Newly developed mRNA vaccines induce immune responses in Litopenaeus vannamei shrimps during primary vaccination.

White spot syndrome virus (WSSV) causes highly destructive infection in crustacean aquaculture, often resulting in 100% mortality within a week. However, there is lack of studies addressing the safety issues of WSSV vaccines in shrimps. In this study, WSSV VP28 mRNA vaccines were developed using codon deoptimization approach. These vaccines were administered to Litopenaeus vannamei shrimps at various dosages to access their safety and the shrimps' immune responses using quantification PCR (qPCR). The findings of this study indicate that the expression level of codon deoptimized VP28 mRNA vaccines are lower compared to the wild type VP28 vaccines, as observed through a comparison of bioinformatic predictions and experimental results. Additionally, the total haemocyte count (THC) in shrimps injected with codon deoptimized VP28 vaccine was higher than those injected with wild type VP28 vaccines. Furthermore, the expression of immune-related genes differed between codon deoptimized and wild type VP28 vaccines. In summary, the results suggest that 0.01 µg codon deoptimized VP28-D1 mRNA vaccine is the most promising WSSV mRNA vaccine, displaying low pathogenicity and expression in shrimps. To the best of our knowledge, this research represents the first attempt to attenuate WSSV using codon deoptimization method and development of a potential mRNA vaccine for shrimp purpose. The study addresses an important gap in shrimp vaccine research, offering potential solutions for WSSV control in shrimps.

Synthetic biology approach revealed enhancement in haeme oxygenase-1 gene expression by codon pair optimization while reduction by codon deoptimization.

Haem oxygenase-1 (HO-1) is a ubiquitously expressed gene involved in cellular homoeostasis, and its imbalance in expression results in various disorders. To alleviate such disorders, HO-1 gene expression needs to be modulated. Codon usage bias results from evolutionary forces acting on any nucleotide sequence and determines the gene expression. Like codon usage bias, codon pair bias also exists, playing a role in gene expression. In the present study, HO-1 gene was recoded by manipulating codon and codon pair bias, and four such constructs were made through codon/codon pair deoptimization and codon/codon pair optimization to reduce and enhance the HO-1 gene expression. Codon usage analysis was done for these constructs for four tissues brain, heart, pancreas and liver. Based on codon usage in different tissues, gene expression of these tissues was determined in terms of the codon adaptation index. Based on the codon adaptation index, minimum free energy, and translation efficiency, constructs were evaluated for enhanced or decreased HO-1 expression. The analysis revealed that for enhancing gene expression, codon pair optimization, while for reducing gene expression, codon deoptimization is efficacious. The recoded constructs developed in the study could be used in gene therapy regimens to cure HO-1 over or underexpression-associated disorders.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

Construction of pseudorabies virus variant attenuated vaccine: codon deoptimization of US3 and UL56 genes based on PRV gE/TK deletion strain.

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

In this study, we applied bacterial artificial chromosome (BAC) technology with PRVΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRVΔTK&gE-US3deop-1, PRVΔTK&gE-US3deop-2, and PRVΔTK&gE-US3deop-3) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRVΔTK&gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVΔTK&gE-US3deop-1 did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD50 of the AH02LA strain, and PRVΔTK&gE-US3deop-1 provided similar protection to that of the control virus PRVΔTK&gEAH02. Finally, PRVΔTK&gE-US3deop-1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 TCID50. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRVΔTK&gE-US3deop-1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVΔTK&gE-US3deop-1 displays great potential as a vaccine candidate.

Non-adaptive evolution in codon usage of human-origin monkeypox virus.

Monkeypox virus (Mpox) is a zoonotic infectious disease that threatens human and animal health, with a global outbreak of the low-pathogenic Mpox beginning from 2022. In this study, we analyzed the codon usage of Mpox between two clades, Clade-I and Clade-IIb-B, to understand changes in host adaptation. Clade-IIb-B of the Mpox genome underwent non-adaptive evolution making it less adapted to its host than Clade-I. The analysis of individual genes revealed that 48 genes exhibited non-adaptive mutation, while 38 genes underwent adaptive mutations. Genes involved in replication, transcription, and host-modulation exhibited a mix of adaptive and non-adaptive evolutionary patterns. This study also found that the mutations of Mpox led to changes in non-adaptative genes in different organs. Additionally, we found that codon usage of Mpox was less similar to that of up-regulated host genes and more similar to that of down-regulated host genes post-infection, indicating that codon usage affects host gene expression. Overall, the study highlights the non-adaptive changes in codon usage as a potential cause of differences in Mpox virulence and provides insights into the evolutionary and adaptive mechanisms of Mpox and its potential impact on pathogenicity and host adaptation.

Codon pair bias deoptimization of essential genes in infectious laryngotracheitis virus reduces protein expression.

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains.

Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions (i.e., 3'end of P3 region). The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3'end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates.

Insights into Synonymous Codon Usage Bias in Hepatitis C Virus and Its Adaptation to Hosts.

Hepatitis C virus (HCV) is enveloped RNA virus, encoding for a polyprotein that is processed by cellular proteases. The virus is responsible for liver cirrhosis, allograft rejection, and human hepatocellular carcinoma. Based on studies including compositional analysis, odds ratio analysis, parity analysis, skew analysis, relative synonymous codon usage, codon bias, and protein properties, it was evident that codon usage bias in HCV is dependent upon the nucleotide composition. Codon context analysis revealed CTC-CTG as a preferred codon pair. While CGA and CGT codons were rare, none of the codons were rare in HCV-like viruses envisaged in the present study. Many of the preferred codon pairs were valine amino acid-initiated, which possibly infers viral infectivity; hence the role of selection forces appears to act on the HCV genome, which was further validated by neutrality analysis where selection accounted for 87.28%, while mutation accounted for 12.72% force shaping codon usage. Furthermore, codon usage was correlated with the length of the genome. HCV viruses prefer valine-initiated codon pairs, while HCV-like viruses prefer alanine-initiated codon pairs. The HCV host range is very narrow and is confined to only humans and chimpanzees. Based on indices including codon usage correlation analysis, similarity index, and relative codon deoptimization index, it is evident in the study that the chimpanzee is the primary host of the virus. The present study helped elucidate the preferred host for HCV. The information presented in the study paved the way for generating an attenuated vaccine candidate through viral recoding, with finely tuned nucleotide composition and a perfect balance of preferred and rare codons.

Vaccinia Virus Attenuation by Codon Deoptimization of the A24R Gene for Vaccine Development.

Poxviruses have large DNA genomes, and they are able to infect multiple vertebrate and invertebrate animals, including humans. Despite the eradication of smallpox, poxvirus infections still remain a significant public health concern. Vaccinia virus (VV) is the prototypic member in the poxviridae family and it has been used extensively for different prophylactic applications, including the generation of vaccines against multiple infectious diseases and/or for oncolytic treatment. Many attempts have been pursued to develop novel attenuated forms of VV with improved safety profiles for their implementation as vaccines and/or vaccines vectors. We and others have previously demonstrated how RNA viruses encoding codon-deoptimized viral genes are attenuated, immunogenic and able to protect, upon a single administration, against challenge with parental viruses. In this study, we employed the same experimental approach based on the use of misrepresented codons for the generation of a recombinant (r)VV encoding a codon-deoptimized A24R gene, which is a key component of the viral RNA polymerase. Similar to our previous studies with RNA viruses, the A24R codon-deoptimized rVV (v-A24cd) was highly attenuated in vivo but able to protect, after a single intranasal dose administration, against an otherwise lethal challenge with parental VV. These results indicate that poxviruses can be effectively attenuated by synonymous codon deoptimization and open the possibility of using this methodology alone or in combination with other experimental approaches for the development of attenuated vaccines for the treatment of poxvirus infection, or to generate improved VV-based vectors. Moreover, this approach could be applied to other DNA viruses. IMPORTANCE The family poxviridae includes multiple viruses of medical and veterinary relevance, being vaccinia virus (VV) the prototypic member in the family. VV was used during the smallpox vaccination campaign to eradicate variola virus (VARV), which is considered a credible bioterrorism threat. Because of novel innovations in genetic engineering and vaccine technology, VV has gained popularity as a viral vector for the development of vaccines against several infectious diseases. Several approaches have been used to generate attenuated VV for its implementation as vaccine and/or vaccine vector. Here, we generated a rVV containing a codon-deoptimized A24R gene (v-A24cd), which encodes a key component of the viral RNA polymerase. v-A24cd was stable in culture cells and highly attenuated in vivo but able to protect against a subsequent lethal challenge with parental VV. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated VV and/or vaccine vectors.

Scalable live-attenuated SARS-CoV-2 vaccine candidate demonstrates preclinical safety and efficacy.

Successfully combating the COVID-19 pandemic depends on mass vaccination with suitable vaccines to achieve herd immunity. Here, we describe COVI-VAC, the only live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine currently in clinical development. COVI-VAC was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. COVI-VAC was temperature sensitive in vitro yet grew robustly (>107 plaque forming units/mL) at the permissive temperature. Tissue viral loads were consistently lower, lung pathology milder, and weight loss reduced in Syrian golden hamsters (Mesocricetus auratus) vaccinated intranasally with COVI-VAC compared to those inoculated with wild-type (WT) virus. COVI-VAC inoculation generated spike IgG antibody levels and plaque reduction neutralization titers similar to those in hamsters inoculated with WT virus. Upon challenge with WT virus, COVI-VAC vaccination reduced lung challenge viral titers, resulted in undetectable virus in the brain, and protected hamsters from almost all SARS-CoV-2-associated weight loss. Highly attenuated COVI-VAC is protective at a single intranasal dose in a relevant in vivo model. This, coupled with its large-scale manufacturing potential, supports its potential use in mass vaccination programs.

