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Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

Traditional emulsion adjuvants are stabilized by non-ionic surfactants and promote high humoral immunity. However, because of their electroneutrality, they cannot effectively carry negatively charged antigens and enter the cell to elicit cellular immunity. To overcome this problem, we developed an emulsion using chitosan hydrochloride salt (CSCL) as stabilizer. After optimization, CSCL was successfully adsorbed on the surface of the emulsion (CPSE), and stabilized for two months. CPSE could effectively loading the antigen and slow down the rate of the antigen release. We evaluated the effectiveness of CPSE through intramuscular immunization in mice after cytotoxicity assay proved its safety. CPSE not only induced strong antibody-mediated immune responses, but also generated memory T cells in the spleen as a marker of adaptive immunity. Importantly, CPSE showed advantages over the conventional vaccine formulations in promoting IFN-γ secretion. CPSE may act as a promising candidate to enhance the efficacy of vaccines with acceptable biocompatibility and safety.

To generate effective immunity post-vaccination, antigens need to be effectively captured and taken up by antigen-presenting cells (APCs) as a prerequisite. Biomimetic designs that mimic natural pathogen-like properties have provided platforms for antigen delivery. However, the structural dynamic properties of pathogens leading to their efficient internalization have been neglected in most platforms. Herein, we redesigned a special multiple emulsion with chitosan hydrogel nanoparticles inside, mimicking the configurational flexibility and deformational flexibility of pathogens. With the assistance of chitosan-antigen particles, the novel emulsion exhibited amplified deformability and the vaccine-cell contact zone was increased. Additionally, its configurational transitions, which offered sustained exposure of sheltered uptake signals including antigens and stimulator chitosan during endocytosis, resulted in efficient antigen delivery to APCs. Prolonged antigen depot effect, versatile antigen presentation, multiple immunocyte activation, and marked adjuvant-sparing effects were achieved as compared with those in the control groups. As a result, the intracellular emulsion formulation robustly induced both humoral and cellular immunity, especially CTL response, against the foot-and-mouth disease virus (FMDV) with improved biosafety. Our study highlights the positive impact of biomimetic structural dynamic properties on robust vaccine-cell interactions and provides a promising FMDV vaccine candidate.

Parenteral vaccinations are not able to elicit effective systemic and gastrointestinal immune protection simultaneously because the lymphocytes are typically restricted to primed tissues. Although all-trans retinoic acid (atRA) was reported to trigger the gut-homing of immunocytes, the bioavailability and systemic immune responses remain limited for use in robust enteric vaccinations. Here, we show that co-delivery of atRA, CpG oligodeoxynucleotides (CpG), and antigens via engineered polymer/lipid nanoparticles (PLNPs) could exploit the amplifying function of draining lymph nodes (DLNs) for potent gut tropism and immune activations. After intramuscular injection, forming an immune-potentiated environment at the injection site, the PLNPs induced the designated transfer of primed dendritic cells (DCs) to the DLNs instead of the gastrointestinal tissues. Within the DLNs, the immune-potentiated environment markedly amplified the antigen presentation and homing receptor switch among immunocytes, which simultaneously stimulated the preferential dissipation of activated lymphocytes in the peripheral and gastrointestinal tissues, that is, exerted a DLN-amplifying effect. Compared with current atRA-containing formulations, the PLNPs not only boosted potent IgG secretions and T cell activations in the peripheral tissue but also provoked robust T cell homing and antigen-specific IgA levels in the gastrointestinal tracts in both ovalbumin and EV71 vaccinations. These data indicate that exploiting DLN amplification can stimulate potent systemic and gastrointestinal responses for more efficient enteric vaccinations.

To generate effective immunity post-vaccination, antigens need to be effectively captured and taken up by antigen-presenting cells (APCs) as a prerequisite. Biomimetic designs that mimic natural pathogen-like properties have provided platforms for antigen delivery. However, the structural dynamic properties of pathogens leading to their efficient internalization have been neglected in most platforms. Herein, we redesigned a special multiple emulsion with chitosan hydrogel nanoparticles inside, mimicking the configurational flexibility and deformational flexibility of pathogens. With the assistance of chitosan-antigen particles, the novel emulsion exhibited amplified deformability and the vaccine-cell contact zone was increased. Additionally, its configurational transitions, which offered sustained exposure of sheltered uptake signals including antigens and stimulator chitosan during endocytosis, resulted in efficient antigen delivery to APCs. Prolonged antigen depot effect, versatile antigen presentation, multiple immunocyte activation, and marked adjuvant-sparing effects were achieved as compared with those in the control groups. As a result, the intracellular emulsion formulation robustly induced both humoral and cellular immunity, especially CTL response, against the foot-and-mouth disease virus (FMDV) with improved biosafety. Our study highlights the positive impact of biomimetic structural dynamic properties on robust vaccine-cell interactions and provides a promising FMDV vaccine candidate.