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Expressing the immunodominant projection domain of infectious bursal disease virus fused to the fragment crystallizable of chicken IgY in yellow maize for a prospective edible vaccine
Abstract
Keywords: Infectious bursal disease virus (IBDV) Chimeric protein IgY fragment crystallizable (Fc) VP2 projection domain (PD) Transgenic maize Immature embryos Biolistic bombardment A B S T R A C T Control of Infectious bursal disease virus (IBDV) in endemic countries has been based on early immunization of chicks using conventional live or inactivated vaccines that became not fully effectual and have biosafety concerns. This endeavor seeks generating a recombinant chimeric protein merging the projection domain (PD) of IBDV VP2 capsid with the fragment crystallizable (Fc) of avian IgY (FcIgY), in maize as a prospective poultry edible vaccine. The PD sequence was built on the basis of very virulent IBDV isolates circulating in Egypt. After optimization of codon-usage in maize, sequences of PD and FcIgY were effectively expressed in two elites of yellow maize via bombardment transformation in immature embryos. Chimeric protein amount in stable transgenic samples ranged from1.36% to 3.03% of the total soluble protein based on tissue age and maize cultivar. IBDV VP2 coding sequence was amplified from viral RNA, cloned, and expressed in E. coli. A group of Balb/C mice were hyper-immunized with purified recombinant VP2 protein for raising anti-recombinant VP2 antibodies (anti-rVP2 Ab). Proper expression in maize and immunoreactivity of the chimeric protein (PD-FcIgY) to chicken anti-IBDV and anti-rVP2 Ab were confirmed by both direct and indirect double antibody sandwich (DAS)-ELISAs as well as western blotting. Seeds of regenerated transgenic maize will be validated for chickens as edible vaccination in further studies.
... munity level was unacceptable (X.-X. Zhang et al., 2013).Maize is one of the best choices as an expression vehicle as the recombinant proteins might be stored for a longer period in dry seeds without any protuberance and it is one of the major feeding sources for the nourishment of poultry farm birds(Lamphear et al., 2002;Streatfield et al., 2003).Salem et al. (2020), constructed a chimeric protein via the integration of VP2 projection domain (PD) of Infectious Bursal Disease virus (IBDV) into the Fc fragment of avian IgY (FcIgY) and expressed it in yellow maize via bombardment transformation to utilize as edible vaccine for Aves. The qualitative and quantitative measurements as well as the immunore ...
... Zhang et al., 2013).Maize is one of the best choices as an expression vehicle as the recombinant proteins might be stored for a longer period in dry seeds without any protuberance and it is one of the major feeding sources for the nourishment of poultry farm birds(Lamphear et al., 2002;Streatfield et al., 2003).Salem et al. (2020), constructed a chimeric protein via the integration of VP2 projection domain (PD) of Infectious Bursal Disease virus (IBDV) into the Fc fragment of avian IgY (FcIgY) and expressed it in yellow maize via bombardment transformation to utilize as edible vaccine for Aves. The qualitative and quantitative measurements as well as the immunoreactivity of PD-FcIgY were examined through serological tests only, that is, further studies are required for the exploration of transformed yellow maize as an edible vaccine(Salem et al., 2020).In another study, the researchers utilized a fungus named Ustilago maydis that causes tumors in the young seedlings of maize and these tumors are termed as corn smut. U. maydis was genetically modified to act as a vector carrying the ctb gene. ...
Vaccination is the most suitable and persuasive healthcare program for the prohibition of various deadly diseases. However, the higher production cost and purification strategies are out of reach for the developing nations. In this scenario, development of edible vaccine turns out to be the most promising alternative for remodeling the pharmaceutical industry with reduced production and purification costs. Generally, oral route of vaccination is mostly preferred due to its safety, compliance, low manufacturing cost and most importantly the ability to induce immunity in both systemic and mucosal sites. Genetically modified microorganisms and plants could efficiently be used as vehicles for edible vaccines. Edible vaccines are supposed to reduce the risk associated with traditional vaccines. Currently, oral vaccines are available in the market for several viral and bacterial diseases like cholera, hepatitis B, malaria, rabies etc. Herein, the review focuses on the breakthrough events in the area of edible vaccines associated with dietary microbes and plants for better control over diseases.
... One immunized hen can produce approximately 35 g of IgY yearly,~100 mg of which is antigen-specific [11]. Additionally, numerous studies have identified the protective effect of IgY for viral infections in poultry, such as avian influenza virus [12,13], Newcastle disease [14,15], duck viral hepatitis [16], chicken infectious bursal disease [17,18], reovirus [19,20], and duck adenovirus [21]. There are no approved vaccines against GAstV infection so far, IgY could be a promising prophylactic treatment strategy. ...
