RESUMO
Coccidioides is the causative agent of San Joaquin Valley fever, a fungal disease prevalent in the semiarid regions of the Americas. Efforts to develop a fungal vaccine over the last 2 decades were unsuccessful. A candidate antigen, Antigen 2 (Ag2), is notoriously difficult to express in Escherichia coli, and this study sought to accumulate the antigen at high levels in maize. Transformed maize lines accumulated recombinant Ag2 at levels >1 g/kg. Mice immunized with this antigen and challenged with live Coccidioides arthroconidia showed a reduction in the fungal load when Ag2 derived from either E. coli or maize was loaded into glucan chitin particles. A fusion of Ag2 to dendritic cell carrier peptide (DCpep) induced a T-helper type 17 response in the spleen when orally delivered, indicative of a protective immune response. The maize production platform and the glucan chitin particle adjuvant system show promise for development of a Coccidioides vaccine, but further testing is needed to fully assess the optimal method of administration.
Assuntos
Antígenos de Fungos/imunologia , Coccidioides/imunologia , Coccidioidomicose/prevenção & controle , Vacinas Fúngicas/imunologia , Glucanos/imunologia , Zea mays/metabolismo , Adjuvantes Imunológicos , Animais , Quitina/genética , Quitina/imunologia , Coccidioides/genética , Coccidioidomicose/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Glucanos/genética , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Vacinas de Subunidades Antigênicas , Zea mays/genéticaRESUMO
Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.
Assuntos
Antígenos de Superfície da Hepatite B/química , Vacinas contra Hepatite B/química , Zea mays/genética , Administração Oral , Sequência de Aminoácidos , Animais , Cromatografia com Fluido Supercrítico , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/isolamento & purificação , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/genética , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Plantas Geneticamente Modificadas , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Vacinas de Plantas Comestíveis/administração & dosagem , Vacinas de Plantas Comestíveis/química , Vacinas de Plantas Comestíveis/genética , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat-stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.
Assuntos
Antígenos de Superfície da Hepatite B/biossíntese , Vacinas contra Hepatite B/biossíntese , Plantas Geneticamente Modificadas/metabolismo , Sementes/química , Zea mays/imunologia , Antígenos de Superfície da Hepatite B/análise , Extratos Vegetais/análise , Extratos Vegetais/imunologia , Óleos de Plantas/análiseRESUMO
Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.
Assuntos
Engenharia Genética/métodos , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Zea mays/genética , Zea mays/metabolismo , Biomassa , Cruzamento , Carboidratos/análise , Celulase/isolamento & purificação , Celulase/metabolismo , Celulose/metabolismo , Cromatografia Líquida de Alta Pressão , Cruzamentos Genéticos , Eletroforese em Gel de Poliacrilamida , Dosagem de Genes/genética , Hibridização Genética , Plantas Geneticamente Modificadas , Sementes/enzimologia , Especificidade por Substrato , Transgenes/genéticaRESUMO
In Arabidopsis thaliana, XIPOTL1 encodes a phosphoethanolamine N-methyltransferase with a central role in phosphatidylcholine biosynthesis via the methylation pathway. To gain further insights into the mechanisms that regulate XIPOTL1 expression, the effect of upstream open reading frame 30 (uORF30) on the translation of the major ORF (mORF) in the presence or absence of endogenous choline (Cho) or phosphocholine (PCho) was analysed in Arabidopsis seedlings. Dose-response assays with Cho or PCho revealed that both metabolites at physiological concentrations are able to induce the translational repression of a mORF located downstream of the intact uORF30, without significantly altering its mRNA levels. PCho profiles showed a correlation between increased endogenous PCho levels and translation efficiency of a uORF30-containing mORF, while no correlation was detectable with Cho levels. Enhanced expression of a uORF30-containing mORF and decreased PCho levels were observed in the xipotl1 mutant background relative to wild type, suggesting that PCho is the true mediator of uORF30-driven translational repression. In Arabidopsis, endogenous PCho content increases during plant development and affects root meristem size, cell division, and cell elongation. Because XIPOTL1 is preferentially expressed in Arabidopsis root tips, higher PCho levels are found in roots than shoots, and there is a higher sensitivity of this tissue to translational uORF30-mediated control, it is proposed that root tips are the main site for PCho biosynthesis in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Metiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Colina/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Marcação In Situ das Extremidades Cortadas , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilcolina/metabolismo , Mutação PuntualRESUMO
Porcine Epidemic Diarrhea Virus (PEDV) causes severe diarrhea and mortality in piglets. Robust immunity may break the transmission cycle. Expression of antigens in maize grains is a promising method for producing low-cost vaccines. As a first step, we expressed maize constructs containing PEDV S1 spike protein targeted to various cellular locations including the cell wall, endoplasmic reticulum, and vacuole, and fused to carrier proteins E. coli heat labile subunit (LTB) and a dendritic cell (DC) binding peptide, and obtained sufficient antigen for oral immunization. Constructs targeting S1 to the ER or fused to carrier proteins produced high levels of antigen of greater than 20 mg/kg. Oral administration to pigs elicited serum neutralizing antibodies, supporting oral immunization as a practical and cost-effective PEDV vaccine.
