NAb induction against NAb-resistant SIVmac239.

(A) Study design.

(B) Plasma viral loads (SIV gag RNA copies/ml plasma) in NAb non-inducers (left) and inducers (right). Viral loads have been previously partially reported (Yamamoto et al., 2007; Nomura et al., 2012; Takahashi et al., 2013; Nakane et al., 2013; Iwamoto et al., 2014).

(C) Plasma SIVmac239 100% neutralizing end point titers by 10 TCID50 killing assay on MT4-R5 cells. Points on the dotted line show marginally NAb-positive results (< 1:2). In some animals, titers were comparable with our reported results using MT4 cells (Kawada et al., 2007).

Selection of a viral nef mutation (Nef-G63E) before NAb induction.

(A) Viral nef mutations in NAb inducers. A linear schema indicating Nef functional domains is aligned above. Mutations at timepoints with NAb- (indicated by white wedge; mostly before 6 months p.i.), NAb+/- (green wedge) and NAb+) (purple wedge; mostly after 1 year) are shown in individual animals. Black and dark gray represent dominant and subdominant mutations (or deletion in R-360) by direct sequencing, respectively. Red and pink indicate dominant and subdominant G63E detection, respectively.

(B) Schema of SIVmac239 Nef structure and Nef-G63E mutation orientation.

(C) Comparison of frequencies of macaques having Nef-G63E in plasma viruses between NAb non-inducers and inducers. Compared by Fisher’s exact test.

(D) Temporal relationship of Nef G63E frequencies in plasma virus and NAb induction. Black boxes (left Y axis) show log2 NAb titers; red triangles and green diamonds (right Y axis) show percentage of G63E and G63R mutations detected by subcloning (15 clones/point on average), respectively.

Nef-G63E is a CD8+ T-cell escape mutation.

(A) Nef62–70 QW9-specific CD8+ T-cell frequencies and related MHC-I alleles in seven Nab inducers selecting Nef-G63E. Mamu-B*039:01 and Mamu-B*004:01 are known to restrict Nef62–70 QW9 epitope (Evans et al., 1999; Sette et al., 2012) and binding of Nef63–70 peptide to Mamu-A1*032:02 was predicted.

(B) CD8+ T-cell responses specific to wild-type Nef62–70 or mutant Nef62–70.G63E or Nef62–70.G63R peptides. See also Figure S3.

Nef-G63E mutation reduces PI3K/mTORC2 binding and pAkt drive.

(A) Representative surface expression level histograms of CD3, CD4, MHC class I, CXCR4 and BST-2 in CD4loNef+ subpopulations after wild-type (WT) or Nef-G63E mutant SIV infection at multiplicity of infection (MOI) 0.1 on HSC-F cells.

(B) Left: representative histograms of relative pAkt serine (Ser) 473 levels in p27+ subpopulations after WT or Nef-G63E mutant SIV infection at MOI 0.1 on HSC-F cells. Numbers show pAkt Ser473 mean fluorescence intensities (MFIs) for each. Right: Deviation of pAkt Ser473 MFIs in p27+ HSC-F cells compared with mean MFI of uninfected cells. Compared by unpaired t test.

(C) Left: Relative pAkt Ser473 levels (normalized to mean MFI of uninfected controls) in Nef+ HSC-F cells assessed for serum starvation (MOI 0.2, 1 day p.i.). Adjusted P values show results of comparison via Sidak’s post-hoc test of 2-way ANOVA (C, left). Right: Deviation of pAkt Ser473 MFIs in Nef+ HSC-F cells assessed for high-MOI infection (MOI 5, 1-day p.i.). Compared by unpaired t test.

(D) Proximity ligation assay (PLA) of Nef binding with mTOR, GβL/mLST8, PI3Kp85 and PI3Kp110α. MFI-based binding index was calculated as (anti-Nef/anti-partner) - (isotype/anti-partner) - (anti-Nef/isotype) + (isotype/isotype). Histograms for samples and isotype/isotype are representatively shown. Differences in MFI binding indexes can be enhanced compared with comparison of raw MFIs. Compared by paired t tests. Data represent one of two [(A); (B); (C, right)] independent experiments in quadruplicate, four independent experiments performed in triplicate [(C, left)] or four independent single-well comparison experiments pooled for statistical analysis (D). Bars: mean ± SD (B, C right), mean ± SEM (C left).

