Biomedical subjects

Daniel D Pinschewer

Publications and source records attributed to Daniel D Pinschewer.

11 recordsLinked to original sources

PubMed2006

Envelope exchange for the generation of live-attenuated arenavirus vaccines.

Arenaviruses such as Lassa fever virus cause significant mortality in endemic areas and represent potential bioterrorist weapons. The occurrence of arenaviral hemorrhagic fevers is largely confined to Third World countries with a limited medical infrastructure, and therefore live-attenuated vaccines have long been sought as a method of choice for prevention. Yet their rational design and engineering have been thwarted by technical limitations. In addition, viral genes had not been identified that are needed to cause disease but can be deleted or substituted to generate live-attenuated vaccine strains. Lymphocytic choriomeningitis virus, the prototype arenavirus, induces cell-mediated immunity against Lassa fever virus, but its safety for humans is unclear and untested. Using this virus model, we have developed the necessary methodology to efficiently modify arenavirus genomes and have exploited these techniques to identify an arenaviral Achilles' heel suitable for targeting in vaccine design. Reverse genetic exchange of the viral glycoprotein for foreign glycoproteins created attenuated vaccine strains that remained viable although unable to cause disease in infected mice. This phenotype remained stable even after extensive propagation in immunodeficient hosts. Nevertheless, the engineered viruses induced T cell-mediated immunity protecting against overwhelming systemic infection and severe liver disease upon wild-type virus challenge. Protection was established within 3 to 7 d after immunization and lasted for approximately 300 d. The identification of an arenaviral Achilles' heel demonstrates that the reverse genetic engineering of live-attenuated arenavirus vaccines is feasible. Moreover, our findings offer lymphocytic choriomeningitis virus or other arenaviruses expressing foreign glycoproteins as promising live-attenuated arenavirus vaccine candidates.

Animals↗

PubMed2006

"Viral déjà vu" elicits organ-specific immune disease independent of reactivity to self.

Autoimmune diseases are often precipitated by viral infections. Yet our current understanding fails to explain how viruses trigger organ-specific autoimmunity despite thymic tolerance extending to many non-lymphohematopoietic self antigens. Additionally, a key epidemiological finding needs to be explained: In genetically susceptible individuals, early childhood infections seem to predispose them to multiple sclerosis (MS) or type 1 diabetes years or even decades before clinical onset. In the present work, we show that the innate immune system of neonatal mice was sufficient to eliminate an attenuated lymphocytic choriomeningitis virus (LCMV) from most tissues except for the CNS, where the virus persisted in neurons (predisposing virus). Virus-specific cytotoxic T cells (CTLs) were neither deleted nor sufficiently primed to cause disease, but they were efficiently triggered in adulthood upon WT LCMV infection (precipitating virus). This defined sequence of viral infections caused severe CNS inflammation that was histomorphologically reminiscent of rasmussen encephalitis, a fatal human autoimmune disease. Yet disease in mice was mediated by antiviral CTLs targeting an epitope shared by the precipitating virus and the predisposing virus persisting in neurons (déjà vu). Thus the concept of "viral déjà vu" demonstrates how 2 related but independently encountered viral infections can cause organ-specific immune disease without molecular mimicry of self and without breaking self tolerance.

Animals↗

PubMed2006

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA.

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.

Animals↗

PubMed2005

Virus-induced polyclonal B cell activation improves protective CTL memory via retained CD27 expression on memory CTL.

Different viruses elicit distinct phenotypes of memory cytotoxic T lymphocytes (CTL). This is reflected in differential expression of homing receptors and costimulatory molecules like CD27. Memory CTL retained CD27 following lymphocytic choriomeningitis virus (LCMV) infection, but not after immunization with recombinant vaccinia virus or tumor cells expressing LCMV glycoprotein. Stable CD27 expression on memory CTL required ligation by CD70 expressed on polyclonally activated B cells during the contraction phase. The functional consequence of CD27 expressed on virus-specific CTL was analyzed in CD27-deficient mice. LCMV infection of CD27(-/-) mice revealed that primary CTL activation and expansion as well as elimination of the virus were independent of CD27 expression. In contrast, ligation of CD27 on memory CTL upon secondary antigen encounter increased clonal expansion and improved protection against re-infection. This points to novel B cell-CTL interactions during viral infection and to a beneficial role of polyclonal B cell activation that represents a characteristic of murine LCMV, human immunodeficiency virus and human hepatitis B and C virus infection.

Animals↗

PubMed2005

Dual role of the lymphocytic choriomeningitis virus intergenic region in transcription termination and virus propagation.

Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3' ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.

Chloramphenicol O-Acetyltransferase↗

PubMed2004

Kinetics of protective antibodies are determined by the viral surface antigen.

