[Vaccine safety]. / Sicherheit von Impfstoffen.

Vaccinations rank among the most effective preventive measures for protection against infectious diseases. Advances in development, production, and control of vaccines facilitate the increasing standards of vaccine safety and tolerance. Comprehensive pre-clinical and clinical tests as well as modern manufacturing and testing methods ensure that vaccines marketed nowadays are safe. As a rule, clinical trials performed before granting the marketing authorisation identify the most frequent adverse events and these results are used to evaluate the safety of the product. Such trials can identify relatively rare adverse events, which occur with a frequency of 1:1,000 to 1:10,000 of all vaccinated individuals. These adverse events will then be included in the summary of product characteristics (SPC) for the vaccine. Even after comprehensive clinical trials of vaccines, it is possible that very rare adverse events may be observed for the first time during general use of a vaccine. In recent years concern over real and alleged risks of vaccines relative to their benefit has grown in many countries including Germany. One reason for this is the fact that most infections that were previously feared have now faded from memory. This situation can be ascribed in part to the success of vaccination. In recent years an increased awareness of substantiated and assumed risks following immunization has been reported in Germany as well as many other countries. In part this may be due to the absence of infectious disease-related mortality and morbidity and to the fact that the severity of vaccine-preventable diseases is no longer observable. Consequently, rare and hypothetical adverse events attain undue public attention. As vaccination willingness diminishes, a resulting lower vaccination rate renders the population susceptible to the natural wild type infection with concomitant increases in mortality and morbidity of vaccine-preventable diseases. Thus, very rare or even unproven adverse events have attracted public attention. Declining vaccination rates resulting from these fears may result in a renewed increase of vaccine-preventable diseases. Adverse events following immunization (AEFI) need to be recognized and adequately assessed. This review presents the scientific knowledge concerning causality and frequency of several AEFI and hypothetical risks.

Analysis of histone gene expression in adult tissues of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus: tissue-specific expression of sperm histone genes.

We analyzed the histone mRNA population found in several adult tissues of the sea urchin Strongylocentrotus purpuratus and in testis of Lytechinus pictus. Unique species of H1 and H2b mRNAs encoding the sperm-specific histone subtypes can be found exclusively in testis RNA. S. purpuratus contains two distinct testis-specific H1 transcripts, while L. pictus contains one such transcript. Each of these mRNAs is larger than either early or late embryonic H1 mRNAs. Other somatic adult tissues contain transcripts derived from members of the late embryonic H1 histone gene family. S. purpuratus contains one H2b transcript found exclusively in testis, while L. pictus contains two such H2b mRNAs. Similarly, in tissues other than testis, late H2b transcripts were found. While there is no sperm-specific H2a protein, a limited set of late histone H2a genes encoding primarily the H2a-beta subtype is expressed in testis. The majority of the H2a protein found in diploid adult tissues is also the H2a-beta subtype; however, the size of the H2a transcripts differs between testis and other tissues. We conclude that different members of the late H2a gene family are differentially expressed in embryos and adult tissues. We prepared and characterized cDNA clones encoding the sperm-specific H2b protein as well as the H2a-beta protein found in testis.

Stably transmitted triple-promoter retroviral vectors and their use in transformation of primary mammalian cells.

Retroviral vectors were constructed which coexpressed three inserted genes from independent transcriptional promoters in singly infected cells. Several such triple-promoter vectors were constructed with various combinations of oncogenes and selectable drug resistance genes. All expressed three mRNAs of the expected size in infected cells. One vector expressing the v-Ha-ras, v-myc, and neo genes was characterized in detail. This retrovirus did not undergo rearrangement during the process of infection, as judged by Southern analysis, and infection of primary rat embryo fibroblasts demonstrated that ras-myc-cotransformed cells could be selected in G418. This demonstration that retroviral vectors can be used to express three cistrons independently increases their value as gene transfer vehicles, particularly for studies involving oncogene cooperation in primary cells.

Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src.

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.

Interleukin-7 retroviruses transform pre-B cells by an autocrine mechanism not evident in Abelson murine.

