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. 2011 Feb 1;29(6):1248-57.
doi: 10.1016/j.vaccine.2010.11.079. Epub 2010 Dec 8.

Yellow Fever 17D-vectored Vaccines Expressing Lassa Virus GP1 and GP2 Glycoproteins Provide Protection Against Fatal Disease in Guinea Pigs

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Yellow Fever 17D-vectored Vaccines Expressing Lassa Virus GP1 and GP2 Glycoproteins Provide Protection Against Fatal Disease in Guinea Pigs

Xiaohong Jiang et al. Vaccine. .
Free PMC article

Abstract

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa.

Figures

Fig. 1
Fig. 1
Construction of genetically stable YF17D/LASV-GP vaccines. (A) Schematic representation of recombinant YF17D-LASV virus carrying the ΔGPC insert. In the initial constructs [42] the insert (1,365nt) was flanked by homologous E-derived sequences (red box) to insure the proper processing of recombinant polyprotein. In a new set of recombinant viruses, the fusion sequence between GP2 and NS1 (pink box) was substituted with heterologous sequences derived either from WNV or artificially designed (Art) to reduce homology with YF17D. Arrows indicate the sequences designed for PCR amplification of LASV ΔGPC insert and flanking areas to track the insert size during passages in BHK-21J cells. (B) Genetic stability of recombinant YF17D/LASV-ΔGPC. Virus-specific RNA was labeled with 3[H]-Uridine in the presence of actinomycin D and intracellular RNA at passage 1, 5 and 10 was analyzed by gel electrophoresis. The RNA from YF17D-infected cells was used as a control. Arrows indicate recombinant YF17D/LASVΔGPC RNA, 12,227nt; and parental YF17D RNA, 10,862nt. (C), (D) Genetic structure of recombinant YF17D/LASV-GP1 and –GP2 viruses, respectively. Cleavage site between the YF17D E and GP2 protein was designed as described in Materials and Methods. (E) The size of LASV GP inserts after electroporation (Elpo) or after 10 passages in BHK-21J. Two independent passage experiments were performed, Exp1 and Exp2. Arrows indicate recombinant YF17D/LASV-GP1 RNA, 11,609nt; and parental YF17D RNA, 10,862nt. The size of YF17D/LASV-GP2 RNA is 11,672nt.
Fig. 1
Fig. 1
Construction of genetically stable YF17D/LASV-GP vaccines. (A) Schematic representation of recombinant YF17D-LASV virus carrying the ΔGPC insert. In the initial constructs [42] the insert (1,365nt) was flanked by homologous E-derived sequences (red box) to insure the proper processing of recombinant polyprotein. In a new set of recombinant viruses, the fusion sequence between GP2 and NS1 (pink box) was substituted with heterologous sequences derived either from WNV or artificially designed (Art) to reduce homology with YF17D. Arrows indicate the sequences designed for PCR amplification of LASV ΔGPC insert and flanking areas to track the insert size during passages in BHK-21J cells. (B) Genetic stability of recombinant YF17D/LASV-ΔGPC. Virus-specific RNA was labeled with 3[H]-Uridine in the presence of actinomycin D and intracellular RNA at passage 1, 5 and 10 was analyzed by gel electrophoresis. The RNA from YF17D-infected cells was used as a control. Arrows indicate recombinant YF17D/LASVΔGPC RNA, 12,227nt; and parental YF17D RNA, 10,862nt. (C), (D) Genetic structure of recombinant YF17D/LASV-GP1 and –GP2 viruses, respectively. Cleavage site between the YF17D E and GP2 protein was designed as described in Materials and Methods. (E) The size of LASV GP inserts after electroporation (Elpo) or after 10 passages in BHK-21J. Two independent passage experiments were performed, Exp1 and Exp2. Arrows indicate recombinant YF17D/LASV-GP1 RNA, 11,609nt; and parental YF17D RNA, 10,862nt. The size of YF17D/LASV-GP2 RNA is 11,672nt.
Fig. 2
Fig. 2
