Application Of CS-?-PGA Nanogels In HBV Vaccines

Background:According to the latest statistics,there are more than 350 million HBV chronic carriers,which at a high risk of cirrhosis and hepatocellular carcinoma.CHB results in more than 1 million deaths each year.However,CHB lacks effective therapeutic medicine,and easy to relapse after medicine withdrawal.Prophylactic vaccination is the best way to prevent HBV infection.Since the 1980s,prophylactic vaccination against HBV in children has resulted in a significant reduction in the incidence of HBV in China.The current aluminum-adjuvanted HBsAg vaccine(HepB)is very effective.However,there are still some deficiencies of HepB in clinical application.HepB is a recombinant subunit vaccine with weak immunogenicity,requies aluminum adjuvant.In addition,HepB should be administered in three doses over six months,which limits universal vaccination.The immunity of HepB against HBV infection may decline over time.The aluminum adjuvant HepB is only capable of inducing humoral immune and the production of specific antibodies after immunization.Approximately 5-10%of immunized individuals fail to elicit detectable specific antibodies,and are still at risk of HBV infection.Therefore,effective induction of humoral and cellular immunity can provide more effective immunoprotection against HBV than humoral immunity inducted by the existing vaccine.And,some concern has been raised for aluminum adjuvant association with aseptic abscess,eosinophilia,myofasciitis,amyotrophic lateral sclerosis and Alzheimer's disease.Therefore,it is beneficial to design a novel HBsAg vaccine with improved efficiency,ease of use,as well as a reduced dosing frequency and vaccine-related consumption.The use of nanotechnology in vaccinology has been increasing exponentially in the past decade,leading to the birth of "nanovaccinology".An increasing number of nanomaterials are being used as carriers for vaccines and adjuvants.Among them,nanogels are swollen networks composed of amphiphilic or hydrophilic polyionic polymers,with a high antigen loading capacity.Chitosan(CS)and poly-?-glutamic acid(y-PGA)are suitable materials for the preparation of naogels.CS and ?-PGA are natural and resource-rich compounds,which are non-toxic,biodegradable,and non-immunogenic.CS is a positively charged polysaccharide.y-PGA is a negatively charged polyelectrolyte.Commercially available hepatitis B vaccines can not induce antigen-specific cellular immune effectively.Inhibition of the secretion of interleukin-12(IL-12)in DCs by aluminum adjuvant is one of the most important reason.IL-12 can significantly promote the production of large amounts of IFN-? by NK cells and cytotoxic T lymphocytes,and mediates cellular immune.IL-12 can also interact with B cells to promote B cell differentiation and antibody production.Thus,IL-12 has a significant role in promoting cellular and humoral immunity.However,IL-12 is expensive and prone to adverse reactions.The IL-12 plasmid(pIL-12)is structurally stable,relatively easy to construct,and easy to mass produce.The transfection efficiency of pIL-12 can be improved by nanogels.The pIL-12 nanogels are expected to be as an adjuvant for the HBsAg vaccine.Object:First of all,we investigated whether nanogels as a carrier of HBsAg could influence the prophylactic effect of HBsAg vaccine.In the second,we investigated whether pIL-12 could influence the prophylactic effect of HBsAg vaccine.Methods:1.HBsAg nanogels and pIL-12 nanogels were prepared by CS and y-PGA.The physicochemical properties of nanogels were investigated.2.C57BL/6J mice were immunized intramuscularly with different formulations.Blood samples were collected from the tail vein and anti-HBs level was detected at different time points.3.8?g of pAAV/HBV1.2 plasmid in 0.9%NaCl solution was hydrodynamically injected via the mouse tail vein to mimic HBV infection in clinical.Blood samples were collected,and the HBsAg and anti-HBs levels were detected at different time points.4.Lymphocytes were extracted from liver,spleen,lymph nodes and