mRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice

Abstract

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

Keywords: Epstein-Barr virus (EBV); cancer immunotherapies; mRNA vaccines; nasopharyngeal carcinoma (NPC).

Figure 1

Mapping of T‐cell‐epitopes on Epstein–Barr virus (EBV) latent antigens and expression of latent membrane proteins (LMP)−2A, EBV nuclear antigens (EBNA)−1, and EBNA3A truncations in vitro. A) T‐cell epitope maps of LMP2A, EBNA1, and EBNA3A. Epitopes were identified from previous studies (data source: IDEB.org) and are illustrated as vertical red bars, identified by the first three amino acids. Thick red bars represent epitopes reported by at least three references, while thin red bars represent epitopes identified by two references. Full details of epitopes are given in Table S1 (Supporting Information). B) Western blot analysis for the expression level of flag‐tagged LMP2A, EBNA1, and EBNA3A truncations is presented. After mRNA transfection into 293T cells for 12 h, cell lysates were analyzed using western blotting, with anti‐Flag tag and anti‐β‐actin antibodies used as primary antibodies. The mutated nuclear localization sequence (K378A, R379A, K397A, R398A) of EBNA3A protein is denoted as MT. C) Representative confocal images of 293T cells expressing flag‐tagged EBNA1 and EBNA3A truncations. The corresponding mRNA was transfected into 293T cells 12 h before detection. Protein expression was detected with Alexa Fluor 488 Conjugated anti‐flag antibody(green), and the nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Scale Bar = 10 µm.

Figure 2

Characterization of mRNA‐liposome nanoparticle (RNP) and cellular immune response elicited by Trunc‐LMP2A‐RNP, Trunc‐EBNA1‐RNP, and Trunc‐EBNA3A‐RNP. A) Schematic representation of the structure and components of the RNP. The liposomes (LNP) were composed of DOTMA (blue) and DOPE (orange). B,C) The particle size (B and C), polydispersity index (PDI) (C, red dots), and zeta potential (C, yellow bars) of the RNP and LNP (n = 3). D,E) Bioluminescence imaging of BALB/c mice (n = 2) after intravenous (i.v.) injection of 20 µg luciferase RNP. F) Bioluminescence imaging of organs in a BALB/c mouse 2 h after i.v. injection of 20 µg luciferase RNP. G) C57BL/6 mice (n = 6) were intravenously immunized with 20 µg RNPs encoding truncated/full‐length EBV latent antigens (LMP2A, EBNA3A, or EBNA1) or irrelevant RNP control (NC) on day 1, 7, and 14. On day 21, immunized mice were euthanized. The spleens and other major organs were collected for determining T‐cell responses and histological analysis. H–J) Frequencies of interferon (IFN)‐γ releasing antigen‐specific cells demonstrated using ELISpot assay. Mice spleens (n = 6) were removed on day 21, and 2 × 10⁵ splenocytes were co‐incubated with 10 µg mL⁻¹ corresponding peptide pools (LMP2A, EBNA3A, or EBNA1). Phorbol myristate acetate (PMA) plus ionomycin and medium alone served as positive and negative controls, respectively. SFU, spot‐forming units; IFN‐γ, interferon‐gamma. The irrelevant RNP contained mRNA encoding the green fluorescent protein (EGFP). Significance was determined using a one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (H–J). Error bars, mean ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Anti‐tumor activities of Trunc‐LMP2A‐RNP and FL‐LMP2A‐RNP. (A) C57BL/6 mice were injected intravenously with B16‐LMP2A cells (2 × 10⁵ per mouse). Mice were randomly divided into three groups and immunized with 40 µg Trunc‐LMP2A‐RNP (n = 12), FL‐LMP2A‐RNP (n = 12), or irrelevant RNP (n = 14) on days 3, 6, 10, and 15 via intravenous injection. B–F) In vivo bioluminescence imaging of tumor growth. B–D) Individual tumor growth curves, E) average bioluminescent signals, and F) representative in vivo bioluminescence images of mice from the three groups. G) Kaplan–Meier survival curves for tumor‐bearing mice treated with Trunc‐LMP2A‐RNP, FL‐LMP2A‐RNP, or irrelevant RNP. Significance was determined using two‐way ANOVA followed by Dunnett's multiple comparisons test (E) or log‐rank test (G). Error bars, mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Activation of humoral and cellular immune responses by Trunc‐LMP2A‐RNP and LMP2A‐FL‐RNP in tumor‐bearing mice. A,B) T‐cell responses against LMP2A were determined using the IFN‐γ ELISPOT assay. T cells were isolated from tumor‐bearing mice on day 28 in Figure 3 and stimulated with LMP2A peptides. A) Representative figures and B) frequencies are illustrated. Sample size: Irrelevant (n = 6); FL‐LMP2A (n = 8); Trunc‐LMP2A (n = 12). (The sample sizes of the irrelevant and FL‐LMP2A groups were reduced due to mice mortality). C,D) Measurement of total serum anti‐LMP2A antibodies from tumor‐bearing mice immunized with 40 µg Trunc‐LMP2A‐RNP (n = 12), FL‐LMP2A‐RNP (n = 12), or irrelevant RNP (n = 14) on day 20. ELISA was performed by coating 96‐well plates with LMP2A peptides, and the absorbance (optical density, OD) was evaluated at 450 nm. E) Adoptive transfer of vaccine‐elicited T cells and/or antibodies to unimmunized tumor‐bearing mice: Healthy C57BL/6 mice were vaccinated with 40 µg Trunc‐LMP2A RNP or PBS on days 1, 3, and 7, administered three times (n = 30). On day 13, recipient C57BL/6 mice were intravenously injected with 2 × 10⁵ B16‐LMP2A cells (n = 6). On day 14, T cells and antibodies were isolated from the spleen and peripheral blood of vaccinated (Vac) or unvaccinated (NC) mice, respectively, and then transferred into tumor‐bearing mice. T cells and antibodies from unvaccinated mice served as the control (NC) group (n = 6). (T cells: 1 × 10⁷ per mouse; antibodies: 200 µg per mouse). F) In vivo bioluminescence imaging of tumor growth in recipient mice. VAC T: T cells from vaccinated mice, VAC Ab: antibodies from vaccinated mice, VAC T + Ab: both T cells and antibodies from vaccinated mice were injected into recipient mice. NC T: T cells from control mice, NC Ab: antibodies from control mice, NC T + Ab: both T cells and antibodies from vaccinated mice were injected into recipient mice (n = 6). Significance was determined using one‐way ANOVA followed by Tukey's multiple comparisons (B and D) and two‐way ANOVA followed by Dunnett's multiple comparisons test (F). Error bars, mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Inhibition of B16‐EBNA3A tumor growth by Trunc‐EBNA3A‐RNP in mice. A) B16‐EBNA3A cells (2 × 10⁵ per mouse) were injected intravenously into C57BL/6 mice, which were divided randomly into three groups and intravenously treated with Trunc‐EBNA3A‐RNP (n = 16), irrelevant‐RNP (n = 16), or left untreated (n = 8). B,C) In vivo bioluminescence imaging of B16‐EBNA3A tumor growth. B) Average bioluminescent signals of mice from three groups, and C) individual tumor growth curves (C) (left: Trunc‐EBNA3A‐RNP; middle: irrelevant‐RNP; right: untreated). D) Kaplan–Meier survival curve of tumor‐bearing mice in different groups. E) Splenocytes harvested from Trunc‐EBNA3A‐RNP, irrelevant‐RNP vaccinated, or untreated mice were stimulated with 10 µg mL⁻¹ EBNA3A peptides overnight and analyzed using ELISPOT (n = 4). Spots were detected with an anti‐IFN‐γ antibody (1 µg mL⁻¹ R4‐6A2, Mabtech). F) Enzyme‐linked immunosorbent assay (ELISA) detection of anti‐EBNA3A antibody in serum harvested from tumor‐bearing mice treated with Trunc‐EBNA3A‐RNP (n = 16), irrelevant‐RNP (n = 16), or left untreated (n = 8) on day 20. ELISA plates (96 wells) were coated with EBNA3A peptides overnight and blocked with 5% bovine serum albumin. G) Weight change in tumor‐bearing mice was monitored, and no significant difference existed between groups. Significance was determined using two‐way ANOVA followed by Dunnett's multiple comparisons test (B), log‐rank test (C), and one‐way ANOVA followed by Tukey's multiple comparisons (E and F). Error bars, mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

