ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment

(1)

ALT-803 Transiently Reduces Simian Immunodeﬁciency Virus

Replication in the Absence of Antiretroviral Treatment

Amy L. Ellis-Connell,

a

Alexis J. Balgeman,

a

Katie R. Zarbock,

b

Gabrielle Barry,

b

Andrea Weiler,

b

Jack O. Egan,

c

Emily K. Jeng,

c

Thomas Friedrich,

b,d

Jeffrey S. Miller,

e

Ashley T. Haase,

f

Timothy W. Schacker,

g

Hing C. Wong,

c

Eva Rakasz,

b

Shelby L. O’Connor

a,b

a_{Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison,}

Wisconsin, USA

b_{Wisconsin National Primate Research Center, Madison, Wisconsin, USA}

c_{Altor BioScience Corporation, Miramar, Florida, USA}

d_{Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA}

e_{Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis,}

Minnesota, USA

f_{Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA}

g_{Division of Infectious Disease and International Medicine, University of Minnesota, Minneapolis,}

Minnesota, USA

ABSTRACT

Developing biological interventions to control human

immunodeﬁ-ciency virus (HIV) replication in the absence of antiretroviral therapy (ART) could

contribute to the development of a functional cure. As a potential alternative to

ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the

number and function of HIV-speciﬁc CD8

⫹

T and NK cell populations

in vitro

. Four

simian immunodeﬁciency virus (SIV)-positive rhesus macaques, three of whom

pos-sessed major histocompatibility complex alleles associated with control of SIV and all

of whom had received SIV vaccine vectors that had the potential to elicit CD8

⫹

T

cell responses, were given ALT-803 in three treatment cycles. The ﬁrst and second

cycles of treatment were separated by 2 weeks, while the third cycle was

adminis-tered after a 29-week break. ALT-803 transiently elevated the total CD8

⫹

effector

and central memory T cell and NK cell populations in peripheral blood, while viral

loads transiently decreased by

⬃

2 logs in all animals. Virus suppression was not

sus-tained as T cells became less responsive to ALT-803 and waned in numbers. No

ef-fect on viral loads was observed in the second cycle of ALT-803, concurrent with

downregulation of the IL-2/15 common

␥

C and

␤

chain receptors on both CD8

⫹

T

cells and NK cells. Furthermore, populations of immunosuppressive T cells increased

during the second cycle of ALT-803 treatment. During the third treatment cycle,

re-sponsiveness to ALT-803 was restored. CD8

⫹

T cells and NK cells increased again

3-to 5-fold, and viral loads transiently decreased again by 1 3-to 2 logs.

IMPORTANCE

Overall, our data show that ALT-803 has the potential to be used as

an immunomodulatory agent to elicit effective immune control of HIV/SIV

replica-tion. We identify mechanisms to explain why virus control is transient, so that this

model can be used to deﬁne a clinically appropriate treatment regimen.

KEYWORDS

ALT-803, ART-naive, IL-15 superagonist, SIV, SIV treatment, nonhuman

primate, virus

D

espite the ability of modern antiretroviral therapy (ART) to control human

immu-nodeﬁciency virus (HIV) infection, a cure remains elusive. Even with ART, the virus

establishes latent infection in resting CD4

⫹

T cells from which reactivation can occur.

Development of alternative biological interventions that harness the strength of the

Received4 October 2017Accepted6 November 2017

Accepted manuscript posted online8 November 2017

CitationEllis-Connell AL, Balgeman AJ, Zarbock KR, Barry G, Weiler A, Egan JO, Jeng EK, Friedrich T, Miller JS, Haase AT, Schacker TW, Wong HC, Rakasz E, O'Connor SL. 2018. ALT-803 transiently reduces simian

immunodeﬁciency virus replication in the absence of antiretroviral treatment. J Virol 92:e01748-17.https://doi.org/10.1128/JVI

.01748-17.

EditorViviana Simon, Icahn School of Medicine at Mount Sinai

crossm

on November 6, 2019 by guest

http://jvi.asm.org/

(2)

host immune system to control virus replication could contribute to the development

of a functional cure.

Even though ART treatment exhibits phenomenal success for HIV

⫹

patients,

effec-tive non-ART-based clinical approaches to enhance the effeceffec-tiveness of ART are not yet

available. Recently, there has been great interest in developing non-ART-based

thera-pies that can work alone or together with ART. One approach is to use prophylactic

vaccines to boost CD8

⫹

T cell responses, as these cells play a crucial role in the control

of virus replication (1–4). The need to enhance CD8

⫹

T cells during ART was recently

highlighted by Cartwright et al., who found that CD8

⫹

T cells were needed to control

virus replication in SIV-infected nonhuman primates being treated with ART (5).

There has been recent enthusiasm for approaches that increase the transcription of

viral genes from latent reservoirs while boosting both adaptive and innate immune cell

populations to eliminate the newly activated virus-infected cells. For example, both

Toll-like receptor 7 (TLR7) agonists and

␣

4

␤

7 integrin molecules have been used with

some success to promote virologic control in simian immunodeﬁciency virus

(SIV)-positive nonhuman primates (6, 7). While these therapies work by different

mecha-nisms, they demonstrate the enthusiasm for non-ART-based approaches that harness

host immunity to control HIV/SIV replication.

Interleukin-15 (IL-15) agonists, such as IL-15:IL-15R

␣

-Fc complexes, are another class

of compounds that may promote antiviral functions of the host immune response.

These agonists are broad activators of several innate and adaptive immune

popula-tions, in particular CD8

⫹

T cells and NK cells (8, 9). There is some evidence that IL-15

agonists also can act as latency-reversing agents and drive virus transcription in latently

infected CD4

⫹

T cells

in vitro

(10). Although treatment with IL-15 was unable to

suppress plasma viremia (11–14), using IL-15 agonists as molecular adjuvants to vaccine

vectors has led to some virologic beneﬁt (15, 16).

A novel IL-15 superagonist, ALT-803, recently has been shown to boost

in vitro

CD8

⫹

T cell and NK cell responses to HIV infection (10). ALT-803 is a soluble IL-15

superagonist complex (IL-15N72D:IL-15R

␣

SuFc) in which a mutant IL-15 (N72D) is

bound to the sushi domain of IL-15R

␣

fused to the Fc region of IgG1 (17–19).

