Vaccine After several attempts to develop a vaccine against hepatitis E, in a successful phase II trial of a novel HEV vaccine was described [ ]. Conclusions and Recommendations The great majority of HEV infections take a self-limiting, asymptomatic clinical course. Funding This research received no external funding. Conflicts of Interest The authors declare no conflict of interest. References 1. Adlhoch C. Pischke S. Hepatitis e virus: Infection beyond the liver?
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Foodborne zoonosis mainly contact or consumption of inadequately cooked pork shellfish deer Strawberries Vegetables spinach, rocket Blood transfusions. The validity of our determined scores is confirmed by comparing them with functional data available for some of the recovered peptides. In congruence with this, we previously observed that both peptides mediate lung transduction but also strongly transduce liver, heart, and kidney.
The specificity of transgene expression in the pulmonary endothelium correlated with vector distribution, which indicates true specificity in gene delivery as opposed to a mere optimization of postentry vector processing. This unprecedented strength and specificity of transgene expression after systemic administration is composed of both, targeting to the lung and detargeting especially from the liver.
Frequently, AAV2 capsid changes sterically close to the site not only result in liver de-targeting but also augment gene expression in the heart. In view of that it is remarkable, that the tropism of the ESGHGYF clone selected by using the technology described here did not show this pattern and was restricted to the lung. The amino acid sequence of the ESGHGYF peptide does not show any striking similarities with one the different lung-homing peptides that have previously been identified by the phage display technique.
This is not surprising and can most likely be explained by the fundamental differences of the library systems that have been used to identify these peptides. The phage display technique allows the selection of well accessible peptides that are terminally fused to the phage's coat protein without being forced into major structural constraints.
In an AAV display library, on the other hand, the peptides are embedded within a structurally very important area of the viral capsid, in which they may be forced into strong physicochemical constraints. Thus, in an AAV display library it is not necessarily the isolated peptide which is being selected for, more likely it is the modified viral capsid being structurally altered by insertion of this peptide.
Although the receptor targeted by the AAV2-ESGHGYF capsid has not been identified yet, the carbohydrates on the endothelial cell surface presumably are essential for specific targeting, since glycans are used as primary attachment receptors by all AAV serotypes analyzed so far. This would not have been the case if the lung-homing was solely explained by a first pass effect. Although the insertion of the ESGHGYF peptide into the AAV2 capsid does not seem to have an positive influence on the antibody reactivity, the AAV2-ESGHGYF vector might very well be a promising candidate for gene therapy, as there are different potential options to circumvent unwanted immune responses in a clinical setting such as plasmapheresis 61 or blocking of antibodies by applying an excess of empty particles.
Thus, choosing the lung as therapeutically highly relevant target, vectors such as the one presented here might be used in gene therapy for disorders like pulmonary hypertension. Our NGS-guided approach of in vivo selection allows unprecedented exploitation of the high potential of random AAV display peptide libraries and could be used to obtain specific vectors for virtually any given target organ.
Preparation of the random AAV display peptide library. Library plasmids were harvested and purified using Qiagen's Plasmid Preparation Kit. Cells were superinfected with Ad5 at an MOI of five plaque-forming units pfu per cell. The final random peptide AAV display library was harvested from the supernatant after 48 hours.
In vivo screening of the random AAV display peptide library. After 48 hours, mice were sacrificed and organs of interest were removed. The PCR-amplified oligonucleotides were used to produce secondary libraries for four further rounds of selection mice were sacrificed 6 days after library injection in round 2—5. Secondary libraries were produced like the primary library as described earlier but without the extra step of producing transfer shuttles.
Data analysis was performed by a custom script see Supplementary Note 1. Vector production and quantification. Four days after transfection, cells were harvested, lysed, and vectors were purified by iodixanol density-gradient ultracentrifugation as previously described.
The oligonucleotide inserts encoding the modified peptides for the alanine scan were synthesized Metabion and further processed as the library inserts described earlier. Animals and vector administration. The peptide competition experiment and GFP reporter gene experiments for immunohistochemistry were performed in 8—12 weeks old SCID mice.
All experiments involving animals were conducted in accordance with the German Animal Protection Code. The protocol was approved by the responsible ethics review board and the local authorities. Assessment of luciferase reporter gene expression in vivo. At day 14, animals were anesthetized with isoflurane. Subsequently, animals were sacrificed, organs of interest were quickly removed and transgene expression images of single organs were taken immediately.
Three-dimensional reconstructions of in vivo luminescence images were obtained by using the DLIT option of the software Living Image 4. To quantify luciferase expression, organs were homogenized in reporter lysis buffer Promega, Madison, WI using a Precelly's 24 tissue homogenizer Peqlab, Erlangen, Germany according to the manufacturer's instructions. Analysis of vector distribution. For quantification of vector genome copy numbers in the circulation, blood was collected by left ventricular puncture and incubated for 10 minutes at room temperature.
PCR efficiency was controlled by a spiked-in plasmid. Immunohistochemistry and histology. Lung tissues were embedded in paraffin. Two-micrometer sections were dewaxed, rehydrated, and used for immunohistochemistry. After washing in phosphate-buffered saline, the sections were incubated for 30 minutes with a secondary biotinylated goat antirabbit antibody Vector Lab. Louis, MO. Selected sections were counterstained with hemalum. In vivo imaging of luminescence mediated by recombinant AAV2 vectors.
Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Figure S8. Note S1. Bioinformatical data processing. Materials and Methods References. Designed the experiments: J. Performed the experiments: J. Analyzed the data: J. Wrote the manuscript: J. The authors declare that there are no further competing financial interests. Read article at publisher's site DOI : Viruses , 12 4 , 18 Apr Li C , Samulski RJ. Nat Rev Genet , 21 4 , 10 Feb Cited by: 22 articles PMID: Int J Mol Sci , 21 1 , 27 Dec This data has been text mined from the article, or deposited into data resources.
This data has been provided by curated databases and other sources that have cited the article. To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. PLoS One , 4 4 :e, 09 Apr J Gen Virol , 93 pt 10 , 04 Jul Cited by: 7 articles PMID: Nat Biotechnol , 21 9 , 03 Aug Cited by: articles PMID: Michelfelder S , Trepel M.
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Share this article Share with email Share with twitter Share with linkedin Share with facebook. Free full text. Mol Ther. Published online Apr Prepublished online Mar PMID: Author information Article notes Copyright and License information Disclaimer. E-mail: ed. Received Oct 5; Accepted Mar This article has been cited by other articles in PMC.
Go to:. Supplementary Methods. Screening the random AAV-display peptide library for lung-targeted capsids in vivo yields a distinct peptide sequence To select specific tissue-targeted AAV2 capsids, an AAV2-displayed random heptamer peptide library was screened in vivo in mice, choosing the lung as the target of interest. Open in a separate window. Figure 1. Figure 2. AAV2 vectors displaying the ESGHGYF peptide mediate strong and specific gene expression in the lung To analyze the in vivo tropism, targeting and control peptides were incorporated into the capsids of AAV vectors carrying a luciferase reporter gene.
Figure 3. Figure 4. ESGHGYF-mediated targeting is based on lung-specific homing of circulating vectors To investigate if the lung-specific transgene expression of intravenously administered ESGHGYF vectors is based on specific homing, we analyzed the vector distribution as early as 2 minutes after injection Figure 5a. Figure 5. Figure 6. Supplementary Methods Click here for additional data file.
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