Goose astrovirus (GAstV) leads to viscera and joints urate deposition in 1- to 20-day-old goslings, with a mortality rate of up to 50%, posing a severe threat to entire colonies; however, there is no efficient prevention and control method for GAstV infection. This study describes a prophylactic anti-GAstV strategy based on the specific immunoglobulin Y (IgY) from egg yolk. The specific IgY was produced by 22-week-old laying hens intramuscularly immunized with the inactivated GAstV three consecutive times, with 2-week intervals. The egg yolk was collected weekly after the immunization and the anti-GAstV IgY titer was monitored using an agar gel immune diffusion assay (AGID). The results revealed that the AGID titer began to increase on day 7, reached a peak on day 49, and remained at a high level until day 77 after the first immunization. The specific IgY was prepared from the combinations of egg yolk from day 49 to day 77 through PEG-6000 precipitation. Animal experiments were conducted to evaluate the effects of prevention and treatment. The result of the minimum prophylactic dose of the IgY showed that the protection rate was 90.9% when 2.5 mg was administrated. Results of the prevention and the treatment experiments showed prevention and cure rates of over 80% when yolk antibody was administered in the early stages of the GAstV infection. These results suggested that the specific IgY obtained from immunized hens with the inactivated GAstV could be a novel strategy for preventing and treating GAstV infection.
... Such gigantic losses were recorded two years after the identification of the first infected animal, describing the urgency of novel methods for vaccine production. The proof-of-concept for such studies has already been accomplished in previous studies and plants have been found as a suitable host for the expression of edible vaccines for Newcastle disease [8], influenza, Ebola, infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) in chickens [9]. The plant-made ZMapp antibodies against Ebola and influenza were not only encouraging but also paved a way for plants to be further utilized as a production host to combat infectious diseases in cost-effective manner [45]. ...
The production of vaccines in plant cells, termed plant-made pharmaceuticals or molecular farming, is a promising technology for scalable production. Compared to mammalian cell lines, like Chinese Hamster Ovary (CHO) or bacterial cells, plants can be grown with less cost on a large scale to make vaccines antigens and therapeutics affordable and accessible worldwide. An innovative application of this alternative system is the production of vaccines in edible tissues that can be consumed orally to deliver protein antigen without any further processing. In this project, we report stable expression of amino acid sequences corresponding to the TM-1 gene of Mycoplasma gallisepticum as a candidate vaccine antigen against Chronic Respiratory Disease (CRD) in chickens using wheat seed′s tissues as a production host. Molecular and immunoblotting analysis confirmed the ubiquitous expression of a recombinant 41.8-kDa protein with an expression level of 1.03 mg/g dry weight in the endosperm tissues. When orally delivered, the plant-made vaccine was effective in terms of developing antibody response in animal model i.e., chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited the immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrates that plant-made vaccines are not only safe but also scalable and cost-effective with prolonged stability at room temperature.
... As this method is time consuming and requires sophisticated infrastructure, using this technique is difficult, especially in low-income countries. In chicks, for controlling the infectious Bursal disease virus (IBDV), the IBDV VP2 capsid, avian IgY (crystallized), and the transgene bombarded in yellow maize are designed as a prospective edible vaccine (Salem et al. 2020;137). Besides that, recombinant virus particles infect the plant and the expression of viral gene along with the transgene during virus replication (Khan et al., 2020;321). ...
Edible vaccines are subunit vaccinations in which the transgenic plant is induced to produce the encoded proteins once the selected genes are introduced in it. Plants have been identified as a valuable resource for developing promising expression systems for commercial vaccination antigen manufacturing. A number of plant-derived vaccine antigens have been shown to be safe and to elicit a sufficient immune response. As a result, transgenic plants, including edible plant components, are advocated as great options for vaccine production and inexpensive scale-up via culture. Designing methodologies for significant expression of vaccination antigens in plants has been motivated by improved understanding of plant molecular biology, and as a result, refinement of genetic engineering techniques. Potato, banana, lettuce, corn, soybean, rice, and legumes are examples of edible vaccination foods. They are simple to administer, preserve, and give to patients of various ages in a timely and cost-effective manner. Edible vaccinations have great potential for significantly decreasing diseases such as measles, hepatitis B, and cholera, particularly in lower-income nations. This chapter envisions to cover the important characteristics of plant as bio-factories for edible vaccines, including their manufacturing methods, plant species involved, applications, and future prospects.
... As this method is time consuming and requires sophisticated infrastructure, using this technique is difficult, especially in low-income countries. In chicks, for controlling the infectious Bursal disease virus (IBDV), the IBDV VP2 capsid, avian IgY (crystallized), and the transgene bombarded in yellow maize are designed as a prospective edible vaccine (Salem et al. 2020;137). Besides that, recombinant virus particles infect the plant and the expression of viral gene along with the transgene during virus replication 321). ...
... Whole-protein extract from the bacterial culture was separated based on their molecular mass using SDS-PAGE, as described by Salem et al. [27], run on SDS-PAGE, and transferred to polyvinylidene difluoride using a Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked in 2% fat-free milk at 4℃ overnight. ...
