Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: Initial immune characterization of HA DNA-LNP formulations and immunogenicity (A) Schematic of DNA-LNP N/P ratios and immunization regimen. (B–D) Biophysical characterization of DNA-LNPs at different N/P ratios. (B) Particle size; (C) polydispersity index (PDI); (D) zeta potential. (E) Representative fluorescence-activated cell sorting (FACS) plots of GC B cells. (F) Bar plots quantifying frequency of GC B cells. (G) Frequency of CA09 HA-specific GC B cells. (H) Frequency of activated Tfh cells. (I) IFNγ ELISpot of splenocytes. (J) Representative TEM images of HA DNA-LNP (top) and HA mRNA-LNP (bottom). Scale bar 100 nm (K and L) Fold change cytokine induction in DLNs at 4 h (K) and 24 h (L) after immunization quantified using Luminex. (M and N) ELISpot assay measuring IFNα (M) and IFNγ (N) 20 h after stimulation of splenocytes ex vivo with DNA-LNP, plasmid DNA, or DNA-LNP in the presence of chemical inhibitors to the indicated DNA sensors. (O) Schematic of relevant pathways implicated in DNA-LNP sensing. Dots represent individual animals; n = 8–9 (E–H), n = 5 (I, K, and L), or n = 3–4 animals per group (L and M); data pooled or representative from two independent experiments (E–H, M, and N) or from one independent experiment (I–L). Plots show mean with SD (B–D and I) or geometric mean with geometric SD (F–H and K–N). Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (F–H, K, and L) or compared to DNA-LNP control (M and N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: For protein vaccination, 1 μg of recombinant full-length CA09 HA (Sino Biologicals Cat# 11055-VNAB) was formulated 1:1 v/v with Addavax Adjuvant (Cat# vax-adx-10, Invivogen).

Techniques: Zeta Potential Analyzer, Fluorescence, FACS, Enzyme-linked Immunospot, Luminex, Ex Vivo, Plasmid Preparation, Control

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP induces robust GC and serum responses Mice were immunized with HA DNA-LNP (2 μg), HA mRNA-LNP (2 μg), or adjuvanted HA protein (1 μg). GC responses were assessed in the DLNs 14 days post immunization and serum responses longitudinally. (A) Representative FACS plots of activated Tfh cells. (B and C) Bar plots show quantification of frequency (B) and numbers (C) of activated Tfh cells. (D) Representative FACS plots of total GC B cells. (E and F) Bar plots show quantification of frequency (E) and numbers (F) of total GC B cells. (G) Representative FACS plots of CA09 HA-specific GC B cells. (H and I) Bar plots show frequency (H) and numbers (I) of CA09 HA-specific GC B cells. (J) Area under the curve (AUC) of total A/California/04/2009 HA-specific serum IgG ELISA data. (K) Serum endpoint titers at week 8 to various H1N1 HAs. (L) HAI titers at week 8 to A/California/07/2009 X-179A. (M and N) AUC of serum binding antibodies to A/Guangdong-Maonan/SWL1536/2019 HA (M) and A/Victoria/4897/2022 HA (N). (O and P) HAI titers to A/Netherlands/602/2009 (O) and A/New York City/PV63249/2022 (P). Dots represent individual animals (B, C, E, F, H, I, K, and L); n = 9–10 animals per group; data pooled from two independent experiments. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (A–I) or active immunization groups (K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: For protein vaccination, 1 μg of recombinant full-length CA09 HA (Sino Biologicals Cat# 11055-VNAB) was formulated 1:1 v/v with Addavax Adjuvant (Cat# vax-adx-10, Invivogen).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP induces potent memory responses in mice and rabbits (A) Schematic of mouse immunization regimen. (B) IFNγ-secreting cells in splenocytes by ELISpot. (C) IFNγ-secreting effector CD8 + T cells by flow cytometry. (D) CA09 HA-specific ASC responses in bone marrow by ELISpot. (E) Representative FACS plot of CA09 HA-specific MBCs. (F and G) Bar plots show frequency (F) and numbers (G) of CA09 HA-specific MBCs. (H) Schematic of rabbit immunization regimen. (I–K) IFNγ ELISpot on peripheral blood mononuclear cells (PBMCs) at day 42 (I), day 105 (J), and day 202 (K). (L) AUC of total A/California/04/2009 HA-specific serum IgG ELISA data. (M and N) HAI titers to A/Netherlands/602/2009 (M) and A/New York City/PV63249/2022 (N). Dots represent individual animals (C, D, F, and G); n = 9–10 animals per group (B–D, F, and G), n = 5 animals per group (I–N); data pooled from two independent experiments. Plots show mean with SD (B and I–K) or geometric mean with geometric SD (C, D, F, G, and L–N). Unpaired one-way or two-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ANOVA was performed at the final time point for (L–N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: For protein vaccination, 1 μg of recombinant full-length CA09 HA (Sino Biologicals Cat# 11055-VNAB) was formulated 1:1 v/v with Addavax Adjuvant (Cat# vax-adx-10, Invivogen).

