Comparative Viral Fitness Assessment of Congenic Mutations Within an Immunodominant CD8+
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Comparative Viral Fitness Assessment of Congenic Mutations Within an Immunodominant CD8+ T-cell Epitope of HIV. Natasha M. Christie 1 , David O. Willer 2,3 , Michael Lobritz 4 , Alan Cochrane 5 , Mark A. Luscher 2 , Eric J. Arts 4 , Kelly S. MacDonald 1,2,3.

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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

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

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

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

Objective

Investigate naturally occurring

variants of SL9 to determine if viral

replication is affected by amino acid

changes in this epitope that alter

immune recognition.


Natural sl9 variant evolution

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

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 panel

Variant Panel

shows decreased immune recognition


Experimental approach

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


1 department of immunology university of toronto

  • 3/4 variants replicated at wild-type levels

    • Kinetic delay in T8V variant (p<0.005)


1 department of immunology university of toronto

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


Conclusions

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

  • Viral fitnessreplicative capacity

    • Mono-infections not always representative

  • Moving forward with experiments in:

    • Primary cells

    • Competition assays


Interpretation

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

Acknowledgements

SupervisorsBiosafety Level 3 Lab

Dr. Kelly MacDonald Dr. Jun Liu

Dr. Mark Luscher Jennifer Biggs

Construction of SL9 MutationsArts Lab

Dr. David Willer Michael Lobritz

University of Toronto, HIV Program Labs Branch Lab

Dr. Rupert Kaul, Dr. Mario Ostrowski, Dr. Kelly MacDonald Nicole Lund

Funding


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