A bioengineered pseudovirus nanoparticle displaying SARS-CoV 2 RBD fully protects mice from mortality and weight loss caused by SARS-CoV 2 challenge.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable morbidity and mortality worldwide. Although authorized COVID-19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine-elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, a pseudovirus nanoparticle (PVNP) displaying the receptor binding domains (RBDs) of SARS-CoV-2 spike, named S-RBD, was generated and shown it as a promising COVID-19 vaccine candidate. The S-RBD PVNP was produced using both prokaryotic and eukaryotic systems. A 3D structural model of the S-RBD PVNPs was built based on the known structures of the S60 particle and RBDs, revealing an S60 particle-based icosahedral symmetry with multiple surface-displayed RBDs that retain authentic conformations and receptor-binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD-specific IgG and neutralizing antibodies in mice. The S-RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18-hACE2 mice from mortality and weight loss after a lethal SARS-CoV-2 challenge, supporting the S-RBD PVNPs as a potent COVID-19 vaccine candidate. By contrast, a PVNP displaying the N-terminal domain (NTD) of SARS-CoV-2 spike exhibited only 50% protective efficacy. Since the RBD antigens of our PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S-RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, these non-replicating PVNPs offer a flexible platform for a safe, effective COVID-19 vaccine with minimal manufacturing cost and time.

SARS-CoV-2 Omicron Specific Mutations Affecting Infectivity, Fusogenicity, and Partial TMPRSS2-Independency.

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infection's dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicron's unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicron's TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cell-cell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

Computational and experimental validation of phthalocyanine and hypericin as effective SARS-CoV-2 fusion inhibitors.

Phthalocyanine and hypericin have been previously identified as possible SARS-CoV-2 Spike glycoprotein fusion inhibitors through a virtual screening procedure. In this paper, atomistic simulations of metal-free phthalocyanines and atomistic and coarse-grained simulations of hypericins, placed around a complete model of the Spike embedded in a viral membrane, allowed to further explore their multi-target inhibitory potential, uncovering their binding to key protein functional regions and their propensity to insert in the membrane. Following computational results, pre-treatment of a pseudovirus expressing the SARS-CoV-2 Spike protein with low compounds concentrations resulted in a strong inhibition of its entry into cells, suggesting the activity of these molecules should involve the direct targeting of the viral envelope surface. The combination of computational and in vitro results hence supports the role of hypericin and phthalocyanine as promising SARS-CoV-2 entry inhibitors, further endorsed by literature reporting the efficacy of these compounds in inhibiting SARS-CoV-2 activity and in treating hospitalized COVID-19 patients.Communicated by Ramaswamy H. Sarma.

Identification and characterization of 7-azaindole derivatives as inhibitors of the SARS-CoV-2 spike-hACE2 protein interaction.

The COVID-19 pandemic, caused by SARS-CoV-2, has become a global public health crisis. The entry of SARS-CoV-2 into host cells is facilitated by the binding of its spike protein (S1-RBD) to the host receptor hACE2. Small molecule compounds targeting S1-RBD-hACE2 interaction could provide an alternative therapeutic strategy sensitive to viral mutations. In this study, we identified G7a as a hit compound that targets the S1-RBD-hACE2 interaction, using high-throughput screening in the SARS2-S pseudovirus model. To enhance the antiviral activity of G7a, we designed and synthesized a series of novel 7-azaindole derivatives that bind to the S1-RBD-hACE2 interface. Surprisingly, ASM-7 showed excellent antiviral activity and low cytotoxicity, as confirmed by pseudovirus and native virus assays. Molecular docking and molecular dynamics simulations revealed that ASM-7 could stably bind to the binding interface of S1-RBD-hACE2, forming strong non-covalent interactions with key residues. Furthermore, the binding of ASM-7 caused alterations in the structural dynamics of both S1-RBD and hACE2, resulting in a decrease in their binding affinity and ultimately impeding the viral invasion of host cells. Our findings demonstrate that ASM-7 is a promising lead compound for developing novel therapeutics against SARS-CoV-2.

