XMRV was cultured and purified by Advanced Biotechnologies, Inc. (ABI, Columbia, MD). Briefly, XMRV (VP62)-infected DU145 prostate cancer cells  obtained from the Cleveland Clinic (Cleveland, OH) were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 200 units penicillin G and 200 μg/ml streptomycin. Virus from the culture supernatants was purified by sucrose gradient centrifugation.
Animals and virus inoculations
Three young adult rhesus macaques (>3 years old; >6 kg body weight) were selected from the Yerkes National Primate Research Center colony of Emory University. These included two males (RIl-10 and RLq-10) and one female (RYh-10). All three macaques were seronegative for antibodies to Simian immunodeficiency virus (SIV), Simian retrovirus (SRV) and Simian T-cell leukemia (STLV) and lacked cross-reactive antibodies to XMRV based on WB analysis using XMRV viral lysate.
After collection of baseline samples, each of the macaques was inoculated intravenously on day 0 with 10 ml of DU145 cell culture supernatant containing 3.6 × 105 TCID50/ml XMRV. One macaque (RLq-10) was sacrificed on day 144. To ensure persistent infections, 2 macaques (RIl-10 and RYh-10) were re-inoculated on day 158 with 3.6 × 106 TCID50 of purified XMRV virus. These 2 macaques were subsequently immunized on day 275 with 0.308 ml of recombinant XMRV proteins including p15 (2.3 μmole), p12 (1.1 μmole), p30 (3.1 μmole), p10 (2.9 μmole), p15E (3.8 μmole) and p70 (1.4 μmole) in incomplete Freund's adjuvant and sacrificed on day 291.
Blood was collected from each primate on days 0, 4, 5, 7, 9, 11, 14, 18, 21, 28, 35, 42, 56, 74, 95, 115, 134, 144, 158 post 1st inoculation and on days 3, 5, 7, 9, 11, 13, 21, 28, 34, 52, 117 post 2nd inoculation. Blood samples were obtained by venipuncture using tubes containing the anticoagulent EDTA and centrifuged at 250 × g for 10 minutes at room temperature. The plasma samples were collected in 0.5 ml aliquots and stored at -80°C.
Human specimens and MuLV antibodies
A total of 2262 random blood donor specimens (1080 sera and 1182 plasma) were obtained from the Gulf Coast Regional Blood Center (Houston, TX). All samples were non-reactive for bloodborne infectious diseases on donor screening tests; these included: HBsAg, anti-HCV, anti-HBc, anti-HIV-1/HIV-2, HIV-1 NAT, HCV NAT, anti-HTLV-I/II, Syphilis, West Nile Virus, and Chagas. Plasma specimens from 100 HIV-1 seropositive Cameroonian blood donors collected in 2007 in accordance with local country regulations were provided by Drs. Lazare Kaptué (Université des Montagnes, Bangangté, Cameroon) and Lutz Gürtler (Max von Pettenkofer Institute, Ludwig-Maximilian Universität, Munich, Germany). Plasma specimens from 5 HTLV-I and 5 HTLV-II seropositive US blood donors were obtained from the Abbott Diagnostics Specimen Bank (Abbott Park, IL).
Goat polyclonal antibodies included anti-Friend MuLV (anti-MuLV pAb) and anti-Env (gp69/71) of Rauscher MuLV (anti-Env pAb) (ATCC, VR-1537AS-Gt and VR-1521 respectively). Rat monoclonal antibody to gag p30 MuLV was produced at Abbott Diagnostics (Abbott Park, IL) using hybridoma cells (ATCC, CRL-1912). Mouse monoclonal antibody to Histidine (anti-His Mab) was from Abcam plc, (Cambridge, UK).
Western blot analysis
Western blot (WB) strips were produced using sucrose gradient purified XMRV (ABI) or recombinant proteins. Virus was lysed with 10 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl and 0.5% Triton at 100° C for 10 min. The viral lysate (80 μg/gel) or recombinant proteins (40-80 μg/gel) were separated by electrophoresis on a 4-12% NuPAGE Bis-Tris 2-dimension gel (Invitrogen, Carlsbad, CA) in the presence of sodium dodecylsulfate (SDS). The protein bands on the gel were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen) according to the manufacture's instructions.
