Enhancing Effect Of Zinc Hydroxide Combine With Heparan Sulfate Or Manno-oligosaccharide On Rabies Vaccine Humoral Immunity Efficiency

Vaccine adjuvants play an important role in vaccine research and development. Research on new vaccines such as synthetic peptides, recombinant antigen subunit and plasmid DNA has rapidly progressed and greatly promoted the development of the vaccines with the development of modern molecular biotechnology and genetic engineering and applications of immunology. Although new vaccines have many advantages, but they generally have weak immunogenicity, and are difficult to induce effective immune response in vivo. Although traditional inactivated vaccines have the characteristic of the simple fabrication technology, but they have some problem such as, their effective antigenic determinant may be changed during the inactivation, their immune response do not last for too long, they also require repeated inoculation. To enhance the body’s immune response to new vaccine and inactive vaccine, adjuvants are needed to add to the vaccine, which makes adjuvants become the new hotspot in the field of vaccine research.Rabies is a zoonotic infectious disease caused by rabies virus infection. Once onset, the mortality of rabies is100%.The rabies vaccine is currently the sole pharmaceutical agent used to control Rabies. In addition to virus mutation, the improvement of technology and detection methods, there are problems about the immune vaccine efficacy of the antigen and the multiple immunity of antigen in the process of rabies vaccine development. Therefore, looking for new adjuvant to enhance rabies vaccine immune response or to change immune response types of rabies vaccine is one of the strategies to increases the effectiveness and to reduce the immunization times of rabies vaccine. We used Zinc hydroxide (Zn(OH)2), Heparan sulfate (HS), Manno-oligosaccharide (MOS) as new vaccine adjuvants in this study. Zinc hydroxide (Zn(OH)2), an inorganic nanoparticles containing zinc, Under certain conditions Zn(OH)2can exist stably in the colloid form and has the ability to adsorb protein antigens. Zn(OH)2showed better allergenic and safer than aluminum hydroxide in joint application with vaccine. Heparan sulfate (HS) is an endogenous dangerous signal which acts as a TLR-4ligand. HS is a kind of glycosaminoglycans widely distributed in the extracellular matrix. When inflammation or injury happens in extracellular matrix, HS will be split into soluble low molecular weight HS. Base on the danger signal model theory which Pony Matzinger hypothesized in1994.The dangerous signal molecules released from the damaged cell can activate antigen-presenting cells and then further activate T/B cells effectively to produce the corresponding immune response. This also suggests that the dangerous signal molecules HS released from the damaged cells play an important role in the immune response regulation and have potential immune adjuvant effect. Manno-oligosaccharide (MOS) is a type of saccharide molecules which can be recognized by mannose receptor (MR) in vivo and participate in receptor mediated endocytosis and phagocytosis to maintain the stability of internal environment and to link the innate immunity and acquired immunity together to form the immune defense system. These results indicated that MOS can be used as a immune stimulants to induce specific immune response.To investigate the immune enhancing effect of Zn(OH)2plus HS or Zn(OH)2plus MOS compound adjuvant, we prepared the the compound adjuvants of Zn(OH)2plus HS and Zn(OH)2plus MOS respectively, and then rabies vaccine was used together with the compound adjuvants to immunize ICR mice. The group of Zn(OH)2plus HS compound adjuvant was divided into the following subgroups:the saline control group, groups of1,2and3doses of Zn(OH)2, groups of1,2and3doses of HS, groups of1,2and3doses of Zn(OH)2plus HS compound adjuvant, groups of1,2,3and5doses of adjuvant-free rabies vaccine. The group of Zn(OH)2plus MOS compound adjuvant was divided into the following subgroups:the saline control group, groups of3doses of Zn(OH)2, groups of3doses of MOS, groups of3doses of Zn(OH)2plus MOS, groups of3doses and5doses of adjuvant-free rabies vaccine. Serum samples were collected1,2,3,4,8,12and16weeks after the first immunization and the anti-RABV IgG levels were detected by the ELISA method.The results indicated that Zn(OH)2plus HS or Zn(OH)2plus MOS compound adjuvant can enhance humoral immune responses induced by rabies vaccine. After the first immunization, the primary immune response and the duration of IgG level were better in Zn(OH)2plus HS compound adjuvant group than in the adjuvant-free rabies vaccine group with the same does immunizations. The IgG level is higher in the Zn(OH)2plus HS compound adjuvant group with the first, the second and the third immunizations than in the adjuvant-free rabies vaccine group with the same immunizations(P<0.05). After two weeks of the first immunization, the IgG level is higher after the second and third immunizations in the Zn(OH)2plus HS compound adjuvant group than after the third immunization in the adjuvant-free rabies vaccine group with five immunizations(P <0.05).Compared with the IgG level after the fourth immunization in the adjuvant-free rabies vaccine group, the IgG level is significantly higher after the third immunization in the Zn(OH)2plus HS compound adjuvant group (P<0.05). The IgG level after the third immunization is higher in Zn(OH)2plus MOS compound adjuvant group than in the adjuvant-free rabies vaccine group(P<0.05). After two weeks of the first immunization, the IgG level is higher after the third immunizations in the Zn(OH)2plus MOS compound adjuvant group than after the third immunization in the adjuvant-free rabies vaccine group with five immunizations(P<0.05). The results indicated the compound adjuvant Zn(OH)2plus HS or Zn(OH)2plus MOS could significantly enhance the humoral immune response induced by rabies vaccine in the early stage of immunization.Our result indicated that the compound adjuvant Zn(OH)2plus HS or Zn(OH)2plus MOS has significantly elevated the immune enhancement effect of rabies vaccine, and the compound adjuvants have no significantly pathological damage to the heart, liver, spleen, lungs and kidneys. Zn(OH)2plus HS or Zn(OH)2plus MOS compound adjuvant has the protential to be the new kind of adjuvant which use in human being.