Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment.

The Coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated, and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, was generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 is active against rRBD proteins from Omicron variants BA.2 and BA.4.5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptides and hybridomas/mAbs and quantitative antigen-binding data are under the intellectual property management of the Clemson University Research Foundation, and the three CDRs have been submitted as an invention disclosure for further patenting and commercialization.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Differential Pathogenesis of SARS-CoV-2 Variants of Concern in Human ACE2-Expressing Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current pandemic, resulting in millions of deaths worldwide. Increasingly contagious variants of concern (VoC) have fueled recurring global infection waves. A major question is the relative severity of the disease caused by previous and currently circulating variants of SARS-CoV-2. In this study, we evaluated the pathogenesis of SARS-CoV-2 variants in human ACE-2-expressing (K18-hACE2) mice. Eight-week-old K18-hACE2 mice were inoculated intranasally with a representative virus from the original B.1 lineage or from the emerging B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), or B.1.1.529 (omicron) lineages. We also infected a group of mice with the mouse-adapted SARS-CoV-2 (MA10). Our results demonstrate that B.1.1.7, B.1.351 and B.1.617.2 viruses are significantly more lethal than the B.1 strain in K18-hACE2 mice. Infection with the B.1.1.7, B.1.351, and B.1.617.2 variants resulted in significantly higher virus titers in the lungs and brain of mice compared with the B.1 virus. Interestingly, mice infected with the B.1.1.529 variant exhibited less severe clinical signs and a high survival rate. We found that B.1.1.529 replication was significantly lower in the lungs and brain of infected mice in comparison with other VoC. The transcription levels of cytokines and chemokines in the lungs of B.1- and B.1.1.529-infected mice were significantly less when compared with those challenged with other VoC. Together, our data provide insights into the pathogenesis of previous and circulating SARS-CoV-2 VoC in mice.

SARS-CoV-2 Infects Primary Neurons from Human ACE2 Expressing Mice and Upregulates Genes Involved in the Inflammatory and Necroptotic Pathways.

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice.

Neuroinvasion and Encephalitis Following Intranasal Inoculation of SARS-CoV-2 in K18-hACE2 Mice.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system (CNS). Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed on day 3 and declined on days 5 and 6 after infection. By contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals on days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

SARS-CoV-2 Variants of Concern Infect the Respiratory Tract and Induce Inflammatory Response in Wild-Type Laboratory Mice.

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern pose a major threat to public health, due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts, in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared with the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-α, IL-1ß) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. In addition, robust expression of viral nucleocapsid protein and histopathological changes were detected in the lungs of B.1.351-infected mice. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.

The FDA-approved gold drug auranofin inhibits novel coronavirus (SARS-COV-2) replication and attenuates inflammation in human cells.

SARS-COV-2 has recently emerged as a new public health threat. Herein, we report that the FDA-approved drug, auranofin, inhibits SARS-COV-2 replication in human cells at low micro molar concentration. Treatment of cells with auranofin resulted in a 95% reduction in the viral RNA at 48 h after infection. Auranofin treatment dramatically reduced the expression of SARS-COV-2-induced cytokines in human cells. These data indicate that auranofin could be a useful drug to limit SARS-CoV-2 infection and associated lung injury due to its antiviral, anti-inflammatory and anti-reactive oxygen species (ROS) properties. Further animal studies are warranted to evaluate the safety and efficacy of auranofin for the management of SARS-COV-2 associated disease.

New Prototype Screened Doors and Windows for Excluding Mosquitoes from Houses: A Pilot Study in Rural Gambia.

Despite compelling evidence that modern housing protects against malaria, houses in endemic areas are still commonly porous to mosquitoes. The protective efficacy of four prototype screened doors and two windows designs against mosquito house entry, their impact on indoor climate, as well as their use, durability and acceptability was assessed in a Gambian village. A baseline survey collected data on all the houses and discrete household units, each consisting of a front and back room, were selected and randomly allocated to the study arms. Each prototype self-closing screened door and window was installed in six and 12 units, respectively, with six unaltered units serving as controls. All prototype doors reduced the number of house-entering mosquitoes by 59-77% in comparison with the control houses. The indoor climate of houses with screened doors was similar to control houses. Seventy-nine percentage of door openings at night occurred from dusk to midnight, when malaria vectors begin entering houses. Ten weeks after installation the doors and windows were in good condition, although 38% of doors did not fully self-close and latch (snap shut). The new doors and windows were popular with residents. The prototype door with perforated concertinaed screening was the best performing door because it reduced mosquito entry, remained fully functional, and was preferred by the villagers. Screened doors and windows may be useful tools for reducing vector exposure and keeping areas malaria-free after elimination, when investment in routine vector control becomes difficult to maintain.

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue.

Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.

Cell-selective proteomics for biological discovery.

Cells alter the proteome to respond to environmental and developmental cues. Global analysis of proteomic responses is of limited value in heterogeneous environments, where there is no 'average' cell. Advances in sequencing, protein labeling, mass spectrometry, and data analysis have fueled recent progress in the investigation of specific subpopulations of cells in complex systems. Here we highlight recently developed chemical tools that enable cell-selective proteomic analysis of complex biological systems, from bacterial pathogens to whole animals.

Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag.

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(i)-catalysed azide-alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows subsequent quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT), as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation.

Long-lived engineering of glycans to direct stem cell fate.

Glycans mediate many critical, long-term biological processes, such as stem cell differentiation. However, few methods are available for the sustained remodeling of cells with specific glycan structures. A new strategy that enables the long-lived presentation of defined glycosaminoglycans on cell surfaces using HaloTag proteins (HTPs) as anchors is reported. By controlling the sulfation patterns of heparan sulfate (HS) on pluripotent embryonic stem cell (ESC) membranes, it is demonstrated that specific glycans cause ESCs to undergo accelerated exit from self-renewal and differentiation into neuronal cell types. Thus, the stable display of glycans on HTP scaffolds provides a powerful, versatile means to direct key signaling events and biological outcomes such as stem cell fate.

Directing neuronal signaling through cell-surface glycan engineering.

The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function. We report a method to modify cell surfaces with specific sulfated chondroitin sulfate (CS) glycosaminoglycans using chemically modified liposomes. Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth. This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.