第9回千葉県胆膵研究会 11.

Additional file 13: Table S5. Statistical analysis of all experiments separated for transmission pairs with high and low diversity transmitters and for transmission pairs where recipient is closer to ancestral genotype, respectively

CNS-derived Macrophage-tropic viruses show increased infection capacity and gp120 shedding.

<p><b>(A)</b><u>Cell-free virus infectivity is increased in CNS-derived macrophage tropic Envs</u>. Infectivity of Env-pseudotyped cell-free virus stocks was assessed by titration on TZM-bl. Infectivity per unit of p24 capsid was calculated (RLU/ng p24) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s015" target="_blank">S7 Fig</a>) and data expressed as percent infection relative to the patient-paired non-macrophage-tropic Env. Data shown is the mean of two independent assays, error bars are SD. <b>(B)</b><u>CNS-derived macrophage-tropic Envs have improved infectivity during cell-cell transmission.</u> Env and NLinGluc cell-cell transmission reporter expressing 293-T were co-cultured with A3.01-CCR5 cells in the absence of polycation to measure cell to cell transmission capability of the individual envelopes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s015" target="_blank">S7 Fig</a>) as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref095" target="_blank">95</a>]. Data shown is the mean of two independent assays, error bars are SD. <b>(C)</b> <u>CNS-derived macrophage-tropic Envs maintain similar cell-fusion efficacy to non-macrophage tropic Envs</u>: Env and NL-Luc-AM reporter expressing 293-T cells were co-cultured with rhesus Trim5α-expressing TZM-bl target cells to measure fusogenicity of panel envelopes. Data shown is the mean of two independent assays, error bars are SD. <b>(D)</b> <u>CNS-derived macrophage-tropic viruses have increased shedding of gp120</u>. Envelope-pseudoviruses carrying mouse CD4 were treated with 2F5 to induce gp120 shedding and immobilized using magnetic beads. Shed gp120 and non-bound virus was washed away and gp120 and p24 levels measured by ELISA as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref096" target="_blank">96</a>]. The difference between gp120 levels of 2F5 treated and to mock-treated controls is depicted as % gp120 shed. Data shown are the means of three independent assays, error bars are SD.</p

CNS-derived Macrophage-tropic viruses show similar entry pattern with rapid engagement of CD4.

<p><b>(A)</b> to <b>(C)</b> Times to reach 50% resistance to CD4, 50% CCR5, and fusion inhibitors was determined for the shown pairs of patient derived macrophage-tropic and non-macrophage-tropic viruses as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.g006" target="_blank">Fig 6B to 6D</a>. Data for each inhibitor and virus combination were derived from at least eight replicates from four to six independent experiments. <b>(A)</b> For each envelope, one representative time course of infection is shown, normalized to infection at 120min post infection. Data shown is for a replicate representative of the calculated mean of all replicates. <b>(B)</b> Time intervals between four stages of the entry process (synchronized start, CD4 binding, CCR5 attachment, fusion) were compared by Mann-Whitney tests of NAB01 and CD4low viruses and M-tropic and non-M tropic pairs from the analyzed three patients. Only envelopes from the same patient (same principal color) were compared. <b>(C)</b> Data depict the percent of virus already resistant to CD4 blocking following the 30min spinoculation at 4°C. Individual data points are two replicates from each of four to six independent experiments. Horizontal bars depict means. <b>(D)</b> Statistical analysis of entry kinetics. Data points from four to six individual experiments were combined before fitting the curves and averaging individual T½ values. Estimated time intervals between the four stages of the entry process (synchronized start, CD4 binding, CCR5 attachment, fusion) were compared by Mann-Whitney tests. Only envelopes from the same patient (same principal color) were compared. Alternate statistical analysis using paired replicates before curve fitting shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s014" target="_blank">S6 Fig</a>.</p

Gp120 sequence mutation pattern following adaptation to CD4^low targets.

<p><b>(A)</b> Summary of amino acid mutations acquired as a result of adaptation to long term <i>in vitro</i> culture and adaptation to CD4^low. Green shading indicates mutations affecting N-linked glycosylation sites. Red boxes denote mutations that occur in both CD4^low adapted clones, yellow and orange boxes indicate mutations that occurred only in CD4^low.c21 and CD4^low.c24, respectively. <b>(B)</b> Structural representations of mutated residues in NAB01 associated with adaptation to low levels of CD4 mapped onto crystal structure 5fyj of X1193.c2 SOSIP [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref100" target="_blank">100</a>]. Dark gray residue shading indicates limits of non-resolved region of gp120 V4 loop. Missing from the model are the residue at 402, within the non-resolved region of V4, and the glycans at residues 408 and 461 which are not present on this subtype G Env variant. Structure rendered using PyMol version 1.4.1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref101" target="_blank">101</a>].</p

Envelopes adapted to low levels of CD4 require a higher proportion of their available trimers to complete entry.

