Acquired Immunity 2. - Vaccines & Immunological Memory - Wataru Ise. WPI Immunology Frontier Research Center (IFReC) Osaka University.

Transcription

1 Acquired Immunity 2 - Vaccines & Immunological Memory - Wataru Ise WPI Immunology Frontier Research Center (IFReC) Osaka University Outline 1. What is vaccine (vaccination)? 2. What is immunological memory? 3. What are the issues in the research for immunological memory? 1

2 Vaccine If you are prepared, you don t have to worry Prevention is better than cure Vaccine =a biological preparation to prevent infectious diseases Vaccination stimulates immune system to recognize viruses and be ready to combat them Normal medicine Vaccine =cure diseases =prevent diseases Types of vaccines Vaccination Virus infection Protection! 1. Attenuated vaccines (measles, rubella, and mumps) created by reducing the virulence of a pathogen, but still keeping it viable 2. Inactivated vaccines (Influenza, Cholera, and Polio) created by killing the disease-causing microbe with chemicals, heat or radiation. 3. Toxoid (tetanus and diphtheria) bacterial toxin whose toxicity has been inactivated either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained 2

3 Invention of the vaccine People who had already had cowpox would never have the often-fatal disease small pox Hypothesis Infection with cowpox gives protection to smallpox Public domain Wikimedia Commons Edward Jenner ( ) Test Took pus from the hand of a milkmaid with cowpox and scratched it into the arm of an 8-year old boy and six weeks later inoculated the boy with smallpox. Results The boy did not catch smallpox! What happens if you get vaccinated? 3

4 Immune responses against invading pathogens Pathogen Sensing of pathogens First defense agaist pathogens Innate Immunity Dendritic cell attack Pathogens-specific attack Macrophage NK cell Neutrophil Co-stimulatory molecules Antigenic peptide Migration to lymph node Antibody (humoural immunity) Cytotoxic T cell (Cellular Immunity) B cell interaction Cytokine B cell Adaptive Immunity T cells or B cells express antigen receptors Innate immune cells (macrophage or dendritic cells) uptake any kind of antigens and initiate immune responses (no antigen-specificity). Cells in adaptive immune system (T cells or B cells) express antigen receptors on their surface and respond only to the specific antigens. T cells or B cells have an almost infinite range of repertoire by rearrangement of antigen receptor genes. B cells respond to different antigens B cells undergo somatic hypermutation in germinal center and thus affinity or specificity of B cell receptors can be changed. 4

5 Vaccination induces virus-specific antibody, T cells, and B cells Influenza virus-specific T cell Vaccination against influenza virus Dendritic cell Signal Influenza virusneutralizing antibody Stimulation Influenza virus-specific B cell Vaccination establishes immunological memory Slow and weak immune response No vaccination Robust virus proliferation Infection NO Protection Induction of virus-specific Ab, T-cell, and B-cell Vaccination (immunization with Virus components) Infection Protection! Immune system remembers the virus and is ready to respond =Immunological Memory 5

6 What is immunological memory? Immunological memory: Quick & Robust A large amount of antibodies are produced quickly Affinity of antibodies become higher Pathogen Concentration of antibodies Affinity of antibodies Primary immunization Secondary immunization 6

7 Immune memory cells Number of Virus-specific B cells 1,000, ,000 10,000 1, days 60 days Memory B cell Virus infection Memory B cells Number of Virus-specific B cells 1,000, ,000 10,000 1, Virus-specific B cells expand and neutralize virus Most of the virus-specific B cells die. Memory B cell However, small fraction of virus-specific B cells survive. Virus infection 30 days 60 days 7

8 Memory B cells respond quickly and robustly Pathogen ~7 days Naïve B cells Plasma cell Pathogen Memory B cell ~3 days Quicker More plasma cells Higher affinity Does vaccination/ immunological memory work perfectly? Need of flu vaccine every year -Type of influenza viruses often differs year by year. -Influenza viruses can mutate. -Levels of protective antibody start to decline over time. 8

9 Research for immunological memory -What we know and what we don t know about memory B cells- Questions Number of virus specific lymphocytes Virus infection Immune memory cells Time (days) 1. Where are memory B cells in our body? 2. How do memory B cells respond to re-infection? 3. Why can a small fraction of cells survive as memory B cells? How are memory B cells generated? 4. Are memory B cells really long-lived? 5. Are there any ways to induce memory B cells efficiently? 9

10 Question-1 Where are memory B cells present in our body? B cells reside in lymph nodes and circulate in lymph or blood Lymph node efferent lymphatic vessel Lymphocytes return to Blood via the thoracic duct Heart Naïve lymphocytes enter lymph nodes from blood afferent lymphatic vessel Antigens from sites of infection reach lymph nodes via lymphatics Lymph node Infection focus 10

11 Immune responses are elicited in lymph nodes 1. Antigens reach lymph nodes 2. Lymphocyte activation & Germinal center formation 3. Egress of activated lymphocytes Efferent lymphatic vessel B cell Zone T cell Zone Efferent lymphatic vessel Germinal center Antigens Afferent lymphatic vessel Afferent lymphatic vessel Activated T cells or B cells egress from lymph nodes to periphery Memory B cells can be distinguished from other type of B cells Germinal center Naïve B cell IgM/IgD CD38 hi CD138- Pathogen FDC Germinal center CD38 lo B cell CD138- GL7 hi Fas hi Memory B cell T cell Marker of memory B cell -Switched Ig (=not naïve B cell) -CD38hi (=resting) -CD138 negative (=not plasma cell) IgG/IgA/IgE CD38 hi CD138- Plasma cell Ig- CD38 hi CD

