Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Advances in developing ACE2 derivatives against SARS-CoV-2.

Extensive immune evasion of SARS-CoV-2 rendered therapeutic antibodies ineffective in the COVID-19 pandemic. Propagating SARS-CoV-2 variants are characterised by immune evasion capacity through key amino acid mutations, but can still bind human angiotensin-converting enzyme 2 (ACE2) through the spike protein and are, thus, sensitive to ACE2-mimicking decoys as inhibitors. In this Review, we examine advances in the development of ACE2 derivatives from the past 3 years, including the recombinant ACE2 proteins, ACE2-loaded extracellular vesicles, ACE2-mimicking antibodies, and peptide or mini-protein mimetics of ACE2. Several ACE2 derivatives are granted potent neutralisation efficacy against SARS-CoV-2 variants that rival or surpass endogenous antibodies by various auxiliary techniques such as chemical modification and practical recombinant design. The derivatives also represent enhanced production efficiency and improved bioavailability. In addition to these derivatives of ACE2, new effective therapeutics against SARS-CoV-2 variants are expected to be developed.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.

Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice.

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

Effectiveness of a booster dose of COVID-19 vaccines during an outbreak of SARS-CoV-2 Omicron BA.2.2 in China: A case-control study.

Real-world evidence on the effectiveness of COVID-19 vaccines marketed in China against the Omicron BA.2.2 variant remains scarce. A case-control study was conducted to estimate the vaccine effectiveness (VE) of COVID-19 vaccines marketed in China (inactivated vaccines, an Ad5-nCoV vaccine, and a recombinant protein vaccine). There were 414 cases infected with SARS-CoV-2 and 828 close contacts whose test results were consecutively negative as controls during the outbreak of the Omicron variant in Lu'an City, Anhui Province, China, in April 2022. The overall adjusted VE against Omicron BA.2.2 variant infection in the vaccinated group with any COVID-19 vaccine was 35.0% (95% CI: -9.1-61.3%), whereas the adjusted VE for booster vaccination was 51.6% (95% CI: 15.2-72.4%). Subgroup analysis showed that the overall adjusted VE of the Ad5-nCoV vaccine (65.8%, 95% CI: 12.8-86.6%) during the outbreak while any dose of inactivated vaccines and recombinant protein vaccine offered no protection. The adjusted VE of three-dose inactivated vaccines was 48.0% (95% CI: 8.0-70.6%), and the two-dose Ad5-nCoV vaccine was 62.9% (95% CI: 1.8-86%). There is no protection from a three-dose recombinant protein vaccine. COVID-19 vaccines offered 46.8% (95% CI: 9.5-68.7%) protection from infection within six months. There were statistically significant differences between the VEs of heterologous booster (VE = 76.4%, 95% CI: 14.3-93.5%) and homologous booster vaccination (VE = 51.8%, 95% CI: 9.6-74.3%) (P = .036). Booster vaccination of COVID-19 vaccines offered more protection than full vaccination. A booster vaccination campaign for a booster dose after three doses of a recombinant protein vaccine must be urgently conducted.

Structure-based design of oligomeric receptor-binding domain (RBD) recombinant proteins as potent vaccine candidates against SARS-CoV-2.

The receptor-binding domain (RBD) of SARS-CoV-2 S protein is proved to be the major target of neutralizing antibodies. However, on the S protein, only a portion of epitopes in RBD can be effectively displayed with dynamic changes in spatial conformations. Using RBD fragment as antigen can better expose the neutralizing epitopes, but the immunogenicity of RBD monomer is suboptimal. Multimeric display of RBD molecules is a feasible strategy to optimize RBD-based vaccines. In this study, RBD single-chain dimer derived from Wuhan-Hu-1 was fused with a trimerization motif, and a cysteine was also introduced at the C-terminus. The resultant recombinant protein 2RBDpLC was expressed in Sf9 cells using a baculovirus expression system. Reducing/non-reducing PAGE, size-exclusion chromatography and in silico structure prediction indicated that 2RBDpLC polymerized and possibly formed RBD dodecamers through trimerization motif and intermolecular disulfide bonds. In mice, 2RBDpLC induced higher levels of RBD-specific and neutralizing antibody responses than RBD dimer, RBD trimer and prefusion-stabilized S protein (S2P). In addition, cross-neutralizing antibodies against Delta and Omicron VOC were also detected in the immune sera. Our results demonstrate that 2RBDpLC is a promising vaccine candidate, and the method of constructing dodecamers may be an effective strategy for designing RBD-based vaccines.

