Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris.

BACKGROUND: Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. RESULTS: In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris. CONCLUSIONS: Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models.

Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.

Codon pair bias deoptimization of the major oncogene meq of a very virulent Marek's disease virus.

Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.

Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference.

The protein synthesis machineries of two distinct phyla of the Animal kingdom, insects of Arthropoda and mammals of Chordata, have different preferences for how to best encode proteins. Nevertheless, arboviruses (arthropod-borne viruses) are capable of infecting both mammals and insects just like arboviruses that use insect vectors to infect plants. These organisms have evolved carefully balanced genomes that can efficiently use the translational machineries of different phyla, even if the phyla belong to different kingdoms. Using dengue virus as an example, we have undone the genome encoding balance and specifically shifted the encoding preference away from mammals. These mammalian-attenuated viruses grow to high titers in insect cells but low titers in mammalian cells, have dramatically increased LD50s in newborn mice, and induce high levels of protective antibodies. Recoded arboviruses with a bias toward phylum-specific expression could form the basis of a new generation of live attenuated vaccine candidates.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

IMPORTANCE: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

An Evolutionary Perspective of Codon Usage Pattern, Dinucleotide Composition and Codon Pair Bias in Prunus Necrotic Ringspot Virus.

Prunus necrotic ringspot virus (PNRSV) is a significant virus of ornamental plants and fruit trees. It is essential to study this virus due to its impact on the horticultural industry. Several studies on PNRSV diversity and phytosanitary detection technology were reported, but the content on the codon usage bias (CUB), dinucleotide preference and codon pair bias (CPB) of PNRSV is still uncertain. We performed comprehensive analyses on a dataset consisting of 359 coat protein (CP) gene sequences in PNRSV to examine the characteristics of CUB, dinucleotide composition, and CPB. The CUB analysis of PNRSV CP sequences showed that it was not only affected by natural selection, but also affected by mutations, and natural selection played a more significant role compared to mutations as the driving force. The dinucleotide composition analysis showed an over-expression of the CpC/GpA dinucleotides and an under-expression of the UpA/GpC dinucleotides. The dinucleotide composition of the PNRSV CP gene showed a weak association with the viral lineages and hosts, but a strong association with viral codon positions. Furthermore, the CPB of PNRSV CP gene is low and is related to dinucleotide preference and codon usage patterns. This research provides reference for future research on PNRSV genetic diversity and gene evolution mechanism.

Synonymous Codon Pair Recoding of the HIV-1 env Gene Affects Virus Replication Capacity.

Synonymous codon pair deoptimization is an efficient strategy for virus attenuation; however, the underlying mechanism remains controversial. Here, we optimized and deoptimized the codon pair bias (CPB) of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene to investigate the influence of env synonymous CPB recoding on virus replication capacity, as well as the potential mechanism. We found that env CPB deoptimization did not always generate attenuation, whereas CPB optimization attenuated virus replication in MT-4 cells. Furthermore, virus attenuation correlated with reduced Env protein production but not with decreased viral RNA synthesis. Remarkably, in our model, increasing the number of CpG dinucleotides in the 5' end of env did not reduce the replication capacity of HIV-1. These results indicate that factors other than CPB or CpG content may have impacted the viral fitness of the synonymously recoded study variants. Our findings provide evidence that CPB recoding-associated attenuation can affect translation efficiency. Moreover, we demonstrated that an increased number of CpGs in the 5' end of HIV-1 env is not always associated with reduced virus replication capacity.

Altering Compositional Properties of Viral Genomes to Design Live-Attenuated Vaccines.

Live-attenuated vaccines have been historically used to successfully prevent numerous diseases caused by a broad variety of RNA viruses due to their ability to elicit strong and perdurable immune-protective responses. In recent years, various strategies have been explored to achieve viral attenuation by rational genetic design rather than using classic and empirical approaches, based on successive passages in cell culture. A deeper understanding of evolutionary implications of distinct viral genomic compositional aspects, as well as substantial advances in synthetic biology technologies, have provided a framework to achieve new viral attenuation strategies. Herein, we will discuss different approaches that are currently applied to modify compositional features of viruses in order to develop novel live-attenuated vaccines.

