| Vaccines are the cost-effective means of preventing infectious diseases,and the development of safe and efficient new vaccines is the goal of vaccine research.Subunit vaccines and recombinant protein vaccines are highly purified proteins.The safety of these vaccines have been improved,but their immunogenicity is still weak.In order to improve the efficacy of proteinbased vaccines,researchers have been looking for efficient adjuvants to enhance the immunogenicity of the antigen,induce a stronger immune response or alter the type of immune response.Alum salts are the longest and the widest used adjuvants in the world.In recent years,several new adjuvants include MF59,AS03,AS04 and Virosomes,have been approved for the human vaccines.The plant polysaccharide,because of its strong immune regulation function,and biodegradability,low toxicity and other advantages,become a hotspot in adjuvant research field.The polysaccharide PCP-I is a kind of homologous polysaccharide extracted from the traditional Chinese medicine Poria cocos,composed of fucose,mannose,glucose and galactose.In this study,the adjuvant effect of PCP-I was evaluated on the humoral immune response and the cellular immune response by using Bacillus anthracis protective antigen(PA)as a model antigen.The adjuvant effect of PCP-I and CpG combination adjuvant was observed.The ability of PCP-I to enhance the protective effect of PA was studied by using the anthrax toxin challenge model and the spore challenge model.The mechanism of PCP-I adjuvant effect was investigated on three aspects: dendritic cell(DC)maturation,germinal center(GC)reaction and peripheral blood monocyte(PMBC)gene expression difference.Firstly,the dose response of PCP-I in mouse immunization experiment was investigated.0.5 ?g PA was mixed with 50,200,500 ?g PCP-I to immunize BALB/c mice.Blood was collected two weeks after the second immunization to detect anti-PA antibodies and toxin neutralizing antibodies in serum.The results showed that anti-PA antibody(5.38×103)and neutralizing antibody(8.7×101)in 200?g PCP-I group were significantly higher than those in 50?g group(1.54×102,2.65×101),slightly higher than 500?g group(4.15×103,6.13×101).Accordingly,200 ?g of PCP-I was selected for subsequent experiments.To evaluate the adjuvant effect of PCP-I on humoral immunity,PCP-I and aluminum adjuvant(Al)were mixed with 0.5 ?g PA to immunize BALB/c mice,and PA without adjuvant group and PBS group were used as negative and blank control respectively.Mice were immunized three times at 2-week interval and blood was taken two weeks after the third immunization.The anti-PA antibody titer(1.81×104)and antibody affinity(0.81)in PA plus PCP-I group were significantly higher than those in PA group(1.60×103,0.52)and similar to PA plus Al group(2.35×104,0.83).The anti-PA antibody subclass was analyzed and found that the IgG1 of PA plus PCP-I group was significantly higher than that of IgG2a(5.12×104,2.93×103),suggesting that PCP-I was a Th2 bias adjuvant.In toxin neutralization assay(TNA),the neutralizing antibody(2.55×103)in PA plus PCP-I group was significantly higher than that in PA group(1.32×102),and no significant difference with PA plus Al group(1.76×103).To observe the effect of PCP-I on cellular immunity,PCP-I and aluminum adjuvant were mixed with 5 ?g PA to immunize BALB/c mice,and PA group without adjuvant group and PBS group were used as negative and blank control respectively.Spleens were collected for analysis of cellular immunity 2 weeks after the third immunization.The frequency of PA-specific memory B cells in the spleen of PA plus PCP-I mice(4.26%)was significantly higher than that of PA group(0.87%),and no significant difference with PA plus Al group(5.39%),indicating that PCP-I can enhance B cell memory.The proliferation of spleen cells in PA plus PCP-I group(1.37)was significantly higher than that in PA plus Al group(1.05)and PA group(1.08)detected by CCK8,indicating that PCP-I can improve the proliferation of mouse spleen cells after re-exposure to antigen.The frequency of IL-4 and IFN-? secreting cells in spleen cells was detected by ELISPOT.The frequency of IL-4 secreting cells in PA plus PCP-I group(92/106)was higher than that in PA plus Al group(34/106)whereas there was no significant difference in the frequency of IFN-? secreted cells.The levels of cytokines in the supernatant of spleen cells stimulated by PA were detected by MILLIPLEX Multiplex Assay.The levels of IL-2,IL-4 and IL-5 in PA plus PCP-I group(63.5,23.0,1020.8pg/ml)were significantly higher than those in PA plus Al group(17.0,2.6,5.1pg/ml),indicating that PCP-I enhances the ability of memory T cells to secrete corresponding cytokines after exposure to antigens again.To assess the capability of PCP-? to enhance vaccine-specific protection,immunized mice were challenged with anthrax lethal toxin and the survival rate was observed.All of the mice immunized with PBS or PA alone died,and the survival rate of PA plus PCP-I group was 75%.In order to assess the effect of PCP-I and CpG combination adjuvant,BALB/c mice were immunized with 0.5 ?g PA mixed with different adjuvants,and blood was collected two weeks after the third immunization.The PA plus PCP-I and CpG combination group had significantly higher anti-PA antibody titers(1.02×105)than PA plus PCP-I group(1.81×104)and PA plus CpG(3.49×103),whose antibody affinity(0.73)was significantly higher than PA plus CpG group(0.52)but slightly lower than PA plus PCP-I group(0.81).The antibody subclass analysis showed that IgG1 was the same as PA plus PCP-I and CpG combination group and PA plus PCP-I group.