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Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer and Liver Disease

Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer... Hindawi Journal of Oncology Volume 2020, Article ID 8958192, 9 pages https://doi.org/10.1155/2020/8958192 Research Article Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer and Liver Disease 1,2 1 1 1 1 Guangzhi Zhang, Kiandokht Bashiri, Mark Kneteman, Kevan Cave, Youngkee Hong, 3 4 1,5 John R. Mackey, Harvey J. Alter, and Andrew L. Mason Center of Excellence for Gastrointestinal Inflammation and Immunity Research, Division of Gastroenterology, University of Alberta, Edmonton, AB T6G 2E1, Canada National Microbiology Laboratory, Winnipeg, MB R3E 3M4, Canada Department of Medical Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD 20892, USA Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada Correspondence should be addressed to Andrew L. Mason; andrew.mason@ualberta.ca Received 30 May 2019; Revised 7 December 2019; Accepted 2 January 2020; Published 27 January 2020 Guest Editor: Hironori Yoshiyama Copyright © 2020 Guangzhi Zhang et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mouse mammary tumor virus (MMTV) is a betaretrovirus that plays a causal role in the development of breast cancer and lymphoma in mice. Closely related sequences that share 91–99% nucleotide identity with MMTV have been repeatedly found in humans with neoplastic and inflammatory diseases. Evidence for infection with a betaretrovirus has been found in patients with breast cancer and primary biliary cholangitis and referred to as the human mammary tumor virus and the human betaretrovirus (HBRV), respectively. Using the gold standard technique of demonstrating retroviral infection, HBRV proviral integrations have been detected in cholangiocytes, lymph nodes, and liver of patients with primary biliary cholangitis. However, the scientific biomedical community has not embraced the hypothesis that MMTV like betaretroviruses may infect humans because reports of viral detection have been inconsistent and robust diagnostic assays are lacking. Specifically, prior serological assays using MMTV proteins have produced divergent results in human disease. Accordingly, a partial HBRV surface (Su) construct was transfected into HEK293 to create an ELISA. -e secreted HBRV gp52 Su protein was then used to screen for serological responses in patients with breast cancer and liver disease. A greater proportion of breast cancer patients (n � 98) were found to have serological reactivity to HBRV Su as compared to age- and sex-matched control subjects (10.2% versus 2.0%, P � 0.017, OR � 5.6 [1.25–26.3]). Similarly, the frequency of HBRV Su reactivity was higher in patients with primary biliary cholangitis (n � 156) as compared to blood donors (11.5% vs. 3.1%, P � 0.0024, OR � 4.09 [1.66–10.1]). While the sensitivity of the HBRV Su ELISA was limited, the assay was highly specific for serologic detection in patients with breast cancer or primary biliary cholangitis, respectively (98.0% [93.1%–99.7%] and 97.0% [93.4%–98.6%]). Additional assays will be required to link immune response to betaretrovirus infection and either breast cancer or primary biliary cholangitis. plays a direct role in the development of breast cancer in mice 1. Introduction [5]. Indeed, cloned betaretrovirus nucleotide sequences from Breast cancer is the most frequent cancer diagnosis among humans reportedly share between 91% and 99% identity with females and a leading cause of cancer deaths worldwide [1, 2]. various regions of the MMTV genome [6–9]. However, di- Several viruses have been linked with the development of agnostic assays are lacking to reproducibly detect betaretrovirus human breast cancer, but none have been established as having infection in humans [10]. a causal etiology [3, 4]. One such agent resembles mouse MMTV does not encode an oncogene but rather acti- mammary tumor virus (MMTV), a murine betaretrovirus that vates growth pathways by insertional mutagenesis to 2 Journal of Oncology observed between HBRV Env compared to MMTV Env that promote carcinogenesis in mice [11]. -e diagnosis of MMTV infection in mice can be challenging. -e viral may alter antigenicity [6]. In the present study, we expressed the HBRV gp52 surface (Su) protein in human cells to create burden is below the limits of detection in blood, and the agent is encoded as an endogenous retrovirus in most mice; an enzyme-linked immunosorbent assay (ELISA). Herein, therefore, exogenous viral genomic nucleic acid sequences we report the seroprevalence of anti-HBRV gp52 Su reac- cannot easily be distinguished from the endogenous ex- tivity in patients with breast cancer, patients with liver pression of MMTV [12]. Furthermore, inadequate humoral disease, and healthy subjects. responses are made by weanling pups infected via ingestion of MMTV in milk due to the tolerizing effects of neonatal 2. Materials and Methods infection by the oral route [13]. Accordingly, a diagnosis of MMTV infection is made by assessing the skewing of T-cell 2.1. Ethics. -e study protocol was approved by the Human Ethics Review Board from the University of Alberta and receptor V-β subsets to demonstrate the MMTV super- antigen effect [14]. institutional review boards/ethics committees at each site. -e project was conducted in accordance with the Decla- Evidence for human infection first surfaced in 1971, when B-type particles resembling MMTV were observed by ration of Helsinki (1964). electron microscopy in the milk of breast cancer patients [15]. Breast cancer patients were also reported to harbor 2.2.PatientSamples. A serum panel of breast cancer patients betaretrovirus nucleic acid sequences and/or proteins in (n � 98) and age/sex-matched controls (n � 102) was ob- various samples, including milk [16], serum [17], salivary tained from the Alberta Tomorrow Project, a longitudinal glands [18], as well as breast cancer tissue [19], cyst fluid [20], study tracking 55,000 adults in Alberta [40]. Liver disease and breast cancer cells in culture [21, 22]. -ereafter, patient serum was prospectively collected from the hep- betaretrovirus sequences resembling MMTV were PCR- atology outpatients at the Zeidler Clinic, University of cloned from breast cancer tissues derived from various Alberta Hospital from January 2003 to December 2014. countries, and the agent was referred to as the human Serum from 156 patients with PBC, 46 with primary scle- mammary tumor virus [7, 23–27]. rosing cholangitis (PSC), 16 with autoimmune hepatitis In 2003, a human betaretrovirus (HBRV) was charac- (AIH), 25 with nonalcoholic fatty liver disease (steatosis), 8 terized in patients with primary biliary cholangitis (PBC; with alcoholic liver disease (ALD), 19 with viral hepatitis, 6 previously known as primary biliary cirrhosis [28]), an with cryptogenic liver disease, and 19 with miscellaneous inflammatory autoimmune liver disease. -e agent was liver disease. Healthy blood donors’ serum samples (n � 194) predominantly detected in perihepatic lymph nodes and was were provided by the Department of Transfusion Medicine, shown to promote the expression of mitochondrial auto- National Institute of Health, Bethesda, MD. antigens in cocultivation studies with cholangiocytes, a well- characterized PBC disease-specific phenotype [9, 29]. Evi- dence