Low-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low- and middle-income countries. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum-salt (Alhydrogel®, AH; AdjuPhos®, AP) and CpG 1018 adjuvants. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37°C) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro , the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Interestingly, the loss of in vivo potency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivo performance to enable successful formulation development of stable and efficacious subunit vaccines.

The development of vaccines against biothreat toxins like ricin (RT) is considered an integral component of the United States national security efforts. RiVaxÒ is a thermostable, lyophilized RT subunit vaccine adsorbed to aluminum salt adjuvant intended for use by military personnel and first responders. Phase 1 studies indicated that RiVax is safe and immunogenic, while a three dose, intramuscular vaccination regimen in non-human primates elicited protection against lethal dose RT challenge by aerosol. Here we investigated, in a mouse model, the durability of RiVax-induced antibody responses and corresponding immunity to lethal dose RT challenge. Groups of mice were subcutaneously administered 3 or 1 μg of RiVax on days 0 and 21 and challenged with 10 ´ LD50 RT by injection at six different intervals over the course of twelve months. Serum antibody titers and epitope-specific competition assays were determined prior to each challenge. We report that the two-dose, 3 μg regimen conferred near complete protection against RT challenge on day 35 and complete protection thereafter (challenge days 65, 95, 125, 245, and 365). The two-dose, 3 μg regimen was superior to the 1 μg regimen as revealed by slight differences in survival and morbidity scores (e.g., hypoglycemia, weight loss) on challenge days 35 and 365. In separate experiments, a single 3 μg RiVax vaccination proved only marginally effective at eliciting protective immunity to RT, underscoring the necessity of a prime-boost regimen to achieve full and long-lasting protection against RT.

Ricin toxin (RT) is a notorious biothreat, as exposure to even trace amounts via injection or inhalation can induce organ failure and death within a matter of hours. In this study, we advance the preclinical testing of a candidate RT vaccine known as RiVax.

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