Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level.

BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.

Respiratory infections and type 1 diabetes: Potential roles in pathogenesis.

Among the environmental factors associated with type 1 diabetes (T1D), viral infections of the gut and pancreas has been investigated most intensely, identifying enterovirus infections as the prime candidate trigger of islet autoimmunity (IA) and T1D development. However, the association between respiratory tract infections (RTI) and IA/T1D is comparatively less known. While there are significant amounts of epidemiological evidence supporting the role of respiratory infections in T1D, there remains a paucity of data characterising infectious agents at the molecular level. This gap in the literature precludes the identification of the specific infectious agents driving the association between RTI and T1D. Furthermore, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on the development of IA/T1D remains undeciphered. Here, we provide a comprehensive overview of the evidence to date, implicating RTIs (viral and non-viral) as potential risk factors for IA/T1D.

Persistence of a Frameshifting Deletion in SARS-CoV-2 ORF7a for the Duration of a Major Outbreak.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. A 17-nucleotide frameshift-inducing deletion in ORF7a rapidly became represented at the consensus level (Delta-ORF7aΔ17del) in most Australian outbreak cases. Studies from early in the COVID-19 pandemic suggest that frameshift-inducing deletions in ORF7a do not persist for long in the population; therefore, Delta-ORF7aΔ17del genomes should have disappeared early in the Australian outbreak. In this study, we conducted a retrospective analysis of global Delta genomes to characterise the dynamics of Delta-ORF7aΔ17del over time, determined the frequency of all ORF7a deletions worldwide, and compared global trends with those of the Australian Delta outbreak. We downloaded all GISAID clade GK Delta genomes and scanned them for deletions in ORF7a. For each deletion we identified, we characterised its frequency, the number of countries it was found in, and how long it persisted. Of the 4,018,216 Delta genomes identified globally, 134,751 (~3.35%) possessed an ORF7a deletion, and ORF7aΔ17del was the most common. ORF7aΔ17del was the sole deletion in 28,014 genomes, of which 27,912 (~99.6%) originated from the Australian outbreak. During the outbreak, ~87% of genomes were Delta-ORF7aΔ17del, and genomes with this deletion were sampled until the outbreak's end. These data demonstrate that, contrary to suggestions early in the COVID-19 pandemic, genomes with frameshifting deletions in ORF7a can persist over long time periods. We suggest that the proliferation of Delta-ORF7aΔ17del genomes was likely a chance founder effect. Nonetheless, the frequency of ORF7a deletions in SARS-CoV-2 genomes worldwide suggests they might have some benefit for virus transmission.

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern.

BACKGROUND: Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway. METHODS: Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat. FINDINGS: Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat. INTERPRETATION: Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).

Simultaneous monitoring of eight human respiratory viruses including SARS-CoV-2 using liquid chromatography-tandem mass spectrometry.

Diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has primarily been achieved using reverse transcriptase polymerase chain reaction (RT-PCR) for acute infection, and serology for prior infection. Assay with RT-PCR provides data on presence or absence of viral RNA, with no information on virus replication competence, infectivity, or virus characterisation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is typically not used in clinical virology, despite its potential to provide supplemental data about the presence of viral proteins and thus the potential for replication-competent, transmissible virus. Using the SARS-CoV-2 as a model virus, we developed a fast 'bottom-up' proteomics workflow for discovery of target virus peptides using 'serum-free' culture conditions, providing high coverage of viral proteins without the need for protein or peptide fractionation techniques. This workflow was then applied to Coronaviruses OC43 and 229E, Influenza A/H1N1 and H3N2, Influenza B, and Respiratory Syncytial Viruses A and B. Finally, we created an LC-MS/MS method for targeted detection of the eight-virus panel in clinical specimens, successfully detecting peptides from the SARS-CoV-2 ORF9B and nucleoprotein in RT-PCR positive samples. The method provides specific detection of respiratory viruses from clinical samples containing moderate viral loads and is an important further step to the use of LC-MS/MS in diagnosis of viral infection.

Detection of respiratory viruses directly from clinical samples using next-generation sequencing: A literature review of recent advances and potential for routine clinical use.

Acute respiratory infection is the third most frequent cause of mortality worldwide, causing over 4.25 million deaths annually. Although most diagnosed acute respiratory infections are thought to be of viral origin, the aetiology often remains unclear. The advent of next-generation sequencing (NGS) has revolutionised the field of virus discovery and identification, particularly in the detection of unknown respiratory viruses. We systematically reviewed the application of NGS technologies for detecting respiratory viruses from clinical samples and outline potential barriers to the routine clinical introduction of NGS. The five databases searched for studies published in English from 01 January 2010 to 01 February 2021, which led to the inclusion of 52 studies. A total of 14 different models of NGS platforms were summarised from included studies. Among these models, second-generation sequencing platforms (e.g., Illumina sequencers) were used in the majority of studies (41/52, 79%). Moreover, NGS platforms have proven successful in detecting a variety of respiratory viruses, including influenza A/B viruses (9/52, 17%), SARS-CoV-2 (21/52, 40%), parainfluenza virus (3/52, 6%), respiratory syncytial virus (1/52, 2%), human metapneumovirus (2/52, 4%), or a viral panel including other respiratory viruses (16/52, 31%). The review of NGS technologies used in previous studies indicates the advantages of NGS technologies in novel virus detection, virus typing, mutation identification, and infection cluster assessment. Although there remain some technical and ethical challenges associated with NGS use in clinical laboratories, NGS is a promising future tool to improve understanding of respiratory viruses and provide a more accurate diagnosis with simultaneous virus characterisation.

