Goose IFIT5 positively regulates goose astrovirus replication in GEF cells.

Interferon-induced protein with tetratricopeptide repeats (IFITs), a family of proteins strongly induced by type I interferon (IFN-I), are deeply involved in many cellular and viral processes. IFIT5, the sole protein in this family found in birds, also plays a crucial role in regulating virus infection. In this study, goose IFIT5 (gIFIT5) was first cloned from peripheral blood lymphocyte (PBL) and phylogenetic analysis showed that it was highly homologous with duck IFIT5 (dIFIT5), sharing 94.6% identity in amino acid sequence. Subsequently, the expression kinetics of gIFIT5 during goose astrovirus (GAstV) infection and the regulatory effect of gIFIT5 on GAstV proliferation were evaluated. Results showed that the mRNA and protein expression level of gIFIT5 was greatly induced by GAstV infection, especially at 12 hpi. Importantly, gIFIT5 could conversely promote GAstV replication in GEF cells. Virus titers in gIFIT5 overexpression group were significantly higher than those in control group at 12 and 24 hpi. Western blot and quantitative real-time PCR (qRT-PCR) further demonstrated that the production of viral cap protein was significantly facilitated in gIFIT5-transfected group. Collectively, GAstV facilitates self-replication via promoting gIFIT5 expression.

Identification of a conserved B-cell epitope on the capsid protein of porcine circovirus type 4.

Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies. IMPORTANCE: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.

Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.

Background: Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China. Methods: In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice. Results: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators. Conclusion: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.

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.

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.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Serum investigation of antibodies against porcine circovirus 4 Rep and Cap protein in Jiangxi Province, China.

In 2019, a novel porcine circovirus 4 (PCV4) was first identified in Hunan Province, China. The circular PCV4 DNA was detected in both diseased and healthy pigs. Recently, PCV4 prevalence surveys have been analyzed in many provinces in both China and South Korea with low positive rates. However, no serological data has been conducted to investigate the prevalence of PCV4 in pigs from Jiangxi Province. To address this issue, an indirect anti-PCV4 antibody enzyme-linked immunosorbent assay (ELISA) based on Cap and Rep protein as a coating antigen was established and applied to study the serum epidemiology of PCV4 in Jiangxi Province. Purified PCV4-His-tagged Cap and Rep were used as the coating antigen to develop an ELISA detection kit. There was no cross-reaction of the Cap/Rep-based ELISA with antisera against PCV2, TGEV and PRRSV, indicating a high specificity of this ELISA assay. The intra-assay coefficient variations (CVs) of Cap-based were 1.239%-9.796%, Rep-based 1.288%-5.011%, and inter-assay CVs of 1.167%-4.694% and 1.621%-8.979%, respectively, indicating a good repeatability. Finally, a total number of 507 serum samples were collected from Jiangxi Province to test for antibody prevalence of PCV4, and 17 (3.35%) and 36 (7.10%) of the samples were Cap and Rep antibody positive, respectively. In summary, our established ELISA kit could be used to detect PCV4 antibodies in serum with good repeatability and high specificity. In addition, field samples detection results showed that the antibody of PCV4 was poorly distributed in intensive pig farms in Jiangxi Province, China.

Live attenuated Salmonella enterica serovar Choleraesuis vector delivering a virus-like particles induces a protective immune response against porcine circovirus type 2 in mice.

The virus-like particles (VLPs) of porcine circovirus type 2 (PCV2) is an attractive vaccine candidate that retains the natural conformation of the virion but lacks the viral genome to replicate, thus balancing safety and immunogenicity. However, the assembly of VLPs requires cumbersome subsequent processes, hindering the development of related vaccines. In addition, as a subunit antigen, VLPs are defective in inducing cellular and mucosal immune responses. In this study, the capsid (Cap) protein of PCV2 was synthesized and self-assembled into VLPs in the recombinant attenuated S. Choleraesuis vector, rSC0016(pS-Cap). Furthermore, rSC0016(pS-Cap) induced a Cap-specific Th1-dominant immune response, mucosal immune responses, and neutralizing antibodies against PCV2. Finally, the virus genome copies in mice immunized with the rSC0016(pS-Cap) were significantly lower than those of the empty vector control group after challenge with PCV2. In conclusion, our study demonstrates the potential of using S. Choleraesuis vectors to delivery VLPs, providing new ideas for the development of PCV2 vaccines.

