Optimization and Deoptimization of Codons in SARS-CoV-2 and Related Implications for Vaccine Development.

The spread of coronavirus disease 2019 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), has progressed into a global pandemic. To date, thousands of genetic variants have been identified among SARS-CoV-2 isolates collected from patients. Sequence analysis reveals that the codon adaptation index (CAI) values of viral sequences have decreased over time but with occasional fluctuations. Through evolution modeling, it is found that this phenomenon may result from the virus's mutation preference during transmission. Using dual-luciferase assays, it is further discovered that the deoptimization of codons in the viral sequence may weaken protein expression during virus evolution, indicating that codon usage may play an important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccines, it is designed several codon-optimized Omicron BA.2.12.1, BA.4/5, and XBB.1.5 spike mRNA vaccine candidates and experimentally validated their high levels of expression. This study highlights the importance of codon usage in virus evolution and provides guidelines for codon optimization in mRNA and DNA vaccine development.

Designing a Multi-epitope Vaccine against the SARS-CoV-2 Variant Based on an Immunoinformatics Approach.

BACKGROUND: SARS-CoV-2 is a life-threatening virus in the world. Scientific evidence indicates that this pathogen will emerge again in the future. Although the current vaccines have a pivotal role in the control of this pathogen, the emergence of new variants has a negative impact on their effectiveness. OBJECTIVE: Therefore, it is urgent to consider the protective and safe vaccine against all sub-coronavirus species and variants based on the conserved region of the virus. Multi-epitope peptide vaccine (MEV), comprised of immune-dominant epitopes, is designed by immunoinformatic tools and it is a promising strategy against infectious diseases. METHODS: Spike glycoprotein and nucleocapsid proteins from all coronavirus species and variants were aligned and the conserved region was selected. Antigenicity, toxicity, and allergenicity of epitopes were checked by a proper server. To robust the immunity of the multi-epitope vaccine, cholera toxin b (CTB) and three HTL epitopes of tetanus toxin fragment C (TTFrC) were linked at the N-terminal and C-terminal of the construct, respectively. Selected epitopes with MHC molecules and the designed vaccines with Toll-like receptors (TLR-2 and TLR-4) were docked and analyzed. The immunological and physicochemical properties of the designed vaccine were evaluated. The immune responses to the designed vaccine were simulated. Furthermore, molecular dynamic simulations were performed to study the stability and interaction of the MEV-TLRs complexes during simulation time by NAMD (Nanoscale molecular dynamic) software. Finally, the codon of the designed vaccine was optimized according to Saccharomyces boulardii. RESULTS: The conserved regions of spike glycoprotein and nucleocapsid protein were gathered. Then, safe and antigenic epitopes were selected. The population coverage of the designed vaccine was 74.83%. The instability index indicated that the designed multi-epitope was stable (38.61). The binding affinity of the designed vaccine to TLR2 and TLR4 was -11.4 and -11.1, respectively. The designed vaccine could induce humoral and cellular immunity. CONCLUSION: In silico analysis showed that the designed vaccine is a protective multi-epitope vaccine against SARS-CoV-2 variants.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system.

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

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.

Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

Variation in synonymous evolutionary rates in the SARS-CoV-2 genome.

Introduction: Coronavirus disease 2019 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Influential variants and mutants of this virus continue to emerge, and more effective virus-related information is urgently required for identifying and predicting new mutants. According to earlier reports, synonymous substitutions were considered phenotypically silent; thus, such mutations were frequently ignored in studies of viral mutations because they did not directly cause amino acid changes. However, recent studies have shown that synonymous substitutions are not completely silent, and their patterns and potential functional correlations should thus be delineated for better control of the pandemic. Methods: In this study, we estimated the synonymous evolutionary rate (SER) across the SARS-CoV-2 genome and used it to infer the relationship between the viral RNA and host protein. We also assessed the patterns of characteristic mutations found in different viral lineages. Results: We found that the SER varies across the genome and that the variation is primarily influenced by codon-related factors. Moreover, the conserved motifs identified based on the SER were found to be related to host RNA transport and regulation. Importantly, the majority of the existing fixed-characteristic mutations for five important virus lineages (Alpha, Beta, Gamma, Delta, and Omicron) were significantly enriched in partially constrained regions. Discussion: Taken together, our results provide unique information on the evolutionary and functional dynamics of SARS-CoV-2 based on synonymous mutations and offer potentially useful information for better control of the SARS-CoV-2 pandemic.