In this study, we have constructed retroviral vectors expressing the interleukin-7 (IL-7) cDNA and have used infection with these retroviruses to express this cytokine endogenously in an IL-7-dependent pre-B-cell line. Infection with IL-7 retroviruses, but not with a control retrovirus, resulted in the conversion of the cells to IL-7 independence. The frequency at which this occurred, together with data on vector expression levels, indicated that secondary events were required for factor independence in this system. Southern analysis showed that the IL-7-dependent clones harbored unrearranged copies of the vector proviruses. The factor-independent cells produced variable quantities of IL-7 as measured by an IL-7-specific bioassay, and their proliferation could be substantially inhibited by a neutralizing antibody directed against IL-7, indicating that a classical autocrine-mechanism was responsible for their transformation. These IL-7-independent cells were tumorigenic, in contrast to the parental IL-7-dependent cells or those infected with a control vector. These results showed that IL-7 could participate in the malignant transformation of pre-B cells. However, neither of two Abelson murine leukemia virus (A-MuLV)-transformed pre-B-cell lines expressed detectable IL-7 mRNA, at a level of sensitivity corresponding to less than one molecule of mRNA per cell. Moreover, the proliferation of the A-MuLV transformants was unaffected by addition of the IL-7 antisera under conditions in which parallel experiments with IL-7 virus-infected cells resulted in greater than 70% growth inhibition. Thus, transformation of pre-B cells by A-MuLV was not associated with a demonstrable autocrine loop of IL-7 synthesis. These results show that IL-7 can participate in the malignant transformation of pre-B cells and suggest studies aimed at assessing the role of autocrine production of IL-7 in the generation of human leukemias and lymphomas.

An in vitro assay for detection of tetanus neurotoxin activity: using antibodies for recognizing the proteolytically generated cleavage product.

Tetanus neurotoxin (TeNT(1)) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.

Stage-specific transformation of murine B lineage cells by ras and myc.

The retroviral vector delta RM coexpresses the v-Ha-ras and v-mycMC29 oncogenes, under the transcriptional control of the retroviral long terminal repeat and an internal SV40 promoter respectively. In this report, the transforming activity of the delta RM virus on murine pre-B cells has been compared and contrasted with its activity on mature splenic B cells. Infection of primary bone marrow cells, followed by growth in the Whitlock-Witte culture system, resulted in the rapid outgrowth of transformed pre-B cells. These cells grew to high saturation densities and could give rise to immortal, interleukin-7-independent progeny that were able to grow independently of stromal elements. In contrast, infection of mature B cells purified from murine spleen resulted in only a transient increase in proliferation, and no immortal B cell lines were obtained. This inability of delta RM to transform mature B lymphocytes was not due to a low infection frequency, since parallel experiments with ecotropic retroviruses conferring drug resistance showed that the mature B cells were readily infectable. Moreover, Northern analysis showed that the delta RM-infected mature B cells expressed ras and myc mRNAs to higher levels than the delta RM transformed pre-B cells. Thus, coexpression of ras and myc resulted in the transformation of primary pre-B cells but not of the mature B cells. The potential explanations for the stage-specific transforming activity of the delta RM retrovirus are discussed.

Sequence comparisons of non-allelic late histone genes and their early stage counterparts. Evidence for gene conversion within the sea urchin late stage gene family.

We have determined the nucleotide sequence of sea urchin (Lytechinus pictus) late stage H3 and H4 histone genes contained on the clone pLpH3H4 -21 and of the early stage H3 gene contained on the plasmid pLpA . Comparison of these differentially regulated histone genes with each other and with other L. pictus late and early stage histone H3 and H4 genes previously sequenced confirms that members of each histone gene family (early and late) are more homologous to each other than they are to members of other histone gene families. The spacer regions between two late H3-H4 gene pairs on the clones pLpH3H4 -19 and pLpH3H4 -21 have diverged to the point where they are no longer homologous. However, comparative analysis of the 5' flanking DNA has identified a sequence 5'C-T-C-A-T-G-T-A-T-T3' upstream of both late H4 genes and another, 5'A-G-A-T-T-C-A3', upstream of both H3 genes. Except for a short conserved sequence near the initiation codon, the transcribed 5' leaders of the late mRNAs differ in length and sequence in the two non-allelic late histone gene pairs. This divergence contrasts with the 95 to 96% conservation found between late histone gene coding sequences. The results suggest that there is intergenic exchange in the germline among members of the late histone gene family and that the unit of exchange is the individual gene rather than the heterotypic dimer which includes the common spacer DNA.

