Early HIV- specific CD8 + T cell responses in treated and untreated hyper acute HIV infec=on in the FRESH cohort

(1)

Early HIV-‐speciﬁc CD8

+

T cell

responses in treated and

untreated hyper acute HIV

infec=on in the FRESH cohort

Zaza M&ne Ndhlovu, PhD

Instructor in Medicine Ragon

Ins&tute of MGH, MIT and Harvard

Senior Lecturer University of KwaZulu

(2)

•

HIV incidence in young women in KwaZulu-‐Natal and in

sub-‐Saharan Africa

•

The FRESH cohort-‐ a unique cohort to address gaps in

HIV preven&on, pathogenesis and cure research

•

Insights on early CD8 and CD4 T cell responses during

untreated and treated hyperacute HIV infec&on

•

Implica&ons for HIV preven&on and cure strategies in

resource-‐limited seQngs

(3)

KwaZulu-‐Natal and Durban, South Africa

Western Cape Eastern Cape Northern Cape North West Free State Northern Province Gauteng KwaZulu Natal Mpumalanga Durban

(4)

Eastern and southern Africa: high new HIV

infec=ons among young women aged 15-‐24 years

•  There is a need to beRer understand the reasons for high incidence

among young women

•  Current strategies are subop&mal-‐ new biomedical interven&ons

(5)

Acute HIV-‐1 infec=on-‐ what lessons can we learn?

Year 1

Vira l lo

ad

Viral set point is a predictor for:

-‐  Rate of disease progression

-‐ Risk of transmission

Key ques(ons:

•  What behavioural, socioeconomic and biomedical factors are responsible for such

high incidence among young women?

•  What is the nature of the transmiRed/founder virus?

•  What do immune responses in acute HIV-‐1 infec&on look like and why do they

(6)

FRESH study cohort

•

FRESH

:

F

emales

R

ising through

E

duca&on,

S

upport and

H

ealth

•

Recruit women 18 to 23 at very high risk of HIV

infec&on

•

Objec=ves

1.

Provide an intensive empowerment, life-‐skills and

job readiness curriculum.

2.

Iden&fy persons in the earliest stages of acute

infec&on.

–  Study an&viral immune mechanisms

(7)

Study setup and sample collec=on

•

Phase I: Surveillance

–  Twice weekly HIV RNA PCR tes&ng via ﬁnger s&ck blood

draws

–  Quarterly blood and mucosal sampling of the female

genital tract

•

Phase II: Acute Infec&on

WEEK 0 1 2 3 4 6 8 12 24 36 48 100 152 PBMC Plasma X X X X X X X X X X X X X SWABS CVL Cytobrush X X X X X X X X X X X X

(8)

Empowerment Program

!

(9)

Acute infec=ons detected (N=32)

•  Median: 124 days; range 0-‐684

As of Sept 29, 2015:

•  699 enrolled

•  32 acute infec&ons (397 uninfected /

ac&ve)

•  Incidence 8.5 (95% CI=5.8-‐12.0) per 100

(10)

Viral and cellular dynamics

in hyperacute HIV infec&on

0 60 120 180 240 300 -43 2 3 4 5 6 7 8 9 0 200 600 800 1000 1200 1400 400 Days Pl a s m a R N A (l o g 10 c opie s /m l) CD4 Co u n t ( c e lls /mm 3 ) 127-33-0097-079 RNA CD4

(11)

FRESH studies

•

The FRESH study design allows us to address

cri&cal ques&ons in:

•

HIV preven&on research-‐

•

Risk factors for HIV acquisi&on (Anahtar et al,

2015,

Immunity

) (Byrne, Anahtar et al,

Lancet ID

,

in press)

•

Innate immune responses (Kløverpris et al, 2016,

Immunity

•

Early B cell responses

•

Early CD8 T cell responses (Ndhlovu et al, 2015,

Immunity

(12)

Dynamics of T cell responses in primary HIV

infec=on: experimental approach

Longitudinal analysis of HIV induced T cell responses

Recently ac&vated T cells (CD38+, HLA-‐DR+)

Recently s&mulated cycling T cells (Ki67+, Bcl-‐2 nega&ve) HIV an&gen-‐speciﬁc T cells (Elispot, ICS and tetramer)

(13)

HIV infec&on induces massive ac&va&on

(CD38+HLA-‐DR+) of CD8+ T cells

Pre-infection Day10 Log VL 7.1 Day3 Log VL 6.3 Day17 Log VL 5.3 Day56 Log VL 4.2 HLA-DR CD38 0.1 6.8 20.1 42.7 1.8