peripheral blood.The proportion,absolute number and cytokine expression of lymphocytes were analyzed by flow cytometry.Results:Part 1:Single dose HBsAg CS-y-PGA nanogels induce potent protective immune responses against HBV infection.1.Positively charged HBsAg nanogels(HBsAg Ng(+))and negatively charged HBsAg nanogels(HBsAg Ng(-))were prepared by adjusting the CS and y-PGA proportion.The encapsulation efficiency of HBsAg was high in both Ng(+)and Ng(-).2.The anti-HBs levels were very high in mice immunized with three dose of HBsAg(Al),HBsAg Ng(-),and HBsAg Ng(+).In the further studies,the mice were immunized with one dose of different formulations.3.Single-dose immunization with HBsAg Ng(+)induced anti-HBs effectively.HBsAg Ng(+)immunized mice could cleare HBsAg and restored anti-HBs production after pAAV/HBV1.2 plasmid challenge.4.HBsAg Ng(+)could promote maturation and antigen presentation ability of DCs.Single-dose HBsAg Ng(+)induced both humoral and cellular immunity,and could induce effector memory T cells,providing long-term protection against HBV.Therefore,Ng(+)are desirable HBsAg prophylactic vaccine carriers.5.Both HBsAg Ng(+)and HBsAg Ng(-)could significantly promote the clearance of HBsAg,increasing the proportion of IFN-?+CD8+ T cells in CHB mice.However,no HBV-specific antibody induction was observedPart 2:Study on the role of negative charge nanogel loaded IL-12 expression vector as an adjuvant for HBsAg vaccine in inducing immune response against HBV infection1.Ng(-)(pIL-12)intramuscular injection could effectively promote IL-12 secretion and increase serum IL-12p70 levels in mice.2.Three dose immunization with high dose Ng(-)(pIL-12)(75 ?g)combined with HBsAg vaccine induced anti-HBs effectively.However,high dose Ng(-)(pIL-12)adjuvant might be detrimental to induction of cellular immune.Three dose immunization with low dose Ng(-)(pIL-12)(5 ?g)combined with HBsAg vaccine could effectively induce humoral immunity and cellular immunity.3.5 ?g Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine can promote the maturation and antigen presentation ability of CD8a+/CD103+ DCs.Compared with control group,the percentage and the absolute numbers of CD8a+ DCs in spleen was increased in low dose Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine immunized mice,accompanied with the elevation of CD40,CD80 and CD86.A similar phenomenon was observed on CD103+ DCs.4.M2 macrophage polarization was inhibited in spleen in low dose Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine immunized mice,accompanied with the elevation of CD86 and the decline of CD206.Compared with control group,the percentage of MDSCs in spleen was decreased in low dose Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine immunized mice after pAAV/HBV1.2 plasmid challenge.5.Low dose Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine induced the generation of HBV-specific CD11ahi CD49dhi CD4+ T cells.Compared with control group,the percentage and the absolute numbers of CD11ahi CD49dhi CD4+ T cells in spleen was increased in low dose Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine immunized mice after pAAV/HBV1.2 plasmid challenge.6.Compared with control group,the absolute numbers of CD11ahi,CD8?lo T cells in spleen was increased in 5 ?g Ng(-)(pIL-12)adjuvant combined with HBsAg vaccine immunized mice,accompanied with low expression of LAG-3?TIGIT,after pAAV/HBV1.2 plasmid challenge.The proportion of SLECs in CDllahi CD8?lo T cells was increased significantly,accompanied with the high expression of CD107a and low expression of TIGIT.Conclusions and Significance:1.Single dose HBsAg Ng(+)immunization can rapidly induce anti-HBV humoral and cellular immune,and can provide immune protection against HBV infection.2.5 ?g Ng(-)(pIL-12)combined HBsAg vaccine can effectively promote the maturation of APCs,increase the number of antigen-specific CD4+ T cells,CD8+ T cells and facilitate the induction of terminally differentiated effector memory SLECs.3.Our research provides new adjuvant options for clinical HBV vaccines.