Recombinant bacterium capable of eliciting an immune response against Streptococcus pneumoniae
Inventors
Curtiss, III, Roy • Santander-Morales, Javier • Wanda, Soo-Young • Wang, Shifeng • Brenneman, Karen • Shi, Huoying • Xin, Wei • Kong, Qingke
Assignees
National Institutes of Health NIH • Arizona State University ASU
Publication Number
US-9050285-B2
Publication Date
2015-06-09
Expiration Date
2029-10-16
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Abstract
The invention encompasses a recombinant bacterium capable of eliciting an immune response against Streptococcus pneumoniae, a vaccine comprising the bacterium, and methods of using the bacterium.
Core Innovation
The invention encompasses a recombinant bacterium capable of eliciting an immune response against Streptococcus pneumoniae, a vaccine comprising the bacterium, and methods of using the bacterium. Specifically, the bacterium is a recombinant Salmonella Typhi or other Salmonella enterica serovar engineered for regulated expression of at least one nucleic acid encoding S. pneumoniae antigen, regulated attenuation, reduced persistence in a host, and reduced fluid secretion upon administration.
The background identifies problems with existing pneumococcal vaccines, including limited serotype coverage, lack of immunogenicity in young children, and the issue of replacement carriage by non-vaccine serotypes. These problems underscore the need for an effective vaccine against S. pneumoniae that can induce broad immune protection, preferably delivered mucosally and orally, to stimulate cellular and humoral immunity.
The recombinant bacterium is capable of regulated expression of S. pneumoniae antigens by comprising chromosomally integrated nucleic acid sequences encoding a repressor under the control of a regulatable promoter and vectors encoding at least one antigen operably linked to a promoter regulated by the repressor. Regulated attenuation is achieved by replacing native promoters of genes encoding attenuation proteins with regulatable promoters responsive to environmental factors such as sugar levels (e.g., arabinose). Additional mutations affect persistence by disabling factors like the Vi capsular antigen and biofilm components and reduce fluid secretion by mutating genes such as sopB and pagP.
Claims Coverage
The patent includes multiple independent claims detailing a recombinant Salmonella enterica serovar Typhi bacterium with regulated antigen expression, regulated attenuation, and specific mutations reducing persistence and fluid secretion, along with vaccine compositions comprising such bacteria.
Regulated expression of multiple Streptococcus pneumoniae antigens
The bacterium comprises at least one nucleic acid sequence encoding a repressor selected from LacI, C2, and C1 operably linked to a regulatable promoter (PBAD or promoters regulated by maltose, rhamnose, or xylose), and at least one nucleic acid sequence encoding at least two S. pneumoniae antigens operably linked to a promoter regulated by the repressor. Expression of the antigens is repressed during in vitro growth and highly expressed in vivo.
Regulated attenuation via sugar-dependent promoters
The bacterium includes mutations that confer regulated attenuation by repression of crp, fur, or phoPQ gene expression in vivo via regulatable promoters (e.g., ΔPcrp::TT araC PBAD crp, ΔPfur::TT araC PBAD fur, ΔPphoPQ::TT araC PBAD phoPQ). Additionally, it includes mutations affecting LPS O-antigen synthesis such as rfc mutations or loss-of-function mutations in pmi or galE influencing attenuation.
Reduced persistence through biofilm and capsule synthesis mutations
The bacterium harbors mutations reducing persistence in the gastrointestinal tract by possessing a mutation reducing synthesis of the Vi capsular antigen, colanic acid, thin aggregative fimbriae, cellulose, extracellular polysaccharide, or combinations thereof (e.g., ΔtviABCDE, Δ(gmd-fcl), Δ(wza-wcaM), ΔagfBAC).
Reduced fluid secretion mutations
The bacterium includes mutations reducing fluid secretion in the host gastrointestinal tract, comprising sopB or pagP mutations (e.g., ΔsopB1925 or ΔpagP81::Plpp IpxE).
Balanced host-vector system with regulated expression and attenuation mutations
The bacterium may additionally comprise a ΔrelA::araC PBAD lacI TT mutation with antigen expression regulated by LacI, combined with the regulated attenuation mutations and mutations reducing persistence and fluid secretion, enabling an integrated system of controlled gene expression and bacterial attenuation.
The claims cover a recombinant Salmonella Typhi bacterium engineered for tightly regulated in vitro repression and in vivo expression of multiple S. pneumoniae antigens, combined with multiple mutations conferring regulated attenuation, reduced persistence through biofilm and capsule synthesis elimination, and decreased host fluid secretion, suitable for vaccine use.
Stated Advantages
The invention provides a vaccine approach eliciting strong and broad protective immunity against Streptococcus pneumoniae across multiple serotypes.
Regulated expression of antigens ensures that expression is minimized during in vitro growth but maximized in vivo to enhance immune stimulation without compromising bacterial viability in production.
Regulated attenuation and multiple safety mutations reduce virulence and persistence, improving safety and biological containment upon administration.
Mutations reducing fluid secretion lessen adverse diarrheal reactions, enhancing tolerability of oral vaccination.
Balanced host-vector systems and mutations minimizing plasmid recombination enhance plasmid stability and antigen expression.
Documented Applications
Use as an oral vaccine to elicit an immune response against Streptococcus pneumoniae in hosts.
Immunization of warm-blooded animals including humans to prevent pneumococcal disease.
Methods of administering the recombinant bacterium orally, intranasally, or via other mucosal routes to induce mucosal, cellular, and systemic immunity.
Use in prophylactic and therapeutic vaccination against Streptococcus pneumoniae infections.
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