... Till now, variety of vaccines against veterinary diseases have been expressed using plant tissues as bioreactor [5]. The proof-of-concept for such studies has already been accomplished in previous studies and plants have been found suitable host for expression of edible vaccines for Newcastle disease [6], influenza, Ebola, infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) in chickens [7]. The plant made ZMapp antibodies against Ebola and influenza were not only encouraging but also paved a way for plants to be further utilized as bioreactor to combat infectious diseases in cost effective manner. ...
The production of vaccines in plant cells, termed as plant-made pharmaceuticals or molecular farming, is a promising technology. Compared to mammalian cell lines, like HEK293 and CHO as established platform, plants can be grown cost effectively on a large-scale without necessitating any sophisticated technologies. An innovative application of this alternative system is the production of vaccines in edible tissues that can be consumed orally to deliver protein antigen without any further processing. In this project we reported stable expression of TM-1 protein of MG as vaccine candidate antigen against Chronic Respiratory Disease (CRD) in wheat seeds tissues. The molecular and Immunoblotting analysis confirmed the integration of recombinant protein of 41.8 kDa with expression level of 1.03mg/g DW in endosperm tissues. When orally delivered, the plant made vaccine were highly effective in terms of developing antibodies in chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited an immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrated that plant-made vaccines are not only safe but also similarly effective to commercially available vaccines.
... 3AB peptide fused with N-terminal 6xHis-tag was purified from the re-suspended pellet using Ni-NTA agarose resin (Qiagen, Germany) as was described in the manufacturer's instructions. Finally, eluted recombinant 3AB-6xHis containing imidazole was subjected to dialysis against 1x PBS overnight at 4 °C, and its concentration was measured using Bradford protocol as previously described [25,26]. ...
During an ongoing outbreak of Foot-and-Mouth Disease Virus (FMDV), it is crucial to distinguish naturally infected from vaccinated seropositive animals. This would support clinical assessment and punctual vigilance. Assays based on 3ABC non-structural protein as an antigen are reliable for this intention. However, the insolubility and degradation of recombinant 3ABC during expression and purification are serious challenges. In this study, alternatively to expressing the recombinant 3ABC (r3ABC), we expressed the 3AB coding sequence (~672 bp) as a recombinant protein (r3AB) with a molecular mass of ~26 KDa. Analytical data from three-dimensional structure, hydrophilicity, and antigenic properties for 3ABC and 3AB exhibited the 3C protein as a hydrophobic, while 3AB as a hydrophilic and highly antigenic protein. The expressed r3AB was recovered as a completely soluble matter after merely native purification, unlike the full expressed r3ABC. Immunoreactivity of r3AB to anti-FMDV antibody in infected sera with different FMDV serotypes was confirmed by the western blot and indirect ELISA. Besides, the authentic antigenicity of purified r3AB was demonstrated through its ability to induce specific seroconversion in mice. Summarily, the removal of 3C: has influenced neither 3D structure nor antigenic properties of the purified r3AB, overcame insolubility and degradation of the r3ABC, and generated a potential superior antigen (r3AB) for herd screening of animals to any FMDV serotype.
Egg yolk immunoglobulin Y (IgY) is a promising class of antibodies with applications in several areas including food safety, disease diagnosis, prevention and treatment. The technology involving IgY manipulation is relatively simple, yet powerful, due to the properties of the antibodies. Despite the increasing interest in IgY research globally, there has been limited exploration of IgY study in Africa, a continent that bears a disproportionate level of disease burden. Building capacity within the continent could contribute significantly to addressing the healthcare challenges facing the region. This study aimed to investigate the capacity and output of researchers affiliated with African institutions in IgY-related research. Data on IgY-based publications in Africa were sourced from the SCOPUS database, and VoSviewer was used to visualize collaborations and research trends. Additional information was obtained from relevant databases to assess publication quality, and the individual publications were summarised. The results showed that there was low research output from African institutions on IgY-related topics, but the published papers were of good quality and have made significant contributions to the field of IgY research. The majority of IgY research in Africa is being conducted by a small number of research groups, and there is a lack of collaboration between them. Furthermore, intra-Africa collaborations are rare. The main focus of IgY research in Africa is on the diagnosis and treatment of infectious diseases affecting humans and animals, which aligns with the healthcare challenges facing the continent. However, areas of IgY research, such as IgY dosage and delivery formulation, and IgY-based antibody mimetics, are currently underexplored by African researchers. This study highlights the need for more investment in IgY research in Africa and the need for collaboration among research groups and institutions. Future research should focus on exploring new areas of IgY research, including the development of viable forms of IgY delivery and dosage formulations, the exploration of IgY-based antibody mimetics and product development. By doing so, African researchers can contribute significantly to the development of novel therapeutic and diagnostic applications for IgY.
Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.
As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain variable fragments (scFvs) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90% recombination frequency, and great enriching factor reaching 88% and 25% in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.
The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carci-noma colon cancer) with IC 50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.