of human betaretrovirus proviral integrations was 2.3. RecombinantDNAExpressionConstructs. -e HBRV Su subsequently demonstrated in PBC patients by ligation- was derived from HBRV sequences obtained from a PBC mediated PCR and Illumina sequencing, using a bio- patients’ perihepatic lymph node [6]. -e HBRV Su coding informatics pipeline that ensured the exclusion of all se- sequence was cloned into pcDNA3.1 (Invitrogen) vector quences potentially related to murine or HERV sequences. along with a TAP tag at the 3′ terminus of the HBRV Su [41] More than 2,200 unique HBRV integrations were identified, and 4 copies of M-PMV cytoplasmic transport element and the majority of PBC patients were found to have evi- (CTE) downstream [42]. -e expressed HBRV envelope dence of proviral integrations linked with HBRV RNA protein sequence corresponds to amino acids 99 to 455 in the production in cholangiocytes [30]. In clinical trials, PBC surface region that includes the receptor-binding domain, patients on combination antiretroviral therapy have shown which shares 97% and 98% amino acid identity with MMTV biochemical and histological improvement with therapy Su [6] (see supplementary material for HBRV Su and [31–34]. MMTV Su alignment; Supplementary Figure 1). -e hypothesis that a betaretrovirus may be linked with human breast cancer has gained little traction over the years because of the inconsistency of findings in different reports, 2.4. Cell Culture, Transfection, and Stable Cell Line a concern for cross-reactivity with human endogenous Generation. HEK293T cells (ATCC) were routinely main- retroviruses (HERV) and the low level of viral burden tained in Dulbecco’s modified Eagle’s medium supple- [10, 35, 36]. With regard to the potential for a link with mented with 10% fetal bovine serum (Gibco) and 100 μg/ml betaretrovirus infection and PBC, investigators have either noromycin. Transfection of HEK293T was performed using been unable to detect viral infection [37] or to confirm the PEI as described previously [43]. Briefly, 10 cells were specificity of HBRV infection in PBC patients [38]. Fur- seeded in 6-well plates one day before transfection, and 2 μg thermore, serological studies using MMTV preparations as of each plasmid was used for each well. To generate stable substrate have been unable to demonstrate specific antibody HEK293T cell lines harboring pHBRV Su-TAP-4C FW, the reactivity to defined MMTV proteins [37, 39]. While HBRV pHBRV Su-TAP-4C FW-puromycin plasmid was linearized shares between 93% and 97% amino acid identity with the with PvuI and transfected into HEK293T cells. Individual MMTV envelope protein, consistent differences have been clones were selected with puromycin (Invitrogen). Journal of Oncology 3 5′ UTR 3′ UTR gag pol pCMV-Su CMV SP Su TAP poly A pol Proviral DNA env U3 RU5 U3 RU5 pCMV-Su-4c CMV SP Su TAP CTE CTE CTE CTE poly A Env mRNA CMV pCMV-Su-4cr SP Su TAP CTE CTE CTE CTE poly A 99 457 Env protein SP Su Tm p14 gp52 gp36 (a) (b) KDa pCMV-Su-4c (c) Figure 1: (a) -e single spliced mRNA of betaretrovirus Env encodes the signal peptide, surface, and transmembrane proteins. (b) -e HBRV Su construct used for mammalian expression contained the cytomegalovirus immediate early promoter, HBRV SP, and Su, a TAP tag; pCMV-Su-4c contained 4 copies of M-PMV CTE inserted in the downstream of Su-TAP in either the sense (pCMV-Su-4c) or the antisense (pCMV-Su-4cr) orientation. (c) Only the pCMV-Su-4c containing the CTE in the correct orientation produced sufficient HBRV Su protein in the cell pellet and supernatant as shown by the western blot analysis. 2.5. Western Blot Analysis. Secreted HBRV Su protein in proviral integrations and HBRV RNA by the QuantiGene 400 μl supernatant was precipitated with TCA and dissolved assay and in situ hybridization [30]. in PBS. Cell lysates were prepared from transfected and For detection of serological reactivity to HBRV Su, stable cells using RIPA buffer with complete proteinase 100 ng of purified protein was resolved on a 10% SDS-PAGE inhibitor (Roche). Approximately 2 ×10 cells were collected minigel (Bio-Rad) and transferred to nitrocellulose mem- and washed twice with ice-cold PBS, incubated with RIPA brane. -e membrane was cut into 5 mm wide stripes. Each buffer on ice for 30 min, and centrifuged at 20,000 ×g for 30 stripe was incubated with serum from a breast cancer patient minutes. Proteins from cell supernatant and lysate were or a control (1 : 400 dilution) and IRDye goat anti-human quantified using the BCA assay (Bio-Rad), and 50 μg and secondary antibody. 100 μg of total protein from cell lysate and supernatant, respectively, were resolved by 10% sodium dodecyl sulfate- 2.6. Scale-Up of HBRV Su Production and Purification and (SDS-) polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane as previously de- Characterization. Stable cells expressing HBRV Su were expanded to 12 ×15 cm cell cultural dishes in Dulbecco’s scribed (Figure 1) [44]. Western blot analysis was performed using the primary modified Eagle’s medium supplemented with 10% fetal bovine serum. -e medium in each plate was replaced with polyclonal goat anti-MMTV envelope gp52 antibody (kindly provided by Dr. Susan Ross, University of Pennsylvania), 25 ml Pro293 CD serum-free medium (Lonza) when cells reached 95% confluence. -e medium was collected after 5-6 mouse monoclonal anti-Flag antibody (Sigma-Aldrich), and IRDye goat anti-mouse and rabbit anti-goat secondary days of incubation and centrifuged at 3,000 g for 20 min. -e clarified medium was adjusted to pH 8.0 and filtered through antibodies (LI-COR). Reacting membranes were visualized with LI-COR Odyssey infrared imaging system. -e anti- a 0.22 μm filter before purification. MMTV envelope gp52 antibody has demonstrable reactive Purification of HBRV Su was performed on 1 ml Histrap biliary epithelial cells extracted from a liver transplant re- FF crude column and buffers as suggested by the supplier cipients with PBC (Supplementary Figure 2), previously (GE Healthcare) using an AKTA explorer 100 (Amersham Pharmacia Biotech). -e conditioned medium was loaded to shown to have HBRV infection with documented HBRV Mock pCMV-Su pCMV-Su-4cr pCMV-Su-4c Supernatant Pellet 4 Journal of Oncology the equilibrated column at the rate of 1 ml/min, and the (Figure 1(c)). -erefore, an M-PMV cytoplasmic transport column was then washed with 20 ml binding buffer and element (CTE) was incorporated into the construct to in- crease protein expression [42]. To this end, two additional Su eluted into 10 × 0.5 ml fractions using elution buffer. -e peak elution fraction was combined and changed to proteins expression constructs were generated with the 4 copies of storage buffer by ultrafiltration (Millipore, 30 kDa cutoff M-PMV CTE inserted in the downstream of Su-TAP for limit concentrator, 4000 g for 20 min). -e final preparation expression studies. Following expression in HEK293T, in- was aliquoted for storage at − 80 C for ELISA. -e 10 eluted creased production of HBRV Su was observed in cell lysates fractions were assessed by western blot analysis using anti- transfected with the pCMV-Su-Tap-4c but not in cells with MMTV Env antibody or anti-FLAG antibody and 10% SDS- the pCMV-Su-Tap-4cr construct that had the CTE arranged PAGE gels stained with Coomassie R-250 blue stain (Bio- in the antisense orientation. Moreover, we were able to Rad). -e protein concentration was determined by BCA detect secreted Su protein in the medium of the cells assay (Pierce) using bovine serum albumin (BSA) as a transfected with the pCMV-Su-Tap-4c plasmid two days standard. after transfection (Figure 1(c)). 