CMI Techniques Standardization for Vaccine Responses Evaluation

Fondation Mérieux Conference Center “Les Pensieres”

Veyrier du Lac - France

September 15-17, 2008

ScientificCommittee:

• Mark DAVIS

•Catherine DUTEL

•Bernard IVANOFF

•Christophe LONGUET

•Nolwenn NOUGAREDE

•Giuseppe PANTALEO

•Emmanuelle TRANNOY

Welcome Letter

September 15, 2008

Dear Participant,

It is our pleasure to welcome you to the symposium entitled: ‘CMI Techniques Standardization for Vaccine Responses Evaluation’ in Fondation Mérieux’s Conference Center “Les Pensières.” We hope you will enjoy this meeting, which brings together some of the world’s foremost experts. The format of the discussion is intended to generate discussion and interaction among participants and to foster the dissemination of new information on this topic. The conference will provide an opportunity for specialists to exchange their knowledge and experience through collaboration with researchers from around the world.

Over the next three days, the team at Les Pensières will be on hand to help you with any question you may have and to make your stay and conference as comfortable and valuable as possible.

Benoît Miribel Directeur Général Fondation Mérieux

P.S.: Your feedback is valuable and allows us to organize conferences of a better quality so please complete the Conference Evaluation form that will be in your package and return it to the front desk before your departure.

For more information: www.fondation-merieux.org

Background and rationale

Cell mediated immunity (CMI) is a critical component of the immunological responses contributing to the protection against many infections, cancer, and autoimmune diseases. Thescientificcommunityhasfacedandnowrecognizethelimitationsofvaccinesapproaches relying on antibody response only to confer protection against intracellular pathogens such as HIV and HCV. This has brought more attention on the role of CMI in the development of new vaccines. Therecentemergingtherapeuticvaccinefieldtofightcancer,aswellasatopicorautoimmune diseases has further participated to the better understanding of the CMI role in protection. Uptorecently,majortechnicalandlogisticaldifficultieshavepreventedthevaccineIndustrytoim plement solid and extensive CMI monitoring strategies for vaccine development. Most of the technologies were too complex, tedious and time consuming to be applied to thou sand of samples. Those techniques also usually require fresh cells or other tissue samples in large quantitiesandwereverydifficulttostandardizedduetothetechnicalaswellasbiologicalvariabili ties. Over the past 15 years, considerable progresses have been made in deciphering and unders tanding the cellular components and effector phases of the immune system. This together with the emergenceofnewandsimplifiedtechnologiesthatcanbeautomated,startingfromsamplepreser vationtothefinalread-outs,includingsophisticatedanalysissoftwareenablevaccinedevelopers to work on standardization of the immunomonitoring assays and to reconsider the evaluation in the clinical phases of the cellular component of the immune response induced by their candidate vacci nes.

Fondation Mérieux is pleased to organize this meeting to focus on CMI monitoring for vaccine eva luation, covering new technology developments and standardizations and also touching automa tion and High Through Put Screening (HTS). The Foundation welcomes invited scientists, immunologists, vaccinologists, representatives from industry and international institutions to Les Pensiéres Conference Center, in Annecy, France, from the 15th to 17th of September 2008, to attend to «CMI Standardization techniques in evalua tion of vaccine response». Its purpose is to give an update and discuss the current status of CMI technologystandardization,evaluatingprogressesinthefield,identifyingthegapsthatstillneedto befiled,andthepotentialofthesenewtechnologiesforroutineevaluationofthevaccinecandida tes requiring a cellular component.

The Conference’s strategic objectives include: • To review progresses and state of the art in technology development for monitoring CMI. • To focus the discussions on the needs for standardization to comply with ethical and regula tory requirements. • Tofosterdialogueamongdifferentactorsofthescientificanddecisionalcommunitiesinvol ved in CMI technology development and CMI monitoring of vaccine responses.

ScientificProgram

Monday September 15, 2008

Tuesday September 16, 2008

17h30 - 18h30 Registration

18h30 - 18h45 Welcome Address Benoît MIRIBEL

18h45 - 19h15Keynote lectureFuture of CMI monitoring in vaccine development

Mark DAVIS

19h45 Dinner

Session 1 State of the art in CMI technology development Chaired by Mark Davis and Jacques Louis

08h30 - 08h50 Human B cell response to vaccina-tion Mark SLIFKA

08h50 - 09h10 Discussion

09h10 - 09h30Monitoring human memory T cells:whattomeasureandhowtomeasure it

Patrip CHATTOPADHYAY

09h30 - 09h50 Discussion

09h50 - 10h10 Measure of innate immunity Eric VIVIER

10h10 - 10h30 Discussion

10h30 - 11h00 Coffee Break

ScientificProgram

11h00 - 11h20Structural and functional assay to measureantigenspecificCD8Tcell

Giuseppe PANTALEO

11h20 - 11h40 Discussion

11h40 - 12h00 Microarraymonitoringprofiling RafikSEKALY

12h00 - 12h20 Discussion

12h20 - 14h00 Lunch

Session2 Standardization:Regulatoryandethicalrequirement ChairedbyPhilippeKrauseandMichaelPfleiderer

14h00 - 14h20 Monitoring of CMI responses in early life Arnaud MARCHANT

14h20 - 14h40 Intracellular cytokine staining standardization Maria JAIMES

14h40 - 15h00

Results and harmonization guide-lines from two largescale interna-tionalELISPOTproficiencypanelsconducted by the CVC

Sylvia JANETZKI

15h00 - 15h30 Coffee Break

15h30 - 15h50

Harmonization of T cell immuno-monitoring within the CIMT mo-nitoring panel - correct protocol choices and additional sources of variation

Cedrik BRITTEN

16h10 - 16h30 Statistical analysis of CMI data Zoé MOODIE

16h30 - 16h50 FDA perspective on cellular assay standardization and validation Philippe KRAUSE

16h50 - 17h45 Panel discussion

19h00 Dinner

ScientificProgram

Session 3 CMI evaluation in vaccine development Chaired by Brigitte Autran and Marcelo Sztein

08h30 - 08h50 CMI evaluation in Dengue vaccine development Bruno GUY

08h50 - 09h10 Discussion

09h10 - 09h30Correlates of protection in the elderly:measuresofthecellularresponse in cancer vaccination