SMALL MOLECULE INHIBITOR FOR BLOCKING SARS-CoV-2 ENTRY

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel and highly pathogenic coronavirus and is the causative agent of COVID-19, an ongoing pandemic that has posed a serious threat to public health and global economy. Thus, there is a pressing need for therapeutic interventions that target essential viral proteins and regulate virus spread and replication. To invade the host cell, the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein binds to the host cell's ACE2 receptor, followed by cleavage events that allow the Spike protein to fuse with the host cell membrane. Thus, the essential role of Spike protein in ACE2 receptor binding and viral fusion makes it a prime target for therapeutic interventions. Method(s): We performed molecular docking and molecular dynamics (MD) simulation-based virtual screening against SARS-CoV-2 RBD/ACE2 interface using a commercial library of 93,835 drug-like compounds. Compounds with promising docking poses and scores were selected for further MD simulation refinement, from which ten lead compounds were identified. Antiviral potencies of ten lead compounds were evaluated against lentiviral vectors pseudotyped with SARS-CoV-2 Spike to down select to a single lead compound, SAI4. ELISA-based assays were employed to determine the binding affinities of SAI4 to recombinant SARS-CoV-2 RBD. Antiviral potential of SAI4 was validated against genuine SARS-CoV-2 in a BSL3 setting. Result(s): We identified SAI4 as a candidate small molecule, which inhibited SARS-CoV-2 pseudovirus entry with IC50 value of ~18 muM. We determined that SAI4 binds RDB with a Kd of ~20 muM. Using cells engineered to express ACE2 and cells that express physiological levels of ACE2, we found that SAI4 inhibited SARS-CoV-2 pseudovirus entry at both engineered and physiological ACE2 levels. We validated the antiviral potential of SAI4 against genuine SARS-CoV-2 and HCoV-NL63. Lastly, we demonstrated antiviral potential of SAI4 against four SARS-CoV-2 variants of concern (alpha, beta, gamma, and delta). Conclusion(s): Using virtual screening, we identified SAI4 as the promising hit compound which displayed inhibitory activities against SARS-CoV-2 entry and its four variants of concern. Thus, our study will pave the way for further development of small molecules for therapeutic targeting of SARS-CoV-2 entry to combat the COVID-19 pandemic.

On Immunological Studies at Sirius University of Science and Technology.

This short report summarizes the results of recent immunological studies performed at new Sirius University of Science and Technology. The report focuses on studying the features of the immune response to vaccination and revaccination against SARS-CoV-2, as well as on a search of potential agents to prevent infection with this virus.

On Immunological Studies at Sirius University of Science and Technology.

This short report summarizes the results of recent immunological studies performed at the new Sirius University of Science and Technology. The report focuses on studying the features of the immune response to vaccination and revaccination against SARS-CoV-2, as well as on a search of potential agents to prevent infection with this virus.

Endogenous antibody response to SARS-CoV-2 in patients who received monoclonal antibodies against COVID-19: A systematic review

Passive immunization with mAbs has been employed in COVID-19. We performed a systematic review of the literature assessing the endogenous humoral immune response against SARS-CoV-2 in patients treated with mAbs. Administration of mAbs in seronegative patients led to a reduction in both antibody titres and neutralizing activity against the virus.Copyright © 2022

Detection and Diagnosis Technologies for SARS-CoV-2

Detection and diagnosis platforms play key roles in early warning, outbreak control and exit strategy for any pandemic, and they are especially pertinent for the Coronavirus disease 2019 (COVID-19) pandemic. The challenges posed by the speed and extent of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) spread around the globe also offered unprecedented opportunities for the development and deployment of novel strategies and products - not only vaccines and therapeutics, but also diagnostics. This chapter provides a brief summary of the vast array of molecular, serological, cell-based and other diagnostic tools for the specific detection of SARS-CoV-2 infections and immune responses. The focus is on the principles and applications of each platform, while detailed protocols can be found in the cited references. © 2023 by World Scientific Publishing Co. Pte. Ltd.

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection.

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.

Rapid determination of SARS-CoV-2 antibody neutralization titer using Bio-Rad Bio-Plex correlates strongly with pseudovirus-determined neutralization titer.

Accurate and rapid evaluation of SARS-CoV-2 half-maximal neutralizing antibody (nAb) titer (NT50) is an important research tool for measuring nAb responses after prophylaxis or therapeutics for COVID-19 prevention and management. Compared with ACE2-competitive enzyme immunoassays for nAb detection, pseudovirus assays remain low-throughput and labor intensive. A novel application of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 D614G S1 Variant nAb Assay was used to determine NT50 from COVID-19-vaccinated individuals and showed strong correlation to a laboratory-developed SARS-CoV-2 pseudovirus nAb assay. The Bio-Plex nAb assay could provide a rapid, high-throughput, culture-free method for NT50 determination in sera.

Establishment of national standard for anti-SARS-Cov-2 neutralizing antibody in China: The first National Standard calibration traceability to the WHO International Standard.

