Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Abstract

Recombinant adenovirus serotype 5 (Ad5) vector has been widely applied in vaccine development targeting infectious diseases, such as Ebola virus disease and coronavirus disease 2019 (COVID-19). However, the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines. Thus, there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors. Herein, we address this need by utilizing biocompatible nanoparticles to modulate Ad5-host interactions. We show that positively charged human serum albumin nanoparticles ((+)HSAnp), which are capable of forming a complex with Ad5, significantly increase the transgene expression of Ad5 in both coxsackievirus-adenovirus receptor-positive and -negative cells. Furthermore, in charge- and dose-dependent manners, Ad5/(+)HSAnp complexes achieve robust (up to 227-fold higher) and long-term (up to 60 days) transgene expression in the lungs of mice following intranasal instillation. Importantly, in the presence of preexisting anti-Ad5 immunity, complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity. These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Keywords: Adenovirus serotype 5; Nanoparticles; Preexisting immunity; Transgene expression; Vaccine.

Fig. 1

Characterization of the Ad5/(+)HSAnp complex. (a, b) DLS measurements of (a) average sizes and (b) zeta potentials of (+)HSAnp (n = 3). (c) Fluorescence microscopy images of cells treated with FITC labeled as (+)HSAnps for 24 h (scale bar:100 μm). (d, e) DLS measurements of (d) average sizes and (e) zeta potentials of Ad5 and the Ad5/(+)HSAnp complex (n = 3). (f) Representative transmission electron microscopy images of (+)HSAnp-4, Ad5, and Ad5/(+)HSAnp-4 (scale bar: 100 nm). Data are presented as means ± SEM. Statistical differences were determined using one-way ANOVA with Dunn’s multiple comparison test.

Fig. 2

Ad5/(+)HSAnp enhance transgene expressions in vitro. (a) Transgene expressions of Ad5-luc in complex with or without (+)HSAnp in Hela cells, A549 cells, 3T3 cells, and THP-1 (n = 5). (b–d) FCM analysis of Ad5-GFP/(+)HSAnp enhancing transgene expression of GFP in (b) Hela cells, (c) BMDCs, and (d) BMMs (n = 3). (e–g) Fluorescence microscopy of Ad5-GFP/(+)HSAnp-4 enhancing transgene expression of GFP in (e) Hela cells, (f) BMDCs, and (g) BMMs (scale bar: 100 μm). Cell experiments were performed at a MOI of 10. Data are presented as means ± SEM. Statistical differences in parts (a–d) were determined using ANOVA with Dunn’s multiple comparison test. Luc intensity: luciferase bioluminescence intensity; MFI: mean fluorescence intensity.

Fig. 3

Investigation of the cellular mechanism underlying the enhanced transgene expressions of Ad5/(+)HSAnp. (a) Correlation analysis of the transgene expression of Ad5-luc and the cellular internalization of (+)HSAnp-4 in Hela and 3T3 cells (n = 5). (b) Transgene expression of Ad5-luc pre-incubated or complexed with (+)HSAnp in 3T3 cells (n = 4). (c) qRT-PCR analysis of the cellular internalizations of Ad5 and Ad5/(+)HSAnp-4 in Hela and 3T3 cells (n = 3). (d) FCM and (e) fluorescence microscopy analysis of the knock-down of CXADR mediated by RNA interference in A549 cells (scale bar: 10 μm). (f) Absolute and (g) normalized transgene expression of Ad5-luc and Ad5-luc/(+)HSAnp-3 in CXADR-knockdown A549 cells (n = 5). (h) Transgene expression of Ad5-luc and Ad5-luc/(+)HSAnp co-incubated with Ad5-neutralizing serum in Hela cells, A549 cells, 3T3 cells, and THP-1 (n = 5). Ad5-luc and Ad5-luc/(+)HSAnp were mixed with anti-Ad5 serum (diluted at 1:4000) for 1 h at RT before transfection into cells. The serum was obtained from BALB/c mice intranasally immunized with an empty Ad5 vector at 4 weeks post administration. Cell experiments were performed at an MOI of 10. Data are presented as means ± SEM. Two-way ANOVA with Šidák’s multiple comparison test was used to determine the significance in parts (b) and (g). Two-tailed unpaired t-test was used to determine the significance within each cell line in (c). One-way ANOVA with Dunn’s multiple comparison test was used to determine the significance of the indicated comparisons within each group in (f) and (h). ns: no significance.

