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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