Janet MC ELHANEY

09h30 - 09h50 Discussion

09h50 - 10h10Correlation between tumor regression and T cell response in cancer vaccination

Pierre COULIE

10h10 - 10h30 Discussion

10h30 - 11h00 Coffee Break

11h00 - 11h20Monitoring cellular immunity induced by vaccination againstTuberculosis

Willem HANEKOM

11h20 - 11h40 Discussion

11h40 - 12h00 Measure of cellular immune response in HIV clinical trials Brigitte AUTRAN

12h00 - 12h20 Discussion

12h20 - 14h00 Lunch

Wednesday September 17, 2008

ScientificProgram

Session4 Lookinginthefuture:enablingtechnology,automationandHTS Chaired by Gil Bernard and Tobias Kollmann

14h00 - 14h20ImmunID:BandTcellrepertoireanalysis:abiomarkeroftreatmentefficiency

Nicolas PASQUAL

14h20 - 14h40 Discussion

14h40 - 15h00 In vitro biomimetic human immune systemmodels:MIMICsystem William WARREN

15h00 - 15h20 Discussion

15h20 - 15h40

Anovelmultiparametricflowcytometry-based cytotoxicity assay simultaneously immunophe-notype effector cells

Albert DONNENBERG

15h40 - 16h00 Discussion

16h00 - 16h20 Statistical modelling of multi-para-meterflowcytometry Cliburn CHAN

16h20 - 16h40 Discussion

16h40 End of the meeting

Keynotelecture:

Future of CMI monitoring in vaccine development

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

T Cell Recognition and the Challenges Facing HumanImmunology

Mark M. DAVISHoward Hughes Medical Institute - Stanford - USA

Much of my lab’s efforts over the years have focused on gaining a betterunderstandingofantigenspecificTcellrecognition,particu-larly the genetics, biochemistry and, more recently, the cell biology of this process.

WefindthatTcellsareextremelysensitiveinthatevenonemole-cule of the correct peptide-MHC combination can be detected. This degree of acuity is on par with sensory systems such as the eye and considerably more sensitive than B lymphocytes. This sug-gests that T cell recognition is at the core of an adaptive immune response.

We have also had a longstanding interest in human immunology and have been developing technologies here that improve the throughput and sensitivity with which T cells and other cells of the immune system can be monitored. We have been applying these approaches as well as standard technologies to the analyses of influenzavaccineresponsesinyoungadultsversuselderlycohortsand will report on those results and approaches.

Session1:

State of the art in CMI technology development

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Human B cell responses to vaccination

Mark K. SLIFKAVaccine and Gene Therapy Institute – Beaverton - USA

Immunological memory provides the foundation for vaccine-media-ted protection against disease. Humoral immune responses elicited by long-lived plasma cells and memory B cell reactivation often play a critical role in vaccine-mediated immunity and quantitative analy-sis of these B cell subsets has gained increased attention over the last several years.

In this presentation, I will describe current techniques for measuring antigen-specificmemoryBcellsdirectlybyflowcytometryandbylimiting dilution analysis. Next, I will describe features of serolo-gical memory to a select number of human pathogens that were revealed by performing longitudinal analysis for >20 years in some individual subjects. Understanding these mechanisms may provide insight into the requirements for sustaining long-term protective im-munity and improving future vaccine design.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Monitoring Human Memory T-cells

Pratip K. CHATTOPADHYAYNIH - Bethesda - USA

A key challenge facing modern medicine is to predict, before clini-cally evident, which individuals are at greatest risk for an infectious disease. Similarly, upon vaccination, it is critical to distinguish tho-se who generate effective immune responses (that will protect them from infection) from those who do not. In both cases, changes in the immune system may provide early clues to the risk of future illness or vaccine failure.Although we recognize the need to measure early changes to the immune system, it remains unclear precisely what should be measu-red. Changes in the cell surface markers expressed by bulk CD4+ and CD8+ T-cells can be tracked; however, cells express hundreds, ifnotthousandsofproteins,anditisnotknownwhichdefinedistinctsubsets of cells. This is particularly problematic when classifying T-cells into naïve, memory, and effector populations.Moreover,thefrequencyofT-cellsspecificforaparticularantigenislow;thus,changesinanantigen-specificpopulationmaybelostin a sea of irrelevant T-cells with unimportant phenotypes. This begs the question, which antigen-specificities do we measure,and how? By combining peptide-MHC tetramer technology with the immunophenotyping approach described above, we can track changesincellsurfacemarkerswithinantigen-specificpopulations.Polychromaticflowcytometry(using12-17colors)allowsdramaticmultiplexing, such that four different tetramers can be examined simultaneously alongside six phenotypic markers. Recent techno-logy has also allowed us to examine the avidity T-cells have for their antigens, using tetramer reagents that differ in the strength of their interaction with CD8 molecules. In sum, these approaches allow us toresolve,infinerdetail,thecorrelatesofprotectiveimmunity.Still, theseapproachescanonlymeasureafewantigenspecifici-ties, and reveal nothing about cell function. Recent studies have demonstrated the importance of measuring multiple cytokines, as disease protection has been correlated with polyfunctional T-cells. Thisapproachallowsonetoexaminecellsspecifictomultipleepi-topes (using overlapping peptides for stimulation) and assess the quality of responding T-cells. These are notable advantages over tetramer-based approaches.Finally, there remains an important obstacle to the widespread use of such advanced monitoring approaches: they rely heavily on po-lychromatic flow cytometry,which is not commonly available. Inlightofthis,findingsurrogatemarkersforantigen-responsiveCD4+and CD8+ T-cells becomes crucial, so that labs can perform mul-tipledownstreamassaysafterstimulation.Recentfindingsinthisregard will also be discussed.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Measure of innate immunity

Eric VIVIERUniversité de la Méditerranée - Aix/Marseille - France

Abstract not provided

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Structural and functional assay to measure antigen specificCD8Tcell

Giuseppe PANTALEO Centre Hospitalier Universitaire Vaudois - Lausanne - Switzerland

Abstract not provided

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Systems Biology approaches to The understanding of memory T cell homeostasis and correlates of immune protection

RafikSEKALYUniversité de Montréal et Centre de Recherche du CHUM Montréal - Canada