<p><b>(A)</b> Relative infectivity of mixed trimer infection experiments with CD4^low panel viruses using the R508S/R511S dominant-negative Env mutants. Infectivity of pseudotyped virus stocks expressing the indicated ratios of wild type and dominant-negative mutant Envs was measured on TZM-bl reporter cells. Infectivity of virus stocks containing solely the respective WT envelope were set as 100%. Data depict mean from two independent experiments. <b>(B)</b> Experimentally defined mean trimer number per virion measured from four independent assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s008" target="_blank">S8 Table</a>) were used to derive mathematical estimates of the entry stoichiometry T based on data shown in (A) as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref112" target="_blank">112</a>]. <b>(C)</b> Graphical presentation of mean trimer number per virion and estimated stoichiometry of entry as shown in (B). <b>(D)</b> The percentage of infectious virions, i.e. virions with at least T trimers, was calculated for each single viral variant based on trimer numbers distributed according to a discretized B-distribution with the measured mean (Fig 10B) as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref114" target="_blank">114</a>].</p

Adaptation to CD4^low allows efficient infection of macrophages.

<p><b>(A)</b> Two types of macrophages, M-MDM and G-MDM expressing differential CD4 levels (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s012" target="_blank">S4A Fig</a>) were infected with 0.1ng p24 of ultra-centrifuged Env-pseudotyped luciferase reporter virus stocks infection measured on day seven post-infection by quantifying luciferase reporter activity (relative light units (RLU)). Data are means from two individual donors, input of ultracentrifugation purified virus was standardized by p24 content, error bars = SD. <b>(B)</b> Infection of M-MDM and G-MDM by CD4^low adapted viruses relative NAB01-PA (data derived from A. <b>(C)</b> Comparison of M-MDM and G-MDM infectivity. Shown is the relative infectivity of G-MDM compared to M-MDM infection (data derived from A).</p

Directed evolution of HIV-1 to utilize CD4^low target cells.

<p><u>Adaptation of HIV-1 to CD4</u>^<u>low</u><u>targets <i>in vitro</i> (A, B)</u>. <b>(A)</b> Overview of stepwise adaptation to CD4^low PBMC targets and PBMC reversion cultures with normal CD4 levels and the derived envelope clones. <b>(B)</b> <u>Summary of the 18 week adaptation to low CD4 expressing target cells.</u> Stepwise decrease in available cell surface CD4 on PBMC was achieved by dose escalation of the CD4 inhibitor DARPin 57.2 (right axis, grey shaded areas). HIV-1 replication as measured by p24 antigen production in culture supernatant on CD4 inhibitor treated cells (CD4^low culture, red) and control culture (untreated PBMC, blue) are shown. <b>(C)</b> <u>Adaptation to CD4</u>^<u>low</u> <u>targets increases sensitivity to CD4-IgG</u>₂ <u>(PRO542).</u> Mean neutralization sensitivity (IC50) of envelope-pseudotyped viruses on TZM-bl cells derived from two to seven independent assays (error bars = SD) are shown. <b>(D)</b> <u>High sensitivity to CD4-IgG</u>₂ <u>is paired with high binding capacity of CD4-IgG</u>₂ <u>to Env trimer.</u> Simple linear regression analysis of CD4-IgG₂ inhibitory capacity (IC50 values shown in panel C) and binding of CD4-IgG₂ to the envelope of the indicated viruses expressed on 293-T cells. Mean fluorescence intensity = MFI. Data are means of two independent experiments; error bars = SD. <b>(E)</b> Adaptation to CD4^low target cells results in high resistance to CD4 inhibitor compared to wild type HIV-1 isolates. Comparison of IC50 of CD4-blocking DARPin 55.2 against 41 wild-type HIV-1 strains from different clades (black dots, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s003" target="_blank">S3 Table</a> for details on virus panel and individual IC50 values) and the CD4^low adaptation virus panel (colored dots, see legend) probed by Env pseudovirus infection on TZM-bl cells. Data are means of one to three independent experiments.</p

CD4^low adapted viruses need extended time to transition between steps in the entry process.

