Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2.

Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.

Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system.

Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3-△env backbone plasmid HpaI and truncating the C-terminal 21 amino acids of the SARS-CoV-2 spike protein (S), high-titer SARS-CoV-2-Sdel21-AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96-well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS-CoV-2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.

Rational prediction of immunogenicity clustering through cross-reactivity analysis of thirteen SARS-CoV-2 variants.

SARS-CoV-2 breakthrough infections in vaccinated individuals underscore the threat posed by continuous mutating variants, such as Omicron, to vaccine-induced immunity. This necessitates the search for broad-spectrum immunogens capable of countering infections from such variants. This study evaluates the immunogenicity relationship among SARS-CoV-2 variants, from D614G to XBB, through Guinea pig vaccination, covering D614G, Alpha, Beta, Gamma, Delta, BA.1, BA.2, BA.2.75, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB, employing three immunization strategies: three-dose monovalent immunogens, three-dose bivalent immunogens, and a two-dose vaccination with D614G followed by a booster immunization with a variant strain immunogen. Three distinct immunogenicity clusters were identified: D614G, Alpha, Beta, Gamma, and Delta as cluster 1, BA.1, BA.2, and BA.2.75 as cluster 2, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB as cluster 3. Broad-spectrum protection could be achieved through a combined immunization strategy using bivalent immunogens or D614G and XBB, or two initial D614G vaccinations followed by two XBB boosters. A comparison of neutralizing antibody levels induced by XBB boosting and equivalent dosing of D614G and XBB revealed that the XBB booster produced higher antibody levels. The study suggests that vaccine antigen selection should focus on the antigenic alterations among variants, eliminating the need for updating vaccine components for each variant.

Immune escape of BA.2.86 is comparable to XBB subvariants from the plasma of BA.5- and BA.5-XBB-convalescent subpopulations.

The EG.5.1 variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been prevalent since mid-July 2023 in the United States and China. The variant BA.2.86 has become a major concern because it is 34 mutations away from the parental variant BA.2 and >30 mutations from XBB.1.5. There is an urgent need to evaluate whether the immunity of the population and current vaccines are protective against EG.5.1 and BA.2.86. Based on a cohort of two breakthrough-infected groups, the levels of neutralizing antibodies (NAbs) against different subvariants were measured using pseudovirus-based neutralization assays. XBB.1.5, EG.5.1, and BA.2.86 are comparably immune-evasive from neutralization by the plasma of individuals recovered from BA.5 infection (BA.5-convalescent) or XBB.1.9.2/XBB.1.5 infection following BA.5 infection (BA.5-XBB-convalescent). NAb levels against EG.5.1 and BA.2.86 subvariants remained >120 geometric mean titers (GMTs) in BA.5-XBB-convalescent individuals 2 months postinfection but were <40 GMTs in BA.5-convalescent individuals. Furthermore, the XBB-targeting messenger RNA (mRNA) vaccine RQ3033 induced higher levels of NAbs against XBB.1.5, EG.5.1, and BA.2.86 than against BA.5-XBB infection. The results suggest that BA.2.86 and EG.5.1 are unlikely to cause more severe concerns than the currently circulating XBB subvariants and that the XBB.1.5-targeting mRNA vaccine tested has promising protection against EG.5.1 and BA.2.86.

Clarifying the Temperature-Dependent Lithium Deposition/Stripping Process and the Evolution of Inactive Li in Lithium Metal Batteries.

