Kathryn Sykes

ORF-FINDER: a vector for high-throughput gene identification.

We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.

High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.

Linear expression elements: A rapid, in vivo, method to screen for gene functions

The increasing accumulation of genomic sequence information has accentuated the need for new methods to efficiently assess gene function and to prepare reagents to study these functions. Toward solving this general problem in functional genomics, we report a method by which any PCR-amplified open-reading frame (ORF) can be noncovalently linked to a eukaryotic promoter and terminator, and directly injected into animals to produce local gene expression. We also demonstrate that ORFs can be delivered into mice to produce antibodies specific for the encoded foreign protein by simply attaching mammalian promoter and terminator sequences. This technology makes it possible to screen large numbers of genes rapidly for their functions in vivo or to produce immune responses without the necessity of cloning, bacterial propagation, or protein purification.

Genetic Live Vaccines Mimic the Antigenicity But Not Pathogenicity of Live Viruses

The development of an effective HIV vaccine is both a pressing and a formidable problem. The most encouraging results to date have been achieved using live-attenuated immunodeficiency viruses. However, the frequency of pathogenic breakthroughs has been a deterrent to their development. We suggest that expression libraries generated from viral DNA can produce the immunologic advantages of live vaccines without risk of reversion to pathogenic viruses. The plasmid libraries could be deconvoluted into useful components or administered as complex mixtures. To explore this approach, we designed and tested several of these genetic live vaccines (GLVs) for HIV. We constructed libraries by cloning overlapping fragments of the proviral genome into mammalian expression plasmids, then used them to immunize mice. We found that inserting library fragments into a vector downstream of a secretory gene sequence led to augmented antibody responses, and insertion downstream of a ubiquitin sequence enhanced cytotoxic lymphocyte responses. Also, fragmentation of gag into subgenes broadened T-cell epitope recognition. We have fragmented the genome by sequence-directed and random methods to create libraries with different features. We propose that the characteristics of GLVs support their further investigation as an approach to protection against HIV and other viral pathogens.

Immunosignaturing: a critical review

Health is a complex interaction between metabolism, physiology, and immunity. Although it is difficult to define quantitatively, the activity of the humoral immune system provides a reasonable proxy for changes in health. Immunosignaturing is a microarray-based technology that quantitates the dynamics of circulating antibodies. Recent advancements in the field warrant a review of the technology. Here, we provide an introduction to the technique, evaluate the current progress, contrast similar technologies, and suggest applications that immunosignaturing could facilitate.

Evaluation of SIV library vaccines with genetic cytokines in a macaque challenge.

Gene and expression library immunization make it possible to functionally test all the gene-encoded antigens of a pathogen in a host challenge system. This comprehensive method could generate new and better vaccine candidates. We constructed expression libraries from simian immunodeficiency virus (SIV) cDNA and genetically immunize monkeys with the libraries alone or with a low dose of plasmids encoding human IL-12 and GMCSF. Eight of twelve animals in the three test groups showed some anti-SIV immune response, whereas the controls did not. Six months after priming, monkeys were intravenously challenged with virulent SIVmac251. All were infected but animals in two groups vaccinated with SIV libraries showed a trend toward lower viral-loads, mitigated clinical disease, and higher survival rates than controls. Significantly, co-administering the GMCSF and IL-12-encoding plasmids worsened these measures of protection. This preliminary study should encourage further development of library-vaccine strategies and caution the use of cytokines as adjuvants.

Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus) genome

BackgroundThe success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach.ResultsThe genome of Parapoxvirus ovis (Orf virus) was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer.ConclusionA streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration of B2 may provide the opportunity to significantly impact host immunity while being itself only weakly recognized. The functional genomics method used to pinpoint B2 within an ORFeome may be more broadly applicable to screening for other biological activities in an animal.

Protective antigens against glanders identified by expression library immunization

Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens’ proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.

Cationic gold microparticles for biolistic delivery of nucleic acids

Here we report preparation and properties of positively charged gold microparticles, and their use for biolistic DNA delivery. Micron-sized gold microparticles were modified by building self-assembling polyethylenimine monolayers on their surfaces, which enabled their electrostatic interaction with negatively charged molecules such as DNA. One milligram of the surface-modified microparticles was able to bind directly to up to 3.5 microg of DNA, exceeding the 1 microg/mg limit of the conventional protocols. The binding showed no apparent dependency on DNA purity, size, or conformation. The interaction occurred over a broad range of pH values and salt concentrations, and was stable throughout the standard protocol for biolistic cartridge preparation. At the standard 1 microg dose, biological activity of the DNA biolistically delivered on the charge-modified gold was 25% higher than that delivered on conventional microparticles. Loading the charge-modified gold with more DNA stimulated proportionally higher gene expression. The charge-modified gold can be also used for delivery of small biological molecules such as siRNA. Tissue culture cells biolistically transfected with a LUC+ specific siRNA showed 80% reduction of Luc expression relative to those cells transfected with an irrelevant siRNA. Along with its superior properties as a DNA delivery vehicle, charge-modified gold offers a unique opportunity to deliver various DNA formulations in addition to traditional naked DNA.

Progress in the development of genetic immunization

Despite its young life, genetic immunization (GI) has seen both fame and infamy. Initially under the limelight because of its appeal as a simple method of delivering vaccines, some experiments displayed disappointing immune potency and, consequently, excitement dimmed. Newer focus on the flexibility of GI, afforded by its foundation in molecular biology, has recently rekindled activity in the field. Approached as a recombinant DNA technology, deficiencies become addressable. Unlike any other subunit vaccine modality, such as protein or carbohydrate, DNA is chemically simple, stable, consistent, easily amplified and the base material of a vast array of bioconstruction and biocontrol techniques. GI provides scientists with a simple platform for merging other disciplines, such as molecular biology, biochemistry, genetics, chemistry, informatics and microbiology, into the development of superior vaccine products.