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.

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.

Orf virus as an adjuvant enhances the immune response to a PCV2 subunit vaccine.

Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1ß, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells.

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Efficacy evaluation of a bivalent subunit vaccine against classical swine fever virus and porcine circovirus type 2.

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) are two of the most devastating and economically significant pathogens affecting pig populations worldwide. Administration of a combination of vaccines against swine pathogens has been demonstrated to be as efficacious as the administration of single vaccines. In this study, we developed and tested a novel bivalent subunit vaccine against CSFV and PCV2. The safety and efficacy of this vaccine were demonstrated in mice and specific pathogen-free (SPF) piglets. In addition to investigating the serological responses after immunization, challenge studies with both viruses were also conducted. The results showed that this CSFV/PCV2 bivalent vaccine elicited a high level of neutralizing antibodies against both viruses and provided protection in challenge studies. In conclusion, the CSFV/PCV2 bivalent vaccine is safe and effective against CSFV or PCV2 challenge.

From obscurity to urgency: a comprehensive analysis of the rising threat of duck circovirus.

Duck circovirus (DuCV) is a small, nonenveloped, single-stranded DNA virus with immunosuppressive effects on ducks that leads to slow growth and elevated mortality following mixed infections. Its infection manifests as feather loss, slow growth, swelling of respiratory tissue, and damage to immune organs in ducks. Although single infections with DuCV do not cause noticeable clinical symptoms, its ability to compromise the immune system and facilitate infections caused by other pathogens poses a serious threat to duck farming. Given the prevalence of this disease and the increasing infection rates in recent years, which have resulted in significant economic losses in duck farming and related sectors, research and control of DuCV infection have become especially important. The aim of this review is to provide a summary of the current understanding of DuCV, serving as a reference for subsequent research and effective control of the virus. We focus mainly on the genetics and molecular biology, epidemiology, clinical symptoms, and pathology of DuCV. Additionally, topics such as the isolation and culture of the virus, vaccines and antiviral therapies, diagnostics, and preventative measures are discussed.

A novel strategy for an anti-idiotype vaccine: nanobody mimicking neutralization epitope of porcine circovirus type 2.

Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.

Cross-protection of a porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae trivalent vaccine against a dual PCV-2e and Mycoplasma hyopneumoniae challenge.

The purpose of this experimental study was to determine the cross-protection of a new trivalent vaccine containing porcine circovirus types 2a/b (PCV-2a/b) and Mycoplasma hyopneumoniae. Pigs were vaccinated intramuscularly at 21 days of age, then challenged at 42 days of age with a dual PCV-2e and M. hyopneumoniae challenge. Growth performance was significantly improved during the experimental period (21 to 63 days of age) in vaccinated-challenged pigs compared to unvaccinated-challenged pigs. Pigs that were vaccinated and challenged elicited a significant amount of PCV-2e- and M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC) and reduced the levels of PCV-2e viremia and laryngeal shedding. The results of the present study demonstrated that a trivalent vaccine provided cross-protection against a dual PCV-2e and M. hyopneumoniae challenge.

Le but de cette étude expérimentale était de déterminer la protection croisée d'un nouveau vaccin trivalent contenant le circovirus porcin de types 2a/b (PCV-2a/b) et Mycoplasma hyopneumoniae. Les porcs ont été vaccinés par voie intramusculaire à l'âge de 21 jours, puis provoqués à l'âge de 42 jours avec double provocation par PCV-2e et M. hyopneumoniae. Les performances de croissance ont été significativement améliorées au cours de la période expérimentale (21 à 63 jours) chez les porcs vaccinés-provoqués par rapport aux porcs non-vaccinés-provoqués. Les porcs qui ont été vaccinés et provoqués ont produit une quantité importante de cellules sécrétant de l'interféron-γ spécifiques au PCV-2e et à M. hyopneumoniae (IFN-γ-SC) et ont réduit les niveaux de virémie du PCV-2e et d'excrétion laryngée. Les résultats de la présente étude ont démontré qu'un vaccin trivalent offrait une protection croisée contre une double provocation par le PCV-2e et M. hyopneumoniae.(Traduit par Docteur Serge Messier).

Field evaluation of novel plant-derived porcine circovirus type 2 vaccine related to subclinical infection.