Proficiency testing for SARS-CoV-2 whole genome sequencing.

Extensive studies and analyses into the molecular features of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) have enhanced the surveillance and investigation of its clusters and transmission worldwide. The whole genome sequencing (WGS) approach is crucial in identifying the source of infection and transmission routes by monitoring the emergence of variants over time and through communities. Varying SARS-CoV-2 genomics capacity and capability levels have been established in public health laboratories across different Australian states and territories. Therefore, laboratories performing SARS-CoV-2 WGS for public health purposes are recommended to participate in an external proficiency testing program (PTP). This study describes the development of a SARS-CoV-2 WGS PTP. The PTP assessed the performance of laboratories while providing valuable insight into the current state of SARS-CoV-2 genomics in public health across Australia. Part 1 of the PTP contained eight simulated SARS-CoV-2 positive and negative specimens to assess laboratories' wet and dry laboratory capacity. Part 2 involved the analysis of a genomic dataset that consisted of a multi-FASTA file of 70 consensus genomes of SARS-CoV-2. Participating laboratories were required to (1) submit raw data for independent bioinformatics analysis, (2) analyse the data with their processes, and (3) answer relevant questions about the data. The performance of the laboratories was commendable, despite some variation in the reported results due to the different sequencing and bioinformatics approaches used by laboratories. The overall outcome is positive and demonstrates the critical role of the PTP in supporting the implementation and validation of SARS-CoV-2 WGS processes. The data derived from this PTP will contribute to the development of SARS-CoV-2 bioinformatic quality control (QC) and performance benchmarking for accreditation.

Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia.

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

Proficiency testing for SARS-CoV-2 in assuring the quality and overall performance in viral RNA detection in clinical and public health laboratories.

Diagnostic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone significant changes over the duration of the pandemic. In early 2020, SARS-CoV-2 specific nucleic acid testing (NAT) protocols were predominantly in-house assays developed based on protocols published in peer reviewed journals. As the pandemic has progressed, there has been an increase in the choice of testing platforms. A proficiency testing program for the detection of SARS-CoV-2 by NAT was provided to assist laboratories in assessing and improving test capabilities in the early stages of the pandemic. This was vital in quality assuring initial in-house assays, later commercially produced assays, and informing the public health response. The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) offered three rounds of proficiency testing for SARS-CoV-2 to Australian and New Zealand public and private laboratories in March, May, and November 2020. Each round included a panel of five specimens, consisting of positive (low, medium or high viral loads), inconclusive (technical specimen of selected SARS-CoV-2 specific genes) and negative specimens. Results were received for round 1 from 16, round 2 from 97 and round 3 from 101 participating laboratories. Improvement in the accuracy over time was shown, with the concordance of results in round 1 being 75.0%, in round 2 above 95.0% for all samples except one, and for round 3 above 95.0%. Overall, participants demonstrated high capabilities in detecting SARS-CoV-2, even in samples of low viral load, indicating excellent testing accuracy and therefore providing confidence in Australian and New Zealand public and private laboratories test results.

Assessment of Inter-Laboratory Differences in SARS-CoV-2 Consensus Genome Assemblies between Public Health Laboratories in Australia.

Whole-genome sequencing of viral isolates is critical for informing transmission patterns and for the ongoing evolution of pathogens, especially during a pandemic. However, when genomes have low variability in the early stages of a pandemic, the impact of technical and/or sequencing errors increases. We quantitatively assessed inter-laboratory differences in consensus genome assemblies of 72 matched SARS-CoV-2-positive specimens sequenced at different laboratories in Sydney, Australia. Raw sequence data were assembled using two different bioinformatics pipelines in parallel, and resulting consensus genomes were compared to detect laboratory-specific differences. Matched genome sequences were predominantly concordant, with a median pairwise identity of 99.997%. Identified differences were predominantly driven by ambiguous site content. Ignoring these produced differences in only 2.3% (5/216) of pairwise comparisons, each differing by a single nucleotide. Matched samples were assigned the same Pango lineage in 98.2% (212/216) of pairwise comparisons, and were mostly assigned to the same phylogenetic clade. However, epidemiological inference based only on single nucleotide variant distances may lead to significant differences in the number of defined clusters if variant allele frequency thresholds for consensus genome generation differ between laboratories. These results underscore the need for a unified, best-practices approach to bioinformatics between laboratories working on a common outbreak problem.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Immunizations with diverse sarbecovirus receptor binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.