<p><b>(A)</b> Schematic of entry kinetic assay to measure timing of CD4 binding, CCR5 binding, and fusion. Virus is added to TZM-bl in the presence of the polycation DEAE, spinoculated onto cells at 2095g for 30 min at 4°C to limit conformational changes upon CD4 binding. Infection is synchronized by the addition of warmed media and inhibitors targeting CD4 (DARPin 55.2), CCR5 (Maraviroc), and fusion (T-20) added in saturating concentrations at 0, 5, 10, 15, 20, 30, 45, 60, and 120 min post start of infection. <b>(B)</b> For each envelope, one representative time course of infection is shown. Infectivity data are normalized to infection at 120min post infection and all treatment conditions are shown as relative infectivity compared to this 100% level. <b>(C)</b> Definition of transition times required to reach 50% of transition to CD4 bound, CCR5 bound stage and fusion. For each inhibitor and each of at least eight replicate measurements derived from four to six independent experiments, T½ values of infection times were estimated. The mean of these estimates is a proxy for the time required to reach 50% CD4 resistance, 50% CCR5 resistance, and 50% fusion resistance. Error bars denote SD. <b>(D)</b> Percentage of viruses already resistant to CD4 blocking following the 30 minute spinoculation at 4°C. Data points are derived from four to six independent experiments done in replicates. Horizontal bars depict means.</p

CD4^low adapted envelopes show heightened sensitivity to inhibitors targeting the CD4bs, V3 loop, and CD4i epitopes, and patient plasma.

<p><b>(A)</b> and <b>(B)</b> Sensitivity of the CD4^low viruses to inhibitors of CD4 (DARPin 55.2) and CCR5 (PSC RANTES, Pro140, Maraviroc). IC50 values are shown relative to the IC50 of NAB01-PA in <b>(A)</b> free virus entry on TZM-bl and <b>(B)</b> 293T-TZMbl^TRIM5α cell fusion. Individual IC50 values are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s006" target="_blank">S6 Table</a>. <b>(C)</b> Sensitivity of CD4^low virus panel to heterologous plasma neutralization. Data are medians derived from neutralization titer on TZM-bl cells of patient plasmas from 24 individuals with different HIV-1 subtype chronic infections (eleven subtype B, four subtype A, and three of each subtype C, 01_AE, and 02_AG). <b>(D)</b> Sensitivity of CD4^low virus panel to neutralizing antibodies and Env targeting inhibitors (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s001" target="_blank">S1 Table</a>). IC50 values were derived in a standard pseudovirus neutralization assay on TZM-bl cells. Darker shading indicates higher sensitivity. Data shown in A, B, and D are mean values from at least two independent assays for each inhibitor.</p

Adaptation to CD4^low targets reduces free virus infectivity despite high fusogenicity.

<p><b>(A)</b><u>Cell-free virus infectivity is reduced upon adaptation to CD4</u>^<u>low</u><u>targets</u>. Infectivity of Env-pseudotyped cell-free virus stocks was assessed by titration on TZM-bl (left) and PBMC (right). Infectivity per unit of p24 capsid was calculated (RLU/ng p24) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s011" target="_blank">S3 Fig</a>) and data expressed as percent infection relative to the parental clone NAB01-PA. Data represent the mean of two to three independent TZM-bl titrations, and the mean of three independent experiments on PBMC using different donor batches of three-way stimulated PBMCs and freshly produced virus stocks. Error bars depict standard deviation (SD). <b>(B)</b><u>CD4</u>^<u>low</u><u>adapted viruses maintain infectivity during cell-cell transmission.</u> Env and NLinGluc cell-cell transmission reporter expressing 293-T were co-cultured with A3.01-CCR5 cells in the absence of polycation to measure cell to cell transmission capability of the individual envelopes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.s011" target="_blank">S3 Fig</a>) as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006255#ppat.1006255.ref095" target="_blank">95</a>]. Data shown is the mean of two independent assays, error bars are SD. <b>(C)</b> <u>CD4</u>^<u>low</u> <u>adapted viruses have high cell-fusion efficacy</u>: Env and NL-Luc-AM reporter expressing 293-T cells were co-cultured with rhesus Trim5α-expressing TZM-bl target cells to measure fusogenicity of panel envelopes. Data shown are the means of three independent assays, error bars are SD. <b>(D)</b> <u>CD4</u>^<u>low</u> <u>adapted viruses are not prone to shedding of gp120</u>. Gp120 shedding from Envelope-pseudoviruses in response to treatment with the MPER nAb was assessed as the percentage of gp120 content after 2F5 treatment relative to mock-treated controls normalized to p24 input. Data shown are the means of three independent assays, error bars are SD.</p