Blood derivative therapy

There are three main components manufactured from whole blood: red blood cells (RBCs), plasma, and platelets. Plasma contains a multitude of different proteins, peptides, and biologic substances. Approximately 53 million liters of plasma was collected in the United States in 2019. Following collection, plasma is frozen and manufactured into plasma-derived medicinal products (PDMPs). During the manufacture process, several thousand plasma units are pooled for Cohn fractionation, which is based upon cold ethanol precipitation of proteins. The PDMPs are further prepared using ion exchange or affinity chromatography and additional steps to inactivate and remove infectious diseases such as viruses. Almost 20 different therapeutic plasma proteins are purified from plasma via these multi-step manufacturing processes. Interestingly, the demand for pharmaceutical plasma products, particularly intravenous immunoglobulin (IVIG) products, has been increasing. The manufacture and therapeutic role of blood derivatives particularly immunoglobulin therapy, Rh immunoglobulin (RhIG), COVID-19 convalescent plasma (CCP) and hyperimmune globulins, albumin, clotting factors, fibrin sealants, and platelet rich plasma will be described.Copyright © 2022 AME Publishing Company. All Rights Reserved.

Research on the 6XS6 of the SARS-CoV-2 Spike Protein

The 6XS6 is the structure of the SARS-CoV-2 spike protein. The physiological role of the spike protein is relative to the respiratory syndrome coronavirus and has a stronger infect on the human body than the ancestor virus. The purification of the 6XS6 is in the homo sapiens cell by the affinity chromatography, PBS supplemented and Size Exclusion chromatography. At last, using the Cryo-Electron Microscopy to see the structure. This paper is using the D614G mutation to illustrate the structure of the 6XS6. The N-terminal domain and C-terminal domain of the 6XS6 protein are ALA27 and VAL1137. Furthermore, the mutation doesn't have the hydrogen bond because the Asp614 is substituted by the Gly614, and the molecule that interacts with the Ala 647 may occur. While the 6XS6 structure has lots of non-covalent and disulfide bonds. Comparing the structure of the 6XS6 and 6VXX, both are glycoproteins, have three monomers, have two subunits, and have the same category of expression and classification. The different conformations of the two structures can affect the binding ability with the ACE2. This paper can help the researchers to further understand the structure and function of the 6XS6 which can be used in future experiments. © 2023 SPIE.

Identification of host factors that bind to the 5 ' end of the MERS-CoV RNA genome

Plain language summaryMERS-CoV is a virus that causes a severe illness in the nose, mouth and throat of humans. It is a zoonotic virus, which means that it can spread from animals to humans. MERS-CoV was first found in Saudi Arabia in 2012 and continues to pose a threat to public health. Interactions between the virus and human cells and proteins are important to establishing infection. Understanding these interactions is important for the development of drugs to treat viral infections. Here, we have identified some proteins that interact with MERS-CoV. Tweetable A proteomic approach for the identification of cellular proteins that interact with the 5 '-terminal region of MERS-CoV RNA genome. #MERS-CoV #RNA_viruses. Aim: The aim of this study was to identify host factors that interact with the 5 ' end of the MERS-CoV RNA genome. Materials & methods: RNA affinity chromatography followed by mass spectrometry analysis was used to identify the binding of host factors in Vero E6 cells. Results: A total of 59 host factors that bound the MERS-CoV RNA genome in non-infected Vero E6 cells were identified. Most of the identified cellular proteins were previously reported to interact with the genome of other RNA viruses. We validated our mass spectrometry results using western blotting. Conclusion: These data enhance our knowledge about the RNA-host interactions of coronaviruses, which could serve as targets for developing antiviral therapeutics against MERS-CoV.

Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.

Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different E. coli strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of E. coli, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2.

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2.

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2.

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.

A natural goldmine of binding proteins and soluble receptors simplified their translation to blockbuster drugs, all in one decade.

Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the "triumph". My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNß proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn's disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.

Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system.

Background and Objectives: The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system. Materials and Methods: A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting. Results: Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC). Conclusion: Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.

