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. 2020 Aug;43(4):375-387.
doi: 10.1016/j.bj.2020.06.003. Epub 2020 Jun 6.

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

Sheng-Wen Huang  1 , Ching-Hui Tai  2 , Yin-Mei Hsu  3 , Dayna Cheng  2 , Su-Jhen Hung  1 , Kit Man Chai  2 , Ya-Fang Wang  2 , Jen-Ren Wang  4
Affiliations

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

Sheng-Wen Huang et al. Biomed J. 2020 Aug.

Abstract

Background: Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses.

Methods: We developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses.

Results: The result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses.

Conclusion: Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

Keywords: Antibody; Antigen; Avian influenza virus; Pseudovirus; Pseudovirus system; SARS-CoV-2.

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Conflict of interest statement

Conflicts of Interest The authors have declared that there is no conflict of interest.

Figures

Fig. 1
Fig. 1
Lentiviral pseudovirus system of SARS-CoV or SARS-CoV-2 and avian influenza H5. Structural protein genes, including S protein of SARS-CoV or SARS-CoV-2 and HA/NA protein of avian influenza H5, were subcloned into envelope expression plasmid derived from pMD.G vector. To generate SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses, we co-transfected the structural protein expressing either S protein or HA and NA vectors, a package vector, and a reporter vector into HEK-293T cells. Generated SARS-CoV or SARS-CoV-2 and avian influenza H5Nx pseudoviruses were harvested and transduced into Vero-E6 or MDCK cells, respectively.
Fig. 2
Fig. 2
Immunoblotting of S protein of SARS-CoV or SARS-CoV-2 and HA protein of avian influenza H5. (A) S proteins of SARS-CoV and SARS-CoV-2 were immunoblotted with mouse anti-SARS-CoV S protein antibody and mouse anti-HA tag protein antibody, respectively. (B) HA proteins of avian influenza H5 were immunoblotted with mouse anti-influenza virus H5 HA protein antibody. As the antibody recognized the HA2 epitope, both of HA0 and HA2 protein were detected by the immunoblotting.
Fig. 3
Fig. 3
Pseudovirus transduction of SARS-CoV or SARS-CoV-2 and avian influenza H5Nx. Generated (A) SARS-CoV or SARS-CoV-2 and (B) avian influenza H5Nx pseudoviruses were transduced into Vero-E6 or MDCK cells, respectively. Red fluorescence indicated the cells transduced by the indicated pseudoviruses with RFP reporter gene. (C) Transduction titers of avian influenza H5Nx pseudoviruses were determined according to the numbers of cells expressing red fluorescence.
Fig. 4
Fig. 4
Transduction optimization of SARS-CoV and SARS-CoV-2 pseudoviruses. Generated SARS-CoV and SARS-CoV-2 pseudoviruses were transduced into Vero-E6 cells. Different transduction medium with (A) 2% FBS or (B) 2.5 μg/ml trypsin. Using transduction medium with 2% FBS showed higher transduction rate for SARS-CoV and SARS-CoV-2 pseudoviruses. Using transduction medium with 2.5 μg/ml trypsin obviously reduced transduction rate, especially for SARS-CoV pseudoviruses.
Fig. 5
Fig. 5
Dose-dependent transduction rates of SARS-CoV-2 pseudoviruses. Generated SARS-CoV-2 pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV-2 was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV-2 pseudovirus was quantified as 2.36 × 106 transduction unit.
Fig. 6
Fig. 6
Dose-dependent transduction rates of SARS-CoV pseudoviruses. Generated SARS-CoV pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of SARS-CoV was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of SARS-CoV pseudovirus was quantified as 2.33 × 105 transduction unit.
Fig. 7
Fig. 7
Dose-dependent transduction rates of VSV-G pseudoviruses. Generated VSV-G pseudoviruses were serially diluted and then transduced into Vero-E6 cells. Transduction rate of VSV-G was gradually reduced in a dose-dependent manner. According to the transduction rate curve, the titer of VSV-G pseudovirus was quantified as 3.85 × 106 transduction unit.
Fig. 8
Fig. 8
Antigenic cartography of avian influenza virus H5Nx pseudoviruses. Antigenic cartography displays the antigenic properties of avian influenza virus H5Nx pseudoviruses. The viruses are shown in color and the antisera as open shapes. Distances between each subtype and antiserum on the map represent the corresponding neutralization assay titers. Both the vertical and horizontal dimensions represent antigenic distance; only the relative positions of antigens and antisera can be determined, i.e., the map can be freely rotated. Each grid line represents a unit of antigenic distance, corresponding to a 2-fold dilution of antiserum in the neutralization table.
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