Ch. Kunz

Safety and immunogenicity of an inactivated hepatitis A candidate vaccine in healthy adult volunteers

The reactogenicity and immunogenicity of a formaldehyde-inactivated hepatitis A vaccine have been investigated. Three different dose levels of vaccine (180, 360 and 720 ELISA units) were administered to healthy volunteers according to a 0, 1, 2 and 12 month schedule. The vaccine was safe and well tolerated. Reactions observed following vaccination were essentially mild and were not dependent upon the quantity of antigen administered. All subjects had measurable titres of anti-HAV antibodies after the full vaccination course; the immune response to the vaccine was dose-related. Antibody titres in vaccinees at month 13 were between 60- and 190-fold higher than those observed in a group of subjects given anti-HAV immunoglobulin.

Simultaneous vaccination against hepatitis A and B: results of a controlled study.

Hepatitis A and hepatitis B are endemic in many countries and must be considered as serious health risks for large parts of the world population. Simultaneous or combined vaccination against these two diseases would therefore be most advantageous. In order to investigate possible interactions between these vaccines with respect to their tolerability and immunogenicity, we conducted a randomized prospective study comparing single and simultaneous administration of the two vaccines. Three groups of healthy volunteers, each with 55 persons, were included in the study. All were negative for hepatitis A and hepatitis B markers and had normal serum liver enzyme values. Group I received hepatitis A vaccine (720 ELISA units) into the left deltoid muscle, group II received hepatitis B vaccine (20 micrograms) into the right deltoid muscle and group III received hepatitis A vaccine into the left, and hepatitis B vaccine into the right deltoid muscle. Three doses of the vaccines were administered at 0, 1 and 6 months. Local and systemic reactions were monitored by means of questionnaires. Blood samples for determination of antibody to hepatitis A virus (anti-HAV) and antibody to hepatitis B surface antigen (anti-HBs) and of serum SGOT and SGPT levels were drawn at months 0, 1, 2, 6 and 7. There were no serious general and only mild local reactions. The mean serum SGOT and SGPT values remained in the normal range in all groups. The seroconversion rates and mean geometric titres of the anti-HAV and anti-HBs antibodies were similar when the vaccines were administered separately or simultaneously. There were no significant differences between the compared groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of Polymeric Glycoprotein Complexes from a Flavivirus: Tick-borne Encephalitis Virus

Treatment of tick-borne encephalitis (TBE) virus with Triton X-100 (TX-100), octylglucoside (OG) or cetyltrimethylammonium bromide (CTAB) caused dissociation of the virus envelope into dimers or monomers of the glycoprotein V3. By centrifugation into detergent-free sucrose density gradients, these subunits were found to reassociate and to form haemagglutinating homogeneous glycoprotein complexes sedimenting at 15 to 16, 16 to 18 and 11 to 23S after TX-100, OG and CTAB treatment, respectively. Glycoprotein complexes obtained after TX-100 solubilization contained less than 1% lipid and detergent by weight.

Detectability of IgM antibodies against TBE virus after natural infection and after vaccination

