Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants (preprint)

The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show using clinical ORF8 deficient viral isolates, virus like particles (VLPs) and viral replicons that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle reducing function to its N terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data support the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production.

Evaluation of the Safety and Immunogenicity of Different COVID-19 Vaccine Combinations in Healthy Individuals: Study Protocol for a Randomized, Subject-blinded, Controlled Phase 3 Trial [PRIBIVAC] (preprint)

Trials guidance: The Abstract should not exceed 350 words. Please minimize the use of abbreviations and do not cite references in the abstract. The abstract must include the following separate sections:• Background: Over 2021, COVID-19 vaccination programs worldwide focused on raising population immunity through the primary COVID-19 vaccine series. In Singapore two mRNA vaccines (BNT162b2 and mRNA-1273) and the inactivated vaccine CoronaVac are currently authorized under the National Vaccination Programme for use as the primary vaccination series. More than 90% of the Singapore population has received at least one dose of a COVID-19 vaccine as of December 2021. With the demonstration that vaccine effectiveness wanes in the months after vaccination, and the emergence of Omicron which evades host immunity from prior infection and/or vaccination, attention in many countries has shifted to how best to maintain immunity through booster vaccinations. • Methods: The objectives of this phase 3, randomized, subject-blinded, controlled clinical trial are to assess the safety and immunogenicity of heterologous boost COVID-19 vaccine regimens (intervention groups 1-4) compared with a homologous boost regimen (control arm) in up to 600 adult volunteers. As non-mRNA vaccine candidates may enter the study at different time points depending on vaccine availability and local regulatory approval, participants will be randomized at equal probability to the available intervention arms at the time of randomization. Eligible participants will have received two doses of a homologous mRNA vaccine series with BNT162b2 or mRNA-1273 at least six months prior to enrolment. Participants will be excluded if they have a history of confirmed SARS or SARS-CoV-2 infection, are immunocompromised or pregnant. Participants will be monitored for adverse events and serious adverse events by physical examinations, laboratory tests and self-reporting. Blood samples will be collected at serial time points [Pre-vaccination/screening (Day -14 to Day 0), Day 7, Day 28, Day 180, Day 360 post-vaccination] for assessment of antibody and cellular immune parameters. Primary endpoint is the level of anti-SARS-CoV-2 spike immunoglobulins at Day 28 post-booster, and will be measured against wildtype SARS-CoV-2 and variants of concern. Comprehensive immune profiling of the humoral and cellular immune response to vaccination will be performed. • Discussion: This study will provide necessary data to understand the quantity, quality and persistence of the immune response to homologous and heterologous third booster dose of COVID-19 vaccines. This an important step in developing COVID-19 vaccination programs beyond the primary series. Trial registration: ClinicalTrials.gov NCT05142319, registered on 2 Dec 2021.

Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early prognostic marker for severe COVID-19 (preprint)

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients revealed a dramatic increase in the number of immature neutrophils. This increase strongly correlated with disease severity and was associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts was observed for CD8 T-cells and VD2 {gamma}{delta} T-cells, which both exhibited increased differentiation and activation. ROC analysis revealed that the count ratio of immature neutrophils to CD8 or VD2 T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.

Potent neutralizing antibodies in the sera of convalescent COVID-19 patients are directed against conserved linear epitopes on the SARS-CoV-2 spike protein (preprint)

The ongoing SARS-CoV-2 pandemic demands rapid identification of immunogenic targets for the design of efficient vaccines and serological detection tools. In this report, using pools of overlapping linear peptides and functional assays, we present two immunodominant regions on the spike glycoprotein that were highly recognized by neutralizing antibodies in the sera of COVID-19 convalescent patients. One is highly specific to SARS-CoV-2, and the other is a potential pan-coronavirus target.