Freel, S.A., et al.

Supplemental Material Figure Legends and Tables

Table S1. Cohort Characteristics: Virus Controller Cohort, Chronic HIV-1+ Cohort and DNA prime/rAd5 boost Vaccinees. Demographics of the three cohorts are shown. For each cohort, the percent having the known protective alleles (HLA-B*57, B*27 or B*5801) are indicated. The median CD4 count, viral load and estimated time since HIV-1+ diagnosis are indicated for the HIV-1 infected subjects. The chronic subjects were all on therapy as indicated: RTI, reverse transcriptase inhibitor; PI, protease inhibitor; INI, integrase inhibitor.

Table S2. Sequence Similarity between Reporter Virus Envelopes and Vaccine Envelopes. Amino acid sequences of the vaccine strains, VRC A, VRC B, and VRC C, gp145 envelopes were each compared to the amino acid sequence of gp160 envelopes of CH040, CH058, CH077, NL4-3, WEAU and WITO. Percent amino acid identity and percent amino acid similarity are shown. Approximately 10% of the differences in the alignments are due to gaps from comparison of the gp145 used in the vaccine with the gp160 envelope sequence used in the virus. This is indicated for each comparison. The virus sequence (NL4-3) with the greatest similarity to the vaccine strain (VRC clade B Env) is indicated in bold.

Table S3. Suppressive activity correlated with Env and Gag-specific MIP-1 monofunctional cells and polyfunctional cells expressing CD107a and/or MIP-1 cells. Pearson’s correlation coefficient (r) is shown for 16 HIV-1+ virus controllers. Data is shown for the Env and Gag responses as they correlate to antiviral activity against the lab-adapted NL4-3. Significant positive correlations are shown in bold (p < 0.05). All other functionally-defined subsets showed no correlation with viral inhibitory activity.

Figure S1: Vaccine and virus sequence homology. HIV-1 envelope peptide sequence alignments are shown for the 6 viruses that were used to assess CD8-mediated virus inhibition and the 3 VRC vaccine components. Homology to the NL4-3 reference strain is highlighted in grey. Blue boxes indicate known HLA B27, B57, or B58-restricted CTL epitopes. Homology to published epitopes sequences (Los Alamos Database) are indicated in bold.

Figure. S2. Characterization of the quality and phenotype of HIV-1-specific T cells. Following ex vivo stimulation with overlapping pools of peptides from HIV-1 antigens, cells were stained with multiple markers to identify CD4 and CD8+ T cells specific for HIV-1. Shown is a typical “gating tree” to identify and characterize those cells. (Top row) Initially, samples are gated to remove highly-fluorescent aggregates; then a gate is used to eliminate doublets. Within the singlet events, live T cells are selected by gating on CD3+ events that do not show CD14 or CD19 expression and exclude ViViD. A lymphocyte gate is followed by gates on CD4 or CD8 to identify the T cell lineages. (Middle rows) Antigen-specific cells are identified as those expressing any of the five functions. This figure is from HIV-1 virus controller, cells stimulated with Gag peptides. Boolean gating was used to identify all possible combinations of the individual functions. (Bottom rows) Progressive gating with CD28, CD45RA, and CCR7 is used to identify Naïve T cells, and memory T cells termed central memory (CM), transitional memory (TM), effector memory (EM), or terminal effector memory (TE), within either CD4 or CD8 lineages. The same gates can be applied to the antigen-specific T cells (i.e., those producing one or more functions upon stimulation) to define the phenotype of HIV-1 specific T cells.

Figure S3. Quality of the CD8+ T cell response for vaccinees with antiviral activity. The expression of each of 5 functions (CD107a, MIP-1, IFN, IL-2, TNF-) was measured in CD8+ cells from 40 vaccinees. The fraction of cells expressing each marker independently or in combination following Env (A) or Gag (B) stimulation was compared between vaccinees with (red symbols) or without (blue) potent antiviral activity. * denotes significant differences (p < 0.05, Wilcoxon Rank).

Figure S4. Quality of the CD8+ T cell response for HIV-1+ subjects with antiviral activity. The expression of each of 5 functions (CD107a, MIP-1, IFN, IL2, TNF-) was measured in CD8+ cells from 14 virus controllers (red symbols), 15 HIV-1+ chronic donors (blue), and 20 vaccinees (green) with positive antiviral activity. The fraction of cells expressing each marker independently or in combination following Env (A), Gag (B), or Nef (C) stimulation is shown.

Figure S5. Sorting of CD8+ T cell subsets for viral inhibition assays. In three separate experiments, cells from vaccinees or virus controllers were stained with antibodies to identify T cell subsets. Slightly different approaches were taken to identify the subsets in each experiment; in Experiment #2 we sorted an early memory population that we identified in individuals, whereas in Experiments #1 and 3 we sorted naïve T cells. Central, effector, and terminal effector populations were sorted in all experiments. Shown are the approximate gating trees used to identify the sorted populations; the final sorted populations are shown in with red names. Arrows indicate hierarchical gating.

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Freel, S.A., et al.

Tables

Table S1

Sample Type / Gender / Race / Age
[median] / HLA-B57*, 27*, 58, 5801 / CD4 count/ml [median (range)] / Viral load/ml [median (range)] / Estimated time since diagnosis [median (range)] / ART at time of sampling
Virus Controller (n=16) / 37%Male / 75%Black
25%Caucasian / 45 / 40% / 764 (389-1865) / 957 (<48-2,950) / 5 (1-18) years* / Naïve
Chronic (n=21) / 87%Male / 33%Black
62%Caucasian
5%Hispanic / 49 / 43% / 685 (258-1532) / 121 (<48-16,000) / 13 (4-25) years* / 100%RTI, 63%PI, 18%INT
Vaccinee (n=40) / 65%Male / 12%Black
83%Caucasian
5%Asian / 30 / 20% / N/D / N/A / N/A / N/A

Table S2

Vaccine envelope (gp145) / Virus envelope (gp160) / % aa identity / % aa similarity / % gaps
VRC A / CH040, CH058, CH077, NL4-3, WEAU, WITO / 67-68% / 77-78% / 10-12%
VRC B / CH040, CH058, CH070, WEAU, WITO
NL4-3 / 70-74%
84% / 78-80%
86% / 10-11%
9%
VRC C / CH040, CH058, CH077, NL4-3, WEAU, WITO / 62-65% / 73-75% / 10-13%

Table S3

CD107a / + / - / + / + / - / - / + / + / + / - / + / -
MIP-1 / + / + / + / + / + / + / - / + / + / + / - / +
IFN / + / + / + / + / + / + / + / - / + / - / - / +
IL-2 / + / + / - / + / - / + / + / + / - / + / + / -
TNF / + / + / + / - / + / - / - / - / - / - / - / -
ENV / correlation / 0.765 / 0.697 / 0.762 / 0.777 / 0.774 / 0.821 / 0.813 / 0.791 / 0.762 / 0.709 / 0.727 / 0.812
p-value / <0.001 / 0.004 / 0.001 / <0.001 / <0.001 / <0.001 / <0.001 / <0.001 / 0.001 / 0.003 / 0.002 / <0.001
GAG / correlation / 0.155 / 0.742 / -0.093 / 0.782 / -0.062 / 0.799 / 0.429 / 0.776 / 0.568 / 0.829 / 0.645 / 0.634
p-value / 0.581 / 0.002 / 0.741 / <0.001 / 0.826 / <0.001 / 0.111 / <0.001 / 0.027 / <0.001 / 0.009 / 0.011

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