Fig. 4

Transgene expression of Ad5/(+)HSAnp in vivo. (a) In vivo BLI of BALB/c mice at 1 day and 10 days post intranasal administration with Ad5-luc or Ad5-luc/(+)HSAnp (n = 5). (b, c) Biodistribution analysis of Ad5-luc/(+)HSAnp at 10 days post intranasal administration (n = 5). (d) Charge-dependence of (+)HSAnp in enhancing the transgene expression of Ad5-luc/(+)HSAnp in mice lungs at 10 days post intranasal administration (n = 5). (e) Dose-dependence of (+)HSAnp-4 in enhancing the transgene expression of Ad5-luc/(+)HSAnp-4 in mice lungs at 10 days post intranasal administration (n = 5). (f) Time-course analysis of the transgene expression of Ad5-luc/(+)HSAnp in mice lungs in the absence of PEI (n = 5). (g) Quantitative transgene expressions of Ad5-luc administrated 2 h before, 2 h after, or complexed with (+)HSAnp-4 (n = 4) at 10 days post intranasal administration. (h) Time-course analysis of the transgene expression of Ad5-luc/(+)HSAnp in mice lungs in the presence of PEI (n = 5). (i) Representative image of BALB/c mice at 5 days post intranasal administration in the presence of PEI (n = 5). Data are presented as means ± SEM. The bioluminescent signals in parts (b), (d), and (e) were measured after lung tissue harvest and are shown as RLU (photons·s⁻¹). The bioluminescent signals in parts (f), (g), and (h) were measured in vivo and are shown as RLU (photons s⁻¹·cm·^–2·sr^–1). The statistical differences in parts (d), (e), and (g) were determined using ANOVA with Dunn’s multiple comparison test. Statistical differences in (f) and (h) were determined using a two-way ANOVA with Šidák’s multiple comparison test.

Fig. 5

Complexed Ad5-based Ebola and COVID-19 vaccines overcome PEI. (a–d) BALB/c mice (n = 10) were immunized intranasally with a single dose of 5 × 10⁶ IFU of Ad5-EBOV or Ad5-EBOV complexed with 1 μg (+)HSAnp-4 (1:1.2 × 10⁴ molar ratio) in the absence (naïve) or presence of PEI. (a) Ad5-specific and (b) GP-specific serum IgG titers were measured at specific time points post vaccination. BALF was collected at week 14 post vaccination and assessed for GP-specific (c) IgG and (d) IgA titers. (e–m) In the presence of PEI, BALB/c mice were immunized intranasally with a single dose of 5 × 10⁶ IFU of Ad5-nCoV or Ad5-nCoV complexed with 1 μg (+)HSAnp-4 (1:1.2 × 10⁴ molar ratio). (e) Ad5-specific serum IgG titers were measured before vaccination. (f) SARS-CoV-2 S-specific IgG serum concentrations were assessed at 2, 4, 8, and 12 weeks post vaccination. BALF was collected at week 12 post vaccination and assessed for (g) S-specific IgG and (h) IgA titers. (i) A biochemical assay was used to measure the serum inhibition of RBD-hACE2 interactions. (j–o) Serum neutralizing antibody titers against (j, m) SARS-CoV-2 WT, (k, n) delta variant, and (l, o) omicron variant were measured by means of pseudovirus neutralization assays at 4 weeks post vaccination. Data are presented as means ± SEM. Statistical differences in parts (b), (f), and (i) were determined using a two-way ANOVA with Šidák’s multiple comparison test. Statistical differences in (c) and (d) were determined using ANOVA with Dunn’s multiple comparison test. Statistical differences in parts (g), (h), (m), (n), and (o) were determined using a two-tailed unpaired t-test.

Fig. 6

Evaluation of the safety profile of Ad5/(+)HSAnp. (a) BALB/c mice (n = 5) were intranasally administrated with a single dose of PBS, 1 μg of (+)HSAnp-4, 5 × 10⁶ IFU of Ad5-nCoV, or 5 × 10⁶ IFU of Ad5-nCoV complexed with 1 μg of (+)HSAnp-4 (1:1.2 × 10⁴ molar ratio). (a) Blood tests and (b) histological analysis of lungs performed at 1 day and 7 days post administration (scale bar: 1000 μm). Data are presented as means ± SEM. Statistical differences were determined using ANOVA with Dunn’s multiple comparison test.