Gene transfer using a novel fusion protein, GAL4/invasin.

The delivery of DNA to target cells using simple, defined, nonviral systems has become an area of intense interest in gene therapy. We describe here the development and characterization of one such novel system. A recombinant, bifunctional, fusion protein was expressed and purified from Escherichia coli. This protein consists of the DNA-binding domain of the yeast transcription factor GAL4 fused to the cell binding, internalization domain of the Yersinia pseudotuberculosis inv gene product, invasin. This protein, GAL4/Inv, together with poly-L-lysine, formed complexes with a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains eight repeats of the GAL4 consensus recognition sequence. These complexes were shown to transfect target cells in an invasin receptor-dependent manner, resulting in transient CAT expression. A simple, targeted DNA delivery vehicle, as we describe here, represents a viable approach to nonviral gene delivery.

Cardiovascular effects, pharmacokinetics, and converting enzyme inhibition of enalapril after morning versus evening administration.

The cardiovascular effects and pharmacokinetics of once-daily enalapril were studied after single-dose and subchronic treatment in eight patients with hypertension by use of ambulatory blood pressure monitoring. Enalapril, 10 mg, was given at either 7 AM or 7 PM in a randomized crossover design. In addition, inhibition of serum converting enzyme was studied. Subchronic treatment at 7 AM significantly reduced blood pressure during the day but was less effective at night. Subchronic dosing at 7 PM significantly further decreased nighttime blood pressure followed by a slow increase during the day, with no effect on elevated afternoon values. Peak concentrations of enalaprilat were found 3.5 hours (morning) and 5.6 hours (evening) after drug intake (p < 0.05), whereas peak effects occurred 7.4 hours (morning) and 12 hours (evening) after drug administration. In conclusion, 24-hour blood pressure profiles in patients with hypertension were significantly influenced by the time of enalapril dosing. Differences in effect profiles could not be attributed to similar changes in pharmacokinetics or to different time courses of angiotensin converting enzyme inhibition.

Efficient gene transfer and expression in primary B lymphocytes.

In this study we have used a panel of vectors expressing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of different regulatory elements to optimize gene transfer and expression in primary B lymphocytes. The Moloney murine leukemia virus long terminal repeat (MoMLV LTR) and the SV40 early region promoters, while functional in transfected plasmacytoma cell lines, did not give rise to detectable CAT activity following transfection into primary activated mouse or human B lymphocytes. In contrast, the human cytomegalovirus immediate-early (HCMV-IE) enhancer/promoter functioned in both established and primary B cells. The highest expression levels in the primary cells were obtained with vectors containing the Adenovirus 2 major late promoter or the HCMV-IE enhancer/promoter in combination with the Adenovirus 2 tripartite leader and VA genes. These latter expression cassettes were placed in a retroviral vector with the aim of combining their capacity for high-level gene expression with the efficient stable gene transfer afforded by retroviral infection. Several retroviral constructs were made, some of which were able to generate high virus titers. However all of these underwent deletions during the process of retroviral infection, as judged by Southern analysis of infected cells, indicating that they were not optimal gene transfer vectors. The HCMV enhancer/promoter, which was the most active of the other expression cassettes tested in the primary B cells, was inserted into a retroviral vector which also expressed the hph gene under the transcriptional control of the retroviral LTR. This vector did not undergo rearrangement during the process of retroviral infection, as judged by Southern analysis. The CAT gene was inserted downstream of the HCMV promoter in this vector, and a high-titer retroviral stock was generated. Primary B lymphocytes infected with this vector gave high levels of CAT activity, under conditions in which parallel experiments with the hph drug resistance marker showed that one in 20 of the cells were infected. These experiments demonstrate efficient gene transfer and expression in primary B lymphocytes in vitro.

The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.

The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.

In vitro determination of specific toxicity in tetanus vaccines.

