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Masern sind eine sehr kontagiöse virale Infektion, die in der Regel im Kindesalter auftritt. Typische Symptome sind unter anderem Fieber, Entzündung der Schleimhäute und die charakteristischen roten Hautflecken. Die subakute sklerosierende Panenzephalitis (SSPE) ist eine besondere Spätkomplikation, wobei es zu Entzündungen im Gehirn auftreten kann und diese langfristig schädigt. Eine hochwirksame Mischimpfung gegen Masern, Mumps und Röteln (MMR) ist am Markt vorhanden. Im Rahmen der Bachelorarbeit wollen wir einen Masernimpfstoff mit Hilfe von Mikroträgern herstellen, auf denen die MRC-5 Zellen in einem 50L Bioreaktorsystem adhärent wachsen können. Diese Arbeit befasst sich mit dem ersten und wichtigsten Prozessschritt des Upstreams, der Inokulumvorbereitung für den Bioreaktor. Es wurde eine Ablaufdarstellung von der Entnahme der Stickstoffampulle bis zum Fermenter, die dazugehörige Gute Herstellungspraxis (GMP)-Dokumente, wie Standardvorgehensweise (SOP) und Master Batch Record (MBR) erstellt. Measles is a highly contagious viral infection that usually occurs in childhood. Typical symptoms are fever, inflammation of the mucous membranes and characteristic red skin spots. Subacute sclerosing panencephalitis (SSPE) is a particular late complication, with inflammation occurring in the brain and gradual decay. Measles, mumps and rubella (MMR) is highly effective mixed vaccine. The aim of this bachelor thesis is to produce a measles vaccine using microcarriers where the MRC-5 cells can grow adherently in a 50L stirred bioreactor. My part focuses on the first and most important process step of the upstream processing, the inoculum preparation for the bioreactor. For that I created a flow chart from nitrogen vial up to the fermenter, associated Good Manufacturing Practice (GMP) documents such as Standard Operating Procedure (SOP) and Master Batch Record (MBR).

