Abstract:VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-n… Show more
“…Sera were isolated and pooled, and neutralizing activity was determined through an in vitro LV neutralization assay previously described (Figure 1). 20 VSVind.G-LV infection was blocked in a dose-dependent manner; however, a similar effect was also observed for LVs pseudotyped with the unrelated feline endogenous retrovirus RD114-derived (RDpro) envelope (Figure 1A, dotted lines). This unspecific neutralizing activity suggested that the inhibition of LV infection might be due to antibodies directed against proteins on the vector surface acquired from the producer cells during the LV production, namely, host-cell proteins.…”
Section: Resultsmentioning
confidence: 99%
“…Here we demonstrate that while close phylogenetic relatives of VSVind.G, MARAV.G and COCV.G, share its receptor usage, the more distant PIRYV.G does not. The phylogenetic closeness of PIRYV.G and Chandipura G 20 implies that vesiculoviruses might be split into at least two groups regarding their primary receptor for cell entry.Figure 5PIRYV.G Does Not Interact with sLDLR(A) VesG- and RDpro-expressing HEK293T cells were incubated with (left) VSV-Poly and anti-RD114 antiserum to determine envelope expression and (right) sLDLR in parallel. The cells were then probed with the respective secondary antibodies to determine envelope expression and anti-6XHis-tag antibody for sLDLR binding.…”
Section: Resultsmentioning
confidence: 99%
“…MARAV.G and COCV.G are two of the closest relatives of VSVind.G, with 78% and 72% homology on the amino acid level, respectively (Figure S2). 20 In vitro neutralization assays performed using the sera isolated from terminal bleeds of IC-VSVind.G displayed weak but neutralizing cross-reactivity against these two pseudotypes (Figure 3). This implied that these three closely related G proteins share immunodominant epitopes, which neutralizing antibodies recognize.…”
Section: Discussionmentioning
confidence: 99%
“…Briefly, 2 × 10 7 cells were seeded in 15-cm plates. Then 24 h later, they were transfected using FuGene6 (Promega, Madison, WI, USA) with the following plasmids: 3.75 μg pCCL.Fluc.2A.EGFP (firefly luciferase and EGFP-expressing vector plasmid), 46 2.5 μg p8.91 (Gag-Pol and Rev expression plasmid), 45 and 2.5 μg VesG expression plasmids previously described 20, 25. Then 4 h after transfection, medium was changed to OptiMEM (Gibco), supplemented with 2 mM L-Glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco).…”
Vesicular stomatitis virus Indiana strain glycoprotein (VSVind.G) mediates broad tissue tropism and efficient cellular uptake. Lentiviral vectors (LVs) are particularly promising, as they can efficiently transduce non-dividing cells and facilitate stable genomic transgene integration; therefore, LVs have an enormous untapped potential for gene therapy applications, but the development of humoral and cell-mediated anti-vector responses may restrict their efficacy. We hypothesized that G proteins from different members of the vesiculovirus genus might allow the generation of a panel of serotypically distinct LV pseudotypes with potential for repeated
in vivo
administration. We found that mice hyperimmunized with VSVind.G were not transduced to any significant degree following intravenous injection of LVs with VSVind.G envelopes, consistent with the thesis that multiple LV administrations would likely be blunted by an adaptive immune response. Excitingly, bioluminescence imaging studies demonstrated that the VSVind-neutralizing response could be evaded by LV pseudotyped with Piry and, to a lesser extent, Cocal virus glycoproteins. Heterologous dosing regimens using viral vectors and oncolytic viruses with Piry and Cocal envelopes could represent a novel strategy to achieve repeated vector-based interventions, unfettered by pre-existing anti-envelope antibodies.
“…Sera were isolated and pooled, and neutralizing activity was determined through an in vitro LV neutralization assay previously described (Figure 1). 20 VSVind.G-LV infection was blocked in a dose-dependent manner; however, a similar effect was also observed for LVs pseudotyped with the unrelated feline endogenous retrovirus RD114-derived (RDpro) envelope (Figure 1A, dotted lines). This unspecific neutralizing activity suggested that the inhibition of LV infection might be due to antibodies directed against proteins on the vector surface acquired from the producer cells during the LV production, namely, host-cell proteins.…”
Section: Resultsmentioning
confidence: 99%
“…Here we demonstrate that while close phylogenetic relatives of VSVind.G, MARAV.G and COCV.G, share its receptor usage, the more distant PIRYV.G does not. The phylogenetic closeness of PIRYV.G and Chandipura G 20 implies that vesiculoviruses might be split into at least two groups regarding their primary receptor for cell entry.Figure 5PIRYV.G Does Not Interact with sLDLR(A) VesG- and RDpro-expressing HEK293T cells were incubated with (left) VSV-Poly and anti-RD114 antiserum to determine envelope expression and (right) sLDLR in parallel. The cells were then probed with the respective secondary antibodies to determine envelope expression and anti-6XHis-tag antibody for sLDLR binding.…”
Section: Resultsmentioning
confidence: 99%
“…MARAV.G and COCV.G are two of the closest relatives of VSVind.G, with 78% and 72% homology on the amino acid level, respectively (Figure S2). 20 In vitro neutralization assays performed using the sera isolated from terminal bleeds of IC-VSVind.G displayed weak but neutralizing cross-reactivity against these two pseudotypes (Figure 3). This implied that these three closely related G proteins share immunodominant epitopes, which neutralizing antibodies recognize.…”
Section: Discussionmentioning
confidence: 99%