WB analysis was performed using WesternBreeze kit reagents (Invitrogen) per the manufacturer's instructions. After blocking, the PVDF membrane was cut into 2 mm strips. WB strips were incubated with macaque sera or human blood donor samples diluted 1:250 (or as specified) overnight at 2-8°C. Goat anti-MuLV pAb and anti-Env pAb were diluted 1:1000 and incubated with WB strips at room temperature for 1 hour. After removal of unbound antibodies, WB strips were incubated with appropriate alkaline phosphatase conjugated secondary antibody for 30 minutes at room temperature. The strips were washed as described and chromogenic substrate solution was added. Goat anti-human IgM and IgG (Southern Biotech, Birmingham, AL) alkaline phosphatase conjugated secondary antibodies were used to individually detect IgM and IgG responses to XMRV infection in the macaque sera.
Competitive inhibition of macaque sera, anti-MuLV pAb or anti-Env pAb binding to WB strips was performed by pre-incubation of the samples with appropriate recombinant XMRV proteins at room temperature for 30 minutes to block specific antibodies. WB analysis was then performed with the pre-absorbed samples as described above.
For comparing antibody responses, WB strips were prepared using recombinant proteins (90 pmole/protein/strip): gp70 (4.4 μg/strip), p15E (4.1 μg/strip) and p30 (3.1 μg/strip). Western blot analysis was then performed with the primate samples diluted 1:200 as described.
Recombinant proteins derived from XMRV gp70, p15E, p30, p15, p12 and p10 were expressed in E. coli or HEK 293 mammalian cells (Invitrogen). For gp70 recombinant protein, a fragment containing amino acids (aa) 1-413 of the surface unit was cloned into an Abbott Laboratories vector selected for use in a mammalian protein expression system (Abbott Bioresearch Center, Worcester, MA) and expressed in DH5α cells (Invitrogen). Purified plasmid DNA was then used to transfect HEK293 cells (Invitrogen). The same sequence was also cloned into CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CKS) expression vector, pJO200 (Abbott Diagnostics), and expressed in XL1-Blue cells (Stratagene, La Jolla, CA) to produce the p70 recombinant protein which lacks glycosylation. For p15E, two versions of the TM protein were expressed. One version (aa 1-201) was cloned into PET expression vector pET-28b (+) (Novagen, Madison, WI) and expressed in E. coli BL21 (DE3) cells (Novagen). Another version with 14 aa deletion in the transmembrane region was cloned into pJO200 and expressed in XL1-Blue cells. For p30, the entire CA (aa 1-263) was cloned into both pJO200 and PL expression vector, pKRR826 (Abbott Diagnostics), and expressed in XL1-Blue cells and DH5α cells, respectively. p15 (aa 1-129 of MA), p12 (aa 1-84 of p12), and p10, (aa 1-56 of NC) were cloned into pJO200 and expressed in XL1-Blue cells. All recombinant proteins, except p15E cloned into the pJO200 vector, contained a 6-histidine tag.
Recombinant proteins produced as insoluble inclusion bodies within E. coli were solubilized in a solution of 6 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0 and 0.1% β-mercaptoethanol, clarified by centrifugation and purified by Sephacryl S-200 size exclusion chromatography (Pharmacia, Piscataway, NJ). All His tag recombinant proteins were further purified by His-Bind Nickel Affinity Chromatography (Novagen). The pJO200 expressed p15E was purified by Sephacryl S-200 size exclusion chromatography. Purity of all recombinant proteins was estimated at >90% by scanning densitometry.