A scalable and high yielding SARS-CoV-2 spike protein receptor binding domain production process.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein is of interest for the development of vaccines and therapeutics against COVID-19. Vaccines are designed to raise an immune response against the spike protein. Other therapies attempt to block the interaction of the spike protein and mammalian cells. Therefore, the spike protein itself and specific interacting regions of the spike protein are reagents required by industry to enable the advancement of medicines to combat SARS-CoV-2. Early production methods of the SARS-CoV-2 spike protein receptor binding domain (RBD) were labor intensive with scalability challenges. In this work, we describe a high yielding and scalable production process for the SARS-CoV-2 RBD. Expression was performed in human embryonic kidney (HEK) 293 cells followed by a two-column purification process including immobilized metal affinity chromatography (IMAC) followed by Ceramic Hydroxyapatite (CHT). The improved process showed good scalability, enabling efficient purification of 2.5 g of product from a 200 L scale bioreactor.

LLAMA (Lama glama) ANTISERUM AGAINST SARS-CoV-2

The receptor-binding domain (RBD) of SARS-CoV-2 Spike protein constitutes the key access for the virus inside the host cell. A positive correlation between titers of anti-RBD immunoglobulin G and antibodies (Ab) capable of neutralizing the virus has been demonstrated. In this context, passive immunotherapies such as convalescent plasma and hyperimmune equine serum have gained relevance as therapies against COVID-19. Another promising alternative is the use of polyclonal Ab from llamas (Lama glama), because of their unique features. For this reason, we aimed to obtain recombinant RBD as an immunogen to generate anti-RBD Ab in llamas. To achieve this, HEK 293 cells were transfected and transduced with the RDB encoding sequence, resulting in higher yields with this last method. The RBD was purified by affinity chromatography. An immunization schedule was designed and evaluated on two male animals, which were initially inoculated with RBD, followed by periodical boosters. Exploratory bleedings were performed in order to evaluate the reached titers, and larger bleedings in order to obtain enriched plasma with anti-RBD Ac. Ac quantification was accomplished by an in-house ELISA. Results showed that the immunization scheme was successful, achieving a maximum titer of 168000 at 28 days post-immunization. The results lay the foundations for the production of polyclonal anti-RBD Ab.

Clinical evaluation and application of detection of IgM and IgG antibodies against SARS-CoV-2 using a colloidal gold immunochromatography assay

The pandemic of the novel coronavirus (SARS-CoV-2) infection has seriously threated global public health, a rapid and easy operated method for coronavirus disease (COVID-19) diagnosis is needed. To evaluate the clinical application efficacy of the colloidal gold rapid test kit for detection of the IgM/IgG antibodies to SARS-CoV-2, a total of 304 clinical diagnosed case, 138 health donor (of which 114 showed SARS-CoV-2) viral RNA negative and 64 other fever patients with respiratory symptoms were selected for the study and the plasma or serum samples were tested for both IgM and IgG with the kit. The comparison of the detection coincidence of the samples from whole blood and plasma or serum were also performed;Furthermore, the time distribution of SARS-CoV-2 viral RNA and IgM/IgG antibodies detections were analyzed. The results showed that, of the 304 clinical diagnosed cases, 105 cases were positive for viral RNA detection, among which the detection sensitivity of IgM and IgG antibodies to SARS-CoV-2 by colloidal gold rapid assay was 76.2% (80/105) and 86.6% (91/105), respectively, and the overall coincidence rate of IgM/IgG antibody was 96.1% (101/105);and 73 cases were negative for both nucleic acid and antibody detection. Of the remaining 126 clinical diagnosed cases, the positive rate of IgM and IgG was 69.2% (87/126) and 98.3% (125/126), respectively, and the overall coincidence rate of IgM/IgG antibody was 100% (126/126). In detections for healthy and other fever patients, the specificity of IgM and IgG was 99% (200/202) and 98% (198/202), respectively, and the total coincidence rate of antibody detection results of homologous whole blood and plasma samples was 99%, indicating a high degree of consistency. In this study, the detection assay of SARS-CoV-2 antibodies using colloidal gold method showed satisfactory detection effect, and it could be used for clinical auxiliary diagnosis and epidemiological investigation, which could be applied in a wide range of scenarios and play a valuable role in the prevention and control of SARS-CoV-2 pandemic.

