Low Dose Mucosal SIV Infection Restricts Early Replication Kinetics and Transmitted Virus Variants in

Rhesus Monkeys

Jinyan Liu October 5, 2010

The Earliest Virologic Events Following Intrarectal SIV Infection in Rhesus Monkeys

• Defining the earliest virologic events following HIV-1 transmission may be critical for the design of vaccine strategies that block acquisition of HIV-1 infection

• A prophylactic vaccine must block infecting viruses in the mucosa to prevent systemic infection

• In particular, the length of the eclipse phase and the number of transmitted virus variants are critical virologic parameters that define the window in which a prophylactic vaccine must act

• However, studying the eclipse phase of HIV-1 infection in humans is very difficult

The Earliest Virologic Events Following Intrarectal SIV Infection in Rhesus Monkeys

• The RV144 study achieved 31% protection against acquisition of HIV-1 infection (Rerks-Ngarm NEJM 2009 361:2209)

• Although this study was not powered for subgroup analyses, the point estimate of protective efficacy appeared to differ by risk group– 40-48% protective efficacy in low and medium risk subjects– 4% protective efficacy in high risk subjects

• Is there a biologic mechanism by which a vaccine may protect better against low risk as compared with high risk transmission?

• Aim: To assess the impact of the virus inoculum dose on key virologic parameters following intrarectal SIV infection of rhesus monkeys

Study Outline

• 24 rhesus monkeys (N=6/group)

• Inoculated once IR with 1:1, 1:10, 1:100, or 1:1000 dilution (109, 108, 107, or 106 SIV RNA copies) of our SIVmac251 challenge stock

• Bled on days 0, 1, 2, 4, 7, 10, 14, 21, 28 for a fine resolution analysis of the eclipse and acute phases of infection

• Assessments:– Infection status – SIV RNA– Length of the eclipse phase, replication kinetics – SIV RNA– Numbers of transmitted/founder viruses – SGA sequencing– Innate immune responses – Luminex cytokine assays– Adaptive immune responses – ELISPOT, ICS, binding antibodies

Low Dose IR SIV Infection Reduces Infectivity but Does Not Impact SIV RNA Levels on Day 21

4

6

8

Lo

g S

IV R

NA

Day 21

Virus Dilution

0.0

0.2

0.4

0.6

0.8

1.0

1:1 1:10 1:100 1:1000

Infe

ctio

n R

ate

Virus Dilution

6/65/6 5/6

2/6

Low Dose IR SIV Infection Lengthens the Eclipse Phase (Time to First Positive Plasma SIV RNA)

2

4

6

8

0 5 10 15

Log

SIV

RN

A

Days Following SIV Infection

1:1

1:10

1:100

1:1000

Individual Monkeys

Log

SIV

RN

A

2

4

6

8

0 5 10 15

Log

SIV

RN

ADays Following SIV Infection

1:1

1:10

1:100

1:1000

Group Medians

Log

SIV

RN

A

Low Dose IR SIV Infection Lengthens the Eclipse Phase (Time to First Positive Plasma SIV RNA)

Virus Dilution Infectivity Eclipse Phase

1:1 6/6 (100%) 4 days

1:10 5/6 (83%) 4 days

1:100 5/6 (83%) 7 days*

1:1000 2/6 (33%) 8.5 days*

* Significantly longer median eclipse phase for lower dose groups as compared with higher dose groups (P = 0.01, Wilcoxon rank-sum test)

1:1 Dilution>10 Median T/F Viruses

1:10 Dilution>10 Median T/F Viruses

1:100 Dilution2 Median T/F Viruses

1:1000 Dilution1 Median T/F Viruses

Low Dose IR SIV Infection Restricts Transmitted/Founder Virus Variants

(B. Keele, H. Li, B. Hahn, G. Shaw)

Virus Dilution Infectivity T/F Variants

1:1 6/6 (100%) >10

1:10 5/6 (83%) >10

1:100 5/6 (83%) 2*

1:1000 2/6 (33%) 1*

* Significantly fewer median T/F virus variants for lower dose groups as compared with higher dose groups (P = 0.0002, Wilcoxon rank-sum test)

Low Dose IR SIV Infection Reduces Innate Serum Cytokine and Chemokine Levels on Day 10

(S. Keating, P. Norris)

0

10

20

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0 10 20 30

pg/m

l

0

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0 10 20 30

Days Following SIV Infection

0

1000

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3000

0 10 20 30

Days Following SIV Infection

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0 10 20 30

pg/m

l

Days Following SIV Infection

0

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20

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0 10 20 30

0

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100

150

0 10 20 30

IFN-γ IL-1Rα IL-15

IFN-α MCP-1 IL-18

1:1

1:10

1:100

1:1000

pg/m

lpg

/ml

Low Dose IR SIV Infection Does Not Impact Cellular Immune Responses but Reduces Central Memory CD4+ T Lymphocyte Depletion on Day 28

20

40

60

% C

D4

CM

Central Memory CD4 Cells

Virus Dilution

Uninfected monkey

0

5000

10000

15000

SFC

/ 10

^6 P

BM

C

Nef

PolEnvGag

1:1 1:10 1:100 1:1000

IFN-g ELISPOT

Virus Dilution

1

Low Dose IR SIV Infection Does Not Impact Humoral IgG Immune Responses

(G. Tomaras)

1

3

5

7

0 10 20 30 40 50 60 70

Log

Env

Tite

r

Days Following SIV Infection

1:1

1:10

1:100

1:1000

Env gp140

Log

ENV

Tite

r

1

3

5

7

0 10 20 30 40 50 60 70

Log

Gag

Tite

rDays Following SIV Infection

1:1

1:10

1:100

1:1000

Gag p55

Log

Gag

Tite

r

• Low dose IR SIV infection resulted in a longer eclipse phase, fewer transmitted/founder virus variants, and reduced innate immune activation as compared with high dose IR SIV infection

• These parameters may critically impact the capacity of a vaccine to block acquisition of infection because:– The eclipse period defines the window of time in which vaccine-

elicited immune responses must act– The number of transmitted/founder virus variants defines the

diversity of viruses that must be blocked

• It would therefore presumably be considerably easier for a vaccine to block a single transmitted virus in a longer timeframe than a swarm of genetically diverse viruses in a shorter timeframe

Conclusions

• High risk HIV-1 transmission in humans is characterized not only by increased frequency of virus exposure but also by a higher virus dose per exposure as well as increased transmitted/founder virus variants (Powers Lancet Infect Dis 2008 8:553; Boily Lancet Infect Dis 2009 9:118; Shaw 2010, Plos Pathogens in press)

• Our data therefore suggest a mechanism by which it may be considerably easier for a vaccine to protect against low risk as compared with high risk HIV-1 transmission

– For example, if vaccine-elicited antibodies could neutralize a fraction of viruses, then such a vaccine may be far more effective against a single virion than against multiple diverse virus variants

– These findings therefore have implications in the design and interpretation of HIV-1 vaccine efficacy studies in humans

Conclusions

• Beth Israel Deaconess Med Ctr, Harvard Medical School– Dan Barouch– Norman Letvin– Hualin Li

• SAIC Frederick, NCI– Brandon Keele

• University of Alabama– Beatrice Hahn– Hui Li– George Shaw

• New England Primate Res Ctr– Angela Carville– Keith Mansfield

• Blood Sys Res Institute, UCSF– Sheila Keating– Philip Norris

• Duke University– Bart Haynes– Georgia Tomaras

Acknowledgements