Autonome Parvoviren

Oncosuppressive properties and a low pathogenicity in adult animals make the two rodent parvoviruses MVMp and H1 promising vectors for cancer gene therapy. Recombinant vectors were developed from genomes of these viruses in which a part of the capsid-protein-coding sequence was replaced by a transgene. These recombinant viruses can replicate their genome and express their transgene, but they are unable to produce progeny viruses. Such vectors are produced by cotransfection of the recombinant virus genome and a helper plasmid which provides the capsid proteins. During this procedure, RCVs (Replication Competent Viruses) can be generated, probably by homologous recombination. As a consequence, RCVs can express their capsid proteins and produce progeny viruses. In the present work, second-generation H1- and MVMp- based recombinant virus genomes were developed in order to minimise RCV levels in the recombinant virus preparations. By constructing chimeras and pseudotypes between MVMp and H1 viruses, virus preparations with high titres were obtained which did not show any detectable RCVs in plaque-assays. This corresponds to a 100-17000fold decrease of RCVs for the chimeras and pseudotypes. In this work we also characterised the host-cell-tropism of the chimeras and the pseudotypes. The chimeric vectors and Pseudotypes allowed us to show that the restriction for viral replication of the rat H1 genome in murine A9 cells is not due to the H1 capsids, but can be ascribed to a 1800 bp long DNA region of the H1 genome, located in the NS coding sequence. To our knowledge this is the first report that convincingly shows that the tropism of a rodent parvovirus is determined by the viral genome. Since a recombinant MVMp genome packaged with either H1 or MVMp capsids led to viral DNA replication in A9 cells, virus entry is not responsible for the lack of recombinant H1 vector replication in these cells. After transfection of A9 cells with H1 viral DNA, replication and viral protein expression took place, whereas after H1 infection it did not. Thus we can conclude that the cause for the restricted H1 virus replication has to be found after virus entry but before the beginning of viral gene transcription. It is usually accepted that viral gene transcription needs the conversion of single-stranded Summary 134 DNA into double-stranded monomeric replicative form. Most probably this conversion reaction is disordered in A9 cells for the H1 genome. A defective double-stranded conversion might be due to an interaction of VP proteins with the viral single stranded DNA sequence. Another possibility is an interaction of a cellular protein with the viral single stranded DNA which then leads to an inhibition of the viral DNA conversion. It might be also possible that a cellular factor which is needed for the conversion reaction can not bind to the H1 genome in A9 cells. Interestingly, in most of the tested murine cell lines the tropism was determined by the MVMp genome, while for all human cell lines the tropism was determined by the virus capsid. The second part of this work focused on the effect that H1 virus infection on cellular gene expression. For this purpose, the cDNA array technique was used. It was previously shown that parvovirus H1 infection leads to the activation of caspase 3 and apoptotic cell death of the human monocytic U937 cell, in a way similar to the apoptotic cell death induced by TNFα. Moreover, the c-myc gene, overexpressed in these cells, is down-regulated during H1 infection. In agreement with these data, we were able to show -by comparing the gene expression profiles between buffer treated and H1 infected synchronised U937 cells- genes coding for caspases 2, 4 and 8 were upregulated after H1 infection. In addition, different genes associated with the TNFα complex were found to be up-regulated, as well as the mad4 gene, which is able to inhibit the transcriptional activity of c-myc by binding its main partner max. Moreover, 2 genes involved in oestrogen-synthesis were found to be up-regulated after infection. Oestrogen was shown to have some protective effect against TNFα induced apoptosis in U937 cells. Induction of these genes might reflect a defence mechanism of the cell. Also, a gene encoding for a subunit of the 26s proteosome and different genes involved in differentiation processes or in apoptosis were up-regulated in infected cells. Few genes were shown to be down-regulated after infection, such as the adenylate cyclase and cAMP dependent transcription factor 1. Altogether, our data give a better understanding of the molecular pathways which are used by the parvovirus and enlighten the diversity of cellular responses to a H1 virus infection in U937 cells.Onkosuppressive Eigenschaften sowie geringe Pathogenität in adulten Tieren machen die beiden Nagerparvoviren MVMp und H1 zu attraktiven Vektoren für den Einsatz in der Tumor-Gentherapie. Daher wurden parvovirale Vektoren entwickelt, die an Stelle der Kapsidproteingene ein Transgen tragen können. Diese rekombinanten Vektoren können ihr Genom replizieren und das eingebrachte Transgen exprimieren, aber keine Nachkommenviren bilden. Zur Produktion solcher Vektoren werden das rekombinante Virusgenom und Helferplasmide zur Expression der Kapsidproteine kotransfiziert. Dabei kommt es durch homologe Rekombination zur Entstehung von replikationsfähigen Revertanten, RCVs (replication competent viruses). Diese RCVs sind in der Lage, ihre Kapsidproteine zu exprimieren und so Nachkommenviren zu bilden. Für die Anwendung von Vektoren in der Gentherapie muss der RCV-Anteil minimiert oder