Proteomic and Metabolomic Signatures Associated With the Immune Response in Healthy Individuals Immunized With an Inactivated SARS-CoV-2 Vaccine.

CoronaVac (Sinovac), an inactivated vaccine for SARS-CoV-2, has been widely used for immunization. However, analysis of the underlying molecular mechanisms driving CoronaVac-induced immunity is still limited. Here, we applied a systems biology approach to understand the mechanisms behind the adaptive immune response to CoronaVac in a cohort of 50 volunteers immunized with 2 doses of CoronaVac. Vaccination with CoronaVac led to an integrated immune response that included several effector arms of the adaptive immune system including specific IgM/IgG, humoral response and other immune response, as well as the innate immune system as shown by complement activation. Metabolites associated with immunity were also identified implicating the role of metabolites in the humoral response, complement activation and other immune response. Networks associated with the TCA cycle and amino acids metabolic pathways, such as phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glycine, serine and threonine metabolism were tightly coupled with immunity. Critically, we constructed a multifactorial response network (MRN) to analyze the underlying interactions and compared the signatures affected by CoronaVac immunization and SARS-CoV-2 infection to further identify immune signatures and related metabolic pathways altered by CoronaVac immunization. These results help us to understand the host response to vaccination of CoronaVac and highlight the utility of a systems biology approach in defining molecular correlates of protection to vaccination.

Microbial determinants of effective donors in faecal microbiota transplantation for UC.

OBJECTIVE: Faecal microbiota transplantation (FMT) has variable efficacy in treating UC. Recently, oral lyophilised FMT was found to induce remission in patients with UC, with one donor having 100% efficacy compared with a second donor (36% efficacy). We characterised differences in the gut microbiota of these two donors with the aim of improving FMT donor selection. DESIGN: Faecal samples from the two donors were collected over a period of 44 (donor 1) or 70 (donor 2) weeks. The microbiome and metabolome were profiled using shotgun metagenomics and untargeted metabolomics RESULTS: Gut microbiome long-term stability was highly evident in the effective donor. Donor microbiota species evenness was a robust feature associated with clinical efficacy across two clinical trials of FMT in UC, leading to increased donor species engraftment in patients. Alpha diversity and beta diversity of donor gut microbiotas significantly differed. 90 bacterial species and one archaeon were differentially abundant between donors, 44 of which were >0.1% in relative abundance. 17/44 species were enriched in the effective donor, 11 of which (64.7%) were assembled into high-quality genomes that were prevalent (≥75% samples) in that donor, and six showed evidence of engraftment in patients. Taxonomic differences between donors translated to substantial microbial functional differences that were validated using metabolomics. CONCLUSION: Donor microbiota stability and species evenness were identified as novel metrics that were associated with therapeutic efficacy in UC, beyond individual microbial species or metabolites. These metrics may represent community resilience that translates to better engraftment in the host. TRIAL REGISTRATION NUMBER: ACTRN12619000611123.

Single-cell transcriptomic atlas reveals distinct immunological responses between COVID-19 vaccine and natural SARS-CoV-2 infection.

To control the ongoing coronavirus disease-2019 (COVID-19) pandemic, CoronaVac (Sinovac), an inactivated vaccine, has been granted emergency use authorization by many countries. However, the underlying mechanisms of the inactivated COVID-19 vaccine-induced immune response remain unclear, and little is known about its features compared to (Severe acute respiratory syndrome coronavirus 2) SARS-CoV-2 infection. Here, we implemented single-cell RNA sequencing (scRNA-seq) to profile longitudinally collected PBMCs (peripheral blood mononuclear cells) in six individuals immunized with CoronaVac and compared these to the profiles of COVID-19 infected patients from a Single Cell Consortium. Both inactivated vaccines and SARS-CoV-2 infection altered the proportion of different immune cell types, caused B cell activation and differentiation, and induced the expression of genes associated with antibody production in the plasma. The inactivated vaccine and SARS-COV-2 infection also caused alterations in peripheral immune activity such as interferon response, inflammatory cytokine expression, innate immune cell apoptosis and migration, effector T cell exhaustion and cytotoxicity, however, the magnitude of change was greater in COVID-19 patients, especially those with severe disease, than in immunized individuals. Further analyses revealed a distinct peripheral immune cell phenotype associated with CoronaVac immunization (HLA class II upregulation and IL21R upregulation in naïve B cells) versus SARS-CoV-2 infection (HLA class II downregulation and IL21R downregulation in naïve B cells from severe disease individuals). There were also differences in the expression of important genes associated with proinflammatory cytokines and thrombosis. In conclusion, this study provides a single-cell atlas of the systemic immune response to CoronaVac immunization and revealed distinct immune responses between inactivated vaccines and SARS-CoV-2 infection.