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet:

Article Snippet: For protein vaccination, 1 μg of recombinant full-length CA09 HA (Sino Biologicals Cat# 11055-VNAB) was formulated 1:1 v/v with Addavax Adjuvant (Cat# vax-adx-10, Invivogen).

Techniques: Virus, Recombinant, Lysis, Reporter Gene Assay, Cell Stimulation, Electron Microscopy, Luminex, Enzyme-linked Immunospot, Luciferase, Plasmid Preparation, Software, Synthesized

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: Initial immune characterization of HA DNA-LNP formulations and immunogenicity (A) Schematic of DNA-LNP N/P ratios and immunization regimen. (B–D) Biophysical characterization of DNA-LNPs at different N/P ratios. (B) Particle size; (C) polydispersity index (PDI); (D) zeta potential. (E) Representative fluorescence-activated cell sorting (FACS) plots of GC B cells. (F) Bar plots quantifying frequency of GC B cells. (G) Frequency of CA09 HA-specific GC B cells. (H) Frequency of activated Tfh cells. (I) IFNγ ELISpot of splenocytes. (J) Representative TEM images of HA DNA-LNP (top) and HA mRNA-LNP (bottom). Scale bar 100 nm (K and L) Fold change cytokine induction in DLNs at 4 h (K) and 24 h (L) after immunization quantified using Luminex. (M and N) ELISpot assay measuring IFNα (M) and IFNγ (N) 20 h after stimulation of splenocytes ex vivo with DNA-LNP, plasmid DNA, or DNA-LNP in the presence of chemical inhibitors to the indicated DNA sensors. (O) Schematic of relevant pathways implicated in DNA-LNP sensing. Dots represent individual animals; n = 8–9 (E–H), n = 5 (I, K, and L), or n = 3–4 animals per group (L and M); data pooled or representative from two independent experiments (E–H, M, and N) or from one independent experiment (I–L). Plots show mean with SD (B–D and I) or geometric mean with geometric SD (F–H and K–N). Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (F–H, K, and L) or compared to DNA-LNP control (M and N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The construct encoding the full-length SARS-CoV-2 spike glycoprotein (with the D614G mutation, which arose early during the COVID-19 pandemic) has been described previously., , Full length CA09 HA was cloned into a pVAX1 vector behind an IgE leader sequence and obtained from Genscript. mRNA vaccines encoding the full-length wild-type SARS-CoV-2 spike glycoprotein were synthesized as described in Laczkó et al. CA09 HA mRNA-LNPs were described previously.

