HIV pathogenesis and persistence in the CNS: The stage is ...cfar.med.miami.edu/documents/Serena_Spudich,_Yale_University,_USA.pdfHIV pathogenesis and persistence in the CNS: The stage

Serena S. Spudich, MDDepartment of Neurology

Yale University

28 January 20192019 Miami Winter SymposiumMiami, USA

HIV pathogenesis and persistence in the CNS: The stage is set in primary infection

What is the early natural history of HIV infection and injury in the CNS?

Does early ART impact long term CNS HIV pathogenesis and persistence?*

Does HIV persist in the CNS during ART?

*And how do we test this?





Garvey et al, AIDS. 2014; Anthony et al, J Neuropath Exp Neur, 2005; Dahl et al, AIDS 2014.

Positron emission tomography (PET): Abnormal brain uptake of ligand specific for activated microglial cells (>3 years of ART vs HIV-).

Brain autopsy: Increased CD68+ cells (macrophages/microglia) in > 1.5 years ART vs HIV-.

Abnormal macrophage/microglial activation detected during ART with plasma viral suppression

CSF: Elevated CSF neopterin, marker of macrophage activation, associates with detectable CSF HIV RNA by single copy assay (> 10 years of ART).

/ HIV-

Estes et al, Nature Medicine, 2017

SIV & SHIV RNA detected in macaque brain after > 20 weeks of suppressive ART

LamivudineAbacavirLopinavir/r

hand tremor, ataxia, slurred speech, aphasia

Latest CD4 308 cells/ul; nadir CD4 60 cells/ul

Peluso et al., AIDS, 2012.

CNS HIV replication during ART with plasma viral suppression: CSF HIV�escape�

CSF HIV RNA

CSF WBC = 26 cells/ul



PI: I13V, K20R, M36I,

I54V, L63P, V82A

RT: M41L, E44D, D67N, V118I,

M184V, L210W,

T215Y

Zidovudine Possible Resistance

Didanosine Resistance

Lamivudine ResistanceStavudine Possible Resistance

Abacavir ResistanceTenofovir Resistance

Nevirapine No Evidence of ResistanceDelavirdine No Evidence of Resistance

Efavirenz No Evidence of Resistance

Saquinavir Resistance

Indinavir ResistanceRitonavir Resistance

Nelfinavir ResistanceAmprenavir Resistance

Lopinavir/r Possible Resistance

Atazanavir Possible ResistanceAtazanavir/r Possible Resistance

Tipranavir/r Possible Resistance

AbacavirLamivudine Darunavir/r Nevirapine Zidovudine

Resolution of signs and symptoms



CSF HIV escape variants that persist over time suggest a replicating source in the CNS

S. Joseph et al., CID 2018





SEARCH clinic, Bangkok

Real-time screening of 285,674 samples in Thailand

Acute HIV infection(n=544 enrolled/681 detected)

Immediate ART(n=539)

RV254/SEARCH010: Acute infection with early ART

Data as of 31 May 2018Overall study director: Jintanat Ananworanich

Identification of recent (<12 months) infection by

nucleic acid testing or recent negative Ab test

Primary infection enrolled - San Francisco (n=109)

- Phlebotomy

- Lumbar puncture

Gothenburg (n=27)

Milan (n=18)

Sydney (n=6)

Follow-up: 6 weeks, each

6 months thereafter

(n=136, total visits=711)

PISCES: Primary infection stage CNS events study

Observational study;

participants could

elect to start ART

Collaborators: R. Price, F. Hecht, C. Pilcher, M. Gisslen, L. Hagberg, P. Cinque, B. Brew

- Phlebotomy

- Neuropsychological testing

- MRI/MRS

- Lumbar puncture

ART total visits=379 No ART total visits=332

Acute and Primary HIV Study ParticipantsRV254/Acute HIV

n = 539PISCES/Primary HIV

n = 160Age 26 (21 - 36) 37 (29 – 45)Male, n (%) 512 (95%) 150 (94%)Estimated duration of infection, days 19 (14 - 25) 88 (52-149)Fiebig I/II, n (%) 206 (38%) NAHIV Subtype 81% CRF01_AE 90% presumed BDuration of infection prior to ART 21 days 229 days

Values are number (%) or median (IQR)

Acute HIV Thailand - Ananworanich, ValcourPrimary HIV US, Europe, Australia - Price, Hecht, Gisslen, Hagberg, Cinque, Brew

HIVRNALevels

0 30 60 90 120 150 180 210 240 270 300 330 360

1

2

3

4

5

6

7

8

log₁₀copies/m

l

DaysPostHIVTransmission

Plasma CSF

HIV RNA in CSF parallels plasma in early infection

Spudich et al. JID 2011; Chan et al. JID 2018

CSF Neopterin

0 30 60 90 120 150 180 210 240 270 300 330 3600

20

40

60

80

100

nmol

/L

Days Post HIV Transmission

Neopterin, biomarker of macrophage activation, is elevated in the CSF compared to HIV negative (dotted line) throughout early infection.

