Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5' region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5' end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5' end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression.

Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins--transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators--all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression.

The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization.

One of the main advantages of de novo gene synthesis is the fact that it frees the researcher from any limitations imposed by the use of natural templates. To make the most out of this opportunity, efficient algorithms are needed to calculate a coding sequence, combining different requirements, such as adapted codon usage or avoidance of restriction sites, in the best possible way. We present an algorithm where a "variation window" covering several amino acid positions slides along the coding sequence. Candidate sequences are built comprising the already optimized part of the complete sequence and all possible combinations of synonymous codons representing the amino acids within the window. The candidate sequences are assessed with a quality function, and the first codon of the best candidates' variation window is fixed. Subsequently the window is shifted by one codon position. As an example of a freely accessible software implementing the algorithm, we present the Mr. Gene web-application. Additionally two experimental applications of the algorithm are shown.

Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli.

The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells.

Promiscuous behavior of HPV16E6 specific T cell receptor beta chains hampers functional expression in TCR transgenic T cells, which can be restored in part by genetic modification.

BACKGROUND: T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRalphabeta open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms. METHODS: We isolated the HVP16E6 specific TCRalpha and TCRbeta open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRalpha and TCRbeta chains. RESULTS: Careful analysis of recipient T cells carrying the HPV16E6 TCRbeta and endogenous TCR chains revealed the transgenic TCRbeta chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-gamma production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay. CONCLUSION: Promiscuous behavior of the HPV16E6 specific TCRbeta chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains.

Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B.

In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.

A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts.

We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays.

Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells.

Expression of native transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks. Here we show that codon modification of an HPV16E7-specific T cell receptor (TCR), together with omission of mRNA instability motifs and (cryptic) splice sites, leads to a dramatic increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The codon-modified TCRs have been tested in three different configurations in the retroviral vector LZRS: (1) TCRalpha-IRES-GFP in combination with TCRbeta-IRES-NGFR, (2) TCRalpha-IRES-TCRbeta, and (3) TCRalpha-2A-TCRbeta. T cells carrying the codon-modified TCRs are functionally active against target cells loaded with relevant peptide, model tumor cells expressing the specific epitope as well as cervical carcinoma cells. The significant improvements we report here in the functional expression of specific human TCRs will hopefully expedite clinical application of TCR transfer-based immunotherapy.

Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from an optimized synthetic gene for NMR studies.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis in all organisms. This study was conducted to obtain a high amount of pure, soluble, and stable PPAT from the hyperthermophilic archaeon Pyrococcus abyssi with the aim of investigating its structural characterization by NMR. Production of this enzyme from its natural gene in the Escherichia coli classical expression strain (BL21(DE3)) was not possible, most likely due to the presence of a high number of E. coli rare codons. Only a low amount of P. abyssi PPAT was previously obtained in two E. coli strains encoding tRNAs that recognize these rare E. coli codons and only by using a very rich growth medium. It was not possible to use this strategy to prepare labelled samples for the NMR study, thus another solution had to be found. Therefore, a synthetic gene encoding P. abyssi PPAT was constructed for which not only the rare codons were changed but which was also optimized to avoid other expression-limiting factors such as internal ribosome entry sites, RNA secondary structures, and DNA repeats. Gene optimization strongly increased the yield of P. abyssi PPAT in E. coli BL21(DE3) and allowed us to start the structural characterization of the enzyme. Circular dichroism and 2D NMR experiments indicate the presence of a well-ordered structure for P. abyssi PPAT and also confirm the existence of this enzyme as a monomer in solution.

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.

Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA.

Many of the problems related with mammalian gene expression, such as low translation efficiency and mRNA halflife, can be solved by means of a rational gene design, based on modern bioinformatics, followed by the de novo generation of a synthetic gene. Moreover, high expression rates and prolonged mRNA stability are not only crucial for heterologous mammalian expression, but, in particular, are important for the generation of effective DNA vaccines. In this chapter we show that an optimized synthetic gene encoding the HIV-1 Pr55gag outperforms wild-type gene driven expression by several orders of magnitude. RNA analysis revealed that this positive effect was mostly due to increased mRNA stability of the optimized transcripts. Moreover, mice vaccinated with the optimized gag gene elicited a much stronger immune response against Pr55gag than the control groups immunized with the respective wild-type gene.

Co-influence of transgene expression in mammalian cells. Mutual influence of transgenes on their expression in mammalian cells.

It becomes increasingly clear that therapeutic gene delivery should provide not only for the sustained high level of gene expression but also, in most cases, for the regulated expression of transgenes as much as it occurs under natural conditions. Over the past few years a variety of different systems have been developed in order to regulate the amounts of transcribed RNA upon administration of exogenous agents, or in autoregulated manner. While efforts were focused on optimizing gene expression at the transcriptional level, other levels are still overlooked. In the meantime, regulation of gene expression is not restricted to transcription, but is also executed at the post-transcriptional level, i.e. mRNA stability, processing, transport, translation, protein stability, and modification. Codon usage is considered to be one of the critical factors that limit the expression rate of heterologous genes in different organisms at the posttranscriptional level. HIV-1 structural genes gag, pol, and env represent one of the most extensively utilized models for studying codon usage-mediated effects on transgene expression. In the current work we demonstrate that the codon content affects not only CMV-driven HIV-1 gag expression but also the expression of luciferase reporter gene transcribed independently from the SV40 promoter. The expression levels of both transgenes co-transfected into the human H1299 were inversely co-dependent. The observed phenomenon may be described as sequence-independent post-transcriptional gene silencing, which reflects the existing limitation of transgene expression in mammalian cells at the post-transcriptional level. Optimization of the codon usage may provide for the additional level of regulation of transgene expression in gene transfer experiments in order to maintain the concentration of the protein at the therapeutic levels.

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.

Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation.

Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.