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Immune Monitoring
1. Immune Monitoring in Vaccine Trials
Jill Gilmour
Exec Director IAVI HIL
Imperial College, London
Symposium on Innovations in Vaccine R+D
Wellcome Trust , London Sept 5th 2012
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2. Immune Monitoring in Vaccine
Trials: Outline
1. Introduction and background
2. Context for immune monitoring on clinical trials
3. Available assays
4. Challenges
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3. HIV Variability: The problem has a solution…….
A globally effective HIV vaccine will likely need to elicit:
• Broadly Neutralizing Antibodies: Prevent infection of any HIV strain
• Broadly Reactive T cells (CMI): Control Infection of any HIV strain
Neutralizing Abs
Cell Mediated
Immunity (CMI)
AIM to elicit both in a final product
Phase IIB testing of vaccine candidates that elicit either broad cellular or broadly neutralizing
antibodies will yield valuable information for field re-distinct immunological spaces.
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4. An Introduction
The Human Immunology Laboratory (HIL)
Mission
Deliver high quality clinical trial data to prioritize and develop HIV
vaccine candidates for IAVI and the field
Coordinate and support IAVI’s global network of clinical
laboratories
Inform the next generation of HIV vaccine design and assessment
through translational clinical research on the African epidemic
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5. IAVI R&D Network Global outlook
IAVI
Development
Design and IAVI Human
Development Lab Immunology Lab
IAVI NAC
at Scripps
Innovation Fund grants
IAVI India Lab
Immune
Neutralizing Vectors correlates/
Antibody Consortium LAC
Consortium
Clinical research
centers
IAVI collaborations with CAVD, HVTN, MVI and UK-HVC.
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6. Immune Monitoring in Clinical Trials
Context
• Regulatory environment
– In EU member states, laboratories performing testing on clinical trial
samples should be accredited e.g. GCLP, and are subject to audit by
regulatory bodies
– Quality systems to ensure integrity of data
– Maybe a requirement to validate assays
• Standardization and method validation
– Need to pool data from multiple clinical centers and compare data
across products, regimens, sites etc
– Ensures studies are powered to meet primary objectives
• Go/ No-Go must be based on robust and scientifically sound data
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7. Immune Monitoring in Clinical Trials
Context Cont’d
Stage of product development
• Phase I/II Trials:
– Primary assays: Accurately assess response rate, kinetics,
magnitude to optimize dose and regimen. Hypothesis testing.
– Research assays: Characterize immunogenicity and test/generate
additional hypothesis
• Efficacy Trials
– Primary assays similar to phase I/II with some additional validation
and streamlining
– Test a proportion of volunteers. Important for manufacturing e.g.
ensure potency between lots.
– Research assays to assess correlate or mechanism of efficacy.
– Implications for vaccine design and manufacturing e.g. optimizing
dose, monitoring vaccine potency as product released to market etc
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8. Correlates of protection after vaccination;
defined quantitative measures. S. Plotkin, Clin
Inf Dis. 2008
For many vaccines we don’t know exact correlates of
protection; HIV, TB, Malaria
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9. What have we learned from HIV vaccine efficacy
studies to date?
• Vaxgen gp 120 (No efficacy)
– Induced Env-specific non-neutralizing antibodies and tier 1 neuts
• STEP Merck Ad5 gag-pol-nef (No efficacy)
– Induced “poly-functional” CD8 T cells
– Limited CD8 breadth
– Evidence of protection against vaccine matched viral strains in vaccine recipients in vivo
and in vitro
• RV144 Canarypox + gp 120 (31% reduction of HIV-1 acquisition with no viral load effect)
– Induced Env-specific non-neutralizing antibodies and tier 1 neuts
– Predominantly CD4 Env-specific T cells
– Correlates analysis (V2 antibody binding)
• DNA prime + Ad5 boost: gag-pol-nef, env A, B and C
– Data expected 3-4Q 2013
– Induces “polyfunctional” T cells
– Antibody and CD4 env responses
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10. Immunogenicity Go/No-go Criteria:
Cellular and Antibody
General Criteria (not product specific)
Modeling suggests a public health benefit with a 50% partially effective vaccine
Go > 60% response rate
Demonstrate superiority either qualitatively and quantitatively to candidates
which have been, or are in efficacy testing
PLUS
Product Specific Criteria-test rationale
Enhanced breadth, depth of coverage
o Evidence of coverage in developing world
Enhanced magnitude and quality of response
o Anti-viral activity, proliferation, CD4 and CD8, avidity, affinity, B cell memory
Induction of effective, boostable, memory response
Mucosal targeting
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11. Outline
1. Introduction and background
2. Context for immune monitoring on clinical trials
3. Available assays
4. Challenges for the future
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12. Primary and secondary assays in common use to
assess vaccine immunogenicity:
