Development of haemagglutination assay for titration of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) was one of the most economically important viral pathogens in all the swine-producing countries and often resulted in tremendous economic losses for the swine industry. As PCV2 could not cause cytopathogenic effects while propagated in infected cells, many complicated experiments should be performed to titrate its virus titer. In this study we developed a simple and effective hemagglutination assay for titration of virus titer of PCV2. To develop the hemagglutination assay, a recombinant bispecific nanobody (BsNb) against PCV2 and chicken red blood cells (cRBCs) was constructed based on two nanobodies (NbPCV11 and NbRBC48) which were selected from the non-immunized nanobody library, respectively. The hemagglutination assay was used to titrate the virus titer of PCV2 propagated in cell culture by simple naked-eye observation within 30 min, with the detection limit of 104.09 tissue culture infective dose 50 (TCID50)/mL, excellent specificity and reproducibility. Therefore, the hemagglutination assay had potential to be a rapid, reliable, cost-effective, user-friendly qualitative and semi-quantitative tool for titration of virus titer of PCV2 during the vaccine manufacturing process.

Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris.

Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

CTA1: Purified and display onto gram-positive enhancer matrix (GEM) particles as mucosal adjuvant.

The A1 subunit of cholera toxin (CTA1) retains the adjuvant function of CT, without its toxic side effects, making the molecule a promising mucosal adjuvant. However, the methods required to obtain a pure product are both complicated and expensive, constricting its potential commercial applicability. Here, we fused the peptidoglycan binding domain (PA) to the C-terminus of CTA1, which enabled the fusion protein to be expressed by Bacillus subtilis, and secreted into the culture medium. CTA1 was then purified and displayed on GEM particles using a one step process, which resulted in the formation of CTA1-GEM complexes. Next, the CTA1-GEM complexes were used as an adjuvant to enhance the immune responses of mice to the influenza subunit vaccine. It was observed that the CTA1-GEM complexes enhanced specific systemic (IgG) and mucosal (IgA) immune responses against antigen, and induced cellular immune responses as well. The data presented here suggests that CTA1-GEM complexes can serve as a viable mucosal adjuvant.

Surface-displayed porcine reproductive and respiratory syndrome virus from cell culture onto gram-positive enhancer matrix particles.

Vaccine immunization is now one of the most effective ways to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. Impurity is one of the main factors affecting vaccine safety and efficacy. Here we present a novel innovative PRRSV purification approach based on surface display technology. First, a bifunctional protein PA-GRFT (protein anchor-griffithsin), the crucial factor in the purification process, was successfully produced in Escherichia coli yielding 80 mg/L of broth culture. Then PRRSV purification was performed by incubation of PA-GRFT with PRRSV and gram-positive enhancer matrix (GEM) particles, followed by centrifugation to collect virions loaded onto GEM particles. Our results showed that most of the bulk impurities had been removed, and PA-GRFT could capture PRRSV onto GEM particles. Our lactic acid bacteria-based purification method, which is promising as ease of operation, low cost and easy to scale-up, may represent a candidate method for the large-scale purification of this virus for vaccine production.

Enhanced soluble production of cholera toxin B subunit in Escherichia coli by co-expression of SKP chaperones.

The cholera toxin B subunit (CTB) is a nontoxic portion of the cholera toxin that retains mucosal adjuvant properties. Expression of CTB in Escherichia coli is difficult as CTB aggregates and accumulates as insoluble inclusion bodies. To remedy this problem, the periplasmic chaperone, SKP, was investigated as possible co-expression partner to increase the solubility of recombinant CTB (rCTB) in E. coli. The result showed co-expression of SKP enhanced the soluble expression of rCTB in E. coli. Moreover, soluble rCTB was successfully expressed and secreted into the periplasmic space through the direction of the LTB leader signal. rCTB in periplasm was purified using an immobilized d-galactose resin; GM1-ELISA experiments showed that rCTB retains strong GM1 ganglioside-binding activity. Intranasal administration of ovalbumin (OVA) with rCTB significantly induced both mucosal and humoral immune responses specific to OVA. These data indicate that co-expression of the molecular chaperone SKP with CTB increased the solubility of rCTB while maintaining its function.

