Nipah pseudovirus system enables evaluation of vaccines in vitro and in vivo using non-BSL-4 facilities - PubMed (original) (raw)

Nipah pseudovirus system enables evaluation of vaccines in vitro and in vivo using non-BSL-4 facilities

Jianhui Nie et al. Emerg Microbes Infect. 2019.

Abstract

Because of its high infectivity in humans and the lack of effective vaccines, Nipah virus is classified as a category C agent and handling has to be performed under biosafety level 4 conditions in non-endemic countries, which has hindered the development of vaccines. Based on a highly efficient pseudovirus production system using a modified HIV backbone vector, a pseudovirus-based mouse model has been developed for evaluating the efficacy of Nipah vaccines in biosafety level 2 facilities. For the first time, the correlates of protection have been identified in a mouse model. The limited levels of neutralizing antibodies against immunogens fusion protein (F), glycoprotein (G), and combination of F and G (FG) were found to be 148, 275, and 115, respectively, in passive immunization. Relatively lower limited levels of protection of 52, and 170 were observed for immunogens F, and G, respectively, in an active immunization model. Although the minimal levels for protection of neutralizing antibody in passive immunization were slightly higher than those in active immunization, neutralizing antibody played a key role in protection against Nipah virus infection. The immunogens F and G provided similar protection, and the combination of these immunogens did not provide better outcomes. Either immunogen F or G would provide sufficient protection for Nipah vaccine. The Nipah pseudovirus mouse model, which does not involve highly pathogenic virus, has the potential to greatly facilitate the standardization and implementation of an assay to propel the development of NiV vaccines.

Keywords: Nipah; animal model; neutralization; pseudo virus.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Optimization of the in vitro neutralization assay. (a) Schematic drawing of NiV pseudovirus production. Identification of the optimal backbone plasmid (b), transfection reagent (c), ratio of F and G plasmids (d), ratio of backbone and envelope plasmids (e), western blotting for F, G and p24 in pseudovirus (f), NiV pseudovirus under electronic microscopy (g), target cell (h). Non-transfected cells were included as negative controls in (b), (c), (d), (e) and (h). Culture supernatants from cells expressing different combinations of NiV proteins were filtered (0.45-μm pore size), then were pelleted through 25% sucrose cushion by ultracentrifugation at 100,000 g for 2.5 hr. The resulting viral pellets were resuspended in PBS and probed with FG immunized guinea pig serum and HIV-1 positive serum samples. As demonstrated by the highest luminescent signals, pSG3.Δenv.Fluc and Lipofectamine 3000 were determined to be the optimal backbone plasmid and transfection reagent, respectively. The optimal ratio for pcDNA3.1.F and pcDNA3.1.G was determined to be 1:2. The optimal ratio for the outer membrane protein genes and the backbone plasmids was found to be 1:4. (i) Effect of truncation of F and G on pseudovirus titre. Truncation of F and G could enhance titres of pseudovirus for about 3.6 fold. For Figure 1(b–e), h and i each experiment was performed twice (two replicates for each run) independently in different days. Mean with SD was shown for every condition.

Figure 2.

Figure 2.

Development of in vivo imaging mouse model for NiV pseudovirus. (a) Optimization of the infection routes. The mice were inoculated with NiV pseudovirus (2.5 × 107 TCID50) and the luminescent signals were detected at 2 dpi. Three challenge routes were investigated, including intraperitoneal (IP), intrathoracic (IC), and intravenous (IV) injections; the IT route yielded the highest signals compared with the other two routes. (b) Biodistribution of the NiV pseudovirus in mice. Various organs of the mice challenged via the IT route (in Figure 2(A)) were investigated using the luminescence detector. The total flux for each organ was collected from three mice, and the images of organs from one mouse are presented. Organs from non-infected mouse were included as negative control. High levels of flux were observed in the spleen and lung, the cast of which could also be found on the surface of the live mouse. (c) Identification of the optimal time point for detection. Flux signals for each mouse (3 mice in total) following inoculation with 4 × 106 TCID50 were recorded every 24 h for up to 5 days. The optimal detection point was determined as 3 dpi, which showed the highest signal level. Non-infected mouse were included as negative control. (d) Determination of the animal infectious dose. Fivefold serially diluted NiV pseudoviruses were injected IT into five groups of mice (5 mice/group) at an initial dose of 2.5 × 107 TCID50. The 50% animal infectious dose (AID50) for the pseudovirus was calculated to be 8.8 × 104 TCID50, and the pseudovirus dose was determined to be 50 AID50 for the in vivo infection assay, which is equivalent to 4.4 × 106 TCID50.

