) Mediated by both CD8 + (dominant) and CD4 + cells CD8+ CTL activity restricted by: Classical class Ia molecules (HLA-A, B, C) Non-classical class-ib molecules (HLA-E)

9 Key Effector CMI to S. Typhi in orally immunized subjects (2) Proliferation and predominant type-1 cytokine responses to soluble S. Typhi antigens (e.g., flagella) IFNγ, TNFα, IL-10 in the absence of IL-4, IL-5 & IL-6 IFNγ produced predominantly by CD4 + cells Homing to mucosal and non-mucosal tissues: IFN-γ production by central and effector memory T subsets that express, or not, the gut homing molecule integrin α 4 /β 7 Presence of long-term multifunctional HLA-E-restricted CD8 + cells co-expressing IFN-γ, TNF-α and CD107

10 Detailed CMI studies to S. Typhi-infected autologous cells in Ty21 vaccinees 1. Define the kinetics of CMI to S. Typhi-infected autologous cells elicited by Ty21a in humans 2. Study IL-17A responses elicited in Ty21a vaccinees IL-17A is a pro-inflammatory cytokine produced by CD4+ and CD8+ T cells. Recently shown to play a key role in mucosal immunity Measure the multi-functionality of T cell responses following Ty21a immunization

11 CMI to S. Typhi-infected autologous cells in Ty21 vaccinees: Multi-functional T cells Why study multi-functionality of the T cell responses following Ty21a immunization? Studies have shown that multifunctional T cells, those producing 2 or more cytokines simultaneously, might be critical effectors in protection from infection in animals and humans (e.g., HIV, Mtb) Technological advances and unsupervised flow ctyometry analysis packages enable the study of all possible combinations of many cytokines to define multi-functional CD8+ T cell subsets

12 Experimental Design: In vitro stimulation with S. Typhi-infected targets Target Cells Autologous EBV-LCL, AEH, blasts Effector cells Ex vivo PBMC collected before and 1, 2, 4, 8, 10, 14, 21, 28, 60, 90, 180, 360 days after Ty21a immunizaiton CD8+ T effectors (CTL, cytokines) CD4+ T effectors (cytokines) S. Typhi infection Others? + γ-irradiation hrs Flow cytometry (Tm subsets, CD107a, IFN-γ, TNF-α, MIP-1β, IL-17A, IL-2, etc)

13 Experimental Design: Flow cytometry gating TABLE 1. Cell phenotypes Phenotype "Dump" channel Dead cells Monocytes B cells EBV-LCL T cell subset Marker Yevid CD14 CD19 CD45 Naïve CD45RA + CD62L + Central memory (T CM ) CD45RA - CD62L + Effector memory (T EM ) CD45RA - CD62L - Effector memory (T EMRA ) CD45RA + CD62L - SSC-A Sequential gating strategy Lymphocytes Singlets No Gate Gate: R1 R1 : 19% R1: 43.89% FSC-A FSC-A R2: 95.28% R2 : 92% FSC-H Live T cells Gate: R1&R2 R201: 75.70% R3 : 75% Yevid-CD14-CD19-CD45 Degranulation (CTL) LAMP-1 Cytokines/chemokines CD107a IFN-γ TNF-α IL-2 IL-17A MIP-1β CD8 CD8 PerCP-Cy5-5-A live CD8+ T cells Gate: R1&R2&R3 R4: 23.47% R4 : 24% CD3 BV650-A CD3 CD62-L CD8+ subsets Gate: R1&R2&R3&R4 R6: 13.17% R7: 56.02% R8: 15.53% R9: 8.56% T CM T N T EM T EMRA CD45-RA CD107a/b IFN-γ TNF-α IL-2 IL-17A MIP-1β

14 IL-17A PerCP-Cy5-5-A TNFa APC-Alexa700-A Tcm 0.18% TNFa APC-Alexa700-A Tn R36: 0.01% Tn 0.01% TNFa APC-Alexa700-A MIP1b APC-A Temra 9.60% IL-2 PE-Cy7-A TNFa APC-Alexa700-A R33: 14.33% Temra 14.33% IFN-γ IFNg APC-Cy7-A MIP1b APC-A R38: 15.70% Temra 15.70% IL-2 Tem R28: 9.60% IFNg APC-Cy7-A Temra 6.40% R37: 26.81% 26.81% Tem 23.39% Tem 14.55% MIP1b APC-A Tcm IFNg APC-Cy7-A R35: 0.18% 0.00% MIP1b APC-A IFNg APC-Cy7-A IL-2 PE-Cy7-A IL-17A Tn 0.00% 0.06% R34: 8.51% total8.51% Temra 2.29% IFNg APC-Cy7-A IL-10 FITC-A Tem 9.53% IL-17A PerCP-Cy5-5-A IL-10 IL-2 PE-Cy7-A R29: 7.42% total 7.42% R27: 14.55% 0.30% Temra R13: 0 Tn0.06% Tem 3.88% Tcm 0.08% IL-17A PerCP-Cy5-5-A R17: 3.88% IL-10 FITC-A IL-2 PE-Cy7-A 0.22% Tem Tcm 0.05% R16: 0.00% R12: 0 IL-17A PerCP-Cy5-5-A 0.00% Tn IL-10 FITC-A IL-2 PE-Cy7-A 0.00% R11: 0 total 4.65% IL-17A PerCP-Cy5-5-A Tcm 0.02% IL-10 FITC-A CD69 Tn CD8+ TEMRA R10: % CD8+ TEM Tcm CD8+ TN IL-10 FITC-A CD8+ TCM 0.08% total 3.04% total 1.24% Total CD8+ R9: 0.0 total Cytokines/chemokines are secreted primarily from the CD8+ TEM & TEMRA subsets TNFa APC-Alexa700-A TNF-α MIP1b APC-A MIP-1β

