Antibody levels to hantavirus in inhabitants of western Santa Catarina State, Brazil

Hantavirus cardiopulmonary syndrome (HCPS) is an infectious disease caused by hantaviruses of the family Bunyaviridae, and is transmitted by aerosols of excreta of infected rodents. The aim of the present study was to determine antibody levels to hantavirus in the population that lives at frontier of Brazil and Argentina. Participated of the study 405 individuals living in the municipalities of Bandeirante, Santa Helena, Princesa and Tunapolis, state of Santa Catarina, Brazil. IgG antibodies to hantavirus were analyzed in sera by an ELISA that uses a recombinant N protein of Araraquara hantavirus as antigen. The results were also confirmed by immunofluorescent test. Eight individuals showed antibodies to hantavirus (1.97% positivity), with serum titers ranging from 100 to 800. Six seropositives were males, older than 30 years and farmers. Our results reinforce previous data on hantavirus circulation and human infections in the southern border of Brazil with Argentina.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (â^ž), NLR (0.017), and Ef (99%).

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Antibody responses in Plasmodium falciparum malaria and their relation to protection against the disease

Protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the P. Falciparum infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to P. falciparum infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure. Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an in vitro assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes. The P. falciparum parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated P. falciparum parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses. Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area. There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against P. falciparum asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice)

Expression of antibodies in plant against essential viral proteins could provide an alternative approach to engineered viral resistance. Engineered single chain Fv antibodies scFV are particularly suitable for expression in plant because of their small size and the lack of assembly requirements. RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of viral replicases required for the replication of all positive strand RNA viruses. By using Phage technology we selected scFvs from a phage library using purified E.coli expressed TBSV(Tomato bushy stunt virus) replicase as antigen. The scFvs mediated-inhibition of RdRp activity was studied in vitro and in planta. In vitro experiments showed the inhibition of CNV(Cucumber necrosis virus) and TCV(Turnip crinkle virus) RdRp. Transient in planta assays based on agroinfiltration and an infectious clone of TBSV demonstrated the inhibition of the replication of TBSV(Tomato bushy stunt virus). Epitope mapping showed that the selected scFvs target the motif E of RdRp which is involved in template binding.Moreover T1 plants of transgenic lines of N. benthamiana expressing different scFvs either in the cytoplasm or the ER (endoplasmic reticulum) showed a high level of resistance against infection with TBSV and RCNMV(Red clover necrotic mosaic virus) upon inoculation with virus particles. This is the first report that scFvs against a RdRp of a plant viruses can inhibit viral replication in vivo. The resistance is even efficient against viruses belonging to different virus families.

Use of E. coli OMVs as delivery system to elicit neutralizing antibodies against Chlamydia infectivity: the HtrA protein example

The obligate intracellular pathogen Chlamydia trachomatis is a gram negative bacterium which infects epithelial cells of the reproductive tract. C. trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and a vaccine against this pathogen is highly needed. Many evidences suggest that both antigen specific-Th1 cells and antibodies may be important to provide protection against Chlamydia infection. In a previous study we have identified eight new Chlamydia antigens inducing CD4-Th1 and/or antibody responses that, when combined properly, can protect mice from Chlamydia infection. However, all selected recombinant antigens, upon immunization in mice, elicited antibodies not able to neutralize Chlamydia infectivity in vitro. With the aim to improve the quality of the immune response by inducing effective neutralizing antibodies, we used a novel delivery system based on the unique capacity of E. coli Outer Membrane Vesicles (OMV) to present membrane proteins in their natural composition and conformation. We have expressed Chlamydia antigens, previously identified as vaccine candidates, in the OMV system. Among all OMV preparations, the one expressing HtrA Chlamydia antigen (OMV-HtrA), showed to be the best in terms of yield and quantity of expressed protein, was used to produce mice immune sera to be tested in neutralization assay in vitro. We observed that OMV-HtrA elicited specific antibodies able to neutralize efficiently Chlamydia infection in vitro, indicating that the presentation of the antigens in their natural conformation is crucial to induce an effective immune response. This is one of the first examples in which antibodies directed against a new Chlamydia antigen, other than MOMP (the only so far known antigen inducing neutralizing antibodies), are able to block the Chlamydia infectivity in vitro. Finally, by performing an epitope mapping study, we investigated the specificity of the antibody response induced by the recombinant HtrA and by OMV-HtrA. In particular, we identified some linear epitopes exclusively recognized by antibodies raised with the OMV-HtrA system, detecting in this manner the antigen regions likely responsible of the neutralizing effect.

Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Multiple pathogen detection using nanoplasmonic sensing

Opportunistic diseases caused by Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) is an omnipresent global challenge. In order to manage these epidemics, we need to have low cost and easily deployable platforms at the point-of-care in high congestions regions like airports and public transit systems. In this dissertation we present our findings in using Localized Surface Plasmon Resonance (LSPR)-based detection of pathogens and other clinically relevant applications using microfluidic platforms at the point-of-care setting in resource constrained environment. The work presented here adopts the novel technique of LSPR to multiplex a lab-on-a-chip device capable of quantitatively detecting various types of intact viruses and its various subtypes, based on the principle of a change in wavelength occurring when metal nano-particle surface is modified with a specific surface chemistry allowing the binding of a desired pathogen to a specific antibody. We demonstrate the ability to detect and quantify subtype A, B, C, D, E, G and panel HIV with a specificity of down to 100 copies/mL using both whole blood sample and HIV-patient blood sample discarded from clinics. These results were compared against the gold standard Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). This microfluidic device has a total evaluation time for the assays of about 70 minutes, where 60 minutes is needed for the capture and 10 minutes for data acquisition and processing. This LOC platform eliminates the need for any sample preparation before processing. This platform is highly multiplexable as the same surface chemistry can be adapted to capture and detect several other pathogens like dengue virus, E. coli, M. Tuberculosis, etc.

90 Nb: potential radionuclide for application in immuno-PET : development of appropriate production strategy and first in vivo evaluation of 90 Nb-labeled monoclonal antibody

Die Nuklearmedizin ist ein modernes und effektives Werkzeug zur Erkennung und Behandlung von onkologischen Erkrankungen. Molekulare Bildgebung, die auf dem Einsatz von Radiopharmaka basiert, beinhaltet die Einzel-Photonen-Emissions-Tomographie (SPECT) und Positronenemissions¬tomographie (PET) und ermöglicht die nicht-invasive Visualisierung von Tumoren auf nano-und picomolarer Ebene.rnDerzeit werden viele neue Tracer für die genauere Lokalisierung von kleinen Tumoren und Metastasen eingeführt und hinsichtlich ihrer Eignung untersucht. Die meisten von ihnen sind Protein-basierte Biomoleküle, die die Natur selbst als Antigene für die Tumorzellen produziert. Dabei spielen Antikörper und Antikörper-Fragmente eine wichtige Rolle in der Tumor-Diagnostik und Behandlung. Die PET-Bildgebung mit Antikörpern und Antikörperfragmenten bezeichnet man als immuno-PET. Ein wichtiger Aspekt hierbei ist, dass entsprechende Radiopharmaka benötigt werden, deren Halbwertszeit mit der Halbwertszeit der Biomoleküle korreliert ist.rnIn neueren Arbeiten wird 90Nb als potenzieller Kandidat für die Anwendung in der immuno-PET vorgeschlagen. Seine Halbwertszeit von 14,6 Stunden ist geeignet für die Anwendung mit Antikörperfragmenten und einige intakten Antikörpern. 90Nb hat eine relativ hohen Anteil an Positronenemission von 53% und eine optimale Energie für die β+-Emission von 0,35 MeV, die sowohl eine hohe Qualität der Bildgebung als auch eine niedrige Aktivitätsmenge des Radionuklids ermöglicht.rnErsten grundlegende Untersuchungen zeigten: i) dass 90Nb in ausreichender Menge und Reinheit durch Protonen-Bombardierung des natürlichen Zirkonium Targets produziert, ii) aus dem Targetmaterial in entsprechender radiochemischer Reinheit isoliert und iii) zur Markierung des monoklonalen Antikörpers (Rituximab) verwendet werden kann und iv) dieser 90Nb-markierte mAb eine hohe in vitro Stabilität besitzt. Desweiteren wurde eine alternative und schnelle Abtrennungsmethode entwickelt, die es erlaubt 90Nb, mit einer geeigneten radiochemischen und radionuklidischen Reinheit für eine anschließende Markierung von Biomolekülen in einer Stunde zu aufzureinigen. Schließlich wurden erstmals 90Nb-markierte Biomolekülen in vivo untersucht. Desweiteren wurden auch Experimente durchgeführt, um den optimalen bifunktionellen Chelatbildner (BFC) für 90Niob zu finden. Mehrere BFC wurden hinsichtlich Komplexbildung mit NbV untersucht. Desferrioxamin (Df) erwies sich als geeignetster Chelator für 90Nb. Der monoklonale Antikörper Bevacizumab (Avastin®) wurde mit 90Nb markiert und eine Biodistributionsstudie und eine PET-Untersuchung durchgeführt. Alle diese Ergebnisse zeigten, dass 90Nb ein vielversprechendes Radionuklid für die Immuno-PET ist, welches sogar für weitere kommerzielle Anwendungen in der klinischen Routine geeignet zu sein scheint.rn

