A single dose of an HIV-A single dose of an HIV-specific, vaginally-applied specific, vaginally-applied microbicide, PSC-RANTES microbicide, PSC-RANTES (PSC) can select for a drug (PSC) can select for a drug

resistant SHIV in a resistant SHIV in a macaque model macaque model

Dawn Moore-DudleyDawn Moore-Dudley

Department of Molecular biology and Department of Molecular biology and MicrobiologyMicrobiology

IntroductionIntroduction Resistance to many antiretroviral drugs can Resistance to many antiretroviral drugs can

emerge in an HIV-1 infected individual with emerge in an HIV-1 infected individual with even a single treatment. The best example even a single treatment. The best example being the emergence of NVP resistance in being the emergence of NVP resistance in mothers or infants given a single dose of NVP mothers or infants given a single dose of NVP to prevent perinatal transmissionto prevent perinatal transmission

NVP resistance NVP resistance maymay also be selected during also be selected during transmission from mother to infants as transmission from mother to infants as suggested by different NVP resistance suggested by different NVP resistance mutations (K103N and Y181C) emerging in the mutations (K103N and Y181C) emerging in the mother and their infant. There is still no direct mother and their infant. There is still no direct evidence that drug resistance can be selected evidence that drug resistance can be selected in a drug-treated recipient from the in a drug-treated recipient from the innoculating wild type virus delivered by the innoculating wild type virus delivered by the donor. donor.

Use of anti-HIV microbicide would be an Use of anti-HIV microbicide would be an example of this scenario. example of this scenario.

Introduction (2)Introduction (2)Use of macaque models for testing Use of macaque models for testing

anti-HIV-1 microbicidesanti-HIV-1 microbicides Progesterone treated macaques are successfully Progesterone treated macaques are successfully

infected through a vaginal route with CCR5-infected through a vaginal route with CCR5-tropic SHIVP3tropic SHIVP3

Several CCR5 inhibitors and entry inhibitors, Several CCR5 inhibitors and entry inhibitors, when applied vaginally, can block SHIVP3 when applied vaginally, can block SHIVP3 infection of macaques in a post-coital modelinfection of macaques in a post-coital model

Potential drawbacks to this model are, however,Potential drawbacks to this model are, however, An almost exclusive use of SHIVP3 for these testsAn almost exclusive use of SHIVP3 for these tests The relative low diversity of SHIVP3 for macaque The relative low diversity of SHIVP3 for macaque

exposures as compared to the genetic diversity of the exposures as compared to the genetic diversity of the HIV-1 innoculum delivered from donor to recipientHIV-1 innoculum delivered from donor to recipient

Too many to discuss but not as relevant to this Too many to discuss but not as relevant to this presentationpresentation

PSC-RANTES as potential anti-HIV-1 microbicide

• A derivative of the natural chemokine RANTES

• ~8 kDa protein• May be the most potent anti-HIV

compound with an ability to inhibit at sub-nanomolar concentrations

• Inhibition is mediated by both CCR5 downregulation on the cell surface and competitive binding with virus

• Has a higher binding affinity for CCR5 than RANTES

• Does not induce CCR5-mediated inflammation like its natural counterpart.

Wilken J. et al. Chemistry and Biology. 1999.

RANTES

AOP

PSC-RANTES blocks gp120 from binding to CCR5 as well as down-regulates and

sequesters CCR5 in the cell.

PSC-RANTES did not induce vaginal irrational or inflammation in macaques

PSC-RANTES effectively down-regulates CCR5 on the surface of macaque PBMCs

30 female progesterone-treated rhesus macaques were vaginally pre-treated with various concentrations of PSC-RANTES or PBS and then challenged with SHIVSF162.

Testing PSC-RANTESas a microbicide

Lederman et al. Science 2004

Viral load of macaques pre-treated with PSC-RANTES Viral load of macaques pre-treated with PSC-RANTES and then vaginally infected with SHIVSF162and then vaginally infected with SHIVSF162

Lederman et al. Science 2004

Amino acid sequence Amino acid sequence alignment surrounding the alignment surrounding the

mutation in V3mutation in V3

SHIV P3 1 VVIRSENFTD NVKTIIVQLK ESVEINCTRP NNNTRKSIPI GPGKAFYATG DIIGDIRQAH CNISGEKWNN TLKQIVTK 78BI32 5-27 330uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78BI32 4-28 330uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78M584 6-2 100uM PSC 1 .......... .......... .......... .......... ...R...... .......... .......... ........ 78M584 6-2 100uM PSC 1 .......V.. .......... .......... .......... ...R...... .......... .......... ........ 78M584 5-5 100uM PSC 1 .......... .......... .......... .......... ...R...... .......... .......... ........ 78M584 5-5 100uM PSC 1 .......... .......... .......... .......... ...R...... .......... .......... ........ 78M584 5-23 100uM PSC 1 .......... .......... .......... .......... ...R...... .......... .......... ........ 78BH53 4-28 100uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78DT58 4-28 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78DT58 5-12 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78DT58 5-5 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78DT59 5-5 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78DT59 4-28 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78N400 12-30 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78BA93 12-30 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78BH49 4-28 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78BH49 6-2 10uM PSC 1 I......... .......... .......... .......... .......... .......... .......... ........ 78BH49 5-12 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78T121 5-5 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78R323 12-30 1uM PSC 1 .......... .......... .......... .......... .......... .......... .......... ........ 78AL37 1-8 PBS 1 .......... .......... .......... .......... .......... .......... .......... ........ 78T314 4-28 PBS 1 .......... .......... .......... .......... .......... .......... .......... ........ 78T314 5-27 PBS 1 ....X..... .......... .......... .......... .......... .......... .......... ........ 78T619 5-27 PBS 1 .......... .......... .......... .......... .......... .......... .......... ........ 78T619 4-28 PBS 1 .......... .........X .......... .......... .......... .......... .......... ........ 78

