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-10774334-B2
Publication Date
2020-09-15
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 making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest. The key innovation is a recombinant bacterium comprising at least one chromosomally integrated nucleic acid sequence encoding a repressor operably linked to a regulatable promoter, and a vector comprising a nucleic acid sequence encoding at least one antigen of interest, which is operably linked to a promoter regulated by said repressor. The invention also provides that the bacterium may further comprise a mutation such as ΔrelA197::araC PBAD lacl TT to enable controlled expression.
The background identifies the need for improved live vaccine strains. Traditional live recombinant vaccines can be limited by the toxicity of overexpressed antigens, loss of plasmid vectors encoding foreign antigens, or hyperattenuation that reduces the ability of the bacteria to colonize and persist in lymphoid tissues, ultimately resulting in poor immunogenicity. Therefore, methods to balance bacterial colonization/growth with antigen expression and to regulate the attenuation phenotype are essential.
This invention solves these problems by genetically engineering bacteria to tightly regulate expression of both antigen and attenuation factors, using inducible systems (such as araC PBAD, responsive to arabinose), repressors (such as Lacl), and promoter modifications to optimize control. In vitro, the antigen expression is repressed, avoiding toxicity and enhancing bacterial fitness; after administration to a host (where no inducer is present), repression is lifted, enabling high-level antigen production and/or induction of attenuation for safety. This system optimizes both vaccine immunogenicity and safety.
Claims Coverage
The independent claim of the patent defines one main inventive feature structured around the specific genetic design of a recombinant bacterium that enables regulated antigen expression using a modified repressor-regulatable promoter system and a specific chromosomal mutation.
Regulated antigen expression system in recombinant bacterium
This inventive feature is a recombinant bacterium capable of regulated expression of at least one nucleic acid sequence encoding an antigen of interest through the following components: - The bacterium includes at least one chromosomally integrated nucleic acid sequence encoding a repressor, which is operably linked to a regulatable promoter. The nucleic acid sequence encoding the repressor and/or the promoter has been modified from the wild-type nucleic acid sequence so as to optimize the expression level of the nucleic acid sequence encoding the repressor. - The bacterium further contains a vector comprising at least one nucleic acid sequence encoding an antigen of interest, which is operably linked to a promoter regulated by the repressor in such a way that expression of the antigen is repressed during in vitro growth, but the bacterium is capable of high-level expression of the antigen in a host. - Additionally, the bacterium carries the chromosomal mutation ΔrelA197::araC PBAD lacl TT, integrating a tightly regulated araC PBAD lacl cassette at the relA locus. The claims further specify that the repressor can be Lacl, C2, or C1, that the repressor’s sequence can include a modified Shine-Dalgarno sequence and optimized codons, and that the antigen-encoding gene on the vector can be linked to a Ptrc promoter. The antigen may be toxic; the vector may encode a secretion signal; and the system can be used to elicit a protective immune response. The claims also allow for the presence of more than one means of attenuation.
In summary, the claims cover a genetically engineered bacterium with a regulated antigen expression and attenuation system, including a chromosomally integrated, optimized repressor under a regulatable promoter, a repressor-regulated antigen vector, and specifically the ΔrelA197::araC PBAD lacl TT mutation, enabling tightly controlled vaccine design.
Stated Advantages
The invention minimizes the negative effects of antigen expression on bacterial host fitness during in vitro growth, allowing for enhanced growth and colonization of lymphoid tissues.
The system enables high-level antigen production in host tissues, leading to stronger and more protective immune responses.
Regulated attenuation strategies provide improved safety by ensuring the bacterium becomes attenuated only after colonization of host lymphoid tissues, reducing risk of disease symptoms.
The system enhances genetic stability and minimizes plasmid loss after immunization, maintaining antigen production and intended immune responses.
The invention enables biological containment via programmed lysis, preventing environmental shedding and unintentional immunization.
Flexibility of promoter/repressor systems and genetic modifications allows optimization for different antigens or attenuation functions.
Documented Applications
Use of recombinant bacteria as live vaccine strains to produce immune responses against antigens of interest in vertebrate hosts.
Administration of such vaccines to induce protective immune responses against bacterial, viral, mycotic, and parasitic pathogens.
Oral, intranasal, or aerosolized delivery of live attenuated vaccine strains for immunization of mammals and birds.
Use of the recombinant bacteria for programmed lysis and biological containment to enhance environmental safety of live vaccines.
Use in human clinical trials for safety and immunogenicity assessment, specifically in S. typhi-based vaccines targeting antigens such as Streptococcus pneumoniae PspA.
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