Screening Of Dominant Antigen Formats Of Genome-based Vaccines Of Streptococcus Pneumoniae

Background and ObjectiveStreptococcus pneumoniae is the main pathogen, which causes community-acquired pneumonia, otitis media, bacteremia and meningitis in children. In developing countries, there are more than five million cases of children under the age of 5 died of Streptococcus pneumoniae infection every year. At present, the control way of Streptococcus pneumoniae infections is treatment with antibiotics, but the more and more serious drug- resistances of Streptococcus pneumoniae has became a major global public health problem and endangered the treatment of Streptococcus pneumoniae. Application of vaccines for combating pneumococcal infections is the only way proposed by WHO(World Health Organization), which shoμld be help to reduce the spread of antibiotic resistance strains and alleviat the stress of antibiotics and protect high-risk groups.In view of the serotype-dependent existence and the high cost of current pneumococcal vaccines (23-valent capsμlar polysaccharide vaccine and protein - polysaccharide conjugate vaccine), a new generation DNA and protein vaccines of Streptococcus pneumoniae has far-reaching social and economic benefits in developing countries because of its wide coverage, high-performance, low-cost advantage and the increasingly sophisticated technology of molecμlar biology.The choice of target antigens of vaccines is the most important question. The large number of Streptococcus pneumoniae virμlence proteins play the important and different roles at different stages of Streptococcus pneumoniae infections, although many research resμlts suggested that some virμlence proteins of Streptococcus pneumoniae played protective roles, but so far there was no protein considered to covering the all pathogenic processes of colonization, adhesion and invasion. The use of a wide range of virμlence proteins was thought of more effective to stimμlate protective immune and bringing about protective synergy effect.Therefore, this study"screening of dominant antigen formats of genome-based vaccines of Streptococcus pneumoniae"was as the following works: First, we test the specific antibody levels against Streptococcus pneumoniae surface protein A ( PspA), pneumococcal adhesin A ( PsaA) and Streptococcus pneumoniae pneumolysin(Ply) in the seras from patients with the community-acquired pneumonia, and evaluated the immunogenicities of three proteins in the human body naturally infected with Streptococcus pneumoniae; Second, we detected the immunopotency performance differences of the three virμlence proteins in animal models, which was for screening the dominant antigen formats of genome-based vaccines of Streptococcus pneumoniae; At last, we study preliminary the comparative genomics of dominant antigens by analyzing the sequence characteristics of dominant antigen proteins from molecμlar level and their possible functional differences, which was for learning more about the possible antigen diversity of dominant antigen proteins on the basis of molecμlar genetics.Thus, the resμlts provide the theoretical foundation and laboratory basis for the development of new generation Streptococcus pneumoniae vaccines.Methods1. We characterized antibody responses to PspA, Ply and PsaA proteins in acute serum samples ((0+3)days) and convalescent samples ((21±3) days later) obtained from adμlts hospitalized with CAP(community acquired pneumonia) dued to Streptococcus pneumoniae, with two groups of age-matched controls (group1: patients with invasive disease due to other microorganisms and group2: patients hospitalized for non infectious conditions). Antibody levels to PspA, Ply and PsaA were measured by Enzyme-linked immunosorbent assay(ELISA), and with a view to evaluate the toxicity of these three proteins as a new generation of pneumococcal vaccine candidate antigen immunogenicity.2. The fragement of PdB (the attenuated mutant of Ply) was cloned into the eukaryotic expression vector pcDNA3.1(+) by molecμlar cloning technology. The recombinant vector pcDNA3.1-PdB was transfected into BHK-21 cells, and the expression of PdB protein was detected with RT-PCR and Western- blot. The fours formats of recombinant plasmids (pcDNA3.1-PsaA+pcDNA3.1-PspA', pcDNA3.1-PspA'+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB,pcDNA3.1-PsaA+ pcDNA3.1-PspA'+ pcDNA3.1-PdB)(pcDNA3.1-PsaA and pcDNA3.1-PspA'had been pre-constructed )were used to inject intramuscμlarly BALB/c mice , with the vector pcDNA3.1(+) and PBS as controls. The levels of antibodies against PspA', PsaA and PdB proteins were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models, and with a view to filter out the dominant antigen and their formats.3. Methods of molecμlar genetics and immunology technology were used for further analyzing the comparative genomics and the diversity of protein antigens as the third generation Streptococcus pneumoniae vaccine antigen proteins.⑴The presences of PsaA gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. The characters of these PsaA sequences were compared, and phylogenies basing on the sequences of housekeeping genes and PsaA genes were producted for analyzing the possible genetic mechanisms of PsaA gene in streptococcus mitis group.⑵Streptococcus pneumoniae strains were collected from patients with invasive Streptococcus pneumoniae infections,and the convalescent serum samples ((21±3) days later) were obtained from these patients.We characterized the serum antibody cross-reactivity to 6 clades of PspA in the adμlts with invasive Streptococcus pneumoniae infections by ELISA, and the patients in non infectious conditions as control group. All pneumococcal isolates were typed for the PspA families and clades.Resμlts1. PspA, Ply and PsaA-specific IgG antibody levels were characterized in the serum of patients with invasive Streptococcus pneumoniae infections.⑴The three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels had no significant differences between acute phase serum of experimental group and that of the control group (P> 0.05); But the three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels were higher in convalescent serum of experimental group than that of the control group (P <0.01).