Auxotrophic Lactobacillus Expressing Porcine Rotavirus VP4 Constructed Using CRISPR-Cas9D10A System Induces Effective Immunity in Mice

Abstract

Porcine rotavirus (PoRV) mainly causes acute diarrhea in piglets under eight weeks of age and has potentially high morbidity and mortality rates. As vaccine carriers for oral immunization, lactic acid bacteria (LAB) are an ideal strategy for blocking PoRV infections. However, the difficulty in knocking out specific genes, inserting foreign genes, and the residues of antibiotic selection markers are major challenges for the oral vaccination of LAB. In this study, the target gene, alanine racemase (alr), in the genome of Lactobacillus casei strain W56 (L. casei W56) was knocked out to construct an auxotrophic L. casei strain (L. casei Δalr W56) using the CRISPR-Cas9D10A gene editing system. A recombinant strain (pPG-alr-VP4/Δalr W56) was constructed using an electrotransformed complementary plasmid. Expression of the alr-VP4 fusion protein from pPG-alr-VP4/Δalr W56 was detected using Western blotting. Mice orally immunized with pPG-alr-VP4/Δalr W56 exhibited high levels of serum IgG and mucosal secretory immunoglobulin A (SIgA), which exhibited neutralizing effects against PoRV. Cytokines levels in serum detected using ELISA, indicated that the recombinant strain induced an immune response dominated by Th2 cells. Our data suggest that pPG-alr-VP4/Δalr W56, an antibiotic-resistance-free LAB, provides a safer vaccine strategy against PoRV infection.

Keywords: CRISPR/Cas9D10A; Lactobacillus; nutritional deficient; oral immunization; porcine rotaviruses.

Figure 1

Schematic diagram of the construction of DNA plasmids. (A) The sgRNAs of alr were fused with the upper and lower homologous arms of the alr gene, and the sequence was inserted into the pLCNICK gene editing vector, which resulted in the pLCNICK-alr gene editing vector. (B) The constitutive cell surface expression plasmid pPG-T7g10-PPT (left). The alr gene was amplified from L. casei W56, cleaved using SacI and ApaI, and was inserted into the expression plasmid pPG-T7g10-PPT, named pPG-alr (middle). The VP4 gene was amplified from the PoRV JL94 strain, cleaved with KpnI and ApaI, and was inserted into pPG-alr, obtaining the recombinant plasmid pPG-alr-VP4 (right).

Figure 2

Construction of alr-auxotrophic mutant L. casei Δalr W56. (A) Schematic diagram of the preparation of L. casei Δalr W56 using the CRISPR/Cas9D10A system. (B) Identification of alr gene depletion in the genomes of L. casei W56 and L. casei Δalr W56 by PCR using alr-F/R as primers. L. casei Δalr W56 was serially passaged 50 times and the genome extracted every 10 passages was identified by PCR and DNA sequencing. (C) Strains L. casei Δalr W56, pPG/Δalr W56, and pPG-alr-VP4/Δalr W56 were cultivated on MRS solid plates with and without D-Alanine. (D) Strains L. casei Δalr W56, pPG/Δalr W56, and pPG-alr-VP4/Δalr W56 were cultivated in MRS liquid medium with and without D-Alanine.

Figure 3

Biological characterization of L. casei Δalr W56. (A) Determination of growth curves of L. casei W56 and L. casei Δalr W56. Colony Forming Units (CFU) per milliliter of L. casei W56 and L. casei Δalr W56 cultures were calculated by Standard Plate Count (SPC) every 2 h respectively. (B) The morphological characteristics of L. casei W56 and L. casei Δalr W56 were identified by gram staining under the ordinary light microscope, and the magnification of view was 1000×. (C) Morphological characteristics of L. casei W56 and L. casei Δalr W56 observed by scanning electron microscope.

Figure 4

Analysis of the inherent stability in recombinant strains expressing alr-VP4. (A) Strains pPG-alr-VP4/Δalr W56, L. casei Δalr W56, and pPG/Δalr W56 were lysed and the protein expression levels of alr-VP4 were analyzed using Western blotting with mouse anti-His monoclonal antibody. (B) Strains pPG-alr-VP4/Δalr W56, pPG/Δalr W56, and L. casei Δalr W56 were continuously transferred for 50 generations and were lysed every 10 (pPG-alr-VP4/Δalr W56) or 50 (L. casei Δalr W56 and pPG/Δalr W56) generations. The genome was extracted, and alr-VP4 was amplified by PCR. (C) Strains pPG-alr-VP4/Δalr W56, pPG/Δalr W56, and L. casei Δalr W56 were continuously transferred for 50 generations and were lysed every 10 (pPG-alr-VP4/Δalr W56) or 50 (pPG/Δalr W56 and L. casei Δalr W56) generations, and the protein expression levels of alr-VP4 were analyzed using Western blotting with mouse anti-His monoclonal antibody.

Figure 5

Levels of anti-PoRV-specific SIgA and IgG in immunized mice. (A) Schematic of oral immunization program and schedule of sampling genital tract fluid, intestinal mucus, nasal fluid, feces, and sera. The immunization program was immunization each time for three consecutive days, once every two weeks, for a total of three times. Samples were taken every 7 days until day 63. (B–E) Specific anti-PoRV SIgA levels in genital tract (B), intestinal mucus (C), nasal fluid (D), and feces (E) of mice were determined post-immunization with strains L. casei Δalr W56, pPG/Δalr W56, pPG-alr-VP4/Δalr W56, or PBS. (F) Detecting the level of anti-PoRV specific IgG antibody in serum. (G) The neutralizing activity of anti-PoRV specific IgG antibody in serum and SIgA in intestinal mucosa were detected. Three parallel groups of each sample were sampled each time, and the samples from each group were analyzed as one sample in triplicate. n = 3, mean ± SD, Student’s test, ns, not significant, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 6

Cytokine secretion level in immunized mice. The mice were immunized thrice with strains L. casei Δalr W56, pPG/Δalr W56, pPG-alr-VP4/Δalr W56 or PBS. (A) The serum was collected and the cytokine for IFN-γ or IL-4 was detected. (B) The ratio of IL4/IFN-γ was analyzed. (C) The serum was collected and the cytokines for IL-2, IL-10, IL-12, and IL-17 were detected. n = 3, mean ± SD, Student’s test, ns, not significant, *, p < 0.05, **, p < 0.01.