ALT-803 promotes rapid expansion of CD8

⫹

T cell and NK cell populations

in vitro

in human donors and mice and

in vivo

in nonhuman primates (20). In contrast to

recombinant IL-15, ALT-803 has superior

in vivo

lymph tissue retention in mouse

biodistribution studies and more potent antitumor activity (20). Previous data of

ALT-803 in several mouse models of cancer suggest broad therapeutic applications

in hematologic and solid tumors (21–23). Furthermore, clinical trials evaluating

ALT-803 are under way for treatment of hematologic and solid cancers and HIV

(ClinicalTrials registration no. NCT02191098).

Given that ALT-803 has such a potentiating effect on cellular immunity, we tested

the hypothesis that ALT-803 modulation of cellular immunity could suppress SIV

replication in nonhuman primates. We treated ART-naive chronic-phase SIV

⫹

rhesus

macaques weekly with 0.1 mg/kg of body weight of ALT-803 subcutaneously for four

consecutive weeks. We observed a dramatic 1- to 2-log decline to levels below the limit

of detection in plasma viremia during the ﬁrst 7 to 14 days of treatment. The effect was

transient, such that virus loads rebounded concomitantly with IL-15 receptor

internal-ization and changes in the sequences of the virus population. Sensitivity to ALT-803

returned after the animals received a 29-week break from treatment. This study

provides evidence that treatment with the IL-15 superagonist ALT-803 can suppress SIV

replication in the absence of ART in nonhuman primates.

RESULTS

Initial subcutaneous ALT-803 treatment transiently reduces viremia of SIV

ⴙ

progressor rhesus macaques.

Four rhesus macaques infected with SIVmac239 for a

minimum of 1.5 years were selected for this study. All animals had been part of previous

studies and had received a variety of vector vaccines expressing SIV antigens that could

or could not elicit CD8

⫹

T cell responses (24). Even though they all initially controlled

February 2018 Volume 92 Issue 3 e01748-17 jvi.asm.org 2

on November 6, 2019 by guest

http://jvi.asm.org/

(3)

viremia to nearly undetectable levels (24), their plasma viral loads ranged from 10

3

to

10

4

viral copy equivalents (CEQ)/ml at the time of this study (Fig. 1A). Three of these

animals expressed

Mamu-B

*

08

and 1 animal expressed

Mamu-A

*

01

major

histocom-patibility complex (MHC) class I allele (Table 1).

To test the hypothesis that ALT-803 modulation of cellular immunity can suppress

SIV replication in nonhuman primates, we treated all animals subcutaneously with 0.1

mg/kg ALT-803 weekly for 4 weeks. This dose had been previously used to boost

cellular immunity in SIV-naive macaques with no adverse side effects (20). Plasma SIV

gag

viral loads were measured biweekly during treatment. During the ﬁrst 7 to 10 days

of ALT-803 treatment, we observed a precipitous decline in the SIV viral loads (Fig. 1A),

such that the viral loads in all 4 animals dropped below the limit of detection on day

10. Unfortunately, suppression of virus replication was transient and the duration was

variable across animals.

-28 -21 -14 -7 0 3 7 10 14 17 21 24 28 35 1

2 3 4 5

Days post ALT-803 treatment

Log

10

V

iral Copy

Equivalents/mL

(CEQ/mL)

-28 -21 -14 -7 0 7 14 21 28 35 0

500 1000 1500 2000 2500 3000 3500

T

otal cells/μL

blood

-28-21-14 -7 0 7 14 21 28 35 0

500 1000 1500 2000 2500

T

otal cells/μL

blood

Total CD4 T cells

Days post ALT-803 treatment

-28 -21 -14 -7 0 7 14 21 28 35 0

500 1000 1500

Total NK cells

T

otal cells/μL

blood

Days post ALT-803 treatment Days post ALT-803 treatment

Total CD8 T cells

Log10 Viral load

Log

10

CD8 T cell counts

R=-0.6668 p≤0.01

Log10 Viral load

Log

10

N

K

c

e

ll c

o

unt

s _R=-0.5436

p≤0.01

Log

10

CD4 T cell counts

Log10 Viral load R=-0.4346 p=0.04

r09089 r11021

r08054

r08016 =Day of ALT803 delivery

A

B

C

D

E

F

G

2 3 4 5

2.0 2.5 3.0 3.5

2 3 4 5

1.5 2.0 2.5 3.0 3.5

2 3 4 5

2.0 2.5 3.0 3.5

FIG 1ALT-803 treatment alters lymphocyte cell populations and viral loads in SIV⫹ _{macaques. (A) Log}

10 virus copy

equivalents/ml in plasma were measured as described in Materials and Methods for each animal. Arrowheads mark days on

which animals received ALT-803. (B and C) Total CD8⫹_{and CD4}⫹_{T lymphocyte populations per}_␮_{l of blood were determined}

as described in Materials and Methods using antibodies described in Table 4 and using complete blood counts determined for each time point. (D) Flow cytometry to measure NK cell populations present in PBMC was performed according to Materials and Methods using antibodies described in Table 2. The percentage of NK cells was determined as indicated in Materials and

Methods. Total NK cell counts per␮l of blood then were calculated based on complete blood counts for each time point. (E

to G) Pearson’s correlation coefﬁcients (R) were calculated as previously described in Materials and Methods by comparing the

log10viral loads/ml plasma to the log10total cell counts for CD8⫹(E), CD4⫹(F), and NK (G) cells.

on November 6, 2019 by guest

http://jvi.asm.org/

[image:3.585.45.442.75.488.2]

(4)

Increasing absolute number of CD8

ⴙ

T cells and NK cells correlates with

decreasing SIV viral loads.

Previously, ALT-803 was reported to increase the absolute

numbers of T cells and NK cells in healthy cynomolgus macaques (20), but this reagent

had not yet been studied in SIV

⫹

animals. We measured the absolute number of T cell

and NK cell populations prior to and during ALT-803 treatment in SIV

⫹

animals.

Consistent with data obtained from healthy animals (20), we observed an increase in

both CD8

⫹

T cells and NK cells after the ﬁrst dose of ALT-803 (Fig. 1B and D), but there

were no marked effects on the total number of CD4

⫹

T cells (Fig. 1C). We performed

Spearman’s correlation analyses to determine if the frequency of CD8

⫹

T cells or NK

cells correlated with changes in viral load. The absolute cell counts for CD8

⫹

, CD4

⫹

, and

NK cells, as well as the viral loads, were converted to log-scale numbers for each time

point before and during ALT-803 treatment. We observed a signiﬁcant negative

cor-relation between CD8

⫹

(

R

⫽ ⫺

0.67) and NK (

R

⫽ ⫺

0.54) cell numbers and viral load

(Fig. 1E and G, respectively). In contrast, the correlation observed between CD4

⫹

T cells

and viral load yielded a low spearman correlation

R

value (

R

⫽ ⫺

0.43; Fig. 1F) and

therefore was not signiﬁcant.

