Spike Ectodomain Research Articles

Insect cell expression and purification of recombinant SARS-COV-2 spike proteins that demonstrate ACE2 binding.

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).

Advances in purification of SARS-CoV-2 spike ectodomain protein using high-throughput screening and non-affinity methods

The spike (S) glycoprotein of the pandemic virus, SARS-CoV-2, is a critically important target of vaccine design and therapeutic development. A high-yield, scalable, cGMP-compliant downstream process for the stabilized, soluble, native-like S protein ectodomain is necessary to meet the extensive material requirements for ongoing research and development. As of June 2021, S proteins have exclusively been purified using difficult-to-scale, low-yield methodologies such as affinity and size-exclusion chromatography. Herein we present the first known non-affinity purification method for two S constructs, S_dF_2P and HexaPro, expressed in the mammalian cell line, CHO-DG44. A high-throughput resin screen on the Tecan Freedom EVO200 automated bioprocess workstation led to identification of ion exchange resins as viable purification steps. The chromatographic unit operations along with industry-standard methodologies for viral clearances, low pH treatment and 20 nm filtration, were assessed for feasibility. The developed process was applied to purify HexaPro from a CHO-DG44 stable pool harvest and yielded the highest yet reported amount of pure S protein. Our results demonstrate that commercially available chromatography resins are suitable for cGMP manufacturing of SARS-CoV-2 Spike protein constructs. We anticipate our results will provide a blueprint for worldwide biopharmaceutical production laboratories, as well as a starting point for process intensification.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

The SARS-CoV-2 spike protein reversibly samples an open-trimer conformation exposing novel epitopes

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we have uncovered that in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This conformation—an open trimer—contains easily accessible RBDs and exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes.

An automated chemiluminescent immunoassay (CLIA) detects SARS-CoV-2 neutralizing antibody levels in COVID-19 patients and vaccinees

ObjectivesA specific and sensitive automated chemiluminescent immunoassay (CLIA) was developed to detect neutralizing antibody (NAb) levels. This assay can be used for the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, treatment and vaccine evaluation.MethodsThe SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike ectodomain as antigens were detected by CLIA. Sera NAb titers and concentrations from 860 SARS-CoV-2 vaccinees, 232 SARS-CoV-2 convalescent patients and 675 healthy individuals were tested by microneutralization test (MNT) and CLIA, respectively. Mathematical models were established to evaluate the relationship between two variables in different groups.ConclusionsWith the RBD-based CLIA protocol, CLIA can be used to replace MNT to test SARS-CoV-2 NAb. Vaccine effectiveness, protectiveness and durability can be evaluated effectively by mathematical models. It isResultsAnalysing the relationship between NAb titers and concentrations, R2 for the decision-making tree was 0.870 and that of progressive linear fitting was 0.821. The receiver operating characteristic curve indicated specificity of 78.1%, sensitivity of 87.4%, cut-off value of 6.43 AU/mL and borderline range of 5.79–7.07 AU/mL for CLIA. Three-quarters (75.4%) of vaccinees were found to be NAb positive, and 5.35% vaccinees had NAb protective capability. The half-life of NAb in vaccinees was 10–11 weeks.for vaccinees to take a NAb assay periodically.

Sars-Cov-2 Antibody Decay Monitoring in mRNA Vaccinated Multiple Myeloma Patients

