Cloning, expression and identification of truncated spike gene of IBV (Sf200) and chicken granulocyte- macrophage colony stimulating factor (GM-CSF)

The aim of this study was to clone, express and identify the truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken. Firstly, two genes were amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The truncated S1 gene designated as Sf200 containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa) 291-398 and (aa) 497-543 and GM-CSF were then amplified from the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis. The in vitro expression of truncated Sf200 and GM-CSF constructs were later expressed in E. coli BL21 with a molecular mass of approximately 38 kDa and 29 kDa respectively as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polyclonal antibodies were developed by injecting E. coli expressed Sf200 and GM-CSF into the SPF mice and were used to identify the recombinant proteins by Western blot analysis. These findings indicated that the polyclonal antibodies produced in mice could be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.

Research progress of herpes zoster vaccine

Herpes zoster is a cluster blister disease that often occurs on the skin of the elderly, immunocompromised individuals and patients with chronic diseases, which is caused by the reactivation of varicella zoster virus dormant in neurons. The persistent pain of herpes zoster tortures patients and is responsible for serious mental and economic burden. With no effective drug treatment for patients, vaccination becomes more important in disease prevention. Now live attenuated vaccine and recombinant protein vaccine with adjuvant are clinically available abroad. Based on the clinical data, recombinant protein vaccine with adjuvant is the preferred one recommended by CDC.In China, many enterprises perform clinical studies on the two vaccines. However, due to the limitation of adjuvant supply, recombinant protein vaccines have not been widely provided. With the global pandemic of COVID-19, mRNA vaccine becomes a hotspot worldwide. Domestic biological enterprises should actively develop new herpes zoster vaccine to fill the vaccine vacancies in China and to enhance market competitiveness. This article briefly reviews the research progress and development of herpes zoster vaccine in various vaccine platforms.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.

The Future of Epidemic and Pandemic Vaccines to Serve Global Public Health Needs.

This Review initiates a wide-ranging discussion over 2023 by selecting and exploring core themes to be investigated more deeply in papers submitted to the Vaccines Special Issue on the "Future of Epidemic and Pandemic Vaccines to Serve Global Public Health Needs". To tackle the SARS-CoV-2 pandemic, an acceleration of vaccine development across different technology platforms resulted in the emergency use authorization of multiple vaccines in less than a year. Despite this record speed, many limitations surfaced including unequal access to products and technologies, regulatory hurdles, restrictions on the flow of intellectual property needed to develop and manufacture vaccines, clinical trials challenges, development of vaccines that did not curtail or prevent transmission, unsustainable strategies for dealing with variants, and the distorted allocation of funding to favour dominant companies in affluent countries. Key to future epidemic and pandemic responses will be sustainable, global-public-health-driven vaccine development and manufacturing based on equitable access to platform technologies, decentralised and localised innovation, and multiple developers and manufacturers, especially in low- and middle-income countries (LMICs). There is talk of flexible, modular pandemic preparedness, of technology access pools based on non-exclusive global licensing agreements in exchange for fair compensation, of WHO-supported vaccine technology transfer hubs and spokes, and of the creation of vaccine prototypes ready for phase I/II trials, etc. However, all these concepts face extraordinary challenges shaped by current commercial incentives, the unwillingness of pharmaceutical companies and governments to share intellectual property and know-how, the precariousness of building capacity based solely on COVID-19 vaccines, the focus on large-scale manufacturing capacity rather than small-scale rapid-response innovation to stop outbreaks when and where they occur, and the inability of many resource-limited countries to afford next-generation vaccines for their national vaccine programmes. Once the current high subsidies are gone and interest has waned, sustaining vaccine innovation and manufacturing capability in interpandemic periods will require equitable access to vaccine innovation and manufacturing capabilities in all regions of the world based on many vaccines, not just "pandemic vaccines". Public and philanthropic investments will need to leverage enforceable commitments to share vaccines and critical technology so that countries everywhere can establish and scale up vaccine development and manufacturing capability. This will only happen if we question all prior assumptions and learn the lessons offered by the current pandemic. We invite submissions to the special issue, which we hope will help guide the world towards a global vaccine research, development, and manufacturing ecosystem that better balances and integrates scientific, clinical trial, regulatory, and commercial interests and puts global public health needs first.