Amino acid sequence alignment of gp41 region

SHIVP3#1 1 TTAVPWNASW SNKSLDQIWN NMTWMEWERE IGNYTNLIYT LIEESQNQQE KNEQELLELD KWASLWNWFD ISKWLWYIKI FIMIVGGLV 89BI32 5-27 330uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89BI32 4-28 330uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89M584 6-2 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89M584 6-2 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89M584 5-5 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89M584 5-5 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89M584 5-23 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89M584 5-12 100uM PSC 1 .......... .......... .......... .....D.... .......... .......... .......... .......... ......... 89BH53 4-28 100uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89DT58 4-28 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89DT58 5-12 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89DT58 5-5 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89DT59 5-5 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89DT59 4-28 33uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89N400 12-30 33uM PSC 1 .......T.. .......... .......... .......... .......... .......... .......... .......... ......... 89N400 12-30 33uM PSC 1 .......T.. .......... .......... .......... .......... .......... .......... .......... ......... 89BA93 12-30 33uM PSC 1 .......... .......... ....I..... .......... .......... .......... .......... .......... ......... 89BA93 12-30 33uM PSC 1 .......... .......... ....I..... .......... .......... .......... .......... .......... ......... 89BH49 4-28 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ....I.... 89BH49 6-2 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ....I.... 89BH49 5-12 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ....I.... 89BH49 4-28 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ....I.... 89T121 5-5 10uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89R323 12-30 1uM PSC 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89AL37 1-8 PBS 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89T314 4-28 PBS 1 .......... .......... .......... .......... .......... .......... .......... .......... ......... 89T314 5-27 PBS 1 ..T....T.. .......... .......... .......... .......... .......... .......... .......... ......S.. 89T619 5-27 PBS 1 .......... .......... .......... .......... ...K...... .......... .......... .......... ......... 89T619 4-28 PBS 1 .......... .......... .......... .......... ...K...... .......... .......... .......... ......... 89

COOHFP N-HR C-HR TM

511 523532 585 620 665

676 697intracellularextracellular

YTNLI

640

GIV

(M584)(100uM)

(538-540)

gp41

V1 V2C1

V3C2 C3

V4C4

V5C5 FP

1126 149159 193 294 328 388 405 450 462 511 523

GPGKAF

315

NH2

(M584)(100uM)

Detecting the low frequency mutations in the inoculating Detecting the low frequency mutations in the inoculating SHIVSHIVSF162SF162 versus virus versus virus

Percentage of K315R Mutation

0.1

1

10

100

100

Virus

Per

cen

tag

e o

f P

op

ula

tio

n

Parental SHIVSF162

100uM PSC 5/5

100uM PSC 6/2

100uM PSC 6/30

33uM PSC

10uM PSC

PBS

PBS

PBS

Lower levelof sensitivity

Percentage of N640D Mutation

0.1

1

10

100

1

Virus

Perc

en

tag

e o

f P

op

ula

tio

n Parental SHIVSF162

100uM PSC 5/5

100uM PSC 6/2

100uM PSC 6/30

33uM PSC

10uM PSC

PBS

PBS

PBS

Lower levelof sensitivity

COOHFP N-HR C-HR TM

511 523532 585 620 665

676 697intracellularextracellular

YTNLI

640

GIV

(M584)(100uM)

(538-540)

gp41

V1 V2C1

V3C2 C3

V4C4

V5C5 FP

1126 149159 193 294 328 388 405 450 462 511 523

GPGKAF

315

NH2

(M584)(100uM)

K315R

3% of inoculating SHIVSF162

>25-fold enrichment

N640D

<0.4% of inoculating SHIVSF162

>250-fold enrichment

Percentage of K315R Mutation

0.1

1

10

100

100

Virus

Per

cen

tag

e o

f P

op

ula

tio

n

Parental SHIVSF162

100uM PSC 5/5

100uM PSC 6/2

100uM PSC 6/30

33uM PSC

10uM PSC

PBS

PBS

PBS

Lower levelof sensitivity

Percentage of N640D Mutation

0.1

1

10

100

1

Virus

Perc

en

tag

e o

f P

op

ula

tio

n Parental SHIVSF162

100uM PSC 5/5

100uM PSC 6/2

100uM PSC 6/30

33uM PSC

10uM PSC

PBS

PBS

PBS

Lower levelof sensitivity

Detecting the low frequency mutations in the inoculating Detecting the low frequency mutations in the inoculating SHIVSHIVSF162SF162 versus virus versus virus

