The views expressed are those of the presenter and should not be construed to represent the positions of the

U.S. Army or Department of Defense

Mark Manak, PhD Department of Laboratory Diagnostics and Monitoring

US Military HIV Research ProgramHenry M Jackson Foundation, Silver Spring, MD,

Walter Reed Army Institute of Research, Silver Spring, MD

HIV Diagnostic Conference,CDC, Atlanta GA

Mar 28, 2019

No Conflict of Interest to Declare

Performance of HIV Ag/Ab Assays on Samples from Individuals Initiating Antiretroviral

Therapy During Acute HIV Infection

CDC guidelines: initiation of antiretroviral therapy (ART)as soon as possible after first HIV diagnosis• Minimize risk of forward transmission• Disrupt establishment of latent reservoirs• Better sustained virologic control• Enhance overall health outcomes

Issue: ART initiation during Acute HIV Infection (AHI) may• Reduce HIV Ag stimulation below threshold for immune response• Block/delay emergence HIV serological diagnostic markers

2

Background

Acute HIV Infection Studies LeveragedRV254/SEARCH010: Bangkok, Thailand HIV-1 viremic volunteers initiated ART immediately post

diagnosis

Stage of HIV infection at time of ART initiation • Fiebig I (N=23) HIV RNA+, p24 Ag-, HIV Ab-• Fiebig II (N=39) HIV RNA+, p24 Ag+, HIV Ab-• Fiebig III (N=13) HIV Ab+, WB-• Fiebig IV (N=9) HIV Ab+, WB Indeterminate

Samples acquired:• Initial visit prior to ART: • 1, 12, and 24 weeks post ART

3SEARCH: South East Asia Research Collaboration with Hawaii

Acute HIV Infection Studies Leveraged

RV217 Early Cohort HIV: Kenya, Uganda, Tanzania, and Thailand Untreated HIV Infected Controls (N=30) Twice weekly testing of at risk populations

• Infection identified by APTIMA HIV-1 RNA reactivity Intensive serial sampling of incident cases

• ~3 day intervals for first 6 weeks; monthly thereafter

4

Study Assays

4th Gen Immunoassays• Bio-Rad GS HIV Combo Ag/Ab EIA (BRC) • Bio-Rad BioPlex® 2200 HIV Ag-Ab Combo (BPX)• Abbott Architect HIV Ag/Ab Combo (ARC)

3rd Gen Immunoassay• Bio-Rad 3rd Gen GS HIV-1/2/ Plus O (1/2/O)

p24 Antigen Assay• Bio-Rad Genscreen HIV-1 p24 Ag assay RUO (Ag)

Supplemental, Confirmatory Assays • Bio-Rad HIV-1 Western blot (WB) • Bio-Rad Geenius™ HIV-1/2 (Geenius)

5

Serological Detection After HIV InfectionRV 217 - No ART

4th Gen Bio-Rad Ab/Ag Combo 3rd Gen Bio-Rad 1/2O (Ab Only)Ave. 6.5 +/- 2.8 days Ave. 15.0 +/- 4.8 days

6

N = 30

0.1

1

10

100

cut-off

s/c

o

0 1 2 3 4 5 6 7 0 7 14 21 28 35 42 490.1

1

10

100

0 1 2 3 4 5 6 7

Weeks after first APTIMA Reactive

Time Course of Serological Markers

7

1 / 2 / O

W B

0 7 1 4 2 1 2 8 3 5 4 2 4 90 . 1

1

1 0

1 0 0

1 0

1 0 0

1 , 0 0 0

p2

4 A

g (u

g/m

l)

B R C

1 / 2 / O

c u t - o f f

0 1 2 3 4 5 6 7

Av

e.

s/c

o

p 2 4 A g

W e e k s a f t e r f i r s t A p t i m a

% W

B P

ositiv

e

1 0 0 %

8 0 %

6 0 %

4 0 %

2 0 %

Weeks After First APTIMA

Time Course of Serological Markers

8

1 / 2 / O

W B

0 7 1 4 2 1 2 8 3 5 4 2 4 90 . 1

1

1 0

1 0 0

1 0

1 0 0

1 , 0 0 0

p2

4 A

g (u

g/m

l)

B R C

1 / 2 / O

c u t - o f f

0 1 2 3 4 5 6 7

Av

e.

s/c

o

p 2 4 A g

W e e k s a f t e r f i r s t A p t i m a

% W

B P

ositiv

e

1 0 0 %

8 0 %

6 0 %

4 0 %

2 0 %

Fiebig Stage I II III IVART Treatment

Weeks After First APTIMA

4th Gen Immunoassays 24 Weeks After ART

BRC reaches max signal at S/CO 14 ARC and BPX continue to higher S/CO to >100 ARC and BPX signal highly correlated

R2 = 0.8404

9

0.1 1 10 100 10000.1

1

10

100

1000

BR

C s

/co

ARC s/co

ARC vs BRC

cut-off

cut-o

ff

0.1 1 10 100 10000.1

1

10

100

1000

BPX s/co

BPX vs BRC

BR

C s

/co

0.1 1 10 100 10000.1

1

10

100

1000

BPX s/co

BPX vs ARC

R2 = 0.8404

AR

C s

/co

S/CO at 24 Weeks After Early ART

FI S/CO remains close to cut-off, most NR FII Increased S/CO compared to FI, Fewer NR FIII/IV Low, but Reactive S/CO, Very few NR

