Systemic and mucosal immune responses elicited by intranasal immunization with a pneumococcal bacterium-like particle-based vaccine displaying pneumolysin mutant Plym2.

Pneumolysin (Ply) is an important virulence factor in pneumococcal infection and a conserved cholesterol-binding cytotoxin expressed by all serotypes of Streptococcus pneumoniae. We previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two amino acids (C428G and W433F), which lost cytotoxicity but retained the ability to induce neutralizing antibodies. In the present work, we applied bacterium-like particles (BLPs) as a carrier and immunostimulant for the development of a Plym2 intranasal vaccine, in which the Plym2 protein was displayed on the surface of BLPs. Intranasal immunization of mice with BLP-Plym2 not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies in lung lavages. Antiserum induced by the BLP-Plym2 vaccine elicited high-titer neutralization activity which could inhibit the hemolysis of wild-type Ply. In conclusion, the BLP-Plym2 vaccine was demonstrated to be a promising strategy for intranasal immunization to enhance both systemic and mucosal immune responses.

Detoxified pneumolysin derivative Plym2 directly protects against pneumococcal infection via induction of inflammatory cytokines.

Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.