A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi.

Here we present an examination of type IV pilus genes associated with competence and twitching in the bacterium Acinetobacter baylyi (strain ADP1, BD413). We used bioinformatics to identify potential competence and twitching genes and their operons. We measured the competence and twitching phenotypes of the bioinformatically-identified genes. These results demonstrate that competence and twitching in A. baylyi both rely upon a core of the same type IV pilus proteins. The core includes the inner membrane assembly platform (PilC), a periplasmic assemblage connecting the inner membrane assembly platform to the secretin (ComM), a secretin (ComQ) and its associated pilotin (PilF) that assists with secretin assembly and localization, both cytoplasmic pilus retraction ATPases (PilU, PilT), and pilins (ComP, ComB, PilX). Proteins not needed for both competence and twitching are instead found to specialize in either of the two traits. The pilins are varied in their specialization with some required for either competence (FimT) and others for twitching (ComE). The protein that transports DNA across the inner membrane (ComA) specializes in competence, while signal transduction proteins (PilG, PilS, and PilR) specialize in twitching. Taken together our results suggest that the function of accessory proteins should not be based on homology alone. In addition the results suggest that in A. baylyi the mechanisms of natural transformation and twitching are mediated by the same set of core Type IV pilus proteins with distinct specialized proteins required for each phenotype. Finally, since competence requires multiple pilins as well as both pilus retraction motors PilU and PilT, this suggests that A. baylyi employs a pilus in natural transformation

Reactive design patterns

<p><b>Twitching zones on (a) soft vs. (b) hard agar for multiple twitching phenotypes.</b> On soft agar (0.5%) in panel (a), mutants <i>fimU</i> and <i>comA</i> twitch a comparable amount to the wildtype, while <i>comP</i> and <i>comE</i> are substantially impaired in agreement with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g003" target="_blank">Fig 3</a>. Panel (b) shows the same mutants but now on hard agar (1.5%). In this case all diameters are comparable.</p

Alignment of FimU and FimT pilins from ADP1.

<p>A. The length of the primary sequence is indicated above with Pfam motifs found in both proteins diagrammed below. N = PF07963.11; GspH = PF12019.7. Such motifs are identified using multiple sequence alignments. B. Pairwise alignment of FimU and FimT. Identical amino acids are indicated by a vertical line connecting them while: and. represent higher and lower degrees of chemical similarity between the two amino acids, respectively. Boldface amino acids are part of the Pfam motifs diagrammed in A.</p

Model of the ADP1 type IV pilus with associated competence and signal transduction proteins.

<p>Components of the model are predicted by homology from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.t001" target="_blank">Table 1</a> and are supported by data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g003" target="_blank">Fig 3</a>.</p

Protein motif diagrams of the type IV pilin proteins encoded by ADP1.

<p>Length in amino acids is indicated along the top, with pilins diagrammed below. Pilins absolutely required for both twitching and competence are in boldface. Pfam motifs are indicated by filled, labeled boxes. The Pfam motif names are: N = PF07963.11 Prokaryotic N-terminal methylation motif; Pilin = PF00114.18 Pilin (bacterial filament); GspH = PF12019.7 Type II transport protein GspH; PilW = PF16074.4 Type IV Pilus-assembly protein W; PilX_N = PF14341.5 PilX N-terminal; ComP_DUS = PF16732.4 Type IV minor pilin ComP, DNA uptake sequence receptor.</p

Competence and twitching phenotypes of null mutations.

<p>All data in this figure were taken using complex media and incubating at 37°C. Both competence and twitching assays were performed on agar: 1.5% for competence and 0.5% for twitching. Color scheme is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182139#pone.0182139.g001" target="_blank">Fig 1</a>. Like symbols indicate that genes are part of the same operon. All data points contain multiple measurements for both competence and twitching. Error bars represent the standard deviation of multiple measurements. For the x-axis standard deviation is given by where x = log and ΔTE is defined as the standard deviation from the mean transformation efficiency, . The detection limit for competence is 10⁻⁹ and for twitching is 0.4. Data points that fall below either detection limit appear on the graph in a “below detection limit” region. Their position within this region has no physical interpretation beyond indicating that they fall below this limit.</p

Type IV pilus protein homologs in ADP1.

<p>Type IV pilus protein homologs in ADP1.</p