Recombinant Spike Protein Research Articles

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.

Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to a continued need for diagnostic and therapeutic tools to monitor and respond to new variants. Glycosylation of the spike protein is critical for the protein's functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma incorporating HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site, was observed in the CHO-S derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO-S expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants in order to devise updated diagnostic tests and therapeutic vaccine designs.

MERS-CoV Infection and Its Impact on the Expression of TSLP Cytokine and IgG Antibodies: An In Vivo and In Vitro Study.

Thymic stromal lymphopoietin (TSLP) is a proinflammatory cytokine produced by epithelial cells that is involved in the activation of allergic disorders. To date, no study has examined TSLP induction during Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Herein, we aimed to study the effects of the recombinant spike protein of MERS-CoV on TSLP production. Additionally, the effects of recombinant human TSLP (rhTSLP) on B cell survival and antibody production were investigated. B cells were separated using the Human B Cell Enrichment Kit, and B cell survival was measured using the WST-1 Assay Kit. Enzyme-linked immunosorbent assay (ELISA) was used to measure TSLP levels in the sera of both MERS-CoV-infected (n=4; median age, 53 years) and healthy individuals (n=5; median age, 35 years). We showed that the group of infected patients had significantly higher levels of TSLP than healthy controls (37.6 pg/mL vs 19.8 pg/mL, *p<0.05). The levels of TSLP in A549 cells were remarkably increased after 48h of stimulation with recombinant full-length spike protein (rSP) (32.2 pg/mL, p=0.01). B cell survival was greatly enhanced by rhTSLP alone or in combination with rSP (0.02 vs 0.046, and 0.045; **p<0.01, respectively). Our data also showed a significant synergistic effect of rhTSLP and rSP on the augmented response of IgG antibodies against the spike protein of MERS-CoV compared with unstimulated cells (0.156 vs 0.22; *p<0.05). TSLP production is induced in vivo after MERS-CoV infection and in vitro after treatment with the rSP of MERS-CoV, which has a significant effect on the survival of B cells. Our data suggest that TSLP can be used as a strong mucosal adjuvant for vaccine development against MERS-CoV infection. However, further investigation is required to study the functional role of TSLP in MERS-CoV infection.

Effects of age and gender on immunogenicity and reactogenicity of SpikoGen recombinant spike protein vaccine: a post-hoc analysis

SpikoGen® COVID-19 vaccine is based on the spike protein extracellular domain of the ancestral Wuhan-Hu-1 strain modified by removal of the furin cleavage site and addition of stabilising mutations expressed as a recombinant protein in insect cells. It is formulated with Advax-CpG55.2™ adjuvant to ensure optimal immunogenicity. In this study, data from several SpikoGen® clinical trials was retrospectively analysed to assess for any effect of gender or age on seroconversion, neutralizing antibody levels or the incidence of adverse events. Following the 1st dose, older age was associated with a reduced rate of fatigue (RR 0.97, p < 0.001), headache (RR 0.98, p = 0.034) and myalgia (RR 0.97, p=0.016), following the 2nd dose, the rate of fatigue (RR 0.98, p = 0.017) but following the 3rd dose no effect of age on adverse events was evident. Similarly, following the 1st dose, men reported a 19% lower incidence of fatigue, 36% lower incidence of headache and 28% lower incidence of myalgia when compared to women. Interestingly, there was no relationship between age or gender and serum neutralizing antibody levels, although after each vaccine dose there was a consistent trend to women having a higher seroconversion rate. There was no correlation between neutralizing antibody levels and adverse events. Unlike what is seen with mRNA vaccines, reactogenicity trended lower after each subsequent SpikoGen® dose. Overall, SpikoGen® exhibited positive immunogenicity and low reactogenicity, indicating that a low incidence of adverse events does not equate to poor immunogenicity. SpikoGen® remains a promising protein-based vaccine platform for COVID-19 protection.

