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1.
Journal of Medical Virology ; n/a(n/a), 2022.
Article in English | Wiley | ID: covidwho-1819379

ABSTRACT

Here we report prolonged viral shedding for over 29 days of SARS-CoV-2 Delta variant (B.1.617.2) in six patients with severe coronavirus disease (COVID-19) admitted to the intensive care unit (ICU). One patient remained culture positive 33 days after onset of disease. These features, including the persistence of high viral load (Ct < 30) in these patients, present new uncertainties concerning the safe discharge of patients from isolation into the general inpatient population. Prolonged high-level shedding and culture positivity of SARS-CoV-2 Delta variant for more than 29 days in hospitalized patients with severe COVID-19 presents new diagnostic and management problems. Viral culture results remain indicative of ongoing risk of transmission in the absence of functional humoral immune responses.This article is protected by copyright. All rights reserved.

3.
Cell ; 185(6): 945-948, 2022 03 17.
Article in English | MEDLINE | ID: covidwho-1748152

ABSTRACT

Long-term protection against SARS-CoV-2 requires effective and durable immunity. In this issue of Cell, two papers closely examine germinal centers, the physiological birthplace of adaptive immunity, to quantify the specificity, breadth, magnitude, and persistence of systemic and local humoral immune responses following natural infection with, or vaccination against, SARS-CoV-2.


Subject(s)
COVID-19 , Immunity, Humoral , Antibodies, Neutralizing , Antibodies, Viral , Germinal Center , Humans , SARS-CoV-2
4.
Cell Rep ; 38(6): 110345, 2022 02 08.
Article in English | MEDLINE | ID: covidwho-1654153

ABSTRACT

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 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.


Subject(s)
Broadly Neutralizing Antibodies/metabolism , SARS-CoV-2/immunology , Adult , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Australia , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , COVID-19/immunology , Cohort Studies , Female , Humans , Immunity/immunology , Immunity, Humoral/immunology , Male , SARS-CoV-2/pathogenicity , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology
5.
Viruses ; 14(2)2022 01 19.
Article in English | MEDLINE | ID: covidwho-1625191

ABSTRACT

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.


Subject(s)
Computational Biology/standards , Consensus , Genome, Viral , Laboratories/standards , Public Health , SARS-CoV-2/genetics , Australia , Computational Biology/methods , Humans , Phylogeny , SARS-CoV-2/classification , Whole Genome Sequencing
6.
Immunity ; 54(12): 2908-2921.e6, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1521063

ABSTRACT

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.


Subject(s)
Betacoronavirus/physiology , COVID-19 Vaccines/immunology , Coronavirus Infections/immunology , SARS Virus/physiology , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Conserved Sequence/genetics , Evolution, Molecular , Humans , Immunization , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
7.
Immunity ; 2021.
Article in English | EuropePMC | ID: covidwho-1489418

ABSTRACT

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.

8.
Front Immunol ; 12: 752003, 2021.
Article in English | MEDLINE | ID: covidwho-1468344

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have become a major concern in the containment of current pandemic. The variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta) have shown reduced sensitivity to monoclonal antibodies, plasma and/or sera obtained from convalescent patients and vaccinated individuals. Development of potent therapeutic monoclonal antibodies (mAbs) with broad neutralizing breadth have become a priority for alleviating the devastating effects of this pandemic. Here, we review some of the most promising broadly neutralizing antibodies obtained from plasma of patients that recovered from early variants of SARS-CoV-2 that may be effective against emerging new variants of the virus. This review summarizes several mAbs, that have been discovered to cross-neutralize across Sarbecoviruses and SARS-CoV-2 escape mutants. Understanding the characteristics that confer this broad and cross-neutralization functions of these mAbs would inform on the development of therapeutic antibodies and guide the discovery of second-generation vaccines.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Viral/blood , Antibody Specificity , Binding Sites, Antibody , Broadly Neutralizing Antibodies/blood , COVID-19/blood , COVID-19/virology , Cross Reactions , Host-Pathogen Interactions , Humans , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
9.
MAbs ; 13(1): 1922134, 2021.
Article in English | MEDLINE | ID: covidwho-1240862

ABSTRACT

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS Virus/immunology , SARS-CoV-2/immunology , Antibody Specificity , Cross Reactions , Humans , Mutagenesis, Site-Directed
10.
Cell Rep Med ; 2(4): 100228, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1195472

ABSTRACT

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.


Subject(s)
Antibodies, Neutralizing/blood , COVID-19/pathology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/immunology , Asymptomatic Diseases , COVID-19/immunology , COVID-19/virology , Female , Humans , Limit of Detection , Male , Middle Aged , Neutralization Tests , Protein Domains/immunology , SARS-CoV-2/isolation & purification , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Time Factors , Young Adult
11.
Sci Rep ; 11(1): 3934, 2021 02 16.
Article in English | MEDLINE | ID: covidwho-1087497

ABSTRACT

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct < 30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Coinfection/diagnosis , Coinfection/virology , SARS-CoV-2/genetics , Sequence Analysis, DNA , Virome/genetics , Australia/epidemiology , Coinfection/epidemiology , Computational Biology , Genome, Viral , Humans , Open Reading Frames/genetics , Reproducibility of Results , Whole Genome Sequencing
12.
Viruses ; 13(2)2021 02 04.
Article in English | MEDLINE | ID: covidwho-1063429

ABSTRACT

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.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay , Neutralization Tests , SARS-CoV-2/immunology , COVID-19/diagnosis , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , ROC Curve , Sensitivity and Specificity
13.
Nat Commun ; 11(1): 6272, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-965783

ABSTRACT

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.


Subject(s)
Nanopore Sequencing/methods , SARS-CoV-2 , Whole Genome Sequencing/methods , COVID-19/diagnosis , COVID-19/virology , Genome, Viral , Humans , RNA, Viral , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
14.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-910292

ABSTRACT

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.


Subject(s)
Hepacivirus/physiology , Viral Envelope Proteins/metabolism , Antibody Affinity , Gene Knockdown Techniques , HEK293 Cells , Hepacivirus/immunology , Hepatitis C/virology , Hepatitis C Antibodies/immunology , Hepatitis C Antigens/immunology , Hepatitis C Antigens/metabolism , Humans , Mannose/chemistry , Polysaccharides/chemistry , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , Tetraspanin 28/genetics , Tetraspanin 28/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/immunology
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