mRNA vaccines encoding membrane-anchored receptor-binding domains of SARS-CoV-2 mutants induce strong humoral responses and can overcome immune imprinting (preprint)

To address the limitations of whole-spike COVID vaccines, we explored mRNA vaccines encoding membrane-anchored receptor-binding domain (RBD-TMs), each a fusion of a variant RBD, the transmembrane (TM) and cytoplasmic tail (CT) fragments of the SARS-CoV-2 spike protein. In naive mice, RBD-TM mRNA vaccines against ancestral SARS-CoV-2, Beta, Delta, Delta-plus, Kappa, Omicron BA.1 or BA.5, all induced strong humoral responses against the target RBD. Multiplex surrogate viral neutralization (sVNT) assays indicated broad neutralizing activity against a range of variant RBDs. In the setting of a heterologous boost, against the background of exposure to ancestral whole spike vaccines, sVNT studies suggested that RBD-TM vaccines were able to overcome the detrimental effects of immune imprinting. Omicron BA.1 and BA.5 RBD-TM booster vaccines induced serum antibodies with 12 and 22-fold higher neutralizing activity against the target RBD than their equivalent whole spike variants. Boosting with BA.1 or BA.5 RBD-TM provided good protection against more recent variants including XBB and XBB.1.5. Each RBD-TM mRNA is 28% of the length of its whole-spike equivalent. This advantage will enable tetravalent mRNA vaccines to be developed at well-tolerated doses of formulated mRNA.

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

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 alpha-Galactosylceramide, or MF59 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 immunoevasive beta variant (N501Y, E484K, K417N). This beta variant RBD vaccine, combined with MF59 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 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 (preprint)

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 or immunisation, 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. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (R 2 =0.75, p<0.0001), and that fingerprick whole blood samples are sufficient for this test. Accordingly, the COVID-19 NAb-test™ device 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 (preprint)

ABSTRACT 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.

Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection (preprint)

Summary SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/ .