Durable reprogramming of neutralising antibody responses following breakthrough Omicron infection (preprint)

SARS-CoV-2 breakthrough infection of vaccinated individuals is increasingly common with the circulation of highly immune evasive and transmissible Omicron variants. Here, we report the dynamics and durability of recalled spike-specific humoral immunity following BA.1 or BA.2 breakthrough infection, with longitudinal sampling up to 8 months post-infection. Both BA.1 and BA.2 infection robustly boosted neutralisation activity against the infecting strain while expanding breadth against other Omicron strains. Cross-reactive memory B cells against both ancestral and Omicron spike were predominantly expanded by infection, with limited recruitment of de novo Omicron-specific B cells or antibodies. Modelling of neutralisation titres predicts that protection from symptomatic reinfection against antigenically similar strains will be remarkably durable, but is undermined by novel emerging strains with further neutralisation escape.

Rapid recall and de novo T cell responses during SARS-CoV-2 breakthrough infection (preprint)

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable levels of expansion. Strikingly, high levels of SARS-CoV-2-specific CD8+ T cell activation at baseline and peak were strongly correlated with reduced peak SARS-CoV-2 RNA levels in nasal swabs and accelerated clearance of virus. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Assessment of twenty-two SARS-CoV-2 rapid antigen tests against SARS-CoV-2: A laboratory evaluation study (preprint)

Background Rapid antigen testing is widely used as a way of scaling up population-level testing. To better inform antigen test deployment in Australia, we evaluated 22 commercially available antigen tests, including an assessment of culture infectivity. Methods Analytical sensitivity was evaluated against SARS-CoV-2 B.1.617.2 (Delta), reported as TCID50/mL, cycle threshold (Ct) value and viral load (RNA copies/mL). Specificity was assessed against non-SARS-CoV-2 viruses. Clinical sensitivity and correlation with cell culture infectivity was assessed using the Abbott PanBio COVID-19 Ag test. Results Nineteen kits consistently detected SARS-CoV-2 antigen equivalent to 1.3x10^6 copies/mL (5.8x10^3 TCID50/mL). Specificity for all kits was 100%. Compared to RT-PCR the Abbott PanBio COVID-19 Ag test was 52.6% (95% CI, 41.6% to 63.3%) sensitive, with a 50% detection probability for infectious cell culture at 5.9 log10 RNA copies/mL (95% CI, 5.3 to 6.5 log10 copies/mL). Antigen test sensitivity was 97.6% (95% CI, 86.3% to 100.0%) compared to positive infectious in cell culture. Conclusions Antigen test positivity correlated with positive viral culture, suggesting antigen test results may determine SARS-CoV-2 transmission risk. Sensitivity varied considerably between test kits and highlights the need for ongoing systematic post-market evaluation, providing valuable information to help guide antigen test selection and deployment.

Multi-Site Assessment of Rapid, Point-of-Care Antigen Testing for the Diagnosis of SARS-CoV-2 Infection in a Low-Prevalence Setting: A Validation and Implementation Study (preprint)

Background: In Australia, COVID-19 diagnosis relies on RT-PCR testing which is relatively costly and time-consuming. To date, no studies have assessed the performance and implementation of rapid antigen-based SARS-CoV-2 testing in a setting with a low prevalence of COVID-19 infections, such as Australia. Methods: This study recruited participants presenting for COVID-19 testing at three Melbourne metropolitan hospitals during a period of low COVID-19 prevalence. The Abbott PanBioTM COVID-19 Ag point-of-care test was performed alongside RT-PCR. In addition, participants with COVID-19 notified to the Victorian Government were invited to provide additional swabs to aid validation. Implementation challenges were also documented. Findings: The specificity of the Abbott PanBioTM COVID-19 Ag test was 99.96% (95% CI 99.73 - 100%). Sensitivity amongst participants with RT-PCR-confirmed infection was dependent upon the duration of symptoms reported, ranging from 78.9% (duration 1 to 33 days) to 100% in those within 7 days of symptom onset. A range of implementation challenges were identified which may inform future COVID-19 testing strategies in a low prevalence setting. Interpretation: Given the high specificity, antigen-based tests may be most useful in rapidly triaging public health and hospital resources while expediting confirmatory RT-PCR testing. Considering the limitations in test sensitivity and the potential for rapid transmission in susceptible populations, particularly in hospital settings, careful consideration is required for implementation of antigen testing in a low prevalence setting. Funding: This work was funded by the Victorian Department of Health and Human Services. The funder was not involved in data analysis or manuscript preparation.Declaration of Interests: All authors: no conflicts.Ethics Approval Statement: Ethics review and study approval was provided by Monash Health Human Research and Ethics Committee (RES-20-0000-678A) and local Governance approval was provided by Melbourne Health and Austin Health Offices for Research.

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/ .

Direct RNA sequencing and early evolution of SARS-CoV-2 (preprint)

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.