B and T cell vaccine responses : what a vaccinologist should know

and T cell vaccine responses : what a vaccinologist should know Claire-Anne Siegrist Center for Vaccinology and Neonatal Immunology University of Geneva Switzerland

= Vaccine immunology

What? How? When? Why? Why not? So what? = Vaccine immunology

What happens first after immunization?

What happens first after immunization? Detection of danger signals PAIN! 1. Innate responses Injection-site innate activation = Cell recruitment and activation = inflammatory focus

What happens first after immunization? Detection of danger signals PAIN! 1. Innate responses Injection-site innate activation = Cell recruitment and activation = inflammatory focus Cell migration to the draining LN

Migration of towards the draining lymph nodes 1. Non live vaccines uptake and transport by Antigen-presenting cells and/or freefluid diffusion of soluble : Deltoid axillary Thigh inguinal Mostly local and unilateral lymph node activation! Which are the administration rules for multiple vaccines to be given during the same visit?

Migration of towards the draining lymph nodes 1. Non live vaccines uptake and transport by Antigen-presenting cells and/or freefluid diffusion of soluble : Deltoid axillary Thigh inguinal Mostly local and unilateral lymph node activation! 2. Live vaccines Minimal local retention / reaction Replication dissemination - pathogen-specific pattern Multifocal lymph node activation stronger responses!

How do vaccine induce protection?

How do vaccine induce protection? 2. Antibody production 1. Innate responses 3. T cell responses Adaptative responses

How do vaccine induce protection? 1. Vaccine antibodies : inding to the enzymatic active sites of toxins or preventing their diffusion Neutralizing viral replication, e.g. preventing viral entry into cells Promoting opsonization and phagocytosis by macrophages and neutrophils Activating the complement cascade - Early reduction of pathogen load - Clearance of extracellular pathogens

Essential role for Antibodies in most current vaccines!

Vaccines as seen by the immune system cell epitopes and/or T cell epitopes and Danger signals activating the innate immune system initiating and shaping and T cell responses

Exposure to cell epitopes induces a wave of short-lived Ab-secreting plasma cells 1 PS Lymph nodes arrival by lymphatic vessels into the marginal sinus capture by subcapsular sinus macrophages and translocation into the marginal cell zone cell zone T cell zone T cell zone

Exposure to cell epitopes induces a wave of short-lived Ab-secreting plasma cells 1 PS Lymph nodes arrival by lymphatic vessels into the marginal sinus entrance into the marginal cell zone inding to cells having specific surface receptors (cr) cell activation and proliferation

Exposure to cell epitopes induces a wave of short-lived Ab-secreting plasma cells 1 Spleen / lymph nodes arrival by lymphatic vessels into the marginal sinus PS entrance into the marginal cell zone cell activation and proliferation IgM Differentiation in short-lived plasma cells lood inding to cells having specific surface receptors (cr) IgM, IgA Rapid differentiation of mostly marginal zone cells into short-lived (weeks-months) plasma cells = extrafollicular response Modest + transient Ab responses

Antibody Titer (, log10) Induction of primary Ab responses to cell epitopes 2 1 3 4 0 10d 30d time Antigen exposure 1. cell response (extrafollicular) 2. Peak titers ~ 2-4 weeks after immunization. 3. Short PC life span in lymph nodes (weeks): rapid decline of Ab titers. 4. Rapid return (months) to baseline levels. No induction of immune memory

What happens after repeat exposure to the same cell epitopes? Lymph nodes 1 2 PS Lymph nodes PS??? lood??? IgM 1 IgM

What happens after repeat exposure to the same cell epitopes? Lymph nodes 1 2 PS Lymph nodes PS lood 2 IgM IgM 1 IgM 1) A second primary response, similar to the first one (no induction of memory)...

