[Show abstract][Hide abstract]ABSTRACT: Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
[Show abstract][Hide abstract]ABSTRACT: The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3 to 9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1M resulted in a small increase of the α-helical content and corresponding decrease in the random coil content. Secondary structure predictions based on the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophillic domain is predicted to contain a long disordered region of 50 to 60 residues, suggesting that the increase of alpha helical content at high ionic strength could arise due to electrostatic interactions involving the disordered region. CapA is shown to be an inner membrane protein and predicted to play a key cellular role in the assembly of polysaccharides.
[Show abstract][Hide abstract]ABSTRACT: The 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.
The 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.
A 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.
[Show abstract][Hide abstract]ABSTRACT: No detectable antibody response to B2 is elicited by mice co-immunized with rHBsAg and rB2. Sera from the same groups of mice (4 to 5 per group) assayed for HBsAg-reactivity in Figure 4b were assayed for reactivity to B2. The ELISA was conducted in duplicate as described in Material and Methods and Figure 4b except plates were coated with rB2 (50 μl/well) instead of HBsAg.
[Show abstract][Hide abstract]ABSTRACT: The B2 protein of ORFV strain NZ2 also displays immune-cell accumulating activity following murine skin inoculation. Mice were injected i.d into the abdomen with PBS (left panel) or 50 ng rB2 protein (right panel) in triplicate. After 4 days, immune cell densities were measured in harvested skin by 2G9 immunostaining, as described in Figures 2 and 3. Scale bars are 40 micron.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text Article · Nov 2011 · Frontiers in Microbiology
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text Article · Oct 2010 · Nucleic Acids Research
[Show abstract][Hide abstract]ABSTRACT: Identification of highly immunogenic antigens is critical for the construction of an efficacious subunit vaccine against Chlamydia pneumoniae infections. A previous project used a genome-wide screen to identify 12 protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al. ). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the Escherichia coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these 12 genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.
[Show abstract][Hide abstract]ABSTRACT: An inbred A/J mouse respiratory challenge model was validated for vaccine testing against Chlamydia (C.) pneumoniae and used to screen the C. pneumoniae genome for vaccine candidates by expression library immunization (ELI). Biolistic delivery of genetic vaccine constructs elicited Th2-like immunity that was associated with inefficient elimination of C. pneumoniae. Delivery by injection elicited protective Th1-like responses. Since biolistic delivery of pools of ORFs was used in first round screening, the screen presumably selected against potent immunogens. Nevertheless, it was sufficiently accurate to identify three weakly protective antigens among all putative C. pneumoniae ORFs. The results suggest ELI discovery of highly protective C. pneumoniae vaccine candidates requires tight control of the Th1 immunity elicited by the genetically delivered library of test antigens.
[Show abstract][Hide abstract]ABSTRACT: We report the results of a general protocol that was used to screen the whole genome of Chlamydophila abortus, type strain B577 (formerly Chlamydia psittaci strain B577), in a mouse pneumonia model. Genetic immunization was used to functionally test the genes of C. abortus as vaccines in a mouse challenge system. Nine gene fragments were isolated that conferred protection, with five protecting as effectively as the live-vaccine positive control. Bioinformatics approaches were unable to reconstruct isolation of these antigens. These results suggest that pathogen genomes can be functionally screened for vaccine candidate antigens in a mouse model to reveal new classes of vaccine candidate antigens that may have therapeutic efficacy across host species, disease manifestations, and delivery platforms.
[Show abstract][Hide abstract]ABSTRACT: The ability to deliver antigens and immunomodulators specifically to Langerhans cells (LCs) in the skin could impact vaccine development. However, cell-specific targeting of therapeutic molecules remains a challenge in biomedicine. Using phage display technologies, we have developed a protocol that identifies peptides that mediate uptake into target cell types. Employing this approach, we have isolated a 20-mer peptide that mediates specific uptake by immunopotent LCs. The peptide is functional outside the context of the phage and is able to deliver a nanoparticle to LCs in vitro. Although selected on cells in vitro, the peptide is able to direct antigens and genes to LCs in vivo. Liposomes bearing the LC targeting peptide are able to deliver a transcriptionally active gene to LCs in a mouse model. Furthermore, we demonstrate that a low-dose injection into mice of phage bearing the LC-targeting peptide yields faster and higher immune responses against phage-associated antigens than control-phage injections.
Full-text Article · Dec 2004 · DNA and Cell Biology
[Show abstract][Hide abstract]ABSTRACT: Discovery of ligands specific to receptor(s) on a surface of a cancer cell could impact clinical issues including functional diagnosis and cell-specific drug delivery. Using a phage display approach, we have isolated 20-mer peptide ligands that bind to 3 different human lung tumor cell lines, NCI-H1299, NCI-H2009, and A549. The panning protocol is unbiased with no selection pressure towards binding a particular cellular receptor. The isolated phage bind to their target cells 24-300 times better than a control phage. Furthermore, the isolated peptides display remarkable cell-specificities and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. The cell-specificities are not coincident with tumor classes indicating that the peptides are able to recognize cell-surface features that are not represented within the classification of tumor type. The isolated peptides are functional outside of the context of the phage and multimerization of the peptide increases its affinity for its given cell type, thus expanding their utility in clinical situations.
[Show abstract][Hide abstract]ABSTRACT: Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.
Article · Jan 2003 · Journal of Medical Primatology
[Show abstract][Hide abstract]ABSTRACT: : Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.
Article · Dec 2002 · Journal of Medical Primatology
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: 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.
[Show abstract][Hide abstract]ABSTRACT: Genetic vaccines have progressed significantly since the first demonstration of the technology in 1992. When Sanford and Johnston first developed the idea, two applications were envisaged. One was as a new, simple, possibly more effective, method for delivering vaccines. The other was as a new tool to explore the immune system and to discover new vaccines. As there has been relatively little emphasis on the latter, we provide three examples of the potential uses of genetic immunization for discovery/manipulation. One of these technologies may have important implications for the safety of the vaccines. Finally, we propose that the clinical application of genetic vaccines may be limited by inadequate delivery systems and propose the characteristics of an ideal system.
[Show abstract][Hide abstract]ABSTRACT: 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.