(14)

The timing and strength of the initial CD8+ T

cell responses impact viral load set point

3.0 3.5 4.0 4.5 5.0 5.5 0 10 20 30 40 Spearman r= 0.8 p=0.003

Log₁₀ viral load set point

Days to peak CD8+ T cell activation 3.0 3.5 4.0 4.5 5.0 5.5 20 40 60 80 100 Spearman r= !0.8 p=0.006

Percent acitvated CD8+T

(15)

Hyperacute CD8+ T cells are highly prolifera&ve

and pro-‐apopto&c (Ki67+BCL2low)

Pre-infection Day10 Log VL 7.1 Day3 Log VL 6.3 Day17 Log VL 5.3 Day56 Log VL 4.2 BCL-2 Ki 67 0.2 0.6 98.1 1.2 6.0 1.4 85.1 7.2 2.1 69.9 13.8 14.4 1.6 45.2 27.1 26.3 1.6 84.6 14.4 0.2 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 036 186 102 039 267 309 093 271 198 268 208 DFOPV Percent Ki67 high BCL-2 low 3.5 4.5 5.5 10 20 30 40 50 Spearman r= !0.7 p=0.03

Percent Ki67

high

BCL-2

low

(16)

Paucity of IFN-‐

γ

produc&on

in hyperacute infec&on

2/22/16 0 Ki67+ BCL2- IFN-γ 16 10 20 30 *** Proportion of responding CD8+ T cells _HIV-‐ speciﬁc?

(17)

Tetramer staining CD8+ T cells

at peak ac&va&on

2/22/16 17 43.8 68.9 70.2 3.8 3.9 10.5 32.4 56.0 8.6 9.2 HLA-DR Te tra me r CD38 CD8

(18)

HIV specific CD8 T cells are defective in

IFN-gamma secretion during acute HIV infection

HIV CMV 0 20 40 60 80 100 P=0.01

% of teramer+ cells secreting

(19)

Assessment of bystander activation during

hyperacute HIV infection

HIV CMV EBV Flu

0 20 40 60 80 100 Kruskal-Wallis p=0.001 CD38+HLA-DR+T etramer+ CD8 T cells HLA-DR CD8 Te tra m er _CD38 HIV-specific CMV-specific Flu-specific Donor093 8.6 0.5 0.1 62.6 25.3 3.0 CD 38 CD 38 HLA-DR HLA-DR

(20)

HIV-specific CD8 T cells fail to upregulate

survival molecules during acute HIV infection

HIV CMV EBV Flu

0 20 40 60 80 100 Kruskal-Wallis p=0.001 P=0.01 P=0.02 P=0.01 CD127+T etramer+ CD8 T cells

(21)

Marked apoptosis of HIV-specific CD8 T cells

during hyperacute HIV infection

Pre infec Peak activ Post activ

0 10 20 30 40 P=0.02 P=0.03 % AnnexinV+PI+ CD8 T cells HIV-specific H IV te tra m er Annexin V CD8 2.1 50.0 CMV-specific CM V te tra m er 1.2 15.2 Apoptosis

(22)

CD8 responses in hyperacute HIV Infection

10.2 Te tra m er CD8 64.4 HLA-DR CD 38 Activation 3.0 3.5 4.0 4.5 5.0 5.5 0 10 20 30 40 Spearman r= 0.8 p=0.003

Days to peak CD8+ T cell activation 3.0 3.5 4.0 4.5 5.0 5.5 20 40 60 80 100 Spearman r= !0.8 p=0.006

Percent acitvated CD8+T

cells

Impact on viral replication

HIV CMV EBV Flu

0 20 40 60 80 100 Kruskal-Wallis p=0.001 P=0.01 P=0.02 P=0.01 CD127+T etramer+ CD8 T cells Poor survival HIV-specific H IV te tra m er Annexin V CD8 2.1 50.0 CMV-specific CM V te tra m er 1.2 15.2 Apoptosis

HIV CMV EBV Flu

0 20 40 60 80 100 Kruskal-Wallis p=0.001 CD38+HLA-DR+T etramer+ CD8 T cells Bystander Activation Ndhlovu et al Immunity 2015

(23)

CD4 T cell responses

(24)

CD4+ T cell ac&va&on

during acute HIV infec&on

0 102 103 104 105 HLA-DR 101 102 103 104 CD38 0.992 0 102 103 104 105 101 102 103 104 3.31 0 102 103 104 105 101 102 103 104 4.3 0 102 103 104 105 101 102 103 104 1.53 0 102 103 104 105 101 102 103 104 1.32 Pre-infection Day 3

