Download Lecture 16 Tues 5-23-06

Host Responses to Viruses: Overview
1. Background Slides:
A.
B.
C.
D.
E.
F.
G.
Levels of immune protection
Cytokines, chemokines
Complement
Patterns of viral infection (Acute vs. Chronic)
CD8+ CTL: mechanism of cytolytic action
What are neutralizing antibodies?
RNA viruses and error catastrophe
2. Viral evasion mechanisms
A.
B.
C.
D.
E.
F.
G.
H.
Viral mimicry of cytokines, chemokines
Complement evasion
Inhibition of Natural killer cells
Antigenic variation
Escape from CD8+ CTLs
Escape from Neutralizing antibodies (Influenza, HIV)
Host susceptibility to virus infection
Persistence in immunologically privileged sites (e.g. neurons)
3. Immunological techniques to explore host responses to viruses
4. References
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Learning Objectives:
1. What are the major mechanisms used by viruses to
evade innate and adaptive immunity?
2. What is the association between types of viral
infection (acute and chronic infection) and modulation
of immune responses?
3. How does HIV trick the various arms of the immune
system?
4. What are the major methods used to measure
adaptive immunity?
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Levels of immune protection
Complement
NK Cells
Source: http://mil.citrus.cc.ca.us/cat2courses/bio104/ChapterNotes/Chapter39notesLewis.htm
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Cytokine, Chemokines
Cytokines are small soluble proteins secreted by one cell
that can alter the behavior or properties of the cell
itself or of another cell. They are released by many
cells in addition to those of the immune system.
Cytokines, such as interferons (IFNs) and tumor-necrosis
factor (TNF), induce intracellular pathways that
activate an anti-viral state or apoptosis, and thereby
limit viral replication.
Janeway, Immunobiology 5th Edition; Fig 1.12
Chemokines are chemoattractant cytokines that regulate
trafficking and effector functions of leukocytes, and so
have an important role in inflammation and immune
surveillance
See Flint Table 14.6
Blocking interferon action
Chemokines: divided into 4 classes, CC-, CXC-, C-, CX3CExpression of specific chemokines, together with
differential expression of respective chemokine
receptors by leukocytes, determines which immune
cells migrate during inflammation
HIV utilizes chemokine receptors CCR5, CXCR4 for entry into target cells
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Viral Mimicry of cytokines, chemokines and their receptors
Alacami A. Nat Rev Immunol. 2003 Jan;3(1):36-50.
Properties of poxvirus-encoded soluble cytokine receptors or binding proteins.
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Complement
Interacting set of enzymes (proteases) that
upon activation give rise to cascade of
reactions culminating in the destruction of
pathogens and infected cells.
Three pathways:
Factor H
C3bH
Classical: implicated in controlling HIV, HTLV,
CMV infected cells
Factor I
C4d + C4c
(inactive)
Mannan binding lectin (MBL) : similar to
classical; lyses HBV, HCV and infuenza
infected cells
Alternative pathway: default pathway;
spontaneous and indiscriminate
deposition of complement factor C3b on
host cell surface or foreign particles;
complement activation will proceed unless
downregulated by complement regulators
CD59, DAF
+
CD46
CR1
+
Factor I
+
CD46
CR1
+
iC3b
C3c
(inactive)
Factor S
CD59
Favoreel HW., et al., J Gen Virol. 84: 1-15 (2003).
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Complement Evasion
1.
Remove Ag:Ab complexes; express Fc receptors
on the infected cells
HSV gE-gI glycoprotein complex binds non-immune IgG
and sterically hindered access of virus specific
effector cells or neutralizing antibodies
PRV gE cytoplasmic domain has two tyr residues that
internalizes through clathrin mediated endocytosis,
followed by degradation
2.
Viral mimicry of complement regulators
Poxvirus and -Herpesvirus infected cells secrete soluble
viral encoded factors that are genetically similar to
complement regulators; inhibit by inactivating C3b
and C4b, C3 convertase
-herpesvirus (HSV) gC glycoprotein acts as
complement receptor for C3b, C5 and P factor
3.
