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Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

Supplementary Figure 6 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Tables 1-4 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Methods and Materials from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure 2 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure 3 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure Legends 1-6 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure 1 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure 5 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Supplementary Figure 4 from Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Background Unavailability of vaccines endangers the overall goal to protect individuals and whole populations against infections. Methods The German notification system includes the publication of vaccine supply shortages reported by marketing authorisation holders (MAH), information on the availability of alternative vaccine products, guidance for physicians providing vaccinations and an unavailability reporting tool to monitor regional distribution issues. Aim This study provides a retrospective analysis of supply issues and measures in the context of European and global vaccine supply constraints. Results between October 2015 and December 2020, the 250 notifications concerned all types of vaccines (54 products). Most shortages were caused by increased demand associated with immigration in Germany in 2015 and 2016, new or extended vaccine recommendations, increased awareness, or changes in global immunisation programmes. Shortages of a duration up to 30 days were mitigated using existing storage capacities. Longer shortages, triggered by high demand on a national level, were mitigated using alternative products and re-allocation; in a few cases, vaccines were imported. However, for long lasting supply shortages associated with increased global demand, often occurring in combination with manufacturing issues, few compensatory mechanisms were available. Nevertheless, only few critical incidents were identified: (i) shortage of hexavalent vaccines endangering neonatal immunisation programmes in 2015;(ii) distribution issues with influenza vaccines in 2018; and (iii) unmet demand for pneumococcal and influenza vaccines during the coronavirus disease (COVID)-19 pandemic. Conclusion Vaccine product shortages in Germany resemble those present in neighbouring EU states and often reflect increased global demand not matched by manufacturing capacities.

Multiple preventive COVID-19 vaccines have been developed during the ongoing SARS coronavirus (CoV) 2 pandemic, utilizing a variety of technology platforms, which have different properties, advantages, and disadvantages. The acceleration in vaccine development required to combat the current pandemic is not at the expense of the necessary regulatory requirements, including robust and comprehensive data collection along with clinical product safety and efficacy evaluation. Due to the previous development of vaccine candidates against the related highly pathogenic coronaviruses SARS-CoV and MERS-CoV, the antigen that elicits immune protection is known: the surface spike protein of SARS-CoV-2 or specific domains encoded in that protein, e.g., the receptor binding domain. From a scientific point of view and in accordance with legal frameworks and regulatory practices, for the approval of a clinic trial, the Paul-Ehrlich-Institut requires preclinical testing of vaccine candidates, including general pharmacology and toxicology as well as immunogenicity. For COVID-19 vaccine candidates, based on existing platform technologies with a sufficiently broad data base, pharmacological–toxicological testing in the case of repeated administration, quantifying systemic distribution, and proof of vaccination protection in animal models can be carried out in parallel to phase 1 or 1/2 clinical trials. To reduce the theoretical risk of an increased respiratory illness through infection-enhancing antibodies or as a result of Th2 polarization and altered cytokine profiles of the immune response following vaccination, which are of specific concern for COVID-19 vaccines, appropriate investigative testing is imperative. In general, phase 1 (vaccine safety) and 2 (dose finding, vaccination schedule) clinical trials can be combined, and combined phase 2/3 trials are recommended to determine safety and efficacy. By applying these fundamental requirements not only for the approval and analysis of clinical trials but also for the regulatory evaluation during the assessment of marketing authorization applications, several efficacious and safe COVID-19 vaccines have been licensed in the EU by unprecedentedly fast and flexible procedures. Procedural and regulatory–scientific aspects of the COVID-19 licensing processes are described in this review.

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion, but also fusion-from-without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during Covid-19 can proceed at low levels of S protein and may not be effectively prevented by antibodies., Graphical Abstract

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir. Electronic supplementary material The online version of this article (10.1007/s00430-020-00692-0) contains supplementary material, which is available to authorized users.

