STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS Immunopathology Unit
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STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS Immunopathology Unit Instituto de Salud Carlos III (Madrid) Introducción AIDS Pandemic http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/2008/ Introducción p2 MA CA p1 NC p6 tat rev 5’LTR vif gag pol nef vpu 3’LTR vpr env IN gp 120 (SU) RT p51 PR RT p66 RNasa H gp 41 (TM) HIV-1 Lentivirus (Family Retroviridae) Size 80-110 nm Outer lipid envelope Inner matrix Core (p24): 2 copies of RNA(9,8 Kb) - PR, RT, IN Entrada Formación y maduración ARN proteínas ARN Retrotranscripción RT ARN ARN ADN Transcripción RT provirus ADN Transporte e Integración Isolated R5 V I R A L Isolated R5X4 (homologous and mixed population) CCR5 E N T R Y CD4 CXCR4 CXCL12 (SDF-1) RANTES MIP-1 α y β Inoculation Primo- and chronic infection R5+X4 Selection of R5 strains Isolated X4 Advanced infection Persistence of R5 strains X4 R5X4 Change in 50% of patients Introducción Assaying Resistance Genotypic Methods Detect mutations in the HIV-1 genome associated with resistance Phenotypic Methods Direct measure of resistance Measure the concentration of pharmaceutical necessary to inhibit viral replication in cell culture - based on hybridization - relies on sequencing - classic: Isolation and quantification of HIV using peripheral blood lymphocytes (PBL) - Labor-intensive -Difficult to reproduce -Selection of viral subpopulations - viral recombination Xba I gag LTR vif pol NotI tat vpu vpr nef R5-1 LTR rev Xho pNL4-3 Resultados Renilla pNL4-3Ren pNL4-3 gag vif pol LTR vpr tat vpu nef env LTR rev Xho I Not I MT2 10000000 PBMC 1000000 NL4-3Ren NL4-3 1000000 1000000 100000 1000 1000 100 10 100 1 2 3 4 5 Days after Infection 6 7 10000 10000 RLUs 10000 p24 (pg/ml) 100000 RLUs p24 (pg/ml) 100000 10000 100000 NL4-3Ren NL4-3 1000 1000 100 10 100 1 2 3 4 5 Days after infection 6 7 Resultados Strategy for generating recombinant virus RT-PCR Plasma HIV RNA PCR DNA pol env Ligation Renilla Cloning 200 colonies Religated < 5% gag LTR PR lac Z ApaI pol vif vpr tat vpu Age I lac env Z nef LTR rev Xho I Not I XbaI TRANSFECCIÓN 293T INFECCIÓN MT-2 RLUs % Inhibition 100 80 60 40 20 0 0.01 0.1 IC50wt + 1 10 [pharmaceutical] [DRUG] Fold resistance IC50patient IC50patient IC50wt Resultados Renilla pNL4-3Ren pNL4-3 gag pol LTR ApaI vif vpr tat vpu env nef LTR rev Xho I Not I EcoRI N155H Q148K Site-directed mutagenesis Raltegravir 140 NL4.3-Renilla NL4.3-155-Renilla NL4-3-148-Ren 120 % RLUs 100 80 60 40 20 0 -6 -5 -4 -3 -2 -1 0 inhibitor (µM) 1 2 Transfection in 293T pNL4.3 gag vif pol pol LTR tat vpu vpr gag GFP gag CherryFP nef env LTR rev BamHI Not I Infection of Hela P4C5 Cells Control NL4.3 NL+gag-GFP Dramatic Declines in Mortality Rates With HAART* 100 Deaths 30 80 25 60 20 40 15 10 20 5 Use of HAART 0 0 1994 1995 1996 1997 1998 1999 Source: Palella. N Engl J Med 1998;338:853. Update: Palella. Personal Communication, 1999. HAART, % patient-days Deaths per 100 person-years 35 Introducción Goal of HAART Suppress maximum replication of virus High genetic variability of HIV-1 - High rate of replication (109-1010 particles/day) HAART (suboptimum conditions) - Low fidelity of Retrotranscriptasa (2,5x10-5/nt) Lack of activity of exonuclease 3’-5’ -Bad adherence to regimen - High frequency of recombination -Bad absorption - Enzymatic plasticity -Pharmaceutical Interactions -Loss of drug potency Introducción Mechanism of Entry HIV-1 A) B) Interaction with CD4 and conformation change in gp120 C) Interaction with co receptor gp41 CD4 gp120 Co-receptor D) Release of fusion protein and insertion into lipid membrane E) Formation of bundle structure of six helices and fusion of membranes Generation of viruses resistance to MVC Inhibitors of CCR5: MARAVIROC Maeda y col., J. Biol. Chem., 2006 Patient nr 1 Date Time to Time to CD4 Clones MT-2 test 3 timepoint timepoint SC(months) SI switch (*10 cells/ul) available SI NSI coR usage (U87) 1 R5-1 23/11/1987 26 n.a. 1,05 >3 X n.d. 2 08/03/1989 42 n.a. 0,7 >3 X n.d. 3 27/11/1991 74 n.a. 0,49 >3 X n.d. 4 R5-10 29/08/1994 107 n.a. 0,19 >3 X n.d. Since start of ART, increasing survival rates for people with HIV Estimated survival for HIV patients from age 25 years Hepatitis C coinfected patients excluded • 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 1.00 Probability of survival • 0.75 0.50 0.25 0.00 25 30 35 40 45 population controls Early HAART (1997–1999) Adapted from Lohse N et al. 16th IAC 2006, Toronto, Canada. Abstract MOPE0310 50 55 60 65 Late HAART (2000–2005) Pre HAART (1995–1996) 70 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 SMR (CI) 6 5 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 Prognosis of late presenters 0.15 0.30 Probability of Death Probability of AIDS or Death 0.40 0.20 0.10 0.00 0 1 2 3 4 5 0.10 0.05 0.00 6 0 • BL CD4 cell count (cells/mm3) 0 – 50 51 – 150 151 – 250 251 – 350 351 – 450 451 – 550 1 2 3 4 5 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 6 Patient 1: Neutralizing HIV Antibody Titers of Sequential Plasma Specimens against Autologous Virus Plasma (months) Dates 9/2/99 11/29/99 2/29/00 5/31/00 8/30/00 11/22/00 2/14/01 5/30/01 9/11/01 Virus (months) 0 3 6 9 12 15 18 21 25 0 3 6 9 12 15 18 21 25 26 29 27 36 19 29 42 41 42 219 179 35 67 48 43 65 66 62 675 1024 78 82 36 64 61 82 56 1403 2151 358 200 64 76 152 84 62 2670 3733 1769 795 76 90 117 85 85 2089 3152 1939 1078 166 119 134 113 77 2190 2808 2247 1371 556 374 122 78 55 2363 2953 3112 2208 937 721 289 107 61 2411 3086 4345 3375 1407 1234 526 296 95 17 24 138 37 294 35 956 60 1172 87 953 97 1584 105 1868 152 2143 209 Control NL43 JRCSF (D.Richman Rapid evolution of the neutralizing antibody response following primary HIV infection. XIV International AIDS Conference . [Abstract nº1051]) Tipos de anticuerpos neutralizantes. Dominios de neutralización dominio interacción coreceptores dominios variables b12 2G12 gp120 dominio interacción CD4 dominio de fusión CD4bs gp41 Viral envelope (Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201]) 2F5 4E10/Z13 Structure of b12 neutralising antibody (Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201])
