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