Acquired Immunity 2 - Vaccines & Immunological Memory

©2015 Osaka University. All rights reserved.
Wataru Ise
WPI Immunology Frontier Research Center (IFReC)
Osaka University
Acquired Immunity 2
- Vaccines & Immunological Memory -
Outline
1. What is vaccine (vaccination)?
2. What is immunological memory?
3. What are the issues in the research
for immunological memory?
1

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
=cure diseases
=prevent diseases
Normal medicine
Vaccine
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

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
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!
Edward Jenner
(1749-1823)
Public domain | Wikimedia Commons
What happens if you get vaccinated?
3

NK cell
Pathogen
Immune responses against invading pathogens
Neutrophil
Dendritic cell
Sensing of pathogens
First defense agaist pathogens
Macrophage
Innate Immunity
Migration to lymph node
attack Antigenic
peptide
Adaptive Immunity
Cytokine
B cell
B cell
Cytotoxic T cell
(Cellular Immunity)
Antibody
(humoural immunity)
Pathogens-specific
attack
interaction
Co-stimulatory
molecules
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

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

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

Immune memory cells
1,000
100,000
10,000
100
1,000,000
Memory B cell
Virus infection
30 days 60 days
NumberofVirus-specificBcells
Memory B cells
1,000
100,000
10,000
100
1,000,000
Memory B cell
Virus infection
30 days 60 days
NumberofVirus-specificBcells
Most of the virus-specific B cells die.
However, small fraction of
virus-specific B cells survive.
Virus-specific B cells expand
and neutralize virus
7

Memory B cells respond quickly and robustly
Naïve B cells
Memory B cell
Plasma cell
Pathogen
~7 days
~3 days More plasma cells
Higher affinity
Quicker
Pathogen
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

Research for immunological memory
-What we know and what we don’t know about memory B cells-
Questions
Virus infection
Numberofvirusspecificlymphocytes
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

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
Infection focus
Naïve lymphocytes enter
lymph nodes from blood
Lymphocytes return to
Blood via the thoracic duct
Heart
Antigens from sites of infection
reach lymph nodes via lymphatics
efferent
lymphatic vessel
afferent
lymphatic vessel
Lymph node
10

Immune responses are elicited in lymph nodes
Germinal center
1. Antigens reach lymph nodes
Afferent lymphatic vessel
Efferent lymphatic vessel
2. Lymphocyte activation
& Germinal center formation 3. Egress of activated lymphocytes
Activated T cells or B cells egress from
lymph nodes to periphery
Antigens
Efferent lymphatic vessel
Afferent lymphatic vessel
T cell
Zone
B cell
Zone
Pathogen
IgM/IgD
CD38hi
CD138-
IgG/IgA/IgE
CD38hi
CD138-
Ig-
CD38hi
CD138+
CD38lo
CD138-
GL7hi
Fashi
Memory B cells can be distinguished from other type of B cells
Germinal center
T cell
FDC
Germinal center
B cell
Naïve B cell
Memory B cell
Plasma cell
Marker of memory B cell
-Switched Ig
(=not naïve B cell)
-CD38hi
(=resting)
-CD138 negative
(=not plasma cell)
11

Immunity. 2001 Feb;14(2):181-92.
Takahashi Y, Ohta H, Takemori T.
NP-binding
IgG
CD38
IgG
CD38
IgG
NP-binding
IgG
GC B cells
Memory B ells
30 days
60 days
How to detect memory B cells
Memory B cells (IgG+, CD38hi)
GC B cells (IgG+, CD38lo)
Spleen of NP-CGG immunized mice
B cells reside in lymph nodes and circulate in lymph or blood
Lymph node
Infection focus
Naïve lymphocytes enter
lymph nodes from blood
Lymphocytes return to
Blood via the thoracic duct
Heart
Antigens from sites of infection
reach lymph nodes via lymphatics
efferent
lymphatic vessel
afferent
lymphatic vessel
Lymph node
12

IgG RFP
CD38
Where do memory B cells localize in the secondary lymphoid tissues?
Germinal Center
Germinal Center
Follicle
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)
Day 60 after
immunization
CD4+ T cells reside close to IgG+ memory B cells
High magnification
B cell follicle
T cell area
IgG1+ cells
CD38
CD4
IgG1
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

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

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

Generation of conditional knock-out mice
X
ERT2-cre mice
(Tamoxifen treatment can induce Cre-recombinase expression)
A B X Y
A B X Y
A B X Y
CreTamoxifen
Gene X is deleted
loxp loxp
Breeding
Generation of genetically-modified mice（2）
Function of gene X at specific time point can be analyzed
1,000
100,000
day30
10,000
100
1,000,000
Delete Gene X
Memory B cell
Research of memory B cells with gene targeting mice
day60
1,000
100,000
10,000
100
1,000,000
Delete Gene Y
Memory B cell
day60day30
NumberofBcell
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

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
Syk Btk
BLNK
PLC-2
P3K
IP3
DAG
+
PKC
ER
Ca2+
Ca2+
IP3
receptor
Ca2+
Ca2+
CRACPIP2
PIP2 PIP3PIP3PIP3
17

PLC-2 is required for maintenance of memory B cells
1,000
100,000
10,000
100
1,000,000
Number of memory B cells was decreased
Memory B cell
Deletion of PLC-2
PLC-2 deletion after memory B cell generation
#ofTcell#ofmemoryBcell
day60day30
Hikida et al. J. Exp. Med. (2009)
Question-3 (2)
Are memory B cells really long-lived?
18

Can memory B cells survive for a long time?
Immunization of protein Ag (PE)
Number of Ag (PE)-specific
Memory B cells
Memory B cells appear to survive long-period time
without antigen re-stimulation
FrequencyofPE-binding
splenicB-cells(x10-4)
Days after priming
2.0
1.5
1.0
0.5
７ 35 70 105 140 175 196
Schittek et al. Nature (1990)
Pathogen
T cellFDC
Germinal center
B cell
Naïve B cell
IgM+ memory B cell
(IgM+)
IgG+ memory B cellIgM+ memory B cell
・Germinal center-independent
・Low affinity
・Germinal center-dependent
・High affinity
19

IgM+ memory B cells are long-lived
Pape et al. Science (2011)
Long-term survival of IgM vs switched (IgG+IgA+IgE) memory B cells
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

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
Head
Stem
HA：Hemagglutinin, a glycoprotein found on the surface of the influenza viruses
Antibodies to HA Head are easily induced
Mutations are frequently induced in HA Head
→Antibodies to HA Head are not efficacious
to different influenza subtypes
Antibodies to HA Stem are not easily induced
HA Stem is not mutated and highly conserved
→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
Influenza Hemagglutinin
21

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