Incorporation or up-regulation of cellular
complement control factors
Enveloped viruses like HIV, Ebola, influenza incorporate
complement control proteins CD46, CD55, CD59
(enriched in lipid rafts) during virus release
HCMV upregulates expression of CD59 in infected cells
leading to suppression of alternate pathway.
Favoreel HW., et al., J Gen Virol. 84: 1-15 (2003).
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Natural Killer Cells
•
•
•
•
NK cells perform immune surveillance
Lymphocytes that do not undergo genetic
recombination events to increase their affinity for
particular ligands (effectors of the innate immune
system)
Capable of killing without previous stimulation
Characterized by the absence of conventional
receptors for antigen (TCR); display CD3- CD16+
phenotype
CD16 (FcγRIII) is a low affinity receptor for IgG and is
involved in Antibody Dependent Cell mediated
Cytotoxicity (ADCC)
Janeway, Immunobiology 5th Edition; Fig 8.19
ADCC of NK cells
•
NK cell activating receptors:
A.
B.
•
Inhibitory receptors:
A.
B.
C.
•
•
Human natural cytotoxicity receptors NKp30, NKp44, NKp46
LFA-1 and CD2 family
receptors for MHC class I belonging to killer cell inhibitory
receptor (KIR) family; KIR receptors recruit phosphatases (Src
domain containing protein tyrosine phosphatase 1) to
transduce the inhibitory signal
Immunoglobulin-like inhibitory receptors (ILT)
Lectin like heterodimer CD94-NKG2A
NK cell responses are also coordinated and regulated
by cytokines such as IFN-, IFN-, IL-2, IL-12, IL-15 and
IL-18
Spontaneously active, unless they are inhibited by
self-MHC molecules.
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Janeway, Immunobiology 5th Edition; Fig 9.34
Well-suited to perform
immunosurveillance for nonself MHCbearing targets (eg, transplanted cells,
tumors, virally-modified cells)
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Viral mechanisms for evading NK cells.
A.
Viral homologs of MHC class I
Binds to inhibitory NK cell class I receptors
B.
Selective modulation of MHC class I allele expression
Increase relative expression of HLA-C, HLA-E by down
regulating HLA-A, HLA-B; NK cells inhibited through class
I CD94-NKG2A and KIR
HIV Nef causes endocytosis of Class I, except HLA-C, HLA-E
HCMV UL40 enhances expression of HLA-E
C.
Inhibition of activating receptor function
(i) Viral cytokine binding proteins (ii) viral homologs (iii)
induce host cell secretion of NK cell inhibitory cytokines
HIV Tat blocks NK cell activation by specifically binding and
blocking Ca2+ influx channel important for NK Cell
cytotoxicity
KSHV K5 protein ubiquitinates, and decreases surface
expression of ICAM-1, B7-2  inhibits NK cell cytotoxicity
D.
Secrete antagonists of NK cell receptor-infected cell ligand
interactions
KSHV vMIP-I, vMIP-II acts as a chemokine antagonist and
intereferes with NK cell trafficking
HPV E6, E7 binds IL-18 receptor and inhibits IFN- production
by NK cells
E.
Orange JS. Et al., Nat Immunol. 3: 1006-12 (2002).
Directly infects NK cells or ligates NK cell inhibitory
receptors through envelope glycoproteins
HIV in vitro infects NK cells through an unknown receptor
HCV E2 glycoprotein binds CD81 on NK cells and sends
inhibitory signal to NK cells
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General patterns of viral infection
Acute Infection
CTL
Ab titers
Virus is cleared
Memory responses established
•
•
•
Rhinovirus
Rotavirus
Influenza virus
Persistent Infection
CTL
Ab
•
Lymphocytic
choriomeningitis virus
Latent, Reactivating infection
•
HSV
Slow virus infection
•
Lentivirus (HIV)
Source: Flint, Principles of Virology 4th Ed Fig: 16.1
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HIV: drives the host defenses into exhaustion, and
ultimately failure
A.
