VACCINE AGAINST BETA-HERPESVIRUS INFECTION AND USE THEREOF
20190117766 ยท 2019-04-25
Inventors
- Christian Thirion (Munich, DE)
- Ulrich Koszinowski (Feldafing, DE)
- Christian A. Mohr (Munchen, DE)
- Zsolt Ruzsics (Diessen am Ammersee, DE)
Cpc classification
C12N2710/16121
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
C12N2710/16152
CHEMISTRY; METALLURGY
C12N2710/16134
CHEMISTRY; METALLURGY
C12N2710/16511
CHEMISTRY; METALLURGY
C12N2710/16111
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention is related to a beta-herpesvirus, wherein the beta-herpesvirus is spread-deficient.
Claims
1. A recombinant beta-herpesvirus, wherein the beta-herpesvirus is spread-deficient.
2. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is endotheliotropic and/or has a wild type-like virion surface.
3. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is endotheliotropic and has a wild type-like virion surface.
4. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is suitable to or capable of inducing an immune response, wherein the immune response comprises neutralizing antibodies against beta-herpesvirus and CD4.sup.+ and CD8.sup.+ T-cells directed against epitopes of beta-herpesvirus.
5. The beta-herpesvirus according to claim 4, wherein the immune response comprises induction of neutralizing antibodies against a wild type beta-herpesvirus, wherein said antibodies are capable of inhibiting said wild type beta-herpesvirus from infecting endothelial cells and/or epithelial cells.
6. The beta-herpesvirus according to claim 5, wherein beta-herpesvirus which is prevented from infecting endothelial cells and/or epithelial cells by the neutralizing antibodies, is preferably a human pathogen.
7. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is a human beta-herpesvirus.
8. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is a cytomegalovirus.
9. The beta-herpesvirus according to claim 7, wherein the beta-herpesvirus is a human cytomegalovirus.
10. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is deficient in at least one gene product involved in primary and/or secondary envelopment.
11. The beta-herpesvirus according to claim 10, wherein the at least one gene product is involved in primary envelopment
12. The beta-herpesvirus according to claim 11, wherein the at least one gene product is encoded by a gene selected from the group comprising UL50 and UL 53 and homologs of each thereof.
13. The beta-herpesvirus according to claim 10, wherein the at least one gene product is involved in secondary envelopment.
14. The beta-herpesvirus according to claim 13, wherein the at least one gene product is encoded by a gene selected from the group comprising UL94 and UL99 and homologs each thereof.
15. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 181652 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 123668 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 181652 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 189192 of the nucleotide sequence according to SEQ ID NO: 20.
16. The beta herpesvirus according to claim 1, wherein the beta-herpesvirus comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 181652 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and a fourth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 34.
17. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 110670 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 131243 to 181652 of the nucleotide sequence according to SEQ ID NO: 20 and a fourth nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 123668 of the nucleotide sequence according to SEQ ID NO: 20, wherein the nucleotide 130670 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 131243 of the nucleotide sequence according to SEQ ID NO: 20 and wherein the nucleotide 181652 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 189192 of the nucleotide sequence according to SEQ ID NO: 20.
18. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 181652 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 181.652 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 189192 of the nucleotide sequence according to SEQ ID NO: 20.
19. The beta-herpesvirus according to claim 1, wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 32, wherein nucleotide 179 of the nucleotide sequence according to SEQ ID NO: 32 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 181652 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 189192 of the nucleotide sequence according to SEQ ID NO: 20.
20. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 63261 to 181652 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 62129 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 63261 of the nucleotide sequence according to SEQ ID NO: 20 and wherein the nucleotide 181652 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 189192 of the nucleotide sequence according to SEQ ID NO: 20.
21. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 63261 to 181652 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and a fourth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 33.
22. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 63261 to 181652 of the nucleotide sequence according to SEQ ID NO: 20, a fourth nucleotide sequence represented by nucleotides 189192 to 233681 of the nucleotide sequence according to SEQ ID NO: 20, a fifth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 32 and a sixth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 33.
23. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus comprises human cytomegalovirus.
24. The beta-herpesvirus according to claim 1, wherein the beta herpesvirus comprises the nucleotide sequence according to SEQ ID NO: 23.
25. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus is deficient in at least one gene product encoded by an immune evasive gene.
26. The beta-herpesvirus according to claim 25, wherein the at least one gene product encoded by an immune evasive gene is selected from the group comprising gene products regulating MHC class I presentation and gene products regulating NK cell response.
27. The beta-herpesvirus according to claim 26, wherein the at least one gene product encoded by an immune evasive gene is a gene product regulating MHC class I presentation.
28. The beta-herpesvirus according to claim 27, wherein the gene product regulating MHC class I presentation comprises human cytomegalovirus.
29. The beta-herpesvirus according to claim 26, wherein the at least one gene product encoded by an immune evasive gene is a gene product regulating NK cell response.
30. The beta-herpesvirus according to claim 29, wherein the gene product regulating NK cell response is selected from the group comprising gene products encoded by the genes UL40, UL16 and UL18.
31. The beta-herpesvirus according to claim 1, wherein the beta-herpesvirus encodes a heterologous nucleic acid.
32. The beta-herpesvirus according to claim 34, wherein the heterologous nucleic acid is a functional nucleic acid, preferably selected from the group comprising antisense molecules, ribozymes and RNA interference mediating nucleic acids.
33. The beta-herpesvirus according to claim 31, wherein the nucleic acid is a nucleic acid coding for a peptide, oligopeptide, polypeptide or protein.
34. The beta-herpesvirus according to claim 33, wherein the peptide, oligopeptide, polypeptide or protein comprises at least one antigen.
35. The beta-herpesvirus according to claim 34, wherein the antigen is an antigen selected from the group comprising viral antigens, bacterial antigens and parasite antigens.
36. The beta-herpesvirus according to claim 1, for use in a method for the treatment of a human subject and/or for use in a method for the vaccination of a human subject.
37. The beta-herpesvirus according to claim 36, wherein the subject is a mammal.
38. The beta-herpesvirus according to claim 36, wherein the beta-herpesvirus is human cytomegalovirus.
39. The beta-herpesvirus according to claim 36, wherein the subject is suffering from a disease or is at risk of suffering from a disease.
40. The beta-herpesvirus according to claim 36, wherein the vaccination is a vaccination against a disease.
41. The beta-herpesvirus according to claim 39, wherein the disease is a disease or condition which is associated with human cytomegalovirus infection.
42. The beta-herpesvirus according to claim 41, wherein the disease or condition comprises congenital inclusion disease.
43. The beta-herpesvirus according to claim 36, wherein the subject is defined as a female with the ability to reproduce.
44. The beta-herpesvirus according to claim 43, wherein the treatment is or is suitable for or capable of preventing the transfer of a beta-herpesvirus, preferably human cytomegalovirus, from the female to a fetus and/or to an embryo carried or to be carried in the future by the female.
