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-11920139-B2
Publication Date
2024-03-05
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 creating recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest. The core innovation centers on engineering bacteria, particularly pathogenic Enterobacteriaceae such as Salmonella, with precise genetic components: at least one chromosomally integrated nucleic acid sequence encoding a repressor (operably linked to a regulatable promoter), and a vector (commonly a plasmid) encoding an antigen of interest under the control of a promoter regulated by the repressor.
The invention addresses a key problem in the field of live recombinant vaccines: constitutive or unregulated expression of antigens can harm bacterial viability, reduce their ability to colonize target lymphoid tissues, and decrease vaccine immunogenicity. Existing approaches using inducible promoters are limited by weak or leaky in vivo expression or are affected by the mode of attenuation. There is a pressing need for systems that enable high-level antigen expression in vivo, but minimal or repressed antigen synthesis during in vitro growth, and similarly, for attenuation traits that are only expressed after efficient tissue colonization to optimize both safety and immunogenicity.
The invention solves these problems by combining regulated expression and regulated attenuation: in vitro, the presence of an inducer (such as arabinose) triggers the production of a repressor protein, which suppresses antigen expression and/or the function of key attenuation proteins. Upon entry into a host (where the inducer is absent), repressor levels drop as bacteria divide, derepressing antigen expression and/or activating attenuation phenotypes only after colonization of lymphoid tissues. This dynamic control ensures robust colonization and effective immune stimulation, while preventing disease symptoms and allowing for highly immunogenic and safe vaccine development.
Claims Coverage
The claims in this patent cover several inventive features, with a primary independent claim describing a recombinant bacterium engineered for regulated expression of at least one nucleic acid sequence encoding a protein of interest, using chromosomally integrated regulatory elements and an optimized repressor system. Additional features, including methods of use, compositions, and specific genetic modifications, are described.
Recombinant bacterium with regulated expression of a protein of interest
A recombinant bacterium comprising at least one chromosomally integrated nucleic acid sequence encoding a repressor operably linked to a regulatable promoter, where the repressor and/or promoter have been modified from the wild-type nucleic acid sequence to optimize repressor expression. - The bacterium is capable of regulated expression of at least one nucleic acid sequence encoding a protein of interest. - The repressor controls a promoter for a gene of interest, and modifications (such as Shine-Dalgarno sequence/codon optimization) may be present. - The system allows repression during in vitro growth and high-level expression in a host environment.
Recombinant bacterium with multiple means of attenuation and antigen expression
The recombinant bacterium may further include genetic deletions or mutations for attenuation, such as a deletion in asd, a ΔasdA::TT araC PBAD c2 mutation, and optionally additional deletions (e.g., in pabA and/or pabB). - The bacterium may also possess more than one means of attenuation.
Use of optimized genetic elements for repressor expression
The nucleic acid sequence encoding the repressor can include a modified Shine-Dalgarno sequence and optimized codons, enhancing control over expression levels of the repressor for fine-tuned regulation.
Plasmid-based vector for regulated antigen expression
The vector used is a plasmid, which supports regulated expression of the antigen-encoding gene, with repression during in vitro growth and derepression in vivo.
Promoter regulated by repressor is a Ptrc promoter
The promoter responsible for controlling the gene of interest is specified as a Ptrc promoter, which is regulated by the repressor.
Capability of eliciting a protective immune response
The recombinant bacterium is capable of eliciting a protective immune response in the host due to its regulated antigen expression.
Combination of repressor, plasmid vector, and Ptrc promoter system
A system wherein the repressor is LacI, the vector is a plasmid, and the promoter regulated by the repressor is a Ptrc promoter, providing a specific genetic circuit for regulation.
Compositions and methods for therapeutic and immunological use
Compositions comprising the recombinant bacterium are claimed, along with methods for ameliorating symptoms of disease and eliciting an immune response in a host by administering an effective amount of the composition.
In summary, the claims broadly cover recombinant bacteria with a modular genetic architecture for regulated antigen expression and attenuation, describe specific genetic modifications to optimize function, and claim their use in therapies and immunizations.
Stated Advantages
The invention enables high-level antigen expression in vivo while repressing antigen synthesis during in vitro growth, improving both safety and immunogenicity of live vaccines.
Regulated attenuation allows vaccine strains to colonize lymphoid tissues effectively before display of attenuation, eliminating disease risk while enhancing immune stimulation.
Balanced regulation prevents over-attenuation, ensuring vaccine strains reach effector lymphoid tissues in sufficient numbers and duration to generate robust protective immune responses.
Plasmid loss after immunization is minimized, ensuring strong antigen-specific immune responses rather than responses against the carrier bacterium.
Genetic modifications (e.g., Shine-Dalgarno, codon optimization) allow fine-tuning of repressor and antigen expression for different antigens and applications.
Biological containment is provided through regulated, programmed lysis systems, preventing unintended survival or environmental spread of vaccine strains.
Documented Applications
Use of recombinant bacteria as live vaccines to elicit protective immune responses against various pathogens in vertebrate hosts, including humans and animals.
Administration of the recombinant bacterium in a composition (vaccine) to a host for prophylactic or therapeutic purposes to induce a mucosal, humoral, or cellular immune response.
Oral, intranasal, intravenous, intramuscular, subcutaneous, or other parenteral administration of the vaccine composition for immunization.
Delivery of bacterial, viral, fungal, or parasitic antigens, allergens, tumor antigens, or gamete-specific antigens for immune modulation, tolerance induction, or antitumor therapy.
Use in kits for vaccination, optionally lyophilized for easy reconstitution and administration.
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