Delayed and weak virus neutralizing antibody (nAb) responses represent a hallmark correlating not only with the establishment of persistent infection but also with unsuccessful vaccine development. Using a reverse genetic approach, we evaluated possible underlying mechanisms in 2 widely studied viral infection models. Swapping the glycoproteins (GPs) of lymphocytic choriomeningitis virus (LCMV, naturally persisting, noncytolytic, inefficient nAb inducer) and vesicular stomatitis virus (VSV, nonpersisting, cytolytic, potent nAb inducer) transferred the only target of nAb's from either virus to the other. We analyzed the nAb response to each of the 2 recombinant and parent viruses in infected mice and found that nAb kinetics were solely determined by the viral surface GP and not by the virus backbone. Moreover, the slowly and poorly nAb-triggering LCMV virion was a potent immunogenic matrix for the more antigenic VSV-GP. These findings indicate that the viral GP determines nAb kinetics largely independently of the specific viral infection context. They further suggest that structural features of viral GPs or coevolutionary adaptation of the virus's GP to the host's naive B cell repertoire, or both, may critically limit nAb kinetics and improvement of vaccine efficacy.

Animals↗

PubMed2003

Rapid induction of autoantibodies against Nogo-A and MOG in the absence of an encephalitogenic T cell response: implication for immunotherapeutic approaches in neurological diseases.

Vaccinations against various antigens of the central nervous system (CNS) are gaining increasing interest as a therapeutic approach in a variety of neurological diseases such as spinal cord injury, ischemic stroke, Alzheimer disease, or spongiform encephalopathy. In the present work, the time window after spinal cord injury allowing potentially therapeutic antibody to penetrate the damaged blood-brain barrier (BBB) was measured by intravenous injection of a monoclonal anti-Nogo-A antibody. Although an influx of Nogo antibodies at the lesion site was detectable up to 2 wk after injury, a significant decrease in BBB permeability was noticed within the first week. Clearly, therefore, a vaccination protocol with a rapid antibody response is required for acute therapeutic interventions after CNS trauma. We designed a conjugate vaccine paradigm with particular focus on the safety and the kinetics of the antibody response. As antigen targets, we used Nogo-A and the strongly encephalitogenic myelin-oligodendrocyte glycoprotein (MOG). Intrasplenic autoimmunization of rats with a Nogo-A-specific region fused to the Tetanus toxin C-fragment (TTC) resulted in a fast IgM response against Nogo-A. A specific switch to IgG was observed as soon as 4-7 days after intrasplenic immunization in TTC-primed animals. In spite of the induction of a specific IgG response after intrasplenic immunization, no signs of experimental autoimmune disease (EAE) or inflammatory infiltrates on histological examinations were observable. In contrast to subcutaneous immunization with MOG, in vitro cytokine secretion assays (IL-2, IL-10, and IFN-gamma) did not reveal activation of MOG-specific T cells after intrasplenic immunization. Our findings have critical implications for future strategies in the development of safe and efficient therapeutic vaccines for neurological diseases.

Animals↗

PubMed2003

Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein.

A recombinant S segment RNA (Sr) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) where the glycoprotein of vesicular stomatitis virus (VSVG) was substituted for the glycoprotein of LCMV (LCMV-GP) was produced intracellularly from cDNA under the control of a polymerase I promoter. Coexpression of the LCMV proteins NP and L allowed expression of VSVG from Sr. Infection of transfected cells with WT LCMV (LCMVwt) resulted in reassortment of the L segment of LCMVwt with the Sr at low frequency. Isolation of recombinant LCMV (rLCMV) expressing VSVG (rLCMV/VSVG) was achieved by selection against LCMVwt by using a cell line deficient in the cellular protease S1P. This approach was based on the finding that processing of LCMV-GP by S1P was required for virus infectivity. Characterization of protein and RNA expression of rLCMV/VSVG in infected cells confirmed the expected virus genome organization. rLCMV/VSVG caused syncytium formation in cultured cells and grew to approximately 100-fold lower titers than WT virus but, like the parent virus, it persisted in neonatally infected mice without clinical signs of disease.

Animals↗

PubMed2003

Role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and RNA replication.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has a bisegmented negative-strand RNA genome. Each segment carries two viral genes in opposite orientation and separated by an intergenic region (IGR). The RNA-dependent RNA polymerase (RdRp) L of LCMV produces subgenomic mRNA and full-length genomic and antigenomic RNA species in two different processes termed transcription and replication, respectively. It is widely accepted that intracellular nucleoprotein (NP) levels regulate these two processes. Intracellular NP levels increase during the course of the infection, resulting in the unfolding of secondary RNA structures within the IGR. Structure-dependent transcription termination at the IGR is thereby attenuated, promoting replication of genome and antigenome RNA species. To test this hypothesis, we established a helper-virus-free minigenome (MG) system where intracellular synthesis of an S segment analogue from a plasmid is driven by RNA polymerase I. Cotransfection with two additional plasmids expressing the minimal viral trans-acting factors L and NP under control of RNA polymerase II allowed for RNA synthesis mediated by the intracellularly reconstituted LCMV polymerase. Both processes, transcription and replication, were strictly dependent on NP. However, both were equally enhanced by incrementally increasing amounts of NP up to levels in the range of those in LCMV-infected cells. Our data are consistent with a central role for NP in transcription and replication of the LCMV genome, but they do not support the participation of NP levels in balancing the two processes.

Animals↗

PubMed2002

Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles.

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral trans-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.

Animals↗

PubMed2002

Effects of promyelocytic leukemia protein on virus-host balance.

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.

Animals↗