Adjuvant Effects Of Momordica Cochinchinensis Seed Saponin And Its Mixture With Mineral Oil

Vaccination is one of the most important methods in prevention of infectious disease in animals. Adjuvants have a crucial contribution in the improvement and development of vaccine immunogenicity. Many substances have been found to have adjuvant activity but only few are permitted. Among those, a large numbers of adjuvants have the strong immunological effects however toxicity is the major obstacle in the use of those adjuvants. To explore new potent adjuvants with minimal toxicity, extraction of cochinchina momordica seed (ECMS) was evaluated for its adjuvant effects on humoral and cellular immune responses. Mice, guinea pig, swine and poultry were used to evaluate the adjuvant effect of ECMS on the immune responses to the vaccination against ovalbumin (OVA), foot-and-mouth disease (FMD), newcastle disease (ND).1.Preparation of ECMS and evaluation of its phy-chemical property and LD50. In present study, crude extraction of the seeds was obtained by ethanol extraction method. The average yield of ECMS is 2.077g/kg.The part of the seed centralized with ECMS is nut. The main component of ECMS is triterpenoid saponin determinded by Liebermann-Burchard reaction, thin layer chromatography analyze experiment, surfactivity experiment and UV spectrum experiment. Endotoxin level was measured by a gel clot Limulus amebocyte lysate assay. The endotoxin level was less than 0.5 EU(endotoxin units)/ml which excluded the influence caused by endotoxin in the following experiments. The lowest dose of ECMS to hemolysis on the blood of swine, sheep and cattle is 125μg/ml. Median lethal dose of ECMS in mice with vein injection route is 108.05mg/kg, intramuscle route is 178.09mg/kg.2. Adjuvant effect of ECMS on the immune responses to ovalbumin in mice.First experiment was conducted for the evaluation of adjuvant effect of ECMS on the immune response to ovalbumin in mice.Thirty mice were distributed in 5 groups (n=6) and immunized with OVA alone serving as control or mixed with 10μg of or 50μg of ECMS or100μg of ECMS or 50μg of aluminum on days 1 and 21.Blood samples were collected on day 14 after boosting. Levels of serum immunoglobulin (Ig) G and subclasses were measured by enzyme-linked immunoabsorbent assay (ELISA). Splenocytes proliferation assay was performed for the detection of cellular immune response. The total IgG antibody titer was significant increased (P< 0.01) in 100μg /dose of ECMS group. The IgG1 level was also significant increased (P< 0.05) in 100μg/dose of ECMS group. The IgG2 level was significant increased (P< 0.01) in 50 and 100μg/dose of ECMS group and aluminum group. There is a significant difference between 50μg/dose of ECMS group and aluminum group in cellular immune response to the stimulation with Con A (P< 0.05).3. Adjuvant effect of ECMS on the immune responses to FMDV in guinea pig.The adjuvant effect of ECMS was also studied in 30 guinea pigs (n=6) immunized with FMDV (O type) alone or containing 100μg/dose of ECMS or containing 100μg/dose of ECMS mixed with mineral oil or containing mineral oil alone. Humoral immune responses were evaluated by sandwich ELISA and VP1 assay on day 14,28 and 42 after boosting:-The IgG antibody level against FMDV serotype O was significantly higher in the animals received vaccine containing ECMS mixed with mineral oil or vaccine containing mineral oil (P<0.05) on day 14,28 and 42 compared with other groups. The IgGl antibody level against FMDV serotype O was significantly higher in the animals received vaccine containing ECMS mixed with mineral oil on day 28 and 42 compared with other groups. The IgG2 antibody level against FMDV serotype O was significantly higher in the animals received vaccine containing ECMS mixed with mineral oil on day 28 compared with other groups (P< 0.05). On day 14 and 28, the anti-VP1 antibodies in ECMS-mineral oil group was significantly higher (P< 0.05) compared with other groups. The anti-VP1 antibody subclass IgG2 in ECMS-mineral oil group was significantly higher(P< 0.05) compared with other groups on day 28. It is concluded that there is a synergistic activity between ECMS and mineral oil and it could improve the immune response against FMDV (type O) in guinea pig.4.Improvement of a commercial FMDV vaccine by supplement of Quil A.The study also evaluated the immunological effect of Quil A on the immune response to FMDV in mice and pigs. In model animal experiment