Using Dual Toll-like Receptor Agonism to Drive Th1-Biased Response in a Squalene- and α-Tocopherol-Containing Emulsion for a More Effective SARS-CoV-2 Vaccine

Abstract

A diversity of vaccines is necessary to reduce the mortality and morbidity of SARS-CoV-2. Vaccines must be efficacious, easy to manufacture, and stable within the existing cold chain to improve their availability around the world. Recombinant protein subunit vaccines adjuvanted with squalene-based emulsions such as AS03™ and MF59™ have a long and robust history of safe, efficacious use with straightforward production and distribution. Here, subunit vaccines were made with squalene-based emulsions containing novel, synthetic toll-like receptor (TLR) agonists, INI-2002 (TLR4 agonist) and INI-4001 (TLR7/8 agonist), using the recombinant receptor-binding domain (RBD) of SARS-CoV-2 S protein as an antigen. The addition of the TLR4 and TLR7/8 agonists, alone or in combination, maintained the formulation characteristics of squalene-based emulsions, including a sterile filterable droplet size (<220 nm), high homogeneity, and colloidal stability after months of storage at 4, 25, and 40 °C. Furthermore, the addition of the TLR agonists skewed the immune response from Th2 towards Th1 in immunized C57BL/6 mice, resulting in an increased production of IgG2c antibodies and a lower antigen-specific production of IL-5 with a higher production of IFNγ by lymphocytes. As such, incorporating TLR4 and TLR7/8 agonists into emulsions leveraged the desirable formulation and stability characteristics of emulsions and can induce Th1-type humoral and cell-mediated immune responses to combat the continued threat of SARS-CoV-2.

Co-Delivery of a Novel Lipidated TLR7/8 Agonist and Hemagglutinin-Based Influenza Antigen Using Silica Nanoparticles Promotes Enhanced Immune Responses

“…In both immunization studies, with 14-day and 28-day regimens, mice were immunized with 1 µg of influenza A H7 trimer adsorbed onto A-SNP (50 and 200 nm) with a low or high density of a dose of 10 nmol of INI-4001. The INI-4001 dose was selected based on previous dose escalation studies of its emulsion formulation [ 18 ].…”

“…INI-4001 was synthesized and processed to over 99% purity as described previously [ 18 , 29 ]. Hemagglutinin antigen trimer (H7) was obtained from Dr. Florian Krammer at the Icahn School of Medicine at Mount Sinai [ 30 , 31 ].…”

“…INI-4001 acts through both human TLR7 and TLR8 receptors and elicits IFN-α as well as pro-inflammatory cytokines from human PBMCs. Further, INI-4001 results in a strong humoral and Th1-biased cellular immune response when used as a vaccine adjuvant in mice [ 18 ].…”

Regulation of Ethanol Assimilation for Efficient Accumulation of Squalene in Saccharomyces cerevisiae

“…Squalene is a linear triterpenoid widely distributed in plants and animals, including humans. It has significant anticancer and antitumor benefits, assists the transport of nucleoside antitumor drugs, the treatment of skin diseases, , and the preparation of vaccines. − Squalene was initially isolated from shark liver oil and more recently from plant sources . However, increasing demand may lead to overfishing and other environmental problems.…”

Co-Delivery of Novel Synthetic TLR4 and TLR7/8 Ligands Adsorbed to Aluminum Salts Promotes Th1-Mediated Immunity against Poorly Immunogenic SARS-CoV-2 RBD

“…Our lab has previously demonstrated that co-delivery of TLR7/8 and TLR4 ligands within a liposome increased IgG2a antibody titers and skewed the immune response to an influenza antigen towards Th1 [ 28 ]. Additionally, we showed that TLR agonists, INI-4001 and INI-2002, could similarly enhance and skew the immune response from Th2 to Th1-dominated when added to an emulsion-based adjuvant in the context of vaccination against SARS-CoV-2 spike RBD [ 29 ]. In line with these results, we developed subunit SARS-CoV-2 vaccines using RBD as the antigen, co-delivered with an aluminum salt alone or in combination with a TLR7/8 agonist (INI-4001) and/or a TLR4 agonist (INI-2002).…”