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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. HIV-1. Lentivirus ( Family Retroviridae ) - PowerPoint PPT Presentation
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STUDY OF PHENOTYPIC CHARACTERISTICS OF HIV THROUGH RECOMBINANT VIRUS
Immunopathology UnitInstituto de Salud Carlos III (Madrid)
Introducción
AIDS Pandemic
http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/2008/
Introducción
HIV-1
gag
envvpr
vif vpunef
tat
rev
p2 p1
NCCAMA p6
pol
IN
RT p51
RNasa HRT p66PR gp 41 (TM)gp 120 (SU)
3’LTR5’LTR gag
envvpr
vif vpunef
tat
rev
p2 p1
NCCAMA p6
pol
IN
RT p51
RNasa HRT p66PR gp 41 (TM)gp 120 (SU)
3’LTR5’LTR
Lentivirus (Family Retroviridae)Size 80-110 nmOuter lipid envelopeInner matrixCore (p24): 2 copies of RNA(9,8 Kb)
- PR, RT, IN
ARN
ARN
RT
ADN
RT
ADN
ARN proteínas
provirus
ARN
Entrada
Retrotranscripción
Transporte e Integración
Formación y maduración
Transcripción
ARN
ARN
RT
ADN
RT
ADN
ARN proteínas
provirus
ARN
Entrada
Retrotranscripción
Transporte e Integración
Formación y maduración
Transcripción
VIRAL
ENTRY
VIRAL
ENTRY
Isolated R5 Isolated X4
Isolated R5X4(homologous and mixed population)
CCR5CD4
CXCR4
RANTESMIP-1 α y β
CXCL12 (SDF-1)
X4R5X4
Change in 50% of patients
R5+X4 Selection of R5 strains Persistence of R5 strains
InoculationPrimo- and chronic infection Advanced infection
Assaying Resistance
Introducción
Genotypic MethodsDetect mutations in the HIV-1 genome associated with resistance
- based on hybridization
- relies on sequencing
Phenotypic MethodsDirect measure of resistanceMeasure the concentration of pharmaceutical necessary to inhibit viral replication in cell culture
- classic: Isolation and quantification of HIV using peripheral blood lymphocytes (PBL)
- Labor-intensive-Difficult to reproduce-Selection of viral subpopulations
- viral recombination
NotI
Xho
gagpol
vifvpr
tatR5-1
rev
nefLTR LTR
vpu
Xba I
Xho
gagpol
vifvpr
tat
rev
nefLTR LTR
vpupNL4-3
Resultados
1 2 3 4 5 6 7
10
100
1000
10000
100000
1000000
10000000
100
1000
10000
100000
1000000
1 2 3 4 5 6 7
10
100
1000
10000
100000
1000000
100
1000
10000
100000
PBMCMT2
NL4-3NL4-3Ren
NL4-3NL4-3Ren
p2
4 (
pg
/ml)
RLU
s
p2
4 (
pg
/ml)
RLU
s
Days after Infection Days after infection
Not IXho I
gag vifvpr
tatenv
rev
nefLTR LTR
vpu
Renilla
pol
pNL4-3pNL4-3Ren
Resultados
TRANSFECCIÓN 293T
RLUs
INFECCIÓN MT-2TRANSFECCIÓN 293T
RLUsRLUs
INFECCIÓN MT-2
Not IXho I
gag vifvpr
tatenv
rev
nefLTR LTR
vpu
Renilla
pol
ApaI Age I
PR lac Z
RT-PCR
Plasma HIV RNA
Cloning
pol
PCRDNA
Ligation
200 colonies
Religated < 5%
Strategy for generating recombinant virus
IC50wt
IC50patientFold
resistance
0.01 0.1 1 100
20
40
60
80
100
IC50wt IC50patient
% Inhib
itio
n
[pharmaceutical]
[DRUG]++
env
lac Z
XbaI
Resultados
Raltegravir
-6 -5 -4 -3 -2 -1 0 1 2
0
20
40
60
80
100
120
140NL4.3-RenillaNL4.3-155-RenillaNL4-3-148-Ren
inhibitor (µM)
% R
LU
s
Not IXho I
gag vifvpr
tatenv
rev
nefLTR LTR
vpu
Renilla
pol
pNL4-3pNL4-3Ren
Q148KN155H
ApaI EcoRI
Site-directed mutagenesis
Transfection in 293T
gagpol
vifvpr
tatenv
rev
nefLTR LTR
vpugagpol
vifvpr
tat
rev
nefLTR LTR
vpu
pNL4.3 gag GFP
gag CherryFP
Not IBamHI
Control NL+gag-GFP NL4.3
Infection of Hela P4C5 Cells
100
80
60
40
20
0
De
ath
s p
er 1
00
pe
rso
n-y
ear
s
35
30
25
20
15
10
5
0
Deaths
Use of HAART
HA
AR
T, %
pa
tien
t-day
s
Source: Palella. N Engl J Med 1998;338:853. Update: Palella. Personal Communication, 1999.
