A TLR4 agonist liposome formulation effectively stimulates innate immunity and enhances protection from bacterial infection.

Stimulation of innate immunity can protect against infectious insult and could be used in combination with other therapies. Since antibiotic resistance is an increasing concern, strategies to reduce the dose or eliminate the need for these drugs are warranted. Lipo-CRX is a formulation in which the TLR4 agonist CRX-527 is incorporated into lipid membranes in liposomes. Lipo-CRX is less inflammatory than either CRX-527 or LPS, but retains unique capacity to enhance host defense responses. We compared lipo-CRX to other agonists in vitro using mammalian cells and in vivo in mice, and assessed indicators of innate immune responses and protection from bacterial infection. Lipo-CRX is similar to E. coli LPS in its capacity to activate bovine γδ T cells and to recruit neutrophils into mouse lungs, but with less reactivity in the LAL assay. However, lipo-CRX uniquely induced the production of systemic innate immune cytokines. In the mouse model of brucellosis, delivery of lipo-CRX to the lungs reduced the dissemination of B. abortus. While lipo-CRX or the antibiotic ampicillin alone did not alter B. abortus burdens in the lung, the combination had a synergistic beneficial effect. Our data suggest that stimulating the innate immune system with lipo-CRX, either alone or when combined with antibiotics, can enhance bacterial clearance in the mouse model of brucellosis.

An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model.

Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM17) is a protease that cleaves ectodomains of transmembrane proteins, including that of ACE2 and the proinflammatory cytokine TNF-α, from cell surfaces upon cellular activation. We hypothesized that blockade of ADAM17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM17 using the monoclonal antibody MEDI3622 in the K18-hACE2 transgenic mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly lower lung pathology than saline-treated mice. However, the viral burden in the lungs of MEDI3622-treated mice was significantly increased. Thus, ADAM17 appears to have a critical anti-viral role, but also may promote inflammatory damage. Since the inflammatory cascade is ultimately the reason for adverse outcomes in COVID-19 patients, there may be a therapeutic application for the MEDI3622 antibody.

Severe Acute Respiratory Syndrome Coronavirus 2 Is Detected in the Gastrointestinal Tract of Asymptomatic Endoscopy Patients but Is Unlikely to Pose a Significant Risk to Healthcare Personnel.

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

Titers, Prevalence, and Duration of SARS-CoV-2 Antibodies in a Local COVID-19 Outbreak and Following Vaccination.

Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor-binding domain (RBD)-specific antibodies, their duration, and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest-lasting response, up to 12 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.

Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/µL for a single guide RNA to 106 copies/µL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/µL and is completed using patient samples in less than 30 min.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncation of ORF7a that limits immune suppression.

Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7aΔ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response.

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

SARS-CoV-2 genomic surveillance identifies naturally occurring truncations of ORF7a that limit immune suppression.

Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We have isolated one of these mutant viruses from a patient sample and used viral challenge experiments to demonstrate that Δ115 mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response.

Getting "Inside" Type I IFNs: Type I IFNs in Intracellular Bacterial Infections.

Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial "sensing" mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

Amphotericin B stimulates γδ T and NK cells, and enhances protection from Salmonella infection.

Amphotericin B (AmB) is a commonly used antifungal drug, with well-documented effects on cellular immune responses. We determined that AmB-stimulated γδ T-cell activation and proliferation in vitro at very low concentrations. AmB also enhanced IFN-γ production by NK cells in combination with IL-18. AmB had a greater effect on IFN-γ production in cells isolated from very young animals. Although innate immunostimulatory aspects of AmB have been defined, AmB has not been extensively applied in non-fungal infection settings. Given that γδ T cells are increased and activated in Salmonella infection in cattle, we assessed the effects of AmB in protection from Salmonella enterocolitis in calves. One injection of AmB, at approximately one-tenth of the concentration used in human patients to counter fungal infection, or saline control, was delivered intravenously to calves prior to infection with Salmonella. This single injection caused no adverse effects, reduced disease symptoms from Salmonella enterocolitis and significantly reduced Salmonella bacteria shed in feces of infected animals. Our findings suggest that AmB may be an inexpensive and readily available prophylactic approach for the prevention of bacterial infection in calves.

Oral delivery of oligomeric procyanidins in Apple Poly® enhances type I IFN responses in vivo.

Type I IFN signaling is a central pathway that provides critical innate protection from viral and bacterial infection and can have regulatory outcomes in inflammatory settings. We determined previously that OPCs contained in the dietary supplement APP enhanced responses to type I IFN in vitro. Here, we confirm that OPCs from two different sources significantly increased pSTAT1, whereas a monomeric form of procyanidin did not. We hypothesized that similar responses could be induced in vivo following ingestion of APP. Ingestion of APP before injection of polyI:C enhanced in vivo responses to type I IFNs in mice. When human subjects ingested APP, enhanced responses to type I IFN and enhanced pSTAT1 ex vivo were detected, whereas ingestion of RES, a monomeric polyphenol, induced minimal such changes. Polyphenols are best known for induction of anti-inflammatory and antioxidant responses; however, our findings suggest a unique, nonantioxidant aspect of OPCs that is broadly applicable to many disease settings. The capacity of oral OPCs to enhance type I IFN signaling in vivo can augment innate protection and may, in part, contribute to the noted anti-inflammatory outcome of ingestion of OPCs from many sources.

Solute carrier 11A1 is expressed by innate lymphocytes and augments their activation.

Solute carrier 11A1 (SLC11A1) is a divalent ion transporter formerly known as the natural resistance-associated macrophage protein (NRAMP1) and the Bcg/Lsh/Ity locus. SLC11A1 was thought to be exclusively expressed in monocyte/macrophages and to have roles in phagosome maturation and cell activation. We characterized the expression of SLC11A1 in the majority of human and bovine γδ T cells and NK cells and in human CD3(+)CD45RO(+) T cells. Consistent with a role for iron-dependent inhibition of protein tyrosine phosphatases, SLC11A1(+) lymphocytes were more prone to activation and retained tyrosine phosphorylation. Transfection of SLC11A1 into a human γδ T cell-like line rendered the cells more prone to activation. Nonadherent splenocytes from wild-type mice expressed significantly greater IFN-γ compared with cells from Sv/129 (SLC11A1(-/-)) mice. Our data suggest that SLC11A1 has a heretofore unknown role in activation of a large subset of innate lymphocytes that are critical sources of IFN-γ. SLC11A1(+) animals have enhanced innate IFN-γ expression in response to Salmonella infection compared with SLC11A1(-) mice, which include commonly used inbred laboratory mice. Expression of SLC11A1 in innate lymphocytes and its role in augmenting their activation may account for inconsistencies in studies of innate lymphocytes in different animal models.