Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60

Abstract

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Keywords: RHDV vaccines; Rabbit dendritic cells; fusion expression; recombinant Lactobacillus; targeting.

Figure 1.

Schematic diagram of the immunization, challenge, and cohabitation of rabbits. Twenty rabbits were divided into five groups for immunization. Twelve rabbits were divided into three groups for challenge. The box indicates rabbits in the orally immunized KC1-VP60/L. reuteri (Dark Pink), PBS (blue), and rabbits infected with RHDV (green) were raised in the same cage.

Figure 2.

Typical morphology and molecular phenotype of raMoDCs(a) morphology of imraMoDCs (Day 6) and mraMoDCs (Day 7, treated with LPS for 24 h for maturation) observed through optical microscopy and scanning electron microscopy. (b) Immunostaining of imraMoDCs and mraMoDCs for MHC-II (green), CD86 (red), and nuclei stained with DAPI (blue). (c) Flow cytometry analysis showing the expression of surface markers (CD172a, MHC-II, CD86, and CD14) on imraMoDCs and mraMoDCs. (d) Flow cytometry assessment of the phagocytic ability of raMoDCs toward FITC-labeled glucans. The data reflect % abundance for positive populations. Brightness, contrast, or color balance adjustments were uniformly applied to all microscopy images.

Figure 3.

Analysis of phage binding ability to raMoDCs by ELISA. (a) Sequences of 12-mer peptides. (b) Cell-ELISA assessing the binding selectivity to raMoDCs of eight phage clones from the last round of biopanning. M13 wild-type phage without any displayed peptide served as a negative control. Data are presented as the mean ± SD of three independent experiments. Significant differences are denoted by different letters (a vs. b, a vs. c, b vs. c) at the same time point (p < 0.01).

Figure 4.

Binding ability of dc-targeting peptides to raMoDCs. (a) Fluorescence microscopy images showing the binding of FITC-labeled HS, KC1, MY, and negative control (NC) peptides to raMoDCs. (b) Flow cytometry analysis of the binding ability of the peptides to raMoDCs. Red indicates rabbit DCs expressing CD86, green represents FITC-labeled peptides, and blue represents nuclei stained with DAPI. Changes in brightness, contrast, or color balance were applied uniformly to all pixels in the microscopy image.

Figure 5.

Characterization of expressed proteins in L. reuteri. (a) Schematic representation of the construction of recombinant plasmids: pPG-HS-VP60, pPG-KC1-VP60, pPG-MY-VP60, and pPG-VP60. (b) Verification of expressed VP60 protein in L. reuteri through western blotting. The primary antibody used was mouse anti-Flag monoclonal antibody. (c) Visualization of expressed proteins in Lactobacillus via fluorescence microscopy. The fluorescence signals confirm the presence of HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri.

Figure 6.

The ability of raMoDCs to recognize and capture recombinant Lactobacillus was evaluated using scanning electron microscopy and the analysis of toll-like receptor and cytokine mRNA levels in raMoDCs (106 cells/mL) in response to Vector/L. reuteri, VP60/L. reuteri, HS-VP60/L. reuteri, KC1-VP60/L. reuteri, MY-VP60/L. reuteri, and LPS (2 µg/mL) stimulation. (a) Scanning electron microscopy images showing the morphology of raMoDCs capturing recombinant Lactobacillus at 30, 60, 120 min. (b) Recombinant Lactobacillus-incubated raMoDCs at a ratio of 1:10 (DCs: recombinant Lactobacillus). RaMoDCs were stimulated by recombinant Lactobacillus and LPS for 12 h. Unstimulated raMoDCs were used as a control. Different letters (a vs. b, a vs. c, b vs. (c) indicate significant differences (p < 0.01) at the same time point.

Figure 7.

Detection of anti-RHDV-specific IgG/sIgA antibody levels and rabbit survival after challenge. (a) Specific anti-RHDV IgG and sIgA antibody levels in rabbits orally immunized with PBS, VP60/L. reuteri, HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. Measurement of a specific anti-RHDV IgG antibody in the antisera from immunized rabbits by ELISA using RHDV as the coating antigen. Measurement of specific anti-RHDV SIgA antibody levels in the feces and nasal cavity by ELISA using RHDV as the coating antigen. (b) Rabbit survival after challenge. (c) RHDV load in the liver, spleen, lungs, and kidneys of infected dead rabbits. Different letters (a vs. b, a vs. c, b vs. c) indicate significant differences (p < 0.01) at the same time point.