Enhanced Immune Responses To Attenuated Porcine Pseudorabies And Classical Swine Fever Virus Vaccines By A Herbal Solution

Infectious disease is one of the major threats to pig industry.Pseudorabies(Pr)for example,a highly contagious disease of animals,was caused by Pseudorabies virus(PrV)and characterized by a 100%mortality rate in piglets less than 15d old and about 10-20%in weaned pigs;Classical swine fever(CSF),another highly infectious disease,was caused by classical swine fever virus(CSFV)and results in great economic loss to the development of pig industry of China.Vaccination with attenuated vaccines is a wide used method to Pr and CSF control in China.The attenuated PrV and CSFV vaccines are often supplied in a form of lyophilized powder,which need to be dissolved using saline or PBS solution.However,poor immune response to vaccination often results in incomplete protection.Thus,this study was designed to develop a diluent to improve the efficacy of attenuated PrV and CSFV vaccines.The related research also provided some useful references to clinical application of diluent.1.Study on the formula of diluent for an aPrV vaccineObjective:To develop an appropriate formula of immunopotentiator to an aPrV vaccine.Methods:ICR mice were immunized with an aPrV vaccine,and PrV gB antibody level in sera was measured to evaluate the immunoenhancement of Polysaccharide from the Rhizome of Atractylodes Macrocephala Koidz.(RAMPS)as well as ginseng stem-and-leaf saponins(GSLS);PrV gB?PrV gB IgGI and PrV gB IgG2a level in sera as well as protection of animals after challenged with a lethal dose of field PrV(fPrV)were measured to evaluate the immunoenhancement of the candidate formula.Results:A higher PrV gB antibody response was induced in sera of mice immunized with an aPrV vaccine formulated with GSLS or RAMPS,but no synergistic effect of GSLS and RAMPS displayed in this experiment.While,addition of GSLS and TS promoted the antibody responses obviously with the highest antibody level found in mice received aPrV vaccine formulated with GSLS 6?g plus TS(thimerosal)1?g.In addition,GSLS-TS promoted the antibody of PrV gB?PrV gB IgG1 and PrV gB IgG2a in advance obviously,which were all maitained at a higher level over a long period of time.After mice were performed with a lethal PrV-challenge,70%of animals were survived in the group received PrV vaccine plus GSLS-TS;whereas only 40%mice were protected in the group received PrV vaccine alone.2.Effects of sterilization methods and storage conditions on the adjuvant activity ofGSLS-TSObjective:To see if high temperature processing and several storage conditions affect the adjuvant property of GSLS-TS.Methods:ICR mice were immunized with an aPrV vaccine,and specific antibody level in sera were measured to evaluate the adjuvant activities of GSLS-TS.aPrV vaccines were dissolved using GSLS solution treated with different high temperatures or GSLS-TS solution storaged in different conditions for one year.Results:The adjuvant property of GSLS markedly weakened after heat sterilization.When GSLS-TS solution had been exposed to room temperature and/or natural light for one year,its adjuvant activity was also weakened indicating that the GSLS-TS solution should be kept in a cool and dark place.3.The comparison of GSLS-TS diluent with 6 commercial diluents of aPrV vaccines for their adjuvant capacityObjective:To compare the adjuvant property of GSLS-TS diluent with 6 kinds of common used commercial diluents of aPrV vaccines.Methods:ICR mice were immunized with 6 aPrV vaccines,which were dissolved using GSLS-TS solution,saline or 6 corresponding diluents respectively.Specific antibody level in sera were measured to evaluate the adjuvant activities of GSLS-TS and specific diluents.Results:5 of 6 commercial diluents exhibited weak adjuvant activities to aPrV vaccines,while GSLS-TS solution showed markedly stronger adjuvant activity than both specific diluents and saline.4.The enhanced humoral immunity of GSLS-TS to attenuated PrV and CSFV vaccines.Objective:To see if GSLS-TS could enforce the antibody responses to attenuated PrV and CSFV vaccines in pigs.Methods:Pigs were immunized with PrV and CSFV vaccines respectively;antibody responses in sera were measured to evaluate the enhancement of GSLS-TS to vaccines.Results:Both higher antibody blocking rate of PrV and CSFV,IgG isotypes(IgGl,IgG2)and neutralizing antibody levels were found in pigs co-immunized with GSLS-TS.5.The effects of GSLS-TS on the cell-mediated immunityObjective:To assess the effects of GSLS-TS on the cell-mediated immunity.Methods:Mice were immunized with an aPrV vaccine dissolved using GSLS-TS solution or saline.24 h after prime injection,splenocytes were prepared to examine NK cell activity;sera were taken to test IFN-y production.2 weeks post the boost immunization,splenocytes from mice were measured for lymphocyte proliferation and production of cytokines.Results:In contrast,a supplement of GSLS-TS exhibited significantly higher cytolytic activity and elevated amounts of serum IFN-?;GSLS-TS also induced a significantly higher proliferative response of lymphocyte than control;higher levels of Thl-like cytokine(IFN-?,IL-12)and Th2-like cytokine(IL-5,IL-10)were observed from the group co-immunized with GSLS-TS.6.Research on the mechanisms of immune enhancement of GSLS-TSObjective:To preliminarily study the possible mechanisms of immune enhancement of GSLS-TS.Methods:Murine macrophages(RAW264.7)were co-cultured with GLSL/TS,and the mRNA expression of miR-132,miR-146a,miR-147 and miR-155 in RAW264.7 cells were measured to analyze the possible correlation between this four miRs and the adjuvant activity of GSLS-TS;ICR and C3H/HeJ mice were immunized with an aPrV vaccine,then specific antibody response in sera,lymphocyte proliferative responses,as well as the mRNA expression of cytokines from lymphocytes were measured to analyze the possible relationship between TLR4 receptor and the adjuvant activity of GSLS-TS.Results:The up-regulation mRNA expression of miR-132,miR-146a,miR-147 and miR-155 were found in RAW 264.7 cells when co-cultured with GSLS/TS and the synergistic effect were also found in the group co-immunized with GSLS and TS.Moreover,we found that the adjuvant activity of GSLS-TS was not exerted via TLR4 since GSLS-TS activated the antibody response,lymphocyte proliferative responses,as well as the mRNA expression of Thl-like and Th2-like cytokines from lymphocytes in both ICR and C3H/HeJ mice.In conclusion,GSLS and TS could synergistically enhance the immune responses to an aPrV vaccine,GSLS-TS diluent induced an obviously higher immune response and protection against a lethal PrV-challenge in mice received aPrV vaccine compared with saline solution.The diluent also increased the specific antibody level of sera in pigs immunized with attenuated PrV and CSFV vaccines,which manifested a potential value of clinical application.The immune enhancement of diluent was not mostly mediated by TLR4,but may be positively corrected with miRs in immune cells,and the exact mechanisms deserve further study.