Techniques: Immunopeptidomics, Zeta Potential Analyzer, Fluorescence, FACS, Enzyme-linked Immunospot, Luminex, Ex Vivo, Plasmid Preparation, Control

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP induces robust GC and serum responses Mice were immunized with HA DNA-LNP (2 μg), HA mRNA-LNP (2 μg), or adjuvanted HA protein (1 μg). GC responses were assessed in the DLNs 14 days post immunization and serum responses longitudinally. (A) Representative FACS plots of activated Tfh cells. (B and C) Bar plots show quantification of frequency (B) and numbers (C) of activated Tfh cells. (D) Representative FACS plots of total GC B cells. (E and F) Bar plots show quantification of frequency (E) and numbers (F) of total GC B cells. (G) Representative FACS plots of CA09 HA-specific GC B cells. (H and I) Bar plots show frequency (H) and numbers (I) of CA09 HA-specific GC B cells. (J) Area under the curve (AUC) of total A/California/04/2009 HA-specific serum IgG ELISA data. (K) Serum endpoint titers at week 8 to various H1N1 HAs. (L) HAI titers at week 8 to A/California/07/2009 X-179A. (M and N) AUC of serum binding antibodies to A/Guangdong-Maonan/SWL1536/2019 HA (M) and A/Victoria/4897/2022 HA (N). (O and P) HAI titers to A/Netherlands/602/2009 (O) and A/New York City/PV63249/2022 (P). Dots represent individual animals (B, C, E, F, H, I, K, and L); n = 9–10 animals per group; data pooled from two independent experiments. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (A–I) or active immunization groups (K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The construct encoding the full-length SARS-CoV-2 spike glycoprotein (with the D614G mutation, which arose early during the COVID-19 pandemic) has been described previously., , Full length CA09 HA was cloned into a pVAX1 vector behind an IgE leader sequence and obtained from Genscript. mRNA vaccines encoding the full-length wild-type SARS-CoV-2 spike glycoprotein were synthesized as described in Laczkó et al. CA09 HA mRNA-LNPs were described previously.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet: HA DNA-LNP induces potent memory responses in mice and rabbits (A) Schematic of mouse immunization regimen. (B) IFNγ-secreting cells in splenocytes by ELISpot. (C) IFNγ-secreting effector CD8 + T cells by flow cytometry. (D) CA09 HA-specific ASC responses in bone marrow by ELISpot. (E) Representative FACS plot of CA09 HA-specific MBCs. (F and G) Bar plots show frequency (F) and numbers (G) of CA09 HA-specific MBCs. (H) Schematic of rabbit immunization regimen. (I–K) IFNγ ELISpot on peripheral blood mononuclear cells (PBMCs) at day 42 (I), day 105 (J), and day 202 (K). (L) AUC of total A/California/04/2009 HA-specific serum IgG ELISA data. (M and N) HAI titers to A/Netherlands/602/2009 (M) and A/New York City/PV63249/2022 (N). Dots represent individual animals (C, D, F, and G); n = 9–10 animals per group (B–D, F, and G), n = 5 animals per group (I–N); data pooled from two independent experiments. Plots show mean with SD (B and I–K) or geometric mean with geometric SD (C, D, F, G, and L–N). Unpaired one-way or two-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ANOVA was performed at the final time point for (L–N). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The construct encoding the full-length SARS-CoV-2 spike glycoprotein (with the D614G mutation, which arose early during the COVID-19 pandemic) has been described previously., , Full length CA09 HA was cloned into a pVAX1 vector behind an IgE leader sequence and obtained from Genscript. mRNA vaccines encoding the full-length wild-type SARS-CoV-2 spike glycoprotein were synthesized as described in Laczkó et al. CA09 HA mRNA-LNPs were described previously.

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Modulation of lipid nanoparticle-formulated plasmid DNA drives innate immune activation promoting adaptive immunity

doi: 10.1016/j.xcrm.2025.102035

Figure Lengend Snippet:

Article Snippet: The construct encoding the full-length SARS-CoV-2 spike glycoprotein (with the D614G mutation, which arose early during the COVID-19 pandemic) has been described previously., , Full length CA09 HA was cloned into a pVAX1 vector behind an IgE leader sequence and obtained from Genscript. mRNA vaccines encoding the full-length wild-type SARS-CoV-2 spike glycoprotein were synthesized as described in Laczkó et al. CA09 HA mRNA-LNPs were described previously.