CME Dermatology 22/Answers: Measles - Empidemiology and Clinical Manifestations

Das Masernvirus (MV) gehört zur Familie der Paramyxoviridae und verursacht die Krankheit Masern, welche schwerwiegende Symptome hervorrufen kann. Seit vielen Jahren werden attenuierte Masernimpfviren vom Stamm Edmonston erfolgreich eingesetzt. Eine MV-Infektion bzw. Impfung induziert eine T- und B-Zell- Antwort, die einen lang anhaltenden Schutz vor einer Infektion mit verschiedenen Wildtyp-Virusvarianten vermittelt. Der Hauptanteil der durch die B-Zell-Antwort induzierten neutralisierenden Antikörper (Ak) erkennt das Oberflächenprotein Hämagglutinin (H). In dieser Arbeit wurden neutralisierende Epitope des H-Proteins von aktuellen Wildtyp-Viren im Vergleich zum Impfvirus analysiert. Das H-Protein besteht aus 617 Aminosäuren (AS). Zwischen dem H-Protein untersuchter Impf- und Wildtyp-Viren bestehen Unterschiede in der AS-Sequenz, die sich auch auf die Struktur neutralisierender Epitope auswirken können. Mit Hilfe der Hybridomatechnologie wurden sieben Impf-induzierte, H-spezifische, neutralisierende monoklonale Antikörper (mAk) hergestellt, um die neutralisierenden Epitope zu charakterisieren. Mit Hilfe eines PepStarTM Peptide Microarrays konnte für drei mAk das zugehörige Epitop auf der C-terminalen Rezeptor-Bindedomäne des H-Proteins lokalisiert werden: der mAk RKI-MV-1d bindet an das bereits beschriebene Epitop MV-H244-250 (neutralizing epitope, NE), der mAk RKI-MV-33a an das ebenfalls bekannte Epitop MV-H381-400 (hemagglutinin noose epitope, HNE) und der mAk RKI-MV-34c an das bekannte Epitop MV-H309-315. Ein Vergleich der neutralisierenden Kapazität der erzeugten mAk gegenüber den Impfviren EdmZ und EdmS und den aktuell zirkulierenden Wildtyp-Viren zeigte, dass nur die mAk RKI-MV-12b und RKI-MV- 34c jeweils ein konserviertes Epitop erkennen. Der mAk RKI-MV-1d konnte aufgrund der AS-Veränderung L249 zu P249 im NE nicht an die Virusisolate Duisburg.DEU/10.09 [D4] und Berlin.DEU/47.00 [D6] binden und keine Infektion verhindern. Die AS-Veränderung von K387 zu R387 im HNE verhinderte die Bindung des RKI-MV-33a an das H-Protein von Essen.DEU/12.10 [D8]. Der mAk RKI-MV-32c neutralisierte nur das Wildtyp-Virus Maryland.USA/0.54 vom Genotyp A und die von diesem Virus ausgehenden adaptierten und attenuierten Impfviren effektiv. Dieser mAk könnte für diagnostische Zwecke interessant sein. Die neutralisierende Kapazität der mAk RKI-MV-9c und RKI-MV-13a unterscheidet sich ebenfalls zwischen den untersuchten Wildtyp-Virusvarianten. Diese Ergebnisse zeigen, dass von sieben hier identifizierten Epitopen zwei konservativ und fünf variabel sind. Jeder der fünf mAk, die ein variables Epitop erkennen, zeigte ein individuelles Neutralisationsmuster gegenüber den eingesetzten Wildtyp-Viren. Humane Impf-induzierte Seren neutralisierten die eingesetzten Wildtyp-Viren unabhängig von ihrem Epitopmuster. Dieses Ergebnis zeigt, dass eine MV-Impfung neutralisierende Ak induziert, die vor einer Infektion mit verschiedenen Wildtyp-Virusvarianten schützt. Epidemiologische Erfahrungen zeigen, dass geimpfte Personen sehr selten erkranken. Neu auftretende Wildtyp- Viren mit zusätzlichen Epitopveränderungen im Vergleich zum Impfvirus können zu einer Verringerung der Impfeffizienz führen. Das Schließen von bestehenden Impflücken in der Population würde zu einem Abbruch der MV-Zirkulation führen und das Auftreten und die Zirkulation neuer Virusvarianten mit einem veränderten Epitopmuster verhindern., The measles virus (MV) belongs to the family of paramyxoviruses and causes the disease measles, which can lead to severe complications. For many years attenuated measles vaccines derived from the Edmonston strain have been applied. A measles virus infection or vaccination induces a T- and B-cell response which facilitates long-lasting protection against an infection with different wild-type measles viruses. Most of the B-cell induced neutralizing antibodies recognize the measles surface protein hemagglutinin (H). In this PhD thesis neutralizing epitopes on the H protein of currently circulating measles wild-type viruses were analyzed in comparison to the vaccine virus. The H protein consists of 617 amino acids (aa). Between the vaccine and wild- type viruses investigated are differences in the aa sequence, which can also influence the structure of neutralizing epitopes. By applying the hybridomatechnology seven vaccine-induced, H-specific, neutralizing monoclonal antibodies were generated to analyze the neutralizing epitopes. The epitopes of three monoclonal antibodies were localized with the help of a peptide- microarray. The monoclonal antibody RKI-MV-1d recognized the known epitope MV-H244-250 (neutralizing epitope, NE), RKI-MV-33a the also already known epitope MV-H381-400 (hemagglutinin noose epitope, HNE) and RKI-MV-34c aa MV-H309-315. A comparison of the neutralizing capacity of the monoclonal antibodies with the vaccine viruses EdmZ and EdmS as well as with the wild- type viruses shows that the monoclonal antibodies RKI-MV-12b and RKI-MV-34c recognized a conserved epitope. The monoclonal antibody RKI-MV-1d could not bind to the H protein and thus not neutralize isolates Duisburg.DEU/10.09 [D4] and Berlin.DEU/47.00 [D6], which was due to the aa exchange from L249 to P249. The aa exchange from K387 to R387 prevented the binding of the monoclonal antibody RKI-MV-33a to the H protein of the isolate Essen.DEU/12.10 [D8]. The monoclonal antibody RKI-MV-32c neutralized only the wild-type virus Maryland.USA/0.54 [A] and the adapted, attenuated vaccine viruses effectively. This monoclonal antibody could be useful for diagnostic purposes. The neutralizing capacity of the monoclonal antibodies RKI-MV-9c and RKI-MV-13a differs between the investigated wild-type viruses as well. Human vaccine- induced sera neutralized the investigated wild-type viruses independently of the epitope pattern. This result indicates that the MV-vaccination induces neutralizing antibodies which prevent an infection with different wild-type viruses. Epidemiological data show that vaccinated people become rarely infected. Newly occurring wild-type viruses with additional changes in the epitopes compared with the epitopes of the vaccine might decrease the efficacy of vaccination. Closing existing vaccination gaps in a population could stop the measles virus circulation and avoid the occurrence and the circulation of new virus variants with a modified epitope pattern.

