Signal sequence contributes to the immunogenicity of Pasteurella multocida lipoprotein E

Abstract

Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protective efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vaccine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4⁺ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl cholera and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineering and has potential to be developed as a fowl cholera vaccine.

Keywords: fowl cholera; lipid moiety; lipoprotein E; signal sequence; subunit vaccine.

Figure 1

Hydrophilicity plot of PlpE and gene construction of SP-tPlpE. (A) Kyte-Doolittle plot of PlpE. (B) Schematics of the SP-tPlpE construct in plasmid vector pET32a. (C) Signal sequence of PlpE containing Lipobox.

Figure 2

Protein analysis of recombinant SP-tPlpE and tPlpE. (A) SDS-PAGE analysis of the recombinant proteins. (B) Western blotting of SP-tPlpE 4h after induction. (C) Both SP-tPlpE and tPlpE recombinant proteins were expressed in a soluble form. (D) Protein quantitation using BSA protein standards, showing SP-tPlpE protein production at 289 mg/L after purification.

Figure 3

HEK-Blue-hTLR2 Cells were treated with SP-tPlpE and tPlpE at a final concentration of 12.5 µg/mL. The cells were treated with the inactivated E. coli (2,000 CFU/well) and SP-tPlpE, tPlpE at 37°C and 5% CO₂. Cell activation was determined after 16 h of incubation by measuring SEAP activity at OD₆₂₅ using the HEK-Blue Detection assay. Results are expressed as means ± S.D. of triplicates and represented in three independent experiments. Statistical significance between SP-tPlpE and tPlpE-treated was determined using Student's t test (P < 0.05)

Figure 4

Antigen-specific antibodies of immunized chickens. Chickens (n = 3) were immunized twice with SP-tPlpE + ISA71, tPlpE + ISA71, or PBS + ISA71, and sera were analyzed by indirect ELISA using PlpE as the coating antigen. Data are presented as mean ± SEM. Different superscript letters indicate significant difference (P < 0.05) between vaccine groups at the same time point.

Figure 5

CD4⁺ and CD8⁺ T cell immune response of immunized chickens. Chickens (n = 3) were immunized twice with SP-tPlpE, tPlpE, or PBS and isolated PBMCs were stained with anti-CD4 or -CD8 antibodies for flow cytometric analysis. Data are presented as mean ± SEM. Different superscript letters indicate significant differences between vaccine groups at the same time point (P < 0.05).

Figure 6

Cytokine expression of PBMCs from immunized chickens. Chickens (n = 3) were immunized twice with SP-tPlpE, tPlpE, or PBS and isolated PBMCs were stimulated with PlpE. Relative mRNA expression levels of IL-6, IFN-γ, IL-12, and IL-4 were determined. Data are presented as mean ± SEM. Different superscript letters significantly differ between vaccine groups (P < 0.05).

Figure 7

Survival rate of immunized chickens when challenged with P. multocida A. Chickens (n = 8) were immunized twice with SP-tPlpE, tPlpE, or PBS and challenged with 20 LD₅₀ (3.2 × 10⁵ CFU/dose) P. multocida.

Figure 8

Gross lesions in the livers of chickens challenged with P. multocida Chu01. (A) Liver of group PBS + ISA71, necrotic white foci on the surface; (B) liver of immunized SP-tPlpE + ISA71 group, swollen; (C) liver of non-challenge chicken, no lesion was observed. (D) Confirmation of P. multocida from livers of inoculated chickens using PCR. (E) The primers were used to amplify the hyaD-hyaC gene.