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

V1V2 deletion impairs virus infectivity and is reflected by a high stoichiometry of entry.

<p>(A) Comparison of infectivity of pseudoviruses expressing wt and V1V2-deleted envs upon infection of TZM-bl cells. Data points depict mean values of luciferase reporter activity per µl virus stock measured in 3 independent experiments. The p-value was calculated by a paired t-test. (B and C) Relative infectivity of mixed trimer infection experiments of 10 wt envs and their V1V2 deleted variants using the R508S/R511S (B) and the V513E (C) dominant-negative mutations are shown. Infectivity of pseudotyped virus stocks expressing the indicated ratios of dominant-negative mutant envs was measured on TZM-bl cells. Infectivity of virus stocks containing solely functional envelope were set as 100%. Data depict mean and SD from 2 to 4 independent experiments. (D) Estimates of T for the wt and V1V2-deleted envs derived from mixed trimer experiments. Data points are the mean of the individual estimates of T obtained with the R508S/R511S and V513E dominant-negative mutations. The p-value was calculated by a paired t-test.</p

The entry stoichiometry governs virus population infectivity.

<p>(A) Scheme depicting the influence of the entry stoichiometry on virus population infectivity. Different Ts (exemplified here: T = 1 and T = 7) will determine the minimum number of trimers that a virion requires in order to be infectious. (B) Correlation analysis (Pearson) of virus strain infectivity (measured by infection of TZM-bl reporter cells and expressed in arbitrary relative light units (RLU) per µl of virus stock) and the estimated T (plotted as mean of the independent R508S/R511S and V513E estimates shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat-1004595-g001" target="_blank">Fig. 1C</a>). Virus infectivities are depicted as mean values derived from 3 independent experiments. (C) Mathematical modeling to investigate the influence of entry stoichiometry on virion population infectivity. The data depict how T = 2 and T = 7 translate into different fractions of a virion population being potentially infectious, in dependence on the trimer number distribution across the virion population. As shown in (D), the overall infectivity of a virus population decreases with increasing T. For (C) and (D) we assumed the trimer number distribution across virions to follow a discretized Beta distribution with constant mean 12.95 and variance 45 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595-Magnus1" target="_blank">[15]</a>.</p

HIV-1 strains and mutants.

<p>Eleven HIV-1 strains from subtypes A, B and C were employed in this study. Mean virion trimer numbers and expression levels of the dominant-negative R508S/R511S and V513E mutants of the utilized virus preparations are shown.</p>a<p>Mean virion trimer numbers as estimated by gp120/p24 ELISA of purified HIV-1 pseudoparticle stocks. Data are means of 2 to 3 independent experiments.</p>b<p>The expression level of the dominant-negative mutants is recorded as percentage of the corresponding wt env based on gp120 quantification by ELISA of purified HIV-1 pseudoparticle stocks.</p><p>HIV-1 strains and mutants.</p

HIV-1 strains differ in the number of trimers required for entry.

<p>(A) Theoretical predictions of relative virus infectivity over the fraction of dominant-negative mutant env (f_m) according to our model. Curves for T ranging from 1 to 8 are shown assuming the trimer number distribution across virions to follow a discretized Beta distribution with constant mean 12.95 and variance 45 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595-Magnus1" target="_blank">[15]</a>. (B) Relative infectivity of mixed trimer infection experiments with 11 HIV-1 strains using the R508S/R511S (left) and V513E (right) 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 wt envelope were set as 100%. Data depict mean and SD from 2 to 4 independent experiments. For each virus the individual curve fits resulting from the model to the obtained data were evaluated (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s001" target="_blank">S1 Fig.</a>). (C) Mathematical estimates of T derived from the data shown in (B). The R508S/R511S (black circles) and V513E (open squares) mutations were analyzed individually. Bootstrap analyses demonstrating the robustness of the obtained estimates of T are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s001" target="_blank">S1 Fig.</a> (D and E) Mixed trimer virus stocks for strain JR-FL (D) and SF-162 (E) carrying the V513E mutation were assayed on healthy donor PBMC and compared to data obtained with TZM-bl target cells. Data depict mean and SD from 2 independent experiments.</p

Loss of the N160 glycosylation site in gp120 steers the stoichiometry of entry and virus infectivity.

<p>(A) Titration of CAP88 wt (black) and CAP88 K160N (red) pseudoviruses on TZM-bl reporter cells. Data shown are mean and SD of luciferase reporter activity upon infection measured in 2 independent experiments. Inset: Infectivity comparison of CAP88 wt and K160N depicted as relative light units (RLU) of luciferase reporter activity per µl of pseudovirus stock upon infection of TZM-bl cells. The fold infectivity difference is indicated. (B) Estimates of T for the CAP88 wt and K160N variant. Mean and range of the individual estimates using the R508S/R511S and V513E dominant-negative mutations are shown.</p

Virus entry kinetics correlate with the stoichiometry of entry.