Memory generation and maintenance are the hallmark of the im-mune system. It results from the clonal expansion and differenti-ationofantigen-specificlymphocytesthat,inthecontextofvacci-nations and/or infections, ultimately persist for a lifetime. The fact that, during HIV infection, both memory CD4+, CD8+ and B-cell subsets are impaired, strongly suggest that HIV-driven depletions of memory pool is not effectually only the consequence of HIV cyto-pathic effects, but it could be due to the lack of the delivery of vital signals, that lymphocytes need for the generation and establish-ment of an efficient immune response.Wehave identified usingsystems biology approaches , two major pathways which are re-gulatedbyTCR,PD1andγ-CreceptorcytokinesandwhichareinvolvedintheregulationofmemoryTcellsurvivalandindefiningthe size and diversity of the HIV reservoir . These pathways include the FOXO3A pathway and the stat-5A pathway. Results will be pre-sented showing how these two transcription factors are regulated by external ligands in normal T cell homeostasis and how dendritic cells defects disrupt the activation of these pathways. We will also present data on how systems biology are being used to identify correlates of immune mediated protection . A yellow fever vaccine trial,yellowfevervaccinationbeingamongstthemostefficientvac-cines , was developed to identify correlates of protections ; results willbepresenteddemonstratingthatitisdifficulttoassignonearmof the immune response as the correlate of protection ; rather it is the integration of all arms of the immune response which can pre-dict the protection provide by the yellow fever vaccine . This work was funded by the NIH , CIHR, Genome Quebec and Genome Canada and INSERM

Session2:

Standardization:regulatoryand ethical requirement

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Monitoring of CMI responses in early life

Arnaud MARCHANTInstitute for Medical Immunology and Centre for Research in Vacci-nology, Charleroi, Belgium

Cell-mediated immune responses in early life are qualitatively and quantitatively different from adult responses. Th1 type responses to vaccines and pathogens are generally low in young infants al-though mature responses can be induced by some vaccines like BCG. This defect is associated with a lower production of cytokines, including IL-12, by neonatal dendritic cells. CD8 T cells may be easier to activate and differentiate in early life as mature responses can be detected in a number of infectious models.

SpecificapproachesarerequiredtomonitorCMIresponsesinearlylife that have not yet been standardised. These approaches would take into account the characteristics of the infant immune system and the limited amount of blood that can be collected in this age group.

Asurveywasorganisedtodefinethecurrentmethodsusedbyex-pertsinthefield.Theresultsofthissurveywillbepresentedatthemeeting.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Guidelines for Quality Assurance of Cytokine Flow Cyto-metry Assays

Maria C. JAIMESBD Bioscience - San José - USA

BackgroundCytokineflowcytometry(CFC)isusedtomeasureantigen-specificT cell responses in settings such as experimental HIV and cancer vaccine trials. To directly compare the data generated at different centers, standardization of CFC is critical. CFC Quality Assurance Programs (QAPs) have been developed by the NIH/NIAID/DAIDS, the Cancer Vaccine Consortium (CVC) and the ANRS, all in col-laboration with BD Biosciences. The goal of these programs is to contributetothegenerationofreliableandaccurateflowcytometrydata.

MethodsIFN-□ and IL-2 responses in CD4+ and CD8+ T-cells to peptide mixes (CMV, CMVpp65 or Gag depending on the QAP), were tested using cryopreserved PBMC from CMV+ and/or HIV + donors. The peptide and antibody cocktails were provided lyophilized into 96 well plates since previous data suggested that lyophilized reagents provide increased standardization of the assay. Each site analyzed its data independently and provided their raw data for centralized analysis. After analysis of the data, the central site produced a re-port, providing feedback to the participants.

ResultsWe have been able to determine that instrument setup, event col-lection and analysis (gating strategy) play a critical role in inter-la-boratory variation in CFC assays. After 5 rounds of testing in the NIH/NIAID/DAIDS QAP and by providing feedback to the labs, gui-delines for data analysis, and a protocol and reagents for instru-ment setup we have been able to decrease the variability from an average of 58 % CV in the initial round to an average of 34 % CV in the latest one.

ConclusionsThe data generated in these QAPs has allowed us to quantify the intra-laboratory variability of a 4 and 5 color CFC assay and to de-termine important sources of variability when comparing data from different sites. Critical to decreasing the variability and improving the quality of the data generated is the provision of standard opera-ting procedures, including detailed instrument setup, and centrally supplied reagents. The implementation of QAPs will ensure greater uniformity of data from multiple labs.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Immune assay harmonization and external quality assu-rance:resultsoftheongoingProficiencyPanelProgramof the Cancer Vaccine Consortium

Sylvia JANETZKIZellNet Consulting - Fort Lee - USA

The Cancer Vaccine Consortium (CVC), a program of the Cancer Research Institute, has addressed the need of external validation of bio-assays used for immune monitoring, and their harmonization acrosslaboratoriesbyconductinglargeinternationalproficiencypa-nel programs for Elispot, ICS, multimer and CFSE staining involving laboratories from biotech and pharmaceutical companies as well as academia. Two rounds of Elispot testing led to the formulation of initial Elispot harmonization guidelines. Their usefulness could be demonstrated in the third Elispot panel testing. Now, harmonization guidelines for multimer staining could also be established, based on the panel results from 27 laboratories.

Current efforts of this CVC program focus on the integration of the-se guidelines as well as on the systematic examination of the most common problems observed, one of which is the choice of medium/serum used at all steps of the applied assay. The goal of those ef-fortsistoproviderecommendationstothefieldthatimproveoverallassay performance and further enhance assay harmonization.

In addition, the large body of survey data from panelists led to the initiative to establish guidelines for reporting immune monitoring data in clinical trial settings. The assay harmonization guidelines and guidelines for reporting immune monitoring data have the po-tentialtodramaticallyshapethefieldofimmunotherapy.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Harmonization of T cell immunomonitoring within the CIMT Monitoring Panel – correct protocol choices and additional sources of variation

Cedrik BRITTENC.M. Britten*(1) , C. Gouttefangeas*(2), M.J.P Welters(1), S. Attig(2), A. Mander(3), T.M.S. Køllgaard(4), A. Letsch(5), C. Ottensmeier(3), S.H. van der Burg(6)

Affiliations:(1) Department of Immunhematology and Blood Transfusion, Leiden University Medical Cen-ter, Leiden, Netherlands(2) Department of Immunology, University of Tuebingen, Tuebingen, Germany(3) Cancer Sciences Division, Southampton University Hospitals, Southampton, UK(4) Department of Haematology, Centre for Cancer Immune Therapy, Herlev, Denmark(5) Department of Hematology, Charite, Berlin, Germany(6) Department of Clinical Oncology, Leiden, Netherlands

The “CIMT monitoring panel” is an international working group of 24 labs from 8 European countries aiming to optimize immunomonito-ring assays. The group has already completed two phases of inter-laboratorytestingproject,inwhichthefrequencyofTcellsspecificfor virus-derived epitopes in pre-selected PBMC samples had to be determined by HLA-tetramer staining and ELISPOT. Results from thefirstpanelphasewereused to identifykeyrecommendationsfor both assays and experiments were repeated using optimized protocols in a second phase.