Proteomic and Metabolomic Signatures Associated With the Immune Response in Healthy Individuals Immunized With an Inactivated SARS-CoV-2 Vaccine

CoronaVac (Sinovac), an inactivated vaccine for SARS-CoV-2, has been widely used for immunization. However, analysis of the underlying molecular mechanisms driving CoronaVac-induced immunity is still limited. Here, we applied a systems biology approach to understand the mechanisms behind the adaptive immune response to CoronaVac in a cohort of 50 volunteers immunized with 2 doses of CoronaVac. Vaccination with CoronaVac led to an integrated immune response that included several effector arms of the adaptive immune system including specific IgM/IgG, humoral response and other immune response, as well as the innate immune system as shown by complement activation. Metabolites associated with immunity were also identified implicating the role of metabolites in the humoral response, complement activation and other immune response. Networks associated with the TCA cycle and amino acids metabolic pathways, such as phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glycine, serine and threonine metabolism were tightly coupled with immunity. Critically, we constructed a multifactorial response network (MRN) to analyze the underlying interactions and compared the signatures affected by CoronaVac immunization and SARS-CoV-2 infection to further identify immune signatures and related metabolic pathways altered by CoronaVac immunization. These results help us to understand the host response to vaccination of CoronaVac and highlight the utility of a systems biology approach in defining molecular correlates of protection to vaccination.

Lyophilised oral faecal microbiota transplantation for ulcerative colitis (LOTUS): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Faecal microbiota transplantation (FMT) delivered via colonoscopic infusion or enemas have been shown to induce remission in a proportion of patients with active ulcerative colitis. Whether orally administered FMT is effective in ulcerative colitis is unknown. We aimed to assess the efficacy of oral lyophilised FMT for the treatment of active ulcerative colitis. METHODS: A double-blind, randomised, placebo-controlled trial was conducted at two centres in Australia. Eligible patients were aged 18-75 years with active ulcerative colitis (defined as clinical and endoscopic active ulcerative colitis, with a total Mayo score of 4-10, and a Mayo endoscopic subscore ≥1). After 2 weeks of amoxicillin, metronidazole, and doxycycline, patients were randomly assigned in a 1:1 ratio to receive either oral lyophilised FMT or placebo capsules for 8 weeks, using a prespecified computer-generated randomisation list with a permuted block size of 8. The primary outcome was corticosteroid-free clinical remission with endoscopic remission or response (total Mayo score ≤2, all subscores ≤1, and ≥1 point reduction in endoscopic subscore) at week 8. At week 8, FMT responders were randomly assigned (in a 1:1 ratio, permuted block size of 8) to either continue or withdraw FMT for a further 48 weeks. Analyses were done by modified intention-to-treat, including all patients who received at least one study dose. This trial is registered with Australian New Zealand Trial Registry, number ACTRN 12619000611123; this is the final report of the trial. FINDINGS: Between May 20, 2019, and March 24, 2020, 35 patients were randomly assigned: 15 to receive FMT and 20 to receive placebo. Recruitment was terminated early due to the COVID-19 pandemic. At week 8, eight (53%) of 15 patients in the FMT group were in corticosteroid-free clinical remission with endoscopic remission or response, as were three (15%) of 20 patients in the placebo group (difference 38·3%, 95% CI 8·6-68·0; p=0·027; odds ratio 5·0, 95% CI 1·8-14·1). Adverse events occurred in 10 (67%) patients in the FMT group and 17 (85%) of those in the placebo group during the 8-week induction period, and were generally mild and self-limiting gastrointestinal complaints. Serious adverse events included worsening ulcerative colitis (two in the FMT group, one in the placebo group) and per-rectal bleeding (one in the placebo group). Ten patients in the FMT group who achieved a clinical or endoscopic response entered the maintenance phase and were randomly assigned to continue open-label FMT (n=4) or withdraw therapy (n=6). All four (100%) patients who continued FMT were in clinical, endoscopic, and histologic remission at week 56 compared with none of the patients who had FMT withdrawn. INTERPRETATION: Antibiotics followed by orally administered FMT was associated with the induction of remission in patients with active ulcerative colitis. Continuing FMT was well tolerated and appeared to demonstrate clinical, endoscopic, and histological efficacy. Oral FMT could be a promising and feasible treatment option for patients with ulcerative colitis. FUNDING: St Vincent's Clinic Foundation, Gastroenterological Society of Australia, Gutsy Group.

A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.

Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid amplification methods for SARS-CoV-2 detection.

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.