Foot-and-mouth disease virus (FMDV) is one of the most devastating animal viruses that affect livestock worldwide. The 1B capsid of FMDV has been widely used to detect and confirm the infection. In the present study, the sequence coding for 1B subunit of FMDV capsid was expressed in insect cells using the baculovirus expression system under the polyhedrin (polh) promoter. The expression of 1B capsid protein was validated in the culture filtrate of insect cells using SDS-PAGE and western blotting. The culture filtrate containing recombinant 1B capsid (r1B) was used as a coated antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The antigenicity and specificity of r1B against SAT 2 serotype-specific antibodies were assessed. Our results revealed that a protein concentration as low as 25 ng could detect SAT 2-specific antibodies in ELISA. The results highlight the application of insect cells developed r1B protein in the detection of FMDV. Further studies are required to determine the ability of r1B to detect other FMDV serotypes.
Foot-and-mouth disease virus (FMDV) is one of the most devastating viral pathogens of cloven-hoofed animals. The detection of antibodies (Ab) against FMDV structural proteins (SP) using virus neutralization test (VNT) and liquid-phase blocking ELISA (LPBE) is the standard procedure in use for monitoring seroconversion in animals post vaccination, the prevalence of infection-surveillance, proving clinical cases and seronegative status of FMDV-free/naïve-animals prior transportation. However, due to variations within SP of FMDV serotypes, each serotype-specific Ab should be detected separately which is laborious and time-consuming. Accordingly, it is crucial to develop a sensitive, rapid, and accurate test capable of detecting FMDV-specific Ab, regardless its serotype. This study describes the heterologous expression of VP2 protein in E. coli, and its evaluation as a capture antigen in a simple indirect ELISA for serotype-independent detection of anti-FMDV Ab. Sequence analysis revealed that the VP2-coding sequence is considerably conserved among FMDV serotypes. The recombinant VP2 (rVP2), a 22 kDa polypeptide, was purified to near homogeneity by affinity chromatography under native conditions. Immunoreactivity of the rVP2 was confirmed by using a panel of positive sera including sera from animals vaccinated with the local trivalent vaccine and guinea pig FMDV antiserum, which is routinely used as tracing/detecting Ab in LPBE testing. The results obtained from the VP2-based ELISA were comparable to those determined by VNT and LPBE standard diagnostic assays. Specificity and sensitivity of rVP2 in capturing anti-FMDV Ab were 98.3% and 100%, respectively. The developed VP2-ELISA is proved reliable and time-efficient assay for detection of FMDV seropositive animals, regardless the FMDV serotype that can be implemented in a combination with VNT and/or LPBE for rapid diagnosis of an ongoing FMDV infection.
Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp) was cloned, sequenced and sub-cloned into pET-30(+) expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa) and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work.
DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.
We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.
We describe a novel nanobiotechnology platform based on subviral particles derived from Infectious bursal disease virus (IBD-SVPs). The major virus coat proteinVP2 assembles into spherical, 23 nm SVPs when expressed as a heterologous protein in the yeast Pichia pastoris. We recovered up to 38 mg of IBD-SVPs at > 95% purity from 1 L of recombinant yeast culture. The purified particles were able to tolerate organic solvents up to 20% concentration (ethanol or dimethylsulfoxide), they resisted temperatures up to 65 °C and remained stable over a wide pH range (2.5–9.0). We achieved bioconjugation to the aminegroups of lysine residues and to the carboxyl groups of aspartic and glutamic acid residues, allowing the functionalization of IBD-SVPs with biotin. The accessibility of surface aminegroups was measured using Alexa Fluor 488N-hydroxysuccinimide (NHS) ester, an amine-selective fluorescent dye, revealing that approximately 60 dye molecules were attached to the surface of each particle. IBD-SVPs can therefore be exploited as a robust and versatile nanoscaffold to display diverse functional ligands.
We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces.
An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.
A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system
proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency
(42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability
of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed
with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots.
Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with
pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed
by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants
by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the
full-length genes in some of the transgenics.
A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally,
four media combinations of 15 or 30 µm dicamba with or without 88 µm AgNO3 were used to study the effect of dicamba and AgNO3 on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations
BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The
production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 µm and when AgNO3 was added to the medium. A synergistic effect between 88 µm AgNO3 and 30 µm dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best
tissue culture performance and plant regeneration capacity.
Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus
derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos.
In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation
efficiency were investigated. Reducing gold particle size from 1.0 μm or 0.6 μm resulted in a significant increase in the
rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic,
early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility
achieved for Type II callus are summarized and compared to those achieved for greenhouse- and field-derived immature zygotic
embryos.
Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses.
A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given
the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces,
FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting
cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa
and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local
and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against
an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG
vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important
strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.
Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8(+) T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8(-)CD11b(+) DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8(+) T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8(+) T cells. These studies thus demonstrate that cross-presentation in CD8(-)CD11b(+) DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8(+) T-cell responses.