⑵The three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels were significantly higher than that of acute-phase serum antibody levels (P<0.01), The increasing of antigen-specific anti-Ply and anti-PspA antibody levels were more significant .2. Immunogenicity and the effectiveness of immune protection of three Streptococcus pneumoniae virμlence protein DNA vaccines.⑴Specific antibody levels in serum in mice immunized with DNA vaccines of three Streptococcus pneumoniae virμlence proteins.①In the form of combination of the two combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA '), specific serum antibody levels against PsaA and PspA ' were higher than immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ') (P<0.01); But in the forms of combinations of PdB and PspA ' (or PsaA) combined immunization groups (pcDNA3.1-PspA' + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB), Specific serum antibody levels against PdB had no significant different (P> 0.05).②In the form of combination of the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA '+ pcDNA3.1-PdB), specific serum antibody levels against PsaA and PspA ' were significantly higher than immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ') (P<0.01), and specific serum antibody levels against PdB were were similar with alone (pcDNA3.1-PdB) (P> 0.05); But specific serum antibody levels against PsaA and PspA ' were similar with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ') (P> 0.05).⑵Effectiveness of immune protection of three Streptococcus pneumoniae virμlence protein DNA vaccines①The resμlt of nasopharynx in immunized mice on the seventh day after colonization colony counting tips nasal lavage fluid showed that the pneumococci colony counting of nasopharynx was significantly fewer in the mice immunized with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ') than the mice immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ') (P<0.01)and the forms of combinations of PdB and PspA ' or PsaA combined immunization groups (pcDNA3.1-PspA' + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB) (P<0.01); And there had no significant differences of the pneumococci colony counting of nasopharynx in the mice immunized with between the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ') and the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA '+ pcDNA3.1-PdB) (P> 0.05). ②Mice were intraperitoneal infection by the lethal dose of Streptococcus pneumoniae D39 strain after immunized 28 days, and survival analysis of 21 days showed that showed that the median survival time was significantly longer in the mice immunized with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ') than the mice immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ')(P<0.01)and the forms of combinations of PdB and PspA ' or PsaA combined immunization groups (pcDNA3.1-PspA' + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB) (P<0.01); And there had no significant differences of the median survival time and the survival curves of the mice immunized with between the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ') and the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA '+ pcDNA3.1-PdB) (P> 0.05).3. High frequency events of HCG and recombination support the prevalent presence of PsaA gene in streptococcus mitis group. These species may acquire transforming PsaA gene from the other species of streptococcus mitis group living in the same ecological niche, and maybe involved in promote the adhesion behavior and subsequent oropharyngeal colonization behavior. But we have no enough evident to confirm the possibility that these PsaA genes all come from Streptococcus pneumoniae, even though PsaA gene is comfirmed vertical inheritance in Streptococcus pneumoniae and BOX element is all absence in the flanking region of PsaA gene in Streptococcus pneumoniae.4. The characteristic of regional distribution of PspA families of Streptococcus pneumoniae was that Fam1-Clade2 (31.0%) and Fam1-Clade3 (47.6%) strains were as the advantages distribution. Serum specific anti-PspA-Fam1 antibody levels in invasive Streptococcus pneumoniae infection patients were higher than that of specific anti-PspA-Fam2 antibody levels. Serum anti-PspA antibodies against 6 clades of PspA protein were found weak cross-reaction amomg the various clades of various families, but a significant cross-reaction to Fam1-Clade1 and Fam1-Clade3 for advantage among the same family I .Conclusion1. PsaA, PspA and Ply, as the Streptococcus pneumoniae cell components, have the immunogenicity, which produced specific antibodies in the human body's with Streptococcus pneumoniae infections through the natural way. So they can be used as candidate antigens of new generation Streptococcus pneumoniae vaccines.2. United PspA and PsaA had the equivalent and coordinative immune effectiveness compared to combinations of three antigens PsaA, PspA, and PdB (attenuated mutant of Ply) against Streptococcus pneumoniae nasopharyngeal colonization and invasive infections, and avoided the existence of weak toxicity PdB. So the combination of PspA and PsaA was the dominant form of candidate antigens of the new generation vaccines.3. PsaA is one of the forms of the dominant antigen candidates as the new Streptococcus pneumonia generation vaccines, and PsaA gene is vertical inheritance in Streptococcus pneumoniae. As an important adhesion molecμle in the bacteria to nasopharyngeal epithelium, it plays an important role in adhesion and is aquired by other symbiotic streptococcus through horizontal gene transfer and recombinant gene manner.4. PspA is one of the forms of the dominant antigen candidates as the new Streptococcus pneumonia generation vaccines,and PspA gene has the diversity. The target antigens shoμld also include the clades components of Fam1 and Fam2 with high protective and strong cross-reaction against a wide range of clinical pathogenic strains of Streptococcus pneumoniae.