Ineffectual Targeting of HIV-1 Nef by Cytotoxic T Lymphocytes in Acute Infection Results in No Functional Impairment or Viremia Reduction

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. Importance: These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia

HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART

HIV-1 Nef Sequence and Functional Compartmentalization in the Gut Is Not Due to Differential Cytotoxic T Lymphocyte Selective Pressure

<div><p>The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef’s function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef’s sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary <i>nef</i> quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env⁻ Vpu⁻ pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of <i>nef</i> sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.</p></div

Amount of selective pressure correlates with the CTL response regardless of location.

<p>Regression analysis shows a statistically significant correlation between the total mean number of SFC and the estimated global dN/dS from all samples, both blood and gut, for each subject (A). The correlation between CTL and dN/dS from all samples is more significant if only Nef-specific SFCs are considered (B). Finally, there is a significant correlation between the total mean number of SFCs and the number of positive peptide pools (C).</p

Summary of MHC Class I and CD4 downregulation.

<p>The mean and standard deviation of HLA-A*02 (A) and CD4 (B) downregulation by control and subject-derived <i>nef</i> alleles from at least three separate infections is shown. Difference from % downregulation by NL4-3 Nef was evaluated with a two-tailed t test, and differences with a p-value <0.05 are marked with an *. Differences between gut and plasma quasispecies from the same subject were also evaluated with a two-tailed t test, and pairs with significant differences are marked with a horizontal bar with the p-value indicated above the bar.</p

MHC Class I and CD4 downregulation by plasma and gut <i>nef</i> quasispecies.

<p>VSVg pseudotyped Env⁻ Vpu⁻ recombinant mCD24 reporter viruses were used to infect CEM T1 cells. On day 3 post-infection levels of CD4 and HLA-A*02 were measured on all mCD24 reporter positive cells. Open histograms are Delta Nef control viruses and the filled histograms are the experimental viruses. A.) For each subject 4 histogram plots are shown: HLA-A*02 downregulation by gut and plasma quasispecies on the left panels (upper-gut and lower-plasma) and CD4 downregulation on the right panels (upper-gut and lower-plasma). B.) Downregulation of HLA-A*02 (left) and CD4 (right) by control viruses carrying NL4-3 Nef, Nef LL>AA specifically deficient in CD4 downregulation, and Nef M20A specifically deficient in MHC Class I downregulation.</p

Measures of selective pressure on blood and gut <i>nef</i> quasispecies.

<p>A.) The global non-synonymous to synonymous substitution rate ratio (dN/dS) with 95% confidence intervals (CI) was calculated for all 234 sequences in the dataset, all gut-derived sequences, all plasma-derived sequences, and for all individual pairs of gut and plasma sequences. The range of the 95% CI for the dN/dS estimate for all sequences is highlighted in grey so that those estimates with a 95% CI entirely outside this range (542 gut, 648 gut and plasma, 650 gut) can be easily identified. B.) The diversity among all sequences from plasma and gut as well as among blood and gut isolates from each subject was calculated with a s.e.m. from 500 bootstrap replicates. The difference in diversity between plasma and gut sequences was evaluated with a two-tailed t test and pairs with significant differences are marked with a bar with the p-value indicated above it.</p

Neighbor-joining phylogenetic tree of plasma and gut-derived <i>nef</i> quasispecies.

<p>234 full-length <i>nef</i> sequences were aligned with consensus B and NL4-3. A neighbor-joining tree was constructed, rooted on consensus B, and evaluated with 1000 bootstrap replicates. Clusters of sequences from each subject all had 100% bootstrap support and other significant bootstrap values >70% within each subject cluster are indicated on the tree. Sequences from plasma are indicated with grey triangles; gut, black circles. Filled circles are sequences from biopsy #1 and open circles, from biopsy #2.</p

Study subject information.

<p>Study subject information.</p