SummaryIgM antibodies againsttick-borne encephalitis (TBE)virus were investigated by means of a three-layer ELISA (antigen bound to the solid phase) and a four-layer ELISA (anti-μ-serum bound to the solid phase) after natural infection as well as after vaccination. In general, the four-layer ELISA detected IgM antibodies more often and for a longer period of time than the three-layer test. In some patients, IgM antibodies were detected for as long as eight months after the disease with the four-layer test but for only six months with the three-layer test. In addition, after the second TBE vaccination, IgM antibodies were found for as long as eight months in 24 sera which were taken within eight months after the second vaccination. Five were positive in the three-layer ELISA and 16 in the four-layer test. IgM antibodies were never detected in specimens taken later than ten months after the second and third vaccination. The results are of diagnostic importance in infections of the CNS which are not caused by TBE virus and which have been preceded by a possibly silent TBE virus infection or by a TBE vaccination.ZusammenfassungIgM-Antikörper gegen dasFrühsommermeningoenzephalitis (FSME)-Virus wurden mittels Drei-Schichten-ELISA (Antigen an die feste Phase gebunden) und Vier-Schichten-ELISA (Anti-μ-Serum an die feste Phase gebunden) nach überstandener Erkrankung sowie nach Impfung untersucht. Generell wurden mit dem Vier-Schichten-ELISA IgM-Antikörper häufiger und für längere Zeit gefunden als mit dem Drei-Schichten-ELISA. Mit ersterem Test wurden bei einigen Patienten IgM-Antikörper bis zu acht, mit letzterem nur bis zu sechs Monaten nach der Erkrankung nachgewiesen. Auch nach zwei FSME-Impfungen wurden IgM-Antikörper bis zu acht Monaten nachgewiesen. Von 24 Sera, die innerhalb von acht Monaten nach der zweiten Impfung abgenommen worden waren, waren fünf im Drei-Schichten-ELISA, aber 16 im Vier-Schichten-ELISA positiv. In Proben, die später als zehn Monate nach der zweiten Impfung abgenommen worden waren, sowie nach der dritten FSME-Impfung konnten keine IgM-Antikörper gefunden werden. Die Befunde haben große Bedeutung bei nicht durch das FSME-Virus hervorgerufenen Infektionen des ZNS, denen eine eventuell stumme FSME-Infektion oder auch Impfung vorangegangen ist.

Experimentelle Untersuchungen über die Aufnahme von Eiweiss, Viren und Bakterien während der Embryogenese des Kaninchens☆

Zusammenfassung 1. 1. Beim Kaninchen tritt 7d12h post coitum Eiweiss in die Fruchtblasenhohle ein und ist wenige Stunden spater hier in gleicher Konzentration wie im Serum der Mutter vorhanden. Dieses Eiweiss stimmt qualitativ mit dem des mutterlichen Blutplasmas uberein. 2. 2. Auch Antikorper, die im mutterlichen Blut zirkulieren, stromen mit ein. 3. 3. Viren, die der Mutter intravenos oder intrauterin appliziert werden, konnen zwar schon 6d16h post coitum in die Fruchtblasenhohle ubertreten, aber nicht fruher. Von diesem Zeitpunkt ab sind die Blastozysten nicht mehr unzerstort ausspulbar. 4. 4. Bakterien verhalten sich wie Viren, allerdings mussen sie intrauterin appliziert werden. 5. 5. Es mussen also Veranderungen der Hullmembran (Zona pellucida und Mukoproteidschicht), die wir als Diskontinuitaten bezeichnen, eingetreten sein. 6. 6. Auf die Moglichkeit, dass durch das Eindringen von Fremdstoffen die Embryonen geschadigt werden konnen, wird hingewiesen. Weitere Untersuchungen sind im Gange. 7. 7. Die Frage, ob durch den Eiweissubergang zur Zeit der Nidation evtl. eine immunologische Toleranz ausgelost werden kann, wird angeschnitten.

Protease treatment and chemical crosslinking of a Flavivirus: Tick borne encephalitis virus

SummaryTick-borne encephalitis virus was treated with pronase or thermolysin. The resulting particles were banded in sucrose gradients and analyzed for polypeptide composition. Both enzymes caused a reduction in particle density from 1.19 to 1.15–1.16 g/cm3. No loss of viral lipid or nucleic acid could be observed. SDS-polyacrylamidegel electrophoresis showed that only the core protein V2 was unchanged whereas the envelope proteins V3 and V1 had disappeared from their original positions in the PAGE profile. Instead a new peptide(s) with molecular weight of 4000–6000 was found in which hydrophobic amino-acids were enriched.Crosslinking by dimethyl-3.3′-dithiobispropionimidate (DTBP) made the virus resistent to solubilization of the envelope proteins by TX-100. This could be interpreted by the formation of a dense envelope protein network around the nucleocapsid preventing its liberation by TX-100. Some data however indicate that direct crosslinking of at least one of the envelope proteins with the core cannot be excluded.

[A new influenza subunit vaccine: reactogenicity and antigenicity in comparison to split and whole virus vaccines (author's transl)].

