Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.
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ABSTRACT:
SUBMITTER: Giritch A
PROVIDER: S-EPMC1566189 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e.ResultsIn this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30% of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus.ConclusionsThis study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.
| S-EPMC4446962 | biostudies-literature
Project description:Duchenne muscular dystrophy (DMD) is the most common lethal muscle disorder in children. It is caused by mutations of the dystrophin gene. Adeno-associated virus (AAV)-mediated gene replacement therapy has been actively pursued to treat DMD. However, this promising therapeutic modality has been challenged by the small packaging capacity of the AAV vector. The size of the full-length dystrophin cDNA is >11?kb, while an AAV virus can carry only a 5?kb genome. Innovative high-capacity AAV vectors may offer an opportunity to express the full-length dystrophin coding sequence. Here we describe several sets of tri-AAV vectors for full-length human dystrophin delivery. In each set, the full-length human dystrophin cDNA was split into three fragments and independently packaged into separate recombinant AAV vectors. Each vector was engineered with unique recombination signals for directional recombination. Tri-AAV vectors were coinjected into the tibialis anterior muscle of dystrophin-deficient mdx4cv mice. Thirty-five days after injection, dystrophin expression was examined by immunofluorescence staining. Despite low reconstitution efficiency, full-length human dystrophin was successfully expressed from the tri-AAV vectors. Our results suggest that AAV can be engineered to express an extra-large (up to 15 kb) gene that is approximately three times the size of the wild-type AAV genome. Further optimization of the trivector strategy may expand the utility of AAV for human gene therapy.
| S-EPMC4064726 | biostudies-literature
Project description:Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and ?-LA were prepared and successfully employed for detecting ?-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.
| S-EPMC5159798 | biostudies-other
Project description:Extensive research on generating an efficient HIV vaccine is ongoing. A major aim of HIV vaccines is the induction of long-lasting, broadly neutralizing antibodies (bnAbs) that can confer sterile immunity for a prolonged period of time. Several strategies have been explored to reach this goal, i.e. protein immunization, DNA, or viral vectors, or a combination thereof. In this review, we give an overview of approaches using viral vectors for the induction of HIV-specific bnAbs. Many pre-clinical studies were performed using various replication-competent and -incompetent vectors. Amongst them, poxviral and adenoviral vectors were the most prevalent ones. In many studies, viral vectors were combined with a DNA prime or a protein boost. However, neutralizing antibodies were mainly induced against the homologous HIV-1 vaccine strain or tier 1 viruses, and in rare cases, against tier 2 viruses, indicating the need for improved antigens and vaccination strategies. Furthermore, we also review next generation Env antigens that are currently being used in protein vaccination approaches and point out how they could be utilized in viral vectors.
| S-EPMC6789710 | biostudies-literature
Project description:B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.
| S-EPMC4552657 | biostudies-literature
Project description:Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV-adenovirus, AAV-herpesvirus, and AAV-baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1?x?10(14) genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.
| S-EPMC2957274 | biostudies-other
Project description:Background and aimsThe study of rapid evolution in invasive species has highlighted the fundamental role played by founder events, emergence of genetic novelties through recombination and rapid response to new selective pressures. However, whether rapid adaptation of introduced species can be driven by punctual changes in genome organization has received little attention. In plants, variation in genome size, i.e. variation in the amount of DNA per monoploid set of chromosomes through loss or gain of repeated DNA sequences, is known to influence a number of physiological, phenological and life-history features. The present study investigated whether change in genome size has contributed to the evolution of greater potential of vegetative growth in invasive populations of an introduced grass.MethodsThe study was based on the recent demonstration that invasive genotypes of reed canarygrass (Phalaris arundinacea) occurring in North America have emerged from recombination between introduced European strains. The genome sizes of more than 200 invasive and native genotypes were measured and their genome size was related to their phenotypic traits measured in a common glasshouse environment. Population genetics data were used to infer phylogeographical relationships between study populations, and the evolutionary history of genome size within the study species was inferred.Key resultsInvasive genotypes had a smaller genome than European native genotypes from which they are derived. This smaller genome size had phenotypic effects that increased the species' invasive potential, including a higher early growth rate, due to a negative relationship between genome size and rate of stem elongation. Based on inferred phylogeographical relationships of invasive and native populations, evolutionary models were consistent with a scenario of genome reduction by natural selection during the invasion process, rather than a scenario of stochastic change.ConclusionsPunctual reduction in genome size could cause rapid changes in key phenotypic traits that enhance invasive ability. Although the generality of genome size variation leading to phenotypic evolution and the specific genomic mechanisms involved are not known, change in genome size may constitute an important but previously under-appreciated mechanism of rapid evolutionary change that may promote evolutionary novelties over short time scales.