Blood transfusion is one of the most frequently used therapies worldwide and is associated with benefits, risks, and costs.To develop a set of evidence-based recommendations for patient blood management (PBM) and for research.The scientific committee developed 17 Population/Intervention/Comparison/Outcome (PICO) questions for red blood cell (RBC) transfusion in adult patients in 3 areas: preoperative anemia (3 questions), RBC transfusion thresholds (11 questions), and implementation of PBM programs (3 questions). These questions guided the literature search in 4 biomedical databases (MEDLINE, EMBASE, Cochrane Library, Transfusion Evidence Library), searched from inception to January 2018. Meta-analyses were conducted with the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) methodology and the Evidence-to-Decision framework by 3 panels including clinical and scientific experts, nurses, patient representatives, and methodologists, to develop clinical recommendations during a consensus conference in Frankfurt/Main, Germany, in April 2018.From 17 607 literature citations associated with the 17 PICO questions, 145 studies, including 63 randomized clinical trials with 23 143 patients and 82 observational studies with more than 4 million patients, were analyzed. For preoperative anemia, 4 clinical and 3 research recommendations were developed, including the strong recommendation to detect and manage anemia sufficiently early before major elective surgery. For RBC transfusion thresholds, 4 clinical and 6 research recommendations were developed, including 2 strong clinical recommendations for critically ill but clinically stable intensive care patients with or without septic shock (recommended threshold for RBC transfusion, hemoglobin concentration7 g/dL) as well as for patients undergoing cardiac surgery (recommended threshold for RBC transfusion, hemoglobin concentration7.5 g/dL). For implementation of PBM programs, 2 clinical and 3 research recommendations were developed, including recommendations to implement comprehensive PBM programs and to use electronic decision support systems (both conditional recommendations) to improve appropriate RBC utilization.The 2018 PBM International Consensus Conference defined the current status of the PBM evidence base for practice and research purposes and established 10 clinical recommendations and 12 research recommendations for preoperative anemia, RBC transfusion thresholds for adults, and implementation of PBM programs. The relative paucity of strong evidence to answer many of the PICO questions supports the need for additional research and an international consensus for accepted definitions and hemoglobin thresholds, as well as clinically meaningful end points for multicenter trials.

Highlights • The RTS,S Plasmodium falciparum malaria vaccine has received a positive regulatory assessment by EMA. • The CYD-TDV dengue vaccine has been licensed in some middle-income countries. • A Phase 3 HIV vaccine trial is due to be initiated in South Africa during 2016–2017. • The RSV vaccine pipeline is in Phase III clinical development and may reach the licensure submission stage within 4 years. • Group B streptococcal vaccine development is advancing, as is the maternal immunisation agenda., There are more vaccines in development, against a greater number of pathogens, than ever before. A challenge with this exceptional level of activity and investment is how to select and resource the most promising approaches to have the most significant impact on public health. The WHO Product Development for Vaccines Advisory Committee (PDVAC) was established in 2014 to provide strategic advice and recommendations to WHO for vaccines in clinical development that could have a significant impact on public health in low and middle income countries. On 7–9th September 2015, PDVAC was convened for the second time, when the committee reviewed vaccine developments in 24 disease areas. This report summarises the key recommendations from that consultation.

Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection.

Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum-free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T-flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated-batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T- and spinner flasks at 37°C (∼107 TCID50 mL−1, where TCID50 is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000-fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 107 TCID50 mL−1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large-scale production.

Differences in fusion activity between measles virus (MV) attenuated, oncolytic strain MVNSe and pathogenic MVwt323 are reflected in amino acid 94 of the fusion (F) proteins. A valine 94 in FNSe (naturally) or Fwt323 (introduced) correlated with enhanced cell–cell fusion activity during transient glycoprotein expression or recombinant MV infections irrespective of the strains’ targeted receptors, whereas the reverse effect was found for methionine 94. Enhanced fusogenicity was determined by weaker glycoprotein interaction and correlated positively with cytotoxicity in both virus strains. Amino acid 94 of F can be used to tailor fusogenicity and cytotoxicity of recombinant MV, while the cellular receptor triggering fusion is not decisive.

