Journal: bioRxiv

Article Title: Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mouse and non-human primates

doi: 10.1101/2021.01.05.422952

Figure Lengend Snippet: (A) Several RBD-binding antibody isotype titers (IgG, IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) measured weekly in 6-10 week-old BALB/c (n=10, 5 females and 5 males) treated IM with two doses of AC1 and AC3. (B) Ratio of RBD-binding IgG2a and IgG1 antibody titers in serum samples harvested 28 days after vaccination of BALB/c mice as described in A. The Geometric Mean Titer (GMT) is shown above each group. (C and F) Cytokine concentration (pg/mL) in supernatants harvested from splenocytes stimulated for 48h with peptides spanning SARS-CoV-2 Spike protein. Splenocytes were extracted from BALB/c (C) and C57BL/6 (F) animals 4 and 6 weeks, respectively, after vaccination with 10 11 gc of AC1 or AC3. (D-E) Spot forming units (SFU) detected by IFN-γ (D) or IL-4 (E) ELISpot in splenocytes extracted from BALB/c animals 4 weeks after vaccination with 10 11 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. (G-H) Spot forming units (SFU) detected by IFN-γ (G) or IL-4 (H) ELISpot in splenocytes extracted from C57BL/6 animals 6 weeks after vaccination with 10 10 gc of AC1 or AC3 and stimulated with peptides spanning SARS-CoV-2 Spike protein for 48h. For (B-H) data are represented as mean ± SD and groups were compared by Kruskal Wallis and Dunn’s post-test.

Article Snippet: Subsequently, the plates were washed and incubated with biotin-conjugated mouse IFN-γ ELISPOT Detection Antibody (BD Biosciences Cat# 551881, RRID:AB_2868948) and 4 μg/ml biotin-conjugated mouse IL4 detection antibody (BD Biosciences Cat# 551878, RRID:AB_2336921) at room temperature for 3 hours and followed by streptavidin-HRP (dilution 1:1000, Sigma-Aldrich, Cat# 18-152) for 45 minutes.

Techniques: Binding Assay, Concentration Assay, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 1. Most—but not all—healthy donors demonstrate preexisting T-cell immune memory to phosphopeptides displayed on cancer cells. Summary response data for CD8þCD45ROþ T cells from healthy donors (HD) stimulated once in vitro with peptide-pulsed autologous mDCs and cultured for 14 days, measured in triplicate wells in an IFNg ELISpot assay. Data represent a minimum of 3 experiments for each peptide to which a response was observed in a donor. Peptides to which responses were not observed initially may have been analyzed in fewer experiments. A, Responses of HLA-A2þ donors to the indicated HLA-A2–restricted peptides. B, Responses of HLA-B7þ donors to HLA-B7–restricted peptides. The bottom portion of B presents data for 5 HLA-B7–restricted phosphopeptides that were analyzed for HD43,HD44, HD67, and HD89 because of limited sample availability. Insufficient PBMCs were available from HD67 and HD89 to assess responses against the larger cohort of HLA-B7–restricted phosphopeptides.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 2. T-cell responses to viral epitopes define two response patterns that distinguish recent or ongoing antigen exposure. A–D, The indicated CD8þ T-cell subsets were enriched by cell sorting, and analyzed in an IFNg ELISpot assay either after one in vitro stimulation with the indicated viral peptide–pulsed autologous DCs and a 14- day culture (cultured), or immediately ex vivo (direct). Responses were normalized for the expansion of cultured cells over 14 days and CD8þ T-cell subset percentages in the donor’s blood, as described in Materials and Methods. Responses in each subset are reported both as the number of IFNgþ cells per 106 CD8þ T cells (left y-axis) and the subset percentage of the total measured response (right y-axis). Responses to influenza M1 were measured in PBMCs collected before the donor received the annual flu vaccine and in the absence of illness (pre-vaccine) or 16 to 17 days after receiving an influenza vaccine (post-vaccine). C, Due to low cell numbers, no data are available for TSCM responses in HD44. The color coding for each subset is based on Supplementary Fig. S1. Most plots are representative of more than 1 experiment, except HD44 cultured and direct responses to EBV BBLF2/3, HD44 cultured response to CMV pp65495–503, HD43 direct response to influenza pre- vaccine, and cultured and direct response post-vaccine. HD, healthy donor.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: FACS, Enzyme-linked Immunospot, In Vitro, Cell Culture, Ex Vivo

