The unexplained association between infection and autoimmune disease is strongest for hepatitis C virus-induced cryoglobulinemic vasculitis (HCV-cryovas). To analyze its origins, we traced the evolution of pathogenic rheumatoid factor (RF) autoantibodies in four HCV-cryovas patients by deep single-cell multi-omic analysis, revealing three sources of B cell somatic mutation converged to drive the accumulation of a large disease-causing clone. A method for quantifying low-affinity binding revealed recurring antibody variable domain combinations created by V(D)J recombination that bound self-immunoglobulin G (IgG) but not viral E2 antigen. Whole-genome sequencing revealed thousands of somatic mutations, numerically comparable to chronic lymphocytic leukemia and normal memory B cells, but with 1-2 corresponding to driver mutations found recurrently in B cell leukemia and lymphoma. V(D)J hypermutation created autoantibodies with compromised solubility in complex with self-IgG. In this virus-induced autoimmune disease, infection promotes a catastrophic confluence of somatic mutagenesis in the descendants of a single B cell.

The unexplained association between infection and autoimmune disease is strongest for hepatitis C virus-induced cryoglobulinemic vasculitis (HCV-CV). We traced the evolution of the pathogenic rheumatoid factor (RhF) autoantibodies in four HCV-CV patients by deep single cell multi-omic analysis, revealing three sources of B cell somatic mutation converged to drive accumulation of a large disease causing clone. A sensitive method for quantifying low affinity binding revealed three recurring heavy/light chain combinations created by V(D)J recombination bound self IgG but not viral E2 antigen. Whole genome sequencing revealed accumulation of thousands of somatic mutations, at levels comparable to CLL and normal memory B cells, but with 1-2 corresponding to driver mutations found recurrently in B cell leukemia/lymphoma. V(D)J hypermutation created autoantibodies with compromised solubility. In this virus-induced autoimmune disease, infection promotes a perfect storm of somatic mutagenesis in the descendants of a single B cell.### Competing Interest StatementThe authors have declared no competing interest.

Background and aimsIn individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection.MethodsBroad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes.ResultsBnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of antigen imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection.ConclusionsMBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development.

Early neutralising antibodies against Hepatitis C virus (HCV) and CD8+T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus yet undefined additional anti-viral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions particularly, antibody-dependent cellular phagocytosis (ADCP) was shown to offer immune protection against several RNA viruses. However, its development, and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelop 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralisation function also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralisation function regardless of clinical outcome and genotype of infecting virus while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titre in patients with chronic disease while maintained in clearers due the quality (affinity) of their anti-E2 antibodies despite having lower antibody titres.

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.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen–specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)–specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80 , FOS , CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80 hi TNFAIP3 hi and CD11c hi CD95 hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at similar to 2 months and similar to 4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-alpha) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80(hi) TNFAIP3(hi) and CD11c(h)(i) CD95(hi) at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Identifying the molecular mechanisms that promote optimal immune responses to coronavirus disease 2019 (COVID-19) vaccination is critical for future rational vaccine design. Here, we longitudinally profile innate and adaptive immune responses in 102 adults after the first, second, and third doses of mRNA or adenovirus-vectored COVID-19 vaccines. Using a multi-omics approach, we identify key differences in the immune responses induced by ChAdOx1-S and BNT162b2 that correlate with antigen-specific antibody and T cell responses or vaccine reactogenicity. Unexpectedly, we observe that vaccination with ChAdOx1-S, but not BNT162b2, induces an adenoviral vector-specific memory response after the first dose, which correlates with the expression of proteins with roles in thrombosis with potential implications for thrombosis with thrombocytopenia syndrome (TTS), a rare but serious adverse event linked to adenovirus-vectored vaccines. The COVID-19 Vaccine Immune Responses Study thus represents a major resource that can be used to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.