Antibody response to SARS-CoV-2 infection and Sputnik V vaccination

Antibodies to the SARS-CoV-2 S-protein and its RBD fragment are considered the primary immune response markers against the SARS-CoV-2. We analyzed the content of antibodies against the Sprotein and RBD of SARS-CoV-2 in the blood of employees of the SB RAS Institute of Chemical Biology and Fundamental Medicine. In 2020, some researchers were infected with COVID-19;in January- April of 2021, more than 80% of the Institute staff were vaccinated with Sputnik V. In May 2021, blood samples from 396 donors were collected. Using ELISA, we analyzed the presence of antibodies against the S- and N-proteins of the SARS-CoV-2 in the donor's blood plasma of four groups: COVID-19 convalescents, vaccinated with Sputnik V (group I);patients vaccinated with Sputnik V that were not infected with COVID-19 (group II);not vaccinated COVID-19 convalescents (group III);patients that were not vaccinated and infected with SARS-CoV-2 (group IV). The blood of the group I patients had the highest amount of antibodies to the S- and N-proteins of the SARS-CoV-2. The level of antibodies against S-protein in group II was higher than in group III. Next, electrophoretically homogeneous preparations of IgG were isolated from blood plasma by affinity chromatography on Protein-GSepharose. Further, using affinity chromatography on sorbents containing immobilized recombinant SARS-CoV-2 S-protein and RBD, we isolated antibody subfractions possessing affinity to these antigens (S-IgG and RBD-IgG, respectively). The content of RBDIgG in the blood plasma of convalescent and vaccinated donors is about 1%. Antibodies of this subfraction have an affinity for other domains of the S-protein than RBD. The amount of these antibodies was less than 1%. In further studies, we plan to characterize SIgG and RBD-IgG preparations and compare them with the total pool of IgG in vaccinated and/or recovered donors.

ACE2 and SARS-CoV-2-Main Protease Capillary Columns for Affinity Chromatography: Testimony of the Binding of Dexamethasone and its Carbon Nanotube Nanovector.

In this paper, each of the two following proteins, the angiotensin-converting enzyme 2 (ACE2) and the Main protease (Main pro) of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) were grafted for the first time on homemade neutravidin poly(GMA-co-EDMA) capillary columns for the research of their ligands. The effect of the column diameter on the quantity of immobilized biotinylated protein was studied. For a capillary length of 40 mm, when its internal diameter varied from 75 to 25 µm, the grafted quantity of ACE2 decreased by 85% (from 1.50 to 0.24 µg). Among all the studied ligands, a particular vigilance has been given for dexamethasone, a widely used molecule today for adult patients hospitalized with SARS-CoV-2. Competition experiments were performed with SARS-CoV-2 Receptor Binding Domain used as reference molecule with the ACE2 affinity column to assess the orthosteric binding site of dexamethasone (Dex) on ACE2. This ligand was then immobilized on Multiwall Carbon Nanotubes (Dex/MWCNT). By comparison of the normalized breakthrough curves measured for Dex and Dex/MWCNT on both the ACE2 and Main pro affinity columns, it was showed for the first time that nanovectorisation of Dex with MWCNT enhanced and stabilized its binding to both ACE2 and Main pro. This last result reinforced the use of Dex and the interest of MWCNT for boosting immune health against COVID 19.