| S-EPMC2794072 | biostudies-literature
Project description:Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.
| S-EPMC9779006 | biostudies-literature
Project description:Rapid and inexpensive immunodiagnostic assays to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion are essential for conducting large-scale COVID-19 epidemiological surveillance and profiling humoral responses against SARS-CoV-2 infections or immunizations. Herein, a colorimetic serological assay to detect SARS-CoV-2 IgGs in patients' plasma was developed using short antigenic epitopes conjugated to gold nanoparticles (AuNPs). Four immunodominant linear B-cell epitopes, located on the spike (S) and nucleocapsid (N) proteins of SARS-CoV-2, were characterized for their IgG binding affinity and used as highly specific biological motifs on the nanoparticle to recognize target antibodies. Specific bivalent binding between SARS-CoV-2 antibodies and epitope-functionalized AuNPs trigger nanoparticle aggregation, which manifests as a distinct optical transition in the AuNPs' plasmon characteristics within 30 min of antibody introduction. Co-immobilization of two epitopes improved the assay sensitivity relative to single-epitope AuNPs with a limit of detection of 3.2 nM, commensurate with IgG levels in convalescent COVID-19-infected patients. A passivation strategy was further pursued to preserve the sensing response in human plasma medium. When tested against 35 clinical plasma samples of varying illness severity, the optimized nanosensor assay can successfully identify SARS-CoV-2 infection with 100% specificity and 83% sensitivity. As the epitopes are conserved within the circulating COVID-19 variants, the proposed platform holds great potential to serve as a cost-effective and highly specific alternative to classical immunoassays employing recombinant viral proteins. These epitope-enabled nanosensors further expand the serodiagnostic toolbox for COVID-19 epidemiological study, humoral response monitoring, or vaccine efficiency assessment.
| S-EPMC8231658 | biostudies-literature
Project description:The main targets for neutralizing anti-hepatitis C virus (HCV) antibodies (HCV-nAbs) are the E1 and E2 envelope glycoproteins. We have studied the characteristics of HCV-nAbs through a retrospective study involving 29 HIV/HCV-coinfected patients who achieved sustained virological response (SVR) with peg-IFNα + ribavirin anti-HCV therapy. Plasma samples at baseline and week 24 after SVR were used to perform neutralization assays against five JFH1-based HCV recombinant viruses coding for E1 and E2 from genotypes 1a (H77), 1b (J4), 2a (JFH1), 3a (S52) and 4a (ED43). At baseline, the majority of plasma samples neutralized 1a, 1b, 2a, and 4a, but not 3a, genotypes. Twenty-four weeks following SVR, most neutralizing titers declined substantially. Furthermore, titers against 3a and 2a were not detected in many patients. Plasma samples with high HCV-nAb titers neutralized all genotypes, and the highest titers at the starting point correlated with the highest titers at week 24 after SVR. In conclusion, high titers of broad-spectrum HCV-nAbs were detected in HIV/HCV-coinfected individuals, however, those titers declined soon after SVR.
| S-EPMC6704069 | biostudies-literature