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.

Robust SARS-CoV-2 antibody and T cell immunity following three COVID-19 vaccine doses in inflammatory bowel disease patients receiving anti-TNF or alternative treatments (preprint)

BACKGROUND AND AIMS: Vaccine-mediated immune responses in patients with inflammatory bowel disease (IBD) may be influenced by IBD therapies. We investigated in-depth humoral and T-cell responses to SARS-CoV-2 vaccination in IBD patients following three COVID-19 vaccine doses. METHODS: Immune responses of 100 SARS-CoV-2-uninfected IBD patients on varying treatments were compared to healthy controls (n=35). Anti-S1/2 and anti-RBD SARS-CoV-2-specific antibodies, CD4+ and CD8+ T-cell responses were measured at baseline and at five time-points after COVID-19 vaccination. RESULTS: Anti-S1/2 and anti-RBD antibody concentrations at ~1 month after second dose vaccination were significantly lower in anti-TNF-treated patients compared to non-TNF IBD patients and healthy controls (126.4 vs 262.1 and 295.5, p<0.0001). Anti-S1/2 antibodies remained reduced in anti-TNF treated patients before and after the third dose (285.7 vs 365.3, p=0.03), although anti-RBD antibodies reached comparable titres to non-TNF patients. Anti-RBD antibodies were higher in the vedolizumab group than controls after second dose (4.2 vs 3.6, p=0.003). Anti-TNF monotherapy was associated with increased CD4+ and CD8+ T-cell activation compared to combination anti-TNF patients after second dose, but comparable after third dose. Overall, IBD patients demonstrated similar CD4+/CD8+ T-cell responses compared to healthy controls regardless of treatment regimen. CONCLUSIONS: Anti-TNFs impaired antibody concentrations when compared to non-TNF patients and controls after two vaccine doses. These differences were not observed after the third vaccine dose. However, vaccine induced SARS-CoV-2-specific T cell responses are robust in anti-TNF-treated patients. Our study supports the need for timely booster vaccination particularly in anti-TNF treated patients to minimise the risk of severe SARS-CoV-2 infection.

Dynamics of immune recall following SARS-CoV-2 vaccination or breakthrough infection (preprint)

Vaccination against SARS-CoV-2 results in protection from acquisition of infection as well as improved clinical outcomes even if infection occurs, likely reflecting a combination of residual vaccine-elicited immunity and the recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and T cell immunity after vaccination of seropositive individuals, and after breakthrough infection in vaccinated individuals. Intensive and early longitudinal sampling reveals the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titres. In breakthrough infections, the delayed kinetics of humoral immune recall provides a mechanism for the lack of early control of viral replication but likely underpins accelerated viral clearance and the protective effects of vaccination against severe COVID-19.

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.

Evaluation of six commercial SARS-CoV-2 Enzyme-Linked Immunosorbent assays for clinical testing and serosurveillance. (preprint)

Background: Serological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. As we move into the era of vaccines, the detection of neutralising antibody will become increasingly important. Many serological tests have been developed under emergency use authorization, but their reliability remains unclear. Methods: We evaluated the performance of six commercially-available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test, for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or neutralising antibodies and a subset of results were compared to microneutralisation. Results: For sera collected > 14 days post-symptom onset the Wantai total Ab performed best with highest sensitivity 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript Surrogate Virus Neutralization Test, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA and 83.3% for Wantai IgM. Specificity for the best performing assay was 99.5% and for the lowest 97.1%. Conclusion: Wantai ELISA, detecting total immunoglobulins against SARS-CoV-2 receptor binding domain, had the best performance. Antibody target, timing and longevity of the immune response, and the objectives of testing should be considered in test choice. ELISAs should be used within a confirmatory testing algorithm to ensure reliable results. ELISAs provide high quality results, with flexibility for test numbers without the need for manufacturer specific analyzers. Key words: SARS-CoV-2, COVID-19, serology, ELISA, antibody, neutralising.

The Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework: An Implementation Science Approach to Evaluating Pathogen Whole Genome Sequencing in Public Health (preprint)

Background Pathogen whole genome sequencing (WGS) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding: (i) the optimal models for integration of genomic data into epidemiological investigations, and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. Methods We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis and SARS-CoV-2. Results The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. Conclusions Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts. 

Implementation and evaluation of a novel real-time multiplex assay for SARS-CoV-2: In-field learnings from a clinical microbiology laboratory (preprint)

The unprecedented scale of testing required to effectively control the coronavirus disease (COVID-19) pandemic has necessitated urgent implementation of rapid testing in clinical microbiology laboratories. To date, there are limited data available on the analytical performance of emerging commercially available assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and integration of these assays into laboratory workflows. Here, we performed a prospective validation study of a commercially available assay, the AusDiagnostics Coronavirus Typing (8-well) assay. Respiratory tract samples for SARS-CoV-2 testing were collected between 1st March and 25th March 2020. All positive samples and a random subset of negative samples were sent to a reference laboratory for confirmation. In total, 2,673 samples were analyzed using the Coronavirus Typing assay. The predominant sample type was a combined nasopharyngeal/throat swab (2,640/2,673; 98.8%). Fifty-four patients were positive for SARS-CoV-2 (0.02%) using the Coronavirus Typing assay; 53/54 (98.1%) positive results and 621/621 (100%) negative results were concordant with the reference laboratory. Compared to the reference standard, sensitivity of the Coronavirus Typing assay for SARS-CoV-2 was 100% [95% CI 93.2%-100%], specificity 99.8% [95% CI 99.1%-100%], positive predictive value 98.1% (95% CI 90.2%-99.7%] and negative predictive value 100% [95% CI 99.4%-100%]. In many countries, standard regulatory requirements for the introduction of new assays have been replaced by emergency authorizations and it is critical that laboratories share their post-market validation experiences, as the consequences of widespread introduction of a sub-optimal assay for SARS-CoV-2 are profound. Here, we share our in-field experience, and encourage other laboratories to follow suit.