Tetanus vaccine is prepared from detoxified tetanus neurotoxin. To ensure the absence of residual toxin activity or to exclude the reversion to toxicity reliable control testing is based on in vivo methods, because no in vitro assay provides the required specificity and sensitivity. Tetanus neurotoxin is a 150 kDa protein produced by Clostridium tetani. The 50 kDa light chain of this neurotoxin belongs to the family of zinc metalloproteases. It cleaves synaptobrevin, a small synaptic vesicle protein, which is involved in neuroexocytosis, at the single Q76-F77 peptide bond. To develop a sensitive in vitro assay capable of quantifying the proteolytic activity of this toxin, we used as substrate a recombinant fragment of synaptobrevin2 (1-97). For detecting the cleavage products a peptide antibody raised against the N-terminal cleavage site was used. In Western Blot analysis only the cleaved substrate was detected while the uncleaved substrate showed no signal. In different approaches, recombinant synaptobrevin was either (i) bound to a microtitre plate, reduced toxin was added and the N-terminal cleavage product was detected by a specific antibody or (ii) the cleavage was performed in test tubes, the samples were transferred to a microtitre plate and immobilised cleavage products were detected. When toxoid or crude toxin is used, non-specific cleavage of synaptobrevin substrate occurs. Depending on the toxoid used different patterns of degradation of substrate are visible in Western Blots. Different protease inhibitors and reaction conditions seem to have an effect on the inhibition of this non-specific cleavage.

Measurements of serum ACE activity in vitro after administration of enalapril in man cannot reflect inhibition of the enzyme in vivo.

We previously demonstrated the dependency of serum angiotensin converting enzyme (ACE) inhibition by the inhibitor enalaprilat in vitro on the concentration of substrate and enzyme present in the assay, according to a competitive tight-binding mechanism. In the present study, the relevance of these findings for ex vivo measurements after administration of enalapril has been confirmed in serum samples of four patients which were assayed twice using different substrate concentrations in vitro. The measured extent of ACE inhibition in the samples was markedly different depending on the substrate concentration added in vitro (in relation to its Km value: S/Km, respectively), suggesting lower inhibition over time when higher S/Km was used. When the dilution factor and the added S/Km were taken into account the measured values could successfully be predicted from the respective serum enalaprilat concentrations by means of the concentration-effect model previously evaluated for the in vitro relationship (Emax tight model). Considering conditions which probably better reflect the situation in vivo (no dilution, physiological substrate concentrations far below Km) we simulated a time course of in vivo serum ACE activity in these patients which suggests almost complete inhibition of serum ACE over time in contrast to the in vitro measurement. Thus, we conclude that the usual ex vivo measurements of ACE activity lead to an underestimation of the extent of inhibition because of sample dilution and high exogenous substrate in vitro, and therefore must fail to reflect enzyme inhibition in vivo.

[Adverse drug reactions following immunization in Germany pursuant to the German Infection Protection Act and the German Medicinal Products Act from January 1, 2004 to December 31, 2005]. / Verdachtsfälle von Impfkomplikationen nach dem Infektionsschutzgesetz und Verdachtsfälle von Nebenwirkungen (von Impfstoffen) nach dem Arzneimittelgesetz vom 1. 1. 2004 bis zum 31. 12. 2005.

