Presentation: 0300Poster # 1623

CROI 2019 March 4-7

Seattle, WA

Effect of Telmisartan given at ART Initiation in AHI on Immune Cells in Lymph Nodes Supranee Buranapraditkun*1,2,3, Hiroshi Takata2,3, Eugène Kroon4, Suthat Chottanapund4 , Carlo P. Sacdalan4, Somporn Tipsuk4, Khunthalee Benjapornpong4, Bessara Nuntapinit5, Jodi Anderson6, Timothy Schacker6, Nicolas Chomont7, Merlin L. Robb2,3,Jintanat Ananworanich2,3,4, Serena Spudich8, Lydie Trautmann2,3, on behalf of RV408/SEARCH018, RV254/SEARCH010 and RV304 Study groups

1Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA; 3U.S. Military HIV Research Program, Walter ReedArmy Institute of Research, Silver Spring, MD, USA; 4SEARCH, The Thai Red Cross AIDS Research Center, Bangkok, Thailand; 5Armed Forces Research Institute of Medical Sciences in Bangkok, Bangkok,Thailand; 6University of Minnesota, MN, USA; 7Centre de Recherche du CHUM (CRCHUM), Université de Montréal, Canada; 8Department of Neurology, Yale University, CT, USA

Rapid and progressive deposition of collagen within the T-cell zones (TZ) of lymphoidtissue occurs within four weeks of HIV or SIV infection. TZs are organized to enable theinteraction of naive and memory T cells with antigen-presenting cells and to provide asource of growth factors and cytokines, facilitating the development and maintenanceof antigen responses and the CD4+ T cell population. Therefore, collagen deposition inlymphoid tissue may prevent CD4+ T cells from interacting with proliferative cytokinesand encountering antigen-presenting cells. HIV infection induces lymph node (LN)fibrosis and persistent inflammation even during successful antiretroviral therapy(ART). Telmisartan (T), an angiotensin II receptor type I antagonist, blocker, has anti-inflammatory and anti-fibrotic effects. It could prevent fibrosis and inflammation inindividuals with HIV when administered at time of ART initiation in acute HIV infection(AHI). In addition, Telmisartan has numerous potential benefits in HIV-infectedpatients, as it has been shown to inhibit TGF-β, interferon-g, tumor necrosis factor(TNF), and IL-6 production and signaling. We assessed telmisartan effect on preventingHIV-mediated damage and on HIV reservoirs in lymph node tissues in peoplerandomized during AHI to telmisartan+ART (T+ART) versus ART alone.

BACKGROUND

MATERIALS AND METHODS

Cryopreserved lymph node mononuclear cells (LNMCs) at time of AHI and 48 weeksafter were included from RV408/SEARCH018 participants randomized to T+ART (n=7)versus ART alone (n=4). Additionally, LNMC samples from other RV254/SEARCH010participants at acute HIV infection (AHI) (n=26) and post-ART at wk48 (n=13), tocompare the RV408 randomized group to a larger group. Nine uninfected Thaiparticipants from RV304/SEARCH013 study were also included. B cells, CD4 and CD8 Tcells were characterized by flow cytometry when sufficient cells permitted. Total HIVDNA levels in CD4 T cells were quantified by ultrasensitive PCR; collagen, TGFb andTNFa were measured by immunohistochemistry. All these participants fulfilleddemographic and clinical data are included in Table 1. This cohort is under theChulalongkorn University (Bangkok, Thailand) and Walter Reed Army Institute ofResearch (WRAIR; Silver Spring, MD, USA) IRB approved clinical study. *p < 0.05; **p <0.005; ***p < 0.0005; ****p < 0.00005 (Kruskal-Wallis test)

STUDY PARTICIPANTS

Table 1. Clinical characterization of study participants.

Study Cohorts ART Number of participants

Median CD4+ T cells (cells/cu.mm.)