Functional evolution of SARS-CoV-2 spike protein: Maintaining wide host spectrum and enhancing infectivity via surface charge of spike protein.

The SARS-CoV-2 virus, which causes the COVID-19, is rapidly accumulating mutations to adapt to the hosts. We collected SARS-CoV-2 sequence data from the end of 2019 to January 2023 to analyze for their evolutionary features during the pandemic. We found that most of the SARS-CoV-2 genes are undergoing negative purifying selection, while the spike protein gene (S-gene) is undergoing rapid positive selection. From the original strain to the alpha, delta and omicron variant types, the Ka/Ks of the S-gene increases, while the Ka/Ks within one variant type decreases over time. During the evolution, the codon usage did not evolve towards optimal translation and protein expression. In contrast, only S-gene mutations showed a remarkable trend on accumulating more positive charges. This facilitates the infection via binding human ACE2 for cell entry and binding furin for cleavage. Such a functional evolution emphasizes the survival strategy of SARS-CoV-2, and indicated new druggable target to contain the viral infection. The nearly fully positively-charged interaction surfaces indicated that the infectivity of SARS-CoV-2 virus may approach a limit.

The efficacy and safety of SARS-CoV-2 vaccines mRNA1273 and BNT162b2 might be complicated by rampant C-to-U RNA editing.

The SARS-CoV-2 RNA vaccines are smartly designed to increase the synonymous codon usage by introducing multiple U-to-C mutations. This design would elevate the translation efficiency of vaccine RNAs. However, we found evidence to reason that the designed cytidines might be converted to uridines again by C-to-U RNA deamination in host cells. This C-to-U mechanism might be a main factor that affects the efficacy and safety of RNA vaccines.

Utilizing the codon adaptation index to evaluate the susceptibility to HIV-1 and SARS-CoV-2 related coronaviruses in possible target cells in humans.

Comprehensive identification of possible target cells for viruses is crucial for understanding the pathological mechanism of virosis. The susceptibility of cells to viruses depends on many factors. Besides the existence of receptors at the cell surface, effective expression of viral genes is also pivotal for viral infection. The regulation of viral gene expression is a multilevel process including transcription, translational initiation and translational elongation. At the translational elongation level, the translational efficiency of viral mRNAs mainly depends on the match between their codon composition and cellular translational machinery (usually referred to as codon adaptation). Thus, codon adaptation for viral ORFs in different cell types may be related to their susceptibility to viruses. In this study, we selected the codon adaptation index (CAI) which is a common codon adaptation-based indicator for assessing the translational efficiency at the translational elongation level to evaluate the susceptibility to two-pandemic viruses (HIV-1 and SARS-CoV-2) of different human cell types. Compared with previous studies that evaluated the infectivity of viruses based on codon adaptation, the main advantage of our study is that our analysis is refined to the cell-type level. At first, we verified the positive correlation between CAI and translational efficiency and strengthened the rationality of our research method. Then we calculated CAI for ORFs of two viruses in various human cell types. We found that compared to high-expression endogenous genes, the CAIs of viral ORFs are relatively low. This phenomenon implied that two kinds of viruses have not been well adapted to translational regulatory machinery in human cells. Also, we indicated that presumptive susceptibility to viruses according to CAI is usually consistent with the results of experimental research. However, there are still some exceptions. Finally, we found that two viruses have different effects on cellular translational mechanisms. HIV-1 decouples CAI and translational efficiency of endogenous genes in host cells and SARS-CoV-2 exhibits increased CAI for its ORFs in infected cells. Our results implied that at least in cases of HIV-1 and SARS-CoV-2, CAI can be regarded as an auxiliary index to assess cells' susceptibility to viruses but cannot be used as the only evidence to identify viral target cells.

