William Rawlinson - Academia.edu (original) (raw)

Papers by William Rawlinson

Immunology and Cell Biology, Dec 1, 2022

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)... more 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-a) 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.

Placenta, Dec 1, 2018

Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylationregulated kinase... more Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylationregulated kinases during placental replication,

Cell Reports, Feb 1, 2022

Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for predicting protect... more Understanding the long-term maintenance of 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 neutralising antibody breadth against the majority of the variants of concern, including the Delta variant. Modelling these immune responses against vaccine efficacy data indicate 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 neutralisation breadth and RBD-specific MBC levels at 12 months post-infection.

Immunity, Dec 1, 2021

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second gener... more 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 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.

Viruses, Feb 13, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

medRxiv (Cold Spring Harbor Laboratory), Sep 9, 2022

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta vari... more 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 frameshiftinducing deletion in ORF7a that truncated the peptide sequence. The variant rapidly became represented at the consensus level (Delta-ORF7a Δ17del) 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 Δ17del mutation by far the most common. Approximately 99.05% of Delta-ORF7a Δ17del 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 Δ17del than with a contemporaneous Delta variant without ORF7a Δ17del (Delta-ORF7a intact), and the proportion was also measurably higher than an early SARS-CoV-2 strain (A.2.2). These results showed that Delta-ORF7a Δ17del potentially has a slight growth advantage compared to Delta-ORF7a intact. Delta-ORF7a Δ17del 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 Δ17del genomes during the Australian Delta outbreak was likely not a result of an intrinsic benefit of the ORF7a Δ17del 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 cladelevel survey of ORF7a deletions will be a useful resource for future studies into this gene region.

Research Square (Research Square), Nov 18, 2020

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respirator... more 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 panviral 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, >10-fold 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 dualfunctionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.

Nature Communications, Feb 8, 2023

The coronavirus SARS-CoV-2, the causative agent of the global COVID-19 pandemic, has resulted in ... more The coronavirus SARS-CoV-2, the causative agent of the global COVID-19 pandemic, has resulted in the death of over 6 million people worldwide 1. Upon SARS-CoV-2 infection, the human adaptive immune response generates antibodies against the viral spike surface glycoprotein 2. Most of the neutralizing antibodies bind the spike receptor binding domain (RBD), and in particular class 1 and 2 epitopes within the receptor binding motif (RBM), directly blocking interaction with the human angiotensin converting enzyme receptor 2 (ACE2) 3-6. Such RBD-and RBM-targeted antibodies are also generated upon vaccination 7 , but often lack neutralization potential against emerging

Nature Communications, Dec 9, 2020

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution... more 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.

Journal of Immunology, Oct 15, 2022

,* and on behalf of the COSIN Study Group 1 Phagocytic responses by effector cells to opsonized v... more ,* and on behalf of the COSIN Study Group 1 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 proteincoated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 7795% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 1860% 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.

Viruses, Jan 19, 2022

Transplant Infectious Disease, Apr 22, 2012

Expansion of the donor pool may lead to utilization of donors with risk factors for viral infecti... more Expansion of the donor pool may lead to utilization of donors with risk factors for viral infections. Donor laboratory screening relies on serological and nucleic acid testing (NAT). The increased sensitivity of NAT in low prevalence populations may result in false-positive results (FPR) and may cause unnecessary discard of organs.We developed a screening algorithm to deal, in real time, with potential FPR. Three NAT assays: COBAS AmpliScreen assay (CAS), AmpliPrep Total Nucleic Acid Isolation/CAS, and AmpliPrep/TaqMan assays, were validated and used in parallel for prospective screening of increased-risk donors (IRD), and the probability of FPR was calculated. The lower limit of detection of this algorithm was 9.79, 21.02, and 4.31 IU/mL for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, respectively, with an average turn-around-time of 7.67 h from sample receipt to result reporting. The probability that a donor is potentially infectious with two NAT concordant results was &amp;gt;90%. NAT screening of 35 IRD within 18 months resulted in transplantation of 102 additional organs that without screening would either not be used or used with restrictions in Australia. Using a parallel testing algorithm, real-time confirmation of seropositive donors allows use of organs from IRD and safer expansion of the donor pool.

Social Science Research Network, 2021

Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for prediction of prot... more 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.

Knowing the prevalence of potential aetiologic agents of non-gonococcal and non-chlamydial cervic... more Knowing the prevalence of potential aetiologic agents of non-gonococcal and non-chlamydial cervicitis is important for improving efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs) designated VDL05, VDL06, VDL07 and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis including: Cytomegalovirus (CMV), Enterovirus (EV), Epstein Barr Virus (EBV), Varicella Zoster virus (VZV), Herpes simplex virus-1 (HSV-1) and Herpes simplex-2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A-E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual health clinic in Sydney Australia, during 2006-2007. The agents detected alone or in combination in all cervical swabs (percentage total swabs) include: CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and Group B streptococci (0.4). Adenovirus species A-E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

bioRxiv (Cold Spring Harbor Laboratory), Apr 6, 2021

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented 1-3... more Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented 1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-19 4,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

Microorganisms

Serological diagnostic assays are essential tools for determining an individual’s protection agai... more Serological diagnostic assays are essential tools for determining an individual’s protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed ei...

The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started... more 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 related sub-lineages. Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants at two 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 strigently curated vaccine and convalescent cohorts. 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 emerg...

Reviews in Medical Virology

The first dominant SARS‐CoV‐2 Omicron variant BA.1 harbours 35 mutations in its Spike protein fro... more The first dominant SARS‐CoV‐2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS‐CoV‐2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co‐circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan‐Hu‐1 and Delta variant. However,...