Characterization of XMRV pseudovirus and single-round neutralization assay.

<p>(A) Comparison of XMRV and control HIV-1 pseudoviruses in yield (p24 accumulation) and infectivity (IU/ml on TZM-bl cells). (B) Detection of antibody specificity to XMRV and HIV-1 pseudoviruses. Pseudoviruses were tested in the neutralization assay with mAb 83A25 that recognizes a shared epitope of MLV Env glycoprotein and with mAb b12 that recognizes HIV-1 Env glycoprotein. (C) Neutralization of the XMRV and HIV-1 pseudoviruses showing a broad range of sensitivity and specificity of the assay using polyclonal antibodies (anti Friend-MuLV).</p

Characterization of antibodies purified from immune and control mouse sera.

<p>Total immunoglobulin pool was affinity purified from immune or control sera collected at the peak time point of neutralizing activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">figure 3B</a>). The ELISA-binding (A) and NAb (B) activities were then measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">Figure 3</a> and showed that immunization elicited an immune response leading to the production of anti-XMRV immunoglobulins.</p

Expression of XMRV Env, Gag and VLP.

<p>(A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.</p

Detection of XMRV-specific antibody production in mouse sera.

<p>Time course of the production of (A) ELISA-binding antibodies and (B) NAb in Balb/C mice (10 animals in each group) immunized with pDP1-XMRV<i>envgag</i> (first arrow; P), Ad5-XMRV (second and third arrows; A) and XMRV VLP (fourth arrow; V). Determination of (C) endpoint dilution and (D) serum neutralizing titers at the peak time point indicated by asterisks in Panels A and B, respectively. The arrow indicates endpoint dilution. (E) The specificity of the serum neutralizing activity was determined by comparing XMRV and HIV-1 pseudoviruses and showed that the primary target for neutralization is the XMRV Env.</p