Der Begriff individualisierte oder auch personalisierte Medizin wird haufig gleichgesetzt mit dem Begriff der stratifizierten Medizin. Dies geschieht zu Unrecht, denn es wird vergessen, dass insbesondere bei biologischen Arzneimitteln andere Aspekte der Individualisierung zum Tragen kommen, die uber die Stratifizierung hinausgehen. Die Prinzipien der stratifizierten Medizin kommen bei biologischen Arzneimitteln schon seit vielen Jahren zur Anwendung. Historisches Beispiel ist das Diphtherie-Antitoxin vom Pferd, aktuelle Beispiele sind die Transfusion von roten Blutkorperchen und die Faktor-VIII-Gabe bei Hamophilie A. Die stratifizierenden Aspekte dieser Arzneimittel ergeben sich aus folgenden Uberlegungen: Die Diphtherie-Antitoxin-Gabe erfolgte nur bei Diagnose einer Diphtherie in der Abgrenzung von anderen Formen der Angina tonsillaris, die Transfusion von roten Blutkorperchen sollte nur bei bestatigter Blutgruppengleichheit erfolgen, und die Faktor-VIII-Gabe erfolgt nur bei Hamophilie A in Abgrenzung zu anderen angeborenen oder erworbenen Erkrankungen des Gerinnungssystems. Die Besonderheiten der biologischen Arzneimittel, vor allem die inharente Variabilitat des Arzneistoffs, ist besonders bei den autologen, zellularen Therapien relevant. Neben der erwarteten Variabilitat des biologischen Ausgangsmaterials kommt die interindividuelle Variabilitat der Patienten als Donoren der Zellen hinzu, die die Festlegung von Grenzen und Kriterien fur pharmazeutische Qualitat und Wirksamkeitstest erschwert. Die Therapie mit modifizierten autologen Zellen, eine der haufigen und wichtigen Anwendungen aus dem Bereich der Arzneimittel fur neuartige Therapien, ist exemplarisch fur die Besonderheiten bei der Charakterisierung der pharmazeutischen Qualitat, des Wirkungsmechanismus und der Beurteilung der toxikologischen Eigenschaften des biologischen Arzneimittels. Die klinische Prufung von Arzneimitteln fur neuartige Therapien stellt wegen der Komplexitat der Therapie, die haufig invasive Interventionen beinhaltet, eine besondere Herausforderung, insbesondere fur den Beleg von Wirksamkeit und Sicherheit, dar. Die Entwicklung von Biomarkern beschleunigt den Prozess hin zur stratifizierten bzw. individualisierten Medizin. Damit gehen aber auch hohere Anforderungen an therapiebegleitende Diagnostika („companion diagnostics“) einher. Durch Fortschritte der Analytik und Biotechnologie ist zu erwarten, dass der Grad der Individualisierung zunehmen wird, moglicherweise bis hin zu Arzneimitteln, die fur jeden Patienten individuell produziert werden. Herkommliche Prinzipien der Arzneimittelprufung und -zulassung konnen hier moglicherweise nur eingeschrankt angewendet werden, da es kein eigentliches Arzneimittel mehr gibt, das wiederholt hergestellt wird. Die Beurteilung des Prozesses, beispielhaft an mehreren unterschiedlichen Arzneimitteln des gleichen Prozesses vorgenommen, wird moglicherweise die Basis fur die Beurteilung darstellen. Fur die Risikoabschatzung fur die klinische Prufung mussen neue Konzepte erwogen werden, dies kann durch die Interaktion von regulatorischen Behorden und Entwicklern erleichtert werden.

