Ruth Elena Soria-Guerra

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.

Methodological ReviewAn overview of bioinformatics tools for epitope prediction: Implications on vaccine development

Exploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned.

Ectopic Expression of Apple F3′H Genes Contributes to Anthocyanin Accumulation in the Arabidopsis tt7 Mutant Grown Under Nitrogen Stress

Three genes encoding flavonoid 3′-hydroxylase (F3′H) in apple (Malus × domestica), designated MdF3′HI, MdF3′HIIa, and MdF3′HIIb, have been identified. MdF3′HIIa and MdF3′HIIb are almost identical in amino acid sequences, and they are allelic, whereas MdF3′HI has 91% nucleotide sequence identity in the coding region to both MdF3′HIIa and MdF3′HIIb. MdF3′HI and MdF3′HII genes are mapped onto linkage groups 14 and 6, respectively, of the apple genome. Throughout the development of apple fruit, transcriptional levels of MdF3′H genes along with other anthocyanin biosynthesis genes are higher in the red-skinned cv Red Delicious than that in the yellow-skinned cv Golden Delicious. Moreover, patterns of MdF3′H gene expression correspond to accumulation patterns of flavonoids in apple fruit. These findings suggest that MdF3′H genes are coordinately expressed with other genes in the anthocyanin biosynthetic pathway in apple. The functionality of these apple F3′H genes has been demonstrated via their ectopic expression in both the Arabidopsis (Arabidopsis thaliana) transparent testa7-1 (tt7) mutant and tobacco (Nicotiana tabacum). When grown under nitrogen-deficient conditions, transgenic Arabidopsis tt7 seedlings expressing apple F3′H regained red color pigmentation and significantly accumulated both 4′-hydrylated pelargonidin and 3′,4′-hydrylated cyanidin. When compared with wild-type plants, flowers of transgenic tobacco lines overexpressing apple F3′H genes exhibited enhanced red color pigmentation. This suggests that the F3′H enzyme may coordinately interact with other flavonoid enzymes in the anthocyanin biosynthesis pathway.

Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge

Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10xa0μg of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10xa0μg-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.

Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin

Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated.

Expression profiles of differentially regulated genes during the early stages of apple flower infection with Erwinia amylovora

To identify genes involved in the response to the fire blight pathogen Erwinia amylovora in apple (Malus×domestica), expression profiles were investigated using an apple oligo (70-mer) array representing 40, 000 genes. Blossoms of a fire blight-susceptible apple cultivar Gala were collected from trees growing in the orchard, placed on a tray in the laboratory, and spray-inoculated with a suspension of E. amylovora at a concentration of 108 cfu ml−1. Uninoculated detached flowers served as controls at each time point. Expression profiles were captured at three different time points post-inoculation at 2, 8, and 24 h, together with those at 0 h (uninoculated). A total of about 3500 genes were found to be significantly modulated in response to at least one of the three time points. Among those, a total of 770, 855, and 1002 genes were up-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively; while, 748, 1024, and 1455 genes were down-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively. Over the three time points post-inoculation, 365 genes were commonly up-regulated and 374 genes were commonly down-regulated. Both sets of genes were classified based on their functional categories. The majority of up-regulated genes were involved in metabolism, signal transduction, signalling, transport, and stress response. A number of transcripts encoding proteins/enzymes known to be up-regulated under particular biotic and abiotic stress were also up-regulated following E. amylovora treatment. Those up- or down-regulated genes encode transcription factors, signaling components, defense-related, transporter, and metabolism, all of which have been associated with disease responses in Arabidopsis and rice, suggesting similar response pathways are involved in apple blossoms.

Gene Expression is Highly Regulated in Early Developing Fruit of Apple

An oligonucleotide-based microarray for apple was developed consisting of ~40,000 sequences, along with positive and negative controls, obtained from 34 cDNA libraries constructed from both vegetative and reproductive tissues at different stages of development, varying genotypes, and under different biotic and abiotic stresses. This apple microarray was used to investigate global gene expression profiles in early developing fruit of three apple genotypes, including “Golden Delicious”, “Gala”, and “Fuji”. A set of 3,348 genes, exhibiting significant differential expression profiles among the three different genotypes, was identified. This set primarily included genes encoding enzymes involved in metabolism and genes related to cell cycle. Differentially expressed genes were grouped into 17 functional categories. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) confirmed differential expression for most genes detected in the microarray analysis, particularly those involved in cell division, cell expansion, and cell enlargement. Among those genes investigated, EF-1 alpha and PRP exhibited differential expression in different apple genotypes and demonstrated a regulatory role during early fruit development. Moreover, a total of 12 differentially expressed genes were identified during early fruit development in three genotypes of apple. These included genes encoding for aminotransferase family protein, DnaJ heat shock, histone, rubisco activase, and tetratricopeptide (TPR) repeat containing protein. This genome-wide analysis suggested that genes engaged in early fruit development among different genotypes of apple are highly regulated.

Transcriptome analysis of resistant and susceptible genotypes of Glycine tomentella during Phakopsora pachyrhizi infection reveals novel rust resistance genes

Soybean rust, caused by Phakopsora pachyrhizi, is a destructive foliar disease in nearly all soybean-producing countries. To identify genes controlling resistance to soybean rust, transcriptome profiling was conducted in resistant and susceptible Glycine tomentella genotypes triggered by P. pachyrhizi infection. Among 38,400 genes monitored using a soybean microarray, at 5% false discovery rate, 1,342 genes were identified exhibiting significant differential expression between uninfected and P. pachyrhizi-infected leaves at 12, 24, 48, and 72xa0h post-inoculation (hpi) in both rust-susceptible and rust-resistant genotypes. Differentially expressed genes were grouped into 12 functional categories, and among those, large numbers relate to basic plant metabolism. Transcripts for genes involved in the phenylpropanoid pathway were up-regulated early during rust infection. Similarly, genes coding for proteins related to stress and defense responses such as glutathione-S-transferases, peroxidases, heat shock proteins, and lipoxygenases were consistently up-regulated following infection at all four time points. Whereas, subsets of genes involved in cellular transport, cellular communication, cell cycle, and DNA processing were down-regulated. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) on randomly selected genes from the different categories confirmed these findings. Of differentially expressed genes, those associated with the flavonoid biosynthesis pathway as well as those coding for peroxidases and lipoxygenases were likely to be involved in rust resistance in soybean, and would serve as good candidates for functional studies. These findings provided insights into mechanisms underlying resistance and general activation of plant defense pathways in response to rust infection.

Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague

Yersinia pestis is a pathogenic agent that causes the bubonic and pneumonic plague. The development of an efficient and low-cost oral vaccine against these diseases is highly desirable. In this study, the immunogenic fusion protein F1-V from Y. pestis was introduced into lettuce via Agrobacterium-mediated transformation, and putative transgenic lines were developed. The presence of the transgene in these putative transgenic lines was determined using polymerase chain reaction (PCR), and transgene integration and transgene copy number were confirmed following Southern blot analysis. The presence of specific F1-V transcripts was confirmed by reverse-transcriptase (RT)-PCR. Using monoclonal antibodies, ELISA and western blot analysis revealed that the expected antigenic F1-V protein was successfully expressed in transgenic lines. Mice immunized subcutaneously with lettuce expressing the F1-V antigen developed systemic humoral responses as ‘proof of concept’ of using lettuce as a production platform for the F1-V immunogen that could be used as a candidate plant-based vaccine against plague.

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacteriumxa0diphtheriae, Bordetellaxa0pertussis, and Clostridiumxa0tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.