Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA expressed in vitro . (A) Schematic diagram of the TriStim-E6/E7 mRNA construct. (B) The expression of CD80-E7 fusion protein (~60 kDa) was detected in mRNA-transfected HEK293 cells by western blotting. (C) The expression of E6 was detected in mRNA-transfected HEK293 cells by immunofluorescence microscopy. (D) The expression of CD80, 4-1BBL and CD70 was detected in mRNA-transfected HEK293 cells by flow cytometry. Data are representative of three independent experiments.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: In Vitro, Construct, Expressing, Transfection, Western Blot, Immunofluorescence, Microscopy, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA induced a robust antigen-specific cellular immune response in mice. (A) Scheme of vaccination. Mice were intramuscularly vaccinated with one, two, or three doses of 25 μg TriStim-E6/E7 mRNA-LNP or control empty LNPs, and the cellular immune responses were assessed. (B, C) IFNγ-producing CD8 + T cells and TNFα-producing CD8 + T cells were measured using flow cytometry in splenocytes stimulated with HPV16 E6 and E7 peptide pools. (D-F) E7 tetramer + CD8 + T cells and E6 tetramer + CD8 + T cells in peripheral blood of immunized mice were measured using flow cytometry. Representative flow cytometry plots of E7 tetramer + CD8 + T cells (D) and quantification of E7 tetramer + CD8 + T cells (E) and E6 tetramer + CD8 + T cells (F) are presented. (G) Splenic T cells from vaccinated mice induced cytotoxicity against TC-1 tumor cells. Data are presented as the mean ± standard deviation, with n = 4 (B, C, E, F) and n = 3 (G) . Statistical analysis was conducted using an unpaired, two-tailed Student’s t-test; ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Control, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA inhibited tumor growth in a syngeneic TC-1 tumor-bearing mouse model. Mice bearing subcutaneous TC-1 tumors were treated with 1, 3 or 5 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and their tumor volumes (A) and body weights (C) were measured, and Kaplan–Meier survival curves (B) were generated. Data are presented as the mean ± standard error of the mean (n = 6). Statistical analysis for tumor volumes was conducted with two-way ANOVA; *P ≤ 0.05, ***P ≤ 0.001. Survival curves were analyzed with a Log-rank (Mantel-Cox) test; **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Generated

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA induced anti-tumor immune memory. (A) C57BL/6 mice were subcutaneously implanted with TC-1 tumor and followed by the treatment of 25 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks. Forty days after tumor implantation, mice cured by TriStim-E6/E7 mRNA vaccination were rechallenged with an autologous TC-1 tumor, and their tumor volume was determined. Naïve mice were challenged with the same tumor at the same time as controls. (B) C57BL/6 mice were intramuscularly vaccinated with 5 or 25 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks. Mice were then challenged three times with TC-1 cells at 21, 56, and 125 days after the first vaccination, respectively. Their tumor volume was determined. Naïve mice were challenged with the same tumor at the same time as controls. (C) Peripheral blood of mice in (B) was collected before and after the third tumor challenge (i.e., at 124 and 131 days post-first vaccination) and analyzed for E7 tetramer + CD8 + cells by flow cytometry. Blue lines (left panel) indicate data generated from mice vaccinated with 5 μg of TriStim-E6/E7 mRNA, while the red lines (right panel) indicate data generated from mice vaccinated with 25 μg of TriStim-E6/E7 mRNA. Data (A, B) are presented as the mean ± standard error of the mean (n = 6). Statistical analysis was conducted using an unpaired, two-tailed Student’s t-test; *P ≤ 0.05, **P ≤ 0.01.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Tumor Implantation, Flow Cytometry, Generated, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA induced superior anti-tumor potency. Mice bearing subcutaneous TC-1 tumors were treated with 5 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and their tumor volumes were measured. Treatment with the same dose of E6/E7-P2P16-MITD mRNA (A) , E6/E7-mut mRNA (B) , Ubiquitin-E6/E7 mRNA (C) , combination of E6/E7-mut mRNA and CD40L/TLR4/CD70 mRNA (D) , E6/E7-CD80/4-1BBL mRNA (E) , E6/E7-CD80/OX40L/CD70 mRNA (E) , E6/E7-CD80/4-1BBL/OX40L mRNA (E) was used for comparison. Data are presented as the mean ± standard error of the mean (n = 6).