<p>(A) Entry kinetic curves for JR-FL wt and JR-FL ΔV1V2. Synchronized pseudovirus infection of TZM-bl cells following spinoculation was terminated by addition of T-20 at the indicated timepoints. Infectivity reached after 120 minutes was set as 100% and all data were normalized relative to this value. Data are mean and SD from 3 independent experiments. (B) Half maximal entry time for wt and V1V2-deleted envs was calculated from kinetic profiles shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat-1004595-g004" target="_blank">Fig. 4A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s007" target="_blank">S7B Fig.</a> Time (in minutes) required to reach 50% entry into target cells is depicted. Data shown are means derived from 2 to 4 independent experiments. The p-value was calculated by a paired t-test. (C) Correlation analysis (Pearson) of wt (black symbols) and V1V2-deleted env (red symbols) half-maximal entry time and estimated T.</p

Point mutations in JR-FL dictate virus infection efficacy and entry stoichiometry.

<p>(A) Infectivities of JR-FL wt and indicated point mutant envs were determined by titration of virus stocks on TZM-bl reporter cells and are shown normalized to JR-FL wt. Data depict mean and SD from 4 independent experiments. (B) Relative infectivity of mixed trimer infection experiments with the specified JR-FL variants using the R508S/R511S and V513E dominant-negative mutations are shown. Infectivity of pseudotyped virus stocks expressing the indicated ratios of dominant-negative mutant envs was measured on TZM-bl cells. Infectivity of virus stocks containing solely functional envelope were set as 100%. Data depict mean and SD from 2 independent experiments. (C) Mathematical analyses of the data shown in (B) yielded estimates of T, shown here as mean and range of the individual T estimates obtained with the R508S/R511S and V513E dominant-negative mutations. (D) Analysis of virus entry kinetics for the four JR-FL variants were performed as shown <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004595#ppat.1004595.s007" target="_blank">S7A Fig.</a></p

Immunogenicity and efficacy of VLP-immunisation against LCMV-challenge in young and old mice with/without Tx or latent MCMV-infection.

<p>Young (5 months old, 3 months p.i.) and old C57BL/6 mice (18 months old, 15 months p.i.) with or without Tx and latent MCMV-infection were immunised with 10 µg VLP-GP33 s.c. Thereafter, the GP33-specific CD8⁺ T cell response was measured on day 7 (A) by tetramer staining and the Qb-specific IgG titres were quantified by ELISA on day 10 (E) and day 20 (F) in the blood. On day 21, all mice were challenged with 2×10³ pfu LCMV-WE i.v. Eight days after LCMV-challenge the GP33-specific CD8⁺ T cell response was determined by ICS in the lung (B) and LCMV-titres were determined in the spleen (C) and in the lung (D) by plaque assay. Circles indicate individual mice, horizontal lines represent the median (A–D). Vertical bars show the mean of an experimental group, error bars correspond to the SEM (E, F). The dotted line shows the detection limit of the assay. 4–5 mice per group were included. ANOVA followed by Bonferroni post-analysis was preformed to test for significant differences (* <i>p</i><0.05; ** <i>p</i><0.01; ns = not significant).</p

Long-term analysis of the CD8⁺ T cell compartment after MCMV infection and/or thymectomy in young, middle-aged and old mice.

<p>C57BL/6 mice were thymectomised at age of 4–5 weeks. Two to 3 weeks later, Tx (right panel) and non-Tx wild type (WT, left panel) mice were infected with 10⁷ pfu MCMV-Δ157 i.v. The absolute number of CD8⁺ T cells was quantified in the blood and phenotypically characterised by polychromatic flow cytometry using CD44 and CD62L. Quantitative analysis of total CD8⁺ T cell numbers (A), naïve CD8⁺ T cell numbers (B; CD8⁺CD44⁻CD62L⁺), effector memory (Tem) CD8⁺ T cell numbers (C; CD8⁺CD44⁺CD62L⁻) and central memory (Tcm) CD8⁺ T cell numbers (D; CD8⁺CD44⁺CD62L⁺). Data were pooled from three independent experiments. Circles indicate values of individual mice, horizontal bars correspond to the median of an experimental group. Significance was assessed by ANOVA followed by Bonferroni post-analysis (* <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001; ns = not significant).</p