Incapacitated whole-cell immunogenic bacterial compositions produced by recombinant expression
The present invention features incapacitated whole-cell bacterial immunogenic compositions and methods of their production, which compositions are useful to deliver antigens in a manner resembling the live infectious organism in terms of elicitation of a robust immune response, but with reduced risk or no risk of disease. The compositions of the invention are produced by rendering a bacterium bacteriostatic through expression of a recombinant promoter in the bacterial cell, which promoter can be operably linked to a polynucleotide encoding a recombinant gene product. In one embodiment, where the bacterium is a gram negative host, the recombinant gene product provides for reduced toxicity of LPS. In one embodiment, the gene product is a bacteriophage protein, such as endolysin, holin, or ndd.
1. A method of producing an immunogenic bacterial composition, comprising the steps of: producing incapacitated bacteria by introducing an expression vector into the bacteria, the expression vector comprising a bacteriophage promoter operably linked to a polynucleotide encoding a gene product wherein the bacteria are recombinantly modified to express a bacteriophage RNA polymerase for transcription from the bacteriophage promoter, wherein expression of the gene product is at a level sufficient to render the bacteria incapacitated; and
optionally adding a pharmaceutically acceptable excipient, thereby producing an immunogenic bacterial composition;
wherein
(i) the bacteriophage promoter is a T7 bacteriophage promoter and the bacteriophage RNA polymerase is T7 RNA polymerase,
(ii) the bacteriophage promoter is a SP6 bacteriophage promoter and the bacteriophage RNA polymerase is SP6 RNA polymerase,
(iii) the bacteriophage promoter is a T3 bacteriophage promoter and the bacteriophage RNA polymerase is T3 RNA polymerase, or
(iv) the bacteriophage promoter is a T7 bacteriophage promoter and the bacteriophage RNA polymerase is T7 RNA polymerase.
2. The method of claim 1 , wherein the immunogenic bacterial composition further comprises an adjuvant.
3. The method of claim 1 , wherein the polynucleotide encodes a lipopolysaccharide binding protein.
4. The method of claim 1 , wherein the polynucleotide encodes a cytokine.
5. The method of claim 1 , wherein said polynucleotide encodes an adjuvant.