Study On Genome-based Streptococcus Pneumoniae Vaccines

Background and ObjectiveStreptococcus pneumoniae (S. pneumoniae) is the main pathogen of community- acquired pneumonia, otitis media. There are more than one hundred million cases of children under the age of 5 died of S. pneumoniae infection every year worldover. At present, the more and more serious drug- resistances of S. pneumoniae have became a major global public health problem and endangered the treatment of S. pneumoniae. Application of vaccines for combating pneumococcal infections is the only way proposed by World Health Organization, which should be help to reduce the spread of antibiotic resistance strains and alleviat the stress of antibiotics and protect high-risk groups.More and more domestic and foreign clinical studies found the shortcomings of current pneumococcal vaccines (23-valent capsular polysaccharide vaccine and protein - polysaccharide conjugate vaccine), which are the serotype-dependent existence, the high cost, and non-reducing S. pneumoniae nasopharyngeal colonization. The study of new genome-based vaccines of S. pneumoniae has far-reaching social and economic benefits.The large numbers of S. pneumoniae virulence proteins play the important and different roles at different pathogenic stages of S. pneumoniae infections. There is growing interest in S. pneumoniae DNA vaccine researches as the third vaccines, not only because of its low production cost, the non-serotype-dependent existence, but also the advantage Th1 immune responses.The choices of target antigens and edge vector systems and ideal inoculations of vaccines are the most important questiones. Preliminary studies by our group have confirmed that the combination of pneumococcal surface protein A (PspA ) and pneumococcal surface adhesion A (PsaA) were the most forms. Since pneumococcal nasopharyngeal colonization is the native mucosal infective route that leads to invasive infection diseases, a DNA vaccine with adhesion protein genes against S. pneumoniae nasopharyngeal colonization should ideally produce protective secretory IgA antibodies in the respiratory mucosa as well as systemic IgG antibodies, in addition to Th1 cellular immune responses, which is hoped to reduce the transmission of antibiotic-resistant strains and prevent further pneumococcal invasive diseases. But a major drawback of the DNA vaccines is its poor mucosal protective efficiency in the nasopharynx, which is inherent in the way that vaccines are currently delivered. An approach that incorporates the advantages of both genetic immunization and mucosal immunization of S. pneumoniae DNA vaccine is thus required.Therefore, this study"study on genome-based Streptococcus pneumoniae vaccines"was as the following works: First, S. pneumoniae virulence protein multivalent DNA vaccines were constructed based pspA'(N-terminal fragment of Streptococcus pneumoniae R6) and psaA, and the immunogenicity and protection effects were evaluated based the animal models of S. pneumoniae nasopharyngeal carriage and intraperitoneal challenges through the way of naked plasmid intramuscular vaccination. The second, we constructed a recombinant salmonella-based balanced-lethal host-vector system of the avirulent Salmonella strainχ4550(Asd-) with eukaryotic expression Asd+ vectors pcDNA3.1001 and pcDNA3.1002 expressing psaA gene or pspA'gene. The stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice immunized with the antigen delivery systems with combination of PspA and PsaA for intranasal vaccinations, comparing to S. pneumoniae virulence protein multivalent DNA vaccines. The last, we study preliminary the comparative genomics of dominant antigens by analyzing the sequence characteristics of dominant antigen proteins from molecular level and their possible functional differences, which was for learning more about the possible antigen diversity of dominant antigen proteins on the basis of molecular genetics.Thus, the results provide the theoretical foundation and laboratory basis for the development of new generation Streptococcus pneumoniae vaccines.Methods1. The pspA'(N-terminal fragment of Streptococcus pneumoniae R6) and psaA genes were cloned into the eukaryotic expression vector pcDNA3.1(+) by molecular cloning technology. The recombinant vectors PsaA-pcDNA3.1 and PspA'-pcDNA3.1 were transfected into BHK-21 cells, and the expression of PsaA and PspA'proteins was detected with RT-PCR and Western- blot. The three formats of recombinant plasmids (PspA'-pcDNA3.1, PsaA-pcDNA3.1 and PsaA-pcDNA3.1 + PspA'-pcDNA3.1) were used to inject intramuscularly BALB/c mice. The levels of IFN-γ, IL-4 and antibodies against PspA', PsaA 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.2. Oral DNA vaccines encoding psaA and pspA'with Salmonella-based balanced-lethal host- eukaryotic vector system as carriers were reconstructed using molecular biology techniques. And the stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice models.