Preferential targeting of lymph node Env-specific B cells by Nef in vivo.

(A) Representative gating of triple noise-cancellation in vivo Nef staining in B cells analyzed by ImageStreamX MKII. Pre-experimental damaged cells are first excluded with Live/Dead from focused/centroid/singlet image-acquired CD19+ B cells (first lane right/R4 gated on “Focused”). Following Nef+ gating (second lane left/R5 gated on R4), a second step of Nef noise cancellation (second lane middle/R7 on R5) comprises double-negative removal of post-experimental stochastic irregular staining building a disparate intracellular staining gradient (X axis, Nef signal pixel Haralick variance mean) and post-experimental batch overt cellular staining (Y axis, Nef signal pixel intensity threshold). This outputs a B-cell population with a fine-textured pericellular Nefint-lo staining, biologically concordant with Nef membrane-anchoring. Probing of anti-Env BCR (αEnv) by recombinant SIV Env (second lane, right) is combined, resulting in a 2-D panel of intracellular Nef versus αEnv for noise-cancelled Nef+ B cells (R7) plus Nef- B cells (R8) (third lane left/“R7+R8”). pAkt Ser473 expression (third lane right) and cellular morphology (B) was further analyzed. DN, double-negative.

(B) Typical images of Nef-transferred Env-specific B cells defined αEnv+-intracellular Nef+-Nef Haralick variance meanlo-Nef Intensity thresholdlo-Live/Dead--CD19+ cells [“Nef+aEnv+” population of lower left panel in (A), gated on “R7, R8”]. Note the pericellular pAkt Ser473 upregulation in these cells (Ch 03/yellow). Data on inguinal lymph node lymphocytes of macaque R10-007 at week 62 post-SIVmac239 infection are shown in (A) and (B).

(C) Comparison of pAkt Ser473 median fluorescence (medFI) intensity levels in Nef- B cells (R8) versus noise-cancelled Nef+ B cells (R7). Analyzed by paired t test.

(D) Comparison of Nef-positive cell frequencies in non-Env-specific (left) versus Env-specific (right) B cells in lymph nodes of persistently SIV-infected macaques (n = 6). Analyzed by paired t test.

Enhanced SIV Env-specific B-cell output up to NAb induction following Nef-G63E selection.

(A) Changes in SIV Env gp140-specific plasmablast (PB) frequencies in viremic NAb non-inducers (left, n = 12) and Nef-G63E-selecting NAb inducers (right, n = 6). Available samples of twelve viremic NAb non-inducers (including ten with ≥ 1-year survival) and six NAb inducers were tracked. In the right panel, the frequencies in NAb non-inducers are shown in background (gray) for comparison.

(B) Comparison of Env gp140-specific PB frequencies between NAb non-inducers and inducers by Mann Whitney U tests. In the right, the frequencies at NAb induction were compared with those at year 1 in NAb non-inducers with ≥ 1-year survival (n = 10). Bars: mean ± SD.

(C) Left: vector chart of Env gp140-specific memory B cell (Bmem) and PB levels. Legends for each animal correspond to the ones in (B). Right: NAb non-inducer vectors empirically define a polygonal GC output attractor area Dn (gray area surrounded with dotted lines) on which they converge by and beyond year 1 p.i. The NAb inducer vectors (shown up to the time of NAb induction) remained outside of Dn. At the moment of NAb induction they converged on a second GC output attractor area Di (red area surrounded with dotted lines), mutually exclusive with Dn (P < 0.0001 by Fisher’s exact test on NAb inducer vector convergence frequency within Dn). Legends for year 1 p.i. in the NAb non-inducers and moment of NAb induction in the NAb inducers are specified.

Profile of SIVmac239-NAb non-inducer subgroup cohort.