Repeat exposure to cell epitopes may induce similar or lower repeat than primary responses Lymph nodes 1 2 PS IgM Lymph nodes PS IgM lood 2) A weaker response ( cell hyporesponsiveness) by exhaustion of the -specific cell reservoir - only slowly reconstituted after its mobilization 1 2 IgM

Vaccines as seen by the immune system cell epitopes polysaccharides and/or + T cell epitopes proteins and Danger signals activating the innate immune system initiating and shaping and T cell responses

Repeat exposure to polysaccharides may induce lower than primary responses Hyporesponsiveness to MenC polysaccharides by prior immunization with MenC PS Poolman J Expert Rev Vaccines 2011; 10(3), 307;

Influence of prior pneumococcal PS immunization on responses to a conjugate vaccine (PCV13) Fold increase of antibody concentrations Fold increase of antibody concentrations Papadatou I, Antigen-specific -cell response to 13-valent PCV in asplenic individuals with β-thalassemia previously immunized with 23-valent pneumococcal polysaccharide vaccine. CID 2014 59(6):862-5.

Influence of prior pneumococcal PS immunization on responses to a conjugate vaccine (PCV13) All serotypes but various magnitudes Duration in years Number of PPS23 mc Number of PPS23 IgM mc Mechanism: induction of cell apoptosis by large dose of PS binding cells without sufficient activation signals (mouse: rynjolfsson SF J Inf Dis 2012) Time since PPS23 mc Time since PPS23 IgM mc Papadatou I, Antigen-specific -cell response to 13-valent PCV in asplenic individuals with β-thalassemia previously immunized with 23-valent pneumococcal polysaccharide vaccine. CID 2014 59(6):862-5.

Polysaccharide vaccines (S. pneumoniae, N. meningitidis) Recruit PS-specific memory cells (elicited by prior colonisation or immunization) Drive their differentiation into short-lived Ab secreting PCs short-lived antibody responses transient protection limited benefit, no population impact (UK) ooster recommended in many countries (example : splenectomy, hemoglobinopathies) 3-5 years 3-5 years 3-5 years 3-5 years ut NO induction of immune memory AND risks of cell depletion need for better pneumococcal vaccines

Providing T cell help to cells - to improve cell functional capacities cell survival, proliferation, plasma cell differentiation, affinity maturation, Ig switch, etc.

ringing together cells, T cells and antigen Lymph nodes cell zone T T T T cell zone T T T How to organize a get-together party?

Responses to protein-containing vaccines : 1. Antigen delivery to cells + T cells Lymph nodes cell zone FDC FDC FDC FDC FDC T cell zone 1. Soluble antigens drain from the site of injection to the capsular sinus of the lymph nodes and are translocated (by specific cells) to follicular dendritic cells ( antigen depot in the cell zone)

Responses to protein-containing vaccines : 1. Antigen delivery to cells + T cells Lymph nodes cell zone DC T Tfh FDC FDC FDC FDC FDC T cell zone 2. Antigens are captured at the site of injection by circulating monocytes dendritic cells which migrate to the border of the T: cell zone

Responses to protein-containing vaccines : 2. Activation of APC T cells + cells Lymph nodes cell zone DC T Tfh FDC FDC FDC FDC FDC lood T cell zone IgM IgM -transporting Dendritic cells activate -specific cells + -specific CD4 T helper cells cell proliferation differentiation = extrafollicular response

Responses to protein-containing vaccines : 3. Development of a Germinal Center reaction cell zone T Lymph nodes Germinal centers DC Tfh FDC Tfh FDC FDC FDC FDC lood Germinal centers = cell factory T cell zone IgM Activated -specific cells and CD4 follicular T cells (Tfh) are attracted by -bearing Follicular dendritic cells + FDCs + cells + Tfh cells induction of Germinal Centers IgM

Responses to protein-containing vaccines : 3. Development of a Germinal Center reaction cell zone T Lymph nodes DC Tfh Tfh FDC Germinal centers IgA IgE lood T cell zone IgM Proliferation and Ig switch IgM *, IgA, IgE * isotype cytokines produced by Tfh (IFNg / IL-4)