Dramatic Declines in Mortality Rates With HAART*
1994 1995 1996 1997 1998 1999
POBLACION VIRALPRESION
FARMACOLOGICA
VARIANTE RESISTENTE
Suppress maximum replication of virus
Goal of HAART
Introducción
High genetic variability of HIV-1
- High rate of replication (109-1010 particles/day)
- Low fidelity of Retrotranscriptasa (2,5x10-5/nt) Lack of activity of exonuclease 3’-5’
- High frequency of recombination
- Enzymatic plasticity
HAART (suboptimum conditions)
-Bad adherence to regimen
- Loss of drug potency
-Bad absorption
-Pharmaceutical Interactions
Introducción
Formation of bundle structure of six helices and fusion of
membranes
Release of fusion protein and insertion into lipid membrane
CD4
Co-receptor
gp41
gp120
A) B) C)
D) E)
Mechanism of Entry HIV-1
Interaction with CD4 and conformation change in
gp120
Interaction with co receptor
Maeda y col., J. Biol. Chem., 2006
Generation of viruses resistance to MVCGeneration of viruses resistance to MVC
Inhibitors of CCR5:
MARAVIROC
Date Time to Time to CD4 ClonesPatient nr timepoint timepoint SC(months) SI switch (*103 cells/ul) available SI NSI coR usage (U87)
1 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.
MT-2 test
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
Adapted from Lohse N et al. 16th IAC 2006, Toronto, Canada. Abstract MOPE0310
0.00
0.25
0.50
0.75
1.00
25 7030 35 40 45 50 55 60 65
population controlsEarly HAART (1997–1999)
Late HAART (2000–2005)Pre HAART (1995–1996)
Pro
babi
lity
of
surv
ival
Estimated survival for HIV patients from age 25 yearsHepatitis C coinfected patients excluded
Aumento de la Supervivencia
Lewden C, et al. J Acquir Immune Defic Syndr 2007;46(1):72−77
CD4 count ≥500mm3 is associated with standard mortality ratio (SMR) similar to general population1
8
SM
R (
CI)
7
6
5
4
3
2
1
0
0 1 2 3 4 5 6 7
Time of truncation after initiation of cART (years)
8CD4: 350 to 499/mm3
CD4 500/mm3
• 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
Prognosis of late presentersP
roba
bili
ty o
f AID
S o
r D
eath
Years Since Start of cART
0.40
0.30
0.20
0.10
0.00
0 1 2 3 4 5 6
0.15
0.10
0.05
0.00
0 1 2 3 4 5 6
Pro
babi
lity
of
Dea
th
Years Since Start of HAART
0 – 50
51 – 150
151 – 250251 – 350
351 – 450451 – 550
BL CD4 cell count (cells/mm3)
Patient 1: Neutralizing HIV Antibody Titers of Sequential Plasma Specimens against
Autologous Virus
DatesVirus
(months) 0 3 6 9 12 15 18 21 25
9/2/99 0 26 219 675 1403 2670 2089 2190 2363 241111/29/99 3 29 179 1024 2151 3733 3152 2808 2953 30862/29/00 6 27 35 78 358 1769 1939 2247 3112 43455/31/00 9 36 67 82 200 795 1078 1371 2208 33758/30/00 12 19 48 36 64 76 166 556 937 140711/22/00 15 29 43 64 76 90 119 374 721 12342/14/01 18 42 65 61 152 117 134 122 289 5265/30/01 21 41 66 82 84 85 113 78 107 2969/11/01 25 42 62 56 62 85 77 55 61 95
Control
NL43 17 138 294 956 1172 953 1584 1868 2143JRCSF 24 37 35 60 87 97 105 152 209
Plasma (months)
(D.Richman Rapid evolution of the neutralizing antibody response following primary HIV infection.XIV International AIDS Conference . [Abstract nº1051])
Viral envelope
gp41
2G12
2F5
CD4bs
gp120
b12
4E10/Z13
(Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201])
Tipos de anticuerpos neutralizantes. Dominios de neutralización
dominio interacción coreceptores
dominio de fusión
dominio interacción CD4
dominios variables
(Burton D. Human neutralizing antibodies and a vaccine for HIV-1. XIV International AIDS Conference [Abstract nº201])
Structure of b12 neutralising antibody