Polymeric Nanoparticles as Oral and Intranasal Peptide Vaccine Delivery Systems: The Role of Shape and Conjugation

Abstract

Mucosal vaccines are highly attractive due to high patient compliance and their suitability for mass immunizations. However, all currently licensed mucosal vaccines are composed of attenuated/inactive whole microbes, which are associated with a variety of safety concerns. In contrast, modern subunit vaccines use minimal pathogenic components (antigens) that are safe but typically poorly immunogenic when delivered via mucosal administration. In this study, we demonstrated the utility of various functional polymer-based nanostructures as vaccine carriers. A Group A Streptococcus (GAS)-derived peptide antigen (PJ8) was selected in light of the recent global spread of invasive GAS infection. The vaccine candidates were prepared by either conjugation or physical mixing of PJ8 with rod-, sphere-, worm-, and tadpole-shaped polymeric nanoparticles. The roles of nanoparticle shape and antigen conjugation in vaccine immunogenicity were demonstrated through the comparison of three distinct immunization pathways (subcutaneous, intranasal, and oral). No additional adjuvant or carrier was required to induce bactericidal immune responses even upon oral vaccine administration.

Keywords: Group A Streptococcus; conjugates; intranasal delivery; mucosal immunology; nanoparticles; oral delivery; peptides; physical mixture; vaccines.

Figure 1

Polymeric nanoparticle-based vaccine candidates. Three components were used to produce vaccine candidates: B-cell epitope (J8), T-helper epitope (PADRE), and polymeric units. These were combined either by (a) conjugation reaction to produce rods–PJ8, worms–PJ8, spheres–PJ8, and tadpoles–PJ8, or (b) physical mixing of antigen and polymeric units to produce rods + PJ8, worms + PJ8, spheres + PJ8, and tadpoles + PJ8. The symbolization for conjugates is ‘–’, whereas that for physical mixers is ‘+’. The antigen PJ8 is shown in a blue font.

Figure 2

Immune responses following oral immunization of C57BL/6 mice (n = 5 per group) with PBS (negative control), PJ8 + CTB (adjuvanted control), and experimental conjugates and physical mixtures. J8-specific serum (a–c) and saliva (d) IgG titers in individual mice are shown by each point, and the average antigen-specific IgG titers are shown by the bars. (e,f) Average bactericidal activity of immunized mouse sera against different GAS strains (GC2203 and D3840). One-way ANOVA with Tukey’s multiple comparison test was used for statistical analysis. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. The statistical analysis is presented in comparison to PBS (upper line) and sphere–PJ8 (lower line).

Figure 3

Immune responses in C57BL/6 mice (n = 5 per group) after intranasal injection of water (negative control), PJ8+CTB (adjuvanted control), and experimental conjugates and physical mixers. J8-specific serum (a–c) and saliva (d) IgG titers. An individual mouse is represented by each point, and average antigen-specific IgG titers are shown by the bars. (e,f) Average percentage of bactericidal activity against two different GAS strains (GC2 203 and D3840) based on serum collected 41 days after the initial intranasal vaccination. One-way ANOVA with Tukey’s multiple comparison test was used for statistical analysis. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. The statistical analysis is presented in comparison to PBS (upper line) and sphere + PJ8 (lower line).

Figure 4

The relationship between anti-J8 IgG titers in mouse sera and bactericidal activity against cultured GAS bacteria (D3840 strain). The data were fitted to a sigmoidal relationship (R² = 0.94). Red circles denote subcutaneous immunization [12], while black circles denote intranasal and oral immunization. Dashed arrows represent the interpolated IC₅₀ and IC₉₀ titer values from the fitted curve.

Figure 5

In vitro proteolysis stability of antigen (PJ8) alone, in physical mixtures, and in conjugates against bovine trypsin.