Figure 3.

Figure 3.

Identification of the protective correlates for passive immunization. Four groups of Balb/c mice (6 mice/group) were passively inoculated with 3-fold serially diluted anti-sera for immunogens F (a), G (b), and F and G combined (c), respectively. One hour after passive transfusion, each mouse was bled for serum collection. Then, the mice were inoculated with the NiV pseudovirus and the luminescent signals were detected at 3 dpi. Flux signals and NAb titres of the serum samples were detected for each mouse. Mice transfused with sera from non-immunized guinea pigs were included as control. The flux signals and NAb titres of each group were compared with the control group using student’s t test (*p < .05, **p < .01, ***p < .001, ****p < .0001). The correlation of the log-transformed values for the flux and NAb titres were analysed for each immunogen. Good linear correlations were found between the log-transformed total flux and the ID50 values for each immunogen. The limited full protection levels of NAbs were identified as 148, 275, and 115 for immunogens F, G, and FG, respectively. For Figure 3(a–c), all the three experiments were performed simultaneously with the same pseudovirus control.

Figure 4.

Figure 4.

Identification of the protective correlates for active immunization. (a) For F antigen, six groups of mice (6 mice/group) were actively inoculated with serially diluted DNA vaccines (3-fold serial dilutions with an initial amount of 50 µg/mouse) for immunogens F (a), G (b), and F and G combined (c), respectively. Fourteen dpi, mice were bled for serum collection and challenged with the NiV pseudovirus. Luminescent signals were detected 3 days after infection. The flux signals and NAb titres were detected for each mouse. Mice with non-immunization were included as control. The flux signals and NAb titres of each group were compared with the control group using student’s t test (*p < .05, **p < .01, ***p < .001, ****p < .0001). The median effective dose (ED50) values were determined to be 3.79 μg (95% confidence interval: 1.13–13.13 μg), 4.00 μg (95% CI: 0.82–28.71 μg), and 3.38 μg (95% CI: 0.88–12.00 μg) for the DNA vaccines expressing F, G, and FG, respectively. The correlation between the log-transformed values for the flux and NAb titres were analysed. Significant linear correlations were found between the log-transformed total flux and the ID50 values for each immunogen. The limited protection levels of NAbs were identified as 52, 170, and 123 for immunogens F, G, and FG, respectively. For the F immunogen study, one mouse in the fifth group died after anesthetization just before bioluminescence detection, so the data for this animal were not included in the analysis. Data for Figure 4(a–c) were generated in one big experimental set-up.

Similar articles

Cited by

References

    1. Chua KB, et al. . Nipah virus: a recently emergent deadly paramyxovirus. Science. 2000;288:1432–1435. doi: 10.1126/science.288.5470.1432 - DOI - PubMed
    1. Hsu VP, Hossain MJ, Parashar UD, et al. . Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis. 2004;10:2082–2087. doi:10.3201/eid1012.040701. - DOI - PMC - PubMed
    1. Lo MK, Lowe L, Hummel KB, et al. . Characterization of Nipah virus from outbreaks in Bangladesh, 2008–2010. Emerg Infect Dis. 2012;18:248–255. doi:10.3201/eid1802.111492. - DOI - PMC - PubMed
    1. Luby SP, Hossain MJ, Gurley ES, et al. . Recurrent zoonotic transmission of Nipah virus into humans, Bangladesh, 2001–2007. Emerg Infect Dis. 2009;15:1229–1235. doi:10.3201/eid1508.081237. - DOI - PMC - PubMed
    1. Sazzad HMS, Hossain MJ, Gurley ES, et al. . Nipah virus infection outbreak with nosocomial and corpse-to-human transmission, Bangladesh. Emerg Infect Dis. 2013;19:210–217. doi:10.3201/eid1902.120971. - DOI - PMC - PubMed

MeSH terms

Substances

Grants and funding

The study was supported by the National Science and Technology Major Projects of Infectious Disease [grant number 2017ZX10304402].

LinkOut - more resources