Ultradeep pyrosequencing detects complex patterns of CD8+ T-lymphocyte escape in simian immunodeficiency virus-infected macaques

Abstract

Human and simian immunodeficiency viruses (HIV/SIV) exhibit enormous sequence heterogeneity within each infected host. Here, we use ultradeep pyrosequencing to create a comprehensive picture of CD8(+) T-lymphocyte (CD8-TL) escape in SIV-infected macaques, revealing a previously undetected complex pattern of viral variants. This increased sensitivity enabled the detection of acute CD8-TL escape as early as 17 days postinfection, representing the earliest published example of CD8-TL escape in intrarectally infected macaques. These data demonstrate that pyrosequencing can be used to study the evolution of CD8-TL escape during immunodeficiency virus infection with an unprecedented degree of sensitivity.

FIG. 1.

Sequencing of Tat_28-35SL8 in rhesus macaques. The Tat_28-35SL8 regions from four Mamu-A1*001-positive rhesus macaques were sequenced using both pyrosequencing and conventional Sanger sequencing. (A) Consensus sequence from each sample with any change indicated as X. The pyrosequencing consensus (left) contains any mutation present in 1% or more of the total reads. Nonsynonymous and synonymous mutations are colored according to prevalence, indicated by the color key in the upper right. The consensus sequence obtained from conventional Sanger sequencing is displayed on the right. (B) Pyrosequencing consensus sequence from a Mamu-A1*001-negative animal, showing all mutations present at greater than 1%. (C) Percentage of each variant observed by pyrosequencing at day 21 within Tat_28-35SL8. Only variants present at 1% or greater in at least one animal are shown. All other variants are expressed as “Other.” Dashes indicate that a variant was not present in that sample. (D) Similar to panel C, showing variants at week 20.

FIG. 2.

Sequencing of Nef_103-111RM9 in MCMs. The Nef_103-111RM9 regions from four Mafa-A1*063-positive MCMs were sequenced using both pyrosequencing and conventional Sanger sequencing. A SIVmac239 inoculum was also sequenced by pyrosequencing. (A) Consensus sequence from each sample with any change indicated as X. The pyrosequencing consensus (left) contains any mutation present in 1% or more of total sequence reads. An average of 1,008 reads was obtained for each sample, with greater than 350 reads obtained from all samples except for cy0161 at day 17 (203 reads) and cy0162 at week 20 (237 reads). Nonsynonymous and synonymous mutations are colored according to prevalence, indicated by the color key in the upper right. The consensus sequence obtained from conventional Sanger sequencing is displayed on the right. (B) Percentage of each variant observed by pyrosequencing at day 17 within Nef_103-111RM9. Only variants present at 1% or greater in at least one animal are shown. All other variants are expressed as “Other.” Dashes indicate that a variant was not present in that sample. (C) Similar to panel B, showing variants at week 20.

FIG. 3.

Data represent the percentage of tetramer-positive cells within the CD3⁺ CD8⁺ cell gate. The limit of detection was 0.02% as determined by day 0 postinfection tetramer staining.

FIG. 4.

Sequencing of Gag_181-189CM9 in Indian rhesus macaques. The Gag_181-189CM9 region from four Mamu-A1*001-positive Indian rhesus macaques was sequenced by pyrosequencing. (A) Consensus sequence from each sample with any change indicated as X. The pyrosequencing consensus (left) contains any mutation present in 1% or more of total reads. Nonsynonymous and synonymous mutations are colored according to prevalence, indicated by the color key in the upper right. (B) Pyrosequencing consensus sequence from four Mamu-A1*001-negative animals and a SIVmac239 inoculum, showing all mutations present at greater than 1%. (C) Percentage of each variant observed by pyrosequencing at week 32 within Gag_181-189CM9. Only variants present at 1% or greater in at least one animal are shown. All other variants are expressed as “Other.” Dashes indicate that a variant was not present in that sample. (D) Similar to panel C, showing variants at week 57. (E) Table showing the linkage between Gag_181-189CM9 variants and known compensatory mutations at amino acid positions 161 and 206 for animal rh2122. (F) Similar to panel E, showing data for rh2127.