Mucosal Immunization with a pH-Responsive Nanoparticle Vaccine Induces Protective CD8+ Lung-Resident Memory T Cells

Abstract

Tissue-resident memory T cells (T_RM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective T_RM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting T_RM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8⁺ T_RM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8⁺ T_RM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting T_RM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8⁺ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8⁺ T_RM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of T_RM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8⁺ T_RM cells in the lungs.

Keywords: influenza; intranasal; lungs; nanoparticle; nucleic acid adjuvant; subunit vaccine; tissue-resident memory T cells.

Figure 1.. Intravascular staining is used to determine localization of CD8⁺ T cells after intranasal delivery to the lungs.

(A) Schematic of the experimental timeline. (B) Flow cytometry was used to identify antigen-specific (Tet⁺) CD8⁺ T cells in distinct lung compartments (airway, AW; interstitium, IST; marginated vascular, MV) and the spleen. BAL was collected to discriminate AW vs. IST cells, and i.v. staining with αCD45 antibody discriminated IST (CD45⁻) vs. MV (CD45⁺) cells. Samples were stained with PE-labeled SIINFEKL/MHC-I tetramer to identify antigen-specific CD8⁺ T cells. After gating out CD11b⁺, CD11c⁺, B220⁺, and CD4⁺ cells (“dump”), CD8α⁺CD45⁻Tet⁺ events in AW and IST, CD8α⁺CD45⁺Tet⁺ events in MV, and CD8α⁺Tet⁺ events in the spleen were quantified. Dot plots are representative of the gating strategy used in multiple experiments (see Figures S3A–S3C). (C) In conjunction with i.v. staining, microscopy was used to visualize fluorescent OVA-NP conjugates in the lower airways 24 h after immunization. Lungs were stained with αCD45 antibody, which labels vascular leukocytes, and tomato lectin, which binds to capillary endothelial cells and allows for visualization of lung structure. Purple: OVA-NP; blue: vascular leukocytes; green: lung vasculature. Scale bar = 100 μm. Immunization dose: 25 μg NP, 7.5 μg OVA.

Figure 2.. Intranasal antigen delivery with pH-responsive nanoparticle enhances lung-resident CD8⁺ T cell response.

Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (A) AW, (B) IST, (C) MV, and (D) spleen were enumerated on d13 after i.n. administration of OVA-NP_pH or OVA-NP_ctrl. Representative dot plots are gated on viable CD8⁺ T cells. Immunization dose: 25 μg NP, 7.5 μg OVA. Data are mean ± SEM and representative of two independent experiments, with n = 6 per group. Limits of detection: 1 cell (AW), 5 cells (IST/MV), 25 cells (spleen). *p<0.05, **p<0.01 by unpaired t-test. ns, not significant. See also Figures S3A–S3C.

Figure 3.. Intranasal dual-delivery of antigen and adjuvant via nanoparticle vaccine enhances magnitude and functionality of lung-resident CD8⁺ T cell response.

(A) Mice were immunized on d0 with the NP vaccine or control formulations, and lungs, spleens, and/or BAL were collected on d13 for analysis of the immune response by tetramer staining or ICCS. (B-E) Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (B) AW, (C) IST, (D) MV, and (E) spleen were enumerated on d13 after i.n. administration of OVA-NP/CpG or control formulations. (F) ICCS was used to identify % CD8⁺ T cells positive for IFNγ and/or TNFα after ex vivo restimulation of lungs and spleen with SIINFEKL peptide. Statistical differences are shown for IFNγ⁺TNFα⁺ group only. Data are mean ± SEM and representative of one to three independent experiments, with (B-E) n = 4–7 per group and (F) n = 2–4 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg CpG. Limits of detection for (B-E): 1 cell (AW), 5 cells (IST/MV), 25 cells (spleen). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by (B-E) ordinary one-way ANOVA with Tukey multiple comparisons test or (F) ordinary two-way ANOVA with Tukey multiple comparisons test. See also Figures S3A–S3D.