Summary of a SIVmac239-NAb non-inducer subgroup cohort (n = 19), composed from previously partially characterized, MHC class I haplotype-balanced naïve animals. Symbols A+, B+, E+, J+ and D+ represent possession of MHC-I haplotypes 90-120-Ia, 90-120-Ib, 90-010-Ie, 90-088-Ij and 90-010-Id, respectively. For the titers, - represents negative; +/- represents neutralization in three out of four wells in a quadruplicate test. Gray shadings indicate euthanasia due to AIDS progression. Mamu-A and Mamu-B alleles of interest in this study are listed. These NAb non-inducers were previously partially reported for their plasma viral loads and MHC-I alleles (Nomura et al., 2012; Takahashi et al., 2013; Nakane et al., 2013; Iwamoto et al., 2014). R01-011 was previously partially reported for NAb titers measured with the same method using MT4 cells as targets (Kawada et al., 2007), which derived comparable results.

IgG class-switched NAbs in the inducers.

Titration of SIVmac239-neutralizing IgGs in the NAb inducers. SIVmac239-specific IgGs, purified from pools of plasma with SIVmac239-specific NAb titers, were obtained from each animal as described (Yamamoto et al., 2007) and examined for their neutralizing activity by 10 TCID50 SIVmac239 killing on MT4-R5 cells. NAb-negative anti-SIV IgG and control IgG were prepared from a representative animal (R01-011) with NAb-negative plasma and pooled plasma of uninfected rhesus macaques, respectively.

Anti-SIV binding IgG profiles in NAb inducers and non-inducers.

(A) Temporal changes in SIVmac251 Env gp120-specific antibody titers in the NAb non-inducers without rapid progression [NAb-negative non-rapid progressors, NAb(-) non-RPs] (left, n = 11) and NAb inducers [NAb(+)] (right, n = 9). NAb non-inducers showing rapid progression (euthanized with AIDS onset within approximately 1 year) were not included except for macaque R10-001 (shown in the left). Samples for non-RPs R06-001 and R10-002 were unavailable.

(B) Unpaired t test of log-transformed anti-SIV Env Ab titers in NAb(-) non-RPs and NAb inducers. Bars represent geometric mean absorbance within each group.

(C) Lysed SIVmac virion linear antigen-binding at year 1 p.i. in NAb(-) non-RPs and NAb inducers. Plasma anti-SIVmac251 IgGs were detected using a commercial western blotting system against the parental strain SIVmac251 (ZeptoMetrix). White arrowheads indicate Env gp120-specific bands. Experiments were performed twice with comparable results.

Env sequence variation pattern is not a major characteristic of NAb induction.

SIVmac239 Env sequence variations before and after NAb induction. For each animal, the upper time point shows pre-induction and the lower time point (in purple) shows post-induction (first or second time point with NAb titer > 1:2). Dominant residue changes are shown as follows: gp120 (gray), variable regions V1-V5 (blue), and gp41 (blue gray). Subdominant mutations are uncolored. Unlabeled gray and black shadings show subdominant and dominant deletion of residues. Parental SIVmac239 sequence is listed for V1-V5. Residues mutated in multiple animals within V1-V5 are shown in red.

Selection of Nef-G63E mutant SIV is linked with Nef62-70-specific CD8+ T-cell response positivity.

(A) Nef62-70-specific CD8+ T-cell responses around week 20 post-SIV challenge in seven viremic NAb non-inducers possessing either of the MHC-I alleles Mamu-B*039:01, Mamu-B*004:01 or Mamu-A1*032:02.

(B) Temporal supernatant SIV p27 concentrations after wild type (WT) or Nef-G63E mutant SIV infection at multiplicity of infection (MOI) 0.001 on HSC-F cells. Data represent one of two independent experiments performed in triplicate. Bars: mean ± SEM.

(C) Comparison of plasma viral loads in Nef-G63E-selecting NAb non-inducers (n = 7) versus non-inducers at indicated time points post-SIV challenge. Bars represent geometric means. log10-transformed values are compared by unpaired t tests.

Decreased Akt hyperactivation properties of Nef-G63E mutant SIV.

(A) Plasma ferritin levels (ng/ml) in examined NAb non-inducers versus Nef-G63E mutant SIV-selecting NAb inducers around year 1 post-infection. Bars represent mean in each group. Compared by unpaired t test.

(B) Relative levels of pAkt Ser473 (left panel) and threonine (Thr) 308 (right panel) in SIV Nef+ HSR5.4 cells (MOI 0.1, 4 days p.i.).

