Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2.

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages.

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

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

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.

Development of a label-free photoelectrochemical immunosensor for novel astrovirus detection.

Since 2017, an infectious goose gout disease characterized by urate precipitation in viscera, mainly caused by novel goose astrovirus (GoAstV) infection, has emerged in the main goose-producing region of China. The current challenge in managing goose gout disease is largely due to the absence of a rapid and efficient detection method for the GoAstV pathogen. Notably, the potential application of immunosensors in detecting GoAstV has not yet been explored. Herein, a label-free PEC immunosensor was fabricated by using purchased TiO2 as the photoactive material and antibody against GoAstV P2 proteins as the specific recognition element. First, we successfully expressed the capsid spike domain P2 protein of ORF2 from GoAstV CHSH01 by using the pET prokaryotic expression system. Meanwhile, the polyclonal antibody against GoAstV capsid P2 protein was produced by purified protein. To our knowledge, this is the first establishment and preliminary application of the label-free photoelectrochemical immunosensor method in the detection of AstV. The PEC immunosensor had a linear range of 1.83 fg mL-1 to 3.02 ng mL-1, and the limit of detection (LOD) was as low as 0.61 fg mL-1. This immunosensor exhibited high sensitivity, great specificity, and good stability in detecting GoAstV P2 proteins. To evaluate the practical application of the immunosensor in real-world sample detection, allantoic fluid from goose embryos was collected as test samples. The results indicated that of the eight positive samples, one false negative result was detected, while both negative samples were accurately detected, suggesting that the constructed PEC immunosensor had good applicability and practical application value, providing a platform for the qualitative detection of GoAstV.

Comparative immunogenic and immunoprotective activities of PCV2d Cap and Rep antigens delivered by an efficient eukaryotic expression system engineered into a Salmonella vaccine vector.

Porcine circovirus type 2 (PCV2) stands as a predominant etiological agent in porcine circovirus-associated diseases. To manage the spread of the disease, it is necessary to develop a next-generation vaccine expressing PCV2 antigens that target the prevailing genotype such as PCV2d. A bacterial-mediated vaccine delivery by live-attenuated Salmonella has attracted interest for its low-cost production and highly effective vaccine delivery. Thus, in this study, we utilized the advantages of the Salmonella-mediated vaccine delivery by cloning PCV2d cap and rep into a eukaryotic expression plasmid pJHL204 and electroporation into an engineered live-attenuated Salmonella Typhimurium JOL2500 (Δlon, ΔcpxR, ΔsifA, Δasd). The eukaryotic antigen expression by JOL2995 (p204:cap) and JOL2996 (p204:rep) was confirmed in vitro and in vivo which showed efficient antigen delivery. Furthermore, vaccination of mice model with the vaccine candidates elicited humoral and cell-mediated immune responses as depicted by high levels of PCV2-specific antibodies, CD4+ and CD8+ T cells, and neutralizing antibodies, especially by JOL2995 (p204:cap) which correlated with the significant decrease in the viral load in PCV2d-challenged mice. Interestingly, JOL2996 (p204:rep) may not have elicited high levels of neutralizing antibodies and protective efficacy, but it elicited considerably higher cell-mediated immune responses. This study demonstrated Salmonella-mediated vaccine delivery system coupled with the eukaryotic expression vector can efficiently deliver and express the target PCV2d antigens for strong induction of immune response and protective efficacy in mice model, further supporting the potential application of the Salmonella-mediated vaccine delivery system as an effective novel approach in vaccine strategies for PCV2d.

The synergy of recombinant NSP4 and VP4 from porcine rotavirus elicited a strong mucosal response.

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP486-175aa (NSP4*) and VP426-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine.

Detection of avian reovirus (ARV) by ELISA based on recombinant σB, σC and σNS full-length proteins and protein fragments.

Introduction. Avian reovirus (ARV) is associated with arthritis/tenosynovitis and malabsorption syndrome in chickens. The σC and σB proteins, both exposed to the virus capsid, are highly immunogenic and could form the basis for diagnostic devices designed to assess the immunological status of the flock.Gap Statement. Commercial ARV ELISAs cannot distinguish between vaccinated and infected animals and might not detect circulating ARV strains.Aim. We aimed to develop a customized test to detect the circulating field ARV strains as well as distinguish between vaccinated and unvaccinated animals.Methodology. We developed ELISA assays based on recombinant (r) σB, σC and the nonstructural protein σNS and tested them using antisera of vaccinated and unvaccinated chickens as well as negative controls. Fragments of σB and σC proteins were also used to study regions that could be further exploited in diagnostic tests.Results. Vaccinated and unvaccinated birds were positive by commercial ELISA, with no difference in optical density values. In contrast, samples of unvaccinated animals showed lower absorbance in the rσB and rσC ELISA tests and higher absorbance in the rσNS ELISA test than the vaccinated animals. Negative control samples were negative in all tests. Fragmentation of σB and σC proteins showed that some regions can differentiate between vaccinated and unvaccinated animals. For example, σB amino acids 128-179 (σB-F4) and σC amino acids 121-165 (σC-F4) exhibited 85 and 95% positivity among samples of vaccinated animals but only 5% and zero positivity among samples of unvaccinated animals, respectively.Conclusion. These data suggest that unvaccinated birds might have been exposed to field strains of ARV. The reduction in absorbance in the recombinant tests possibly reflects an increased specificity of our test since unvaccinated samples showed less cross-reactivity with the vaccine proteins immobilized on ELISAs. The discrepant results obtained with the protein fragment tests between vaccinated and unvaccinated animals are discussed in light of the diversity between ARV strains.

