Thumbi Ndung’u, BVM, PhDAfrica Health Research Institute and

HIV Pathogenesis Programme (HPP), University of KwaZulu-Natal

International Workshop on HIV Transmission Rockville, MD, USA 12 December 2019

Acute HIV infection studies in African women reveal opportunities and challenges for prevention and cure strategies

High HIV prevalence and incidence

Wong et al, in preparation

Lessons from acute HIV-1 infection

Key questions:• Characteristics of the transmitted/founder virus• Immune responses in acute HIV-1 infection• Impact of early treatment• Ultimate goal: immune-based prevention or cure of HIV

Year 1

Fiebig I/II Fiebig III-VI

Fiebig I/II: anti-HIV humoral immune responses undetectable, viral load on upward trajectory

≥ Fiebig III: humoral immune responses detectable, peak viremia or past

FRESH study cohort

• FRESH: Females Rising through Education, Support and Health

• Recruit women 18 to 23 at very high risk of HIV infection

– Provide an intensive HIV prevention (including PrEP and POC viral load testing), empowerment, life-skills and job readiness curriculum that coincides with the sample collection protocol (Ndung’u et al, Sci Imm, 2018).

• Empowerment and training program coincides with twice per week HIV RNA testing for 9 months. Serial pre- and post-infection samples (blood, FGT and lymph nodes) are collected.

• Study host and viral factors associated with acquisition and disease progression

PrEP experience among young women in Durban

64% - PrEP uptake13 groups (~500 women)1 year (May 2018 – Apr 2019)

83 acute infections detected

As of June 2019:

• 2,053 enrollees• Incidence 7.9 (95% CI=5.8-12.0) per 100 p/y• 14 untreated, 69 treated early• 81% Fiebig I• Median 4 days since last negative HIV RNA • ART started within median 1 day

cART started in Fiebig stage I blunts peak viremia and preserves CD4 T cells

Dong et al, Lancet HIV, 2018

Participants treated in Fiebig stage I do not seroconvert*

*WB- BioRad GS kit

-500 0 50 10

015

020

025

030

035

050

0

1000

1500

2000

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Days after detection of plasma viremia

Acu

te P

ati

en

t N

um

ber

Late-treated

Early-treated

*

#

*

##

Negative

Impact of immediate ART on western blot after detection of AHI

Indeterminate

Positive*#

Fiebig III

Fiebig V

-500 0 50 10

015

020

025

030

035

050

0

1000

1500

2000

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Days after detection of plasma viremia

Acu

te P

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um

ber

Late-treated

Early-treated

*

#

*

##

Negative

Impact of immediate ART on western blot after detection of AHI

Indeterminate

Positive*#

Fiebig III

Fiebig V

-500 0 50 10

015

020

025

030

035

050

0

1000

1500

2000

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Days after detection of plasma viremia

Acu

te P

ati

en

t N

um

ber

Late-treated

Early-treated

*

#

*

##

Negative

Impact of immediate ART on western blot after detection of AHI

Indeterminate

Positive*#

Fiebig III

Fiebig V

Almost all Fiebig I treated individuals developed low magnitude but highly functional HIV-specific CD8+ T cell responses

Treated Untreated0

5

10

15p=0.01

Fre

quency o

f sin

gle

tetr

am

er+

cells

(%of to

tal C

D8+

PB

MC

)

Treated Untreated0

20

40

60

80

100p=0.002

% o

f te

tram

er+

IFN

-g+

HIV

-specific

CD

8+

T c

ells

Frequency of HIV-specific T-cells IFN-γ+ HIV-specific T-cells

Overall, almost all participants who started treatment in Fiebig I developed CTL responses:• 69% by IFN-γ ELISPOT• 80% by tetramer staining• 90% by CFSE proliferation

Ndhlovu et al, Sci Trans Med, 2019

Early cART preserves proliferation of HIV-specific CD4 T cells20180916 Workspace.jo Layout

9/16/18 2:35 PM Page 1 of 1 (FlowJo v9.9.4)

0 102

103

104

105

0

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103

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5.4

0 102

103

104

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0

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104

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21.8

0 102

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0

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0 102

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24.6

0 102

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0

102

103

104

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3.6

0 102

103

104

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0

102

103

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0.1

Tx Fiebig I

Untreated

CFSE dilution

Day 28 Day >250

Fiebig

I-II

Fiebig

III-V

Un

-Tx

0

20

40

60

80

% p

rolif

era

ting C

D4

+ T

cells

of th

e tota

l C

D4

+ T

cells

P=0.005

P=0.03

P=NS

Day>250

Fiebig

I-II

Fiebig

III-V

Un

-Tx

0

20

40

60

80

% p

rolif

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ting C

D4

+ T

cells

of th

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P=0.05

P=NS P=NS

Day 14-42

20180916 Workspace.jo Layout

9/16/18 2:35 PM Page 1 of 1 (FlowJo v9.9.4)

0 102

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Day 28 Day >250

0 1 4 36 0 1 4 36

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Weeks from detection

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pg

/ml)

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******

***

00.