Fiebig I Day 10Fiebig I Day 56Fiebig V1

LogVL:4.1 LogVL:6.9 LogVL:6.11 LogVL:5.3

Day 17 Fiebig I 0 20 40 60 80 100100 150 200 0 4 8 12 036 186 102 039 267 309 093 271 198 268 208

Days following onset of plasma viremia (DFOPV)

Percent activated CD4+ cells

(CD38+HLA

(25)

CD4+ T prolifera&on

during hyperacute HIV infec&on

0 102 103 104 105 BCL-2 0 102 103 104 105 Ki67 0.79 0.79 87 11.4 0 102 103 104 105 0 102 103 104 105 3.33 0.274 77.6 18.8 0 102 103 104 105 0 102 103 104 105 6.17 0.346 72.1 21.2 0 102 103 104 105 0 102 103 104 105 1.99 0.773 72.4 24.8 0 102 103 104 105 0 102 103 104 105 0.972 0.505 83.7 14.6

LogVL:4.1 LogVL:6.9 LogVL:6.11 LogVL:5.3

Day 56 Fiebig VI Day 17 Fiebig I Day 10 Fiebig I Day 3 Fiebig I Pre-infection 0 20 40 60 80 100100 150 200 0 2 4 6 8 036 186 102 039 267 309 093 271 198 268 208

Days following onset of plasma viremia (DFOPV)

Percent Ki67

high

BCL-2

low

(26)

Dynamics of HIV-specific CD4+ T cell

responses during acute HIV infection

1-6 _7-13 14-2 0 21-2 7 28-3 4 35-5 6 57-8 4 0.00 0.05 0.10 0.15 0.20

Days post Fiebeg 1

% IFN-γ + CD4+ T cells Gag Nef Env

(27)

Comparison between activated CD4+ T

cells and gamma secreting cells

Activated IFN-_γ+ICS 0 2 4 6 8 10 Proportion of responding CD4+ T cells

(28)

Number of HLA-DRB1 variants that

can present each peptide

Fr eque nc y of r esponde rs (% ) 80 HLA DRB 1*11:01 HLA DRB 1*13:01 HLA DRB 1*03:01 119 HLA DRB 1*13:01 83 HLA DRB 1*13:01 163 HLA DRB 1*11:02 HLA DRB 1*13:01 37 HLA DRB 1*11:02 HLA DRB 1*03:01 HLA DRB 1*03:02 HLA DRB 1*11:01 38 HLA DRB 1*11:02 41 HLA DRB 1*13:01 HLA DRB 1*11:01 HLA DRB 1*07:01 HLA DRB 1*01:01 HLA DRB 1*03:01 HLA DRB 1*03:02 HLA DRB 1*13:03 59 HLA DRB 1*11:02 40 HLA DRB 1*13:01 HLA DRB 1*03:01 64 HLA DRB 1*13:01 32 HLA DRB 1*07:01 5 HLA DRB 1*13:01 10 HLA DRB 1*07:01 HLA DRB 1*13:01 HLA DRB 1*15:01 4 HLA DRB 1*01:01 HLA DRB 1*13:03 6 HLA DRB 1*11:01 HLA DRB 1*03:01 HLA DRB 1*13:01 369 HLA DRB 1*11:01 25 HLA DRB 1*03:01 HLA DRB 1*07:01 63 HLA DRB 1*03:01 79 HLA DRB 1*03:01 81 HLA DRB 1*03:01 292 HLA DRB 1*03:01 301 HLA DRB 1*03:01 353 HLA DRB 1*03:01 365 HLA DRB 1*03:01 HLA DRB 1*11:01 55 HLA DRB 1*03:01 46 HLA DRB 1*07:01 HLA DRB 1*03:01 ₂₉₀ HLA DRB 1*13:01 289 HLA DRB 1*15:01 92 HLA DRB 1*13:03

(29)

Class II tetramer staining

0 102 103 104 105

<Alexa Fluor 700-A>: CD4

0 102 103 104 105 <PE-A>: DRB1101- DV16 0.1 0 102 103 104 105

0 102 103 104 105 <APC-A>: DRB1101- DV16 0.1 0 102 103 104 105 <APC-A>: DRB1101- DV16 0 102 103 104 105 <PE-A>: DRB1101- DV16 0.0 0.1 0.0 99.9

(30)

Activation profile of HIV-specific CD4 T

cells

0 102 103 104 105 <Alexa Fluor 700-A>: CD4 0 102 103 104 105 <APC-A>: DRB1101- DV16 0.03 0 102 103 104 105 <APC-H7-A>: HLADR 0 102 103 104 105 <PE-Cy7-A>: CD38 0 102 103 104 105