3.2. Large-Scale Production and Purification of HBRV Su. 2.7. HBRV Su ELISA. ELISA was performed at room tem- Since abundant HBRV Su protein was secreted from perature with all sera in duplicate using high-binding 293Tcells transfected with the pCMV-Su-Tap-4c plasmids, a microplates (Greiner, Monroe, USA). Briefly, wells were strategy was developed to purify the protein directly from a coated with 100 μl of 2 ng/μl purified HBRV Su in PBS for 18 large-scale cell culture medium (Figure 2(a)). Stable hours and blocked with 1% BSA in PBS for 3 hours. Serum 293T cell lines were generated following transfection with was incubated at 100 μl/well at a 1 : 400 dilution in PBS with the pCMV-Su-Tap-4c plasmid and the cells with the highest 1% BSA (Sigma) for 1 hour. A serial dilution of polyclonal Su secretion in the culture medium were expanded to anti-MMTV Env was included on each plate as a standard 12 ×15 cm cell culture dishes using DMEM supplemented and then incubated with 100 μl/well donkey anti-human and with 10% FBS. When cells reached 90–95% confluence, the donkey anti-goat secondary antibodies (Jackson Immuno- medium was replaced with serum-free medium and incu- Research Lab) for 1 hour. -e plate was washed 3 × 5 min bated for another 5 days before collection. Approximately after each step using PBS with 0.5% Tween. Plates were 300 ml was obtained for each batch, which was then purified developed with 100 μl/well tetramethylbenzidine substrate with chromatography to derive 150–200 μg HBRV Su (TMB, Sigma) for 20 min and then stopped with 50 μl/well protein. SDS-PAGE revealed that the purified Su protein was 2N H SO . -e absorbance at 450 nm and 540 nm (back- 2 4 homogeneous and devoid of other contaminants. Western ground) was measured with EMAX Plus Microplate Reader blot analysis with polyclonal anti-MMTV Env confirmed (Molecular Devices, USA) and the cutoff level was estab- that the purified protein was HBRV Su along with select lished using the reactivity of control samples by adding the serum from seropositive and negative breast cancer and mean background level to 3 × S.D. Two-tailed Fisher’s exact control samples (Figures 2(b) and 2(c)). test was used to assess significant differences in frequency between different groups, followed by calculation of the odds ratio (Baptista–Pike methodology) along with sensitivity, 3.3. Detection of Anti-HBRV Su Protein Antibodies by ELISA. specificity, positive predictive value, negative predictive -e ELISA protocol was established using 200 ng/well of value, and likelihood ratio (Wilson Brown methodology) purified HBRV Su. -e antibody response was calculated by using Prism 8 software. converting the optical density reading to the equivalent ng/ ml reactivity of the positive control, polyclonal anti-MMTV Env antibody. -e background reactivity was calibrated 3. Results using the serum samples from the age/sex-matched healthy 3.1. HBRV Su Expression in HEK 293T Cells. A mammalian controls used as a comparison group for the breast cancer expression system was employed to express the HBRV Su patients. -e cutoff level (mean background + 3 × S.D.) was because prior attempts to express multiple constructs calculated as 61 ng/mL and samples found to be greater than this were considered positive (Figure 3). Accordingly, a expressing HBRV Env protein in bacteria and baculovirus systems were not sufficiently productive. MMTV Env greater proportion of breast cancer patients (10.2%) were found to have serological reactivity to HBRV Su versus 2.0% protein is encoded by a single spliced mRNA in mice, which produces a signal peptide (SP p14), surface (Su gp52), and of age- and sex-matched control subjects (Figure 3: P � 0.017, OR � 5.6 [1.25–26.3]). transmembrane domain (TM gp36) (Figure 1(a)); the Su protein is generated by removal of the signal peptide by -e seroprevalence of HBRV Su reactivity in patients signal peptidase and cleavage of the transmembrane domain with PBC was comparable to that observed in patients with by cellular Furin. -erefore, a mammalian expression vector breast cancer (Figure 3: 11.5% vs. 10.2%). -e frequency of pCMV Su-Tap was constructed, using the cytomegalovirus HBRV Su reactivity was significantly higher in PBC patients immediate early promoter to drive protein expression and a vs. blood donors (11.5% vs. 3.1%, P � 0.0024, OR � 4.09 TAP tag to enable protein purification (Figure 1(b)). Using [1.66–10.1]). In prior studies using the gold standard methodology of detecting HBRV integrations in patients’ the pCMV-Su-TAP construct, very little HBRV Su protein was detected in lysates from transfected HEK293T cells cholangiocytes, subjects with cryptogenic liver disease and Journal of Oncology 5 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 CMV SP SU TAP CTE CTE CTE CTE poly A pCMV-Su-4c KDa Transfection Stable cell line selection Cell expansion Coomassie blue staining Supernatant collection Anti-gp52 His-tag column loading Washing and elution (a) (b) (c) Figure 2: (a) Schematic showing large scale HBRV Su protein purification from the supernatant of HEK293T cells using a His-tag column. (b) Coomassie blue staining and western blot analysis demonstrate the purity of the HBRV gp52 protein using anti-MMTV gp52 Su in sequential elutions. (c) Western blot confirmation of ELISA positive and negative samples demonstrates reactivity using select breast cancer, PBC, and blood donor control samples. -e breast cancer serum sample used in lane 7 is positive by western blot and negative by ELISA. AIH were found to harbor infection, and in this study, breast cancer and PBC, respectively (98.0% [93.1%–99.7%] isolated reactivity was observed in subjects with cryptogenic and 97.0% [93.4%–98.6%]). Accordingly, the positive liver disease (16.7%) and AIH (6.3%), whereas other subjects predictive values (83.3% [55.2%–97.0%] and 75.0% [55.1%– with liver disease were universally negative (Figure 3(b)). 88.0%]) were diagnostically more useful that the negative predictive values (53.2% [46.1%–60.2%] and 57.7% [52.3%– While reactivity in healthy blood donors was incre- mentally higher than the healthy age/sex-matched com- 62.9%]) for patients in the breast cancer and the liver parison group for the breast cancer patients, the difference disease study groups. was not found to be significant (3.1% vs. 2.0%; P � 0.72). -e sensitivity of the HBRV Su ELISA was limited in 4. Discussion detecting reactivity in patients with breast cancer and PBC as compared to their respective control groups (10.2% -is is the first report using an HBRV ELISA for assessing [5.6%–17.8%] and 11.5% [7.4%–17.5%]), whereas the assay the seroprevalence of infection in patients. Approximately was highly specific for serologic detection in patients with 10% of breast cancer and PBC patients had detectable anti- Positive control Negative control Breast cancer (ELISA +ve) PBC (ELISA –ve) PBC (ELISA –ve) PBC (ELISA +ve) Breast cancer (ELISA –ve) Blood donor(ELISA –ve) Blood donor(ELISA +ve) PBC (ELISA +ve) AIH (ELISA +ve) PSC (ELISA –ve) PBC (ELISA –ve) 6 Journal of Oncology 200 800 0 0 (a) (b) Figure 3: (a) A higher