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.

Sample Pooling is a Viable Strategy for SARS-CoV-2 Detection in Low-Prevalence Settings (preprint)

BACKGROUNDThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly increased demand on laboratory throughput and reagents for nucleic acid extraction and polymerase chain reaction (PCR). Reagent shortages may limit the expansion of testing required to scale back isolation measures. AIMTo investigate the viability of sample pooling as a strategy for increasing test throughput and conserving PCR reagents; to report our early experience with pooling of clinical samples. METHODSA pre-implementation study was performed to assess the sensitivity and theoretical efficiency of two, four, and eight-sample pools in a real-time reverse transcription PCR-based workflow. A standard operating procedure was developed and implemented in two laboratories during periods of peak demand, inclusive of over 29,000 clinical samples processed in our laboratory. RESULTSSensitivity decreased (mean absolute increase in cycle threshold value of 0.6, 2.3, and 3.0 for pools of two, four, and eight samples respectively) and efficiency increased as pool size increased. Gains from pooling diminished at high disease prevalence. Our standard operating procedure was successfully implemented across two laboratories. Increased workflow complexity imparts a higher risk of errors, and requires risk mitigation strategies. Turnaround time for individual samples increased, hence urgent samples should not be pooled. CONCLUSIONSPooling is a viable strategy for high-throughput testing of SARS-CoV-2 in low-prevalence settings.

Validation of a Single-step, Single-tube Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2 RNA (preprint)

2.IntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 L of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65{degrees}C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals. ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD {+/-}7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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.