Enhancement Of Immune Responses To Vaccination Against Foot-and-Mouth Disease By Oral Administration Of Extract Made From Cochinchina Momordica Seeds

Foot and mouth disease (FMD) is considered one of the most contagious diseases affecting economically important livestock species such as cattle, sheep and piglets. FMD vaccines play a key role in control campaigns and eradication of FMD. However some FMD vaccines have been reported to induce poor immune responses. Many antigens are weakly immunogenic and require an adjuvant to achieve a satisfactory immune response. To explore new potent adjuvants with minimal toxicity, extract of Cochinchina momordica seed (ECMS) has been evaluated for its adjuvant effects on humoral and cellular immune responses. The seed of Cochinchina momordica is a traditional Chinese medicine and has been used for more than one thousand years. According to previous studies from our laboratory, medicinal and therapeutic use of the seed is well documented however no reports have been found on its oral adjuvant properties. For the purposes of the present study, oral adjuvant potentials of ECMS have been explored. Crude seed extraction obtained by extraction with ethanol extraction method. ECMS was diluted with normal saline for the study on laboratory animals. Mice were used to evaluate the oral adjuvant effect of ECMS on the immune responses to the vaccination against foot-and-mouth disease (FMD).First experiment was conducted determination the effects of oral administration with Quil A and extract of Cochinchina momordica seeds (ECMS) on body weight of mice challenged with FMDV antigen (Asia 1). Mice (n= 40) were allocated to treatment (ten mice per treatment). Factors were oral administration with or without Quil A (200μg/mice) and ECMS (200μg/mice). Diets and water fed ad libitum. Mice were weighted at before (d 0) and after d 7,14,28 and 42 after FMD antigen-challenged. Treatment had no effect on mice growth. The body weight was similar for all treatments between d 0 and 42 of the trial. Oral administration saponin and FMD antigen-challenged had no influence on any measured aspect of mice body weight.Second experiment was conducted for investigation of the dose effect for oral administration of Quil A and extraction of Cochinchina momordica seeds (ECMS) on the immune responses to FMDV antigen (serotypes O) in mice. Forty two ICR mice were randomly divided into 7 groups with each consisting of 6 animals. Mice in groups 2 to 4 were orally administered ECMS (200μg in 250μL saline) one, two and three days respectively or 250μL saline in group 1, or orally administered with Quil A (200μg in 250μL saline) in groups 5 to 7 one, two and three days respectively. Forty-eight hours later, the animals in groups 2 to 7 were SC immunized with 200μL of FMDV antigen (serotypes O). Mice in group 1 received injection of 200μL saline only. Three weeks after the first immunization, ECMS or Quil A and boosting immunization were administered in the same manner. After 2 weeks, blood samples were collected for measurement of FMDV (serotypes O) specific IgG levels and isotype IgGl, IgG2a, IgG2b and IgG3 responses. Results indicated that one day oral administration of ECMS and Quil A had higher responses of serum specific IgG and most of IgG isotypes to immunization of a FMDV (serotypes O) antigen in mice than two or three day administration but no significant differences (P>0.05). Considering the safety, immunomudulatory effect, and the frequent also does not required of ECMS and Quil A in mice demonstrated in present study, this extract deserves further investigation to evaluate its potential improvement of FMD vaccination for one day oral administartion.Later Experiments to study the effects of oral administration of saponins extracted from the bark of Quilaja saponaria Molina (Quil A) and Momordica cochinchinensis (Lour.) Sprenger seeds (ECMS) on the immune responses of mice to vaccination against foot-and-mouth disease (FMD) was investigated. ICR mice were immunized twice with inactivated FMDV antigen at two week intervals. Twenty four hours before immunization, the mice had already been orally administered Quil A (200μg in 250μL saline) or ECMS (200μg in 250μL saline). Blood samples were collected 2 weeks after boosting injection. Results indicated that serum IgG level in mice immunized with FMDV antigen after oral administration of Quil A, and particularly serum IgG and the