Screening and Stability Evaluation of Freeze-Dried Protective Agents for a Live Recombinant Pseudorabies Virus Vaccine.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k-. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2-8 °C for 24 months, and a predicted shelf life of 6.61 years at 2-8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- vaccines.

The High Immunity Induced by the Virus-Like Particles of Foot-and-Mouth Disease Virus Serotype O.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious and economically devastating viral disease of cloven-hoofed animals worldwide. In this study, the coexpression of small ubiquitin-like modifier (SUMO)-fused capsid proteins of FMDV serotype O by single plasmid in Escherichia coli was achieved with an optimal tandem permutation (VP0-VP3-VP1), showing a protein yield close to 1:1:1. After SUMO removal at a low level of protease activity (5 units), the assembled FMDV virus-like particles (VLPs) could expose multiple epitopes and have a size similar to the naive FMDV. Immunization of pigs with the FMDV VLPs could induce FMDV-specific humoral and cellular immune responses effectively, in a dose-dependent manner. These data suggested that the stable FMDV VLPs with multiple epitope exposure were effective for the induction of an immune response in pigs, which laid a foundation for the further development of the FMDV subunit vaccine.

[Co-expression, purification and bioassay of three avian viral antigens].

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Establishment and application of an indirect ELISA for porcine circovirus 3.

Porcine circovirus type 3 (PCV3) was initially reported in 2016 in the United States of America. Since then, the virus has been detected on swine farms in Poland, South Korea, and China using PCR. However, a serological survey of PCV3 in pig populations has never been conducted. In this study, for the first time, we established an indirect enzyme-linked immunosorbent (ELISA) assay and performed a national retrospective serological survey for PCV3. Our results showed that the PCV3-postive rate increased from 22.35% to 51.88% between 2015 and 2017. The above results suggest PCV3 has spread widely in China with increased positive rates since 2015.

Establishment and application of a competitive enzyme-linked immunosorbent assay differentiating PCV2 antibodies from mixture of PCV1/PCV2 antibodies in pig sera.

BACKGROUND: Porcine cirovirus type 1 (PCV1) and type 2 (PCV2) are circulating in Chinese pig herds and the infected pigs develop antibodies to both viruses. Current commercial available ELISA kits cannot differentiate PCV2-specific antibodies from the mixtures of PCV1 and PCV2 antibodies in PCV1/2-infected or PCV2-vaccinated pigs. Therefore, the need for developing PCV2-specific ELISA methods is urgent to evaluate PCV2 antibody level in exclusion of PCV1 antibody interference after PCV2 vaccination. RESULTS: Virus-like particles (VLPs) of PCV2 based on the recombinant Cap protein were expressed in Escherichia coli. A competing ELISA was established by using the VLPs as coating antigen and a PCV2-specific monoclonal antibody as the competing antibody. The competing ELISA was compared with the results obtained by using an immunoperoxidase monolayer assay on 160 serum samples. The sensitivity and specificity of this competing ELISA were determined as 96.5 and 96.0 %, at 2 standard deviation from the mean or 91.8 and 100 % at 3 standard deviations from the mean. Next, a serological survey of 1297 vaccinated serum samples collected from commercial pig herds in Beijing, Hunan and Henan provinces in China was conducted. The results showed that 85.9 % of sera having positive PCV2 antibodies. CONCLUSIONS: The competing ELISA we developed in this study was both sensitive and specific to PCV2 and was suitable for large-scale PCV2 antibody monitoring in exclusion of PCV1 antibody interference after PCV2 vaccination.

Development of a Monoclonal Antibody Against Porcine Circovirus2 Cap Protein.

Female BALB/c mice were immunized with a commercial PCV2 vaccine, and a monoclonal antibody (MAb) designated as 3H11 was achieved by hybridoma techniques. The MAb specifically reacted with Cap protein of PCV2, which has been identified by western blot. Immunoperoxidase monolayer assay results showed that 3H11 did not cross-react with PCV1-infected cells. Therefore, this work suggested that 3H11 could be a useful tool as a specific diagnostic reagent for PCV2 research.

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

BACKGROUND: Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. RESULTS: In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. CONCLUSIONS: Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

A novel soluble ß-1,3-D-glucan salecan reduces adiposity and improves glucose tolerance in high-fat diet-fed mice.

Salecan is a recently identified water-soluble viscous extracellular ß-1,3-D-glucan polysaccharide from an Agrobacterium species. It is a high-molecular-mass polymer (about 2 × 106 Da) and composed of a linear chain of glucosyl residues linked through a repeat unit of seven ß-(1,3) and two α-(1,3) glucosidic bonds. In the present study, we examined the effects of dietary Salecan fed at 2 and 5 % in a high-fat diet (64 % energy) in C57BL/6J mice. After 6 weeks, mice fed 2 and 5 % Salecan had significantly lower body weight, fat mass and percentage of body fat mass compared with those fed a high-fat cellulose (control) diet. Both the Salecan groups significantly and dose-dependently improved glucose tolerance, with a 9 and 26 % reduction of glucose AUC, respectively. Liver and adipose tissue weights were also significantly decreased by the Salecan treatment. Supplementation with 5 % Salecan led to lower serum TAG, total cholesterol (TC) and HDL-cholesterol (52, 18 and 19 %, respectively) and lower hepatic TAG by 56 % and TC by 22 % compared with the high-fat cellulose control group. Dietary Salecan intake caused an obvious elevation of fat in the faeces. Supplementation with Salecan disturbed bile acid-promoted emulsification and reduced the size of emulsion droplets in vitro. These results indicate that Salecan decreases fat absorption, improves glucose tolerance and has biologically important, dose-related effects on reducing high-fat diet-induced obesity.

The mPlrp2 and mClps genes are involved in the hydrolysis of retinyl esters in the mouse liver.

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5'-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.

The environmental light influences the circulatory levels of retinoic acid and associates with hepatic lipid metabolism.

Environmental light is involved in the regulation of photochemical reaction in mouse retina. It remains unclear whether light-mediated increase in all-trans retinoic acid (ATRA) synthesis in retina will result in altering the circulatory levels of ATRA and regulating downstream gene expression and physiological function. Here we showed circulatory levels of ATRA decreased in mice under constant darkness and elevated by light exposure. Fat gene pancreatic lipase-related protein 2 (mPlrp2) and its partner procolipase (mClps), but not hepatic lipase (mHl), activated in livers for responding to lack of light illuminating. Light-triggered alterations in circulatory ATRA levels regulated ecto-5'-nucleotidase gene expression by retinoic acid receptor retinoic acid receptor-alpha and modulated 5'-AMP levels in blood and were associated with mPlrp2 and mClps expression in the livers. Mice deficient in adenosine receptors displayed mPlrp2 and mClps expression in livers under 12-h light, 12-h dark cycles. Caffeine blocked adenosine receptors and induced hepatic mPlrp2 and mClps expression in wild-type mice. Mice activated in hepatic mPlrp2 and mClps expression lowered hepatic and serum lipid levels and markedly elevated circulatory levels of all-trans retinol. Our results suggest environmental light influence hepatic lipid homeostasis by light-modulated retinoic acid signaling associated with mPlrp2 and mClps gene expression in livers.