Anakinra-An Interleukin-1 Receptor Antagonist for COVID-19.

BACKGROUND: Coronavirus disease (COVID-19) caused by SARS-CoV-2 virus caused a global pandemic in 2019. There are limited pharmacologic options available. The Food and Drug Administration initiated an emergency use authorization process to expedite pharmacologic agents to treat COVID-19. There are several agents available through the emergency use authorization process, ritonavir-boosted nirmatrelvir, remdesivir, and baricitinib. Anakinra is an interleukin (IL)-1 receptor antagonist that exhibits properties in fighting against COVID-19. MECHANISM OF ACTION, PHARMACODYNAMICS, AND PHARMACOKINETICS: Anakinra is a recombinant IL-1 receptor antagonist. The epithelial cell damage that may occur with COVID-19 enhances the release of IL-1, which plays a central role in severe cases. Thus, drugs that inhibit the IL-1 receptor may be beneficial in the management of COVID-19. Anakinra has good bioavailability after subcutaneous injection and a half-life of up to 6 hours. CLINICAL TRIALS: The SAVE-MORE, double-blind, randomized controlled trial, phase 3 evaluated the efficacy and safety of anakinra. Anakinra 100 mg was given subcutaneously daily for up to 10 days in patients with moderate and severe COVID-19 and plasma suPAR ≥6 ng/mL. Anakinra group had a 50.4% fully recovered with no viral RNA detected on day 28 versus 26.5% for placebo, and more than 50% of relative decrease in mortality. A significantly decreased risk of worse clinical outcome was observed. THERAPEUTIC ADVANCE: COVID-19 causes global pandemic and a serious viral disease. There are limited therapy options to combat this deadly disease. Anakinra is an IL-1 receptor antagonist and shown to be effective for the treatment of COVID-19 in some trials but not others. Anakinra, the first in this class, seems to have a mix result for the treatment of COVID-19.

Enhancement of immunogenicity and neutralizing responses against SARS-CoV-2 spike protein using the Fc fusion fragment.

AIMS: Vaccination has played an important role in protecting against death and the severity of COVID-19. The recombinant protein vaccine platform has a long track record of safety and efficacy. Here, we fused the SARS-CoV-2 spike S1 subunit to the Fc region of IgG and investigated immunogenicity, reactivity to human vaccinated sera, and neutralizing activity as a candidate protein vaccine. MATERIALS AND METHOD: We evaluated the immunogenicity of CHO-expressed S1-Fc fusion protein and tag-free S1 protein in rabbits via the production of S1-specific polyclonal antibodies. We subsequently compared the neutralizing activities of sera from immunized rabbits and human-vaccinated individuals using a surrogate Virus Neutralization Test (sVNT). KEY FINDINGS: The results indicate that S1-specific polyclonal antibodies were induced in all groups; however, antibody levels were higher in rabbits immunized with S1-Fc fusion protein than tag-free S1 protein. Moreover, the reactivity of human vaccinated sera against S1-Fc fusion protein was significantly higher than tag-free S1 protein. Lastly, the results of the virus-neutralizing activity revealed that vaccination with S1-Fc fusion protein induced the highest level of neutralizing antibody response against SARS-CoV-2. SIGNIFICANCE: Our results demonstrate that the S1 protein accompanied by the Fc fragment significantly enhances the immunogenicity and neutralizing responses against SARS-CoV-2. It is hoped that this platform can be used for human vaccination.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants.

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40-60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients' sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

In vitro immunogenic profile of recombinant SARS-CoV2 S1-RBD peptide in murine macrophage and microglial cells.

