The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2–RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID₅₀, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG₁ F_c-fusion protein, and an mRNA encoding a membrane-anchored RBD. 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 μg, N = 32), mRNA vaccine (10, 20, or 50 μg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. govNCT05272605. No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4⁺ and CD8⁺ T cell activation. There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.

Existing mRNA COVID-19 vaccines have shown efficacy in reducing severe cases and fatalities. However, their effectiveness against infection caused by emerging SARS-CoV-2 variants has waned considerably, necessitating the development of variant vaccines. Ideally, next-generation vaccines will be capable of eliciting broader and more sustained immune responses to effectively counteract new variants. Additionally, in vitro assays that more closely represent virus neutralization in humans would greatly assist in the analysis of protective vaccine-induced antibody responses. Here, we present findings from a SARS-CoV-2 VLP vaccine encompassing three key structural proteins: Spike (S), Envelope (E), and Membrane (M). The VLP vaccine effectively produced neutralizing antibodies as determined by surrogate virus neutralization test, and induced virus-specific T-cell responses: predominantly CD4⁺, although CD8⁺ T cell responses were detected. T cell responses were more prominent with vaccine delivered with AddaVax compared to vaccine alone. The adjuvanted vaccine was completely protective against live virus challenge in mice. Furthermore, we utilized air–liquid-interface (ALI)-differentiated human nasal epithelium (HNE) as an in vitro system, which authentically models human SARS-CoV-2 infection and neutralization. We show that immune sera from VLP-vaccinated mice completely neutralized SARS-CoV-2 virus infection, demonstrating the potential of ALI-HNE to assess vaccine induced Nab.