subclasses IgG1, IgG2a, IgG2b and IgG3 levels, as well as production of IFN-y and IL-5 by splenocytes in mice immunized with FMDV antigen after oral administration of ECMS were significantly higher than those in the control mice orally administered physiological saline solution. This suggested that one day oral administration of ECMS and Quil A significantly (P<0.05) pre-conditioned mice, and increased both Thl and Th2 immune responses to vaccination against FMD.The oral adjuvant effect of ECMS on vaccination of bivalent commercial vaccine against FMDV (serotypes O and Asia 1) was studied in mice. Thirty-two ICR mice were randomly divided into 4 groups with each consisting of 8 animals. The animals were then orally administered ECMS (200μg in 250μL saline) in groups 3 or 250μL saline in group 2, or SC injected with ECMS (50μg in 100μL saline) in groups 4. After that, the animals in groups 2 to 4) were SC immunized with 100μL of oil-adjuvanted bivalent FMDV vaccine (serotypes O and Asia 1). Mice in group 1 received injection of 100μL saline only. Three weeks after the first immunization, ECMS and boosting immunization were administered in the same manner. After 2 weeks, blood samples were collected for measurement of FMDV (serotypes O and Asia 1) specific IgG levels and isotype IgG1, IgG2a, IgG2b and IgG3 responses. Results indicated that one day oral administration or SC injection of ECMS significantly (P<0.05) augmented responses of serum specific IgG and most of IgG isotypes to immunization of a commercial FMDV (serotypes O and Asia 1) vaccine in mice. Considering the safety and immunomudulatory effect of ECMS in normal mice demonstrated in present study, this extract deserves further investigation to evaluate its potential improvement of FMD vaccination in farm animals such as pigs, sheep and cattle. The oral adjuvant effect of ECMS on vaccination of bivalent commercial vaccine against FMDV (serotype Asia I and O) was also studied in immunosuppressive mice. Forty ICR mice were randomly divided into 5 groups with each consisting of 8 animals. Mice in groups 3 to 5 were subcutaneously (SC) injected with 0.5 mg of dexamethasone daily for 4 days to induce immunosuppression. The animals were then orally administered ECMS (200μg in 250μL saline) in groups 4 or 250μL saline in group 2, or SC injected with ECMS (50μg in 100μL saline) in groups 5 or 100μL saline in group 3. After that, the animals in groups 2 to 5 were SC immunized with 100μL of oil-adjuvanted bivalent FMDV vaccine (serotypes O and Asia 1). Mice in group 1 received injection of 100μL saline only. Three weeks after the first immunization, ECMS and boosting immunization were administered in the same manner. After 2 weeks, blood samples were collected for measurement of FMDV (serotypes O and Asia 1) specific IgG levels and isotype IgGl, IgG2a, IgG2b and IgG3 responses. Results indicated that one day oral administration of ECMS significantly (P<0.05) increased and subcutaneous injection of ECMS tended to enhance serum specific IgG and IgG isotype responses to immunization of a commercial FMDV (serotypes O and Asia 1) vaccine in immunosuppressed mice by SC injection of dexamethasone. Therefore, oral administration ECMS could be an alternative approach to improving animal FMD vaccination when the animal is under immunosuppression to induce an effective immune response.Therefore, these results suggest that (1) oral administration of ECMS and Quil A had no influence on the body weight of mice; (2) one day oral administration of ECMS and Quil A had higher responses of serum specific IgG and most of IgG isotypes to immunization of a FMDV (serotypes O) antigen in mice than two or three day administration but no significant differences (P>0.05); (3) one day oral administration of ECMS and Quil A significantly (P<0.05) pre-conditioned mice, and increased both Thl and Th2 immune responses to vaccination against FMD; (4) one day oral administration or SC injection of ECMS significantly (P<0.05) augmented responses of serum specific IgG and most of IgG isotypes to immunization of a commercial FMDV (serotypes O and Asia 1) vaccine in mice; (5) one day oral administration of ECMS significantly (P<0.05) increased and subcutaneous injection of ECMS tended to enhance serum specific IgG and IgG isotype responses to immunization of a commercial FMDV (serotypes O and Asia 1) vaccine in immunosuppressed mice by SC injection of dexamethasone.