RESUMO
Infectious diseases continue to be a significant cause of morbidity and mortality, and although efficacious vaccines are available for many diseases, some parenteral vaccines elicit little or no mucosal antibodies which can be a significant problem since mucosal tissue is the point of entry for 90% of pathogens. In order to provide protection for both serum and mucosal areas, we have tested a combinatorial approach of both parenteral and oral administration of antigens for diseases caused by a viral pathogen, Hepatitis B, and a fungal pathogen, Coccidioides. We demonstrate that co-administration by the parenteral and oral routes is a useful tool to increase the overall immune response. This can include achieving an immune response in tissues that are not elicited when using only one route of administration, providing a higher level of response that can lead to fewer required doses or possibly providing a better response for individuals that are considered poor or non-responders.
RESUMO
BACKGROUND: Upstream open reading frames (uORFs) are elements found in the 5'-region of an mRNA transcript, capable of regulating protein production of the largest, or major ORF (mORF), and impacting organismal development and growth in fungi, plants, and animals. In Drosophila, approximately 40% of transcripts contain upstream start codons (uAUGs) but there is little evidence that these are translated and affect their associated mORF. RESULTS: Analyzing 19,389 Drosophila melanogaster transcript annotations and 666,153 dipteran EST sequences we have identified 44 putative conserved peptide uORFs (CPuORFs) in Drosophila melanogaster that show evidence of negative selection, and therefore are likely to be translated. Transcripts with CPuORFs constitute approximately 0.3% of the total number of transcripts, a similar frequency to the Arabidopsis genome, and have a mean length of 70 amino acids, much larger than the mean length of plant CPuORFs (40 amino acids). There is a statistically significant clustering of CPuORFs at cytological band 57 (p = 10-5), a phenomenon that has never been described for uORFs. Based on GO term and Interpro domain analyses, genes in the uORF dataset show a higher frequency of ORFs implicated in mitochondrial import than the genome-wide frequency (p < 0.01) as well as methyltransferases (p < 0.02). CONCLUSION: Based on these data, it is clear that Drosophila contain putative CPuORFs at frequencies similar to those found in plants. They are distinguished, however, by the type of mORF they tend to associate with, Drosophila CPuORFs preferentially occurring in transcripts encoding mitochondrial proteins and methyltransferases. This provides a basis for the study of CPuORFs and their putative regulatory role in mitochondrial function and disease.
Assuntos
Sequência Conservada/genética , Dípteros/genética , Drosophila melanogaster/genética , Genoma de Inseto/genética , Genômica , Fases de Leitura Aberta/genética , Peptídeos/genética , Animais , Sequência de Bases , Dípteros/classificação , Etiquetas de Sequências Expressas , Duplicação Gênica , Genes de Insetos/genética , Genoma de Planta/genética , Proteínas de Insetos/genética , Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Família Multigênica , Mapeamento Físico do Cromossomo , Estrutura Terciária de Proteína/genética , Vocabulário ControladoRESUMO
BACKGROUND: Upstream open reading frames (uORFs) can mediate translational control over the largest, or major ORF (mORF) in response to starvation, polyamine concentrations, and sucrose concentrations. One plant uORF with conserved peptide sequences has been shown to exert this control in an amino acid sequence-dependent manner but generally it is not clear what kinds of genes are regulated, or how extensively this mechanism is invoked in a given genome. RESULTS: By comparing full-length cDNA sequences from Arabidopsis and rice we identified 26 distinct homology groups of conserved peptide uORFs, only three of which have been reported previously. Pairwise Ka/Ks analysis showed that purifying selection had acted on nearly all conserved peptide uORFs and their associated mORFs. Functions of predicted mORF proteins could be inferred for 16 homology groups and many of these proteins appear to have a regulatory function, including 6 transcription factors, 5 signal transduction factors, 3 developmental signal molecules, a homolog of translation initiation factor eIF5, and a RING finger protein. Transcription factors are clearly overrepresented in this data set when compared to the frequency calculated for the entire genome (p = 1.2 x 10(-7)). Duplicate gene pairs arising from a whole genome duplication (ohnologs) with a conserved uORF are much more likely to have been retained in Arabidopsis (Arabidopsis thaliana) than are ohnologs of other genes (39% vs 14% of ancestral genes, p = 5 x 10(-3)). Two uORF groups were found in animals, indicating an ancient origin of these putative regulatory elements. CONCLUSION: Conservation of uORF amino acid sequence, association with homologous mORFs over long evolutionary time periods, preferential retention after whole genome duplications, and preferential association with mORFs coding for transcription factors suggest that the conserved peptide uORFs identified in this study are strong candidates for translational controllers of regulatory genes.