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5 2 4 12

0

200

400

600

Weeks from detection

CX

CL

13 (

pg

/ml)

Untreated Early-treated**

********

Treatment in Fiebig stage I abrogates the cytokine* storm

*11 of 11 cytokines with post-infection significant change Muema et al, submitted

0.0

0.2

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no

cyte

s (

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9/L

)

P<0.001 P=0.004

P<0.001

Pre-infection

Fiebig I-II Fiebig III-V Chronic HIV

Acute HIV

0.0

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P=0.003

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Pre-infection

Fiebig I-II Fiebig III-V Chronic HIV

Acute HIV

HIV infection is associated with immune cell subset changes even in FiebigI treated participants

• Eosinophils, basophils and neutrophils significantly reduced post-infection

Muema et al, submitted

What about sensitivity of transmitted/founder virus to bNAbs?

V1V2 (glycan dependent) – PG9, PG16, PGT141-145,

CH01-04, CAP256-VRC26, PGDM1400CD4 binding site

b12, VRC01, 3BNC117, HJ16, NIH45-46, CH31, CH103, 12A12

MPER – 4E10, 2F5,

z13, 10E8

C3/V3 (glycan dependent) –

2G12, PGT128, PGT121,PGT135, 1010-74

Adapted from Burton et al, Science, 2012

gp120-gp41 interface (glycan dependent)

PGT151, 35O22,

CAP248 30.2B

Clonal sequenceSGA sequence

Most env viral sequences are identical indicative of a single transmitted/founder virus

BDA1

A2A2

036

079

186

208

268

267

271

318

0.03

VRC07-523LS and CAP256 show excellent coverage of T/F clade C viruses

T/F Env pseudotyped viruses were assayed in the TZM-BL luciferase assay

T/F virus PGT151 10E8 CAP256.VRC26.08VRC01 PGDM1400 PGT121 3BNC117 10-1074 VRC07-523LS

093 0,06 0,64 0,01 0,59 0,09 <0.005 >10 0,01 0,02

208 0,02 1,31 0,46 >10 0,3 0,04 0,06 0,23 0,13

268 0,03 0,51 >10 >10 9,29 0,06 >10 nd nd

079 0,02 1,4 <0.005 2,18 <0.005 >10 >10 nd nd

318 0,2 0,52 <0.005 0,92 >10 0,02 0,06 <0.005 0,05

036 <0.005 0,91 5,48 0,23 <0.005 <0.005 0,07 <0.005 0.01

271A 0,06 1,05 0,19 0,32 >10 >10 0,55 >10 0,01

271B 0,04 2,43 <0.005 6,37 <0.005 >10 >10 >10 <0.005

267 1,52 0,17 <0.005 1,33 <0.005 0,09 0,14 >10 0,03

186 0,01 0,5 <0.005 0,4 0,01 0,02 0,1 nd nd

198 0,01 0,76 <0.005 0,85 0,02 0,02 0,06 0,06 <0.005

201 2,12 1,72 <0.005 >10 0,08 0,05 >10 0,02 >10

498 >10 5,11 0,16 >10 1,36 >10 >10 >10 0,26

499 >10 0,96 0,95 3,30 0,81 >10 0,67 >10 0,05

559 >10 0,17 0,67 0,55 0,12 >10 >10 >10 <0.005

1388 0.03 >10 0.01 0.9 1.14 0.02 >10 0.02 0.01

1439 0.02 3.39 <0.005 5.01 0.01 >10 0.17 >10 0.02

% Breadth (10ug/ml) 82% 94% 94% 71% 88% 59% 53% 50% 94%

% Breadth (0.1ug/ml) 65% 0% 59% 0% 53% 59% 29% 35% 71%

IC50 Titers (µg/ml)

<0,1 µg/ml

0,9 - 0,1

3,0 - 0,91

10,0 -3,1

>10

nd not done

An opportunity to identify the most effective bNAbs against transmitted/founder viruses