45. The beta-herpesvirus according to claim 43, wherein the treatment is for or is suitable for the generation of or capable of generating an immune response in the female body or the immune response in the female body, whereby preferably such immune response confers protection to a fetus and/or to an embryo carried or to be carried in the future by the female against beta-herpesvirus, preferably human cytomegalovirus, and/or a disease or condition associated with beta-herpesvirus infection, preferably human cytomegalovirus infection.
46. Use of a beta-herpesvirus according to claim 1, for the manufacture of a medicament.
47. Use according to claim 46, wherein the medicament is for the treatment and/or prevention of beta-herpesvirus infection.
48. Use according to claim 46, wherein the medicament is for the treatment and/or prevention of a disease or condition associated with beta-herpesvirus infection, preferably human cytomegalovirus infection.
49. Use of a beta-herpesvirus according to claim 1, for the manufacture of a vaccine.
50. Use according to claim 49, wherein the vaccine is for the treatment and/or prevention of beta-herpesvirus infection.
51. Use according to claim 50, wherein the vaccine is fin the treatment and/or prevention of a disease or condition associated with beta-herpesvirus infection, preferably human cytomegalovirus infection.
52. Use according to claim 49, wherein the vaccine is or is suitable for the administration to a subject, whereby the subject is selected form the group comprising a pregnant female, a female of reproductive age, a donor of a transplant, a recipient of a transplant and a subject being infected with HIV or being at risk of being infected with HIV.
53. Use according to claim 52, wherein the donor is a potential donor and/or the recipient is a potential recipient.
54. A nucleic acid coding for a beta-herpesvirus according to claim 1.
55. A vector comprising the nucleic acid according to claim 54.
56. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 123688 of the nucleotide sequence according to SEQ ID NO: 20.
57. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 34.
58. The vector according to claim 57, wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 34 and wherein nucleotide 252 of the nucleotide sequence according to SEQ ID NO: 34 is covalently linked to nucleotide 123668 of the nucleotide sequence according to SEQ ID NO: 20.
59. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 130670 of the nucleotide sequence according to SEQ. ID NO: 20, a third nucleotide sequence represented by nucleotides 131243 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 123668 of the nucleotide sequence according to SEQ ID NO: 20 and wherein the nucleotide 130670 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 131243 of the nucleotide sequence according to SEQ ID NO: 20.
60. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 122630 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 123668 to 130670 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 131243 to 233681 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 34 and a fourth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 35
61. The vector according to claim 60, wherein nucleotide 122630 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 34, wherein nucleotide 252 of the nucleotide sequence according to SEQ ID NO: 34 is covalently linked to nucleotide 123668 of the nucleotide sequence according to SEQ ID NO: 20, wherein nucleotide 130670 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 35 and wherein nucleotide 67 of the nucleotide sequence according to SEQ ID NO: 35 is covalently linked to nucleotide 131243 of the nucleotide sequence according to SEQ ID NO: 20
62. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the :nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20.
63. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 32.
64. The vector according to claim 63, wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 32 and wherein nucleotide 179 of the nucleotide sequence according to SEQ ID NO: 32 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20.
65. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 63261 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 62129 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 63261 of the nucleotide sequence according to SEQ ID NO: 20.
66. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 63261 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and a third nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 33
67. The vector according to claim 66, wherein nucleotide 62129 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 33 and wherein nucleotide 38 of the nucleotide sequence according to SEQ ID NO: 33 is covalently linked to nucleotide 63261 of the nucleotide sequence according to SEQ ID NO: 20.
68. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 63261 to 233681 of the nucleotide sequence according to SEQ ID NO: 20 and wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20 and wherein the nucleotide 62129 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to the nucleotide 63261 of the nucleotide sequence according to SEQ ID NO: 20.
69. A vector comprising the nucleic acid according to claim 55, wherein the vector comprises a nucleotide sequence, wherein the nucleotide sequence comprises a first nucleic acid sequence represented by nucleotides 1 to 58442 of the nucleotide sequence according to SEQ ID NO: 20, a second nucleotide sequence represented by nucleotides 59623 to 62129 of the nucleotide sequence according to SEQ ID NO: 20, a third nucleotide sequence represented by nucleotides 63261 to 233681 of the nucleotide sequence according to SEQ ID NO: 20, a fourth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 32 and a fifth nucleotide sequence comprising a nucleotide sequence according to SEQ ID NO: 33.
70. The vector according to claim 69, wherein nucleotide 58442 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 32, wherein nucleotide 179 of the nucleotide sequence according to SEQ ID NO: 32 is covalently linked to nucleotide 59623 of the nucleotide sequence according to SEQ ID NO: 20, wherein nucleotide 62129 of the nucleotide sequence according to SEQ ID NO: 20 is covalently linked to nucleotide 1 of the nucleotide sequence according to SEQ ID NO: 33 and wherein nucleotide 38 of the nucleotide sequence according to SEQ ID NO: 33 is covalently linked to nucleotide 632161 of the nucleotide sequence according to SEQ ID NO: 20.
Description
[0145] The present invention is now further illustrated by the following figures and examples from which further features, embodiments and advantages may be taken.
[0146] More specifically,
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[0148]
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[0163]
EXAMPLE 1
Spread Assay
[0164] The spread assay described herein may be used in connection with the characterization of a beta-herpesvirus and a human cytomegalovirus so as to determine whether such virus is spread-deficient.
[0165] Primary fibroblast cell lines MRC5 for human CMV and NIH/3T3 for mouse CMV and complementing cell lines TCL94/99-BP and NTM94-7, respectively, are plated and infected at an MOI of about 0.25 for 1 h and then washed twice with D-PBS. Cells are incubated for 6 h and afterwards washed four times with D-PBS. Equal numbers of non-infected cells were stained with 5 M CFSE for 8 min and blocked by 2% FCS/D-PBS, then washed twice with 2% FCS/D-PBS and subsequently seeded on top of the unstained but infected cells.
EXAMPLE 2
Assay for Determining Whether a Virus is Endotheliotropic
[0166] The assay described herein is used for determining whether a virus is endotheliotropic.
[0167] As to determine whether a human CMV is endotheliotropic a primary human fibroblast cell line, a complementing cell line which complements the product of the gene in relation to which the HCMV of the invention is deficient, and a human endothelial cell line are plated and infected at an MOI of about 0.1 with HCMV wild type or the virus of the present invention. 24 hours after infection immediate early staining is performed by incubating fixed cells with a monoclonal antibody against immediate early gene product of the beta-herpesvirus of the invention, more specifically CMV 1E 1/2 monoclonal Antibody CH160 (Plachter et al. supra), commercially available from Virusys Co. in 3% BSA/D-PBS. After three D-PBS washes, cells are incubated with an Alexa Fluor 555-coupled secondary antibody directed against the monoclonal antibody against human immediate early 1 of HCMV in 3% BSA/D-PBS. Finally cells are washed three times and imaged by UV microscopy. Cells infected with wild type HCMV are used as positive control and counted immediate early 1- and CFSE-positive cells using the ImageJ Cell Counter plugin (Rasband supra).
