Development Of Improved Liposomal Vaccines Against Group A Streptococcus

Objective: Group A Streptococcus(GAS),also known as Streptococcus Pyogenes,is one of the most pathogenic streptococci which can cause a variety of diseases,including non-invasive and invasive infections.Untreated infections can also induce post-infection autoimmune diseases,causing serious clinical consequences.Although penicillin is still the first-line medication for GAS infection,the rate of treatment failure is eminent,and the incidence of invasive GAS(i GAS)infection has been increasing year by year,which becomes a huge medical burden worldwide.GAS vaccine is the most effective way to fight against diseases.However,there are many bacterial serotypes and a possibility of cross-reactions with normal tissues,which pose a great challenge to vaccine development.No vaccine has yet been licensed.M protein,the major virulence factor of GAS,contains a highly conserved C region.Theoretically,vaccine candidates targeted on this region can against most of GAS strains.Studies have shown that J8 i is the minimal B cell epitope within C3 region and become immunogenic when inserted into the yeast DNA-binding protein GCN4.Previously,we have successfully formulated J8-Lipo-DT using liposome as a carrier and self-adjuvanted system.Moreover,we found a new peptide termed as p*17,which has the same parent peptide as J8.In this study,based on J8-Lipo-DT,we aim to design and improve GAS vaccines by three aspects: peptides,adjuvants,and immunization regimes,so that we can not only screen out an ideal formulation of GAS vaccines,but also determine a reasonable administration route.Maximizing the immunogenicity and minimizing the toxicity can accelerate the progress of GAS vaccines into human clinical trials.Methods:(1)J8-Lipo-DT was formulated by standardized thin film hydration method.Mice were immunized with J8-Lipo-DT intranasally to determine its immunogenicity and protection against upper respiratory infection with GAS M12 strain.Interleukin(IL)-1? knockout mice were used to investigate the role of IL-1? in liposome vaccine-induced immunity.Subsequently,improve vaccine formulations by adding another adjuvant PHAD or replacing J8 to p*17 in order to screen out the most promising candidates.(2)A head-to-head study comparing the immunogenicity and protection of p * 17-DT / Lipo-PHAD and p * 17-DT / CAF01 conjugate vaccine was performed.In vitro,serum bacterial killing assay and the binding of vaccine anti-sera to GAS detected by flow cytometry were carried out respectively as well.(3)As GAS is a human restricted organism,h CD4.IAE-/-.DR3-DQ2.B6 humanized mice were immunized with p * 17-DT + K4S2-DT / CAF01 combination vaccine using prime-pull immunization strategy,to broaden the scope of cov R/S GAS mutant strain and evaluate its immunogenicity and protection against emm75 GAS strain.Another cohort of mice was immunized with the same vaccine and rested until the antibody titer dropped.They were given a low dose of bacteria or antigen to boost their immune response again.Antibody levels,inflammatory cytokines and specific antibodies secreted B cells were then measured by ELISA,CBA and ELISPOT respectively.Results:(1)J8-Lipo-DT intranasally immunized mice induced serum and salivary J8 specific antibodies.The bioburden of all tissues or organs in the vaccinated group decreased significantly after challenge with p M12 strain via upper respiratory tract.After IN challenge,higher bioburden was observed in vaccinated IL-1?-/-mice compared to C57BL/6 wild-type mice.Moreover,the level of IL-17 A secreted by splenocytes in vitro was also lower than that of wild-type mice.When adding PHAD to the liposome delivery system,whether they were induced by J8-Lipo-DT PHAD or J8-DT/Lipo-PHAD,antibody titers were considerably higher than J8-Lipo-DT.GAS bioburden was significantly reduced in both PHAD related vaccine groups when challenged intranasally.On the other hand,we formulated p*17-Lipo-DT using another peptide from M protein.Antigen-specific antibodies of p * 17-Lipo-DT started to increase after one single immunization,which was much faster than that of J8-Lipo-DT.The protection against upper respiratory tract infection of p*17-Lipo-DT and J8-Lipo-DT were comparable.Similar to the J8-based liposomal vaccines,p * 17-Lipo-DT PHAD and p * 17-DT/Lipo-PHAD are more immunogenic than p * 17-Lipo-DT,which can protect against GAS infection in the upper respiratory tract.However,they still could not provide effective protection against skin infection.(2)A head-to-head study comparing of p * 17-DT/Lipo-PHAD and p * 17-DT/CAF01 was performed.Prime-pull(subcutaneous injection twice followed by one intranasal immunization)immunization regime can significantly increase the level of antigen-specific antibodies.The immunogenicity and protection of p * 17-DT/ CAF01 immunized mice were significantly higher than those of p * 17-DT / Lipo-PHAD group,which can protect against both the upper respiratory tract and skin infections.Pre-vaccination of DT/Alum did not affect the immunogenicity of p * 17-DT/CAF01 conjugate vaccine.In vitro study showed that p * 17-DT/CAF01 anti-sera had higher binding ability to GAS than other compound adjuvanted GAS vaccine anti-sera.(3)h CD4.IAE-/-.DR3-DQ2.B6 humanized mice were immunized with p * 17-DT + K4S2-DT/CAF01,using prime-pull(two intramuscular injections followed by one intranasal immunization).All vaccinated mice induced high titers of p * 17 and K4S2 specific antibodies.p * 17-DT + K4S2-DT / CAF01 immunized mice showed some protection against URT infection of emm75 GAS strain.The antibody titers dropped quicker in no pre-exposure of DT vaccinated group than those with DT pre-vaccinated group.Immunized mice were re-boosted with low dose of cov R/S mutant GAS strain intranasally when rested until the levels of serum antibodies dropped.More p*17 specific Ig G secreting cells were observed in salivary glands compared to na?ve group.TNF,IL-2,IL-6 and IL-17 A secreted by spleen cells of vaccinated mice were significantly higher na?ve group,with no difference to those in the newly immunized mice.Conclusions:(1)J8-Lipo-DT administrated intranasally can protest against the upper respiratory tract infection of GAS M12 strain.Apart from neutralizing antibodies,IL-1? may participate in liposomal vaccine induced immunity by enhancing the production of IL-17.p*17-Lipo-DT produced antigen specific antibodies quicker than J8-Lipo-DT and has a comparable protection against upper respiratory tract infection as J8-Lipo-DT,which indicated p*17 might be better than J8 as a peptide used in GAS vaccines development.PHAD can significantly enhance the efficacy of liposomal vaccines.Yet,liposomal vaccines administrated intranasally are not capable to induce protection against skin infection.(2)Prime-pull immunization of p*17-DT/CAF01 can protect against both upper respiratory tract infection and skin infection,which indicated prime-pull strategy can amplify the protection of GAS vaccines.It might be an ideal immunization regime for GAS mucosal vaccines.p*17-DT/CAF01 showed a significant better protection than p*17-DT/Lipo-PHAD in both in vivo and in vitro studies.(3)In combination of K4S2-DT,p * 17-DT + K4S2-DT / CAF01 vaccinated mice showed a protection against emm75 intranasally to some extent.This vaccine candidate can induce long term memory.Pre-exposure of DT may help maintaining the immunogenicity of the p * 17-DT + K4S2-DT / CAF01 conjugate vaccine.Antigen specific Ig G plays a key role in fighting against URT infection.Moreover,TNF,IL-2,IL-6 and IL-17 A are important cytokines involved in vaccine mediated immune responses.