[Comparison of the binding activity of Lactococcus lactis peptidoglycan protein anchor with different number of motifs].

OBJECTIVE: The aim of the present study was to compare the binding activity of Lactococcus lactis peptidoglycan protein anchor (PA) with different number of motifs. METHODS: L. lactis PA gene sequences with 2 and 3 lysin motifs (LysM) were obtained by PCR amplification. Then, the recombinant plasmid of pET-32a(+) containing PA gene was constructed and transformed into E. coli BL21 (DE3) , and induced to express the fusion protein PA2 and PA3. The purified and refolded PA2 and PA3 were incubated with gram positive enhancer matrix (GEM). The binding activities of PA2 and PA3 were identified and compared by Western blot, TEM and SDS-PAGE. RESULTS: The PA2 and PA3 could bind to GEM. The anchoring activity of PA3 was obviously superior to the PA2. CONCLUSION: The data indicated that PA with 3 LysMs had a better binding capacity compared with 2 LysMs. The results provided foundations to further improve the design of GEM-PA display system.

Conventional dendritic cells 2, the targeted antigen-presenting-cell, induces enhanced type 1 immune responses in mice immunized with CVC1302 in oil formulation.

Multifunctional CD4+ T helper 1 (Th1) cells, producing IFN-γ, TNF-α and IL-2, define a correlate of vaccine-mediated protection against intracellular infection. In our previous study, we found that CVC1302 in oil formulation promoted the differentiation of IFN-γ+/TNF-α+/IL-2+Th1 cells. In order to extend the application of CVC1302 in oil formulation, this study aimed to elucidate the mechanism of action in improving the Th1 immune response. Considering the signals required for the differentiation of CD4+ T cells to Th1 cells, we detected the distribution of innate immune cells and the model antigen OVA-FITC in lymph node (LN), as well as the quantity of cytokines produced by the innate immune cells. The results of these experiments show that, cDC2 and OVA-FITC localized to interfollicular region (IFR) of the draining lymph nodes, inflammatory monocytes localized to both IFR and T cell zone, which mainly infiltrate from the blood. In this inflammatory niche within LN, CD4+ T cells were attracted into IFR by CXCL10, secreted by inflammatory monocytes, then activated by cDC2, secreting IL-12. Above all, CVC1302 in oil formulation, on the one hand, targeted antigen and inflammatory monocytes into the LN IFR in order to attract CD4+ T cells, on the other hand, targeted cDC2 to produce IL-12 in order to promote optimal Th1 differentiation. The new finding will provide a blueprint for application of immunopotentiators in optimal formulations.

Analysis of CVC1302-Mediated Enhancement of Monocyte Recruitment in Inducing Immune Responses.

Monocytes (Mos) are believed to play important roles during the generation of immune response. In our previous study, CVC1302, a complex of PRRs agonists, was demonstrated to recruit Mo into lymph nodes (LNs) in order to present antigen and secret chemokines (CXCL9 and CXCL10), which attracted antigen-specific CD4+ T cells. As it is known that Mos in mice are divided into two main Mo subsets (Ly6C+ Mo and Ly6C- Mo), we aimed to clarify the CVC1302-recruiting Mo subset and functions in the establishment of immunity. In this study, we found that CVC1302 attracted both Ly6C+ Mo and Ly6C- Mo into draining LNs, which infiltrated from different origins, injection muscles and high endothelial venule (HEV), respectively. We also found that the numbers of OVA+ Ly6C+ Mo in the draining LNs were significantly higher compared with OVA+ Ly6C- Mo. However, the levels of CXCL9 and CXCL10 produced by Ly6C- Mo were significantly higher than Ly6C+ Mo, which plays important roles in attracting antigen-specific CD4+ T cells. Under the analysis of their functions in initiating immune responses, we found that the ability of the Ly6C+ monocyte was mainly capturing and presenting antigens, otherwise; the ability of the Ly6C- monocyte was mainly secreting CXCL9 and CXCL10, which attracted antigen-specific CD4+ T cells through CXCR3. These results will provide new insights into the development of new immunopotentiators and vaccines.