Measles are a systemic infectious disease caused by a single stranded ribonucleic acid virus (measles virus) from the paramyxovirus family. Typically, the disease is characterized by a two-phase course. After an average incubation period of 8 to 11 days, initial symptoms such as fever, cough, coryza and conjunctivitis appear. Two thirds of the patients shows a white-marked enanthema on the buccal mucosa (Koplik's spots). After disappearance of these symptoms, a second increase of temperature and the typical measles exanthema, a brownish-red maculopapular rash, appear. Infection with measles virus induces transient immunodeficiency that favours the formation of several complications. Some of them, e. g. encephalitic diseases, are severe and associated with a high mortality. Measles are world-wide distributed and belong to the ten most frequent infectious diseases in some less developed countries. The disease is associated with a high mortality in some African and South-East Asian countries, in particular in children aged less than 12 months. Of particular note, measles are the most important cause of blindness in children in population with borderline vitamin A status. In Germany, the number of reported measles cases has been declined dramatically since the introduction of a vaccine more than four decades ago. However, regional outbreaks or small epidemics still occur. Because there is no specific antiviral treatment, therapy of measles is symptomatic and depends on the manifestation of the disease. The most important prevention strategy is immunization with a life-attenuated vaccine that can be applied as monovaccination or in combination with mumps and rubella virus (MMR vaccination) or mumps, rubella and varicella virus (MMRV vaccination).

During the last twenty years the incidence of measles and mumps decreased after introduction of vaccinations in the industrial nations. The vaccination rate of the population in Germany lies currently under the required elimination rate. The epidemiological situation has changed altogether. The illness age has on the one hand moved up, teenager and adult suffer more frequent these so-called children's diseases with an increased complication rate. On the other hand illness cases in infancy seeming to increase. It was aim of this study to examinate the current serological situation for measles and mumps antibody status in women in childbed and their healthy newborns at birth time and during the first seven to nine life months.237 healthy newborns, born in 1999 in Bochum, West-Germany and their mothers were included. The mothers were asked if and when they did suffer from measles and mumps or were questioned to active vaccinations against these diseases. Immediately antepartal from all mothers was taken a venous blood sample. Immediately postpartal from all newborn childs umbilical cord blood was taken and again a blood sample in the age of six to eight months. In the serum tests the measle and mumps IgG antibody concentrations were examined quantitatively with ELISA methods. In cases with negative or borderline positive values the plaque neutralization test was also used for measuring of the measle antibodies.Between the maternal and neonatal titers results the knownly positive correlation. Maternal lending immunity against measle and mumps lasts more less into infancy as generally suspected. An earlier first active immunization against measles and mumps could be discussed. The strict realisation of the present vaccinating recommendations should be operated as a matter of priority to lead to the desired measles and mumps eradication.

Subacute sclerosing panencephalitis is a rare progressive neurological disorder of childhood and early adolescence caused by persistent measles virus. The diagnosis is based upon characteristic clinical manifestations, periodic EEG discharges, raised antibody titre against measles/SSPE in the plasma and cerebrospinal fluid and increase of gamma-globulins in the cerebrospinal fluid. Histopathological examination shows neuronal loss, astrogliosis, demyelination, infiltration of inflammatory cells, and intranuclear inclusions in neurons, oligodendrocytes and astrocytes. In most cases nucleocapsids are detected by electron microscopy. Although treatment is still undetermined, combination of intrathecal high-dose interferon-alpha and intravenous ribavirin administered at an early stage of SSPE seems to be effective.

