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Expression and immunogenicity of nsp10 protein of porcine epidemic diarrhea virus.

ABSTRACT: Porcine epidemic diarrhea virus (PEDV), a swine enteropathogenic coronavirus, causes lethal watery diarrhea to the piglets, which poses significant economic losses and public health concerns. The nsp10 protein of PEDV is essential regulatory subunits that are critical for virus replication. Since PEDV nsp10 is a crucial regulator of viral RNA synthesis, it is promising that nsp10 might become anti-virus drugs target or candidate for rapid diagnosis of PEDV infection. In this study, the PEDV nsp10 was inserted into pMAL-c2x-MBP / pET-28a vector, efficiently and stably expressed in E.coli system. Then the purified nsp10 protein was found to mediate potent antibody responses in immunized mice. The antibodies of immunized mice and PEDV infection swine strongly recognized purified nsp10 protein from cell lysates. Furthermore, cytokines test revealed that the expression of IL-2, IL-4, IL-10, TNF-α, IFN-γ were significantly higher than those in control group, indicated that purified nsp10 protein induce the cellular immune response mechanism in mice. Using modified seroneutralization test, we also demonstrated that sera from nsp10-immunized mice inhibited PEDV replication to some extent. These findings suggest that nsp10 has a high immunogenicity. This study may have implications for future development of PEDV detection or anti-virus drugs for swine.

SUBMITTER: Zhu L

PROVIDER: S-EPMC8721950 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.

Project description:BackgroundPorcine epidemic diarrhea virus (PEDV) causes diarrhea in all ages of pigs with 50-100% mortality rates in neonatal piglets. In the United States, inactivated and subunit PEDV vaccines for pregnant sows are available, but fail to induce sufficient protection in neonatal piglets farrowed from PEDV naïve sows. A safe and efficacious live attenuated vaccine that can prime mucosal immune responses is urgently needed. In this study, we evaluated the safety and efficacy of two attenuated PEDV vaccine candidates, the emerging non-S INDEL PEDV strain PC22A at the 100th cell culture passage level - Clone no. 4 (P100C4) and at the 120th passage level (P120), in weaned pigs.ResultsFour groups of 40-day-old weaned pigs were inoculated orally with PEDV PC22A-P3 (virulent), -P100C4, -P120, and mock, respectively, and challenged with the P3 virus at 24 days post-inoculation (dpi). After inoculation, P3 caused diarrhea in all pigs with a high level of fecal viral RNA shedding. P100C4 and P120 did not cause diarrhea in pigs, although viral RNA was detected in feces of all pigs, except for one P100C4-inoculated pig. Compared with the P120 group, P3- and P100C4-inoculated pigs had higher serum PEDV-specific IgG and viral neutralizing (VN) antibody (Ab) titers at 14 dpi. After the challenge, no pigs in the P3 group but all pigs in the P100C4, P120, and mock groups had diarrhea. Compared with the P120 group, pigs in the P100C4 group had a more rapid decline in fecal PEDV RNA shedding titers, higher titers of serum PEDV-specific IgG, IgA, and VN Abs, and higher numbers of intestinal IgA Ab-secreting cells.ConclusionsPEDV PC22A P100C4 and P120 were fully attenuated in weaned pigs but failed to elicit protection against virulent P3 challenge. P100C4 induced higher PEDV-specific antibody responses than P120 post inoculation resulting in a greater anamnestic response post challenge. Therefore, P100C4 potentially could be tested as a priming vaccine or be further modified using reverse genetics. It also can be administered in multiple doses or be combined with inactivated or subunit vaccines and adjuvants as a PEDV vaccination regimen, whose efficacy can be tested in the future.

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Expression and immunogenicity of nsp10 protein of porcine epidemic diarrhea virus.

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