Plaque morphology of YF17D/LASV-GP viruses. YF17D/LASV-GP2 (A) and YF17D (B) viruses recovered after electroporation were plated on confluent monolayers of BHK-21J cells in 6-well plates and viruses were titrated by plaque assay as previously described [42]. The transfected cells were incubated for 96 hours, fixed with 8% formaldehyde and stained with crystal violet. The final titer was 1 × 108 and 2 × 107 PFU/ml for YF17D and YF17D/LASV-GP2, respectively.
Fig. 3
Fig. 3
Immunofluorescence staining of transfected cells. Cells transfected with YF17D/LAS-GP1 were fixed at 24 hours post electroporation, followed by staining for YF17D NS1 (green) and LASV GP1 (red). Nuclei were stained with Hoechst (blue). Magnification: × 40.
Fig. 4
Fig. 4
Pulse-chase labeling of YF17D proteins. Cells infected with either YFV17D or the recombinant viruses were radioactively labeled for 4 hours, or pulsed 30 min, followed by chasing for either 1 hour or 3.5 hours. Cell lysates were subjected to immunoprecipitation with mouse hyperimmune ascetic fluid to YF17D (ATCC). The precipitates were separated on 12.5% SDS-PAGE gel, and analyzed by phosphor-imaging. 1, label 4 hours; 2, pulse-labeling, 30 minutes; 3, 1 hour chase; 4, 3.5 hours chase. Viral proteins: NS5 (103 kDa), NS3 (70 kDa), E (50 kDa), NS1 (46 kDa), NS4B (27 kDa), PrM (26 kDa), NS2A (22 kDa), and NS2B (14 kDa) could be seen. Molecular weight markers are indicated on the left side.
Fig. 5
Fig. 5
Western-blot analysis of LASV-specific glycoproteins in cells infected with recombinant YF17D/LAS-GP1 and –GP2 viruses. The analysis was carried out with a 1:2,000 dilution of anti-GP1 and anti-GP antibodies as previously described [42]. Markers in kilodaltons are indicated on the side. The arrow in (A) indicates GP1 and in (B) indicates GP2 glycoproteins. The recombinant YF17D/LASV-GP2BglII contained natural YFV E – NS1 cleavage site with Leu/Asp mutation in the first amino acid of GP2. The recombinant YF17D/LASV-GP2fus contained a sequence encoding the first 14 aa of LASV GP1 inserted between YF17D E and LASV GP2 (see Materials and Methods for details).
Fig. 6
Fig. 6
Evaluation of immunogenicity of LASV vaccine candidates in CBA/J mice. (A) Vaccination control group: activated CD3+CD8+ cells peaked on day 14 after immunization. (B) Mapping of GPC H2k-restricted epitopes: on day 14 after vaccination splenocytes were stimulated with a peptide library consisting of overlapping 21-mer peptides derived from LASV GPC. Stimulated splenocytes were incubated in wells of the ELISPOT plates pre-coated with Abs against mouse IFN-γ and washed away. Cytokine-secreting cells or spot-forming cells (SFC) cells were detected and enumerated according to the manufacturer’s instructions for mouse IFN-γ ELISPOT (U-CyTech biosciences, Utrecht, The Netherlands). Responses to peptides derived from GP1 and GP2 are indicated by black bars. (C) CD8+ T cell responses in mice immunized with recombinant YF17D/LAS-GP1 and –GP2 viruses. Splenocytes from immunized mice were stimulated with peptide cocktails derived from full GPC (black boxes) or from GP1 or GP2 subunits (sparse boxes).
Fig. 7
Fig. 7
YF17D/LASV-GP1&GP2 vaccination protects guinea pigs from fatal outcome but does not prevent LASV infection. (A) Survival rate and clinical scores (insert). Clinical scoring was based on evaluation of responsiveness, edema, dehydration, rash, appearance, petechia, feed consumption, anorexia, stool, urine, fluid uptake (0, normal; 1, mild; 2, moderate; 3, severe. (B) Temperature (°F) was monitored by sensor-implants and recorded twice per day for each animal. (C) Weight of animals on day of challenge was assayed as 100%. Open circles, animals vaccinated with YF17D/LASV-GP1&GP2; open squares, animals vaccinated with ML29 (positive control); closed squares, negative control (animals inoculated with media). (D) Viral loads (LASV RNA copies) in vaccinated-challenged animals. Black boxes, animals challenged with LASV (no vaccine); white boxes, animals vaccinated with YF17D/LASV-GP1&GP2; spare boxes, animals vaccinated with ML29 (positive control).

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