ZusammenfassungEine neue, praktisch nur Hämagglutinin und Neuraminidase enthaltende Influenza-Subunit-Vakzine wurde im Hinblick auf Verträglichkeit und Immunogenität am Menschen untersucht. Zum Vergleich wurden ein im Handel befindlicher adjuvanshältiger Tween-Äther Spaltimpfstoff (Dosierung 800 IE) und eine Ganzvirusvakzine ohne Adjuvans (Dosierung 2100 IE) herangezogen. Zwei Dosierungen (700 und 2100 IE) der Subunit-Vakzine ohne Adjuvans wurden verglichen. Alle Impfstoffe enthielten die von der WHO für die Saison 1975/76 empfohlenen Virusstämme.Die Impfung wurde als Doppelblindstudie durchgeführt, wobei die 399 Probanden den vier Vakzinen randomisiert zugeordnet wurden. Die Probanden wurden 24 und 48 Stunden nach der Impfung im Hinblick auf Nebenwirkungen vom Impfarzt untersucht. Die hämagglutinationshemmenden Antikörper wurden vor und vier Wochen nach der Impfung bestimmt. Der niedrig dosierte Subunit-Impfstoff führte zu signifikant weniger lokalen Nebenreaktionen als die vergleichbar dosierte Spaltvakzine. Die Antikörperbildung war aber hinsichtlich der antikörperstimulierenden Wirkung gegen die in der Vakzine enthaltenen A-Stämme deutlich überlegen. Beim Vergleich der beiden hoch dosierten Vakzinen fiel besonders die gute Verträglichkeit der Subunit-Vakzine auf. Die Impfung mit der Ganzvirusvakzine hatte bei mehr als 15% der Probanden systemische Nebenreaktionen, wie Fieber und Kopfschmerz, zur Folge. In der Induktion von Antikörpern weist die Subunit-Vakzine leichte aber nicht signifikante Vorteile auf. Die Erhöhung auf das Zwei- bis Dreifache der derzeit in Europa üblichen Antigendosierung in der Subunit-Vakzine führt zu einer hoch signifikanten Wirksamkeitssteigerung.SummaryThe reactogenicity and immunogenicity of a new influenza subunit vaccine essentially containing only haemagglutinin and neuraminidase was studied in man. The vaccine was compared to commercially available vaccines, an adjuvant containing tween-ether split vaccine (800 IU per dose), and a fluid whole-virus vaccine (2100 IU per dose). Two dosages (700 and 2100 IU) of the fluid subunit vaccine were compared. All vaccines contained the virus strains recommended by the WHO for the 1975/76 season.In a double-blind study 399 volunteers were randomly selected to receive one of the four vaccines. The volunteers were examined for side-effects 24 and 48 hr after vaccination. Antibodies inhibiting haemagglutination were determined prior to and four weeks after vaccination. The subunit vaccine at 700 IU per dose caused significantly fewer local side effects than the comparable split vaccine, and resulted in significantly higher antibody titers against both influenza A strains. A comparison of the subunit and whole virus vaccines containing high dosages (2100 IU) showed striking differences in reactogenicity. Subunit vaccine was very well tolerated, whereas whole virus vaccine caused systemic reactions, including fever and headache, in 15% of the volunteers. No significant reactogenicity was seen with a high dosage of subunit vaccine (2100 IU) although this is a three-fold increase on the currently used European dosage. Antibody titers were significantly enhanced however.The reactogenicity and immunogenicity of a new influenza subunit vaccine essentially containing only haemagglutinin and neuraminidase was studied in man. The vaccine was compared to commercially available vaccines, an adjuvant containing tween-ether split vaccine (800 IU per dose), and a fluid whole-virus vaccine (2100 IU per dose). Two dosages (700 and 2100 IU) of the fluid subunit vaccine were compared. All vaccines contained the virus strains recommended by the WHO for the 1975/76 season. In a double-blind study 399 volunteers were randomly selected to receive one of the four vaccines. The volunteers were examined for side-effects 24 and 48 hr after vaccination. Antibodies inhibiting haemagglutination were determined prior to and four weeks after vaccination. The sudunit vaccine at 700 IU per dose caused significantly fewer local side effects than the comparable split vaccine, and resulted in significantly higher antibody titers against both influenza A strains. A comparison of the subunit and whole virus vaccines containing high dosages (2100 IU) showed striking differences in reactogenicity. Subunit vaccine was very well tolerated. whereas whole virus vaccine caused systemic reactions, including fever and headache, in 15% of the volunteers. No significant reactogenicity was seen with a high dosage of subunit vaccine (2100 IU) although this is a three-fold increase on the currently used European dosage. Antibody titers were significantly enhanced however.

