Redirecting Lentiviral Vectors Pseudotyped with Sindbis Virus-Derived Envelope Proteins to DC-SIGN by Modification of N-Linked Glycans of Envelope Proteins

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Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelope's natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of "stemness" (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (ϳ10 6 IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5F⌬N3). T5F⌬N3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2؉ cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5F⌬N3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2 ؉ populations in vivo or in vitro.

mentioning

confidence: 99%

Nipah Virus Envelope-Pseudotyped Lentiviruses Efficiently Target ephrinB2-Positive Stem Cell Populations In Vitro and Bypass the Liver Sink When Administered In Vivo

Palomares

Vigant

Handel

et al. 2013

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“…All lentiviral vectors were produced in 293T cells by a previously described calcium phosphate transfection method (38). Enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors pseudotyped with various Envs were produced by transfecting 293T cells with an Env expression vector (Sindbis, 2.2, 2.2 1L1L, VSV envelope G protein [VSV-G], gp64, or RRV), the packaging plasmid PAX2 (Addgene), and a lentiviral vector (cppt2e) containing EGFP as its transgene (39). The 2.2 1L1L, VSV-G, and gp64 pseudotypes labeled with EGFP were generated by transfecting 293T cells with PAX2 and p-gagEGFP (NIH AIDS Research and Reference Reagent Program, Germantown, MD), in addition to expression vectors for each Env (40).…”

Section: Methodsmentioning

confidence: 99%

Role of Phosphatidylserine Receptors in Enveloped Virus Infection

Morizono

Chen

2014

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154

178

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. IMPORTANCEEnvelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

“…Furthermore, the lentivectors' envelopes are well suited for engineering, enabling the design of targeted lentivectors. Several methods have been described to redirect lentivectors to specific antigen-presenting cells (9)(10)(11)(12). However, to our knowledge subset-specific delivery of transgenes has not been described.…”

mentioning

confidence: 99%

Targeting of Human Antigen-Presenting Cell Subsets

Goyvaerts

Dingemans

Groeve

et al. 2013

dAntigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets. Since the discovery of dendritic cells (DCs) in 1973, these antigen-presenting cells have been at the center of attention in vaccine development (1). In the past decade, an extra dimension of complexity was introduced by revealing an unforeseen diversity of DC subsets with distinct functions and locations (2-4). Since DC subsets induce distinct immune responses, it might be beneficial to develop strategies to target particular DC subsets and as such exploit the immune system to its full potential (5).Numerous animal studies have proven the efficiency and safety of lentivectors (LVs) as vaccination moieties (6, 7). As lentivectors are intrinsically immunogenic, they deliver both antigens and activation signals to antigen-presenting cells (8). Furthermore, the lentivectors' envelopes are well suited for engineering, enabling the design of targeted lentivectors. Several methods have been described to redirect lentivectors to specific antigen-presenting cells (9-12). However, to our knowledge subset-specific delivery of transgenes has not been described.Targeting myeloid DCs could be advantageous as they are considered to be important mediators of antigen-specific immunity. They are able to induce proper and oriented stimulation of CD4 ϩ T helper 1 and CD8 ϩ cytotoxic T cells. In addition, targeting may reduce the risk of adverse reactions such as autoimmune responses or induction of tolerance due to transgene expression and presentation by non-antigen-presenting cells or tolerogenic DC subtypes. Finally, as myeloid DCs have a limited life span, their targeting should result in a natural clearance of the lentivector and as such in a reduction of the risk of insertional mutagenesis.We recently delivered a proof of concept on the use of nanobodies (Nbs) to target lentivectors to antigen-presenting cells (10). Nbs or V H H fragments are antibody fragments of about 12 to 25 kDa that are engineered from heavy-chain-only antibodies found in Camelidae. Because of their size and target affinity, they are of particular interest as targeting moieties. In the present study, we further refined the transduction profile of lentivectors, targeting them to human myeloid DCs using Nbs.Two Nb libraries, derived from peripheral blood lymphocytes of llamas that were immunized with immature or lipopolysaccharide-stimulated murine bone marrow-derived DCs, were at our disposal (13). These were screened for cross-reactivity with hu-