The evolution of SARS-CoV-2 poses challenges for vaccines and immunotherapies

A CAR-T-cell recipient developed severe COVID-19, intractable RNAemia, and viral replication lasting >2 months. Pre-mortem endotracheal aspirate contained 2x10 10 SARS-CoV-2 RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intra-host virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

Head and neck squamous cell carcinoma (HNSCC) is a devastating disease for which new treatments, such as immunotherapy are needed. Synthetic double-stranded RNAs, which activate toll-like receptor 3 (TLR3), have been used as potent adjuvants in cancer immunotherapy by triggering a proapoptotic response in cancer cells. A better understanding of the mechanism of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis could lead to improvements in current treatment. Using paired, autologous primary and metastatic HNSCC cells we previously showed that metastatic, but not primary tumor-derived cells, were unable to activate prosurvival NF-κB in response to p(I):p(C) resulting in an enhanced apoptotic response. Here, we show that transcriptional downregulation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in metastatic HNSCC cells causes a loss of TLR3-mediated NF-κB signaling, resulting in enhanced apoptosis. Loss of RIPK1 strongly correlates with metastatic disease in a cohort of HNSCC patients. This downregulation of RIPK1 is possibly mediated by enhanced methylation of the RIPK1 promoter in tumor cells and enhances protumorigenic properties such as cell migration. The results described here establish a novel mechanism of TLR3-mediated apoptosis in metastatic cells and may create new opportunities for using double stranded RNA to target metastatic tumor cells.

Hepatitis C virus (HCV) entry is a multiple-step process involving a number of host factors and hence represents a promising target for new antiviral drug development. In search of novel inhibitors of HCV infection, we found that a human apolipoprotein E (apoE) peptide, hEP, containing both a receptor binding fragment and a lipid binding fragment of apoE, specifically blocked the entry of cell culture grown HCV (HCVcc) at sub-micromolar concentrations. hEP caused little cytotoxicity in vitro and remained active even if left 24 hours in cell culture. Interestingly, hEP inhibited neither HIV-HCV pseudotypes (HCVpp) nor HIV and Dengue virus (DENV) infection. Further characterization mapped the anti-HCV activity to a 32-residue region that harbors the receptor binding domain of apoE, but this fragment must contain a cysteine residue at the N-terminus to mediate dimer formation. The anti-HCV activity of the peptide appears to be dependent on both its length and sequence and correlates with its ability to bind lipids. Finally, we demonstrated that the apoE-derived peptides directly blocked the binding of both HCVcc and patient serum-derived virus to hepatoma cells as well as primary human hepatocytes. Conclusion apoE peptides potently inhibit HCV infection and suggest a direct role of apoE in mediating HCV entry. Our findings also highlight the potential of developing apoE mimetic peptides as novel HCV entry inhibitors by targeting HCV-host interactions.

Defensive droplets from glandular hairs of the pupa of the Mexican bean beetle, Epilachna varivestis, contain a group of structurally novel alkaloids, the azamacrolides. The major constituent of this secretion, epilachnene, is shown to be (5Z)-11-propyl-12-azacyclotetradec-5-en-14-olide. The secretion also contains an epilachnadiene and trace amounts of three closely related components. (Fig. 1). Suspecting this fluid to be defensive, we exposed pupae to ant attacks in a series of laboratory presentations. The pupae were individually placed on the bottom of small Petri dishes that served as the foraging arenas for laboratory colonies of the ants (Leptothorax longispinosus). The results, which were videotaped, were consistent and dramatic. The moment an individual ant, in an exploratory approach to the pupa, contacted some of the glandular hairs, it backed away and cleansed itself. While retreating, it repeatedly dragged its mouthparts and/or antennae against the substrate. None of the pupae (n = 10; each in a different arena) were injured during the 10-min presentations, although each was contacted by dozens of ants during this period (all pupae eventually gave rise to adults). We proceeded to collect the secretory droplets for analysis and here report on the chemicals isolated from the fluid (5). MATERIALS AND METHODSSample Collection. Secretion from the glandular hairs, collected with microcapillaries, was sealed directly in glass tubes (1.8 x 20 mm) or extracted with ether or dichloromethane (100 ul). For NMR spectroscopy, a sample (500 pupae) was taken up in 400 ul of C2HC13 (2H, 99.96%).Analytical Procedures. Samples were analyzed by gas chromatography (GC) on a Hewlett-Packard (HP) 5890 instruThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. ment equipped with a split/splitless injector, a flame ionization detector, and an HP 3396A integrator. Undiluted samples in glass capillaries were injected by a solid sampling technique (6, 7); solutions were introduced by splitless injection. Low-resolution mass spectrometry (MS) was carried out with an HP 5890 gas chromatograph linked to a Finnigan ion-trap detector (ITD 800) or an HP mass selective detector. Chemical ionization mass spectra were obtained on the ITD with methane as the reagent gas. High-resolution mass spectra were obtained on a Kratos 890 instrument. Gas-phase IR spectra were obtained with an HP 5890 gas chromatograph linked to an HP 5965A IR detector. Unless otherwise noted, 1H and 13C NMR spectra were obtained in C2HC13 on a Varian XL-400 instrument.Derivatization Techniques. (i) Microhydrogenation. A small sample of secretion in ether (50 ,ul) was placed in a glass vial and =0.5 mg of 10%o Pd on activated charcoal was added. A balloon filled with hydrogen was attached to the vial. After 10 hr, 25 ul of ether was added and the supernatant was withdrawn and examined by GC/MS.(ii) A...

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients. IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.

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Current population genotyping methods for resistance monitoring are high cost and low throughput. NGS, combined with simpler sample collection and storage matrices (e.g. dried blood spots), has considerable potential to broaden global surveillance and patient monitoring for HIVDR. Recent adaptions of NGS to identify integration sites of HIV in the human genome and to characterize the integrated HIV proviruses are likely to facilitate investigations of the impact of experimental 'curative' interventions on HIV reservoirs.