Towards antibody-mediated targeting of dendritic cells for cancer immunotherapy with multivalent polymer-antigen conjugates

Der Fokus dieser Arbeit lag auf der definierten Synthese multifunktioneller Polymer-Konjugate zur Anwendung in der Krebs-Immunotherapie. Durch gezielte Variation der Kon-jugationsbedingungen wurde Zusammensetzung, Größe und Aggregationsverhalten in Zell-medium sowie in humanem Serum untersucht. Nach definierter physikalisch-chemischer Charakterisierung wurde dann die induzierte Antigen-Präsentation zur Aktivierung der T-Zellproliferation analysiert.rnDafür wurden zwei verschiedene polymere Carrier-Systeme gewählt, lineares Poly-L-lysin und eine Polylysinbürste (PLL-Bürste). Es wird vermutet, dass die PLL-Bürste aufgrund der anisotropen Form eine bessere Verteilung im Körper und eine verlängerte Zirkulationsdauer zeigen wird. Die zu konjugierenden biologisch aktiven Komponenten waren der antiDEC205-Antikörper (aDEC205) für die gezielte Adressierung CD8-positiver dendritischer Zellen (DC), ein Ovalbumin (OVA)-spezifisches Antigen mit der Kernsequenz SIINFEKL für die Spezifität der Immunantwort gegen Krebszellen, die dieses Antigen tragen, und ein immunaktivieren-der TLR9-Ligand, CpG1826. Die Effizienz dieses Konjugates dendritische Zellen zu aktivieren, welche wiederum eine Immunantwort gegen OVA-exprimierende Krebszellen induzieren, wurde durch die Konjugation aller Komponenten am identischen Trägermolekül deutlich höher erwartet.rnLineares Poly-L-lysin diente als Modellsystem um die Konjugationschemie zu etablieren und dann auf die zylindrische Polylysinbürste zu übertragen. Anhand dieser polymeren Träger wurde das Verhalten der verschiedenen Topologien des Knäuels und der Bürste im Hinblick auf den Einfluss struktureller Unterschiede sowohl auf Konjugationsreaktionen als auch auf das in situ und in vitro Verhalten untersucht.rnFluoreszenzmarkiertes Antigen und der CpG Aktivator konnten jeweils aufgrund einer Thiol-Modifizierung an die Thiol-reaktive Maleimidgruppe des heterobifunktionellen Linkers Sulfo-SMCC an PLL-AlexaFluor48 konjugiert werden. Anschließend wurde aDEC205-AlexaFluor647 an PLL gekoppelt, entweder durch Schiff Base-Reaktion des oxidierten Antikörpers mit PLL und anschließender Reduzierung oder durch Click-Reaktion des PEG-Azids modifizierten An-tikörpers mit Dicyclobenzylcyclooctin (DIBO)-funktionalisiertem PLL. Die Konjugation der biologisch aktiven Komponenten wurde mit Durchflusszytometrie (FACS) und konfokaler Laser Scanning Mikroskopie (CLSM) untersucht und die Zusammensetzung des Konjugatesrnmittels UV/Vis-Spektroskopie bestimmt. Die PLL-Bürste alleine zeigte eine hohe Zytotoxizität bei HeLa und JAWS II Zelllinien, wohingegen lineares PLL und PLL-Konjugate sowie die PLL Bürsten-Konjugate keine ausgeprägte Zytotoxizität aufwiesen. Die Polymer-Konjugate wie-sen keine Aggregation in Zellmedium oder humanem Serum auf, was mittels winkelabhängi-ger dynamischer Lichtstreuung bestimmt wurde. CLSM Aufnahmen zeigten Kolokalisation der an die einzelnen Komponenten gebundenen Fluoreszenzfarbstoffe in dendritischen Zel-len, was die erfolgreiche Konjugation und Internalisierung der Konjugate in die Zellen bele-gen konnte. FACS Messungen ergaben eine geringfügig erhöhte Aufnahme des adressierten PLL-Antigen-Antikörper-Konjugates verglichen mit dem PLL-Antigen-Konjugat. Experimente mit dem „Specific Hybridization Internalization Sensor“ (SHIP) zeigten jedoch nur Aufnahme der PLL-Konjugate in CD8+ unreife DC, nicht in reife DC, die nicht mehr unspezifisch, sondern nur noch über Rezeptoren internalisieren. Dies bewies die unspezifische Aufnahme des Kon-jugates, da Antikörper-Konjugation keine Rezeptor-vermittelte Endozytose in reife DC indu-zieren konnte. T-Zell-Proliferationsassays ergaben eine Aktivierung von CD8+ T-Zellen indu-ziert durch Antigen-tragende Konjugate, wohingegen Konjugate ohne Antigen als Negativ-kontrollen dienten und keine T-Zell-Proliferation erzielten. Es konnte jedoch kein Unter-schied zwischen adressierten und nicht adressierten Konjugaten aufgrund der unspezifischen Aufnahme durch das Polymer beobachtet werden. Lösliches SIINFEKL alleine bewirkte schon bei geringeren Konzentrationen eine T-Zell-Proliferation.rnEs war somit möglich, drei biologischen Komponenten an einen polymeren Träger zu konju-gieren und diese Konjugate im Hinblick auf Zusammensetzung, Größe, Internalisierung in dendritische Zellen und Aktivierung der T-Zell-Proliferation zu untersuchen. Außerdem wur-de die Konjugationschemie erfolgreich von dem Modellsystem des linearen PLL auf die PLL-Bürste übertragen. Die Polymer-Konjugate werde unspezifisch in DC aufgenommen und in-duzieren T-Zellproliferation, die mit Antigen-Präsentationsassays nachgewiesen wird. Es konnte jedoch durch Konjugation des Antikörpers keine Rezeptor-vermittelte Aufnahme in CD8+ DC erzielt werden.rnDiese Studien stellen einen erfolgsversprechenden ersten Schritt zur Entwicklung neuer Na-nomaterialien für die Anwendung in Krebs-Immuntherapie dar.

