Journal: bioRxiv

Article Title: Polymer-lipid hybrid nanoparticle enhances mRNA delivery and T cell-mediated immunity

doi: 10.64898/2026.01.22.701138

Figure Lengend Snippet: (a) Schematic illustration of the design of LNP and PLNP. (b) Left: sizes of LNP and PLNP measured by dynamic light scattering (DLS). Right: TEM images of LNP and PLNP. Scale bars: 100 nm. (c) Polydispersity index (PDI) of LNP and PLNP. (d) mRNA encapsulation efficiencies of LNP and PLNP. (e) Spike mRNA translation in RAW 264.7 macrophages after incubation with spike mRNA-loaded LNP or PLNP for 48 hours. Spike expression were measured by flow cytometry. Dose: 1 µg mRNA/mL. n = 3. (f) FLuc mRNA translation in RAW 264.7 macrophages on day 1, 2, 3, and 5 after incubation with FLuc mRNA-loaded LNP or PLNP. Dose: 1 µg mRNA/mL. n = 3. The FLuc signals were normalized to the day-1 signal of LNP-treated group. (g) Left: Representative flow plots of CD11c + DCs expressing SIINFEKL/H-2K b after incubation with OVA mRNA-loaded LNP or PLNP for 48 hours. Right: The percentages of CD11c + SIINFEKL/H-2K b+ DCs in total DCs. Dose: 1 µg mRNA/mL. n = 3. (h) Intracellular cytokine levels in RAW 264.7 macrophages after incubation with spike mRNA-loaded LNP, spike mRNA-loaded PLNP, empty LNP, or empty PLNP for 24 hours. Left: IFN-γ. Middle: TNF-α. Right: IL-2. Dose: 1 µg mRNA/mL. n = 3. (i) Cytokine secretion from human PBMCs after incubation with spike mRNA-loaded LNP, spike mRNA-loaded PLNP, empty LNP, or empty PLNP for 24 hours. Dose: 1 µg mRNA/mL. n = 3. (j) Schematic illustration of PLNP enhancing antigen presentation and proinflammatory cytokine generation in APCs. Data are presented as mean ± s.e.m. Statistical significance was determined using two-sided unpaired t-test (d, e, g), one-way ANOVA with Tukey post hoc test for multiple comparisons (h), or two-way ANOVA with Tukey post hoc test for multiple comparisons ns: no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and ****P < 0.0001.

Article Snippet: Wild-type SARS-CoV-2 spike encoding plasmid was obtained from the MacMaster lab. Omicron spike (B.1.1.529), influenza A H1N1 (A/Puerto Rico/8/1934) hemagglutinin (HA), and FLuc sequences were obtained from commercially available omicron spike, HA, and FLuc Gene ORF cDNA clone expression plasmids (Sino Biological #VG40835-UT, Sino Biological #VG11684-UT, and Addgene #101156) and inserted into the same plasmid backbone using Gibson assembly.

Techniques: Encapsulation, Incubation, Expressing, Flow Cytometry, Immunopeptidomics

Journal: bioRxiv

Article Title: Polymer-lipid hybrid nanoparticle enhances mRNA delivery and T cell-mediated immunity

doi: 10.64898/2026.01.22.701138

Figure Lengend Snippet: (a) Left: In vivo luminescence images of BALB/c mice treated with 1×PBS, FLuc mRNA-loaded LNP, or FLuc mRNA-loaded PLNP at 4, 24, 48, 72, and 96 hours post administration. Right: In vivo luminescence signals measured by integration of total flux for each mouse. Dose: 5 µg mRNA/mouse. N = 5. (b) Left: Fluorescence images of lymph nodes collected from C57BL/6 mice at 24 h post vaccination with DiD-labeled spike mRNA-loaded LNP or PLNP. Right: Quantification of LNP and PLNP accumulation in lymph nodes at 24 h post vaccination. Dose: 10 µg mRNA/mouse. n = 4. (c) Innate immune cell activation (CD86 + ) at 7 days post boost vaccination. C57BL/6 mice were vaccinated with spike mRNA-loaded LNP or PLNP on day 0 and boosted with same doses on day 21. Dose: 10 µg mRNA/mouse. n = 5. Left: DCs. Middle: Macrophages. Right: Monocytes. (d) Schematic illustration of PLNP enhancing lymph node targeting and innate immune cell activation. Data are presented as mean ± s.e.m. Statistical significance was determined using two-way ANOVA with Tukey post hoc test for multiple comparisons (a) or one-way ANOVA with Tukey post hoc test for multiple comparisons (b, c). ns: no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, and ****P < 0.0001.