percentage of reactivity to HBRV Su was observed in breast cancer patients’ sera versus age/sex-matched healthy controls (10/98 vs. 2/102; P � 0.017). (b) Anti-HBRV reactivity was highest in patients with PBC (18/156) and found in AIH (1/16), cryptogenic liver disease (1/6), and healthy blood donors (6/194), whereas reactivity was not observed in patients with PSC, steatosis (NAFLD), ALD, or miscellaneous liver disease (PBC vs. blood donors 11.5% vs. 3.1%, P � 0.0024, OR � 4.09 [1.66–10.1]). HBRV Su, and the test was found to be highly specific for was attributed to autoreactivity with the antimitochondrial both disorders. -e likelihood ratio for having breast cancer antibody, which is found in up to 95% of patients and used with HBRV Su reactivity was 5.2 and for having PBC with for diagnosing PBC [29], whereas similar MMTV western HBRV Su reactivity was 3.7; the difference in likelihood blot studies employing mitochondrial proteins to remove the autoantibodies from PBC patients’ serum demonstrated ratios probably reflects the chosen control groups for each disorder. Notably, the breast cancer control subjects were the presence of signal to the betaretrovirus gp52 surface mainly middle-aged women and therefore a more suitable protein [48]. As the purified antimitochondrial antibody has control group for the PBC patients, who are also predom- no reactivity with HBRV Su, we can conclude that humans inantly female; whereas the blood donors were more of an do make humoral responses to HBRV based on our ELISA. admixture of both sexes. -e healthy comparison groups A second issue to be addressed is that the prevalence of revealed a sizeable population seroprevalence of ∼2-3%. infection detected by the HBRV Su ELISA was somewhat -ese data are in keeping with the hypotheses that HBRV lower than other reports using different techniques to di- infection may only be disease related in genetically pre- agnose disease. Indeed, our western blots (Figure 2(c)) show disposed individuals [10, 45]. reactivity to one breast cancer sample that was negative by Prior seroprevalence studies using MMTV proteins have the ELISA, suggesting that our cutoff level may have been too stringent. Using nonserological techniques, a meta- been widely inconsistent. For example, an ELISA-based study using MMTV proteins demonstrated serological re- analysis of molecular epidemiological studies reported a activity in 26% of breast cancer patients and 8% of healthy prevalence of 40% HBRV infection in Western countries controls [46]. A similar study found no difference between based on PCR detection of betaretrovirus sequences in breast breast cancer patients and their respective controls [47] and cancer samples [49]. An even higher prevalence of infection a study using 4 strains of MMTV reported only nonspecific has been reported in PBC patients based on the presence of reactivity in breast cancer patients, although reactivity proviral HBRV integrations detected by ligation-mediated consistent with the molecular weights of viral proteins was PCR and Illumina sequencing, with provirus found in 58% observed in individual strains of MMTV [39]. In studies of of cholangiocytes from patients with PBC as compared to 7% patients with liver disease, MMTV western blot reactivity of nonautoimmune liver disease controls [30]. -e Anti-HBRV Su levels (ng/mL) Breast cancer Age-matched controls Anti-HBRV Su levels (ng/mL) PBC PSC AIH Steatosis ALD Viral hep. Crytogenic Liver disease misc. Blood donors Journal of Oncology 7 discrepancy of a higher frequency of viral infection in tissue patients participating in the Alberta Tomorrow Project who as compared to a lower seroprevalence of anti-HBRV Su subsequently developed breast cancer. Accordingly, we will reactivity may be partly explained by observations from be able to study whether anti-HBRV Su predates the de- neonatal mouse infection. Weanling pups have a high risk of velopment of disease and may act as a biomarker for breast developing breast cancer from MMTV infection because cancer. they become immunotolerant to viral infection. -is occurs because MMTV is taken up in the gut-associated lymphoid Data Availability tissue along with bacterial lipopolysaccharide, which triggers -e data used to support the findings of this study are in- a cascade of events. -e lipopolysaccharide/viral complex cluded within the supplementary information files and engages Toll-like receptor 4 that in turn triggers an IL-4- and available on request from the corresponding author. IL-6-dependent production of IL-10, which renders the mouse unresponsive to MMTV Su and prevents the for- Conflicts of Interest mation of neutralizing antibodies [13]. It is currently un- known whether a similar immunological process may occur -e authors declare that there are no conflicts of interest in humans with HBRV infection. Notably, the cellular regarding the publication of this paper. immune response to HBRV peptides is more prevalent in patients with liver disease [50]. Authors’ Contributions Our overall goal was to derive a reliable and repro- ducible diagnostic ELISA to investigate the frequency of AM designed and coordinated the study with JRM and HJA. HBRV infection. In prior experiments, we used bacterial or KB, GZ, and AM interpreted data and wrote the article. GZ, baculovirus expressed proteins but failed to generate suf- MK, KC, and YH performed the majority of the experi- ficient amounts of pure viral protein. We also generated ments. GZ generated the constructs. All authors read and serological data using the bacterially expressed Gag pro- approved the final manuscript. teins, and while a higher seroprevalence was observed in our PBC population as a whole, no significant differences were Acknowledgments found between patients and controls with liver disease. Notably, cross reactivity with retroviral Gag (Group Anti- We thank the Alberta Cancer Foundation, Alberta Innovates Gen) is a common occurrence in patients with any viral Health Solutions, Canadian Institutes for Health Research, infection due to the positively charged antigenic determi- and Canadian Liver Foundation for supporting this project nants in capsid and core proteins surrounding the viral as well as Michael Houghton and Michael Sakalian for their genome [51, 52]. For this ELISA, a novel strategy for large- assistance. scale production of purified and secreted HBRV Su protein was developed using HEK 293T cells. -ree factors con- Supplementary Materials tributed to the production of HBRV Su sufficient for HBRV Su expression construct and coding sequences; multiple ELISAs: these included (i) using multiple copies of alignment of HBRV Su and MMTV Su proteins as well as CTE downstream of the Su coding region to enhance HBRV anti-MMTV gp52 Su reactivity to biliary epithelial cells Su expression and secretion; (ii) ensuring the stable ex- cultured from a PBC patient’s resected liver following liver pression of HBRV Su protein in human cells; and (iii) transplantation. (Supplementary Materials) replacing the FBS containing medium with serum-free medium to remove a source of protein contamination and References ensure the high purity of protein after chromatography purification. We can also speculate that the use of HBRV [1] A. Jemal, F. Bray, M. M. 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Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer and Liver Disease