The deposition/stripping behavior of lithium metal is intriguing, and the associated formation of inactive lithium at various temperatures remains elusive, which hinders the practical application of lithium metal batteries. Here, utilizing the variable-temperature operando solid-state nuclear magnetic resonance (SS NMR) technique, we reveal the temperature effects on the lithium microstructure evolution in a carbonate-based electrolyte system. In addition, the mass spectrometry titration (MST) method is used to quantify the evolution of inactive lithium components, including dead lithium, solid electrolyte interface (SEI), and lithium hydride (LiH). Combined SS NMR and MST results show that the morphology of lithium metal is reasonably correlated to the amount of inactive Li formed. At low/ambient temperature, the lithium microstructure has a similar evolution pattern, and its poor morphology leads to a large amount of dead lithium, which dominates capacity loss; however, at high temperature large and dense lithium deposits form with less dead Li detected, and the intensified electrolyte consumption in SEI formation is the major cause for capacity loss. Our phase-field simulation results reveal that the compact lithium deposition formed at higher temperature is due to the more uniformly distributed electric field and Li+ concentration. Lastly, two strategies in forming a dense Li deposit are proposed and tested that show performance-enhancing results.

Development of an automated, high-throughput SARS-CoV-2 neutralization assay based on a pseudotyped virus using a vesicular stomatitis virus (VSV) vector.

ABSTRACTThe global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z' factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

Development of a Bioluminescent Imaging Mouse Model for SARS-CoV-2 Infection Based on a Pseudovirus System.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains widely pandemic around the world. Animal models that are sensitive to the virus are therefore urgently needed to evaluate potential vaccines and antiviral agents; however, SARS-CoV-2 requires biosafety level 3 containment. To overcome this, we developed an animal model using the intranasal administration of SARS-CoV-2 pseudovirus. As the pseudovirus contains the firefly luciferase reporter gene, infected tissues and the viral load could be monitored by in vivo bioluminescent imaging. We used the model to evaluate the protective efficacy of monoclonal antibodies and the tissue tropism of different variants. The model may also be a useful tool for the safe and convenient preliminary evaluation of the protective efficacy of vaccine candidates against SARS-CoV-2, as well as the treatment efficacy of anti-viral drugs.

Sera from breakthrough infections with SARS-CoV-2 BA.5 or BF.7 showed lower neutralization activity against XBB.1.5 and CH.1.1.

From December 2022 to January 2023, SARS-CoV-2 infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) spread in China. It is urgently needed to evaluate the protective immune responses in the infected individuals against the current circulating variants to predict the future potential infection waves, such as the BQ.1.1, XBB.1.5, and CH1.1 variants. In this study, we constructed a panel of pseudotyped viruses for SARS-CoV-2 for the past and current circulating variants, including D614G, Delta, BA.1, BA.5, BF.7, BQ.1.1, XBB.1.5 and CH.1.1. We investigated the neutralization sensitivity of these pseudotyped viruses to sera from individuals who had BA.5 or BF.7 breakthrough infections in the infection wave of last December in China. The mean neutralization ID50 against infected variants BA.5 and BF.7 are 533 and 444, respectively. The highest neutralizing antibody level was observed when tested against the D614G strain, with the ID50 of 742, which is about 1.52-folds higher than that against the BA.5/BF.7 variant. The ID50 for BA.1, Delta, and BQ.1.1 pseudotyped viruses were about 2-3 folds lower when compared to BA.5/BF.7. The neutralization activities of these serum samples against XBB.1.5 and CH.1.1 decreased 7.39-folds and 15.25-folds when compared to that against BA.5/BF.7. The immune escape capacity of these two variants might predict new infection waves in future when the neutralizing antibody levels decrease furtherly.

Pseudotyped Virus for Henipavirus.

The genus Henipavirus (HNV) includes two virulent infectious viruses, Nipah virus (NiV) and Hendra virus (HeV), which are the focus of considerable public health research efforts and have been classified as priority infectious diseases by the World Health Organization. Both viruses are high risk and should be handled in biosafety level 4 laboratories. Pseudotyped viruses containing the envelope proteins of HNV viruses have the same envelope protein structure as the authentic viruses; thus, they can mimic the receptor-binding and membrane fusion processes of authentic viruses with host cells and can be handled in biosafety level 2 laboratories. These characteristics enable pseudotyped viruses to be widely used in studies of viral infection mechanisms (packaging, budding, virus attachment, membrane fusion, viral entry, and glycosylation), inhibitory drug screening assays, and monoclonal antibody neutralization characteristics. This review will provide an overview of the progress of research concerning pseudotyped virus packaging systems for NiV and HeV.