Prostate secretory protein 94 inhibits sterol binding and export by the mammalian CAP protein CRISP2 in a calcium-sensitive manner.

Members of the CAP protein superfamily are present in all kingdoms of life and have been implicated in many different processes, including pathogen defense, immune evasion, sperm maturation, and cancer progression. Most CAP proteins are secreted glycoproteins and share a unique conserved αßα sandwich fold. The precise mode of action of this class of proteins, however, has remained elusive. Saccharomyces cerevisiae has three CAP family members, termed pathogen related in yeast (Pry). We have previously shown that Pry1 and Pry2 export sterols in vivo and that they bind sterols in vitro. This sterol binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with prostate secretory protein of 94 amino acids (PSP94), another major protein component in the seminal plasma. Here we examine whether the interaction between CRISP proteins and PSP94 affects the sterol binding function of CAP family members. We show that coexpression of PSP94 with CAP proteins in yeast abolished their sterol export function and the interaction between PSP94 and CAP proteins inhibits sterol binding in vitro. In addition, mutations that affect the formation of the PSP94-CRISP2 heteromeric complex restore sterol binding. Of interest, we found the interaction of PSP94 with CRISP2 is sensitive to high calcium concentrations. The observation that PSP94 modulates the sterol binding function of CRISP2 in a calcium-dependent manner has potential implications for the role of PSP94 and CRISP2 in prostate physiology and progression of prostate cancer.

Truncated Diphtheria Toxin DT390 Enhances the Humoral Immunogenicity of Porcine Circovirus Type 2 Capsid Antigen in Mice.

Porcine circovirus type 2 (PCV2) is the causative agent of PCV-associated disease, which harms the swine industry worldwide. Open reading frame 2 of PCV2 encodes the principal immunogenic capsid (Cap) protein, which induces neutralizing antibodies and protective immunity. Cap has been developed as a subunit vaccine against PCV2 infection, although its use is hindered by low immunogenicity. Here, we hypothesized that the truncated diphtheria toxin DT390 might enhance the immunogenicity of Cap. To verify this hypothesis, we fused Cap with DT390, which was expressed using the unique diphtheria toxin-resistant Pichia pastoris expression system. We assessed the immunogenicity of DT390-Cap using BALB/c mice. DT390-Cap induced significantly higher Cap-specific and neutralizing antibodies than Cap alone with or without the ISA201 adjuvant. DT390-Cap with ISA201 adjuvant induced production of more Cap-specific antibodies and neutralizing antibodies than Ingelvac CircoFLEX (positive control). DT390-Cap induced slightly higher Th2-associated interleukin-4 production than Cap alone but did not affect Th1-associated interferon-γ production. The protection study demonstrated that DT390-Cap induced more effective protective immunity than Cap alone, when challenged with PCV2. The viral loads in the lungs, liver, and thymus in mice immunized using DT390-Cap were significantly lower than in those immunized with the corresponding Cap with or without the ISA201 adjuvant. Taken together, the engineered DT390 effectively enhanced the immunogenicity and protective immunity of Cap in mice. Thus, DT390-Cap is a promising novel vaccine candidate against PCV2 infection.

Genetic Diversity and Prevalence of Porcine Circovirus Type 2 in China During 2000-2019.

As the major pathogen for porcine circovirus-associated disease (PCVAD), porcine circovirus type 2 (PCV2) is no longer treated as an emerging virus anymore. The wide distribution of PCV2 infection in China causes huge economic losses in the swine industry. Currently, it is generally believed that PCV2 has eight genotypes (PCV2a to PCV2h), with PCV2a, PCV2b, and PCV2d being widely distributed. To comprehensively explore the genetic diversity and prevalence of PCV2 in China, PCV-2 sequences submitted from China in the GenBank database were retrieved. With a total of 714 PCV2 strains were retrieved, we found that early-submitted PCV2 sequences were mainly collected from coastal provinces in the southeast part of China, which may indicate PCV2 was initially circulating in those regions. From 2002 to 2008, PCV2b was the dominant prevalent genotype in those retrieved sequences. From 2009, PCV2d became the dominant genotype in those sequences, dropping a hint that a potential shift of PCV2b to PCV2d might occur in 2009, which is similar to the patterns at the global level. In addition to the PCV2a, PCV2b, and PCV2d genotypes, novel strains were also characterized. We further revealed that the amino acid sequences consistency of PCV2a Cap is higher than those in other genotypes. Together, this study provided clues for the possible prevalent genotypes and dynamics of genetic diversity in China from 2000 to 2019.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin.

