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Background: Virus-like particles (VLPs) are supramolecular structures composed of multiple protein subunits and resemble natural virus particles in structure and size, making them highly immunogenic materials for the development of next-generation subunit vaccines. The orderly and repetitive display of antigenic epitopes on particle surface allows efficient recognition and cross-link by B cell receptors (BCRs), thereby inducing higher levels of neutralizing antibodies and cellular immune responses than regular subunit vaccines. Here, we present a novel multiple antigen delivery system using SpyCatcher/Spytag strategy and self-assembled VLPs formed by porcine circovirus type 2 (PCV2) Cap, a widely used swine vaccine in solo., Results: Cap-SC, recombinant Cap with a truncated SpyCatcher polypeptide at its C-terminal, self-assembled into 26-nm VLPs. Based on isopeptide bonds formed between SpyCatcher and SpyTag, classical swine fever virus (CSFV) E2, the antigen of interest, was linked to SpyTag and readily surface-displayed on SpyCatcher decorated Cap-SC via in vitro covalent conjugation. E2-conjugated Cap VLPs (Cap-E2 NPs) could be preferentially captured by antigen presenting cells (APCs) and effectively stimulate APC maturation and cytokine production. In vivo studies confirmed that Cap-E2 NPs elicited an enhanced E2 specific IgG response, which was significantly higher than soluble E2, or the admixture of Cap VLPs and E2. Moreover, E2 displayed on the surface did not mask the immunodominant epitopes of Cap-SC VLPs, and Cap-E2 NPs induced Cap-specific antibody levels and neutralizing antibody levels comparable to native Cap VLPs., Conclusion: These results demonstrate that this modularly assembled Cap-E2 NPs retains the immune potential of Cap VLP backbone, while the surface-displayed antigen significantly elevated E2-induced immune potency. This immune strategy provides distinctly improved efficacy than conventional vaccine combination. It can be further applied to the development of dual or multiple nanoparticle vaccines to prevent co-infection of PCV2 and other swine pathogens., (© 2022. The Author(s).)

Background: Dendritic cell (DC) targeted antigen delivery is a promising strategy to enhance vaccine efficacy and delivery of therapeutics. Self-assembling peptide-based nanoparticles and virus-like particles (VLPs) have attracted extensive interest as non-replicating vectors for nanovaccine design, based on their unique properties, including molecular specificity, biodegradability and biocompatibility. DCs are specialized antigen-presenting cells involved in antigen capture, processing, and presentation to initiate adaptive immune responses. Using DC-specific ligands for targeted delivery of antigens to DCs may be utilized as a promising strategy to drive efficient and strong immune responses., Methods: In this study, several candidates for DC-binding peptides (DCbps) were individually integrated into C-terminal of porcine circovirus type 2 (PCV2) Cap, a viral protein that could self-assemble into icosahedral VLPs with 60 subunits. The immunostimulatory adjuvant activity of DC-targeted VLPs was further evaluated in a vaccine model of PCV2 Cap., Results: With transmission electron microscopy (TEM), E. coli expressed Cap-DCbp fusion proteins were observed self-assembled into highly ordered VLPs. Further, in dynamic light scattering (DLS) analysis, chimeric VLPs exhibited similar particle size uniformity and narrow size distribution as compared to wild type Cap VLPs. With a distinctly higher targeting efficiency, DCbp3 integrated Cap VLPs (Cap-DCbp3) displayed enhanced antigen uptake and increased elicitation of antigen presentation-related factors in BM-DCs. Mice subcutaneously immunized with Cap-DCbp3 VLPs exhibited significantly higher levels of Cap-specific antibodies, neutralizing antibodies and intracellular cytokines than those with other DCbp integrated or wild type Cap VLPs without any DCbp. Interestingly, Cap-DCbp3 VLPs vaccine induces robust cellular immune response profile, including the efficient production of IFN-γ, IL-2 and IL-10. Meanwhile, the improved proliferation index in lymphocytes with Cap-DCbp3 was also detected as compared to other VLPs., Conclusion: This study described the potential of DC-binding peptides for further improved antigen delivery and vaccine efficacy, explainning nanovaccine optimization in relation to a range of emerging and circulating infectious pathogens., Competing Interests: The authors declare no competing financial interests and no conflicts of interest for this work., (© 2022 Lu et al.)

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.

Porcine circovirus type 2 (PCV2) has recently been reported to elicit the unfolded protein response (UPR) via activation of the PERK/eIF2α (RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α) pathway. This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein. By transient expression, we found that both replicase (Rep) and capsid (Cap) proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eIF2α-ATF4 (activating transcription factor 4)-CHOP (CCAAT/enhancer-binding protein homologous protein) axis. Cap expression, but not Rep, significantly reduced anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage, possibly due to increased expression of CHOP. Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression, caspase-3 cleavage, and apoptotic cell death possibly by partially rescuing Bcl-2 expression, we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eIF2α/ATF4/CHOP/Bcl-2 pathway. This study, together with our earlier studies, provides insight into the mechanisms underlying PCV2 pathogenesis.

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.