Immunogenicity and safety of SARS-CoV-2 recombinant spike protein vaccine in South African people living with and without HIV-1 infection: A phase 2 randomised trial

BackgroundResponse data for COVID-19 vaccines in immunosuppressed individuals are typically limited to standard dosing in small populations. Adjusting number or interval of doses may impact immune responses based on HIV status. MethodsThis phase 2 randomised, observer-blinded, placebo-controlled South African study (2019nCoV-505/NCT05112848) enrolled medically stable people living with HIV (PLWH) and HIV-uninfected participants aged 18–65 years. PLWH were randomised 1:1:1 to receive NVX-CoV2373 on day 0 (D0) and either D21 (2-DoseD0/D21) or D70 (2-DoseD0/D70), or on D0, D21, and D70 (3-Dose). HIV-uninfected participants were randomised 1:1 to each 2-Dose regimen. PLWH were stratified into well-controlled and less–well-controlled subgroups. The primary immunologic endpoint included serum IgG and neutralising antibody responses (per geometric mean fold rise [GMFR] in titre and seroconversion rate) to ancestral SARS-CoV-2 at D35 (2-DoseD0/D21) and D84 (2-DoseD0/D70 and 3-Dose). The primary safety endpoints were participants with an unsolicited adverse event through D84, at D120, and at D180, or reactogenicity ≤7 days post-vaccination. ResultsOf 288 PLWH, 98, 96, and 94 were randomised into the 2-DoseD0/D21, 2-DoseD0/D70, and 3-Dose groups, respectively; 96 HIV-uninfected participants were randomised to the 2-DoseD0/D21 (n=47) or 2-DoseD0/D70 (n=49) regimens. Most (>85%) of the population were SARS-CoV-2 positive at baseline. Ancestral anti-spike IgG GMFRs in PLWH and HIV-uninfected participants, respectively, were 12·4 and 12·9 (D35) and 12·2 and 13·6 (D84). Comparable outcomes occurred across dosing regimens and in well-controlled and less–well-controlled PLWH. Microneutralization GMFRs at D84 in PLWH and HIV-uninfected participants, respectively, were: 6·9 and 10·1 (2-DoseD0/D21), 11·0 and 11·3 (2-DoseD0/D70), and 17·2 (PLWH 3-Dose). Antibody responses against BA.1 trended similar to those against the ancestral virus. Safety outcomes were comparable among PLWH and HIV-uninfected participants. ConclusionThis study demonstrated that NVX-CoV2373 produced consistent immunogenicity responses to SARS-CoV-2 among PLWH and HIV-uninfected participants, with no new safety signals. FundingNovavax, Inc.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA)

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Interaction Between SARS-CoV-2 Spike Protein S1 Subunit and Oyster Heat Shock Protein 70.

There is growing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminates the marine environment and is bioaccumulated in filter-feeding shellfish. Previous study shows the Pacific oyster tissues can bioaccumulate the SARS-CoV-2, and the oyster heat shock protein 70 (oHSP70) may play as the primary attachment receptor to bind SARS-CoV-2's recombinant spike protein S1 subunit (rS1). However, detailed information about the interaction between rS1 and oHSP70 is still unknown. In this study, we confirmed that the affinity of recombinant oHSP70 (roHSP70) for rS1 (KD = 20.4nM) is comparable to the receptor-binding affinity of rACE2 for rS1 (KD = 16.7nM) by surface plasmon resonance (SPR)-based Biacore and further validated by enzyme-linked immunosorbent assay (ELISA). Three truncated proteins (roHSP70-N/C/M) and five mutated proteins (p.I229del, p.D457del, p.V491_K495del, p.K556I, and p.ΣroHSP70) were constructed according to the molecular docking results. All three truncated proteins have significantly lower affinity for rS1 than the full-length roHSP70, indicating that all three segments of roHSP70 are involved in binding to rS1. Further, the results of SPR and ELISA showed that all five mutant proteins had significantly lower affinity for rS1 than roHSP70, suggesting that amino acids at these sites are involved in binding to rS1. This study provides a preliminary theoretical basis for the bioaccumulation of SARS-CoV-2 in oyster tissues or using roHSP70 as the capture unit to selectively enrich virus particles for detection.

Post-Hoc Analysis of Potential Correlates of Protection of a Recombinant SARS-CoV-2 Spike Protein Extracellular Domain Vaccine Formulated with Advax-CpG55.2-Adjuvant.