We further examined changes in the frequency of CD8

⫹

T, CD4

⫹

T, and NK cell

subpopulations (Fig. 2). Consistent with previous IL-15 and ALT-803 studies (20, 25), the

majority of the upregulated CD8

⫹

T cells had an effector memory (EM) phenotype (Fig.

2B), with minor increases in central memory (Fig. 2A) and naive (Fig. 2C) CD8

⫹

T cells

as well. For the NK cells, the majority of the upregulated cells consisted of CD16

⫹

cells,

with minor increases in both CD16

⫺

CD56

⫺

and CD56

⫹

populations (Fig. 2G to I). There

were no signiﬁcant changes in CD4

⫹

T cell subsets (Fig. 2D to F).

Sensitivity to ALT-803 returns after an extended break from treatment.

We

wanted to determine if suppression of SIV viremia would return in the presence of

ongoing ALT-803 treatment or if a break from ALT-803 was needed to restore sensitivity

to the drug. Three of the animals, two of which were

Mamu-B

*

08

⫹

and one of which

was

Mamu-A

*

01

⫹

, were included in this part of the study. The fourth animal had to be

removed for unrelated reasons but otherwise appeared healthy. These three animals

were given an additional 4-week course of ALT-803 immediately after the ﬁrst cycle of

treatment (deﬁned as cycle 2) (Fig. 3A), the animals were given a break for 29 weeks,

and then they received a ﬁnal 4-week course of ALT-803 (deﬁned as cycle 3) (Fig. 3A).

We found that there was no suppression of SIV viremia during cycle 2, but remarkably,

sensitivity to ALT-803 returned during cycle 3 and plasma SIV viral loads were again

suppressed, although less substantially than during cycle 1 (Fig. 3B).

CD8

ⴙ

T and NK cell numbers are still boosted during subsequent ALT-803

treatment cycles in the peripheral blood.

One possibility for the lack of suppression

of plasma viremia observed during cycle 2 of ALT-803 is that CD8

⫹

T cells and NK cells

did not expand in the peripheral blood. Therefore, we assessed whether CD8

⫹

T cell

and NK cell populations increased in the blood during cycles 2 and 3. While there were

still increased numbers of CD8

⫹

T cells (Fig. 3C) and NK cells (Fig. 3D) in the peripheral

blood relative to pretreatment levels, the increases were more modest than those we

observed during the initial ALT-803 treatment (Fig. 1). We then assessed the

relation-ship between the number of CD8

⫹

T cells and NK cells and plasma viral loads. We did

not observe a negative relationship between CD8

⫹

T cells or NK cells and viral load in

the second cycle of ALT-803 treatment (Fig. 3G and H), but the negative correlation

[image:4.585.45.544.83.147.2]

between CD8

⫹

T cells and viral loads was restored when ALT-803 treatment resumed

TABLE 1Animal information for animals involved in study

Animal

Age (at time of ALT-803), yr

Known MHC allele

expression Infecting virus

Dose (TCID50; IRa administration)

Years post-SIV infection (at time of ALT-803), yr

r11021 5 Mamu-A*01 SIVmac239 200 1.5

r08016 8 Mamu-B*08 SIVmac239 200 3

r08054 6 Mamu-B*08 SIVmac239 200 3

r09089 7 Mamu-B*08 SIVmac239 200 3

a_{IR, intrarectal.}

on November 6, 2019 by guest

http://jvi.asm.org/

(5)

after a 29-week break (cycle 3) (Fig. 3G). There were no other differences between the

number of naive or memory subpopulations of CD8

⫹

T cells or the cytotoxic or

cytokine-producing NK cells across the three treatment cycles (Fig. 4).

To determine if the increased numbers of peripheral CD8

⫹

T cells and NK cells were

due to redistribution from tissues or proliferation in the blood, we measured changes

in the population of cells expressing the proliferation marker ki-67. Relative to

pre-treatment controls, CD8

⫹

T cells and NK cells expressed signiﬁcantly higher ki-67 levels

during the ﬁrst cycle of ALT-803 (Fig. 3E and F), suggesting that these populations of

cells proliferated in the peripheral blood rather than redistributed from the tissues. This

increase was transient and consistent with previous ﬁndings that use other IL-15

agonists in macaques (11, 25). ki-67 expression was markedly attenuated during the

second cycle of ALT-803 for both cell populations, whereas transient upregulation of

ki-67 was restored for CD8

⫹

T cells but not for NK cells for the third cycle of ALT-803

treatment.

No signiﬁcant changes in peripheral CD4

ⴙ

T cells during ALT-803 treatment.

We explored the possibility that the number of CD4

⫹

T cells changed during additional

cycles of ALT-803 treatment, thus altering the number of target cells for SIV replication.

-28 -21 -14 -7 0 7 14 21 28 35 0

100 200 300 400 500

Days post ALT-803 treatment

T

otal cells/μL

blood

CD8 CM (CD28+CD95+)

-28 -21 -14 -7 0 7 14 21 28 35 0

500 1000 1500 2000 2500

T

o

tal cells/μL

blood

CD8 EM (CD28-CD95+)

-28 -21 -14 -7 0 7 14 21 28 35 0

100 200 300 400 500

Days post ALT-803 treatment CD8 Naїve (CD28+CD95-)

T

o

tal cells/μL

blood

-28 -21 -14 -7 0 7 14 21 28 35 0

200 400 600 800

r09089 r11021

r08054 r08016

CD4 CM (CD28+CD95+)

T

otal cells/μL

blood

-28 -21 -14 -7 0 7 14 21 28 35 0

50 100 150

T

o

tal cells/μL

blood

Days post ALT-803 treatment CD4 EM (CD28-CD95+)

-28 -21 -14 -7 0 7 14 21 28 35 0

500 1000 1500 2000

T

o

tal cells/μL

blood

Days post ALT-803 treatment CD4 Naїve (CD28+CD95-)