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Copyright © 2020 Guangzhi Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2020, Article ID 8958192, 9 pages https://doi.org/10.1155/2020/8958192 Research Article Seroprevalence of Human Betaretrovirus Surface Protein Antibodies in Patients with Breast Cancer and Liver Disease 1,2 1 1 1 1 Guangzhi Zhang, Kiandokht Bashiri, Mark Kneteman, Kevan Cave, Youngkee Hong, 3 4 1,5 John R. Mackey, Harvey J. Alter, and Andrew L. Mason Center of Excellence for Gastrointestinal Inflammation and Immunity Research, Division of Gastroenterology, University of Alberta, Edmonton, AB T6G 2E1, Canada National Microbiology Laboratory, Winnipeg, MB R3E 3M4, Canada Department of Medical Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD 20892, USA Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada Correspondence should be addressed to Andrew L. Mason; andrew.mason@ualberta.ca Received 30 May 2019; Revised 7 December 2019; Accepted 2 January 2020; Published 27 January 2020 Guest Editor: Hironori Yoshiyama Copyright © 2020 Guangzhi Zhang et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mouse mammary tumor virus (MMTV) is a betaretrovirus that plays a causal role in the development of breast cancer and lymphoma in mice. Closely related sequences that share 91–99% nucleotide identity with MMTV have been repeatedly found in humans with neoplastic and inflammatory diseases. Evidence for infection with a betaretrovirus has been found in patients with breast cancer and primary biliary cholangitis and referred to as the human mammary tumor virus and the human betaretrovirus (HBRV), respectively. Using the gold standard technique of demonstrating retroviral infection, HBRV proviral integrations have been detected in cholangiocytes, lymph nodes, and liver of patients with primary biliary cholangitis. However, the scientific biomedical community has not embraced the hypothesis that MMTV like betaretroviruses may infect humans because reports of viral detection have been inconsistent and robust diagnostic assays are lacking. Specifically, prior serological assays using MMTV proteins have produced divergent results in human disease. Accordingly, a partial HBRV surface (Su) construct was transfected into HEK293 to create an ELISA. -e secreted HBRV gp52 Su protein was then used to screen for serological responses in patients with breast cancer and liver disease. A greater proportion of breast cancer patients (n � 98) were found to have serological reactivity to HBRV Su as compared to age- and sex-matched control subjects (10.2% versus 2.0%, P � 0.017, OR � 5.6 [1.25–26.3]). Similarly, the frequency of HBRV Su reactivity was higher in patients with primary biliary cholangitis (n � 156) as compared to blood donors (11.5% vs. 3.1%, P � 0.0024, OR � 4.09 [1.66–10.1]). While the sensitivity of the HBRV Su ELISA was limited, the assay was highly specific for serologic detection in patients with breast cancer or primary biliary cholangitis, respectively (98.0% [93.1%–99.7%] and 97.0% [93.4%–98.6%]). Additional assays will be required to link immune response to betaretrovirus infection and either breast cancer or primary biliary cholangitis. plays a direct role in the development of breast cancer in mice 1. Introduction [5]. Indeed, cloned betaretrovirus nucleotide sequences from Breast cancer is the most frequent cancer diagnosis among humans reportedly share between 91% and 99% identity with females and a leading cause of cancer deaths worldwide [1, 2]. various regions of the MMTV genome [6–9]. However, di- Several viruses have been linked with the development of agnostic assays are lacking to reproducibly detect betaretrovirus human breast cancer, but none have been established as having infection in humans [10]. a causal etiology [3, 4]. One such agent resembles mouse MMTV does not encode an oncogene but rather acti- mammary tumor virus (MMTV), a murine betaretrovirus that vates growth pathways by insertional mutagenesis to 2 Journal of Oncology observed between HBRV Env compared to MMTV Env that promote carcinogenesis in mice [11]. -e diagnosis of MMTV infection in mice can be challenging. -e viral may alter antigenicity [6]. In the present study, we expressed the HBRV gp52 surface (Su) protein in human cells to create burden is below the limits of detection in blood, and the agent is encoded as an endogenous retrovirus in most mice; an enzyme-linked immunosorbent assay (ELISA). Herein, therefore, exogenous viral genomic nucleic acid sequences we report the seroprevalence of anti-HBRV gp52 Su reac- cannot easily be distinguished from the endogenous ex- tivity in patients with breast cancer, patients with liver pression of MMTV [12]. Furthermore, inadequate humoral disease, and healthy subjects. responses are made by weanling pups infected via ingestion of MMTV in milk due to the tolerizing effects of neonatal 2. Materials and Methods infection by the oral route [13]. Accordingly, a diagnosis of MMTV infection is made by assessing the skewing of T-cell 2.1. Ethics. -e study protocol was approved by the Human Ethics Review Board from the University of Alberta and receptor V-β subsets to demonstrate the MMTV super- antigen effect [14]. institutional review boards/ethics committees at each site. -e project was conducted in accordance with the Decla- Evidence for human infection first surfaced in 1971, when B-type particles resembling MMTV were observed by ration of Helsinki (1964). electron microscopy in the milk of breast cancer patients [15]. Breast cancer patients were also reported to harbor 2.2.PatientSamples. A serum panel of breast cancer patients betaretrovirus nucleic acid sequences and/or proteins in (n � 98) and age/sex-matched controls (n � 102) was ob- various samples, including milk [16], serum [17], salivary tained from the Alberta Tomorrow Project, a longitudinal glands [18], as well as breast cancer tissue [19], cyst fluid [20], study tracking 55,000 adults in Alberta [40]. Liver disease and breast cancer cells in culture [21, 22]. -ereafter, patient serum was prospectively collected from the hep- betaretrovirus sequences resembling MMTV were PCR- atology outpatients at the Zeidler Clinic, University of cloned from breast cancer tissues derived from various Alberta Hospital from January 2003 to December 2014. countries, and the agent was referred to as the human Serum from 156 patients with PBC, 46 with primary scle- mammary tumor virus [7, 23–27]. rosing cholangitis (PSC), 16 with autoimmune hepatitis In 2003, a human betaretrovirus (HBRV) was charac- (AIH), 25 with nonalcoholic fatty liver disease (steatosis), 8 terized in patients with primary biliary cholangitis (PBC; with alcoholic liver disease (ALD), 19 with viral hepatitis, 6 previously known as primary biliary cirrhosis [28]), an with cryptogenic liver disease, and 19 with miscellaneous inflammatory autoimmune liver disease. -e agent was liver disease. Healthy blood donors’ serum samples (n � 194) predominantly detected in perihepatic lymph nodes and was were provided by the Department of Transfusion Medicine, shown to promote the expression of mitochondrial auto- National Institute of Health, Bethesda, MD. antigens in cocultivation studies with cholangiocytes, a well- characterized PBC disease-specific phenotype [9, 29]. Evi- dence of human betaretrovirus