Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies Against Delta and Omicron in Mice

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We have previously developed highly thermo-tolerant monomeric and trimeric receptor binding domain derivatives that can withstand 100{degrees}C for 90 minutes and 37{degrees}C for four weeks, and help eliminate cold chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations, and 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for the upcoming Phase I human clinical trials, and that there is potential for increasing efficacy with vaccine matching to improve responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions which show that while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible, and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

At least three doses of leading vaccines essential for neutralization of SARS-CoV-2 Omicron variant

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.

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

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 3UTR 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/.

Tracking the COVID-19 pandemic in Australia using genomics

BACKGROUNDWhole-genome sequencing of pathogens can improve resolution of outbreak clusters and define possible transmission networks. We applied high-throughput genome sequencing of SARS-CoV-2 to 75% of cases in the State of Victoria (population 6.24 million) in Australia. METHODSCases of SARS-CoV-2 infection were detected through active case finding and contact tracing. A dedicated SARS-CoV-2 multidisciplinary genomic response team was formed to enable rapid integration of epidemiological and genomic data. Phylodynamic analysis was performed to assess the putative impact of social restrictions. RESULTSBetween 25 January and 14 April 2020, 1,333 COVID-19 cases were reported in Victoria, with a peak in late March. After applying internal quality control parameters, 903 samples were included in genomic analyses. Sequenced samples from Australia were representative of the global diversity of SARS-CoV-2, consistent with epidemiological findings of multiple importations and limited onward transmission. In total, 76 distinct genomic clusters were identified; these included large clusters associated with social venues, healthcare facilities and cruise ships. Sequencing of sequential samples from 98 patients revealed minimal intra-patient SARS-CoV-2 genomic diversity. Phylodynamic modelling indicated a significant reduction in the effective viral reproductive number (Re) from 1.63 to 0.48 after the implementation of travel restrictions and population-level physical distancing. CONCLUSIONSOur data provide a comprehensive framework for the use of SARS-CoV-2 genomics in public health responses. The application of genomics to rapidly identify SARS-CoV-2 transmission chains will become critically important as social restrictions ease globally. Public health responses to emergent cases must be swift, highly focused and effective.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Direct RNA sequencing and early evolution of SARS-CoV-2

Fundamental aspects of SARS-CoV-2 biology remain to be described, having the potential to provide insight to the response effort for this high-priority pathogen. Here we describe the first native RNA sequence of SARS-CoV-2, detailing the coronaviral transcriptome and epitranscriptome, and share these data publicly. A data-driven inference of viral genetic features and evolutionary rate is also made. The rapid sharing of sequence information throughout the SARS-CoV-2 pandemic represents an inflection point for public health and genomic epidemiology, providing early insights into the biology and evolution of this emerging pathogen.

Breadth of concomitant immune responses underpinning viral clearance and patient recovery in a non-severe case of COVID-19

We report the kinetics of the immune response in relation to clinical and virological features of a patient with mild-to-moderate coronavirus disease-19 (COVID-19) requiring hospitalisation. Increased antibody-secreting cells, follicular T-helper cells, activated CD4+ and CD8+ T-cells and IgM/IgG SARS-CoV-2-binding antibodies were detected in blood, prior to symptomatic recovery. These immunological changes persisted for at least 7 days following full resolution of symptoms, indicating substantial anti-viral immunity in this non-severe COVID-19.