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Ubiquitin Proteomics, Comparison

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA induced anti-tumor activity was mediated by antigen-specific CD8 + T cells. (A, B) Mice bearing subcutaneous TC-1 tumors were treated with 5 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and 200 μg of anti-CD4 or anti-CD8 monoclonal antibodies twice a week for 3 consecutive weeks (on day 0, 3, 7, 10 and 14 after grouping), and their tumor volumes (A) were measured, and Kaplan–Meier survival curves (B) were generated. (C, D) Mice immunized with 3 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks, were subcutaneously implanted with TC-1 tumor 21 days after the first immunization. Tumors were resected 2 days later for TILs analysis. Representative flow cytometry plots of E7 tetramer + CD8 + T cells (C) and quantification of E7 tetramer + CD8 + T cells (D) were presented. Data in (A) are presented as the mean ± standard error of the mean (n = 6). Data in (D) are presented as the mean ± standard deviation (n = 5). Statistical analysis for tumor volumes (A) was conducted through two-way ANOVA with Sidak’s multiple comparison test; ns, not significant, *P ≤ 0.05. Survival curves (B) were analyzed with a Log-rank (Mantel-Cox) test; ns, not significant, ***P ≤ 0.001. Statistical analysis for E7 tetramer + CD8 + T cells (D) was conducted using an unpaired, two-tailed Student’s t-test; ***P ≤ 0.001.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Activity Assay, Bioprocessing, Generated, Flow Cytometry, Standard Deviation, Comparison, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: TriStim-E6/E7 mRNA induced T cell activation and proliferation in vitro . (A, B) HEK293 cells co-transfected with 0.25 μg of mb-α-CD3 mRNA and 5 μg of TriStim-E6/E7 mRNA, were used to stimulate mouse splenocytes. The expression of CD69 (A) and CD25 (B) in splenic T cells was assessed by flow cytometry analysis. Control groups included HEK293 cells transfected with empty LNPs, 0.25 μg of mb-α-CD3 mRNA alone, or combination of 0.25 μg of mb-α-CD3 mRNA and 5 μg of E6/E7-P2P16-MITD mRNA. (C, D) HEK293 cells co-transfected with 0.25 μg of mb-α-CD3 mRNA and 5 μg of TriStim-E6/E7 mRNA, were treated with mitomycin C before co-cultured with mouse splenocytes. The proliferation of CFSE-prestained mouse splenic T cells were assessed by flow cytometry. Control groups included HEK293 cells transfected with empty LNPs, 0.25 μg of mb-α-CD3 mRNA alone, or combination of 0.25 μg of mb-α-CD3 mRNA and 5 μg of E6/E7-P2P16-MITD mRNA. Data are presented as the mean ± standard deviation (n = 3). Statistical analysis was conducted using an unpaired, two-tailed Student’s t-test; **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Activation Assay, In Vitro, Transfection, Expressing, Flow Cytometry, Control, Cell Culture, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: Combination treatment with TriStim-E6/E7 mRNA and immune checkpoint inhibitors improved anti-tumor efficacy. Mice bearing subcutaneous TC-1 tumors were treated with 3 μg of TriStim-E6/E7 mRNA once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and 200 μg of anti-PD-L1 monoclonal antibody twice a week for 3 consecutive weeks (on day 0, 3, 7, 10 and 14 after grouping), and their tumor volumes (A) were measured. E7 tetramer + CD8 + T cells (B) in peripheral blood of mice were measured using flow cytometry, 18 days after the first dose. Data in (A) are presented as the mean ± standard error of the mean (n = 6). Data in (B) are presented as the mean ± standard deviation (n = 4).

