Immune response in piglets orally immunized with recombinant Bacillus subtilis expressing the capsid protein of porcine circovirus type 2.
ABSTRACT: BACKGROUND:Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap). METHODS:Electroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9). RESULTS:We evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P?
Project description:In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-?-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection.
Project description:Porcine circovirus type 2 (PCV2) capsid protein (Cap) is a unique structure protein that plays pivotal roles in the process of viral replication and pathogenesis. Herein, we characterized a putative porcine Makorin RING finger protein 1 (pMKRN1) variant, an N-terminal-truncated variant of putative full-size porcine MKRN1 which has a unique expression pattern resulting from the porcine mkrn1 gene and which interacts with PCV2 Cap. A domain mapping assay showed that the C terminus of pMKRN1 and fragments (amino acids 108 to 198) of Cap are required for this interaction. PCV2 transiently upregulated pMKRN1 in PK-15 cells, but persistent viral infection downregulated pMKRN1 in major pathological tissues of PCV2-infected piglets. Overexpression of pMKRN1 significantly inhibited the generation of progeny PCV2 via ubiquitination and degradation of Cap, whereas knockout of pMKRN1 blocked Cap degradation and promoted progeny virus replication. pMKRN1 specifically targeted PCV2 Cap lysine residues 164, 179, and 191 to induce polyubiquitination and subsequent degradation. Mutation of either of the three lysine residues in the Cap protein or mutation of the histidine at residue 243 within the RING finger domain of pMKRN1 abrogated the E3 ligase activity of pMKRN1, rendering cells incapable of inducing Cap ubiquitination and degradation. Consistent with this finding, a Cap ubiquitination-deficient PCV2 strain showed enhanced virus replication and produced severe histological lesions in the lung and lymph node tissues compared with wild-type PCV2. Taken together, the results presented here suggest that PCV2 downregulates the pMKRN1 variant to avoid pMKRN1-mediated Cap ubiquitination and degradation, thus promoting viral replication and pathogenesis in its targeted tissues.IMPORTANCE Porcine circovirus type 2 is the pathogen to which pigs are the most susceptible, causing immense economic losses in the global swine industry, but whether host cells have developed some strategies to prevent viral replication is still unclear. Here, we found that porcine MKRN1 (pMKRN1) was upregulated in the early stage of PCV2 infection and mediated the polyubiquitination and degradation of Cap protein to block PCV2 replication, yet persistent PCV2 infection downregulated pMKRN1 levels to avoid degradation, promoting viral replication and pathogenesis in its targeted tissues. These data present new insight into the molecular mechanisms underlying the antiviral effects of pMKRN1 E3 ligase during PCV2 infection and also suggest potential new control measures for PCV2 outbreaks.
Project description:Porcine circovirus type 2 (PCV2) causes immunosuppression in pigs. One causative factor is an imbalance in cytokine levels in the blood and lymphoid tissues. Many studies have reported changes in cytokine production, but the regulatory mechanisms involved have not yet been elucidated. In this study, we investigated alteration and regulation of IL-4 and IL-12 production in lymphocytes following incubation with PCV2 in vitro. The levels of IL-4 decreased and levels of IL-12 increased in lymphocyte supernatants, and the DNA-binding activity of NF-?B and the expression of p65 in the nucleus and p-I?B in the cytoplasm of lymphocytes increased after incubation with PCV2. However, these effects were reversed when lymphocytes were coincubated with PCV2 and the NF-?B inhibitor BAY11-7082. In addition, the expression of MyD88 protein increased and the expression of mRNA for the toll-like receptors (TLRs) TLR2, TLR3, TLR4 and TLR9 was upregulated when lymphocytes were incubated with PCV2. However, no change was seen in TLR7 and TLR8 mRNA expression. In conclusion, this study showed that PCV2 induced a decrease in IL-4 and an increase in IL-12 production in lymphocytes, and these changes were regulated by the TLR-MyD88-NF-?B signal pathway.