Techniques: Virus, Recombinant, Lysis, Reporter Gene Assay, Cell Stimulation, Electron Microscopy, Luminex, Enzyme-linked Immunospot, Luciferase, Plasmid Preparation, Software, Synthesized

Journal: NPJ Vaccines

Article Title: Effects of repeated influenza vaccination and infection on durable seroprotection in healthcare workers

doi: 10.1038/s41541-025-01259-x

Figure Lengend Snippet: During 2010/11–2013/14, the circulating viruses in Norway matched the vaccine viruses included in the trivalent seasonal influenza vaccine (TIV): A/California/7/2009(H1N1pdm09) (CA09), A/Perth/16/2009(H3N2) (PE09) or A/Victoria/361/2011(H3N2) (VI11), and B viruses belonging to B/Victoria (B/Vic) or B/Yamagata (B/Yam) lineage. A Study population. Healthcare workers (HCWs) were either infected (A/H1N1pdm09 n = 31, A/H3N2 PE09 n = 14 or VI11 n = 14, B/Vic n = 29, B/Yam n = 26) or vaccinated with TIV for the first time (First TIV, n = 51) in any of the 4 seasons. See Table and Fig. for details. A subgroup of infected HCWs (Hybrid; A/H1N1pdm09 n = 5, any A/H3N2 n = 6, and any B n = 6) and first-TIV vaccinated HCWs (Second TIV, n = 16) chose to be vaccinated in the subsequent season. B Comparisons of the magnitude and the durability of HI antibodies after infection or the first TIV. The geometric mean HI titres (GMTs) with 95% confidence interval (CI) are shown for pre- and post-season, and 12–36 months (M) later. The dotted line indicates the protective threshold HI titre of 40. C Comparisons of HI antibody responses to subsequent TIV in HCWs with hybrid immunity or vaccination immunity alone. The GMTs with 95% CI are shown for pre- and post-season of infection or first vaccination, and pre- day (D)0 and post-subsequent TIV at 21 days, 3–12 months (M). The effect of exposure (Infection vs. First TIV, Hybrid vs. Second TIV) on log-transformed HI titres was analysed in mixed-effect models, followed by post-hoc tests comparing estimated means with Holm-Sidak’s multiple comparison adjustment (Supplementary Tables and ). Within each group (*), the estimated means at pre-season time points were compared to post-season, 12-, 24-, and 36-month time points (B) and the estimated means at day 0 were compared to post-subsequent TIV time points (C). The estimated means at each time point were also compared between groups ( # ) (Infection vs. First TIV (B), Hybrid vs. Second TIV (C)). Levels of significance are shown above the graph with colours referring to groups. * ,# p < 0.05. ** ,## p < 0.01. ***,### p < 0.001.

Article Snippet: The 2009 AS03-adjuvanted monovalent pandemic inactivated vaccine contained 3.75 μg hemagglutinin (HA) of A/California/7/2009(H1N1pdm09) (CA09) virus (Pandemrix, GlaxoSmithKline, Belgium).

Techniques: Infection, Transformation Assay, Comparison

Journal: NPJ Vaccines

Article Title: Effects of repeated influenza vaccination and infection on durable seroprotection in healthcare workers