Responses to protein-containing vaccines : 3. Development of a Germinal Center reaction cell zone T T cell zone Lymph nodes DC Tfh IgM Tfh FDC Germinal centers Plasma cells Proliferation and Ig switch IgM Differentiation into plasma cells IgA IgE lood IgM, IgA, IgE Ab production / cell

Responses to protein-containing vaccines : 3. Development of a Germinal Center reaction cell zone T Lymph nodes DC Tfh Tfh FDC Germinal centers Plasma cells IgA IgE lood T cell zone IgM Proliferation and Ig switch IgM Differentiation into plasma cells Mutations + clonal selection IgM, IgA, IgE Ab production / cell Selection of the best PC!

Responses to protein-containing vaccines : 4. Induction and differentiation of plasma cells cell zone T T cell zone Lymph nodes DC Tfh IgM Tfh FDC Germinal centers Plasma cells lood IgM one Marrow Antigen-specific plasma cells in the blood after 10-14 days Peak Ab titers 4 weeks after injection. IgA IgE

Responses to protein-containing vaccines : 4. Induction and differentiation of plasma cells cell zone T T cell zone Lymph nodes DC Tfh IgM Tfh FDC Germinal centers Plasma cells lood Shortlived PC IgM one Marrow Antigen-specific plasma cells in the blood after 10-14 days Peak Ab titers 4 weeks after injection. Early cell death (short-lived plasma cells) rapid Ab fall! IgA IgE

Responses to protein-containing vaccines : 5. Survival of plasma cells into survival niches cell zone T T cell zone Lymph nodes DC Tfh IgM Tfh FDC Germinal centers Plasma cells lood Shortlived PC IgM PC homing to survival niches in the bone marrow are rescued from apoptosis long-lived PC : prolonged Ab persistence! IgA IgE one Marrow SC SC SC SC SC Long-lived PC

Antibody Titer (, log10) Induction of primary Ab responses to protein antigens 2 3 1 4 0 10d 30d time Antigen exposure 1. Initial early extrafollicular response 2. Higher peak of Ab titers (GCs), ~ 4 wks after immunization. 3. Short life span of PCs in spleen / nodes: rapid initial Ab decline. 4. Long-lived PC in the M: slower decline of Ab titers (years).

Organizing a successful party Define the best party place Enthusiastically invite all participants Create a hot-spot the bar! Generate an intimate environment Provide goodies for all to remain at the bar At some point, plan to attract participants to other places / events!! The cell follicle Cell activation and attraction (chemokines) The Germinal Center Follicular Dendritic Cells (FDCs) as madams Antigen and survival signals by FDC Change of receptors cell egress from the draining lymph nodes

Determinants of peak primary Ab responses T Lymph nodes Germinal centers DC Tfh FDC Plasma cells lood Shortlived PC one Marrow SC SC SC SC SC Which are the essential factors that control peak Ab responses / Ab titers?

Determinants of peak primary Ab responses T Lymph nodes Germinal centers DC Tfh FDC Plasma cells lood Shortlived PC one Marrow SC SC SC SC SC Antigen nature (epitopes) Antigen dose / T cell repertoire Vaccine antigen Genetics APCs: activation status Tfh cells : control GC responses Innate immunity PAMPs, adjuvants

How may adjuvants enhance peak antibody responses? y increasing : delivery to cells + DC / Tfh / cell activation GC responses Antibody responses Plasma cells Ab titers

Determinants of the duration of antibody responses to protein-containing vaccines : T Lymph nodes Germinal centers DC Tfh FDC Plasma cells lood Shortlived PC one Marrow SC SC SC SC SC Which are the essential factors that control the persistence of Ab responses / titers?

