siv viral variation; implications for vaccines and transmission - mars stone phd

SIV Viral Variation; Implications for Vaccines and Transmission

Mars Stone, Ph.D.California National Primate Research Center

University of California, Davis

PART 1 Viral diversity at mucosal transmission

-determine if vaginal SIV inoculation of rhesus macaquesrecapitulates HIV-1 variant transmission

PART 2 Viral diversity in vaccine setting

-Characterize the replication levels and anatomic distribution of vaccine (SHIV 89.6) and challenge (SIVmac239) virus in

monkeys prior to and after challenge.

-Characterize evolution of SIV env population complexity of SIV DNA in PBMC of SHIV immunized and control animals.

Error in Reverse Transcriptionleads to Viral Population Complexity

Reverse transcriptase synthesizes viral DNA from viral RNA

Error rate of 1.7x105 nucleotide incorporations

Host RNA polymerase II transcribes the proviral DNA into RNA which will be packed into virions.

Mutation can occur during one or all of these replication steps

~1 error per replication cycle

RNA viruses exist as a quasispecies

Raul Andino, PLoS Pathog. 2010

Every round of replication mutations are generated, constantly introducing variation as

population expands.

Important findings:Studied 5 seroconverters, 2 linked transmission partners

• Homogeneous HIV env populations within newly infected patients

• No common signature sequence among transmitted variants

• Transmitted sequence represented only minor variant in complex population of chronically infected transmitting partner

They conclude that the transmitted virus should be the target of vaccines

3 proposed models of transmission bottleneck

1. Limited variability from transmitter

3. Selective amplification

2. Selective transmission

HomogeneousSystemic infection

Single Genome Amplification

Methods were developed to generate and sequence amplicons derived from a single template,

avoiding artifacts common to basic cloning and sequencing approach

Single Genome Amplification

• Proportional representation of variants

• Excludes PCR induced misincorporation error

• Eliminates PCR-mediated recombination

Why env?

• Env is primary determinant of cellular tropism and selective transmission would likely involve selection among env variants

• Is the most variable gene in the HIV quasispecies

Is the SIVenv variant population transmitted by vaginal inoculation

• Homogeneous?• Heterogeneous?

SIVmac251stock

intravaginal inoculation

7 animals

Plasma collected from earliest vRNA+

45 SGAs >20 SGAs / animal

Variant Transfer in Mucosal InfectionTest this observation in a controlled experiment using SGA techniques

Ma ZM, Stone M et al J Virol. 2009

Variant transmission is not related to inoculum dose

Stone, Keele et al JVI 2010

Purpose:

1. Determine the number and identity of SIVmac251 env variants in stock

2. Determine the number and identity of SIVmac251 env variants transmitted by vaginal inoculation

3. Determine if signature sequence is selected for by

vaginal inoculation

SIVmac251 Stock

Stone, Keele et al JVI 2010

Single transmission event

Fig 2A

Multiple Transmission Events

Fig 3

Composite NJ tree of

SIVmac251 stock and

transmitted variants

-No two low diversity lineages were identical

-each lineage distributed throughout the tree

Composite Highlighter plot of

SIVmac251variants and recipient variants

One 251 variant transmitted

unchanged to 3 different animals!

Variant transmission is not related to inoculum dose

Stone, Keele et al JVI 2010

Low dose

high dose

Number of founder variants in blood of infected animals

3 proposed models of transmission bottleneck

1. Limited variability from transmitter

3. Selective amplification

2. Selective transmission

Systemic infection

Complex inoculum!

HomogeneousSystemic infection

No common signature sequence

Model of Mucosal Infection (B. Keele)

Model of Early Diversification (B. Keele)

Conclusions 1:

-Rhesus macaque/SIV model of HIV-1 vaginal transmission recapitulates human infection.

• Relatively few genetic variants establish systemic infection even when exposed to complex inoculum

• A specific viral variant was not consistently transmitted by i.vag. Inoculations

PART 1 Viral diversity at mucosal transmission

-Recapitulate HIV-1 variant transmission in rhesus macaque model of vaginal SIV infection.

-Explore possible host factors affecting variant transmission

PART 2 Viral diversity in vaccine setting

-Characterize the replication levels and distribution of vaccine (SHIV 89.6) and challenge (SIVmac239) virus in monkeys prior to and after challenge.

-Characterize the population complexity of SIV in PBMC vDNA of SHIV immunized and control animals over time.

PART 2 Viral diversity in vaccine setting

• HIV is primarily transmitted mucosally, and a vaccine to prevent mucosal transmission is the best opportunity to stop the AIDS pandemic

• Live attenuated vaccines have demonstrated the best protection from pathogenic vaginal SIV challenge

• Live attenuated vaccines are not likely to be used due to safety concerns, but they do provide a good model to understand the nature of immune protection.

• In unprotected animals it is important to know if there are specific anatomic sites that are resistant to vaccine-induced immune control of challenge virus replication.

Immunization withnonpathogenic

SHIV89.6

IV

pathogenicSIVmac239

IVAG

6-12 month immunization period 6 month follow up period

Nx

Live-attenuated SHIV89.6 / IVAG SIVmac239 challenge

system

gag1303-1381

SIVenv7298-7389 (C3)

Primer Binding Sites for RT-PCR Viral Detection and Differentiation

HIVenv6955-7053

Intravaginal SIVmac239 challenge outcome in SHIV89.6 vaccinated female macaques

Prior SHIV89.6 infection “protects” 60% of rhesus monkeys from vaginal challenge with SIVmac239

Abel et al. J Virol, 2003

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Goals:

AIM 1• Characterize the replication levels and distribution of vaccine (SHIV

89.6) and challenge (SIVmac239) virus in monkeys prior to and after challenge.

