PCV2-specific IgG Research Articles

Multi-walled carbon nanotube polysaccharide modified Hericium erinaceus polysaccharide as an adjuvant to extend immune responses

In recent years, the utilization of CS-MWCNT as targeted drug carriers has attracted considerable attention. Hericium erinaceus polysaccharide (HEP) has been reported as an immunostimulant to improve immune responses. This study was focussed on developing CS-MWCNT encapsulating HEP (CS-MWCNT-HEP). Using in mice peritoneal macrophages, we found the immune response could be effectively regulated by CS-MWCNT-HEP, promoted the expression of the MHCII, CD86, F4/80 and gp38. Moreover, the mice immunized with CS-MWCNT-HEP nanoparticles significantly extended PCV2-specific IgG immune response and the levels of cytokines. The results demonstrated that CS-MWCNT-HEP may be a promising drug delivery system for immuno-enhancement.

Deletion of a decoy epitope in porcine circovirus 2 (PCV2) capsid protein affects the protective immune response in mice.

The swine pathogen porcine circovirus type 2 (PCV2) causes significant economic damage worldwide. The PCV2 capsid (CP) residues 169-STIDYFQPNNKR-180 have been identified as a decoy epitope that diverts the host immune response away from protective epitopes. However, the decoy epitope may include important linear or conformational protective epitopes against PCV2. In this study, we used the baculovirus system to express recombinant complete CP (1-233) and mutant CP (Δ169-180), in which the decoy epitope was deleted, and evaluated the immune response to these in mice. Immunization with mutant CP (Δ169-180) protein, which formed very low level of virus-like particles (VLPs), elicited significantly lower levels of PCV2 CP-specific IgG antibodies and a slightly lower neutralizing activity than immunization with the complete CP (1-233) protein. This finding suggests that the complete CP is important for efficient VLP assembly and induction of PCV2-specific IgG antibodies and neutralizing antibodies in mice. This study may provide useful information for next-generation vaccine design for PCV2 control.

Interaction between biological sex and early life adversity on gastrointestinal (GI) immune development

Abstract Early postnatal life represents a critical window of GI immune development when lifelong immune function is shaped. Yet, little is known on how early life events impact long-term immune development in animals and people. Based on our previous research, we investigated how biological sex and early weaning (EW) stress interact to shape long-term GI immune development in the pig. Female (F), male intact (MI) and male castrated (MC) piglets (Yorkshire-cross) were weaned at 17 d (EW) or at 26 d of age (Later weaning; LW). At 77 d of age, tissue samples were harvested for RNA-seq, qPCR, and immunoassays. Serum cytokine and ileal mucosal IgG responses were measured following oral and intramuscular vaccination for Lawsonia intracellularis (LI) and PCV2, respectively. Compared with LW pigs, EW MC and MI pigs had reduced body weight gain (P<0.05). The impact of biological sex and EW were observed in vaccine responses. Serum from F showed higher PCV2-specific IgG titers and LI-antibody levels compared with MC and MI pigs. EW F had higher IgG titers in response to vaccination compared with LW F. In contrast, EW MC had suppressed antibody titers compared with LW MC. Pathway analysis of RNA-seq data from ileal mucosa revealed enrichment of genes associated with increased in immune-related pathways (e.g. inflammation) in EW F. In contrast, EW MC exhibited enrichment for genes associated with a down-regulation of immune response (e.g. immune cell migration). Together, these studies showed that biological sex and EW have a major impact on GI immune development in the pig. An understanding of the role and underlying sex-specific mechanisms driving altered immune development in EW pigs could reveal new targets for enhancing early and long-term immune development.

Immune response in piglets orally immunized with recombinant Bacillus subtilis expressing the capsid protein of porcine circovirus type 2

BackgroundPorcine 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).MethodsElectroporation 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).ResultsWe 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 < 0.05 or P < 0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P < 0.01), intestinal intraepithelial lymphocytes (IELs) (P < 0.01), CD3+ T lymphocytes (P < 0.01) and CD4+ T lymphocytes (P < 0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P < 0.01), and induced the secretion of cytokines IL-1β, IL-6, interferon-γ, and β-defensin 2 (P < 0.01).ConclusionsWe constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs.Graphical abstract

Polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide as an adjuvant to enhance immune responses

Nanoparticle delivery systems have been widely investigated as new vaccines strategy to enhance the immune responses to antigens against infectious diseases. The positively charged nanoparticles could efficiently improve the immune responses due to targeting and activating the antigen-presenting cells. In this study, the immunopotentiator Angelica sinensis polysaccharide (ASP) was encapsulated into Poly (lactic-co-glycolic acid) (PLGA) nanoparticles, and the polyethylenimine, one of the cationic polymers, was used to coat nanoparticles to develop a new nanoparticle delivery system (ASP-PLGA-PEI) with positively charged. The ASP-PLGA-PEI nanoparticles significantly activated macrophages, and promoted the expression of the MHCII and CD86 and the production of IL-1β and IL-12p70 cytokines of macrophages. Furthermore, the antigen adsorbed on the surface of the ASP-PLGA-PEI nanoparticles enhanced the antigen uptake by macrophages. Moreover, the mice immunized with PCV2 antigen adsorbed ASP-PLGA-PEI nanoparticles significantly enhanced PCV2-specific IgG immune response and the levels of cytokines, induced a mixed Th1/Th2 immune response with Th1 bias compared with other groups. These findings demonstrate that the positively charged nanoparticles (ASP-PLGA-PEI) have the potential to serve as an effective vaccine delivery and adjuvant system to induce vigorous and long-term immune responses.

Are PCV2-Cattle Infections at an Early Evolutionary Virus Adaption Stage?

