| With the extensive abuse of antibiotics, bacterial resistance becomes very serious. Vaccine developments against various pathogenic bacteria have attracted much attention. Subunit vaccine has become a focus in the research of new bacteria vaccines for its safety, specificity and easy production. By conjugation of adjuvant to antigen or different adjuvant into a unified whole, chemical conjugation technology can enhance the immune protective effect of subunit vaccine. In Neisseria meningitidis polysaccharide conjugate vaccine, we prepared conjugate vaccines containing different spacer arms. Then, the effect of PEG as a spacer arm on immunogenicity of conjugate vaccine was investigated. In Mycobacterium tuberculosis subunit vaccine research, we prepared new adjuvant AG-P by coupling poly(I:C) to arabinogalactan (AG). In application of a co-delivery system by conjugating Mtb Ag85B-HspX (AH) fusion protein with AG-P, we developed a highly efficient vaccine (AH-AG-P). The detailed research conclusions are as follows:(1) Neisseria meningitidis is a life-threatening pathogen that causes meningitis and other clinical manifestations. As a key virulence determinant, meningococcal capsular polysaccharide (PS) can be used as an immunogen for vaccine development. Conjugation to a carrier protein can turn PS from a T cell independent antigen into a T cell dependent antigen, significantly improve its immunogenicity, induce memory response and as a result can provide immune protection for infants and young children. However, as a biomacromolecule carrier protein may cause steric shielding effect on important antigenic PS epitopes leading to lower the immunogenicity of PS. Here, a heterobifunctional polyethylene glycol (PEG) was used as a spacer arm to conjugate meningococcal group Y capsular PS with tetanus toxoid (TT). Heterobifunctional PEG can avoid self-crosslink of PS and increase the PS/TT ratio of the vaccine. Significant structural change in TT and PS was not observed upon conjugation. As compared to the vaccine without PEG, immunization with the vaccine using PEG as the spacer arm led to a 3.0-fold increase in the PS-specific IgG titers and a prolonged immune persistence. As a non-immunogenic hydrophilic polymer, PEG has been widely used to couple therapeutic protein for prolonging its circulatory time and decreasing its immunogenicity. But in polysaccharide conjugate vaccine PEG as a spacer arm can improve the immunogenicity of PS by fully decreasing the steric shielding effect of TT on antigenic epitopes of PS and suppressing the immunogenicity of TT. In addition, PEG can prolong the immune persistence of the conjugate vaccine. Thus, PEG can be used as a spacer arm to develop effective polysaccharide conjugate vaccine.(2) Mycobacterium tuberculosis (Mtb) is one of the main pathogens that can cause pulmonary tuberculosis in the world. Protein-based vaccine is promising to improve or replace BCG vaccine for its specificity, safety and easy production. However, protein-based vaccine calls for potent adjuvants and improved delivery systems to provide effective immune protection for human beings. Polyinsinic-polycytidylic acid (poly(I:C)) is a synthetic analog of viral double-stranded RNA produced by polyinosinic acid and polycytidylic acid through base pairing. Poly(I:C) can strengthen human immune response against intracellular Mtb by signaling via TLR3. Arabinogalactan (AG) is a biocompatible polysaccharide that can increase splenocyte proliferation, stimulate macrophages and improve the secretion of cytokines such as IFN-y and TNF-a. The AG-poly(I:C) conjugate (AG-P) could play a potent immunostimulatory activity through a synergistic adjuvant interaction of AG and poly(I:C). Ag85B and HspX are two important virulent protein antigens expressed by Mtb at actively replicating and latent stages, respectively. Ag85B-HspX fusion protein (AH) was prepared by recombinant fusion expression of the two antigen genes. An antigen-adjuvant co-delivery system (AH-AG-P) was developed by conjugation of AH with AG-P to insure that both AH and AG-P reach and stimulate the antigen presenting cells simultaneously. Immunological evaluation revealed that AH-AG-P could elicit high AH-specific IgG titers mostly in the form of IgG2c and stimulate lympho-cyte proliferation. AH-AG-P could also increase high splenic CD8+ T cell proportion by promoting cross-presentation of antigen and provoked the secretion of Th1-type cytokines (IFN-y, TNF-a and IL-2). Pharmacokinetic analysis revealed that conjugation with AG-P could prolong half-life of AH in serum and persistently stimulate the immune system. Pharmacodynamic analysis suggested that AH-AG-P led to a rapid and intense production of AH-specific IgG.Thus, co-delivery of AH with a new potent adjuvant AG-P is an effective strategy to develop an efficacious and protective vaccine against Mtb infection. |
