Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice

Figure 1. Coomassie brilliant blue-stained SDS-PAGE (top) and anti-His6 Western blot analysis (bottom) illustrating the expression, solubility, and inclusion-body preparation of K18(SU) from E. coli Rosetta (DE3). (a) Specific protein expression (E) in transformed bacterial cultures induced with 1 mM IPTG can be observed in comparison to non-induced control samples (N). (b) Analysis of pellet (P) and supernatant (S) after cell lysis and centrifugation revealed that K18(SU) was expressed in insoluble form. (c) Enrichment of K18(SU) through sequential washing steps (S: supernatants, P: pellets after centrifugation) during inclusion-body preparation can be observed in comparison to the bacterial lysate (L). M: Protein ladder (Coomassie blue-stained gels) or molecular weight marker standards (Western blots). Arrows point to the target protein.

Figure 2. Purification of HERV-K18(SU) from bacterial inclusion bodies using nickel nitrilotriacetic acid (NTA) chromatography. (a) The chromatogram shows absorbance at 280 nm and electric conduction, as well as the fractions collected during the purification process (F: flow through; W1 and W2: wash with 25 mM imidazole; E: elution with 250 mM imidazole). (b) Coomassie brilliant blue-stained SDS-PAGE of the load (L) and the fractions depicted in (a). M: protein ladder. (c) Anti-His6 Western blot of the load (L) and the fractions depicted in (a). M: molecular weight marker standard. Arrows point to the target protein. (d) Mass spectrometric analysis of the elution fraction. The protein band corresponding to the target protein was excised from the polyacrylamide gel in the elution fraction, trypsin-digested and analyzed with mass spectrometry for matches (blue lines) with the template sequence. The sequence coverage was 55%.

Figure 3. Antibody and T-cell response to recombinant HERV-K18(SU). (a,b) Six week old female Balb/c mice received two subcutaneous immunizations (indicated by arrows) with 50 µg HERV-K18(SU) (n = 3) or a vehicle (n = 2) in IFA with a four-week interval. Plasma IgG titers were analyzed at three time points using HERV-K18 Env-specific ELISA. As ELISA plates could not be coated with insoluble recombinant HERV-K18(SU), cell extracts that did (target) or did not (background) contain HERV-K18 Env were used instead. Graphs in (a,b) show original absorbance data and the background subtracted values, respectively. Representative results of one of two independent experiments are shown as the mean ± SEM. Statistics: unpaired t-test (K18(SU) versus vehicle). (c,d) Balb/c mice (n = 5) were immunized intraperitoneally with 10 µg HERV-K18(SU) daily for 3 × 5 days. Plasma IgG titers were analyzed using HERV-K18 Env-specific ELISA as described above. Graphs in (c) and (d) show original absorbance data and the background subtracted values, respectively. Statistics: unpaired t-test (K18(SU) versus vehicle). (e,f) Isolated splenocytes (e) or MACS-sorted T cells (f) from immunized mice were isolated and…

Figure 4. HERV-K18(SU) does not provoke an immune reaction in a model of EAE. (a) Clinical score (bar graph) and relative body weight (line graph) over 30 days for mice immunized with an emulsion containing either Freund’s complete adjuvant (CFA, n = 10), Freund’s incomplete adjuvant (IFA, n = 5), or Freund’s incomplete adjuvant and 10 µg or 50 µg K18(SU) (n = 10, respectively). Data are represented as means with SE, except for the mean relative body weight of the groups receiving IFA, 10 µg K18(SU) and 50 µg K18(SU) for reasons of clarity. (b) Percentage distribution of clinical scores for CFA-immunized mice. Note that one mouse showed a biphasic disease course with a second peak at day 29.

Figure 5. Luxol fast blue staining of representative cross-sections of the spinal cord of immunized mice for visualization of myelin sheaths. Treatment is indicated in the corner of each large image. Counter-stains with cresyl violet acetate shown in the left panel indicate staining of cell nuclei. (A–H) Magnifications of the areas labeled in the IFA and CFA sections to show intact white matter (A,B,E,F) and demyelinated areas (C,D,G,H) with putative infiltrated immune cells ((C,D,G,H), dotted lines). Scale bar: 200 µm.