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 3. Longitudinal cultured response patterns to phosphopeptides by healthy donor 44 (HD44). A and B, CD8þ T-cell subsets isolated by FACS from blood samples collected at different time points were stimulated once in vitro with peptide-pulsed autologous mDCs and cultured for 14 days, and then responses to the indicated phosphopeptides were measured in triplicate wells using an IFNg ELISpot assay. Responses were normalized for the expansion of cultured cells and subset percentages in the donor’s blood, as described in Materials and Methods. Responses in each subset are reported both as the number of IFNgþ cells per 106 CD8þ T cells (left y-axis) and the subset percentage of the total measured response (right y-axis). The color coding for each subset is based on Supplementary Fig. S1. Each time point reflects one analysis of PBMCs harvested at that time point. A, Longitudinal responses measured in the first collected blood sample (initial) and in samples collected at the indicated times in relation to the initial sample. B, The N þ SCM subset was sorted using CD95 to evaluate responses from na€ve or memory stem cells. Responses shown are from 5 months (pWWTR186–94), 10 months (pPEG10248–259 and pPEG10248–258), or 13 months (pLSP1249–258, pCHEK1461–471, pSRP72466–473, and pCDC25B38–46) and are representative of at least 2 examined time points. C, Responses to the peptides in each box were considered related to Response Pattern 2 and evidence of active immunogenic exposure at the indicated time points.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: Cell Culture, Isolation, In Vitro, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 4. Longitudinal cultured response patterns to phosphopeptides by healthy donor 43 (HD43). A and B, CD8þ T-cell subsets isolated by FACS from blood samples collected at different time points were stimulated once in vitro with peptide-pulsed autologous mDCs and cultured for 14 days, and then responses to the indicated phosphopeptides were measured in triplicate wells using an IFNg ELISpot assay. Responses were normalized for the expansion of cultured cells and subset percentages in the donor’s blood, as described in Materials and Methods. Responses in each subset are reported both as the number of IFNgþ cells per 106 CD8þ T cells (left y-axis) and the subset percentage of the total measured response (right y-axis). The color coding for each subset is based on Supplementary Fig. S1. Each time point reflects one analysis of PBMCs harvested at that time point. A, Longitudinal responses measured in the first collected blood sample (initial) and in samples collected at the indicated times in relation to the initial sample. B, The N þ SCM subset was sorted using CD95 to evaluate responses from na€ve or memory stem cells. Responses shown are from 9 months (pPEG10248–259), 13 months (pCDC25B38–46), or 16 months (pCHEK1461–471 and pSRP72466–473) and are representative of at least 2 examined time points. C, Responses to the peptides in each box were considered related to Response Pattern 2 and evidence of active immunogenicexposure atthe indicated time points.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: Cell Culture, Isolation, In Vitro, Enzyme-linked Immunospot

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 5. Direct responses to phosphopeptides in healthy donors (HD) are largely consistent with cultured response patterns. Responses of CD8þ T-cell subsets isolated by FACS from HD44 (A) and HD43 (B) to the indicated phos- phopeptides were measured directly ex vivo and normalized for the subset percentages in the donor’s blood, as described in Materials and Methods. Responses in each subset are reported both as the number of IFNgþ cells per 106 CD8þ T cells (left y-axis) and the subset percentage of the total measured response (right y-axis). The color coding for each subset is based on Supplementary Fig. S1. Each time point reflects one analysis of PBMCs harvested at that time point.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: Cell Culture, Isolation, Ex Vivo

