Fig 1.
Generation and Characterization of infectivity of R-PV-GFP.
A. Scheme showing generation of Rabies pseudovirus expressing GFP reporter (R-PV-GFP) using rVSV (G*ΔG-GFP) and full-length Rabies virus G protein expressing plasmid pHMC122 (Generated using Biorender; https://www.biorender.com/). B. Immunofluorescence analysis of Rabies virus G protein expression in HEK293 producer cells used for pseudovirion generation. Cells transfected with full-length G protein expressing plasmid were immunostained with 17C7 monoclonal antibody conjugated with Alexafluor 488, 24h post-transfection. Nuclei were counter-stained with DAPI and cells were imaged under a Leica Stellaris 5 confocal microscope equipped with 40x objective (3x Zoom). C. Supernatants (100 μl) containing pseudovirions generated from HEK293 producer cells transfected either with Rabies G coding plasmid (pHMC122) (2 × 104 FFU R-PV-GFP pseudovirion particles) or mock plasmid (pCDNA3.1), were used for infecting the target cells. Monolayers of Vero and HEK293T cells grown in 96-well plates were used as target cells to find the permissiveness to R-PV-GFP infection at a MOI of 1. Cells were imaged 24h post-infection.
Fig 2.
Susceptibility of R-PV-GFP to neutralization by anti-rabies G monoclonal antibody.
Indicated two-fold dilutions of the anti-Rabies monoclonal antibody 17C7 were preincubated with the R-PV-GFP pseudovirions (2 × 104 FFU) for 1hr at 37 °C; and were used to infect HEK293T target cells. Similar dilutions of a control antibody (4G2) was used as the negative control. Live cells were imaged 12 h post-infection using Zeiss inverted fluorescence microscope equipped with FITC excitation-emission filter sets.
Fig 3.
Susceptibility of R-PV-GFP to neutralization by immune serum from vaccine recipient.
Indicated two-fold dilutions of the serum from an individual who received post-exposure prophylactic vaccination in 100 μl of 2% FBS containing DMEM were pre-incubated with an equal volume of the medium containing 2 × 104 R-PV-GFP pseudovirion particles for 1hr at 37 °C; and were used to infect monolayers of HEK293T target cells cultured in 96-well plates. Similar dilutions of serum from an unvaccinated individual was used as a negative control. Live cells were imaged 12 h post-infection using Zeiss inverted fluorescence microscope equipped with FITC excitation-emission filter sets.
Fig 4.
Generation and evaluation of R-PV-SEAP.
A. Scheme showing generation of Rabies pseudovirus expressing secreted alkaline phosphatase as reporter (R-PV-SEAP) using G*ΔG-SEAP rVSV and full-length Rabies virus G protein expressing plasmid pHMC122 (Generated using Biorender; https://www.biorender.com/). B. Immunofluorescence analysis of R-PV-SEAP infection on HEK 293T cells. The panel represents the infection of target cells with 2500 TCID50 Rabies G*(∆G- SEAP) rVSV pseudovirion (top panel) and VSV G*(∆G- SEAP) rVSV pseudovirion (bottom panel) respectively. The infected cells were fixed for immunofluorescence with 17C7 mAb (primary antibody) followed by anti- human Alexa fluor 488 secondary antibody. Pseudoviruses on the surface and within the cells are stained green and nuclei stained blue with DAPI. Scale bar, 20 μM. C. SEAP activity in the culture supernatants HEK293T target cells upon infection with increasing concentrations of R-PV-SEAP pseudovirions 24h post-infection. Each value is a mean ± SD of absorbance from two independent experiments (N = 2). D. Neutralization efficiency of anti-Rabies virus G monoclonal antibody (17C7) and a negative control antibody (4G2) at 1:64 dilution against varying concentrations of R-PV-SEAP pseudovirions. The antibodies were pre-incubated with the R-PV-SEAP pseudovirions for 1hr at 37 °C; and were used to infect HEK293T target cells. Cell culture supernatants were collected at 24h post-infection and subjected to SEAP assay. Each value is a mean ± SD of absorbance from two independent experiments (N = 2).
Fig 5.
Neutralization efficiency of R-PV-SEAP by reference antibodies and human serum samples with varying RFFIT titre.
A. 100TCID50 of R-PV-SEAP pseudovirions were subjected to neutralization by varying dilutions of the 17C7 anti-Rabies G monoclonal antibody and a non-neutralizing negative control antibody (4G2) by incubating at 37 °C for 1hr; and then infecting HEK293T target cells. At 12h post-infection, the culture supernatants were subjected to SEAP assay and percentage neutralization was calculated against pseudovirus infected controls. Each value represents a mean ± SD of absorbance from three independent experiments. B. A representative colorimetric reaction plate. 100TCID50 of the R-PV-SEAP pseudovirions were subjected to neutralization by incubating with varying dilution of a WHO reference serum (2IU/mL) for 1hr at 37 °C; and subsequently used to infect target HEK293T cells. Media controls and cells infected with un-neutralized pseudovirus were kept as controls. Visualization of the SEAP activity in the culture supernatants after 12h are shown. C. Neutralization efficiency of human serum samples from rabies vaccinated individuals with varying levels of Rabies Fluorescent Focus Inhibition Test (RFFIT) positivity against 100 TCID50 of R-PV-SEAP pseudovirions. Each data point represents mean ± SD of absorbance from two independent experiments.
Table 1.
Comparison of PVNT and RFFIT results of human serum samples at 1:64 serum dilution.
Fig 6.
Concordance of Pseudovirus neutralization titres (PVNT) with RFFIT titers.
Neutralization efficiency of human serum samples from 71 rabies vaccine recipients with known RFFIT titres were evaluated against 100 TCID50 of R-PV-SEAP and plotted. A. Number of samples showing positivity at 50% neutralization efficiency cut-off. Border-line positive samples are indicated by the rectangular box. B. Number of samples showing positivity at 75% neutralization efficiency cut-off. Individual values calculated as mean from reading in duplicates are represented as data points. C. Correlation of pseudovirus neutralization titres (PVNT) and RFFIT titres of 71 human serum samples. PVNT values were converted to International Units with respect to the WHO reference serum neutralization values and plotted. A few of the data points represent multiple samples having identical neutralization titers. D. Receiver operator characteristics (ROC) of the PVNT assay. Sensitivity and specificity values of the PVNT Assay using the 71 samples at varying positivity cut-offs were calculated and plotted (Table 2). A few of the data points represent multiple samples having identical values.
Table 2.
PVNT Assay performance of RFFIT validated human serum samples (n = 71) at different neutralization cut-offs.
Fig 7.
Overall Scheme of R-PV-SEAP pseudovirion production and pseudovirus neutralization test (PVNT).
(Generated using Biorender; https://www.biorender.com/).