B.
C.
D.
E.
F.
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High rate of viral evolution
Early escape from CTL
Escape from neutralizing antibodies
Down regulation of MHC
CD4+ T helper cell destruction
Integration and reactivation
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Immune responses to HIV
1. Control of primary HIV-1
infection coincides with the
appearance of virus-specific
Cytotoxic T lymphocytes
(CTLs)
2. Antibodies form early;
neutralizing antibodies arise
later in infection
Ferrantelli F., et al., Curr Opin Immunol. 14: 495 (2002).
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Strong immune responses to HIV
infection control it for many
years but ultimately host dies
after erosion of T cells
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HIV: Antigenic Escape, Quasispecies, Error
Threshold
Domingo E. Virology 270, 251-253 (2000)
RNA Viruses:
Genome size ~104 nucleotides
Error rate of RNA replicases is 10-4
1 error with every replication
RNA Viruses
E. coli:
Genome size 4.6 x 106 bp or ~107 bp
Error rate in DNA replication is 10-10
1 error for every 1000 genome
replications
HIV and other RNA viruses thus exist as dynamic distributions of nonidentical but related genomes – Quasispecies
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DNA Distance
HIV intrapatient viral evolution
0
2
4
6
8
10
12
0
2
4
6
8
10
12
Years post seroconversion
Shankarappa et al., J. Virol. 73:10489 (1999)
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Viral Escape from CD8+ Cytotoxic T
lymphocytes (CTLs)
Flint Table 15.4 and Figure 15.7
*RSV--less MHC class I transcription
*HIV--Tat, Vpu interfer with MHC class I synthesis
*Adenovirus--E3 and E1a reduce MHC in ER and transcription
*CMV, HSV, EBV--Tap transporter inhibition, retention of
peptides in ER
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CD8+ CTLs: Mechanism of cytolytic
action
Potent defenders against virus infection and intracellular
pathogens
Mediates apoptotic death through Fas-FasL interactions
Upon interaction with an infected cell, an “immunological
synapse” between the CTL and the target cell is formed
Targeted release of effector molecules
1. Perforin: forms pore in the target cell (release of
intracellular contents from infected cell); translocation
channel for granzymes
2.
Granzyme B: belongs to family of serine proteases that
mediate apoptotic cell death through the (i) direct
cleavage of pro-caspase-3 or, (ii) indirectly, through
caspase-8, (iii) cleavage of BID resulting in its
translocation, with other members of the pro-apoptotic
BCL2-family such as BAX, to the mitochondria, (iv) direct
activation of DFF40/CAD (DNA fragmentation 40/caspaseactivated deoxynuclease) — which damages DNA and
leads to cell death — by granzyme-B-mediated
proteolysis of the inhibitor ICAD.
CD8+ CTLs have non-cytolytic action too (today’s discussion
paper)
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Pabio552 Lecture 17; NL Haigwood Barry M., et al., Nat Rev Immunol. 2: 401-9 (2002). 16
CD8+ CTL control of HIV infection
Evidence
•
Control of high viral loads associated with the acute phase of HIV infection is
coincident with the appearance of anti-HIV CTLs
•
CTLs can be found at sites of HIV replication in vivo
•
The quantity of anti-HIV CTLs, as measured by MHC-I tetrameric complexes,
correlates inversely with viral load (data conflicting)
•
When SIV infected macaques were immunodepleted for CD8+ T cells, a dramatic
increase in SIV replication was observed
•
HIV vaccine strategies targeting CTL responses show decrease in viral load in SIV/
SHIV infected macaques
•
CTLs from Long-term nonprogressors secrete higher levels of perforin, granzyme B
•
BUT, vaccines that elicit only CTL cannot protect from infection, only control, and
patients can be superinfected with another HIV-1 isolate, even if CTL are present
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Impairment of the cellular immune response
•
•
•
Interference with proteosomal degradation
Peptide transport associated with Ag presentation
Retention of MHC I molecules in subcellular compartments
A. Destruction of MHC class I heavy chains
•
•
•
•
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Induction of functional paralysis of DCs
Downregulation of MHC Class II expression
Circumvent NK-cell mediated killing
Antigenic variation of T cell epitopes
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Viral load is critical to the control of disease
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Viral Escape from Neutralizing
Antibodies
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What are Neutralizing antibodies?
