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Feasibility of Monitoring Cell Mediated
Immunity during Vaccine Trials

FIRN – 2009
July 2009, Kona HI, USA

Dr. Thomas O. Kleen
Director, Business and Technology Development
Cellular Technology Limited (C.T.L.)
www.immunospot.com

The views expressed are those of the author and do not necessarily correspond with the current views of CTL orSilversword agencies
Haleakalā any regulatory (1998)
© Dr. Thomas O. Kleen, Jul-09 CONTACT: Thomas.Kleen@immunospot.com C.T.L. CONFIDENTIAL ( www.immunospot.com ) 1

Immune Monitoring and Biomarker Screening
during Vaccine Development is
Good Science and Good Business
• Accelerates time to market and save costs:
• Potentially adds weeks, months or more to a product
lifecycle under IP protection and facilitates the ability to
enter the market ahead of a competing product

• Go/no go decisions:
• Enables to quickly refocus on alternative approaches if
immune monitoring data indicate a lack of efficacy or
should safety concerns arise during proof of concept,
animal studies or any clinical phase of the development

Objectives for Immune Monitoring and
Biomarker Screening during Vaccine
Development

• Delivery of solid comparison data:
• Immune monitoring and biomarker screening can be
implemented during all pre-clinical and clinical phases of
vaccine development

• Increase chances of a successful clinical trial:
• High resolution, GLP compliant immune monitoring
provides regulatory acceptable data


Rationale for Monitoring Cell Mediated
Immunity during Vaccine Trials
• Cell Mediated Immunity (CMI) is a critical component during most
immunological responses; involved in infectious diseases,
cancer, and autoimmunity

• Basic research into correlates of protection has exposed the
limitations of vaccine approaches that rely solely on antibody
responses to confer protection against pathogens (e.g., HIV,
HCV, TB, Smallpox, …..)

• The emerging field of therapeutic vaccines has further
highlighted the need to induce or modulate CMI in order to fight
cancer, as well as autoimmune diseases and allergic responses


Current Challenges facing Broad
Implementation of CMI Monitoring

• Monitoring technologies with sufficient high resolution are
often perceived to be too costly, complex, and time
consuming to be routinely utilized with hundreds or even
thousands of samples.

• This applies in particular to technologies that aim to
detect crucial low frequency T-cell responses

• Common misconceptions surrounding regulatory
acceptance of CMI and biomarker data have to be
overcome


Current Challenges facing Broad
Implementation of CMI Monitoring (cont)

• Limited awareness exists in certain segments of the
industry regarding recent advances in:

• Cell sample cryo-preservation and thawing procedures

• Cost effective, sensitive, single cell-based, high
throughput capable assay technologies

• Validation procedures for cell-based assay systems

• Standardization reagents and procedures for cell-based
assay and test systems

Required Characteristics of Assays to Monitor
CMI in Regulated Environments
• Measures a physiological and clinically relevant response

• Is a reproducible, reliable assay for testing serial samples

• Lends itself to validation (possibility of determining Accuracy,
Precision, Specificity, Linearity and Limits of Detection)

• Has a meaningful sensitivity that is able to detect low frequency
cells (Memory T-cells are frequently rare as 1 in 100,000 or less)

• Available data analysis equipment must be able to be integrated
in 21 CFR Part 11 compliant laboratory settings (i.e., system
validation, computer generated audit trails)


Desirable Features of Assays for Monitoring
CMI in Regulated Environments
• Performs identical with fresh and previously frozen samples

• Can be standardized; to enable, for example, inter-study
comparisons to make data more robust for multicenter or large
clinical trials

• Uses the least amount of cells and clinical sample material

• Capability to be run in high throughput mode to accommodate
large-volume testing with hundreds of samples a day

• Availability of automated, high throughput capable data read-out
equipment including data analysis software to insure objectivity


Widely Used Assay Technologies to Evaluate
Cytokine Production and CMI
• Enzyme-Linked Immunosorbent Assay (ELISA)
Supernatant based
• Luminex & Cytometric Bead Arrays (CBA)

• Fluorescence-Activated Cell Sorter (FACS)

• Intracytoplasmatic Cytokine Staining (ICS)

• Tetramer staining
Single Cell based

• Pentamer staining

• Surface marker staining

• Enzyme-Linked Immunospot Assay (ELISPOT)

Requirement for the Ability to Evaluate Multiple
Functions, Molecules or Cytokines


Advantage of Using Single Cell Cytokine
Secretion by T-cells as Biomarker
• Establishes the quality of an immune response, i.e., the
type of products T-cells secrete in response to a vaccine
candidate, allowing the differentiation between
Th1/Th2/Th5/Th17/Thpp (CD4+ T-cells) and Tc1/Tc2 (CD8+
T-cells) based on cytokine signatures

• Establishes the quantity (amount) of the secreted cytokine
product in response to an antigen or vaccine

• Establishes the frequency (clonal sizes) of the responding
cells to an antigen or vaccine, giving an indication about
scale or potency of the immune response (or potential
adverse effects)