Plants as Biofactories for Therapeutic Proteins and Antiviral Compounds to Combat COVID-19.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had a profound impact on the world's health and economy. Although the end of the pandemic may come in 2023, it is generally believed that the virus will not be completely eradicated. Most likely, the disease will become an endemicity. The rapid development of vaccines of different types (mRNA, subunit protein, inactivated virus, etc.) and some other antiviral drugs (Remdesivir, Olumiant, Paxlovid, etc.) has provided effectiveness in reducing COVID-19's impact worldwide. However, the circulating SARS-CoV-2 virus has been constantly mutating with the emergence of multiple variants, which makes control of COVID-19 difficult. There is still a pressing need for developing more effective antiviral drugs to fight against the disease. Plants have provided a promising production platform for both bioactive chemical compounds (small molecules) and recombinant therapeutics (big molecules). Plants naturally produce a diverse range of bioactive compounds as secondary metabolites, such as alkaloids, terpenoids/terpenes and polyphenols, which are a rich source of countless antiviral compounds. Plants can also be genetically engineered to produce valuable recombinant therapeutics. This molecular farming in plants has an unprecedented opportunity for developing vaccines, antibodies, and other biologics for pandemic diseases because of its potential advantages, such as low cost, safety, and high production volume. This review summarizes the latest advancements in plant-derived drugs used to combat COVID-19 and discusses the prospects and challenges of the plant-based production platform for antiviral agents.

The role of membrane and circulating forms of ACE 2 in pathological processes in COVID-19 infection

The review analyzes milestone information about the function and pathogenic significance of human angiotensin-converting enzyme 2 (ACE 2). ACE 2 is involved in the development of diseases such as hypertension, malabsorption of certain amino acids in the intestine, and a new type of pneumonia COVID-19 caused by the SARS-CoV-2 virus. Based on the latest literary sources, an assessment is made of the role of differential expression of receptor and soluble forms of this protein in the functioning of the renin-angiotensin-aldosterone system, as well as the mechanisms of ACE 2 participation in the sequential chemical conversion of angiotensin II and its effect on the function of the cardiovascular system. The role of ACE 2 in the development of inflammatory processes in the intestine and its effect on the composition of the intestinal microbiota are also discussed. In addition, the review presents most general data on the proteolytic activation of the S-glycoprotein of the SARS-CoV-2 virus and its participation, together with ACE 2, in the process of virus introduction into the host cell. In conclusion, the hypothesis about autoimmune complications of COVID-19 associated with the formation of the S-glycoprotein-ACE 2 immune complex and the production of autoantibodies is considered. Copyright © 2021 All-Russian Public Organization Antihypertensive League. All rights reserved.

Recombinant vaccines in 2022: a perspective from the cell factory.

The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80's, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.

Recombinant expression of SARS-CoV-2 receptor binding domain (RBD) in Escherichia coli and its immunogenicity in mice.

Objectives: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), giving rise to the coronavirus disease 2019 (COVID-19), has become a danger to wellbeing worldwide. Thus, finding efficient and safe vaccines for COVID-19 is of great importance. As a basic step amid contamination, SARS-CoV-2 employs the receptor-binding domain (RBD) of the spike protein to lock in with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. SARS-CoV-2 receptor-binding domain (RBD) is the main human antibody target for developing vaccines and virus inhibitors, as well as neutralizing antibodies. A bacterial procedure was developed for the expression and purification of the SARS-CoV-2 spike protein receptor-binding domain. Materials and Methods: In this research study, RBD was expressed by Escherichia coli and purified with Ni-NTA chromatography. Then it was affirmed by the western blot test. The immunogenicity and protective efficacy of RBD recombinant protein were assessed on BALB/c mice. Additionally, RBD recombinant protein was tested by ELISA utilizing sera of COVID-19 healing patients contaminated with SARS-CoV-2 wild type and Delta variation. Results: Indirect ELISA was able to detect the protein RBD in serum of the immunized mouse expressed in E. coli. The inactive SARS-CoV2 was detected by antibodies within the serum of immunized mice. Serum antibodies from individuals recovered from Covid19 reacted to the expressed protein. Conclusion: Our findings showed that RBD is of great importance in vaccine design and it can be used to develop recombinant vaccines through induction of antibodies against RBD.

Molecular farming using transgenic rice endosperm.

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.