Increases in Cellular Immune Responses Due to Positive Effect of CVC1302-Induced Lysosomal Escape in Mice.

This study found a higher percentage of CD8+ T cells in piglets immunized with a CVC1302-adjuvanted inactivated foot-and-mouth disease virus (FMDV) vaccine. We wondered whether the CVC1302-adjuvanted inactivated FMDV vaccine promoted cellular immunity by promoting the antigen cross-presentation efficiency of ovalbumin (OVA) through dendritic cells (DCs), mainly via cytosolic pathways. This was demonstrated by the enhanced levels of lysosomal escape of OVA in the DCs loaded with OVA and CVC1302. The higher levels of ROS and significantly enhanced elevated lysosomal pH levels in the DCs facilitated the lysosomal escape of OVA. Significantly enhanced CTL activity levels was observed in the mice immunized with OVA-CVC1302. Overall, CVC1302 increased the cross-presentation of exogenous antigens and the cross-priming of CD8+ T cells by alkalizing the lysosomal pH and facilitating the lysosomal escape of antigens. These studies shed new light on the development of immunopotentiators to improve cellular immunity induced by vaccines.

Reduction of Postweaning Multisystemic Wasting Syndrome-Associated Clinical Symptoms by Virus-Like Particle Vaccine Against Porcine Parvovirus and Porcine Circovirus Type 2.

The porcine circovirus type 2 (PCV2) capsid (Cap) protein and porcine parvovirus (PPV) VP2 protein have been studied in vaccines to control postweaning multisystemic wasting syndrome (PMWS). Virus-like particle (VLP) vaccines are nonreplicative vectors that deliver epitopes and induce immune responses. However, most VLP vaccines are recombinant proteins expressed in eukaryotic systems and are expensive and complex. In this study, the full-length PCV2-Cap and PPV-VP2 proteins were expressed in Escherichia coli, which self-assembled into VLPs. The highly soluble proteins were purified using Ni-chelating affinity chromatography. The proteins self-assembled into VLPs of ∼20 nm (Cap VLP) and 25 nm (VP2 VLP) in diameter. The immunogenicities of Cap VLP and VP2 VLP were determined in piglets coinfected with PPV and PCV2 postimmunization. The results suggested that Cap VLP and VP2 VLP did not antagonize each other. The combined vaccine induced stronger humoral and cellular immune responses and provided the best protection against PPV and PCV2 coinfection. On a farm containing PMWS-infected pigs, the combined Cap VLP and VP2 VLP vaccine significantly improved piglet growth indices; the average daily weight gains were significantly higher than those of the Cap VLP vaccine and nonimmunized groups. Thus, Cap and VP2 protein expression in E. coli is feasible for large-scale VLP vaccine production. The combined vaccine may be a promising candidate vaccine for better preventing PMWS-associated diseases coinfected with PCV2 and PPV.

Development of GEM-PA-nanotrap for purification of foot-and-mouth disease virus.

Vaccination is the primary preventative measure against outbreaks of foot-and-mouth disease (FMD). The efficacy of inactivated FMD vaccines is mainly determined by the integrity of foot-and-mouth disease virus (FMDV) particles (referred to as 146S particles), and impurities in the inactivated vaccines could result in side effects. In this study, we developed an effective affinity purification method for the purification of FMDV from cellular lysates, referred to as GEM-PA-nanotrap. To develop the GEM-PA-nanotrap, a nanobody (Nb205) against FMDV vaccine strain O/MYA98/BY/2010 146S particles was selected from a non-immunized library and fused to a peptidoglycan-binding protein anchor (PA). The PA-Nb205 fusion protein was non-covalently coupled to the surface of Gram-positive enhancer matrix (GEM) particles, which were prepared from the non-living, non-genetically modified, Gram-positive, food-grade Lactococcus lactis bacteria. The GEM-PA-nanotrap was used to purify FMDV from cellular lysates through a simple incubation and centrifugation step. The FMDV recovery rate was more than 99%, the efficiency of nonviral protein removal was about 98.3%, and the purification process had almost no effect on the integrity and immunogenicity of 146S particles. Therefore, the GEM-PA-nanotrap has potential as an effective method for the recovery and purification of FMDV during the vaccine manufacturing process.

