A Polysaccharide-Based Oral-Vaccine Delivery System and Adjuvant for the Influenza Virus Vaccine

Abstract

Influenza virus enters the host body through the mucosal surface of the respiratory tract. An efficient immune response at the mucosal site can interfere with virus entry and prevent infection. However, formulating oral vaccines and eliciting an effective mucosal immune response including at respiratory mucosa presents numerous challenges including the potential degradation of antigens by acidic gastric fluids and the risk of antigen dilution and dispersion over a large surface area of the gut, resulting in minimal antigen uptake by the immune cells. Additionally, oral mucosal vaccines have to overcome immune tolerance in the gut. To address the above challenges, in the current study, we evaluated inulin acetate (InAc) nanoparticles (NPs) as a vaccine adjuvant and antigen delivery system for oral influenza vaccines. InAc was developed as the first polysaccharide polymer-based TLR4 agonist; when tailored as a nanoparticulate vaccine delivery system, it enhanced antigen delivery to dendritic cells and induced a strong cellular and humoral immune response. This study compared the efficacy of InAc-NPs as a delivery system for oral vaccines with Poly (lactic-co-glycolic acid) (PLGA) NPs, utilizing influenza A nucleoprotein (Inf-A) as an antigen. InAc-NPs effectively protected the encapsulated antigen in both simulated gastric (pH 1.1) and intestinal fluids (pH 6.8). Moreover, InAc-NPs facilitated enhanced antigen delivery to macrophages, compared to PLGA-NPs. Oral vaccination studies in Balb/c mice revealed that InAc-Inf-A NPs significantly boosted the levels of Influenza virus-specific IgG and IgA in serum, as well as total and virus-specific IgA in the intestines and lungs. Furthermore, mice vaccinated with InAc-Inf-A-NPs exhibited notably higher hemagglutination inhibition (HI) titers at mucosal sites compared to those receiving the antigen alone. Overall, our study underscores the efficacy of InAc-NPs in safeguarding vaccine antigens post-oral administration, enhancing antigen delivery to antigen-presenting cells, and eliciting higher virus-neutralizing antibodies at mucosal sites following vaccination.

Keywords: influenza A nucleoprotein; influenza virus vaccine; inulin acetate; mucosal vaccine; subunit vaccine.

Figure 1

Characterization of InAc-Inf-A-NPs: (A) the mean particle size distribution was measured using DLS; (B) Zeta potential shows the surface charge of InAc-Inf-A-NPs a slightly negative or neutral (−0.9 ± 0.2 mV); (C) the morphology of InAc-Inf-A-NPs were spherical particles with a diameter of ~500 nm as shown by scanning electron microscopy (SEM).

Figure 2

Efficacy of InAc-NPs in preventing premature release of the encapsulated antigen. InAc-NPs containing Fluoresceine Sodium dye as the encapsulated antigen were dispersed in DI Water, Simulated Gastric Fluid (SGF), or Simulated Intestinal Fluid (SIF). Suspension was incubated in an orbital shaker at a speed of 100 rpm at 37 °C for 24 h. Fluorescein concentration in the supernatant solution at different time points was measured by fluorimeter and % cumulated release was calculated by comparing its fluorescent intensity with 100% release of Fluoresceine Sodium from NPs dissolved in 100% acetone or DMF.

Figure 3

InAc-FITC-Ova-NPs uptake by murine macrophages. The InAc-FITC-Ova-NPs or PLGA-FITC-Ova-NPs each with 25 µg equivalent to FITC-Ova were incubated with wild-type macrophages. After 1 h incubation, the cells were analyzed by flow cytometry for the number of cells having the antigen (FITC-Ova, green fluorescence) and the relative amount of antigen per cell by mean fluorescent intensity (MFI).

Figure 4

Fold change in Inf-A specific IgG (panel (A)) and IgA (panel (B)) in the serum following oral vaccination. BALB/c mice were vaccinated by oral administration of saline, Influenza A peptide alone in saline, or Influenza A peptide encapsulated in InAc-NPs (InAc-Inf-A-NPs). Two doses were given at one-week intervals. Blood was collected on day 0, day 7, and day 35 post-first vaccination. Panel (A) shows fold change in Inf-A-specific IgG tier at day 0, day 7-, and 35 days post-first vaccination while Panel (B) shows fold change in Inf-A-specific IgA tiers in serum at 35 days post-first vaccination. * Shows a significant difference at a 95% level of significance (p < 0.05).

Figure 5

The concentration of total IgA (panel (A)) and Inf-A specific IgA (panel (B)) in the tissues following oral vaccination. BALB/c mice were orally vaccinated with two doses of saline, Influenza A peptide alone in saline, or InAc-Inf-A-NPs one week apart. Following five weeks of the first vaccination, the mice were sacrificed, and the tissues such as ileum (small intestine), lungs, and spleen were collected. Collected tissue samples were homogenized in protease inhibitor and normalized for equal protein concentration followed by measuring the concentration of total IgA (panel (A)) and influenza virus A specific IgA (panel (B)) by sandwich ELISA. * shows a significant difference at a 95% level of significance (p < 0.05).

Figure 6

Hemagglutination inhibition (HI) titer following oral vaccination. BALB/c mice were orally vaccinated with two doses of saline, Influenza A peptide alone in saline, or InAc-Inf-A-NPs one week apart. After five weeks of the first vaccination, mice were sacrificed, and tissues were collected. The tissue samples were homogenized in protease inhibitor and supernatants of these homogenates were analyzed for the functionality of Influenza A virus-specific antibodies using HI assays. * shows a significant difference at a 95% level of significance (p < 0.05 in HI titer in tissue homogenates.