(C) Relative pAkt Ser473 levels in Nef+ HSC-F cells without stimuli and transiently pulsed with IFN-γ, IL-2 or SIVmac Env gp130 after infection (MOI 0.2, 1 day).

(D) Gene expression significantly differing between WT and Nef-G63E SIV infection (MOI 5, 1 day). Black bars show potentially Akt-related genes. Asterisks show dynamics concordant with the Nef-G63E phenotype (PI3K-pAkt Ser473 downstream downregulation or inhibition upregulation).

(E) Representative flow cytometric gating (NAb inducer R01-012, wk 30 p.i.) of peripheral blood CXCR3-CXCR5+PD-1+ memory follicular CD4+ T cells. Cells were gated as CD3+CD8-CD4+CD95+CXCR5+PD-1+CXCR3- live singlet PBMCs. Available samples of six NAb inducers showing peak Nef-G63E mutant selection around week 30 p.i. and thirteen viremic NAb non-inducers were tracked.

(F) Comparison of peripheral CXCR3-CXCR5+PD-1+ memory Tfh cell frequencies between pre-SIV infection and around week 30 post-infection in NAb non-inducers developing disease before week 60 (left: n = 5) and alive more than 70 weeks (middle: n = 8) and Nef-G63E-detected NAb inducers alive more than 70 weeks (right: n = 6). Analyzed by Wilcoxon signed-rank tests.

MFIs relative to those in uninfected controls (%) are shown. **: P < 0.01, ***: P < 0.001, n.s.: not significant between WT and Nef-G63E mutant by unpaired t tests (B-C). Data represent one of two (B, C) independent experiments performed in triplicate (C: unstimulated) or quadruplicate (B, C: PI3K-pulsed). Bars: mean ± SEM.

Machine learning-verified morphological gating of Nef-invaded B cells in vivo.

(A) Representative gating strategy plus schematic of inguinal lymph node Nef+CD19+ B cells of an SIVmac239-infected rhesus macaque analyzed by ImageStreamX MK II imaging flow cytometry (R10-012, week 160 p.i.). First-step incorporation of Live/Dead staining gates out CD19+ B cells staining positive for Nef potentially due to pre-experimental membrane permeation (e.g., image #246896, #248952 and #71859). Second-step dual gating for intracellular Nef signal pixel Haralick variance mean (H Variance mean, X axis), a textural feature calculated from a gray level co-occurrence matrix (GLCM) for each image representing adjacent pixel signal strength heterogeneity and Nef signal intensity threshold (Y axis) gates out CD19+ B cells showing potentially non-specific, strong intracellular binary clustered staining pixels for Nef deriving a large variance (X axis), and/or cells stained overtly strong for Nef and hence becoming void for feasible image verification (high threshold) (Y axis), both likely originating stochastically from post-experimental rupturing procedures including membrane permeation (e.g. image #18541). This two-step noise cancellation results in acquisition of a Live/Dead--Nef signal Haralick variance meanlo-Nef signal intensity thresholdlo-Nef+CD19+ B-cell population (e.g. image #116318, #116250 and #132927) with a pericellular Nefint-lo staining, biologically concordant with Nef membrane anchoring, resembling Nef+ cells experimentally generated in vitro and showed the highest score in IDEAS6.3 machine learning module scoring for segregation of typical void vs. valid images (upper right). This gating was implemented for counting and analysis of Env-specific B-cell Nef invasion in vivo (Figure 5).

(B) Detection of contact-dependent Nef invasion from SIV-infected HSC-F CD4+ T cells to cocultured Ramos B cells in vitro. Two typical images of an intercellular conduit (SSC, purple) with Nef (green) protrusions from infected HSC-F cells to CD19+ (red) Ramos B cells from two independent experiments are shown.

Gating strategy of SIVmac239 Env gp140-specific B-cell responses.

Representative gating (R02-004, week 32 p.i.) of SIV Env gp140-specific memory B cells (Bmem) and plasmablasts (PBs). Two panels for PB gating are shown in lower resolution for visibility. Bmem staining was performed separately and gatings are merged with PB panels (second row, panels 2–4). For lineage-specific exclusion (first row, panel 4), CD10 was stained independently for Bmem (see parenthesis).