Formulation development and comparability studies with an aluminum-salt adjuvanted SARS-CoV-2 Spike ferritin nanoparticle vaccine antigen produced from two different cell lines

Abstract

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ~100%, ~40% or ~10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates

“…To do so, multiple copies of a codon-optimized DCFHP gene were inserted into CHO-K1 cells using a Leap-In transposase at ATUM 47,48 , a bioengineering company (see Methods). As anticipated 49 , N-glycosylation profiles of these proteins expressed transiently in Expi293 versus stably in CHO cells differ 32 but they are similarly immunogenic in mice, as measured in pseudoviral neutralization assays 32 . Following preliminary analysis of expression in pooled cells, 125 single-cell clones were evaluated for high levels of cell growth and productivity, resulting in 24 lead clones (SI Fig.…”

“…Our vaccine formulation, DCFHP-alum, consists of DCFHP antigen formulated with aluminum hydroxide (Alhydrogel™, referred to herein as alum) as the only adjuvant 32 . Aluminum salt adjuvants are the most commonly used adjuvant in human vaccines licensed by the FDA and regulatory agencies worldwide, and have been administered to billions of individuals over the past 90 years [33][34][35] .…”

Decreased efficacy of a COVID-19 vaccine due to mutations present in early SARS-CoV-2 variants of concern

“…VOCs and SARS-CoV-1. Our results suggest that DCFHP-alum has potential to be used as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use, including in infants 1, 3 .…”

“…Nanoparticle vaccines optimally facilitate increased uptake by dendritic cells and promote B-cell receptor clustering, leading to enhanced immunogenicity compared to isolated subunit vaccines 5,6 . We previously produced our vaccine candidate DCFHP by engineering the Wuhan-1 strain of SARS-CoV-2 spike protein and displaying it on a ferritin nanoparticle scaffold 1, 3,7 . DCFHP presents the engineered spike protein at the three-fold axes of symmetry on the ferritin nanoparticle, enabling assembly of eight spike trimers from the constituent 24 monomers.…”