Official batch control of influenza vaccines: Is it still useful?

INTRODUCTION: The governmental quality control of human vaccines is a long established tradition in many European countries. In Germany, vaccines have been controlled by a governmental agency since 1935. In the beginning, vaccine production and control was a purely national activity. However, that changed fundamentally in 1994 when the so-called Official Control Authority Batch Release Network (OCABR) was implemented shortly after the establishment of the European Union. Today, Official Medicinal Control Laboratories (OMCLs) are part of the European OCABR Network. In many European countries, OMCLs experimentally test every batch of human vaccines before they enter the market. We wanted to gain insights into the benefits of batch release by the Network and address the question whether batch release is still useful. This question was investigated in the context of influenza vaccines. METHODS: Notifications on influenza vaccines circulated from 2006 to 2016 within the OCABR network were compiled and organized into 32 cases. The impact of these findings was evaluated, and the communication pathways between companies and respective European control laboratories were examined. RESULTS: Approximately 5850 batches were tested by the OMCL network between 2006 and 2016. Among these, notifications belonging to 32 cases were observed. The predominant proportion of the circulated notifications related to manufacturing issues. In most cases, the manufacturer itself had withdrawn the batches before they entered the market. However, in three cases, batches of insufficient quality were detected by the respective European Control Laboratory leading to withdrawal of 13 batches. CONCLUSION: 13 batches which did not meet the specifications of influenza vaccine were detected by the OMCL network between 2006 and 2016 which would not have been identified by the manufacturer. This demonstrates the impact of governmental batch release. Together with the intrinsic values of the OCABR system and keeping in mind that vaccines are given to healthy often young individuals, governmental batch release of influenza vaccines is still justified.

[Falsified medicines in parallel trade]. / Arzneimittelfälschungen im Parallelhandel.

The number of falsified medicines on the German market has distinctly increased over the past few years. In particular, stolen pharmaceutical products, a form of falsified medicines, have increasingly been introduced into the legal supply chain via parallel trading. The reasons why parallel trading serves as a gateway for falsified medicines are most likely the complex supply chains and routes of transport. It is hardly possible for national authorities to trace the history of a medicinal product that was bought and sold by several intermediaries in different EU member states. In addition, the heterogeneous outward appearance of imported and relabelled pharmaceutical products facilitates the introduction of illegal products onto the market. Official batch release at the Paul-Ehrlich-Institut offers the possibility of checking some aspects that might provide an indication of a falsified medicine. In some circumstances, this may allow the identification of falsified medicines before they come onto the German market. However, this control is only possible for biomedicinal products that have not received a waiver regarding official batch release. For improved control of parallel trade, better networking among the EU member states would be beneficial. European-wide regulations, e. g., for disclosure of the complete supply chain, would help to minimise the risks of parallel trading and hinder the marketing of falsified medicines.

Interaction of human immunodeficiency virus type 1 Vif with APOBEC3G is not dependent on serine/threonine phosphorylation status.

The human immunodeficiency virus type 1 accessory protein Vif is important for viral infectivity because it counteracts the antiviral protein APOBEC3G (A3G). ³²P metabolic labelling of stimulated cells revealed in vivo phosphorylation of the control protein, whereas no serine/threonine phosphorylation was detected for Vif or the A3G protein. These data were confirmed by in vitro kinase assays using active recombinant kinase. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 efficiently phosphorylated its target ELK, but failed to phosphorylate Vif. Putative serine/threonine phosphorylation point mutations in Vif (T96, S144, S165, T188) using single-round infection assays demonstrated that these mutations did not alter Vif activity, with the exception of Vif.T96E. Interestingly, T96E and not T96A was functionally impaired, indicating that this residue is critical for Vif-A3G physical interaction and activity. Our data suggest that Vif and A3G are not serine/threonine phosphorylated in human cells and phosphorylation is not linked to their functional activities.

Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease.

BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene.

APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.

APOBEC3 proteins inhibit human LINE-1 retrotransposition.

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

An inducible T7 RNA polymerase-dependent plasmid system.

RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.

IL-2 induction by simian immunodeficiency virus involves MAP kinase signaling but is independent of calcineurin/NF-AT activity.

The major T cell growth factor interleukin-2 (IL-2) is secreted by activated T cells in response to antigenic stimulation. This requires signal transduction via the CD3/TCR complex and the CD28 coreceptor, leading to activation of mitogen-activated protein kinase (MAPK) and calcineurin/NF-AT signaling pathways. We observed that simian immunodeficiency virus derived from African green monkeys (SIVagm3) is a potent activator of IL-2 gene expression. IL-2 promoter studies in A3.01 T cells demonstrated that SIVagm3 induced an up to 38-fold increased transcriptional activation of the IL-2 promoter. Inhibition of MAPK signaling pathways using inhibitors of MEK, JNK or p38 abolished SIVagm3-induced IL-2 activation in a dose-dependent manner. In contrast, the immunosuppressive drug cyclosporin A (CyA), a classical IL-2 inhibitor that blocks calcineurin activity, had no effect. Consistent with this finding, the nuclear factor of activated T cells (NF-AT), which is activated by calcineurin, was not induced by SIVagm3. Analyzing further transcription factor binding sites located on the IL-2 promoter we found that SIVagm3 did mainly promote transcriptional activation of the CD28/AP-1 and NF-kappaB responsive elements. These DNA elements were also induced by the viral transactivator protein (Tat) and expression of Tat was sufficient to activate IL-2 induction in stimulated cells. Our results show that SIVagm3 is capable of stimulating IL-2 gene expression via molecular mechanisms different from those induced during classical T cell activation.

The antiretroviral activity of APOBEC3 is inhibited by the foamy virus accessory Bet protein.

Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet-deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counter-acting proteins.