Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Significance Bovine meat and milk factors (BMMF) are routinely found in bovine sera and dairy products, predominantly of Eurasian dairy cattle. BMMF DNA and proteins are demonstrated in tissues of colon cancer patients, specifically interstitial macrophages of peritumor tissues. BMMF represent plasmid-like, zoonotic infectious agents with an indirect role in cancer formation by inducing chronic inflammation leading to oxidative stress and DNA mutation in nearby replicating cells, which may develop into polyps as progenitors for colon cancer. Detection of BMMF during long latency periods prior to symptoms developing allows for specific preventive and early therapeutic measures. Detection of BMMF might offer a prognostic tool for prediction of patient survival, preventive approaches, and therapy success.

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Materials and Methods
Supporting material on production and characterization of BMMF monoclonal antibodies Figures S1 to S14 Tables S1 to S4 (1). The supernatants were screened by ELISA (with purified, full-length Rep antigen or peptide 1 or 2), as well as by WB and IF. Validated mother clones were subcloned by limited dilution to obtain monoclonal cell clones.  Therefore, an additional analysis was performed based on a peptide chip with cyclic peptides with lengths of 7, 10, and 13 aa respectively (with 6, 9, 12 aa overlap, in duplicates) to cover the full Rep (staining procedure as described for chip with linear peptides). In addition, monoclonal antibodies were also tested in WB for detection of specific regions of the H1MSB.1. A human codon-optimized full-length co-immunodetection (2). Differences between the cohorts were analyzed using Mann-Whitney U test (GraphPad Prism 8).  Cloning of PCR-products and sequencing: Extracted PCR products were T/A-cloned into pCR2.1 (pCR2.1 TA cloning kit, Invitrogen). Ligation reaction (2 µl) transformed into One Shot TOP10F

Production of BMMF-specific monoclonal antibodies:
A set of mouse monoclonal antibodies was produced for immunodetection of BMMFs. Two exceptionally conserved and immunogenic amino acid regions were identified in amino acid alignments of known  (Table S1 and Fig. S2).

Characterization of monoclonal antibodies:
Different approaches were followed for mapping and validating each antibody against the BMMF1 Rep protein. The predicted (Fig 1B) protein structure of H1MSB.1 Rep was described previously (6,9). In  (aa 1 to aa 229), WH2+C-terminus (aa137 to aa324), and C-terminus (aa230 to aa324), as well as the Rep of the additional BMMF1 isolates, H1MSB.2 and C1MI.1. Results indicated varying reactions for each antibody in the respective assay (Tab. S1). All antibodies were suitable for use in both WB and IF, with the exception of AB13, which is only suitable for use in IF.

WB analyses:
The full set of 14 mouse monoclonal antibodies was tested against over-expressed H1MSB.1 Rep in WB (Fig. 1A). Antibodies generated by immunization with the specific peptides 1 and 2, located in WH2 and WH1, respectively, also tested positive for these regions -AB1 in WH2 and AB2/5/14/15 all in WH1. In addition, AB8 and AB11 obtained after immunization with the Rep protein, stained positive for the WH1 region and detected the purified C-terminal (with weaker intensity).
AB3/9/10 strongly detected the C-terminal of the Rep protein (with AB9 also detecting the WH1).
Antibodies AB4/6/7 stained the full-length Rep protein, but were negative in the sub-localizations. The full-length Rep staining of AB13 was inconclusive.
Linear and cyclic epitopes: Sequences of the specific epitopes were determined for each antibody by peptide chip staining (PEPperPRINT) ( Table S1). The epitope sequences of antibodies (AB2/5/14/15) generated with consensus peptide 1, cover the terminal part of the peptide and differ in length between each other (EARETGKGINANDPLTVH). The epitope sequence of AB1 generated with peptide 2 (KQINEHTDITASYEOHKKGRT) covers the central part of the peptide sequence. Antibodies raised against the full-length Rep protein led to varying results. No distinct epitope sequence was determined for AB11, although it proved positive in WB and IF analyses. A possible explanation is that protein modification was involved during immunization which is not accounted for on the peptide chip. Similarly, no clear linear epitope was defined for AB8. Several epitopes were determined for both AB6 and AB7 partially overlapping between them, with the main epitope (aa 97 to aa 209, QINEHTDITASYE) being identical. Interestingly, despite these two antibodies being generated using full-length Rep protein, their main epitope overlaps with 12 aa in the consensus peptide 2 which was used to generate AB1. Initially no linear epitopes were detected for AB3 and AB10. Additional analysis based on a peptide chip with cyclic peptides however identified a major epitope stretch (aa313 to aa323) for each of these antibodies (Table S1).

Antibody specificity:
The specificity of antigen detection was verified by quantitative correlation of Repspecific IHC detection in the presence of differing amounts of overexpressed H1MSB.1 Rep protein.
Rep antigen expression was controlled by decreasing the amounts of expression plasmid applied in transient transfection (100/50/25/0% DNA transfection) and measured by subsequent WB-detection with anti-HIS antibodies (Fig. S3A). In addition, Rep-expressing HEK293TT cells were detached and paraffinembedded prior to IHC bright-field staining with Diaminobenzidine (DAB) (Fig. S3B).       * the corresponding main epitope is suggested by PEPperPRINT epitope mapping, but additional epitopes exist, † identification by WB analysis with WH1, WH2, or C-terminal Rep domain, ‡ H1MSB.1-specific C-terminal main epitope is suggested by PEPperPRINT epitope mapping, but an additional (conserved) epitope is suggested by WB, § no epitope identified by PEPperPRINT epitope mapping and WB, but putative conformational epitope allocated by IF in Rep WH1, ¶ These antibodies did not detect H1MSB.2 and C1MI.1 Rep in experimental tests, but the suggested epitopes are conserved for other BMMF1 Reps which might allow detection.

Table S3 -BMMF detection by Immunohistochemistry (IHC)
Summary of IHC analyses of colorectal cancer tissues (peritumor (P) and tumor (T)) and polyps analyzed with individual anti-Rep antibodies. Tissues with strong antibody staining throughout the full tissue section are indicated in dark blue color. Heavily, but only regionally stained tissues, as well as medium intensity staining over larger tissue areas are indicated in light blue color. Tissues with staining occurring in <5 single tissue regions were indicated as negative together with tissues with no detectable staining (grey).

Table S4 -List of antibodies and staining kits
List of primary and secondary antibodies used for immunodetection including commercial staining kits (* performed according to manufacturer's instructions).