Controlledmodificationoftheprotocolssuccessfullyincreasedthesensitivity and reduced the inter-centre variability of the results. The totalnumberofCD8+Tcellsanalyzedintheflowcytometerwasthemost important parameter influencing sensitivity the detection ofspecificCD8+Tcellsbytetramerstaining.IntheELISPOTassay,ahigher sensitivity was obtained by labs using a high cell input-num-ber, a resting phase after thawing, and no addition of allogeneic an-tigen presenting cells. These observations have been incorporated intothefirstCIMTguidelinesfortheseassays.Inaddition,itbeca-meclearthatthevariationofreportedfrequenciesofspecificTcellsobtained by ELISPOT or HLA-peptide tetramer staining does not solelydependonprotocoldetailsbutcanalsobeinfluencedby(1)the quality of cells used, (2) the way data is acquired and analysed, (3) the way acceptance criteria are set and data is interpreted and (4) the specific education of all people involved in themeasure-ment. Therefore, new panel phases for ELISPOT, tetramer staining and intracellular cytokine staining are ongoing and are aiming to improveonthesestepsthatinfluenceassayperformance.

Guidelinesdeducedfromtheresultsoftheproficiencypanelswillhelp to transform commonly used immunomonitoring assays into valuable biomarkers.The quantification of distinct immunologicalsignatures induced by immunotherapy regimens will be a major pre-requisite to use such tests as surrogate markers for clinical en-dpoints.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Statistical Analysis of HIV-induced Cell-Mediated Immunity Data

Zoé MOODIEFred Hutchinson Cancer Research Center - Seattle - USA

In the past decade, HIV vaccine clinical trials have focused on HIV-specificTcellresponsesasmeasuredbytheELISpotassayandmore recently, the intracellular cytokine staining (ICS) assay. Ini-tially, binary responses (positive/negative) played a primary role in distinguishing candidate vaccines. Recent advances both in vaccine candidates evoking stronger, broader responses and im-provements in the standardization and validation of multicolor ICS assays have led to a richer description of HIV-vaccine induced T cell responses that includes CD4+ and CD8+ T cell responses to multiple cytokines.

Thistalkwillbrieflydescribestatisticalmethodsfordeterminingpo-sitivity in the context of the ELISpot and ICS assays. I will then des-criberecentdevelopmentsintheanalysisofpolyfunctionalprofilesarising from higher dimensional ICS data. These allow for a much more detailed evaluation of candidate vaccines and the potential to reveal differences not captured by earlier approaches.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

FDA perspective on cellular assay standardization and validation

Philip KRAUSEFDA - CBER - Bethesda - USA

Although cellular immunity is known to play a substantial role in pre-vention of illness, the development of cellular assays that predict clinical outcomes of vaccination has been an elusive goal. While CMI responses to vaccines can often be measured, it is more dif-ficulttoshowthattheseresponsescorrelatewithvaccineefficacy.In addition to the need to identify a measurable CMI parameter that correlateswithvaccineefficacy,assaysforthatparametermustbevalidated, which can sometimes place practical limitations on the parameters that can be considered. This presentation will sum-marize FDA regulatory experience with CMI assays in the context of several vaccines, and describe issues in assay validation, with special attention to CMI assays.

Session3:

CMI evaluation in vaccine development

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Evaluation of cell-mediated immunity induced by dengue vaccine candidates

Bruno GUYSanofiPasteur-Marcyl’Etoile-France

Humoral and innate/adaptive cellular immune responses following infection by any of the four dengue (DEN) virus serotypes have been linked to both protective immunity and immunopathology. Therefore, it is important to monitor the cellular responses triggered by dengue vaccine candidates along with antibody levels in order toestablishtheprofileoftheinducedresponse.Characterizingtheprofilewillhelpdetermineiftheresponseislikelytobeprotective.

Assays have been developed to measure the responses in three cell types (dendritic cells [DC], T cells and B cells) following in vitro orinvivostimulation/immunizationwithsanofipasteurvaccinecan-didates. Candidates so far tested are live-attenuated vaccines ob-tained by cell passage, and chimeric vaccines based on the Yellow Fever (YF) 17D vaccine replication engine; the latter candidates will soonenterlarge-scaleefficacytrials.

Innate immunity is evaluated in vitro in human peripheral blood mo-nonuclear cells (PBMC) and monocyte-derived DC by comparing immune changes induced by dengue vaccines or by the parental virus strains using assays including intracellular cytokine staining (ICS), cytometric bead array (CBA), ELISA, and gene arrays. In addition, early innate responses are investigated in clinical trials byquantifyingserumcytokineandviruslevelsduringthefirsttwoweeks after vaccination: it is hypothesized that a dengue vaccine-inducedincreaseinseruminflammatorycytokineslevelsshouldbeminimal and comparable to that induced by YF 17D vaccination; an effective,widelyusedflavivirusvaccine.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Suite

Adaptive immune responses are investigated in trial subjects by studyingdengueserotype-specificandcross-reactiveCD4/CD8Tcell responses to the E protein from each serotype, and to the YF 17D and DEN non-structural NS3 proteins. Analyses are performed on the day of each immunization and 28 days after. Secreted Th1/Th2cytokinelevels(IL-2,IL-4,IL-5,IL-10,IFN-γ,TNF-α)arequan-tified incellsupernatants following invitrostimulationwith liveorlive-attenuated vaccines. A library of 15-mer peptide pools span-ning the complete YF 17D and DEN3 NS3 coding sequence is also usedtostimulateCD4andCD8specificresponsestoNS3;ICSisthenusedtodetecttheinducedCD3/CD8/CD4/IFN-γ/TNF-αcells.Controlsforcytokinedetection(IFN-γandTNF-α+activated,fixedand lyophilized PBMCs) and permeabilization (Perm-a-sure™) are used to ensure assay consistency. Based on the previously iden-tifiedcriteriaofabeneficialadaptivecellularresponse,apredomi-nantTh1/Tc1profile,IL-2secretion,andIFN-γ>TNF-αwaslookedfor.