Gastroenterological oncology requires new strategies with new mechanisms of action and without cross-resistance to currently available treatment regimes. Virotherapy which is based on the employment of replication-competent viral vectors exhibiting strong oncolytic properties is such an approach currently under preclinical/clinical investigation. Techniques of molecular virology are required for further improvement of current vectors, particularly with respect to oncolytic activity, tumour selectivity, tumour spread capacity, and safety.

Measles, mumps, rubella and varicella zoster virus (VZV) infections are regarded as typical diseases of childhood: They are normally clinically mild and result in lifelong immunity. Severe clinical disease is known in immunocompromised patients; rubella virus infections during pregnancy often result in congenital rubella syndrome. All these diseases are preventable by vaccination which is recommended in Germany, recently vaccination against VZV for teenager without immunity since July 2001. In the following study we screened for immunity against the four viruses.Serum samples were obtained at the Institute of Medical Virology Frankfurt/Main from January 1999 until December 2000. We tested for specific antibodies against measles (n = 915), against mumps (n = 857), against rubella (n = 1886) and against VZV (n = 2291). Seroprevalences were determined in different age groups.Altogether the highest rate of seronegatives is detected in younger children. VZV-seronegativity rates decrease from 74 % to 32 % in younger children. Against rubella also in this age group rate of seronegatives is found in 40 % and less than 10 % by teenagers. From this age group also immunity against rubella is found approximately in 80 % of seropositives.The following study shows that high seronegative rates are detectable, and here specially against VZV-specific antibodies. For seronegative teenagers, vaccination against VZV is now recommended in Germany. Immunization rates of at least 95 % in childhood would be effective in avoiding severe courses of disease and giving protection in pregnancy.

In 1996 the effects on the immunity profile of a Swiss population exposed to MMR vaccination, which has been recommended since 1985, were evaluated with an age-stratified seroprevalence study for measles, mumps and rubella (MMR). At the age of 1.5-2.5 years, seroprevalence attained 76% for measles and rubella, which is respectively 17% and 24% above the values observed in 1992. The seroprevalence for mumps attained only 55% at the same age, which could reflect the poor immunogenicity of this component of the MMR vaccine. The seroprevalence for measles IgG showed a slow but steady increase from vaccination age to adulthood, attaining nearly 100%. The concentrations of measles IgG were about 700 IU/l into adolescence and rose to a plateau at about 1500 IU/l during young adulthood. These observations are compatible with low endemic activity of measles in the last 20 years and a predominance of vaccine-induced immunity up to about 20 years of age. This corresponds to the time period when measles vaccines--single or as MMR--have been in use. In 1992, at the peak of epidemic activity, seroprevalence for mumps rose substantially faster than in 1996. In addition, the rapid increase in quantitative values during preschool age mirrors the ongoing wild virus circulation with minimal vaccine effect. In the vaccine cohort (2-12 years of age) the seroprevalence of rubella IgG reached 70-80%. That there is no rise in the curve during school age shows that the recommended catch-up vaccinations before or during school age have been neglected. The median concentrations of rubella IgG were about 65 IU/ml at vaccination, declined to 40-50 IU/ml during preschool age, and rose again during school age, suggesting wild virus circulation. These data show that the MMR vaccine cover in Swiss children is insufficient to interrupt virus circulation, and administration of a second dose of MMR for catch-up immunisation has been omitted. The poor efficacy of the mumps component of the MMR vaccine that has mainly been used in Switzerland is also evident. The average age at infection is therefore expected to rise, thus involving a risk of increasing age-dependent complications. Efforts to implement the MMR vaccination program in Switzerland should be improved.