In individuals highly exposed to hepatitis C virus (HCV), reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. Broad neutralising antibodies (BnAbs) and Envelope 2 (E2)-specific memory B cell (MBCs) responses were examined longitudinally in 15 subjects with varied reinfection outcomes. BnAb responses were associated with MBC recall, but not with reinfection clearance. Strong evidence of immune imprinting was found, and the B cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single cell transcriptomic analyses showed that persistent reinfections featured an inhibitory transcriptomic profile in HCV-specific B cells that failed to expand upon reinfection. MBC quality is important for protection against antigenically diverse variants which is encouraging for HCV vaccine development.

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) immunity is critical for predicting protection against reinfection. In an age-and gender-matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralizing antibody breadth against the majority of the variants of concern, including the Delta variant. Modeling these immune responses against vaccine efficacy data indicate a 45%-76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralization breadth and RBD-specific MBC levels at 12 months post-infection.

We longitudinally profiled immune responses in 102 adults who received BNT162b2 (Pfizer-BioNTech) or ChAdOx1-S (Oxford-AstraZeneca) as their primary vaccinations. Bloods were collected pre-vaccination, 1-7 days after the 1st, 2nd and 3rd doses (BNT162b2 or mRNA-1273) to assess innate and early adaptive responses, and ∼28 days after the 2nd and 3rd doses to assess immunogenicity. Using a multi-omics approach including RNAseq, cytokine multiplex assay, proteomics, lipidomics, and flow cytometry we identified key differences in the immune responses induced by the ChAdOx1-S and BNT162b2 vaccines that were correlated with subsequent antigen-specific antibody and T cell responses or vaccine reactogenicity. We observed that vaccination with ChAdOx1-S but not BNT162b2 induced a memory-like response after the first dose, which was correlated with the expression of several proteins involved in complement and coagulation. The COVID-19 Vaccine Immune Responses Study (COVIRS) thus represents a major resource to understand the immunogenicity and reactogenicity of these COVID-19 vaccines. ### Competing Interest Statement V.I., P.M. and C.M. received funding from AstraZeneca to undertake the proteomics component of this study. J.Z. is an employee of, and holds or may hold stock, in AstraZeneca. All other authors declare no competing interests ### Funding Statement This work was funded by BioPlatforms Australia, Flinders Foundation and EMBL Australia Group Leader funding awarded to DJL. The proteomics component of this study was funded by AstraZeneca. This work was also supported in part by a MRFF Coronavirus research response grant (APP2015305) and The Hospital Research Foundation Group COVID-19-SA grant. We thank SA Pathology for help with sample collection and the South Australian Genomics Centre (SAGC) for help with RNA sequencing. The SAGC is supported by the National Collaborative Research Infrastructure Strategy (NCRIS) via BioPlatforms Australia and by the SAGC partner institutes. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of CALHN Human Research Ethics Committee, Adelaide, Australia gave ethical approval for this work (Approval No. 14778). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Multi-omics datasets and R code are available via the Lynn Laboratory BitBucket (https://bitbucket.org/lynnlab/covirs). RNASeq data have been deposited in the Gene Expression Omnibus (GEO) under accession GSE199750. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036608 Multi-omics datasets and R code are available via the Lynn Laboratory BitBucket (https://bitbucket.org/lynnlab/covirs). RNASeq data have been deposited in the Gene Expression Omnibus (GEO) under accession GSE199750. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036608