Median plasma HIV-RNA (copies/ml)

RV408 gr.1 wk.0 No 7374

(148-577)686,329

(3,385-2,830,592)

RV408 gr.1 wk.48 T+ART 7625

(274-954)20

(20-20)

RV408 gr.2 wk.0 No 4251

(231-457)582,561

(146,194-10,902,900)

RV408 gr.2 wk.48 ART 4833

(529-1,121)20

(20-20)

RV254 wk.0 No 24328

(158-794)1,379,268

(24,663-31,650,200)

RV254 wk.48 ART 12577

(336-795)20

(20-20)

RV304A HIV negative

9- -

RESULTSI. Alteration of the B cells in LN

Figure 1. B regulatory cells, resting memory B cells, tissue-likememory B cells and intermediate memory B cells of all participants(A) B cells gating strategy. Co-expression of CD21 and CD27 in IgG+ Bcells were used to identify the following four groups of memory B cells.B regulatory cells (Breg) were gated as CD38hiCD24hiCD19+. (B)Frequencies of Bregs were slightly increased during AHI in LNcompared to HIV negative but decreased after ART to lower levels thanin HIV negative except in the T+ART group. (C) Frequencies of restingmemory B cells (RM; CD21+CD27+IgG+CD19+) were not different inAHI compared to HIV negative but were decreased after ART in theRV408 ART and RV254 but maintained at similar levels as HIV negativein the T+ART group. (D, E) Frequencies of tissue-like memory B cells(TLM; CD21-CD27-IgG+CD19+) and intermediate memory B cells (IM;CD21+CD27-IgG+CD19+) were significantly lower in the T+ART groupcompared to the RV408 ART group as well as the RV254 group.

II. HIV Reservoir in LN

Figure 2. The HIV DNA reservoir level in LN (A, B) The total HIV DNA reservoir (A) and integrated proviral DNA (B) at each time point studied. Frequencies of total and integrated HIV DNA in AHI were evaluated in LNMC at AHI and decreased after ART. No differences were observed between the T+ART group and the placebo or RV254 group after ART.

A. B.

Disclaimers: The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the Department of Defense.

ACKNOWLEDGEMENTSWe are grateful for the RV408/SEARCH018, RV254/SEARCH010 and RV304/SEARCH013 participants; the staff at SEARCH Thailandand the Thai Red Cross AIDS Research Center; the Cellular Immunology Section and the Flow Core at U.S. MHRP and support throughthe International Neuro HIV Cure Consortium (INHCC net). This research was funded by the following sources: NIH grantsR01AI108433 and R01MH106466 (LT), and a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundationfor the Advancement of Military Medicine, Inc., and the U.S. Department of Defense (DoD) and the National Institutes of MentalHealth. Thai Pharmaceutical Organization, Merck, ViiV Healthcare and Gilead. The views expressed are those of the authors andshould not be construed to represent the positions of the U.S. Army or the Department of Defense.

IV. Collagen type 1 and TNFalpha in LN

B. C.

A.

Figure 4. Collagen1 and TNFaplpha in LN (A) 20x images of Collagen type 1 (brown) in the differentgroups. (B) Percentage of collagen1 were significantly increased during AHI and after treated ART withand without telmisartan compared to HIV negative. (B) TNFalpha per gram of tissue: no difference inLN between the groups.

HIV negative Telmisartan wk.0 Telmisartan wk.48 ART wk.0 ART wk.48

III. Proliferating and activated T cells in LN

Figure 3. Proliferating and activated CD4 and CD8 Tcells of all participants(A) CD8 T cells gating strategy. Proliferating T cellswere gated as Ki67+ Bcl2lo, activated T cells weregated as CD38+HLADR+ (B, C) Percentage ofproliferating CD4 (B) and CD8 (C) T cells at eachgroup. No difference was observed between the twogroups in proliferating CD4 and CD8 T cells in LN afterART. (D, E) Percentage of activated CD4 (D) and CD8(E) T cells at each group. No difference was observedbetween the two groups in activated CD4 and CD8 Tcells in LN after ART.

A.

