ABSTRACT
We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.
Subject(s)
Antibodies, Bacterial , Bacterial Proteins , Immunoglobulin G , Recombinant Proteins , Streptococcus pneumoniae , Streptolysins , Streptolysins/immunology , Streptolysins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/genetics , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/genetics , Animals , Recombinant Proteins/immunology , Recombinant Proteins/genetics , Antibodies, Bacterial/immunology , Antibodies, Bacterial/blood , Immunoglobulin G/immunology , Immunoglobulin G/blood , Mice , Pneumococcal Infections/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Infections/microbiology , Adjuvants, Immunologic , Aluminum Hydroxide/immunology , Aluminum Hydroxide/administration & dosage , Pneumococcal Vaccines/immunology , Pneumococcal Vaccines/administration & dosage , FemaleABSTRACT
We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.
Subject(s)
Bacterial Proteins , Recombinant Proteins , Streptococcus pneumoniae , Streptolysins , Streptolysins/toxicity , Streptolysins/genetics , Animals , Bacterial Proteins/toxicity , Bacterial Proteins/genetics , Chlorocebus aethiops , Mice , Vero Cells , Streptococcus pneumoniae/drug effects , Humans , Recombinant Proteins/toxicity , Recombinant Proteins/genetics , A549 CellsABSTRACT
A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.
Subject(s)
Escherichia coli , Streptococcus pneumoniae , Streptococcus pneumoniae/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Streptolysins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.
Subject(s)
Streptococcus pneumoniae , Streptolysins , Toll-Like Receptor 4 , Mice , Animals , Streptococcus pneumoniae/genetics , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Bacterial Proteins/metabolism , Dendritic CellsABSTRACT
Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized intraperitoneally with recombinant pneumolysin sorbed on Al(OH)3 (200 µg per mouse). In 2 weeks after immunization, the isotypes of antibodies to recombinant pneumolysin in the serum of immunized mice were determined by ELISA. The animals were infected with Streptococcus pneumoniae serotype 3. Immunization with recombinant pneumolysin induced the production of anti-pneumolysin antibodies, mainly of IgG1 subisotype. On day 21 after intraperitoneal infection with S. pneumoniae serotype 3 in a dose of 106 microbial cells, the survival rate of animals immunized with recombinant pneumolysin in a dose of 25 µg/mouse was 67% vs. 0% in the control (p<0.001). Recombinant pneumolysin could be considered as a promising protective antigen for inclusion in the serotype-independent vaccine against S. pneumoniae.