Secreted antiviral entry inhibitors (iSAVE) for gene therapy of HIV infection

Die Gentherapie bietet eine interessante alternative Behandlungsoption bei der Therapie der HIV-Infektion und könnte langfristig die Standardmedikation mit antiretroviralen Substanzen ergänzen oder ersetzen. Antivirale Genprodukte, die frühe Schritte im HIV-Replikationszyklus hemmen, bevor sich das Virus in das Genom der Zielzelle integriert hat, sind dabei besonders vielversprechend. Hierzu zählen insbesondere die von der C-terminalen heptad repeat Region des HIV-Hüllglykoproteins gp41 abgeleiteten C-Peptide, die hochwirksame Inhibitoren des Viruseintritts sind. Während des HIV-Eintrittsprozesses interagieren sie mit den viralen gp41 N-Helices und verhindern somit die Ausbildung des zur Fusion von viraler und zellulärer Membran erforderlichen Sechs-Helix-Bündels. Die Sekretion antiretroviraler C-Peptide durch genmodifizierte T-Lymphozyten in vivo birgt großes therapeutisches Potential: Nach Freisetzung in den extrazellulären Raum können die Peptide nicht nur genmodifizierte sondern auch unbehandelte Nachbarzellen vor HIV-Infektion schützen (Bystander-Effekt). Somit könnte selbst mit den heute zur Verfügung stehenden Methoden, mit denen lediglich ein Teil aller potentiellen HIV-Zielzellen modifiziert werden kann, die Virusreplikation effektiv unterdrückt werden. Im Rahmen der vorliegenden Arbeit wurden daher C-Peptid-basierte in vivo sezernierte antivirale Eintrittsinhibitoren (iSAVE) für die HIV-Gentherapie entwickelt. Kurze Peptide, wie die antiviralen C-Peptide, werden von eukaryotischen Zellen aufgrund von Größenbeschränkungen beim Eintritt in den Sekretionsweg jedoch nur schlecht sezerniert. Um die effiziente Sekretion von iSAVE-Peptiden durch genmodifizierte humane Zellen zu erreichen, wurde das C-Peptid daher verlängert. Hierbei wurde das therapeutische Peptid einerseits um nicht antiviral aktive Gerüstelemente ergänzt. Andererseits wurden Concatemer-Konstrukte generiert, in denen zwei C-Peptide jeweils über einen flexiblen oder proteolytisch spaltbaren Linker verbunden sind. Die unterschiedlichen iSAVE-Peptid-Varianten wurden in vitro in transfizierten und transduzierten Zelllinien und in primären humanen T-Lymphozyten charakterisiert. Hierbei wurden Sekretionseffizienz und Prozessierung sowie antivirale Aktivität und Bystander-Inhibition der sezernierten Peptide untersucht. Dabei zeigte sich, dass die Effizienz der C-Peptidsekretion stark mit der Peptidlänge korreliert, so dass durch Sequenzverlängerungen die Sekretion deutlich gesteigert werden konnte. Darüber hinaus waren N-Glykane für die effiziente Sekretion der C-Peptide unerlässlich. Die antiretrovirale Aktivität hingegen reduzierte sich mit zunehmender Peptidlänge dramatisch und wurde auch durch N-Glykane leicht beeinträchtigt, so dass weder die durch Gerüstelemente verlängerten C-Peptide, noch die ungespaltenen C-Peptid-Concatemere antiretrovirale Wirkung zeigten. Durch die Generierung proteolytisch spaltbarer C-Peptid-Concatemere konnten die strukturellen Erfordernisse für effiziente Sekretion mit hoher inhibitorischer Aktivität vereinbart werden. Die Prozessierung der Concatemere durch die Proprotein-Convertase Furin war allerdings nicht einfach zu erreichen. Nur das Einfügen eines flexiblen Linkers mit optimierter Furinerkennungssequenz zwischen den beiden C-Peptiden erlaubte die effiziente Spaltung in monomere Peptide mit hoher antiretroviraler Aktivität. Therapeutisch wirksame Peptidkonzentrationen dieser optimierten iSAVE-Peptide wurden sowohl von transfizierten und transduzierten Zelllinien als auch von primären humanen T-Zellen sezerniert. Nach Freisetzung in den extrazellulären Raum konnten die Peptide nicht nur genmodifizierte sondern auch unbehandelte Nachbarzellen in vitro vor HIV-1 Eintritt und Infektion schützen. Die generierten iSAVE-Peptide bilden damit eine hervorragende Grundlage für die weitere präklinische und klinische Entwicklung eines neuen Gentherapieansatzes zur Behandlung der HIV-Infektion.Gene therapy is an interesting alternative treatment option for the therapy of HIV infection and could potentially overcome the limitations of current antiretroviral drug therapy. Antiviral gene products that inhibit early steps in the viral life cycle prior to integration of the proviral DNA into the host cell genome are most promising. So-called C peptides derived from the C-terminal heptad repeat region of the viral glycoprotein gp41 are highly efficient inhibitors of virus entry. During the HIV entry process the C peptides interact with the viral gp41 N-helices, thereby preventing the six-helix bundle formation and subsequent membrane fusion. The secretion of antiviral C peptides from gene-modified T lymphocytes in vivo has great therapeutic potential, as peptides secreted into the extracellular space could protect the genetically modified cells as well as non-modified neighboring cells from HIV infection (bystander effect). Thus, virus replication could be effectively suppressed even if only a small fraction of cells is genetically modified. In the present study, C peptide-based in vivo secreted antiviral entry inhibitors (iSAVE) were developed for the gene therapy of HIV infection. However, short peptides like the antiviral C peptides are only inefficiently secreted from eukaryotic cells. In order to achieve efficient iSAVE peptide secretion the therapeutic C peptide was elongated either by scaffold elements without antiviral activity, or by the generation of peptide concatemers. The latter consist of two C peptides connected by a flexible or proteolytically cleavable linker. The different iSAVE peptide variants were characterized in vitro in transfected and transduced cell lines as well as in primary human T lymphocytes. Here, secretion efficacy and antiviral activity of the peptides were analyzed. The efficacy of peptide export correlated with peptide length, thus, elongation significantly improved secretion rates. Furthermore, N-glycans were required for efficient secretion. However, elongation and glycosylation dramatically decreased the antiviral activity and neither the the elongated peptides nor the non-cleaved concatemers inhibited virus entry. The generation of proteolytically cleavable C peptide concatemers allowed combination of the structural requirements for efficient secretion and a high antiviral activity. However, it was difficult to achieve processing of the concatemers by the proprotein convertase furin. Only after introduction of a flexible and optimized furin recognition sequence between the two C peptides efficient processing in monomers with high antiretroviral activity was observed. Therapeutic concentrations of these optimized iSAVE peptides were secreted from transfected and transduced cell lines as well as from primary human T cells. After secretion into the extracellular space the peptides exerted a clear bystander effect and protected genetically modified as well as non-modified cells from HIV infection. Therefore, the iSAVE peptides generated here provide an excellent basis for the further preclinical and clinincal development of a novel gene therapy approach for the treatment of HIV infection