Understanding the failure process of sulfide-based all-solid-state lithium batteries via operando nuclear magnetic resonance spectroscopy.

The performance of all-solid-state lithium metal batteries (SSLMBs) is affected by the presence of electrochemically inactive (i.e., electronically and/or ionically disconnected) lithium metal and solid electrolyte interphase (SEI), which are jointly termed inactive lithium. However, the differentiation and quantification of inactive lithium during cycling are challenging, and their lack limits the fundamental understanding of SSLMBs failure mechanisms. To shed some light on these crucial aspects, here, we propose operando nuclear magnetic resonance (NMR) spectroscopy measurements for real-time quantification and evolution-tracking of inactive lithium formed in SSLMBs. In particular, we examine four different sulfide-based solid electrolytes, namely, Li10GeP2S12, Li9.54Si1.74P1.44S11.7Cl0.3, Li6PS5Cl and Li7P3S11. We found that the chemistry of the solid electrolyte influences the activity of lithium. Furthermore, we demonstrate that electronically disconnected lithium metal is mainly found in the interior of solid electrolytes, and ionically disconnected lithium metal is found at the negative electrode surface. Moreover, by monitoring the Li NMR signal during cell calendar ageing, we prove the faster corrosion rate of mossy/dendritic lithium than flat/homogeneous lithium in SSLMBs.

Olivine-Type MgMn0.5 Zn0.5 SiO4 Cathode for Mg-Batteries: Experimental Studies and First Principles Calculations.

Magnesium driven reaction in olivine-type MgMn0.5 Zn0.5 SiO4 structure is subject of study by experimental tests and density functional theory (DFT) calculations. The partial replacement of Mn in Oh sites by other divalent metal such as Zn to get MgMn0.5 Zn0.5 SiO4 cathode is successfully developed by a simple sol-gel method. Its comparison with the well-known MgMnSiO4 olivine-type structure with (Mg)M1 (Mn)M2 SiO4 cations distribution serves as the basis of this study to understand the structure, and the magnesium extraction/insertion properties of novel olivine-type (Mg)M1 (Mn0.5 Zn0.5 )M2 SiO4 composition. This work foresees to extend the study to others divalent elements in olivine-type (Mg)M1 (Mn0.5 M0.5 )M2 SiO4 structure with M = Fe, Ca, Mg, and Ni by DFT calculations. The obtained results indicate that the energy density can be attuned between 520 and 440 W h kg-1 based on two properties of atomic weight and redox chemistry. The presented results commit to open new paths toward development of cathodes materials for Mg batteries.

Human antibody BD-218 has broad neutralizing activity against concerning variants of SARS-CoV-2.

As SARS-CoV-2 variants of concern (VOC) reduce the effectiveness of existing anti-COVID therapeutics, it is increasingly critical to identify highly potent neutralizing antibodies (nAbs) that bind to conserved regions across multiple variants, especially beta, delta, and omicron variants. Using single-cell sequencing with biochemical methods and pseudo-typed virus neutralization experiments, here we report the characterization of a potent nAb BD-218, identified from an early screen of patients recovering from the original virus. We have determined the cryo-EM structure of the BD-218/spike protein complex to define its epitope in detail, which revealed that BD-218 interacts with a novel epitope on the receptor-binding domain (RBD) of the spike protein. We concluded that BD-218 is a highly effective and broadly active nAb against SARS-CoV-2 variants with promising potential for therapeutic development.

Insights into the Reaction Mechanisms of Nongraphitic High-Surface Porous Carbons for Application in Na- and Mg-Ion Batteries.

