Robust induction of TRMs by combinatorial nanoshells confers cross-strain sterilizing immunity against lethal influenza viruses

Abstract

Antigen-specific lung-resident memory T cells (T_RMs) constitute the first line of defense that mediates rapid protection against respiratory pathogens and inspires novel vaccine designs against infectious pandemic threats, yet effective means of inducing T_RMs, particularly via non-viral vectors, remain challenging. Here, we demonstrate safe and potent induction of lung-resident T_RMs using a biodegradable polymeric nanoshell that co-encapsulates antigenic peptides and TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) in a virus-mimicking structure. Through subcutaneous priming and intranasal boosting, the combinatorial nanoshell vaccine elicits prominent lung-resident CD4⁺ and CD8⁺ T cells that surprisingly show better durability than live viral infections. In particular, nanoshells containing CpG-ODN and a pair of conserved class I and II major histocompatibility complex-restricted influenza nucleoprotein-derived antigenic peptides are demonstrated to induce near-sterilizing immunity against lethal infections with influenza A viruses of different strains and subtypes in mice, resulting in rapid elimination of replicating viruses. We further examine the pulmonary transport dynamic and optimal composition of the nanoshell vaccine conducive for robust T_RM induction as well as the benefit of subcutaneous priming on T_RM replenishment. The study presents a practical vaccination strategy for inducing protective T_RM-mediated immunity, offering a compelling platform and critical insights in the ongoing quest toward a broadly protective vaccine against universal influenza as well as other respiratory pathogens.

Keywords: T cell vaccine; influenza virus; nanoshell; peptide vaccine; resident memory T cell; respiratory infection; universal influenza vaccine.

Graphical abstract

Figure 1

Induction of robust antigen-specific T cell responses with minimal systemic adverse effects by combinatorial nanoshell vaccines (A) Schematic representation of a combinatorial nanoshell vaccine containing MHC class I and class II- restricted peptide antigens and CpG. (B and C) CryoEM visualizations of empty PLGA nanoshells (B) and peptide- and CpG-loaded nanoshells (C). (D) Dynamic light scattering measurement of nanoparticles loaded with OVA_I/II peptides and CpG (NS(OVA_I/II + CpG)). (E) IFN-γ production of CD8⁺ and CD4⁺ cells in mice receiving different immunization formulas. WT (Thy1.2) mice were s.c. immunized with PBS control, empty nanoshell control, different doses of simple mixture of NP_I/II (L: 5 μg/5 μg), NP_I/II (H: 10 μg/10 μg), NP_I/II + CpG (L: 5 μg/5 μg + 2.5 μg), and NP_I/II + CpG (H: 10 μg/10 μg + 50 μg), and indicated doses of NS (NP_I/II + CpG). At day 7 post immunization, mice were sacrificed for analysis of IFN-γ production of CD8⁺ and CD4⁺ cells in the spleen and lymph node (n ≥ 5 mice per group compiled from 2 independent experiments). ∗∗∗∗p ≤ 0.0001 (one-way ANOVA). (F) The proportional body weight changes of recipient WT mice were monitored at the indicated days post immunization. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 (Student’s t test). (G and H) At day 7 post-harvested spleens were measured for organ weight (G) and were also photographed (H). Data were pooled from 2~3 independent experiments. Individual organ weight of immunized mice for spleens with means plus SE (n ≥ 4 mice per group). ∗∗∗p ≤ 0.001 (one-way ANOVA).

Figure 2

The immunogenicity and protectivity of combinatorial nanoshells with NP_366–374/NP_311–325 (A) Schematic illustration of vaccination strategy with NS(NP_I/II + CpG) to induce protective T cell immunity against heterosubtypic IAV infection. Mice were immunized by s.c. priming (500 μg/mouse) and i.n. boosting (300 μg/mouse) with the indicated vaccine formulas and then infected by different strains of influenza virus (PR8, WSN, HKx31, and VN^HA,NA (H5N1)). (B and C) The body weight (B) and survival rates (C) of PR8-infected (110 PFU) mice immunized by indicated vaccine formulas. A body weight reduction of 25% or more was defined as the experimental end point, and mice were then euthanized at day 7 post infection. (D) Lung viral loads of mice immunized by different vaccine formulas were analyzed at day 7 post PR8 infection (n ≥ 4 mice per group compiled from 2 independent experiments). (E and F) The viral kinetics (E) and T cell immunity (F) of nanoshell vaccine-immunized mice after challenge by lethal PR8 infection. (F) Individual percentages with means plus SE of NP_I-specific and NP_II-specific IFN-γ-producing CD8⁺ and CD4⁺ T cells, respectively, in lungs at day 7 post-PR8 infection (n ≥ 5 mice per group compiled from 2 independent experiments). (G–I) Mice were immunized by s.c. priming and i.n. boosting with the either empty nanoshells or NS(NP_I/II + CpG) and then infected by lethal doses of WSN (1 × 10⁴ PFU/mouse) (G), HKx31 (1 × 10⁵ PFU/mouse) (H), or VN^HA,NA (H5N1) (400 PFU/mouse) (I) following the experimental protocol shown in (A). Survival rates (top), body weight loss (middle), and lung viral loads (bottom) were analyzed at day 7 post infection of WSN (G), HKx31 (H), and VN^HA,NA (H5N1) (I) (n ≥ 5 mice per group compiled from 2 independent experiments). ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 (Fisher’s exact test for survival rate, Student’s t test for viral kinetics, and one-way ANOVA for lung viral load and percentages of IFN-γ production).

