Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples

<div><p>Introduction</p><p>A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC).</p><p>Results</p><p>The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.</p><p>Conclusion</p><p>Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.</p></div

Summary of the immunohistochemistry results of selected CRC cases.

<p>Summary of the immunohistochemistry results of selected CRC cases.</p

Examples of Gag-H immunohistochemistry in two representative clinical MSS cases.

<p>The upper row represents the tumor and the bottom row the normal tissue of the cases. Both cases: No. 6 (A-F) and No. 13 (G-L) showed strong cytoplasmic immunostaining of Gag-H in tumor and in normal tissue (B, E, H, K by x20 objective; C, F, I, L by x40 objective). Negative immunostaining was achieved with the control antibody 3D8C6E7 (A, D, G, and J by x20 objective).</p

Examples of Gag-H immunohistochemistry in two representative clinical MSI cases.

<p>The upper row represents the tumor and the bottom row the normal tissue of the cases. Both cases: No. 8 (A-F) and No. 11 (G-L) showed strong cytoplasmic immunostaining of Gag-H in tumor and in normal tissue (B, E, H, K by x20 objective; C, F, I, L by x40 objective). Negative immunostaining was achieved with the control antibody 3D8C6E7 (A, D, G, and J by x20 objective).</p

Epitope mapping.

<p>Representative bar charts for epitope mapping are depicted. Upper left: clone 1B3H7, upper right: clone 1D7D11, lower left: clone 14H11G1 and lower right: clone 2H2D6.</p

ELISA analyses.

<p>ELISA analyses.</p

Gag-H protein expression with anti-Gag-H clone 14H11G1.

<p>Overlay of histograms obtained by flow cytometric analyses of 11 CRC cell lines with no primary antibody (filled light grey surface), irrelevant antibody (anti-His; 2G2B3; grey line) and anti-Gag-H antibody (clone 14H11G1; black line) are presented in the figure.</p