Fully autonomous domino capillaric circuit for instrument-free, quantitative detection of SARS-CoV-2 in saliva

Rapid, sensitive, quantitative and patient-friendly diagnostic tools have yet to be developed for COVID-19 continued monitoring at the point-of-care. Here, we present an instrument-free capillary microfluidic chip coupled to a lateral flow module that is compatible with a smartphone application for quantitative detection of SARS-CoV-2 from saliva samples. The microfluidic chip is fully autonomous, and performs aliquoting, sample metering, and sequential delivery of reagents. The limit of detection is 0.07 ng/mL for recombinant nucleocapsid protein in saliva. This rapid antigen test provides results in less than 1 hour, without sacrificing analytical sensitivity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Immunogenicity of genetic vaccine based on recombinant hepatitis B Core antigen and B cell multi-epitopes of Porcine epidemic diarrhea virus spike glycoprotein

In present study, in order to develop a new and effective porcine epidemic diarrhea virus (PEDV)vaccine, three B cell epitopes and the truncated S1 gene of PEDV spike protein were combined and inserted into the immunodominant region of the HBcAg. Then the constructed recombinant plasmid HBcAg-PE was transformed to E. coli BL21 (DE3) for expression. After purification and identification by Western-blot, the expressed recombinant proteins HBPE were injected into BALB/c mice as vaccine antigen with different doses through intramuscular injection and its immune effect were preliminary evaluated. The results showed that the recombinant proteins HBPE was expressed as precipitation form and it could reacted specifically with PEDV-positive serum after purification and renaturation. Besides, the RH could induce anti-PEDV specific antibodies and the related Thl and Th2 cytokines in mice. The above results indicate that the recombinant compound epitope antigen of PEDV was successfully constructed. and its immunogenicity as a new vaccine candidate was evaluated in the mice in this study. The results of this study provided a new idea for the development of PEDV genetic engineering vaccine in the future.

Preparation of monoclonal antibody against porcine saperovirus VP1 protein and identification of its antigenic epitope

As a member of the family Picornaviridae, porcine sapelovirus (PSV) is often infected with porcine epidemic diarrhea virus, teschovirus and so on. In recent years, PSV has been isolated from porcine in many provinces of China. It suggests that it is necessary to strengthen the research on PSV. In this study, according to the sequence of PSV HuN2 strain, VP1 gene was inserted into the pGEX-6 P-1 vector, and expressed the recombinant protein. BALB/c mice aged 6-8 weeks were immunized according to the standard procedure. After the third immunization, the mouse orbital blood was collected to identify the antibody level. The highly positive mouse spleen cells were selected for cell fusion. The positive hybridoma cells and two subclones were screened by IFA method, and then a PSV VP1 monoclonal antibody was obtained, named as 33-2 A. The results of IFA showed that PSV could be recognized by 33-2 A MAb, and specific green fluorescence appeared in the cytoplasm;The results of WB and IP showed that PSV infected porcine cell could specifically bind to 33-2 A, and there was a specific band at 32 ku. We also identified the B-cell antigen epitope of 33-2 A, it was at amino acids 40-46 of PSV VP1 protein, and the polypeptide sequence was 40PALTAAE46. The results showed that the monoclonal antibody can react with PSV VP1 protein. The epitope was analyzed with the PSV sequences uploaded in NCBI, 33-2 A antibody can react with most PSV strains and has a certain universal to PSV. This study laid a foundation for the study of the etiology and pathogenesis of PSV.

Design and assembly of plant-based COVID-19 candidate vaccines: reсent development and future prospects.

An outbreak of a new variant of the coronavirus infection, known as COVID-19, occurred at the end of 2019 in China, in the city of Wuhan. It was caused by the SARS-CoV-2 virus. This variant of the virus is characterized by a high degree of variability and, as the current situation with its spread across different regions of the globe shows, it can lead to a progressive spread of infection among the human population and become the cause of a pandemic. The world scientific community is making tremendous efforts to develop means of protection,prevention and treatment of this disease based on modern advances in molecular biology, immunology and vaccinology. This review provides information on the current state of research in the field of vaccine development against COVID-19 with an emphasis on the role of plants in solving this complex problem. Although plants have long been used by mankind as sources of various medicinal substances, in a pandemic, plant expression systems become attractive as biofactories or bioreactors for the production of artificially created protein molecules that include protective antigens against viral infection. The design and creation of such artificial molecules underlies the development of recombinant subunit vaccines aimed at a rapid response against the spread of infections with a high degree of variability. The review presents the state of research covering a period of just over two years, i. e. since the emergence of the new outbreak of coronavirus infection. The authors tried to emphasize the importance of rapid response of research groups from various scientific fields towards the use of existing developments to create means of protection against various pathogens. With two plant expression systems - stable and transient - as examples, the development of work on the creation of recombinant subunit vaccines against COVID-19 in various laboratories and commercial companies is shown. The authors emphasize that plant expression systems have promise for the development of not only protective means under conditions of rapid response (subunit vaccines), but also therapeutic agents in the form of monoclonal antibodies against COVID-19 synthesized in plant cells.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Effects of recombinant porcine Lactobacillus reuteri secreting expression of bovine lactoferrin peptides on growth performance of newborn piglets and protective effect against diarrhea virus infection

