Microbiology & Immunology - Research Publications
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ItemImpaired Th17 immunity in recurrent C. difficile infection is ameliorated by fecal microbial transplantation( 2020-06-10)Background & Aims: Clostridioides difficile is a leading cause of infectious diarrhea and an urgent antimicrobial resistant threat. Symptoms are caused by its toxins, TcdA and TcdB, with many patients developing recurrent C. difficile infection (CDI), requiring fecal microbiota transplant (FMT). Antibody levels have not been useful in predicting patient outcomes, which is an unmet need. We aimed to characterize T cell-mediated immunity to C. difficile toxins and assess how these responses were affected by FMT. Methods: We obtained blood samples from patients with newly acquired CDI, recurrent CDI (with a subset receiving FMT), inflammatory bowel disease with no history of CDI, and healthy individuals (controls). Toxin-specific CD4+ T cell responses were analysed using a whole blood flow cytometry antigen-induced marker assay. Serum antibodies were measured by ELISA. Tetramer guided mapping was used to identify HLA-II-restricted TcdB epitopes and DNA was extracted from TcdB-specific CD4+ T cells for TCR repertoire analysis by Sanger sequencing. Results: CD4+ T cell responses to C. difficile toxins were functionally diverse. Compared to controls, individuals with CDI, or inflammatory bowel disease had significantly higher frequencies of TcdB-specific CD4+ T cells. Subjects with recurrent CDI had reduced proportions of TcdB-specific CD4+ Th17 cells, FMT reversed this deficit and increased toxin-specific antibody production. Conclusions: These data suggest that effective T cell immunity to C. difficile requires the development of Th17 cells. In addition, they show that an unknown aspect of the therapeutic effect of FMT may be enhanced T and B cell-mediated immunity to TcdB.
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ItemNo Preview AvailableReprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance( 2020)
ABSTRACT
Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses. -
ItemEvaluation of serological tests for SARS-CoV-2: Implications for serology testing in a low-prevalence setting( 2020)
Background
Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little pre-market validation.Methods
Performance characteristics for five PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralisation test (sVNT).Results
Sensitivities for PoCT ranged from 51.8% (95% CI 43.1 to 60.4%) to 67.9% (95% CI 59.4–75.6%), and specificities from 95.6% (95% CI 89.2–98.8%) to 100.0% (95% CI 96.1–100.0%). Overall ELISA sensitivity for either IgA or IgG detection was 67.9% (95% CI 59.4–75.6), increasing to 93.8% (95% CI 85.0–98.3%) for samples > 14 days post symptom onset. Overall, sVNT sensitivity was 60.9% (95% CI 53.2–68.4%), rising to 91.2%% (95% CI 81.8–96.7%) for samples collected > 14 days post-symptom onset, with a specificity 94.4% (95% CI 89.2–97.5%),Conclusion
Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in the reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings. -
ItemImmune responses to SARS-CoV-2 in children of parents with symptomatic COVID-19( 2020)Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have mild or asymptomatic infection, but the underlying immunological differences remain unclear. We describe clinical features, virology, longitudinal cellular and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who were repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children were similar to their parents at all timepoints. All family members had salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincided with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child had IgG antibody detected against the S1 protein and virus neutralising activity ranging from just detectable to robust titers. Using a systems serology approach, we show that all family members demonstrated higher levels of SARS-CoV-2-specific antibody features than healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological evidence of infection. This raises the possibility that despite chronic exposure, immunity in children prevents establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may therefore not identify exposed children, with implications for epidemiological and clinical studies across the life-span.
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ItemNo Preview AvailableTranscriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection.( 2020-12-22)SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/ .
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ItemSystematic analysis of key parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria( 2020-09-25)Background: Pairwise single nucleotide polymorphisms (SNPs) are a cornerstone for genomic approaches to multidrug-resistant organisms (MDROs) transmission inference in hospitals. However, the impact of key analysis parameters on these inferences has not been systematically analysed. Methods: We conducted a multi-hospital 15-month prospective study, sequencing 1537 MDRO genomes for comparison; methicillin-resistant Staphylococcus aureus , vancomycin-resistant Enterococcus faecium , and extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae . We systematically assessed the impact of sample and reference genome diversity, masking of prophage and regions of recombination, cumulative genome analysis compared to a three-month sliding-window, and the comparative effects each of these had when applying a SNP threshold for inferring likely transmission (≤15 SNPs for S. aureus , ≤25 for other species). Findings: Across the species, using a reference genome of the same sequence type provided a greater degree of pairwise SNP resolution, compared to species and outgroup-reference alignments that typically resulted in inflated SNP distances and the possibility of missed transmission events. Omitting prophage regions had minimal impacts, however, omitting recombination regions a highly variable effect, often inflating the number of closely related pairs. Estimating pairwise SNP distances was more consistent using a sliding-window than a cumulative approach. Interpretation: The use of a closely-related reference genome, without masking of prophage or recombination regions, and a sliding-window for isolate inclusion is best for accurate and consistent MDRO transmission inference. The increased stability and resolution provided by these approaches means SNP thresholds for putative transmission inference can be more reliably applied among diverse MDROs. Funding: This work was supported by the Melbourne Genomics Health Alliance (funded by the State Government of Victoria, Department of Health and Human Services, and the ten member organizations); an National Health and Medical Research Council (Australia) Partnership grant (GNT1149991) and individual grants from National Health and Medical Research Council (Australia) to NLS (GNT1093468), JCK (GNT1008549) and BPH (GNT1105905).