Neutralizing antibody (NtAb) levels are key indicators in the development and evaluation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. Establishing a unified and reliable WHO International Standard (IS) for NtAb is crucial for the calibration and harmonization of NtAb detection assays. National and other WHO secondary standards are key links in the transfer of IS to working standards but are often overlooked. The Chinese National Standard (NS) and WHO IS were developed by China and WHO in September and December 2020, respectively, the application of which prompted and coordinated sero-detection of vaccine and therapy globally. Currently, a second-generation Chinese NS is urgently required owing to the depletion of stocks and need for calibration to the WHO IS. The Chinese National Institutes for Food and Drug Control (NIFDC) developed two candidate NSs (samples 33 and 66-99) traced to the IS according to the WHO manual for the establishment of national secondary standards through a collaborative study of nine experienced labs. Either NS candidate can reduce the systematic error among different laboratories and the difference between the live virus neutralization (Neut) and pseudovirus neutralization (PsN) methods, ensuring the accuracy and comparability of NtAb test results among multiple labs and methods, especially for samples 66-99. At present, samples 66-99 have been approved as the second-generation NS, which is the first NS calibrated tracing to the IS with 580 (460-740) International Units (IU)/mL and 580 (520-640) IU/mL by Neut and PsN, respectively. The use of standards improves the reliability and comparability of NtAb detection, ensuring the continuity of the use of the IS unitage, which effectively promotes the development and application of SARS-CoV-2 vaccines in China.

Characterisation of the Antibody Response in Sinopharm (BBIBP-CorV) Recipients and COVID-19 Convalescent Sera from the Republic of Moldova.

The early availability of effective vaccines against SARS-CoV-2, the aetiologic cause of COVID-19, has been at the cornerstone of the global recovery from the pandemic. This study aimed to assess the antispike RBD IgG antibody titres and neutralisation potential of COVID-19 convalescent plasma and the sera of Moldovan adults vaccinated with the Sinopharm BBIBP-CorV vaccine. An IgG ELISA with recombinant SARS-CoV-2 spike RBD and two pseudovirus-based neutralisation assays have been developed to evaluate neutralising antibodies against SARS-CoV-2 in biosafety level 2 containment facilities. A significant moderate correlation was observed between IgG titres and the overall neutralising levels for each neutralisation assay (ρ = 0.64, p < 0.001; ρ = 0.52, p < 0.001). A separate analysis of convalescent and vaccinated individuals showed a higher correlation of neutralising and IgG titres in convalescent individuals (ρ = 0.68, p < 0.001, ρ = 0.45, p < 0.001) compared with vaccinated individuals (ρ = 0.58, p < 0.001; ρ = 0.53, p < 0.001). It can be concluded that individuals who recovered from infection developed higher levels of antispike RBD IgG antibodies. In comparison, the Sinopharm-vaccinated individuals produced higher levels of neutralising antibodies than convalescent plasma.

Evaluation of immune evasion in SARS-CoV-2 Delta and Omicron variants.

Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibits immune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Cross-Reactivity of Antibodies in Intravenous Immunoglobulin Preparation for Protection against SARS-CoV-2.

Severe cases of COVID-19 continue to put pressure on medical operations by prolonging hospitalization, occupying intensive care beds, and forcing medical personnel to undergo harsh labor. The eradication of SARS-CoV-2 through vaccine development has yet to be achieved, mainly due to the appearance of multiple mutant-incorporating strains. The present study explored the utility of human intravenous immunoglobulin (IVIG) preparations in suppressing the aggravation of any COVID-19 infection using a SARS-CoV-2 pseudovirus assay. Our study revealed the existence of IgG antibodies in human IVIG preparations, which recognized the spike protein of SARS-CoV-2. Remarkably, the pretreatment of ACE2/TMPRSS2-expressing host cells (HEK293T cells) with IVIG preparations (10 mg/mL) inhibited approximately 40% entry of SARS-CoV-2 pseudovirus even at extremely low concentrations of IgG (0.16-1.25 mg/mL). In contrast, the antibody-dependent enhancement of viral entry was confirmed when SARS-CoV-2 pseudovirus was treated with some products at an IgG concentration of 10 mg/mL. Our data suggest that IVIG may contribute to therapy for COVID-19, including for cases caused by SARS-CoV-2 variants, since IVIG binds not only to the spike proteins of the virus, but also to human ACE2/TMPRSS2. An even better preventive effect can be expected with blood collected after the start of the COVID-19 pandemic.

Inhibitory activity of a sulfated oligo-porphyran from Pyropia yezoensis against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has spread around the world at an unprecedented rate. A more homogeneous oligo-porphyran with mean molecular weight of 2.1 kD, named OP145, was separated from Pyropia yezoensis. NMR analysis showed OP145 was mainly composed of →3)-ß-d-Gal-(1 â†’ 4)-α-l-Gal (6S) repeating units with few replacement of 3,6-anhydride, and the molar ratio was 1:0.85:0.11. MALDI-TOF MS revealed OP145 contained mainly tetrasulfate-oligogalactan with Dp range from 4 to 10 and with no more than two 3,6-anhydro-α-l-Gal replacement. The inhibitory activity of OP145 against SARS-CoV-2 was investigated in vitro and in silico. OP145 could bind to Spike glycoprotein (S-protein) through SPR result, and pseudovirus tests confirmed that OP145 could inhibite the infection with an EC50 of 37.52 µg/mL. Molecular docking simulated the interaction between the main component of OP145 and S-protein. All the results indicated that OP145 had the potency to treat and prevent COVID-19.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean red ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean red ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.