-28 -21 -14 -7 0 7 14 21 28 35 0

250 500 750 1000

-28 -21 -14 -7 0 7 14 21 28 35 0

20 40 60

-28 -21 -14 -7 0 7 14 21 28 35 0

100 200 300 400

T

otal cells/μL

blood

T

o

tal cells/μL

blood

T

o

tal cells/μL

blood

Days post ALT-803 treatment CD16+CD56- NK cells

CD16-CD56+ NK cells

CD16-CD56- NK cells

=Day of ALT803 delivery

A

B

C

D

E

F

G

H

I

FIG 2Assessment of memory CD4⫹_{and CD8}⫹_{T cell populations and NK cell populations during cycle 1 of ALT-803} treatment. (A to F) Fresh PBMC were isolated from whole-blood samples and were stained as described in Materials and

Methods and Table 4, and central memory (CM; CD28⫹_CD95⫹_CCR7⫹_{) (A and D), effector memory (EM; CD28}⫺_CD95⫹

CCR7⫺_{) (B and E), and naive (CD28}⫹_CD95⫺_CCR7⫹_{) (C and F) CD4}⫹_{and CD8}⫹_{T cells were deﬁned from the CD3}⫹_CD4⫹

and CD3⫹_CD8⫹_{parent populations, respectively. Total cell numbers were determined based on complete blood counts.}

(G to I) Fresh PBMC were isolated as described for panels A to F and were stained as indicated in Materials and Methods

and Table 2. CD16⫹_CD56⫺_{(G), CD56}⫹_CD16⫺_{(H), and CD16}⫺_CD56⫺_{(I) NK cells were deﬁned from the CD3}⫺_CD8⫹

NKG2A⫹_{parent population. Total cell numbers were determined based on complete blood counts.}

on November 6, 2019 by guest

http://jvi.asm.org/

[image:5.585.42.430.73.430.2]

(6)

Log

10

V

irus Copy

Equivalents/mL

(CEQ/mL)

Days post-ALT803 treatment

Tot

a

l CD8+ T c

e

lls

/μL blood

Tot

a

l NK c

e

lls

/μL blood

Days post-ALT803 treatment Days post-ALT803 treatment

r09089

r11021

r08016

=Day of ALT803 delivery

Log

10

CD8

+ T cells/μL

blood

Log10 Virus CEQ/mL

R=-0.33 p=N.S. R=-0.73 p≤0.01 R=0.55 p≤0.05 R=-0.13 p=N.S.

B

-8

4

-2

8

-2

1

-1

4 -7 0 3 7

10 14 17 21 24 28 31 35 38 42 45 49 52 56 59 63 70 77 84 96 110

224 231 238 245 252 259 261 266 269 273 276 280 283 287 294

1

2

3

4

5

-2 8 -2 1 -1

4 _-7 ₀ ₇

14 21 28 35 42 49 56 63 70 77 84

224 231 238 245 252 259 266 273 280 287 294

0 100 200 300 400 500 500 1000 1500 2000 2500 -2 8 -2 1 -1 4

-7 0 7 14 21 28 35 42 49 56 63 70 77 84 ₂₂₄ ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ ₂₉₄ 0

500 1000 1500

Cycle 2 Cycle 3

C

Pre

ALT-803

Cycle 1

Cycle 2

Cycle 3

Post ALT-803 2 week break 29 week break

A

Cycle 2 Cycle 3 Cycle 1 Cycle 2 Cycle 3

D

Fold change in

% ki-67

+CD8 + T cells

Fold change in % ki-67 +NK cells

* * * * *

Days post ALT-803 Days post ALT-803

-2 8 -2 1 -1 4

-7 0 7 14 21 28 35 42 49 56 63 70 77 84 ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ ₂₉₄

0 2 4 6 8 10 12 14 -2 8 -2 1 -1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇ ₈₄

231 238 245 252 259 266 273 280 287 0 5 10 15 20 25 294 Pre

treat Cycle 1 Cycle 2 Break Cycle 3 Post treat

Pre

treat Cycle 1 Cycle 2 Break Cycle 3 Post treat Pre

treat Cycle 1 Cycle 2 Break Cycle 3 Post treat Log 10 NK cells/μL blood

Log10 Virus CEQ/mL

E

F

G

H

2 3 4 5 2.0

2.5 3.0 3.5

2 3 4 5 2.0

2.5 3.0 3.5

2 3 4 5 1.5

2.0 2.5 3.0

2 3 4 5 1.5

2.0 2.5 3.0

FIG 3Subsequent cycles of ALT-803 treatment have a similar ability to boost cell populations but differential effects on viral loads. (A)

Schematic of ALT-803 treatment. Days of ALT-803 delivery are indicated with arrowheads. (B) Plasma viral loads (log10virus copy

equivalents/ml) determined at each time point. (C and D) Total CD8⫹_{cells (C) or NK cells (D) present in PBMC were determined as}

indicated in Materials and Methods and Fig. 1. Total CD8⫹_{T cells or NK cells per}_␮_{l of blood were calculated based on complete blood}

counts. (E and F) The ki-67⫹_{subpopulations of CD8}⫹_{T cells (CD3}⫹_CD8⫹_NKG2A⫺_{) (E) or NK cells (CD3}⫺_CD8⫹_NKG2A⫹_{) (F) from PBMC}

(Continued on next page)

on November 6, 2019 by guest

http://jvi.asm.org/

[image:6.585.42.477.69.684.2]

(7)

No signiﬁcant changes were observed in total CD4

⫹

T cells throughout the study (Fig.

5A). When we examined subpopulations of CD4

⫹

T cells, we found no signiﬁcant

changes in central memory (Fig. 5B), effector memory (Fig. 5C), or naive (Fig. 5D) CD4

⫹

T cell subpopulations during treatment with ALT-803.

Ex vivo

virus-speciﬁc CD8

ⴙ

T cell and NK cell function do not correlate with

ALT-803-dependent virus suppression.

Both CD8

⫹

T cells and NK cells play key roles

in control of SIV replication (26–28). Therefore, we wanted to determine if virus-speciﬁc

CD8

⫹

T cells exhibited an ALT-803-dependent increase in their capacity to produce the

cytokines gamma interferon (IFN-

␥

) and tumor necrosis factor alpha (TNF-

␣

) as well as

increased surface expression of CD107a as a marker of degranulation. We performed

peripheral blood mononuclear cell (PBMC) intracellular cytokine staining (ICS) assays at

several time points after ALT-803 treatment in the presence or absence of SIV peptides

FIG 3Legend (Continued)

were stained according to Table 2. The fold change in ki-67 relative to the pretreatment level was calculated for each time point. Student’s

unpairedttest analysis was performed as indicated in Materials and Methods; signiﬁcance relative to all pretreatment time points (*,Pⱕ

0.05;**,Pⱕ0.01) is shown. (G and H) Pearson correlation coefﬁcients (R) were calculated as indicated in Materials and Methods to

determine the relationship between plasma viral loads (log10CEQ/ml) and log10CD8⫹T cells/␮l plasma (G) or log10NK cells/␮l plasma

(H) during ALT-803 cycle 2 (left) and cycle 3 (right). N.S., not signiﬁcant.

