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Vaccines and Antiviral Agents

Expression of MIP-1α (CCL3) by a Recombinant Rabies Virus Enhances Its Immunogenicity by Inducing Innate Immunity and Recruiting Dendritic Cells and B Cells

Ling Zhao, Harufusa Toriumi, Hualei Wang, Yi Kuang, Xiaofeng Guo, Kinjiro Morimoto, Zhen F. Fu
Ling Zhao
1Departments of Pathology
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Harufusa Toriumi
1Departments of Pathology
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Hualei Wang
1Departments of Pathology
2College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, China
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Yi Kuang
1Departments of Pathology
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Xiaofeng Guo
1Departments of Pathology
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Kinjiro Morimoto
3Yasuda Women's University, Hiroshima 731-0153, Japan
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Zhen F. Fu
1Departments of Pathology
4Infectious Diseases, University of Georgia, Athens, GA 30602
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  • For correspondence: zhenfu@uga.edu
DOI: 10.1128/JVI.00326-10
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  • FIG. 1.
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    FIG. 1.

    Construction and characterization of recombinant RABV with the MIP-1α gene cloned but without the protein expressed. (A) rHEP-MIP1α(−) was constructed by introducing two stop codons near the N terminus of the MIP-1α gene as described previously. le, leader region; N, nucleoprotein; P, phosphoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. (B) Virus growth curves were determined by infecting mouse neuroblastoma (NA) cells with either the rHEP, rHEP-MIP1α, or rHEP-MIP1α(−) virus at a multiplicity of infection (MOI) of 0.01. At days 1, 2, 3, 4, and 5 after infection, the culture supernatants were harvested, and viral titers in NA cells were determined with fluorescein isothiocyanate (FITC)-conjugated antirabies antibodies (FujiRab, Melvin, PA). Antigen-positive foci were counted under a fluorescence microscope (Zeiss, Germany), and viral titers were calculated as FFU per milliliter. All titrations were carried out in quadruplicate. (C) Expression of MIP-1α was determined by infecting NA cells with either the rHEP, rHEP-MIP1α, or rHEP-MIP1α(−) virus at an MOI of 0.001, 0.01, 0.1, or 1. After a 24-h incubation at 34°C, the culture supernatants were harvested, and the amounts of MIP-1α were determined by a murine MIP-1α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacture's protocol. The positive control (MIP-1α) was supplied with the ELISA kit. Checkered bar, rHEP; horizontally striped bar, rHEP-MIP1α; vertically striped bar, rHEP-MIP1α(−). (D) The pathogenicity of recombinant rHEP-MIP1α(−) was determined by inoculating BALB/c mice (6 to 8 weeks of age) i.c. with 105 FFU of either the rHEP, rHEP-MIP1α, or rHEP-MIP1α(−) virus or with medium alone (mock infection). Body weight was monitored daily. Data were obtained from 10 mice in each group and presented as mean values ± standard errors (SEs). The asterisk indicates a significant difference (P < 0.05) in results among the indicated experimental groups, as calculated by one-way analysis of variance (ANOVA) with the Holm-Sidak method.

  • FIG. 2.
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    FIG. 2.

    Immunogenicity of recombinant RABVs expressing chemokines. Groups of 10 ICR mice were immunized by the i.m. route with serial 10-fold dilutions of rHEP, rHEP-MIP1α, rHEP-MIP1α(−), rHEP-RANTES, or rHEP-IP10. (A) At day 20 after immunization, blood was obtained, and the sera were used to determine VNA titers, using the RFFIT as described previously (27). Titers were normalized to IU, using the WHO standard. GMT, geometric mean titer. (B) Mice were then challenged i.c. with 50 LD50 of CVS-24 and observed daily for 2 weeks. The numbers of survivors were recorded and compared. Data were analyzed with SigmaStat software (Systat Software, Inc., San Jose, CA). (A and B) Asterisks indicate significant differences (P < 0.05) in results among the experimental groups, as calculated by one-way ANOVA with the Holm-Sidak method.

  • FIG. 3.
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    FIG. 3.

    Effects of MIP-1α expression on the recruitment of DCs and B cells in the draining lymph nodes and the peripheral blood. Female BALB/c mice of 6 to 8 weeks of age were inoculated i.m. with 105 FFU of one of the recombinant RABVs [rHEP, rHEP-MIP-1α, or rHEP-MIP-1α(-)] or with medium alone (sham infection). At days 3, 6, and 9 p.i., single-cell suspensions were prepared from the draining (inguinal) lymph nodes or the peripheral blood and stained with antibodies to B cell (CD19 and CD40) or DC (CD11c and CD80) markers. Data collection and analysis were performed with a BD LSR II flow cytometer and BD FACSDiva software (BD Pharmingen). (A and B) Representative flow cytometric plots of the infiltration of mature B cells (CD19+/CD40+) (A) or DCs (CD11c+) or activated DCs (CD11c+/CD80+) (B) into the inguinal lymph nodes at day 6 p.i. are shown. (C and D) The percentages of B cells (C) or DCs (D) in the inguinal lymph nodes and peripheral blood at different time points were quantified from the results for 4 mice in each group and presented as mean values ± standard errors. Asterisks (*) indicate significant differences (*, P < 0.05; **, P < 0.01) in results between the indicated experimental groups, as calculated by one-way ANOVA with the Holm-Sidak method.

  • FIG. 4.
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    FIG. 4.

    Virus replication and chemokine expression at the inoculation site. BALB/c mice (6 to 8 weeks of age) were inoculated i.m. in the hind leg with 105 FFU of one of the recombinant viruses [rHEP, rHEP-MIP-1α, or rHEP-MIP-1α(−)] or with medium alone. The hind leg muscles of 4 mice from each group were removed at days 3 and 6 p.i. Total RNA was extracted from the muscle tissue, and viral genomic RNA (A), MIP-1α mRNA (B), CD19 mRNA (C), CD11c mRNA (D), or IL-4 mRNA (E) was analyzed by QRT-PCR. For absolute quantities of viral genomic RNA, a standard curve was generated from a serially diluted, in vitro-transcribed RNA, using a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to that of 1 μg of total RNA. For MIP-1α, CD19, CD11c, and IL-4 expression, mRNA copy numbers were normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Levels of gene expression in a test sample are presented as the fold increase over that detected in sham-infected controls. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) between the indicated experimental groups, as calculated by one-way ANOVA with the Holm-Sidak method.

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Expression of MIP-1α (CCL3) by a Recombinant Rabies Virus Enhances Its Immunogenicity by Inducing Innate Immunity and Recruiting Dendritic Cells and B Cells
Ling Zhao, Harufusa Toriumi, Hualei Wang, Yi Kuang, Xiaofeng Guo, Kinjiro Morimoto, Zhen F. Fu
Journal of Virology Aug 2010, 84 (18) 9642-9648; DOI: 10.1128/JVI.00326-10

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Expression of MIP-1α (CCL3) by a Recombinant Rabies Virus Enhances Its Immunogenicity by Inducing Innate Immunity and Recruiting Dendritic Cells and B Cells
Ling Zhao, Harufusa Toriumi, Hualei Wang, Yi Kuang, Xiaofeng Guo, Kinjiro Morimoto, Zhen F. Fu
Journal of Virology Aug 2010, 84 (18) 9642-9648; DOI: 10.1128/JVI.00326-10
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KEYWORDS

B-Lymphocytes
Chemokine CCL3
dendritic cells
Immunity, Innate
Rabies Vaccines
rabies virus

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