Finally, B cell responses and antibody function are assessed by an antibody dependant enhancement (ADE) assay using the K562 cell line, and by measuring PRNT levels.

The results of these assays from four independent clinical trials will be presented.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Correlatesofprotectionintheelderly:measuresofthecellularresponsetoinfluenzavaccination

Janet MC ELHANEYJanet E. McElhaney1,2, Catherine Ewen3, Kevin P. Kane3, Alison Kleppinger1, R. Chris Bleackley4

Affiliations:1Department of Immunology, University of Connecticut School of Medicine, 2Department of Medicine, University of British Columbia, 3Department of Microbiology and Immunology, 4Department of Biochemistry, University of Alberta.

Influenzaremainsaseriousillnessintheelderlyinspiteofwides-preadinfluenzavaccinationprogramsinthispopulation.Thelimi-tedefficacyofcurrentsplit-virusvaccinesinolderadultsiswidelyrecognized but the reliance on antibody titers as a sole measure of vaccineefficacypresentsachallengeto thedevelopmentofnewvaccines for the older adult population.

Our work has shown that measures of the T-cell response are bet-ter correlates of protection against influenza in older people. Inperipheral blood mononuclear cell (PBMC) cultures obtained from vaccinatedolderadultsandstimulatedwithliveinfluenzavirus,theratio of the T helper type 1 (Th1):Th2 cytokines measured by the interferon-:interleukin-10 ratio is diminished in older adults who subsequentlydevelopinfluenzaillness.Also,alowlevelofthecy-tolytic mediator, granzyme B, in these virus-stimulated PBMC also correspondstoincreasedriskforinfluenzaillness.

Because older adults demonstrate an ability to generate an effec-tive memory response to natural influenza infection, the declinein vaccine efficacywith agingmay be a limitation of the currentsplit-virus (killed) vaccine formulations, rather than an inability of the aged immune system to mount a robust cellular immune res-ponses. Ongoing studies may shed light on mechanisms that could betargetedintheformulationofnewinfluenzavaccinesthatofferenhance protection in the older adult population.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Correlation between tumor regressions and anti-vaccine T cell responses in cancer vaccination ?

Pierre G. COULIEDuve Institute - Brussels - Belgium

MAGE antigens have been used for therapeutic vaccination trials of metastatic melanoma patients, either as antigenic peptides, pro-tein, a pox family recombinant virus carrying a MAGE-3 sequence, or peptide-pulsed dendritic cells. Tumor regressions were observed in only 10-20% of the patients. Recent results suggest that MAGE vaccination of lung carcinoma patients is associated with a better prognosis.

Our work focused on the mechanisms of the tumor regressions that were occasionally observed in vaccinated melanoma patients. We found an anti-vaccine CTL response in 5 out of 10 regressor patients and in 2 out of 18 progressors, suggesting a correlation between the occurrence of these CTL responses and the tumor regressions. However many regressor patients had undetectable or very low frequencies of anti-vaccine T cells.

Searching for other effector mechanisms, we observed that all me-tastatic melanoma patients had frequencies of anti-tumor CTL, re-cognizingtumor-specificantigensdifferentfromthoseofthevacci-nes, ranging from 10-4 to 3 x 10-3 of the blood CD8 T cells, i.e. 10 to 10,000-fold higher than the frequencies of anti-vaccine CTL in the same patients.

Frequencies of similar magnitude were already present prior to vaccination, suggesting that these anti-tumor lymphocytes became inefficientatrejectingthetumor.Whenweanalysedregressingtu-mors, we found high enrichments of anti-tumor but not of anti-vac-cine T cells, suggesting that anti-vaccine CTL may exert their main effect by triggering in the tumor a stimulation of other anti-tumor CTL which destroy the tumor cells.

These results help us to reformulate what can or cannot be expec-ted from the immunological monitoring of cancer vaccine clinical trials.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Monitoring cellular immunity induced by vaccination against Tuberculosis

Willem HANEKOMSouth African Tuberculosis Vaccine Initiative -Cape Town South Africa

Future TB vaccination strategies are likely to include BCG as a pri-me vaccine at birth, followed by a boost vaccine, given within the EPI schedule. At SATVI, we are characterising immunity induced by BCG, and by boost vaccines in phase I/IIa trials. Complementary studies focus on delineating immune correlates of BCG-induced protection against TB, and on the best age to give BCG and boost vaccines.

Flow cytometric assessment of T cell-associated cytokine produc-tion, following incubation of whole blood with peptide pools of vacci-ne antigens, has proven most useful. This assay allows delineation of different patterns of responses, even though participant popula-tions differed.

Newborn BCG vaccination potently induced complex and variable patterns of IFN-γ, IL-2 and/or TNF-α expression in CD4T cells;singleIFN-γ-expressingcellsweremostcommon.Incontrast,po-lyfunctionalCD4Tcells,whichexpressed IFN-γ, IL-2andTNF-αtogether, were most commonly induced by boost vaccines. The se-cond most common population induced by one boost vaccine ex-pressed IL-17 together with these 3 Th1 cytokines, whereas IL-17 was not induced by another boost vaccine.

BCG induced lower frequencies of CD8 T cells, whereas one of the boost vaccines induced potent CD8 responses in some recipients. Another boost vaccine induced a CD8 response only at the highest dosetestedintrials.IFN-γ-producingcellspredominateinthissub-set,whilesomecellsalsoexpressTNF-α.

Effector memory T cells were by far the most common phenotype of vaccination-induced T cells, regardless of vaccination strategy. Longitudinally, very few cells differentiated into classical central me-mory T cells.