Figure 4.. Pulmonary immunization via intranasal administration is optimal for generating a lung-resident CD8⁺ T cell response.

Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (A) AW, (B) IST, (C) MV, and (D) spleen were enumerated on d13 after i.n. or s.c. administration of OVA-NP/CpG. Data are mean ± SEM, with n = 4–5 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg CpG. Limits of detection: 1 cell (AW), 5 cells (IST/MV), 25 cells (spleen). **p<0.01, ***p<0.001, ****p<0.0001 by unpaired t-test.

Figure 5.. Nanoparticle-mediated dual-delivery enhances co-localization and retention of cargo in pulmonary APCs and expression of activation markers.

(A) Lungs and spleens were imaged at 24, 48, and 72 h post-immunization to quantify uptake of OVA₆₄₇. Representative images of lungs and spleens from each treatment group at each timepoint (left) and quantification of OVA₆₄₇ fluorescence in lungs and spleens over time (right). (B) Flow cytometry was used to quantify the # of pulmonary APCs positive for both OVA₆₄₇ and CpG₄₈₈ (OVA⁺CpG⁺) at each time point (“cell count”). Cell counts were also multiplied by either OVA MFI or CpG MFI to determine “relative uptake” of each cargo (MFI × cell count). Bar graphs for CD103⁺ DC and CD11b⁺ DC are replicated for visibility. (C) Expression of CD86 was measured for several cell subsets in the lungs. Data are mean ± SEM and representative of four independent experiments, with n = 3–4 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg CpG. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by (A) unpaired t-test at each timepoint comparing OVA-NP/CpG vs. OVA+CpG, with Holm-Sidak multiple comparisons test, (B) ordinary two-way ANOVA with Sidak’s multiple comparisons test, or (C) ordinary two-way ANOVA with Tukey multiple comparisons test (significance shown for OVA-NP/CpG vs. OVA+CpG). See also Figure S5.

Figure 6.. Acute cytokine response to nanoparticle vaccine is localized, transient, and supportive of lung-resident CD8⁺ T cells.

Cytokines associated with CD8⁺ T cells (IFNγ, IL-1β, IL-6, IL-12p70) and T_RM generation (TNFα, IFNβ, IL-33, IFNα) were measured in lungs, BAL, and serum obtained 6 h, 24 h, 48 h, or 7 d after i.n. administration OVA-NP/CpG or OVA+CpG. Data are mean ± SEM and representative of two independent experiments, with n = 4–5 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg CpG. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by ordinary two-way ANOVA with Tukey’s multiple comparisons test. Statistical differences shown are for comparison of OVA-NP/CpG vs. OVA+CpG. See also Figure S6.

Figure 7.. OVA-specific CD8⁺ T cells are maintained at memory timepoints and express T_RM markers CD69 and CD103.

(A) Mice were immunized i.n. with OVA-containing formulations on d0 and lungs and spleens were analyzed on d30 or d60 via tetramer and surface marker staining. CXCR3 was used as a marker of AW residence; CD103 and CD69 were used as markers of tissue residency. (B-E) Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (B) AW, (C) IST, (D) MV, and (E) spleen were enumerated on d30 after i.n. administration of OVA-NP/CpG, OVA-NP, or OVA+CpG. (F-I) Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (F) AW, (G) IST, (H) MV, and (I) spleen were enumerated on d60 after i.n. administration of OVA-NP/CpG, OVA-NP, or OVA+CpG. (J) Flow cytometry was used to quantify Tet⁺ CD8⁺ T cells expressing T_RM markers (CD103, CD69) in the airway (CXCR3^hi) and lung interstitium (CXCR3^lo). (K-L) Number (#) of Tet⁺ CD8⁺ T cells expressing CD69±CD103 in AW were enumerated on (K) d30 or (L) d60 after immunization. (M-N) Number (#) of Tet⁺ CD8⁺ T cells expressing CD69±CD103 in IST were enumerated on (M) d30 or (N) d60 after immunization. Data are mean ± SEM and representative of two to four independent experiments, with n = 5–6 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg. CpG Limit of detection: 1 cell (AW), 5 cells (IST/MV), 25 cells (spleen). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by (B-I) ordinary one-way ANOVA or (K-N) ordinary two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant. Statistical comparisons are shown for OVA-NP/CpG only.

