Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine

The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. Here, we report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid -Galactosylceramide, or MF59(R) squalene oil-in-water adjuvant. Each formulation drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. We have also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the highly immuno-evasive beta variant (N501Y, E484K, K417N). This beta variant RBD vaccine, combined with MF59(R) adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a third dose booster vaccine following priming with whole spike vaccine, anti-sera from beta-RBD-Fc immunised mice increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1 and BA.2. These results demonstrated that an RBD-Fc protein subunit/MF59(R) adjuvanted vaccine can induce high levels of broad nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain Spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial.

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2

As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus; whether through infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure SARS-CoV-2 immunity, ideally with rapid turnaround and without the need for laboratory-based testing. Current rapid point-of-care (POC) tests measure antibodies (Ab) against the SARS-CoV-2 virus, however, these tests provide no information on whether the antibodies can neutralise virus infectivity and are potentially protective, especially against newly emerging variants of the virus. Neutralising Antibodies (NAb) are emerging as a strong correlate of protection, but most current NAb assays require many hours or days, samples of venous blood, and access to laboratory facilities, which is especially problematic in resource-limited settings. We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibodies from whole blood, with a result that can be determined by eye (semi-quantitative) or on a small instrument (quantitative), and results show high correlation with microneutralisation assays. This assay also provides a measure of total anti-RBD antibody, thereby providing evidence of exposure to SARS-CoV-2, regardless of whether NAb are present in the sample. By testing samples from immunised macaques, we demonstrate that this test is equally applicable for use with animal samples, and we show that this assay is readily adaptable to test for immunity to newly emerging SARS-CoV-2 variants. Accordingly, the COVID-19 NAb-test test described here can provide a rapid readout of immunity to SARS-CoV-2 at the point of care.

Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay

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 that immune escape can occur through two mechanisms, antibodies that fail to recognize mutations, along with antibodies that have reduced inhibitory capacity due to enhanced variant RBD-ACE2 affinity. A competitive approach where antibodies simultaneously compete with ACE2 for binding to the RBD may therefore more accurately reflect the physiological dynamics of infection. 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.

Immunogenic profile of SARS-CoV-2 spike in individuals recovered from COVID-19

The rapid global spread of SARS-CoV-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. Multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. However, the immunogenic properties of the spike protein in humans are unresolved. To address this, we undertook an in-depth characterisation of humoral and cellular immunity against SARS-CoV-2 spike in humans following mild to moderate SARS-CoV-2 infection. We find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ACE2/RBD interaction. Expanded populations of spike-specific memory B cells and circulating T follicular helper cells (cTFH) were detected within convalescent donors, while responses to the receptor binding domain (RBD) constitute a minor fraction. Using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific cTFH subsets. Thus both qualitative and quantitative features of B and T cell immunity to spike constitute informative biomarkers of the protective potential of novel SARS-CoV-2 vaccines.