Vergleichende Untersuchung verschiedener genetischer Impfstoffvektoren in vivo

Impfstoffe gelten als effektive und günstige Arzneimittel. Jedoch stehen für viele Infektionskrankheiten wie AIDS, Tuberkulose, Hepatitis C oder Malaria keine oder keine wirksamen Impfmöglichkeiten zur Verfügung. Um gegen intrazelluläre Erreger oder auch Tumore wirksam zu sein, muß ein potentieller Impstoff eine humorale und eine starke zelluläre Immunantwort induzieren. Diese Vorraussetzung kann vor allem durch genetische Immunisierungen mit DNA oder viralen Vektoren erfüllt werden. Vor diesem Hintergrund wurde im Rahmen dieser Arbeit das Potential einer DNA-Vakzine und eines HSV-1-basierten Vektors untersucht, Mäuse vor Infektionen mit dem intrazellulären Bakterium Listeria monocytogenes zu schützen. Zunächst wurde die Immunantwort untersucht, welche durch Immunisierung mit einem OVA-kodierenden Plasmid induziert wurde. Dabei konnte erstmals in vivo gezeigt werden, daß die Form (sezerniert oder membrangebunden) des exprimierten Antigens nach einer DNA-Immunsierung mittels Gene Gun sowohl das Ausmaß der humoralen Immunantwort beeinflußt, als auch die Expansion antigenspezifischer CD4- und CD8-T-Zellen. Desweiteren stellte sich heraus, daß replikationsinkompetente, rekombinante HSV-1- (rHSV-1) Vektoren und HSV-1-Amplikons in der Lage sind, eine sehr starke CTL-Antwort, aber nur eine schwache Antikörper- und CD4-T-Zellantwort zu induzieren. Eine starke, antigenspezifische CTL-Antwort konnte ebenso durch Immunisierung mit rekombinanten MVA-Vektoren generiert werden. In Vakzinierungsexperimenten konnte nachgewiesen werden, daß mit rHSV-1 immunisierte Mäuse vor Infektionen mit hohen Dosen an rekombinanten Listerien geschützt waren, wohingegen DNA-immunisierte Mäuse nur vor Infektionen mit einer mittleren, aber dennoch letalen Dosis geschützt waren. Der Immunschutz war in beiden Fällen von langer Dauer. Im Rahmen dieser Arbeit konnte demonstriert werden, daß die DNA-Immunisierung per Gene Gun eine einfach anzuwendende, verläßliche und effektive Methode ist, eine humorale und zelluläre protektive Immunantwort in Mäusen zu induzieren. Besonders im Hinblick auf neue Vakzinierungsstrategien gegen intrazelluläre Erreger, die vor allem durch starke zytotoxische T-Zellantworten bekämpft werden können, erscheinen Immunisierungen mit rHSV-1-Vektoren als sehr geeignete Methode, die es gilt, sorgfältig zu untersuchen und weiterzuentwickeln

Adoptive immunotherapy induces CNS dendritic cell recruitment and antigen presentation during clearance of a persistent viral infection

Given the global impact of persistent infections on the human population, it is of the utmost importance to devise strategies to noncytopathically purge tissues of infectious agents. The central nervous system (CNS) poses a unique challenge when considering such strategies, as it is an immunologically specialized compartment that contains a nonreplicative cell population. Administration of exogenously derived pathogen-specific memory T cells (referred to as adoptive immunotherapy) to mice burdened with a persistent lymphocytic choriomeningitis virus (LCMV) infection from birth results in eradication of the pathogen from all tissues, including the CNS. In this study, we sought mechanistic insights into this highly successful therapeutic approach. By monitoring the migration of traceable LCMV-specific memory CD8+ T cells after immunotherapy, it was revealed that cytotoxic T lymphocytes (CTLs) distributed widely throughout the CNS compartment early after immunotherapy, which resulted in a dramatic elevation in the activity of CNS antigen-presenting cells (APCs). Immunotherapy induced microglia activation as well as the recruitment of macrophages and dendritic cells (DCs) into the brain parenchyma. However, DCs emerged as the only CNS APC population capable of inducing memory CTLs to preferentially produce the antiviral cytokine tumor necrosis factor-α, a cytokine demonstrated to be required for successful immunotherapeutic clearance. DCs were also found to be an essential element of the immunotherapeutic process because in their absence, memory T cells failed to undergo secondary expansion, and viral clearance was not attained in the CNS. These experiments underscore the importance of DCs in the immunotherapeutic clearance of a persistent viral infection and suggest that strategies to elevate the activation/migration of DCs (especially within the CNS) may facilitate pathogen clearance

