The Use of Flow Cytometry To Study Acute HIV-1 Infection

Transcription

1 The Use of Flow Cytometry To Study Acute HIV1 Infection 5th INTEREST Workshop Dar es Salaam, Tanzania 113 May 211 Guido Ferrari, MD Duke University Medical Center Center for AIDS Research (CFAR) Duke Human Vaccine Institute (DHVI) International AIDS Training Research Program (IATRP) Duke Global Health Institute (DGHI) Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

2 Flow Cytometry is a Quantitative Method That Measures Single Events (Cells) Light Amplification by Stimulated Emission of Radiation Lasers generate intense beams of coherent light. y_systems/support/training/online Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

3 Basic Aspect of a Flow Cytometer Analog Digital Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

4 Flow Cytometry: A Quantitative Assay Platform Frequency of the Cellular Subset 1 Level of Expression Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

5 The Achilles Heel of Flow Cytometry: Signal Spillover Non Compensated Parameter 1 Compensated Parameter 2 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

6 The Achilles Heel of Flow Cytometry: Signal Spillover Non Compensated Parameter 1 Compensated Parameter 2 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

7 The Achilles Heel of Flow Cytometry: Signal Spillover Non Compensated Parameter 1 Compensated Parameter 2 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

8 Flow Cytometry Applications in Acute HIV1 Infection Clinical Applications Research Applications Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

9 Clinical Application: Patient Management 1 Assay for CD4 Count and Rx Adherence CD4 Count: Clinical Stage Glencross et al. CD8/CD38 activation yields important clinical information of effective antiretroviral therapy. Cytometry (28) vol. 74B (S1) pp. S131S14 CD8 Activation Virus Load Virus Load Rx Adherence CD8 Activation Rx Adherence Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

10 Please see Posters P_1: Moodley K. et al.: CD38 Activation (MFI) Assessment by Flow Cytometry in HIV and HIV Patients with or without TB CoInfection Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

11 What is the level of CD8 activation during acute HIV1 infection? How does early initiation of HAART impact the activation of CD8 T cells? Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

12 Levels of Total CD8 T cells Activation during Acute HIV1 Infection (n=61) % of parental CD3 Gay C., Ferrari G., et al. AIDS (211) vol. 25 (7) pp Time from infection Does not Reset Following Initiation of HAART Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

13 Flow Cytometry Applications in Acute HIV1 Pathogenesis Clinical Applications Research Applications: 1. Frequency of Immune Cellular Subsets (T, B, NK, DC subsets); 2. Memory Phenotype and Maturation Stage of Cellular Susbsets; 3. Cellular Functions: Cytokine production, Cytotoxic Activity, Ab production; 4. Proliferative Capability of Agspecific and nonagspecific Cells. Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

14 Basic Aspect of a Flow Cytometer Analog Digital Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

15 Detection of 8 parameters to identify all possibility for 4 combined parameters: 4color Cytometer = 25 tubes = 25 million cells = 25 ml of blood 8color Cytometer = 1 tube = 1 million cell = 1 ml of blood 488nm Blue Laser (2mW, Solid State) Duke LSRII Rm 23 FITC CFSE GFP Calcein 635nm Red Laser (25mW, Diode) Duke LSRII Alexa 68 Alexa 7 515/ 2 71/ 5 532nm Green Laser (15mW, Solid State) PE Gre en SS C PE Cy 5 71/ 5 PE Blu e PerCP Cy5.5 Duke LSRII APC Alexa 647 PerCPCy5.5 PE Cy5.5 PETR Alexa 61PE 45nm Violet Laser (5mW, Diode) Am Cya n QD ot Duke LSR II QDot PE QD QD ot Cy ot Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania QD ot /4 535 LP QDot 585 APCCy7 APCHL75 Alexa 75 Qdot 545 PacOrange Cascade Blue vamine PacBlue

