Transcript Transient Reversions in MHC

Dynamic immune responses maintain
cytotoxic T lymphocyte epitope
mutations in transmitted simian
immunodeficiency virus variants
Barouch et al.,2005
Nat. Imm. 6(3): 247-252
Hane Hiim, Rebecca Powell, Sacha Bhinder
The HIV (Retroviral) Lifecycle
Reverse Transcription
CD8+
CD4+
-Virus-specific CD8+ CTL response
critical for control of HIV-1 replication
in humans and SIV replication in
rhesus monkeys
-Stimulating this response with a
vaccine is a potential preventative
strategy
-Mamu-A*01-positive monkeys
vaccinated with plasmids encoding
epitopes of SIV proteins known to be
dominantly recognized by their
CTLs
-initially control viral replication
after challenge
-Long term studies show
breakthroughs of viral replication &
disease progression
-Correlates with SIV ‘escape’
mutations in these dominant epitopes
How genetically stable are these viral
variants?
Are these variants pathogenic?
Is there an evolutionary interplay
between viral fitness and immunological
pressure?
Infected naïve
Mamu-A*01-positive and
negative rhesus monkeys with SIV
variants
Assessing SIV Variant Pathogenicity
• Challenge with SIV
viral epitope variants
– Variants escape MamuA*01-restricted CTL
Gag p11C epitope
• Infect naïve monkeys
with three SIV
variants
Assessing SIV Variant Pathogenicity
• Infected naïve
monkeys with three
SIV variants
– p11C mutations: T182
to S, I, or A.
– Peak viral RNA 107108 copies/ml
• 7/9 monkeys died due to
disease progression
Plasma Viral RNA
Broad Cellular Response
Mamu-A*01 Positive
Env ELISPOT
Pol ELISPOT
Gag ELISPOT
Broad Humoral Response
Mamu-A*01 Positive
Gag ELISA Titers
SIV Neutralizing Ab Titer
Variable Gag-specific Cellular
Response
• Mamu-A*01 positive
monkey
– Gag-specific cellular
response showed
considerable variation.
– Variable despite stable
viral RNA
– Following 1º viremia
resolution
Gag ELISPOT
• Secondary peaks of Gagspecific cellular
Plasma Viral RNA
Variable Gag-specific Cellular
Response
• Mamu-A*01 negative monkey
• Stable Gag response
• Stable Env and Pol in positive and negative
Gag ELISPOT
Variable Gag-specific Cellular
Response
• Mamu-A*01 negative monkey
• Stable Gag response
• Stable Env and Pol in positive and negative
Env ELISPOT
Pol ELISPOT
Transient
Reversions
in
MHC-matched
Hosts
Method:
Identification & Quantification
of p11C-specific CD8+ T-cells
The MHC:peptide tetramer is made
from recombinant Mamu-A*01-p11C complexes,
bound to streptavidin via biotin, labeled with
phycoerythrin
T cell
Used in conjuction with FITC-labeled mAb to human CD8
and allophycocyanin-labeled mAb to rhesus monkey CD3
- 5 x 105 PBMCs from each monkey stained
- Analysed by FACS for triple stain
Figure 2
- No p11C-specific responses during acute infection (0-10 wks)
- Distinct but transient expansions of specific T-cells in 3/4 monkeys,
- Correlates with increased Gag-specific ELISPOT (fig 1)
Wild-type
Figure 2
- Data suggests that viruses containing wild-type p11C
epitope rapidly stimulated expansion of specific CTLs
=>Exerted immunological pressure on virus to reselect
SIV mutants
= escape CTL recognition
-p11C-specific CTL function confirmed by chromium-release
assay (data not shown)
-Magnitude of specific CTL response at 0.3-0.5% of cells;
- ~ 10% of typical response
- likely reflects transient p11C antigen stimulus
compared to innoculation with wild-type virus
Permanent
Reversions
In
MHC-mismatched
Hosts
Figure 3:
Percentage of viral clones with wild-type p11C sequences
Results demonstrate
fitness advantage of
wild-type SIV in the
absence of
immunological
selection pressure
(MHC mismatch)
Time to
reversion
suggests de
novo
mutations
(possible
wild-type
species in
innoculum)
Replicative Capacity in vitro of
Mutant SIV
• Assessed the
replicative capacity of
WT SIV and natural
mutant SIV variants in
the p11C epitope
• in vitro infection model
– SIV Gag (p27) by
ELISA
Replicative Capacity in vitro of
Mutant SIV
• Similar kinetics of
replication between
WT and natural
mutants
– Small mutation cost
does not result in
replication defect for
natural mutants
Replicative Capacity in vitro of
Mutant SIV
• Introduction of natural
SIV mutations into WT
SIV resulted in a
replicative defect in vitro
Replicative Capacity in vitro of
Mutant SIV
• Introduction of natural
SIV mutations into WT
SIV resulted in a
replicative defect in vitro
• Natural viral variants
display higher replication
rate than engineered
variants
– Engineered variants slower
in replication
Replicative Capacity in vitro of
Mutant SIV
• Limited replicative
capacity of initial CTL
epitope mutants
• Compensatory viral
mutation restore
replicative capacity.
– Great mutant shift vs.
transition to replicative
mutant
The Great Escape…
• Nature of SIV and HIV result in generation
of mutation
– Escape CTL detection through epitope
mutation
– Selective advantage for escapees results in
population-level dominance of epitope-mutant
variants
…but do they last
• Immunological selection results in a fine balance between viral
replicative potential and CTL avoidance
• Epitope mutants persist in MHC-matched hosts
– Reversion to WT transient in face of CTL expansion and
pressure for mutant selection
John & Mallal, Nature, 6(3), 2005
Expansion and Oscillation
• Maximize Fitness--Select for highest replication
rate with avoidance of a suppressive anti-wild-type
CTL response
– Transmission changes immunological constraints
John & Mallal, Nature, 6(3), 2005
From SIV to HIV
• HIV partially contained following infection—
limited success of antiviral response
• Mutation of epitopes provides an explanation for
viral escape from CTL generation
– Escape constrained by MHC diversity
• MHC diversity enhances CTL epitope specificity
and viral inhibition
• However, HIV has high rate of mutation, replication, and
adaptation
HIV Vaccine Constraints
• Ideally, vaccine epitopes cover all epitopes
for all population MHC genotypes
– Maximally restrict escapee generation
• Diversity of epitopes exploits MHC
diversity—counter HIV mutability and
adaptability