Background The Envelope (Env) protein of the human endogenous retrovirus H (HERV-H) was shown to be overexpressed in different kinds of cancer tissue, especially in colon cancer and head and neck cancer (1). Therefore, it is a potential target for cancer immunotherapy. Herein we aimed towards analyzing the potential impact of modifications in the HERV-H Env on its expression and immunogenicity with the ultimate goal to instruct the generation of diagnostic tools and gather information for future immune intervention.

Materials and Methods Derivatives of the full-length HERV-H env gene were codon-optimized and synthesized based on annotated sequences. The wild type (WT) sequence was modified regarding the transmembrane-spanning domain including the cytoplasmic tail (TM+CT). For modified membrane tethered variants, the autologous TM+CT sequence was exchanged for a heterologous sequence. In addition, variants lacking the TM+CT or variants deprived of the complete transmembrane subunit were designed, resulting in the expression of a soluble secreted Env trimer or of the monomeric Env (SU). Expression of the HERV-H Env protein was analyzed by western blot and flow cytometry. Groups of 6 Balb/c mice were immunized with DNA vaccine constructs encoding the WT and modified HERV-H Env proteins on day 0 and 14 (prime), respectively, and boosted twice on day 42 and 70 with an adjuvanted formulation of a recombinant trimeric HERV-H Env protein. Antibody responses were monitored by ELISA against various HERV-H Env (trimer, SU and extracellular domain of TM).

Results Western blot and flow cytometry analysis showed proper expression of membrane-bound Env proteins with no significant enhancement of cell surface display for the TM modified protein. Antibody responses against all Env variants could be shown already after two DNA immunizations and were elevated after boosting with the recombinant Env protein. Except for the group immunized with the DNA vaccine encoding the SU, which showed significant higher antibody titers compared to all others, no differences could be seen between the groups following the protein boost.

Conclusions The results prove the antigenicity and immunogenicity of the HERV-H Env protein and its derivatives. All engineered HERV-Env derivatives were able to prime Env specific antibody responses in Balb/c mice. Booster immunization with adjuvanted Env trimer yielded comparable antibody titers, except for SU, which showing superior responses in terms of magnitude. Data and reagents described herein provide a valuable foundation for the development of diagnostic tools for tumor stratification, antibody-based intervention as well as therapeutic vaccination strategies. 1. Zhang, M, Liang, JQ, Zheng, S. Expressional activation and functional roles of human endogenous retroviruses in cancers. Rev Med Virol. 2019; 29:e2025. https://doi.org/10.1002/rmv.2025

Disclosure Information J. Gille: None. I. Skandorff Pedersen: None. C. Thirion: None. P.J. Holst: None. R. Wagner: None.