BACKGROUND: The objective of this field trial was to evaluate the efficacy of a new plant-based porcine circovirus type 2a (PCV2a) vaccine. This vaccine was a recombinant capsid subunit PCV2a vaccine based on the Nicotiana benthamiana expression system. METHODS: Three farms were selected for the study based on their history of subclinical PCV2 infection. A total of 40 18-day-old pigs were randomly allocated to either vaccinated or unvaccinated groups (20 pigs per group; 10 = male and 10 = female). Pigs received a 2.0-mL dose of the plant-based PCV2a vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate buffered-saline at the same age. RESULTS: Vaccination had a positive effect on pig growth performance compared to that of unvaccinated pigs on all three of the farms. Vaccination of pigs with a plant-based PCV2a vaccine induced high levels of neutralizing antibodies titres against PCV2d and PCV2d-specific interferon-γ secreting cells which resulted in the reduction of PCV2d viral load and reduced lymphoid lesions severity. CONCLUSIONS: The results of this field trial demonstrated cross-protection of PCV2d by a plant-based PCV2a vaccine and a positive effect of pig growth performance with vaccination.

Recombinant Orf virus induced antibody production against capsid protein of porcine circovirus type 3 in mice.

The emerging worldwide distributed porcine circovirus type 3 (PCV3) infection poses a serious threat to swine herds. An important means of preventing and controlling PCV3 infection is the development of the vaccine, while, the inability to cultivate in vitro has become the biggest obstacle. Orf virus (ORFV), the prototypic member of the Parapoxviridae, has been proven to be a novel valid vaccine vector for preparing various candidate vaccines. Here, recombinant ORFV expressing capsid protein (Cap) of PCV3 was obtained and proved its favorable immunogenicity inducing antibody against Cap in BALB/c mice. Based on the enhanced green fluorescent protein (EGFP) as a selectable marker, the recombinant rORFVΔ132-PCV3Cap-EGFP was generated. Then, recombinant ORFV expressing Cap only, rORFVΔ132-PCV3Cap, was obtained based on rORFVΔ132-PCV3Cap-EGFP using a double homologous recombination method by screening single non-fluorescent virus plaque. Results of the western blot showed that the Cap can be detected in rORFVΔ132-PCV3Cap infected OFTu cells. The results of immune experiments in BALB/c mice indicated that a specific antibody against Cap of PCV3 in serum was induced by rORFVΔ132-PCV3Cap infection. The results presented here provide a candidate vaccine against PCV3 and a feasible technical platform for vaccine development based on ORFV.

Effect of duck interferon-α and an anti-cap protein polyclonal antibody against duck circovirus.

Duck circovirus (DuCV) is one of the most prevalent viruses in the duck breeding industry, and causes persistent infection and severe immunosuppression. Currently, there is a serious lack of prevention and control measures and no commercial vaccine against DuCV. Therefore, effective antiviral drugs are important for treating DuCV infection. Interferon (IFN) is an important component of antiviral innate immunity, but it remains unclear whether duck IFN-α has a clinical effect against DuCV. Antibody therapy is an important way to treat viral infections. The DuCV structural protein (cap) is immunogenic, and it remains to be determined whether an anti-cap protein antibody can effectively block DuCV infection. In this study, the duck IFN-α gene and the DuCV structural protein cap gene were cloned, expressed and purified in Escherichia coli to prepare duck recombinant IFN-α and the cap protein. Then, rabbits were immunized with the recombinant cap protein to prepare a rabbit polyclonal antibody. This study investigated the antiviral effect of duck recombinant IFN-α and the anti-cap protein antibody and their combined effect on Cherry Valley ducks infected with DuCV. The results showed that the treatment significantly alleviated the clinical symptoms of immune organ atrophy and immunosuppression compared with the control. The histopathological damage of the target organs was alleviated, and replication of DuCV in the immune organs was significantly inhibited. The treatment also reduced the damage caused by DuCV to the liver and immune function, and increased the level of the DuCV antibody in the blood, thereby improving antiviral activity. Notably, the combination of duck IFN-α and the polyclonal antibody completely blocked DuCV infection after 13 days under the experimental conditions, showing a better inhibitory effect on DuCV infection than single treatments. These results showed that duck recombinant IFN-α and the anti-cap protein antibody can be used as antiviral drugs to clinically treat and control DuCV infection, particularly the vertical transmission of the virus in breeding ducks.

Evaluation of cross-immunity among major porcine circovirus type 2 genotypes by infection with PCV2b and PCV2d circulating strains.