Introduction: Amid the current COVID-19 pandemic, the highest mortality rates are among the elderly and immunocompromised. Multiple myeloma (MM) patients are immunocompromised and often are elderly. Not only do MM patients develop more frequent infections, but it is one of the leading causes of death for these patients. This population develops more severe COVID-19 due to various mechanisms that impair their ability to fight infection. This also reduces their ability to generate immunity from vaccination, as has been demonstrated by their diminished responses to vaccines for various respiratory illnesses. It follows that while phase III trial results for mRNA1273 and BNT162b2 COVID-19 vaccines showed an efficacy of 94-95% against even mild infection with this virus, the efficacy has been shown to be lower among MM patients. We recently evaluated patients' antibody responses to vaccination for COVID-19 by measuring anti-spike IgG levels in patient serum from before vaccination (baseline) and two weeks after dose 2 (D2W2) of vaccination with mRNA-1273 or BNT162b2, and found that most MM patients have impaired responses (Stampfer et al., Leukemia 2021). Patients who received mRNA-1273 vaccine had higher antibody levels than those who were vaccinated with BNT162b2, and specific clinical and myeloma-related characteristics predicted vaccine responsiveness. In the current study, we are monitoring anti-spike IgG antibody levels at Dose 2 Week 8 (D2W8) and Dose 2 Week 16 (D2W16) post-mRNA vaccination among these patients and in age-matched healthy controls to investigate antibody decay.Methods: Participants in the trial included MM patients (n=91) at the Berenson Cancer Center, and age-matched healthy controls (n=27). Healthy subjects were not known to be immunocompromised or currently receiving immunosuppressive therapy. Vaccination was done outside of the clinic and subjects provided copies of their CDC-issued COVID-19 vaccination cards to confirm dosing dates. Sera from vaccinated individuals were drawn at baseline (0-60 days prior to first vaccine dose) and at intervals following their second dose (14-21, 56-70, and 112-126 days post-vaccination). Background levels were determined from the clinic's serum bank of healthy subjects drawn pre-April 2019. Using an ELISA-based assay that detects IgG antibodies to SARS-CoV-2 spike protein, we determined Anti-SARS-CoV-2 spike ectodomain serum antibody levels and quantified them in IU/mL based on the WHO International Standard 20/136.Results: We analyzed the patient and control populations, and specifically for those classified as responders from our original published study (D2W2 >50 IU/mL). All controls but only 70% of patients (64/91) were classified as responders, so this responder-specific analysis was only relevant for patients. There was a significant decline in anti-SARS-CoV-2 spike antibodies from D2W2 to D2W8 for both patients and controls; D2W16 results are pending. Median values dropped from 283.1 IU/mL to 90.9 IU/mL among patients, and 893.6 IU/mL to 354.4 IU/mL among controls. Median antibody levels among patients classified as responders dropped from 482.9 IU/mL to 145.5 IU/mL (p <0.0001). We also found that the D2W8 value for patients was significantly lower than that of controls (90.9 IU/mL vs 354.4 IU/mL, p<0.0001), as well as among the responder group (145.5 IU/mL vs 354.4 IU/mL, p=0.0005). A cutoff of <147 IU/mL has previously been associated with an increased risk for breakthrough infections. Spike antibody levels of 50% of patients who responded and 64.8% of all patients were <147 IU/mL at D2W8, whereas only 7.4% of controls were <147 IU/mL (p<0.0001).Conclusions: The markedly lower anti-spike antibody levels among MM patients compared to healthy controls at week 8 post-vaccination indicate that they remain at high risk for breakthrough infections. Combined with their rapid decline in anti-spike antibody levels over only a 6-week period, this indicates that even MM patients who responded initially to vaccination are likely to require boosters sooner than healthy individuals. This immunocompromised population remains at high risk even following vaccination and should continue to maintain social distancing precautions during periods of high local SARS-CoV-2 transmission. DisclosuresNo relevant conflicts of interest to declare.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

A Serological Snapshot of COVID-19 Initial Stages in Israel by a 6-Plex Antigen Array.

ABSTRACTThe first case of SARS-CoV-2 was discovered in Israel in late February 2020. Three major outbreaks followed, resulting in over 800,000 cases and over 6,000 deaths by April 2021. Our aim was characterization of a serological snapshot of Israeli patients and healthy adults in the early months of the COVID-19 pandemic. Sera from 55 symptomatic COVID-19 patients and 146 healthy subjects (early-pandemic, reverse transcription-quantitative PCR [qRT-PCR]-negative), collected in Israel between March and April 2020, were screened for SARS-CoV-2-specific IgG, IgM, and IgA antibodies, using a 6-plex antigen microarray presenting the whole inactivated virus and five viral antigens: a stabilized version of the spike ectodomain (S2P), spike subunit 1 (S1), receptor-binding-domain (RBD), N-terminal-domain (NTD), and nucleocapsid (NC). COVID-19 patients, 4 to 40 days post symptom onset, presented specific IgG to all of the viral antigens (6/6) in 54 of the 55 samples (98% sensitivity). Specific IgM and IgA antibodies for all six antigens were detected in only 10% (5/55) and 4% (2/55) of the patients, respectively, suggesting that specific IgG is a superior serological marker for COVID-19. None of the qRT-PCR-negative sera reacted with all six viral antigens (100% specificity), and 48% (70/146) were negative throughout the panel. Our findings confirm a low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population prior to the COVID-19 outbreak. We further suggest that the presence of low-level cross-reacting antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals.IMPORTANCE A 6-plex protein array presenting the whole inactivated virus and five nucleocapsid and spike-derived SARS-CoV-2 antigens was used to generate a serological snapshot of SARS-CoV-2 seroprevalence and seroconversion in Israel in the early months of the pandemic. Our findings confirm a very low seroprevalence of anti-SARS-CoV-2 antibodies in the Israeli adult population. We further propose that the presence of low-level nonspecific antibodies in naive individuals calls for a combined, multiantigen analysis for accurate discrimination between naive and exposed individuals enabling accurate determination of seroconversion. The developed assay is currently applied to evaluate immune responses to the Israeli vaccine during human phase I/II trials.