Macrophage activation in the CNS is triggered in early infection

Peluso et al., JID 2013; Sanford et al, CID 2018

Neuronal injury begins during primary infection

CSF NFL

0

1000

2000

3000

ng/L

HIV- AHI PHI CHI

HIV- Acute Primary ChronicHIV HIV HIV

Neurofilament light chain (NFL) = specific CSF biomarker of axonal injury

Thalamus volume declines in early infection prior to ART (red line)

Acute HIV = median 19 days post infectionPrimary HIV = median 90 days post infection

HIVRNALevels

0 30 60 90 120 150 180 210 240 270 300 330 360

1

2

3

4

5

6

7

8

log₁₀copies/m

l


CSF

When does CNS HIV compartmentalization start during the course of infection?

ì

HXB2

Tovanabutra et al., submitted

0.02

036

HXB2

002

024

036 004 CM244

032029

023

019

034

*

*

PlasmaCSF

* Multiple T/F

*

016

No CNS compartmentalization detected during acute HIV by single genome amplification of env

Phylogenetic tree of plasma & CSF HIV env sequences

0.02

Median duration of infection = 17 days

ì

Varia

nt fr

eque

ncy

15 23 19 42 50 42

Frequency of major, minor#1, and recombinantsin protease (PR) and reverse transcriptase (RT)

15% 23% 19% 42% 50% 42%

Enrichment of minor variants in CSF during acute infection detected by deep sequencing

IonTorrent PGM deep sequencing platform, n = 17 acute HIV participants

HaplotypeRegion

Sirijatuphat, et al, CROI 2017; Tovanabutra et al., submitted

HIVRNALevels

0 30 60 90 120 150 180 210 240 270 300 330 360

1

2

3

4

5

6

7

8

log₁₀copies/m

l


CSF

When does CNS HIV compartmentalization start during the course of infection?

Eq (-) Int Eq (+) Comp2

3

4

5

6

CSF

HIV-

1 RN

A (lo

g 10c

opie

s/m

L)

Not Analyzed Equilibrated (-) Equilibrated (+) Intermediate Compartmentalized

0-4 months(n=44)

5-12 months(n=70)

13-24 months(n=30)

68% 61% 67%14%

16%

2%

13%

6%

7%13%

20%

7%

3%3%A

P < 0.0001r = 0.48

Limit of Detection

P = 0.0006

P = 0.02C D

B

36%

40%

9%

15%

CSF Pleocytosis(n=33)

Eq (-) Int Eq (+) Comp2

3

4

5

6

CSF

HIV-

1 RN

A (lo

g 10c

opie

s/m

L)Not Analyzed Equilibrated (-) Equilibrated (+) Intermediate Compartmentalized

0-4 months(n=44)

5-12 months(n=70)

13-24 months(n=30)

68% 61% 67%14%

16%

2%

13%

6%

7%13%

20%

7%

3%3%A

P < 0.0001r = 0.48

Limit of Detection

P = 0.0006

P = 0.02C D

B

36%

40%

9%

15%

CSF Pleocytosis(n=33)

Variable compartmentalization of env sequences between plasma and CSF in primary infection

Sturdevant C., PloS Pathogens 2015

BloodCSF

Compartmentalized evolution of CSF HIV variants over first two years of infection

CSF HIV RNA0.001

0.001

Sub. 9040Overall TMRCA: 209 days

A

****

* **

******

***

*** *

****

*

**** **

**

**

* ***

* *****

P/Cdays p.i.165352644918

TMRCA:134 days


P/Cdays p.i.140341

TMRCA:102 days

TMRCA:55 days

0.001


P/Cdays p.i.156

TMRCA:85 days

TMRCA:134 days

**

*

**

**

*

*

****

*

**

** * * **

** * *

***

**

*

*

B C

Sturdevant C., PloS Pathogens 2015





CSF HIV RNA

0

2

4

6

8

log

10 c

/ml

AHI6 mo ART

n = 62

AHInaive

n = 117

AHI24 mo ART

n = 35

p<0.0001

p<0.0001

ART initiated in acute HIV effectively reduces CSF HIV RNA

CSF HIV RNA

0

2

4

6

8

log

10 c

/ml

AHI6 mo ART

n = 62

AHInaive

n = 117

AHI24 mo ART

n = 35

p<0.0001

p<0.0001

Months post estimated infection

Handoko et al., CROI 2019.