Cellular Antibody
Response rate ELISPOT ELISA
1o Kinetics Magnitude
Target antigens
Flow cytometry Antibody class / subclass
Phenotype CD8 CD4 Memory CD4 B Memory
2o
Function Viral Inhibition Assay Neutralisation Assay
Proliferation, Cytokines…… Non-neutralising: ADCC, ADCVI, V2
Breadth ELISPOT: Epitope mapping Epitope mapping
Viral Inhibition Assay Neutralization Assay
3o Location GI for containment of viral reservoir GI for containment of viral reservoir
(mucosal GU for containment of infection GU for containment of infection
sampling)
• Research assays: novel technologies e.g. Proteomics, genomics, DNA
microarrays, RNA-sequencing, single cell multiplexing e.g. fluidym, nano-string,
• Antibody gene sequencing 12
13. Example primary and secondary T cell immunogenicty
assessment for an Ad35 GRIN-Env vaccine regimen
IFN-g ELISPOT – Gag peptide pool >18 peptides mapped /
3000
2000
1000 Map Gag 8 unique regions in 8
SFC/106 PBMC
500
400
Epitopes individuals
300 1-3 epitopes / vaccinee
200
100
0
Low Mid High GRIN
Conserved vs variable epitopes
Log Inhib
Polychromatic Flow Cytometry
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14. Limitations of Most T Cell Assays
High concentrations
High concentrations Indirect measurements
ofof exogenous peptide
synthetic antigen (peptide) (e.g. cytokines, peforins)
New methods of assessing CTL
function need to be developed Limited to
peripheral
blood
Irrelevant Targets Do not measure antiviral function
(B-cell lines) (MHC tetramer stain)
Adapted from Watkins 2008
15. Alternative Assay: Viral Inhibition Assay
High concentrations Indirect measurements
Whole HIV Direct Antiviral Effects
of synthetic antigen (peptide)
CD8
The Cell
The in vivo
Antigen New methods of assessing CTL
e.g. VIA Assess
mucosal
responses
The site
CD4
HIV infected
Autologous CD4
T Cells
XCD4
Measure antiviral activity
The outcome
The in vivo target
17. CD8 mediated inhibition of and HIV-1IIIB
correlates with in vivo virus control HIV-1
Spentzou et al (2010) JID 201: 720-729
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18. In VIA Volunteers vaccinated with MRK-AD5 gag-pol-
nef inhibit only viruses matched to vaccine insert:
As predicted by efficacy data
Total vaccinees = 16. Efficient inhibition > 1.5 log10 inhibition.
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19. VIA activity correlates with viral control in HIV
and vaccinated NHP controlling SIV challenge
■ 1st generation VIA correlates to in vivo virus control and excellent
specificity in HIV vaccine trials (Spentzou et al. JID. 2010)
■ Higher inhibition in the VIA correlates to lower viral loads in vivo. (Julg
et al. JVI 2010)
■ VIA inhibition correlates with viral load dynamics over time (CHAVI 001)
(Freel et al. JVI 2012)
■ VIA in DNA-Ad5 vaccinated NHP correlate with lowered peak and set
point viral loads (Yamamoto et al. J.Virol. 2012)
■ VIA like assay (ICS) results correlate with in vivo control (LTNP)
(Migueles et al. PLoS Path. 2011)
■ VIA in LN cells correlates with protection against live-attenuated SIV
(Picker et al. unpublished data) 19
20. Location, location, location. S. Plotkin, Clin Inf Dis. 2008
HIV – Mucosal sampling is critical
Over 50% of all T cells reside in the gut
The gut is the initial site of
High-level HIV replication
Massive CD4T cell depletion
--occurs within the first two weeks after infection
--observed after intravenous, intra-rectal, oral and
vaginal challenges
Limiting the initial viral replication in the gut could be crucial for
an AIDS vaccine
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21. Mucosal Assay Development
• Assess the feasibility of various mucosal sampling
methods in clinical trial setting
• Develop clinical sampling methods and assays to assess
vaccine induced mucosal immune responses in UK and
Africa, focused on two assays
(a) cellular responses in the gut
(b) antibody responses in genital secretions
Apply into IAVI sponsored/collaborative clinical trials
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22. CD8 T cell responses to gag in
blood compared with gut
Differences in phenotype and function between blood and gut,
and between colon and duodenum also observed
23. KAVI developing as a Centre of Excellence in
Mucosal Immunology in East Africa
Supported by and IAVI and HIL, KAVI has
embarked on a number of studies on
mucosal immunology:
- 2009 – initiated study to develop mucosal
sampling methods and assays to assess
mucosal immune responses in GU tract
- 2011 – initiated pilot study to assess
cellular responses in GALT (colon) using
IAVI-HIL methodologies
- 2011 – initiated mucosal sampling in
vaccine trials
Haas et al Nature 2010 Mar 11; 464(7286):
217-23
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24. Mucosal Specimen Collection and Assay Methods
Sampling Method Assay
Merocel sponge
Antibody (Ab)
(rectal and cervical)
Digene cytobrush
MMC
(rectal and cervical)
Semen MMC and Ab
Rectal biopsies MMC
SoftCup MMC and Ab
Aspirator Ab
Saliva (active and passive) Ab
Oro and naso-pharyngeal swabs Ab 24
25. Other assays for assessment of T cells
For HIV – Makedonas & Betts 2010
TB, Malaria & other
diseases plethora of
possible responses
TCR avidity, affinity, clonotype
‘The degree of polyfunctionality of CD8+ T cells correlates
to the number of functions for which are assayed.’