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ItemUnicycler: resolving bacterial genome assemblies from short and long sequencing reads( 2016-12-22)The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce more complete genome assemblies, but the sequencing is more expensive and error prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler utilises a novel semi-global aligner, which is used to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler .
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ItemNo Preview AvailableGene expression profiling of malaria parasites reveals common virulence gene expression in adult first-time infected patients and severe cases( 2020)Sequestration of Plasmodium falciparum -infected erythrocytes to host endothelium through the parasite-derived Pf EMP1 adhesion proteins is central to the development of malaria pathogenesis. Pf EMP1 proteins have diversified and expanded to encompass many sequence variants conferring each parasite a similar array of human endothelial receptor binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum infected adult travelers returning to Germany. Patients were categorized into either malaria naïve (n=15) or pre-exposed (n=17), and into severe (n=8) or non-severe (n=24) cases. For differential expression analysis of Pf EMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with Pf EMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding Pf EMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic Pf EMP1 variants are more common in patients with a naïve immune status and/or adverse inflammatory host responses to first infections favors growth of EPCR-binding parasites.
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ItemDecay of Fc-dependent antibody functions after mild to moderate COVID-19( 2020)The capacity of antibodies to engage with innate and adaptive immune cells via the Fc region is important in preventing and controlling many infectious diseases, and is likely critical in SARS-CoV-2 infection. The evolution of such antibodies during convalescence from COVID-19 is largely unknown. We developed novel assays to measure Fc-dependent antibody functions against SARS-CoV-2 spike (S)-expressing cells in serial samples from a cohort of 53 subjects primarily with mild-moderate COVID-19, out to a maximum of 149 days post-infection. We found that S-specific antibodies capable of engaging dimeric FcγRIIa and FcγRIIIa decayed linearly over time. S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declined linearly as well, in line with the decay of S-specific IgG. Although there was significant decay in S-specific plasma ADCC and ADP activity, they remained readily detectable by all assays in 94% of our cohort at the last timepoint studied, in contrast with neutralisation activity which was only detectable in 70% of our cohort by the last timepoint. Our results suggest that Fc effector functions such as ADCC and ADP could contribute to the durability of SARS-CoV-2 immunity, particularly late in convalescence when neutralising antibodies have waned. Understanding the protective potential of antibody Fc effector functions is critical for defining the durability of immunity generated by infection or vaccination.
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ItemRapid and lasting generation of B-cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 disease and convalescence( 2020)
ABSTRACT
Background
Lasting immunity to SARS-CoV-2 following infection is questioned because serum antibodies decline in convalescence. However, functional immunity is mediated by long-lived memory T and B (Bmem) cells.Objective
To determine the longevity and immunophenotype of SARS-CoV-2-specific Bmem cells in COVID-19 patients.Methods
Recombinant spike receptor binding domain (RBD) and nucleocapsid protein (NCP) were produced for ELISA-based serology, and biotinylated for fluorescent tetramer generation to identify SARS-CoV-2-specific Bmem cells by flow cytometry with a panel of 13 mAbs. 36 blood samples were obtained from 25 COVID-19 patients (11 paired) between 4-242 days post-symptom onset for detection of neutralizing antibodies, IgG serology and flow cytometry.Results
The recombinant RBD and NCP were specifically recognized by serum IgG in all patients and reactivity declined >20 days post-symptom onset. All patients had detectable RBD- and NCP-specific Bmem cells at 8.23-267.6 cells/ml of blood (0.004-0.13% of B cells) regardless of sampling time. RBD- and NCP-specific Bmem cells predominantly expressed IgM or IgG1, with the latter formed slightly later than the former. RBD-specific IgG + Bmem were predominantly CD27 + , and numbers significantly correlated with circulating follicular helper T cell numbers.Conclusion
RBD- and NCP-specific Bmem cells persisted for 8 months, indicating that the decline in serum antibodies after 1 month does not indicate waning of immunity but a contraction of the immune response. Flowcytometric detection of SARS-CoV-2-specific Bmem cells enables detection of long-term functional immunity following infection or vaccination for COVID-19.