Virol J. 2012 Jan 25;9:32. doi: 10.1186/1743-422X-9-32.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

Ou W, Delisle J, Jacques J, Shih J, Price G, Kuhn JH, Wang V, Verthelyi D, Kaplan G, Wilson CA.

Source

Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Bldg, 29B, Room 5NN22, 8800 Rockville Pike, Bethesda, MD 20892, USA.

Abstract

BACKGROUND:

The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system.

METHODS:

To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs.

RESULTS:

Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced.

CONCLUSION:

Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

PMID:: 22273269; [PubMed - indexed for MEDLINE]
PMCID:: PMC3284443

Free PMC Article

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Figure 1

Antigens used for immunization. A. Schematic diagram of the full-length GP_1,2of Ebola virus (EBOV GP_1,2, GenBank# NC_002549) that encodes a polyprotein which upon cleavage yields two subunits, GP₁and GP₂, linked together through a disulfide bond. GP₁contains the receptor binding site (RBS) and the highly variable, highly glycosylated, and dispensable mucin-like domain (MLD). GP₂contains an extracellular domain (ECD), a transmembrane domain (TM) and a cytoplasmic tail (CT). The numbers above the diagram represent the amino acid residue numbers. B. Schematic diagram of the resulting protein expressed by plasmids used for immunization studies or to derive in vitro VLPs. EBOV GPΔMLD was deleted in the MLD domain and the cleavage site between GP₁and GP₂[40], so the resulting protein expressed is a single molecule. C. Production scheme for VLPs. Supernatant from HEK 293T cells containing VLPs was centrifuged through a 20% sucrose cushion to concentrate and partially purify the VLP, which was further purified to remove endotoxin. D. Verification of EBOV GPΔMLD incorporation into VLPs. The VLP product was resolved through a NuPAGE 4-12% Bis-Tris gel, the total protein was evaluated with silver staining, and EBOV GPΔMLD was detected by western blot.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 3

Antibody response. Two weeks after the third immunization, sera were collected and pooled for each group. The anti-GP_1,2antibody titers for the DNA group (A) and DNA/VLP group (B) were determined using a sandwich ELISA. GP_1,2-Fc or the negative control Fc was captured to the ELISA plate using an anti-Fc antibody. Antibody titer was defined as the highest dilutions where the OD of the sample was higher than 0.15 and at least two times that of the control. The same assay was used to determine the dynamics of anti-GP_1,2antibodies for the DNA group (C, all diluted at 1:64) and DNA/VLP group (D, all diluted at 1:2,000). The syringe symbol indicates the time points when mice were injected with the indicated immunogen. (E) Sera from mice immunized with DNA/VLP or VLP were collected 2 weeks after the third immunization and analyzed by western blot against lysates from cells transiently expressing full-length GP_1,2(E, EBOV) or from cells transfected with no plasmid DNA (M, mock)

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 5

Neutralization of rVSV-ZEBOVGP. Either wild-type VSV or recombinant VSV with its envelope G protein replaced with EBOV GP_1,2[rVSV-ZEBOVGP] was pre-incubated with the pooled sera, as indicated from each group at the final concentration of 1:25 dilution in the presence (A and C) or absence (B and D) of complement. Vero cells were exposed to the virus/serum mixture, and the virus titer was quantified by counting the number of plaques. The data shown represent analyses of pooled sera collected from mice in each group at week 40: VLP (n = 5), DNA (n = 6), DNA/VLP (n = 7), or serum from single animals for each of the following groups, PBS, CpG, and CpG/PBS. E, the data represent analysis of the sera collected from the indicated individual mice at week 37 prior to the last boosting. Shown here are the average titers +/- standard deviation (SD) of the triplicate samples tested. The titer was normalized to the inoculation titer, which was calculated from transduction in the absence of any mouse serum. Representative data from two or more experiments are shown.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 7