Determinants of the duration of antibody responses to protein-containing vaccines : T Lymph nodes Germinal centers DC Tfh FDC Plasma cells lood Shortlived PC one Marrow SC SC SC SC SC 1. Numbers of long-lived plasma cells 2. PC persistence in M survival niches

The persistence of Hs vaccine antibodies may be predicted based on initial Ab titers Health care workers Fujisawa, T et al, JPGN. 23(3):270-274,1996. 3 doses of Hs peak serology after the 3rd dose 10-99 100-999 1000 Proportion with HsAb >10 UI/l with time elapsed after adult immunization Antibodies eventually return to baseline

How may vaccines confer long term Ab-mediated protection? 1 2 1. Induction of long-lived bone-marrow plasma cells Reactivation of memory cells +

ooster doses are recommended to maintain protective antibody titers

-specific Ab level (, log10) Comparison of primary and booster Ab responses to protein-containing vaccines Primary response ooster response 0 10d 30d 0 10d 30d 6-12 mo time Initial antigen exposure ooster antigen exposure Which differences between primary and booster responses?

-specific Ab level (, log10) Comparison of primary and booster Ab responses to protein-containing vaccines Primary response ooster response 0 10d 30d 0 10d 30d 6-12 mo time Initial antigen exposure ooster antigen exposure ooster Ab responses are faster (peak ~ at day 7), stronger (Ab titers), more prolonged (years) and of a higher neutralizing capacity (increased affinity).

Which vaccines induce memory?

Which vaccines induce memory? Induction of memory PS vaccines 0 Protein-containing vaccines! Diphtheria + Tetanus + Pertussis + Poliomyelitis + Hib + Pneumo conj. + MenC conj. + Influenza + Hepatitis A + Hepatitis + HPV + TE/FSME + MMR + VZV + Yellow fever + Rabies +

1- Induction of memory cells by proteincontaining vaccines T DC M IgM Memory cells Tfh FDC M Plasma cells lood M IgM one Marrow SC SC SC Induction in the same -induced Germinal Centers as PCs Exit of GC, migration through blood return to the cell zones of lymph nodes where expect a new encounter SC SC

Reactivation of memory cells upon contact with their specific antigen -specific memory cells are only reactivated when exposed to their specific : natural microbial exposure (colonization, infection) mimicry of microbial exposure (ex: PS injection) booster vaccine doses Kinetics of responses : rapid (4-7 days) and strong increase of serum

Kinetics of HI responses to PS in children vaccinated as infants with conjugate vaccines Day 3 Day 7 Day 4-5 Day 10-14 Pichichero M, Ped Inf Dis J 1999 4-7 days necessary no early protection!

Affinity maturation of antigen-specific cells T DC M Germinal centers Tfh FDC Memory cells undergo affinity maturation during several months This increases the affinity for of their surface When reactivated, m cells thus produce antibodies of higher affinity e.g. higher NT capacity M 1. Random mutations in cell Ig genes: - most deleterious - a few conferring better binding capacity 2. Limited availability of : competition among cells for binding! 3. FDC survival signals only to -bound cells SURVIVAL OF THE FITTEST!

Induction and magnitude of memory responses T DC M Tfh FDC Germinal centers M Which are the essential factors that control the induction and magnitude of memory responses?

Induction and magnitude of memory responses T DC M Tfh FDC Germinal centers M Antigen nature (epitopes) Vaccine antigen Antigen dose / T cell repertoire DC : state of activation Tfh cells : control GC responses Germinal center responses Genetics Innate immunity PAMPs, adjuvants + vaccine schedule ( time for affinity maturation)!

1 Influence of the vaccine schedule on the reactivation of memory responses 2 M Memory cells M lood one Marrow Tfh M T DC FDC SC SC SC SC SC T Plasma cells DC Short interval (weeks) btwn 2 doses: 2 nd wave of primary responses (memory cells, plasma cells)

1 Influence of the vaccine schedule on the reactivation of memory responses 2 T DC M M M M M M M M M M PC Memory cells Tfh FDC PC PCPC PC PC PC PC M Plasma cells PC PC lood M one Marrow SC SC SC SC SC SC SC SC Long interval ( 4 months) btwn 2 doses: affinity maturation of memory cells enhanced capacity to respond to!

