Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge

Abstract

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Keywords: CD4 T cells; Trm; airway T cells; influenza A; influenza virus challenge; lung parenchyma vasculature; mucosal immunology; polyfunctional T cells; rational vaccine design; tissue resident memory.

FIG 1

Potentiating lung localized CD4 T cell immunity via intranasal immunization. (A) Schematic representation of immunization regimen. Naive C57BL/6 mice were immunized with monovalent live attenuated influenza virus (LAIV), a 6:2 reassortant of caA/Ann Arbor/6/60 and A/New Caledonia/99. Responses were allowed to contract for 30 days prior to intranasal boost with NLP. NLP immunizations were composed of 5 μg recombinant influenza NP and 5 μg CpG conjugated to the NLP at a molar ratio of 1:2.4:18.4, NLP to NP to CpG, respectively. Responses were analyzed at 10 days post boost. (B) Schematic demonstrating the NLP self-assembly process. Purified components (including lipids DGS-NTA-Ni and DOPC, with apolipoprotein) are solubilized with surfactant and mixed in aqueous solution. Removal of the surfactant initiates self-assembly of the functionalized NLPs. Conjugation of adjuvants and antigens is achieved by reacting with cognate NLP surface functionalities (e.g., His tag with chelated nickel) or through anchoring of lipidic moieties featured on amphiphilic cargo molecules (e.g., cholesterol-tagged CpG). (C) His-tagged NP antigen can be conjugated to nickel-chelating NLPs at ratios from 2 to 16 antigens per NLP. Size exclusion chromatography (SEC) is used to monitor the conjugation, evidenced by a dose-dependent decrease in retention time and an increase in NLP absorbance intensity, indicating successful conjugation of antigen to NLP. (D) SEC demonstrates the successful incorporation of both NP protein and cholesterol-modified CpG in the final vaccine assembly, evidenced by a shift in retention time and increase in absorbance intensity.

FIG 2

Live-attenuated virus establishes memory. (A to C) Naive C57BL/6 mice were primed with live-attenuated influenza virus to establish influenza virus-specific memory. The CD4 T cell response was assayed in lung (A), mLN (B), and spleen (C) at 7 days postinfection via IL-2 and IFN-γ cytokine ELISpot to identify immunodominant CD4 T cell epitopes across hemagglutinin (HA), nonstructural protein 1 (NS1), nucleoprotein (NP), and neuraminidase (NA) viral proteins. Results are presented as the mean number of cytokine-producing spots per million CD4 T cells with the standard deviation shown. The mean is representative of three independent experiments of five pooled mice each.

FIG 3

Intranasal NLP:NP boost elicits a higher frequency of NP-specific CD4 T cells in the lung than does an LAIV boost. (A to F) Thirty days postimmunization with LAIV, CD4 T cell responses were assayed in lung, mLN, and spleen via IFN-γ (A to C) and IL-2 (D to F) cytokine ELISpot to characterize the immune response prior to intranasal boost with NLP:NP. At 10 days post intranasal immunization with NLP:NP, the extent of CD4 T cell boosting in lung (A and D), mLN (B and E), and spleen (C and F) was quantified by IL-2 and IFN-γ cytokine ELISpot. Results are presented as the mean number of cytokine-producing spots per million CD4 T cells with the standard deviation shown. The mean is representative of three to five independent experiments of five pooled mice each.

