Immature NL4-3 virions improved the elicited cellular immune response.

<p>(A) Electron microscopy analysis of purified NL4-3 virions treated with AT-2 and APV. Different virion morphologies were observed; most were immature and eccentric. (B) 52 electron-micrographs were analysed for virus morphologies. The APV incubation reduces mature virions but increases immature and eccentric forms. Error bars represent standard deviaations. (C) Twenty-one cryopreserved PBMCs from HIV seropositive individuals were assessed by ELISPOT to detect IFN-γ production. PBMCs were pulsed with 200 ng/ml p24 equivalents in all cases. Magnitude response was significantly lower against WT inactivated virions (WT+AT-2) than against WT+APV+AT-2 (*p<0.05). Averaged values from duplicate wells normalized to SFC/10⁶ PBMCs are shown for the stimulation conditions indicated. Plots represent mean ± standard error of the mean (SEM). Positivity threshold for each construct or antigen was defined as at least 50 SFC/10⁶ PBMC and at least twice that of the control medium.</p

Analysis of virion morphologies

<p><b>by electron microscopy.</b> (A) Electron micrographic analysis of 293-T cells transfected with pNL4-3. Mature virions (black arrows) show their characteristic core (white arrow heads). Budding particles (black arrow heads) are also present. (B) Electron micrograph showing immature viral particles budding from 293-T cells transfected with pNL4-3/ΔRT (black arrows). (C) Electron micrographic analysis of pNL4-3 purified viruses showing mature, immature and eccentric virion forms. (D) Electron micrograph of pNL4-3/ΔRT purified virions in which only immature virion forms can be observed. (E and F) NL4-3 and NL4-3/ΔRT virion magnifications respectively are shown. (G) Virion form measurements of NL4-3 <i>versus</i> NL4-3/ΔRT. NL4-3/ΔRT virion conformation was mostly immature and no mature virions were observed. On the other hand, NL4-3 virions were mainly mature. Error bars represent standard deviations. Samples were imaged using a Tecnai Spirit Twin electron-microscope.</p

NL4-3/ΔRT plasmid renders virions infectious but non-replicative.

<p>(A) PBMC infection was monitored using NL4-3 (WT) and NL4-3/ΔRT (ΔRT) virions labelled with a Gag fusion protein (Gag-EGFP). Samples were imaged using a Leica SP2 confocal microscope. We observed an accumulation of Gag-EGFP in the cells infected with NL4-3 and NL4-3/ΔRT (arrows) but not in cells infected with Gag-EGFP. Cellular nuclei are stained in blue (DAPI), F-actin in red (Phalloidine) and the fusion protein Gag in green (Gag-EGFP). (B) NL4-3 and NL4-3/ΔRT virion replication measurements by luciferase activity in TZM-bl cells. The NL4-3 virions showed a decrease in luciferase activity directly proportional to the p24 quantity, while for NL4-3/ΔRT virions, luciferase activity was under the background independently of the p24 quantity.</p

NL4-3/ΔRT plasmid graphic scheme.

<p>NL4-3 (WT) plasmid was used to generate the NL4-3/ΔRT plasmid by deleting an 892 bp fragment, which codes for the retrotranscriptase (RT) and is inside the <i>pol</i> gene. These plasmids were used to transfect 293-T cells in order to obtain viruses. Numbers refer to pNL4-3.</p

Virological failure rates over time in different resource limited settings.

2<p> <b>Ferradini et al. Lancet 2006; 367:1335–42. ³ Ferradini et al. AIDS 2007, 21:2293–2301.</b></p

RAMS among patients with virological failure (VL>5.000 copies/ml) amplified and evaluated by genotyping.

<p>RAMS among patients with virological failure (VL>5.000 copies/ml) amplified and evaluated by genotyping.</p

Patients with VL >5.000 copies/ml (N = 55).

<p>Patients with VL >5.000 copies/ml (N = 55).</p

Patient's disposition.

<p>Patient's disposition.</p

NNRTI's resistance mutations prevalence.

<p>NNRTI's resistance mutations prevalence.</p