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 6. Immune responses to viral and phosphorylated peptides by ovarian cancer patients. CD8þ T-cell subsets (TCMþ TN and TEM þ TEMRA þ TSCM; Supplementary Fig. S2) isolated by FACS from 4 identified ovarian cancer patients (Sup- plementary Table S3) were stimulated once in vitro with the indicated viral or phosphorylated peptide–pulsed autologous mDCs and irradiated CD4CD8 PBLs as antigen-presenting cells and cultured for 14 days, and then responses to the same peptides were measured in triplicate wells in an IFNg ELISpot assay. Responses were normalized for the expansion of cultured cells and subset percentages in the donor’s blood, as described in Materi- als and Methods. Each datapoint reflects one analysis of PBMCs or TILs harvested at the identified time point, and is the sum of responses from both sorted subsets. T, phosphopeptide expressed on the patient’s tumor as identified by mass spectrometry.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: Isolation, In Vitro, Irradiation, Cell Culture, Enzyme-linked Immunospot, Phospho-proteomics, Mass Spectrometry

Journal: Cancer Immunology Research

Article Title: Characteristics of immune memory and effector activity to cancer-expressed MHC class I phosphopeptides differ in healthy donors and ovarian cancer patients

doi: 10.1158/2326-6066.cir-21-0111

Figure Lengend Snippet: Figure 7. Phosphopeptide expression on patient tumor is associated with an active immune response. CD8þ T-cell subsets (TCM þ TN and TEM þ TEMRA þ TSCM; Supplementary Fig. S2) isolated by FACS from ovarian cancer patients VTB241 (A) and VTB239 (B; Supplementary Table S3) were stimulated once in vitro with the indicated viral or phosphorylated peptide–pulsed autologous mDCs and irradiated CD4CD8 PBLs as antigen-presenting cells and cultured for 14 days, and then responses to the same peptides were measured in triplicate wells in an IFNg ELISpot assay. Responses were normalized for the expansion of cultured cells and subset percentages in the donor’s blood, as described in Materials and Methods. Responses in each subset are reported both as the number of IFNgþ cells per 106 CD8þ T cells (left y-axis) and the subset percentage of the total measured response (right y-axis). The color coding for each subset is based on Supplementary Fig. S2. Each graph represents one analysis of PBLs or TILs.

Article Snippet: IFNg Single-Color ELISpot Plates (Cellular Technology Limited; cat #hIFNg-1M/2) were activated with 70% ethanol and incubated with primary antibody at 4 C overnight.

Techniques: Phospho-proteomics, Expressing, Isolation, In Vitro, Irradiation, Cell Culture, Enzyme-linked Immunospot

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐E7P therapeutic vaccine induces antigen‐specific immune responses and potent antitumor protection in TC‐1 tumor model. (A) Schematic of the adenoviral‐based therapeutic vaccine. (B) Experimental schema for evaluating the antitumor efficacy of Ad‐E7P in the TC‐1 tumor model. C57BL/6 mice were subcutaneously inoculated with 1 × 10 6 TC‐1 tumor cells in the right flank, followed by weekly intramuscular (i.m.) immunizations with 10 9 viral particles (VP) of Ad‐E7P or the empty vector (Adv) for a total of two doses when tumor volumes reached approximately 50 mm 3 . Mice in the negative control group received PBS. On Day 17, lymph nodes and spleens were harvested for immune cell analysis. (C) Tumor growth curves of mice treated with Ad‐E7P, Adv, or PBS ( n = 5 mice per group). (D and E) Flow cytometric analysis of DCs, total T cells, and CD8+ T cells in the lymph nodes (D), and CD8+ T cells in the spleen (E) following immunization ( n = 5 mice per group). (F) On Day 17, antigen‐specific T cell activation in splenocytes was assessed by ELISpot assay following in vitro stimulation with the E7 49‐57 peptide. SFU, spot‐forming unit ( n = 5 mice per group). (G) Treatment schedule for Ad‐E7P vaccination and correlative immune kinetics analysis. (H and I) Flow cytometric analysis of the percentages of CD8+ T cells (H) and ELISpot analysis of IFN‐γ–producing cells (I) in the spleen at different time points following Ad‐E7P vaccination ( n = 3 per group). Data are presented as the means ± SD. One‐way analysis of variance (ANOVA) with Tukey's multiple comparisons test was performed for all comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Plasmid Preparation, Negative Control, Cell Analysis, Activation Assay, Enzyme-linked Immunospot, In Vitro