Inhibition of B16‐EBNA1 tumor growth by Trunc‐EBNA1‐RNP in mice. A) Schematic of the immune study in tumor‐bearing mice. C57BL/6 mice (6–8‐week‐old) were inoculated with B16‐EBNA1 cells (2 × 10⁵ per mouse) intravenously on day 0 and immunized i.v. with Trunc‐EBNA1‐RNP or irrelevant RNP on days 3, 6, 10, and 15 (n = 15). B) Kaplan–Meier survival curve of B16‐EBNA1‐bearing mice treated with Trunc‐EBNA1‐RNP or irrelevant RNP. C,D) Bioluminescence imaging was performed to monitor B16‐EBNA1 tumor growth in vivo. C) The average bioluminescent signals of mice from the two groups are shown in, and D) the tumor growth curves of individual mice are shown in (upper: irrelevant RNP; lower: Trunc‐EBNA1‐RNP). E) ELISPOT was used to detect IFN‐γ‐secreting splenocytes specific to EBNA1 peptides. Splenocytes harvested from vaccinated mice were stimulated with 10 µg mL⁻¹ EBNA1 peptides overnight, and the medium alone served as the negative control (n = 5). F) Detection of EBNA1‐specific antibody in mice sera (n = 15). Levels of anti‐EBNA1 antibodies were measured using ELISA. G) Body weight of tumor‐bearing mice was monitored (n = 15). Although the body mass increased marginally in the vaccinated mice, no significant difference was found between the two groups. Significance was determined using the log‐rank test (B), two‐way ANOVA and Dunnett's multiple comparisons tests (C and F), and Mann–Whitney U test (E). Error bars, mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.