Lothar Stitz

De novo generation of a transmissible spongiform encephalopathy by mouse transgenesis.

Most transmissible spongiform encephalopathies arise either spontaneously or by infection. Mutations of PRNP, which encodes the prion protein, PrP, segregate with phenotypically similar diseases. Here we report that moderate overexpression in transgenic mice of mPrP(170N,174T), a mouse PrP with two point mutations that subtly affect the structure of its globular domain, causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease was reproduced with 100% attack rate by intracerebral inoculation of brain homogenate to tga20 mice overexpressing WT PrP, and from the latter to WT mice, but not to PrP-deficient mice. Upon successive transmissions, the incubation periods decreased and PrP became more protease-resistant, indicating the presence of a strain barrier that was gradually overcome by repeated passaging. This shows that expression of a subtly altered prion protein, with known 3D structure, efficiently generates a prion disease.

Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection

Despite substantial improvements, influenza vaccine production—and availability—remain suboptimal. Influenza vaccines based on mRNA may offer a solution as sequence-matched, clinical-grade material could be produced reliably and rapidly in a scalable process, allowing quick response to the emergence of pandemic strains. Here we show that mRNA vaccines induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice and that the vaccine remains protective upon thermal stress. This vaccine format elicits B and T cell–dependent protection and targets multiple antigens, including the highly conserved viral nucleoprotein, indicating its usefulness as a cross-protective vaccine. In ferrets and pigs, mRNA vaccines induce immunological correlates of protection and protective effects similar to those of a licensed influenza vaccine in pigs. Thus, mRNA vaccines could address substantial medical need in the area of influenza prophylaxis and the broader realm of anti-infective vaccinology.

Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA

Stabilized synthetic RNA oligonucleotides (ORN) and protected messenger RNA (mRNA) were recently discovered to possess an immunostimulatory capacity through their recognition by TLR 7 and 8. We wanted to find out whether this danger signal is capable of triggering anti‐tumor immunity when injected locally into an established tumor. Using the mouse glioma tumor cell line SMA‐560 in syngenic VM/Dk mice, we were able to show that intra‐tumor injections of protamine‐stabilized mRNA do indeed induce tumor regression and long‐term anti‐tumor immunity. Residual RNA‐injected tumors show CD8 infiltration. Distant injections of protamine‐protected mRNA and intra‐tumor injection of naked mRNA also result in anti‐tumor immunity. Although they are strong danger signals, RNA are labile molecules with a short half‐life: they do not trigger side effects such as long‐term, uncontrolled immunostimulation evidenced by splenomegaly in CpG DNA‐treated mice. In conclusion, RNA molecules are potent and safe danger signals that are relevant for active immunotherapy strategies aimed at the eradication of solid tumors.

Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies

OBJECTIVES To establish an animal model to study transmissible spongiform encephalopathy using hamsters and steel wires contaminated with infectious brain materials as transfer vehicles, and, based on this model, to test decontamination procedures against the infectious prion proteins on the steel wires as a near real situation bioassay. DESIGN Infectious brain materials were given to healthy hamsters intracerebrally either as a suspension or as dried materials on the surface of steel wires. The animals were observed for 18 months. During this period, animals showing definitive clinical signs were euthanized. Decontamination studies were performed by reprocessing contaminated steel wires with different disinfection agents and procedures before implantation. RESULTS Pathological prion proteins were able to bind to the steel wires and caused disease after the contaminated wires were implanted in the brains of hamsters. When the contaminated wires were treated with different reprocessing procedures before implantation, infectivity was reduced, which was manifested directly by prolonged survival time of the test animals. These results show that this model can be used as a bioassay to validate reprocessing procedures for surgical instruments. CONCLUSIONS At the time of submission of this article, only the group of hamsters incubated with wires reprocessed with an alkaline detergent, followed by sterilization with a modified cycle in a hydrogen peroxide gas plasma sterilizer (4 injections), showed no clinical signs of disease and remained alive. Two animals from the group receiving sodium hydroxide followed by autoclaving (at 134 degrees C for 18 minutes) died. Furthermore, the tested enzymatic cleaning agent seemed to have no positive effect.

Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity

ABSTRACT The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-β) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.

Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.

No Borna disease virus-specific RNA detected in blood from psychiatric patients in different regions of Germany

In Germany and other parts of Europe, infections with Borna disease virus (BDV) cause behavioural disturbances and meningoencephalitis in horses and sheep. Based on serological data, it has been suggested that BDV might contribute to the pathogenesis of psychiatric disorders in human beings. BDV-specific RNA has been detected in necropsy brain specimens of patients with hippocampal sclerosis,1 bipolar affective disorder,2 and schizophrenia.2 BDV-specific RNA was also reported to be present in peripheral blood mononuclear cells of a high proportion of psychiatric patients from Germany.

Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice.

ABSTRACT Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the naïve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4+ or CD8+ T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4+ T cells exhibited reduced protection, whereas animals lacking CD8+ T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.

Lung-specific expression of active Raf kinase results in increased mortality of influenza A virus-infected mice.

Alterations in signalling via the Raf/MEK/ERK pathway interfere with influenza A virus replication in cell culture. While virus yields are reduced in cells expressing dominant-negative Raf or ERK, virus propagation is enhanced upon expression of constitutively active Raf or MEK. To study the impact of active Raf on influenza virus propagation in vivo, we investigated transgenic mice expressing an activated mutant of c-Raf (Raf-BxB) in the main target tissue of influenza virus, the lung. Raf-BxB expression results in multicentric alveolar adenomas. Influenza virus A infection of Raf-BxB mice results in increased disease symptoms and higher mortality rates. The immune response against viral pathogens in transgenic animals did not differ from wild-type mice as determined by the use of a Pseudorabies virus (PRV) as a model for a viral infection not affecting the lung. No significant differences of influenza virus titers in the lung of Raf-BxB and wild-type mice were observed. However, immunohistology revealed increased numbers of influenza NP-positive cells in the alveolar linings of Raf-BxB mice, demonstrating the strong tropism of influenza virus for cells expressing active Raf. These findings disclose the possibility to use modified influenza virus for the therapy of tumors with an activated Ras/Raf signalling pathway.

The Expanded Octarepeat Domain Selectively Binds Prions and Disrupts Homomeric Prion Protein Interactions

Insertion of additional octarepeats into the prion protein gene has been genetically linked to familial Creutzfeldt Jakob disease and hence to de novo generation of infectious prions. The pivotal event during prion formation is the conversion of the normal prion protein (PrPC) into the pathogenic conformer PrPSc, which subsequently induces further conversion in an autocatalytic manner. Apparently, an expanded octarepeat domain directs folding of PrP toward the PrPSc conformation and initiates a self-replicating conversion process. Here, based on three main observations, we have provided a model on how altered molecular interactions between wild-type and mutant PrP set the stage for familial Creutzfeldt Jakob disease with octarepeat insertions. First, we showed that wild-type octarepeat domains interact in a copper-dependent and reversible manner, a “copper switch.” This interaction becomes irreversible upon domain expansion, possibly reflecting a loss of function. Second, expanded octarepeat domains of increasing length gradually form homogenous globular multimers of 11–21 nm in the absence of copper ions when expressed as soluble glutathione S-transferase fusion proteins. Third, octarepeat domain expansion causes a gain of function with at least 10 repeats selectively binding PrPSc in a denaturant-resistant complex in the absence of copper ions. Thus, the combination of both a loss and gain of function profoundly influences homomeric interaction behavior of PrP with an expanded octarepeat domain. A multimeric cluster of prion proteins carrying expanded octarepeat domains may therefore capture and incorporate spontaneously arising short-lived PrPSc-like conformers, thereby providing a matrix for their conversion.