16 1. Stimulate ICS Methodology 2. Viability&Surface Staining 3. Permeabilize 4. Stain Brefeldin A EDTA 6 h Wash Wash Wash cytokine lymphocyte erythrocyte A g Ag presenting cell T cell.. CD8 PE IFN FITC Brefeldin A IFN CD3 APC Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania 5. Acquisition 6. Analysis Horton et al. Optimization and validation of an 8color intracellular cytokine staining (ICS) assay to quantify antigenspecific T cells induced by vaccination. Journal of Immunological Methods (27) vol. 323 (1) pp. 3954

17 Gating Strategy for the ICS Assay Analysis I. Live Lymphocytes Subsets FSCH 25K 2K 15K 1K Singlets 77.9 Viability Live CD3 SSCA 25K 2K 15K 1K Lymphocytes 97.7 CD K K 1 2 5K 1K 15K 2K 25K FSCA CD8 Naive CD3 CD CD8 Memory K 1K 15K 2K 25K CD57 51 FSCA CD8 CD CD CD Memory CD45RO CD45R CD45R CD45R memory CD8 Functions CD17a 1 3 IFN 1 3 IL2 IL2 1 3 MIP1 1 3 CD17a IFN TNF TNF TNF TNF Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

18 What is the level of Agspecific CD4 and CD8 activation during acute HIV 1 infection? Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

19 Antigenspecific CD4 cells were significantly more activated, as per CD38 expression, when compared to the total memory compartment in both acute and chronic infection Riou C., Gray C. et al. submitted Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

20 Multiparameter ICS to Identify Functional CD8 Memory Subsets memory CD8 Functions CD17a 1 3 IFN 1 3 IL2 IL2 1 3 MIP1 1 3 CD17a IFN TNF TNF TNF TNF Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

21 Boolean Logic in Flow Cytometry The Boolean logic in Flow Cytometry allows to identify populations that fell in different gates according to the And, Or, or Not principles. Subset 1 Not 2 Subset 1 And 2 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

22 Multiparameter ICS to Identify Functional CD8 Memory Subsets memory CD8 Functions CD17a 1 3 IFN 1 3 IL2 IL2 1 3 MIP1 1 3 CD17a IFN TNF TNF TNF TNF CD17 IFN IL2 MIP1 TNF Boolean Analysis CD17a IFN IL2 MIP1 TNF Functional Family Pies Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

23 Comparison of Ontogeny of Functional CD4 and CD8 Memory T Cell Responses following Acute HIV Infection CD4 Responses Freq. Funct Freq. Funct. CD8 Responses Week 13 Week 48 Week 1216 Week 2834 G. Ferrari, Gray CM, et al. Unpublished Data Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania Week 5478

24 What functionality is displayed by the Agspecific CD8 driving escape during acute HIV1 infection? Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

25 We ranked the mapped epitopes within each patients according to the order of appearance of escape mutants Ferrari G, Korber B. et al. PLoS Pathog (211) vol. 7 (2) pp. e11273 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

26 MIP1 production dominates early responses EarlyM LateM NonM EarlyM LateM NonM Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

27 Frequency of Epitopespecific MIP1 Functional CD8 T cells Correlates with order of Appearance of Escape Mutants Earliest Positive ICS Total Freq Fxn 1 Fxn 25 MIP1 p = Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

28 Flow Cytometers in Africa by just 1 company Flow Cytometry does not have to be highly complex and can be implemented in researchlimited setting if focused and aimed to study any of the parameters thus far presented. Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

29 What is needed to maximize the utilization of Flow Cytometry in limited resource setting? Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

30 Way forward: 1. To mentor young scientists 2. Create a network of young scientists 3. To implement education of clinical staff Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

31 Participants We received 11 applications from the countries indicated on the map. Twentyfour (24) applicants were selected for the symposia and 12 more for the African Flow Cytometry Workshops that followed the Symposium Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

32 Organizing Committee * Scientific Committee and Faculty * * * * ** * * * * * * Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

33 29 Infectious Diseases in Africa: Measurement of Immune Responses & 3rd African Flow Cytometry Workshop NICD, Johannesburg 1117 November 27 Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

