Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-02
2002-07-02
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100, C530S300000, C530S350000, C530S403000
Reexamination Certificate
active
06413768
ABSTRACT:
TABLE OF CONTENTS
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
1.2 Description of Related Art
1.2.1 Bacterial Live Vector Vaccines
1.2.2 Attenuated Salmonella typhi as a live vector strain
1.2.3 Plasmid Instability
1.2.4 Plasmid Stabilization Systems
1.2.5 Antibiotic Resistance
1.2.6 Segregational Plasmid Maintenance Functions
1.2.7 Post-Segregational Killing (PSK) Functions
1.2.7.1 Proteic Maintenance System: The phd/doc System
1.2.7.2 Antisense Maintenance System: The hok-sok System
1.2.7.3 Balanced Lethal Systems
2. SUMMARY OF THE INVENTION
3. DEFINITIONS
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Suicide Vectors
5.2 Plasmid-based Expression of Heterologous Antigens
5.3 Balanced Lethal Systems
5.4 Segregation Limitations
5.5 Catalytic Activity Limitations
5.6 The Non-Catalytic SSB PSK Function
5.7 Expression Plasmids and Self-Contained Genetic Cassettes
5.8 Components of the Antigen Expression and Replication Cassette
5.8.1 Promoter
5.8.2 Origin of Replication
5.8.3 Expressed Protein or Peptide
5.8.4 Heterologous Antigens
5.8.4.1 The Shiga Toxin Family
5.8.5 Site-Specific Mutagensis of Shiga Toxins
5.9 Pharmaceutical Formulations
6. EXAMPLES
6.1 pGen Structure
6.2 P
OmpC
Promoter
6.3 Modified OmpC Promoter
6.4 Origins of Replication and Selection Cassettes
6.5 The Hok-Sok Antisense Post-Segregational Killing Locus
6.6 Complementation-Based Killing System
6.7 Conclusions
7. REFERENCES
THE CLAIMS
ABSTRACT OF THE DISCLOSURE
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to expression plasmids stabilized by a Plasmid Maintenance System (as defined herein) capable of expressing a protein or peptide, such as an antigen for use in a live vector vaccine, and methods for making and using the stabilized plasmids. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on catalytic balanced lethal maintenance systems; and (2) incorporating a plasmid partition system to prevent random segregation of expression plasmids, thereby enhancing inheritance and stability.
1.2 Description of Related Art
Set forth below is a discussion of art relevant to the present invention.
1.2.1 Bacterial Live Vector Vaccines
Bacterial live vector vaccines represent an important and promising strategy in the field of vaccine development. These vaccines deliver antigens to a host immune system by expressing the antigens from genetic material contained within a bacterial live vector. The genetic material is typically a replicon, such as a plasmid. The antigens may include a wide variety of proteins and/or peptides of bacterial, viral, parasitic or other origin.
Among the bacterial live vectors currently under investigation are attenuated enteric pathogens (e.g.,
Salmonella typhi,
Shigella,
Vibrio cholerae
), commensals (e.g., Lactobacillus,
Streptococcus gordonii
) and licensed vaccine strains (e.g., BCG). For the reasons discussed below,
S. typhi
is a particularly attractive candidate for human vaccination.
1.2.2 Attenuated
Salmonella typhi
as a Live Vector Strain
S. typhi
is a well-tolerated live vector that can deliver multiple unrelated immunogenic antigens to the human immune system.
S. typhi
live vectors have been shown to elicit antibodies and a cellular immune response to expressed antigen. Examples of antigens successfully delivered by
S. typhi
include the non-toxigenic yet highly immunogenic fragment C of tetanus toxin and the malaria circumsporozoite protein from
Plasmodium falciparum.
S. typhi
is characterized by enteric routes of infection, a quality which can enable oral vaccine delivery.
S. typhi
also infects monocytes and macrophages and can therefore target antigens to professional APCs.
Expression of an antigen by
S. typhi
generally requires incorporation of a recombinant plasmid encoding the antigen. Consequently, plasmid stability is a key factor in the development of high quality attenuated vaccines with the ability to consistently express foreign antigens.
Attenuated
S. typhi
vaccine candidates for use in humans should possess at least two well separated and well defined mutations that independently cause attenuation, since the chance of in vitro reversion of such double mutants would be negligible.
The attenuated vaccine candidate
S. typhi
CVD908 possesses such properties. CVD908 contains two non-reverting deletion mutations within the aroC and aroD genes. These two genes encode enzymes critical in the biosynthetic pathway leading to synthesis of chorismate, the key precursor required for synthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Chorismate is also required for the synthesis of p-aminobenzoic acid; after its conversion to tetrahydrofolate, p-aminobenzoic acid is converted to the purine nucleotides ATP and GTP.
1.2.3 Plasmid Instability
Plasmidless bacterial cells tend to accumulate more rapidly than cells containing active plasmids. Summers,
The Biology of Plasmids,
65-91, 1996 (incorporated herein by reference). One reason for this increased rate of accumulation is that the transcription and translation of plasmid genes imposes a metabolic burden which slows cell growth and gives plasmidless cells a competitive advantage. Furthermore, foreign plasmid gene products are sometimes toxic to the host cell.
Stable inheritance of plasmids is desirable in the field of attenuated bacterial live vector vaccines to ensure successful continued antigen production, as well as in commercial bioreactor operations in order to prevent bioreactor takeover by plasmidless cells.
Stable inheritance of a plasmid generally requires that: (1) the plasmid must replicate once each generation, (2) copy number deviations must be corrected, and (3) upon cell division, the products of replication must be distributed to both daughter cells. Summers,
The Biology of Plasmids,
65-91, 1996 (the entire disclosure of which is incorporated herein by reference).
Although chromosomal integration of foreign genes confers stability to such sequences, the genetic manipulations involved can be difficult, and the drop in copy number of the heterologous gene often results in production of insufficient levels of heterologous antigen to ensure an optimal immune response. Introduction of heterologous genes onto multicopy plasmids maintained within a live vector strain is a natural solution to the copy number problem. Genetic manipulation of such plasmids for controlled expression of such heterologous genes is straightforward; however, resulting plasmids can become unstable in vivo, resulting in loss of these foreign genes.
1.2.4 Plasmid Stabilization Systems
In nature bacterial plasmids are often stably maintained. See Gerdes et al.
Annu. Rev. Genet.,
31:1-31, 1997 (incorporated herein by reference). In some circumstances, stable maintenance may simply result from a high copy number. However, many proteins, such as antigens, which may be desirably produced by bacterial cells are toxic if produced in large amounts per cell. Therefore, it is desirable to provide stable lower copy number plasmids for use in bacterial cells.
Stable inheritance of naturally occurring lower copy number plasmids can depend on the presence of certain genetic systems which actively prevent the appearance of plasmid-free progeny. A recent review of plasmid stabilization systems can be found in Jensen et al.
Molecular Microbiol.
17:205-210, 1995 (incorporated herein by reference).
1.2.5 Antibiotic Resistance
One means for stabilizing plasmids is to provide an antibiotic resistance gene on the plasmid and to grow the cells in antibiotic-enriched media. However, this method is subject to a number of difficulties. The antibiotic resistance approach is expensive, requiring the use of costly antibiotics and perhaps, more importantly, the use of antibiotics in conjunction with in vivo administration of vaccine vectors may promote the growth of antibiotic-resistant bacteria and is currently forbidden by the U.S. Food and Drug Administ
University of Maryland
Yucel Remy
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