Reciprocal Inhibition of Immunogenic Performance in Mice of Two Potent DNA Immunogens Targeting HCV-Related Liver Cancer

Abstract

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells

“…The infiltration of tumor cells into mouse organs characterizing the motility of tumor cells in vivo was assessed using BLI of mouse organs at the experimental endpoint as was described by us earlier [49]. The most affected organs were the lungs and to a lesser extent the liver; spleen was not involved (Figure 8A-C, Supplementary Figure S6).…”

“…The average BLI signal of 21,000 ± 6000 p/s in the latter groups significantly exceeded the background level, indicating the presence of at least three tumor cells in the lungs of each mouse. The infiltration of tumor cells into mouse organs characterizing the motility of tumor cells in vivo was assessed using BLI of mouse organs at the experimental endpoint as was described by us earlier [49]. The most affected organs were the lungs and to a lesser extent the liver; spleen was not involved (Figure 8A-C, Supplementary Figure S6).…”

“…Prior to implantation, PR20.2 cells were resuspended in RPMI-1640 at a concentration of 2 × 10 5 cells/mL; 50 µL of the suspension (containing 10 5 cells) was subcutaneously injected into the BALB/c mice at two sites to the right and to the left of the base of the tail, at least 1 cm over the site of DNA immunization. Tumor growth was assessed by regular bioluminescent imaging (BLI) on IVIS Spectrum (Perkin Elmer, Waltham, MA, USA) following the protocols described by us earlier [49,50]. For the quantification of photon emission, a frame was created of the size sufficient to enclose all photon emissions from the mice throughout the entire course of the study (region of interest, ROI); this ROI frame was applied to all images.…”

The regulations of telomerase reverse transcriptase (TERT) in cancer

“…To date, GV1001 single-use [182] or combined with HR2822 [183], UV1 [184] singleuse or combined with Ipilimumab (checkpoint inhibitor) [185], Vx-001 [186], and Gx-301 [187] have undergone clinical trials in patients with different types of cancer. Based on CD4 + /CD8 + T cell recognition, the TERT vaccine presents a promising therapeutic efficacy in a proportion of cancer, although its safety and universal applicability in different types of cancer requires further research and verification [153,188,189].…”

Targeting Telomere Dynamics as an Effective Approach for the Development of Cancer Therapeutics

“…Jansons et al have elucidated that a DNA vaccine derived from rat TERT contributed to the antitumor responses by inducing the release of IFN-γ/IL-2/TNF-α from CD4 + and CD8 + T cells. 105 For the purpose of improving the antigenspecific antitumor immunity of DNA vaccines, bringing in the adjuvants has gained extensive attraction. The DNA vaccine (pCCL21-Te-Fc) was based on plasmids and constructed by linking human CCL21 and IgG Fc to two hTERT fragments.…”