ELISPOT Assay’s Unique Qualification for
Cytokine-Based Immune Monitoring
100.00
• Sensitivity: Routine detection limits of 1 in

% IFN-γ positive cells
FACS
10.00

500,000 events or better. Meaning in day to 1.00

0.10

day laboratory operations several to 0.01
101 102 103 104 105 106
0.8
hundred fold more sensitive than ELISA, 0.6
ELISA

OD405
CBA, ICS, Tetramer, etc. 0.4

0.2

0.0

101 102 103 104 105

• Direct ex vivo cell frequencies: Measures

# of IFN-γ Spots/Well
500
400
ELISPOT

the physiological magnitude of T-cell 300
200

immunity, with no need for in vitro expansion 100
0
0 100 200 300 400 500 600
(not provided by ELISA, CBA, *ICSlow, Number of Cells

*Tetramerlow, etc.)
*(only applies to direct ex vivo low frequency T cell responses)


Cytokine-Based Immune Monitoring (cont)
• Samples not pharmacologically treated: No use of
secretion inhibitors or cell permeable agents as with ICS
and FACS

• Robustness: Cell samples can be freeze thawed while
maintaining highly reproducible results (with the right
protocols)

• Validation Capabilities: Assays can be validated under
GLP with multiple cytokines and test systems (e.g., CTL
Laboratories has validated the assay for its clients in
human, mouse, monkey, and pig test systems)

Cytokine-Based Immune Monitoring (cont.)
• Scalable high throughput capability: In trained and
appropriately setup up laboratory environments, up to 450
clinical samples can be cost-effectively tested per week (e.g.,
CTL tested 10,000 individual samples in less then 6 months)

• Available 21 CFR Part 11 integration enabled equipment: For
example, CTL’s ImmunoSpot® ELISPOT analysis equipment and
software solutions are designed to be integrated into Part 11
compliant laboratory settings

• Possibilities of standardization: Ranging for methods and
reagents for clinical sample preservation to post trial final data
read-outs the implementation of assay standardization is feasible

Fresh versus frozen PBMC in ELISPOT

Cytokine secretions are
insensitive to a freeze IFN-g IL-2
thaw cycle:
• ELISPOT assays with
human PBMC measuring
IFN-g (A), IL-2 (B), IL-4 (C),
or IL-5 (D) IL-4 IL-5

• PBMC were tested either
fresh (black circles) or
thawed, 1 week after
freezing (gray circles)
Kreher et al., J. Immunol. Methods, (2003), 278:79-93

The Choice of Test-system and Cytokines to
monitor is Vital to Detect Relevant Responses in
CD4 cells
• Use of Adjuvants with CD4
cells: The choice of adjuvant
during vaccination (i.e. IFA
versus CFA) can switch peptide
specific CD4+ recall responses
form Th1 (IFN-g) to Th2 (IL-5)

Yip et. Al,. J. Immunology, (1999) 162:3942-3949

Use of Adjuvants with CD4 cells
E.g. immunization with PLP 139-151 peptide plus CFA as
adjuvant induces similar numbers of IFN-g and IL-2 producing
CD4 cells, but IL-17 producing CD4 cells are induced by CFA
only

Tigno et al., J. Immunother, (2009), 32:389-398

The Choice of Cytokines or Effector function to
monitor is Vital to Detect Relevant Responses of
CD8 Cells
• Use of Adjuvants with CD8 cells : The choice of
adjuvant during vaccination (i.e. CFA versus CPG) can
switch peptide specific CD8+ recall responses between
mainly DTH-mediating or mainly cytotoxic responses
• Results in mice after immunization with class I restricted peptides in different
type 1 adjuvants:

IFN-g IL-2 IL-17 Killing DTH

CFA +++ +++ +++ - +++

CpG + + - +++ -


Use of Adjuvants with CD8 cells
Immunization with “Uty” or SIINFEKL peptides plus CFA as
adjuvant induce higher numbers of IFN-g and IL-2 producing
CD8 cells than immunizations with CpG


Use of Adjuvants with CD8 cells (cont)
Immunization with “Uty” or SIINFEKL peptides plus CFA as
adjuvant induce IL-17 producing CD8 cells while immunization
with CpG does not

SFU per 2 x 105 CD8 cells plated
SFU per 1 x 106 splenocytes

150 120
IL - 17 p < 0.01 IL - 17 p < 0.05
125 100

100 * 80

75 60
*
50 40

25 20

0 * 0 *
PBS Uty PBS Uty PBS SIINFEKL

Uty Uty SIINFEKL

Bulk response CD8 response

Tigno et al., J. Immunother, (2009), 32:389-398 n=6

Use of Adjuvants with CD8 cells (cont)
Immunization with Uty or SIINFEKL peptides plus CpG as
adjuvant induces stronger in vivo killing than immunization
with CFA


The Choice of Test-system and Cytokines or
Molecule to monitor is Vital to Detect Relevant
Responses
• Example HIV: In humans HIV-peptide specific recall responses show a
dissociated production of IFN-g and GzB (up to 40% of peptides only induce
GzB) Patients recall to representative HIV peptides