The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient
unidirectional transport of IgG, because FcRn binds IgG at
pH 6.0–6.5 but not at pH 7 or more. As milk passes through
the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport
vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a nonperturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features
of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking.
Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.
Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.
The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.
The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.
Recombinant plant expression systems offer a means to produce large quantities of selected antigens for subunit vaccines. Cereals are particularly well-suited expression vehicles since the expressed proteins can be stored at relatively high concentrations for extended periods of time without degradation and dry seed can be formulated into oral vaccines suitable for commercial applications. A subunit vaccine candidate directed against porcine transmissible gastroenteritis virus and expressed in corn seed has been developed for oral delivery to swine. Here, we show that this vaccine, when administered to previously sensitized gilts, can boost neutralizing antibody levels in the animals' serum, colostrum and milk. Thus, this vaccine candidate is effective at boosting lactogenic immunity and is appropriate to pursue through large-scale field trials preceding commercialization.
As a step towards developing a safe and effective edible vaccine against Newcastle disease virus (NDV), we have explored the use of plants genetically engineered to express viral proteins. We report the construction of transgenic potato plants expressing the genes coding for immunogenic proteins of NDV under the regulation of CaMV 35S promoter and its immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund adjuvant produced specific anti-NDV antibodies. Animals fed with transgenic leaves showed a specific response against NDV. Detection of IgA released from in vitro-cultured intestinal tissue fragments indicated the presence of IgA-secreting cells in the gut.
Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic diversity among its serotypes, requiring several anti-FMDV antibodies for its laboratory diagnosis, which complicated the used techniques. To conquer this cumbersome, we developed a new panel of different single-chain fragment variable (scFv) for serotype-independent detection of FMDV. The recombinant VP2 capsid protein, as a relatively conserved protein among FMDV serotypes, was expressed in E. Coli, and injected in mice. Spleen's RNA was extracted for isolating the coding sequences of IgG variable domains that were assembled into repertoires of scFv. Phage library displaying scFv was constructed with ∼1.9 × 108plaque forming units. Characterization of the library showed eight of unique scFvs, which were expressed as bacterial periplasmic proteins with apparent molecular weight of ∼27 kDa. Our data revealed the broad-spectrum binding affinity of the eight scFvs as both coating and tracing antibodies to FMDV serotypes A, O, and SAT 2.
p>Multiple functions have recently been identified for the neonatal Fc receptor FcRn. In addition, a human homolog of the rodent forms of FcRn has been identified and characterized. This major histocompatibility complex class I-related receptor plays a role in the passive delivery of immunoglobulin (Ig)Gs from mother to young and the regulation of serum IgG levels. In addition, FcRn expression in tissues such as liver, mammary gland, and adult intestine suggests that it may modulate IgG transport at these sites. These diverse functions are apparently brought about by the ability of FcRn to bind IgGs and transport them within and across cells. However, the molecular details as to how FcRn traffics within cells have yet to be fully understood, although in vitro systems have been developed for this purpose. The molecular nature of the FcRn-IgG interaction has been studied extensively and encompasses residues located at the CH2-CH3 domain interface of the Fc region of IgG. These Fc amino acids are highly conserved in rodents and man and interact with residues primarily located on the α2 domain of FcRn. Thus, it is now possible to engineer IgGs with altered affinities for FcRn, and this has relevance to the modulation of IgG serum half-life and maternofetal IgG transport for therapeutic applications.</p
A distinctive infectious bursal disease (IBD) virus genotype (ITA) was detected in IBD-live vaccinated broilers in Italy without clinical signs of IBD. It was isolated in specific-pathogen-free eggs and molecularly characterized in the hypervariable region of the virus protein (VP) 2. Phylogenetic analysis showed that ITA strains clustered separately from other homologous reference sequences of IBDVs, either classical or very virulent, retrieved from GenBank or previously reported in Italy, and from vaccine strains. The new genotype shows peculiar molecular characteristics in key positions of the VP2 hypervariable region, which affect charged or potentially glycosylated amino acids virtually associated with important changes in virus properties. Characterization of 41 IBDV strains detected in Italy between 2013 and 2014 showed that ITA is emergent in Italian densely populated poultry areas being the 68% of the IBDV detections made during routine diagnostic activity over a two-year period, in spite of the immunity induced by large-scale vaccination. Four very virulent strains (DV86) and one classical strain (HPR2), together with eight vaccine strains, were also detected. The currently available epidemiological and clinical data do not allow defining the degree of pathogenicity of the ITA genotype. Only in vivo experimental pathogenicity studies conducted in secure isolation conditions, through the evaluation of clinical signs and macro/microscopic lesions, will clarify conclusively the virulence of the new Italian genotype.
Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.
