Identify VSV-G positive lentivirus with NanoSight Pro F-NTA

Introduction 

In viral vector formulation development, it is often assumed that the purification process is effective and that all vectors are formed as expected. However, this is not always the case. What if specific vector components could be tagged to detect and confirm their presence?

This application note demonstrates how a specific, two-step labeling process can be used to detect VSV-G positive lentivirus within a purified sample using NanoSight Pro’s fluorescence mode. By targeting VSV-G on the surface, this study showcases the NanoSight Pro, utilizing Nanoparticle Tracking Analysis (NTA) technology, to characterize a lentivirus* formulation.

Targeting VSV-G on the surface 

Specific labeling of lentivirus targeting the VSV-G protein was performed to isolate and characterize lentivirus from impurities and other components in the sample. A two-step labeling process, utilizing primary and secondary antibodies. Anti-VSV-G primary antibody (Leander details) binds to the surface VSV-G receptor of the lentivirus. Next secondary antibody with conjugated fluorophore links to primary antibody (Figure 1).

Figure 1: Schematic of lentivirus 2-step labeling with primary antibody and secondary fluorescent antibody

Method

First, lentivirus was incubated for 30 minutes at room temperature with a primary anti-VSV-G antibody. Next, the secondary antibody linked with Alexa 488 fluorophore, was added to the labeling mixture and incubated further for 20 minutes at room temperature (Figure 1). Both the labeled sample and the unlabeled controls were analyzed with NanoSight Pro equipped with a 488nm laser and 500LP fluorescence filter as the best configuration for Alexa488 excitation and fluorescence signal collection. 

The data was collected using NS Xplorer 1.1 software in both the light scatter and fluorescence mode for the labeled sample, the lentivirus controls with only the primary and only the secondary antibodies and the unlabeled control. The output in the format of size distribution of labeled sample and unlabeled control was compared and is presented in Figure 2.

Results

Figure 2: The overlay of the size distributions of the labeled lentivirus and unlabeled control

The labeling resulted in the majority of particles being VSV-G positive, with a labeling efficiency of 74% for 50 nm - 150 nm size range, indicating the proportion of specifically tagged vectors (Figure 2, green line). Unlabeled particles in the sample are unlikely to contain VSV-G component and may represent co-purified extracellular vesicles, proteins, or other impurities. 

The light scatter size distribution profiles and titer/concentration (Table 1) of the labeled and unlabeled samples (Figure 2, orange line) remained similar (Figure 2, blue line) indicating that the labeling process did not change the physicochemical properties of the lentivirus. 

To make sure the labeling was specific, relevant controls of lentivirus with primary and secondary antibodies were run at te same conditions as the labeled sample. The comparison of the controls' size distributions is presented in Figure 3 below. 

Figure 3: The overlay of the size distributions of the lentivirus controls

The size distribution of lentivirus controls with the primary (Figure 3, red line) and the secondary antibodies (Figure 3, yellow line) matched unlabeled and labeled samples in the light scatter. The overall sample concentration remained also similar for unlabeled and antibody controls (Table 1). For both antibody controls, no NTA fluorescence signal was detected; this further rejects the possibility of unspecific binding and confirms the successful binding of primary-secondary antibodies to the VSV-G target presented on the surface of the lentivirus particles. 

Table 1: Light scatter data summary of lentivirus unlabeled control, lentivirus with primary ab and secondary ab controls and a labeled sample. 

Summary 

The two-step labeling was successful, enabling tagging of a specific component on the lentivirus surface. NanoSight Pro rapidly confirmed overall sample characteristics in light scatter mode. It also distinguished VSV-G positive lentivirus subpopulation in fluorescence mode. This specific labeling and fluorescence characterization brings us a step closer to viral vector identification. 

Further reading

This application is part of a five-part series. Explore the rest of the series to dive deeper into this topic:

• Quick viral vector purity assessment with NanoSight Pro 
• Viral vector stability and storage assessment by NanoSight Pro 
• Identify viral vectors with NanoSight Pro F-NTA

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*Lentivirus and MVA samples along with formulation buffers were kindly supplied by one of Malvern Panalytical’ s collaborators.