A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization - PubMed (original) (raw)

A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization

Renqiang Liu et al. Antiviral Res. 2018 Feb.

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) has been a highly threatening zoonotic pathogen since its outbreak in 2012. Similar to SARS-CoV, MERS-CoV belongs to the coronavirus family and can induce severe respiratory symptoms in humans, with an average case fatality rate of 35% according to the World Health Organization. Spike (S) protein of MERS-CoV is immunogenic and can induce neutralizing antibodies, thus is a potential major target for vaccine development. Here we constructed a chimeric virus based on the vesicular stomatitis virus (VSV) in which the G gene was replaced by MERS-CoV S gene (VSVΔG-MERS). The S protein efficiently incorporated into the viral envelope and mediated cell entry through binding its receptor, human DPP4. Knockdown of clathrin expression by siRNA drastically abrogated the infection of VSVΔG-MERS in Vero cells. Furthermore, in animal studies, the recombinant virus induced neutralizing antibodies and T cell responses in rhesus monkeys after a single intramuscular or intranasal immunization dose. Our findings indicate the potential of the chimeric VSVΔG-MERS as a rapid response vaccine candidate against emerging MERS-CoV disease.

Copyright © 2017 Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

Generation of VSVΔG-MERS and VSVΔG-eGFP-MERS and expression of S protein. Schematic representation of the recombinant viruses (A). S protein expression in VSVΔG-MERS and VSVΔG-eGFP-MERS infected Vero E6 cells by indirect immunofluorescence staining (B) and Western blot (C).

Fig. 2

Fig. 2

Growth properties of recombinant viruses in Vero E6 cells. VSV, VSVΔG-MERS and VSVΔG-eGFP-MERS were inoculated on Vero E6 cells on 6-well plate at a MOI = 0.01, supernatant was taken out from 12 h to 96 h at 12-h intervals. The titer was expressed as the reciprocal of the highest dilution titer (fluorescence forming unit, FFU).

Fig. 3

Fig. 3

Incorporation of S protein into viral particles. S protein efficiently incorporated into the viral particles as indicated by electron and immunoelectron microscopy (A). VSV-MERS, a recombinant VSV virus has MERS-CoV S gene inserted between VSV M and G gene as an additional transcription unit (unpublished work). VSV-MERS has both G and S protein on viral surface. Long arrows indicate S protein; short stealth arrows indicate VSV G protein.

Fig. 4

Fig. 4

VSVΔG-MERS and VSVΔG-eGFP-MERS utilize human DPP4 as receptor. BHK-21 cells and human DPP4-transfected BHK-21 cells were infected with VSVΔG-eGFP-MERS (A). Vero E6 cells transfected with DPP4 siRNA, irrelevant siRNA, VSV L siRNA and mock siRNA were infected with VSVΔG-eGFP-MERS (B).

Fig. 5

Fig. 5

Clathrin is important for VSVΔG-eGFP-MERS infection. Vero E6 cells were transfected with clathrin siRNA, VSV L siRNA, mock siRNA and irrelevant siRNA to observe the impact of clathrin-knockdown on VSVΔG-eGFP-MERS (I A) or VSV-eGFP (I B) infection. The infection ratio (mean ± SD) and statistical analysis is presented (II A and B); significant differences between conditions is designated with (a) in panel II A and (b) in panel II B; p < .01.

Fig. 6

Fig. 6

Humoral responses of VSVΔG-MERS-immunized mice. 10 mice were intramuscularly immunized with 1 × 106 FFU VSVΔG-MERS, mice were observed and weighed daily for 14 days (A). At 21 days after the first dose, mice were given the booster dose. S protein specific IgG (B) and neutralizing antibody (C) was analyzed. (a), (b) p < .01.

Fig. 7

Fig. 7

Humoral immune responses of rhesus monkeys to VSVΔG-MERS vaccination. Monkeys were immunized with 2 × 107 FFU of recombinant virus intramuscularly (i.m) or intranasally (i.n). Blood samples were collected at the indicated time-points. Serum IgG (A) and neutralizing antibody (B) were determined. The neutralizing antibody from both routes at the same time-point was compared. (a) p < .01.

Fig. 8

Fig. 8

T cell responses of immunized rhesus monkeys to VSVΔG-MERS vaccination. PBMCs from immunized monkeys were tested for MERS-CoV S peptide-specific T cell responses by ELISPOT. An S protein overlapping peptide pool which contained 269 peptides (15-mers designed for CD8+ T cells) were used to stimulate the monkey PBMCs. Ex-vivo IFN-γ ELISPOT assay was performed to determine the active cells. Six peptides that yielded the most spots were selected for presentation. No statistical difference was observed in the peptides between the i.m and i.n groups.

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