-2

8

-2

1

-1

4 -7 0 7 14 21 28 35 42 49 56 63 70 77

224 231 238 245 252 259 266 273 280 287 294

0 100 200 300 400

-2

8

-2

1

-1

4 -7 0 7 14 21 28 35 42 49 56 63 70 77 ₂₂₄ ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ ₂₉₄

0 100 200 300 400 500 500 1000 1500 2000 2500

-2

8

-2

1

-1

4 -7 0 7 14 21 28 35 42 49 56 63 70 77 ₂₂₄ ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ ₂₉₄

0 50 100 150 200

-2

8

-2

1

-1

4

-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇

224 231 238 245 252 259 266 273 280 287 294

0 250 500 750 1000

-2

8

-2

1

-1

4 _-7 ₀ ₇

14 21 28 35 42 49 56 63 70 77 ₂₂₄ ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ ₂₉₄

0 20 40 60 80

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇

224 231 238 245 252 259 266 273 280 287 294

0 50 100 150 200 250

CM CD8+ T cells

T

o

tal cells/uL

blood

EM CD8+ T cells

T

o

tal cells/uL

blood

Naїve CD8+ T cells To

tal cells/uL

blood

CD16+CD56- NK cells T

o

tal cells/uL

blood

CD56+CD16- NK cells T

o

tal cells/uL

blood

CD16-CD56- NK cells To

tal cells/uL

blood

r09089 r11021

r08016 =Day of ALT-803 delivery

A

B

C

D

E

F

FIG 4Assessment of memory CD8⫹_{T cell populations and NK cell populations during cycles 1 to 3 of ALT-803 treatment. (A to C) Fresh PBMC}

were isolated from whole-blood samples and were stained as described in Materials and Methods and Table 4, and central memory (CM; CD28⫹

CD95⫹_CCR7⫹_{) (A), effector memory (EM; CD28}⫺_CD95⫹_CCR7⫺_{) (B), and naive (CD28}⫹_CD95⫺_CCR7⫹_{) (C) CD8}⫹_{T cells were deﬁned from the}

CD3⫹_CD8⫹_{parent population. Total cell numbers were determined based on complete blood counts. (D to F) Fresh PBMC were isolated as}

described for panels A to C and were stained as indicated in Materials and Methods and Table 2. CD16⫹_CD56⫺_{(D), CD56}⫹_CD16⫺_{(E), and CD16}⫺

CD56⫺_{(F) NK cell populations were deﬁned from the CD3}⫺_CD8⫹_NKG2A⫹_{parent population. Total cell numbers were determined based on}

complete blood counts.

on November 6, 2019 by guest

http://jvi.asm.org/

[image:7.585.43.501.77.429.2]

(8)

known to be presented by either

Mamu-B

*

08

(Env

573–581

KL9, Nef

137–146

RL10, and

Vif

123–131

RL9 [29–31]) or

Mamu-A

*

01

(Gag

181–189

CM9, Tat

28 –35

SL8, and Vif

100 –109

VL10

[32, 33]). The percentages of CD107a, TNF-

␣

, and IFN-

␥

⫹

CD8

⫹

T cells were measured

and normalized to a no-peptide control for each time point. We saw no consistent

evidence of increased CD8

⫹

T cell function during cycle 1, when there was the greatest

virus suppression (Fig. 6A to C). We observed occasional increases in CD8

⫹

T cell

function in individual animals that coincided with each ALT-803 treatment cycle (i.e.,

upregulation of CD107a in animal r11021 at day 7 compared to pretreatment for

Vif

100 –109

VL10 peptide); however, no statistically signiﬁcant common trend was

ob-served in cytokine or degranulation marker upregulation among all animals (data not

shown). These data suggest that there was no clear correlation between

in vivo

virus

suppression and the function of the peripheral CD8

⫹

T cells speciﬁc for the peptides

that we tested. It is possible that CD8

⫹

T cells targeting entirely different peptides

exhibited ALT-803-dependent increased function or that CD8

⫹

T cells in the tissues

were more effective, but we were unable to explore these possibilities in this study.

-2

8

-2

1

-1

4 _{-7 0 7 14 21 28 35 42 49 56 63 70 77}

224 231 238 245 252 259 266 273 280 287 294

0 200 400 600

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇

224 231 238 245 252 259 266 273 280 287 294

0 50 100 150

-2

8

-2

1

-1

4 _{-7 0 7 14 21 28 35 42 49 56 63 70 77}

224 231 238 245 252 259 266 273 280 287 294

0 200 400 600 800 1000

CM CD4+ T cells

T

o

tal cells/uL

blood

EM CD4+ T cells

T

o

tal cells/uL

blood

Naїve CD4+ T cells To

tal cells/uL

blood

B

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇

224 231 238 245 252 259 266 273 280 287 294

0 500 1000 1500

T

o

tal CD4+ T cells/uL

blood

A

C

D

r09089 r11021

r08016 =Day of ALT803 delivery Pre

FIG 5CD4⫹_{T cell populations are minimally altered during ALT-803 treatment. (A to D) Peripheral blood}

samples were stained as indicated in Table 4. Total CD4⫹_{T cells (A), central memory (CM) CD4}⫹_{T cells}

(CD28⫹_CD95⫹_CCR7⫹_{) (B), effector memory CD4}⫹_{T cells (CD28}⫺_CD95⫹_CCR7⫺_{) (C), and naive CD4}⫹_T

cells (CD28⫹_CD95⫺_CCR7⫹_{) (D) in the peripheral blood were determined as indicated in Materials and}

Methods and calculated based on complete blood counts.

on November 6, 2019 by guest

http://jvi.asm.org/

[image:8.585.98.316.70.480.2]

(9)

We also measured ALT-803 enhancement of NK cell function at the same time points

using a method described previously (34) (Fig. 6D). Although ALT-803 appeared to

transiently increase the function of NK cells, there was no statistically signiﬁcant

correlation between NK cell activity and virus suppression across the three cycles of

ALT-803 treatment.

The amino acid composition of a subset of CD8

ⴙ

T cell epitopes is altered by

ALT-803 treatment.

