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. 2023 Jan 9:13:1103327.
doi: 10.3389/fmicb.2022.1103327. eCollection 2022.

Orally administered recombinant Lactobacillus expressing African swine fever virus antigens that induced immunity responses

Hongliang Zhang  1 , Saisai Zhao  1   2 , Haojie Zhang  1 , Yu Shen  1 , Peijun Zhang  1 , Hu Shan  1 , Xiulei Cai  1
Affiliations

Orally administered recombinant Lactobacillus expressing African swine fever virus antigens that induced immunity responses

Hongliang Zhang et al. Front Microbiol. .

Erratum in

Abstract

African swine fever (ASF) is a highly contagious, acute, febrile disease caused by the African swine fever virus (ASFV), with morbidity and mortality rates approaching 100% in domestic and wild swine, resulting in massive economic losses to the pig industry worldwide. This study aimed to express the p30, p54, and p72 proteins encoded by ASFV in vitro using the Lactobacillus lactis (L. lactis) expression system. Here, six new functional recombinant L. lactis were constructed, and the expression of the p30 protein, p54 protein, p72 protein, p30-LTB (heat-labile enterotoxin B, LTB) fusion protein, p54-LTB fusion protein, and the p72-LTB fusion protein was successfully detected by Western blot analysis. Following oral immunization of rabbits with recombinant L. lactis, serum IgG, intestinal mucosal sIgA, cytokines (IL-4 and INF-γ), and splenocyte viability were higher than in the control group via ELISA. Notably, without the LTB adjuvant group, humoral and Th1 cellular immunity were promoted, whereas, with the LTB adjuvant group, local mucosal immunity, humoral immunity, and Th2 cellular immunity were promoted, providing new insights into the design and development of an ASFV subunit vaccine.

Keywords: African swine fever virus; Lactococcus lactis; enterotoxin B subunit; immunogenicity evaluation; oral immunization; recombinant expression.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Samples collection of immunizated rabbits.
Figure 2
Figure 2
Construction and identification of the recombinant L. lactis. (A) A map of the plasmid pMG36e-p30-His, the plasmid size is 4,279 bp; (B) A map of the plasmid pMG36e-p54-His, the plasmid size is 4,249 bp; (C) A map of the plasmid pMG36e-p72-His, the plasmid size is 4,096 bp; (D) A map of the plasmid pMG36e-p30-LTB-His, the plasmid size is 4,741 bp; (E) A map of the plasmid pMG36e-p54-LTB-His, the plasmid size is 4,711 bp; (F) A map of the plasmid pMG36e-p72-LTB-His, the plasmid size is 4,558 bp. (G) Identification results of recombinant L. lactis by colony PCR, MG1363/pMG36e (lane 1); MG1363/pMG36e-p30-His (lane 2); MG1363/pMG36e-p54-His (lane 3); MG1363/pMG36e-p72-His (lane 4); MG1363/pMG36e-p30-LTB-His (lane 5); MG1363/pMG36e-p54-LTB-His (lane 6); MG1363/pMG36e-p72-LTB-His (lane 7); Negative control (lane 8).
Figure 3
Figure 3
All six constructs, denoted as p30, p54, p72, p30-LTB, p54-LTB, and p72-LTB, were inserted into expression plasmid pMG36e and expressed in Lactobacillus. (A) The expressions of the recombinant L. lactis ASFV p30-His protein and p54-His protein were verified by Western blot, lane 1, 3–4: Negative control, lane 2: p30-His, lane 5: p54-His. (B) The expression of the recombinant L. lactis ASFV p30-LTB-His fusion protein was verified by Western blot, lane 1: Negative control, lane 2: p30-LTB-His. (C) The expression of the recombinant L. lactis ASFV p54-LTB fusion protein was verified by Western blot, lane 1–2: Negative control, lane 3: p54-LTB-His. (D) The expressions of the recombinant L. lactis ASFV p72-His protein and p72-LTB-His fusion protein were verified by Western blot, lane 1: Negative control, lane2: p72-His, lane 3: p72-LTB-His.
Figure 4
Figure 4
Determination of intestinal mucosa sIgA levels on 17 and 34 dpv by ELISA. “**” in the figure means the difference is extremely significant p < 0.01, “*” indicates a significant difference p < 0.05, and “ns” indicates no significant difference p > 0.05.
Figure 5
Figure 5
The levels of cytokines IL-4 and IFN-γ in serum were measured using ELISA on 17 and 34 dpv. (A) The levels of cytokines IL-4 in sera on 17 and 34 dpv were determined using ELISA. (B) The levels of cytokines IFN-γ in sera on 17 and 34 dpv were measured via ELISA. “**” in the figure means the difference is extremely significant p < 0.01, “*” indicates a significant difference p < 0.05, and “ns” indicates no significant difference p > 0.05.
Figure 6
Figure 6
The cell viability of splenocytes after the first immunization on 17 and 34 dpv by CCK-8. “**” in the figure means the difference is extremely significant p < 0.01, “*” indicates a significant difference p < 0.05, and “ns” indicates no significant difference p > 0.05.
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