Project description:To develop an effective, safe, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI) H5N1, we have constructed a recombinant Bacillus subtilis strain (B.S.-HA) expressing the hemagglutinin (HA) protein. Then we evaluated the immune function in chicken bone marrow derived dendritic cells (BM-DCs), and the immune response after oral immunization. Our results show that the recombinant Bacillus subtilis B.S.-HA could be sampled by BM-DCs in vitro and increase the BM-DCs major histocompatibility complex (MHC) II phenotype. The weight, height of the small intestine villus, and lymphoid tissue area of the ileum increased significantly in B.S.-HA immunized chickens (P?<?0.05 or P?<?0.01). B.S.-HA induced the secretion of cytokines and the expression of Toll-like receptors in the trachea and small intestine (P?<?0.05 or P?<?0.01). In addition, B.S.-HA elevated the specific IgA titers in the trachea, IgG and HI antibody titers in serum (P?<?0.05 or P?<?0.01). Therefore, B.S.-HA provides a potential novel strategy and approach for developing an H5N1 vaccine.
Project description:<h4>Background</h4>Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets.<h4>Results</h4>The high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets.<h4>Conclusions</h4>Above all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.
Project description:Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.
Project description:<h4>Background</h4>Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide.<h4>Objectives</h4>A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy.<h4>Methods</h4>In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) <i>in vitro</i> and <i>in vivo</i> after gene transfer.<h4>Results</h4>The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-? showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2.<h4>Conclusions</h4>The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.
Project description:The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-?-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.
Project description:BACKGROUND:Enteritis is one of the most frequently reported symptoms in piglets infected with porcine circovirus type 2 (PCV2), but the immunopathogenesis has not been reported. OBJECTIVES:This study examined the effect of a PCV2 infection on the intestinal mucosal immune function through morphological observations and immune-related molecular detection. METHODS:Morphological changes within the ileum of piglets during a PCV2 infection were observed. The expression of the related-molecules was analyzed using a gene chip. The immunocyte subsets were analyzed by flow cytometry. The secretory immunoglobulin A (SIgA) content was analyzed by enzyme-linked immunosorbent assay. RESULTS:The PCV2 infection caused ileal villus damage, intestinal epithelial cells exfoliation, and an increase in lymphocytes in the lamina propria at 21 days post-infection. Differentially expressed genes occurred in the defense response, inflammatory response, and the complement and coagulation cascade reactions. Most of them were downregulated significantly at the induction site and upregulated at the effector site. The genes associated with SIgA production were downregulated significantly at the induction site. In contrast, the expression of the Toll-like receptor-related genes was upregulated significantly at the effector site. The frequencies of dendritic cells, B cells, and CD8?T cells were upregulated at the 2 sites. The SIgA content decreased significantly in the ileal mucosa. CONCLUSIONS:PCV2 infections can cause damage to the ileum that is associated with changes in immune-related gene expression, immune-related cell subsets, and SIgA production. These findings elucidated the molecular changes in the ileum after a PCV2 infection from the perspective of intestinal mucosal immunity, which provides insights into a further study for PCV2-induced enteritis.
Project description:Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Pigs with PMWS are often infected with a variety of other pathogens, including bacteria, viruses and mycoplasm, in addition to porcine circovirus type 2 (PCV2). PCV2 and Haemophilus parasuis serovar 4 (HPS4) coinfection remain epidemic in China.Here we report construction of a three-week-old naturally farrowed, colostrum-deprived (NFCD) piglet's infection model and demonstrate that PCV2-infected piglets with the HPS4 coinfection increased the virulence of PCV2 and these pathogens interact acquired PMWS.All the single infected piglets were transiently bacteremic or viremic. All the PCV2/HPS4 coinfected piglets developed PMWS, characterized by dyspnea, anorexia, prostration and lose weight severely. Co-infection with PCV2 and HPS4 resulted in an increased amount of virus in serum and tissues, presented a slower generation and lower levels of antibodies against PCV2. Co-infection with PCV2 and HPS4 resulted in further reductions in total and differential peripheral blood leukocyte counts. Meantime, PCV2/ HPS4 coinfection potentiated the severity of lung and lymphoid lesions by PCV2-associated, increased the virulence of PCV2-antigen and enhanced the incidence of PMWS in piglets.Co-infection with PCV2 and HPS4 induce the exacerbation of system injuries and enhance the pathogenicity of PCV2 in piglets.