Broadly active intranasal influenza vaccine with a nanocomplex particulate adjuvant targeting mast cells and toll-like receptor 9

Abstract

Flumist is the only FDA-approved intranasal influenza vaccine. Although it has recently been approved for at-home use, it has significant limitations. These include reduced effectiveness in generating a protective immune response in patients with extensive influenza exposure, safety concerns due to its live attenuated virus formulation, and reduced efficacy due to viral drift/shift. To address this limitation, we have developed a nanocomplex comprised of a mast cell (MC) agonist and toll-like receptor 9 (TLR9) ligand to adjuvant a broadly acting influenza antigen. The newly reported MC agonist was identified by screening mastoparan-7 analogs for MC degranulation activity, which led to a more active peptide analog, MP12W. Positively charged MP12W spontaneously forms nanoparticulate complexes (NPs) with CpG 1826 that were then used to intranasally vaccinate mice with a computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) protein. The NPs were further optimized by substituting CpG 1826 with CpG 55.2, a TLR-9 agonist identified by machine learning to be more active in humans. MP12W-CpG 1826 NPs showed an increased pro-inflammatory response and decreased cytotoxicity in vitro compared to M7 complexes, translating into a safer profile in a model of increased hypersensitivity, collaborative cross mice 027 (CC027). Intranasal vaccination with this complex and broadly reactive HA resulted in higher mucosal antibody concentration and increased cytokine production with antigen recall. These responses were enhanced with MP12W-CpG 55.2 NP vaccination. MP12W-CpG NPs provided similar protection in an influenza challenge model. This study demonstrates the potential of this novel intranasal nanocomplex for vaccination.

Keywords: Broadly active influenza vaccine; CpG 55.2; Intranasal vaccination; MP12W; Self-assembled nanoparticles.

Figure 1.. M7 derivatives, their MCD50 and adjuvant activity

. (A) Table showing the name and sequence of the top 7 M7 analogs ordered by their capacity to in vitro degranulate 50% of mast cells (MCD50). (B) C57BL/6 mice (n=3) were i.n. immunized on day 0 and 14 with Spike protein (2.5 μg) alone or combined with a normal dose (5 nmol) or a low dose (0.5 nmol) of M7, MP12W, MP12L, or MP12F. Serum was collected on day 21 and tested for anti-Spike IgG titers. Data is shown as mean ± SD. Significant difference for a one-way ANOVA test compared versus “no adjuvant” group * p≤0.001.

Figure 2.. Evaluation of cytotoxicity and induction of proinflammatory cytokines of MP12W-CpG 1826 NPs.

(A) SEM image showing nanocomplex particles (NPs) when MP12W and CpG 1826 are combined. DC 2.4 cells were stimulated with M7 or MP12W (64 μM), CpG 1826 (8 μM), and M7-CpG 1826 NPs or MP12W-CpG 1826 NPs (64 μM + 8 μM). Supernatants were tested for (B) LDH release, (C) TNF-α and (D) IL-6. Data is shown as mean ± range. Significant differences were determined using a one-way ANOVA test with Tukey’s multiple comparison ** p≤0.01, *** p≤0.005, **** p≤0.001.

Figure 3.. Assessment of body temperature and specific IgE titers of MP12W-CpG1826 in allergy-prone mice.

CC027 mice (n=5) were intranasally immunized on days 0, 21, and 35 with OVA alone (non-adjuvanted) or OVA combined with M7-CpG 1826 NPs, MP12W-CpG 1826 NPs, CTB once a week (CTB 1X) or CTB thrice a week (CTB 3X). Rectal temperature was recorded at different time points (0–5 h) post-immunization during the (A) prime, (B) first boost, and (C) second boost. (D) Area under the curve of the rectal temperatures obtained on A-C. (E) Day 42 sera was tested for OVA-specific IgE titers. Data is shown as mean ± SD. (A-C) Significant differences were determined using a two-way ANOVA test with Tukey’s multiple comparisons vs. OVA alone groups at the same time point * p≤0.05, ** p≤0.01, *** p≤0.005. (D-E) Significant differences were determined using a one-way ANOVA test with Tukey’s multiple comparison * p≤0.05.

Figure 4.. Analysis of mucosal antibody titers and T cell responses of M7/MP12W and CpG 1826 NPs.

C57BL/6 mice (n=5) were intranasally immunized on day 0, 21 and 35 with PBS, OVA alone (non-adjuvanted), or OVA combined with CpG 1826, M7, MP12W, M7-CpG 1826 NPs or MP12W-CpG 1826 NPs. (A) Anti-OVA IgG titers on BAL and (B) Anti-OVA IgA titers on nasal washes from the immunized mice at day 42. Spleen and LNs from the different groups of mice were collected and processed at day 42, splenocytes were stimulated with OVA for 36 h. (C) IFN-γ-producer cells were detected by ELISPOT and counted. Total counts of central memory CD4+ T cells on (D) spleen and (E) LN by flow cytometry. Data is shown as mean ± range. Significant differences were determined using a one-way ANOVA test with Tukey’s multiple comparisons * p≤0.05, ** p≤0.01.

Figure 5.. Evaluation of sera IgM, HAI titers and protective response of MP12W-CpG 55.2 NPs.

(A) SEM image showing nanocomplex particles (NPs) when CpG 55.2 and MP12W are combined. C57BL/6 mice (n=5) were intranasally immunized on days 0, 21, and 35 with PBS (Saline) or OVA combined with CpG 1826, CpG 55.2, CpG 1826-M7 NPs, CpG 1826-MP12W NPs, CpG 55.2-M7 or CpG 55.2-MP12W NPs. (B) Sera was collected on days 14, 28, and 42 and run for anti-OVA IgM titers. (C) Splenocytes from the immunized mice were stimulated on day 42 with OVA for 36 h, and IL-2-producer cells were detected and counted using ELISPOT. C57BL/6 mice (n=5) were intranasally immunized on day 0, 21 and 35 with PBS (Saline), or COBRA J4 combined with CpG 1826, CpG 55.2, CpG 1826-MP12W NPs or CpG 55.2-MP12W NPs. (D) On day 42 post-immunization sera were collected and tested for anti-SW/13 HAI titers and (E) on day 56 mice were intranasally challenged with 2.5K pfu of A/Hong Kong/1/68 influenza virus and survival was analyzed for 14 days post-challenge. Data is shown as mean ± range (A-D) or survival percentage (E). Significant differences were determined using a one-way ANOVA test with Tukey’s multiple comparison ** p≤0.01, *** p≤0.005, **** p≤0.001.