Indirect format chemiluminescent immunoassays
The indirect prototype p15E, p70 and p30 antibody assays were developed on the high-throughput (200 tests/hour) and fully automated ARCHITECT® instrument system (Abbott Diagnostics, Dallas, TX). They are two-step chemiluminescent immunoassays (CMIAs) that utilize an indirect (anti-human) assay format (Figure 5A). Capture antigens were prepared by coating the E. coli-expressed recombinant proteins (p15E, p70 or p30) onto polystyrene paramagnetic microparticles (Varian, Inc. Palo Alto, CA). Detection conjugate was prepared by labelling a goat anti-human IgG (KPL, Inc. Gaithersburg, MD) with a chemiluminescent compound, N-hydroxysuccinimide ester of 10-sulfopropyl-n-tosyl-n-(2-carboxyethyl)-9-acridinium carboxamide trifluoromethyl sulfonate (SPCP-acridinium, Abbott Diagnostics). The acridinium labeled conjugate was purified with high-performance liquid chromatography (HPLC) using Bio-Sil SEC-250 gel filtration column (Bio-Rad Laboratories, Hercules, CA). In the first step of the assay (Additional file 2, section B1), a serum or plasma sample (10 μL) was mixed with specimen diluent buffer (90 μL) and antigen coated paramagnetic microparticles (50 μL) and incubated at room temperature (r.t.) for 18 minutes. During the incubation, the XMRV-specific antibodies present in the sample were captured on paramagnetic microparticles. Following incubation, the microparticles were washed to remove unbound proteins. In the second step, acridinium-labeled anti-human IgG conjugate (50 μL) was added and incubated at r.t. for 4 minutes. Following an additional wash cycle, alkaline hydrogen peroxide solution was added to release acridinium chemiluminescence signal. The intensity of the chemiluminescence, measured as relative light units (RLU) is proportional to the amount of specific antibody captured by the recombinant proteins.
Direct format chemiluminescent immunoassays
The prototype direct gp70, p15E and p30 antibody assays are CMIAs developed for the automated ARCHITECT® instrument system (assay format shown in Figure 5B). The capture antigens were prepared by individually coating recombinant proteins (E. coli-expressed p15E, p30 or mammalian-expressed gp70) onto polystyrene paramagnetic microparticles (Varian). For direct p15E and p30 CMIAs, detection antigens were prepared by labeling the p15E protein with N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide pentaflurophenyl ester (SPSP-acridinium, Abbott Diagnostics) and the p30 protein with SPCP-acridinium. Both conjugates were purified with HPLC using Bio-Sil SEC-250 gel filtration column (Bio-Rad Laboratories). The p15E and p30 CMIAs utilized a two-step assay protocol as depicted in Scheme B2 (Additional file 2). In the first step of the assay, a serum or plasma sample (100 μL) was mixed with specimen diluent buffer (50 μL) and p15E or p30 antigen coated paramagnetic microparticles (50 μL) and incubated at r.t. for 18 minutes. During the incubation, the XMRV-specific antibodies present in the sample were captured on paramagnetic microparticles. Following the incubation, the microparticles were washed to remove unbound proteins. In the second step, 50 μl of acridinium-labeled p15E or p30 was added and incubated at r.t. for 4 minutes. Following an additional wash cycle, alkaline hydrogen peroxide solution was added to release acridinium chemiluminescence signal. The intensity of the chemiluminescence, measured as relative light units (RLU) is proportional to the amount of specific antibody captured by the recombinant proteins p15E or p30.
For the direct format gp70 CMIA, avidin-biotin complex (ABC) was used to enhance gp70 conjugate potency. The gp70 protein was labeled with NHS-LC-LC-Biotin (Thermo Fisher Scientific Inc. Rockford, IL) and Streptavidin (Leinco Technologies, Inc. St Louis, Missouri) was labeled with SPCP-acridinium. After purification by HPLC using Bio-Sil SEC-250 gel filtration column (Bio-Rad Laboratories), the purified biotinylated gp70 protein was mixed with purified acridinylated streptavidin at 1.3:1 molar ratio to form the ABC gp70 conjugate. A one-step assay protocol was utilized to provide a longer incubation time for the ABC gp70 conjugate with anti-gp70 antibodies. In the one-step protocol (Additional file 2, section B3) a serum or plasma sample (100 μL), ABC gp70 conjugate (50 μL) and gp70 coated paramagnetic microparticles (50 μL) were combined and incubated at r.t. for 22 minutes. During incubation, anti-gp70 antibodies present in the sample simultaneously bound to the ABC gp70 conjugate and the gp70 antigen-coated paramagnetic microparticles. Following the incubation, the microparticles were washed to remove unbound proteins and ABC gp70 conjugate. Chemiluminescent signal measurement was performed as described above.