SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Sprayable copper and copper–zinc nanowires inks for antiviral surface coating

Copper alloys are known for their high antimicrobial efficacy. Retrofitting high-touch surfaces in public space with solid copper components is expensive and often impractical. Directly coating copper onto these high-touch surfaces can be achieved with hot or cold spray, but the procedure is complicated and requires special equipment. This article reports on the development of sprayable copper and copper–zinc nanowire inks for antiviral surface coating applications. Our results show that copper nanowires inactivate the SARS-CoV-2 virus faster than bulk copper. And a trace amount of zinc addition has a significant effect in enhancing the virucidal effect. More importantly, these nanowire inks are sprayable. They can be easily applied on high-touch surfaces with a spray can. When combined with common chemical disinfectants, the copper-based nanowire ink spray may prolong the disinfecting effect well after application. SEM and TEM images of copper and copper–zinc nanowires that are sprayable for antiviral surface coating.

Effective antiviral coatings for deactivating SARS-CoV-2 virus on N95 respirator masks or filters.

The application of antiviral coatings to masks and respirators is a potential mitigating step toward reducing viral transmission during the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. The use of appropriate masks, social distancing, and vaccines is the immediate solution for limiting the viral spread and protecting people from this virus. N95 respirator masks are effective in filtering the virus particles, but they cannot kill or deactivate the virus. We report a possible approach to deactivating SARS-CoV-2 by applying an antimicrobial coating (Goldshield 75) to masks and respirators, rendering them suitable for repeated use. Masks coated with Goldshield 75 demonstrated continuous inactivation of the Alpha and Beta variants of the SARS-CoV-2 over a 3-day period and no loss of inactivation when stored at temperatures at 50 °C.

Automated Flow Synthesis of Peptide–PNA Conjugates

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5′ untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 μM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents. A fully automated flow instrument is used to manufacture cell-penetrating peptide-conjugated PNAs in a single shot. Using this technology, novel synthetic PPNAs were designed and found to reduce SARS-CoV-2 viral titer by >95%.

Enhanced apoptosis as a possible mechanism to self-limit SARS-CoV-2 replication in porcine primary respiratory epithelial cells in contrast to human cells.

The ability of SARS-CoV to infect different species, including humans, dogs, cats, minks, ferrets, hamsters, tigers, and deer, pose a continuous threat to human and animal health. Pigs, though closely related to humans, seem to be less susceptible to SARS-CoV-2. Former in vivo studies failed to demonstrate clinical signs and transmission between pigs, while later attempts using a higher infectious dose reported viral shedding and seroconversion. This study investigated species-specific cell susceptibility, virus dose-dependent infectivity, and infection kinetics, using primary human (HRECs) and porcine (PRECs) respiratory epithelial cells. Despite higher ACE2 expression in HRECs compared to PRECs, SARS-CoV-2 infected, and replicated in both PRECs and HRECs in a dose-dependent manner. Cytopathic effect was particularly more evident in PRECs than HRECs, showing the hallmark morphological signs of apoptosis. Further analysis confirmed an early and enhanced apoptotic mechanism driven through caspase 3/7 activation, limiting SARS-CoV-2 propagation in PRECs compared to HRECs. Our findings shed light on a possible mechanism of resistance of pigs to SARS-CoV-2 infection, and it may hold therapeutic value for the treatment of COVID-19.

Automated Flow Synthesis of Peptide-PNA Conjugates.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5' untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 µM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Induction of Potent and Durable Neutralizing Antibodies Against SARS-CoV-2 Using a Receptor Binding Domain-Based Immunogen.

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.