0 102 103 104 105 <APC-A>: DRB1101- DV16 0.06 0 102 103 104 105 <APC-H7-A>: HLADR 0 102 103 104 105 <PE-Cy7-A>: CD38 Acute HIV Chronic HIV

(31)

Phenotypic profile of HIV-specific CD4

+

T cells in

acute and chronic HIV infection

0 102 103 104 105 <FITC-A>: CXCR5 0 102 103 104 105 <Pacific Blue-A>: PD-1 0 102 103 104 105 <FITC-A>: CXCR5 0 102 103 104 105 <Pacific Blue-A>: PD-1

(32)

Conclusions

•  CD8+ T cell immune responses during acute HIV-‐1 infec&on is

broader than previously thought (Ndhlovu et al, 2015,

Immunity)

–  Acute HIV infec&on rapidly induces massive ac&va&on and

prolifera&on of CD8+ and CD4+T cells

–  The magnitude and &ming of CD8+ T cell ac&va&on impact viral load

set point

–  A high propor&on of ac&vated CD8+ cells are HIV-‐speciﬁc

•  HIV-‐speciﬁc CD8+ and CD4+ T cells are defec&ve for cytokine

secre&on, memory genera&on and prone to apoptosis during acute HIV infec&on following ex-‐vivo s&mula&on

•  There is measurable expansion of HIV-‐speciﬁc peripheral TfH

(33)

-5 0 0 5 0 1 0 0 1 02 1 03 1 04 1 05 1 06 1 07 1 08 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 F R E S H A c u te 1 D a y s P la s m a R N A ( c o p ie s /m l) C D 4 C o u n t (U /L ) R N A C D 4 -5 0 0 5 0 1 0 0 1 02 1 03 1 04 1 05 1 06 1 07 1 08 0 5 0 0 1 0 0 0 D a y s P la s m a R N A ( c o p ie s /m l) 3 5 0 C D 4 C o u n t (U /L ) F R E S H R x P a tie n t 1 P la sm a R N A C D 4 C o u n t

The impact of early treatment

on generation and maintenance of

HIV-specific T cell responses?

(34)

Treatment during hyperacute phase blunts peak

viremia

(35)

Raltegravir intensiﬁca=on reduces =me to full

suppression

(36)

Impact of early ART on T cell responses

Inves&gate if very early ART treatment

suppresses the subsequent development of HIV

speciﬁc CD8 cell responses by removing the

an&genic s&mulus, OR.

If early ART treatment might help preserve HIV

speciﬁc CD4 to CD8 and B cell responses.

(37)

Very early ART limit CD4 T cell loss

Preinfection Nadir CD4 Rebound CD4

0 500 1000 1500 2000 CD4 Count (cell/mm 3 ) p=0.01 ns ns

Preinfection Nadir CD4 Rebound CD4

0 500 1000 1500 CD4 Count (cell/mm 3) p=0.0001 p=0.001 p=0.01

(38)

CD8

+

T cell responses in early treated subject

0 102 103 104 105 HLA-DR 0 102 103 104 105 CD38 Gate % CD8 23.1 TetPE 3.56e-3 0 102 103 104 105 0 102 103 104 105 Gate % CD8 46.3 TetPE 5.67 0 102 103 104 105 0 102 103 104 105 Gate % CD8 43.1 TetPE 9.8 0 102 103 104 105 0 102 103 104 105 Gate % CD8 34.4 TetPE 5.7 0 102 103 104 105 0 102 103 104 105 5.7 Day 56 Log VL <2.0 0 102 103 104 105 0 102 103 104 105 9.8 Day 21 Log VL <2.0 Day 4 Log VL 3.5 Pre-infection 0 102 103 104 105 0 102 103 104 105 5.7 0 102 103 104 105 CD8 0 102 103 104 105 Tetramer 0.0 -150 -75 00 25 50 75 100 125 150 2 3 4 5 6 7 8 0 200 400 600 800 1000 1200 1400 127-33-0628-451 RNA CD4 Days Pl a s m a R N A (l o g10 c opie s /m l) CD4 Co u n t ( c e ll/mm 3 ) Treatment initiation

(39)

Frequency of individual tetramers

+

CD8

+

cells in early

treated and untreated subjects

Treated Untreated

0 5 10

15 p=0.01

Frequency of single tetramer+ cells

(40)

Increased CD127 expression on CD8+_{T cells during early treated}

hyperacute HIV infection

Treated Untreated 0 20 40 60 80 100 p=0.0001 CD127 + Te tr am er + CD8 + cells

(41)

