Options for Inactivation,Adjuvant, and Route of Topical Administration of a Killed,Unencapsulated Pneumococcal Whole-Cell Vaccine

We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae, given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4⁺ T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone—all used without postinactivation washing—produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 μg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.Streptococcus pneumoniae (pneumococcus) imparts a major disease burden among children in low-income countries (27). Capsular polysaccharide conjugate vaccine provides type-specific protection but has the disadvantages of limited serotype coverage, serotype replacement, and high cost of production, storage, and injection (12, 29). Therefore, potentially more economical serotype-independent vaccines based upon species-common protein antigens are being pursued (32). In one such approach, we have studied killed cells of noncapsulated pneumococci, with the intent of maximizing the exposure of species-common subcapsular antigens. This antigen preparation, designated “whole-cell vaccine” (WCV), when formulated with a suitable adjuvant, is intended for mucosal administration to reduce pneumococcal colonization. Killing the cells with 70% (vol/vol) ethanol at 4°C produces a more immunogenic antigen (WCE) than traditional methods of inactivation such as heat, formalin, or UV radiation (21) (unpublished data). Intranasal (i.n.) application had been examined initially, since this route is effective for inducing both systemic and mucosal immunity. Vaccination i.n. with WCE plus cholera toxin (CT) as a mucosal adjuvant prevents fatal serotype 3 pneumonia in rats and reduces nasopharygeal (NP) and middle ear colonization in mice by strains of serotype 6B or 23F (22, 23). Although the levels of serum antibodies are raised by the i.n. vaccination, protection against colonization is induced in mice in the absence of antibodies by a CD4⁺ T-cell-dependent, interleukin 17A (IL-17A)-mediated mechanism (20, 24). As few as 10⁷ cells (ca. 10 μg of protein) of WCE, given thrice sequentially, are protective in the colonization model (34). This potency and the potential low cost of manufacture were motivation for a partnership with PATH and Instituto Butantan (São Paolo, Brazil) for further development of the WCV.Previous studies used strain Rx1AL expressing native pneumolysin, a potent cytolysin. Here, anticipating human studies, a derivative of pneumolysin (PdT), with mutations W433F, D385N, and C428G (which render the molecule nonhemolytic and unable to activate complement [5] but maintain its TLR4 agonistic properties [21]), was used. Previously, cells were grown in Todd-Hewitt-yeast broth, which contains beef heart infusion. Here, to avoid any hazard of bovine components, cells grown in a soy-based medium (19) were examined for protection. The challenge of safe handling and disposal of large volumes of ethanol led to the evaluation of three alternative agents of inactivation that are bactericidal at low concentration: chloroform, trichloroethylene, and beta-propiolactone. Since chloroform and trichloroethylene are both highly volatile and beta-propiolactone is inactivated by warming, these agents may be removed without postinactivation washing, which permitted convenient examination of soluble components released from the killed cells. Soluble components produced by sonication also were examined.The possible side effects of enterotoxins as adjuvants (25) and other problems with intranasal vaccination prompted the consideration of genetically detoxified enterotoxin derivatives and of the buccal and sublingual routes of administration. Transdermal immunization was also examined with sonicated antigen preparations. These varied immunization procedures were surveyed for protection against colonization, evident as acceleration of nasopharyngeal clearance after intranasal challenge with a strain of serotype 6B (20). The results indicate that the cells can be inactivated by several agents to generate a potent whole-cell antigen that could be given in a variety of ways to accommodate preferences of a particular vaccination program.