Reverse and structural vaccinology approach to design a highly immunogenic multi-epitope subunit vaccine against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a pathogen that resides in the upper respiratory tract of healthy individuals, maintaining a commensal relationship with its host. However, the virulent form may be the etiology of pneumonia, meningitis, bacteremia, and other respiratory tract infections. Streptococcal diseases are preventable by vaccination; but currently available vaccines have some drawbacks, especially due to the high capsule variability of streptococci strains. Thus, an effective prevention strategy continues to be the focus of extensive research. In our work, several bioinformatics tools were used to identify immunogenic peptides from a selected pool of 46 conserved proteins from Streptococcus pneumoniae. In silico analysis showed that 10 proteins had epitopes with affinity for B and T lymphocytes, which were present in at least 26 different pathogens serotypes and were considered promiscuous. The multi-epitope protein, designated HC44, was designed based on these epitopes and specific linkers to improve stability and exposure to T lymphocytes. The recombinant HC44 protein was expressed in E.coli and Swiss-Webster mice were immunised by intraperitoneal injection. Immunisation with the multi-epitope HC44 protein resulted in the production of very high levels of IgG with title superior to 1/1.200.000. However, subtype IgG was highly unbalanced toward IgG1 and no protection was afforded after challenge with S.pneumoniae in a sepsis model. Thus, our strategy has been effective in constructing a highly antigenic protein but novel immunisation strategies should be investigated to reorient the immune system toward a protective response.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.