Women suffer from otosclerosis 1.6 times more often than males. Histologically, otosclerotic foci can be found in temporal bones of females 1.9 times more often than in those of males. Characteristic topographic regions are the oval window, round window niche and promontory. Otosclerosis can also occur principally in any area of the enchondral/periosteal layer of the otic capsule. Evidence is presented that otosclerosis is an inflammatory tissue reaction associated with macrophages, T- and B-lymphocytes, HLA-DR positive cells and plasma cells. Dependent on the stage of the osteolytic bone disease present deposits of complement and immunoglobulins (IgG, IgA) can be found. These immunoglobulins have been identified as antibodies to measles virus proteins. Using the polymerase chain reaction we were successful in demonstrating RNA sequences of measles viruses in otosclerotic bone from footplates removed during stapes surgery. Since most of the otosclerotic lesions were in direct contact to the perilymphatic space, it may be expected that the endolymphatic sac--as the immune competent organ of the inner ear--specifically reacts to antigens delivered from the otosclerosis focus into the perilymph. Perilymph samples from patients were collected during stapes surgery and their antibody titers against measles were compared with that in corresponding blood serum. All samples revealed a significantly elevated-specific anti-measles IgG amount which was significantly higher than in the corresponding serum. In contrast, antibody titer in the perilymph against herpes simplex or cytomegalo viruses did not differ from that of the serum. These findings indicate that otosclerosis is a measles virus associated inflammatory osteolytic disease of the temporal bone. Since women suffer from severe measles virus infections more often than males, it can be hypothesized that females have a higher susceptibility of their cochleo-vestibular tissues to these infections (organotropism). In addition, estrogens are well-known stimulators of osteocytic activity and may play a dominant role during ossification of an otospongeotic bone lesion. This may explain the onset of a conductive hearing loss due to otosclerosis during pregnancy.

The respiratory tract represents both the port of entrance of the measles virus (MV) and an important target tissue for measles complications. Therefore, mucosal immunization could be an attractive alternative to the current subcutaneous route. We studied the virus-specific IgA and IgG antibody response in the serum after intranasal and intragastric immunization with MV in the mouse model under non-replicating conditions. Hemagglutination-inhibiting antibodies, mostly without virus-neutralizing activity were found after intranasal immunization. No virus-specific antibodies were detected after intragastric immunization.

Paramyxoviruses have been described in the osteoclasts in Paget's disease and it has been suggested that the disease was caused by this virus. There is considerable similarity of the histological features of active otosclerosis and Paget's disease and the probable presence of viral antigens in the otosclerotic footplate of the stapes of 15 patients has been investigated by means of immunohistochemical methods. The active otosclerotic lesions of all 15 patients studied displayed a high concentration of antibodies of the IgG-class which was less pronounced in the cytoplasm of the cells of the adjacent enchondral layer. Using polyclonal antibodies against measles and rubella viruses, both antigens could be shown expressed by the chondrocytes of the enchondral layer and by occasional osteocytes and osteoclasts of the otosclerotic lesion. IgG antibodies or viral antigens were absent in the normal bone of the stapes specimens. The interpretation and significance of these findings is discussed, with particular reference to the pathogenesis of otosclerosis.

A child with acute infantile lymphoblastic leukaemia in remission after cytostatic therapy contracted apparently uncomplicated measles. 8 weeks later, an acute neurological syndrome developed, which led to death within 2 weeks. At autopsy, a peculiar type of inclusion body "encephalitis" almost without signs of inflammatory infiltration was found. Electron microscopic examination showed changes of paramyxovirus infection, which were attributable to slow measles virus disease of the CNS. There was evidence of disturbed nucleocapside synthesis, which is considered to be one of the basic defects in the development of slow-virus processes and their altered viral maturation. Possible pathogenetic factors are discussed in the light of our case.

U cells (a permanent, human amnion cell line) were protected against infection with Semliki Forest Virus (SFV) by human interferon (HIF) from peripheral leukocytes. Despite the usual genus-specific action of interferons, mouse L929 cultures (a permanent mouse fibroblast cell line) were also protected by HIF. The antiviral action of HIF in six other primate cell cultures was also examined. It is of interest that two lymphoblastoid cell lines, RPMI1788 and Kaplan, were insensitive to HIF. The sensitivity of ten different viruses against HIF in primary African green monkey kidney cell cultures was compared. Among the viruses tested SFV was the most sensitive whereas two strains of vaccinia virus were barely inhibited. In contrast, type 1 herpes simplex virus was relatively sensitive to the action of HIF.