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Generation and immunogenicity assessment of ELPylated virus-like particles of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). METHODS: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. RESULTS: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. CONCLUSION: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

The yeast cell wall protein Pry3 inhibits mating through highly conserved residues within the CAP domain.

Members of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization.This article has an associated First Person interview with the first author of the paper.

Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

Oral vaccination with the porcine circovirus type 2 (PCV-2) capsid protein expressed by Lactococcus lactis induces a specific immune response against PCV-2 in mice.

AIMS: Porcine circovirus type 2 (PCV2) can cause postweaning, multisystemic wasting syndrome in pigs, which leads to enormous losses in the swine industry worldwide. Here, a genetically engineered Lactococcus strain expressing the main protective antigen of PCV2, the Cap protein, was developed to act against PCV2 infection as an oral vaccine. METHODS AND RESULTS: Expression of the Cap protein was confirmed via western blot, ELISA and fluorescence microscopy. Over 90% of the recombinant pAMJ399-Cap/MG1363 survived a simulated gastrointestinal transit. It also survived the murine intestinal tract for at least 11 days. Then, the safety and immunogenicity of pAMJ399-Cap/MG1363 in orally immunized mice was evaluated. The levels of the sIgA, IgG and cytokines (IL-4 and IFN-γ) obtained from the mice immunized with pAMJ399-Cap/MG1363 were significantly higher than those in the control groups. CONCLUSIONS: pAMJ399-Cap/MG1363 can survive in the gastrointestinal transit and effectively induce mucosal, cellular and humoral immune response against PCV2 infection via oral administration. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of the genetically engineered Lactococcus lactis as a candidate for an oral vaccine against PCV2.

Quantitative protein detection using single molecule imaging enzyme-linked immunosorbent assay (iELISA).

Protein detection is a key step in molecular biology research and is required for pathogen and protein marker testing for disease diagnostics. Here, single molecule imaging enzyme-linked immunosorbent assay (iELISA) is proposed to quantitatively measure the porcine circovirus type 2 (PCV2) Cap protein. The monoclonal antibody against PCV2 Cap protein indirectly immobilized on a polyethylene glycol (PEG) passivated slide by biotin-streptavidin interaction is used to capture the PCV2 Cap protein, and the PCV2 Cap protein can be detected in single molecule level according to the fluorescein isothiocyanate (FITC)-labeled secondary antibody using total internal reflection fluorescence microscopy. The single molecule iELISA measurements can be finished within 1 h skipping the time-consuming sample preparation procedures; moreover, it also exhibits excellent protein selectivity and anti-interference capability. With the proposed single molecule iELISA, linear relation between the fluorescent signals and logarithm of target protein concentrations is obtained with the detection limit of 7 ng/mL. Considering its high accuracy in target protein detection with simple procedures and fast speed, it is believed single molecule iELISA can be potentially adopted in fast trace protein detection.

Production of a monoclonal antibody against Porcine circovirus type 3 cap protein.

Porcine cirvovirus type 3 (PCV3) is a newly emerging swine virus with worldwide distribution. The pathogenesis of PCV3 remains unknown due to failures in propagating and isolating the virus on successive cell lines. The virus cap protein is the only structural protein of PCV3 and no monoclonal antibodies against it are available. Although antisera are available from PCV3-infected pigs, they are not suitable for immunoassays such as immunohistochemical staining of infected tissues due to high non-specific background. Developing monoclonal antibodies against the cap protein is indispensable for elucidating PCV3 biological properties. In this study, a monoclonal antibody (mAb 1H1) with a titer of 1/25,600 that recognized the PCV3 cap protein was developed. Western-blot results showed that mAb 1H1 reacted strongly with the recombinant PCV3 cap protein preparation but not with the PCV2 cap protein. MAb 1H1 was also applied successfully for the detection of the cap protein in PCV3-infected pig tissues using immunochemistry staining. In conclusion, mAb 1H1 has significant potential uses in PCV3 diagnosis as well as the study of PCV3 biology.