Die elektronenmikroskopische Darstellung des Virus der Frühsommer-Meningoencephalitis (FSME) in HeLa-Zellen

1. Das Virus der Frühsommer-Meningoencephalomyelitis wird erstmalig in HeLa-Zellen dargestellt. Es ist 25 mμ, groß, rund, und besitzt einen dichten Innenkörper sowie eine helle von einer Membran begrenzte Außenzone. 2. Die Infektion mit dem Virus führt in HeLa-Zellen zu einer Hyperplasie und Hypertrophie der Strukturen des Golgifeldes und besonders zum Auftreten bis zu 80 mμ großer, dicht gelagerter Bläschen. Daneben finden sich in wechselnder Menge intraplasmatische Fibrillen, die als unspezifische Differenzierungen gedeutet werden. Ort und Ausdehnung des veränderten Golgifeldes entsprechen den fluoreszenz-serologisch nachweisbaren Antigenzonen. 3. Viruselementarkörperchen sind ausschließlich im Bereich der paranucleären Verdichtungszonen nachweisbar. Die Anzahl der Viruspartikel bleibt auch bei schwersten cytopathologischen Erscheinungen auffallend niedrig. 4. Größere Säcke und Schläuche mit granulärem Inhalt im Bereich des Golgifeldes werden als Bildungsort der Virusinnenkörper diskutiert. Das Virus der Frühsommer-Meningoencephalomyelitis wird erstmalig in HeLa-Zellen dargestellt. Es ist 25 mμ, groß, rund, und besitzt einen dichten Innenkörper sowie eine helle von einer Membran begrenzte Außenzone. Die Infektion mit dem Virus führt in HeLa-Zellen zu einer Hyperplasie und Hypertrophie der Strukturen des Golgifeldes und besonders zum Auftreten bis zu 80 mμ großer, dicht gelagerter Bläschen. Daneben finden sich in wechselnder Menge intraplasmatische Fibrillen, die als unspezifische Differenzierungen gedeutet werden. Ort und Ausdehnung des veränderten Golgifeldes entsprechen den fluoreszenz-serologisch nachweisbaren Antigenzonen. Viruselementarkörperchen sind ausschließlich im Bereich der paranucleären Verdichtungszonen nachweisbar. Die Anzahl der Viruspartikel bleibt auch bei schwersten cytopathologischen Erscheinungen auffallend niedrig. Größere Säcke und Schläuche mit granulärem Inhalt im Bereich des Golgifeldes werden als Bildungsort der Virusinnenkörper diskutiert. 1. The virus of spring-summer meningoencephalomyelitis has been demonstrated in HeLa cells for the first time. It is 25 mμ in size, round-shaped with a dense inner-body and a transparent outer zone surrounded by a surface membrane. 2. The infection of HeLa cells induces hyperplasia and hypertrophy of the Golgi structures and especially the appearance of tightly packed vesicles, up to 80 mμ in size. Besides them a varying number of intraplasmatic fibrils are observed which are regarded, however, as nonspecific structures. The location and extension of the modified Golgi apparatus correspond to antigen zones demonstrable by means of fluorescent antibodies. 3. Virus elementary bodies are found only in the condensed paranuclear areas. The number of virus particles is remarkably low, even in cells which show the most severe cytopathic changes. 4. Larger bags and tubules with granular contents in the region of the Golgi apparatus are discussed as possible sites of formation of the virus inner-bodies. The virus of spring-summer meningoencephalomyelitis has been demonstrated in HeLa cells for the first time. It is 25 mμ in size, round-shaped with a dense inner-body and a transparent outer zone surrounded by a surface membrane. The infection of HeLa cells induces hyperplasia and hypertrophy of the Golgi structures and especially the appearance of tightly packed vesicles, up to 80 mμ in size. Besides them a varying number of intraplasmatic fibrils are observed which are regarded, however, as nonspecific structures. The location and extension of the modified Golgi apparatus correspond to antigen zones demonstrable by means of fluorescent antibodies. Virus elementary bodies are found only in the condensed paranuclear areas. The number of virus particles is remarkably low, even in cells which show the most severe cytopathic changes. Larger bags and tubules with granular contents in the region of the Golgi apparatus are discussed as possible sites of formation of the virus inner-bodies.

