Frank Notka

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.

Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase.

An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) activity was isolated from porcine liver, and sequences derived from trypsinized peptides indicated homology to liver carboxylesterase. By use of degenerate primers, human cDNA clones were identified, which were identical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately threefold increase in cellular ACAT activity. This was accompanied by an approximately 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained numerous lipid droplets that were not present in control CHO cells. Expression of an antisense cDNA in HepG2 cells reduced cellular ACAT activity by 35% compared with control. To further investigate the role of the enzyme in cellular cholesterol homeostasis, regulation of the mRNA was investigated in 7-day cultured human mononuclear phagocytes (MNPs). When these cells were incubated in lipoprotein-deficient serum for 18 hours, the mRNA for ACAT/carboxylesterase was almost not detectable on Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evidence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxylesterase is relevant for cellular cholesterol esterification in vivo. The regulation in MNPs indicates that the enzyme is also involved in foam cell formation during early atherogenesis.

Inhibition of wild-type human immunodeficiency virus and reverse transcriptase inhibitor-resistant variants by Phyllanthus amarus

Substantial progress has been made in research on natural products which effectively inhibit HIV-1 replication. Many active compounds were isolated from traditionally used medicinal plants including Phyllanthus species. This study shows that aqueous as well as alcohol-based Phyllanthus amarus extracts potently inhibit HIV-1 replication in HeLa CD4(+) cells with 50% effective concentration (EC(50)) values ranging from 0.9 to 7.6 microg/ml. A gallotannin enriched fraction showed enhanced activity (0.4 microg/ml), and the purified gallotannins geraniin and corilagin were most active (0.24 microg/ml). HIV-1 replication was also blocked in CD4(+) lymphoid cells with comparable EC(50) values. Applying a cell-based internalization assay, we could demonstrate 70-75% inhibition of virus uptake at concentrations of 2.5 microg/ml for the water/alcohol extract and geraniin. In addition, a concentration-dependent inhibition of HIV-1 reverse transcriptase (RT) could be demonstrated in vitro. The 50% inhibitory concentration (IC(50)) values varied from 1.8 to 14.6 microg/ml. The ability to inhibit replication of a variety of RT inhibitor-resistant HIV-1 strains points to the potential of P. amarus extracts, as natural products, in the chemotherapy of HIV infections.

The impact of intragenic CpG content on gene expression

The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1α). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

Accelerated clearance of SHIV in rhesus monkeys by virus-like particle vaccines is dependent on induction of neutralizing antibodies

Recombinant, insect cell derived SIV Pr56(gag) virus-like particles (VLPs) have been modified either by inserting HIV-1 Gp160 derived peptides into the Pr56(gag) precursor or by integrating the complete HIV-1 gp120 in the particle membrane. To investigate the protective efficacy of these particulate antigens, rhesus macaques were immunized with VLPs both adjuvant-free or adsorbed to alum. In addition, recombinant Semliki Forest viruses (SFV) expressing proteins corresponding to the VLP constructs were established and administered as live vaccines in combination with particulate antigens. Vaccination induced specific humoral responses irrespective of the immunization regimen. However, in contrast to Pr56(gag)-specific antibodies, Env-specific antibody titers could not be increased by booster immunizations in this study. Cell-mediated immune responses were detected following vaccination with VLP-preparations as well as recombinant SFVs. A tendency towards stimulating both enhanced cell mediated as well as humoral immune responses was observed following priming with recombinant SFVs. Upon challenge with SHIV-4 all vaccinated monkeys became infected. However, animals, that were vaccinated with VLPs presenting the complete gp120, managed to clear virus faster than nonimmunized controls. The observed virus elimination significantly correlated with an anamnestic antibody response and an accelerated appearance of neutralizing antibodies postchallenge.

Safety and Immunogenicity of Recombinant Human Immunodeficiency Virus-Like Particles in Rodents and Rhesus Macaques

Data from long-term non-progressing human immunodeficiency virus (HIV)-infected individuals and populations at high risk suggest that an early cytolytic T cell response rather than the humoral immune response might be involved in controlling disease progression. These observations may be used as a guide to the type of response that a vaccine should induce. To clarify the role of different arms of the immune system in conferring protection, the candidate vaccine should allow a regulated, selective induction of different immune responses. Based on a better understanding of the molecular mechanisms regulating the morphogenesis of HIV, we developed an autologous, non-replicating and safe antigen delivery system. This system takes advantage of molecular characteristics of the HIV group-specific antigens (gag) to self-assemble to highly immunogenic virus-like particles (VLP). The immunogenicity of the gag-derived VLP was expanded either by replacing defined domains by selected HIV-1 cytotoxic T lymphocyte (CTL) epitopes (type 1 VLP) or by stable anchoring derivatives of the HIV-1 envelope protein on the surface of the VLP (type 2 VLP). In complete absence of adjuvants, type 1 and type 2 VLP stimulated CD8+ CTL in BALB/c mice, which specifically recognised HIV sequences. In contrast to type 1 VLP, generating an HIV-specific CTL response in the absence of env-specific antibodies, type 2 VLP induced both arms of the immune system including reasonable levels of neutralising antibodies. Initial studies performed in rhesus macaques confirmed these results. Thus, depending on the type and formulation of the VLP, the proposed antigen delivery system allows either the induction of a CTL response (1) in the absence and (2) the presence of an envelope-specific antibody response. A comparison of these approaches in appropriate animal models might contribute to further define the correlates of protection.

Fusion of Epstein-Barr Virus Nuclear Antigen-1-Derived Glycine–Alanine Repeat to Trans-Dominant HIV-1 Gag Increases Inhibitory Activities and Survival of Transduced Cells In Vivo

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

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.