Novel technologies: e.g. Proteomics, genomics, DNA/RNA microarrays,
RNA-sequencing, single cell multiplexing e.g. fluidigm, nano-string,
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26. Gene expression profiles induced by YF-17D vaccination in
the European and Ugandan population
A. YF-17D vaccination induces different
gene expressions in Lausanne and
Entebbe. (A) Number of genes at 5% FDR
with a threshold of 2 or 3 fold change. The
magnitude of gene expression changes
was higher in Lausanne compared to
Entebbe. (B) Fold changes of the top 100
genes in Lausanne and Entebbe at 3, 7,
14, 56 and 84 days after a first
vaccination. The expression changes were
observed at days 3 and 7 after YF-17D
vaccination. The expression changes of
individuals genes was different in
VGTI (Rafick Sekaly), UVRI and IAVI Lausanne and Entebbe.
B.
27. Single Cell Genomics
Just like flow cytometry, this technology provides us with two
independent pieces of information:
– How many cells express a gene?
– How much do these cells express?
Standard (bulk) analysis confounds these two measurements to
generate an average
Single cell analysis allows us then to answer another question:
– What is the co-expression of genes?
Slide courtesy Mario Roederer and CAVD VIMC-T cell consortium
28. Fluidigm Technology
Dispense cDNA into Microfluidics
Sample cDNAChip
sample vessels
Primers & probes into
reagent vessels
Primers & Probes
Microfluidics mixes all
combinations in
nanoliter-sized
chambers
40 Cycle RT-PCR
Monitor fluorescence
from each chamber
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29. Why is Single Cell Important?
BLIMP1
CD84 (SLAMF5) TNFR-1
BLIMP1
CD84 (SLAMF5) TNFR-1
Single cell analysis reveals a completely different picture of
regulation of these genes!
30. Fluidigm Analysis Summary
• On Single Cells:
– Distribution of gene expression
– Coordinate regulation of genes
– Reveals further heterogeneity (subsets) – potential correlates for
vaccine or disease analysis
• On Bulk Populations (~100 cells)
– Remarkable precision & sensitivity (RT-PCR)
– Economical, directed micro-array-like analysis
• Needs enormous bioinformatics support!
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32. Assays to Monitor the Humoral Response
Research Assays
• B Cells
– B cell Elispot (memory B cells)
– B cell phenotyping
• Antibody
– Deep sequencing/moAb isolation to characterize engagement &
mutation of Ab genes.
– Isolation and characterization of Env-specific monoclonal
antibodies (IgG and IgA) from B memory cells and plasmablasts
sorted according to homing receptors in the systemic and
mucosal compartments
– Use antibodies to explore anti-viral functions e.g. aggregation,
inhibition of trancytosis, mucus inhibition
– Epitope mapping
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33. Challenges
• Need a pipeline of immunogens for clinical testing
• Lack of human challenge models and efficacy trial and data
– E.g. Need to define what an effective broadly cross-
reactive antibody or anti-viral T cell response
• Bio-informatics and data management are not keeping up
with technology and data bases not tranlsational
• Define targets-functional or structural constraints
• Standardization and qualification in vaccine trials
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34.
35. Developing Assays Defining Immunogenicity
Go/No-go Criteria
•Viral control is possible
•Effective immune responses
HIV •Target immunogens
Pathogenesis •Transmission
NHP •Host and viral genetics
Efficacy
• Protection is possible
•Optimize dose, regimen,
e.g. passive antibodies Clinical Trial
•Safety, experience with product
• Viral control possible Data
•Immunogenicity e.g response rate,
e.g. CMV, Live attenuated,
kinetics, function, breadth
Ad26/MVA mosaic,
•Test strategy e.g. Increased breadth,
DNA/IL12/Ep+Ad5
function, location
• Immune correlates
e.g. LAV-VIA in lymph-node,
CMV broad TEM, DNA+Ad5 VIA
Efficacy Trials Correlates of protection
Mechanism of protection
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36. Assay Validation and Qualification
• Primary Immunogenicity Assays
– Validate and document fit for purpose
– Hypothesis testing
– Good precision, accuracy, rigorous and robust
– Ideally low cost and high throughput
– Critical as progress to large scale manufacturing
• Secondary
– Qualified
– Characterize immunogenicity, test secondary hypothesis, generate
new hypothesis
• Exploratory
– R+D
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37. Immune Monitoring in Clinical Trials
Context Cont’d
• Data from the field
– e.g. Step trial and RV144
– Advancements in systems biology, deep sequencing, and other
technologies enable us to look at immune responses in exquisite
detail
• Post RV144 and STEP
– Tendency to assess every cellular and humoral immune response
possible –may not feasible for every trial
• Partnerships, collaborations, multiple clinical centers, study
populations and funders / sponsors required
– Complex operations, reporting, coordination, compromise,
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