The Decade of Vaccines Collaboration and development of the Global Vaccine Action Plan provides a catalyst and unique opportunity for regulators worldwide to develop and propose a global regulatory science agenda for vaccines. Regulatory oversight is critical to allow access to vaccines that are safe, effective, and of assured quality. Methods used by regulators need to constantly evolve so that scientific and technological advances are applied to address challenges such as new products and technologies, and also to provide an increased understanding of benefits and risks of existing products. Regulatory science builds on high-quality basic research, and encompasses at least two broad categories. First, there is laboratory-based regulatory science. Illustrative examples include development of correlates of immunity; or correlates of safety; or of improved product characterization and potency assays. Included in such science would be tools to standardize assays used for regulatory purposes. Second, there is science to develop regulatory processes. Illustrative examples include adaptive clinical trial designs; or tools to analyze the benefit-risk decision-making process of regulators; or novel pharmacovigilance methodologies. Included in such science would be initiatives to standardize regulatory processes (e.g., definitions of terms for adverse events [AEs] following immunization). The aim of a global regulatory science agenda is to transform current national efforts, mainly by well-resourced regulatory agencies, into a coordinated action plan to support global immunization goals. This article provides examples of how regulatory science has, in the past, contributed to improved access to vaccines, and identifies gaps that could be addressed through a global regulatory science agenda. The article also identifies challenges to implementing a regulatory science agenda and proposes strategies and actions to fill these gaps. A global regulatory science agenda will enable regulators, academics, and other stakeholders to converge around transformative actions for innovation in the regulatory process to support global immunization goals.

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.

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCE The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MV vac2 genome, and the respective viruses were rescued (MV vac2 -CoV-S and MV vac2 -CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MV vac2 -CoV-S in Vero cells turned out to be comparable to that of the control virus MV vac2 -GFP (encoding green fluorescent protein), while titers of MV vac2 -CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo , immunization of type I interferon receptor-deficient (IFNAR −/− )-CD46Ge mice with 2 × 10 5 50% tissue culture infective doses of MV vac2 -CoV-S(H) or MV vac2 -CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.

International audience; Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

The R peptide in the cytoplasmic tail (C-tail) of γ-retroviral envelope proteins (Env) prevents membrane fusion before budding. To analyse its role in the formation of replication competent, infectious particles, we developed chimeric murine leukaemia viruses (MLV) with unmodified or R-peptide deleted Env proteins of the gibbon ape leukaemia virus (GaLV). While titres of these viruses were unaffected, R-peptide deficiency led to strongly impaired spreading. Most remarkably, we isolated an escape mutant which had restored an open reading frame for a C-terminal extension of the truncated C-tail. A reconstituted virus encoding this escape C-tail replicated in cell culture. In contrast to R-peptide deficient Env, particle incorporation of the escape Env was effective due to an enhanced protein expression and restored intracellular co-localisation with Gag proteins. Our data demonstrate that the R peptide not only regulates membrane fusion but also mediates efficient Env protein particle incorporation in γ-retrovirus infected cells.

Gentherapie = Therapie mit Genen: Wie kann das denn funktionieren? Gene sind ja in jeder unserer Korperzellen und im Erbgut der Keimzellen befinden sich die Gene, die wir vererben. Sind diese betroffen? So oder ahnlich durften nicht nur die Laien, sondern auch die Forscher reagiert haben, als sie Ende der 1980er Jahre in den USA und Anfang der 1990er Jahre in Deutschland von den ersten klinischen Gentherapievorhaben horten.

Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro , suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3 . The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo , thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation.

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Background Lentiviral vectors allow stable gene transfer into nonreplicating cells and are increasingly used in clinical gene therapy approaches. Vectors derived from different origins can show distinct target cell transduction properties. Therefore, the construction of modern vector systems of different viral origin remains desirable. The generation of safe and efficient lentivirus-derived transfer vectors by gradual enhancing cloning steps is a time-consuming process that depends on the presence of suitable restriction sites. Multiple-step cloning protocols also enhance the risk of acquisition of mutations or other genetic instabilities. Methods We constructed novel HIV-2 and SIVsmmPBj-derived transfer vectors by amplification of three essential segments of the viral genome [5′-long terminal repeat (LTR), rev responsive element, ΔU3-3′-LTR] on the template of the lentiviral full-length genome by a highly flexible three-step fusion polymerase chain reaction approach. Further necessary vector elements, as well as a multiple cloning site, were included into the resulting vector by extension of the primer sequences. The respective vesicular stomatitis virus G pseudotyped lentiviral vector particles were generated and analysed. Results Two novel transfer vectors of different lentiviral origin were successfully generated. Titers for the corresponding SIVsmmPBj- and HIV-2-derived vectors reached up to 9.9 × 107 transforming units (TU)/ml and 1.2 × 108 TU/ml, respectively. The specific capacity to transduce primary human monocytes was maintained in both newly-generated vector systems. Conclusions We anticipate that this novel and fast way of generating any lentiviral transfer vector will improve the generation of such vectors. The HIV-2- and SIVsmmPBj-derived vectors described will prove valuable for future gene therapy strategies. Copyright © 2010 John Wiley & Sons, Ltd.