⑴To construct the Asd+ eukaryotic vector pcDNA3.1000 contained in the Salmonella-based balanced-lethal host-eukaryotic vector system, we reconstructed the eukaryotic vector pcDNA3.1 (+), which is under transcriptional control of the human CMV promoter. First, a 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori and 1.9 kb fragment of Pcmv-MCS- BGHpA-f1ori were retried through digesting pcDNA3.1(+) with BspHI and XmnI restriction enzymes. Then the pUCori fragment was obtained with SalI restriction enzymes through digesting the 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori, while the 1.07 kb asd gene fragment was amplified from Streptococcus mutans UA159 by PCR with an N-terminal XhoI site and a C-terminal SalI site. The reaction was carried out. Thus, the 1.07 kb fragment of the asd gene was obtained, and then the fragments of pUCori, asd and Pcmv-MCS-BGHpA-f1ori were connected with T4 DNA ligase. Thus the fragments of Neor and AMP gene sequences were deleted from pcDNA3.1(+), and the asd gene sequence was obtained, and then the recombinant vector pcDNA3.1000 (Asd+) was amplified intoχ4550(Asd-).⑵The new oral DNA vaccines encoding the two most promising candidates antigens psaA and pspA'genes with Salmonella-based balanced-lethal host-vector system as carriers were constructed using molecular cloning technology. And the recombinant vectors pcDNA3.1001 and pcDNA3.1002 were transfected into BHK-21 cells, and the expression of PsaA and PspA'proteins was detected with RT-PCR and Western- blot. The three formats of oral DNA vaccines (pcDNA3.1001x, pcDNA3.1002x and pcDNA3.1001x +pcDNA3.1002x) were used to orally deliver to BALB/c mice, with the vector pcDNA3.1000x and PBS as controls. Comparing to vector DNA vaccines (pcDNA3.101, PsaA-pcDNA3.102 and pcDNA3.101 +pcDNA3.102), the levels of IFN-γ, IL-4 and antibodies against PspA', PsaA 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.3. Methods of molecular 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.⑴Presence 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 genes 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.⑵Presence of pspA'gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. Phylogenies basing on the sequences of pspA'genes from Streptococcus pneumoniae strains were typed for the PspA families and clades.Results1. The immunogenicity and protection effects in mice immunized intramuscularly with naked plasmid Streptococcus pneumoniae multivalent DNA vaccines.⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of PspA'-pcDNA3.1 and PsaA-pcDNA3.1 in BHK-21 cells.⑵Immune responses in mice immunized with naked plasmid Streptococcus pneumoniae DNA vaccines PspA'-pcDNA3.1 and PsaA-pcDNA3.1.①Cell immune responses. PsaA and PspA'antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA'-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 21th day after last immunization than that on day 0, and significantly higher than the control plasmid group; PsaA and PspA 'antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA'-pcDNA3.1 + PsaA-pcDNA3.1 were significantly higher than that with PspA'-pcDNA3.1 or PsaA-pcDNA3.1 alone on 21th day after last immunization; PsaA and PspA 'antigen-specific cytokine IL-4 in spleen cells supernatant of mice group immunized with PspA'-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, and plamids contral group were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.②Humoral immune responses. PsaA and PspA'antigen-specific IgG antibodies in serum of mice immunized with PspA'-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 28th day after last immunization; PsaA and PspA'antigen-specific IgG antibodies in serum of mice immunized with PspA'-pcDNA3.1+ PsaA-pcDNA3.1 was higher than that with PspA'-pcDNA3.1 or PsaA-pcDNA3.1 alone.⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with naked plasmid S. pneumoniae multivalent DNA vaccines PspA'-pcDNA3.1 and PsaA-pcDNA3.1.①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with naked plasmid S. pneumoniae multivalent DNA vaccines PspA'-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, than the contral plasmid and PBS groups. And the effect of immunization on carriage with mixed DNA vaccine plasmids was highly significant versus with one of them alone.