15 IL-17A is secreted by CD8+ T EM cells in response to Ty21a immunization in humans 5 53s 5 54s % positive cells % positive cells Days post immunization Days post immunization 5 50s % positive cells IL-17A Days post immunization

16 Kinetics of net CD8+ T EM responses to stimulation with S. Typhi-infected autologous cells in Ty21a vaccinees % positive cells s IL-10 IL-17A IL-2 IFN-γ TNF-α MIP-1β Days post immunization Immunization

17 IL-17A+ multifunctional CD8+ T EM following exposure to S. Typhi-infected autologous cells 4 53s IL-17A+ IFN-γ+ TNF-α+ IL-17A+ IL-2+ IFN-γ+ TNF-α+ IL-17A+ IFN-γ+ TNF-α+ MIP-1β+ IL-17A+ IL-2+ IFN-γ+ TNF-α+ MIP-1β+ % positive cells Days post immunization

18 Conclusions (I) Ty21a-immunized volunteers exhibited CD8+ T EM and T EMRA cell responses following stimulation with S. Typhi-infected autologous EBV-LCL. Multifunctional CD8+ T cells are present in response to Ty21a immunization. Multifunctional T cells have been reported to correlate with lack of disease progression in HIV, have been identified as a potential correlate of protection for Leishmania major immunization in a mouse model, etc.

19 Conclusions (II) This is the first demonstration of IL-17A production in response to Ty21a immunization IL-17A secreting cells have been shown to play a role in mucosal immunity IL-17A is secreted from multifunctional cells that coproduce IL-2, IFN-γ, TNF-α, and/or MIP-1β There is heterogeneity in the kinetics of the immune responses to Ty21a immunization. Bi-phasic & tri-phasic responses to S. Typhi-infected autologous EBV-LCL are typical. Thus, evaluating a single time point may fail to accurately evaluate responsiveness

20 S. Typhi Peyer s Patch CD8 M Ag uptake & presentation Mesenteric lymph nodes T H 1 T & B activation M cells B act ASC Ab B M T act DC T H 2 GALT Lumen Epithelium T CD8 H 1 Proliferation PC T H 2 Th1 T H 1 cytokine B pattern: IFN-γ,TNF-α, IL-10, IFN-γ IL-17, T H 2 B FDC IL-4, IL-5, IL-6 Lamina CD8 T PC B H 1 CTL [classical class Ia- and Propria class Ib (HLA-E)-restricted] B T M [ T EM T IL-4 IL-5 IL-6 Cytokines EMRA T (e.g., CM ] IFN-γ, TNF-α) Other Chemokines tissues Biliary tract, other organs? Thoracic duct Bacteremia Immune cells path? S. Typhi path Peripheral circulation IELs CD8 CD8 T EM T EMRA T CM expressing [or not] integrin α4/β7 M S-IgA LPLs Common Mucosal Immune System? RES spleen, liver, bone marrow, LN, intracellular replication

21 The question remains: Which of these complex and heterogeneous CMI responses, if any, are associated with protection from typhoid fever?

22 To answer this question, and to better understand typhoid disease, we recently initiated a collaboration with Dr. Pollard and his team at Oxford who have reestablished a human challenge model with wild-type S. Typhi

23 Multiphasic kinetics of CMI responses by CD8+ T EM to S. Typhi-infected AEH-cells (HLA-E-restriction) This volunteer (Ox6) did not develop typhoid fever (No TD) CD107a IFN-γ TNF-α ABX 5 ABX 8 ABX % positive cells IL-2 IL-17 MIP-1β ABX 0.25 ABX 6 ABX Days post-challenge ABX : antibiotics

24 Multifunctional CD8+ T EM to S. Typhi-inf B- LCL in wild-type S. Typhi-challenged subjects Peak d No TD: Ox4 Ox6 Ox7 Ox8 Ox12 mean TD: Ox3 Ox5 Ox9 Ox10 Ox11 mean % positive cells % positive cells CD107a IFN-γ TNF-α IL-2 IL-17A MIP-1β CD107a IFN-γ TNF-α IL-2 IL-17A MIP-1β

25 Mass cytometry Mass cytometry (CyTOF), a transformational flow cytometry technology to measure human immune responses by simultaneously evaluating >35 parameters/cell

26 S. Typhi appears to be such an effective pathogen, at least in part, by being exquisitely stealth. Thus, identifying the effective immunological CoP among a sea of nonprotective or downregulatory immunity might hold the key for the development of more effective vaccines.

27 Identification of the precise immunological correlates of protection (either mechanistic or non-mechanistic), can significantly advance the development of broad spectrum vaccines for enteric fevers (e.g., S. Paratyphi A, S. Paratyphi B)

28 Acknowledgements Center Vaccine Development, UMB Immunology Group Monica McArthur Stephanie Fresnay Franklin Toapanta Rosangela Mezghanni Rezwan Wahid Cathy Storrer Regina Harley Clinical studies Myron Levine Robin Barnes CVD Oxford Team Andrew J Pollard Claire Jones Claire Waddington Thomas Darton Christoph Blohmke Gordon Dougan Jeremy Farrar Paul Langford Brian Angus Derrick Crook Stephen Lockhart Supported by grants U19 AI (UMB-CCHI) and R01 AI-36525