The role of this assay, as well a novel whole blood and PBMC-based assays of lymphoproliferation and cytokine producing poten-tial of induced cells, will be discussed.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Measure of cellular immune response in HIV clinical trials

Brigitte AUTRANHôpital Pitié-Salpêtrière - Paris - France

Developing new therapies or immune-based interventions against HIV requires careful monitoring evaluating either the immune re-constitution and recovery from immune defects or the restoration of anefficientimmunityagainstHV.Such immune monitoring involves innovative CMI methodologies and has to adapt to the multicenter clinical trials by developing ca-reful standardisation. Different effort at evaluating standardisation of CMI techniques within national or international networks will be presented. Results from immune monitoring of clinical trials testing novel anti-retroviral strategies or therapeutic immunisation will be presented and discussed.

Session4:

Lookinginthefuture:enabling technology, automation and HTS

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

ImmunID:BandTcellrepertoireanalysis:abiomarkeroftreatmentefficiency

Nicolas PASQUALCEO - ImmunID Technologies - Grenoble - France

BackgroundImmunID has developed a new approach based on TCR rearran-gements detection using multiplex PCR on genomic DNA and co-vering the large majority of V, D and J genes on both TCR and BCR loci (http://www.immunid.com). These approaches named Immun-TraCkeR® and Immun’Ig® have the advantages to be exhaustive in one shot, qualitative and sensitive. Working on DNA allows stability of the material (adapted for multicentric trials) and quantitative ana-lysis because of the direct relation: one cell = one rearrangement. This technology gives the most exhaustive and integrated view of the TCR and BCR repertoire until now. More importantly, this ap-proach allows at the same time the detection of clonal expansions related to a functional state or an anatomical region and the evalua-tion of the repertoire quality for protection against infectious risks. In that way, ImmunID provides an integrated view of lymphocytes combinatorial diversity in order to delineate immune signatures of patients.

Perspectives in vaccination Immunomonitoring of combinatorial diversity provides insight into normal biological and pathological processes before and after treat-ment (i.e. : epitope or adjuvant selection, dose escalation, vaccine efficacy),withthehopeofpredictingindividualizedcoursesofthe-rapy. Thus the ability to obtain qualitative & quantitative information fromaT(andB)cellcombinatorialdiversityprofileisofinteresttogenerate databases relevant to stratify responding and non respon-ding patients.

Methods & ResultsOur recents invivo and invitro results, based on T cell repertoire selection and combinatorial diversity assessment, indicate that Im-munTraCkeR solution is helpful to compare and select improved antigen (both peptides and complex antigen construction were eva-luated). These approaches produce new data on the T cell reper-toire quality in patients before and after vaccination, and will be of importance to understand why a number of patients are not respon-ding to a vaccine.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Suite

Features of the process and quality controlNo gold standard is available to analyse the immune repertoire, ImmunID is working to meet this need by developing the original NDL® “risk management evaluation concept” that scores “healthy” combinatorial repertoire, which can be compared to “treated” or “pathological” repertoires. Therefore, samples are tested in Immu-nID facilities (ISO 9001 and ISO 13485 certified, and complyingwith GLP requirements) with a number of rigorous in-process qua-lity controls that assure consistent optimal quality until generation of finalresultsinterpretation.

Bibliography

ImmunogeneticandMolecularBiologyFundamentalfield-Bosco N, Hung HC, Pasqual N, Jouvin-Marche E, Marche PN, Gascoigne NR, Ceredig R. Abstract Role of the T cell receptor alpha chain in the development and phenotype of natu-rally arising CD4+CD25+ T cells.Mol Immunol. 2006 Feb;43(3):246-54.

- Pasqual N, Gallagher M, Aude-Garcia C, Loiodice M, Thuderoz F, Demongeot J, Ceredig R, Marche PN, Jouvin-Marche E, Quantitative and qualitative changes in V-J alpha rearrange-ments during mouse thymocytes differentiation: implication for a limited T cell receptor alpha chain repertoire, J Exp Med. 2002 Nov 4;196(9):1163-73.

BioinformaticImmunogenetic database- Baum TP, Hierle V, Pasqual N, Bellahcene F, Chaume D, Lefranc MP, Jouvin-Marche E, Marche PN, Demongeot J. IMGT/GeneInfo: T cell receptor gamma TRG and delta TRD ge-nes in database give access to all TR potential V(D)J recombinations. BMC Bioinformatics. 2006 Apr 26;7:224.

- Baum TP, Pasqual N, Thuderoz F, Hierle V, Chaume D, Lefranc MP, Jouvin-Marche E, Marche PN, Demongeot J. IMGT/GeneInfo: enhancing V(D)J recombination database ac-cessibility. Nucleic Acids Res. 2004 Jan 1;32(Database issue):D51-4.

Hematology-Fuschiotti P, Pasqual N, Hierle V, Borel E, London J, Marche PN, Jouvin-Marche E. Analysis oftheTCRalpha-chainrearrangementprofileinhumanTlymphocytes.MolImmunol.2007Jul;44(13):3380-8 .- Garban F, Laurin D, Hannani D, Pasqual N, Bulabois CE, Pernollet M, Cahn JY, Functional Approach of the alloreactivity and monitoring of the immune response after allogreffe of he-matopoietic cells, Poster 2006- Mi JQ, Manches O, Wang J, Perron P, Weisbuch S, Marche PN, Renversez JC, Bensa JC, Sotto JJ, Cahn JY, Leroux D, Bonnefoix T. Development of autologous cytotoxic CD4+ T clones in a human model of B-cell non-Hodgkin follicular lymphoma. Br J Haematol. 2006 Nov;135(3):324-35.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

InVitroBiomimeticHumanImmuneSystemModels:MIMIC System+

William WARRENVaxDesign Corporation - Orlando - USA

Despite the promise of high-throughput screening, genomics, and proteomics, the number of new drugs reaching the market has not increased. A challenge is the translation from test systems (ani-mal or cell culture) to human immunology. The successful transfer between human biology and traditional test systems requires an intricate understanding of disease pathogenesis and cellular and humoral immunological responses at all levels; innate, adaptive, functional. This talk will provide data on the development of the in vitro MIMIC system to assess drug, biologics, vaccine and adjuvant candidates using tissue engineered constructs to mimic human im-munophysiology. We will focus on the MIMIC model to induce both antigen-specificCD4andCD8Tcellresponsesbyexaminingboththe quantity and the quality of the in vitro T cell responses. The MIMICTM model is based on a multidimensional interrogation of human leukocytes. It is composed of isolated human cells which are placed into engineered tissue constructs that are functionally equivalent to the physiological environment of the human immune system.TheMIMICsystemismodular;thefirstmodule(Vaccina-tion Site) simulates the innate responses via antigen presentation andinflammatoryresponses;thesecondmodule(LymphoidTissueEquivalent) simulates the adaptive responses of antigen specificT and B cells; and the third module, a functional assay or disease model, uses the products of the other two modules together with a pathogen to measure the effect of the immune response on the disease, e.g., neutralizing antibody responses. Data will be shown that these in vitro modules reproduce the conditions that exist in a human body, such as the spatial segregation of immune cells and the temporal dynamics that bring different immune cells together in asequentialorder thataccurately reflects interactions ina lymphnode.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Suite

