Development and application of a high-throughput microneutralization assay: lack of xenotropic murine leukemia virus-related virus and/or murine leukemia virus detection in blood donors

Abstract

Background: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses--with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses.

Study design and methods: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization.

Results: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed.

Conclusion: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism.

Fig. 1. Detection of XMRV Env neutralizing antibodies in positive controls

MLV-luc(XMRV Env) pseudovirus infection of 293T/17 and prostate LNCaP cells was neutralized by sera from both mice (A) and rhesus macaques (B) challenged with XMRV, whereas no clear neutralization was observed with pre-immune sera, or HIV-luc(VSV G) pseudoviruses. (C) MLV-luc(MLV-E Env) pseudoviruses were neutralized by sera from rhesus macaques challenged with XMRV in mCAT-1 expressing CHO cells (CERD9 cells), but no clear neutralization of LacZ encoding MLV-P or HIV-luc(VSV G) pseudoviruses was observed in 293T/17 cells. Infection of pseudoviruses with firefly luciferase reporter was detected with Bright-Glo^™ Luciferase Assay System (Promega), whereas infection of LacZ encoding MLV-P was measured using Galacto-Light Plus System for detection of β-Galactosidase (Applied Biosystems). Absolute values for the no sera controls were: MLV-luc(XMRV Env) gave 55810 RLU on 293T cells and 20213 RLU on LNCaP cells; HIV-luc(VSV-G) gave 65961 RLU on 293T cells and 51677 RLU on CHO cells; MLV-P gave 32356 RLU on 293T cells and MLV-luc(MLV-E Env) gave 41771 RLU on CHO cells. Results are presented as percentage of neutralization and shown as mean ± S.D. of triplicate measurements. A representative experiment of at least two experiments is shown.

Fig. 2. XMRV Env neutralizing antibody in blood donor sera using a cell-based XMRV micro-neutralization assay system

Shown is an example screen of 80 donor serum samples (80-fold dilutions) for XMRV neutralization with virus combinations of MLV-luc(XMRV Env) and HIV-ren(Lassa GP) in a 96-well plate format. Three (B37, B58, and B80) out of a total of over 80 donor sera showed approximately 50% reduction in XMRV Env-, but not Lassa GP-, mediated viral infection in 293T/17 cells.

Fig. 3. Dose response curves with selected blood donor sera

Neutralization of infection of HIV-luc(XMRV Env) and HIV-luc(VSV G) pseudoviruses with serially diluted donor sera samples were detected in 293T/17 cells and HIV-luc(MLV-E Env) in mCAT-1 expressed CHO cells (CERD9 cell). Results are presented as percentage of neutralization and shown as mean ± S.D. of triplicate measurements. A representative of at least two experiments is shown.

Fig. 4. Absence of XMRV/MLV antibodies in blood donor sera by Western blot (WB) analysis

Purified, denatured XMRV antigen from XMRV-infected DU145 prostate cells (C7) was used for WB detection of anti-XMRV/MLV antibodies in selected donor sera samples. Results of positive control anti-sera to purified XMRV antigen and 24 normal donor sera samples (B58, B80, E6, E8, E10, D17, D40, C5, C20, C30, C33, C35, C45, C47, C49, C50, C51, C67, 3, 4, 5, 6, 7, 8, from left to right) are shown; locations of reactivity to specific viral proteins are indicated. Env (gp69/71), envelope; TM (p15E), transmembrane; Gag (pr68); MA (p15), matrix; CA (p30), capsid. Molecular weight markers (kD) are provided on the left of the WB.

Fig. 5. Absence of XMRV gag sequences in blood donor plasma by nested RT-PCR

A representative result of 12 donor samples is shown with positive controls containing 1 to 100 copies/μl of a plasmid harboring a cloned fragment of XMRV gag and negative water controls. First round PCR amplification used primer pair 419F and 1154R and second round PCR amplification used primer pairs Gag-I-F and Gag-I-R, or NP116 and NP117. GAPDH RNA and DNA PCR results for the same samples are shown in the bottom two panels.