Directed vaccination against pneumococcal disease

Abstract

Immunization strategies against commensal bacterial pathogens have long focused on eradicating asymptomatic carriage as well as disease, resulting in changes in the colonizing microflora with unknown future consequences. Additionally, current vaccines are not easily adaptable to sequence diversity and immune evasion. Here, we present a "smart" vaccine that leverages our current understanding of disease transition from bacterial carriage to infection with the pneumococcus serving as a model organism. Using conserved surface proteins highly expressed during virulent transition, the vaccine mounts an immune response specifically against disease-causing bacterial populations without affecting carriage. Aided by a delivery technology capable of multivalent surface display, which can be adapted easily to a changing clinical picture, results include complete protection against the development of pneumonia and sepsis during animal challenge experiments with multiple, highly variable, and clinically relevant pneumococcal isolates. The approach thus offers a unique and dynamic treatment option readily adaptable to other commensal pathogens.

Keywords: S. pneumoniae; biofilm; pneumococcal; vaccine; virulence.

Fig. 1.

S. pneumoniae pathogenesis outcomes and infectious disease statistics in the United States (1998–2013). (A) S. pneumoniae colonizes the human nasopharynx and produces a bacterial biofilm with an accompanying extracellular matrix capable of providing protection from external and host challenges. External triggers such as viral infection prompt the active release of virulent pneumococci that disseminate to secondary sites and cause disease. (B) Leading vaccination strategies [polysaccharide conjugate vaccines (PCVs), such as the Prevnar family] mediate protection against certain bacterial serotypes by promoting clearance of pneumococci before biofilm establishment. Clearing all bacteria opens the niche to colonization by nonvaccine serotypes or other bacterial species. (C) The strategy featured in this work mediates clearance of only virulent biofilm-released bacteria while maintaining the presence of the preexisting biofilm. (D) The annual infection rate per 100,000 people for the total population (blue) and children under the age of 5 y (red), 1998–2013. The first years following the introduction of Prevnar 7 and 13 are marked with dotted lines. (E) Prevalence of infectious pneumococcal strains, 1998–2013 (29). Strains are grouped into those covered by Prevnar 7 (blue), those covered by Prevnar 13 (red), and nonvaccine types (NVT; green). (F) The reduction in the annual infection rate in children under the age of 5 y from 1998–2008 relative to 1998–1999. The dashed line corresponds to the division between Prevnar 7 vaccine and nonvaccine type strains in 1999–2000 (35). (G) The reduction in the annual infection rate in children under the age of 5 y, 2008–2013. The dashed line corresponds to the division between Prevnar 13 vaccine and nonvaccine type strains in 2008–2009 (35).

Fig. 2.

Antigen identification and S. pneumoniae conditioning through an in vitro biofilm model. (A) S. pneumoniae were seeded on epithelial cells, and the biofilm structure was investigated using SEM. Visible in these images are the extracellular matrix, water channels, tower formations, and the honeycomb structure that pneumococci form with larger biofilms. (B and C) Mouse bacterial burden was determined after i.p. injections (sepsis model) (B) or aspiration with anesthesia (pneumonia model) (C) using broth-grown (Planktonic), biofilm-associated (Biofilm), or biofilm heat-released (Heat) S. pneumoniae strain EF3030. Each dot in the graphs represents an individual mouse. The dotted line represents the limit of detection for bacterial counts. (D) Time-to-death assessment of mice inoculated with biofilm heat-released bacteria; mice that were inoculated with either planktonic or biofilm-associated bacteria did not die in any challenge model. (E–G) Mice were immunized with various antigens and challenged with biofilm heat-released EF3030 in sepsis (E and F) and pneumonia (G) models. ***P < 0.001, compared with planktonic and biofilm samples (B and C) and PspA (E).

Fig. S1.

Characterization of Co-PoP liposomal delivery device. (A and B) HPLC analysis of Co-PoP (A) and the resulting mass spectrum (B). (C) Liposome-binding analysis for all proteins used in the study. (D and E) Particle diameter (D) and zeta potential (E) of liposomes formulated with 0, 5, and 15 μg of PspA.

Fig. 3.

Directed clearance of biofilm-released bacteria and protection against mouse-passaged challenge strains. (A) Bacterial burden at various anatomical sites was determined daily in unimmunized (filled circles) and GlpO + PncO immunized (open circles) mice. Mice were inoculated intranasally without anesthesia with planktonic or biofilm heat-released EF3030 (Upper) or D39 (Lower) bacteria. (B) Comparative analysis of the planktonic (black) and biofilm-released (red) EF3030 clinical isolate data from A. A value of 100% represents no difference between immunized and unimmunized subjects; values below 100% indicate a directed response. A full statistical comparison is presented in Table S5. (C and D) Protective capabilities of GlpO + PncO immunization were evaluated further in sepsis (C) and pneumonia (D) models with established mouse-passaged pneumococcal bacteria. Dotted lines represent the limit of detection for bacterial counts.

Fig. S2.

Characterization of biofilm-released D39 bacterial virulence and vaccine protective capabilities. (A and B) Bacterial burden was determined for pneumococcal populations after i.p. injections (sepsis model) (A) or intranasal aspiration after anesthesia (pneumonia model) (B). Mice were inoculated with broth-grown (Planktonic), biofilm-associated (Biofilm), or biofilm heat-released (Heat) D39. Each dot in the graphs represents an individual mouse; an “x” represents a mouse that became moribund and was killed before the end of the experiment. The dotted line represents the limit of detection for bacterial counts. (C and D) Time-to-death assessment of mice inoculated with heat-released bacteria from the sepsis (C) and pneumonia (D) models after immunization with PspA or GlpO + PncO.

Fig. S3.

Bacterial burden associated with protection against mouse-passaged pneumococcal challenge strains. Mice were immunized with GlpO + PncO and challenged with planktonic D39 (A), A66.1 (B), WU2 (C), and TIGR4 (D) cells in either a sepsis or pneumonia model. Dotted lines represent the limit of detection for bacterial counts.

Fig. S4.

Characteristics of directed clearance and protection against an expanded set of strains. (A) Directed clearance of biofilm-released bacteria in passively immunized mice. Bacterial burden at various anatomical sites was determined daily in unimmunized (filled circles) and GlpO + PncO passively immunized (open circles) mice. Mice were inoculated intranasally without anesthesia with planktonic (red) or biofilm heat-released (blue) EF3030. Dotted lines represent the limit of detection for bacterial counts. (B) Protective capabilities of GlpO + PncO immunization were evaluated further in the sepsis model using an expanded set of mouse pneumococcal strains. *P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant.

Fig. 4.

Bacterial dissemination and time-to-death assessment of mice stably colonized with pneumococci and triggered with IAV. (A and B) Bacterial burden of EF3030 or D39 in unimmunized or GlpO + PncO immunized mice was measured at 1 (A) or 5 (B) d postinfection with IAV. The dotted line represents the limit of detection for bacterial counts. (C and D) Protective capabilities of traditionally or passively GlpO + PncO immunized mice against in vivo IAV-mediated bacterial release of EF3030 (C) or D39 (D). *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Fig. S5.

Schedule of immunization and bacterial challenge.