Identification of a norovirus GII-specific antigenic epitope.

Noroviruses (NoVs) are the chief cause of acute viral gastroenteritis worldwide. By employing the major capsid protein VP1 of a GII.6 NoV strain as an immunogen, we generated two monoclonal antibodies (mAbs) with wide-spectrum binding activities against NoV genogroup II (GII) VP1 proteins. One mAb (10G7) could bind to native and denatured GII-specific VP1 proteins. The other mAb (10F2) could bind to all tested native GII VP1 proteins, but not to denatured GII.3, GII.4, GII.7, or GII.17 VP1 proteins. Using GII.6/GII.4 fusion proteins, the mAb 10F2 binding region was confirmed to be located in the C-terminal P1 domain. An enzyme-linked immunosorbent assay based on peptides covering the P domain did not detect any binding. Using a panel of VP1 proteins with swapped regions, deletions, and mutations, the mAb 10F2 binding region was determined to be located between residues 496 and 513. However, the residue(s) responsible for its varied binding affinity for different denatured GII VP1 proteins remain to be identified. In summary, two NoV GII-specific cross-reactive mAbs were generated, and their binding regions were determined. Our results might facilitate the detection and immunogenic study of NoVs.

Antigenic epitope analysis of Pakistani G3 and G9 rotavirus strains compared to vaccine strains revealed multiple amino acid differences.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains RotarixTM and RotaTeqTM, multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

Epitope mapping and establishment of a blocking ELISA for mAb targeting the p72 protein of African swine fever virus.

The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20-39 aa, 8A9 and 5E10 are recognized at 263-282 aa, which is consistent with the reported 265-280 aa epitopes. Conserved analysis revealed 20-39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. KEY POINTS: • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody.

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines.

Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Characteristics of Norovirus capsid protein-specific CD8+ T-Cell responses in previously infected individuals.

Norovirus (NV) infection causes acute gastroenteritis in children and adults. Upon infection with NV, specific CD8+ T cells, which play an important role in anti-infective immunity, are activated in the host. Owing to the NV's wide genotypic variability, it is challenging to develop vaccines with cross-protective abilities against infection. To aid effective vaccine development, we examined specific CD8+ T-cell responses towards viral-structural protein (VP) epitopes, which enable binding to host susceptibility receptors. We isolated peripheral blood mononuclear cells from 196 participants to screen and identify predominant core peptides towards NV main and small envelope proteins using ex vivo and in vitro intracellular cytokine staining assays. Human leukocyte antigen (HLA) restriction characteristics were detected using next-generation sequencing. Three conservative immunodominant VP-derived CD8+ T-cell epitopes, VP294-102 (TDAARGAIN), VP2153-161 (RGPSNKSSN), and VP1141-148 (FPHIIVDV), were identified and restrictively presented by HLA-Cw * 0102, HLA-Cw * 0702, and HLA-A *1101 alleles, separately. Our findings provide useful insights into the development of future vaccines and treatments for NV infection.

Loop PDE of viral capsid protein is involved in immune escape of the emerging novel variant infectious bursal disease virus.

Infectious bursa disease (IBD) is an acute, highly contactable, lethal, immunosuppressive infectious disease caused by the Infectious bursa disease virus (IBDV). Currently, the emerged novel variant IBDV (nVarIBDV) and the sustainedly prevalent very virulent IBDV (vvIBDV) are the two most prevalent strains of IBDV in China. The antigenic properties of the two prevalent strains differed significantly, which led to the escape of nVarIBDV from the immune protection provided by the existing vvIBDV vaccine. However, the molecular basis of the nVarIBDV immune escape remains unclear. In this study, we demonstrated, for the first time, that residues 252, 254, and 256 in the PDE of VP2 are involved in the immune escape of the emerging nVarIBDV. Firstly, the IFA-mediated antigen-antibody affinity assay showed that PBC and PDE of VP2 could affect the affinity of vvIBDV antiserum to VP2, of which PDE was more significant. The key amino acids of PDE influencing the antigen-antibody affinity were also identified, with G254N being the most significant, followed by V252I and I256V. Then the mutated virus with point or combined mutations was rescued by reverse genetics. it was further demonstrated that mutations of V252I, G254N, and I256V in PDE could individually or collaboratively reduce antigen-antibody affinity and interfere with antiserum neutralization, with G254N being the most significant. This study revealed the reasons for the widespread prevalence of nVarIBDV in immunized chicken flocks and provided innovative ideas for designing novel vaccines that match the antigen of the epidemic strain.

Lactiplantibacillus plantarum surface-displayed VP6 (PoRV) protein can prevent PoRV infection in piglets.

Porcine rotavirus (PoRV) poses a threat to the development of animal husbandry and human health, leading to substantial economic losses. VP6 protein is the most abundant component in virus particles and also the core structural protein of the virus. Firstly, this study developed an antibiotic-resistance-free, environmentally friendly expression vector, named asd-araC-PBAD-alr (AAPA). Then Recombinant Lactiplantibacillus plantarum (L. plantarum) strains induced by arabinose to express VP6 and VP6-pFc fusion proteins was constructed. Subsequently, This paper discovered that NC8/Δalr-pCXa-VP6-S and NC8/Δalr-pCXa-VP6-pFc-S could enhance host immunity and prevent rotavirus infection in neonatal mice and piglets. The novel recombinant L. plantarum strains constructed in this study can serve as oral vaccines to boost host immunity, offering a new strategy to prevent PoRV infection.