No ART Established Infection; S/CO BRC 13.5-15.0; ARC 800-1200: BPX >200

10

No ART Established Infection

0

1

10

100

1,000

BRC ARCBRC ARC BPX0.1

1

10

100

1000

cut-off

s/co

FI

BRC ARC BPX0.1

1

10

100

1000FII

BRC ARC BPX0.1

1

10

100

1000FIII/IV

Time Course of BPX Reactivity After ART

Transient increase in signal in some individuals at week 1 Decrease to low level and little increase thereafter Seroreversion observed in some individuals

11

>10

Reactivity of BPX at 24 weeks after Early ART

12

0%

20%

40%

60%

80%

100%

FI FII FIII/IV0%

20%

40%

60%

80%

100%

FI FII FIII/IV

Week 12 Week 24FI FII FIII/IV FI FII FIII/IV

NR 69.6% 12.8% 9.1% NR 52.2% 7.7% 4.5%S/CO 10 8.7% 53.8% 59.1%

Total 100.0% 100.0% 100.0% Total 100.0% 100.0% 100.0%

Evolution of Western Blot Ag Reactivity

13

MWgp160gp120

p65p51/p55gp41p40

p34

p24

MWgp160gp120

p65p51/p55

gp41p40

p34

p24

12 24 12 24 12 24 Weeks After Start of ART

Hi Lo N 4 7 17 24 29 35 42 53 77 150 Day0 1 2 3 4 5 6 7 12 24 Week

Weeks after First RNA

No Treatment Treated at Acute HIV InfectionControls FI FII FIII/IV

Geenius and WB Ag at 24 Weeks After Early ART

14

Geenius and WB have comparable sensitivity for gp41 and gp160 Geenius is less sensitive for p24 (early Ag) Both assays have low sensitivity for p31 (late Ag)

0%

20%

40%

60%

80%

100%

GN WB GN WB GN WB GN WB

p24 p31 gp41 gp160 %

Rea

ctiv

e A

ntig

ens

FIFIIFIII/IV

Geenius and WB Reactivity at 12-24 Weeks After ART

15

0%

20%

40%

60%

80%

100%

FI FII FIII/IV

0%

20%

40%

60%

80%

100%FI FII FIII/IV

Week 12 Week 24

WB Geenius WB Geenius WB Geenius WB Geenius WB Geenius WB GeeniusR 25.0% 25.0% 65.8% 44.7% 72.7% 59.1% 30.4% 26.1% 73.7% 50.0% 59.1% 45.5%IND 37.5% 20.8% 31.6% 52.6% 27.3% 40.9% 21.7% 21.7% 21.1% 44.7% 36.4% 45.5%NR 37.5% 54.2% 2.6% 2.6% 0.0% 0.0% 47.8% 52.2% 5.3% 5.3% 4.5% 9.1%

100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Fiebig III/IVFiebig I Fiebig II Fiebig III/IV Fiebig I Fiebig II

Conclusions I Evolution of serological markers in untreated individuals

develops rapidly with time post infection Reduction in immune response by early ART can confound

the ability of serological assays to correctly classify HIV infectious status

At 24 Weeks of therapy:• 52.2% of those initiating ART at FI, 7.7% at FII, and 4.5% at FIII/IV

remain 4th Gen NR; 36-39% of samples demonstrated low S/CO (

Conclusions II These results have implications in monitoring individuals

who initiate ART in acute infection or participate in programs such as:

• Treatment as Prevention (TasP)• Pre-Exposure Prophylaxis (PrEP)• Post Exposure Prophylaxis (PEP)• HIV Cure studies.

Consider a contextual algorithm for testing of ART treated populations

Alternative approaches such as testing for cell-associated HIV RNA/DNA may be required to rule out HIV-1 infection (Jagodzinski, J Clin Micro 2019)

17

Acknowledgements

Funding from DAIDS, US Army, DOD, Thai Red Cross18

WRAIR - Maryland o Sheila Peelo Jennifer Maliao Linda Jagodzinski

MHRP HJF – Marylando Jintanat Anaworanicho Leigh Anne Ellero Merlin Robbo Ashley Shutto Dejha Browno Jason Ouellette

MHRP/AFRIMS - Thailando Mark de Souzao Siriwat Akapirat

Bio-Rad – Benicia, CAo Mike Leos

Thai Red Cross AIDS Research Centreo Donn Colbyo Nittaya Phanuphako Praphan Phanuphako James Fletchero Nitiya Chomchey

RV254/SEARCH010o SE Asia Research Collaboration with Hawaii

RV217 Siteso AFRIMS-Thailando MUWRP-Ugandao MMRP-Tanzaniao WRP-Kericho

Volunteers

Slide Number 1Slide Number 2Acute HIV Infection Studies LeveragedAcute HIV Infection Studies LeveragedSlide Number 5Serological Detection After HIV Infection�RV 217 - No ARTTime Course of Serological MarkersTime Course of Serological Markers4th Gen Immunoassays 24 Weeks After ARTS/CO at 24 Weeks After Early ARTTime Course of BPX Reactivity After ARTReactivity of BPX at 24 weeks after Early ARTEvolution of Western Blot Ag ReactivityGeenius and WB Ag at 24 Weeks After Early ARTGeenius and WB Reactivity at 12-24 Weeks After ARTConclusions IConclusions IIAcknowledgements