This experiment was conducted to investigate the effects of recombinant porcine Lactobacillus reuteri secreting bovine lactoferrin peptide (LFCA) on growth performance of newborn piglets and the protective effect on porcine transmissible gastroenteritis virus (TGEV)infection which caused piglet diarrhea. Experiment 1:thirty-six one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 3 groups, which were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group and control group, each group with 12 piglets. Piglets in each group were orally administered recombinant porcine Lactobacillus reuteri expressing LFCA pPG-LFCA/LR-CO21, containing empty vector plasmid PPG/LR-CO21 and equal volume phosphate buffer (PBS);oral administration continued for 3 days, and the observation time after oral administration was 14 d. During the period, piglets were fed freely, and the changes of body weight and diarrhea were recorded. Experiment 2:thirty one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 5 groups and given TGEV with a half tissue culture infection dose (TCID50) of 10-7.50/mL by oral administration of 1, 3, 6, 9 and 12 mL, respectively. The observation period of 7 d was set to analyze the conditions of half lethal dose. Experiment 3:another thirty-two newborn piglets with an average body weight of about 1.5 kg were selected as experimental animals and randomly divided into 4 groups, with 8 piglets in each group. The groups were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group, control group and TGEV infect group. There were 8 replicates in each group and 1 piglet in each replicate. Each head of the experimental group was orally fed ppG-LFCA/LR-CO21, pPG/LR-CO21 and equal volume of PBS at a dose of 2..1010 CFU per day for 1 consecutive week. At 8 days of age, TGEV was infected by oral administration at half lethal dose, and samples were collected after 7 days of infection. The weight change and diarrhea of each group of piglets were recorded;hematoxylin-eosin staining was used to detect the length of intestinal villi and the depth of crypts;enzyme linked immunosorbent assay (ELISA) was used to determine total serum total immunoglobulin G (IgG) and total secretory immunoglobulin A (sIgA) antibody contents. RT-qPCR was used to detect the mRNA relative expression levels of Claudin-1, Occludin, tight junction protein-1 (ZO-1), inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-P (IFN-P), tumor necrosis factor-a (TNF-a) and Toll-like receptor 2 (TLR2). The flora structure of the contents of the piglet's cecum was analyzed. After oral recombinant porcine Lactobacillusreuteri, compared with the control group, the average daily gain of newborn piglets in the pPG-LFCA/LR-CO21 group was significantly increased (P < 0.01), while the diarrhea rate was significantly decreased (P < 0.01). Compared with TGEV infection group, the average daily gain of piglets in pPG-LFCA/LR-CO21 group was increased and diarrhea rate was decreased, and the differences were significant (P < 0.05). Villus height and the ratio of villus height to crypt depth in jejunum and ileum were significantly increased (P < 0.05). The contents of total IgG and intestinal mucosal total sIgA antibody in serum of piglets were significantly increased (P < 0.05);the mRNA relative expression levels of tight junction protein-related genes Claudin-1, Occludin and ZO-1 in intestinal mucosal tissue were extremely significantly increased (P < 0.01), and the serum TNF-a content was extremely significantly decreased (P < 0.01). Serum IFN-P, IL-6, IL-8 and TLR2 contents were significantly increased (P < 0.01), and the survival rate of piglets was improved. The analysis of the bacterial diversity in the contents of the piglets' cecum showed that the proportion of normal intestinal flora of piglets decreased after TGEV infection. Compared with the TGEV infect group, the proportion of pathogenic bacteria Bacteroides in piglet's intestinal flora decreased by o

Preparation of recombinant antigen and monoclonal antibody of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein

Objective: To prepare the recombinant antigen and monoclonal antibody (mAb) of nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARA-CoV-2).

