Epitope-Speciﬁc CD8 (cid:1) T Lymphocytes Cross-Recognize Mutant Simian Immunodeﬁciency Virus (SIV) Sequences but Fail To Contain Very Early Evolution and Eventual Fixation of Epitope Escape Mutations during SIV Infection (cid:1) †

Human immunodeﬁciency virus (HIV) and simian immunodeﬁciency virus (SIV) evade containment by CD8 (cid:1) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true represen- tation of the plasticity of these viruses or the intensity of CD8 (cid:1) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8 (cid:1) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8 (cid:1) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8 (cid:1) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-speciﬁc CD8 (cid:1) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-speciﬁc CD8 (cid:1) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and ﬁxation of the mutant epitope sequences in the viral quasispecies. (cid:1) T controlling (YSSGPGIRY) class I tetramer construction. To construct Mamu-A*02 tetrameric complexes with variant p199RY epitope peptides, a Mamu-A*02 fusion gene plasmid containing a small portion of the (cid:3) 3 domain of Mamu-A*01 (24) was modiﬁed to increase biotinylation efﬁciency. A 3-amino-acid linker between the BirA substrate peptide and the Mamu-A*02/A*01 fusion sequence was introduced by PCR mutagenesis using the Stratagene QuikChange II kit (Agilent Technologies) and the oligonucleotide primers pMN1insertF (5 (cid:4) -CCTGAAAT and pMN1insertR -CCAGAATATGATGCAGGGATCCCTGGGAAGACGGC according to the manufacturer’s directions. The ex- pressed protein was refolded in vitro with human (cid:5) 2 -microglobulin in the presence of WT or mutant p199RY epitope and the were biotinylated as previously Monomers streptavidin at to lymphocyte chronically infected with SIVmac251 were separated from EDTA-preserved whole blood by Ficoll density gradient centrifugation. PBMCs were incubated at 37°C in a 5% CO 2 environment in the presence of RPMI 1640–10% FCS and 1 (cid:6) g/ml of WT p199RY peptide for 10 days, receiving fresh interleukin-2 (IL-2) (Hoffman-LaRoche) at 20 U/ml on days 3, 5, 7, and 9 of culture. Live cells were separated