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and-independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs. The Journal of Immunology, 2022, 209: 1499-1512.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. Whole-genome sequencing of virus from an early case revealed a sub-consensus level of sequencing reads supporting a 17-nucleotide frameshift-inducing deletion in ORF7a that truncated the peptide sequence. The variant rapidly became represented at the consensus level (Delta-ORF7a{Delta}17del) in most of the outbreak cases in Australia. Retrospective analysis of ORF7a deletions in all GISAID clade GK Delta genomes showed that of 4,018,216 genomes, 134,751 (~3.35%) possessed a deletion in ORF7a, with the ORF7a{Delta}17del mutation by far the most common. Approximately 99.05% of Delta-ORF7a{Delta}17del genomes on GISAID originated from the Australian Delta outbreak, and comprised 87% of genomes in the outbreak. In vitro comparison of lineages in cell culture showed a significantly greater proportion of cells were infected with Delta-ORF7a{Delta}17del than with a contemporaneous Delta variant without ORF7a{Delta}17del (Delta-ORF7aintact), and the proportion was also measurably higher than an early SARS-CoV-2 strain (A.2.2). These results showed that Delta-ORF7a{Delta}17del potentially has a slight growth advantage compared to Delta-ORF7aintact. Delta-ORF7a{Delta}17del viruses still produced ORF7a protein, but significantly less than A.2.2, in a different cellular distribution with a more diffuse expression throughout the cytoplasm of infected cells. These data suggest that the proliferation of Delta-ORF7a{Delta}17del genomes during the Australian Delta outbreak was likely not a result of an intrinsic benefit of the ORF7a{Delta}17del mutation, but rather a chance founder effect. Nonetheless, the abundance of different ORF7a deletions in genomes worldwide suggests these have some benefit to virus transmission. IMPORTANCE Deletions in the ORF7a region of SARS-CoV-2 have been noted since early in the COVID-19 pandemic, but are generally reported as transient mutations that are quickly lost in the population. Consequently, ORF7a deletions are considered disadvantageous to the virus through possible loss-of-function effects. In constrast to these earlier reports, we present the first report of a SARS-CoV-2 variant with an ORF7a deletion that dominated for the entirety of a protracted outbreak, and found no associated fitness disadvantage or advantage in cell culture. The relatively common rise and fall of ORF7a deletion variants over time likely represent chance founder events followed by proliferation until a more fit variant(s) is introduced to the population. Our global clade-level survey of ORF7a deletions will be a useful resource for future studies into this gene region.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays have formed the backbone of COVID-19 diagnosis since the start of the SARS-CoV-2 pandemic. Assay performances have remained relatively stable over the course of the pandemic to date, but any genomic variants in the primer binding or probe regions of assays have the potential to compromise performance. One notable example is the spike protein H69/70del mutation, associated with the emergence of the alpha variant (B.1.1.7).1Galloway S.E. Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020–January 12, 2021.MMWR Morb Mortal Wkly Rep. 2021; 70: 95-99Crossref PubMed Google Scholar H69/70del results in false negative S-gene results in the Applied Biosystems TaqPath COVID-19 assay (ThermoFisher Scientific, USA), but the two other gene targets of the assay are not affected. This altered assay performance became a proxy for detection of the alpha variant.1Galloway S.E. Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020–January 12, 2021.MMWR Morb Mortal Wkly Rep. 2021; 70: 95-99Crossref PubMed Google Scholar Surveillance for mutations causing diagnostic assay failures for specific gene targets is crucial, especially if only one gene target is used in a particular assay. There have been relatively few COVID-19 cases in Australia relative to global case numbers, but there is currently (1) a sustained outbreak of COVID-19 infections stemming from community transmission of the delta variant of concern (VOC) in June (Winter) 2021, and (2) rising case numbers of the omicron VOC since early December 2021. The Xpert Xpress SARS-CoV-2 assay ('GX'; Cepheid, USA) is one of several diagnostic assays currently used for detection of SARS-CoV-2 in Australia and globally. The GX assay has two targets within the envelope (E) and nucleocapsid (N) genes of SARS-CoV-2. If a sample is positive, the expected behaviour is for both gene targets to be detected with similar cycle threshold (Ct) values.1Galloway S.E. Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020–January 12, 2021.MMWR Morb Mortal Wkly Rep. 2021; 70: 95-99Crossref PubMed Google Scholar A change in the performance of the GX assay was recently observed across multiple assay cartridges from different cartridge lots, with large differences in cycle threshold (Ct) values between the E and N gene targets in a subset of delta SARS-CoV-2 samples. Samples were classified as possessing an altered GX profile (GXalt) if there was a >3 Ct value difference between the E and N gene targets of the assay. A single category change to presumptive positive was adopted when a sample was E-gene positive (Ct value >30) but was N-gene negative. The altered GX profile was first observed in August of 2021 in 9.5% (10/105) of cases, increasing to 20% (18/88) and 15% (3/20) of cases in September and October, respectively. Retrospective analysis of all positive cases from July 2021 confirmed that 0% (0/110) of cases exhibited the altered GX profile (GXalt). Although the altered GX profile was defined as any sample with a greater than three Ct value difference between E-gene and N-gene targets, the differential was often far greater (median 10 Ct, IQR 9–11 Ct). Throughout the period of testing, no false negatives were observed. Whole genome sequencing of isolates was undertaken as part of routine genomic sequencing, as previously described.2Bull R.A. Adikari T.N. Ferguson J.M. et al.Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis.Nat Commun. 2020; 11: 6272Crossref PubMed Scopus (115) Google Scholar A candidate causative SNP for the GXalt profile was identified at genomic position 29179 of the SARS-CoV-2 genome (G29179T) through comparative analyses. This SNP was not observed in any non-GXalt samples. Most commercial assays use closed-source, proprietary primer sequences. However, definitively linking SNPs to altered assay performance requires knowing the primer sequences, or precise target locations on the reference genome. Accordingly, we cloned the GX assay primers into a pcDNA3.1V5 His plasmid backbone (#K480001; ThermoFisher Scientific Australia), then sequenced purified clone products using Sanger sequencing. Thus, we confirmed that the G29179T point mutation is located 5 nt upstream from the 3′ end of the N-gene target region of the GX assay (Fig. 1). In correspondence, Cepheid agreed that 'the data appear to support this (altered assay performance)' (personal communication). At the time of carrying out this study, the G29179T SNP had not yet been linked to assay instability. However, during the editorial process, three studies from Western Australia, South Korea, and the United States of America have found that the G29179T SNP leads to N-gene target detection failure in the GX assay,3Miller S. Lee T. Merritt A. Pryce T. Levy A. Speers D. Single-point mutations in the N gene of SARS-CoV-2 adversely impact detection by a commercial dual target diagnostic assay.Microbiol Spectr. 2021; 9e0149421Crossref Scopus (12) Google Scholar, 4Hong K.H. In J.W. Lee J. et al.Prevalence of a single-nucleotide variant of SARS-CoV-2 in Korea and its impact on the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay.Ann Lab Med. 2022; 42: 96-99Crossref PubMed Google Scholar, 5Leelawong M. Mitchell S.L. Fowler R.C. et al.SARS-CoV-2 N gene mutations impact detection by clinical molecular diagnostics: reports in two cities in the United States.Diagn Microbiol Infect Dis. 2021; 101: 115468Crossref PubMed Scopus (11) Google Scholar strongly and independently supporting our findings in the present manuscript. Instability of the GX assay caused by different point mutations has also been observed previously. For example, previously described mutations have resulted in GX N-target failure, with explanation centred on the primers/probes closely resembling the Centers for Disease Control and Prevention (CDC) primer and probe set.6Shu B. Kirby M.K. Davis W.G. et al.Multiplex real-time reverse transcription PCR for influenza A virus, influenza B virus, and severe acute respiratory syndrome coronavirus 2.Emerg Infect Dis. 2021; 27: 1821-1830Crossref PubMed Scopus (16) Google Scholar, 7Ziegler K. Steininger P. Ziegler R. Steinmann J. Korn K. Ensser A. SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene.Eurosurveillance. 2020; 25: 2001650Crossref Scopus (91) Google Scholar, 8Fox-Lewis S. Fox-Lewis A. Harrower J. et al.Lack of N2-gene amplification on the Cepheid Xpert Xpress SARS-CoV-2 assay and potential novel causative mutations: A case series from Auckland, New Zealand.IDCases. 