Immune Requirements of Post-Exposure Immunization with Modified Vaccinia Ankara of Lethally Infected Mice

Current prophylactic vaccines work via the induction of B and T cell mediated memory that effectively control further replication of the pathogen after entry. In the case of therapeutic or post-exposure vaccinations the situation is far more complex, because the pathogen has time to establish itself in the host, start producing immune-inhibitory molecules and spread into distant organs. So far it is unclear which immune parameters have to be activated in order to thwart an existing lethal infection. Using the mousepox model, we investigated the immunological mechanisms responsible for a successful post-exposure immunization with modified vaccinia Ankara (MVA). In contrast to intranasal application of MVA, we found that intravenous immunization fully protected mice infected with ectromelia virus (ECTV) when applied three days after infection. Intravenous MVA immunization induced strong innate and adaptive immune responses in lethally infected mice. By using various gene-targeted and transgenic mouse strains we show that NK cells, CD4 T cells, CD8 T cells and antibodies are essential for the clearance of ECTV after post-exposure immunization. Post-exposure immunization with MVA is an effective measure in a murine model of human smallpox. MVA activates innate and adaptive immune parameters and only a combination thereof is able to purge ECTV from its host. These data not only provide a basis for therapeutic vaccinations in the case of the deliberate release of pathogenic poxviruses but possibly also for the treatment of chronic infections and cancer

Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host ' s inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8(+) T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment. Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8(+) T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA. Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8(+) T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory

Clearance of an immunosuppressive virus from the CNS coincides with immune reanimation and diversification

<p>Abstract</p> <p>Once a virus infection establishes persistence in the central nervous system (CNS), it is especially difficult to eliminate from this specialized compartment. Therefore, it is of the utmost importance to fully understand scenarios during which a persisting virus is ultimately purged from the CNS by the adaptive immune system. Such a scenario can be found following infection of adult mice with an immunosuppressive variant of lymphocytic choriomeningitis virus (LCMV) referred to as clone 13. In this study we demonstrate that following intravenous inoculation, clone 13 rapidly infected peripheral tissues within one week, but more slowly inundated the entire brain parenchyma over the course of a month. During the establishment of persistence, we observed that genetically tagged LCMV-specific cytotoxic T lymphocytes (CTL) progressively lost function; however, the severity of this loss in the CNS was never as substantial as that observed in the periphery. One of the most impressive features of this model system is that the peripheral T cell response eventually regains functionality at ~60–80 days post-infection, and this was associated with a rapid decline in virus from the periphery. Coincident with this "reanimation phase" was a massive influx of CD4 T and B cells into the CNS and a dramatic reduction in viral distribution. In fact, olfactory bulb neurons served as the last refuge for the persisting virus, which was ultimately purged from the CNS within 200 days post-infection. These data indicate that a functionally revived immune response can prevail over a virus that establishes widespread presence both in the periphery and brain parenchyma, and that therapeutic enhancement of an existing response could serve as an effective means to thwart long term CNS persistence.</p

A new Type Line-Array for Acoustic Source Localization at Drive-By Tests of Trains

Abstract/Kurzfassung: The application of microphone arrays for the allocation of sound sources at objects moving along a known trajectory is of special interest in case of the investigation of noise-sources at trains. Especially in case of high-speed trains it is a challenging task, since the angular velocity of the sound sources is high if the recording takes place at close distances of around 10 m from the track. Different techniques exist for the localization and quantification of sound sources. Examples are elliptical acoustic mirrors and phased microphone arrays. In case of microphone-arrays the focussing process is carried out by means of summing up signals from different sensors with defined phase delay. A short overview of the history of microphone array measurements for train investigations is provided by Michel 2006. In order to detect a flat or even spatial distribution of sound sources with a directional microphone with a concave acoustic mirror, the concave mirror has to be moved with the microphone at its focal point for scanning the volume of interest. This implies a fixed position of the sound sources of interest. When using a microphone-array, the focus point can be varied as desired in the post-processing of the recorded signals, leading to much smaller acquisition times compared to measurements with acoustic mirrors. However, a high spatial resolution requires a large number of microphones distributed on a 2-dimensional mesh. In the following a new type of microphone-array combining a line array with a two-dimensional elliptical acoustic mirror is described. The combination of both techniques allows a three-dimensional reconstruction of the source distribution. The design was successfully tested on a high speed train track in Germany, which is used by different type of Inter City Express and other passenger trains. It was possible to improve the quality of the results signifficantly and individual wheel sets and aeroacoustic noise caused by roof structures can be identified in the source maps

Simultaneous multiplane PIV and microphone array measurements on a rod-airfoil configuration