Human interaction targets of SARS-COV-2 spike protein: A systematic review

Objectives: The development of effective targeted therapy and drug-design approaches against the SARS-CoV-2 is a universal health priority. Therefore, it is important to assess possible therapeutic strategies against SARS-CoV-2 via its most interaction targets. The present study aimed to perform a systematic review on clinical and experimental investigations regarding SARS-COV-2 interaction targets for human cell entry. Methods: A systematic search using relevant MeSH terms and keywords was performed in PubMed, Scopus, Embase, and Web of Science (ISI) databases up to July 2021. Two reviewers independently assessed the eligibility of the studies, extracted the data, and evaluated the methodological quality of the included studies. Additionally, a narrative synthesis was done as a qualitative method for data gathering and synthesis of each outcome measure. Results: A total of 5610 studies were identified, and 128 articles were included in the systematic review. Based on the results, spike antigen was the only interaction protein from SARS-CoV-2. However, the interaction proteins from humans varied including different spike receptors and several cleavage enzymes. The most common interactions of the spike protein of SARS-CoV-2 for cell entry were ACE2 (entry receptor) and TMPRSS2 (for spike priming). A lot of published studies have mainly focused on the ACE2 receptor followed by the TMPRSS family and furin. Based on the results, ACE2 polymorphisms as well as spike RBD mutations affected the SARS-CoV-2 binding affinity. Conclusion: The included studies shed more light on SARS-CoV-2 cellular entry mechanisms and detailed interactions, which could enhance the understanding of SARS-CoV-2 pathogenesis and the development of new and comprehensive therapeutic approaches.

Specific anti-SARS-CoV-2 S1 IgY-scFv is a promising tool for recognition of the virus

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, a series of vaccines, antibodies and drugs have been developed to combat coronavirus disease 2019 (COVID-19). High specific antibodies are powerful tool for the development of immunoassay and providing passive immunotherapy against SARS-CoV-2 and expected with large scale production. SARS-CoV-2 S1 protein was expressed in E. coli BL21 and purified by immobilized metal affinity chromatography, as antigen used to immunize hens, the specific IgY antibodies were extracted form egg yolk by PEG-6000 precipitation, and the titer of anti-S1 IgY antibody reached 1:10,000. IgY single chain variable fragment antibody (IgY-scFv) was generated by using phage display technology and the IgY-scFv showed high binding sensitivity and capacity to S1 protein of SARS-CoV-2, and the minimum detectable antigen S1 protein concentration was 6 ng/µL. The docking study showed that the multiple epitopes on the IgY-scFv interacted with multiple residues on SARS-CoV-2 S1 RBD to form hydrogen bonds. This preliminary study suggests that IgY and IgY-scFv are suitable candidates for the development of immunoassay and passive immunotherapy for COVID-19 to humans and animals.

DEVELOPMENT of COVID-19 MONOCLONAL ANTIBODIES and RECOMBINANT PROTEINS AS REAGENTS for BIOMEDICAL RESEARCH and DIAGNOSTIC TESTS

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.

Preparation and characterization of immobilized 5-HT1A receptor stationary phase for high throughput screening of the receptor-binding ligands from complex systems like Curcuma wenyujin Y. H. Chen et C. Ling extract.

The incidence of depression has increased significantly during the COVID-19 pandemic. This disease is closely associated with serotonin 1A (5-HT1A) receptor and often treated by complex prescription containing Curcuma wenyujin Y. H. Chen et C. Ling. Therefore, we hypothesized that this herb contains bioactive compounds specially binding to the receptor. However, the rapid discovery of new ligands of 5-HT1A receptor is still challenging due to the lack of efficient screening methods. To address this problem, we developed and characterized a novel approach for the rapid screening of ligands by using immobilized 5-HT1A receptor as the chromatographic stationary phase. Briefly, haloalkane dehalogenase was fused at the C-terminal of 5-HT1A receptor, and the modified 5-HT1A receptor was immobilized on amino-microspheres by the reaction between haloalkane dehalogenase and 6-chlorohexanoic acid linker. Scanning electron microscope and X-ray photo-electron were used to characterize the morphology and element of the immobilized receptor. The binding of three specific ligands to 5-HT1A receptor was investigated by two different methods. Moreover, we examined the feasibility of 5-HT1A receptor colume in high throughput screening of new ligands from complex systems as exemplified by Curcuma wenyujin Y. H. Chen et C. Ling. Gweicurculactone, 2-hydroxy-1-(3,4-dihydroxybenzene)-7-(4'-hydroxybezene)-heptane and curcuminol F were identified as the ligands of 5-HT1A receptor with the binding energies of -7.06 kcal/mol, -7.77 kcal/mol and -5.26 kcal/mol, respectively. Collectively, these results indicated that the immobilized 5-HT1A receptor was capable of screening bioactive compound from complex system, providing an effective methodology for high throughput screening.