[0168] As to determine whether a mouse CMV is endotheliotropic a primary mouse fibroblast cell line, a complementing cell line which complements the product of the gene in relation to which the MCMV of the invention is deficient, and a mouse endothelial cell line are plated and infected at an MOI of about 0.1 with MCMV wild type or the virus of the present invention. 24 hours after infection immediate early staining is performed by incubating fixed cells with a monoclonal antibody against immediate early gene product of the beta-herpesvirus of the invention, more specifically Croma 101 designated as antibody 6/20/1 in Keil et al. (Keil et al., supra) in 3% BSA/D-PBS. After three D-PBS washes, cells are incubated with an Alexa Fluor 555-coupled secondary antibody directed against the mouse monoclonal antibody against immediate early 1 of mouse CMV in 3% BSA/D-PBS. Finally cells are washed three times and imaged by UV microscopy. Cells infected with wild type mouse CMV are used as positive control and counted immediate early 1 positive cells using the ImageJ Cell Counter plugin (Rasband supra).
EXAMPLE 3
Materials and Methods
[0169] Cells and Mice
[0170] The fibroblast cell line NIH/3T3 and BALB/c derived murine embryonic fibroblasts (MEF) were cultured as described in Cicin-Sain et al., (Cicin-Sain et al. 2005 J Virol 79:9492-9502.). C57BL/6 (B6) mice, B6.SJL-Ptpr.sup.c (Ptpr.sup.c) mice and 129.IFNR.sup./ mice were purchased from Elevage Janvier (Le Genest Saint isle, France), Jackson Laboratories (Bar Harbor, Me., USA) and B&K Universal Limited (Grimston, England), respectively. 129.IFNR.sup./ mice (Muller et al. 1994 Science 264:1918-1921,) were backcrossed on the B6 background (B6.IFNR.sup./). T cell receptor transgenic mice OT-I (Hogquist et al. 1994 Cell 76:17-27.) and OT-II (Barnden et al. 1998 Immunol Cell Biol 76:34-40.) were backcrossed to Ptpr.sup.c (CD45.1) or Thy1.1 (CD90.1) congenic mice, respectively. Alb-cre (Postic et al. 1999 J Biol Chem 274:305-315.) and Tie2-cre (Constien et al. 2001 Genesis 30:36-44) were maintained on the B6 background. Mice were kept under specified pathogen free conditions. Animal experiments were approved by the responsible office of the state of Bavaria (approval no. 55.2-1-54-2531-111-07) or by the Ethics Committee at the University of Rijeka.
[0171] Generation of the Trans-Complementing Cell Line NT/M94-7
[0172] The conditional trans-complementing cell line NT/M94-7 was generated according to (Lotzerich et al. supra). Briefly, the M94 ORE was amplified from pSM3fr (Sacher et al. 2008 Cell Host Microbe 3:263-272.) using primers HAM94for (SEQ.ID.No.1) and M94rev (SEQ.ID.No.2) thereby introducing an HA tag at the N-terminus. The PCR product was digested with BamHI and XbaI and inserted into the BamHI- and NheI-cleaved pTRE2Hyg vector (BD Biosciences Clontech, Heidelberg, Germany), resulting in pTRE-HAM94 (SEQ.ID.NO:22) putting HAM94 expression, the HAM94 protein is depicted in SEQ.ID.NO:31, under the control of the tetracycline (tet) inducible promoter. Stable NIH/3T3 transfectants harboring pTRE-HAM94 were selected with 50 g/ml Hygromycin B. The deletion virus MCMV-M94 was reconstituted by transfecting different NT/M94 cell clones with the respective BAC. The most productively infected trans-complementing cell line NT/M94-7 was subcloned using limiting dilution. The trans-complementing cell line was deposited under the Budapest Treaty with the DSZM, Germany on May 5, 2010.
[0173] Generation of Recombinant Viruses
[0174] Recombinant mouse CMV (MCMV) mutants were derived from the MCMV bacterial artificial chromosome (BAC) clone pSM3fr, originated from Smith strain (Messerle et al. 1997 Proc Natl Acad Sci USA 94:14759-14763). Nucleotide positions are given according to Rawlinson et al. (Rawlinson et al, supra). The 1.4 kilo base pair (bp) SmaI fragment of pCP15 carrying the FRT flanked kanamycin resistance gene (Kan.sup.r) was introduced into the BssHII site of pCR3 (Invitrogen, Basel, Switzerland) resulting in pCR3-FRT-Kan.sup.r-FRT. A fragment containing an ATG start codon and a loxP site was generated by annealing the oligonucleotides ATGlox1 (SEQ.ID.No.3) and ATGlox2 (SEQ.ID. No.4). This fragment was inserted into the EcoRI and XhoI site positioned between the major immediate early promoter of HCMV (IEP) and the polyA signal of the bovine growth hormone of pCR3-FRT-Kan.sup.r-FRT to obtain pCR3-FRT-Kan.sup.r-FRT-ATG-loxP. The ovalbumin gene (ova) was synthesized as contained in pBSK-OVA (SEQ.ID.NO: 21) introducing GGAA after nt position 9 resulting in a BspEI restriction site for further cloning. Ova was inserted in frame using BspEI and NotI of pCR3-FRT-Kan.sup.r-FRT-ATG-loxP resulting in a full length ova with inserted loxP site after the initial ATG under control of IEP named pCR3-FRT-Kan.sup.r-FRT-ATG-loxP-ova. To obtain a construct with Cre inducible ovalbumin (OVA) expression (SEQ.ID.NO: 24) a floxstop cassette (Sacher et al. supra) was inserted into the EcoRI and BspEI sites of pCR3-ATG-loxP-ova resulting in pCR3-ATG-flox-step-ova. Using these constructs as templates and oligonucleotides 5-m157-pCR3-FRT-Kan.sup.r-FRT (SEQ.ID.No.5)(nt position 216243 to 216290) and 3-m157-flox-egfp (SEQ.ID.No.6) (nt position 216885 to 216930) as primers a linear DNA fragment containing the IEP-ova cassette, the FRT flanked Kan.sup.r, and the viral homology sequences to the MCMV genome target site m157 was generated. In a similar procedure the firefly luciferase gene (luc) was cloned under control of the IEP into pCP15 carrying the FRT flanked Kan.sup.r. These fragments were introduced into m157 of pSM3fr as described (Sacher et al. supra) resulting in pSM3fr-m157-ova, pSM3fr-m157-flox-ova and pSM3fr-m157-luc. For excision of the FRT flanked Kan.sup.r FLP recombinase was transiently expressed from plasmid pCP20.