There are three main genotypes of porcine circovirus type 2 (PCV2), namely PCV2a, PCV2b and PCV2d, of which PCV2b and PCV2d are currently the most common. There are antigenic differences between these different genotypes. To explore the effect of PCV2 antigen differences on the immune protection provided by vaccines, a cross-immune protection test was carried out in pigs. Three genotype strains, PCV2a-CL, PCV2b-MDJ and PCV2d-LNHC, were inactivated and emulsified to prepare inactivated vaccines to immunize pigs, who were then challenged with the circulating strains PCV2b-BY and PCV2d-LNHC. Immunoperoxidase monolayer assays (IPMAs) and micro-neutralization assays were used to detect antibodies against the three different genotypes of PCV2. The results showed that the three genotype vaccines induced pigs to produce antibodies against the same and different genotypes of PCV2, but the levels of IPMA and neutralizing antibodies against the same genotype were higher than those against different genotypes. Quantitative Polymerase Chain Reaction (qPCR), virus titration and immunohistochemistry were used to detect PCV2 genomic DNA, live virus and antigen, respectively, in inguinal lymph nodes of experimental pigs. Following challenge with the PCV2b-BY strain, the viral DNA load in the inguinal lymph nodes of pigs immunized with the three genotype vaccines was reduced by more than 99 % compared to the unimmunized group. Following challenge with the PCV2d-LNHC strain, the viral DNA loads in the inguinal lymph nodes of pigs immunized with PCV2a, PCV2b and PCV2d genotype vaccines were reduced by 93.8 %, 99.8 % and 98.3 %, respectively, compared to unimmunized controls. In addition, neither live PCV2 virus nor antigen were detected in the inguinal lymph nodes of pigs immunized with any of the genotype vaccines (0/18), but both were detected in the lymph nodes of experimental pigs in the unimmunized control group (6/6). These findings suggest that, although the antigenic differences of the three genotype strains induce significant differences in antibody levels, they seem to have little effect on cross-protection between different genotypes.

Encapsidated-CpG ODN enhances immunogenicity of porcine circovirus type 2 virus-like particles.

A DNA fragment containing CpG motifs (CpG ODN) is one of the potent immunopotentiators used to improve vaccine efficacy. It can enhance a protective immunity by stimulating both innate and adaptive immune responses. In this study, we designed and constructed a recombinant plasmid carrying the combined CpG ODN to generate an immunopotentiator for boosting the immunogenicity of porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The capsid protein of PCV2b was expressed in insect cells and purified by affinity chromatography. The purified capsid protein was incubated with the CpG ODN in the reaction that allowed VLPs formation and encapsidation of the CpG ODN to occur simultaneously. Morphology of the reassembled VLPs was similar to the PCV2 virions as observed using an electron microscope. When the CpG ODN-encapcidated VLPs was treated with DNase I, the VLPs could protect the packaged CpG ODN from the enzyme digestion. Moreover, we immunized mice subcutaneously with VLPs, CpG ODN-loaded VLPs, or phosphate buffer saline for three times at two-week intervals. The results showed that the CpG ODN-loaded VLPs could elicit significantly higher levels of PCV2-specific neutralizing antibodies and interferon gamma (IFN-γ) expression in the immunized mice compared to those conferred by the VLPs alone. Conclusively, we have proved that the CpG ODN incorporated in VLPs can serve as a potent immunopotentiator for PCV2 vaccine development.

Immunogenicity and Immunoprotection of PCV2 Virus-like Particles Incorporating Dominant T and B Cell Antigenic Epitopes Paired with CD154 Molecules in Piglets and Mice.

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine.

An evaluation of intradermal all-in-one vaccine based on an inactivated recombinant Mycoplasma hyopneumoniae strain expressing porcine circovirus type 2 (PCV2) capsid protein against Korean stains of PCV2d and M. hyopneumoniae challenge.

This study evaluates the efficacy of intradermal all-in-one vaccine (MHYOSPHERE® PCV ID, Laboratorios Hipra S.A. Amer, Spain) based on the strain Nexhyon, an inactivated recombinant M. hyopneumoniae strain with an embedded/integrated PCV2a capsid protein thereof, as the single active substance. Pigs were administered the vaccine intradermally at 21 days of age with 0.2 mL, then challenged at 49 days of age with either M. hyopneumoniae (intratracheal route), PCV2d (intranasal route), or both. Upon dual challenge, growth performance was improved when the intradermal all in one vaccine was administered compared to the unvaccinated group. In pigs receiving single or dual challenge, vaccination increased neutralizing antibodies against PCV2d and specific interferon-γ secreting cells for each pathogen. In contrast, viral load of PCV2d in the blood, M. hyopneumoniae load in the larynx, and the severity of pulmonary and lymphoid lesions were decreased. Vaccination provided good protection against challenge with M. hyopneumoniae and PCV2d.

Carboxyl-Terminal Decoy Epitopes in the Capsid Protein of Porcine Circovirus Type 2 Are Immunogenicity-Enhancers That Elicit Predominantly Specific Antibodies in Non-Vaccinated Pigs.