SpikoGen® vaccine is a subunit COVID-19 vaccine composed of an insect cell expressed recombinant spike protein extracellular domain formulated with Advax-CpG55.2™ adjuvant. A randomized double-blind, placebo-controlled Phase II clinical trial was conducted in 400 adult subjects who were randomized 3:1 to receive two intramuscular doses three weeks apart of either SpikoGen® vaccine 25 μg or saline placebo, as previously reported. This study reports a post hoc analysis of the trial data to explore potential immune correlates of SpikoGen® vaccine protection. A range of humoral markers collected pre- and post-vaccination, including spike- and RBD-binding IgG and IgA, surrogate (sVNT), and conventional (cVNT) virus neutralization tests were compared between participants who remained infection-free or got infected over three months of follow-up. From 2 weeks after the second vaccine dose, 21 participants were diagnosed with SARS-CoV-2 infection, 13 (4.2%) in the SpikoGen® group and 8 (9%) in the placebo group. Those in the vaccinated group who experienced breakthrough infections had significantly lower sVNT titers (GMT 5.75 μg/mL, 95% CI; 3.72-8.91) two weeks after the second dose (day 35) than those who did not get infected (GMT 21.06 μg/mL, 95% CI; 16.57-26.76). Conversely, those who did not develop SARS-CoV-2 infection during follow-up had significantly higher baseline sVNT, cVNT, spike-binding IgG and IgA, and RBD-binding IgG, consistent with a past SARS-CoV-2 infection. SpikoGen® further reduced the risk of re-infection (OR 0.29) in baseline seropositive (previously infected) as well as baseline seronegative participants. This indicates that while SpikoGen vaccine is protective in seronegative individuals, those with hybrid immunity have the most robust protection.

The PARP inhibitor rucaparib blocks SARS-CoV-2 virus binding to cells and the immune reaction in models of COVID-19.

To date, there are limited options for severe Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 virus. As ADP-ribosylation events are involved in regulating the life cycle of coronaviruses and the inflammatory reactions of the host; we have, here, assessed the repurposing of registered PARP inhibitors for the treatment of COVID-19. The effects of PARP inhibitors on virus uptake were assessed in cell-based experiments using multiple variants of SARS-CoV-2. The binding of rucaparib to spike protein was tested by molecular modelling and microcalorimetry. The anti-inflammatory properties of rucaparib were demonstrated in cell-based models upon challenging with recombinant spike protein or SARS-CoV-2 RNA vaccine. We detected high levels of oxidative stress and strong PARylation in all cell types in the lungs of COVID-19 patients, both of which negatively correlated with lymphocytopaenia. Interestingly, rucaparib, unlike other tested PARP inhibitors, reduced the SARS-CoV-2 infection rate through binding to the conserved 493-498 amino acid region located in the spike-ACE2 interface in the spike protein and prevented viruses from binding to ACE2. In addition, the spike protein and viral RNA-induced overexpression of cytokines was down-regulated by the inhibition of PARP1 by rucaparib at pharmacologically relevant concentrations. These results point towards repurposing rucaparib for treating inflammatory responses in COVID-19.

Novel Competitive ELISA Utilizing Trimeric Spike Protein of SARS-CoV-2, Could Identify More Than RBD-RBM Specific Neutralizing Antibodies in Hybrid Sera.

Since the initiation of the COVID-19 pandemic, there has been a need for the development of diagnostic methods to determine the factors implicated in mounting an immune response against the virus. The most promising indicator has been suggested to be neutralizing antibodies (nAbs), which mainly block the interaction between the Spike protein (S) of SARS-CoV-2 and the host entry receptor ACE2. In this study, we aimed to develop and optimize conditions of a competitive ELISA to measure serum neutralizing titer, using a recombinant trimeric Spike protein modified to have six additional proline residues (S(6P)-HexaPro) and h-ACE2. The results of our surrogate Virus Neutralizing Assay (sVNA) were compared against the commercial sVNT (cPass, Nanjing GenScript Biotech Co., Nanjing City, China), using serially diluted sera from vaccinees, and a high correlation of ID50-90 titer values was observed between the two assays. Interestingly, when we tested and compared the neutralizing activity of sera from eleven fully vaccinated individuals who subsequently contracted COVID-19 (hybrid sera), we recorded a moderate correlation between the two assays, while higher sera neutralizing titers were measured with sVNA. Our data indicated that the sVNA, as a more biologically relevant model assay that paired the trimeric S(6P) with ACE2, instead of the isolated RBD-ACE2 pairing cPass test, could identify nAbs other than the RBD-RBM specific ones.