Porcine circovirus type 2 (PCV2) has a major economic impact on pig production. Vaccination against the virus appears to keep PCV2 disease (PCVD) manifestations in check. Nevertheless, PCV2 has not been eliminated. Several reports indicate that cattle also seem to be susceptible to PCV2 infection. In this study, we detected increased PCV2 cross-species transmission and suggest a strategy for identifying hidden PCV2 infection in cattle. We compared a postweaning multisystemic wasting syndrome (PMWS) distressed-farm with a subclinically-infected farm and investigated how differences in virus concentration would affect PCV2 transmission to a susceptible host such as cattle. The pig population of farm 1 had recurrent PMWS cases among weaners. The 34 cattle in proximity to the PMWS-affected pigs included 14 cattle (41%) that carried a PCV2 load of on average 2.5 x 105 virus genomes per ml blood. Among these was a 7 years old cow with chronic mastitis that was infected with the highest PCV2 load of 1.3 x 106 virus genomes per ml blood. These numbers contrasted with farm 2 that housed PCV2-subclinically infected pigs in proximity to 31 cattle of which six (19%) were infected with an average of 1.7 x 105 virus genome per ml blood. No PCV2-specific humoral response was measured in these cattle. Additionally, we encountered the problem of how to measure PCV2 latent/persistent infections. In pigs, after a one-shot vaccination, we have previously observed a direct anti-PCV2 IgG response instead of the expected IgM response. This indicated that the pig immune system was already primed with PCV2. We therefore hypothesized that cattle may also be PCV2- primed. Although the cattle’s humoral immune system hardly responded to vaccination, we detected PCV2-specific IgG antibody in one cow after the first vaccination shot. This, taken together with the PCV2 transmission rate in hybrid farms indicated sporadic infections in the cattle population. Thus, it can be concluded that PCV2 infections were not well established in cattle in comparison to the situation in swine.

Antibody Titers from Finisher Populations with Persistent/Latent PCV2 Infection Before, During and After the PCV2-SD Epizootic

Porcine circovirus type 2 (PCV2) diseases (PCVDs) have affected pig production worldwide over the last three decades. Since the advent of mass vaccination, manifestations of PCVDs have dramatically decreased. Nevertheless, persistent/latent PCV2 infections linger in the pig population. Therefore, we investigated whether conclusions can be drawn regarding the health status of a population based on humoral responses to a persistent pathogen such as PCV2. We examined the Swiss finisher population because in this population, time points associated with major events have been well documented. We measured PCV2- specific antibody titers of finishers before the Swiss epizootic in 1996-97 and compared these titers with antibody titers in 2006, during the peak of the epizootic, and in 2011, three years after the start of the mass vaccination of piglets. Two hundred serum samples from finishers were analyzed for each of these time periods, which correspond to before, during and after the Swiss epizootic. PCV2-specific IgG antibody titers were low to modestly positive during the pre-epizootic and post-epizootic periods. At the peak of the epizootic in 2006, almost all serum samples were positive, with higher average IgG concentrations than those detected in pre-epizootic and post-epizootic samples. Moreover, IgM antibodies were analyzed for 60 randomly chosen finishers in each time period. Of these 180 samples, only two serum samples from 2006 contained PCV2-specific IgM antibodies. Mass vaccination against PCV2 reduced PCV2-specific antibody titers in pigs with persistent/latent infection to pre-epizootic levels. Our data show prevalence for and concentrations of IgG-specific PCV2 antibodies, which appear to be correlated with PCVD. This result is counterintuitive because one would typically expect higher antibody concentrations to be associated with less disease; a possible interpretation is that elevated concentrations of immature anti-PCV2 antibodies provided pigs with a certain level of protection against PCVDs.

Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets.

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-γ-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.

The immunological activity of Lycium barbarum polysaccharides liposome in vitro and adjuvanticity against PCV2 in vivo

In previous researches, the results showed that Lycium barbarum polysaccharides (LBP) encapsulated with liposome could enhance the immune activity of LBP. Therefore, the present study was designed to investigate the effects of LBPL on spleen lymphocytes and macrophages of mice in vitro and evaluate the immunological adjuvant activity of PCV2 vaccine in vivo. The results showed that LBPL could significantly promote splenocyte proliferation synergistically with PHA or LPS, increase the ratio of CD4+ to CD8+ T cells and promote the cytokine secretion of macrophages; enhance PCV2-specific IgG antibody responses, promote Th1 cytokines (IFN-γ and TNF-a) and Th2 cytokine (IL-4) secretion. The histomorphological observation of spleen demonstrated that LBPL as a vaccine adjuvant also has good improvement and stimulating effect on the immune organ.

Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice.

The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases.

Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

BackgroundPorcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.ResultsIn this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.ConclusionsWe have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.

Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus

The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity.

Expression of the capsid protein of porcine circovirus type 2 in Lactococcus lactis for oral vaccination

Diseases associated with porcine circovirus type 2 (PCV2) infections are becoming a major problem for the swine industry worldwide. The capsid protein (Cap) of PCV2 is an antigen important for both early diagnosis and development of vaccines. In this study, Lactococcus lactis was used as vehicle to deliver the PCV2 antigen in an attempt to develop oral vaccine. A cap gene with a deleted nuclear localization signal sequence ( dcap) was cloned into an Escherichia coli/ L. lactis shuttle vector pSEC: LEISS under the control of a nisin promoter. Intracellular and extracellular expression of the dCap was confirmed by Western blot analysis. Significantly higher levels of PCV2-specific IgG in the sera of mice were observed upon oral administration of strain cultures expressing the PCV2 antigen. These results suggest that it is feasible to use L. lactis as an antigen delivery vehicle for developing oral vaccines against PCV2 infection.