CD4
CCR5/ CXCR4
Target cell
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What are Neutralizing antibodies?
CD4 binding induces a
conformational change in envelope
leading to exposure of the
coreceptor binding site
Target cell
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What are Neutralizing antibodies?
Binding to CCR5 exposes the
fusion domain leading to
subsequent viral core entry
Target cell
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What are Neutralizing antibodies?
Most antibodies however, bind
the virus but do not neutralize
Target cell
Neutralizing antibodies: Block
virus infection in the target
cells by directly binding to
virion and prevent their
interaction with receptor on
the target cells
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NAbs act at different levels
Burton DR. Nat Rev Immunol. 2: 706 (2002).
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Envelope modifications enable NAb escape
Influenza Virus
No cross-protective immunity
Escape from neutralizing
antibodies
Original antigenic sin: Abs
made only to epitopes present
on the initial viral variant when
infected with a second variant
Source: http://www.gak.co.jp/FCCA/glycoword/GD-A06/GD-A06_E.html
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HIV: Escape from Antibody-mediated Neutralization
Reasons
1.
Schematic of envelope trimer
Transient exposure of neutralizing
epitopes only at critical moments
during the entry process
CCR5 binding site is highly protected and is
exposed only after conformation changes has
occurred upon binding CD4
CD4 independent viruses are highly
neutralization sensitive
2.
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CD4 binding
site
CCR5
binding site
Occlusion of conserved domains
within the oligomer
Pabio552 Lecture 17; NL Haigwood
Wyatt R., et al., Nature 393: 705 - 711 (1998)
27
HIV: Escape from Antibody mediated Neutralization
Reasons
Schematic of envelope trimer
3.
Extrusion of variable domains from the
exposed surface of the oligomeric
complex
Serves as an antigenically variable shield and
covers the more conserved envelope core
Removal of V2 loop opened up the CD4 binding
site rendered the virus susceptible to
neutralization from other clades, and also
resulted in high titer neutralizing antibodies in a
SHIV infected macaque and reduced viral burden
(Stamatatos et al; J Virol. 1998. 72:7840)
CD4 binding
site
CCR5
binding site
Wyatt R., et al., Nature 393: 705 - 711 (1998)
Land A., et al., Biochimie. 83: 783-90 (2001).
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HIV: Escape from Antibody mediated Neutralization
Reasons
4. Extensive glycosylation of the
envelope proteins
HIV/ SIV envelope contains 28-30 potential N-linked
glycosylation sites that account for 50% of the mass
of gp120
Position of the glycans modulate antibody
neutralization specificity
Removal of N-linked glycan in V3 loop increased
HIV’s sensitivity to neutralizing antibodies
Longitudinal analyses indicate that the number of
glycans remain fairly constant, while positions vary,
with indels, and most commonly a shift by a couple
of aa’s
Land A., et al., Biochimie. 83: 783-90 (2001).
Evolving Glycan shield
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HIV: Escape from Antibody mediated Neutralization
Evidence
Viral variants are resistant to plasma
(NAbs) from the same time point
During chronic HIV infection, there
are high levels of NAbs,
however they are not directed
towards autologous virus from
the patient at the same time
point
NAbs that recognize heterologous
isolates arise late in infection
(Broad)
The development of heterologous
NAb hinders the development of
autologous NAb responses that
may control infection
Richman DD., et al. PNAS. 100(7): 4144. (2003)
Despite these changes, Env is by necessity relatively
conserved in CD4, co-receptor binding regions
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Broad NAbs do arise in some patients
Ferrantelli F., et al., Curr Opin Immunol. 2002 Aug;14(4):495-502.
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Passive immunization studies
Combination of broad human monoclonal NAbs act synergistically to
confer protection against SHIV and SIV
1.