②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with the mixed DNA vaccines plasmids PspA'-pcDNA3.1+ PsaA-pcDNA3.1 had significantly longer median survival times than the group that received alone with one of them.2. The immunogenicity and protection effects in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system.⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of pcDNA3.1001 and pcDNA3.1002 in BHK-21 cells. All of these plasmids(pcDNA3.1000, pcDNA3.1001, pcDNA3.1002) were 100% stably maintained for 25 generations in S. typhimurium (Asd-) hosts grown in the presence or absence of DAP.⑵Immune responses in mice immunized with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.①Cell immune responses. LPS antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, but there had no significantly difference among all groups; PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x + pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, and PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with oral DNA vaccines(pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x ) were significantly higher than DNA vaccine plamids(pcDNA3.101, pcDNA3.102 or pcDNA3.101+pcDNA3.102); PsaA and PspA'antigen-specific cytokine IL-4 in spleen cells supernatant of all mice group immunized were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.②Humoral immune responses. The antibody responses to Salmonella LPS and to the foreign antigen PspA'or PsaA in the sera and the nasal wash of the immunized mice were measured. Responses reached a maximal anti-LPS, -PsaA, and–PspA'IgG levels on 28th day after last immunization and all IgG antibody responses in vaccine groups were significantly stronger than the control pcDNA3.1 (+) and pcDNA3.1000x groups. Anti-LPS IgG antibody levels were similar in the mice of groups immunized by all oral DNA vaccines. IgG antibody levels against foreign antigens (PspA'or PsaA) in the mice of groups immunized by the oral multiantigen DNA vaccines ( pcDNA3.1001x+ pcDNA3.1002x)were significantly higher than that by the multiantigen DNA vaccine plasmids (i.m) (pcDNA3.101+ pcDNA3.102). IgG antibody levels against PspA'or PsaA in the mice of groups immunized by the multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x or pcDNA3.101+ pcDNA3.102 )were significantly higher than that by the univalent DNA vaccines encoding either one of pspA'and psaA (including pcDNA3.1001x, pcDNA3.1002x and pcDNA3.101, pcDNA3.102).③Mucosal immune responses. Secretory IgA antibodies to PspA'and PsaA were detected in the nasal washes of mice immunized by oral DNA vaccines, and negligible secretory IgA elicited by the DNA vaccine plasmids (i.m.) group. On 28th day after last immunization, the antibody leves reached peak, and higher IgA antibody levels against either PspA'or PsaA were observed in the groups of mice immunized with oral multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than in mice immunized with one of them alone (pcDNA3.1001x, pcDNA3.1002x) and the control group pcDNA3.1000x.⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). The effect of immunization on carriage with mixed oral DNA vaccine pcDNA3.1001x +pcDNA3.1002x was highly significant versus with one of them alone pcDNA3.1001x or pcDNA3.1002x.②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). And the mice immunizaed with mixed oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with one of them alone.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.4. PspA gene diversity of the region N terminal antigen genomic analysis suggested that the trend of regional distribution of PspA protein family: PspA-Fam1-Clade2 (31.0%) and PspA-Fam2-Clade3 (47.6%) as the predominant strains, and differences in foreign reports.ConclusionSuccessfully, oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x induced significant immune responses versus the DNA vaccine plasmids pcDNA3.101 and pcDNA3.102 intramuscular injected (i.m.), containing mucosa, cellular and humoral immune responses, and enhanced protection against nasopharyngeal colonization and protection against the intraperitoneal challenge. The studies also confirmed the cooperating, additive protection with multiantigen DNA vaccines against S. pneumoniae nasopharyngeal colonization and infection. The results provided the potential of using the salmonella-based balanced-lethal host-eukaryotic vector system as carriers to deliver S. pneumoniae protective antigens.PsaA and PspA are the best forms of dominant antigen candidates of the new Streptococcus pneumonia generation vaccines. PsaA gene is vertical inheritance in Streptococcus pneumoniae, which may be aquired by other symbiotic streptococcus through horizontal gene transfer and recombinant gene manner. PspA gene has the diversity, and the target antigens should 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.