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dc.contributor.advisor Stahl-Hennig, Christiane Dr.
dc.contributor.author Hotop, Sven-Kevin
dc.date.accessioned 2014-10-02T08:26:05Z
dc.date.available 2014-10-02T08:26:05Z
dc.date.issued 2014-10-02
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-0023-98F2-2
dc.language.iso deu de
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/
dc.subject.ddc 570 de
dc.title Feinkartierung humoraler Immunantworten von Makaken nach Immunisierung und/oder viraler Infektion mittels Peptid - Microarray de
dc.type doctoralThesis de
dc.title.translated Mapping humoral immune responses of macaques after immunization and/or viral infection using peptide-microarrays de
dc.contributor.referee Kramer, Wilfried PD Dr.
dc.date.examination 2014-09-22
dc.description.abstracteng The aim of this study was to map humoral immune responses in rhesus macaques (RM) which had either been immunized and/or infected with different viruses. This was achieved by screening serum samples using peptide microarrays. Further goals were the optimization of the analysis and the minimization of the required sample volumes. For the readout, an independent computer-assisted algorithm was formulated which provided an objective method to optimize peptide microarray production and data mining. In order to screen large numbers of serum samples, new tools were developed that allowed a safe handling of up to four samples per slide simultaneously. In polyclonal serum samples the length of closely related epitopes could not be determined precisely due to the design of the array, i.e. tiled peptides with an offset of three amino acids. Thus, the more fitting term “antibody target region” (ATR) was given to describe antibodies that recognize such regions on the antigen. In using the peptide microarray technology, 27 distinct ATRs were identified on the envelope spike glycoprotein (gp) 160 precursor of the simian immunodeficiency virus (SIV). The respective serum samples that were used for analysis had been collected during different AIDS-macaque model studies. In one of the studies, performing multiple low dose viral exposures which however did not lead to a productive infection, one particular ATR was detected. This might be involved in the protection through antibodies against the constant region five of gp120. Remarkably, no qualitative differences in the antibody reaction pattern against linear epitopes between SIV-infected long-term survivors (LTS) and animals progressing to AIDS-like disease were observed. Furthermore, antibodies directed against the N-terminal end of SIVgp120 were identified as a new early seromarker of SIV-infection. Irrespective of the inoculated SIV strain those antibodies reacted exclusively with the amino acid sequence of SIVmac251. The sequencing of viral RNA in serial plasma samples of an animal infected with SIVmac239 demonstrated the lack of amino acid exchanges in the region of interest. The predicted structure of this epitope based on a known 3D-structure of the human immunodeficiency virus type 1 (HIV-1) revealed a sharp bend at the original binding site. The above mentioned data imply that this identified ATR might represent a conformation-dependent epitope, which has not been seen with previous methods. Analysis of sera from herpes B virus (BV) infected monkeys with a peptide microarray containing peptides covering the amino acid sequences of the two glycoproteins B and D (gB and gD) revealed 17 new ATRs and confirmed one previously described epitope. Interestingly, antibodies against one ATR on the BV gD were detected in an animal, which was considered uninfected due to negative testing through a cross-reactive herpes simplex virus type 1 (HSV-1)-ELISA. Antibodies against this ATR could block the main entry receptor Nectin-1 on human cells. Since BV poses an occupational hazard for animal keepers and others dealing with macaques this result could pave the way for the development of a peptide-based vaccination strategy. Another detected ATR in the crown domain of gB seems to be conformation-dependent and was further characterized by adopting the 3D-structure of HSV-1 and by alanine scan. The highly immunogenic endodomain of gD revealed at least two distinguishable epitopes. In this case, the previously described C-terminal epitope was mapped to the last two amino acids. Through the establishment of a peptide-based ELISA the BV-peptide microarray data were confirmed. This ELISA was used to prove the serological relevance of the C-terminal end of gD in BV infection of RM. The longitudinal analysis of RM from a breeding group suggested that BV is more likely transmitted via sexual contacts rather than bites or scratches. The lack of the repeated rise in antibody levels of animals kept under experimental conditions may indicate that recurrence is a rare event during the infection with BV. The results obtained in this thesis demonstrate that peptide microarrays are useful and powerful tools for initial screening of serological markers. When standardized synthetized peptides are used the above mentioned results allow the establishment of low cost serological tests. To date, the screening of conformation-dependent epitopes is done through random peptide libraries but this technique is prone to errors. The results may lead to the development of a new generation of biochips, which allow inexpensive screening for conformational epitopes. However, a detailed assessment of the established method has indicated some limitations: In case of antibodies binding to “open” end structures, such as the gD C-terminal end, linked- or conjugated peptides may fail to detect such antibodies. A validation of antibody binding to unlinked C-terminal end containing peptides is therefore essential. de
dc.contributor.coReferee Pöhlmann, Stefan Prof. Dr.
dc.subject.eng Antibodies; epitope mapping; B Virus; SIV; Peptide microarrays de
dc.identifier.urn urn:nbn:de:gbv:7-11858/00-1735-0000-0023-98F2-2-1
dc.affiliation.institute Biologische Fakultät für Biologie und Psychologie de
dc.subject.gokfull Biologie (PPN619462639) de
dc.identifier.ppn 797927301

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