Construction and immunological characterisation of a non-toxic form of pneumolysin for use in pneumococcal vaccines

Abstract

There are 90 different serotypes of Streptococcus pneumoniae and current pneumococcal vaccines are somewhat limited in their protection against invasive pneumococcal disease. The adult pneumococcal vaccine is composed of capsule polysaccharide from 23 of the most common disease causing serotypes. Infants are the major 'at risk' group from pneumococcal disease; however, polysaccharide-based vaccines are not protective in this age group. This has led to the development of a paediatric conjugate vaccine, which is composed of polysaccharide from seven of the most prevalent disease causing serotypes individually conjugated to a carrier protein. Although the conjugate vaccine is highly efficacious, it only elicits protection against disease caused by the seven homologous pneumococcal serotypes. Serotype specific immunisation is only a short-term solution to combating pneumococcal disease. Problems with serotype replacement have already arisen within five years since licensure of the paediatric vaccine, with non-vaccine serotypes replacing the eliminated vaccine serotypes. A solution to this is the development of pneumococcal vaccines containing a species wide pneumococcal protein to elicit nonserotype specific protection, Pneumolysin, the pore-forming toxin produced by S. pneumoniae, may have an application in fixture pneumococcal vaccines as it is produced by all invasive isolates, is a major virulence factor and has been demonstrated to confer non-serotype specific protection against pneumococcal disease. The toxicity of pneumolysin is problematic in terms of vaccine use and existing pneumolysin mutants possess residual cytotoxicity. By mutating a region involved in protein oligomerisation, pneumolysin pore formation was abolished. This resulted in a non-toxic form of pneumolysin that retained the immunogenicity of wild type pneumolysin without the associated effects such as hypothennia, inflammatory cytokine production and vascular leakage following intranasal administration to mice. Vaccination of mice with this pneumolysin toxoid elicited high titres of neutralising antibody, which significantly protected animals from pneumococcal infection. Conjugation of the pneumolysin toxoid to capsule polysaccharide from serotype 4 S. pneumoniae elicited full protection against infection from the homologous serotype. This indicated that the pneumolysin toxoid was as effective as the carrier protein used in the current pneumococcal conjugate vaccine. Active vaccination with free pneumolysin toxoid significantly increased survival times in mice following challenge with a non-vaccine serotype. This research implies that combining conjugated and free pneumolysin toxoid may be more efficacious than current vaccines in protection against pneumococcal disease. The importance of the pore-forming property of pneumolysin in pathogenesis of pneumococcal disease was investigated by construction of a pneumococcal strain carrying the same mutation used to construct the pneumolysin toxoid for vaccination. Pore formation was found not to be important for pathogenesis during murine pneumococcal pneumonia. This was further supported by the identification of serotype 1 strains from clinical disease that expressed non pore-forming pneumolysin yet were isolated from patients with invasive pneumococcal disease