FIG 4

Intranasal NLP:NP vaccination establishes larger tissue-resident CD4 T cell populations in the lung than does an LAIV boost. (A) Analysis of lung localization among CD4 T cells, depicting representative flow plots. (B) Frequency of lung tissue-localized CD4 T cells. (C) Absolute abundance of CD4 T cells in the intravascular labeled CD45⁺ lung vasculature and unlabeled CD45⁻ lung tissue. (D) CD4 T cells were assessed for their expression of CD44 and CD62L. (E and F) The frequency (E) and absolute abundance (F) of effector CD44⁺ CD62L⁻ CD4 T cells was assessed in the lung tissue and vasculature. (G) CD44⁺ CD62L⁻ effector CD4 T cells were subsequently gated on expression of CD11a and CD69. (H and I) The frequency (H) and abundance (I) of CD11a⁺ CD69^+/− was calculated among effector CD4 T cells in the lung tissue. (J) Expression of memory-precursor marker Ly6C was assessed among CD44⁺ CD62L⁻ effector CD4 T cells. (K and L) The frequency (K) and abundance (L) of Ly6C⁺ and Ly6C⁻ CD4 T cells was assessed among lung tissue localized effector CD4 T cells. Results are presented as the mean number of the respective population with the standard deviation and individual mice shown. The mean is representative of three independent experiments of five individual mice each. With the exception of (B), significance (***, P < 0.001; ****, P < 0.0001) for comparisons of cellular frequency and abundance between LAIV and LAIV+NLP boost cohorts was determined by two-way ANOVA with Tukey’s correction for multiple comparisons.

FIG 5

Intranasal NLP:NP immunization boosts antigen-specific CD4 T cells which localize to the lung tissue. (A) Representative plot of NP-specific CD4 T cells detected by I-A^b pMHC-II NP45 tetramer staining in LAIV and LAIV+NLP immunized murine lung. (B) Frequency of vasculature-localized CD45⁺ and tissue-localized CD45⁻ pMHC-II NP45 tetramer⁺ cells. (C and D) Quantification of the total number of NP45-specific CD4 T cells (C) and those localized to lung tissue and vasculature (D) of LAIV and LAIV+NLP immunized mice. Results are presented as the mean number of pMHC-II NP45 tetramer⁺ cells per lung with standard deviation and individual replicates shown. The mean is representative of 10 individual mice analyzed as pools of two lungs each. Cell numbers from pooled lungs are divided by two to reflect the cellularity of a single lung. Significance (***, P < 0.001) for comparisons of cellular abundance between LAIV and LAIV+NLP boost cohorts was determined by two-way ANOVA with Tukey’s correction for multiple comparisons.

FIG 6

Codelivery of antigen and adjuvant on NLP:NP enhances CD4 T cell responses to intranasal vaccination. (A) Schematic representation of the immunization regimen, where mice were primed with LAIV and subsequently boosted with NLP:NP or soluble NP+CpG, and responses were assayed at 10 days post intranasal boost. The CD4 T cell response to NLP:NP and soluble NP+CpG immunization was compared via IFN-γ and IL-2 cytokine ELISpot assay in lung (B and C), mLN (D and E), and spleen (F and G). Results are presented as the mean with standard deviation of two independent experiments of five pooled mice each. Statistical significance was determined by unpaired, two-tailed t test with Welch’s correction.

FIG 7

Intranasal vaccination with NLP:NP elicits a higher frequency of antigen-specific CD4 T cells than peripheral vaccination. (A) Schematic representation of the immunization regimen used to compare the effect of intranasal (i.n.) versus subcutaneous (S.Q.) immunization with NLP:NP. (B to D) Comparison of the frequency of lung- (B), mLN- (C), and spleen-localized (D) CD4 T cell responses following i.n. or S.Q. immunization as determined by cytokine ELISpot. (F to G) Assessment of the abundance of antigen-experienced CD4 T cells within the airway (F), lung tissue (G), and lung vasculature (H) following i.n. or S.Q. NLP:NP immunization. Results are presented as the mean with standard deviation of four individual mice per cohort. One cohort was assessed by cytokine ELISpot and a second cohort was assessed by flow cytometry. Statistical significance was determined by unpaired, two-tailed t test with Welch’s correction.