HIV

-specific CD8

+

T cells in early treated subjects

have a more more functionally competent

0 102 ₁₀3 ₁₀4 ₁₀5 0 102 103 104 105 3.5 0 102 ₁₀3 ₁₀4 ₁₀5 0 102 103 104 105 1.6 3.0 2.0 93.3 0 102 ₁₀3 ₁₀4 ₁₀5 0 102 103 104 105 1.3 0 102 ₁₀3 ₁₀4 ₁₀5 0 102 103 104 105 1.2 0.3 0.4 98.1 Treated Untreated IFN-‐gamma IFN-‐gamma CD8 CD8 Te tr am er Te tr am er Te tr am er Te tr am er

(42)

Conclusions

•  Very early treated individuals have measurable T cell responses that

have a pro-‐survival phenotype

•  Prompt an&gen withdrawal results in stable HIV-‐speciﬁc CD8 T cell

clonal repertoire.

(43)

(44)

The Ad26 mosaic vaccine yielded many more epitope-‐ speciﬁc responses than did the Ad26 M consensus, clade B + clade C, or op&mal natural clade C vaccines

Vaccinate FRESH subjects with Ad26 mosaic vaccine

Rhesus Monkeys

(45)

Proteinase K Lysis Cells

Pre-‐ampliﬁca=on from cell lysate

Single well pre-‐ampliﬁca=on of HIV and CD3

5’LTR gag pol env 3’LTR

Alu Alu

Total HIV DNA Integrated HIV DNA

U5 U3 gag pol en v 2-‐LTR circles HIV CD3

Real-‐=me nested PCR of pre-‐ampliﬁed products TaqMan Probes

(sensi=vity: 1 HIV DNA copy)

Frequency of cells harbouring HIV DNA

HIV copy number 2 X CD3 copy number

Assays to measure HIV Persistence

adapted from

(46)

Conclusions I

•

The FRESH study design allows us to address cri&cal

ques&ons in HIV preven&on research-‐

–  such as the impact of cervicovaginal microbiota on female

genital inﬂamma&on (Anahtar et al, 2015, Immunity) and impact of IPC on HIV acquisi&on risk (Byrne, Anahtar et al, Lancet ID, in press)

•

FRESH study par&cipants ini&ated on cART during

hyperacute HIV infec&on may oﬀer new insights on long-‐

term viral remission

•

Understanding of immune responses following cART

(47)

Acknowledgements

•

Ragon Ins=tute

–  Dr. Bruce Walker –  Galit Alter –  Xu Yu

•

HIV Pathogenesis

Programme/UKZN

–  Prof. Thumbi Ndung’u

–  Krista Dong –  Amber Moodley –  Nikoshia Mewalal –  Karyn Pretorius –  Philomena Kamya –  Sannie Mahungela –  Nasreen Ismael

–

FRESH Par&cipants

2/22/16

Early HIV- specific CD8 + T cell responses in treated and untreated hyper acute HIV infec=on in the FRESH cohort

Early HIV-­‐speciﬁc CD8

sub-­‐Saharan Africa

resource-­‐limited seQngs

Acute HIV-­‐1 infec=on-­‐ what lessons can we learn?

Vira l lo

FRESH study cohort

infec&on.

Phase II: Acute Infec&on

As of Sept 29, 2015:

Viral and cellular dynamics

Lancet ID

HIV infec&on induces massive ac&va&on

Hyperacute CD8+ T cells are highly prolifera&ve

at peak ac&va&on

HIV CMV 0 20 40 60 80 100 P=0.01

Assessment of bystander activation during

Marked apoptosis of HIV-specific CD8 T cells

CD4 T cell responses

LogVL:4.1 LogVL:6.9 LogVL:6.11 LogVL:5.3

CD4+ T prolifera&on

LogVL:4.1 LogVL:6.9 LogVL:6.11 LogVL:5.3

Dynamics of HIV-specific CD4+ T cell

can present each peptide

Phenotypic profile of HIV-specific CD4

The impact of early treatment

suppresses the subsequent development of HIV

T cell responses in early treated subject

have a more more functionally competent

Vaccinate FRESH subjects with Ad26 mosaic vaccine

(sensi=vity: 1 HIV DNA copy)

Assays to measure HIV Persistence

FRESH study par&cipants ini&ated on cART during

FRESH Par&cipants

Early HIV-‐speciﬁc CD8

sub-‐Saharan Africa

resource-‐limited seQngs

Acute HIV-‐1 infec=on-‐ what lessons can we learn?