Measles antibody determinations in kindergarten and primary school children in Basle-City showed that today just under 10% of 10-year-olds and roughly 25% of 5-year-olds have no immunity to measles. In the 10-year-old primary school children the effects of measles immunization are not yet markedly apparent, whereas almost half the kindergarten children already owe their measles immunity to immunization. No measles antibodies could be detected in 13,7% of the subjects who, according to their official vaccination certificates, had previously been immunized against measles. Most of these failures are probably due to careless routine immunization techniques.

The serological diagnostics of suspicions of measles is of ever growing importance with adequately decreasing morbidity. The utilization of the complement binding reaction is compared with results of the haemagglutination test, the IgG enzyme immunoassay, the IgG and IgM immunofluorescence technique and the haemadsorption immunosorbent test for IgM prove of measles. The virus-specific methods of evidence prove to be favourable ones. The present possibilities of the decentralized routine diagnostics in district laboratories make the complement binding reaction a reliable method.

In our opinion there is no specific germ for multiple sclerosis. The pathogenic process is developed after a clinically "silent" period and results in a demyelinization process. Partially genetical aspects have to take into account for certain disease processes. Up to now both, the theory of infection as well as the neuroallergetic theory are of importance.

Neutralizing antibodies against measles, poliomyelitis, rubella, herpes simplex, influenza and vaccinia viruses were measured in batches of a representative selection of human immunoglobulins for intravenous use licensed in the Federal Republic of Germany. Rubella antibodies were also examined by haemagglutination-inhibition tests. Where available, reference preparations were used, and antibody levels expressed in I. U. All preparations contained significant amounts of the examined antibodies, with relatively low variation from one batch to the other. In most cases activity data specified by the manufacturers could be confirmed. However, differences between the various preparations were sometimes important, some of them relating mainly to individual antibodies, others more or less to the whole range. Some striking findings are emphasized with consideration of manufacturing and control problems.

A study was undertaken to assess the viral antibody spectrum in human immunoglobulin preparations from four different manufacturers for intravenous and/or intramuscular application. Studies of the kind seem to be reasonable for calculating the expected therapeutic effect of the administered immunoglobulin. The investigated 16 gammaglobulin batches showed almost the same variety of viral antibodies and only slightly differring antibody titers. It is interesting to note, that antibodies to vaccinia virus could not be demonstrated. Quantitative determination of viral antibody titers in immunoglobulin preparations for standardization purposes should include the testing of antibodies to viruses against which a therapy with gammaglobulins is reasonable.

A newly developed ion exchange chromatography is reported; it is a simple and quick method to separate a highly purified IgM fraction from blood serum. The immunofluorescence test was used to demonstrate that these fractions contain only virus-specific IgM antibodies, but no demonstrable IgG antibodies, which can often block the specific IgM reactivity by competitive inhibition. The specificity of the reaction was tested on sera from 145 persons: the IgM test was always positive in clinically proven viral infections; the reaction was negative in all patients whose infection had occurred some time in the past. A series of suspected virus infections could be verified by the demonstration of specific IgM antibodies.

Serum concentrations of immunoglobulins A, M and G, of alpha 2-macroglobulin, complement component C 3 and of specific antibodies against streptolysin O, staphylolysin, influenza/parainfluenzavirus and measles virus were measured from 323 German and 167 alien pregnant women. Except from a statistically significant increase of IgG and alpha 2-macroglobulin in the alien group, no measurable differences could be seen. It is concluded that the evaluation of all measured parameters can be equal for both populations.

We tested the effect of lysozyme on the nasal mucosa in a controlled double-blind in vivo study. The administration of non-pathogenic vaccine strains of living measles virus and of living Vivotif bacteria (attenuated Salmonella typhi) to 14 healthy subjects showed that lysozyme possesses a clear antimicrobial and antiviral effect.