34 211 Infectious Diseases in Africa: Measurement of Immune Responses & 4th African Flow Cytometry Workshop University of Cape Town, Cape Town 1113 November 211 Visit: Topics of the Symposium: 1. Faculty Seminars on Adaptive and Innate Immune Responses to HIV, Malaria, and TB; Presentations by Young Investigators on HIV, Malaria, and TB; 2 will be reinvited from the previous event because were the top performers 3. Workshops: 1. Challenges in writing a research proposal. 2. How to present your data. Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

35 211 Infectious Diseases in Africa: Measurement of Immune Responses & 4th African Flow Cytometry Workshop University of Cape Town, Cape Town 1113 November 211 Topics of the Workshop: TBD 1. Instrument optimization for BD Calibur and LSR II 2. Panel Optimization 3. Assay standardization 4. Software training for DIVA, FlowJo, PESTLE, and SPICE Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

36 211 Infectious Diseases in Africa: Measurement of Immune Responses & 4th African Flow Cytometry Workshop University of Cape Town, Cape Town 1113 November 211 Evaluation of the event s impact: 1. Daily questionnaire reviewing the topic of the day. Qs&As were discussed by different groups the following day (8 attendees with 23 faculty); 2. Final questionnaire to review all of the topics with scores; 3. The scores were used to identify the recipients of two fellowships to attend an International Meeting or Technical Workshop. Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

37 Way forward: 1. To mentor young scientists 2. Create a network of young scientists 3. To implement education of clinical staff: open clinical/research laboratory to medical student Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

38 Medical Education Partnership Initiative (MEPI) Goal to sustainably enhance and support medical education through partnerships between African and US schools of medicine Jointly sponsored by NIH and PEPFAR Eleven awards announced October 21 Kilimanjaro Christian Medical CollegeDuke University School of Medicine awarded $1,, over 5 years (Prof. Moshi and Dr. Bartlett P.I.) Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania

39 The Patients. Acknowledgements CLINICAL CORE Mike Cohen (UNC, US) Marybeth McCauley (FHI, US) Joe Eron (UNC) Charles Hicks (Duke) Cindy Gay (UNC) Kara McGhee (Duke) Ian Williams (St Mary,UK) Perry Pellegrino (St Mary, UK) T CELL CORE Michael Liu Nilu Goonetilleke Emma Turnbull Stephen Moore Rachel Tanner Kati Digleria Tim Rostron McM CHAVI LAB Persephone Borrow Andrew McMichael (Oxford, UK) Christine Marking Ralph Picking Kent Weinhold (Duke, US) Mandla Mlotshwa Catherine Riou Clive Gray (NICD, S.Africa) Vaccine Research Center/NIH Mario Roederer U. of Pennsylvania Michael Betts VIRUS CORE Bette Korber (LANL, US) Jesus Salazar Maria GonzalezSalazar Brandon Keele Beatrice Hahn George Shaw (UAB, US) Jennifer Kirchner Chunlai Jiang Feng Gao (Duke, US) Florette Treurnicht Carolyn Williamson (UCT, S. Africa) MANAGEMENT CORE Kelly Soderberg Tom Denny Liz Petzold Bart Haynes (Duke, US) Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania CHAVI SUPPORT TEAMS CHAVI REPOSITORIES FHI Duke Management team SCHARP Steve Self Natalie Hawkins

40 ACKNOWLEDGMENTS Coorganizer of IDA and Workshop Clive Gray (NICD, South Africa) The Members of the Organizing and Scientific Committees. The Senior and Junior Faculty and the Participants. Sponsored by: African AIDS Vaccine Initiative; National Institute of Health and Office For AIDS Research. Supported by: National Institute Communicable Disease (NICD); Duke University CFAR, IATRP, and GHI; BD Bioscience; TreeStar Inc.; InVitrogen. Presented at the 5th INTEREST workshop 1 13 May 21, DaresSalaam, Tanzania