(7.90×104,5.12×104),while the IgG2 a was significantly higher than that of the latter(5.12×104,2.93×103).The neutralizing antibody(1.38×104)in PA plus PCP-I and CpG combination group was significantly higher than that in PA plus PCP-I group(2.55×103)and PA plus CpG group(1.62×103).These results suggest that the adjuvant can enhance the anti-PA antibody in a balanced manner with Th1/Th2,and the ability to neutralize toxins of serum was significantly increased.The mice were challenged with lethal toxin(LT)to observe the survival rate.The survival rate of PA plus PCP-I and Cp G combination group was the same as that of PA plus PCP-I group(75%),slightly higher than PA plus CpG group(50%).To directly evaluate the ability of combination adjuvant to enhance the protective effect of PA,0.5 ?g of PA was used to mix different adjuvant to immunize A/J mice which was susceptible to anthrax spores.The blood was collected 2 weeks after the third immunization and mice were challenged subcutaneous(s.c)with the A16 R strain of B.anthracis 4 weeks after the third immunization.The results showed that anti-PA antibody and neutralizing antibody(7.18×103,2.42×102)in PA plus PCP-I group were lower than PA plus Al group(3.23×104,3.49×103),while and that in plus PCP-I and CpG combination(3.23×104,2.29×103)was comparable to the PA plus Al group.After the challenge,the groups immunized with PA plus PCP-I and CpG combination and PA plus Al survived,and the survival rate of PA plus PCP-I group was 83%.The adjuvant effect of PCP-I alone was not as good as that of BALB / c mice in A/J mice.It was presumed that the adjuvant effect of PCP-I may be related to the activation complement pathway because A/J mice were complement molecule C5-deficient mice.And the addition of CpG could compensate for the adverse effect of C5 deletion on PCP-I adjuvant effect to a certain extent.In order to explore the mechanism of PCP-I,a preliminary study was carried out on three aspects: DC maturation,GC reaction and PBMC gene expression difference.Bone marrowderived DCs were stimulated with PA plus PCP-I,PA plus Al and PA,respectively.The expression of CD80 and MHC-II was detected by flow cytometry.The percentage of CD80 positive cells in DC of PA plus PCP-I group(82.2%)was significantly higher than that in PA group(51.7%),which was slightly higher than that in PA plus Al group(65.1%).The percentage of MHC-II positive cells in PA plus PCP-I group(74.9%)was significantly higher than that in PA group(46.8%)and PA plus Al group(56.1%).These results suggest that PCP-I can induce DC up-regulation expression of CD80 and MHC-II and promote DC maturation.BALB / c mice were immunized with 5 ?g PA and mixed with PCP-I and aluminum adjuvant respectively.The mice were immunized twice at intervals of two weeks.The spleen cells were collected at 7 days after the second immunization and GC B cells and follicular helper T cells(Tfh)was detected by flow cytometry.The frequency of GC B cells(7.73%)in PA plus PCP-I group was significantly higher than that in PA group(6.30%),and the frequency of Tfh cells was slightly higher than that of the latter(4.97% vs 4.20%),suggesting that PCP-I promote GC response.The gene expression of PBMCs was observed by transcriptome analysis after 3 days of immunization with PA plus PCPI,PA plus Al and PA.Through the data analysis,66 different genes were screened between PA plus PCP-I group and PA group,among which 53 genes were overlapped with that between PA plus Al group and PA group.13 genes were PCP-I specific differences gene.It was found that Il1r2,Clec4 e,Stab1 and C5ar1 were associated with the immune response,in which C5ar1 encodes C5 aR,participates in the regulation of specific immune response of complement C5 a.This result is consistent with the hypothesis that the adjuvant effect of PCP-I is related to C5.In summary,as a vaccine adjuvant,the plant polysaccharide PCP-I can enhance humoral immune response as good as aluminum adjuvant,however,with a higher cellular immune response.And when PCP-I combined with CpG,the humoral immune response was further enhanced.Preliminary mechanism analysis showed that PCP-I could enhance the antigen-specific immune response by promoting DC maturation and enhancing GC reaction,and its mechanism may also be related with complement C5 and C5 a R. |
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