A, ICR mice were immunized with ovalbumin (OVA) adjuvanted with QuilA or mineral oil or their combination (Quil A-mineral oil). In model animal experiment B, ICR mice were immunized with foot-and-mouth disease virus (FMDV) antigens or with a commercially available oil adjuvanted foot-and-mouth disease (FMD) vaccine (type O) alone or mixed with QuilA. After that, serum samples were collected to analyze specific IgG and IgG subclasses IgGl, IgG2a, IgG2b, andIgG3. In experiment C, pigs were immunized with FMD (type O) vaccine alone or together with Quil A. Serum samples were collected before and 4 weeks after immunization to analyze indirect haemagglutination (IHA) titers. Results from experiment A indicated a synergistic effect of QuilA and oil on IgG and the subclass responses. Experiment B revealed that supplement of QuilA in FMD vaccine significantly increased IgG and the subclass responses in mice.Experiment C demonstrated that supplement of QuilA in the FMD vaccine significantly enhanced humeral immune responses (as determined by IHA test) in pigs. It is concluded that supplement of QuilA in FMD vaccine can significantly enhanced immune responses and could be an alternative way to improve FMD vaccinationin pigs.5. Enhancement of immune responses to foot-and-mouth disease vaccine by a supplement ECMS.The adjvuant effect of ECMS was also studied in 108 pigs. In experiment A/B,24 pigs with the low maternal antibody (IHA titer<27/>27)aged 40 days were divided into four groups. Each pig was immunized with FMDV vaccine(O type) mixed with 0,1,2 or 4 mg ECMS. Blood samples were collected before immunization and 3 weeks after immunization. Humoral immune responses were evaluated by IHA test. In experiment C,36 pigs with the low maternal antibody (IHA titer<2') aged 40 days were divided into two groups. Each pig was immunized with FMDV vaccine (O type)mixed with 0 or 4 mg ECMS. Blood samples were collected before immunization and 3 or 7 weeks after immunization. Humoral immune responses were evaluated by IHA test. Cellular immune response were also evaluated 7 weeks after immunization. In experiment D,24 pigs with the low maternal antibody (IHA titer<27) aged 40 days were divided into two groups. Each pig was immunized with FMDV vaccine(O type) mixed with 0 or 4 mg ECMS. Blood samples were collected before immunization and 3 or 6 weeks after immunization. Humoral immune responses were evaluated by indirect ELISA assay for VP1 total IgG and subclass. Results from experiment A, C, D indicated ECMS can enhance the humoral immune responses in pigs immunized with FMDV vaccine,4mg/dose was the best dose. Four mg/dose ECMS could improve both the humoral and cellular immune response in pigs immunized with FMDV vaccine.6. Enhancement of immune responses to newcastle disease vaccine by a supplement ECMS.The study also evaluated the immunological effect of ECMS on the immune response to ND in chickens. Fourty eight chicken were divided to 4 groups (n=12). Each chicken was immunized with ND vaccine mixed with 0μg or 20μg or 40μg or 80μg ECMS on day 35. Blood samples were collected on day 0,7,14,21,28,35 after immunization. Humoral and cellular immune responses were evaluaed by indirect ELISA assay and MTT method. Results indicated that humoral immune response was enhance by ECMS 14 days after immunization. Eighty microgramme of ECMS showed the best dose with the ND vaccine and was significant difference with other groups 21 days after immunization. No significance difference was found in cellular immune response while 80μg ECMS group was average higher than other groups 35 days after immunization. No sideffect was found on the growth performance during the experiment.Above all, Crude extraction of the seeds was obtained by a modified ethanol extraction method. The lowest dose of ECMS to hemolysis on the blood of swine, sheep and cattle is 125μg/ml. Median lethal dose of ECMS in mice with vein injection route is 108.05mg/kg, intramuscle route is 178.09mg/kg. It is safe for muscle injection with ECMS. ECMS has the adjuvant activity, it can improve the immune response to OVA in-mice. When co-administered with ECMS in mice, OVA can induce significantly higher production of IgG1 and IgG2a, but favor IgG2a. There is a synergistic activity between Quil A or ECMS with mineral oil. Adjuvant activity of their combination is higher than used alone seperately. Immune response to the commercially FMDV or ND vaccine could be enhanced by supplement ECMS or Quil A.