The accessory protein Vpx is encoded by lentiviruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage. It is packaged into virions and is indispensable in early steps of monocyte infection. HIV-1, which does not encode Vpx, is not able to infect human monocytes, but Vpx enables infection with HIV-1. The underlying mechanism is not completely understood. In this work, we focus on Vpx-mediated intracellular postentry events as counteraction of host cell proteins. We found that Vpx binds to apolipoprotein B mRNA-editing catalytic polypeptide 3 family member A (APOBEC3A; A3A), a member of the family of cytidine deaminases, present in monocytes. This interaction led to a reduction of the steady-state protein level of A3A. A single-point mutation in Vpx (H82A) abrogated binding to A3A and single-round infection of monocytes by HIV-1. Taken together, our data indicate that lentiviral Vpx counteracts A3A in human monocytes.

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however low vector concentration in batch cultures and high temperature-dependent decay limit sufficiently large-scale production. The ability of commercial ceramic asymmetric ultrafiltration membranes with a cut-off of 20 kDa to purify retroviral pseudotype vectors derived from the murine leukemia virus (MLV) carrying the HIV-1 envelope protein MLV (HIV-1) was studied. Experimental study was carried out in order to analyse the impact of inline tangential flow filtration (TFF) combined with different harvest temperatures in continuous production mode compared to batch filtration. Retroviral pseudotype vectors were produced using a 200 ml fixed bed reactor for high cell density cultivation on macro porous carriers up to 400 hours. By tangential flow ultrafiltration combined with cooling down the harvest supernatant to 4°C, the vector concentration was increased 12-fold with an average recovery of 78.7% of the initial infective capacity.

Arzneimittel fur neuartige Therapien („advanced therapy medicinal products“, [ATMP]) unterliegen, wie alle anderen Biotechnologie-Arzneimittel, dem zentralisierten Zulassungsverfahren, welches eine einmalige, zentrale wissenschaftliche Bewertung durch die Europaische Arzneimittelbehorde (European Medicines Agency [EMEA]) in London, UK, beinhaltet. Fur ATMP gelten die gleichen regulatorischen Prinzipien wie fur andere Biotechnologie- Arzneimittel. Die technischen Anforderungen, insbesondere Art und Umfang der fur den Nachweis der Qualitat, Unbedenklichkeit und Wirksamkeit des Produkts erforderlichen Daten, konnen sich jedoch z.T. deutlich von den Anforderungen fur konventionelle chemische Arzneimittel unterscheiden. Dem tragt der aktualisierte Anhang I der Verordnung 2001/83/EG Rechnung. Die Entwicklung von ATMP wird durch eine Reihe von europaischen Richtlinien u.a. zu Qualitat, Praklinik und klinischen Aspekten von Gen- bzw. Zelltherapeutika unterstutzt. Die klinische Anwendung dieser Produkte kann zu verschieden Risiken fur den Patienten, das medizinische Fachpersonal und die allgemeine Bevolkerung fuhren. Die Abschatzung der unterschiedlichen Risiken ist bereits zu Beginn der Produktentwicklung vorzunehmen. Kriterien fur die Risikoanalyse von ATMP sind bereits in dem Entwurf des Annex I der revidierten Verordnung 2001/83/EG genannt und konnen bei der Einschatzung des Gesamtrisikos fur das Produkt herangezogen werden. Ein weiterer wichtiger Aspekt der Vorschriften klinischer Prufungen mit Arzneimitteln fur neuartige Therapien ist das Erfordernis langfristiger Nachuntersuchungen der behandelten Patienten. Ziel dieser Nachuntersuchungen ist es, Zeichen verzogerter oder mit der Zeit zuruckgehender Arzneimittelwirkungen aufzuspuren, die klinischen Folgen solcher Arzneimittelreaktionen zu verhindern bzw. eine zeitnahe Behandlung zu garantieren sowie Informationen uber die langfristige Unbedenklichkeit und Wirksamkeit der Masnahme zu erlangen.