Evolutionary Patterns of Codon Usage in Major Lineages of Porcine Reproductive and Respiratory Syndrome Virus in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is economically important and characterized by its extensive variation. The codon usage patterns and their influence on viral evolution and host adaptation among different PRRSV strains remain largely unknown. Here, the codon usage of ORF5 genes from lineages 1, 3, 5, and 8, and MLV strains of type 2 PRRSV in China was analyzed. A compositional property analysis of ORF5 genes revealed that nucleotide C is most frequently used at the third position of codons, accompanied by rich GC3s. The effective number of codon (ENC) and codon pair bias (CPB) values indicate that all ORF5 genes have low codon bias and the differences in CPB scores among four lineages are almost not significant. When compared with host codon usage patterns, lineage 1 strains show higher CAI and SiD values, with a high similarity to pig, which might relate to its predominant epidemic propensity in the field. The CAI, RCDI, and SiD values of ORF5 genes from different passages of MLV JXA1R indicate no relation between attenuation and CPB or codon adaptation decrease during serial passage on non-host cells. These findings provide a novel way of understanding the PRRSV's evolution, related to viral survival, host adaptation, and virulence.

mSphere of Influence: Experimental Evolution of RNA Viruses.

Gonzalo Moratorio works in the field of experimental evolution of viruses. In this mSphere of Influence article, he reflects on how the papers "Virus attenuation by genome-scale changes in codon pair bias" by Coleman et al. (Science 320:1784-1787, 2008, https://doi.org/10.1126/science.1155761) and "Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus" by Lauring et al. (Cell Host Microbe 12:623-632, 2012, https://doi.org/10.1016/j.chom.2012.10.008) made an impact on his thinking about how to employ synthetic biology to study experimental evolution of viruses.

A codon-pair deoptimized live-attenuated vaccine against respiratory syncytial virus is immunogenic and efficacious in non-human primates.

Despite a critical need for a respiratory syncytial virus (RSV) vaccine and decades of development efforts, a vaccine to protect infants, elderly, and other at-risk populations from RSV infection remains elusive. We have previously generated a new, live-attenuated vaccine candidate against RSV using rational, computer-aided gene design and chemical synthesis through a process termed viral gene "deoptimization." In this study, we assessed the attenuation, immunogenicity, and efficacy of this synthetic, live-attenuated RSV vaccine candidate, RSV-MinL4.0, in African Green Monkeys. RSV-MinL4.0 was produced under good-manufacturing-practice (GMP) in Vero cells. Vaccination with RSV-MinL4.0 resulted in minimal virus shedding after vaccination, generation of robust humoral and cellular immune responses (despite the presence of baseline RSV neutralizing antibodies in one animal) that were comparable to a wildtype infection, and protection from virus shedding post-challenge with wildtype RSV. These findings demonstrate the promise of RSV-MinL4.0 as a live-attenuated vaccine which will undergo clinical trials to test its ability to safely and effectively protect pediatric and elderly populations from infection with RSV.

Mechanism of Virus Attenuation by Codon Pair Deoptimization.

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.

COSMO: A dynamic programming algorithm for multicriteria codon optimization.

Codon optimization in protein-coding sequences (CDSs) is a widely used technique to promote the heterologous expression of target genes. In codon optimization, a combinatorial space of nucleotide sequences that code a given amino acid sequence and take into account user-prescribed forbidden sequence motifs is explored to optimize multiple criteria. Although evolutionary algorithms have been used to tackle such complex codon optimization problems, evolutionary codon optimization tools do not provide guarantees to find the optimal solutions for these multicriteria codon optimization problems. We have developed a novel multicriteria dynamic programming algorithm, COSMO. By using this algorithm, we can obtain all Pareto-optimal solutions for the multiple features of CDS, which include codon usage, codon context, and the number of hidden stop codons. User-prescribed forbidden sequence motifs are rigorously excluded from the Pareto-optimal solutions. To accelerate CDS design by COSMO, we introduced constraints that reduce the number of Pareto-optimal solutions to be processed in a branch-and-bound manner. We benchmarked COSMO for run-time and the number of generated solutions by adapting selected human genes to yeast codon usage frequencies, and found that the constraints effectively reduce the run-time. In addition to the benchmarking of COSMO, a multi-objective genetic algorithm (MOGA) for CDS design was also benchmarked for the same two aspects and their performances were compared. In this comparison, (i) MOGA identified significantly fewer Pareto-optimal solutions than COSMO, and (ii) the MOGA solutions did not achieve the same mean hypervolume values as those provided by COSMO. These results suggest that generating the whole set of the Pareto-optimal solutions of the codon optimization problems is a difficult task for MOGA.

Codon and Codon-Pair Usage Tables (CoCoPUTs): Facilitating Genetic Variation Analyses and Recombinant Gene Design.