Identification of mechanisms conferring an enhanced immune response in mice induced by CVC1302-adjuvanted killed serotype O foot-and-mouth virus vaccine.

The adjuvant CVC1302 was previously shown to efficiently enhance the immunogenicity of killed foot-and-mouth disease virus (FMDV) in mice and piglets. However, the underlining mechanism of action of CVC1302 remains unclear, especially at local injection sites and draining lymph nodes. Since the FMDV vaccine is administrated intramuscularly in field settings, we studied local immune responses to FMDV following intramuscular injection in mice, and found that CVC1302-adjuvanted killed FMDV (KV-CVC1302) induced secretion of several chemokines in murine muscle tissues, including MCP-1, MIP-1α, and MIP-1ß. The number of monocytes recruited to the site of injection was significantly higher in mice immunized with KV-CVC1302 compared with mice immunized with killed FMDV alone (KV). iTAQ-based quantitative proteomic assays were additionally employed to explore the molecular mechanisms of CVC1302 action in the draining lymph nodes. A total of 35 proteins were identified as being differentially expressed among the control group, KV-immunized group and KV-CVC1302-immunized group at 10 days post immunization (dpi). Proteins exhibiting differential expression were mainly involved in signal transduction, apoptosis, endocytosis and innate immune responses. Pathway analysis demonstrated that AMPK, phospholipase D, cAMP, Rap1, and MAPK signaling pathways were potentially induced by the immunopotentiator CVC1302. Understanding the local mechanism of CVC1302 action at injection sites and draining lymph nodes will provide new insights into the development of FMDV vaccines.

The immunopotentiator CVC1302 enhances immune efficacy and protective ability of foot-and-mouth disease virus vaccine in pigs.

The immunological enhancement characteristics of the immunopotentiator CVC1302 were evaluated for foot-and-mouth disease virus (FMDV) inactivated vaccine in pigs. Eight-week-old piglets were vaccinated with the foot-and-mouth disease (FMD) vaccine alone (FMD-vaccine group) or with the addition of CVC1302 (FMD-CVC1302 group), and the serum liquid phase blocking ELISA (LPB-ELISA) antibody titers, IgG1 and IgG2 levels, and the levels of four cytokines secreted by peripheral blood lymphocytes were measured at 28 days post vaccination (dpv). In the FMD-CVC1302 group, the LPB-ELISA antibody titers, IgG1, and IgG2 titers, and IL-2, IL-4, IL-6, and IFN-γ levels at 28 dpv were significantly higher than those in the FMD-vaccine group. The FMD-CVC1302 group had long-lasting antibody titers (>7.8 log2), lasting for at least 6 months. In addition, piglets were vaccinated with or without addition of CVC1302 to the FMD vaccine at three different doses (1, 1/3, and 1/9 of the standard vaccine dose) and the serum LPB-ELISA antibody and serum neutralizing (SN) antibody titers were detected at 28 dpv. Then all pigs were challenged with virulent FMDV for PD50 value, and the levels of FMDV-specific RNA copies for the two full-dose groups at 3 and 10 days post challenge (dpc) were measured. The LPB-ELISA and SN antibody titers for the three doses in the FMD-CVC1302 groups were significantly higher than those in the FMD-vaccine groups at the same doses (p < 0.05). Post-virus challenge, the FMDV-specific RNA copy number in the FMD-CVC1302 group was lower than that in the FMD-vaccine group at 3 and 10 dpc. The PD50 value was 15.85 for the FMD-CVC1302 group, which was obviously higher than that for the FMD-vaccine group (10.96), and in the 1/9-dose of FMD-vaccine group only 3/5 pigs were protected. These results indicate that CVC1302 can enhance the immune efficacy and protective ability of the FMD vaccine in pigs.

[Design and immunogenicity evaluation for the bacteria-like particle vaccine against swine type O foot-and-mouth disease virus].

Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 µg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.

Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets.