Strategy to enhance dendritic cell-mediated DNA vaccination in the lung

Abstract

We here introduce a new paradigm to promote pulmonary DNA vaccination. Specifically, we demonstrate that nanoparticles designed to rapidly penetrate airway mucus (mucus-penetrating particle or MPP) enhance the delivery of inhaled model DNA vaccine (i.e. ovalbumin-expressing plasmids) to pulmonary dendritic cells (DC), leading to robust and durable local and trans-mucosal immunity. In contrast, mucus-impermeable particles were poorly taken up by pulmonary DC following inhalation, despite their superior ability to mediate DC uptake in vitro compared to MPP. In addition to the enhanced immunity achieved in mucosal surfaces, inhaled MPP unexpectedly provided significantly greater systemic immune responses compared to gold-standard approaches applied in the clinic for systemic vaccination, including intradermal injection and intramuscular electroporation. We also showed here that inhaled MPP significantly enhanced the survival of an orthotopic mouse model of aggressive lung cancer compared to the gold-standard approaches. Importantly, we discovered that MPP-mediated pulmonary DNA vaccination induced memory T-cell immunity, particularly the ready-to-act effector memory-biased phenotype, both locally and systemically. The findings here underscore the importance of breaching the airway mucus barrier to facilitate DNA vaccine uptake by pulmonary DC and thus to initiate full-blown immune responses.

Keywords: adaptive immunity; airway mucus; nanoparticle; pulmonary DNA vaccination.

Figure 1.. In vivo mucus penetration and DC uptake of different DNA-loaded nanoparticles carrying fluorescently labeled pOVA following intratracheal administration.

Representative confocal images demonstrating penetration of pOVA-loaded nanoparticles (magenta), including (A) pOVA-MPP and (B) pOVA-CP, through mouse lung airway mucus 1 hour after the administration (Left; scale bar = 200 𝜇m). DAPI staining represents cell nuclei (blue). The areas enclosed by white boxes are zoomed in (Right; scale bar = 50 𝜇m). (C) Representative flow cytograms demonstrating pOVA-MPP and pOVA-CP uptake by pulmonary DC at 16-hour post-administration. Data shown as mean ± SEM. (D) Percentages of pulmonary DC that took up pOVA-MPP and pOVA-CP at 16-hour post-administration (n = 6). Data shown as mean ± SEM. ***p < 0.0005 by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test.

Fig 2.. In vitro characterization of pOVA-MPP formulations with or without adjuvant co-packaging.

(A) Transmission electron micrographs of pOVA-MPP, p(I:C)/pOVA-MPP and CpG/pOVA-MPP. Scale bars = 200 nm. (B) Changes in hydrodynamic diameters (Left) and polydispersity index (PDI) values (Right) of pOVA-MPP, p(I:C)/pOVA-MPP and CpG/pOVA-MPP in PBS over 6 hours (n = 3 – 9). Data shown as mean ± SEM. (C) Electrophoretic analysis showing the ability of p(I:C)/pOVA-MPP (Left) and CpG/pOVA-MPP (Right) to protect respective nucleic acid payloads in presence of DNase and/or RNase. Red and black arrows indicate pOVA and adjuvants, respectively. Leftmost lanes denote the 1 kb plus DNA ladders. (D) Median MSD of pOVA-MPP, p(I:C)/pOVA-MPP and CpG/pOVA-MPP in freshly collected human airway mucus samples (n = 3 – 8) at a timescale of 1 s. The MSD values are directly proportional to particle diffusion rates. Data shown as median ± SEM. p < 0.05, and **p < 0.005 by one-way ANOVA with Kruskal-Wallis multiple comparison test. (E) Percentage of CD11c+ DC co-expressing MHC-II+ and CD86+ following a 6-hour incubation with various adjuvants or particle formulations (n = 4 – 6). Data shown as mean ± SEM. *p < 0.05, **p < 0.005 and ***p < 0.0005 by one-way ANOVA with Dunnett’s multiple comparison test.

Figure 3.. Pulmonary DC uptake, LN trafficking and antigen-specific CD8+ T-cell response following intratracheal administration of CpG/pOVA-MPP.

(A) Experimental immunization schedule. (B) Representative confocal image showing uptake (white arrows) of CpG/pOVA-MPP (magenta) by pulmonary DC (CD11c+; yellow). Scale bar = 20 𝜇m. (C) Representative confocal image showing CpG/pOVA-MPP (magenta) localization in mediastinal LN. Scale bar = 200 𝜇m. Cell nuclei are stained with DAPI (blue). (D) Percentage of CD8+ T-cells (CD3ε+ CD8+) expressing OVA-specific MHC-I peptide (SIINFEKL) in lung, respective draining LN and spleen following pOVA-mediated DNA vaccination via different administration routes (n = 5 – 12). ID: intradermal; IM-EP: intramuscular electroporation; IT; intratracheal. Data shown as mean ± SEM. *p < 0.05 and ***p < 0.0005 by one-way ANOVA with Dunnett’s multiple comparison test.

Figure 4.. Trans-mucosal CD8+T-cell responses following pulmonary immunization with CpG/pOVA-MPP.

Percentage of CD8+ T-cells (CD3ε+CD8+) expressing OVA-specific MHC-I peptide (SIINFEKL) in mesenteric LN, Payer’s patch and vagina (A) with (n =4 – 5) or (B) without (n = 3 – 5) adoptive T-cell transfer. CD8+ T cells from OT-I mice were adoptively transferred into C57BL/6 mice one day prior to immunization with CpG/pOVA-MPP and OVA-specific T-cell response was quantified 3 days after the immunization. ID: intradermal; IM-EP: intramuscular electroporation; IT; intratracheal. Data shown as mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005 and ****p < 0.00005 by (A) one-way ANOVA with Dunnett’s multiple comparison test or (B) two-sided Student’s t-test.

Figure 5.. Memory T-cell responses following pulmonary immunization with CpG/pOVA-MPP.

(A) Percentage of CD8+ T-cells (CD3ε+CD8+) expressing OVA-specific MHC-I peptide (SIINFEKL) in lung (Left), mediastinal LN (Middle) and spleen (Right) 70 days after the immunization (n = 4 – 5). ***p < 0.0005 by one-way ANOVA with Dunnett’s multiple comparison test. Data shown as mean ± SEM. (B) Percentage of OVA-specific central memory T cells (T_CM) co-expressing CD44^hi and CD62L^hi and effector memory T cells (T_EM) co-expressing CD44^hi and CD62L^lo in lung (Left), mediastinal LN (Middle) and spleen (Right) 70 days after the immunization (n = 4 – 8). Data shown as mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005 by two-sided Student’s t-test with Holm-Sidak multiple comparison test.

Figure 6.. Survival of an orthotopic mouse model of OVA-expressing lung cancer following pulmonary immunization with CpG/pOVA-MPP.

(A) Experimental schedule. C57BL/6 mice were immunized as described in Figure 3A and OVA-LLC cells were intratracheally inoculated into the lung 7 days after the boost. Mice received CpG/pOVA or CpG/OVA-MPP via different administration routes. (B) Kaplan-Meier survival curve (n = 5 – 10). ID: intradermal; IM-EP: intramuscular electroporation; IT; intratracheal. *p < 0.05 and ***p < 0.0005 by log rank test with Gehan-Breslow-Wilcoxon test.