Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge

Abstract

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime--boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8(+) specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.

Figure 1. A graphical scheme with a timeline of the mice experiments.

Figure 2. Detection of serum antibodies to the E-protein.

IgG1 (A) and IgG2a (B) titers of mice that were ID (4 mice), IM (5 mice), Topically (5 mice) or not primed (−/bE, 5 mice) with WNV-DermaVir nanoparticles and boosted either with WNV-DermaVir nanoparticles (IM) or E-protein combined with Matrix-M1 adjuvant (s.c.). IgG1 (A) and IgG2a (B) titers were determined by ELISA. Abbreviations: bE: boost E-protein, bDNA: boost WNV-DermaVir nanoparticles.

Figure 3. Detection of serum antibody to DIII of the E-protein.

IgG1 (A) and IgG2a (B) titers of mice primed ID (4 mice), IM (5 mice), Topically (5 mice) or not primed (−/bE, 5 mice) with WNV-DermaVir nanoparticles and boosted either with WNV-DermaVir nanoparticles (IM) or E-protein combined with Matrix-M1 adjuvant (s.c.). IgG1 (A) and IgG2a (B) titers were determined by ELISA against recombinant DIII. Abbreviations: bE: boost E-protein, bDNA: boost WNV-DermaVir nanoparticles.

Figure 4. Detection of virus neutralizing antibodies.

Five mice were vaccinated IM or topically with WNV-DermaVir nanoparticles and boosted either with WNV-DermaVir nanoparticles (IM) or E-protein combined with Matrix-M1 adjuvant (s.c.). Virus neutralization titers were determined by a focus reduction neutralization assay. Abbreviations: bE: boost E-protein, bDNA: boost WNV-DermaVir nanoparticles.

Figure 5. Induction of WNV-E specific IL-4 response by vaccination with WNV-DermaVir nanoparticles followed by a WNV-E protein boost.

WNV-E specific IL-4 responses were determined by IL-4 ELISPOT. Splenocytes obtained two weeks after the boost were stimulated with WNV-E protein and the numbers of cells producing IL-4 per 3×10⁵ cells were determined in triplicate. Mice were primed ID (4 mice), IM (5 mice), topically (top, 5 mice) or not (−/, 5 mice) with WNV-DermaVir nanoparticles and boosted s.c. with E-protein combined with Matrix-M1.

Figure 6. Intracellular IFN-γ production in CD4⁺ (A) and CD8⁺ (B) splenocytes.

Five mice were IM or Topically primed with WNV-DermaVir nanoparticles and boosted IM with WNV-DermaVir nanoparticles or s.c. with WNV-E protein combined with Matrix-M1. Splenocytes were pooled per group. Cells were stained for cytokines and surface markers (as indicated in the Methods) and analyzed by flow cytometry. Abbreviations: bE: boost E-protein, bDNA: boost WNV-DermaVir nanoparticles.

Figure 7. Heterologous prime (WNV-DermaVir nanoparticles) - boost (WNV-E protein) protect mice against a lethal WNV challenge.

Two groups of 10 BALB/c mice were vaccinated IM or topically (top) with WNV-DermaVir nanoparticles and boosted with WNV-E protein combined with Matrix-M1 four weeks later. Nine control mice were vaccinated with WNV-DermaVir nanoparticles containing an egfp control plasmid. Two weeks after the boost, the mice were challenged i.p. with a lethal dose of WNV. Abbreviations: bE: boost E-protein.

Figure 8. Heterologous prime (WNV-DermaVir nanoparticles) - boost (WNV-E protein) protect mice against morbidity after a lethal WNV challenge.

Two groups of 10 BALB/c mice were vaccinated IM or topically (top) with WNV-DermaVir nanoparticles and boosted with WNV-E protein combined with Matrix-M1 four weeks later. In addition, nine control mice were vaccinated with WNV-DermaVir nanoparticles containing an egfp control plasmid. Two weeks after the boost, the mice were challenged i.p. with a lethal dose of WNV. Individual illness scores of mice vaccinated with (A) egfp control plasmid, (B) IM with and (C) topically with WNV-DermaVir nanoparticles. (D) Body weight in percent of individual baseline mean after challenge. Abbreviations: bE: boost E-protein.

Figure 9. Macro- and microscopical analysis of the skin after topical application of WNV-DermaVir nanoparticles.

Panel A shows the macroscopic lesions observed on the back of two representative mice one day after the topical application of the WNV-DermaVir nanoparticles. Panel B and C are H&E- stained skin biopsies of a control animal (B) and an animal that received WNV-DermaVir nanoparticles via topical application. The skin biopsies were taken from the treated area 14 day after topical application of the WNV-DermaVir nanoparticles. The different structures of the skin are noted : epidermis (e), dermis (d), panniculus adiposus (pa) and the panniculus carnosus (pc).