A Codon-Pair Bias Associated With Network Interactions in Influenza A, B, and C Genomes.

A new codon-pair bias present in the genomes of different types of influenza virus is described. Codons with fewer network interactions are more frequency paired together than other codon-pairs in influenza A, B, and C genomes. A shared feature among three different influenza types suggests an evolutionary bias. Codon-pair preference can affect both speed of protein translation and RNA structure. This newly identified bias may provide insight into drivers of virus evolution.

Betacoronaviruses genome analysis reveals evolution toward specific codons usage: Implications for SARS-CoV-2 mitigation strategies.

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly widespread worldwide becoming one of the major global public health issues of the last centuries. Currently, COVID-19 vaccine rollouts are finally upon us carrying the hope of herd immunity once a sufficient proportion of the population has been vaccinated or infected, as a new horizon. However, the emergence of SARS-CoV-2 variants brought concerns since, as the virus is exposed to environmental selection pressures, it can mutate and evolve, generating variants that may possess enhanced virulence. Codon usage analysis is a strategy to elucidate the evolutionary pressure of the viral genome suffered by different hosts, as possible cause of the emergence of new variants. Therefore, to get a better picture of the SARS-CoV-2 codon bias, we first identified the relative codon usage rate of all Betacoronaviruses lineages. Subsequently, we correlated putative cognate transfer ribonucleic acid (tRNAs) to reveal how those viruses adapt to hosts in relation to their preferred codon usage. Our analysis revealed seven preferred codons located in three different open reading frame which appear preferentially used by SARS-CoV-2. In addition, the tRNA adaptation analysis indicates a wide strategy of competition between the virus and mammalian as principal hosts highlighting the importance to reinforce the genomic monitoring to prompt identify any potential adaptation of the virus into new potential hosts which appear to be crucial to prevent and mitigate the pandemic.

Vaccine candidates generated by codon and codon pair deoptimization of enterovirus A71 protect against lethal challenge in mice.

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease (HFMD) in young children. It is associated with severe neurological complications and death. This study aims to develop a live-attenuated vaccine by codon deoptimization (CD) and codon-pair deoptimization (CPD) of EV-A71. CD is generated by introducing the least preferred codons for amino acids while CPD increases the presence of underrepresented codon pairs in the specific genes. CD and CPD chimeras were generated by synonymous mutations at the VP2, VP3, VP1 and 2A gene regions, designated as XYZ. All twelve deoptimized viruses were viable with similar replication kinetics, but the plaque sizes were inversely proportional to the level of deoptimization. All the deoptimized viruses showed attenuated growth in vitro with reduced viral protein expression at 48 h and lower viral RNA at 39 °C. Six-week-old ICR mice were immunized intraperitoneally with selected CD and CPD X and XY vaccine candidates covering the VP2-VP3 and VP2-VP3-VP1 genes, respectively. All vaccine candidates elicited high anti-EV-A71 IgG levels similar to wild-type (WT) EV-A71. The CD X and CPD X vaccines produced robust neutralizing antibodies but not the CD XY and CPD XY. On lethal challenge, offspring of mice immunized with WT, CD X and CPD X were fully protected, but the CD XY- and CPD XY-vaccinated mice had delayed symptoms and eventually died. Similarly, active immunization of 1-day-old suckling mice with CD X, CPD X and CD XY vaccine candidates provided complete immune protection but CPD XY only protected 40% of the challenged mice. Histology of the muscles from CD X- and CPD X-vaccinated mice showed minimal pathology compared to extensive inflammation in the post-challenged mock-vaccinated mice. Overall, we demonstrated that the CD X and CPD X elicited good neutralizing antibodies, conferred immune protection and are promising live-attenuated vaccine candidates for EV-A71.

A comparative genomics-based study of positive strand RNA viruses emphasizing on SARS-CoV-2 utilizing dinucleotide signature, codon usage and codon context analyses.

The novel corona virus disease or COVID-19 caused by a positive strand RNA virus (PRV) called SARS-CoV-2 is plaguing the entire planet as we conduct this study. In this study a multifaceted analysis was carried out employing dinucleotide signature, codon usage and codon context to compare and unravel the genomic as well as genic characteristics of the SARS-CoV-2 isolates and how they compare to other PRVs which represents some of the most pathogenic human viruses. The main emphasis of this study was to comprehend the codon biology of the SARS-CoV-2 in the backdrop of the other PRVs like Poliovirus, Japanese encephalitis virus, Hepatitis C virus, Norovirus, Rubella virus, Semliki Forest virus, Zika virus, Dengue virus, Human rhinoviruses and the Betacoronaviruses since codon usage pattern along with the nucleotide composition prevalent within the viral genome helps to understand the biology and evolution of viruses. Our results suggest discrete genomic dinucleotide signature within the PRVs. Some of the genes from the different SARS-CoV-2 isolates were also found to demonstrate heterogeneity in terms of their dinucleotide signature. The SARS-CoV-2 isolates also demonstrated a codon context trend characteristically dissimilar to the other PRVs. The findings of this study are expected to contribute to the developing global knowledge base in countering COVID-19.

