1. Vaccine potential of dendritic cells pulsed with autologous virus in anti-HIV immune response Prof. Dr. Alberto José da Silva Duarte DERMATOLOGY DEPARTMENT FACULDADE DE MEDICINA DA USP

2. -Leukapheresis acquisition of PBMC, DC differentiation and stimulation with inactivated autologue HIV -18 subjects chronically infected with HIV without HAART -3x10 7 DCs inoculated (4 subcutaneous injections) -2 boosters (2nd and 4th weeks)

5. To evaluate the abillity of DC to potentialize specific cellular immune response anti-HIV in vitro in chronically HIV-1 infected subjects : I. Näive II. With successful use of HAART III. Using HAART but with therapeutic failure GENERAL AIM

6. -To investigate the expression profile of co-stimulatory molecules and chemokine and cytokine production in DCs of HIV+ pacients pulsed in vitro with inactivated autologous HIV -To evaluate the effect of the co-culture of the DCs pulsed with inactivated HIV on T cell proliferation, and perforin and IFN-  production -To analyze the global transcriptome of DC pulsed by autologous HIV utilizing microarray technology -Based on the in vitro results described above, to be able to predict which patients would benefit from the dendritic cell vaccination SPECIFIC AIMS

7. -15 patients chronically infected by HIV not using HAART; TCD4+ cell count above 350 cell/mm 3; viral load above 5.000 copies/mL -15 patients successfully using HAART; TCD4 count above 350 cells/mm 3 and viral load under 1.500 copies/mL of viral RNA -15 patients using HAART but with therapeutic failure; TCD4 count above 350 células/mm 3 and viral load above or equal to 10.000 copies/mL of viral RNA, after at least 6 months of treatment STUDY POPULATION * Patients with significant variations (25% of total numbers of TCD4+ and ≥ 0,5 log viral load) in the last 6 months will be excluded

8. -Separation of PBMCs from total blood by Ficoll -Viral expansion using previously activated PBMCs from healthy donors in the presence of IL-2 -Viral inactivation with aldrithiol-2 VIRAL CULTURE, EXPANSION AND INACTIVATION

9. nucleocapsid - AT-2 inhibits replication but maintains intact viral conformation AT-2 MECHANISM OF ACTION

10. Peripheral blood DIFFERENTIATION OF DCs FROM PERIPHERAL BLOOD MONOCYTES

11. Magnetic beads linked to anti-CD14+ antibodies SEPARATION OF CD14+ MONOCYTES USING BEADS

12. Monocytes CD14 + Immature dendritc cells CD14 - CD11c + CD1a + 5-7 days DIFFERENTIATION OF DCs FROM PERIPHERAL BLOOD MONOCYTES IL-4 + GM-CSF

13. 2 h TNF-alpha IL-1 beta IL-6 TNF-alpha IL-1 beta IL-6 + Immature dendritc cells Mature dendritc cells Autologous Inactivated HIV STIMULATION OF DCs WITH AUTOLOGOUS VIRUS AND MATURATION INDUCTION 1) Immature dendritc cells 48 h 2)

14. CULTURE OF ACTIVATED DCs

15. ANALYSIS OF ACTIVATED DENDRITIC CELLS DCs phenotyping – CD11c, CD1a, CD80, CD86, HLADR, DC-SIGN Cytokine production: IL-12, IL-10 Global expression profile by microarray Capacity to induce specific cellular responses

16. -Cell Proliferation -Cytokine production: IFN-gama and IL-2 -Perforin expression COCULTURE DC:T LYMPHOCYTES

17. Mo Mo-DC imMo-DC mat GM-CSF/IL-4 Day 0Day 6Day 8 pro- inflammatory cytokines + HIV-AT2 T0T1T2 MICROARRAY

18. PROJECT TEAM COORDINATOR: Prof. Dr. Alberto J.S. Duarte – LIM 56 ASSESSORS: Dr. Jorge Casseb – LIM 56 Dr.João Bosco Oliveira- lim 56 Dr. Ulisses Braga – LAVITE / FIOCRUZ Dr. Claudio Panutti - IMT / USP RESEARCHERS INVOLVED: Dra. Telma Miyuki Oshiro – LIM 56 Biomedica Noemia Mie Orii – LIM 56 Biomedico Rafael Martins – LIM 56 Biomedico Luciano Lopes – LIM 56 Biomedica Claudia Finazzo – LIM 56 Técnico Wilton Santos Freire – LIM 52

19. PROJECT TEAM COORDINATOR: Prof. Dr. Alberto J.S. Duarte – LIM 56 ASSESSORS: Dr. Jorge Casseb – LIM 56 Dr.João Bosco Oliveira- LIM 56 Dr. Ulisses Braga – LAVITE / FIOCRUZ Dr. Claudio Panutti - IMT / USP RESEARCHERS INVOLVED: Dra. Telma Miyuki Oshiro LIM 56 Biomedica Noemia Mie Orii LIM 56 Biomedico Rafael Martins LIM 56 Biomedico Luciano Lopes LIM 56 Biomedica Claudia Finazzo LIM 56 Técnico Wilton Santos Freire LIM 52

20. CENTRAL DOGMA OF MOLECULAR BIOLOGY Genomics Transcriptomic Proteomic TranscriptionTranslation Replication MICROARRAY