proviral integrations was 2.3. RecombinantDNAExpressionConstructs. -e HBRV Su subsequently demonstrated in PBC patients by ligation- was derived from HBRV sequences obtained from a PBC mediated PCR and Illumina sequencing, using a bio- patients’ perihepatic lymph node [6]. -e HBRV Su coding informatics pipeline that ensured the exclusion of all se- sequence was cloned into pcDNA3.1 (Invitrogen) vector quences potentially related to murine or HERV sequences. along with a TAP tag at the 3′ terminus of the HBRV Su [41] More than 2,200 unique HBRV integrations were identified, and 4 copies of M-PMV cytoplasmic transport element and the majority of PBC patients were found to have evi- (CTE) downstream [42]. -e expressed HBRV envelope dence of proviral integrations linked with HBRV RNA protein sequence corresponds to amino acids 99 to 455 in the production in cholangiocytes [30]. In clinical trials, PBC surface region that includes the receptor-binding domain, patients on combination antiretroviral therapy have shown which shares 97% and 98% amino acid identity with MMTV biochemical and histological improvement with therapy Su [6] (see supplementary material for HBRV Su and [31–34]. MMTV Su alignment; Supplementary Figure 1). -e hypothesis that a betaretrovirus may be linked with human breast cancer has gained little traction over the years because of the inconsistency of findings in different reports, 2.4. Cell Culture, Transfection, and Stable Cell Line a concern for cross-reactivity with human endogenous Generation. HEK293T cells (ATCC) were routinely main- retroviruses (HERV) and the low level of viral burden tained in Dulbecco’s modified Eagle’s medium supple- [10, 35, 36]. With regard to the potential for a link with mented with 10% fetal bovine serum (Gibco) and 100 μg/ml betaretrovirus infection and PBC, investigators have either noromycin. Transfection of HEK293T was performed using been unable to detect viral infection [37] or to confirm the PEI as described previously [43]. Briefly, 10 cells were specificity of HBRV infection in PBC patients [38]. Fur- seeded in 6-well plates one day before transfection, and 2 μg thermore, serological studies using MMTV preparations as of each plasmid was used for each well. To generate stable substrate have been unable to demonstrate specific antibody HEK293T cell lines harboring pHBRV Su-TAP-4C FW, the reactivity to defined MMTV proteins [37, 39]. While HBRV pHBRV Su-TAP-4C FW-puromycin plasmid was linearized shares between 93% and 97% amino acid identity with the with PvuI and transfected into HEK293T cells. Individual MMTV envelope protein, consistent differences have been clones were selected with puromycin (Invitrogen). Journal of Oncology 3 5′ UTR 3′ UTR gag pol pCMV-Su CMV SP Su TAP poly A pol Proviral DNA env U3 RU5 U3 RU5 pCMV-Su-4c CMV SP Su TAP CTE CTE CTE CTE poly A Env mRNA CMV pCMV-Su-4cr SP Su TAP CTE CTE CTE CTE poly A 99 457 Env protein SP Su Tm p14 gp52 gp36 (a) (b) KDa pCMV-Su-4c (c) Figure 1: (a) -e single spliced mRNA of betaretrovirus Env encodes the signal peptide, surface, and transmembrane proteins. (b) -e HBRV Su construct used for mammalian expression contained the cytomegalovirus immediate early promoter, HBRV SP, and Su, a TAP tag; pCMV-Su-4c contained 4 copies of M-PMV CTE inserted in the downstream of Su-TAP in either the sense (pCMV-Su-4c) or the antisense (pCMV-Su-4cr) orientation. (c) Only the pCMV-Su-4c containing the CTE in the correct orientation produced sufficient HBRV Su protein in the cell pellet and supernatant as shown by the western blot analysis. 2.5. Western Blot Analysis. Secreted HBRV Su protein in proviral integrations and HBRV RNA by the QuantiGene 400 μl supernatant was precipitated with TCA and dissolved assay and in situ hybridization [30]. in PBS. Cell lysates were prepared from transfected and For detection of serological reactivity to HBRV Su, stable cells using RIPA buffer with complete proteinase 100 ng of purified protein was resolved on a 10% SDS-PAGE inhibitor (Roche). Approximately 2 ×10 cells were collected minigel (Bio-Rad) and transferred to nitrocellulose mem- and washed twice with ice-cold PBS, incubated with RIPA brane. -e membrane was cut into 5 mm wide stripes. Each buffer on ice for 30 min, and centrifuged at 20,000 ×g for 30 stripe was incubated with serum from a breast cancer patient minutes. Proteins from cell supernatant and lysate were or a control (1 : 400 dilution) and IRDye goat anti-human quantified using the BCA assay (Bio-Rad), and 50 μg and secondary antibody. 100 μg of total protein from cell lysate and supernatant, respectively, were resolved by 10% sodium dodecyl sulfate- 2.6. Scale-Up of HBRV Su Production and Purification and (SDS-) polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane as previously de- Characterization. Stable cells expressing HBRV Su were expanded to 12 ×15 cm cell cultural dishes in Dulbecco’s scribed (Figure 1) [44]. Western blot analysis was performed using the primary modified Eagle’s medium supplemented with 10% fetal bovine serum. -e medium in each plate was replaced with polyclonal goat anti-MMTV envelope gp52 antibody (kindly provided by Dr. Susan Ross, University of Pennsylvania), 25 ml Pro293 CD serum-free medium (Lonza) when cells reached 95% confluence. -e medium was collected after 5-6 mouse monoclonal anti-Flag antibody (Sigma-Aldrich), and IRDye goat anti-mouse and rabbit anti-goat secondary days of incubation and centrifuged at 3,000 g for 20 min. -e clarified medium was adjusted to pH 8.0 and filtered through antibodies (LI-COR). Reacting membranes were visualized with LI-COR Odyssey infrared imaging system. -e anti- a 0.22 μm filter before purification. MMTV envelope gp52 antibody has demonstrable reactive Purification of HBRV Su was performed on 1 ml Histrap biliary epithelial cells extracted from a liver transplant re- FF crude column and buffers as suggested by the supplier cipients with PBC (Supplementary Figure 2), previously (GE Healthcare) using an AKTA explorer 100 (Amersham Pharmacia Biotech). -e conditioned medium was loaded to shown to have HBRV infection with documented HBRV Mock pCMV-Su pCMV-Su-4cr pCMV-Su-4c Supernatant Pellet 4 Journal of Oncology the equilibrated column at the rate of 1 ml/min, and the (Figure 1(c)). -erefore, an M-PMV cytoplasmic transport column was then washed with 20 ml binding buffer and element (CTE) was incorporated into the construct to in- crease protein expression [42]. To this end, two additional Su eluted into 10 × 0.5 ml fractions using elution buffer. -e peak elution fraction was combined and changed to proteins expression constructs were generated with the 4 copies of storage buffer by ultrafiltration (Millipore, 30 kDa cutoff M-PMV CTE inserted in the downstream of Su-TAP for limit concentrator, 4000 g for 20 min). -e final preparation expression studies. Following expression in HEK293T, in- was aliquoted for storage at − 80 C for ELISA. -e 10 eluted creased production of HBRV Su was observed in cell lysates fractions were assessed by western blot analysis using anti- transfected with the pCMV-Su-Tap-4c but not in cells with MMTV Env antibody or anti-FLAG antibody and 10% SDS- the pCMV-Su-Tap-4cr construct that had the CTE arranged PAGE gels stained with Coomassie R-250 blue stain (Bio- in the antisense orientation. Moreover, we were able to Rad). -e protein concentration was determined by BCA detect secreted Su protein in the medium of the cells assay (Pierce) using bovine serum albumin (BSA) as a transfected with the pCMV-Su-Tap-4c plasmid two days standard. after transfection (Figure 1(c)). 