The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene.

Lentiviral vectors are potent gene transfer vehicles frequently applied in research and lately also in clinical applications. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, which now allow targeting of cell entry to any cell population of interest, as well as the transduction of quiescent cells of the haematopoietic system. We propose that measles virus envelope glycoproteins are especially well suited for this purpose because they can mediate pH-independent cell entry at the cell surface membrane and can induce cytoskeleton rearrangements that facilitate the transport of lentiviral core particles to the cell nucleus. Lentiviral vectors pseudotyped with measles virus glycoproteins are expected to improve the safety and efficacy of gene transfer to human cells.

The APOBEC3 cytidine deaminases are part of the intrinsic defense of cells against retroviruses. Lentiviruses and spumaviruses have evolved essential accessory proteins, Vif and Bet, respectively, which counteract the APOBEC3 proteins. We show here that Bet of the Prototype foamy virus inhibits the antiviral APOBEC3C activity by a mechanism distinct to Vif: Bet forms a complex with APOBEC3C without inducing its degradation. Bet abolished APOBEC3C dimerization as shown by coimmunoprecipitation and cross-linking experiments. These findings implicate a physical interaction between Bet and the APOBEC3C. Subsequently, we identified the Bet interaction domain in human APOBEC3C in the predicted APOBEC3C dimerization site. Taken together, these data support the hypothesis that Bet inhibits incorporation of APOBEC3Cs into retroviral particles. Bet likely achieves this by trapping APOBEC3C protein in complexes rendering them unavailable for newly generated viruses due to direct immobilization.

Purpose: Given the multiple escape mechanisms of tumor cells, immunotherapy targeting tumor-dependent stroma may be an effective cancer treatment strategy. Animal models indicate that inducing immunity to tumor endothelia engenders potent antitumor effects without significant pathology. Recently, the first human tumor endothelial antigen vascular endothelial growth factor receptor-2 (VEGFR-2) recognized by HLA class I–restricted CD8+ T cells has been characterized. In this study, we sought to investigate specific recognition of this molecule by human CD4+ T cells.Experimental Design: To identify HLA-DR–restricted antigenic peptides on VEGFR-2 recognized by CD4+ T cells of healthy donors and cancer patients.Results: Nine candidate VEGFR-2 peptides with high binding probability to six common HLA-DRB1 alleles were synthesized using the SYFPEITHI algorithm. One 15-mer peptide (EKRFVPDGNRISWDS), mapping to the 167-181 region of VEGFR-2, stimulated CD4+ T cells in association with several HLA-DR alleles, including DR4 and DR7. Importantly, the epitope could be naturally processed and presented both by HLA-DR–matched antigen-expressing proliferating endothelial cells and by dendritic cells loaded with the native antigen. Furthermore, circulating VEGFR-2–specific CD4+ T cells were detected in 4 of 10 healthy donors and 12 of 40 cancer patients even after single-round peptide stimulation in short-term culture. Patient's T cells could recognize antigen-expressing proliferating endothelial cells in a HLA-DR–restricted fashion.Conclusion: These findings indicate an important role for the 167-181 region of VEGFR-2 in the stimulation of CD4+ T cell responses to VEGFR-2 protein, and may be instrumental both for the development and monitoring of upcoming antitumor vessel vaccines against different cancers based on VEGFR-2 immunogens.