Cellular Immune Response. Splenocytes of each mouse in the VLP group (n = 5), DNA group (n = 6) and the DNA/VLP (n = 7) were stimulated with three EBOV GP_1,2peptides (ZGP-1, ZGP-4 and ZGP-5) or no peptide as a negative control. Three replicates were used for each combination. Splenocytes from a single mouse were used for each negative control group: CpG, CpG/PBS, and PBS. The numbers of IFN-γ secretion cells (spots) were counted using an ELISPOT reader (Axio Imager M2, Zeiss, Thornwood, NY). Representative data from two experiments are shown.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 2

Immunization and bleeding scheme. Mice were divided into six groups, ranging from 3 to 10 mice per group: CpG (n = 3), CpG/PBS (n = 3) and PBS (n = 4) served as the negative controls for DNA (n = 10), DNA/VLP (n = 10) and VLP (n = 10) groups, respectively. Immunization dose and route of administration for each group is as follows: CpG (16 μg CpG/100 μl/mouse/i.m. injection), PBS (100 μl endotoxin-free PBS/mouse/i.p. injection), DNA (50 μg of pVR1012-EBOVGPΔMLD + pMLV-GagPol at a 2:1 ratio + 16 μg CpG/100 μl/mouse/i.m. injection), VLP (33 μg retroVLP/100 μl/mouse/i.p. injection). The DNA, VLP, and CpG were formulated with endotoxin-free PBS. Immunization and blood collection schedules are shown. Mice were killed (†) at week 39 and 40. The symbols of syringes and blood drops represent immunization and blood collection, respectively.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 4

Cross-species reactivity of anti-EBOV GPΔMLD antibodies. Shown here are western blots using lysates from mock-transfected cells or from cells transfected with plasmids encoding full-length GP_1,2of EBOV, SUDV or TAFV, blotted with the pooled sera collected 2 weeks after the third immunization of the DNA/VLP group at 1:2000 dilution (A), the VLP group at 1:2000 dilution (B), or a control anti-GP_1,2rabbit polyclonal antibody R.F88-2 at 1:10,000 dilution [42] (C)

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Figure 6

Neutralization of MLV pseudotyped virus bearing envelopes from different viruses. β-galactosidase-expressing MLV pseudotypes bearing the GP_1,2of EBOV, SUDV, TAFV, or BDBV or the G protein of VSV were pre-incubated with the pooled sera of each group (VLP (n = 5), DNA (n = 6), DNA/VLP (n = 7)), which were collected at week 40, at the final concentration of 1:25 dilution. The control group represents a pool of sera collected from one mouse in each of the negative control groups: CpG, CpG/PBS, and PBS. Vero cells were exposed to virus/antibody mixtures and the virus titer was quantified by counting the number of blue forming units (BFU) under microscope. Two or three replicates were used in each experiment. For each pseudotype, the titer observed in the presence of sera pooled from negative control mice was normalized to 100%, and all other titers for that particular pseudotype are reported as relative to that value. Representative data from two or more experiments are shown.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

Virol J. 2012;9:32-32.

Publication Types, MeSH Terms, Substances

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Research Support, Non-U.S. Gov't

MeSH Terms

Animals
Antibodies, Neutralizing/blood
Antibodies, Viral/blood*
Cross Reactions*
Ebola Vaccines/administration & dosage
Ebola Vaccines/genetics
Ebola Vaccines/immunology*
Ebolavirus/immunology*
Female
Glycoproteins/genetics
Glycoproteins/immunology
Immunity, Cellular
Mice
Mice, Inbred BALB C
Protein Structure, Tertiary
Retroviridae/genetics
Retroviridae/immunology*
Vaccines, Synthetic/administration & dosage
Vaccines, Synthetic/genetics
Vaccines, Synthetic/immunology
Vaccines, Virosome/administration & dosage
Vaccines, Virosome/genetics
Vaccines, Virosome/immunology
Viral Proteins/genetics
Viral Proteins/immunology*
Virosomes/genetics
Virosomes/immunology*

Substances

Antibodies, Neutralizing
Antibodies, Viral
Ebola Vaccines
Glycoproteins
Vaccines, Synthetic
Vaccines, Virosome
Viral Proteins
Virosomes

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Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

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Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

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