AIM 2• Determine relationship between SIV population diversity and viral

replication in control animals and animals that eventually fail vaccine protection

Vaccine or Challenge virus?

In other attenuated lentivirus vaccine models it is unclear if “vaccine failure” is due to replication of the vaccine virus, the challenge virus,

or both

The Lancet

Experimental Design: Acute infection

3 days 7 days 14 days

vaccinationSHIV89.6

IV

pathogenicSIVmac239

IVAG6 months

Nx time points for 21 SIV controls

Nx time points for 30 SHIV-vaccinated animals

Stone et al, Virology 2009

Fig. 1

Fig. 1

Challenge outcomePlasma vRNA

7 days PC 14 days PC

Stone et al, Virology 2009

Fig. 1

Fig. 1

Challenge outcomePlasma vRNA

7 days PC 14 days PC

Which virus is where?Digging deeper

11 Tissues

-cervix-vagina (3)-Obturator LN-Inguinal LN-Iliac LN-Axillary LN-Spleen-Mesenteric LN-Colon

3 Targets

SIVgag

SIVenv

HIVenv

HIVenv vRNA levels in tissues

SHIV immunized animals

SIVenv vRNA levels in tissues

SHIV immunized animals

SIV control animals

Nx time point for 5 SHIV-immunized animals,

CD8 depleted animals

anti-CD8 (cM T807; 50mg/kg)

Experimental Design: Acute infection

3 days 7 days 14 days

vaccinationSHIV89.6

IV

pathogenicSIVmac239

IVAG6 months

Nx time points for 21 SIV controls

Nx time points for 30 SHIV-vaccinated animals

Genescà et al J Virology

What role do CD8+ play in vRNA levels and distribution?

CD8+ DepletedPlasma vRNA levels

after vaginal SIV challenge

SIVgag

SIVenv

Stone et al, Virology 2009

7 days PC 14 days PC

SIV replication in tissues 14 days post SIV challenge

Conclusions 2:

• Pathogenic challenge virus SIVmac239 is responsible for Vaccine failure– Although vaccine virus persists in some tissues, it is not responsible for vaccine failure in

this model.– No anatomic sites the immune system can’t reach to control SIV replication

• In vaccinated animals that control virus replication, dissemination of SIV beyond the genital lymph nodes is limited

• CD8+ depletion abrogates protective effect of SHIV immunization– There is increased SIV replication in CD8- SHIV vaccinated animals in the female genital

tract consistent with an increase in target cells

Goals:AIM 1• Characterize the replication levels and distribution of vaccine (SHIV

89.6) and challenge (SIVmac239) virus in monkeys prior to and after challenge.

AIM 2• Determine relationship between SIV population diversity and viral

replication in control animals and animals that eventually fail vaccine protection

IVAG SIVmac239 challenge outcome in SHIV89.6 vaccinated female macaques

Apply SGA methods to determine if increase in population diversity precedes increase in viral replication in animals that

eventually fail vaccine protection

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SHIV89.6 Vaccinated SIVmac239 challenged

Rhesus macaques

Why env?

• Env has appropriate properties of molecular biology and immunology for serving as a marker of genetic diversity

– Tolerates variability without change in biological properties

– There is no vaccine – induced immune pressure acting on env in immunized animals, vaccine and challenge virus are heterologous.

SIVmac239 stock

Early/late diversity in SIV infected animals

Early/late diversity in SIV infected animals

Early/late diversity in SHIV vaccinated SIV infected animals

Early/late diversity in SHIV vaccinated SIV infected animals

27578 30474

239

w9

Uncoupling of

replication and

diversity

239

w9

Viral load,but purifying

selection keeps diversity

Viral load,but lack of competition for target cells allows diversity

SIVenv genetic diversity and divergence in vaccinated and control rhesus macaques

A.

B.

Model of Mucosal Infection (B. Keele)

Model of Early Diversification (B. Keele)

Model of Mucosal Infection with Pre-existing Immune selection pressures (modified from B. Keele)

R0=1

CD8

CD8

CD8

CD8

CD8

CD8

CD8

CD8

CD8CD8

CD8

CD8

CD8

CD8

CD8

CD8

CD8

CD8

Conclusions 3:– Although plasma vRNA not detected by our assays, some replication must

be occurring to provide substrate that allows generation of breakthrough variants

– Competition between parental and mutant variants for target cells leads to purifying selection that accounts for relatively low levels of diversity in animals with high viral replication

– Conversely, lack of competition between parental and mutant variants for target cells in animals with low replication levels allows diversity to accumulate

Regardless of levels of replication, diversity increases over time in all animals ...so a vaccine must block transmission and prevent establishment of systemic infection after which the viral quasispecies becomes a complex moving target.

Thanks to:

Chris Miller

Mike McChesneyMeritxell GenescaZhong-Min Ma

Linda Fritts Vero deSilvaJoe Dutra

Ding LuTracy RourkeLili Guo

Primate Services Unit

NIH/NCIBrandon Keele

UABGeorge ShawBeatrice Hahn

University of NottinghamLiz Bailes

University of California-Davis