Prevention of SHIV transmission to macaque monkeys when IgG1 b12 was
mucosally applied
2.
Post-exposure prophylaxis with human monoclonal antibodies prevented
SHIV89.6P infection or disease in neonatal macaques
3.
Potent cross-group neutralization (Clades A, C, D, E, and F) of primary HIV isolates
with monoclonal antibodies - IgG1b12 (b12), F105, 2G12, 2F5, 4E10, F424, and
Clone 3 (CL3)
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Host susceptibility to viral infection
HLA haplotypes influences HIV disease progression
1.
2.
3.
Associated with slower disease progression: A*11, B*27, B*57, B858, Cw*02, Cw*14
Associated with rapid disease: B*35, B*53, Cw*04
Maternally acquired protective alleles were no longer protective against disease
progression in vertically infected infants
Mutant forms/ Polymorphisms of coreceptor modulates progression to AIDS
1.
2.
3.
4.
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Individuals homozygous for a 32-bp deletion (32) in CCR5 are resistant to infection by
HIV-1
HIV-1-infected individuals heterozygous for CCR5 32 (-/+) show a slightly slower disease
progression than CCR5+/+ homozygotes
Common polymorphism in the 3'-untranslated region of the stromal derived factor gene
(SDF-1; natural ligand for CXCR4) associated with delayed disease progression from
some cohorts. In others, the same homozygous genotype has been associated with rapid
progression to AIDS, but with prolonged survival after diagnosis of AIDS
Polymorphism in 3’ UTR SDF-1 is associated with increased risk of vertical transmission
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Immunological techniques
Not an exhaustive list!
Humoral Immunity (B cells)
A. ELISA; Radioimmunoassay : checks for the development of virus-specific antibodies in plasma,
other body fluids
B. Ab competitive inhibition assay
C. Antibody-dependent cellular cytotoxicity (ADCC)
D. How are monoclonal antibodies generated?
E. What is Biacore? What does it measure?
F. Neutralization assay – viral infectivity on target cells in the presence or absence of Ab
Cellular immunity (CD4, CD8 T cells)
A. How do you check for the presence of cytokine secreted by a T cell?
B. Antigen proliferation assay (also called lymphocyte proliferation assay, thymidine uptake assay)
C. MHC Tetramer Assay: Ag specific T cell staining reagent
Functional Assays
A. Cytokine production: ELIspot, intracellular cytokine staining
B. B- cell activation: What is an ELIspot?
C. Target Cell Killing: Chromium release assay: what does it measure?
D. Antibody dependent cell-mediated cytotoxicity (ADCC)
E. Chemotaxis assay
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References
Reviews
1.
2.
3.
4.
5.
6.
7.
Alcami A. Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol. 3: 36-50 (2003).
Favoreel HW., et al. Virus Complement evasion strategies. J. Gen. Virol. 84: 1-15 (2003).
Orange JS., et al. Viral evasion of natural killer cells. Nat. Immunol. 3: 1006 (2002).
Vossen MTM., et al. Viral Immune evasion: am masterpiece of evolution. Immunogenetics. 54: 527 (2002).
Domingo E. Viruses at the edge of adaptation. Virology. 270: 251 (2000)
Barry M., et al. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2: 401 (2002).
Ferrantelli F, et al., Neutralizing antibodies against HIV -- back in the major leagues? Curr Opin Immunol. 14:
495 (2002).
8. Land A., et al., Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the
endoplasmic reticulum. Biochimie. 83: 783 (2001).
9. Burton DR. Antibodies, viruses and vaccines. Nat Rev Immunol. 2: 706-13 (2002).
10. Wyatt R. et al., The antigenic structure of the HIV gp120 envelope glycoprotein. Nature. 393: 705-11 (1998).
Primary papers
1.
2.
3.
4.
5.
6.
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Schlender et al. Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human
plasmacytoid dendritic cells by respiratory syncytial virus and measles virus. J Virol. 2005 May;79(9):550715.
Richman DD., et al. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. PNAS. 100:
4144 (2003).
