STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS - PowerPoint PPT Presentation

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STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS

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  1. STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS Immunopathology Unit Instituto de Salud Carlos III (Madrid)

  2. Introducción AIDS Pandemic http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/2008/

  3. Introducción

  4. HIV-1 Lentivirus(FamilyRetroviridae) Size 80-110 nm Outerlipidenvelope Innermatrix Core (p24): 2 copies of RNA(9,8 Kb) - PR, RT, IN

  5. Isolated R5X4 (homologous and mixedpopulation) Isolated R5 Isolated X4 CD4 CCR5 CXCR4 CXCL12 (SDF-1) RANTES MIP-1 α y β V I R A L E N T R Y Inoculation Primo- and chronic infection Advanced infection Selection of R5 strains X4 R5X4 R5+X4 Persistence of R5 strains Change in 50% of patients

  6. Introducción AssayingResistance PhenotypicMethods GenotypicMethods Directmeasure of resistance Measuretheconcentration of pharmaceuticalnecessarytoinhibit viral replication in cell culture - classic: Isolation and quantification of HIV usingperipheralbloodlymphocytes (PBL) - Labor-intensive -Difficultto reproduce -Selection of viral subpopulations - viral recombination Detectmutations in the HIV-1 genomeassociatedwithresistance - basedonhybridization - reliesonsequencing

  7. Xba I NotI gag gag vif vif tat tat vpu vpu nef nef pNL4-3 pol pol vpr vpr R5-1 LTR LTR LTR LTR rev rev Xho Xho

  8. Resultados pNL4-3Ren Renilla pNL4-3 gag vif tat vpu nef env pol vpr LTR LTR rev Xho I Not I MT2 PBMC NL4-3Ren NL4-3Ren NL4-3 NL4-3 p24 (pg/ml) p24 (pg/ml) RLUs RLUs Days after infection Days after Infection

  9. Resultados env pol 100 80 60 40 IC50patient Foldresistance 20 IC50wt 0 0.01 0.1 1 10 IC50wt IC50patient Strategyforgeneratingrecombinant virus RT-PCR PCR Plasma HIV RNA DNA • Ligation • 200 colonies • Religated < 5% Cloning Renilla gag vif tat vpu nef PR vpr env pol LTR LTR lac Z lac Z rev Xho I XbaI ApaI Age I Not I % Inhibition [pharmaceutical] [DRUG] + +

  10. Resultados pNL4-3Ren Renilla pNL4-3 gag vif tat vpu nef env pol vpr LTR LTR rev Xho I Not I ApaI EcoRI N155H Site-directed mutagenesis Q148K % RLUs

  11. Transfection in 293T pNL4.3 gag GFP gag gag vif vif tat tat vpu vpu nef nef pol pol vpr vpr env LTR LTR LTR LTR rev rev gag CherryFP BamHI Not I Infection of Hela P4C5 Cells Control NL4.3 NL+gag-GFP

  12. 100 80 60 40 20 0 35 30 25 20 15 10 5 0 Deaths HAART, % patient-days Deaths per 100 person-years Use of HAART Dramatic Declines in Mortality Rates With HAART* 1994 1995 1996 1997 1998 1999 Source: Palella. N Engl J Med 1998;338:853. Update: Palella. Personal Communication, 1999.

  13. Introducción Goal of HAART Suppressmaximumreplication of virus • High geneticvariability of HIV-1 • - High rate of replication (109-1010particles/day) • Lowfidelity of Retrotranscriptasa(2,5x10-5/nt) Lack of activity of exonuclease 3’-5’ • High frequency of recombination • - Enzymaticplasticity • HAART (suboptimumconditions) • -Badadherencetoregimen • Loss of drugpotency • -Badabsorption • -PharmaceuticalInteractions

  14. Introducción

  15. Mechanism of Entry HIV-1 A) B) C) Interaction with CD4 and conformation change in gp120 Interaction with co receptor gp41 CD4 gp120 Co-receptor D) E) Release of fusion protein and insertion into lipid membrane Formation of bundle structure of six helices and fusion of membranes

  16. Generation of virusesresistance to MVC Inhibitors of CCR5: MARAVIROC Maeda y col., J. Biol. Chem., 2006

  17. Since start of ART, increasing survival rates for people with HIV • Survival from age 25 years: Cumulative survival curve for HIV-infected individuals and general-population controls • HIV-infected individuals are divided into three calendar periods of observation Estimated survival for HIV patients from age 25 yearsHepatitis C coinfected patients excluded 1.00 0.75 Probability of survival 0.50 0.25 0.00 25 30 35 40 45 50 55 60 65 70 population controls Late HAART (2000–2005) Early HAART (1997–1999) Pre HAART (1995–1996) Adapted from Lohse N et al. 16th IAC 2006, Toronto, Canada. Abstract MOPE0310

  18. Aumento de la Supervivencia CD4 count ≥500mm3 is associated with standard mortality ratio (SMR) similar to general population1 CD4: 350 to 499/mm3 CD4 500/mm3 8 7 6 5 SMR (CI) 4 3 2 1 0 0 1 2 3 4 5 6 7 8 Time of truncation after initiation of cART (years) Lewden C, et al. J Acquir Immune Defic Syndr 2007;46(1):72−77

  19. Prognosis of late presenters BL CD4 cell count (cells/mm3) 0 – 50 151 – 250 351 – 450 51 – 150 251 – 350 451 – 550 0.40 0.15 0.30 0.10 0.20 Probability of AIDS or Death Probability of Death 0.05 0.10 0.00 0.00 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Years Since Start of HAART Years Since Start of cART • Based on 24,444 patients from 15 cohort studies • 808 deaths and 2366 events in 81,071 person-years of follow-up Adapted from Sterne J, et al 16th CROI; Montreal, Canada; February 8-11, 2009. Abstract 72LB

  20. Patient 1: Neutralizing HIV Antibody Titers of Sequential Plasma Specimens against Autologous Virus Plasma (months) Virus (months) 0 3 6 9 12 15 18 21 25 Dates 0 26 219 675 1403 2670 2089 2190 2363 2411 9/2/99 3 11/29/99 29 179 1024 2151 3733 3152 2808 2953 3086 6 2/29/00 27 35 78 358 1769 1939 2247 3112 4345 9 5/31/00 36 67 82 200 795 1078 1371 2208 3375 12 8/30/00 19 48 36 64 76 166 556 937 1407 15 11/22/00 29 43 64 76 90 119 374 721 1234 18 2/14/01 42 65 61 152 117 134 122 289 526 21 5/30/01 41 66 82 84 85 113 78 107 296 25 9/11/01 42 62 56 62 85 77 55 61 95 Control NL43 17 138 294 956 1172 953 1584 1868 2143 JRCSF 24 37 35 60 87 97 105 152 209 (D.Richman Rapid evolution of the neutralizing antibody response following primary HIV infection. XIV International AIDS Conference . [Abstract nº1051])

  21. Tipos de anticuerpos neutralizantes. Dominios de neutralización  dominio interacción coreceptores  dominios variables 2G12 b12 gp120  dominio interacción CD4 CD4bs 2F5 gp41  dominio de fusión 4E10/Z13 Viral envelope (Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201])

  22. Structure of b12 neutralising antibody (Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201])