Marcus Graf

Characterization of a Virtually Full-Length Human Immunodeficiency Virus Type 1 Genome of a Prevalent Intersubtype (C/B′) Recombinant Strain in China

ABSTRACT A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B′)-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the gag-pol coding region, (ii) in vpr and at the 3′ end of the vpu gene, and (iii) in thenef open reading frame. (B′)-sequences therefore include (i) several insertions in the gag-pol coding region; (ii) 3′-vpr, the complete vpu gene, and the first exons of tat and rev; and (iii) the 5′ half of the nef gene. Breakpoints located in thevpr/vpu coding region as well as in the nefgene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B′ recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B′ chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.

Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein

ABSTRACT We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by essentialcis-acting sites such as the 5′-untranslated region and 3′-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with thesyngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wtgag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of thesyngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of thesyngag DNA vaccine.

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.

Concerted Action of Multiple cis-Acting Sequences Is Required for Rev Dependence of Late Human Immunodeficiency Virus Type 1 Gene Expression

ABSTRACT Based on the human immunodeficiency virus type 1 (HIV-1)gag gene, subgenomic reporter constructs have been established allowing the contributions of differentcis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimizedgag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

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.

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.

Computer aided multi-parameter gene design: impact of synthetic dnas on protein expression enhancement

Protein production in cells is dependent on various factors including the underlying nucleotide sequence. Gene optimization is dedicated to improve the expression properties of transgenes by codon adaptation to the individual host, increasing RNA production, stability and nuclear export. However, most gene optimization strategies depend on codon usage adaptation only, whereas RNA optimization relies on optimization of many different parameters such as removal of RNA secondary structures, adjustment of CG-values, avoidance of splice sites and elimination of instability elements. With the help of a multi-parameter optimizing algorithm, degeneration of the genetic code provides a powerful tool to identify, analyze as well as utilize parameters and respective motifs to increase and/or adjust expression yields or other important properties of a gene such as its safety or genetic stability.