Characterisation of Antibody Interactions with the G Protein of Vesicular Stomatitis Virus Indiana Strain and Other Vesiculovirus G Proteins

Abstract

29 Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly 30 used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and 31 clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), 32 for example Cocal virus, have been proposed as alternative LV envelopes with 33 possible advantages compared to VSVind.G. Well-characterised antibodies that 34 recognise VesG will be useful for vesiculovirus research, development of G protein-35 containing advanced therapy medicinal products (ATMPs), and deployment of 36 VSVind-based vaccine vectors. Here we show that one commercially available 37 monoclonal antibody, 8G5F11, binds to and neutralises G proteins from three strains 38 of VSV as well as Cocal, and Maraba viruses, whereas the other commercially 39 available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralises only 40 VSVind.G. Using a combination of G protein chimeras and site-directed mutations, 41 we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds 42 close to the receptor binding site and competes with soluble low-density lipoprotein 43receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralisation. 44In contrast, 8G5F11 binds close to a region known to undergo conformational changes 45 when the G protein moves to its post-fusion structure, and we propose that 8G5F11 46 cross-neutralises VesGs by inhibiting this. 47 IMPORTANCE 48 VSVind.G is currently regarded as the gold-standard envelope to pseudotype lentiviral 49 vectors. However, recently other G proteins derived from vesiculoviruses have been 50 proposed as alternative envelopes. Here, we investigated two anti-VSVind.G 51 monoclonal antibodies for their ability to cross-react with other vesiculovirus G 52 proteins, and identified the epitopes they recognise, and explored the mechanisms 53 behind their neutralisation activity. Understanding how cross-neutralising antibodies 54 interact with other G proteins may be of interest in the context of host-pathogen 55 interaction and co-evolution as well as providing the opportunity to modify the G 56 proteins and improve G protein-containing medicinal products and vaccine vectors.57 58 59The rhabdovirus, vesicular stomatitis virus Indiana stain (VSVind), has been used 60 ubiquitously as a model system to study humoral and cellular immune responses in 61 addition to being a promising virus for oncolytic virotherapy against cancer (1-3). 62Furthermore, its single envelope G protein (VSVind.G) is the most commonly used 63 envelope to pseudotype lentiviral vectors and serves as the gold-standard in many 64 experimental and clinical studies (4-6). Both receptor recognition and membrane 65 fusion of the wild-type virus, as well as the pseudotyped particles, are mediated by this 66 single transmembrane viral glycoprotein that homotrimerises and protrudes from the 67 viral surface (7-9). Recently G proteins derived from other vesiculovirus subfamily 68 members, namely, Cocal, Piry, and ...

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

“…This differential sensitivity allowed us to map the region of the envelope responsible by constructing chimeras between VSVind.G and COCV.G; these were designed so that the protein junctions were made in regions of homology between the two G proteins (Figure 5B). These chimeras were expressed as detected by flow cytometric analysis of 8G5F11 (Kerafast, Boston, MA) immunostaining, an extracellular anti-VSVind.G mAb, and could produce infectious LVs at levels comparable with VSVind.G following transient transfection) 34 . LVs expressing the chimeric G proteins 4A, 1B, and 2B were resistant to human serum inactivation, suggesting that the region of VSVind.G between amino acids 136 and 370 confers complement sensitivity (Figure 5C).…”

“…These chimeras were expressed as detected by flow cytometric analysis of 8G5F11 (Kerafast, Boston, MA) immunostaining, an extracellular anti-VSVind.G mAb, and could produce infectious LVs at levels comparable with VSVind.G following transient transfection). 34 LVs expressing the chimeric G proteins 4A, 1B, and 2B were resistant to human serum inactivation, suggesting that the region of VSVind.G between amino acids 136 and 370 confers complement sensitivity ( Figure 5 C). On the crystal structure of VSVind.G, this region corresponds to most of the pleckstrin homology domain and parts of the fusion, trimerization, and lateral domains ( Figure S1 ).…”

Lentiviral Vector Bioprocessing

“…Unlike VSV-G, Cocal-G is not inactivated by human serum and can be expressed constitutively allowing the potential for stable expression in vector producing cell lines [36,78], although this does lead to superinfection and cell instability [37]. Additional vesiculovirus family glycoproteins have been examined for stable producer cell line generation, such as those from PIRY, Chandipura and VSV-New Jersey, which displayed titres of 10 5 to 10 6 TU/mL and robustness during concentration and freeze-thaw [37,79]. In addition, the viral envelope protein RD114 has been applied in vector production.…”