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: Human TriStim-E6/E7 mRNA inhibited tumor growth in a humanized mouse model. (A, B) . CD28/4-1BB/CD27 triple-humanized mice were intramuscularly vaccinated with 20 μg hTriStim-E6/E7 mRNA-LNP or control empty LNPs once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and the cellular immune responses were assessed. IFNγ-producing CD8 + T cells (A) and TNFα-producing CD8 + T cells (B) were measured using flow cytometry in splenocytes stimulated with HPV16 E6 and E7 peptide pools. (C, D) CD28/4-1BB/CD27 triple-humanized mice bearing subcutaneous TC-1 tumors were treated with 3 μg or 20 μg of hTriStim-E6/E7 mRNA once a week for 3 consecutive weeks (on day 0, 7 and 14 after grouping), and their tumor volumes (C) were measured. E7 tetramer + CD8 + T cells (D) in peripheral blood of mice were measured using flow cytometry, 21 days after the first dose. Data in (A, B) are presented as the mean ± standard deviation (n = 4). Data in (C) are presented as the mean ± standard error of the mean (n = 8). Data in (D) are presented as the mean ± standard deviation (n = 8). Statistical analysis in (A, B, D) was conducted using an unpaired, two-tailed Student’s t-test; ns, not significant, *P ≤ 0.05, ***P ≤ 0.001. Statistical analysis for tumor volumes (C) was conducted through two-way ANOVA with Sidak’s multiple comparison test; *P ≤ 0.05, ***P ≤ 0.001.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Control, Flow Cytometry, Standard Deviation, Two Tailed Test, Comparison

Journal: Frontiers in Immunology

Article Title: A novel mRNA-based therapeutic vaccine elicits robust anti-tumor immunity against HPV-associated malignancies

doi: 10.3389/fimmu.2026.1823374

Figure Lengend Snippet: Human TriStim-E6/E7 mRNA did not induced cytokine release syndrome in an immune system humanized mouse model. (A) M-NSG mice were engrafted with human PBMCs. Twenty days post-engraftment, reconstitution of human leukocytes was confirmed by flow cytometry analysis of mouse peripheral blood samples using an anti-human CD45 antibody. (B) On day 21, PBMC-reconstituted mice were intramuscularly vaccinated with 20 μg of hTriStim-E6/E7 mRNA-LNP or control empty, or intravenously injected with 2 mg/kg of TGN1412 and their serum cytokines (IFNγ, IL-10, IL-6, IL-2, IL-4, and TNFα) were determined by cytometric bead array analysis 6 and 24 h post-treatment. Data are presented as the mean ± standard deviation (n = 5). Data are representative of two independent experiments.

Article Snippet: For human TriStim-E6/E7 mRNA immunization, CD28/4-1BB/CD27 triple-humanized mice from Shanghai Model Organisms Center, Inc. were used.

Techniques: Flow Cytometry, Control, Injection, Standard Deviation

Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: LINE-1 RNAs are primarily chromatin-associated nascent transcripts. A, Subcellular localization of LINE-1 RNAs in cancer cells. B, Immunoblot of LINE-1 ORF1p expression across 10 human cancer cell lines. GAPDH was used as a loading control. C, Relative abundance of RNAs in cytosolic, nuclear soluble, or chromatin fractions as assessed by qRT-PCR. The relative target sites of four LINE-1–specific qPCR primers (A, B, C, and D) are schematized along the L1HS consensus sequence. 45S rRNA is analyzed as a control for chromatin RNA. Exonic GAPDH is a control for cytosolic RNA. Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated using an unpaired two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. D, Representative fluorescent micrographs of four human cancer cell lines probed with smRNA-FISH oligos conjugated with Cy5 (pink pseudo-colored, L1HS 5′UTR) or Cy3 (yellow pseudo-colored, β-actin). DAPI staining of nuclei is shown as blue pseudo-color. Quantification of the relative number of cytosolic versus nuclear foci per transcript per cell. N = 30 cells were quantified per cell line. P values were calculated using an unpaired two-sided Student t test. E, ChIRP-seq to assess genomic binding sites of LINE-1 5′UTR-containing transcripts. Scatter plot shows correlation of read counts from two ChIRP-seq biological replicates. Proportion of HOMER annotations overlapping ChIRP-seq 5′UTR peaks are shown as a pie chart. F, Top: binding sites for Northern blotting oligo probes A and B are depicted along the L1HS 5′UTR consensus sequence. Bottom: representative Northern radiograms with L1HS 5′UTR A or B vs. β-actin probes. Relative positions for 28S (5.8 kb) and 18S (1.8 kb) rRNAs were marked based on ethidium bromide staining signal during gel electrophoresis and alkaline transfer. G, Relative polyadenylation of transcripts was assessed by qRT-PCR of the enriched or unbound fraction after oligo-dT bead isolation. The relative target sites of LINE-1 qPCR primers (A, B, C, and D) are schematized in C . Results are shown as mean ± SD ( N ≥ 3 replicates). P values were calculated by an unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Left: Experimental scheme of 4sU pulse-chase experiments in MCF7 cells . Right: Chromatin dissociation kinetics of various RNAs. SRY is a control for cytosol-localized noncoding RNA, whereas α-satellite RNA is a control for chromatin-associated noncoding RNA. Chr, chromatin bound; Cyto, cytosolic; LTR, long terminal repeat; Nuc, nuclear soluble or chromatin released; RPM, reads per million.