Low rate of CSF escape after treatment in acute HIV (n=1/89, 1% vs ~10% reported after treatment in chronic HIV).

ART initiated in acute HIV normalizes CSF myeloid activation (neopterin) & chemokines

Hellmuth et al, JID, 2019.

CSF Neopterin

0

10

20

30

CSF

Neo

pter

in, n

mol

/L

AHI cARTnaive

AHIcART

6m

AHIcART24m

HIVnegative

*p<0.001*p<0.001

p=0.101

*p<0.001

0

1000

2000

3000

4000

CS

F M

CP

-1 p

g/m

L

AHI cARTnaive

AHIcART

6m

AHIcART24m

HIVnegative

*p=0.004

*p=0.010

*p=0.008

p=0.697CSF CCL2/MCP-1

ART was initiated at a median of 225 days post-infection.

Months on cART=13.3 (5.7, 35.3)

Log 1

0QAl

b

Months following cART initiation (t=0)

Parameter Estimate P-valueDays following cART initiation (t)

-1E-5 0.083

Rahimy et al., JID 2017

ART initiated in primary HIV does not reverse blood brain barrier injury

ART initiated in primary HIV attenuates but does not reverse accumulated brain inflammation

N= 26ART initiation WPI = 28 (12, 73)Follow-up weeks = 121 (38, 171)

Young et al., Neurology 2014

Compartmentalized CSF HIV detected by single

genome sequencing despite ART in primary infection

Dahl et al., JID, 2014.

Blood CSF

0.0005

Tree rooted with pre-ART plasma sequence.

Grey/black – pre-ART sequences

Colors – on ART sequences: interval 1, 2

Participant 9058

Treated in primary Infection

HIV-specific CD8 T cells in CSF after ART in acute HIV

Subra et al. CROI 2019

V1 V13 V19 V1 V13 V190

5

10

15

Tota

l fre

quen

cy o

f C

D8+

T ce

ll re

spon

se (%

) - a

ll pr

otei

ns

Total Frequency of CD8+ T cell response

Fiebig I-II Fiebig III-V

Presence of CSF CD8 T cells pre-ART (V1), and after 6 (V13) and 24 months (V19) of ART.

ChronicTreated

EarlyUntreated

EarlyTreated

p24 MA RT gp41gp120INT PRTime

RXt Time

Serum CSF

Time

Relative fold increaseabove uninfected

RXt7226 10 days 330 days 7226 10 days 330 days7227 12 days 270 days 7227 12 days 270 days5043 14 days 54 days 5043 14 days 54 days5043 14 days 1735 days 5043 14 days 1735 days52375 23 days 23 days 52375 23 days 23 days52375 23 days 766 days 52375 23 days 766 days52488 30 days 30 days 52488 30 days 30 days52488 30 days 752 days 52488 30 days 752 days5552 30 days 30 days 5552 30 days 30 days5552 30 days 2794 days 5552 30 days 2794 days56583 35 days 28 days 56583 35 days 28 days56583 35 days 118 days 56583 35 days 118 days5146 36 days 132 days 5146 36 days 132 days9026 62 days 46 days 9026 62 days 46 days9026 62 days 753 days 9026 62 days 753 days9015 78 days 1269 days 9015 78 days 1269 days

50287* 148 days 35 days 50287* 148 days 35 days50287* 148 days 247 days 50287 * 148 days 247 days9056* 256 days 45 days 9056* 256 days 23 days9056* 256 days 1194 days 9056* 256 days 1194 days

Berlin 4 yr Stem 16.3 yr Berlin 4 yr Stem 16.3 yr

5071 >12 years 5071 >12 years5136 >12 years 5136 >12 years5170 >12 years 5170 >12 years5212 >12 years 5212 >12 years5222 >12 years 5222 >12 years5240 >12 years 5240 >12 years5272 >12 years 5272 >12 years5278 >12 years 5278 >12 years5285 >12 years 5285 >12 years5317 >12 years 5317 >12 years

5383 20 days 5383 20 days5383 778 days 5383 778 days9025 26 days 9025 26 days9025 51 days 9025 51 days507 28 days 507 28 days

9104 30 days9094 30 days

5561 32 days 5561 32 days5561 1487 days 5561 1487 days

9117 35 days9113 34 days 9113 35 days9113 384 days 9113 913 days

9039 37 days9042 42 days

p24 MA RT gp41gp120INT PRTime

HIV-specific antibodies

Burbelo et al. JID 2019

No detectable CSF HIV RNA1

(<80 copies/ml) during post-TI rebound plasma viremia.