Figure 8.. Mice immunized with nanoparticle vaccine exhibit less weight loss and lower viral burden after intranasal challenge with recombinant vaccinia virus.

(A) Mice immunized on d0 were challenged i.n. with recombinant SIINFEKL-expressing vaccinia virus (sublethal dose of 1×10⁷ pfu/mouse) either 30 or 60 d post-immunization. Mice were weighed daily and lungs were harvested on d6 post-inoculation (p.i.). (B-C) Percent (%) weight loss in mice challenged on (B) d30 or (C) d60 after i.n. administration. Left: weight loss over time. Right: weight at d6 p.i. expressed as % initial body weight. (D-E) Lungs of mice challenged on (D) d30 or (E) d60 post-immunization were harvested on d6 p.i. for quantification of viral load. Dots show titers for individual animals. Data are mean ± SEM, with (B) n = 12–23 per group, (C) n = 5–6 per group, and (D-E) n = 5 per group. Immunization dose: 25 μg NP, 7.5 μg OVA, 1.4 μg CpG. Data are pooled from one to four independent experiments. Limit of detection for plaque assay = 6 pfu. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by (B-C, left) repeated measures two-way ANOVA with Tukey multiple comparisons test or (B-C, right; D-E) ordinary one-way ANOVA with Tukey multiple comparisons test.

Figure 9.. Flu-specific CD8⁺ T cells are maintained at memory timepoints and express T_RM markers CD69 and CD103.

(A) Mice were immunized i.n. with Flu-containing formulations on d0 and lungs and spleens were analyzed on d30 or d60 via Tet and surface marker staining. CXCR3 was used as a marker of AW residence; CD103 and CD69 were used as markers of tissue residency. (B-E) Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (B) AW, (C) IST, (D) MV, and (E) spleen were enumerated on d30 after i.n. administration of Flu-NP/CpG, Flu-NP, or Flu+CpG. (F-I) Number (#) and frequency (%) of Tet⁺ CD8⁺ T cells in (F) AW, (G) IST, (H) MV, and (I) spleen were enumerated on d60 after i.n. administration of Flu-NP/CpG, Flu-NP, or Flu+CpG. (J-M) Number (#) of Tet⁺ CD8⁺ T cells expressing CD69±CD103 in AW were enumerated on (J) d30 or (K) d60 after immunization. (L-M) Number (#) of Tet⁺ CD8⁺ T cells expressing CD69±CD103 in IST were enumerated on (L) d30 or (M) d60 after immunization. Data are mean ± SEM, with n = 3–6 per group, and representative of two independent experiments. Immunization dose: 25 μg NP, 9.5 μg Flu, 1.4 μg CpG. Limit of detection: 1 cell (AW), 5 cells (IST/MV), 25 cells (spleen). *p<0.05, **p<0.01, ***p<0.001, by (B-I) ordinary one-way ANOVA or (J-M) ordinary two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant.

Figure 10.. Mice immunized with nanoparticle vaccine exhibit improved survival after intranasal challenge with influenza A H1N1 virus.

(A) Mice immunized on d0 were challenged i.n. with influenza A H1N1 PR8 virus (lethal dose of 200 FFU/mouse) either 30 d or 60 d post-immunization. Mice were weighed daily and evaluated for morbidity/mortality. (B-C) Percent (%) weight loss in mice challenged on (B) d30 or (C) d60 after i.n. administration. (D-E) Survival of mice challenged on (D) d30 or (E) d60 post-immunization. Mice that exceeded 30% weight loss were considered deceased. Data are mean ± SEM, with n = 4–6 per group, from two independent experiments. Immunization dose: 25 μg NP, 9.5 μg Flu, 1.4 μg CpG. Statistical significance in survival curves was determined with a Mantel-Cox log-rank test (**p<0.01; ns, not significant).