1-Department of Immunology, University of Toronto

Comparative Viral Fitness Assessment of Congenic Mutations Within an Immunodominant CD8+

T-cell Epitope of HIV

Natasha M. Christie1, David O. Willer2,3, Michael Lobritz4, Alan Cochrane5, Mark A. Luscher2, Eric J. Arts4, Kelly S. MacDonald1,2,3

1-Department of Immunology, University of Toronto 2-Department of Medicine, University of Toronto 3-Department of Microbiology, Mt. Sinai Hospital, Toronto 4-Department of Molecular Biology and Microbiology,Case Western Reserve University 5-Department of Medical Genetics, University of Toronto

CD8+ T-cell Epitope Escape

• Conflicting pressures on CD8+ T cell epitope sequences within the HIV-1 genome– Immune pressure Diversification– Viral Replication Conservation

• Understanding these pressures is crucial for best selection of epitopes during vaccine design

SLYNTVATL (SL9)

• Located in the matrix (p17) subunit of the Gag polyprotein– amino acid positions 77-85 in HIV genome

• Response is immunodominant and sequence is highly conserved – Restricted by HLA-A2

• Multiple amino acid changes required to decrease immune recognition– Iversen A et al., Nat. Immunol., 2006

Questions…

• In the face of such an immunodominant response, why is the epitope sequence conserved and CTL escape limited?

• Is the constraint of this epitope sequence dictated by viral replication requirements?

Objective

Investigate naturally occurringvariants of SL9 to determine if viral replication is affected by amino acid

changes in this epitope that alter immune recognition.

Natural SL9 Variant Evolution• Phylogenetic

evidence of stepwise accumulation of mutations in A2+ individuals

• Immunological evidence pointing to diminished recognition of triple mutant (Y3F/V6I/T8V)

Iversen A et al. Nat. Immunol. 2006

Construction of SL9 Mutants

• Focus on epitope variants defined as steps in the evolution of escape mutant sequences

• Congenic mutations made in plasmid backbone of pLAI.2 using overlapping PCR– Commonly utilized infections clone of IIIB

type virus; X4 tropism

Variant PanelWild type

pLAI.2

S L Y N T V A T L

TCA TTA TAT AAT ACA GTA GCA ACC CTC

Y3F - - F - - - - - -

TCA TTA TTC AAT ACA GTA GCA ACC CTC

T8V - - - - - - - V -

TCA TTG TAT AAT ACG GTC GCA GTC TTG

V6I/T8V - - - - - I - V -

TCA TTG TAT AAT ACG ATC GCA GTC TTG

Y3F/V6I/T8V - - F - - I - V -

TCA TTG TTC AAT ACG ATC GCA GTC TTG

shows decreased immune recognition

Experimental Approach• Plasmids transfected into 293T cells• Transfection supernatant passaged on

U87.CD4.CXCR4 cells

• TCID50 was determined using Karber method

• Mono-infection assays in CEM-T4 cells using equal Multiplicity of Infection(MOI) of virus

• p24 ELISA used to monitor viral replication

• 3/4 variants replicated at wild-type levels– Kinetic delay in T8V variant (p<0.005)

HIV-1 capsid protein (p24) production during CEM-T4 infection timecourse

(MOI 0.01)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

3 5 7 10 13 16

days post infection

p24 c

oncentr

ati

on(ug/m

L))

media

LAI

Y3F

T8V

V6I /T8V

Y3F/V6I /T8V

• Higher levels of infecting virus do not overcome T8V kinetic delay

HIV-1 capsid protein(p24) production during CEM-T4 infection timecourse (MOI 0.1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

3 5 7 10 13

days post infection

p2

4 c

on

cen

trati

on

(u

g/m

L)

media

LAI

Y3F

T8V

V6I /T8V

Y3F/V6I /T8V

Conclusions• Naturally occurring variants of SLYNTVATL

that diminish immune recognition do not necessarily correlate with decreased replicative capacity

• T8V variant delayed kinetically but replicates at wild-type levels later in time course

• No evident replication defect associated with Y3F/V6I/T8V variant

Viral Fitness

• Viral fitness replicative capacity– Mono-infections not always representative

• Moving forward with experiments in:1) Primary cells2) Competition assays

Interpretation

• Restriction of variation in this sequence is not solely dictated by viral requirements

• Balance of evolutionary pressures on this immunodominant epitope may be misunderstood– Conservation due to lack of immune pressure

rather than viral importance?

Acknowledgements

Supervisors Biosafety Level 3 LabDr. Kelly MacDonald Dr. Jun LiuDr. Mark Luscher Jennifer Biggs

Construction of SL9 Mutations Arts LabDr. David Willer Michael Lobritz

University of Toronto, HIV Program Labs Branch LabDr. Rupert Kaul, Dr. Mario Ostrowski, Dr. Kelly MacDonald Nicole Lund

Funding