We wanted to determine if ALT-803-dependent expansion of

CD8

⫹

T cells preceded changes in the sequences of the replicating virus population. We

deep sequenced virus populations in duplicate from two of the

Mamu-B

*

08

⫹

animals

at two time points before and two time points after treatment with ALT-803. We

quantiﬁed the frequency of different amino acid sequences of epitopes that are

restricted by

Mamu-B

*

08

.

For animal r09089, we found that the composition of amino acid sequences in the

epitopes Nef

137–146

RL10 (Fig. 7A), Nef

8 –16

RL9 (Fig. 7B), and Vif

123–131

RL9 (Fig. 7C) were

different before and after ALT-803 treatment. For animal r08016, there were

ﬂuctua-tions in the frequencies of variants in Nef

8 –16

RL9 (Fig. 7F), and we detected a new

variant in the Vif

172–179

RL8 epitope (Fig. 7H) on day 56. The other epitopes for this

animal displayed no dramatic changes (Fig. 7E and G). Unfortunately, we cannot

determine if these changes in the virus populations were a consequence of latent virus

reactivation or selection of new virus populations, but it does indicate that perturbing

the homeostasis of the CD8

⫹

T cell population corresponded to an effect on the

sequences of the replicating virus population.

ALT-803 promotes upregulation of inhibitory markers CD39 and PD-1 on CD8

ⴙ

T cells and the development of regulatory CD4 T cells.

We next wanted to

deter-mine if there were changes in the phenotypes of CD8

⫹

T cell and NK cell populations

Fold Change in % CD107a

Fold change in %TNFα

Fold change in %

IF

Nγ

Days post ALT-803

A

B

r08016 r09089 r11021 -7 7 21 42 56 70 231 266 280 294 0 2 4 6 8 10 12 14

-7 7 21 42 56 70 231266 280 294 0 2 4 6 8 10

-7 7 21 42 56 70 231 266 280 294 0 2 4 6 8 10 12 14 -7 7 21 4256 70 231 266 280 287 294

0 2 4 6 8 10 12

-7 7 21 42 56 70 231 266 280 287 294 0

2 4 6 8

-7 7 21 42 56 70 231 266 280 287 294 0 2 4 6 8 10

-7 7 21 42 56 70 231 266 280 287 294 0.5 1.0 1.5 2.0 2.5 3.0

-7 7 21 42 56 70 231 266 280 287 294 0 2 4 6 8 10

-7 7 21 42 56 70 231 266 280 287 294 0 2 4 6 8 10 12 18 24

-7 7 21 42 56 70 231 266 280 287 294 0 1 2 3 4 5

-7 7 21 42 56 70 231 266 280 287 294 0 1 2 3 4 5 5 10 15 20 25

Gag181-189CM9 Tat28-35SL8 Vif100-109VL10 Env573-581KL9 Nef137-146RL10 Vif123-131RL9

Fold Change in % CD107a

IF Nγ Pre-Treat Cycle 1 Day: 7 21 Cycle 2 Day: 7 21 Break Cycle 3 Day: 7 21 Post-treat

r11021 CD8+_{T cells}

Pre-Treat Cycle 1 Day: 7 21 Cycle 2 Day: 7 21 Break

Cycle 3 Day: 7 21

Post-treat

r08016 CD8+_{T cells}

Pre-Treat Cycle 1 Day: 7 21 Cycle 2 Day: 7 21 Break Cycle 3 Day: 7 21 Post-treat

r09089 CD8+_{T cells}

F

old C

h

a

n

ge in % C

D107a

IF

Nγ

Pre-Treat Cycle 1 Day: 7 21 Cycle 2 Day: 7 21

Break Cycle 3Day: 7 21

Post-treat

Days post ALT-803

Fold Change in % CD

107a

IF

Nγ

C

D

NK cells

Env573-581KL9 Nef137-146RL10 Vif123-131RL9

FIG 6CD8⫹_{T cell responsiveness to peptide in ICS assays is similar in all three cycles of ALT-803 treatment. (A to C) Frozen PBMC were rested overnight, and}

ICS assays were performed as described in Materials and Methods using the indicated A*01-restricted (Gag181–189CM9, Tat28 –35SL8, and Vif100 –109VL10; animal

r11021) (A) or B*08-restricted (Env573–581KL9, Nef137–146RL10, Vif123–131RL9; animals r08016 and r09089) (B and C) peptides or a no-peptide control. Cells were

stained as shown in Table 6, and live CD8⫹_{T cells were deﬁned as CD3}⫹_CD8⫹_CD4⫺_{. Within the CD8}⫹_{T cell gate, the percentages of CD107a}⫹_{(top), TNF-}_␣⫹

(middle), and IFN-␥⫹_{(bottom) were determined individually. The data then were normalized to the no-peptide control for each individual time point. (D) Fresh}

PBMC were used to perform ICS assays as indicated in Materials and Methods. Cells were stained with antibodies according to Table 3. NK cells were deﬁned

as CD3⫺_CD8⫹_NKG2A⫹_{. The percentages of CD107a}⫹_{(top), TNF-}_␣⫹_{(middle), and IFN-}_␥⫹_{(bottom) cells were determined and normalized to a control sample}

(no. 721.221 cells).

on November 6, 2019 by guest

http://jvi.asm.org/

[image:9.585.47.544.73.338.2]

(10)

that may have had an effect on their

in vivo

function. We measured activation (HLA-DR,

CD25, and CD69) and inhibitory/exhaustion (CD39 and PD-1) markers present on CD4

⫹

and CD8

⫹

T cell memory populations and NK populations throughout all three ALT-803

treatment cycles by ﬂow cytometry.