[0175] Generation of Spread-Deficient Virus Mutants
[0176] As shown in
[0177] For generation of the recombinant MCMV lacking the M94 sequence the parental MCMV BACs pSM3fr (MCMV-wt), pSM3fr-m157-ova (MCMV-ova) and pSM3fr-m157-rec-egfp (MCMV-m157-rec-egfp) (Sacher et al. supra) were applied to a second mutagenesis step. Therefore, the plasmid pO6-tTA-mFRT-Kan.sup.r-mFRT was obtained by insertion of the Kan.sup.r, on both sides flanked by mutant 34 bp FRT sites from pO6ic-F5 into pO6-tTA (Lotzerich et al. supra) to express the tTA transactivation gene under control of the IEP necessary for trans-complementation of pM94 (SEQ.ID:NO: 30). A linear DNA fragment containing the tTA cassette, the Kan.sup.r and viral homology sequences to the MCMV genome target site (MCMV upstream-homology: nt position 136189 to 136234 and MCMV downstream-homology: nt position 137256 to 137309) was generated using primer 5M94-pO6-tTA (SEQ.ID.No.7), primer 3-M94-pO6-tTA (SEQ.ID.No.8) and plasmid pO6-tTA-mFRT-Kan.sup.r-mFRT as template. This PCR fragment was inserted into the different parental pSM3fr clones, hereby deleting the M94 gene. Since ORFs of M94 and M93 are overlapping 47 bp of homology had to be left at the 5-end of M94 to keep the M93 ORF intact and 17 bp homology are still present at the former 3-end of M94. Again FLP recombinase was expressed for excision of the Kan.sup.r. Construction of pSM3fr-M94, pSM3fr-ova-M94, pSM3fr-flox-ova-M94 and pSM3fr-m157-rec-egfp-M94 was confirmed by restriction digest analysis and sequencing.
[0178] Viruses were reconstituted from BAC DNA, propagated on NT/M94-7 complementing cells and purified on a sucrose cushion as previously described (Sacher et al. supra). For analysis of virus replication supernatants from infected cells were taken every, 24 h. Quantification of infectious virus was done using TCID.sub.50 (median tissue culture infectious dose) method on NIH/3T3 or complementing NT/M94-7 cells. For the determination of virus replication in vivo virus load was determined by standard plaque assay as plaque forming units (PFU) per gram organ as described (Sacher et al. supra). Spread-deficiency of each virus stock of M94 deficient mutants (MCMV-M94, MCMV-ova-M94, MCMV-flox-ova-M94 and MCMV-m157-rec-egfp-M94) was confirmed by the absence of plaque formation after infection of non-complementing MEF, although CPE of individually infected cells was detectable. The E. coli containing the pSM3fr-M94 BAC of the spread-deficient MCMV-M94 was deposited under the Budapest Treaty with the DSZM on Apr. 28, 2010 as DSM 23561.
[0179] UV Inactivation of Virus
[0180] For in vivo application, a fraction of the MCMV-wt virus preparation used for immunization was inactivated by exposure to 1.5 kJ/cm.sup.2 UV light at a distance of 5 cm in a UV-crosslinker (Stratagene, Amsterdam, Netherlands) at 4 C. Viral infectivity was decreased by factor 2.410.sup.7. The same treatment was sufficient to abolish viral gene expression when MCMV-m157-rec-egfp was subjected to different doses (0.5, 1.0 and 1.5 kJ/cm.sup.2) of UV light and subsequently titrated on MEF. After 4 days post infection (p.i.) EGFP expression was monitored in single infected cells if virus was irradiated with low dose (0.5 kJ/cm.sup.2) of UV and no EGFP expression was seen after strong irradiation (1.5 kJ/cm.sup.2). Untreated MCMV-m157-rec-egfp formed EGFP plaques.
[0181] Immunization and Challenge of Mice
[0182] 8 to 10 weeks old female B6 mice were immunized by intraperitoneal or subcutaneous (s.c.) injection of either MCMV-wt or mutant MCMV. Each mouse received 100 l of virus suspension s.c. or 300 l i.p. C57BL/6 mice were immunized with 110.sup.5 TCID.sub.50 MCMV-wt of MCMV-deltaM94, 129.IFNR.sup./ with 2.510.sup.5 TCID.sub.50 of MCMV-deltaM94 or UV irradiated MCMV-wt, and B6.IFNR.sup./ with 310.sup.5 TCID.sub.50 of MCMV-M94 or MCMV-wt. Mock treated mice received same volumes of PBS. To boost mice, this procedure was repeated 14 days p.i. Sera collected from mice 12 weeks p.i. were used to determine amounts of virus specific antibodies by virus neutralization assay, as described below.
[0183] 28 days or 20 weeks post priming, mice were challenged by intravenous (i.v.) injection of 10.sup.6 PFU of tissue culture derived MCMV-wt. Five days post challenge lungs, liver and spleen were collected under sterile conditions and stored at 80 C. Organ homogenates were analyzed for infectious virus load by standard plaque assay on MEF cells. Salivary glands derived MCMV (sgMCMV-wt) was generated as a homogenate of salivary glands from mice infected with tissue culture derived MCMV-wt as described in Trgovcich et al. (Trgovcich et al, 2000 Arch Virol 145:2601-2518). The isolated sgMCMV-wt is more virulent compared to tissue culture derived MCMV-Wt (Pilgrim et al. 2007 Exp Mol Pathol. 82:269-279). Vaccinated B6.IFNR.sup./ mice were challenged with 210.sup.5 PFU sgMCMV-wt and 129.IFNR.sup./ mice were challenged with 2.510.sup.5 TCID.sub.50 tissue culture derived MCMV-wt.
[0184] Virus Neutralization Assay
[0185] Heat inactivated serum (56 C., 30 min) from 5 immunized mice 12 weeks p.i. were pooled and serially diluted 1:2 in DMEM containing a final concentration of 10% guinea-pig complement. Each dilution was mixed with 50 PFU of MCMV-luc and incubated for 90 min at 37 C. and subsequently added to NIH/3T3 cells in a 96 well format. After 1 h at 37 C. the virus inoculum was removed and NIH/3T3 medium added. The cultures were incubated for 24 h and luciferase activity was determined in cell extracts using the luciferase assay (Promega, Mannheim, Germany) in a luminometer (Berthold, Bad Wildbad, Germany) according to the supplier's and manufacturer's instructions, respectively.
[0186] In Vivo Cytotoxicity Assay
[0187] To evaluate CD8.sup.+ cell effector function in vivo, splenocytes of congenic CD45.1.sup.+ C Ptpr.sup.c mice were incubated with 2 M of the indicated peptide and stained with 2 M, 0.7 M, or 0.1 M carboxyfluorescein succinimidyl ester (CFSE) and PKH26 Red Fluorescent Cell Linker Mini Kit according to the manufacturer's instructions (Sigma-Aldrich). At day 6 p.i., labeled CD45.1.sup.+ cells were transferred into B6 (CD45.2.sup.+) recipients. After 16 h spleens of recipient mice were removed and flow cytometrical analysis of the target cells was performed. Specific cytotoxicity of target cells was calculated using the equation: % spec lysis=(1ratio unprimed/ratio primed)*100; ratio=(% CFSE low/% CFSE high) (Lauterbach et al. 2005 J Gen Virol 86:2401-2410). The OVA derived class I peptide (SEQ.ID.NO.9) and MCMV specific peptides derived from m139 (SEQ.ID.No.10), ie3 (SEQ.ID.No.11), M57 (SEQ.ID.No.12) and M45 (SEQ.ID.No.13) (Snyder et al. 2008 supra) were purchased from Metabion, Germany and were dissolved and stored according to manufacturer's device.
