MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques - PubMed (original) (raw)

. 2017 Feb 21;8(8):12686-12694.

doi: 10.18632/oncotarget.8475.

Xuexing Zheng 2 3 4, Weiwei Gai 2, Yongkun Zhao 2 4, Hualei Wang 2 4, Haijun Wang 2, Na Feng 2 4, Hang Chi 2, Boning Qiu 2, Nan Li 2, Tiecheng Wang 2 4, Yuwei Gao 2 4, Songtao Yang 2 4, Xianzhu Xia 2 4

Affiliations

MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques

Chong Wang et al. Oncotarget. 2017.

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans with a case fatality rate of over 39%, and poses a considerable threat to public health. A lack of approved vaccine or drugs currently constitutes a roadblock in controlling disease outbreak and spread. In this study, we generated MERS-CoV VLPs using the baculovirus expression system. Electron microscopy and immunoelectron microscopy results demonstrate that MERS-CoV VLPs are structurally similar to the native virus. Rhesus macaques inoculated with MERS-CoV VLPs and Alum adjuvant induced virus-neutralizing antibodies titers up to 1:40 and induced specific IgG antibodies against the receptor binding domain (RBD), with endpoint titers reaching 1:1,280. MERS-CoV VLPs also elicited T-helper 1 cell (Th1)-mediated immunity, as measured by ELISpot. These data demonstrate that MERS-CoV VLPs have excellent immunogenicity in rhesus macaques, and represent a promising vaccine candidate.

Keywords: Middle East respiratory syndrome coronavirus; immune response; nonhuman primates; vaccine; virus-like particles.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no competing interests.

Figures

Figure 1

Figure 1. Schematic of the recombinant baculovirus expressing MERS-CoV S, E and M genes

The Tn7 regions, gentamicin resistance gene (Gm), HSV tk polyadenilation signal [Tk p (A)], p10 promoter (p10), polyhedrin promoter (ph), SV40 polyadenylation signal [SV40 p (A)], and MERS-CoV isolate Al-Hasa_15_2013 genes are shown. S, spike protein; E, envelope protein; M, membrane protein.

Figure 2

Figure 2. IFA detection of expression of the triple baculoviruses in Sf9 infected cells

Cells were infected with the recombinant baculoviruses in A, C, E. and were mock infected in B, D, F. Cells were detected with anti-S polyclonal sera in A and B, anti-M polyclonal sera in C and D, and anti-E polyclonal sera in E and F, respectively.

Figure 3

Figure 3. TEM and IEM analysis of MERS-CoV VLPs

A. MERS-CoV VLPs produced by infected Sf9 cells and purified by sucrose gradientwere stained with 1% sodium phosphotungstate. Bar = 100 nm. B. The VLPs were incubated with anti-S polyclonal antibody and probed using a gold-labeled goat anti-mouse IgG antibody.

Figure 4

Figure 4. Detection of expression of S, E and M proteins in MERS-CoV VLPs

MERS-CoV VLPs analyzed by WB using A. rabbit anti-S polyclonal antibody (lane 1 MERS-CoV VLPs; lane 2 lysate from pFastBacDual infected cells served as a control) and B. mouse anti-M polyclonal antibody (lane 1 lysate from pFastBacDual infected cells served as a control; lane 2 MERS-CoV VLPs) and C. mouse anti-E polyclonal antibody (lane 1 lysate from pFastBacDual infected cells served as control; lane 2 MERS-CoV VLPs).

Figure 5

Figure 5. Comparison of spike glycoprotein expression in insect and mammalian cells

Western blot analysis of MERS-CoV VLPs (lane 1) and cell lysate from pcDNA3.1-MERS-S transfected BSR cells at 72 h after transfection (lane 2), lysate from uninfected BSR cells served as controls (lane 3). S protein was analyzed by immunoblotting using a polyclonal anti-S antibody.

Figure 6

Figure 6. NHP immunization procedure, RBD-specific antibody and neutralizing antibodies against MERS-CoV infection

A. Rhesus macaques (n=3) were vaccinated IM with 250 μg of MERS-CoV VLPs and Alum adjuvant (at 0, 2, 4, 6 weeks) and one group was treated with equivalent volumes of PBS as a control (n=3). Blood samples were collected from the femoral vein of monkeys before immunization and two weeks after each immunization. B. ELISA results show that immunized monkeys were able to induce a robust humoral response specific to RBD, with serum IgG titers of up to 1:1,280 after the fourth immunization. C. Rhesus macaque blood samples were collected from the femoral vein of monkeys two weeks after last immunization. MERS-CoV live virus-based inhibition assay were taken in Vero E6 cells in triplicate. The titers were determined as the highest serum dilutions that completely prevent CPE in at least 50% of the wells (NT50) and are expressed as mean +/- SEM. Statistical analysis between the two groups were analyzed by t-test, in which a p-value of less than 0.05 was considered statistically significant (** p<0.05).

Figure 7

Figure 7. Enzyme-linked immunospot assays of IFN-γ and IL-4 secretion in NHPs

PBMCs were isolated from NHPs and stimulated with the purified RBD of the MERS-CoV S protein. PBMCs secreting A. IFN-γ or B. IL-4 were quantitated using ELISpot assay. The data represent the mean +/- standard deviation (SD) of SFCs per million. Statistical analysis between the two groups were analyzed by t-test, in which a p-value of less than 0.05 was considered statistically significant (* p< 0.05).

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