Kleen et al., AIDS, (2004), 18:383-392

The Choice Cytokines or Secreted Molecules to
monitor is Vital to Detect Relevant Responses
CEF peptides do not induce direct ex vivo GzB or perforin in
healthy human PBMC, but CD8 cells start secreting GzB and
perforin 72 h after in vitro restimulation
Ex vivo After 72h pre-activation

Nowacki et al., Cellular Immunol, (2007), 247:36-48

The Choice of Cytokines or Secreted Molecules to
monitor is Vital to Detect Relevant Responses (cont)

Dissociation of IFN-g vs. perforin and granzyme B allows
distinction of memory vs. effector CD8 cells
• Results in humans after recall with nominal peptide:

IFN-g IL-2 GzB Perforin

Memory +++ (+) - -

Effector +++ (+) +++ +++

Nowacki et al., Cellular Immunol, (2007) 247:36-48

Standardization Strategies for CMI monitoring
and ELISPOT
• Harmonize sample collection at clinical sites
• Training and qualification of all clinical sites in proper sample
handling, PBMC processing, cryopreservation and shipping
standard operating procedures (SOPs)

• Harmonize methods across all participating laboratories
• Implementation of detailed SOPs for cell thawing, cell counting,
assay procedures and the analysis of assay results
• Standardize all assay materials, including plates, antibodies,
media and enzymes (always re-qualify new lots!)
• Utilize cell-based reference sample PBMC to optimize assays
and compare performance between laboratories

Use of cryo-preserved reference sample
PBMC
• Existing Reference PBMC Library of 50 healthy, HLA-typed and
immune-characterized individuals with up to 10 billion cells per
individual
• Each sample has an established cytokine secretory reactivity to 32
individual T cell peptide epitopes of Cytomegalo-, Epstein-Barr, and
Flu virus (CEF), as well as to 5 proteins from Candida, Dust Mite,
Mumps, Tetanus, and PPD
• Used for assay validation and protocol development through ulitisation
of authentic, physilogical relevant cell activities without need for
artifical or artifact prone mitogenic stimulation (PHA, ConA, ..…)
• Enables realistic benchmarking of laboratory perfomance and internal
controls during clinical trials without scarifcing precious clinical
samples


Cryopreseved PBMC Reference Sample Show
Low Intra- and Inter-Laboratory Variability of
ELISPOT Results
Intra-laboratory reproducibility of ELISPOT
data.
The three reference samples LP51, LP37 and
LP58 were tested in three independent
experiments with the same SOPs and materials by
a single individual (A)
or by three different individuals, whereby each of
them thawed and processed the cells
independently (B)
In (C), for a separate experiment, the cells were
thawed, processed and counted by a single
individual and subsequently split into 3 identical
aliquots for testing by 3 independent investigators.
The mean and standard deviation for three
replicate wells in each test is shown
Data obtained in cooperation with Stéphanie McArdle during
NEUCAPS multicenter trial

Advantages of serum-free cryopreservation
reagents and assay media
• Use of standardized serum-free cryopreservation reagents for
PBMC yields a <95% post-thaw viability, maximizing clinical
sample utilization (in combination with optimized freeze-thaw
procedures)
• Serum-containing undefined products like Fetal Bovine Serum
(FBS), Fetal Calf Serum (FCS), as well as human ABO serum in
cryo-preservation and assay media can cause significant assay
background noise and artifacts rooted in large intra-batch and
performance variations
• Utilization of bovine products is prohibitive for all clinical trials
involving international shipping of samples, based on strict import
restrictions of many countries due to BSE concerns (mad cow
disease)

Serum Free Versus Serum Containing Test Media (cont)

Serum Free Media

FCS Containing Media

Media CMVpp65 Media CMVpp65

PBMC sample 1 PBMC sample 2

Data obtained by Stéphanie McArdle during NEUCAPS multicenter trial

Serum Free Versus Serum Containing Test Media (cont)

Serum Free Media

Prescreened Batch Tested
human AB Serum
Containing Media

Media CMVpp65 Media CMVpp65

PBMC sample 1 PBMC sample 2

Data obtained by Stéphanie McArdle during NEUCAPS multicenter trial

ELISPOT as Validated Cellular Readout of a Functional Assay
96 well mircotiter plate coated with
A detection antibody

Secreting cell – e.g., antigen-
B stimulated T cell secreting cytokine

Plate-bound secretory product
C
(cytokine) after cells are washed
away
Product-specific detection antibody
D
with linked enzyme

Enzyme catalyzed chromogen
E
spot development or fluorescence
label
PVDF-membrane Antigen-stimulated cell Enzyme-coupled detection antibody

Secreted cytokine Capture antibody Chromogen/Fluorescence label


Validated Computer Assisted Image Acquisition,
Counting and Analysis of Data


Acknowledgements
Dr. Paul V. Lehmann

Dr. Magdalena Mary-Lehmann

Dr. Oleg Targoni

Dr. Wenji Zhang

Dr. Stéphanie McArdle
&
All 11 participating Laboratories during
the NEUCAPS – I multicenter trials

Dr. Stefanie Kuerten
Dr. Justin Tingo


Thank You!

Questions?


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