0 103 104 105

<R780-A>: CD45RA

0

50K

100K

150K

200K

250K

FSC-

H

58.3

0 102 103 104 105

<B515-A>: Bcl-2

0

103

104

105

<UV3

79-A

>: K

i-67

5.73

81.3

CD45RA- Ki67+Bcl2lo

0 103 104 105

<UV515-A>: CD4 BUV515

0

103

104

105

<V78

0-A>

: CD8

BV7

85

63.9

25.2

0 103 104 105

<R730-A>: CD3 AF700

0

103

104

105

<V52

5-A>

: L/D

Aqu

a

71.2

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SSC-A

50.1

Lymphocytes CD3+ Live

CD3+CD4+

CD45RA

FSC

-H

Bcl2

Ki6

7

CD3+CD8+

B. C.

E.

D.

CD38+HLA-DR+

CD38

HLA

-DR

A.

B. C. D.

E.

0 103 104 105

<B710-A>: CD21 PerCP Cy5_5

0

103

104

105

<V45

0-A

>: C

D27

BV

421

1.16 17.3

801.49

RM

IMTLM

AM

CD21

CD

27

Memory B cell (IgG+)

0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SSC-A

40.7

Lymphocytes CD19+ B cells CD19+ Live

0 103 104 105

<R730-A>: CD3 AF700

0

103

104

105

<V78

0-A

>: C

D19

BV7

85

21.6

0 103 104 105

<V525-A>: L/D Aqua

0

103

104

105

<V78

0-A

>: C

D19

BV7

85

97.3

CD

24

0 103 104 105

<G575-A>: CD38PE

0

103

104

105

<R78

0-A>

: CD2

4 AP

C Cy

7

86.8 2.46

3.637.15

CD38

Regulatory B cell

- Addition of telmisartan to ART during AHI did not result in reduced T cell activation, decreased HIV reservoir or collagen deposition in LN.

- Telmisartan + ART induced higher resting memory B cells and Bregs in LN.

These data suggest that adjunctive therapy with telmisartan in AHI over 48 weeks may preserve immune cells in LN especially the B cell compartment.

CONCLUSIONS

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480

1

2

3

4

5

CD

38hi

CD

24hi

(Bre

g, %

)_LN

MC

*** **

*

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480

20

40

60

80

100

CD

21+C

D27

+IgG

+ C

D20

+(R

estin

g M

emor

y B ce

lls)_

LNM

C ***

*

HIV-

RV408_

w0

RV254_

w0

RV408_

T+ART_w48

RV408_

ART_w48

RV254_

ART_w48

0

5

10

15

20

CD

21-C

D27

-IgG

+ (T

issu

e Li

ke M

emor

y B

cells

)_LN

MC

***

****

* **

HIV-

RV408_

w0

RV254_

w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480

20

40

60

CD

21+C

D27

-IgG

+

(Inte

rmed

iate

mem

ory B

cells

)_LN

MC

* **

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w4810-1

100

101

102

103

104

105

Total

HIV

DNA

(c

opies

/106 LN

MC)

** ***

HIV-

RV408_

w0

RV254_

w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480.1

1

10

100

1000

Ki67

+BCl

2lo

CD45

RA-C

D4+

(%)_

LNM

C

******* *

*****

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480.1

1

10

100

1000

Ki67

+BCl

2loCD

45RA

-CD8

+ (%

)_LN

MC

********

*****

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480.01

0.1

1

10

100

1000

CD38

+HLA

-DR+

CD45

RA-C

D4+

(%)_

LNM

C

*************

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480.1

1

10

100

1000

CD38

+HLA

-DR+

CD45

RA-C

D8+

(%)_

LNM

C *********

***** ************ *

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480

10

20

30

40

50

Colla

gen1

(%)

****

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w480

2×107

4×107

6×107

8×107

TNF

alph

a (#

/gra

m)

HIV-

RV408_w0

RV254_w0

RV408_T+ART_w48

RV408_ART_w48

RV254_ART_w4810-1

100

101

102

103

104

105

Integ

rated

HIV

DNA

(c

opies

/106 LN

MC)

**