Dual-Antigen Subunit Vaccine Nanoparticles for Scrub Typhus.

Orientia tsutsugamushi is the causative pathogen of scrub typhus, an acute febrile disease prevalent in the Asia-Pacific region that is spread to people through chigger bites. Despite the emerging threat, there is no currently available vaccine against O. tsutsugamushi. Here, we developed dual-antigen subunit vaccine nanoparticles using recombinant 47 kD and 56 kD proteins, which are immunogenic outer membrane antigens of O. tsutsugamushi. The biocompatible protein vaccine nanoparticles were formed via desolvation of r56 or r47E antigens with acetone, coating with an additional layer of the 56 kD protein, and stabilization with reducible homobifunctional DTSSP and heterobifunctional SDAD crosslinkers. The dual-antigen subunit vaccine nanoparticles significantly improved antigen-specific antibody responses in vaccinated mice. Most importantly, the dual-antigen nanoparticles coated with an additional layer of the 56 kD protein were markedly more immunogenic than soluble antigens or single-antigen nanoparticles in the context of cellular immune responses. Given the significance of cellular immune responses for protection against O. tsutsugamushi, these results demonstrate the potent immunogenicity of dual-layered antigen nanoparticles and their potential as a promising strategy for developing vaccines against scrub typhus.

Rational Design of Antigen Incorporation Into Subunit Vaccine Biomaterials Can Enhance Antigen-Specific Immune Responses.

Peptide subunit vaccines increase safety by reducing the risk of off-target responses and improving the specificity of the induced adaptive immune response. The immunogenicity of most soluble peptides, however, is often insufficient to produce robust and lasting immunity. Many biomaterials and delivery vehicles have been developed for peptide antigens to improve immune response while maintaining specificity. Peptide nanoclusters (PNC) are a subunit peptide vaccine material that has shown potential to increase immunogenicity of peptide antigens. PNC are comprised only of crosslinked peptide antigen and have been synthesized from several peptide antigens as small as 8 amino acids in length. However, as with many peptide vaccine biomaterials, synthesis requires adding residues to the peptide and/or engaging amino acids within the antigen epitope covalently to form a stable material. The impact of antigen modifications made to enable biomaterial incorporation or formation is rarely investigated, since the goal of most studies is to compare the soluble antigen with biomaterial form of antigen. This study investigates PNC as a platform vaccine biomaterial to evaluate how peptide modification and biomaterial formation with different crosslinking chemistries affect epitope-specific immune cell presentation and activation. Several types of PNC were synthesized by desolvation from the model peptide epitope SIINFEKL, which is derived from the immunogenic protein ovalbumin. SIINFEKL was altered to include extra residues on each end, strategically chosen to enable multiple conjugation chemistry options for incorporation into PNC. Several crosslinking methods were used to control which functional groups were used to stabilize the PNC, as well as the reducibility of the crosslinking. These variations were evaluated for immune responses and biodistribution following in vivo immunization. All modified antigen formulations still induced comparable immune responses when incorporated into PNC compared to unmodified soluble antigen alone. However, some crosslinking methods led to a significant increase in desirable immune responses while others did not, suggesting that not all PNC were processed the same. These results help guide future peptide vaccine biomaterial design, including PNC and a wide variety of conjugated and self-assembled peptide antigen materials, to maximize and tune the desired immune response.

Protein and Peptide Biomaterials for Engineered Subunit Vaccines and Immunotherapeutic Applications.

Although vaccines have been the primary defense against widespread infectious disease for decades, there is a critical need for improvement to combat complex and variable diseases. More control and specificity over the immune response can be achieved by using only subunit components in vaccines. However, these often lack sufficient immunogenicity to fully protect, and conjugation or carrier materials are required. A variety of protein and peptide biomaterials have improved effectiveness and delivery of subunit vaccines for infectious, cancer, and autoimmune diseases. They are biodegradable and have control over both material structure and immune function. Many of these materials are built from naturally occurring self-assembling proteins, which have been engineered for incorporation of vaccine components. In contrast, others are de novo designs of structures with immune function. In this review, protein biomaterial design, engineering, and immune functionality as vaccines or immunotherapies are discussed.

Cross-Linked Peptide Nanoclusters for Delivery of Oncofetal Antigen as a Cancer Vaccine.

Peptide subunit vaccines are desirable because they increase control over the immune response and safety of the vaccine by reducing the risk of off-target responses to molecules other than the target antigen. The immunogenicity of most peptides, however, is low. Peptide nanoclusters (PNC) are proposed as a subunit peptide vaccine delivery system made completely of cross-linked peptide antigen that could improve the immunogenicity of a peptide vaccine. Proof of concept is demonstrated with oncofetal antigen (OFA), an immature laminin receptor protein expressed by many hematologic cancer cells but not by healthy cells. Peptide epitopes from this protein, called OFA 1, 2, and 3, were synthesized into PNC as a potential cancer peptide vaccine delivery system. PNC were formed by desolvation and stabilized with disulfide bonds using a trithiol cross-linker. Cysteines were added to the C-terminus of each peptide to assist in this cross-linking step, denoted OFA 1C, 2C, and 3C PNC. OFA 2C was found to form the smallest PNC, 148 ± 15 nm in diameter and stable in solution. This size is in the range where particles are readily internalized by dendritic cells (DCs) and may also passively diffuse to regional lymph nodes. OFA 2C PNC and soluble OFA 2C were internalized similarly by DCs in vitro, but only PNC resulted in significant peptide presentation by DCs. This indicates the potential for PNC to improve immune activation against this antigen. Additionally, PNC displayed higher retention at the intradermal injection site in vivo than soluble peptide, allowing more time to interact with DCs in an area of increased DC activity. While offering traditional nanoparticle benefits such as increased DC recognition, slower diffusion, and potential for multivalent cellular interactions, PNC also maximize antigen delivered per particle while minimizing off-target material delivery because the antigens are the main building blocks of the particle. With these properties, PNC are a delivery system with potential to increase peptide subunit vaccine immunogenicity for OFA and other peptide antigens.

Development of three-dimensional lung multicellular spheroids in air- and liquid-interface culture for the evaluation of anticancer therapeutics.

Three-dimensional (3D) lung multicellular spheroids (MCS) in liquid-covered culture (LCC) and air-interface culture (AIC) conditions have both been developed for the evaluation of aerosol anticancer therapeutics in solution and aerosols, respectively. The MCS were formed by seeding lung cancer cells on top of collagen where they formed spheroids due to the prevalence of cell-to-cell interactions. LCC MCS were exposed to paclitaxel (PTX) in media whereas AIC MCS were exposed to dry powder PEGylated phospholipid aerosol microparticles containing paclitaxel. The difference in viability for 2D versus 3D culture for both LCC and AIC was evaluated along with the effects of the particles on lung epithelium via transepithelial electrical resistance (TEER) measurements. For LCC and AIC conditions, the 3D spheroids were more resistant to treatment with higher IC50 values for A549 and H358 cell lines. TEER results initially indicated a decrease in resistance upon drug or particle exposure, however, these values increased over the course of several days indicating the ability of the cells to recover. Overall, these studies offer a comprehensive in vitro evaluation of aerosol particles used in the treatment of lung cancer while introducing a new method for culturing lung cancer MCS in both LCC and AIC conditions.