A Synthetic Biology Approach for Vaccine Candidate Design against Delta Strain of SARS-CoV-2 Revealed Disruption of Favored Codon Pair as a Better Strategy over Using Rare Codons.

The SARS-CoV-2 delta variant (B.1.617.2) appeared for the first time in December 2020 and later spread worldwide. Currently available vaccines are not so efficacious in curbing the viral pathogenesis of the delta strain of COVID; therefore, the development of a safe and effective vaccine is required. In the present study, we envisaged molecular patterns in the structural genes' spike, nucleoprotein, membrane, and envelope of the SARS-CoV-2 delta variant. The study was based on determining compositional features, dinucleotide odds ratio, synonymous codon usage, positive and negative codon contexts, rare codons, and insight into relatedness between the human host isoacceptor tRNA and preferred codons from the structural genes. We found specific patterns, including a significant abundance of T nucleotide over all other three nucleotides. The underrepresentation of GpA, GpG, CpC, and CpG dinucleotides and the overrepresentation of TpT, ApA, CpT, and TpG were observed. A preference towards ACT- (Thr), AAT- (Asn), TTT- (Phe), and TTG- (Leu) initiated codons and aversion towards CGG (Arg), CCG (Pro), and CAC (His) was present in the structural genes of the delta strain. The interaction between the host tRNA pool and preferred codons of the envisaged structural genes revealed that the virus preferred the codons for those suboptimal numbers of isoacceptor tRNA were present. We see this as a strategy adapted by the virus to keep the translation rate low to facilitate the correct folding of viral proteins. The information generated in the study helps design the attenuated vaccine candidate against the SARS-CoV-2 delta variant using a synthetic biology approach. Three strategies were tested: changing TpT to TpA, introducing rare codons, and disrupting favored codons. It found that disrupting favored codons is a better approach to reducing virus fitness and attenuating SARS-CoV-2 delta strain using structural genes.

Natural selection pressure exerted on "Silent" mutations during the evolution of SARS-CoV-2: Evidence from codon usage and RNA structure.

From the first emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) till now, multiple mutations that caused synonymous and nonsynonymous substitutions have accumulated. Among them, synonymous substitutions were regarded as "silent" mutations that received less attention than nonsynonymous substitutions that cause amino acid variations. However, the importance of synonymous substitutions can not be neglected. This research focuses on synonymous substitutions on SARS-CoV-2 and proves that synonymous substitutions were under purifying selection in its evolution. The evidence of purifying selection is provided by comparing the mutation number per site in coding and non-coding regions. We then study the two forces of purifying selection: synonymous codon usage and RNA secondary structure. Results show that the codon usage optimization leads to an adapted codon usage towards humans. Furthermore, our results show that the maintenance of RNA secondary structure causes the purifying of synonymous substitutions in the structural region. These results explain the selection pressure on synonymous substitutions during the evolution of SARS-CoV-2.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Attenuation and Degeneration of SARS-CoV-2 Despite Adaptive Evolution.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) has followed similar trends as other RNA viruses, such as human immunodeficiency virus type 1 and the influenza A virus. Rapid initial diversification was followed by strong competition and a rapid succession of dominant variants. Host-initiated RNA editing has been the primary mechanism for introducing mutations. A significant number of mutations detrimental to viral replication have been quickly purged. Fixed mutations are mostly diversifying mutations selected for host adaptation and immune evasion, with the latter accounting for the majority of the mutations. However, immune evasion often comes at the cost of functionality, and thus, optimal functionality is still far from being accomplished. Instead, selection for antibody-escaping variants and accumulation of near-neutral mutations have led to suboptimal codon usage and reduced replicative capacity, as demonstrated in non-respiratory cell lines. Beneficial adaptation of the virus includes reduced infectivity in lung tissues and increased tropism for the upper airway, resulting in shorter incubation periods, milder diseases, and more efficient transmission between people.

Development of an in silico multi-epitope vaccine against SARS-COV-2 by précised immune-informatics approaches.