Vaccination with ancestral SARS-CoV-2 spike adjuvanted with TLR agonists provides cross-protection against XBB.1

Many different platforms have been used to develop highly protective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans. However, protection has eroded over time due to the emergence of antigenically diverse viral variants, especially the Omicron subvariants. One successful platform for the generation of SARS-CoV-2 vaccines are recombinant spike protein vaccines, of which two are licensed in the United States and Europe. Typically, purified recombinant protein antigens are poorly immunogenic and adjuvants must be included in the formulation. Here, we adjuvanted recombinant ancestral SARS-CoV-2 Wuhan-Hu-1 spike proteins with an emulsion formulation combined with synthetic Toll-like receptor (TLR) 4 and 7/8 agonists. This combination led to the induction of a Th1-skewed immune response that included high titers of antibodies against Wuhan-Hu-1 spike. These serum antibodies included neutralizing and cross-reactive antibodies that recognized the spike from multiple SARS-CoV-2 variants, as well as the receptor binding domain (RBD) from SARS-CoV-1. Despite an absence of robust cross-neutralization, vaccination against Wuhan-Hu-1 spike in the context of TLR-containing emulsions provided complete cross-protection against disease from a lethal challenge with XBB.1 in a stringent K18-hACE2 mouse model. We believe that the combination of recombinant spike antigens with TLR agonist-based emulsion formulations could lead to the development of next-generation SARS-CoV-2 vaccines that provide significant protection from future emerging variants.

Formulation Development of a COVID-19 Recombinant Spike Protein-Based Vaccine.

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen-deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein.

Development and evaluation of vaccination strategies for addressing the continuous evolution SARS-CoV-2 based on recombinant trimeric protein technology: Potential for cross-neutralizing activity and broad coronavirus response

Given the significant decline in vaccine efficacy against Omicron, the development of novel vaccines with specific or broad-spectrum effectiveness is paramount. In this study, we formulated four monovalent vaccines based on recombinant spike trimer proteins, along with three bivalent vaccines, and five monovalent vaccines based on recombinant spike proteins. We evaluated the efficacy of different vaccination regimens in eliciting neutralizing antibodies in mice through pseudovirus neutralization assays. Following two doses of primary immunization with D614G, mice received subsequent immunizations with Omicron (BA.1, BA.2, BA.4/5) boosters individually, which led to the generation of broader and more potent cross-neutralizing activity compared to D614G boosters. Notably, the BA.4/5 booster exhibited superior efficacy. Following two doses of primary immunization with Omicron (BA.1, BA.2, BA.4/5), mice were subsequently immunized with one dose of D614G booster which resulted in broader neutralizing activity compared to one dose of Omicron (BA.1, BA.2, or BA.4/5). In unvaccinated mice, full-course immunization with different bivalent vaccines induced broad neutralizing activity against Omicron and pre-Omicron variants, with D614G&BA.4/5 demonstrating superior efficacy. However, compared to other variants, the neutralizing activity against XBB.1.5/1.9.1 is notably reduced. This observation emphasizes the necessity of timely updates to the vaccine antigen composition. Based on these findings and existing studies, we propose a vaccination strategy aimed at preserving the epitope repertoire to its maximum potential: (1) Individuals previously vaccinated or infected with pre-Omicron variants should inoculate a monovalent vaccine containing Omicron components; (2) Individuals who have only been vaccinated or infected with Omicron should be inoculated a monovalent vaccine containing pre-Omicron variants components; (3) Individuals without SARS-CoV-2 infection and vaccination should inoculate a bivalent vaccine comprising both pre-Omicron and Omicron components for primary immunization. Additionally, through cross-inoculation of SARS-CoV-2 D614G spike trimer protein and SARS-CoV-1 spike protein in mice, we preliminarily demonstrated the possibility of cross-reaction between different coronavirus vaccines to produce resistance to the pan-coronavirus.

Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination.

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.

Establishment of human post-vaccination SARS-CoV-2 standard reference sera

There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern. ImportanceThe explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.

A portable transistor immunosensor for fast identification of porcine epidemic diarrhea virus

Widespread distribution of porcine epidemic diarrhea virus (PEDV) has led to catastrophic losses to the global pig farming industry. As a result, there is an urgent need for rapid, sensitive and accurate tests for PEDV to enable timely and effective interventions. In the present study, we develop and validate a floating gate carbon nanotubes field-effect transistor (FG CNT-FET)-based portable immunosensor for rapid identification of PEDV in a sensitive and accurate manner. To improve the affinity, a unique PEDV spike protein-specific monoclonal antibody is prepared by purification, and subsequently modified on FG CNT-FET sensor to recognize PEDV. The developed FET biosensor enables highly sensitive detection (LoD: 8.1 fg/mL and 100.14 TCID50/mL for recombinant spike proteins and PEDV, respectively), as well as satisfactory specificity. Notably, an integrated portable platform consisting of a pluggable FG CNT-FET chip and a portable device can discriminate PEDV positive from negative samples and even identify PEDV and porcine deltacoronavirus within 1 min with 100% accuracy. The portable sensing platform offers the capability to quickly, sensitively and accurately identify PEDV, which further points to a possibility of point of care (POC) applications of large-scale surveillance in pig breeding facilities.

Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile.

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

A phase 3 randomized controlled trial of a COVID-19 recombinant vaccine S-268019-b versus ChAdOx1 nCoV-19 in Japanese adults.

We assessed S-268019-b, a recombinant spike protein vaccine with a squalene-based adjuvant, for superiority in its immunogenicity overChAdOx1 nCoV-19 vaccine among adults in Japan. In this multicenter, randomized, observer-blinded, phase 3 study, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naïve participants (aged ≥ 18years, without prior infection or vaccination against SARS-CoV-2) were randomized (1:1) to receive either S-268019-b or ChAdOx1 nCoV-19 as two intramuscular injections given 28days apart. Participants who provided consent for a booster administration received S-268019-b at Day 211. The primary endpoint was SARS-CoV-2 neutralizing antibody (NAb) titer on Day 57; the key secondary endpoint was the seroconversion rate for SARS-CoV-2 NAb titer on Day 57. Other endpoints included anti-SARS-CoV-2 S-protein immunoglobulin (Ig)G antibody titer and safety. The demographic and baseline characteristics were generally comparable between S-268019-b (n = 611) and ChAdOx1 nCoV-19 (n = 610) groups. S-268019-b showed superior immunogenicity over ChAdOx1 nCoV-19, based on their geometric mean titers (GMTs) and GMT ratios of SARS-CoV-2 NAb on Day 57 by cytopathic effect assay (GMT [95% confidence interval {CI}] 19.92 [18.68, 21.23] versus 3.63 [3.41, 3.87]; GMT ratio [95% CI] 5.48 [5.01, 6.00], respectively; two-sided p-values < 0.0001). Additionally, NAb measured using a cell viability assay also showed similar results (GMT [95% CI] 183.25 [168.04, 199.84] versus 24.79 [22.77, 27.00]; GMT ratio [95% CI] 7.39 [6.55, 8.35] for S-268019-b versus ChAdOx1 nCoV-19, respectively; p < 0.0001). The GMT of anti-SARS-CoV-2 S-protein IgG antibody was 370.05 for S-268019-b versus 77.92 for ChAdOx1 nCoV-19 on Day 57 (GMT ratio [95% CI] 4.75 [4.34, 5.20]). Notably, immune responses were durable through the end of the study. S-268019-b elicited T-helper 1 skewed T-cell response, comparable to that of ChAdOx1 nCoV-19. After the first dose, the incidence of solicited systemic treatment-related adverse events (TRAEs) was higher in the ChAdOx1 nCoV-19 group, but after the second dose, the incidence was higher in the S-268019-b group. Headache, fatigue, and myalgia were the most commonly reported solicited systemic TRAEs, while pain at the injection site was the most frequently reported solicited local TRAE following both doses in both groups. No serious treatment-related adverse serious TRAEs events were reported in the two groups. S-268019-b was more immunogenic than ChAdOx1 nCoV-19 vaccine and was well tolerated (jRCT2051210151).

Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

Multivalent and Sequential Heterologous Spike Protein Vaccinations Effectively Induce Protective Humoral Immunity against SARS-CoV-2 Variants.

The emergence of new SARS-CoV-2 variants continues to cause challenging problems for the effective control of COVID-19. In this study, we tested the hypothesis of whether a strategy of multivalent and sequential heterologous spike protein vaccinations would induce a broader range and higher levels of neutralizing antibodies against SARS-CoV-2 variants and more effective protection than homologous spike protein vaccination in a mouse model. We determined spike-specific IgG, receptor-binding inhibition titers, and protective efficacy in the groups of mice that were vaccinated with multivalent recombinant spike proteins (Wuhan, Delta, Omicron), sequentially with heterologous spike protein variants, or with homologous spike proteins. Trivalent (Wuhan + Delta + Omicron) and sequential heterologous spike protein vaccinations were more effective in inducing serum inhibition activities of receptor binding to spike variants and virus neutralizing antibody titers than homologous spike protein vaccination. The higher efficacy of protection was observed in mice with trivalent and sequential heterologous spike protein vaccination after a challenge with a mouse-adapted SARS-CoV-2 MA10 strain compared to homologous spike protein vaccination. This study provides evidence that a strategy of multivalent and sequential heterologous variant spike vaccination might provide more effective protection against emerging SARS-CoV-2 variants than homologous spike vaccination and significantly alleviate severe inflammation due to COVID-19.