The viral vector vaccine VSV-GP boosts immune response upon repeated applications

Background: Vesicular stomatitis virus (VSV) is a potent candidate vaccine vector for various viral diseases (e.g. HIV, HCV, RSV). The biggest limitation of VSV, however, is its neurotoxicity, which limits application in humans. The second drawback is that VSV induces neutralizing antibodies rapidly and is thus ineffective as a vaccine vector upon repeated applications. Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), VSV-GP, is not neurotoxic. The aim of this project was to evaluate the potential of VSV-GP as a vaccine vector. Methods: For this purpose, we used Ovalbumin (OVA) as a model antigen and analyzed immunogenicity of GP-pseudotyped and wildtype VSV containing OVA (VSV-GP-OVA and VSV-OVA) in vitro and in vivo in mouse models. Results: We showed that both vectors infected murine bone marrow-derived dendritic cells (bmDCs) in vitro. These bmDCs were able to activate OVA specific CD8+ and CD4+ T cells. Immunization experiments in mice revealed that both VSV-OVA and VSV-GP-OVA induced functional OVA-specific cytotoxic T cells (CTLs) after a single immunization. In addition, with both viruses, mice generated antibodies against OVA. However, boosting with the same virus was only possible for the GP-pseudotyped virus but not for wild type VSV. The efficacy of repeated immunization with VSV-OVA was most likely limited by high levels of neutralizing antibodies, which we detected after the first immunization. In contrast, no neutralizing antibodies against VSV-GP were induced even after boosting. Conclusion: Taken together, we showed that the non-neurotoxic VSV-GP is able to induce specific T cell and B cell responses against the model antigen OVA to the same level as the wild type VSV vector. However, in contrast to wild type VSV, VSV-GP-OVA boosted the immune response upon repeated applications. Thus, VSV-GP is a promising novel vaccine vector

The effect of age on visuo-spatial short-term memory in family dogs

Decline in the visuo-spatial memory domain may be an early marker for cognitive decline and has a relevant impact on animal welfare. Current research on visuo-spatial memory in family dogs is often limited by factors such as the need of extensive pre-training, limited attention to co-occurring medical conditions, a focus on laboratory dogs, or low sample size. Therefore, we aimed to develop a test that relies on visuo-spatial short-term memory, may be performed in a short time, and does not require explicit training. We tested a large sample of young and old dogs, finding that young dogs were more likely to perform correctly, although performance decreased with consecutive trials in both age groups. However, groups did not vary in the severity of mistakes. This task represents the first measure of dogs’ age-related decline of short-term spatial memory that does not require explicit training. The test could potentially be used in veterinary behaviour contexts to monitor cognitive changes in ageing dogs, utilizing a simple binary measure of success

Secreted Antiviral Entry Inhibitory (SAVE) Peptides for Gene Therapy of HIV Infection

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen