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. 2013 Aug;87(16):9353-64.
doi: 10.1128/JVI.00909-13. Epub 2013 Jun 19.

Acute-phase CD8 T Cell Responses That Select for Escape Variants Are Needed to Control Live Attenuated Simian Immunodeficiency Virus

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Free PMC article

Acute-phase CD8 T Cell Responses That Select for Escape Variants Are Needed to Control Live Attenuated Simian Immunodeficiency Virus

Max Harris et al. J Virol. .
Free PMC article

Abstract

The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select for sequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escape variants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected with m3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔnef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during early m3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These results provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase of infection are needed to establish viral control in vivo.

Figures

Fig 1
Fig 1
Amino acid sequence differences between SIVmac239 and m3KOΔnef. Twenty-three nucleotide mutations that affected 24 amino acid sites were engineered into SIVmac239Δnef to create m3KOΔnef. The SIV proteins and the amino acid positions affected are shown. Dots represent identity, and capital letters represent amino acid differences. The underlined letter in light gray indicates a nucleotide change that was synonymous with respect to that specific protein. (A) Substitutions engineered into epitopes restricted by MHC class I molecules encoded by the M3 MHC haplotype are shown. (B) Additional substitutions outside known epitopes are shown.
Fig 2
Fig 2
Virus replication is not controlled in M3 homozygous MCMs infected with m3KOΔnef. Viral loads were measured for 20 weeks after infection of the following groups: M3 homozygous MCMs infected with m3KOΔnef (blue), MHC-mismatched MCMs infected with m3KOΔnef (green), and MHC-diverse MCMs infected with SIVmac239Δnef (black). All MHC genotypes are shown in Table 1. Log10 viral load trajectories are shown for each individual (thin lines) and each group (smoothed, heavy lines). Viral load set point estimates and 95% confidence intervals for each group are represented by short vertical bars (see Table 2 for statistics). The tall vertical black line is a reference point at 14 weeks after infection.
Fig 3
Fig 3
CD4 T cell counts in MCMs infected with m3KOΔnef. PBMCs were stained during the first 20 weeks after infection with m3KOΔnef with anti-CD3, -CD4, -CD8, -CD95, and -CD28 antibodies. Samples were analyzed by flow cytometry. Absolute counts are shown for the whole CD4 T cell compartment and various CD4 T cell subsets, as indicated.
Fig 4
Fig 4
CD8 T cell responses targeting disrupted epitopes are absent during the acute phase of infection of M3 homozygous MCMs with m3KOΔnef. IFN-γ ELISPOT assays were performed with PBMCs from M3 homozygous MCMs at 3 weeks after infection with m3KOΔnef to measure T cell responses targeting 12 SIV-derived peptide epitopes that are restricted by M3 MHC class I molecules. Samples were plated in duplicate, and the average numbers of SFCs per 106 PBMCs are shown. The Nef194-203LW10 peptide contains the Nef196-203HW8 epitope. ConA, concanavalin A; Stim, stimulation.
Fig 5
Fig 5
CD8 T cells detected in M3 homozygous MCMs during the acute m3KOΔnef infection phase target different epitopes compared to the acute phase of SIVmac239 infection. MHC/peptide tetramers presenting four SIVmac239-derived epitopes were used to quantify the percentage of antigen-specific CD3+ CD8+ T cells in lymph nodes at 3 weeks after infection with the indicated viruses. The specificities examined were Env338-346RF9 (A), Tat42-49QA8 (B), ARF130-40QL11 (C), and Rev59-68SP10 (D). Each point represents a different animal. The monkeys with blue vests represent M3 homozygous MCMs; the monkeys with green-and-white vests represent MHC-mismatched MCMs. Unpaired t tests of the log-transformed data were performed, and the P values are shown.
Fig 6
Fig 6
Accumulation of sequence variants in Env338-346RF9 is more rapid in M3 homozygous MCMs infected with m3KOΔnef than in those infected with SIVmac239. Viruses replicating in M3 homozygous MCMs at 3 weeks after m3KOΔnef infection (A), 12 weeks after m3KOΔnef infection (B), and 12 weeks after SIVmac239 infection (C) were subjected to genome-wide sequencing by Roche/454 pyrosequencing. Amino acid variants in Env338-346RF9 were investigated as described in Materials and Methods. The wild-type sequence and animal identification numbers are shown at the top of each panel. Masked identical bases are represented by dots, and amino acid replacements are represented by capital letters. The frequency of each epitope variant sequence is shown. A variant sequence had to be present in at least one animal at a frequency of 1% or greater to be included in the graphic. The legend indicates the color associated with a given variant frequency. A zero indicates that no reads were detected with that sequence.
Fig 7
Fig 7
Rapid accumulation of nucleotide substitutions in the ARF130-40QL11 epitope at 3 weeks after infection in M3 homozygous MCMs infected with m3KOΔnef. Virus populations were isolated and subjected to genome-wide deep sequencing. Samples came from M3 homozygous MCMs 3 weeks after infection with m3KOΔnef (A), M3 homozygous MCMs 4 weeks after infection with SIVmac239 (B), and M3 homozygous MCMs 12 weeks after infection with SIVmac239 (C). Amino acid variants in ARF130-40QL11 were investigated as described in Materials and Methods. The wild-type sequence and animal identification numbers are shown at the top of each panel. Masked identical bases are represented by dots, and amino acid replacements are represented by capital letters. The frequency of a given epitope variant sequence is shown. A variant sequence had to be present in at least one animal at a frequency of 1% or greater to be included in the graphic. The legend indicates the color associated with a given variant frequency. A zero indicates that no reads were detected with that sequence.

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