[0188] Adoptive Transfer and Flow Cytometrical Analysis
[0189] OVA specific CD8.sup.+ T cells were isolated from spleen and cervical, axillary, brachial and inguinal lymph nodes of OT-I TCR transgenic mice backcrossed to congenic CD45.1.sup.+ mice. OT-I cells were purified by negative selection via the CD8.sup.+T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). 310.sup.5 transgenic T cells were injected i.v. into recipient B6 mice one day prior to i.p. infection with 10.sup.5 TCID.sub.50 MCMV. To follow expansion of the transferred OT-I T cells 100 l blood was taken 3, 6 and 8 days p.i., erythrocytes were lysed (PharmLyse, BD Biosciences, Heidelberg, Germany) and remaining cells were incubated with PE-TexasRed coupled -CD8 (5H10; Caltag, Sacramento, Calif., USA) and PE coupled -CD45.1 antibodies (A20, BD Biosciences Pharmingen). Flow cytometrical acquisition was performed using an Epics XL-MCL (Beckman-Coulter) and data were analyzed using FlowJo software (Tristar, Ashland, Oreg., USA).
[0190] OVA specific CD4.sup.+ T cells were isolated from spleen and cervical, axillary, brachial and inguinal lymph nodes of OT-II TCR transgenic mice backcrossed to congenic CD90.1.sup.+ mice. After lysis of erythrocytes 310.sup.5 transgenic T cells were injected into recipient mice one day prior to infection with 10.sup.5 TCID.sub.50 MCMV. Spleens were removed and splenocytes were incubated with Fc block (2.4G2; BD Biosciences) and subsequently stained with PE conjugated -CD90.1 (HIS51; eBioscience) and PE-Cy5.5 coupled -CD4 (RM 4-5; eBioscience). Flow cytometrical acquisition was performed using a FAGS Calibur (BD Biosciences) and data were analyzed using FlowJo software.
[0191] Quantification of Viral Genomes in Organ Homogenates
[0192] Lungs were removed from mice twelve month after infection. Organs were homogenized and DNA was extracted using the DNeasy Blood & Tissue Kit from Qiagen (Hilden, Germany). Elution was done with 100 l of the supplied elution buffer and genomic DNA concentration of each sample was quantified in duplicates using a NanoDrop ND-1000 UV-Vis Spectrophotometer. To quantify the viral DNA a quantitative realtime PCR specific for the MCMV M54 gene (Cicin-Sain et al. 2005 supra) was performed using a specific Taqman-Probe (SEQ.ID.NO.14) and the Taqman 1000 RXN PCR Core Reagents kit on an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Carlsbad, Calif., USA). To calculate the viral genome copy number, a standard curve of the BAC plasmid pSM3fr containing the M54 gene was included.
Example 4
MCMV-M94 is Spread-Deficient
[0193] The HCMV virion protein pUL94 is essential for virus replication (Dunn et al. supra) and is expressed with late kinetics (Wing et al. supra). It has been found that pM94, the MCMV homolog, is also essential and plays a crucial role in a post nuclear step of virus maturation. In order to trans-complement the essential M94 gene product and reconstitute an M94 deletion mutant the NIH/3T3 derived complementing cell line NT/M94-7 harbouring the M94 gene under control of the TRE promoter was generated. The TRE promoter is only active in the presence of the Tet trans-activator (tTA). To provide the tTA for trans-complementation of pM94 the tTA expression cassette was introduced into pSM3fr (Messerle et al. supra) disrupting M94 generating pSM3fr-M94. MCMV-M94 virus was reconstituted by transfecting NT/MN94-7 cells (
[0194] The results of this Example are shown in
[0195] As shown in
[0196] As shown in
[0197] While MCMV-M94 replicated to MCMV-wt-like titers on NT/M94-7 cells, no infectious virus was detectable in the supernatant of NIH/3T3 cells (
[0198] Complementing NT/M94-7, parental NIH/3T3 fibroblasts and myocardium-derived endothelial cells MHEC5-T were infected with 0.1 TCID50/cell MCMV-M94-m157-rec-egfp (MCMV-M94) or MCMV-m157-rec-egfp (wt). At indicated time points EGFP expressing cells were monitored. Scale bar represents 100 m.
EXAMPLE 5
[0199] MCMV-M94 Does Not Revert to Replication Competent Virus
[0200] A major safety concern is reversion of vaccine strains to replication competent viruses during preparation (Roizman et al. 1982 Dev Biol Stand. 52:287-304) or in the vaccinated patient (Iyer et al. 2009 Ann. Emerg. Med 53:792-795). To exclude acquisition of the M94 gene through recombination via homologous sequences between MCMV-M94 and the complementing cell line homologies were carefully avoided during virus construction. Replication competent virus indicative of recombination between the deletion virus and the M94 gene expressed by NT/M94-7 was never observed. In order to investigate the safety of MCMV-M94 for vaccination studies in a highly susceptible mouse strain, 129.IFNR.sup./ mice were infected with MCMV-wt or MCMV-M94. While all IFNR.sup./ mice died within 14 days upon infection with MCMV-wt, after infection with MCMV-M94 all mice survived with no or only minimal weight loss (
EXAMPLE 6
MCMV-M94 Induces Neutralizing Antibody and T Cell Responses
[0201] Poor induction of neutralizing antibodies that prevent viral entry is a problem in HCMV infection (Landini et al. 1991 Comp Immunol Microbiol Infect Dis 14:97-105). Therefore, the neutralizing antibody response to MCMV-wt and MCMV-M94 was compared 12 weeks post immunization. Serial dilutions of sera were mixed with a luciferase expressing MCMV (MCMV-luc) prior to infection of NIH/3T3. The reduction of the luciferase signal reflected the neutralizing capacity of the antisera. Immunization with MCMV-M94 induced a slightly lower amount of neutralizing antibodies than with MCMV-wt (
[0202] The results of this example are shown in
[0203] In
[0204] In
[0205] Both CD4.sup.+ and CD8.sup.+ T cells play important roles in host defense against CMV. Antiviral CD8.sup.+ T cells are effective in controlling MCMV during acute infection and mediate protection after immunization (Reddehase et al. supra). In addition, CD4.sup.+ T helper cells are required for virus clearance in salivary glands (Jonjic et al. 1989 J Exp Med 169:1199-1212). In order to compare the level of CD4.sup.+ and CD8.sup.+ T cell responses induced by MCMV-wt and MCMV-M94, OVA as a model antigen was chosen to be expressed by the vaccine. B6 mice were infected with MCMV-ova and MCMV-ova-M94 one day after adoptive transfer of OVA specific CD4.sup.+ or CD8.sup.+ T cells. For MCMV-ova the expansion of OVA specific CD4.sup.+ and CD8.sup.+ T cells peaked at day 6 p.i., concordant with published data (Karrer et al, 2004 J Virol 78:2255-2264). Remarkably, MCMV-ova-M94 also stimulated the proliferative response of OVA specific CD8.sup.+ and CD4.sup.+ (
[0206] This observation was to be confirmed for native MCMV antigens. B6 mice were infected with MCMV-M94 or MCMV-wt. At six days p.i., target cells loaded with viral peptides derived from either m139, ie3, M57, or M45 (Snyder et al, 2008 supra) were injected and their cytolysis in vivo was analyzed (
EXAMPLE 7
Role of Viral Target Cell Types in CD8.SUP.+ T Cell Activation
[0207] The strong adaptive immune response against MCMV-M94 was surprising, since MCMV-M94 gene expression is limited to the first target cells. Induction of a specific T cell response is dependent on antigen presentation by infected cells and by professional antigen presenting cells (Villadangos et al, 2008 Immunity. 29:352-361). In order to assess the contribution of infection of different cell types in the generation of an efficient CD8.sup.+ cell response, the replication deficient MCMV was combined with conditional activation of a marker gene (Sacher et al. supra). MCMV-flox-ova-M94 was constructed which expresses OVA only after Cre-mediated recombination.