Untersuchungen zur antitumoralen Wirkungsweise eines bispezifischen MCSP-CD3 Antikörpers in vitro und im humanisierten Tumormausmodell

Humanes MCSP ist ein gut charakterisiertes Tumorantigen, das auf der Mehrzahl aller malignen Melanome hoch exprimiert wird, und stellt somit eine gute Zielstruktur für immuntherapeutische Ansätze dar. rnInnerhalb der vorliegenden Arbeit wurden die Wirkmechanismen eines neuen bispezifischen Antikörpers, der gegen humanes MCSP und CD3 auf T-Zellen gerichtet ist, in vitro und im humanisierten Tumormausmodell in vivo untersucht. In humanen T Zellkokulturen induzierte der bispezifische MCSP-CD3 Antikörper in Gegenwart MCSP-positiver Melanomzellen konzentrationsabhängige T-Zellaktivierung, Sekretion von Zytokinen und effiziente Tumorzelllyse durch CD4- und CD8-positive T-Zellen. Die induzierte Lyse war hierbei unabhängig von der T-Zellrezeptorspezifität sowie kostimulatorischen Molekülen und allein abhängig von der Expression des Tumorantigens sowie CD3 auf den T-Zellen. Wie hier diskutiert, liegt es nahe, dass die Freisetzung lytischer Moleküle (Perforin und Granzym-B) durch CD8- und auch CD4 positiver T-Zellen den Hauptmechanismus in der Lyse der Melanomzellen darstellt. rnUm die Wirksamkeit in vivo testen zu können, wurde ein humanisiertes Tumormausmodell etabliert. Die Injektion humaner hämatopoetischer Stammzellen in neugeborene Rag2-/-gc-/- Mäuse führte zur Entwicklung funktioneller T-Zellen im murinen Thymus, welche lymphatische Organe besiedelten. In vitro induzierten die T-Zellen humanisierter Mäuse in Anwesenheit des bispezifischen MCSP-CD3 Antikörpers ebenfalls konzentrationsabhängige Lyse der Melanomzellen. Wie hier gezeigt, induzierte die Injektion humaner Melanomzellen in humanisierte Mäuse keine messbare Abstoβungsreaktion. Unter Behandlung mit MCSP-CD3 wurde zwar eine erhöhte Anzahl humaner T-Zellen im Tumorgewebe nachgewiesen, jedoch verfügte die verwendete Melanomzelllinie über eine geringe basale T Zellinfiltration, geringe Vaskularisierung und ein noduläres Wachstumsverhalten. Wie innerhalb dieser Arbeit diskutiert, kann durch die Kombination mit Therapien, die eine erhöhte T-Zellinfiltration in das Tumorgewebe ermöglichen, die Wirksamkeit von bispezifischen Antikörpern möglicherweise gesteigert werden. rn

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.