möglichst eliminiert werden. In der vorliegenden Arbeit wurden dazu Strategien erarbeitet und überprüft. Durch die Konstruktion von Chimären und Pseudotypen aus MVMp und H1 konnten Virusstocks produziert werden, die keinen im Plaque-Assay detektierbaren RCVAnteil zeigten. Das entspricht einer 100- bis 17.000-fachen Senkung des RCV-Anteils für die Chimären und Pseudotypen. Dabei wurden Titer erreicht, die im Bereich der Titer der Ausgangsvektoren lagen. Ein weiterer Schwerpunkt der Arbeit ist die Charakterisierung der Chimären und Pseudotypen hinsichtlich ihres Wirtszelltropismus. Durch den Einsatz der Pseudotypen und der Chimären konnte gezeigt werden, dass einzig durch einen 1800 bp langen H1- Genombereich, der im NS-kodierenden Bereich liegt, die Replikation des Ratten-H1-Virus in den murinen A9 Zellen unterbleibt und nicht etwa durch Determinanten im H1- Kapsid. Dies ist der erste Fall, in dem gezeigt wurde, dass der Wirtszelltropismus von Parvoviren nur durch eine nicht für das Kapsid kodierende Sequenz im viralen Genom bestimmt wird. Dabei liegt der Zeitpunkt der Restriktion nach dem Eintritt des Virus`, aber noch vor Beginn der Transkription. Ursache für die gestörte Transkription ist dabei wahrscheinlich eine unvollständige Konversion der einzelsträngigen Virus-DNA in die doppelsträngige monomere replikative Form. Der Grund der gestörten Konversion- Reaktion kann eine Interaktion einzelner Kapsid-Proteine mit dem oben genannten H1- Genomabschnitt sein oder aber die fehlende Assoziation zellulärer Faktoren in eben Zusammenfassung 132 diesem H1-Genomabschnitt, die für die Konversion nötig sind. Denkbar ist auch die Interaktion eines zellulären Faktors mit dem H1-Genom, die dann die Konversion behindert. In allen untersuchten murinen Zelllinien ist bis auf eine Ausnahme das MVMp-Genom, nicht aber das MVMp-Kapsid für den Tropismus ausschlaggebend. In den untersuchten humanen Zelllinien ist der Tropismus immer über das Kapsid determiniert. Im zweiten Teil der Arbeit wird der Effekt einer Infektion mit dem Parvovirus H1 in U937 Zellen auf genregulatorischer Ebene mittles cDNA-Arrays untersucht. Für diese Zellen wurde schon gezeigt, dass eine H1-Infektion durch Aktivierung der Kaspase 3 zum apoptotischen Absterben der Zellen führt. Ebenso ist nach Infektion das sonst überexprimierte c-myc-Gen herabreguliert. Bereits publizierte Daten lassen die Vermutung zu, dass das H1-Virus den TNFα−Rezeptor entweder direkt stimuliert oder aber nachfolgende Schritte im TNFα−Signalweg. In dieser Arbeit wird unter Verwendung von cDNA-Arrays gezeigt, dass neben Kaspase 3, auch die Kaspasen 2, 4 und 8 nach Infektion hochreguliert werden. Ebenso werden mehrere mit TNF-Rezeptoren assoziierte Gene nach Infektion hochreguliert, sowie das in die Regulation der transkriptorischen Aktivität von c-myc involvierte mad4-Gen. Das Mad4-Protein bindet den Hauptinteraktionspartner des c-Myc-Proteines, nämlich das Max-Protein. Desweiteren werden zwei Gene nach Infektion hochreguliert, die in die Östrogensynthese involviert sind. Für Östrogene ist schon gezeigt worden, dass sie einen gewissen protektiven Effekt auf U937 Zellen gegen TNFα−induzierte Apoptose haben. Die Hochregulation dieser Gene kann einen Abwehrmechanismus der U937 Zelle auf die Virusinfektion darstellen. Eine Untereinheit des 26s-Proteosoms sowie mehrere möglicherweise in Differenzierungsvorgänge oder apoptotische Vorgänge involvierte Gene sind ebenfalls durch eine Infektion hochreguliert. Es wurden nur wenige herabregulierte Gene gefunden, hierzu gehören die für die cAMP-Bildung verantwortliche Adenylatzyklase und der cAMP-abhängige aktivierende Transkriptionsfaktor 1. Die Ergebnisse der cDNAArrays stehen im Einklang mit publizierten Daten. Daneben geben sie interessante Hinweise auf weitere mögliche Effekte, zu denen eine H1-Virusinfektion in U937 Zellen führen kann. Diese Ergebnisse müssen in nachfolgenden Arbeiten bestätigt und näher analysiert werden

Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8(+) T cells

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8(+) T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4(+)CD25(+) regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8(+) T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The γ (C) cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of γ (C) cytokine–responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8(+) T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells

miR-155 augments CD8(+) T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic gamma(c) cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic gamma c cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.112922Ysciescopu

Chimeric and Pseudotyped Parvoviruses Minimize the Contamination of Recombinant Stocks with Replication-Competent Viruses and Identify a DNA Sequence That Restricts Parvovirus H-1 in Mouse Cells

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus

Additional file 2: of Toll-like receptor agonist therapy can profoundly augment the antitumor activity of adoptively transferred CD8+ T cells without host preconditioning

One day before ACT, mice were antibody depleted of host CD4 + T cells (0.1 mg/treatment) and subsequently administered every other day for a total of 5 doses or left untreated. The ACT treatment regimen was comprised of the adoptive transfer of 5e5 cultured pmel-1 T cells, fowlpox hgp100 vaccination and hIL-2 or were left untreated. One day after ACT, mice received 2 μg of LPS. The ratio of donor Vβ13 to host CD4 cells are shown from splenocytes on day 5 (mean ± SEM, 5 mice per group). ****P < .0001, unpaired t-test. (PDF 70 kb