Regulated expression of antigen and/or regulated attenuation to enhance vaccine immunogenicity and/or safety
Inventors
Curtiss, III, Roy • Wang, Shifeng • Wanda, Soo-Young • Kong, Wei
Assignees
Washington University in St Louis WUSTL • Arizona State University ASU
Publication Number
US-11180765-B2
Publication Date
2021-11-23
Expiration Date
2028-05-09
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Abstract
The invention relates to compositions and methods for making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest.
Core Innovation
The invention provides compositions and methods for recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigen-encoding nucleic acids. A core aspect of this technology is the use of a chromosomally integrated nucleic acid sequence encoding a repressor, which is operably linked to a regulatable promoter. This enables repression of antigen expression during in vitro growth and high-level expression in a host organism, thus temporally controlling antigen production for optimal vaccine performance.
The problem addressed by this invention involves limitations of current live recombinant vaccines: high-level constitutive expression of antigens can be toxic to the bacteria, reduce their ability to colonize lymphoid tissues, and diminish immunogenicity. Additionally, overproduction of antigen from multicopy plasmids can lead to plasmid instability and loss after immunization, resulting in diminished immune response to the antigen. Furthermore, conventional attenuation often results in bacteria being too susceptible to host stresses, reducing their colonization, persistence, and immunogenic effect. There is a specific need for methods to regulate both antigen expression and display of the attenuated phenotype to achieve balance between safety, colonization ability, and immunogenicity.
The invention introduces systems wherein a repressor (such as LacI, C2, or C1) under control of a regulatable promoter (like araC PBAD) is chromosomally integrated, while a plasmid vector (carrying the antigen-encoding nucleic acid under a corresponding repressor-responsive promoter, such as Ptrc) is provided. In vitro, the presence of an inducer (e.g., arabinose) leads to repressor production and suppression of antigen gene expression, minimizing stress and toxicity. In vivo, where the inducer is absent, new repressor production ceases and its concentration is diluted with cell division, allowing high-level antigen expression at the relevant host site. The system also allows for regulated attenuation by replacing the native promoter of an attenuation protein with a regulatable promoter, further increasing safety and immunogenicity of live vaccine vectors.
The invention is exemplified with Enterobacteriaceae, especially pathogenic Salmonella strains, and demonstrates that regulated antigen expression and attenuation promote efficient colonization and strong immune responses without sacrificing safety. The described methods include genetic modifications such as start codon or Shine-Dalgarno sequence changes to optimize repressor expression, providing tight control and flexibility. The system can be tailored for various attenuating mutations and multiple regulatory cassettes, making it broadly applicable for vaccine strain development.
Claims Coverage
The independent claim covers a recombinant bacterium with three main inventive features related to regulated expression of a protein of interest, repressor system design and optimization, and a specific chromosomal mutation.
Recombinant bacterium with regulated expression system for a protein of interest
A bacterium is engineered to have at least one chromosomally integrated nucleic acid sequence encoding a repressor under the control of a regulatable promoter. The bacterium contains a vector comprising a nucleic acid sequence encoding a protein of interest, linked to a promoter that responds to the repressor. Expression of the protein-encoding nucleic acid is repressed during in vitro growth but can be highly expressed in a host.
Genetic optimization of repressor system
The nucleic acid encoding the repressor and/or the associated promoter is modified from the wild-type sequence to optimize the level of repressor expression. This may include modifications such as altered Shine-Dalgarno sequence or optimized codons.
Specific chromosomal integration mutation ΔrelA198::araC PBAD lacI TT
The recombinant bacterium specifically comprises the ΔrelA198::araC PBAD lacI TT chromosomal mutation, ensuring integration of the repressor system at a defined genomic locus.
The claim as a whole covers a recombinant bacterium with an optimized, integrated regulated expression system for proteins of interest, including a defined genetic locus for system integration and the capacity for high-level expression in a host.
Stated Advantages
The invention enables high-level antigen expression in vivo while minimizing toxicity and metabolic burden during in vitro growth, enhancing the health and colonization ability of the bacterial vaccine strain.
Regulated attenuation allows the live vaccine strain to initially mimic wild-type pathogens, improving colonization of host lymphoid tissues and achieving strong, protective immune responses without causing disease symptoms.
The system improves plasmid stability and ensures strong immune responses against the antigen by coordinating high antigen expression with host colonization.
The invention provides flexibility to optimize repressor and system parameters for different antigens and bacterial backgrounds.
Documented Applications
Use of recombinant bacteria as live vaccines to produce immune responses against bacterial, viral, mycotic, parasitic, tumor, or allergen antigens in vertebrate hosts.
Administration of vaccine compositions by oral, intranasal, injection, or other parenteral routes to induce mucosal, humoral, or cellular immunity.
Modulation of host immune systems for prophylactic or therapeutic purposes, including amelioration of symptoms of infectious diseases.
Application to generate vaccine strains for human and veterinary use, including example vaccines for Streptococcus pneumoniae, Salmonella, and other pathogens.
Use in biological containment systems to prevent persistence and environmental release of recombinant vaccine strains through regulated programmed lysis.
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