Vaccines represent one of the most compelling examples of how biomedical research has improved society by saving lives and dramatically reducing the burden of infectious disease. Despite the importance of vaccinology, we are still in the early stages of understanding how the best vaccines work and how we can achieve better protective efficacy through improved vaccine design. Most successful vaccines have been developed empirically, but recent advances in immunology are beginning to shed new light on the mechanisms of vaccine-mediated protection and development of long-term immunity. Although natural infection will often elicit lifelong immunity, almost all current vaccines require booster vaccination in order to achieve durable protective humoral immune responses, regardless of whether the vaccine is based on infection with replicating live-attenuated vaccine strains of the specific pathogen or whether they are derived from immunization with inactivated, non-replicating vaccines or subunit vaccines. The form of the vaccine antigen (e.g., soluble or particulate/aggregate) appears to play an important role in determining immunogenicity and the interactions between dendritic cells, B cells and T cells in the germinal center are likely to dictate the magnitude and duration of protective immunity. By learning how to optimize these interactions, we may be able to elicit more effective and long-lived immunity with fewer vaccinations.
Infectious bursal disease (IBD) is a viral immunosuppressive disease of chickens attacking mainly an important lymphoid organ in birds [the bursa of Fabricius (BF)]. The emergence of new variant strains of the causative agent [infectious bursal disease virus (IBDV)] has made it more urgent to develop new vaccination strategies against IBD. One of these strategies is the use of recombinant vaccines (DNA and viral-vectored vaccines). Several studies have investigated the host immune response towards IBDV. This review will present a detailed background on the disease and its causative agent, accompanied by a summary of the most recent findings regarding the host immune response to IBDV infection and the use of recombinant vaccines against IBD.
The activity of the 5′-region of the rice actin 1 gene (Act1), covering a region 1.4 kb upstream of the Act1 translation initiation codon, was extensively analyzed in transgenic maize plants. The 5′-region of Act1 fused to the β-glucuronidase (GUS) gene (gus) coding region was co-transformed to maize with the phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene (pin2). One and 29 independent transformation events with expression of both bar and gus were recovered from bombardment of immature embryo-derived embryogenic callus of Hi-II derivative and bombardment of shoot tips of Honey N Pearl and Illinois Golden Extra Sweet, respectively. Expression of gus in tissues of transgenic plants was examined by histochemical assay, immunoblot analysis, and fluorometric GUS specific activity assay. A constitutive expression of the introduced gus was observed throughout the evelopmental stages of the vegetative and reproductive organs in transgenic maize plants. Quantitative analysis of GUS in transgenic plants showed that GUS, as percent of total soluble protein, was as much as 3.1% in leaves and 2.8% in roots. The functional activity of the 5′-region of Act1 was inherited to transgenic progeny. The results indicate that the 1.4-kb 5′-region of Act1 is an efficient and strong promoter for gene expression in stable transgenic maize plants.
The effects of osmotic conditioning on both transient expression and stable transformation were evaluated by introducing plasmid DNAs via particle bombardment into embryogenic suspension culture cells of Zea mays (A188 B73). Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 h prior to and 16 h after bombardment resulted in a statistically significant 2.7-fold increase in transient -glucuronidase expression. Under these conditions, an average of approximately 9,000 blue foci were obtained from 100 l packed cell volume of bombarded embryogenic tissue. Osmotic conditioning of the target cells resulted in a 6.8-fold increase in recovery of stably transformed maize clones. Transformed fertile plants and progeny were obtained from several transformed cell lines. We believe the basis of osmotic enhancement of transient expression and stable transformation resulted from plasmolysis of the cells which may have reduced cell damage by preventing extrusion of the protoplasm from bombarded cells.
Recognition of chlorine as a plant micronutrient has been extended to include ten species. Acute chlorine deficiencies or decreased yields were produced with lettuce, tomato, cabbage, carrot, sugar beet, barley, alfalfa, buckwheat, corn, and beans. Squash plants showed neither loss in yield nor other deficiency symptoms when cultured at the same time and under the same conditions as the aforementioned species. All plants acquired more chlorine during their growth than can be accounted for from seeds, inorganic salts, or water used in the experiments. Plant species least susceptible to injury when cultured upon low chlorine salt solutions were also the ones most capable of acquiring extrinsic chlorine. Of the species studied, lettuce was the most sensitive to minus chlorine culture solutions and squash, the least sensitive. However, the concentration of chlorine in all of the species cultured under limited chlorine supply was not greatly different. It is inferred that plants such as corn, beans, and squash survived the minus chlorine cultures by reason of greater accretion of extrinsic chlorine from the atmosphere. The form of the atmospherically borne chlorine is not known.
We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.
A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling
and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are
the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.
To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests - corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta(TM) contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.
Thesis - Auburn University. Photocopy of typescript.
Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.
