| It is an important task and challenge for development an AIDS vaccine in theworld after30years of the virus has been found. Because of the variability and diversity,there is no vaccine for the AIDS can be put into use so far. The epitopes of broadlyneutralizing antibodies (4E10,2F5,Z13and10E8) from human immunodeficiencyvirus (HIV) envelope protein gp41membrane proximal external region (MPER) as atarget are concerned in HIV vaccine research.When the HIV virus infected cells, MPER occurs a series of conformationalchanges and expose a short epitope recognized by immune system to generate responses.The MPER still is a key part for neutralization to virus. The crystal structure of thelinear polypeptide co-crystallized with4E10antibody displayed the core epitope was-helical. Many researchers tried to constraint the-helical structure to simulate thenative conformational to obtain broadly NAbs, but no successful, so there is not acertain structure information of MPER. In2013, Zhang et al tried to designimmunogens of2F5/4E10epitope from MPER. With a core relative rigid structure oflysine and flexible connection of6-aminocaproic acid provide a dynamic equilibriumstructure in order to obtain broadly neutralizing antibodies, the results showed that the4E10immunogens can successfully induce broadly neutralizing antibodies against toBC (73%positive)、C (93%positive)、B (20%positive) and AE (30%positive) subtypeof HIV virus. In2015, US researchers Matthew R. Gardner gave a report on《Nature》showed that the eCD4-Ig can bind to two constant sites on the surface of HIV andprevent some known strains of HIV. The results provides a new reference direction forHIV vaccine research.Based on quality of4E10multiple antigen peptide immunogen, the pre-performance liquid chromatography, mass spectrometry and small-molecule proteinelectrophoresis were used to retested the purity and molecular weight, while ELISAwas used to confirm the validity of the4E10epitope. In order to obtain the best immuneeffect of4E10multiple antigen peptide immunogen, we chosed immune times、immunedoses and adjuvants for determinining the optimum immune condition and composition of preparation prescription. The results showed that:1)The molecular weight, purity andvalidity of4E10epitope were satisfied with quality standards;2)1M acetic acid candissolve4E10multiple antigen peptide immunogen;3)The highest antibody titers morethan104after the fourth immunization;4) Aluminum hydroxide adjuvant is better thanMF59;5)It is better of50μg dose than low dose and high dose(mice). Although MF59has strong immunomodulatory effects in influenza vaccines, interestingly MF59play amore significant advantage in long-term immunity. Guinea pigs experiments alsoshowed similar results. From the above results,1M acetic acid was dissolution mediumfor4E10multiple antigen peptide immunogen and aluminum hydroxide adjuvant wasa composition of pharmaceutical formulations, the mice (50μg/per) and guinea pigs(100μg/per) immunized four times was the best immune condition.Next part we studied the related quality standards of pharmaceutical formulations,first we inspected the adsorption rate of pH, phosphate, concentration and action timebetween aluminum hydroxide adjuvant and4E10. Followed we tested the effectivecomponents of the preparation, including post-dissociation antigenicity of4E10multiple antigen peptide immunogen and vaccine evaluation (animal test), and finallyexamined the stability of the formulation.The results showed:1)There is no significantimpact of pH to adsorption rate, after0.05M phosphate treatment of adjuvant canincreasea the adsorption rate up to85%, the concentration only1mg can reach tooptimal conditions in2hours;2) The sample was frozen preparations1M acetic acidover four hours can be dissociated, there is no changing in structure and function duringthe dissociation process after Western blotting and ELISA test. After four times ofimmunization of mice, the titer can increase to104;3) It has a good stability after fiveweeks storage at6±2℃.In summary, based on4E10multiple antigen peptide immunogen can inducebroadly neutralizing antibodies in guinea pigs against a known HIV virus, this paperstudied the composition of preparation、optimal immunization program and relatedquality standards of4E10multiple antigen peptide immunogen, the research provide anecessary support for processes and clinical studies of candidate AIDS vaccine. |
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