Self-Assembling Nanovaccine Fused with Flagellin Enhances Protective Effect against Foot-and-Mouth Disease Virus

Abstract

Nanovaccines based on self-assembling nanoparticles (NPs) can show conformational epitopes of antigens and they have high immunogenicity. In addition, flagellin, as a biological immune enhancer, can be fused with an antigen to considerably enhance the immune effect of antigens. In improving the immunogenicity and stability of a foot-and-mouth disease virus (FMDV) antigen, novel FMDV NP antigens were prepared by covalently coupling the VP1 protein and truncated flagellin containing only N-terminus D0 and D1 (N-terminal aa 1-99, nFLiC) with self-assembling NPs (i301). The results showed that the fusion proteins VP1-i301 and VP1-i301-nFLiC can assemble into NPs with high thermal tolerance and stability, obtain high cell uptake efficiency, and upregulate marker molecules and immune-stimulating cytokines in vitro. In addition, compared with monomeric VP1 antigen, high-level cytokines were stimulated with VP1-i301 and VP1-i301-nFLiC nanovaccines in guinea pigs, to provide clinical protection against viral infection comparable to an inactivated vaccine. This study provides new insight for the development of a novel FMD vaccine.

Keywords: flagellin; foot−and−mouth disease; nanoparticles; nanovaccine; self−assembling.

Figure 1

Expression and self−assembly of NPs. (A) Schematic diagram of VP1, VP1−i301 and VP1−i301−nFLiC expression cassettes cloned in the pSMA expression vector. (B) SDS−PAGE and Western blot analyses of purified fusion proteins. (C) TEM image of VP1−i301 (1) and VP1−i301−nFLiC NPs (2). (D) Hydrodynamic diameter and size distribution of VP1−i301 and VP1−i301−nFLiC NPs were analyzed with DLS. (E) The Size, PDI, and Zeta Potential data of VP1−i301 and VP1−i301−nFLiC NPs.

Figure 2

Thermal stability analysis of NPs. (A) NPs remained soluble at high temperatures. (B) Hydrodynamic diameter and size distribution of VP1−i301 NPs at different temperatures were analyzed with DLS. (C) Hydrodynamic diameter and size distribution of VP1−i301−nFLiC NPs at different temperatures were analyzed using DLS. (D) Dot blot analysis of the stability of monomeric VP1, VP1−i301, and VP1−i301−nFLiC NPs stored for 10 days at 37 °C.

Figure 3

Cellular uptake of NPs. Efficiency of the cellular uptake of NPs and control VP1 protein. The BHK−21 (A) and RAW−264.7 (B) cells were stimulated with different protein samples and analyzed for the presence of internal VP1 with Western blot at 1, 2, 4, and 6 h. The original Western blots and relative VP1 levels determined using densitometry are shown. β−actin was used as a loading control for normalization. Experiments were performed in triplicate and data are presented as mean ± SD. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 4

Expression of TLR5 pathway proteins. (A) Schematic diagram of TLR5 pathway molecules. (B) Immature BMDCs were stimulated with PBS, VP1, VP1−i301−nFLiC NPs, or VP1−i301−nFLiC NPs for 8 h. The expression of the TLR5 pathway molecules in BMDCs was detected using Western blot. β−actin was used as a loading control for normalization. Experiments were performed in triplicate and data are presented as mean ± SD. ns, p > 0.05, * p < 0.05; ** p < 0.01.

Figure 5

Detection of surface markers and cytokine expression of BMDCs. Total RNAs were extracted from VP1−, VP1−i301 NP−, or VP1−i301−nFLiC NP−stimulated BMDCs. mRNA levels of surface marker MHC II (A); costimulatory molecules CD80 (B) and CD86 (C); and pro−inflammatory cytokines TNF−α (D), IL−6 (E), and IFN−γ (F) were determined using q−PCR. β−actin was used as a loading control for normalization. Experiments were performed in triplicate and data are presented as mean ± SD. ns, p > 0.05; *, p < 0.05; **, p < 0.01.

Figure 6

Animal experiment and effect evaluation. Blood samples were collected at 7, 14, 21, and 28 dpi (days post−immunization) for detecting the specific antibodies and neutralizing antibodies in guinea pigs. Spleen lymphocytes were isolated to detect the proliferation of T lymphocytes. (A) The procedure of animal experimental immunization. (B) Detection of specific antibodies. (C) Detection of neutralizing antibodies. (D) Detection of T lymphocyte proliferation. (E–G) Total RNAs of guinea pigs were isolated at 28 dpi from the blood, and mRNA levels of TNF−α (E), IFN−γ (F), and IL−12 (G) were determined using q−PCR. GAPDH was used as a loading control for normalization. Experiments were performed in triplicate, and data are presented as mean ± SD. ns, p > 0.05, *, p < 0.05; **, p < 0.01; ***, p < 0.001.