Gene and somatic cell therapy medicinal products as well as tissue engineered products are summarized under the term advanced therapy medicinal products in the EU. The Paul-Ehrlich-Institut has established a specialized division with various sections providing scientific advice and clinical trial authorization to applicants, thereby protecting patients’ health and supporting product development. Gene therapy medicinal products include products suitable for in vivo diagnostics, for the prevention of infectious diseases (such as vaccines) and the treatment of common diseases such as cancer, cardiovascular and infectious diseases, and of rare or orphan diseases such as inherited monogeneic diseases. Clinical trial authorization in Germany is provided by the Paul-Ehrlich-Institut and, if genetically modified organism-containing medicinal products are used, includes deliberate release authorization under consultation with the Federal Office for Consumer Protection and Food Safety (BVL). Firstmarketing authorization applications of gene therapy medicinal products developed for the treatment of cancer have been submitted to the European Medicines Agency (EMEA) and have been or are being reviewed by experts from national medicines agencies. Beneficial gene therapy treatment in clinical trials has been reported for infants suffering from adenosine deaminase deficiency-based severe combined immunodeficiency disease. Human somatic cell therapy medicinal products are mainly being developed for cancer immunotherapy; tissue engineered products contain viable cells able to replace, repair or substitute for human tissues or cells, e.g., after myocardial infarction. Chondrocytes are being used clinically for cartilage repair and an application for EU-wide marketing has been submitted to the EMEA. The main issues for cell-containing medicinal products are cell selection, differentiation, manipulation to obtain specific characteristics and avoiding pre-neoplastic alterations. In summary, special expertise is needed to develop advanced therapy medicinal products and has to be met by scientists specialized in regulatory aspects to support patient safety and product development. Consequently, the EMEA will establish the Committee for Advanced Therapies to review future marketing authorization applications.

Am 1. August 2007 trat in Deutschland das Gewebegesetz in Kraft, welches unter anderem Regelungen hinsichtlich der Qualitat und Sicherheit von Gewebezubereitungen (GWZ) trifft. Klassische GWZ sind Arzneimittel fur die Anwendung am Menschen, a) die menschliche Gewebe und Zellen enthalten oder ausgehend von diesen hergestellt werden, b) deren wesentliche Be- oder Verarbeitungsverfahren in der EU hinreichend bekannt bzw. neu, aber mit bekannten Verfahren vergleichbar sind, und c) deren Wirkungen und Nebenwirkungen aus dem wissenschaftlichen Erkenntnismaterial ersichtlich sind. Dazu gehoren z.B. Herzklappen, Corneae, muskuloskelettale GWZ, Gefase oder Stammzellen aus Knochenmark. Wenn bei einer GWZ die oben genannten Voraussetzungen erfullt sind und diese nicht mit ionisierenden Strahlen behandelt wird, ist fur das Inverkehrbringen eine Genehmigung gemas § 21a Abs. 1 Arzneimittelgesetz (AMG) durch das Paul-Ehrlich-Institut erforderlich. Zur Erleichterung der Antragstellung fur die Genehmigung stehen Formulare auf der Internetseite des Paul-Ehrlich-Instituts zur Verfugung. Schwerwiegende unerwunschte Reaktionen und schwerwiegende Zwischenfalle gemas § 63c AMG mussen von Gewebeeinrichtungen unverzuglich an die zustandige Bundesbehorde gemeldet werden. Des Weiteren besteht fur Gewebeeinrichtungen eine Meldepflicht gemas § 8d Abs. 3 Transplantationsgesetz (TPG) zur Erfassung der verarbeiteten Gewebe, die in Form eines jahrlichen Berichts bis zum 1. Marz des folgenden Jahres an das Paul-Ehrlich-Institut erfolgt.

Fur eine Reihe von Infektionskrankheiten sowohl bakterieller, parasitarer als auch viraler Genese wurde vor allem im 20. Jahrhundert eine Vielzahl von Impfstoffen entwickelt, die Impflinge erfolgreich schutzen konnten. Dies fuhrte zu einer drastisch verminderten Krankheitslast in der Bevolkerung, zu einer deutlich verringerten Sauglingssterblichkeit und zu einer Erhohung der Lebenserwartung. Aktuell werden die globalen Bemuhungen intensiviert, das Poliomyelitis- und das Masern-Virus auszurotten.