Assuntos
Arabidopsis/genética , Evolução Molecular , Genes de Plantas , Fases de Leitura Aberta , Oryza/genética , Peptídeos/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência Conservada , Magnoliopsida/classificação , Magnoliopsida/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Transcrição GênicaRESUMO
BACKGROUND: The hepatitis B surface antigen (HBsAg) has been administered over the last 20 years as a parenteral vaccine against the hepatitis B virus (HBV). Despite high seroconversion rates, chronic infection rates are still high worldwide. Orally delivered vaccines provide a practical alternative to injected vaccines, potentially helping poorly responding populations and providing a viable alternative for populations in remote locations. Anamnestic responses are vital to establishing the efficacy of a given vaccine and have been assessed in this study using a plant-based oral delivery platform expressing HBsAg. METHODS: Long-term immunological memory was assessed in mice injected with a primary dose of Recombivax and boosted with orally-delivered HBsAg wafers, control wafers, or parenterally-delivered commercial vaccine (Recombivax). RESULTS: Mice boosted with HBsAg orally-administered wafers displayed sharp increases in mucosal IgA titers in fecal material and steep increases in serum IgA, whereas mice boosted with Recombivax showed no detectable levels of IgA in either fecal or serum samples following four boosting treatments. Long-term memory in the orally-treated mice was evidenced by sustained fecal IgA, and serum IgA, IgG, and mIU/mL over one year, while Recombivax-treated mice displayed sustained serum IgG and mIU/mL. Furthermore, sharp increases in these same antibodies were induced after re-boosting at 47 and 50 weeks post-primary injection. CONCLUSIONS: Orally-delivered vaccines can provide long-term immune responses mucosally and systemically. For sexually-transmitted diseases that can be acquired at mucosal surfaces, such as HBV, an oral delivery platform may provide added protection over a conventional parenterally administered vaccine.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Hepatite B Crônica/prevenção & controle , Imunidade nas Mucosas , Memória Imunológica , Administração Oral , Animais , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-Hepatite B/análise , Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Hepatite B Crônica/imunologia , Imunização Secundária , Imunoglobulina A/sangue , Imunoglobulina A Secretora/análise , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Vacinação/métodos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Zea mays/genéticaRESUMO
The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 µg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Imunidade nas Mucosas , Plantas Geneticamente Modificadas/metabolismo , Administração Oral , Animais , Cromatografia com Fluido Supercrítico , Anticorpos Anti-Hepatite/sangue , Antígenos de Superfície da Hepatite B/biossíntese , Imunoglobulina A/sangue , Imunoglobulina A Secretora/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologia , Zea mays/genética , Zea mays/metabolismoRESUMO
Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.
Assuntos
Sistemas de Liberação de Medicamentos , Antígenos de Superfície da Hepatite B/administração & dosagem , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/imunologia , Administração Oral , Animais , Anticorpos Antivirais/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Plantas Comestíveis/administração & dosagem , Vacinas de Plantas Comestíveis/imunologia , Zea mays/genética , Zea mays/metabolismoRESUMO
Two recent studies demonstrated a positive correlation between divergence in gene expression and protein sequence in Drosophila. This correlation could be driven by positive selection or variation in functional constraint. To distinguish between these alternatives, we compared patterns of molecular evolution for 1,862 genes with two previously reported estimates of expression divergence in Drosophila. We found a slight negative trend (nonsignificant) between positive selection on protein sequence and divergence in expression levels between Drosophila melanogaster and Drosophila simulans. Conversely, shifts in expression patterns during Drosophila development showed a positive association with adaptive protein evolution, though as before the relationship was weak and not significant. Overall, we found no strong evidence for an increase in the incidence of positive selection on protein-coding regions in genes with divergent expression in Drosophila, suggesting that the previously reported positive association between protein and regulatory divergence primarily reflects variation in functional constraint.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila/genética , Evolução Molecular , Seleção Genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Drosophila/química , Regulação da Expressão Gênica no Desenvolvimento , Alinhamento de SequênciaRESUMO
SUMMARY: Errors are prevalent in cDNA sequences but the extent to which sequence collections differ in frequencies and types of errors has not been investigated systematically. cDNA quality control, or cQC, was developed to evaluate the quality of cDNA sequence collections and to revise those sequences that differ from a higher quality genomic sequence. After removing rRNA, vector, bacterial insertion sequence and chimeric cDNA contaminants, small-scale nucleotide discrepancies were found in 51% of cDNA sequences from one Arabidopsis cDNA collection, 89% from a second Arabidopsis collection and 75% from a rice collection. These errors created premature termination codons in 4 and 42% of cDNA sequences in the respective Arabidopsis collections and in 7% of the rice cDNA sequences.