12 How to detect memory B cells Spleen of NP-CGG immunized mice 30 days NP-binding CD38 Memory B cells (IgG+, CD38hi) GC B cells (IgG+, CD38lo) IgG IgG 60 days NP-binding CD38 Memory B ells GC B cells IgG IgG Immunity Feb;14(2): Takahashi Y, Ohta H, Takemori T. B cells reside in lymph nodes and circulate in lymph or blood Lymph node efferent lymphatic vessel Lymphocytes return to Blood via the thoracic duct Heart Naïve lymphocytes enter lymph nodes from blood afferent lymphatic vessel Antigens from sites of infection reach lymph nodes via lymphatics Lymph node Infection focus 12

13 Where do memory B cells localize in the secondary lymphoid tissues? Germinal Center IgG RFP Germinal Center Follicle CD38 The experiments in which memory B cells are labeled with fluorescence RFP Memory B cells reside close to germinal center Aiba et al. PNAS (2010) CD4 + T cells reside close to IgG + memory B cells B cell follicle T cell area IgG1 + cells CD38 CD4 IgG1 Day 60 after immunization High magnification IgG1 CD4 CD38, CD4, IgG1 Memory B cells can get help from CD4+ T cells efficiently upon re-infection Aiba et al. PNAS (2010) 13

14 Question-2 How are memory B cells generated? Transcription factors that regulate B cell development Pathogen T cell Germinal center FDC Bcl6, Bach2 Naïve B cell Germinal center B cell Blimp1, IRF4 Plasma cell Memory B cell 14

15 Identification of genes specifically expressed in memory B cells Memory B cell Naïve B cell GC B cell Plasma cell Gene chip data Comparison of gene expression pattern Kaji et al. J. Exp. Med. (2012) Identification and functional analysis of the candidate genes Generation of genetically-modified mice 1 Wild-type mice A B X Y Gene X Knock-out mice A B X Y (Function of gene X can be analyzed) Gene X GFP Knock-in mice A B X GFP Y Fluorescent Protein (Expression pattern of gene X can be analyzed) 15

16 Generation of genetically-modified mice 2 Generation of conditional knock-out mice X Breeding ERT2-cre mice (Tamoxifen treatment can induce Cre-recombinase expression) A B X Y loxp loxp Tamoxifen Cre A B X Y Gene X is deleted A B X Y Function of gene X at specific time point can be analyzed Research of memory B cells with gene targeting mice Number of B cell 1,000, ,000 10,000 1, Delete Gene X Memory B cell 1,000, ,000 10,000 1, Delete Gene Y Memory B cell day30 day60 day30 day60 If this is the case, this data suggests that Gene X is required for memory B cell generation If this is the case, this data suggests that Gene Y is required for memory B cell maintenance 16

17 Question-3 (1) What is molecular basis for long-term survival of memory B cells? Identification of signaling molecules that are essential for memory B cell survival B cell receptor Ca 2+ PIP3 PIP3 PIP2 PIP3 PIP2 CRAC PLC- 2 Ca 2+ Syk Btk BLNK P3K Ca 2+ IP3 + DAG PKC IP 3 receptor Ca 2+ ER 17

18 PLC- 2 is required for maintenance of memory B cells PLC- 2 deletion after memory B cell generation 1,000, ,000 10,000 1, Deletion of PLC- 2 Memory B cell # of T cell #of memory B cell day30 day60 Number of memory B cells was decreased Hikida et al. J. Exp. Med. (2009) Question-3 (2) Are memory B cells really long-lived? 18

19 Can memory B cells survive for a long time? Immunization of protein Ag (PE) Number of Ag (PE)-specific Memory B cells Frequency of PE-binding splenic B-cells (x10-4 ) Days after priming Schittek et al. Nature (1990) Memory B cells appear to survive long-period time without antigen re-stimulation IgM + memory B cell Pathogen FDC T cell Germinal center B cell Naïve B cell (IgM+) Germinal center-independent Low affinity IgM+ memory B cell Germinal center-dependent High affinity IgG+ memory B cell 19

20 IgM + memory B cells are long-lived Long-term survival of IgM vs switched (IgG+IgA+IgE) memory B cells Pape et al. Science (2011) IgM + memory B cells are long-lived, whereas IgG + memory B cells are short-lived Heterogeneity in longevity or function among memory B cell subsets Question-4 What kind of memory B cells should be induced for efficient vaccination? 20

21 Important factors 1 High affinity Affinity of antibodies are increased as a result of somatic hypermutation in GC. Thus, a vaccine that sustains GC response is desirable. 2 Longevity If long-term immunological memory is established, repeated vaccination is not needed. 3 Cross-reactivity Influenza virus has many subtypes and is frequently mutated. Pre-existing antibodies may not be protective. Thus, induction of cross-reactive antibodies or memory B cells is the key for the efficient protection. Antibody to Influenza HA HA Hemagglutinin, a glycoprotein found on the surface of the influenza viruses Antibodies to HA Head are easily induced Head Mutations are frequently induced in HA Head Antibodies to HA Head are not efficacious to different influenza subtypes Stem Antibodies to HA Stem are not easily induced HA Stem is not mutated and highly conserved Influenza Hemagglutinin Antibodies to HA Stem are highly efficacious to different influenza subtypes How to generate HA-Stem specific memory B cells is an important issues and extensively studied in the filed 21