Figure 3

Uptake and tracking of nanoshell vaccines in lungs and dLNs (A) The percentages of macrophages (SSC^HighCD11c⁺MHC-II^LowF4/80⁺) and DCs (SSC^LowCD11c⁺MHC-II^HighCD103⁺ and SSC^LowCD11c⁺MHC-II^HighCD11b⁺) in lungs at 12 h, 24 h, and 48 h after i.n. administration of nanoparticles (n = 4 mice per group compiled from 2 independent experiments). (B) t-distributed stochastic neighbor embedding (t-SNE) map of different subset of DCs (AF555⁺) colored by FlowSOM metaclusters in lungs at 24 h. Data are downsampled to 1 × 10⁴ cells/mouse (from 3 mice/group), and representative heatmap statistic is 1 mouse per group. The right and middle t-SNE maps were gated by AF555⁺ cells. The color bar represents the expression levels of CD86 in NS-positive (AF555⁺) cells. (C) The percentages of CD86⁺ cells in NS-positive (AF555⁺) CD11c⁺CD103⁺ and CD11c⁺CD11b⁺ DCs of lungs at 12 h, 24 h, and 48 h after i.n. administration of nanoparticles (n ≥ 3 mice per group compiled from 2 independent experiments). (D) t-SNE map of different subset of DCs (AF555⁺) colored by FlowSOM metaclusters for dLNs. Data are downsampled to 1 × 10⁴ cells/mouse (from 3 mice/group, contour), and representative heatmap statistic is 1 mouse per group. Color bar means the proportion of NS-positive cells. (E) The mean fluorescent (AF555⁺) intensity (MFI) of NS-positive (AF555⁺) CD11c⁻ and CD11c⁺ cells of dLNs at 12 h, 24 h, and 48 h after i.n. administration of nanoparticles (n ≥ 3 mice per group compiled from 2 independent experiments). ∗p ≤ 0.05; ∗∗p ≤ 0.01 (Student’s t test).

Figure 4

T cell proliferation stimulated by combinatorial nanoshell vaccine after depletion of CD11c-positive DCs (A) Schematic representation of the experimental protocol. CD11c-DTR mice received PBS or DT depletion once. On the next day mice were co-transferred with CFSE-stained Thy1.1^+/+ CD8⁺OT-I and Thy1.1/Thy1.2 CD4⁺OT-II cells and then i.n. immunized with NS (OVA_I/II + CpG). (B) The fold change of CD11c⁺ cells in immunized mice at day 5 post PBS or DT treatment. (C and D) Individual percentages (C) and cell numbers (D) of proliferating CD4⁺OT-II and CD8⁺OT-I cells in dLNs in immunized mice at day 5 post PBS or DT treatment. (n ≥ 6 mice per group compiled from 3 experiments). ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 (Student’s t test).

Figure 5

The immunogenicity and protectivity of combinatorial nanoshell vaccines by different vaccination strategies (A) Schematic illustration of the experimental protocol. C57BL/6 mice received primary and secondary immunization by the indicated vaccine formulations and routes in a 28-day interval. The doses used are as follows: s.c. (500 μg of nanoshells) and i.n. (300 μg of nanoshells). (B and C) Mice were then infected with 5 × 10⁵ PFU of HKx31-HA-OVA_I/II at day 56 post primary immunization (28 days after secondary immunization) and monitored for the body weight loss (B) and survival rate (C). (D) Lung viral loads were analyzed at day 5 post-HKx31-HA-OVA_I/II infection. Data are individual viral loads with means plus SE (n ≥ 5 mice per group compiled from 3 independent experiments). (E) Individual percentages of virus-specific IFN-γ-producing CD4⁺ and CD8⁺ T cells in spleens and dLNs with means plus SE at day 5 post-HKx31-HA-OVA_I/II infection (n ≥ 5 mice per group compiled from 3 independent experiments). ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 (log-rank test for the survival rate and one-way ANOVA for lung viral loads and percentages of IFN-γ production).

Figure 6

Elicitation of memory T cell populations by nanoshell vaccines with different vaccination strategies (A) WT (Thy1.2) mice were transferred with naive Thy1.1⁺CD8⁺OT-I cells 1 day before immunization and immunized by the indicated individual protocols. Memory T cells were analyzed at 28 days post secondary immunization. (B) Lung samples were gated on Thy1.1⁺CD3e⁻CD8⁺CD62L⁻KLRG-1⁻ cells and analyzed for the total number of CD69⁺, CD103⁺, and CD69⁺CD103⁺ cells (n ≥ 7 mice per group compiled from 3 independent experiments). Spleen samples were analyzed for the total number of T_EM (CD62L⁻KLRG-1⁺) and T_CM (CD62L⁺KLRG-1⁻) cells (n ≥ 7 mice per group compiled from 3 independent experiments). (C) T_RM cells in lungs and T_EM cells in spleens were measured after depletion of lung-resident CD8⁺ T cells by intratracheal administration with anti-CD8 antibody (n ≥ 5 mice per group compiled from 2 independent experiments) ∗p ≤ 0.05; ∗∗p ≤ 0.01 (Student’s t test).

Figure 7

Schematic summary of the cross-protective T cell immunity against IAVs elicited by the combinatorial nanoshell vaccine After peripheral subcutaneous priming, intranasal boosting with the combinatorial nanoshell vaccine containing two class I and class II MHC-restricted epitope peptides (i.e., NP_I/II) and CpG-ODN led to efficient uptake and maturation of pulmonary CD11c⁺ DCs. Mature CD11c⁺ DCs subsequently migrate to draining lymph nodes, where they present epitope peptides and stimulate cognate T cells recruited from the circulatory memory T cell population. Finally, lung-resident memory T cells and circulatory memory T cells, including T_CMs and T_EMs, are established and act synergistically to provide near-sterilizing cross-protection against IAVs of different strains and subtypes.