One major objective in aeroacoustic research is the identification of flow structures that are involved in the generation mechanisms of aeroacoustic noise sources. The simultaneous measurement of acoustic pressure fluctuations in the far-field together with some near-field quantity has been investigated by various authors to achieve this objective. The idea of this concept is to correlate the measured far-field pressure with the near-field quantity and in doing so identify regularities in the near field fluctuations that are related to the radiated sound field. Previous experiments have shown that synchronized PIV and microphone measurements can be used to obtain the cross-correlation function between a near-field quantity and the acoustic pressure in the far field. The authors have shown that in case of a relatively simple flow configuration (e.g. cylinder wake flow) a reasonable amount of 5000 PIV recordings are sufficient to obtain a significant correlation between the velocity fluctuations and the far field pressure in the whole region of interest measured via PIV. This has not been the case for a more complex configuration (e.g. a slat flow field of a swept wing), probably because of the more three dimensional character of this flow field compared to the cylinder wake flow field. The studies performed previously by the authors have been performed in a closed test section wind-tunnel which is not optimized for acoustic measurements. Besides, the sound field has been recorded by one in-flow microphone only. In the present study measurements are conducted in an aeroacoustic wind tunnel with an open test section in an anechoic chamber. The acoustic data where acquired using a microphone array consisting of 87 microphones. Furthermore, a multi plane PIV system is used, enabling the calculation of temporal derivatives. A rod-airfoil configuration is investigated by means of the described correlation method. In such a configuration the airfoil undergoes a broadband perturbation dominated by the turbulent periodic vortex shedding frequency separating from the rod. Compared to the flow field generated by a single cylinder the radiated noise is increased over a wide range of frequencies

Systematic Comparison of Microphone Array Measurements in Open and Closed Wind Tunnels

The phased microphone array has become a widely used tool in aeroacoustic testing for the localisation and quantification of sound sources. The measurements are often carried out in open or closed wind tunnels using scaled models. Test sections in both types of tunnels have advantages and drawbacks when used for aerodynamic and aeroacoustic testing. The closed-test section wind tunnel is normally preferred for measuring aerodynamic properties due to the reduced uncertainties compared with the open section. Conducting aerodynamic and aeroacoustic measurements simultaneously or at least back-to-back helps to reduce wind tunnel access time and costs. Furthermore, in many cases a better microphone array resolution can be achieved because of the shorter distances between array and model. In-flow measurements in closed test sections using flush mounted microphones in the wind tunnel wall are typically subjected to strong hydrodynamic pressure fluctuations induced by the turbulent boundary layer, which can result in a dramatic decrease of the signal-to-noise ratio (SNR). The reverberations in the closed test section are also of concern, since the beamforming accuracy may be affected. In contrast, the open jet acoustic wind tunnel is not plagued by these particular drawbacks. Nevertheless, other problems will occur and can influence the quality of the measurement results. The sound waves have to pass through the wind tunnel shear layer before reaching the microphone. That results in a spatial coherence loss due to turbu- lent scattering and refraction and potentially leads to degraded source maps and under-estimated source levels. In comparison with the closed wind tunnel, far-field measurements can be normally performed

A new Type of Line-Array for Aeroacoustic and Acoustic Source Localization at Drive-by Tests of Trains

The application of microphone arrays for the allocation of sound sources at objects moving along a known trajectory is of special interest in case of the investigation of noise-sources at trains. Especially in case of high-speed trains it is a challenging task, since the angular velocity of the sound sources is high if the recording takes place at close distances of around 10 m from the track. Different techniques exist for the localization and quantification of sound sources. Examples are elliptical acoustic mirrors and phased microphone arrays. In case of microphone-arrays the focussing process is carried out by means of summing up signals from different sensors with defined phase delay. A short overview of the history of microphone array measurements for train investigations is provided by Michel 2006. In order to detect a flat or even spatial distribution of sound sources with a directional microphone with a concave acoustic mirror, the concave mirror has to be moved with the microphone at its focal point for scanning the volume of interest. This implies a fixed position of the sound sources of interest. When using a microphone-array, the focus point can be varied as desired in the post-processing of the recorded signals, leading to much smaller acquisition times compared to measurements with acoustic mirrors. However, a high spatial resolution requires a large number of microphones distributed on a 2-dimensional mesh. In the following a new type of microphone-array combining a line array with a two-dimensional elliptical acoustic mirror is described. The combination of both techniques allows a three-dimensional reconstruction of the source distribution. The design was successfully tested on a high speed train track in Germany, which is used by different type of Inter City Express and other passenger trains. It was possible to improve the quality of the results signifficantly and individual wheel sets and aeroacoustic noise caused by roof structures can be identified in the source maps