We detected notable changes in the expression of CD39 on CD8

⫹

T cells during the

course of treatment. CD39 is an ectonucleotidase that hydrolyzes extracellular ATP (35),

and CD4

⫹

and CD8

⫹

T cells expressing CD39 can inhibit cytokine production from

[image:10.585.43.440.70.574.2]

Day -21 V a ri a nt Fr e que nc y E... G... RRHRILDIYL K... Other

Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 0.0 0.2 0.4 0.6 0.8 1.0

Day -7 Day 35 Day 56

0.0 0.2 0.4 0.6 0.8 1.0 V ari an t F req u en cy RRHRILDIYL ...T.. Day -21

Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Day -7 Day 35 Day 56

0.0 0.2 0.4 0.6 0.8 1.0 ...K..R.. Other Day -21

0.0 0.2 0.4 0.6 0.8 1.0 Day -21

Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Day -7 Day 35 Day 56 0.0 0.2 0.4 0.6 0.8 1.0 Day -21

0.0 0.2 0.4 0.6 0.8 1.0 Day -21

Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Day -7 Day 35 Day 56 0.0 0.2 0.4 0.6 0.8 1.0 Day -21

V ari an t F req u en c y V ari an t F req u en cy V a ri an t F req u en cy V a ri an t F req u e n cy V ari an t F req u e n c y V ari an t F req u e n c y RRAIRGEQL ..V... ..V....R. RRSRPSGDL ...R. ...K... ...R.. RRDNRRGL .G... KG... G... Other RRDNRRGL ...G.. ...GS. Other RRAIRGEQL ..V... K.V... ..V...Q Other ..V.K.... RRSRPSGDL K... .Q... K..K... ...K... .Q.K... ...KQ.... Other ...T.. K..G...T.. E..K...T.. ...T...T.. ...A...T..

Nef_137-146RL10 Nef_137-146RL10

R09089 R08016

H

G

F

E

A

B

C

D

Nef8-16RL9 Nef8-16RL9

Vif123-131RL9 Vif123-131RL9

Vif_172-179RL8 Vif_172-179RL8

FIG 7Distinct viral variants appear in severalMamu-B*08-restricted epitopes during ALT-803 treatment. (A) Deep-sequencing analysis was performed, as described in Materials and Methods, on plasma samples from animals r09089 and r08016. The time

points shown are relative to the ﬁrst ALT-803 treatment. Viral variant frequencies were determined for Nef137–146RL10 (A and

E), Nef8 –16RL9 (B and F), Vif123–131RL9 (C and G), and Vif172–179RL8 (D and H). Amino acid variation relative to the wild-type

sequence for each epitope is indicated by letters; amino acids that were the same as those of the wild-type sequence are shown as dots.

on November 6, 2019 by guest

http://jvi.asm.org/

(11)

CD39-negative T cells (36–38). High surface expression of CD39 has been linked to

terminal exhaustion of CD8

⫹

T cells in chronic HIV infection (39). We found that CD39

⫹

central memory CD8

⫹

T cells increased most prominently during the second cycle of

ALT-803, whereas effector memory CD8

⫹

T cells increased over the baseline during

each cycle of ALT-803 treatment (Fig. 8A and B). While no signiﬁcant changes were

observed in overall PD-1 expression in either effector memory or central memory CD8

⫹

T cells (data not shown), we did not ﬁnd many changes in CD39/PD-1 double-positive

cells in the central memory CD8

⫹

T cell subpopulations (Fig. 8C); we found signiﬁcant

increases in the effector memory CD8

⫹

T cell populations that coexpressed both CD39

and PD-1 (Fig. 8D). This double-positive population was statistically signiﬁcant during

the second cycle of ALT-803 treatment, coincident with reduced effect of ALT-803 on

viral loads (Fig. 3B). The expression of these markers alone is not necessarily a hallmark

*** ****

**

*

Fold change in

% CD4

+CD25 +CD39 + cells

Fold change in

% CD39

+CD8 +CM cells

Fold change in

% CD39

+CD8 +EM cells

Fold change in

% PD1

+CD39 +CM cells

Fold change in

% PD1

+CD39 +EM cells *

* * * *

*

** ***

**** **

*

*** *

* ****

*

Days post ALT-803 Days post ALT-803

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇ ₈₄

231 238 245 252 259 266 273 280 287 0

2 4 6 8 10

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇

224 231 238 245 252 259 266 273 280 287 294 0

2 4 6 8 10

-2

8

-2

1

-1

4 _-7 ₀ ₇

14 21 28 35 42 49 56 63 70 77 84 ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ 0

2 4 6 8 10 12

-2

8

-2

1

-1

4

-7 0 7 14 21 28 35 42 49 56 63 70 77 84 ₂₃₁ ₂₃₈ ₂₄₅ ₂₅₂ ₂₅₉ ₂₆₆ ₂₇₃ ₂₈₀ ₂₈₇ 0

2 4 6 8 10 12

-2

8

-2

1

-1

4 _-7 0 7 ₁₄ ₂₁ ₂₈ ₃₅ ₄₂ ₄₉ ₅₆ ₆₃ ₇₀ ₇₇ ₈₄

231 238 245 252 259 266 273 280 287 0

1 2 3 4 5

294

294 294

294

A

B

C

D

E

Pre

treat Cycle 1 Cycle 2 Break Cycle 3 Post treat

Pre

treat Cycle 1 Cycle 2 Break Cycle 3 Post treat Pre

FIG 8ALT-803 increases CD39⫹_CD8⫹_{effector memory and central memory T cells and regulatory T cells (T}

regs) (A and B) CD39⫹cells were determined for the

parent central memory (CM; CD3⫹_CD8⫹_CD28⫹_CD95⫹_CCR7⫹_{) and effector memory (EM; CD3}⫹_CD8⫹_CD28⫹_CD95⫹_CCR7⫺_{) CD8}⫹_{T cell populations as}

described in Materials and Methods and Table 4. The fold change in CD39 expression was determined relative to the average of the pretreatment time points.

Student’s unpairedttest analysis was performed as described in Materials and Methods; signiﬁcance relative to all pretreatment time points (*,Pⱕ0.05;**,

Pⱕ0.01) is shown. (C and D) PD-1⫹_{cell levels were determined for each of the CD39}⫹_{CM and EM CD8}⫹_{T cell parent populations. The fold changes in PD-1}⫹

CD39⫹_{for CM and EM CD8}⫹_{T cells were calculated as described for panels A and B. Statistics were performed as described for panels A and B. (E) T}

regswere

deﬁned as CD45⫹_CD3⫹_CD4⫹_{cells that were both CD25 and CD39 positive. The fold change in T}

regswas determined as described for panels A to D. Statistics

were performed as indicated for panels A and B.

on November 6, 2019 by guest

http://jvi.asm.org/

[image:11.585.41.550.75.489.2]

(12)

of immune exhaustion (40). Furthermore, we did not measure peptide-speciﬁc

stimu-lation from this double-positive popustimu-lation of cells; therefore, the CD39

⫹

PD-1

⫹

cells

cannot deﬁnitively be declared exhausted. However, these data suggest that

continu-ous ALT-803 treatment does lead to an increase in a population of CD8

⫹

T cells that

express regulatory markers and inhibit

in vivo

cytokine production.

