Study On Different Adjuvants That Can Increase The Immunogenicity Of The Universal Influenza Vaccine NM2e

Universal influenza vaccine is important for the prevention and control of influenza pandemic. Over the past decades, universal influenza vaccine has made important progress. In this subject, NP and M2e fusion protein NM2e subunit vaccine expressed in E. coli, is preferred for its low production costs, repeated large-scale production and good development prospect. However, due to the weak immunogenicity of this vaccine, it requires adjuvants to induce effective and long-term protective immunity.Aluminum is the most widely used adjuvant in a class of vaccine adjuvants, but it mainly induces Th2-type immune response. CpG is a widely studied Thl-type adjuvant, and it can induce strong cellular immunity, but it has not been approved commercially. It has been reported that some chemical drugs such as Levamisole, Cimetidine, Vardenafil and Poly I:C have immunomodulatory effects. Some of these chemical substances reported in the document have a significant adjuvant effect in the animal test on the hepatitis B antigen. Because these drugs are cheap, and their side effects are very clear in long-term application of human experience, once developed as adjuvants, they will have important applications. Therefore, adjuvant research in the subunit vaccine using these drugs has become a new strategy. Therefore, in this research, we studied the adjuvant effect of Al, CpG, LMS, Cim, Var and Poly I:C using NM2e as antigen.In this research, we first purified a lot of NM2e protein and studied the adjuvant effects of Al and CpG. Then we evaluated the probability of four commercially available immunoregulatory medicines, levamisole, cimetidine, vardenafil, and Poly I:C as the adjuvants of a subunit vaccine of influenza virus fusion protein NM2e through humoral immunity, cellular immunity and the protective effects of post-challenge after immunization. Main results were summarized as follows.1. NM2e protein's best soluble expression conditions are as follows:IPTG=0.1mM, OD6oo=0.8, t=10h, T=25℃. We obtained 70mg NM2e protein from 10L E.coli, and its purities was over 90% using two-step purification process of anion exchange chromatography and gel filtration chromatography. The NM2e protein was identified by Western-blot assay.2. Al adjuvant can significantly improve serum IgG1 titer, and it can increase percent survival of challenged mice to 70%(7/10). CpG adjuvant can enhance serum IgG2a titer, and it can increase percent survival of challenged mice to 30%(3/10). When Al in combination with CpG were used as adjuvants, the titer of IgG1, IgG2a is higher than CpG alone, the titer of IgG2a is higher than Al, and it can increase percent survival of challenged mice to 100%.3. Levamisole didn't show obvious adjuvant effect for subunit vaccine of NM2e protein, however, levamisole plus Al could accelerate weight recovery of virus challenged mice; Cimetidine could clearly increase serum IgG2a titer and percent survival of challenged mice (30%) induced by NM2e protein, however the percent survival was significantly lower than that induced by Al adjuvanted NM2e protein (70%), and it didn't show the synergy between cimitidine and Al; Vardenafil could increase serum IgG1 and IgG2a titers and percent survival induced by NM2e protein (36%), however it inhibited the adjuvant effect of Al; Poly I:C could increase serum IgG titer and percent survival induced by NM2e protein (20%), however the percent survival was significantly lower than that induced by Al adjuvanted NM2e protein (70%), and it showed a little synergy with Al.All the results above suggested that, we purified a lot of NM2e protein; Al and CpG adjuvants alone could increase percent survival of challenged mice and they showed the synergy. Levamisole, cimetidine, vardenafil and Poly I:C showed certain immunoregulatory effects on NM2e protein. Cimetidine, Vardenafil and Poly I:C could improve protective effect induced by NM2e protein in virus challenge experiment. However the protective effects were obviously poorer than that induced by Al adjuvanted NM2e protein, and it didn't show the obvious synergy with Al.