Antigen-adjuvant interactions, stability, and immunogenicity profiles of a SARS-CoV-2 receptor-binding domain (RBD) antigen formulated with aluminum salt and CpG adjuvants

ABSTRACT

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.

KEYWORDS:

• COVID-19
• vaccine
• RBD
• alum
• CpG
• adjuvant
• formulation
• stability
• immunogenicity

Acknowledgments

We gratefully acknowledge the Wadsworth Center’s Veterinary Sciences staff for assistance with animal care, and the Tissue Culture core facility for media preparation. The authors would also like to thank Dr. Dong Yu, Dr. Mattew J. Bottomley, and Dr. Robert L. Coffman at Dynavax Technologies for providing the CpG 1018 adjuvant and reviewing this manuscript.

Abbreviations

AH	=	Alhydrogel®
AP	=	AdjuPhos®

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The dataset generated and/or analyzed during the current study are available in the KU ScholarWorks repository, https://doi.org/10.17161/1808.32758. The data is also available with the corresponding author(s).

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2022.2079346.

Additional information

Funding

This work was supported, in whole or in part, by the Bill & Melinda Gates Foundation [Investment IDs INV-002740 and INV-027417]. Research conducted at the Wadsworth Center was also supported, in part, by the National Cancer Institute of the National Institutes of Health under award number U01CA260508. N.C.D. was supported by the Ludwig Center at MIT’s Koch Institute. RBD and trimeric Spike antigen used for the analysis of mouse sera were kindly provided by MassBiologics (Boston) and produced under a Project Award Agreement from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) and financial assistance award 70NANB20H037 from the U.S. Department of Commerce, National Institutes of Standards and Technologies. We gratefully acknowledge the Wadsworth Center’s Veterinary Sciences staff for assistance with animal care, and the Tissue Culture core facility for media preparation. The authors would also like to thank Dr. Dong Yu, Dr. Mattew J. Bottomley and Dr. Robert L. Coffman at Dynavax Technologies for providing CpG 1018 adjuvant and reviewing this manuscript.