Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here ‘Fusoselect', a universal procedure allowing the identification and engineering of molecular determinants for cell-cell fusion-activity by directed evolution. The system couples cell-cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a γ-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell-cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselec

Fusoselect: cell–cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here ‘Fusoselect’, a universal procedure allowing the identification and engineering of molecular determinants for cell–cell fusion-activity by directed evolution. The system couples cell–cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a γ-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell–cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect

A 5-year look-back at the notification and management of vaccine supply shortages in Germany

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.Peer Reviewe

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

Selection of functional human antibodies from retroviral display libraries

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries

Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

APOBEC3 (A3, Apolipoprotein B mRNA-editing catalytic polypeptide 3) genes in the genome of domestic cat (Felis catus) were identified and characterize

Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj

Background: Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx. Methods and Findings: Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled. Conclusion: Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes