Evaluation of QuantiFERON SARS-CoV-2 interferon-γ release assay following SARS-CoV-2 infection and vaccination.

T cells are important in preventing severe disease from SARS-CoV-2, but scalable and field-adaptable alternatives to expert T-cell assays are needed. The interferon-gamma release assay QuantiFERON platform was developed to detect T-cell responses to SARS-CoV-2 from whole blood with relatively basic equipment and flexibility of processing timelines. Forty-eight participants with different infection and vaccination backgrounds were recruited. Whole blood samples were analysed using the QuantiFERON SARS-CoV-2 assay in parallel with the well-established 'Protective Immunity from T Cells in Healthcare workers' (PITCH) ELISpot, which can evaluate spike-specific T-cell responses. The primary aims of this cross-sectional observational cohort study were to establish if the QuantiFERON SARS-Co-V-2 assay could discern differences between specified groups and to assess the sensitivity of the assay compared with the PITCH ELISpot. The QuantiFERON SARS-CoV-2 distinguished acutely infected individuals (12-21 days post positive PCR) from naïve individuals (P < 0.0001) with 100% sensitivity and specificity for SARS-CoV-2 T cells, whilst the PITCH ELISpot had reduced sensitivity (62.5%) for the acute infection group. Sensitivity with QuantiFERON for previous infection was 12.5% (172-444 days post positive test) and was inferior to the PITCH ELISpot (75%). Although the QuantiFERON assay could discern differences between unvaccinated and vaccinated individuals (55-166 days since second vaccination), the latter also had reduced sensitivity (44.4%) compared to the PITCH ELISpot (66.6%). The QuantiFERON SARS-CoV-2 assay showed potential as a T- cell evaluation tool soon after SARS-CoV-2 infection but has lower sensitivity for use in reliable evaluation of vaccination or more distant infection.

Divergent trajectories of antiviral memory after SARS-CoV-2 infection.

The trajectories of acquired immunity to severe acute respiratory syndrome coronavirus 2 infection are not fully understood. We present a detailed longitudinal cohort study of UK healthcare workers prior to vaccination, presenting April-June 2020 with asymptomatic or symptomatic infection. Here we show a highly variable range of responses, some of which (T cell interferon-gamma ELISpot, N-specific antibody) wane over time, while others (spike-specific antibody, B cell memory ELISpot) are stable. We use integrative analysis and a machine-learning approach (SIMON - Sequential Iterative Modeling OverNight) to explore this heterogeneity. We identify a subgroup of participants with higher antibody responses and interferon-gamma ELISpot T cell responses, and a robust trajectory for longer term immunity associates with higher levels of neutralising antibodies against the infecting (Victoria) strain and also against variants B.1.1.7 (alpha) and B.1.351 (beta). These variable trajectories following early priming may define subsequent protection from severe disease from novel variants.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern.

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Author Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.

Inflammation associated ethanolamine facilitates infection by Crohn's disease-linked adherent-invasive Escherichia coli.

BACKGROUND: The predominance of specific bacteria such as adherent-invasive Escherichia coli (AIEC) within the Crohn's disease (CD) intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is however readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. METHODS: Quantitative RT-PCR (qRT-PCR) to determine expression of genes central to ethanolamine metabolism; transmission electron microscopy to detect presence of bacterial microcompartments (MCPs); in vitro infections of both murine and human macrophage cell lines examining intracellular replication of the AIEC-type strain LF82 and clinical E. coli isolates in the presence of ethanolamine; determination of E. coli ethanolamine utilization (eut) operon transcription in faecal samples from healthy patients, patients with active CD and the same patients in remission following treatment. RESULTS: Growth on the intestinal short chain fatty acid propionic acid (PA) stimulates significantly increased transcription of the eut operon (fold change relative to glucose: >16.9; p-value <.01). Additionally ethanolamine was accessible to intra-macrophage AIEC and stimulated significant increases in growth intracellularly when it was added extracellularly at concentrations comparable to those in the human intestine. Finally, qRT-PCR indicated that expression of the E. coli eut operon was increased in children with active CD compared to healthy controls (fold change increase: >4.72; P < .02). After clinical remission post-exclusive enteral nutrition treatment, the same CD patients exhibited significantly reduced eut expression (Pre vs Post fold change decrease: >15.64; P < .01). INTERPRETATION: Our data indicates a role for ethanolamine metabolism in selecting for AIEC that are consistently overrepresented in the CD intestine. The increased E. coli metabolism of ethanolamine seen in the intestine during active CD, and its decrease during remission, indicates ethanolamine use may be a key factor in shaping the intestinal microbiome in CD patients, particularly during times of inflammation. FUND: This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/K008005/1 & BB/P003281/1 to DMW; by a Tenovus Scotland grant to MJO; by Glasgow Children's Hospital Charity, Nestle Health Sciences, Engineering and Physical Sciences Research Council (EPSRC) and Catherine McEwan Foundation grants awarded to KG; and by a Natural Environment Research Council (NERC) fellowship (NE/L011956/1) to UZI. The IBD team at the Royal Hospital for Children, Glasgow are supported by the Catherine McEwan Foundation and Yorkhill IBD fund. RKR and RH are supported by NHS Research Scotland Senior fellowship awards.

Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

Bacterial-mediated cancer therapy has shown great promise in in vivo tumour models with increased survival rates post-bacterial treatment. Improving efficiency of bacterial-mediated tumour regression has focused on controlling and exacerbating bacterial cytotoxicity towards tumours. One mechanism that has been used to carry this out is the process of bactofection where post-invasion, bacteria deliver plasmid-borne mammalian genes into target cells for expression. Here we utilised the cancer-targeting Salmonella Typhimurium strain, SL7207, to carry out bactofection into triple negative breast cancer MDA-MB-231 cells. However, we noted that post-transformation with the commonly used mammalian expression vector pEGFP, S. Typhimurium became filamentous, attenuated and unable to invade target cells efficiently. Filamentation did not occur in Escherichia coli-transformed with the same plasmid. Further investigation identified the region inducing S. Typhimurium filamentation as being the f1 origin of replication (f1 ori), an artefact of historic use of mammalian plasmids for single stranded DNA production. Other f1 ori-containing plasmids also induced the attenuated phenotype, while removal of the f1 ori from pEGFP restored S. Typhimurium virulence and increased the bactofection capacity. This work has implications for interpretation of prior bactofection studies employing f1 ori-containing plasmids in S. Typhimurium, while also indicating that future use of S. Typhimurium in targeting tumours should avoid the use of these plasmids.

Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published with one of the panels in Figure 5A inserted twice (SL-pEGFP). In Figure 5B there was also a typo. SL-LacZ should have read SL-pEGFP(-f1). Both 5A and 5B are corrected in both the PDF and HTML versions of the Article.

Draft Genome Sequence of the Tumor-Targeting Salmonella enterica Serovar Typhimurium Strain SL7207.

Salmonella enterica serovar Typhimurium strain SL7207 is a genetically modified derivative of strain SL1344, which preferentially accumulates in tumors and can be used as a vehicle for tissue-specific gene delivery in vivo Here, we report the draft genome sequence of SL7207, confirming a purported aroA deletion and four single-nucleotide polymorphisms compared to SL1344.

Draft Genome Sequence of the Commensal Escherichia coli Strain F-18.

Here, we report the draft genome sequence of Escherichia coli strain F-18, originally isolated from the feces of a healthy individual in 1977. The draft genome is 5,246,829 bp, with a G+C content of 50.50%, and it encodes 4,933 predicted coding sequences (CDSs), 10 rRNAs, and 84 tRNAs.