Early treatment blunts peak viral load (HIV RNA) but not peak total HIV DNA

U n t r e a t e d T r e a t e d

1 02

1 03

1 04

1 05

1 06

1 07

1 08

Pe

ak

V

ir

al

Lo

ad

(

co

pi

es

/m

l)

p = 0 . 0 0 3

U n t r e a t e d T r e a t e d

1 0

1 0 0

1 0 0 0

1 0 0 0 0

Pe

ak

T

ot

al

H

IV

D

NA

(c

op

ie

s/1

06

c

el

ls

)

p = 0 . 4

Total cellular DNA Plasma HIV RNA

Early treatment blunts peak viral load (HIV RNA) but not peak total HIV DNA

U n t r e a t e d T r e a t e d

1 02

1 03

1 04

1 05

1 06

1 07

1 08

Pe

ak

V

ir

al

Lo

ad

(

co

pi

es

/m

l)

p = 0 . 0 0 3

U n t r e a t e d T r e a t e d

1 0

1 0 0

1 0 0 0

1 0 0 0 0

Pe

ak

T

ot

al

H

IV

D

NA

(c

op

ie

s/1

06

c

el

ls

)

p = 0 . 4

Total cellular DNA Plasma HIV RNA

Early treatment has much more profound suppression of viral load(HIV RNA) compared to total DNA at 12 months post-infection

Plasma viral load Total DNA reservoir

HIV Gagp24 staining of a LN

tissue from an early treated

subject

19

Gag p24

Nuclei

-500101

102

103

104

105

106

107

108

0 50 100 150 200 250 300 3500

200

400

600

800

1000

1200

1400

1600

1800V

iral lo

ad (

copie

s/m

l)

CD

4 c

ounts

(cells

/µL)

Days since detection

LN excision

Gagp24

Nuclei

BCL-6

Prolonged HIV persistence in lymph nodes despite early treatment

DAPI Nuclei HIV Gagpol RNACD4

HIV RNA persistence in lymph nodes

Baiyegunhi, Mann et al, submitted

Sequence evolution and minor variant populations may shape the reservoir in PBMCs and lymph nodes

Pt 1Pt 2Pt 3

Pl day 0/1 Pl day 0/1 bulkPl day 7Pl day 7 bulkP2 day 14Pl day 14 bulkP3 day 19 bulkLN >1 year bulkPBMC >1 year bulk

Gag SGA, bulk and deep sequencing approaches

Pt 3: No evidence of virus evolution Pt 2: LN sequences identical to T/F minor variant Pt 1: Seq evolution in LN and PBMC

Pt 1

Pl day 0/1 Pl day 0/1 bulkPl day 7Pl day 7 bulkPl day 14Pl day 14 bulkPl day 19 bulkLN >1 year bulkPBMC >1 year bulk

0 100 200 300 400 500

Plasma Day 1

Plasma Day 7

PBMC Day 415

LN Day 403

M – Pt 1

20RLRPGGKKRY20--------L-

76RSLYNTIAVLF76-----I----S

76RSLYNTIAVLF76-----I----S

RY10 (A*30) RY11 (A*30) RY11 (A*30)

Evidence of sequence evolution despite suppressive ART

Conclusions

• Acute HIV infections- novel insights into characteristics of T/F virus, immune responses and reservoir characteristics.

• Early treatment results in incomplete antibody seroconversion, low magnitude but highly functional CD8 T cell immune responses, modulates transcriptional profile of CD8 T cells and abrogates the cytokine storm- more functional responses?

• HIV persists in lymph nodes long after viral suppression in early treated individuals and some patients show evidence of virus evolution on ART

• A unique platform to inform, test and implement HIV prevention and curative strategies

AcknowledgementsHPP/UKZN and AHRI• FRESH study participants• FRESH study team• Krista Dong• Amber Moodley• Zaza Ndhlovu• Kavidha Reddy• Omolara Baiyegunhi• Jaclyn Mann• Jenn Mabuka• Bongiwe Ndlovu• Kamini Gounder• Daniel Muema• Keshni Hiramen• Nasreen Ismael• Denis Chopera

Prince Mshiyeni Hospital• Johann Pansegrouw

Funding

• Bill and Melinda Gates Foundation

• IAVI

• NIH

• South African DST/NRF

• HHMI

• Gilead Sciences, Inc.

• Wellcome Trust

Thai Red Cross/MHRP

• Merlin Robb

• Jintanat Ananworanich

• Eugene Kroon

Harvard/MGH/Ragon

• Bruce Walker

• Douglas Kwon

• Galit Alter

• Mathias Lichterfeld

• Guinevere Lee

• Musie Ghebremichael

• Sam Kazer

• Alex Shalek