In the context of the carboxyl-terminus (C-terminus) of the capsid protein of porcine circovirus type 2a (PCV2a) and PCV2a vaccines, this study aimed to explore its unrevealing cryptic epitope and its relation to PCV2-infected herd immunity. To discover the C-terminus of the capsid protein of PCV2a, monoclonal antibodies (mAbs) were generated in this work. Two mAbs bound the two minimal linear epitopes (229PPLKP233 and 228DPPLNP233 (or 229PPLNP233)), which were located at the C-terminus of the capsid proteins of PCV2a and PCV2b, respectively. One mAb bound to the minimal linear epitope (220QFREFNLK227, peptide P82), but it neither bound the virus-like particle (VLP) of PCV2a nor produced positive staining in PCV2a-infected cells by immunofluorescence assay. Further, the residues 220-227 were not accessible on the surface of the VLP on the three-dimensional model, but the residues 228-231 extend toward the VLP exterior. Immunoassays were conducted in this study to screen anti-viral peptide-specific IgGs, which could differentiate vaccinated pigs from non-vaccinated ones. The data show two 220QFREFNLKDPPLKP233-containing peptides had a significantly higher binding reactivity with sera from PCV2-infected pigs in the control group than with sera from the VLP-vaccine group, particularly seen in sera from swine aged 15 weeks to 24 weeks. However, the peptide P82 had not this phenomenon in that test. This study confirmed that C-terminal epitopes play an important role in PCV2-induced decoy of swine humoral immunity.

A chimeric PCV rescued virus with the immunogenic cap gene of PCV3 cloned into the genomic backbone of the nonpathogenic PCV1 induces specific antibodies but with no pathogenic in pigs.

A chimeric PCV called PCV1-3 with the immunogenic Cap gene of pathogenic PCV type 3(PCV3) cloned into the genomic skeleton of the nonpathogenic PCV1 was rescued and inoculated into PCV3 negative piglets. The results of fluorescence quantitative PCR showed that the PCV1-3 DNA detected in serum and tissues was negative. The pathogenicity of piglets showed that PCV 1-3 did not cause the clinical characteristics and pathological changes. The viral neutralization assay revealed that infected pigs could produce antibodies and neutralize the viral activity. All results showed that chimeric virus induced specific antibodies but with no pathogenic in pigs, which provided new candidate strains for the development of PCV3 vaccine.

DNA Prime and Recombinant Protein Boost Vaccination Confers Chickens with Enhanced Protection against Chicken Infectious Anemia Virus.

Chicken infectious anemia (CIA) is an immunosuppressive disease caused by chicken infectious anemia virus (CIAV) that poses a great threat to the poultry industry worldwide. At present, vaccination is an important way to prevent and control CIA. Apart from a CIAV-attenuated vaccine used in clinical practice, the research and development of a genetically engineered vaccine has good prospects. However, it is difficult to induce a strong protective effect with a single subunit vaccine or DNA vaccine. Therefore, the goal of this study is to develop and evaluate a DNA prime/protein boost vaccine strategy for defense against CIAV infection and spread. In this study, the recombinant proteins of CIAV VP1 and VP2 were prepared using an Escherichia coli (E. coli) expression system, and the eukaryotic expression plasmid pBud-VP1-VP2 was constructed. Subsequently, the effects of the DNA prime/protein boost strategy on antibody production and cellular immunity response were measured. The results showed that combined vaccination could induce a higher antibody titer than those of a DNA vaccine or subunit vaccine alone. In addition, spleen lymphocyte index (SI) and IL-2, IL-4, and IFN-γ levels were also significant in chickens the received the combined vaccination. To further investigate the protective effect of DNA prime/protein boost vaccination, a CIAV challenge experiment was carried out. The results showed that infection with CIAV reduced the hematocrit value (Hct) and thymus index, while vaccination recovered this reduction, and the combined immunization group was the least affected by CIAV infection. Furthermore, the CIAV viral load in the combined immunization group was the lowest, indicating that the combined immunization could provide a better protective efficacy. In conclusion, the DNA prime and recombinant protein boost vaccination can be used as an important anti-CIAV strategy, which can induce both enhanced cellular and humoral immunity responses in chickens and provide a new avenue for CIAV prevention and control.

Porcine Circovirus Type 2 Vaccines: Commercial Application and Research Advances.

Porcine circovirus type 2 (PCV2) infection can lead to porcine circovirus-associated disease (PCVAD), causing great economic losses to the global swine industry. Conventional vaccination programs are a major measure in the prevention and control of this disease. Currently, there are 5 commercially available PCV2 vaccines in the international market and 10 kinds commercially available PCV2 vaccines in the Chinese market that confer good efficacy against this virus by alleviating clinicopathological manifestations and enhancing growth performance in pigs. In addition, diverse experimental PCV2 vaccines with protective efficiency have been developed, including attenuated chimeric, nucleic acid, subunit, multivalent, and viral-vectored vaccines. These experimental vaccines have been shown to be relatively effective in improving the efficiency of pig production and simplifying prevention procedures. Adjuvants can be used to promote vaccines with higher protective immunity. Herein, we review the application of multiple commercial vaccines over the years and research advances in experimental vaccines, which provide the possibility for the development of superior vaccines to successfully prevent and control PCV2 infection in the future.