Protection against pneumococcal infection elicited by immunization with glutamyl tRNA synthetase, polyamine transport protein D and sortase A.

Protein-based vaccines are considered to be the next-generation of pneumococcal vaccines. Here we evaluated the protection elicited by immunization with recombinant glutamyl tRNA synthetase (Gts), polyamine transport protein D (PotD) and sortase A (SrtA) antigens in preclinical mouse models. In mucosal immunization studies, intranasal immunization with either Gts, PotD or SrtA could significantly reduce pneumococcal nasopharyngeal and lung colonization and significantly increase mice survival times following invasive pneumococcal challenge, and combinations of these antigens could enhance this protection. In systemic immunization studies, intraperitoneal immunization with multiple protein antigens also provided better protection against pneumococcal sepsis caused by different pneumococcal strains. Finally, passive immunization studies showed an additive effect by using multiple anti-sera when compared to single anti-sera. Therefore, a multicomponent protein-based pneumococcal vaccine composed of Gts, PotD or SrtA could confer protection against pneumococcal colonization as well as invasive infections in terms of efficacy of protection and serotype coverage.

[Prokaryotic expression and immunization of glutamyl tRNA synthetase of Streptococcus suis].

OBJECTIVE: Glutamyl tRNA Synthetase (Gts) is a protease which catalyzes esterification between tRNA and glutamine. The immunogenicity of Gts was evaluated through immunization and challenge experiment. METHODS AND RESULTS: We cloned gts from the genome of SC19, and inserted it into prokaryotic expression plasmid pET28a-gts. The recombinant vector was transformed into E. coli BL21. Induced by IPTG, one 58 kD protein, was expressed and purified by using Ni - NTA column (Novagen). The purity of rGtS was 93.3% and the concentration of purified protein was 433 microg/mL. We proved the immunogenicity of recombinant protein rGtS by western blot analysis. We immunized Balb/c mice with rGtS and Freund's adjuvant, and after two boost vaccinations, all mice were challenged with 4 times LD50 amount of SC19 (1.2 x 10(9) CFU). The survive rate of vaccination group is 50% (4/8), significantly higher than blank vector control group (1/8). CONCLUSION: These results proved that GtS has certain immunogenicity and can offer partial protection against high dose challenge. Therefore Gts could be a potential candidate of subunit vaccine against Streptococcus suis.

Streptococcus pneumoniae surface-exposed glutamyl tRNA synthetase, a putative adhesin, is able to induce a partially protective immune response in mice.

Glutamyl tRNA synthetase (GtS) has been found to be among the Streptococcus pneumoniae cell wall-derived proteins that have age-dependent immunogenicity in children. Here, GtS was cloned, expressed, and purified and then was used to immunize 7-week-old BALB/c OlaHsd mice. Serum obtained from mice immunized with recombinant (r) GtS cross-reacted with a 55.9-kDa protein, identified as GtS, in the cell wall fraction derived from genetically and capsularly unrelated strains of S. pneumoniae. Surface localization of GtS was further confirmed using flow cytometry analysis. The rGtS and anti-rGtS antiserum significantly inhibited the adhesion of 3 pairs of encapsulated and unencapsulated strains of S. pneumoniae to A549 cells. Thirty-nine percent of rGtS-immunized mice survived a lethal bacterial challenge, whereas no control mice survived. These results suggest that GtS, an age-dependent S. pneumoniae antigen, is a surface-located adhesin that is capable of inducing a partially protective immune response against S. pneumoniae in mice.

Immunoelectron microscopic localization of glutamyl-/ prolyl-tRNA synthetase within the eukaryotic multisynthetase complex.

A high molecular mass complex of aminoacyl-tRNA synthetases is readily isolated from a variety of eukaryotes. Although its composition is well characterized, knowledge of its structure and organization is still quite limited. This study uses antibodies directed against prolyl-tRNA synthetase for immunoelectron microscopic localization of the bifunctional glutamyl-/prolyl-tRNA synthetase. This is the first visualization of a specific site within the multisynthetase complex. Images of immunocomplexes are presented in the characteristic views of negatively stained multisynthetase complex from rabbit reticulocytes. As described in terms of a three domain working model of the structure, in "front" views of the particle and "intermediate" views, the primary antibody binding site is near the intersection between the "base" and one "arm." In "side" views, where the particle is rotated about its long axis, the binding site is near the midpoint. "Top" and "bottom" views, which appear as square projections, are also consistent with the central location of the binding site. These data place the glutamyl-/prolyl-tRNA synthetase polypeptide in a defined area of the particle, which encompasses portions of two domains, yet is consistent with the previous structural model.

Monoclonal antibodies to the components of the high-molecular-mass aminoacyl-tRNA synthetase complex.

Six monoclonal antibodies to the components of the rabbit multienzyme aminoacyl-tRNA synthetase complex were generated and characterised. Two, F7 and F31 were directed against arginyl-tRNA synthetase, two, F8 and F25 against glutamyl-tRNA synthetase, and two, F6 and F12 recognised 38 and 43 kDa polypeptides, respectively. All antibodies were species-specific and failed to affect the activity of the respective enzymes.