Influence of vaccine schedules on booster responses - Cervarix (HPV, GSK) - 3 doses (0-1-6 mo) @ 15-25 years = - 2 doses (0-6 mo) @ 9-14y >>> - 2 double doses (0-2 mo) @ 9-14y (Romanovski., Human Vaccines 2011)

0-1- 6 : the classical immunization schedule! DC T M lood one Marrow Memory cells Tfh FDC Plasma cells Affinity maturation M M IgM, IgA SC SC SC SC SC 1(-2) doses for priming Affinity maturation and clonal selection of the fittest cells 4 months 1 booster dose to reactivate memory cells

0-1- 6 : the classical immunization schedule! DC T M lood one Marrow Memory cells Tfh FDC Plasma cells M M prime, prime, boost!! Affinity maturation IgM, IgA SC SC SC SC SC 1(-2) doses for priming Affinity maturation and clonal selection of the fittest cells 4 months 1 booster dose to reactivate memory cells

0-1- 6 : the classical immunization schedule! M lood one Marrow Memory cells Tfh FDC When may vaccines be used with a 2-dose schedule? SC DC Why SC T may some vaccines be used with a 1-dose schedule? Why does the EPI schedule Plasma include cells 3 primary doses? Why is a 4 th dose needed for accelerated schedules? Affinity maturation M M IgM, IgA SC SC SC 1(-2) doses for priming Affinity maturation and clonal selection of the fittest cells 4 months 1 booster dose to reactivate memory cells

How do vaccine induce protection? 1. Early innate responses T cell responses

How do vaccine induce protection? 2. Vaccine-specific CD4 T cells do not prevent infection but participate in pathogen reduction, control and clearance by: Supporting antibody responses (T-dependent antigens) Producing cytokine / cytolytic activities 3. Vaccine-specific CD8 T cells do not prevent infection but participate in pathogen reduction, control and clearance by Producing cytokine / cytolytic activities Essential preventive role for T cells against complications caused by intracellular pathogens (Mtb CG, )

How do vaccines induce specific T cells? Th1 Th2 Th9 Th17 Th22 Treg Tfh T cells may differentiate into various subsets with distinct functions The number indicates the T cell subset and function This differentiation is essentially mediated by the cytokines present at the time/site of their differentiation by dendritic cell activation. Dendritic cell activation may be influenced by vaccine formulations.

How do we expect vaccines to elicit potent specific T cell responses? Injection-site innate activation Recruitment and activation of professional APCs (DCs) T cell responses

How do vaccines induce specific T cells?

How do we expect vaccines to elicit potent specific T cell responses? Injection-site innate activation Recruitment and activation of professional APCs (DCs) Migration Draining LN T cell responses -specific polarized T cells

How do vaccines induce specific T cells? itreg Th17 Th2 Tfh Th1 cytocd4

Danger signals / adjuvants activate dendritic cells directly or indirectly De Gregorio E, Current Opin Immunol 2009;21:339

How do vaccines induce specific T cells? nature / dose route of exposure DC subpopulation Costimulatory signals Genetic factors Etc

T cell responses and induction of T cell memory Nb 12 8 1 2 4 0 3 0 2 4 6 8 TIME (wks) 1. T CELL DIFFERENTIATION IN THE THYMUS self / non-self education 2. EXPANSION OF EFFECTOR T CELLS driven by (days) 3. DEATH OF EFFECTOR T CELLS (>90%) when Antigen is cleared 4. DIFFERENTIATION OF PRECURSOR MEMORY T CELLS INTO : - Central memory T cells : resting, reservoir in lymph nodes, M, etc. - Effector memory T cells : pre-activated, in tissues

Influence of vaccine schedule on the induction of memory T cells T cell responses are lower if boosters are given too early!

