The Research On Protective Effect And Mechanisms Of Immunization With Attenuated Vaccine Strain SPY1and Adjuvant C48/80in Infant Mice

ObjectiveStreptococcus pneumoniae (S. pn)，a gram-positive pathogen，alwayslocalizes on the mucosal surfaces of human upper respiratory tract，whichmay induce pneumonia, bacteremia, meningitis, otitis media and so on.Currently, Streptococcus pneumoniae is the leading cause of death inchildren on a global scale, especially in developing countries in Asia andAfrica.Pneumococcal vaccines are urgently needed all around the world, butwidely used pneumococcal vaccines on the markets now have significantlimitations, including high cost, serotype replacement and the bad matchbetween included serotype of vaccines and the situation in developingcountries. SPY1, a non-capsular derivative of pneumococcal strain D39wasobtained in our previous study. The survival rate of infant mice infectedintranasally with high dose (1×108CFU per mice) of SPY1was100%. AsSPY1is highly attenuated, we tried to use it as a pneumococcal vaccine. Adult mice could be well protected by SPY1, but the protective effects oninfant mice were limited.The use of adjuvants is an effective means of improving the efficacy ofvaccines. C48/80, a condensation of N-methyl-p-methoxy-phenethylaminewith formaldehyde, is a well known mast cells (MCs) activators. A series ofstudy of anthrax vaccine and influenza vaccine，C48/80showed excellentadjuvanticity. Thereby, This study was carried out to investigate theprotective effect and mechanisms of nasal immunization with SPY1andC48/80, and then provide the basic of theory and practice to inventpneumococcal vaccines for early life.MethodsInfant mice (2-week-old) were administrated intranasally on days0,7,14and28with1dose of SPY1(1×108CFU) in the presence of CT or C48/80or Pam2CSK4, with SPY1alone or with one of selected adjuvants alone in atotal volume10μl. The group administrated intranasally with PBS wasincluded as negative control. One week after the last immunization,antibodies and cytokines levels were measured by Enzyme-LinkedImmunosorbent Assay (ELISA). Two weeks after the last immunization,mice were challenged with pneumococcal serotype19F or D39andprotective effects were observed. To investigate whether B cells, IL-4andIL-17A contributed to the clearance of nasal pneumococcal colonization,nasal colonization levels of deficient mice (μMT mice, IL-4deficient mice and IL-17A deficient mice) administrated with SPY1+C48/80, SPY1,C48/80or PBS were measured and compared. Furthermore, we detected thesafety of SPY1by a survival experiment and evaluated the safety of C48/80by monitoration of body weight，serum IgE and pathological analysis.ResultsAll three adjuvants enhanced antibody (Ab) responses, whereas IgGtiters maintained in a stable level during the3months after the lastimmunization only in SPY1+C48/80group and SPY1+CT group. IgG1,IgG2a and IgG2b levels of all three groups immunized with SPY1plus oneof selected adjuvant were significantly higher than those of SPY1alonggroup (P<0.05or P<0.01or P<0.001), but IgG3levels were too low to detectin all groups. The saliva IgA level of every immunized group was low exceptthat of SPY1+CT group and the IgA levels of nasal washes of all theimmunized groups were limited and similar. Both C48/80and CT inducedcytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes,while Pam2CSK4did not induce increase in level of Th2-type cytokine IL-4.It is worth noting that C48/80showed exceptional ability to promote theclearance of nasal pneumococcal colonization which CT and Pam2CSK4didnot show. We found that B cells and IL-17A are involved in the clearance ofnasal pneumococcal colonization induced by SPY1and the clearance ofnasal pneumococcal colonization depends on B cells and IL-17A can bereinforced by C48/80. Additionally, our data revealed that C48/80, as a mucosal adjuvant, showed greater ability to protect infant mice against lethalpneumococcal infection than CT. In experiments of safety evaluation, wefound intranasal administration of C48/80(20μg per infant mouse) did notlead to weight loss, increase of serum IgE and lung injury of infant mice, butthe situation in CT group was opposite.ConclusionWe found that when nasally co-administrated with SPY1in the infantmice model, C48/80, as an adjuvant, is more effective than Pam2CSK4andhas the ability to stimulate humoral and cellular immunity comparable to CT.Both CT and C48/80may help to induce immune memory, but only C48/80among3selected adjuvants helps to clear nasal pneumococcal colonization.C48/80’sabilityagainstnasalpneumococcalcolonization depends onBcellsand IL-17A. C48/80even has greater protective ability to help mice againstlethal pneumococcal infection compared with CT. Further more, incomparison with CT, C48/80is safer. Therefore, C48/80has a hopefulpotential to be used as adjuvant in human vaccines.