Development of a novel hybrid alphavirus-Nipah virus pseudovirion for rapid quantification of vaccine-induced neutralization antibodies.

Figures

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  Figure 1.. Design, assembly, and infection of Ha-NiV particles. (A) Illustration of the design and assembly of Ha-NiV vector and particles. The alphavirus-based genomic vector contains a RSV promoter that transcribes viral RNA genome to be packaged into Ha-NiV particles. Shown are the 5’ untranslated region followed by open-reading frames coding for nonstructural proteins (nsp) 1–4 from Semliki Forest virus (SFV), viral subgenomic promoters for Luc and GFP reporter expression, the 3’ untranslated region, and a poly(A) tail that is self-cleaved by the hepatitis delta virus ribozyme (RZ). To assemble viral particles, HEK293T cells were co-transfected with the Ha-Niv vector and the vectors expressing the 4 structural proteins of NiV (G, F, M, and N). Ha-NiV particles in the supernatant were harvested at 48 h. (B) Infection of target cells with Ha-NiV(GFP) particles. HEK293T cells were infected with Ha-NiV (GFP) particles. GFP expression was observed 24 h post infection. (C) Rapid reporter expression following Ha-NiV infection. Vero cells were infected with Ha-NiV(Luc) for 6 h, washed, and then lysed and analysed for Luc expression. The addition of virus to cells was defined as time “0.”…

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  Figure 2.. Validation of Ha-NiV(Luc) particles for rapid quantification of neutralizing antibodies. (A) Quantification of neutralizing antibodies with Ha-NiV(Luc) particles. Shown are the concentration-dependent inhibition of Ha-NiV(Luc) by a recombinant monoclonal antibody (12B2) to NiV F glycoprotein. 12B2 was serially diluted and incubated with Ha-NiV(Luc) particles for 1 h at room temperature. The Ha-NiV(Luc)-antibody complex was used to infect HEK293T cells. Neutralization activities were quantified by luciferase assay at 18 h post addition of virus to cells. (B) The IC50 was calculated using the relative percentage of infection with or without 12B2 neutralization.

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  Figure 3.. Validation of Ha-NiV(Luc) for rapid quantification of anti-sera from NiV-infected African green monkeys. (A) Illustration of the procedure for rapid quantification of anti-serum with Ha-NiV(Luc). (B) Quantification of neutralization activities of anti-sera with Ha-NiV(Luc) particles. Shown is the anti-serum concentration-dependent inhibition of Ha-NiV(Luc) by a standard neutralizing antibody (Standard) or the individual anti-sera from 16 NiV-infected animals. Neutralization activities were quantified by luciferase assay at 18 h post addition of the antibody-virus complex to cells. Control serum was from uninfected animal sera (uninfected).

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  Figure 4.. Quantification of anti-sera from NiV-infected African green monkeys using PRNT. For comparison with Ha-NiV nAB assay, the same anti-sera from 16 NiV-infected animals (Figure 3(B)) were quantified for neutralization activities using the plaque reduction neutralization test (PRNT). Shown is the concentration-dependent reduction of NiV plaques by individual anti-sera or a standard neutralizing antibody (Standard). Uninfected animal sera (uninfected) were also used for negative control.

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  Figure 5.. Correlation of serum neutralization activities quantified with Ha-NiV(Luc) and PRNT. (A) The neutralization activities of the anti-sera from 16 NiV-infected animals were quantified with the Ha-NiV(Luc) assay (Figure 3) or PRNT (Figure 4), and the IC50 values were calculated and plotted. (B) The IC50 values from a were plotted for the correlation of IC50 between the Ha-NiV(Luc) assay and PRNT.