2021; 25e01233Crossref PubMed Scopus (6) Google Scholar, 9Hasan M.R. Sundararaju S. Manickam C. et al.A novel point mutation in the N gene of SARS-CoV-2 may affect the detection of the virus by reverse transcription-quantitative PCR.J Clin Microbiol. 2021; 5 (e03278-20)Google Scholar, 10Lu X. Wang L. Sakthivel S.K. et al.US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2.Emerg Infect Dis. 2020; 26: 1654-1665Crossref PubMed Scopus (303) Google Scholar Given the impact of G29179T on the GX assay, and the assay's global popularity for diagnostics, we queried the global phylogeny of SARS-CoV-2 (https://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/, 20 December 2021) to determine the global spread and abundance of G29179T. By querying the phylogeny using matUtils,11McBroome J. Thornlow B. Hinrichs A.S. et al.A daily-updated database and tools for comprehensive SARS-CoV-2 mutation-annotated trees.Mol Biol Evol. 2021; 38: 5819-5824Crossref PubMed Scopus (22) Google Scholar we determined that 12,481 of 3,152,290 (0.4%) sequences in the global alignment from 27 countries (earliest occurrence 15 March 2020) contained the G29179T mutation. Given that the phylogeny is not exhaustive with respect to global sequencing efforts (for example by not including any sequences that are only available on GISAID; https://www.gisaid.org/), the true number of samples with G29179T is likely higher. Samples with G29179T were not monophyletic, emerging independently in multiple lineages, suggesting convergent acquisition of G29179T. The G29179T mutation is most prevalent in delta genomes, and no omicron samples have yet acquired the G29179T mutation. However, the independent acquisition of G29179T in a wide range of lineages suggests that omicron samples may also be affected over time. Our analysis cannot suggest whether the repeated evolution of G29179T is adaptive or neutral, but, regardless, the mutation is clearly important for molecular diagnostics. Given the global distribution of G29179T, and independent confirmation of its negative impact on the GX assay in three different countries,3Miller S. Lee T. Merritt A. Pryce T. Levy A. Speers D. Single-point mutations in the N gene of SARS-CoV-2 adversely impact detection by a commercial dual target diagnostic assay.Microbiol Spectr. 2021; 9e0149421Crossref Scopus (12) Google Scholar, 4Hong K.H. In J.W. Lee J. et al.Prevalence of a single-nucleotide variant of SARS-CoV-2 in Korea and its impact on the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay.Ann Lab Med. 2022; 42: 96-99Crossref PubMed Google Scholar, 5Leelawong M. Mitchell S.L. Fowler R.C. et al.SARS-CoV-2 N gene mutations impact detection by clinical molecular diagnostics: reports in two cities in the United States.Diagn Microbiol Infect Dis. 2021; 101: 115468Crossref PubMed Scopus (11) Google Scholar the global performance of the GX assay and other assays that target the N-gene [e.g., Mira-Mic SARS-CoV-2 (Bio Molecular Systems, Australia) and BD Max COVID-19 (BD Life Sciences, USA)] is likely to be affected.12Navarathna D.H. Sharp S. Lukey J. et al.Understanding false positives and the detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and BD MAX SARS-CoV-2 assays.Diagn Microbiol Infect Dis. 2021; 100: 115334Crossref PubMed Scopus (13) Google Scholar Consequently, obtaining a positive result is reliant on the stability of the alternative target(s) of these assays, with pooling of samples potentially leading to incremental performance instability. Clinicians should be aware of this emerging problem in diagnosing delta SARS-CoV-2 and that ongoing assay performance monitoring is critical in the setting of rapid viral evolution. The authors state that there are no conflicts of interest to disclose.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Background: Duration and quality of immunity to SARS-CoV-2 have significant implications for the management of COVID-19 pandemic. Here, we present a comprehensive set of immunological data from a cohort of individuals (n=43), 12 months after mild COVID-19 disease and in the absence of virus re-exposure.Methods: Serum and PBMC were collected from mild-COVID-19 convalescents 12 months after the COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15). Serum titers of SARS-CoV-2-specific immunoglobulins were quantified by ELISA and virus neutralisation activity was assessed using SARS-CoV-2-Spike pseudovirus particles. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped with an activation-induced marker assay.Findings: In the absence of re-exposure to SARS-CoV-2 Spike- and RBD-specific antibodies were present in 90% of COVID-19 convalescents 12 months post-infection. RBD-specific IgG + memory B cells were maintained in 88.9% of patients, while 62% of patients had serum neutralising activity. Functionally mature memory CD4 + and CD8 + T cells were maintained at frequencies previously reported for earlier time points post-COVID-19, indicating substantial maintenance of durable T cell responses. Interpretations: Immunity to SARS-CoV-2 persists for 12 months in mild COVID-19 convalescent patients that retain high Spike-specific antibody titres, virus neutralisation capacity and circulating RBD-specific memory B cells. Significantly, T cell immunity remained stable 12 months post-infection. This study offers vital information on the duration of natural COVID-19 immunity and its potential protective effect against SARS-CoV-2 reinfection and clinical disease, with clear implications for the ongoing management of the global pandemic. Funding Statement: This work was funded by project grants from The Hospital Research Foundation and Women's and Children's Foundation, Adelaide, Australia. This work has been supported by NIH contract 75N9301900065 (A.S, D.W).Declaration of Interests: A.S. is currently a consultant for Gritstone, Flow Pharma, Arcturus, Epitogenesis, Oxfordimmunotech, Caprion and Avalia. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work.Authors PGV, CMH, MGM, AELY, HB, ZAM, ZAD, AA, DA, JG, CF, SO, EMM, DJL, GM, EJG, BAJR, DS, CKL, MRB, DW, RAB, SCB and BGB declare no conflict of interest.Ethics Approval Statement: Study protocols were approved by the Central Adelaide Clinical Human Research Ethics Committee (#13050) and the Women's and Children's Health Network Human research ethics (protocol HREC/19/WCHN/65), Adelaide, Australia.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions. Author Summary Limited data suggest antibody dependent effector functions including phagocytosis may contribute to the immunological and inflammatory responses in SARS CoV-2 infection, however, their development, maintenance, and clinical significance remain unknown. In this study we show: Patients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma. In convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1. Longitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice.

Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for prediction of protection against reinfection. In a cohort of 24 participants, the association of disease severity and early immunological measurements on the maintenance of humoral immune responses 12 months post-infection were examined. All severely affected participants maintained a stable subset of SARS-CoV-2 receptor-binding domain (RBD) specific memory B cells (MBCs) and good neutralising antibody breadth against the majority of the variants of concern, including the Delta variant. Modelling these immune responses on vaccine efficacy data indicated a level equivalent to a vaccine efficacy of approximately 45-76% against symptomatic reinfection (variant dependent). Overall, these findings indicate durable humoral responses in most participants, provide an estimate of the level of protection and identifies the magnitude and phenotype of baseline antigen-specific CD4+ T cell response as a predictor of maintenance of both antibody neutralisation breadth and RBD-specific MBC levels at 12 months post-infection.Funding: The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication do not necessarily represent the position of the Australian Government. Research reported in this publication was supported by Snow Medical Foundation as an investigator-initiated study. The content is solely the responsibility of the authors. RAB, MM, CR and ARL are fellows funded by National Health and Medical Research Council (NHMRC). MWAC is in part funded by the Research Infrastructure Programme of UNSW.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District and the University of New South Wales, NSW Australia (ETH00520) and was conducted according to the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice (ICH/GCP) guidelines and local regulatory requirements. Written informed consent was obtained from all participants before study procedures.