Which cell responses should be assessed in vaccine studies? Quantification Function -specific /M/A -specific /M/A ELISA, -binding assay Western lot Immunoprecipitation -specific cells FACS analyses ELISPOTs for Absecreting plasma cells ELISPOTs (+ ) for reactivated memory cells Serum neutralization titers Pathogen Serum bactericidal specific! activity Opsonophagocytosis Hemagglutination inhibition (flu) Correlates Avidity of index protection!

Which parameters of T cell responses should be assessed in vaccine studies? Quantitative Nb of CD8 T cells binding to a specific : tetramers T cell proliferation following recognition (3H thymidine, CSFE dye + FACS, others) Pathogen specific! Correlates of protection! Function Nb of cytokine producing CD4/CD8 T cells: ELISPOTs, flow cytometry Cytokine detection in T cell supernatants Cytotoxicity : in vivo (animal), in vitro

System vaccinology : measure everything and then try to understand!

What is elicited by vaccination? Antibodies : magnitude, persistence, functional capacity? T cells : characteristics? persistence? Duration of protection : Long term effectors Memory cells induction? reactivation kinetics? persistence? What is needed for protection? Antibodies? Protective threshold? T cells? Pattern of responses? Long term protection? Effector mechanisms? Pathogen specific! Correlates of immunity!

Siegrist CA, Vaccine immunology, Chapter 2, Plotkin and Orenstein, 6th edition References to review the basics of vaccine immunology

cell responses - references Stephen L. Nutt, Philip D. Hodgkin, David M. Tarlinton & Lynn M. Corcoran. The generation of antibody-secreting plasma cells. Nature Reviews Immunology, 15, 160-171 (2015), doi:10.1038/nri3795 Shane Crotty, A brief history of T cell help to cells. Nature Reviews Immunology, 15, 185-189 (2015), doi:10.1038/nri3803 Crotty S. T follicular helper cell differentiation, function, and roles in disease. Immunity. 2014 Oct 16;41(4):529-42. doi: 10.1016/j.immuni.2014.10.004 Linterman MA, Hill DL. Can follicular helper T cells be targeted to improve vaccine efficacy? F1000Res. 2016 Jan 20;5. pii: F1000 Faculty Rev-88. doi: 10.12688/f1000research.7388.1. Corcoran LM, Tarlinton DM. Regulation of germinal center responses, memory cells and plasma cell formation-an update. Curr Opin Immunol. 2016 Apr;39:59-67. doi: 10.1016/j.coi.2015.12.008. Nilushi S. De Silva & Ulf Klein, Dynamics of cells in germinal centres. Nature Reviews Immunology, 15, 137-148 (2015), doi:10.1038/nri3804 Zhang Y, Garcia-Ibanez L, Toellner KM. Regulation of germinal center -cell differentiation. Immunol Rev. 2016 Mar;270(1):8-19. doi: 10.1111/imr.12396. Doria-Rose NA, Joyce MG. Strategies to guide the antibody affinity maturation process. Curr Opin Virol. 2015 Apr;11:137-47. doi: 10.1016/j.coviro.2015.04.002. Tomohiro Kurosaki, Kohei Kometani & Wataru Ise, Memory cells, Nature Reviews Immunology 15, 149 159 (2015) doi:10.1038/nri3802

T cell respones - references Jinfang Zhu, Hidehiro Yamane, and William E. Paul. Differentiation of Effector CD4 T Cell Populations*. Annual Review of Immunology Vol. 28: 445-489 (April 2009). DOI: 10.1146/annurev-immunol-030409-101212 Zhu J1, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*).Annu Rev Immunol. 2010;28:445-89. doi: 10.1146/annurev-immunol-030409-101212. Scott N. Mueller, Thomas Gebhardt, Francis R. Carbone, and William R. Heath. Memory T Cell Subsets, Migration Patterns, and Tissue Residence. Vol. 31: 137-161 (March 2013). DOI: 10.1146/annurev-immunol-032712-095954 Sarah C Gilbert. T-cell-inducing vaccines what's the future? Immunology. 2012 Jan; 135(1): 19 26. doi: 10.1111/j.1365-2567.2011.03517.x