Usage of sequential codon-pairs is non-random and unique to each species. Codon-pair bias is related to but clearly distinct from individual codon usage bias. Codon-pair bias is thought to affect translational fidelity and efficiency and is presumed to be under the selective pressure. It was suggested that changes in codon-pair utilization may affect human disease more significantly than changes in single codons. Although recombinant gene technologies often take codon-pair usage bias into account, codon-pair usage data/tables are not readily available, thus potentially impeding research efforts. The present computational resource (https://hive.biochemistry.gwu.edu/review/codon2) systematically addresses this issue. Building on our recent HIVE-Codon Usage Tables, we constructed a new database to include genomic codon-pair and dinucleotide statistics of all organisms with sequenced genome, available in the GenBank. We believe that the growing understanding of the importance of codon-pair usage will make this resource an invaluable tool to many researchers in academia and pharmaceutical industry.

Extensive genomic recoding by codon-pair deoptimization selective for mammals is a flexible tool to generate attenuated vaccine candidates for dengue virus 2.

The four serotypes of dengue virus (DENV) are the leading etiologic agent of disease caused by arthropod-borne viruses (arboviruses) in the world, with billions at risk of DENV infection spread by infected mosquitoes. DENV causes illness ranging from dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV proliferates well in two different host systems, an invertebrate mosquito vector and vertebrate primate host, which have a distinct difference in their preference of codon pairs (CP) for translation (different "codon pair bias"). Consequently, arboviruses must delicately balance the use of codon pairs between mammals and arthropods, which presents an Achilles' heel that we have exploited by specifically shifting the codon pair preference in the E and NS3 ORFs away from mammals while keeping the CPB favorable for mosquito ORFs. Here we report that recoding of the ORFs has led to variants that were over-attenuated in rhesus macaques although induction of protective antibodies in animals vaccinated with the smallest recoded ORF (E) was observed. The flexibility of our synthetic vaccine design (by decreasing the number of unfavorable CPs in the E ORF), allowed us to construct two new vaccine candidates (EhminA and EhminB) with intermediate attenuation in cell culture and neonatal mice, a result demonstrating proof of concept. New DENV vaccine candidates are being developed based on selective attenuation by dramatic recoding, with flexibility in balancing the attenuation and immunogenicity by marrying rational design and empirical modification.

Attenuation of Marek's disease virus by codon pair deoptimization of a core gene.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of Gallus gallus, the domesticated chicken. Control strategies rely upon vaccination with live attenuated viruses of antigenically similar avian herpesviruses or attenuated strains of MDV. Recent studies in other viruses have shown that recoding certain viral genes to employ synonymous but rarely-used codon pairs resulted in viral attenuation. We deoptimized two MDV proteins, UL54/ICP27 and UL49/VP22, and demonstrate that the more severely deoptimized variant of UL54 accumulates significantly less gene product in vitro. Using these UL54 deoptimized mutants, we further demonstrate that animals infected with the UL54-recoded recombinant virus exhibited decreased viral genome copy number in lymphocytes, reduced lymphoid atrophy and reduced tumor incidence. This study demonstrates that codon pair deoptimization of a single viral gene can produce attenuated strains of MDV. This approach may be useful as a rational way of making novel live attenuated virus vaccines for MDV.

Attenuation of Viruses by Large-Scale Recoding of their Genomes: the Selection Is Always Biased.

PURPOSE OF REVIEW: This review summarizes the current understanding of virus attenuation by large-scale recoding of viral genomes and discusses what would ultimately be necessary for construction of better and safer live modified virus vaccines. RECENT FINDINGS: It has been shown that codon and codon pair deoptimization are rapid and robust methods that can be used for the development of attenuated vaccine candidates. The viruses attenuated by large-scale recoding have the added value that they are extremely genetically stable. However, the exact mechanisms that lead to viral attenuation by recoding are yet to be determined. SUMMARY: While the advantages of large-scale recoding (speed, simplicity, potency, and universal applicability) have been known for more than a decade, this approach has been only inadequately explored and the attention was focused on a limited number of RNA viruses. Attenuation of viruses by large-scale recoding should be explored to combat known and future viral threats.

Evaluation of the attenuation, immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus, in ferrets.

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA+NA)(Min)), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA+NA)(Min) virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10(6)TCID50/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA+NA)(Min) virus at doses ranging from 10(3) to 10(5) TCID50 led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA+NA)(Min) virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.

Downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza A virus vaccines.

Vaccination represents the best option to protect humans against influenza virus. However, improving the effectiveness of current vaccines could better stifle the health burden caused by viral infection. Protein synthesis from individual genes can be downregulated by synthetically deoptimizing a gene's codon usage. With more rapid and affordable nucleotide synthesis, generating viruses that contain genes with deoptimized codons is now feasible. Attenuated, vaccine-candidate viruses can thus be engineered with hitherto uncharacterized properties. With eight gene segments, influenza A viruses with variably recoded genomes can produce a spectrum of attenuation that is contingent on the gene segment targeted and the number of codon changes. This review summarizes different targets and approaches to deoptimize influenza A virus codons for novel vaccine generation.