We additionally observed increases in CD39

⫹

CD25

⫹

CD4

⫹

T cells in all three cycles

of treatment (Fig. 8E). The coexpression of CD39 with CD25 on CD4

⫹

or CD8

⫹

T cells

is indicative of a T regulatory (Treg) phenotype (36, 41, 42). Thus, continuous ALT-803

treatment boosted the frequencies of regulatory T cells.

Surface expression of the IL-15 receptor declines during the second cycle of

ALT-803 treatment.

ALT-803-dependent cell activation requires that the modiﬁed

IL-15 of the ALT-803 molecule interacts with the IL-15/IL-2

␤

(CD122) and the common

␥

C (CD132) receptors present on target cells. Modulation of the surface expression of

these receptors has been observed in other

in vivo

IL-15 treatment models (25). To

determine if ALT-803 modulated surface expression of the IL-15 receptor, we used ﬂow

cytometry to measure the mean ﬂuorescence intensity (MFI) of these two receptors

during treatment. We found a signiﬁcant increase in surface expression of CD122 (as

indicated by MFI) on CD8

⫹

EM T cells at days 7 and 10 after the ﬁrst ALT-803 treatment

(Fig. 9A, left). However, the surface expression of CD122 returned to pretreatment levels

with continued ALT-803 treatment. We found that the surface expression of CD122 did

not increase during the second phase of treatment but once again transiently increased

during the ﬁrst 7 days of the third cycle of ALT-803 treatment (Fig. 9A, left). We did not

observe any signiﬁcant changes in the expression of CD132 during ALT-803 treatment

in the CD8

⫹

EM T cell population (Fig. 9A, right).

In contrast, we found that CD8

⫹

CM T cells exhibited a transient decline in the surface

expression of both CD122 (Fig. 9B, left) and CD132 (Fig. 9B, right) during the second phase

of ALT-803 treatment. In addition, NK cells exhibited a decline in the expression of CD122

during the second phase of ALT-803 treatment (Fig. 9C).

Overall, the changes in the expression of these receptors are consistent with the

hypothesis that memory CD8

⫹

T cells and NK cells initially respond to ALT-803 but then

surface expression of the receptor declines, likely translating to reduced responsiveness

of these cell populations over time.

DISCUSSION

In this study, we show that treatment of SIV

⫹

animals with the IL-15 superagonist

ALT-803 can suppress virus replication to undetectable levels in the absence of ART. We

selected four SIV

⫹

rhesus macaques who had been infected for more than 1.5 years and

who had plasma viremia ranging from 10

3

to 10

4

copies/ml. Within the ﬁrst 2 weeks

after the initial treatment with ALT-803, SIV plasma viremia reached undetectable levels

in all four animals. Virus suppression was coincident with increased peripheral CD8

⫹

T

cell and NK cell counts. Virus suppression, however, was only transient. Even with ongoing

ALT-803 treatment, plasma viremia rebounded. Remarkably, ALT-803-dependent virus

suppression was partially restored in the three animals that resumed ALT-803 treatment

after a break of 29 weeks. These data together suggest that the host immune response

can be modulated by ALT-803 to control SIV replication in chronically infected

ma-caques, even without ART.

Unfortunately, the speciﬁc mechanism of virus suppression remains unknown. Our

data suggest that the expansion of immune cells in the peripheral blood played a key

role in this suppression. We found that the frequencies of CD8

⫹

T cells and NK cells

were inversely correlated with plasma viremia during the treatment cycles when

animals responded to ALT-803. We did observe signiﬁcant increases in the population

of ki-67

⫹

CD8

⫹

T cells and NK cells in the ﬁrst cycle and then a signiﬁcant increase in

ki-67

⫹

CD8

⫹

T cells during the third cycle of ALT-803 (Fig. 4), suggesting that increased

T cell proliferation in the blood was a factor in improving antiviral immunity. We also

measured the function of SIV-speciﬁc CD8

⫹

T cells by intracellular cytokine assays, but

there was no correlation between increased epitope-speciﬁc cytokine production and

on November 6, 2019 by guest

http://jvi.asm.org/

(13)

suppression of plasma SIV viremia. Additional studies will need to be performed to

elucidate the immunological mechanism explaining how ALT-803 reduced viral loads in

the ﬁrst and third treatment cycles, so that altered treatment regimens can be

identi-ﬁed to sustain virus suppression for a longer period of time.

For an anti-HIV immunomodulatory therapeutic to be effective, it will need to

stimulate host immunity to continuously suppress viremia for a long period of time,

even though mechanisms exist to prevent overactivation of the host immune system.

We chose animals who were ART naive to assess whether immunomodulation, without

ART, could induce and maintain virus suppression in these speciﬁc animals. Not

surprisingly, suppression of virus replication was only transient, as these animals

employed mechanisms to prevent overstimulation. These mechanisms included

down-regulation of the IL-15 receptors (CD122 and CD132) on target cells. We found that

Days post ALT-803 treatment

CD8

+EM

MFI CD122

Days post ALT-803 treatment

CD8

+CM

MFI CD122

Days post ALT-803 treatment

CD8

+ CM

MFI CD132

Days post ALT-803 treatment

NK cells

MFI CD122

Days post ALT-803 treatment * * * * * * * *

A

B

C

-100 -2 1 _-7 7

10 21 35 42 56 70 ₁₀₀ ₂₃₁ ₂₆₆ ₂₈₀ ₂₈₇ ₂₉₄ 150 200 250 300 350 400 450 500 550 -100 -2 1

-7 7 10 21 35 42 56 70 77

100 231 266 280 287 294 200 250 300 350 400 450 500 -100 -2

1 _-7 7 ₁₀ ₂₁ ₃₅ ₄₂ ₅₆ ₇₀ ₇₇

100 231 266 280 287 294 500

1000 1500

-100 -2

1 _-7 7 ₁₀ ₂₁ ₃₅ ₄₂ ₅₆ ₇₀ ₇₇

100 231 266 280 287 294 400

800 1200 1600

-100 -2 1 _-7 7

10 21 35 42 56 70 77 ₁₀₀ ₂₃₁ ₂₆₆ ₂₈₀ ₂₈₇ ₂₉₄ 1000 1200 1400 1600 1800 2000 -100 -2 1 _-7 7

10 21 35 42 56 70 77 ₁₀₀ ₂₃₁ ₂₆₆ ₂₈₀ ₂₈₇ ₂₉₄ 500 1000 1500 2000 Cycle 1 Day:

7 10 21

Cycle 2 Day:

0 7 21