FIG 8

Intranasal NLP:NP immunization elicits distinct populations of NP-specific CD4 T cells localized to the airway, lung tissue, and lung vasculature. CD4 T cells isolated from the indicated tissue were restimulated with cognate antigen and stained intracellularly with IFN-γ, TNF-α, and IL-2 antibodies. Cells producing one or more cytokines were determined using Boolean gating. (A and B) The frequency of cells producing one or more cytokines was quantified among CD4 T cells from LAIV alone (A) or from LAIV+NLP-boosted (B) mice. (C and D) Quantification of antigen-experienced CD4 T cells that underwent degranulation (C), as evidenced by expression of CD107a+ in response to antigen stimulation or those expressing putative cytolytic marker NKG2 (D). Data are presented as the mean of two independent experiments of 15 pooled mice per treatment group (LAIV alone or LAIV+NLP boost) with standard deviation shown. Statistical significance (*, P < 0.05; **, P < 0.01 ***, P < 0.001; and ****, P < 0.0001) for comparisons of cellular frequency between tissues was determined by two-way ANOVA with Tukey’s correction for multiple comparisons.

FIG 9

NP45 peptide-stimulated CD4 T cells produce prototype Th1 cytokines but not IL-4 or IL-17. CD4 T cells isolated from the indicated tissue were restimulated with cognate antigen and stained intracellularly with IFN-γ, TNF-α, IL-2, IL-4, and IL-17 antibodies as described in Fig. 8. (A) The frequency of cells singly producing each cytokine was quantified among CD4 T cells from NLP-boosted mice in the indicated tissues. Data are presented as the mean of five single mice.

FIG 10

Unsupervised clustering highlights polyfunctionality of CD4 T cells elicited by NLP:NP immunization. (A to E) viSNE pseudocolored maps depicting the relative expression of the indicated markers (columns) across CD4 T cells isolated from airway, lung, mLN, and spleen (rows). Features of interested are highlighted in the viSNE pseudocolored maps using circles and letters (A to E) corresponding to descriptions in the results. The scale bar reflects the relative expression difference of each marker indicated at the top of each column from low (blue) to high (red). Cells were stimulated as described in Fig. 7. Manually gated live CD4⁺ and CD8⁺ cells were input into the viSNE clustering algorithm, with 50,000 cells per population input into the clustering algorithm for a total of 200,000 cells. viSNE plots were generated with an iteration number of 3,000 and perplexity of 50 (final KL Divergence of run 3.87).

FIG 11

NLP:NP immunization elicits a population of antigen-specific CD4 T cells that persist in the lung long term. Persistence of antigen-specific CD4 T cells was assessed at 9 months postimmunization to assess durability of responses without confounding effects of immunosenescence. (A to C) The frequency of IL-2- and IFN-γ-producing cells was assessed in lung (A), mLN (B), and spleen (C) by ELISpot assay. Data, presented as cytokine-producing spots per million CD4 T cells, are the mean of three independent experiments of five pooled mice each.

FIG 12

Adoptive transfer of CD4 T cells from NLP:NP-boosted, but not LAIV-boosted, mice is sufficient for protection of naive mice from pathogenic pH1N1 virus challenge. The protective potential of CD4 T cells from NLP:NP-boosted and LAIV-boosted mice was assessed in the context of a lethal influenza virus challenge experiment. Five million purified CD4 T cells were adoptively transferred into naive CD45.1 congenic recipient mice at 1 day prior to challenge with a lethal dose of A/California/09 pH1N1 virus. Mice that lost >20% of their starting body weight were scored deceased and humanely euthanized in accordance with institutional guidelines. (A to D) Weight loss was tracked daily over the course of 15 days. Weight loss (A) and overall survival (B) are shown for mice that received CD4 T cells from NLP:NP-boosted mice. Matched adoptive transfer and viral challenge data showing weight loss (C) and survival (D) of mice that received CD4 T cells from LAIV-boosted mice. Data are presented as the mean of two independent experiments with the standard error of the mean (SEM) shown, where n = 5 to 8 for the lung-derived T cells and n = 18 for the mock control. Statistical significance was determined by one-way ANOVA and log rank (Mantel-Cox) test.