3.2. Large-Scale Production and Purification of HBRV Su. 2.7. HBRV Su ELISA. ELISA was performed at room tem- Since abundant HBRV Su protein was secreted from perature with all sera in duplicate using high-binding 293Tcells transfected with the pCMV-Su-Tap-4c plasmids, a microplates (Greiner, Monroe, USA). Briefly, wells were strategy was developed to purify the protein directly from a coated with 100 μl of 2 ng/μl purified HBRV Su in PBS for 18 large-scale cell culture medium (Figure 2(a)). Stable hours and blocked with 1% BSA in PBS for 3 hours. Serum 293T cell lines were generated following transfection with was incubated at 100 μl/well at a 1 : 400 dilution in PBS with the pCMV-Su-Tap-4c plasmid and the cells with the highest 1% BSA (Sigma) for 1 hour. A serial dilution of polyclonal Su secretion in the culture medium were expanded to anti-MMTV Env was included on each plate as a standard 12 ×15 cm cell culture dishes using DMEM supplemented and then incubated with 100 μl/well donkey anti-human and with 10% FBS. When cells reached 90–95% confluence, the donkey anti-goat secondary antibodies (Jackson Immuno- medium was replaced with serum-free medium and incu- Research Lab) for 1 hour. -e plate was washed 3 × 5 min bated for another 5 days before collection. Approximately after each step using PBS with 0.5% Tween. Plates were 300 ml was obtained for each batch, which was then purified developed with 100 μl/well tetramethylbenzidine substrate with chromatography to derive 150–200 μg HBRV Su (TMB, Sigma) for 20 min and then stopped with 50 μl/well protein. SDS-PAGE revealed that the purified Su protein was 2N H SO . -e absorbance at 450 nm and 540 nm (back- 2 4 homogeneous and devoid of other contaminants. Western ground) was measured with EMAX Plus Microplate Reader blot analysis with polyclonal anti-MMTV Env confirmed (Molecular Devices, USA) and the cutoff level was estab- that the purified protein was HBRV Su along with select lished using the reactivity of control samples by adding the serum from seropositive and negative breast cancer and mean background level to 3 × S.D. Two-tailed Fisher’s exact control samples (Figures 2(b) and 2(c)). test was used to assess significant differences in frequency between different groups, followed by calculation of the odds ratio (Baptista–Pike methodology) along with sensitivity, 3.3. Detection of Anti-HBRV Su Protein Antibodies by ELISA. specificity, positive predictive value, negative predictive -e ELISA protocol was established using 200 ng/well of value, and likelihood ratio (Wilson Brown methodology) purified HBRV Su. -e antibody response was calculated by using Prism 8 software. converting the optical density reading to the equivalent ng/ ml reactivity of the positive control, polyclonal anti-MMTV Env antibody. -e background reactivity was calibrated 3. Results using the serum samples from the age/sex-matched healthy 3.1. HBRV Su Expression in HEK 293T Cells. A mammalian controls used as a comparison group for the breast cancer expression system was employed to express the HBRV Su patients. -e cutoff level (mean background + 3 × S.D.) was because prior attempts to express multiple constructs calculated as 61 ng/mL and samples found to be greater than this were considered positive (Figure 3). Accordingly, a expressing HBRV Env protein in bacteria and baculovirus systems were not sufficiently productive. MMTV Env greater proportion of breast cancer patients (10.2%) were found to have serological reactivity to HBRV Su versus 2.0% protein is encoded by a single spliced mRNA in mice, which produces a signal peptide (SP p14), surface (Su gp52), and of age- and sex-matched control subjects (Figure 3: P � 0.017, OR � 5.6 [1.25–26.3]). transmembrane domain (TM gp36) (Figure 1(a)); the Su protein is generated by removal of the signal peptide by -e seroprevalence of HBRV Su reactivity in patients signal peptidase and cleavage of the transmembrane domain with PBC was comparable to that observed in patients with by cellular Furin. -erefore, a mammalian expression vector breast cancer (Figure 3: 11.5% vs. 10.2%). -e frequency of pCMV Su-Tap was constructed, using the cytomegalovirus HBRV Su reactivity was significantly higher in PBC patients immediate early promoter to drive protein expression and a vs. blood donors (11.5% vs. 3.1%, P � 0.0024, OR � 4.09 TAP tag to enable protein purification (Figure 1(b)). Using [1.66–10.1]). In prior studies using the gold standard methodology of detecting HBRV integrations in patients’ the pCMV-Su-TAP construct, very little HBRV Su protein was detected in lysates from transfected HEK293T cells cholangiocytes, subjects with cryptogenic liver disease and Journal of Oncology 5 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 CMV SP SU TAP CTE CTE CTE CTE poly A pCMV-Su-4c KDa Transfection Stable cell line selection Cell expansion Coomassie blue staining Supernatant collection Anti-gp52 His-tag column loading Washing and elution (a) (b) (c) Figure 2: (a) Schematic showing large scale HBRV Su protein purification from the supernatant of HEK293T cells using a His-tag column. (b) Coomassie blue staining and western blot analysis demonstrate the purity of the HBRV gp52 protein using anti-MMTV gp52 Su in sequential elutions. (c) Western blot confirmation of ELISA positive and negative samples demonstrates reactivity using select breast cancer, PBC, and blood donor control samples. -e breast cancer serum sample used in lane 7 is positive by western blot and negative by ELISA. AIH were found to harbor infection, and in this study, breast cancer and PBC, respectively (98.0% [93.1%–99.7%] isolated reactivity was observed in subjects with cryptogenic and 97.0% [93.4%–98.6%]). Accordingly, the positive liver disease (16.7%) and AIH (6.3%), whereas other subjects predictive values (83.3% [55.2%–97.0%] and 75.0% [55.1%– with liver disease were universally negative (Figure 3(b)). 88.0%]) were diagnostically more useful that the negative predictive values (53.2% [46.1%–60.2%] and 57.7% [52.3%– While reactivity in healthy blood donors was incre- mentally higher than the healthy age/sex-matched com- 62.9%]) for patients in the breast cancer and the liver parison group for the breast cancer patients, the difference disease study groups. was not found to be significant (3.1% vs. 2.0%; P � 0.72). -e sensitivity of the HBRV Su ELISA was limited in 4. Discussion detecting reactivity in patients with breast cancer and PBC as compared to their respective control groups (10.2% -is is the first report using an HBRV ELISA for assessing [5.6%–17.8%] and 11.5% [7.4%–17.5%]), whereas the assay the seroprevalence of infection in patients. Approximately was highly specific for serologic detection in patients with 10% of breast cancer and PBC patients had detectable anti- Positive control Negative control Breast cancer (ELISA +ve) PBC (ELISA –ve) PBC (ELISA –ve) PBC (ELISA +ve) Breast cancer (ELISA –ve) Blood donor(ELISA –ve) Blood donor(ELISA +ve) PBC (ELISA +ve) AIH (ELISA +ve) PSC (ELISA –ve) PBC (ELISA –ve) 6 Journal of Oncology 200 800 0 0 (a) (b) Figure 3: (a) A higher percentage of reactivity to