Generating an HIV-1 Generating an HIV-1 chimeric virus chimeric virus

containing the P3 and containing the P3 and M584 SHIV env gene for M584 SHIV env gene for

phenotypic studiesphenotypic studies

0

50

100

150

200

250

300

350

400

450

U87.CD4.Hu-R5 cells U87.CD4.Rh-R5 cells

nM

chan

gein

Cal

ciu

m

RANTESPSC-RANTES

RANTES-100,000

0100,000

PSC-RANTES-100,000

0100,000

RANTES

200,000

100,000

0

-100,000

PSC-RANTES

200,000

100,000

0

-100,000

U87.CD4.Hu-R5cells U87.CD4.Rh-R5

Development of a stable U87 cell expressing huCD4 and rhCCR5

-RANTES and PSC-RANTES inducea calcium flux response in both U87.CD4.Rh-R5 and U87.CD4.Hu-R5cell lines.

-PSC-RANTES causes CCR5down-regulation in U87.CD4.Rh-R5 cells.

0

Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors

PSC-RANTES

0.01

0.1

1

10

U87.CD4.Hu-R5 U87.CD4.Rh-R5

Mea

n IC

50 (

nM)

P3-4

M584-7

P<0.01

P<0.001

The macaque treated with 100 uM (animal M584) was infected with SHIV clone resistant to PSC-RANTES. This resistance was more pronounced in cells expressing the Rh CCR5 than the Hu CCR5.

Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitors

In the Rh CCR5 cells, PSC-RANTES could not completely inhibit the M584 SHIV whereas the innoculating virus was completely inhibited.

Testing sensitivity of the M584 and the Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitorsinnoculating P3-4 to entry inhibitors

TAK-779

0.001

0.01

0.1

1

10

U87.CD4.Hu-R5 U87.CD4.Rh-R5

Mea

n IC

50 (

uM)

P3-4

M584-7

P=0.15

P<0.001

Cross resistance to TAK-779 was only observed in the Rh CCR5 cells

Testing sensitivity of the M584 and the Testing sensitivity of the M584 and the innoculating P3-4 to entry inhibitorsinnoculating P3-4 to entry inhibitors

The M584 virus is hypersensitive to T20 possibly due to the mutation in the C terminal heptad repeat of gp41

T-20

0.001

0.01

0.1

1

10

U87.CD4.Hu-R5 U87.CD4.Rh-R5

Mea

n IC

50 (

uM)

P3-4

M584-7

P=0.001 P=0.004

Impact of the selection of these Impact of the selection of these mutations in M584 on viral fitnessmutations in M584 on viral fitness

The mutations in M584 have not emerged at a fitness cost to the SHIVP3. Is fitness of M584 greater in the Rh CCR5 cells?

C3 primary isolate competitor

0

0.5

1

1.5

2

2.5

U87.CD4.Hu-R5 U87.CD4.Rh-R5

mea

n r

elativ

e fi

tnes

s

P3-4

M584-7

Env34 chimeric competitor

0

0.5

1

1.5

2

2.5

U87.CD4.Hu-R5 U87.CD4.Rh-R5

mea

n r

elativ

e fi

tnes

s

P3-4

M584-7

Take home question…..Take home question…..

If we can select for a PSC-RANTES If we can select for a PSC-RANTES resistant clone from a SHIVP3 inoculum resistant clone from a SHIVP3 inoculum in even one out of 10 macaques vaginally in even one out of 10 macaques vaginally exposed and treated PSC-RANTES,exposed and treated PSC-RANTES,

That will happen if we treat humans with That will happen if we treat humans with vaginal microbicides where the vaginal microbicides where the inoculating virus could be more diverse inoculating virus could be more diverse by orders of magnitude?by orders of magnitude?

YongGao

ImmaculateNankya

KenNelson

RickGibson

LoraAngelova

KendallKrebs

Angel Reyes-Rodriguez

DawnMoore-Dudley

MikeLobritz

MattLalonde Eric

Arts

Denis-MangaTebit

KenHenry

Collaborators-Ronald S. Veazey-Tulane National Primate Research Center, Covington, LA.-Oliver Hartley-Department of Structural Biology and Bioinformatics Centre Medical Universitaire, Geneva, Switzerland

Measuring the frequency of K315R and N640D in the SHIVSF162P.3 inoculum.

1. Anneal a radiolabeled probe with identical sequence to the mutant template to both templates.

2. Anneal a 5’phosphorylated probe to both templates.

3. Allow thermostabile cycling ligase to ligate together the two probes.

4. Ligation will only occur when there is a perfect match between the probe and the template.

5. Mismatch stalls the enzyme and perhaps kicks it off the template.

6. Detects a mutant present at 0.4% of the population.

Oligonucleotide LigationOligonucleotide LigationAssay (OLA) Assay (OLA)