Journal: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: MPLA+PUUC NPs increase T cell responses in the lung when delivered intranasally with spike protein. On days 0 (1st dose) and 28 (2nd dose), female BALB/c mice were immunized I.N. with unformulated (i.e., soluble) or NP-conjugated spike protein (1 μg) and PLGA-PEI NPs (4 mg) loaded with MPLA (24 μg), PUUC (17 μg), and MPLA+PUUC (20 μg, 17 μg). Mice were euthanized and lungs were collected on day 35, one week after the 2nd dose. Lung cells were restimulated with spike peptide pools for 6 h and stained for analysis by flow cytometry. Percentages of cells expressing A) CD4 + CD44 + out of CD45 + cells, B) IFNγ + out of CD4 + CD44 + cells, C) TNFα + out of CD4 + CD44 + cells, D) CD69 + CD103 + (tissue resident memory T cells) out of CD45 + cells. BAL fluid from vaccinated mice using soluble spike antigen was assayed for anti-spike E) IgG and F) IgA with ELISA. G) Sera were assayed for anti-spike IgG with ELISA (error bars represent the SEM). p values are * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 calculated using A – D) One-way ANOVA with Tukey post-hoc test, E , F) Kruskal-Wallis with Dunn's post-hoc test for nonparametric data, or G) Two-way ANOVA with Tukey post-hoc test.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Staining, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Controlled Release

Article Title: Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

doi: 10.1016/j.jconrel.2022.05.023

Figure Lengend Snippet: PUUC NPs delivered intramuscularly with spike protein enhance humoral responses. Female BALB/c mice were immunized I.M. into both tibialis anterior muscles at day 0 (1st dose) with soluble spike protein at doses of 80 ng, 200 ng, 1000 ng with or without adjuvant-NPs (4 mg) loaded with PUUC (+P, 20 ng PUUC dose). Peripheral blood was sampled on day 26. On day 28, mice received a 2nd dose of protein subunit vaccines. Mice received the same formulations, except for two groups that received 80 ng spike protein as a 1st dose received 1000 ng spike protein as a 2nd antigen dose (80/1000 and 80/1000 +P). Mice were euthanized on day 36 for to collect blood and popliteal LNs. A) Anti-spike IgG in post-1st dose sera at various dilutions measured by absorbance at 450 nm during ELISA assays and B) comparison of area under the curve (AUC). C – D) Anti-spike IgG in post-2nd dose sera measured by absorbance at 450 nm and comparison of AUC. E) ACE-2 signal measured by absorbance at 450 nm in spike protein neutralization assay with post-2nd dose sera. Absorbance was normalized to a blank well in each row of a 384 well plate to correct for plate effects. Lower absorbance values indicate higher spike-neutralizing antibody levels in sera. Percentages of cells expressing F) Bcl6 + out of B220 + cells, G) GL7 + out of B220 + cells and H) CXCR5 + out of B220 − cells from combined popliteal lymph nodes. B,D) Normality was assessed with the Kolmogorov-Smirnov test. Statistical significance was determined with the Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. E – H) Statistical significance calculated with One-Way ANOVA and Tukey post-hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 for all graphs.

Article Snippet: Spike-neutralizing antibodies were quantified using a modified ELISA assay in a 384-well UltraCruz® ELISA high-binding plate (Santa Cruz Biotechnology).

Techniques: Muscles, Adjuvant, Vaccines, Enzyme-linked Immunosorbent Assay, Comparison, Neutralization, Expressing