Article Snippet: The predesigned HCR control probe set targeting human β-actin mRNA was also purchased from Molecular Instruments, Inc. All HCR RNA-FISH experiments were performed following the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Sequencing, Staining, Binding Assay, Northern Blot, Nucleic Acid Electrophoresis, Isolation, Pulse Chase

Journal: Cancer Discovery

Article Title: LINE-1 Locus Transcription Nucleates Oncogenic Chromatin Architecture

doi: 10.1158/2159-8290.CD-25-1085

Figure Lengend Snippet: LINE-1 transcription is necessary and sufficient to form long-range chromatin interactions. A, L1HS transcription unit is schematized along with relative positions of 5′UTR-targeting ASO1, ASO2, and smRNA-FISH probes. Subfamily expression of LINE-1 loci by RNA-seq in MCF7 cells upon ASO treatment. SCR, scrambled control. Results are shown as mean ± SD ( N ≥ 3 replicates). TPM values for individual loci were summed per subfamily and normalized by subfamily full-length copy number. P values were calculated using two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, NS, not significant. B, Expression changes of individual full-length LINE-1 loci upon LINE-1 ASO2 treatment in MCF7 cells. HILLs are marked in large red dots, whereas non-HILLs are marked in small pink dots. C, Representative fluorescent micrographs of smRNA-FISH foci in MCF7 cells treated with SCR or LINE-1 ASO2. Pink pseudo-colors, LINE-1 Cy5 probes. Yellow pseudo-colors, β-actin Cy3 probes. Quantification of the numbers of LINE-1 smRNA-FISH foci from N = 30 cells. P values were calculated using an unpaired Student t test. **, P < 0.01; ***, P < 0.001. D, Representative genome browser view of L1CaP-C interactions in MCF7 cells treated with SCR or LINE-1 ASO2. E, Circos plots depicting intrachromosomal and interchromosomal multi-way chromunities identified by the Chromunity algorithm as orange arcs using MCF7 L1CaP-C data. Green boxes depict positions of full-length LINE-1 loci in hg38. The top 50 genes with the highest number of supporting concatemers are shown. F, Schematic of cell engineering to test sufficiency of LINE-1 transcription in generating de novo chromatin interactions. Donor cassette encodes mScarlet to enrich for knock-in MCF7 cells by FACS. Capture probe was designed to target the 3′ end of the TRE3GS promoter to ensure equal enrichment across conditions and transgenes. G, Top: genotyping validation of knock-in cassettes in enriched MCF7 cells engineered to harbor EV control transgene or a 6-kb LINE-1 reporter. Primers span the Rogi2 locus. “+/−” denotes successful knock-in and “−/−” denotes the empty allele. Bottom: expression analysis of reporter transgenes by qRT-PCR with or without dox induction. H, Violin plots of the distance span ( d ) of L1CaP-C–detected interactions with the reporter locus. P values were calculated based on combined replicates using a Mann-Whitney rank-sum test. ****, P < 0.0001. Cyto, cytosolic; Nuc, nuclear; WT, wild type.

Article Snippet: The predesigned HCR control probe set targeting human β-actin mRNA was also purchased from Molecular Instruments, Inc. All HCR RNA-FISH experiments were performed following the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing, Control, Knock-In, Biomarker Discovery, Quantitative RT-PCR, MANN-WHITNEY