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: The therapeutic vaccine Ad‐E7P induces intratumoral immune activation and reduces tumor recurrence. (A) Representative flow cytometry plots of CD8+ TILs. Numbers indicate the percentage of CD8+ TILs within the gated population. (B) Quantification of CD8+ TILs is shown ( n = 5 per group). (C) Representative hematoxylin and eosin (H&E)–stained images of paraffin‐embedded TC‐1 tumor sections from different treatment groups. TLSs were observed adjacent to tumors in Ad‐E7P‐vaccinated mice and confirmed by both H&E and CD3 immunohistochemical staining. Scale bars: 200 µm (overview) and 50 µm (zoomed‐in view). (D and E) Experimental scheme. TC‐1 tumor‐regressing mice, cured by Ad‐E7P vaccination, were re‐inoculated with the same number of tumor cells in the left flank on day 60, and survival was monitored (D). Untreated mice served as controls. Tumor growth curves (E, left) and corresponding survival kinetics of mice after tumor rechallenge (E, right) (Control, n = 5 mice; Ad‐E7P, n = 10 mice). (F and G) Flow cytometric analysis of splenic CD8+ T cells after tumor rechallenge. Representative plots from Day 7, including a CD8 fluorescence‐minus‐one (FMO) control to identify the positive population (F), and the percentages of CD8+ T cells on Days 7, 14, 21, 30, and 60 (G) ( n = 3 per group). (H) Summary statistics for ELISpot assays on Day 7 and 14 after tumor rechallenge ( n = 5 per group). Data are presented as mean ± SD. r. Statistical analyses were conducted by one‐way ANOVA with Tukey's correction for multiple comparisons in (B), by two‐way ANOVA in (H), and by log‐rank (Mantel‐Cox) test in (E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Activation Assay, Flow Cytometry, Staining, Immunohistochemical staining, Control, Fluorescence, Enzyme-linked Immunospot

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐E7P vaccine combined with the oncolytic virus SKV‐012 inhibits tumor progression and induces a potent antitumor immune response in the TC‐1 tumor‐bearing mouse model. (A) Experimental scheme. C57BL/6 mice were subcutaneously inoculated with 1 × 10 6 TC‐1 tumor cells in the right flank. When tumor volumes reached approximately 50 mm 3 , mice were assigned to receive Ad‐E7P, SKV‐012, a combination of Ad‐E7P and SKV‐012, or PBS as a control. For Ad‐E7P group, mice received two doses of 10 9 VP Ad‐E7P administered once a week. For SKV‐012 group, mice received three intratumoral injections of 10 6 PFU SKV‐012 every 3 days. For combination treatment group, mice received two doses of 10 9 VP Ad‐E7P once a week and three doses of 10 6 PFU SKV‐012 intratumorally every three days. (B) Tumor growth curves ( n = 5 per group). (C and D) Representative IFN‐γ ELISPOT images (C) and summary of ELISPOT results (D) from splenocytes stimulated in vitro with the E7 49‐57 peptide ( n = 5 per group). (E) Proportions of various immune cell populations in lymph nodes and spleens ( n = 3 per group). (F and G) Representative flow cytometric analysis of CD86 expression on dendritic cells in the lymph nodes (F), and quantification of mean fluorescence intensity (MFI) of CD86 expression (G) ( n = 3 per group). (H and I) Representative flow cytometric analysis of IFN‐γ+ CD8+ T cells in both lymph nodes and spleen (H), and statistical analysis of the results (I). ( n = 3 per group). Data are presented as mean ± SD. One‐way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Virus, Control, Enzyme-linked Immunospot, In Vitro, Expressing, Fluorescence