Production of recombinant SARS-coV-2 proteins and diphtheria toxoid crm197-based fusion

The quickly emerged global COVID-19 pandemic raised a desperate need in the development of pro-tecting vaccines targeting this disease. Therefore, a generation of effective producers of recombinant SARS-CoV-2 proteins became an urgent task. Its resolving contributes to the study of functional SarS-CoV-2 properties, as well as will allow developing the domestic COVID-19 vaccine in Ukraine, thus playing an important strategic role in tackling the pandemics. The aim of the study was to generate prokaryotic and eukaryotic producers of recombinant SarS-CoV-2 proteins and to isolate nucleocapsid (N) protein, receptor-binding do-main (RBD) of spike (S) protein, as well as RBD fused to the carrier – diphtheria toxoid CRM197. For this purpose, appropriate genetic constructs, in particular, replication deficient recombinant AdvC5-based adenoviral vectors expressing the SarS-CoV-2 proteins and CRM197-fused conjugate were created through methods of molecular biology and genetic engineering. Restriction analysis and/or dna sequencing confirmed that we created the correct constructs. Immobilized metal affinity chromatography was used to purify the recombinant proteins. Compliance of their properties was confirmed by the results from polyacrylamide gel electrophoresis, Western blotting, immunoenzymatic assay and MALDI-TOF mass spectrometry. As a result, we generated E. coli Rosetta (de3) bacterial strain and HEK293 cell line producing recombinant SARS-CoV-2 proteins and CRM197-based fusion. In addition, pure N protein, RBD of S protein and RBD-crm197 fusion protein were isolated. The obtained recombinant SarS-CoV-2 proteins can be used to study immunogenic and antigenic properties of the SarS-CoV-2 proteins. Cells producing recombinant SarS-CoV-2 proteins and RBD-crm197 fusion protein are able to provide cheap and safe synthesis of the antigenic substances for domestic development and production of immunodiagnostics for COVID-19 and COVID-19 vaccines in Ukraine. © 2021 Krynina O. I. et al.

IgG Study of Blood Sera of Patients with COVID-19.

The COVID-19 pandemic, which began at the end of 2019 in Wuhan, has affected 220 countries and territories to date. In the present study, we studied humoral immunity in samples of the blood sera of COVID-19 convalescents of varying severity and patients who died due to this infection, using native SARS-CoV-2 and its individual recombinant proteins. The cross-reactivity with SARS-CoV (2002) was also assessed. We used infectious and inactivated SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 strain, inactivated SARS-CoV strain (strain Frankfurt 1, 2002), recombinant proteins, and blood sera of patients diagnosed with COVID-19. The blood sera from patients were analyzed by the Virus Neutralization test, Immunoblotting, and ELISA. The median values and mean ± SD of titers of specific and cross-reactive antibodies in blood sera tested in ELISA were mainly distributed in the following descending order: N > trimer S > RBD. ELISA and immunoblotting revealed a high cross-activity of antibodies specific to SARS-CoV-2 with the SARS-CoV antigen (2002), mainly with the N protein. The presence of antibodies specific to RBD corresponds with the data on the neutralizing activity of blood sera. According to the neutralization test in a number of cases, higher levels of antibodies that neutralize SARS-CoV-2 were detected in blood serum taken from patients several days before their death than in convalescents with a ranging disease severity. This high level of neutralizing antibodies specific to SARS-CoV-2 in the blood sera of patients who subsequently died in hospital from COVID-19 requires a thorough study of the role of humoral immunity as well as comorbidity and other factors affecting the humoral response in this disease.

A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens.

The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens.

Potential for Developing Plant-Derived Candidate Vaccines and Biologics against Emerging Coronavirus Infections.

The emerging human coronavirus infections in the 21st century remain a major public health crisis causing worldwide impact and challenging the global health care system. The virus is circulating in several zoonotic hosts and continuously evolving, causing occasional outbreaks due to spill-over events occurring between animals and humans. Hence, the development of effective vaccines or therapeutic interventions is the current global priority in order to reduce disease severity, frequent outbreaks, and to prevent future infections. Vaccine development for newly emerging pathogens takes a long time, which hinders rapid immunization programs. The concept of plant-based pharmaceuticals can be readily applied to meet the recombinant protein demand by means of transient expression. Plants are evolved as an expression platform, and they bring a combination of unique interests in terms of rapid scalability, flexibility, and economy for industrial-scale production of effective vaccines, diagnostic reagents, and other biopharmaceuticals. Plants offer safe biologics to fulfill emergency demands, especially during pandemic situations or outbreaks caused by emerging strains. This review highlights the features of a plant expression platform for producing recombinant biopharmaceuticals to combat coronavirus infections with emphasis on COVID-19 vaccine and biologics development.