doi: 10.1038/s41541-025-01259-x

Figure Lengend Snippet: Healthcare workers (HCWs, n = 250) were vaccinated with the AS03-adjuvanted monovalent pandemic vaccine containing the A/California/7/2009(H1N1pdm09) (CA09) virus in an open-label clinical trial in 2009 and followed up for 5 years (European Clinical Trials Database, EudraCT 2009-016456-43; www.clinicaltrials.gov , NCT01003288 ) , . During four seasons from 2010/11 to 2013/14, HCWs voluntarily chose to be vaccinated with the trivalent seasonal influenza vaccine (TIV), containing the same A/H1N1pdm09 CA09 virus during the whole study period, while A/H3N2 and B viruses changed between seasons with vaccine updates: A/Perth/16/2009(H3N2) (PE09) and B/Brisbane/60/2008 (BR08) of B/Victoria lineage (B/Vic) in 2010/11 and 2011/12; A/Victoria/361/2011(H3N2) (VI11) in 2012/13 and 2013/14; B/Wisconsin/1/2010 (WI10) in 2012/13 and B/Massachusetts/2/2012 (MA12) in 2013/14, both belonging to B/Yamagata lineage (B/Yam). Blood samples were collected at day 21 (D21), 3, 6, and 12 months (3 M, 6 M, 12 M) post-vaccination in TIV-vaccinated HCWs and yearly pre-season in unvaccinated individuals. The dropout shows the total number of HCWs who dropped out from the study in each season, irrespective of seasonal vaccination status. In each season, HCWs were divided into 4 groups: Current & Previous (if they were vaccinated with TIVs in both the previous and the current seasons), Current Only (if they were only vaccinated in the current season), Previous Only (if they were only vaccinated in the previous season), and Unvaccinated (if they were not vaccinated in both the previous and the current seasons). The seasonal vaccination status in 2009 was obtained via questionnaire from the HCWs. See Supplementary Tables – for details. A subgroup of the Current only group who had never been previously vaccinated with TIV was termed First TIV, as this was their first recorded seasonal vaccination.

Article Snippet: The 2009 AS03-adjuvanted monovalent pandemic inactivated vaccine contained 3.75 μg hemagglutinin (HA) of A/California/7/2009(H1N1pdm09) (CA09) virus (Pandemrix, GlaxoSmithKline, Belgium).

Techniques: Virus, Clinical Proteomics

Journal: NPJ Vaccines

Article Title: Effects of repeated influenza vaccination and infection on durable seroprotection in healthcare workers

doi: 10.1038/s41541-025-01259-x

Figure Lengend Snippet: All healthcare workers (HCWs) were vaccinated with the AS03-adjuvanted pandemic vaccine containing the A/California/7/2009(H1N1pdm09) (CA09) virus in 2009. During 2010–14, the trivalent seasonal influenza vaccines (TIVs) included the same A/H1N1pdm09 CA09 strain, while the A/H3N2 and B components changed from A/Perth/16/2009(H3N2) (PE09) and B/Brisbane/60/2008 of B/Victoria lineage (B/Vic) during 2010–12 to A/Victoria/361/2011(H3N2) (VI11) and B/Wisconsin/1/2010 or B/Massachusetts/2/2012 of B/Yamagata lineage (B/Yam) during 2012–14. The dynamics of the pre-vaccination HI titres ( A ), the peak HI titres at 21 days post-vaccination ( B ), and the estimated half-life of the peak titres post-vaccination by months ( C ) are presented by the sequential order of vaccination against the same influenza A viruses or B lineages. They are designated as 1st, 2nd, 3rd, 4th, and 5th vaccination for A/H1N1pdm09 CA09 virus; 1st and 2nd PE09, 1st and 2nd VI11 with previous vaccination against PE09 for A/H3N2 viruses; and 1st and 2nd B/Vic, 1st and 2nd B/Yam with previous vaccination against B/Vic for B viruses. HCWs who were vaccinated with TIV in 2012–14 were designated as 1st and 2nd VI11 only or 1st and 2nd B/Yam only. Symbols with connecting lines represent an individual’s response. The symbols with connecting lines in red indicate the geometric means (GM) of all HCWs. The dotted line represents the protective threshold HI titre of 40. The numbers of HCWs ( n ), the geometric mean titres (GMT), and the GM are reported above the graph. The effect of repeated vaccination on log-transformed HI titres was analysed in mixed-effect models, followed by post-hoc tests comparing estimated means between the sequential order of vaccinations with Holm-Sidak’s multiple comparison adjustment (Supplementary Table ). Levels of significance are shown above the graph. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: The 2009 AS03-adjuvanted monovalent pandemic inactivated vaccine contained 3.75 μg hemagglutinin (HA) of A/California/7/2009(H1N1pdm09) (CA09) virus (Pandemrix, GlaxoSmithKline, Belgium).

Techniques: Virus, Vaccines, Transformation Assay, Comparison