| Vaccination is still an important strategy to prevent infectious diseases.Noticeably, the antibody levels induced by the vaccines vary substantially among individuals. Many factors might contribute to the variety, such as vaccine formulation,gender, age and psychological stress. So it is worth to investigate the possible factors of individuals after inoculation and then improve the low or lack reponse. In the present study, we try to explore the variety among individuals and possible adjuvant types interfering in the dimorphism of antibody production between male and female individuals.This could provide insights for human vaccines personalized or sexualized.1.Impact of emulsions on the antibody response to influenza vaccination in mice and chickenInfluenza is a highly infectious respiratory disease caused by influenza virus. In the 20 th century, four influenza pandemics emerged and killed tens of millions of people. Therefore, it is very important to develop a safe and effective influenza vaccine. To evaluate the disparity among different species on the antibody response,the mice and chicken were chosen.a) Impact of MF59 and ISA206 on the antibody response to FM1 influenza vaccineThe ICR mice were immunized with inactivated FM1 plus MF59 or FM1 plus ISA206 and bled on day 28 and 42 for collecting sera. The anti-FM1 antibodies and neutralization antibody in the sera were detected and then monitor the survival and weight loss of the mice challenged with live FM1 influenza virus. The results showed that both MF59 and ISA206 could promote the antibody production to the FM1 influenza vaccine and raise the survival rate of the mice challenged with live FM1 influenza virus.b) Impact of MF59 and ISA206 on the antibody response to H9N2 influenzavaccineThe chickens were immunized with inactivated H9N2 plus MF59 or H9N2 plus ISA206 and then sera, respiratory tract lavage and alveolus lavage were collected.Neutralization antibodies were detected by HI and the results showed that ISA206, not MF59, could promote neutralization antibody production to the H9N2 influenza vaccine.In summary, mice can be an ideal object to evaluate the assist effect of different forms flu vaccine and chicken is not. This may be because the chicken belongs to oviparous animal whose immune system is different with human. Thus, the mice were selected as subjects in the later experiments.2. Impact of emulsions in the gender dimorphism on antibody productionDuring the process of a novel adjuvant research, we observed that the female ICR mice produced significantly higher levels of antibodies to FMDV than the male ICR mice. To confirm whether the gender dimorphism on antibody production could be influenced by different emulsions, we make a series of research by choosing different mouse strains and antigens.a)Impact of emulsion types in the gender dimorphism on antibody productionFirstly, BALB/c mice were chose to detect whether the gender dimorphism on antibody response can be influenced by ISA35. The results showed that ISA35 but not ISA206 induced gender dimorphism on levels of anti-FMDV specific Ig M antibodies,while when immunized with ISA35 or ISA206, there was no significant difference in levels of anti-FMDV specific Ig G antibodies between the female and male BALB/c mice to inactivated FMDV vaccines. It demonstrated that types of adjuvant may have effects on antibody response.b)Impact of antigen components in the gender dimorphism on antibody productionConsidering the complexity of inactivated FMDV virus components, we have chosen a protein antigen HBs Ag to further confirm the above phenomenon.Unexpectedly, unlike the inactivated FMDV, the female mice immunized with HBs Ag produced significantly higher levels of anti-HBV specific Ig G antibodies than that the male did. However, this gender dimorphism disappeared when the mice were immunized with HBs Ag plus ISA35.This indicates that the antigen components can influence gender dimorphism on antibody production.c)Impact of mice strain on gender dimorphism on antibody productionThe F1( BALB/b ♀×C57BL/6♂) mice were chosen to further confirm the phenomenon. Unlike BALB/c mice, the female F1 mice produced higher levels of specific Ig G antibodies to inactivated FMDV on day 21. The higher levels of specific Ig G antibodies to HBs Ag of the female F1 mice can be reversed when the mice immunized with ISA35 or ISA206 emulsion. It demonstrated that, besides the types of adjuvant and antigen compositions, the strains can also affect the gender dimorphism on antibody production.d)The activation of B cell in the female and male BALB/c mice immunized with Hepatitis B Virus Vaccine or stimulated with HBs Ag in vitro.In order to observe the differences on B cell activation between female and male mice, the ratio of CD69+CD19+ cells isolated from the mice immunized with HBV vaccine were detected by flow cytometry. Results showed that, the ratio of