The generation of adaptive immunity is a highly orchestrated and ti-ghtly regulated process involving numerous cell signaling cascades in distinct tissue sites. Injury or infection in the periphery causes a localized inflammatoryreaction that triggersresidentantigenpre-senting cells (APC) to activate and migrate towards lymph nodes drainingthetissue.WewilldiscusshowthefirstMIMICmodule,theVaccination Site (VS) produces APCs that are essentially identical to those found in human dermal explants. The VS module is found to replicate human innate immune responses to biologics, vaccines and adjuvants in good accord with that found in the literature.

In our second module, the lymphoid tissue equivalent (LTE), we create an environment analogous to that within the secondary lym-phoid tissues. We will show that APCs trigger the differentiation ofnaiveantigen-specificCD4+andCD8+Tcells intohelperandcytotoxic T lymphocytes (CTL), respectively, by measuring lympho-proliferation, effector function and intracellular cytokine production by these T cells. The MIMIC platform is much more sensitive than standard PBMC assays for eliciting primary and recall helper T cell responses against soluble proteins and formulated viral vaccines (both inactivated and live-attenuated). As well, the fact that the ma-gnitudeofantigen-specificCD4+Tcellresponsesdetectedinourin vitro cultures increased following vaccination (individuals donate bloodpre-andpost-vaccination)suggeststhatspecificresponsesgeneratedinMIMICassaysarereflectiveoftheimmunestatusofthe donor.

Considering the possibility that this system could be used to gene-rate T cell populations for cloning or clinical use, we show various mechanisms, such as the removal of regulatory T cells or inclusion of pro-T cell cytokines, to rapidly increase the number of antigen-specifichelperTcellsinMIMICassays.

We demonstrate presence of multifunctional in vitro T cells in res-ponse to vaccine stimulus. Multifunctional CD8 T cell responses wereobservedbymulti-parameterflowinresponsetovaccinesti-mulus for in both a recall challenge (Flumist) and a naïve challenge (YF-VAX) in the in vitro MIMIC system. The magnitude of both total andmultifunctionalnaïveCD8Tcellresponsesweresignificantlyincreased via a second re-stimulation with DCs with the cultured cells, a process that resembles prime/boost vaccination strategies. These in vitro datasets are consistent with previous multi-parame-terflowcytometrystudies++thatdemonstratethedelineationofTcells into distinct functional populations, and hence help assess the quality of the T cell response to vaccination.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Suite

Last,theflexibilityoftheMIMICsystemallowsustoinvestigatehowvarious facets of the immune system (innate stimuli, DC maturation state, Tregs, etc.) impact the induction of cellular responses in hu-mans.

+This work was supported by DARPA/DSO in the Rapid Vaccine Assessment Program and by the IAVI Innovation Fund.

++Robert A. Seder, Patricia A. Darrah, Mario Roederer, Nat. Rev. Immunology 8, 257 (2008)

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Flow cytometry-based cytotoxicity assays for the measu-rement of effector responses

Albert D. DONNENBERGUniversity of Pittsburgh Cancer Institute - USA

Among assays of cell mediated immunity, the measurement of cel-lular cytotoxicity, the ability of an effector cell to kill a target cell in vitro, is the most fundamental. The 4-hour chromium release assay (CRA), a test in which target cells are labeled with radioactive so-dium chromate, quickly became the gold standard because of its quantitative nature. First applied to mixed leukocyte cultures, it was demonstrated that effector lymphocytes sensitized to radiolabe-ledallogeneiccellsinculturebecameefficientalloantigenspecifickillers. The CRA was quickly applied to virus infected targets, provi-dingthefirstassayofcellmediatedvirus-specificresponse.Shortlyafter its development, the CRA played an instrumental role in the discovery of MHC restriction of T-cell responses and in the eluci-dation of HLA unrestricted killing mediated by large granular lym-phocytes (NK cells). Despite the development of surrogate assays suchasantigenstimulatedcytokine release,andflowcytometricmeasurement of effector associated markers, cellular cytotoxicity remains the prototype effector response. Despite its advantages, CRA is essentially a single parameter assay that provides no infor-mation concerning the nature of the effector or target cells. It also requires handling of the radioisotope 51Cr, a potentially hazardous gamma emitter.Toaddresstheseconcernswedevelopedasimplefour-colorflowcytometry-based cytotoxicity (FCC) assay to simultaneously measu-re NK-cell cytotoxicity and NK-cell phenotype (CD3-CD16+CD56+). This assay can easily be extended to T-cell mediated killing and the measurement of additional surface markers on effector or target cells. Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute num-bers of all cells were measured based on light scatter (FSC/SSC), doublet discrimination (peak integral versus height), and fluores-cence. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T)cellratios(50,25,12,and6)confirmedthata3hincubationwasoptimal.Usingimagingflowcytometry,wewereabletodirectlyvisualized NK-target conjugate formation and killing. The FCC as-sayismoresensitivethantheCRA,hasacoefficientofvariation(CV) 8-13% and reliably measures NK cell- or lymphokine-activa-ted killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Suite

The FCC assay can be used to study a range of phenotypic at-tributes, in addition to lytic activity of various subsets of effector cells, cost effectively without the use of radioactive tracers. The FCC assay also has the potential to provide information about the molecular interactions underlying target cell lysis and is thus may have great utility for studies of disease pathogenesis and vaccine responses.

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

...............................................................

Statisticalmodelingofmulti-parameterflowcytometry

Cliiburn CHANDuke University - Durham - USA

The ability to monitor complex immune responses quantitatively is increasingly recognized as essential for the development of vacci-nes, and also in the diagnosis and prognosis of several diseases, most notably cancer and HIV infection.