[0208] One day prior to i.p. injection of 10.sup.5 TCID.sub.50 of MCMV-flox-ova-M94 (M94-flox-ova), MCMV-ova-M94 (M94-ova), MCMV-wt (wt) or PBS 310.sup.5 congenic OT-I CD8.sup.+ T-cells were transferred i.v. into B6, Alb-cre and Tie2-cre mice. At day 6 p.i. a flow cytometrical analysis was performed on PBL for the congenic marker CD15.1 and CD8. Boxes represent the ratio of OT-I cells per CD8.sup.+ cells as a pool of 3 independent experiments and extend from the 25 to the 75 percentile. The lines indicate the median. Whiskers extend to show the extreme values. The P-values were obtained applying a two-tailed Wilcoxon rank sum test, (**, P<0.01; ***, P<0.001). The results are shown in
[0209] Endothelial cells (EC) and hepatocytes (H) are among the first target cells infected by MCMV in vivo (Sacher et al. supra). Whether these cell types contribute to CD8.sup.+ T cell activation was addressed by infecting mice that express Cre recombinase selectively in vascular EC (Tie2-cre) or He (Alb-cre). One day after adoptive transfer of OVA specific CD8.sup.+ T cells mice were infected with 10.sup.5 TCID.sub.50 of spread-deficient MCMV-flox-ova-M94. He are the main producers of infectious virus during the first few days of infection and are highly effective in activating a conditional marker gene by Cre recombinase (Sacher et al. supra). Yet, selective induction of OVA expression in MCMV infected He resulted in only weak proliferation of OVA specific CD8.sup.+ T cells (
[0210] The experimental details in connection with this example were, in addition to the ones outlined in Example 3, as follow and the results of this example are depicted in
[0211] B6 mice (n=5) were immunized (1.sup.st) s.c. or. i.p. with 10.sup.5 TCID.sub.50 MCMV-wt (wt; closed symbols), MCMV-M94 (M94; open symbols), m01-17+m144-158-MCMV (, gray symbols) or PBS (light gray symbols). Virus preparations were UV irradiated before immunization (UV) as indicated. Optionally, mice were boosted (2.sup.nd) two weeks later with the same dose, route and virus. Challenge infection was applied i.v. 20 (A) or four weeks (B) post prime with 10.sup.6 PFU MCMV-wt. Five day post challenge plaque assay was performed. Horizontal bars show the median of each group. Each symbol represents one individual mouse. DL=detection limit.
EXAMPLE 8
MCMV-M94 Protects Against Challenge with MCMV-wt
[0212] In order to test protection of MCMV-M94 against lethal challenge, B6 mice were infected with either spread-deficient MCMV-M94, the attenuated strain m01-17+m144-158-MCMV (Cicin-Sain et al. 2007 J Virol 81:13825-13834) or MCMV-wt. A boost infection was applied 4 weeks later with the same dose. 20 weeks after priming mice were challenged i.v. with 10.sup.6 TCID.sub.50 tissue culture derived MCMV-wt. Most remarkably, already a singular immunization dose of MCMV-M94 was already sufficient to strongly suppress MCMV-wt replication by 10,000 fold in lungs, 1,000 fold in liver and at least 100 fold in spleen, whereas non-immunized controls had high virus loads in all organs tested (all P<0.01;
[0213] It was asked, whether the strong protection after singular administration of MCMV-M94 could also be realized in a short-term vaccination protocol. In addition, the influence of two different application routes was tested. B6 mice were injected either i.p. or s.c. followed by challenge infection with MCMV-wt only 4 weeks later. Here, vaccination with MCMV-M94 resulted in about 100 fold reduction of challenge virus load in liver (P<0.05), lungs (P<0.01) and spleen (P<0.01;
[0214] Summarized, vaccination with the spread-deficient MCMV-M94 was able to efficiently protect immunocompetent mice against challenge with MCMV-wt after vaccination with a singular dose. Remarkably, vaccination with MCMV-M94 was as efficient as vaccination with MCMV-wt concerning long-term vaccination, whereas the use of UV inactivated virus could not compete even after a second application.
EXAMPLE 9
Protection of Severely Immune Compromised Recipients
[0215] Type I interferons are key cytokines in the immune response against CMV and deletion of their receptor results in a mouse (IFNR.sup./) that is severely immunocompromised and at least 1.000-fold more susceptible to MCMV infection than the parental mouse strain (Presti et al. 1998 J Exp Med 188:577-588). Since spread-deficient MCMV-M94 was proven to be well tolerated by IFNR.sup./ mice (
[0216] The results of this Example are shown in
[0217] In
[0218] In
[0219] B6 mice profit from an Ly49H-dependant activation of natural killer cells resulting in a strong innate immune response stimulated by the MCMV protein encoded by m157 (Sun et al. 2008. J. Exp. Med. 205:1819-1828.). 129.IFNR.sup./ mice do not express Ly49H and are even more susceptible to MCMV infection than B6.IFNR.sup./ mice. 129.IFNR.sup./ mice were vaccinated with MCMV-M94 and challenged 4 weeks later with a dose of 2.510.sup.5 TCID.sup.50 tissue culture derived MCMV-wt (
EXAMPLE 10
Maintenance of the MCMV-M94 Genome In Vivo
[0220] One argument against the application of attenuated life vaccines is their ability to establish a latent infection that bears the risk of reactivation (Iyer et al, supra). On the other hand non-productive reactivation episodes might result in endogenous boosts of the antiviral immune response (Snyder et al, 2008 Immunity 29:650-659). Thus, it was intriguing to test whether MCMV-M94 genome is maintained in vaccinated hosts. Quantitative PCR analysis on total DNA extracted from lungs, a key manifestation site of CMV disease (Balthesen et al. 1993 J Virol 67:5360-5366), was performed. Twelve months p.i. genomes of MCMV-M94 could be detected in all mice tested (
[0221] The results of this example are shown in
[0222] B6 mice were infected i.p. with 10.sup.5 TCID.sub.50 MCMV-wt (vet) (n=5) or MCMV-M94 (M94) (n=6). Twelve months p.i. total DNA was extracted from lungs. (
EXAMPLE 11
Vaccination with MCMV-M94 Prevents Replication of Virus in the Respiratory Tract
[0223] From epidemiological studies it was suggested that saliva is an important route of transmission of HCMV (Pass et al. 1986 N. Engl. J Med 314:1414-1418.). To test whether the vaccine MCMV-M94 is able to block virus replication in salivary glands and lungs C57BL/6 mice were immunized with MCMV-M94 or control viruses and received twelve months later a challenge infection with 10.sup.6 PFU MCMV-wt i.v. (
EXAMPLE 12
Discussion
[0224] It is reported herein on the vaccination against a beta-herpesvirus using a spread-deficient vaccine. The vaccine induced a strong adaptive immune response comparable to MCMV-wt conferring protection even in highly immune compromised mice. This means that infection of the first target cells is sufficient for successful vaccination.