Article Snippet: Wild-type SARS-CoV-2 spike encoding plasmid was obtained from the MacMaster lab. Omicron spike (B.1.1.529), influenza A H1N1 (A/Puerto Rico/8/1934) hemagglutinin (HA), and FLuc sequences were obtained from commercially available omicron spike, HA, and FLuc Gene ORF cDNA clone expression plasmids (Sino Biological #VG40835-UT, Sino Biological #VG11684-UT, and Addgene #101156) and inserted into the same plasmid backbone using Gibson assembly.

Techniques: In Vivo, Fluorescence, Labeling, Activation Assay

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron S1 shows reduced immune cells recruitment and expansion compared with the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A) , Tg(mfap4:Tomato) (B) , Tg(nfkb:eGFP) (C) of 2-dpf larvae. Neutrophil (A) and macrophage (B) recruitment and number and Nfkb reporter activity (C) were analyzed at 6, 12, and/or 24 hpi by fluorescence microscopy. Representative photos for each treatment are shown from 24 hpi. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Activity Assay, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron is more proinflammatory than the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of WT (A–F) 2-dpf larvae. The transcript levels of the indicated genes (A–E) were analyzed at 12 hpi by RT-qPCR in larval head and rest of the body and caspase-1 activity was determined at 24 hpi using a fluorogenic substrate (F) . Graphs shown are representative of three independent experiments; technical replicates are displayed in each graph. The means ± SEM for each group is shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. n=45 in (A–E) , n=35 in (F) . ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Quantitative RT-PCR, Activity Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Omicron causes higher neutrophil cell death than the ancestral variant. Recombinant S1WT, S1 Omicron or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A, B) of 2-dpf larvae. Tunel positive neutrophil number (double positive) was counted at 6 hpi in the head and tail of the larvae (A, B) . Representative photos for each treatment are shown. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01 and **** p < 0.0001.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Trimeric ancestral variant induces a weaker immune response than its monomeric form. Recombinant S1WT (monomeric), S1/S2WT-T (trimeric) or vehicle (-) were injected in the hindbrain ventricle (HBV) of Tg(lyz:DsRED) (A) , Tg(mfap4:Tomato) (B) , Tg( nfkb :eGFP) (C) 2-day postfertilization larvae (dpf). Neutrophil (A) and macrophage (B) recruitment and number and Nfkb reporter activity (C) were analyzed at 6, 12, and/or 24 hpi by fluorescence microscopy. Representative photos for each treatment are shown from 24 hpi. Scale bar 500 μm. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Activity Assay, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Dual role of ACE2 in regulating inflammation triggered by Omicron S1 and other SARS-CoV-2 Spike variants

doi: 10.3389/fimmu.2025.1667880

Figure Lengend Snippet: Trimeric ancestral variant induces a weaker proinflammatory response than its monomeric form. Recombinant S1WT, S1/S2WT-T or vehicle (-) were injected in the hindbrain ventricle (HBV) of WT (A–E) 2-day postfertilization larvae (dpf). The transcript levels of the indicated genes (A–D) were analyzed at 12 hpi by RT-qPCR in larval head and rest of the body and caspase-1 activity was determined at 24 hpi using a fluorogenic substrate (E) . Graphs shown are representative of three independent experiments; technical replicates are displayed in each graph. The means ± SEM for each group is shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. n=40 in A-D, n=35 in E. ns, not significant; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** p < 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant His-tagged Spike S1 wild-type (#40591-V08B1), S1 Omicron (#40592-V08H121) or Spike S1/S2 TRIMER wild-type (#40589-V08H8), all from Sino Biological at a concentration of 0.25 mg/ml supplemented with phenol red were injected into the hindbrain (1 nl) of 48 hpf zebrafish larvae.

Techniques: Variant Assay, Recombinant, Injection, Quantitative RT-PCR, Activity Assay, Fluorescence