Development of a Multi-Epitope Vaccine for Mycoplasma hyopneumoniae and Evaluation of Its Immune Responses in Mice and Piglets.

Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing Mycoplasma pneumonia of swine (MPS), brings massive economic losses worldwide. Genomic variability and post-translational protein modification can enhance the immune evasion of Mhp, which makes MPS prone to recurrent outbreaks on farms, even with vaccination or other treatments. The reverse vaccinology pipeline has been developed as an attractive potential method for vaccine development due to its high efficiency and applicability. In this study, a multi-epitope vaccine for Mhp was developed, and its immune responses were evaluated in mice and piglets. Genomic core proteins of Mhp were retrieved through pan-genome analysis, and four immunodominant antigens were screened by host homologous protein removal, membrane protein screening, and virulence factor identification. One immunodominant antigen, AAV27984.1 (membrane nuclease), was expressed by E. coli and named rMhp597. For epitope prioritization, 35 B-cell-derived epitopes were identified from the four immunodominant antigens, and 10 MHC-I and 6 MHC-II binding epitopes were further identified. The MHC-I/II binding epitopes were merged and combined to produce recombinant proteins MhpMEV and MhpMEVC6His, which were used for animal immunization and structural analysis, respectively. Immunization of mice and piglets demonstrated that MhpMEV could induce humoral and cellular immune responses. The mouse serum antibodies could detect all 11 synthetic epitopes, and the piglet antiserum suppressed the nuclease activity of rMhp597. Moreover, piglet serum antibodies could also detect cultured Mhp strain 168. In summary, this study provides immunoassay results for a multi-epitope vaccine derived from the reverse vaccinology pipeline, and offers an alternative vaccine for MPS.

Porcine Epidemic Diarrhea Virus Induces Vero Cell Apoptosis via the p53-PUMA Signaling Pathway.

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

Genomic Variability and Post-translational Protein Processing Enhance the Immune Evasion of Mycoplasma hyopneumoniae and Its Interaction With the Porcine Immune System.

Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is a geographically widespread and economically devastating pathogen that colonizes ciliated epithelium; the infection of Mhp can damnify the mucociliary functions as well as leading to Mycoplasma pneumonia of swine (MPS). MPS is a chronic respiratory infectious disease with high infectivity, and the mortality can be increased by secondary infections as the host immunity gets down-regulated during Mhp infection. The host immune responses are regarded as the main driving force for the disease development, while MPS is prone to attack repeatedly in farms even with vaccination or other treatments. As one of the smallest microorganisms with limited genome scale and metabolic pathways, Mhp can use several mechanisms to achieve immune evasion effect and derive enough nutrients from its host, indicating that there is a strong interaction between Mhp and porcine organism. In this review, we summarized the immune evasion mechanisms from genomic variability and post-translational protein processing. Besides, Mhp can induce the immune cells apoptosis by reactive oxygen species production, excessive nitric oxide (NO) release and caspase activation, and stimulate the release of cytokines to regulate inflammation. This article seeks to provide some new points to reveal the complicated interaction between the pathogen and host immune system with Mhp as a typical example, further providing some new strategies for the vaccine development against Mhp infection.