A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded “down” to an exposed “up” state in order to bind the human ACE2 receptor and infect cells. While snapshots of the “up” and “down” states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be expanding the pandemic disease across the globe. Although SARS-CoV-2 vaccines were rapidly developed and approved for emergency use of vaccination in humans, supply and production difficulties are slowing down the global vaccination program. The efficacy of many different versions of vaccine candidates and adjuvant effects remain unknown, particularly in the elderly. In this study, we compared the immunogenic properties of SARS-CoV-2 full-length spike (S) ectodomain in young adult and aged mice, S1 with receptor binding domain, and S2 with fusion domain. Full-length S was more immunogenic and effective in inducing IgG antibodies after low dose vaccination, compared to the S1 subunit. Old-aged mice induced SARS-CoV-2 spike-specific IgG antibodies with neutralizing activity after high dose S vaccination. With an increased vaccine dose, S1 was highly effective in inducing neutralizing and receptor-binding inhibiting antibodies, although both S1 and S2 subunit domain vaccines were similarly immunogenic. Adjuvant effects were significant for effective induction of IgG1 and IgG2a isotypes, neutralizing and receptor-binding inhibiting antibodies, and antibody-secreting B cell and interferon-γ secreting T cell immune responses. Results of this study provide information in designing SARS-CoV-2 spike vaccine antigens and effective vaccination in the elderly.

DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice

The outbreak of the SARS-CoV-2 virus and its rapid spread into a global pandemic made the urgent development of scalable vaccines to prevent coronavirus disease (COVID-19) a global health and economic imperative. Here, we characterized and compared the immunogenicity of two alphavirus-based DNA-launched self-replicating (DREP) vaccine candidates encoding either SARS-CoV-2 spike glycoprotein (DREP-S) or a spike ectodomain trimer stabilized in prefusion conformation (DREP-Secto). We observed that the two DREP constructs were immunogenic in mice inducing both binding and neutralizing antibodies as well as T cell responses. Interestingly, the DREP coding for the unmodified spike turned out to be more potent vaccine candidate, eliciting high titers of SARS-CoV-2 specific IgG antibodies that were able to efficiently neutralize pseudotyped virus after a single immunization. In addition, both DREP constructs were able to efficiently prime responses that could be boosted with a heterologous spike protein immunization. These data provide important novel insights into SARS-CoV-2 vaccine design using a rapid response DNA vaccine platform. Moreover, they encourage the use of mixed vaccine modalities as a strategy to combat SARS-CoV-2.

Molecular docking study of fatty acids from Pliek U Oil in the inhibition of SARS-CoV-2 protein and enzymes

This study aimed to analyze the fatty acid inPliek U oil andevaluate its inhibitor potential based on the interaction to several proteins and enzymes in SARS-CoV-2 using the in silico approach. Pliek U oil containing capric acid, caprylic acid, lauric acid, linoleic acid, myristic acid, oleic acid, and palmitic acids, with oleic acid as a dominant substance. Molecular docking analysis showed that linoleic acid has the best interaction to the receptors with the lowest binding affinity to 3CLpro (6LU7), Spike protein (6VXX), PLpro (6WX4), RdRp (6M71), E protein (5X29), and Spike Ectodomain Structure (6VYB) of -4.9, -5.8, -4.7, -4.3, -5.3 and -5.5 kcal/mol, respectively. The finding suggests that the binding of linoleic acid to the SARS-CoV-2 protein and enzyme may cause impairment of viral attachments to host cells, thus reducing infectivity in COVID-19 patients.

Newcastle Disease Virus-Like Particles Displaying Prefusion-Stabilized SARS-CoV-2 Spikes Elicit Potent Neutralizing Responses.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Cold sensitivity of the SARS-CoV-2 spike ectodomain.

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations, receptor-accessible “up” or receptor-inaccessible “down” states. Here we report that the commonly used stabilized S ectodomain construct “2P” is sensitive to cold temperature, and this cold sensitivity is abrogated in a “down” state–stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use SARS-CoV-2 S ectodomain.

A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice.

The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

Identification of potential antivirals against SARS-CoV-2 using virtual screening method.

SARS-CoV-2 has triggered a major epidemic among people around the world, and it is the newest in the sequence to become prevalent among other infectious diseases. The drug repurposing concept has been utilized effectively for numerous viral infections. Considering the situation and the urgency, the idea of drug repurposing for coronavirus infection (COVID-19) is also being studied. The molecular docking method was used for the screening of 29 antiviral drugs against primary protease proteins (MPP) of SARS-CoV-2, spike ecto-domain, spike receptor binding domain, Nsp9 RNA binding protein, and HR2 domain. Among these drugs, in terms of least binding energy, Indinavir, Sorivudine, Cidofovir, and Darunavir showed minimum docking scores with all the key proteins. For ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analysis, the ADMET properties of the top 4 drug candidates were retrieved through literature study. This analysis revealed that these drug candidates are well metabolized, distributed, and bioavailable, but have some undesirable effects. Furthermore, some approved structural analogues, such as Telbivudine, Tenofovir, Amprenavir, Fosamprenavir, etc., were predicted as similar drugs which may also be used for treating viral infections. We highly recommend these drug candidates as potential fighters against the deadly SARS-CoV-2 virus, and suggest in vivo trials for experimental validation of our findings.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100−150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.