[0225] An intact immune system usually protects against HCMV disease. Hence, the antigenic capacity of the wild type virus is sufficient for the induction of a protective immune response. The inability of UV inactivated virus to protect efficiently against challenge infection demonstrated the need for viral antigen expression including nonstructural antigens (Cicin-Sain et al. 2007 supra; Gill et al. 2000 J Med Virol 62:127-139). As a consequence an ideal vaccine should exploit the full immunogenic but avoid the pathogenic potential of the wild type virus.
[0226] The alpha-herpesvirus field has pioneered the use of replication defective viruses as vaccines (Dudek et al. supra). These vaccines were generated by the deletion of genes essential for virus replication and are thus apathogenic (Dudek et al. supra). Now, to construct a spread-deficient beta-herpesvirus vaccine deletion of M94 was chosen for the following reasons. First, M94 is essential for spread of MCMV and inferred from studies of HCMV it should he expressed with late kinetics during virus replication (Scott et al. supra; Wing et al. supra). Second, pM94 does not belong to the group of glycoproteins which comprise major targets for the neutralizing antibody response of HCMV. Third, M94 of MCMV is the homolog of UL94 in human CMV (Wing et al, supra) that in principle allows translation to the human pathogen. Finally, the deletion of UL94 of HCMV might even be of advantage because pUL94 induces autoreactive antibodies that are associated with systemic sclerosis (Lunardi et al. 2000 Nat Med 6:1183-1186). The SSc cross-reactive UL94 peptide is depicted in SEQ.ID.:NO: 28. Interestingly, genomes of the spread-deficient MCMV-M94 were detected in lungs after i.p. infection, showing that virus can disseminate either as free particles (Hsu et al. 2009 J Gen Virol 90:33-43) or associated to cells. Monocytes and macrophages were shown to be attracted to the peritoneal cavity after infection and transport the virus in blood (Stoddart et al. 1994 J Virol 68:6243-6253; van der Strate et al. 2003 J Virol 77:11274-11278). These cells could also release virus at distant sites to infect EC or other cell types, a process called trans infection (Halary et al. 2002 Immunity 17:653-664).
[0227] The spread-deficient betea-herpesvirus vaccine presented here, has a strong protective capacity similar to wild type CMV infection. The immune response of the vaccinee controls virus replication in all analysed organs preventing overt CMV-disease. The absence of detectable amounts of infectious virus in salivary glands of long-term vaccinated mice two weeks after challenge implies that also horizontal transmission to other individuals via saliva is abrogated. Because of this it is plausible that such an equivalent vaccine will protect against HCMV-disease, similar to the protective effect of a pre-existing infection. This is supported by the observation that women who were exposed to HCMV were at lower risk to give birth to children with symptomatic disease compared to non-infected women (Fowler et al. 2003 JAMA 289:1008-1011.). The seropositivity of the mother could not prevent infection but pathogenesis of the children. In addition, frequent exposure to different CMV strains could further increase immunity against reinfection (Adler et al. supra). It is therefore again plausible that a spread-deficient human CMV vaccine induces an immune response equal to natural infection which will protect against symptomatic human CMV infection without the risk for reactivation and pathogenesis.
[0228] The immune response to MCMV-M94 reached a level comparable to MCMV-wt. Protection was similar to the recently generated vaccine m01-17+m144-158-MCMV (Cicin-Sain et al. 2007 supra) which lacks 32 viral genes but which is not spread-deficient in vitro. In m01-17+m144-158-MCMV immune evasive genes were deleted to increase the antiviral immune response and thereby to attenuate the virus (Scalzo et al. 2007 Immunol Cell Biol. 85:46-54.).
[0229] It is within embodiments of the present invention that (a) at least one essential gene and (b) at least one immune evasive gene is deleted, whereby it is preferred that the deleted at least one immune evasive gene is selected from the group comprising genes encoding gene products affecting antigen presentation, interaction with cytokines, the complement system and humoral immunity. More preferably, the deleted at least one immune evasive gene is selected from the group comprising genes encoding gene products that down-regulate MHC I to avoid CTL response, gene products that evade the NK cell response, gene products that interfere with MHC II presentation, down-regulate adhesion molecules, gene products that interact with IL-1, gene products that activate TGF-.
[0230] Infection of susceptible IFNR.sup./ mice with spread-deficient MCMV proved the safety of the vaccination concept. Furthermore, IFNR.sup./ mice were protected against otherwise lethal challenge, similar to other infection models (Calvo-Pinilla et al, 2009 PLoS One. 4:e5171; Paran et al. 2009 J Infect Ibis 199:39-48). Although recent work revealed the capacity of MCMV to efficiently induce type I interferon (Hokeness-Antonelli et al. 2007 J Immunol 179:6176-6183), the efficacy of the spread-deficient MCMV vaccine in IFNR.sup./ mice implies that type I interferon-dependent immunity is not essential in the protection conferred by short term vaccination.
[0231] Interestingly, the spread-deficient MCMV induced an adaptive immune response with similar efficiency as MCMV-wt. The CD4.sup.+ and CD8.sup.+ T cell response was on the same level as MCMV-wt and the neutralizing antibody response was only marginally reduced. This slightly lower neutralizing capacity might be caused by the smaller number of infected cells and by the therefore reduced amount of antigen that is released after infection with MCMV-M94. A lower number of antigen-antibody complexes might lead to less efficient affinity maturation creating antibodies of lower neutralizing capacity. Nevertheless, the neutralization of virus appears sufficient to control virus replication.