The Deoptimization of Rabies Virus Matrix Protein Impacts Viral Transcription and Replication.

Rabies virus (RABV) matrix (M) protein plays several important roles during RABV infection. Although previous studies have assessed the functions of M through gene rearrangements, this interferes with the position of other viral proteins. In this study, we attenuated M expression through deoptimizing its codon usage based on codon pair bias in RABV. This strategy more objectively clarifies the role of M during virus infection. Codon-deoptimized M inhibited RABV replication during the early stages of infection, but enhanced viral titers at later stages. Codon-deoptimized M also inhibited genome synthesis at early stage of infection and increased the RABV transcription rates. Attenuated M through codon deoptimization enhanced RABV glycoprotein expression following RABV infection in neuronal cells, but had no influence on the cell-to-cell spread of RABV. In addition, codon-deoptimized M virus induced higher levels of apoptosis compared to the parental RABV. These results indicate that codon-deoptimized M increases glycoprotein expression, providing a foundation for further investigation of the role of M during RABV infection.

Attenuation of Human Respiratory Viruses by Synonymous Genome Recoding.

Using computer algorithms and commercial DNA synthesis, one or more ORFs of a microbial pathogen such as a virus can be recoded and deoptimized by several strategies that may involve the introduction of up to thousands of nucleotide (nt) changes without affecting amino acid (aa) coding. The synonymous recoding strategies that have been applied to RNA viruses include: deoptimization of codon or codon-pair usage, which may reduce protein expression among other effects; increased content of immunomodulatory CpG and UpA RNA, which increase immune responses and thereby restrict viral replication; and substitution of serine and leucine codons with synonymous codons for which single-nt substitutions can yield nonsense codons, thus limiting evolutionary potential. This can reduce pathogen fitness and create potential live-attenuated vaccines that may have improved properties. The combined approach of genome recoding, synthetic biology, and reverse genetics offers several advantages for the generation of attenuated RNA viruses. First, synonymous recoding involves many mutations, which should reduce the rate and magnitude of de-attenuation. Second, increasing the amount of recoding can provide increased attenuation. Third, because there are no changes at the aa level, all of the relevant epitopes should be expressed. Fourth, attenuation frequently does not compromise immunogenicity, suggesting that the recoded viruses have increased immunogenicity per infectious particle. Synonymous deoptimization approaches have been applied to two important human viral pathogens, namely respiratory syncytial virus (RSV) and influenza A virus (IAV). This manuscript will briefly review the use of these different methods of synonymous recoding to generate attenuated RSV and IAV strains. It also will review the characterization of these vaccine candidates in vitro and in animal models, and describe several surprising findings with respect to phenotypic and genetic instability of some of these candidates.

Attenuation of a dengue virus replicon by codon deoptimization of nonstructural genes.

The overwhelming increase of dengue virus (DENV) infections in recent years shows that current strategies to combat dengue do not work. The lack of a highly effective dengue vaccine and the limited effectivity of vector controls exacerbate this situation. To point the way to a novel method of creating DENV vaccine candidates, here we disrupted the codon usage in a DENV-2 reporter replicon to generate variants with different replication characteristics. Six different mutated constructs containing stretches of altered codon usage in the non-structural genes were generated. The mutated sequences were deoptimized to the least favorable codons for human cells. We studied the replication efficiency of these constructs by measuring luciferase reporter activity, relative RNA fold change, and NS1 secretion. Our findings showed that the level of virus attenuation is closely associated with the amount of codon deoptimization. Indeed, replication was completely abolished in extensively-deoptimized constructs D2Rep-6 and D2Rep-5, intermediate with constructs D2Rep-4 (771 bp silent mutations) and D2Rep-3 (756 bp silent mutations) and restored almost to wildtype levels with constructs D2Rep-2 (394 silent mutations) and D2Rep-1 (48 silent mutations). We also determined that the position of codon deoptimization within the genome is crucial to the degree of attenuation observed. Based on our analysis, we propose that the design for an ideal DENV vaccine candidate could include 700-1500 silent mutations within the NS2A and NS3 genes. Our results suggest that codon deoptimization is an ideal strategy that can readily be used to develop a DENV vaccine candidate with "fine-tuned" attenuation.

Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate.

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.