The coronavirus family has been infecting the human population for the past two decades, but the ongoing coronavirus called SARS-CoV-2 has posed an enigmatic challenge to global public health security. Since last year, the mutagenic quality of this virus is causing changes to its genetic material. To prevent those situations, the FDA approved some emergency vaccines but there is no assurance that these will function properly in the complex human body system. In point of view, a short but efficient effort has made in this study to develop an immune epitope-based therapy for the rapid exploitation of SARS-CoV-2 by applying in silico structural biology and advancing immune information strategies. The antigenic epitopes were screened from the Surface, Membrane, Envelope proteins of SARS-CoV-2 and passed through several immunological filters to determine the best possible one. According to this, 7CD4+, 10CD8+ and 5 B-cell epitopes were found to be prominent, antigenic, immunogenic, and most importantly, highly conserved among 128 Bangladeshi and 110 other infected countries SARS-CoV-2 variants. After that, the selected epitopes and adjuvant were linked to finalize the multi-epitope vaccine by appropriate linkers. The immune simulation disclosed that the engineered vaccine could activate both humoral and innate immune responses. For the prediction of an effective binding, molecular docking was carried out between the vaccine and immunological receptors (TLRs). Strong binding affinity and good docking scores clarified the stringency of the vaccines. Furthermore, MD simulation was performed within the highest binding affinity complex to observe the stability. Codon optimization and other physicochemical properties revealed that the vaccine would be suitable for a higher expression at cloning level. So, monitoring the overall in silico assessment, we anticipated that our engineered vaccine would be a plausible prevention against COVID-19.

A Live Attenuated COVID-19 Candidate Vaccine for Children: Protection against SARS-CoV-2 Challenge in Hamsters.

Children are at risk of infection from severe acute respiratory syndrome coronavirus-2 virus (SARS-CoV-2) resulting in coronavirus disease (COVID-19) and its more severe forms. New-born infants are expected to receive short-term protection from passively transferred maternal antibodies from their mothers who are immunized with first-generation COVID-19 vaccines. Passively transferred antibodies are expected to wane within first 6 months of infant's life, leaving them vulnerable to COVID-19. Live attenuated vaccines, unlike inactivated or viral-protein-based vaccines, offer broader immune engagement. Given effectiveness of live attenuated vaccines in controlling infectious diseases such as mumps, measles and rubella, we undertook development of a live attenuated COVID-19 vaccine with an aim to vaccinate children beyond 6 months of age. An attenuated vaccine candidate (dCoV), engineered to express sub-optimal codons and deleted polybasic furin cleavage sites in the spike protein of the SARS-CoV-2 WA/1 strain, was developed and tested in hamsters. Hamsters immunized with dCoV via intranasal or intramuscular routes induced high levels of neutralizing antibodies and exhibited complete protection against the SARS-CoV-2 wild-type isolates, i.e., the Wuhan-like (USA-WA1/2020) and Delta variants (B.1.617.2) in a challenge study. In addition, the dCoV formulated with the marketed measles-rubella (MR) vaccine, designated as MR-dCoV, administered to hamsters via intramuscular route, also protected against both SARS-CoV-2 challenges, and dCoV did not interfere with the MR vaccine-mediated immune response. The safety and efficacy of the dCoV and the MR-dCoV against both variants of SARS-CoV-2 opens the possibility of early immunization in children without an additional injection.

What Can RNA-Based Therapy Do for Monogenic Diseases?

The use of RNA-based approaches to treat monogenic diseases (i.e., hereditary disorders caused by mutations in single genes) has been developed on different fronts. One approach uses small antisense oligonucleotides (ASOs) to modulate RNA processing at various stages; namely, to enhance correct splicing, to stimulate exon skipping (to exclude premature termination codon variants), to avoid undesired messenger RNA (mRNA) transcript degradation via the nonsense-mediated decay (NMD) pathway, or to induce mRNA degradation where they encode toxic proteins (e.g., in dominant diseases). Another approach consists in administering mRNA, which, like gene therapy, is a mutation-agnostic approach with potential application to any recessive monogenic disease. This is simpler than gene therapy because instead of requiring targeting of the nucleus, the mRNA only needs to be delivered to the cytoplasm. Although very promising (as demonstrated by COVID-19 vaccines), these approaches still have potential for optimisation, namely regarding delivery efficiency, adverse drug reactions and toxicity.