Potential and pitfalls in the use of immunoassays for the quantification of live and inactivated virus using tick-borne encephalitis virus as a model.

Competitive radioimmunoassay, four layer enzyme immunoassay and quantitative evaluation of virus peaks in density gradients were applied for the quantification of live or formalin-inactivated Tick-borne encephalitis (TBE) virus. The outcome of immunoassays was extremely dependent on the physical form of the analyzed antigen. Disruption of the virus by detergents resulted in a strongly increased antigenicity, i.e. the same amount of protein present in complete virus yielded only about one-tenth the value obtained after solubilization. This effect could not be demonstrated with formalin-inactivated virus and was shown to be due to the formation of stable covalent crosslinks between structural proteins by formalydehyde thus preventing their solubilization by detergents. These results most likely are relevant also for in vitro quantification of other viral antigens and the important implications with respect to the application of such assays for the standardization of antigen-determinations in inactivated vaccines are discussed. The evaluation of the characteristic peak formed by TBE virus upon density gradient centrifugation is an accurate non-immunological means for the quantification of physically intact TBE virions.

Ein mauspathogener Stamm des „Marburg-Virus“ (Rhabdovirus simiae)

Der für weiße Säuglingsmäuse infektiöe, jedoch nicht pathogene Stamm „F“ des „Marburg-Virus“ (Rhabdovirus simiae) erwarb durch 3 Passagen in Goldhamstern mauspathogene Eigenschaften und führte in neugeborenen Mäusen zu einer tödlichen Enzephalitis. Durch mehrere Mauspassagen konnte die Virulenz sowie die Virusanreicherung im Gehirn gesteigert werden. In der 6. Säuglingsmauspassage war das Virus während der ersten 4 Tagep.i. nur in geringen Mengen nachweisbar; der Virusgehalt stieg aber bis zum 7. Tagp.i. langsam an, erreichte maximal 104,0 LD50 und blieb dann bis zum Tode etwa auf diesem Niveau. Adulte Mäuse erwiesen sich als resistent. Das Virus blieb, wie vor den Hamsterpassagen, weiterhin pathogen für Meerchweinchen und verursachte einen zytopathischen Effekt in humanen embryonalen Lungenfibroblasten (ELF-Zellstamm). Mit der Immunfluoreszenzmethode konnte der neuro-adaptierte Stamm „F“ in Gehirnabtupfen von Mäusen als „Marburg-Virus“ identifiziert werden. Außerdem reagierte ein aus infiziertem Mausgehirn hergestelltes Antigen in der KBR spezifisch mit Meerschweinchenimmun- und Patientensera. After three passages in baby hamsters intracerebral inoculation of Marburg virus (Rhabdovirus simiae) strain F into newborn mice resulted in the occurrence of a fatal terminating encephalitis. During further succesive passages in suckling mice virulence of the pathogenic agent increased and from brains of infected animals maximum amounts of virus (104.5 LD50 and 105.5 LD50 in passage 6 and 8, respectively) were recovered 7 days post inoculation. Adult mice, however, proved to be insusceptible. In both immunofluorescent studies and complement fixation tests the mouse pathogenic virus and Marburg virus showed identical characteristics. Moreover, pathogenicity of strain F for guinea pigs as well as cytopathogenicity of the virus for human embryonic lung fibroblasts (strain ELF) apparently were not changed during passage in hamsters and mice.