CD69+CD19+ cells in the splenocytes and draining lymph node cells was higher in the mice immunized with HBs Ag plus ISA35 than that in the mice immunized with single HBs Ag. This indicates that ISA35 could promote immune response to vaccine. In addition, spleen cells and lymph node cells were isolated from na?ve mice and then co-cultured with HBs Ag or FMDV for 24 h. By using flow cytometry, we found that the ratio of CD19+ cell in the spleen cells stimulated with HBs Ag was higher in female mice than that in the male mice. And the ratio of CD19+ cell in the spleen cells stimulated with FMDV was similar between female and male mice. Thus we canXVI know that gender dimorphism on antibody response was easier observed in the mice immunized with a subunit protein vaccine than inactivated virus vaccine.3. Impact of fighting on antibody response to vaccineThe initial purpose of the present study was to observe whether water in oil emulsion adjuvant could influence sexually dimorphic-antibody responses to vaccine.Unexpectedly, we found an obviously elevated antibody response to HBs Ag vaccines in a group of male BALB/c mice experienced severe fighting. To confirm the finding,we first set out to establish a mouse-fighting model, and then examined the potential associations between fighting and the enhanced antibody response to HBs Ag vaccines in male mice.a)Induction of fighting among male BALB/c mice aroused by C57BL/6 miceFirstly, BALB/c mice were chosen to verify the impact of fighting on antibody production, 6 male BALB/c mice were individually kept for 7 days, and then put together in one cage to arouse fighting which was lasted for about 1 hour. To arouse more intense fighting, one male C57BL/6 mouse was introduced into the cage where the 6 immunized BALB/c male mice were kept. The introduction aroused fighting immediately between the male C57BL/6 mouse and each of male BALB/c mice. The fighting was intermittent with an interval of about 2-5 minutes. The intermittent fighting could last for around 12 hours. The antibodies in the sera were detected by ELISA. The results showed that fighting tended to enhance the HBs Ab response in the male BALB/c mice.b)Induction of fighting among BALB/c mice by separation and cross-meetingTo induce more intense fighting, the BALB/c mice were induced to fight by separation and cross-meeting. To measure the fighting more accurately, an evaluating standard of fighting number was defined as one time when two mice chased and bit each other for more than 3 seconds. The results showed that female BALB/c mice didn’t fight between each other, male BALB/c mice fight less 10 times. On the whole,the fighting between the BALB/c mice was not fierce. Finally, the fighting just tendedto enhance the HBs Ab response in the male BALB/c mice. Together, the results may imply that the intensity of fighting is mild and not strong enough to impact the antibody response to HBV vaccine.c)Induction of fighting among C57BL/6 mice by separation and cross-meetingConsidering the BALB/c mice were relatively mild, we chose aggressive C57BL/6 mice to establish the model. In the model, all of the paired mice fought each other for more than 10 times in an hour, which was considered as intense fighting. The antibodies in the sera were detected by ELISA. The results showed that the HBs Ab levels in the fighting induced male C57BL/6 mice were significantly elevated when comparing with that in no-fighting male C57BL/6 mice. However, no correlation between the fighting number and the level of HBV specific antibody were found,these results indicate that fighting do elevate antibody levels in male C57BL/6 mice.d)Impact of fighting on the activation of immune cellsTo find how fighting could influence antibody response, we tried to detect the activation of immune cells by using flow cytometry. Firstly, the mice were sacrificed for collecting blood and spleen after fighting induction. The results demonstrated that the ratios of CD80+CD11c+ and CD86+CD11C+cells in PBMCs tended to be higher in the male mice experienced the fighting than that in male mice without fighting, and that the ratios of CD80+CD11c+ and CD86+CD11C+ cells in the spleens were similar among the mice in the three groups. Furthermore, the splenocytes collected from the mice in the three groups were cultured with HBs Ag for 24 h and 48 h, respectively. The flow cytometry analysis showed that the ratios of CD80+CD11c+ cells and CD69+CD19+ were significantly increased the spleens of the male mice experienced fighting compared to that in the spleens of the male mice without fighting. And the ratios of CD86+CD11+ cells were tended to be higher in male mice with fighting than that in female mice and male mice without fighting. These results indicate that fighting indeed affect the activation of dendritic cells which may be reason of various antibody response. |
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