The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease that began in 2019 (COVID-19), has been responsible for 1.4 million deaths worldwide as of 13 November 2020. Because at the time of writing no vaccine is yet available, a rapid diagnostic assay is very urgently needed. Herein, we present the development of anti-spike antibody attached gold nanoparticles for the rapid diagnosis of specific COVID-19 viral antigen or virus via a simple colorimetric change observation within a 5 minute time period. For rapid and highly sensitive identification, surface enhanced Raman spectroscopy (SERS) was employed using 4-aminothiophenol as a reporter molecule, which is attached to the gold nanoparticle via an Au-S bond. In the presence of COVID-19 antigen or virus particles, owing to the antigen-antibody interaction, the gold nanoparticles undergo aggregation, changing color from pink to blue, which allows for the determination of the presence of antigen or virus very rapidly by the naked eye, even at concentrations of 1 nanogram (ng) per mL for COVID-19 antigen and 1000 virus particles per mL for SARS-CoV-2 spike protein pseudotyped baculovirus. Importantly, the aggregated gold nanoparticles form "hot spots" to provide very strong SERS signal enhancement from anti-spike antibody and 4-aminothiophenol attached gold nanoparticles via light-matter interactions. Finite-difference time-domain (FDTD) simulation data indicate a 4-orders-of-magnitude Raman enhancement in "hot spot" positions when gold nanoparticles form aggregates. Using a portable Raman analyzer, our reported data demonstrate that our antibody and 4-aminothiophenol attached gold nanoparticle-based SERS probe has the capability to detect COVID-19 antigen even at a concentration of 4 picograms (pg) per mL and virus at a concentration of 18 virus particles per mL within a 5 minute time period. Using HEK293T cells, which express angiotensin-converting enzyme 2 (ACE2), by which SARS-CoV-2 enters human cells, we show that anti-spike antibody attached gold nanoparticles have the capability to inhibit infection by the virus. Our reported data show that antibody attached gold nanoparticles bind to SARS-CoV-2 spike protein, thereby inhibiting the virus from binding to cell receptors, which stops virus infection and spread. It also has the capability to destroy the lipid membrane of the virus.

Aptamer Conjugated Gold Nanostar-Based Distance-Dependent Nanoparticle Surface Energy Transfer Spectroscopy for Ultrasensitive Detection and Inactivation of Corona Virus.

The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been determined to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Effective screening of SARS-CoV-2 neutralizing antibodies in patient serum using lentivirus particles pseudotyped with SARS-CoV-2 spike glycoprotein.

Pseuodotyped particles have significant importance and use in virology as tools for studying the biology of highly pathogenic viruses in a lower biosafety environment. The biological, chemical, and serological studies of the recently emerged SARS-CoV-2 will be greatly aided by the development and optimization of a suitable pseudotyping system. Here, we pseudotyped the SARS-CoV-2 Spike glycoprotein (SPG) on a traditional retroviral (MMLV) as well as a third generation lentiviral (pLV) vector and tested the transduction efficiency in several mammalian cell lines expressing SARS-CoV-2 receptor hACE2. While MMLV pseudotyped the vesicular stomatitis virus G glycoprotein (VSV-G) efficiently, it could not pseudotype the full-length SPG. In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titers of pLV-G particles were produced. Among all the tested mammalian cells, 293Ts expressing hACE2 were most efficiently transduced using the pLV-S system. The pLV-S particles were efficiently neutralized by diluted serum (>:640) from recently recovered COVID-19 patients who showed high SARS-CoV-2 specific IgM and IgG levels. In summary, pLV-S pseudotyped virus provides a valid screening tool for the presence of anti SARS-CoV-2 specific neutralizing antibodies in convalescent patient serum.

The structure-activity relationship of the interactions of SARS-CoV-2 spike glycoproteins with glucuronomannan and sulfated galactofucan from Saccharina japonica.

The SARS-CoV-2 spike glycoproteins (SGPs) and human angiotensin converting enzyme 2 (ACE2) are the two key targets for the prevention and treatment of COVID-19. Host cell surface heparan sulfate (HS) is believed to interact with SARS-CoV-2 SGPs to facilitate host cell entry. In the current study, a series of polysaccharides from Saccharina japonica were prepared to investigate the structure-activity relationship on the binding abilities of polysaccharides (oligosaccharides) to pseudotype particles, including SARS-CoV-2 SGPs, and ACE2 using surface plasmon resonance. Sulfated galactofucan (SJ-D-S-H) and glucuronomannan (Gn) displayed strongly inhibited interaction between SARS-CoV-2 SGPs and heparin while showing negligible inhibition of the interaction between SARS-CoV-2 SGPs and ACE2. The IC50 values of SJ-D-S-H and Gn in blocking heparin SGP binding were 27 and 231 nM, respectively. NMR analysis showed that the structure of SJ-D-S-H featured with a backbone of 1, 3-linked α-L-Fucp residues sulfated at C4 and C2/C4 and 1, 3-linked α-L-Fucp residues sulfated at C4 and branched with 1, 6-linked ß-D-galacto-biose; Gn had a backbone of alternating 1, 4-linked ß-D-GlcAp residues and 1, 2-linked α-D-Manp residues. The sulfated galactofucan and glucuronomannan showed strong binding ability to SARS-CoV-2 SGPs, suggesting that these polysaccharides might be good candidates for preventing and/or treating SARS-CoV-2.