A calreticulin-linked HPV-16 E7 minigene DNA vaccine elicits strong E7-specific CD8+ T-cell immunity and durable antitumor effects in a preclinical model

Abstract

Abstract Calreticulin (CRT) is an endoplasmic reticulum chaperone that facilitates antigen processing and presentation, making it an attractive fusion partner for enhancing tumor-specific T-cell responses in DNA vaccine development. However, the relative efficacy of CRT compared with other antigen-presentation–enhancing strategies has not been fully evaluated. We constructed a DNA vaccine encoding the immunodominant human papillomavirus (HPV)-16 E7 minigene epitope (aa 49–57) fused to the C-terminus of CRT (pcDNA3-CRT-E7(49–57), hereafter CRT-E7(49–57)). This vaccine was compared with DNA vaccines encoding CRT linked to full-length E7 (CRT-E7(1–98)) and other antigen-presentation–enhancing constructs, including ubiquitin linked to E7 minigene (Ub-E7(49–57)) and MHC class I trafficking domain linked to E7 minigene (MITD-E7 (49–57)). We evaluated E7-specific cytotoxic T-cell responses, tumor volume, and survival in both prophylactic and therapeutic TC-1 tumor models. Intramuscular electroporation of C57BL/6 mice with CRT-E7(49–57) elicited robust E7-specific CD8⁺ T-cell responses comparable to those induced by CRT-E7(1–98) and MITD-E7(49–57), but higher than those elicited by Ub-E7 (49–57). CRT-E7(49–57) vaccination provided complete protection against TC-1 tumor challenge, with mice remaining tumor-free and surviving beyond 60 days. Vaccinated mice also exhibited strong immune memory, as tumor rechallenge triggered potent E7-specific CD8⁺ T-cell responses and restricted tumor growth. In therapeutic settings, two doses of CRT-E7(49–57) completely suppressed tumor progression, outperforming Ub-E7(49–57) and conferring superior survival compared with MITD-E7(49–57). The CRT-E7(49–57) DNA vaccine induces durable, high-magnitude E7-specific CD8⁺ T-cell responses and confers effective prophylactic and therapeutic antitumor immunity, underscoring its promise as a versatile platform for cancer immunotherapy.