Influence of complement system genomic variations on the course of COVID-19

Background: The pandemic of SARS-CoV- 2 is a severe worldwide problem increasing morbidity and mortality.1, 2 Severe COVID-19 presents as multiple organ failure caused by systemic inflammation, thrombin generation, and hypofibrinolysis. Diffuse microvascular thrombi and inter-alveolar deposits of complement fragments are observed. The enhanced immunothrombosis is mediated by direct overactivation of complement by virus surface components or damaged cells.3-5 Aims: The study aimed to find whether genetic changes responsible for complement dysregulation known in atypical hemolytic-uremic syndrome (aHUS) can be found in severe COVID-19 patients. Method(s): The study included adult COVID-19 subjects undergoing extracorporeal membrane oxygenation support for severe acute respiratory distress syndrome. Two independent physicians signed informed consent, and the study was approved by a local ethics committee (No. 109/2021) and supported by the University Hospital fund. Next-Generation Sequencing Panel of C3 component, membrane cofactor protein (CD46), complement factor B (CFB), complement factor H (CFH), complement factor H related genes 1-5 (CFHR 1-5), diacylglycerol Kinase Epsilon, thrombomodulin (THBD) and mannose-binding lectin (MBL) genes were performed, with confirmations of positive results by Sanger sequencing. Result(s): Twenty-two patients (13 were male) aged 33 to 65 years were included. No pathogenic gene variants in the C3, CD46, CFB, CFH genes, CFHR 5, CFI, THBD were detected. However, we have shown the presence of modifiers (CFH-H3 haplotype, MCP-GGAAC haplotype, and CFH/CFHR1), which may, together with triggers (infection), increase the severity of the disease (aHUS).6-8 Moreover, we have identified single nucleotide polymorphisms in exon 1 at codon 52 (c.154C>T) and 54 (c.161G>A) of the MBL2 gene promoter associated with low serum levels or dysfunctional MBL and higher incidence of infections. Conclusion(s): We did not detect any complement-related pathogenic gene variants known in aHUS. Thus, It is unlikely that complement dysregulation is the main factor influencing immunothrombosis in a cohort of the most severe COVID-19 patients.

Bioinformatics analysis on structure and function and expression vector construction of SARS-CoV-2 related protein TMPRSS2

Aim Human TMPRSS2 is a transmembrane serine protease.In this paper, the structure and function of the protein were systematically analyzed by bioinformatics, the codon was optimized and the pro- karvotie expression vector was constructed to explore the molecular mechanism of SARS-CoV-2 infecting host cells.Methods The recombinant expression vector pET-22b-TMPRSS2 was generated by molecular cloning technology.The homology, functional sites, subcellular localization, three-dimensional structure and evolutionary characteristics of TMPRSS2 protein were systematically analyzed by using analytical tools such as Protparam, NetPhos3.1, Blast, Clustal X2 and MEGA7.0.Results The prokarvotic expression plas- mid was constructed correctly;TMPRSS2 belongs to medium molecular weight protein, which is composed of 492 amino acid residues.The theoretical isoelectric point is 8.12, the molecular extinction coefficient is 118 145 L * mol~1 * cm"1 , and the half-life is 30 h;TMPRSS2 has 15 potential glycosylation sites and 49 possible phosphorylation sites.It is a transmembrane hydrophilie protein without signal sequenee.In addition, the protein has 13 potential B-cell epitopes and 7 T-eell epitopes.Seeondarv structure analysis showed that random coil accounted for the highest proportion of TMPRSS2 protein ( 0.453 3) , followed by extended strand (0.252 0).Sequence comparison and evolutionary analysis showed that the highest sequence consistency and closest genetic relationship with human TMPRSS2 was Pan troglodytes, followed by gorilla.Conclusions Human-derived TMPRSS2 protein is ev- olutionarilv conserved and functionally important.Hie results of this study can help to reveal the structure and mechanism of action of TMPRSS2 protein, provide ideas for the diagnosis and treatment of COYID-19, and accelerate the research and development process of new drugs targeting TMPRSS2 protein. Copyright © 2022 Publication Centre of Anhui Medical University. All rights reserved.