Ferrantelli F., et al., Potent cross-group neutralization of primary human immunodeficiency virus isolates
with monoclonal antibodies--implications for acquired immunodeficiency syndrome vaccine. J Infect Dis.
189: 71-4 (2004).
Mascola JR., et al., Protection of macaques against vaginal transmission of pathogenic HIV-1/SIV chimeric
virus by passive infusion of neutralizing antibodies. Nat Medicine 6: 207-210 (2000).
Chackerian et al., Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of
simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies. J
Virol. 1997 Oct;71(10):7719-27.
Wei X et al. Antibody neutralization and escape by HIV-1. Nature. 2003 Mar 20;422(6929):307-12.
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Neutralization Assay
• Target cells (Cell line/ PBMC) expressing viral receptors
for entry (For ex, HIV-1 CD4, CCR5) and a reporter gene
(-gal, luciferase)
• Mechanism
Ta
t LTR
- Gal
• Only infected cells produce  Gal  Easy visualization
through X-gal staining
• Neutralization assay: pretreatment of virus with Ab
– Reduction in  Gal positive cells indicates virus
neutralization
– Calculate % neutralization
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Generation of monoclonal antibodies
Janeway, Immunobiology 5th Edition; Fig A.15
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Antigen proliferation assay
2. Ag specific Lymphocytes start
3.
proliferating
1.
After a 5 day incubation period,
pulse with H3thymidine
Incubate PBMCs with Antigen/
Stimulus
8-16 hours
incubation
Antigen: gp120,
peptide pools (Gag, Env)
AT-2 inactivated SHIV89.6
Contols: PHA
SEB
a-CD3 mAb
irrelavant peptide
Plain media
4. Harvest the cells onto filters using
cell harvester
5. Read filters on a scintillation
counter
Calculate Stimulation index = Samplecpm
Irr cpm
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ELIspot
Source: www.bdbiosciences.com
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Tetramer staining
•
Stain antigen-specific T cells
•
MHC:peptide tetramers are formed from recombinant refolded
MHC:peptide complexes containing a single defined peptide
epitope
•
Recombinant MHC heavy chain is linked to a bacterial biotinylation
sequence, a target for the E. coli enzyme BirA, which is used to add
a single biotin group to the MHC molecule
•
Streptavidin is a tetramer, each subunit having a single binding site
for biotin, thus the streptavidin/MHC:peptide complex is a tetramer
of MHC:peptide complexes
•
While the affinity between the T-cell receptor and its MHC:peptide
ligand is too low for a single complex to bind stably to a T cell, the
tetramer, by being able to make a more avid interaction with
multiple MHC:peptide complexes binding simultaneously, is able to
bind to T cells whose receptors are specific for the particular
MHC:peptide complex
•
Streptavidin is coupled to a fluorochrome, so that the binding to T
cells can be monitored by flow cytometry.
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Janeway, Immunobiology 5th Edition; Fig A.32
40
Biacore
1.
Based on Surface plasmon Resonance (SPR)
technology
2.
SPR arises when light is reflected under certain
conditions from a conducting film at the interface
between two media of different refractive index
3.
SPR causes a reduction in the intensity of reflected
light at a specific angle of reflection
4.
Angle varies with the refractive index close to the
surface on the side opposite from the reflected light
(the sample side in Biacore)
5.
When molecules in the sample bind to the sensor
surface, the concentration and therefore the
refractive index at the surface changes and an SPR
response is detected
6.
Plotting the response against time (called
sensorgram) during the course of an interaction
provides a quantitative measure of the progress of
the interaction
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Source: www.biacore.com
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Chromium Release assay
Janeway, Immunobiology 5th Edition; Fig A.38
1. Measures Cytotoxic T-cell activity
2. Target cells are labeled with radioactive chromium as Na251CrO4,
washed to remove excess radioactivity and exposed to cytotoxic
T cells
3. Cell destruction is measured by the release of radioactive
chromium into the medium, detectable within 4 hours of mixing
target cells with T cells.
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