(567c) Virus-like Particle-Templated Silica-Adjuvanted Nanovaccines with Enhanced Humoral and Cellular Immunity

Virus-like particles (VLPs) are self-assembled biomolecules that closely resemble native viruses. VLPs exhibit repetitive and high-density conformational viral epitopes that can elicit enhanced immunostimulating effects. Foreign epitopes could also be included into VLPs, which provide a platform to display multivalent homologous or heterogeneous antigens. Therefore, VLPs are superior antigen forms for vaccine applications. Thus far, VLP-based HBV and HPV vaccines have been authorized for human use. Many others, e.g., respiratory syncytial virus, Norwalk virus, Ebola, SARS-CoV-2, and influenza virus vaccines, are in clinical trials. However, in order to achieve higher immunogenicity and antigen dose-sparing effect, adjuvants are necessarily formulated in VLP vaccines.

Currently, most adjuvants in approved VLP vaccines are aluminum salts. For instance, hepatitis B vaccines, e.g., Engerix-B^® and Recombivax HB^®, were adjuvanted with aluminum hydroxide and amorphous aluminum hydroxyphosphate sulfate (AAHS), respectively. Human papillomavirus vaccines, e.g., Gardasil^® and Cervarix^®, were formulated with AAHS and AS04 (aluminum hydroxide with 3-O-desacyl-4’-monophosphoryl lipid A), respectively. Although aluminum salt-based adjuvants (Alum) are widely used in these authorized vaccines, there are still some existing challenges. Alum are known to induce strong Th2-mediated humoral responses, but not for Th1-mediated cellular immune responses. The biased immunostimulation is not helpful to provide effective immune protection. In addition, antigen adsorption on Alum is required to ensure efficient antigen delivery, however, the amount and strength of antigen adsorption are unpredictable because adsorption is based on various physical or chemical interactions, and there is no universally applicable design rules for adjuvants with high antigen delivery capability for different VLP antigens. It is, therefore, important to construct an efficient antigen delivery system with superior adjuvanticity to induce both humoral and cellular immune responses is required for VLP vaccines.

VLPs are mainly composed of self-assembled viral proteins. They have precisely controlled three-dimensional nanostructures with the display of charged functional groups such as amine and carboxyl groups on the surface. Thus, VLPs could be utilized as biotemplates to synthesize engineering nanomaterials. It has been reported that viruses or VLPs are used to prepare a variety of various organic and inorganic nanomaterials. These facts provide an inspiration to design a VLP-based vaccine platform, in which VLP antigens could be utilized as biotemplates to directly construct engineered nanomaterial-adjuvanted vaccines with high immunostimulating potentials.

Silica nanoparticles have been used as the drug delivery carriers and adjuvants due to their biocompatibility and the capability of enhancing innate and adaptive immunity. Herein, we used HBsAg VLPs and HPV18 VLPs as biotemplates to demonstrate the one-step synthesis of the raspberry-like silica-adjuvanted vaccines (VLP@Silica), and further determined their capability in mediating more balanced humoral and cellular immune responses (Figure 1). By analyzing synthesis dynamics of HBsAg VLP@Silica, the synthetic chemistry and formation mechanism of VLP@Silica were explored. The circular dichroism (CD) spectra and immunogenicity analysis of HBsAg VLPs suggested minimum VLP conformational and immunogenic change in HBsAg VLP@Silica. In bone marrow-derived dendritic cells (BMDCs), VLP@Silica could induce the secretion of both Th1 and Th2 type cytokines, e.g., IFN-γ, TNF-α, IL-12 and IL-6, and promote the lysosomal escape and cytosolic delivery of VLPs. In addition, VLP@Silica enhanced dendritic cell (DC) maturation and antigen migration into draining lymph nodes (DLNs). When VLP@Silica were templated with either HBsAg VLPs or HPV 18 VLPs, they induced coordinated antigen-specific antibodies and T cell-mediated cellular immune responses. Taken together, this design strategy can utilize VLPs derived from a diversity of viruses or their variants as templates to construct both prophylactic and therapeutic vaccines with improved immunogenicity.

Figure 1. The humoral and cellular immune responses induced by VLP@Silica with HBsAg VLPs and HPV18 VLPs as biotemplates. A) The ratio of serum HBsAg-specific IgG_2c to IgG₁. Female C57BL/6 mice (8-week, n = 7) were immunized by HBsAg VLP@Silica via i.m. on day 0 and 14. Each dose contains 2 μg of HBsAg VLPs and 20 μg of Si. HBsAg VLPs and HBsAg VLP_Alum (containing 20 μg of Al) were set as controls. The secretions of (B, D) IFN-γ and (C, E) IL-4 by antigen-stimulated (B-C) CD4⁺ T cells and (D-E) CD8⁺ T cells. The cells isolated from spleens of immunized mice were treated with HBsAg VLPs (2 μg/mL) for 60 h. F) TEM images of HPV 18 VLPs and HPV VLP@Silica. The scale bar is 300 nm. Female C57BL/6 mice (8-week, n = 7) were immunized by HPV VLP@Silica via i.m. on day 0 and 21. Each dose contains 4 μg of HPV VLPs and 40 μg of Si. G) The ratio of serum HPV-specific IgG_2c to IgG₁. After stimulation by HPV VLPs for 60 h, the expressions of CD69 on H) CD4⁺ and I) CD8⁺ T cells were examined by flow cytometry (n = 3). Data are all presented as mean values ± SEM (NS, not significant; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).