“…Briefly, 2 × 10 7 cells were seeded in 15-cm plates. Then 24 h later, they were transfected using FuGene6 (Promega, Madison, WI, USA) with the following plasmids: 3.75 μg pCCL.Fluc.2A.EGFP (firefly luciferase and EGFP-expressing vector plasmid), 46 2.5 μg p8.91 (Gag-Pol and Rev expression plasmid), 45 and 2.5 μg VesG expression plasmids previously described 20, 25. Then 4 h after transfection, medium was changed to OptiMEM (Gibco), supplemented with 2 mM L-Glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco).…”
Vesicular stomatitis virus Indiana strain glycoprotein (VSVind.G) mediates broad tissue tropism and efficient cellular uptake. Lentiviral vectors (LVs) are particularly promising, as they can efficiently transduce non-dividing cells and facilitate stable genomic transgene integration; therefore, LVs have an enormous untapped potential for gene therapy applications, but the development of humoral and cell-mediated anti-vector responses may restrict their efficacy. We hypothesized that G proteins from different members of the vesiculovirus genus might allow the generation of a panel of serotypically distinct LV pseudotypes with potential for repeated
in vivo
administration. We found that mice hyperimmunized with VSVind.G were not transduced to any significant degree following intravenous injection of LVs with VSVind.G envelopes, consistent with the thesis that multiple LV administrations would likely be blunted by an adaptive immune response. Excitingly, bioluminescence imaging studies demonstrated that the VSVind-neutralizing response could be evaded by LV pseudotyped with Piry and, to a lesser extent, Cocal virus glycoproteins. Heterologous dosing regimens using viral vectors and oncolytic viruses with Piry and Cocal envelopes could represent a novel strategy to achieve repeated vector-based interventions, unfettered by pre-existing anti-envelope antibodies.
“…H and F envelope proteins from the measles virus (MV) have been used to retarget various cell types. More recently other proteins have been considered as alternative envelopes for LV pseudotyping such as glycoproteins derived from other vesiculovirus subfamilies, Cocal, Piry and Chandipura viruses or the VSV New Jersey strain as well as the Nipah virus and other rhabdoviruses, for which it was proposed that they could have an advantage over the commonly used VSV-G Indiana strain [37,38]. However, it is difficult to obtain high-titer vectors with some of these glycoproteins, though they transduce efficiently hematopoietic cells (Summary in Table 1).…”
Viruses have been repurposed into tools for gene delivery by transforming them into viral vectors. The most frequently used vectors are lentiviral vectors (LVs), derived from the human immune deficiency virus allowing efficient gene transfer in mammalian cells. They represent one of the safest and most efficient treatments for monogenic diseases affecting the hematopoietic system. LVs are modified with different viral envelopes (pseudotyping) to alter and improve their tropism for different primary cell types. The vesicular stomatitis virus glycoprotein (VSV-G) is commonly used for pseudotyping as it enhances gene transfer into multiple hematopoietic cell types. However, VSV-G pseudotyped LVs are not able to confer efficient transduction in quiescent blood cells, such as hematopoietic stem cells (HSC), B and T cells. To solve this problem, VSV-G can be exchanged for other heterologous viral envelopes glycoproteins, such as those from the Measles virus, Baboon endogenous retrovirus, Cocal virus, Nipah virus or Sendai virus. Here, we provide an overview of how these LV pseudotypes improved transduction efficiency of HSC, B, T and natural killer (NK) cells, underlined by multiple in vitro and in vivo studies demonstrating how pseudotyped LVs deliver therapeutic genes or gene editing tools to treat different genetic diseases and efficiently generate CAR T cells for cancer treatment.
Since cell‐based virus neutralization assays are still the gold standard to assess a patient's immune protection against a given virus, they are of utmost importance for serodiagnosis, convalescent plasma therapy, and vaccine development. Monitoring the emergence and characteristics of neutralizing antibodies in an outbreak situation, confirming neutralizing antibodies as correlates of protection from infection and testing vaccine‐induced potency of neutralizing antibody responses, quests for automated, fast, and parallel neutralization assays. We developed an impedance‐based sensor platform (electric cell‐substrate impedance sensing, ECIS) providing time‐resolved monitoring of the host cell response to viral pseudotypes. For validation, the impedance assay was compared with state‐of‐the‐art quantification of virus‐induced reporter protein expression as an independent indicator of virus infection and neutralization. Vesicular stomatitis virus (VSV) derived pseudoviruses encoding the green fluorescent protein (GFP) as reporter and the autologous G protein (VSV‐G) for the initial binding to the host cell membrane were used for monitoring of HEK293T cell infection and neutralization with both, impedance and optical readout. Virus‐induced cytopathic effects (CPE) were detectable for low pseudotype concentrations (multiplicity of infection 1) in time‐resolved impedance profiles as soon as 5–10 h after infection in a concentration‐dependent manner. Neutralization efficacy of α‐VSV‐G antibodies was determined from impedance time courses and IC50 values compared favorably with fluorescence measurements of virus‐borne GFP expression. Sera of convalescent COVID‐19 patients were tested successfully for SARS‐CoV‐2 neutralizing antibodies by incubating VSV, pseudotyped with the SARS‐CoV‐2 spike protein, with different sera before host cell exposure and impedance recordings. In summary: (i) ECIS monitoring was successfully applied to detect virus‐mediated cell infection and neutralization; (ii) Impedance‐based monitoring allows reducing the assay time to 5–10 h; and (iii) the platform is easily adapted to other virus‐based diseases and scalable to high‐throughput.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