BACKGROUND: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can infect common mice inducing significant pathological lung lesions and inflammatory responses. This substantially mimics coronavirus disease 19 (COVID-19) infection and pathogenesis in humans. OBJECTIVES: To characterise the effects of recombinant SARS-CoV-2 S1 receptor-binding domain (RBD) peptide in murine macrophage and microglial cells' immune activation compared with classical PAMPs in vitro. METHODS: Murine RAW 264.7 macrophages and BV2 microglial cells were exposed to increasing concentrations of the RBD peptide (0.01, 0.05, and 0.1 µg/mL), Lipopolysaccharide (LPS) and Poly(I:C) and evaluated after two and 24 h for significant markers of macrophage activation. We determined the effects of RBD peptide on cell viability, cleaved caspase 3 expressions, and nuclear morphometry analysis. FINDINGS: In RAW cells, RBD peptide was cytotoxic, but not for BV2 cells. RAW cells presented increased arginase activity and IL-10 production; however, BV2 cells expressed iNOS and IL-6 after RBD peptide exposure. In addition, RAW cells increased cleaved-caspase-3, apoptosis, and mitotic catastrophe after RBD peptide stimulation but not BV2 cells. CONCLUSION: RBD peptide exposure has different effects depending on the cell line, exposure time, and concentration. This study brings new evidence about the immunogenic profile of RBD in macrophage and microglial cells, advancing the understanding of SARS-Cov2 immuno- and neuropathology.

Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

Correlation between partial thromboplastin time and thromboelastography in adult critically ill patients requiring bivalirudin for extracorporeal membrane oxygenation.

STUDY OBJECTIVE: Thromboelastography (TEG) offers a more dynamic assessment of hemostasis over activated partial thromboplastin time (aPTT). However, the clinical utility of TEG in monitoring bivalirudin during extracorporeal membrane oxygenation (ECMO) remains unknown. The purpose of this study was to evaluate the correlation between aPTT and TEG in adult ECMO patients anticoagulated with bivalirudin. DESIGN: Multicenter, retrospective, cohort study conducted over a 2-year period. SETTING: Two academic university medical centers (Banner University Medical Center) in Phoenix and Tucson, AZ. PATIENTS: Adult patients requiring ECMO and bivalirudin therapy with ≥1 corresponding standard TEG and aPTT plasma samples drawn ≤4 h of each other were included. The primary endpoint was to determine the correlation coefficient between the standard TEG reaction (R) time and bivalirudin aPTT serum concentrations. MEASUREMENTS AND MAIN RESULTS: A total of 104 patients consisting of 848 concurrent laboratory assessments of R time and aPTT were included. A moderate correlation between TEG R time and aPTT was demonstrated in the study population (r = 0.41; p < 0.001). Overall, 502 (59.2%) concurrent assessments of TEG R time and aPTT values showed agreement on whether they were sub-, supra-, or therapeutic according to the institution's classification for bivalirudin. The 42.2% (n = 271/642) discordant TEG R times among "therapeutic" aPTT were almost equally distributed between subtherapeutic and supratherapeutic categories. CONCLUSIONS: Moderate correlation was found between TEG R time and aPTT associated with bivalirudin during ECMO in critically ill adults. Further research is warranted to address the optimal test to guide clinical decision-making for anticoagulation dosing in ECMO patients.

Novel Affibody Molecules Specifically Bind to SARS-CoV-2 Spike Protein and Efficiently Neutralize Delta and Omicron Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented public health disaster in human history, and its spike (S) protein is the major target for vaccines and antiviral drug development. Although widespread vaccination has been well established, the viral gene is prone to rapid mutation, resulting in multiple global spread waves. Therefore, specific antivirals are needed urgently, especially those against variants. In this study, the domain of the receptor binding motif (RBM) and fusion peptide (FP) (amino acids [aa] 436 to 829; denoted RBMFP) of the SARS-CoV-2 S protein was expressed as a recombinant RBMFP protein in Escherichia coli and identified as being immunogenic and antigenically active. Then, the RBMFP proteins were used for phage display to screen the novel affibody. After prokaryotic expression and selection, four novel affibody molecules (Z14, Z149, Z171, and Z327) were obtained. Through surface plasmon resonance (SPR) and pseudovirus neutralization assay, we showed that affibody molecules specifically bind to the RBMFP protein with high affinity and neutralize against SARS-CoV-2 pseudovirus infection. Especially, Z14 and Z171 displayed strong neutralizing activities against Delta and Omicron variants. Molecular docking predicted that affibody molecule interaction sites with RBM overlapped with ACE2. Thus, the novel affibody molecules could be further developed as specific neutralization agents against SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 and its variants are threatening the whole world. Although a full dose of vaccine injection showed great preventive effects and monoclonal antibody reagents have also been used for a specific treatment, the global pandemic persists. So, developing new vaccines and specific agents are needed urgently. In this work, we expressed the recombinant RBMFP protein as an antigen, identified its antigenicity, and used it as an antigen for affibody phage-display selection. After the prokaryotic expression, the specific affibody molecules were obtained and tested for pseudovirus neutralization. Results showed that the serum antibody induced by RBMFP neutralized Omicron variants. The screened affibody molecules specifically bound the RBMFP of SARS-CoV-2 with high affinity and neutralized the Delta and Omicron pseudovirus in vitro. So, the RBMFP induced serum provides neutralizing effects against pseudovirus in vitro, and the affibodies have the potential to be developed into specific prophylactic agents for SARS-CoV-2 and its variants.

