Formulation of Lipid Nanoparticles with Viral Subunit Antigens for Vaccination

Abstract

Vaccination provides significant advantages over post-infection treatment such as long-lasting protection, prevention of co-morbidities, and a reduction in the dissemination of pathogens. While vaccination has tempered many once virulent pathogens, others remain without effective vaccines. Moreover, the emergence of previously unknown or isolated pathogens is presenting a significant threat to human health. Overpopulation, increased urbanization, and international travel provide continuous sources of naïve hosts, permitting the persistence and spread of pathogens along with an increased potential of pandemics. Here, three projects are presented describing the development and characterization of viral subunit loaded vaccine nanoparticles for the generation of protective humoral immune responses against hepatitis C virus, Ebola virus, and human immunodeficiency virus. In the first project, lipid-based nanoparticles, called interbilayer-crosslinked multilamellar vesicles (ICMVs), were produced with hepatitis C virus (HCV) recombinant antigens E2.661 or E2c.661, displayed average antigen loading efficiencies were 54% and 50%, respectively, and average nanoparticle dimeters between 115-132 nm. The preservation of surface displayed antigens was confirmed by indirect immunofluorescence staining with antigen-specific antibodies, and in vivo vaccination of mice with ICMV formulations generated ~10-fold higher antigen-specific serum IgG titers compared with control vaccine formulations. Immune sera were tested for their neutralization capacities by an in vitro assay, and both ICMV formulations exhibited neutralization of autologous and heterologous HCV virus like particles, with E2c.661 ICMVs displaying a balanced neutralization profile compared to E2.661 ICMVs, indicating E2c.661 as a candidate antigen for a broadly effective vaccine formulation. In a second application, recombinant Ebola envelope glycoprotein (rGP) was formulated with ICMVs or a variant (NTA ICMVs) for concerted display of rGP on ICMV surfaces. Loading efficiencies varied between formulations (15 and 33%), with the addition of NTA approximately doubling rGP loading. The large rGP complex and epitope conformations were preserved throughout nanoparticle synthesis, and both formulations displayed distinct antibody binding profiles. Regardless of the surface antigen display, both nanoparticle formulations generated marked titers of class switched antigen-specific antibodies in mice after vaccination compared to the vehicle or rGP control groups. Four weeks after immunization, mice were challenged with a lethal dose of murine adapted Ebola virus and 100% survival was observed for mice vaccinated with either ICMV formulation as well as the adjuvanted control formulation. While these data demonstrated short-term protection in three of the tested groups, further research is needed to evaluate long-term protection and the epitope specificity of the generated antibodies. Lastly, a new ICMV nanoparticle design was developed for formulation with the recombinant human immunodeficiency virus envelope glycoprotein (SOSIP). The new nanoparticle, called ICMV-NHS, display ~25% loading efficiency of SOSIP, and a mean diameter of ~300 nm. Preliminary studies indicate preservation of the SOSIP protein complex and conformational epitopes, which are necessary to produce protective and broadly neutralizing humoral responses. However, further optimization and characterization of the nanoparticle are needed to enhance antigen loading and evaluate antigen display prior to in vivo immunogenicity studies. The data reported here highlights the complexity of formulating subunit-loaded vaccine nanoparticles. Many factors including antigen design, display, and antigen-nanoparticle interfaces are important considerations and can contribute significantly to strength and specificity of the generated immune response. To bridge this gap of knowledge, in-depth characterization of nanoparticles, like those reported here, can aid in elucidating and correlating in vitro properties of vaccine nanoparticles with in vivo performance.