A panel of two non-neutralizing and six neutralizing monoclonal antibodies (Mabs) were used in antigen-capture enzyme immunoassays (AC-ELISA) to examine the antigenicity of 1301 wild type infectious bursal disease viruses (IBDV) isolated from different poultry flocks throughout the United States over a three year period. Analysis of these isolates with protective, neutralizing Mabs directed against the VP2 structural protein of IBDV showed that four antigenically distinct groups of serotype 1 IBDV could be separated on the basis of the presence or absence of one or more Mab defined, conformation-dependent, multivalent neutralization site. AC-ELISA reactivity patterns of the Mabs with isolates demonstrated that IBDV field populations were relatively antigenically homogeneous per premise isolation. Geographically, various antigenic species were more or less prevalent, or nearly absent. Competition analysis with neutralizing Mabs coupled with AC-ELISA results suggested that neutralization epitopes for IBDV are distinct, spatially arranged, yet closely linked. Of 5 Mab defined neutralization epitopes, shown to be related to protection from virulent challenge by Classic IBDV strains isolated prior to 1985, only two of the epitopes remain unaltered on the most recent emergent variant field strain of IBDV isolated.
Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.
The sequence of 1015 nucleotides from the 3' poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones. This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region. The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV. The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of Mr 30910. The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions. The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses. The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5'-proximal one-fifth of the genes. Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion. A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins. The BYMV 3'-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract.
Statistically significant antigenic differences were detected among serotype I infectious bursal disease viruses (IBDV) using the virus-neutralization test. Eight serotype I commercial vaccine strains, five serotype I field strains, and two serotype II field strains were tested. Hyperimmune guinea pig antisera against heterologous and homologous IBDV strains were used in cross-neutralization tests. Relatedness values were calculated from geometric mean antibody titers based on a minimum of three tests. Six subtypes were distinguished among the 13 serotype I strains tested.
The genome of infectious bursal disease virus (IBDV) strain 002-73 was found to consist of two segments of double-stranded (ds) RNA which were 3400 bp (MW 2.06 X 10(6)) and 2900 bp (MW 1.76 X 10(6)) long, respectively. The ds IBDV RNA could be translated, in vitro, only after extensive denaturation. The small RNA segment was found to code for a single polypeptide of MW 90K, while the large RNA segment coded for three major polypeptides of MW 52K, 32K, and 28K, and two minor polypeptides of MW 41K and 16K. The large RNA segment could encode proteins of MW 125K while the MW of the translated products was 169K suggesting that a precursor-product relationship exists between some of the translation products. A method is described for the synthesis of ds cDNA from large ds RNA molecules. Analyses of recombinant colonies showed that inserts covering the entire IBDV genome had been cloned.
The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein.
Previously, we demonstrated that intranasal (i.n.) but not intraperitoneal (i.p.) immunization with a recombinant adenovirus vector expressing glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) induced mucosal immune responses and conveyed long-term protection to mice against an i.n. challenge with heterologous HSV-2. We now show that i.n. immunization of female mice with this same vector, AdgB8, provides secretory and serum-derived humoral immune responses in the genital tract. Intranasal immunization induced anti-HSVgB IgA and IgG in vaginal washes of mice, whereas i.p. immunization only induced IgG, which appeared to be serum-derived. Interestingly, intravaginal (ivag) immunization with AdgB8 resulted in little or no anti-HSVgB IgA and only low levels of specific IgG in vaginal washes. All three routes of inoculation induced gB-specific serum IgG and IgA; however, i.n. immunized mice demonstrated the highest level of serum anti-HSVgB IgA. Additionally, ivag boosting with AdgB8 did not significantly alter the serum or vaginal wash antibody responses in i.n. or i.p. immunized mice. The IgG to IgA ratios of gB-specific and total antibody titres in the serum and vaginal washes of i.n. immunized mice indicated that the IgA in the vaginal washes was likely to be secretory. Furthermore, the titres of anti-HSVgB IgA relative to total IgA were higher in vaginal washes than sera, suggesting that the gB-specific vaginal wash IgA present in i.n. immunized mice was locally produced.
The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody specific for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, specifically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its specificity for mouse I-Ak. Although mouse monoclonal antibodies specific for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct was able to elicit an A loop-specific, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes.
A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.
Multiple functions have recently been identified for the neonatal Fc receptor FcRn. In addition, a human homolog of the rodent forms of FcRn has been identified and characterized. This major histocompatibility complex class I-related receptor plays a role in the passive delivery of immunoglobulin (Ig)Gs from mother to young and the regulation of serum IgG levels. In addition, FcRn expression in tissues such as liver, mammary gland, and adult intestine suggests that it may modulate IgG transport at these sites. These diverse functions are apparently brought about by the ability of FcRn to bind IgGs and transport them within and across cells. However, the molecular details as to how FcRn traffics within cells have yet to be fully understood, although in vitro systems have been developed for this purpose. The molecular nature of the FcRn-IgG interaction has been studied extensively and encompasses residues located at the CH2-CH3 domain interface of the Fc region of IgG. These Fc amino acids are highly conserved in rodents and man and interact with residues primarily located on the alpha2 domain of FcRn. Thus, it is now possible to engineer IgGs with altered affinities for FcRn, and this has relevance to the modulation of IgG serum half-life and maternofetal IgG transport for therapeutic applications.
