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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