| Streptococcus pneumonia (S.pn) is one of the most important pathogens which could induce serious invasive infection as well as upper respiratory tract infection, it was estimated by WHO that each year about 1.6 million people worldwide died of various diseases caused by Streptococcus pneumoniae, among which, 1.0 million cases were children under the age of 5, and more than 90% of deaths happened in developing countries; With the emergence of more and more resistant strains, the treatment of Streptococcus pneumoniae infection faced more problems, therefore effective vaccines could be an important means for prevention.At present, the choice of immune approaches is becoming the issue for consideration of developping vaccines, compare to the traditional intramuscular immunization, intranasal immunization could stimulate local immune responses as well as systemic immune response, which played important roles for prevention of many infectious diseases, thus WHO put the style of intranasal immunization as the trend of pediatric vaccine development.Pneumolysin (Ply) is one of the important toxins in Streptococcus pneumoniae, which exists in almost all serotypes of S.pn with a good conservative, the latest research indicates that, Ply expresses in the cell surface. Because of its cytotoxic, then Ply must be optimized before as a vaccine. Preliminary studies by our group showed that the deficiency of the 146th amino acid could induce the completely lost of its virulent and hemolytic activity of Ply, and could stimulate the production of protective antibodies by host, therefore, the mutant Ply could be used as a good vaccine candidate protein of Streptococcus pneumoniae, so here we will carry out this research for evaluating the protective efficiency ofΔA146 Ply which was introduced to BALB/C mice intranasally.MethodProkaryotic expression technology was used to express the recombinant protein Ply with the deletion of 146th amino acid (ΔA146 Ply) in vitro, purified protein was introduced to BALB/C mice intranasally to collect theΔA146 Ply specific antisera. SDS-PAGE and Western blot analysis were applied to evaluate the conservative of Ply in the six common pathogenic Streptococcus pneumoniae in the conservative, while indirect ELISA was used to detect titers of theΔA146 Ply-specific antibody in serum and saliva of immunized mice, as well as analyze IgG subtypes. Meanwhile, spleen cells were collected from immunized mice and cultured for detecting levels of IL-17A produced by host. The strain of 19F of S.pn was introduced intranasally to immunized mice, 3 days later, nasal wash and lung homogenates were collected to quantitate colonized units of bacteria. Pathogenic strains of Streptococcus pneumoniae, NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 infected immunized mice intranasally, and evaluation of the protective efficiency by intranasal immunization withΔA146 Ply was preceded. Vitro anti-adhesion experiments were carried out to evaluate whetherΔA146 Ply and its specific antisera could inhibit Streptococcus pneumoniae R6 adhere to A549 cells.ResultsThe re-engineering strain BL21 (DE3) with the sequences ofΔA146 Ply (146 amino acid deletion) of S.pn was induced by IPTG to produce a large number of recombinant protein in a soluble manner, with Ni-NTA affinity chromatography and SDS-PAGE we collected the purified protein with a purity>95%. Otherwise, it was founded by western blot that Ply had a good conservative in the six common pathogenic Streptococcus pneumoniae strains, which included NCTC7466, TIGR4, CMCC (B) 31436, CMCC (B) 31207, CMCC (B) 31614 and CMCC (B) 31693. Recombinant protein was introduced to BALB/C mice intranasally, serum and saliva were collected to detect theΔA146 Ply specific antibodies and we found that IgG titers in serum were up to 5.12×10~5, while IgG and IgA titers in saliva were 4.0×10~3 and 4.8×10~3 respectively; with goat anti-mouse IgG1, IgG2a, IgG2b, IgG3 subtypes from Santa company , we founded that immunized mice withΔA146 Ply intranasally stimulated host to produce IgG1 and IgG2b mainly, which suggested that the immune response generated by host was Th2 type in more extent. The level of IL-17A in the supernatants of spleen cells suggested that immunization withΔA146 Ply intranasally could stimulate high level of this cytokine, the dose was as high as 678.55±189 (pg / ml), and peaked at 48h after stimulation. Anti-colonization experiment with CMCC (B) 31693 showed thatΔA146 Ply intranasal immunity reduced the S.pn units colonizing in nasopharynx and lung obviously, about 10-20 times (p <0.05). Pathogenic S.pn strains, NCTC7466, CMCC (B) 31436, CMCC (B) 31207 and CMCC (B) 31614 were introduced to immunized mice intranasally, the counts colonizing in nasopharynx and lungs showed that, nasopharyngeal colonization of CMCC (B) 31436 and CMCC (B) 31614 decreased 20 times (p<0.05), while lung colonization of NCTC7466, CMCC (B) 314367 and CMCC (B) 31614 decreased 20-30 times (p<0.05). Immune protection experiments showed that intranasal immunization with recombinant proteinΔA146 Ply could prolong the survival time effectively and improve survival rates, the protective efficiency for strains of NCTC7466, CMCC (B) 31436, CMCC (B) 31614 and CMCC (B) 31207 were 50%, 41.7%, 50%, 41.7% respectively. Vitro anti-adhesion experiments showed that, with the dilution of 100, 500, 2,500, 10,000 times,ΔA146 Ply specific antiserum could inhibit R6 strain adhere to A549 cells, and the inhibition rates were 65%, 60%, 55% and 35% respectively; while the inhibition rates were 15%, 20%, 25% and 30% respectively with the concentrations of recombinant protein in 1μg/ml, 10μg/ml, 20μg/ml, 50μg/ml.ConclusionThe results above suggest that, intranasal immunization withΔA146 Ply protein could protect BALB / C mice effectively against nasopharyngeal and lung colonization of Streptococcus pneumoniae, which may be dependent on the high levels of IgA antibodies and IL-17A; at the same time, intranasal immunization withΔA146 Ply protein could protect host effectively against the common four serotypes of pathogenic S.pn, which may be dependent on the produce of specific antibodies activated by humoral immunity stimulation.
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