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein.

Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein.

Engineered soluble ACE2 receptor: Responding to change with change.

SARS coronavirus 2 (SARS-CoV-2) invades the human body by binding to major receptors such as ACE2 via its S-spike protein, so the interaction of receptor-binding sites has been a hot topic in the development of coronavirus drugs. At present, the clinical progress in monoclonal antibody therapy that occurred early in the pandemic is gradually showing signs of slowing. While recombinant soluble ACE2, as an alternative therapy, has been modified by many engineering methods, both the safety and functional aspects are approaching maturity, and this therapy shows great potential for broadly neutralizing coronaviruses, but its progress in clinical development remains stalled. Therefore, there are still several key problems to be considered and solved for recombinant soluble ACE2 to be approved as a clinical treatment as soon as possible.

Recombinant Protein Vaccines against Human Betacoronaviruses: Strategies, Approaches and Progress.

Betacoronaviruses have already troubled humanity more than once. In 2002-2003 and 2012, the SARS-CoV and MERS-CoV, respectively, caused outbreaks of respiratory syndromes with a fatal outcome. The spread of the SARS-CoV-2 coronavirus has become a pandemic. These three coronaviruses belong to the genus Betacoronavirus and have a zoonotic origin. The emergence of new coronavirus infections in the future cannot be ruled out, and vaccination is the main way to prevent the spread of the infection. Previous experience in the development of vaccines against SARS and MERS has helped to develop a number of vaccines against SARS-CoV-2 in a fairly short time. Among them, there are quite a few recombinant protein vaccines, which seem to be very promising in terms of safety, minimization of side effects, storage and transportation conditions. The problem of developing a universal betacoronavirus vaccine is also still relevant. Here, we summarize the information on the designing of vaccines based on recombinant proteins against highly pathogenic human betacoronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2.

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies.

The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.

Seroprevalence of four endemic human coronaviruses and, reactivity and neutralization capability against SARS-CoV-2 among children in the Philippines.

Four endemic human coronaviruses (HCoV), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are closely related to SARS-CoV-2. These coronaviruses are known to infect humans living in temperate areas, including children under 5 years old; however, the seroprevalence of four HCoVs among children in tropical areas, including the Philippines, remains unclear. This study aimed to assess the prevalence of antibodies against four HCoVs and to determine the reactivity and neutralization of these antibodies against SARS-CoV-2 among children in the Philippines. A total of 315 serum samples collected from 2015 to 2018, before the emergence of SARS-CoV-2, in Biliran island, Philippines, were tested for the presence of antibodies against four HCoVs and SARS-CoV-2 using recombinant spike ectodomain proteins by IgG-enzyme-linked immunosorbent assay (ELISA). Reactivity to and neutralization of SARS-CoV-2 were also investigated. The seroprevalence of the four HCoVs was 63.8% for HCoV-229E, 71.4% for HCoV-NL63, 76.5% for HCoV-HKU1, and 83.5% for HCoV-OC43 by ELISA. Age group analysis indicated that seropositivity to all HCoVs reached 80% by 2-3 years of age. While 69/315 (21.9%) of the samples showed reactive to SARS-CoV-2, almost no neutralization against SARS-CoV-2 was detected using neutralization assay. Reactivity of antibodies against SARS-CoV-2 spike protein obtained by ELISA may not correlate with neutralization capability.