Sufficient post-marketing surveillance is necessary for safety monitoring of vaccines. In this respect the spontaneous reporting system of reporting suspected adverse drug reactions (ADR) following vaccination is an essential tool for safety monitoring. The marketing authorization holder and/or pharmaceutical manufacturer has the legal obligation to report suspected adverse drug reactions (German Drug Law and European Regulation). In addition physicians and traditional healers have to report suspected cases of complications after immunizations pursuant to the German Infection Protection Act (Infektionsschutzgesetz, IfSG). The reports are medically assessed and stored in a database at the Paul Ehrlich Institute. For the publication referenced here, all reported suspected cases of adverse drug reactions after immunizations were evaluated for the period from January 1, 2004-December 31, 2005 according to different criteria. In 2004 (2005) a total of 1237 (1393) suspected cases of adverse drug reactions or suspected complications after immunizations were notified. 858 (919) of these adverse drug reactions (ADR) were serious (69 % and 66 %, respectively). 414 (517) of the ADRs (i.e. 33 % and 37 %, respectively) were reported by physicians according to the IfSG; the other reports were from industry and other reporting sources. 251 (229) i.e. 61 % (44 %) of these reactions were serious. The total number of reports divided by the total number of vaccine doses launched on the German market during the observation period (according to the data provided by the pharmaceutical industry) revealed an overall "reporting rate" of approx. 3 reports per 100,000 vaccine doses. The age groups with the highest absolute number of reported cases were infants and young children (0-2 years), and adults (18-59 years) accounting for approx. one third each of the reports. The age distribution of the suspected cases was comparable with that of previous years. In both years, approx. half of all suspected adverse drug reactions following immunization were of transient nature, i.e. there was a complete recovery (restitution ad integrum). In both years, a very small proportion of cases were reported as permanent damage (30 and 34 cases respectively; 2.4 % of all cases) or resulted in death (35 or 23 cases, respectively; 2.8 % or 1.7 %, respectively). With a few exceptions these adverse events were considered to be related to other diseases and unlikely related to vaccination. Overall, the association between vaccination and adverse events was assessed by the PEI as "possible" in 58 % (62 %) of all cases, respectively, as "likely" in 6 % (8 %) of all cases, and as "certain" in 0.4 % (0.6 %) of all cases. In 14 % (13 %) of the cases, the causal relation was stated as "unlikely". In 17 % (15 %) of all cases, a scientific evaluation was not possible on the basis of the data provided. A separate analysis of reports was conducted for all suspected adverse drug reactions following varicella immunization. According to these data varicella vaccination is considered to be well tolerated. With the exception of an increase in local reactions following pneumococcal polysaccharide vaccination no new safety signal has been recognised during the observation period.

[Thiomersal and immunisations]. / Thiomersal und Impfungen.

Thiomersal was used in the 1930s for the first time for the preservation of vaccines to prevent bacterial and fungal contamination. Thiomersal is an organic compound containing 49% mercury (Hg) by weight. It is generally well known that mercury and its compounds, including thiomersal, ethylmercury, and methylmercury, act as nephro- and neurotoxicants, however, at much higher doses than used in vaccines. In the 1990s the question of toxicity of thiomersal in vaccines was reassessed since the numbers of vaccines recommended for routine administration to infants and children, and therefore the cumulative thiomersal dose in children, increased in some countries. Various international committees (European Agency for the Evaluation of Medicinal Products, EMEA, US Public Health Service/American Academy of Pediatrics, Institute of Medicine, IOM) concluded after an extensive risk/benefit analysis that scientific evidence is inadequate to reject or explicitly recommend thiomerosal-containing vaccines for children. However (in line with the global goal of reducing exposure to mercury), they recommended promoting the elimination of thiomerosal from paediatric vaccines. This has largely been achieved in Germany. Today a child in Germany can be immunised in accordance with the official recommendations (STIKO) almost without the administration of thiomerosal (residual amounts). Results of new pharmacokinetic and epidemiological studies are discussed. The evidence available to date does not support the hypothesis of a potential relationship between neurodevelopmental disorders and thiomersal-containing vaccines.

Pharmacokinetics and converting enzyme inhibition after morning and evening administration of oral enalapril to healthy subjects.

Possible circadian changes in the pharmacokinetics and effect on serum angiotensin-converting enzyme (ACE) activity of the ACE inhibitor enalapril have been studied in 8 healthy subjects after oral ingestion of 10 mg enalapril maleate either at 08.00 h or 20.00 h. The time to peak serum concentration (tmax) of enalapril was increased after administration at 20.00 h compared to 08.00 h (2.4 h versus 1.3 h), whereas other kinetic parameters were not significantly altered. The 24 h-kinetics of the active metabolite enalaprilat did not differ significantly between the two treatments, but the area under the curve (AUC (0-24] and the peak serum concentration (Cmax) were slightly higher after intake at 20.00 h. The relationship between the measured serum enalaprilat level and the degree of inhibition of serum ACE was the same after both treatments. Overall, the evening and morning administration of enalapril did not differ markedly in the pharmacokinetics and the time course of ACE inhibition.