One of the most versatile technologies used in immune monitoring isflowcytometry,whichcanbeusedtosimultaneouslytrackphe-notype and effector responses of individual cells in a population. However, robust and accurate quantification of flowdata can bedifficult, andmanual gating for exploringmulti-parameter data isextremelyinefficient.

This talk will describe the development of statistical mixture mo-dels and software for the objective and automated analysis of mul-ti-parameter flow cytometry,with applications to several differentaspects of cell mediated immunity.

Speakers AUTRAN Brigitte JANETSKI Sylvia APHP Zellnet consulting brigitte.autran@psl.ap-hop-paris.fr sylvia@zellnet.com

BENARD Gil KOLLMAN Tobias University of Saõ Paulo BC Children’s Hospital & UBC mahong@usp.br tkollm@dccnet.com

BRITTEN Cedrik KRAUSE Philip LUMC FDA c.m.britten@lumc.nl philip.krause@fda.hhs.gov

CHAN Cliburn LOUIS Jacques Duke University Institut Pasteur cliburn.chan@duke.edu jlouis@pasteur.fr

CHATTOPADHYAY Patrip MARCHANT Arnaud NIH Université Libre de Bruxelles pchattop@mail.nih.gov arnaud.marchant@ulb.ac.be

COULIE Pierre MC ELHANEY Janet Duve Institute Providence Health Care pierre.coulie@uclouvain.be jmcelhaney@providencehealth.bc.ca

DAVIS Mark MOODIE Zoe Stanford University SCHARP mmdavis@stanford.edu zoe@scharp.org

DONNENBERG Albert NOUGAREDE Nolwenn UPMC-Pittsburg SanofiPasteur donnenbergad@upmc.edu nolwenn.nougarede@sanofipasteur.com GUY Bruno PANTALEO Giuseppe SanofiPasteur CHUV bruno.guy@sanofipasteur.com Giuseppe.Pantaleo@chuv.ch

HANEKOM Willem PASQUAL Nicolas University of Cape Town ImmunID Technologies Willem.Hanekom@uct.ac.za npasqual@immunid.com JAIMES Maria PFLEIDERER Michael BD Biosciences Paul Ehrlich Institut Maria_Jaimes@bd.com pflmi@pei.de

Speakers SEKALYRafick Université de Montréal rafick-pierre.sekaly@umontreal.ca

SLIFKA Mark Oregon Health and Science University slifkam@ohsu.edu

SZTEIN Marcelo University of Maryland msztein@medicine.umaryland.edu WARREN William VaxDesign Corporation wwarren@vaxdesign.com

Participants AUGAGNEUR Edith DODET Betty SanofiPasteur DodetBiosciences edith.augagneur@sanofipasteur.com betty.dodet@dodetbiosciences.com

BARTHA Kalman DUBOIS Patrice National Center for Epidemiology Immunovac Consulting bartha.kalman@oek.antsz.edu pmdubois@immunovacc.com

BERTHOUD Tamara DUCLOS Philippe CRESIB WHO tamara.berthoud@cresib.cat pduclos@who.int

BISCEGLIA Hélène DUTEL Catherine SanofiPasteur FondationMérieux helene.bisceglia@sanofipasteur.com catherine.dutel@fondation-merieux.org

BURDIN Nicolas ELLEFSEN LAVOIE Kim SanofiPasteur CHUV nicolas.burdin@sanofipasteur.com kim.ellefsen-lavoie@chuv.ch

CAILLET Catherine GALLI Grazia SanofiPasteur NovartisVaccinesandDiagnostics catherine.caillet@sanofipasteur.com grazia.galli@novartis.com

CASTELLINO Flora GALLICHAN Scott NovartisVaccinesandDiagnostics SanofiPasteur flora.castellino@novartis.com scott.gallichan@sanofipasteur.com

CATERINI Judy GITAU Evelyn SanofiPasteur KemriWellcomeTrust judy.caterini@sanofipasteur.com egitau@kilifi.kemri-wellcome.org

COLER Rhea GODOY Karina IDRI SMI rcoler@idri.org karina.godoy@smi.ki.se

DELORE Valentine GROENEVELD Kees SanofiPasteur HealthCouncil valentine.delore@sanofipasteur.com k.groeneveld1@tip.nl

DOBANO Carlota HARARI Alexandre CRESIB CHUV carlota.dobano@cresib.cat alexandre.harari@chuv.ch

Participants HENDRICKS Jenny PICOT Valentina Crucell Fondation Mérieux jenny.hendricks@crucell.com valentina.picot@fondation-merieux.org

HESSLER Catherine PLANTIER Nadia SanofiPasteur IMMUNID catherine.hessler@sanofipasteur.com nplantier@immunid.com

IVANOFF Bernard ROCHA Katia FondationMérieux LaboratoryofInvestigationinImmunodeficiency bivan@bluewin.ch katamanzza@yahoo.com.br

LANDRY Claire RODRIGUEZ MUNOZ Ariane NIML Crucell claire.landry@umontreal.ca ariane.rodriguez@crucell.com

LANG Pierre-Olivier SCHULLERY Daniel Hôpital des Trois Chênes University of Pennsylvania pierre.o.lang@hcuge.ch sdan@mail.med.upenn.edu

LATHEY Janet SEDEGAH Martha SanofiPasteur USMMVP janet.lathey@sanofipasteur.com martha.sedegah@med.navy.mil

LAUPEZE Béatrice SIEGRIST Claire-Anne GSK Université de Genève beatrice.laupeze@gskbio.com claire-anne.siegrist@medecine.unige.ch

LONGUET Christophe SIX Adrien Fondation Mérieux Université Pierre et Marie Curie christophe.longuet@fondation-merieux.org adrien.six@upmc.fr

MEYER Claudius TRANNOY Emmanuelle UniversityHospitalMainz SanofiPasteur meyer@uni-mainz.edu emmanuelle.trannoy@sanofipasteur.com

MORIS Philippe TUYNDER Marcel GSK Université Libre de Bruxelles philippe.moris@gskbio.com mtuynder@ulb.ac.be PAUL Stéphane VERON Philippe CHU Saint-Etienne Genethon stephane.paul@chu-st-etienne.fr veron@genethon.fr