The fabrication of low-cost carbon materials and high-performance sodium- and magnesium-ion batteries comprising hierarchical porous electrodes and superior electrolytes is necessary for complementing Li-ion energy storage. In this work, nongraphitic high-surface porous carbons (NGHSPCs) exhibited an unprecedented formation of n-stages (stage-1 and stage-2) due to the co-intercalation of sodium (Na(dgm)2C20) with diglyme. X-ray diffraction patterns, Patterson diagram, Raman spectra, and IR spectra suggested the presence of n-stages. This phenomenon implies an increase of the initial capacity (∼200 mAh g-1) and good Na-ion diffusion (2.97 × 10-13 cm2 s-1), employing diglyme as compared to standard electrolytes containing propylene carbonate and fluoroethylene carbonate. Additionally, the current approach is scalable to full Na- and Mg-ion cells by using t-Na5V(PO4)2F2 and MgMnSiO4 cathodes, respectively, reaching 250 and 110 W h kg-1 based on the anode mass. The simultaneous Mg (de)insertion from/into MgMnSiO4 and the adsorption/desorption of bistriflimide ions on the NGHSPC surface is responsible for capacity enhancement.

Cross-reactivity of eight SARS-CoV-2 variants rationally predicts immunogenicity clustering in sarbecoviruses.

A steep rise in Omicron reinfection cases suggests that this variant has increased immune evasion ability. To evaluate its antigenicity relationship with other variants, antisera from guinea pigs immunized with spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were cross-tested against pseudotyped variants. The neutralization activity against Omicron was markedly reduced when other VOCs or VOIs were used as immunogens, and Omicron (BA.1)-elicited sera did not efficiently neutralize the other variants. However, a Beta or Omicron booster, when administered as the 4th dose 3-months after the 3rd dose of any of the variants, could elicit broad neutralizing antibodies against all of the current variants including Omicron BA.1. Further analysis with 280 available antigen-antibody structures and quantification of immune escape from 715 reported neutralizing antibodies provide explanations for the observed differential immunogenicity. Three distinct clades predicted using an in silico algorithm for clustering of sarbecoviruses based on immune escape provide key information for rational design of vaccines.

Mitigating the Surface Reconstruction of Ni-Rich Cathode via P2-Type Mn-Rich Oxide Coating for Durable Lithium Ion Batteries.

Ni-rich materials have received widespread attention as one of the mainstream cathodes in high-energy-density lithium-ion batteries for electric vehicles. However, Ni-rich cathodes suffer from severe surface reconstruction in a high delithiation state, constraining their rate capabilities and life span. Herein, a novel P2-type NaxNi0.33Mn0.67O2 (NNMO) is rationally selected as the surficial modification layer for LiNi0.8Co0.1Mn0.1O2 (NCM811) cathode, which undergoes a spontaneous Na+-Li+ exchange reaction to form an O2-type LixNi0.33Mn0.67O2 (LNMO) layer revealed by combining X-ray diffraction and solid-state nuclear magnetic resonance techniques. Owing to the specific oxygen stacking sequence, O2-type LNMO significantly prevents the initial layered structure of NCM811 from transforming to the spinel or rock-salt phases during cycling, thus effectively maintaining the integral surficial structure and the Li+ diffusion channels of NCM811. Eventually, the NNMO@NCM811 electrode yields enhanced thermal stability, outstanding rate performance, and long cycling stability with 80% capacity retention after 294 cycles at 200 mA g-1, and its life span is further extended to 531 cycles while enhancing the mechanical stability of the bulk material.

Antigenicity comparison of SARS-CoV-2 Omicron sublineages with other variants contained multiple mutations in RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, particularly those with multiple mutations in receptor-binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID-19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster.

Analysis of SARS-CoV-2 variants B.1.617: host tropism, proteolytic activation, cell-cell fusion, and neutralization sensitivity.

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell-cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.