Compositions containing amino acids, phosphate and manganese and their uses
The invention provides methods of producing vaccines directed against microorganisms, with the methods comprising culturing, harvesting and/or suspending the microorganism in the presence of a radiation-protective composition and irradiating the bacteria or viruses with a dose of radiation sufficient to render the microorganism replication-deficient and/or non-infective. The radiation-protective compositions used in the methods of the present invention comprise at least one nucleoside, at least one antioxidant and at least one small peptide. The invention also provides methods of rendering bacteria in culture resistant to ionizing radiation (IR), with these methods comprising culturing the bacteria in the presence of a radiation-protective composition.
1. A method of producing a replication-deficient microorganism, the method comprising
a) culturing, harvesting, and/or suspending a viable microorganism in an aqueous environment comprising a radiation-protective composition, the composition comprising divalent manganese, orthophosphate and (i) a collection of amino acids consisting of Aspartate, Glutamate, Histidine, Glycine, Threonine, Alanine, Valine, Methionine, Leucine, and Lysine, or (ii) a peptide of about 25 residues in length or less, with the peptide consisting of amino acids Aspartate, Glutamate, Histidine, Glycine, Threonine, Alanine, Valine, Methionine, Leucine, and Lysine,
b) irradiating the microorganism in the radiation-protective composition with a dose of ionizing radiation sufficient to render the microorganism replication-deficient.
2. The method of claim 1 , wherein the radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation.
3. The method claim 1 , wherein the composition further comprises at least one nucleoside selected from the group consisting of adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof.
4. The method of claim 3 , wherein the at least one nucleoside is adenosine or uridine.
5. The method of claim 4 , wherein the concentration of the at least one nucleoside is from about 1 mM to about 15 mM.
6. The method of claim 1 , wherein concentration of the divalent manganese is from about 1 mM to about 12.5 mM.
7. The method of claim 1 , wherein the divalent manganese is in a form selected from the group consisting of MnCl 2 and manganous phosphate.
8. The method of claim 1 , wherein the composition further comprises an ultrafiltrate from Deinococcus radiodurans.
9. The method of claim 1 , wherein the dose of radiation is at least about 10 kGy.
10. The method of claim 1 , wherein the microorganism is a bacterium.
11. The method of claim 10 , wherein the bacterium is Staphylococcus.
12. The method of claim 11 , wherein the Staphylococcus is Staphylococcus aureus.
13. The method of claim 1 , wherein the peptide comprises the amino acid sequence of SEQ ID NO:1.
14. The method of claim 1 , wherein the microorganism is a virus.
15. A method of producing a replication-deficient microorganism, the method comprising
a) culturing, harvesting, and/or suspending a viable microorganism in an aqueous environment comprising a radiation-protective composition, the composition comprising divalent manganese at a concentration from about 1 mM to about 12.5 mM, orthophosphate, a peptide of about 25 residues in length or less, with the peptide consisting of amino acids Aspartate, Glutamate, Histidine, Glycine, Threonine, Alanine, Valine, Methionine, Leucine, and Lysine, and at least one nucleoside selected from the group consisting of adenosine, uridine, 3-pseudouridine, and inosine;
b) irradiating the microorganism in the radiation-protective composition with a dose of ionizing radiation sufficient to render the microorganism replication-deficient.
16. The method of claim 15 , wherein the peptide comprises the amino acid sequence of SEQ ID NO:1.
17. The method of claim 15 , wherein the microorganism is a virus.
18. The method of claim 15 , wherein the radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation.
19. The method of claim 15 , wherein the microorganism is a bacterium.
20. The method of claim 19 , wherein the bacterium is Staphylococcus.
21. The method of claim 20 , wherein the Staphylococcus is Staphylococcus aureus.
22. A method of producing a replication-deficient microorganism, the method comprising
a) culturing, harvesting, and/or suspending a viable microorganism in an aqueous environment comprising a radiation-protective composition, the composition comprising divalent manganese, orthophosphate and (i) a collection of amino acids consisting of asparagine, glutamine, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, phenylalanine, isoleucine, lysine, ornithine, leucine, valine and alanine or (ii) a peptide of about 25 residues in length or less, with the peptide consisting of amino acids aspartate, glutamate, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, phenylalanine, isoleucine, lysine, ornithine, leucine, valine and alanine,
b) irradiating the microorganism in the radiation-protective composition with a dose of ionizing radiation sufficient to render the microorganism replication-deficient.
23. The method of claim 22 , wherein the radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation.
24. The method claim 22 , wherein the composition further comprises at least one nucleoside selected from the group consisting of adenosine, uridine, β-pseudouridine, inosine, and mixtures thereof.
25. The method of claim 24 , wherein the at least one nucleoside is adenosine or uridine.
26. The method of claim 25 , wherein the concentration of the at least one nucleoside is from about 1 mM to about 15 mM.
27. The method of claim 22 , wherein concentration of the divalent manganese is from about 1 mM to about 12.5 mM.
28. The method of claim 22 , wherein the divalent manganese is in a form selected from the group consisting of MnCl2 and manganous phosphate.
29. The method of claim 22 , wherein the composition further comprises an ultrafiltrate from Deinococcus radiodurans.
30. The method of claim 22 , wherein the dose of radiation is at least about 10 kGy.
31. The method of claim 22 , wherein the microorganism is a bacterium.
32. The method of claim 31 , wherein the bacterium is Staphylococcus.
33. The method of claim 32 , wherein the Staphylococcus is Staphylococcus aureus.
34. A method of producing a replication-deficient microorganism, the method comprising
a) culturing, harvesting, and/or suspending a viable microorganism in an aqueous environment comprising a radiation-protective composition, the composition comprising divalent manganese at a concentration from about 1 mM to about 12.5 mM, orthophosphate, a peptide of about 25 residues in length or less, with the peptide consisting of amino acids aspartate, glutamate, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, phenylalanine, isoleucine, lysine, ornithine, leucine, valine and alanine, and at least one nucleoside selected from the group consisting of adenosine, uridine, 3-pseudouridine, and inosine;
b) irradiating the microorganism in the radiation-protective composition with a dose of ionizing radiation sufficient to render the microorganism replication-deficient.
35. The method of claim 34 , wherein the microorganism is a virus.
36. The method of claim 34 , wherein the radiation is selected from the group consisting of ultraviolet radiation, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation.
37. The method of claim 34 , wherein the microorganism is a bacterium.
38. The method of claim 37 , wherein the bacterium is Staphylococcus.
39. The method of claim 38 , wherein the Staphylococcus is Staphylococcus aureus.