Recombinant bacterium and methods of antigen and nucleic acid delivery
View Patent ↗The present invention provides a recombinant bacterium and methods of using the recombinant bacterium to induce an immune response.
1. A recombinant Salmonella bacterium, wherein the bacterium comprises:
(a) a mutation selected from the group consisting of a ΔP hilA ::P trcΔlacO hilA mutation and a ΔP hilA ::P hilA hilA mutation, wherein the ΔP hilA ::P hilA hilA mutation comprises a mutation in the hilA promoter that reduces nucleoid-associated protein H-NS binding,
(b) a ΔtlpA mutation and a ΔsseL mutation and, optionally at least one mutation selected from the group consisting of an ampG mutation, an ampD mutation, and a nagE mutation, and
(c) at least one mutation selected from the group consisting of a tlyC mutation, a pld mutation, a ΔsifA mutation and a ΔP sifA ::TT araC P BAD sifA mutation.
2. The recombinant Salmonella bacterium of claim 1 , wherein the bacterium lacks expression of sifA.
3. A recombinant Salmonella bacterium, wherein the bacterium is:
(a) capable of regulated delayed attenuation,
(b) capable of regulated delayed lysis, characterized by a ΔP murA ::TT araC P BAD murA mutation and a ΔasdA::TT araC P BAD c2 mutation,
(c) capable of escape from a host endosomal compartment, characterized by at least one mutation selected from the group consisting of a tlyC mutation and a pld mutation,
(d) capable of hyper-invasion, characterized by hilA operably linked to a constitutive promoter, and
(e) capable of reducing bacterium-induced host programmed cell death, characterized by a ΔtlpA mutation and a ΔsseL mutation and, optionally, at least one mutation selected from the group consisting of an ampG mutation, an ampD mutation, and a nagE mutation.
4. The recombinant Salmonella bacterium of claim 3 , wherein the programmed cell death is pyroptosis or apoptosis.
5. The recombinant Salmonella bacterium of claim 3 , wherein the bacterium further comprises a DNA vaccine encoding a protective antigen.
6. The recombinant Salmonella bacterium of claim 5 , wherein the DNA vaccine comprises a nucleic acid sequence encoding one or more bacterial, viral, protozoan, or fungal antigens.
7. The recombinant Salmonella bacterium of claim 5 , wherein the DNA vaccine comprises a nucleic acid sequence encoding one or more influenza antigens.
8. The recombinant Salmonella bacterium of claim 3 , wherein the bacterium further comprises a single vector for the production of influenza virus.
9. The recombinant Salmonella bacterium of claim 3 , wherein the bacterium escapes from the host endosomal compartment before undergoing regulated lysis.
10. The recombinant Salmonella bacterium of claim 5 , wherein the DNA vaccine comprises a repeated DNA nuclear targeting sequence and a nuclease resistant polyadenylation encoding sequence.
11. The recombinant Salmonella bacterium of claim 10 , wherein the DNA vaccine comprises an artificial NF-kB recognition sequence and or an artificial AP-2 recognition sequence.
12. The recombinant Salmonella bacterium of claim 5 , wherein the DNA vaccine comprises the vector pYA4545.
13. The recombinant Salmonella bacterium of claim 5 , wherein the DNA vaccine comprises an influenza HA antigen-SopE C-terminal fusion.
14. The recombinant Salmonella bacterium of claim 3 , wherein the bacterium further comprises the vector pYA3681.
15. The recombinant Salmonella bacterium of claim 1 , wherein the bacterium comprises a ΔampG mutation, a ΔampD mutation, or a ΔnagE mutation.
16. The recombinant Salmonella bacterium of claim 10 , wherein the nuclease resistant polyadenylation sequence is derived from a DNA virus capable of replicating in a eukaryote.
17. A recombinant Salmonella bacterium, wherein the bacterium is:
(a) capable of regulated delayed attenuation,
(b) capable of regulated delayed lysis, characterized by ΔP murA ::TT araC P BAD murA mutation and a ΔasdA::TT araC P BAD c2 mutation,
(c) capable of escape from a host endosomal compartment, characterized by at least one mutation selected from the group consisting of a tlyC mutation, a pld mutation, a ΔsifA mutation and ΔP sifA ::TT araC P BAD sifA mutation,
(d) capable of hyper-invasion, characterized by hilA operably linked to a constitutive promoter, and
(e) capable of reducing bacterium-induced host programmed cell death, characterized by a ΔtlpA mutation and a ΔsseL mutation and, optionally, a mutation selected from the group consisting of an ampG mutation, an ampD mutation, and a nagE mutation.
18. The recombinant Salmonella bacterium of claim 3 , wherein the hilA operably linked to a constitutive promoter is selected from the group consisting of ΔP hilA ::P trcΔlacO hilA and ΔP hilA ::P hilA hilA mutation, wherein the ΔP hilA ::P hilA hilA mutation comprises a mutation in the hilA promoter that reduces nucleoid-associated protein H-NS binding.
19. The recombinant Salmonella bacterium of claim 17 , wherein the hilA operably linked to a constitutive promoter is selected from the group consisting of a ΔP hilA ::P trcΔlacO hilA mutation and a ΔP hilA ::P hilA hilA mutation, wherein the ΔP hilA ::P hilA hilA mutation comprises a mutation in the hilA promoter that reduces nucleoid-associated protein H-NS binding.
20. A recombinant Salmonella bacterium, wherein the bacterium is:
(a) capable of regulated delayed attenuation,
(b) capable of regulated delayed lysis, characterized by a ΔP murA ::TT araC P BAD murA mutation and a ΔasdA::TT araC P BAD c2 mutation,
(c) capable of escape from a host endosomal compartment, characterized by at least one mutation selected from the group consisting of a tlyC mutation, a pld mutation, a ΔsifA mutation and ΔP sifA ::TT araC P BAD sifA mutation, and
(d) capable of reducing bacterium-induced host programmed cell death, characterized by a ΔtlpA mutation and a ΔsseL mutation and, optionally, a mutation selected from the group consisting of an ampG mutation, an ampG mutation, and a nagE mutation.
21. A vaccine, the vaccine comprising the recombinant Salmonella bacterium of claim 20 .