The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.
The current belief is that the humoral immune response plays the principal role in defense against virulent infectious bursal disease virus (IBDV). In this study we used a model, in which chickens were compromised in functional T cells by neonatal thymectomy and Cyclosporin A (TxCsA) treatment, to demonstrate the role of T cells in protective immunity against IBDV. We demonstrated that T cells were necessary to achieve full protection against virulent IBDV. When T cell compromised TxCsA-treated chickens were vaccinated with an inactivated IBDV (iIBDV) vaccine, 91% were not protected against IBDV challenge in comparison to T cell-intact chickens, which had a protection rate of 91%. The iIBDV vaccine induced virus neutralizing (VN) and ELISA antibodies, respectively, in 65 and 5% of TxCsA-treated, and in 100 and 58% of T cell-intact birds. These observations provide evidence that the stimulation of T helper cells is needed for the production of protective antibody levels in iIBDV-vaccinated chickens. Passive administration of VN anti-IBDV antibodies inducing a circulating antibody level of log(2)8 in chickens revealed that the levels of antibodies that protected T cell-intact chickens against virulent IBDV challenge were not protective for TxCsA chickens. These results indicated that antibody alone was not adequate in inducing protection against IBDV in chickens and that T cell-involvement was critical for protection. We propose that the inability of iIBDV to protect TxCsA chickens was due to compromised T cell immunity, functional T helper cells and most likely also cytotoxic T cells are needed in iIBDV vaccine protection.
To characterise infectious bursal disease viruses (IBDVs) prevalent at major commercial sites throughout Australia and to compare the nucleic acid sequences of local strains of IBDV with those of characterised overseas strains.
Samples of bursae were collected from 20 broiler farms that belonged to different poultry companies in New South Wales (NSW), Queensland (Qld), Victoria (Vic), Westem (WA) and South Australia (SA).
Bursae were collected from broilers between 24 and 35 days of age. Bursal tissue was homogenised and tested for the presence of IBDV antigen using four monoclonal antibodies (Mabs) which detect antigenic variation in IBDV strains. The nucleotide sequences of the hypervariable region (HVR) within the VP2 gene of IBDVs was determined and the deduced amino acid sequences compared with three vaccine strains and six previously characterised Australian IBDV strains. The deduced amino acid sequences were also compared with the published amino acid sequences of overseas strains. The phylogenetic relationships between Australian strains and overseas strains were then determined.
IBDV was detected in birds from 14 out of 20 farms sampled. Typing with four Mabs showed that all viruses from Vic (6) and SA (10) were antigenic variants, whereas all viruses from NSW (29), Qld (4) and WA (5) were classical-like strains. Nucleotide sequencing of one sample from each of the 14 farms on which IBDV was detected confirmed results obtained with Mabs. The amino acid sequences of all Australian viruses differed from the amino acid sequences of foreign IBDV strains. Phylogenetic analysis showed that Australian IBDV viruses belonged to two distinct genetic groups. Very virulent (vv) IBDV strains belonged to a third genetic group, and overseas classical and variant strains belonged to a fourth genetic group.
The results confirmed previous findings that there are two groups of IBDV strains circulating in commercial broilers in Australia. The majority are classical-like strains that are antigenically and genetically similar to vaccine strains 002/73 and V877. These classical strains were prevalent in broilers in three states, NSW, Qld and WA. The second group of strains are antigenic variants that were only found in broilers in two states, Vic and SA. All Australian IBDVs characterised to date are genetically distinct and can be differentiated from all other overseas strains. This enables identification of incursion of any exotic strain into Australian poultry, be it classical, US variant or wIBDV strains.
The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery. Here, we demonstrate that the Lt-B protein of enterotoxigenic E. coli is evenly distributed in defatted corn germ prepared from transgenic grain. Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.
Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.
Induction of immune responses following oral immunization is frequently dependent upon the co-administration of appropriate adjuvants that can initiate and support the transition from innate to adaptive immunity. The three bacterial products with the greatest potential to function as mucosal adjuvants are the ADP-ribosylating enterotoxins (cholera toxin and the heat-labile enterotoxin of Escherichia coli), synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN), and monophosphoryl lipid A (MPL). The mechanism of adjuvanticity of the ADP-ribosylating enterotoxins is the subject of considerable debate. Our own view is that adjuvanticity is an outcome and not an event. It is likely that these molecules exert their adjuvant function by interacting with a variety of cell types, including epithelial cells, dendritic cells, macrophages, and possibly B- and T-lymphocytes. The adjuvant activities of CpG and MPL are due to several different effects they have on innate and adaptive immune responses and both MPL and CpG act through MyD88-dependent and -independent pathways. This presentation will summarize the probable mechanisms of action of these diverse mucosal adjuvants and discuss potential synergy between these molecules for use in conjunction with plant-derived vaccines.