HBRV Su was observed in breast cancer patients’ sera versus age/sex-matched healthy controls (10/98 vs. 2/102; P � 0.017). (b) Anti-HBRV reactivity was highest in patients with PBC (18/156) and found in AIH (1/16), cryptogenic liver disease (1/6), and healthy blood donors (6/194), whereas reactivity was not observed in patients with PSC, steatosis (NAFLD), ALD, or miscellaneous liver disease (PBC vs. blood donors 11.5% vs. 3.1%, P � 0.0024, OR � 4.09 [1.66–10.1]). HBRV Su, and the test was found to be highly specific for was attributed to autoreactivity with the antimitochondrial both disorders. -e likelihood ratio for having breast cancer antibody, which is found in up to 95% of patients and used with HBRV Su reactivity was 5.2 and for having PBC with for diagnosing PBC [29], whereas similar MMTV western HBRV Su reactivity was 3.7; the difference in likelihood blot studies employing mitochondrial proteins to remove the autoantibodies from PBC patients’ serum demonstrated ratios probably reflects the chosen control groups for each disorder. Notably, the breast cancer control subjects were the presence of signal to the betaretrovirus gp52 surface mainly middle-aged women and therefore a more suitable protein [48]. As the purified antimitochondrial antibody has control group for the PBC patients, who are also predom- no reactivity with HBRV Su, we can conclude that humans inantly female; whereas the blood donors were more of an do make humoral responses to HBRV based on our ELISA. admixture of both sexes. -e healthy comparison groups A second issue to be addressed is that the prevalence of revealed a sizeable population seroprevalence of ∼2-3%. infection detected by the HBRV Su ELISA was somewhat -ese data are in keeping with the hypotheses that HBRV lower than other reports using different techniques to di- infection may only be disease related in genetically pre- agnose disease. Indeed, our western blots (Figure 2(c)) show disposed individuals [10, 45]. reactivity to one breast cancer sample that was negative by Prior seroprevalence studies using MMTV proteins have the ELISA, suggesting that our cutoff level may have been too stringent. Using nonserological techniques, a meta- been widely inconsistent. For example, an ELISA-based study using MMTV proteins demonstrated serological re- analysis of molecular epidemiological studies reported a activity in 26% of breast cancer patients and 8% of healthy prevalence of 40% HBRV infection in Western countries controls [46]. A similar study found no difference between based on PCR detection of betaretrovirus sequences in breast breast cancer patients and their respective controls [47] and cancer samples [49]. An even higher prevalence of infection a study using 4 strains of MMTV reported only nonspecific has been reported in PBC patients based on the presence of reactivity in breast cancer patients, although reactivity proviral HBRV integrations detected by ligation-mediated consistent with the molecular weights of viral proteins was PCR and Illumina sequencing, with provirus found in 58% observed in individual strains of MMTV [39]. In studies of of cholangiocytes from patients with PBC as compared to 7% patients with liver disease, MMTV western blot reactivity of nonautoimmune liver disease controls [30]. -e Anti-HBRV Su levels (ng/mL) Breast cancer Age-matched controls Anti-HBRV Su levels (ng/mL) PBC PSC AIH Steatosis ALD Viral hep. Crytogenic Liver disease misc. Blood donors Journal of Oncology 7 discrepancy of a higher frequency of viral infection in tissue patients participating in the Alberta Tomorrow Project who as compared to a lower seroprevalence of anti-HBRV Su subsequently developed breast cancer. Accordingly, we will reactivity may be partly explained by observations from be able to study whether anti-HBRV Su predates the de- neonatal mouse infection. Weanling pups have a high risk of velopment of disease and may act as a biomarker for breast developing breast cancer from MMTV infection because cancer. they become immunotolerant to viral infection. -is occurs because MMTV is taken up in the gut-associated lymphoid Data Availability tissue along with bacterial lipopolysaccharide, which triggers -e data used to support the findings of this study are in- a cascade of events. -e lipopolysaccharide/viral complex cluded within the supplementary information files and engages Toll-like receptor 4 that in turn triggers an IL-4- and available on request from the corresponding author. IL-6-dependent production of IL-10, which renders the mouse unresponsive to MMTV Su and prevents the for- Conflicts of Interest mation of neutralizing antibodies [13]. It is currently un- known whether a similar immunological process may occur -e authors declare that there are no conflicts of interest in humans with HBRV infection. Notably, the cellular regarding the publication of this paper. immune response to HBRV peptides is more prevalent in patients with liver disease [50]. Authors’ Contributions Our overall goal was to derive a reliable and repro- ducible diagnostic ELISA to investigate the frequency of AM designed and coordinated the study with JRM and HJA. HBRV infection. In prior experiments, we used bacterial or KB, GZ, and AM interpreted data and wrote the article. GZ, baculovirus expressed proteins but failed to generate suf- MK, KC, and YH performed the majority of the experi- ficient amounts of pure viral protein. We also generated ments. GZ generated the constructs. All authors read and serological data using the bacterially expressed Gag pro- approved the final manuscript. teins, and while a higher seroprevalence was observed in our PBC population as a whole, no significant differences were Acknowledgments found between patients and controls with liver disease. Notably, cross reactivity with retroviral Gag (Group Anti- We thank the Alberta Cancer Foundation, Alberta Innovates Gen) is a common occurrence in patients with any viral Health Solutions, Canadian Institutes for Health Research, infection due to the positively charged antigenic determi- and Canadian Liver Foundation for supporting this project nants in capsid and core proteins surrounding the viral as well as Michael Houghton and Michael Sakalian for their genome [51, 52]. For this ELISA, a novel strategy for large- assistance. scale production of purified and secreted HBRV Su protein was developed using HEK 293T cells. -ree factors con- Supplementary Materials tributed to the production of HBRV Su sufficient for HBRV Su expression construct and coding sequences; multiple ELISAs: these included (i) using multiple copies of alignment of HBRV Su and MMTV Su proteins as well as CTE downstream of the Su coding region to enhance HBRV anti-MMTV gp52 Su reactivity to biliary epithelial cells Su expression and secretion; (ii) ensuring the stable ex- cultured from a PBC patient’s resected liver following liver pression of HBRV Su protein in human cells; and (iii) transplantation. (Supplementary Materials) replacing the FBS containing medium with serum-free medium to remove a source of protein contamination and References ensure the high purity of protein after chromatography purification. We can also speculate that the use of HBRV [1] A. Jemal, F. Bray, M. M. 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Published: Jan 27, 2020

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