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Combination of Ad‐E7P vaccine and SKV‐012 provides long‐term protection in the TC‐1 tumor model and induces robust antitumor immunity in the mEERL tumor model. (A) Experimental design. Mice cured by the SKV‐012+Ad‐E7P combination treatment were re‐inoculated with 1 × 10 6 TC‐1 tumor cells in the left flank on Day 60, and survival was monitored. Untreated mice served as controls. (B) Survival kinetics of mice after tumor rechallenge (Control, n = 5 mice; SKV‐012+Ad‐E7P, n = 10 mice). (C) On Days 7, 14, 21, 30, 60, and 90 post‐tumor challenge, the proportion of CD8+ T cells in the spleen was assessed by flow cytometry ( n = 3 per group). (D) On Days 7 and 14 post‐tumor challenge, spleens were harvested, and the frequency of IFN‐γ‐producing T cells was assessed using an ELISPOT assay following in vitro stimulation with the E7 peptide ( n = 5 per group). (E and F) Differential expression of KLRG1 and CD127 on spleen T cells. Representative contour plots (E, Day 7) and quantification (F) of KLRG1 and CD127 expression are shown ( n = 5 per group). (G) Experimental Scheme. C57BL/6 mice were subcutaneously implanted with 2 × 10 6 mEERL tumor cells. Mice were treated with Ad‐E7P, SKV‐012, Ad‐E7P + SKV‐012, or PBS (control) on Day 12, when tumor volumes reached approximately 50 mm 3 . For Ad‐E7P group, mice received two doses of 10 9 Ad‐E7P administered once a week. For SKV‐012 group, mice received three intratumoral injections of 10 6 PFU SKV‐012 every three days. For combination treatment group, mice received two doses of 10 9 VP Ad‐E7P once a week and three doses of 10 6 PFU SKV‐012 intratumorally every 3 days. Tumor volume was monitored. (H) Tumor growth curves in mEERL model ( n = 5 per group). (I) Survival kinetics in mEERL model (Control, n = 5 mice; Treatment group, n = 7 mice). Mice from independent experimental cohorts. (J and K) Representative images of IHC staining for CD3 in mEERL tumor tissue sections on Day 20 (K), and quantification of CD3+ cells per tumor area (J) (cells/mm 2 ) ( n = 3 mice per group; N = 3 images/field of view per mouse).

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunospot, In Vitro, Quantitative Proteomics, Expressing, Immunohistochemistry

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐MP triggers antigen‐specific T cell response and enhances antitumor efficacy with SKV‐012 in vitro. (A) Experimental design for assessing the SKV‐012 + Ad‐E7P immune response in vitro. The figure panel was created in BioRender. (B) Representative flow cytometry plots of CD80 and CD86 expression in DCs. DCs were isolated and induced from the peripheral blood of HPV‐related tumor patients and loaded with the Ad‐MP vaccine, and were tracked at Day7. (C and D) Representative flow cytometry plots of activated CD69 expression in T cells (C) and quantification of CD69+CD3+ T cells are shown (D). The Ad‐MP‐loaded DCs were co‐cultured with autologous PBMCs at a ratio of 1:100 for 24 h ( n = 3 per group). Empty‐loaded DC were prepared as a control. (E) The number of IFN‐γSFUin PBMCs was assessed after 24 h of stimulation with single peptides ( n = 3 per group). Before performing the ELISPOT assay, antigen‐loaded DCs were pre‐co‐cultured with autologous PBMCs from HPV‐related tumor patients for 24 h ( n = 3 per group). (F) The concentrations of IL‐12, IFN‐γ, IL‐2, and TNF‐α in the supernatants were measured by ELISA after 48 h of co‐culture of primary tumor cells with autologous PBMCs and DCs. Prior to co‐culturing with activated autologous PBMCs, tumor cells were infected with SKV‐012 at an MOI of 0.01 for 24 h to assess responses ( n = 3 per group). Data are presented as mean ± SD. One‐way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: In Vitro, Flow Cytometry, Expressing, Isolation, Cell Culture, Control, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Infection