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.

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.

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.

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.

Insight into the first multi-epitope-based peptide subunit vaccine against avian influenza A virus (H5N6): An immunoinformatics approach.

The rampant spread of highly pathogenic avian influenza A (H5N6) virus has drawn additional concerns along with ongoing Covid-19 pandemic. Due to its migration-related diffusion, the situation is deteriorating. Without an existing effective therapy and vaccines, it will be baffling to take control measures. In this regard, we propose a revers vaccinology approach for prediction and design of a multi-epitope peptide based vaccine. The induction of humoral and cell-mediated immunity seems to be the paramount concern for a peptide vaccine candidate; thus, antigenic B and T cell epitopes were screened from the surface, membrane and envelope proteins of the avian influenza A (H5N6) virus, and passed through several immunological filters to determine the best possible one. Following that, the selected antigenic with immunogenic epitopes and adjuvant were linked to finalize the multi-epitope-based peptide vaccine by appropriate linkers. For the prediction of an effective binding, molecular docking was carried out between the vaccine and immunological receptors (TLR8). Strong binding affinity and good docking scores clarified the stringency of the vaccines. Furthermore, molecular dynamics simulation was performed within the highest binding affinity complex to observe the stability, and minimize the designed vaccine's high mobility region to order to increase its stability. Then, Codon optimization and other physicochemical properties were performed to reveal that the vaccine would be suitable for a higher expression at cloning level and satisfactory thermostability condition. In conclusion, predicting the overall in silico assessment, we anticipated that our designed vaccine would be a plausible prevention against avian influenza A (H5N6) virus.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines.

The design of Pfizer/BioNTech and Moderna mRNA vaccines involves many different types of optimizations. Proper optimization of vaccine mRNA can reduce dosage required for each injection leading to more efficient immunization programs. The mRNA components of the vaccine need to have a 5'-UTR to load ribosomes efficiently onto the mRNA for translation initiation, optimized codon usage for efficient translation elongation, and optimal stop codon for efficient translation termination. Both 5'-UTR and the downstream 3'-UTR should be optimized for mRNA stability. The replacement of uridine by N1-methylpseudourinine (Ψ) complicates some of these optimization processes because Ψ is more versatile in wobbling than U. Different optimizations can conflict with each other, and compromises would need to be made. I highlight the similarities and differences between Pfizer/BioNTech and Moderna mRNA vaccines and discuss the advantage and disadvantage of each to facilitate future vaccine improvement. In particular, I point out a few optimizations in the design of the two mRNA vaccines that have not been performed properly.

Reactivity of human antisera to codon optimized SARS-CoV2 viral proteins expressed in Escherichia coli.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV2 virus continues to pose a serious threat to public health worldwide. The development of rapid diagnostic kits can assist the Tzu Chi Foundation in supporting global volunteers working to provide relief during the current pandemic. MATERIALS AND METHODS: In this study, nucleotide sequences derived from publicly available viral genome data for several domains of the SARS-CoV2 spike and nucleocapsid (N) proteins were chemically synthesized, with codon optimization for Escherichia coli protein expression. No actual viral particles were involved in these experiments. The synthesized sequences were cloned into an E. coli expression system based on pQE80L, and expressed viral proteins were subsequently purified using Ni-affinity chromatography. Western blotting was conducted using human antiviral sera to assess the response of codon-modified viral proteins to COVID-19 patient sera. RESULTS: N protein was expressed in amounts large enough to support large-scale production. The N-terminal domain, receptor-binding domain (RBD), Region 3, and the S2 domain were expressed in small but sufficient amounts for experiments. Immunoblotting results showed that anti-N IgG and anti-N IgM antibodies were detected in most patient sera, but only 60% of samples reacted with the recombinant RBD and S2 domain expressed by E. coli. CONCLUSION: The results indicated that codon-optimized SARS-CoV2 viral proteins can be expressed in E. coli and purified for rapid antibody detection kit preparation, with the codon-optimized N protein, RBD, and S2 protein demonstrating the most potential.