Vaccination is a common routine for prevention and control of human and animal diseases by inducing antibody responses and cell-mediated immunity in the body. Through vaccinations, smallpox and some other diseases have been eradicated in the past few years. The use of a patho- gen itself or a subunit domain of a protein antigen as immunogens lays the basis for traditional vaccine development. But there are more and more newly emerged pathogens which have expe- rienced antigenic drift or shift under antibody selective pressures, rendering vaccine-induced im- munity ineffective. In addition, vaccine development has been hampered due to problems includ- ing difficulties in isolation and culture of certain pathogens and the antibody-dependent enhancement of viral infection (ADE). How to induce strong antibody responses, especially neu- tralizing antibody responses, and robust cell-mediated immune responses is tricky. Here we re- view the progress in vaccine development from traditional vaccine design to reverse vaccinology and structural vaccinology and present with some helpful perspectives on developing novel vac- cines.
Pseudorabies (PR) outbreaks have devastated many swine farms in several parts of China since late 2011. The outbreak-associated pseudorabies virus (PRV) variant strains exhibited some typical amino acid changes in glycoprotein E (gE), a diagnostic antigen used for discriminating between PRV-infected and vaccinated animals (DIVA). To counteract the potential impact of epitope variations on current serological diagnostics of PRV, we produced monoclonal antibodies (mAbs) against gE protein of one representative PRV variant strain and developed a blocking immunoperoxidase monolayer assay (b-IPMA) for DIVA. The b-IPMA was based on the inhibition of binding between PRV-infected cells and mAb by PRV-specific antibodies present in clinical swine sera and was validated by comparison with a commercial PRV gpI Antibody Test Kit (IDEXX Laboratories, USA). The diagnostic sensitivity, diagnostic specificity and agreement were determined to be 99.25%, 98.18% and 99.02% respectively upon testing 509 serum samples. b-IPMA detected only PRV-specific antibodies and showed no cross- -reactivity with antibodies elicited by gE-deleted vaccine or other common swine pathogens. Thus, b-IPMA has the potential to be used for high-throughput screening of PRV-infected animals in veterinary clinics.
In this study, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed for rapid detection of porcine parvovirus (PPV) 6. Primer pairs targeting the conserved regions of PPV6 Capsid gene were designed. Sensitivity analyses revealed the lowest detection limit of the SYBR Green-based real-time PCR assay to be 47.8 copies/μL, which indicated it was 1000 times higher than that found in the conventional PCR investigations. This assay was specific and showed no cross-species amplification with other six porcine viruses. The assay demonstrated high repeatability and reproducibility; the intra- and inter-assay coefficients of variation were 0.79% and 0.42%, respectively. The positive detection rates of 180 clinical samples with SYBR Green-based real-time PCR and conventional PCR were 12.22% (22/180) and 4.44% (8/180), respectively. Our method is sensitive, specific, and reproducible. The use of SYBR Green-based real-time PCR may be suitable for the clinical detection and epidemiological investigation of PPV6.
Telomerase reverse transcriptase (TERT) vectors were transfected into bone marrow mesen- chymal stem cells (BMSCs) which were then cultured and selected to establish TERT-BMSC cell lines whilst sequencing BMSCs and TERT-BMSCs via transcriptome in this study to explore their regulatory mechanism and effect on osteogenic differentiation after TERT ectopic expres- sion in sheep BMSCs. After sequencing and analysing differential genes, PI3K/Akt signalling pathway related to osteogenic differentiation was investigated. Western blot was used before and after applying the PI3K/Akt signalling pathway inhibitor LY294002 to detect protein expression levels of AKT and p-AKT. On the twenty-first day of osteogenic differentiation, RT-qPCR and Western blot were used to detect mRNA and protein expression levels of RUNX2 and OPN and alizarin red staining was utilised to analyse calcium salt deposition. Results showed that pro- tein expression levels of AKT and p-AKT were significantly up-regulated, mRNA and protein expression levels of RUNX2 and OPN increased and calcium salt deposition increased after ectopic expression of TERT. After applying LY294002, the protein expression of AKT and p-AKT was down-regulated, mRNA and protein expression levels of RUNX2 and OPN were reduced and calcium salt deposition was reduced. These results confirmed the stable integration and expression of the exogenous TERT gene in BMSCs to promote the differentiation of BMSC osteoblasts, which may be mediated by the PI3K/Akt signalling pathway.
We investigated changes in concentrations of ADP (adiponectin), LEP (leptin), BHBA (beta-hydroxybutyric acid), NEFA (non-esterified fatty acid), Glucose (Glu) and INS (insulin) in serum of healthy perinatal dairy cows and cows with ketosis. Twenty-one healthy cows and seventeen cows with ketosis from a herd of a total 60 Holstein cows (near dry period i.e. 56 days antepartum) were selected. Blood was collected through the tail vein every 7 days, from 56 day antepartum to 56 day postpartum. Serum ADP, LEP, BHBA, NEFA, Glu, and INS concentrations were determined, and ketosis was diagnosed through serum BHBA (≥1.2 mmol/L). We showed the concentration of serum adipokines and energy balancing indices were stable during antepar- tum period. However, ADP concentration increased while LEP decreased, and there were a significant increase in cows with ketosis compared to that of in healthy cows. Serum BHBA and NEFA concentrations increased significantly at first, and then gradually decreased in both healthy cows and cows with ketosis. However, cows with ketosis showed higher concentrations of BHBA and NEFA which restored later. The serum concentration of Glu in both healthy dairy cows and cows with ketosis showed a decreasing trend. INS concentration in healthy cows was decreased while it was increased in cows with ketosis. The results reflect the extent of hypo- glycemia and lipid mobilization postpartum, suggest IR exists in cows with ketosis while serum ADP and LEP might play roles in the development of ketosis.
Emerging researches in humans, pigs and mice, highlighted that estrogen plays a pivotal role in self-renewal and differentiation of bone marrow mesenchymal stem cells (BMSCs). The present study aimed at evaluating effects of 17 beta-estradiol (E2) on proliferation and apoptosis of canine-derived bone marrow mesenchymal stem cells (cBMSCs) in vitro. The results showed that E2 supplementation at the concentration of 10-11 M promoted the proliferation of cBMSCs by CCK-8 assay and RT-qPCR analysis for the proliferation-related genes, with proliferating cell nuclear antigen (PCNA), cyclin-D1 (CCND1) being up-regulated and cyclin-dependent kinase inhibitor 1B (CDKN1B) being down-regulated. Contrarily, analysis of fluorescence-activated cell sorting (FACS) and RT-qPCR demonstrated that E2 supplementation above 10-11 M had inhibitory effects on the proliferation of cBMSCs and induced apoptosis. Intriguingly, cBMSCs still possessed the capability to differentiate into osteoblasts and adipocytes with 10-11 M E2 addition. Taken together, this study determined the optimal culture condition of cBMSCs in vitro, and has important implications for further understanding the regulatory effect of E2 on the self-renewal of cBMSCs, which are helpful for the clinical application of BMSCs.