No significant change in:• MRI measures of inflammation

and neuronal integrity.• CSF inflammatory measures

(neopterin, MCP1, IP10, and sCD14).

• Neuropsychological test performance over time.

1Standard Roche COBAS TaqMan HIV-1 Test V2.0.

CSF, n = 4MRS, n = 5Flanker task, n = 8

Pre-TI

Post-TI

0

2

4

6

8

10

nmol

/L

CSF Neopterin

p = 0.34

Pre-TI

Post-TI

0

400

800

1200

1600

pg/m

L

CSF IP-10

p = 0.69

Pre-TI

Post-TI

0

25

50

75

100

125

ng/m

L

CSF sCD14

p = 0.49

Pre-TI

Post-TI

0

400

800

1200

1600

pg/m

L

CSF MCP-1

p = 0.99

Chan et al., CROI 2017

CNS monitoring of ART interruption after treatment during Fiebig I acute HIV

Cell free HIV RNA CA-HIV RNA CA-HIV DNA0

10

20

30

40

50

60

70

80

90

100%

Det

ecta

ble

HIV Persistence Measures in CSFn = 69 samples

Boxes indicate % positive; bars represent 95% confidence intervals

HIV DNA detected in CSF cells during long-term ART initiated in chronic HIV

• Among those with detected CSF CA-HIV DNA, median level is similar to blood: 2.1 (range 0.12 - 7.00) copies/103 cells.

• CSF cell-associated HIV RNA = intracellular transcribed virus.

CA = cell-associated

48%

9% 4%

ACTG 5321

Median ART duration = 8.6 years

A5321 Study Team, submitted

Single cell RNA seq identifies myeloid cell clusters found predominantly in CSF in chronic suppressed HIV

BLOOD 1BLOOD 2CSF 1CSF 2

Myeloid-2

Myeloid-5

Farhadian et al, JCI Insight, 2018.

Myeloid-2 cells – share RNA expression patterns with brain-derived microglial cells

During primary HIV infection: HIV enters the CNS and immune activation is

established.HIV can begin to evolve independently within

the CNS.

During chronic HIV infection: CNS inflammation and viral persistence can be

detected during ART.

Conclusions

ART in early infection: Reduces inflammation in the CNS.May impact long-term viral persistence.

Key questions:

à How early is early enough for ART to prevent neuropathogenesis and establishment of CNS HIV persistence?

à Is adjunctive therapy with immune modulating agents during early HIV needed?

à What is the optimal approach to assess CNS HIV persistence?

UNC:Ron SwanstromSarah JosephChrista SturdevantGretja SchnellKevin RobertsonJoe EronTHINC team

U. Gothenburg:Lars HagbergMagnus GisslenHenrik Zetterberg

Milan: Paola Cinque

Innsbruk: Dietmar Fuchs

Sydney:Bruce Brew

AcknowledgementsYale:Jen ChiarellaMichael KozalBrinda EmuLeah LeShelli FarhadianPayal PatelMichelle ChintanapholTobias KirchweyRyan HandokoAveline LiZaina ZayyadHetal MistryMichael PelusoAndrew YoungJoome SuhIdil KoreElham Rahimy

UCSF/San Francisco:

Victor ValcourRichard W. Price Joanna HellmuthRick HechtChris PilcherUCSF Options Study StaffMagnet/SF AIDS FoundationTeri Liegler/Virology Staff

Study Participants

WRAIR/MHRP:Jintanat AnanworanichSodsai TovanabutraGustavo KijakSuteeraporn PinyakornRobert GramzinskyNelson MichaelMerlin RobbSandy VasanLydie TrautmannLinda JagodzinskiDiane BoltonShelly KrebsBonnie SlikeLisa Reilly

Thai Red Cross AIDS Res Center/SEARCHNittaya PhanuphakPraphan PhanuphakMark de SouzaJames FletcherEugene KroonDonn ColbyCarlo SacdalanDenise HsuPhillip ChanNitiya ChomcheyOrlanda Goh

Study Participants

NIHR21MH110260R21MH099979R01MH081772R01MH095613R01 NS084911K23MH074466W81XWH-11-2-0174; IAA number NIAID Y1-AI502602

Funding Support

Documents

HIV pathogenesis and persistence in the CNS: The stage is ...cfar.med.miami.edu/documents/Serena_Spudich,_Yale_University,_USA.pdfHIV pathogenesis and persistence in the CNS: The stage