[0232] Why did the adaptive immune response to the vaccine reach a level near to MCMV-wt infection despite the inability to spread? MCMV-M94 was able to establish viral genome maintenance as efficient as MCMV-wt. The classical definition of herpesviral latency includes the potential for reactivated gene expression with subsequent release of infectious virus (Roizman et al. 1987 Annu Rev Microbiol 41:543-571.). Although the term latency is formally not applicable to the situation with MCMV-M94 in the absence of productive infection, there is no evidence that pM94 affects reactivation of gene expression. Because the protective effect of MCMV-M94 rather increased than faded over time, the inventors believe that periodic restimulation of the immune response due to reactivation of gene expression contributed to the sustained protection induced by MCMV-M94. Interestingly, virus infected cells are not eliminated by the activated immune response. This means that the first target cells that are infected by the spread-deficient vaccine are resistent to elimination. Similarly, cells infected with a spread-deficient mutant of the gamma herpesvirus MHV-68 were not attacked by the adaptive immune reponse (Tibbetts et al. 2006 Virology 353:210-219). For MCMV-wt it was shown that T cells are activated against a highly antigenic virus epitope of M45 presented by professional APC but the activated T cells did not eliminate infected target cells in organs of C57BL/6 mice (Holtappels et al. 2004 J Exp Med 199:131-136). This protection was caused by m152, that is known to downmodulate MHC class I. The target cells that are protected from CD8.sup.+ T cell elimination were not identified and it could be shown that at least some of these protected cells are first target cells of MCMV.
[0233] Endothelial cells (EC), hepatocytes (He) and macrophages are first target cells for HCMV and MCMV in vivo (Hsu et al. supra; Sacher et al, supra). In addition, EC have recently been identified as sites of virus latency (Seckert et al. 2009 J Virol 83:8869-8884), and at least liver EC are able to directly stimulate a cytotoxic T cell response (Kern et al, 2010 Gastroenterology 138(1):336-46). Using MCMV-M94 constructs for conditional gene expression, substantial differences were noticed in the ability of EC and He to activate a CD8.sup.+ T cell response. In contrast to EC, He one of the most important first targets for MCMV during acute infection (Sacher et al, supra), induced only a poor CD8.sup.+ T cell response. This lack of stimulatory capacity is apparently not compensated by cross presentation through professional antigen presenting cells. Cross presentation was shown to be important for the induction of a T cell response against fibroblasts infected with a single-cycle MCMV (Snyder et al. 2010 PLoS One. 5:e9681). On the other hand, bone marrow derived APC, that are thought to be important cross presenting cells, seem not to be necessary for the activation of a CD8.sup.+ T cell response via cross presentation against MCMV infection (Kern et al. supra). In addition to EC also other cell types seem to contribute to CD8.sup.+ T cell stimulation as antigen expression in most infected cells led to a stronger T cell response than expression in infected EC only. Infected dendritic cells and macrophages were described to activate a T cell response against MCMV in vitro (Mathys et al. 2003 J Infect Dis 187:988-999) and are infected in vivo (Andrews et al. 2001 Nat Immunol 2:1077-1084). Therefore, it suggests itself that infected professional APC contribute to immune stimulation against MCMV in addition to EC. It appears noteworthy that the attenuated human CMV strains such as Towne and AD169 which are characterized by a 20-fold reduction of immunogenicity and the inability to confer immune protection (Adler et al. supra) accumulated mutations resulting in their inability to infect EC, epithelial cells, smooth muscle cells and macrophages (Hahn, G. et al. 2004 J Virol 78:10023-10033). Thus, it appears likely that the restricted cell tropism may in fact represent the cause for their failure as human CMV vaccines.
EXAMPLE 13
Spread-Assay of MCMV-M94
[0234] The phenotype of MCMV-M94 was analyzed in cell-to-cell spread. This was investigated by an in vitro spread assay as essentially described herein in Example 1 with the following mo modifications
[0235] The results of this Example are shown in
[0236] NIH/3T3 and NT/M94-7 cells were plated and infected with MCMV1-16-FRT (del1-16) and MCMVM94tTA () at an MOI of 0.25 for 1 h and then washed twice with D-PBS. Cells were incubated for 6 h and afterwards washed four times with D-PBS. Equal numbers of non-infected cells were stained with 5 M Carboxyfluorescein succinimidyl ester (CFSE) for 8 min and blocked by 2% FCS/D-PBS, then washed twice with 2% FCS/D-PBS, and subsequently seeded on top of the unstained but infected cells. Cells were fixed 48 hours post infection with 4% PEA in D-PBS for 10 min at 37 C. and washed and permeablized with 0.1% Triton X-100 for 10 min. After triple washing cells were blocked with 3% BSA/D-PBS for 1 h. Staining of immediate early gene products was performed by incubating fixed cells with a monoclonal antibody to MCMV immediate-early 1 in 3% BSA/D-PBS. After three D-PBS washes, cells were incubated with an Alexa Fluor 555-coupled anti-mouse secondary antibody (Invitrogen) in 3% BSA/D-PBS. Finally, cells were washed three times and imaged by confocal microscopy using a LSM 510 Meta (Zeiss). Virus transmission was determined by counting immediate-early 1- and CFSE-positive cells using the ImageJ Cell Counter plugin.
[0237]
EXAMPLE 14
Propagation of Spread-Deficient Human CMV
[0238] Generation of the Trans-Complementing Cell Line TCL94/99-BP
[0239] Recombinant lentiviruses expressing a) UL99 coupled with EGFP (encoded by pCB-Ubic-UL99-IRES-gfp; SEQ.ID.No:18), b) UL99 coupled with UL94 mCherry (encoded by pCB-Ubic-UL94-IRES-mChe; SEQ.ID.No:17) and c) beta-lactamase coupled with puromycine resistance gene (encoded by pLV-Ubiqc-BLAS-IRES-Puro; SEQ.ID.No:19) were constructed and propagated by Sirion GmbH using ViraPower lentiviral packaging mix (Invitrogen) in 293FT cells (Invitrogen). 210.sup.6 MRC5 fibroblasts (ATCC CCL-171) were transduced by 5 TDU/cell (transduction units/cell) of each lentivirus by spin infection according to the manufacturer's protocol. The transduced cells were plated out on a 10 cm dish and were selected for 5 days with 20 g/ml puromycin in OPTI-MEM 5% FCS. The tranduced cells were passaged (1:2) one time in the presence of 20 g/ml puromycin and the double positive (mCherry+EGFP) cells were purified by fluorescence associated cell sorting and re-plated at density of 2.510.sup.4 cell/cm.sup.2. 48 h after confluency the cells were passaged (1:5) two more times in the presence of 20 g/ml puromycin and re-sorted as above. After one more passage in OPTI-MEM 5% FCS+20 g/ml puromycin the cells were aliquoted to 0.710.sup.7 cell/vial and were deep frozen OPTI-MEM supplemented with 10% PCS and 10% DMSO.
[0240] Construction of Spread-Deficient Human CMV
[0241] To generate a non-functional UL94 locus pTB40E-BAC4-FRT; SEQ.ID.No:20 (Serivano L, et al., 2011. HCMV spread and cell tropism are determined by distinct virus populations. PLoS. Pathog. 7:e1001256; Sinzger, C. et al., 2008. Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J. Gen. Virol. 89:359-368.) was introduced in GS1783 E. coli strain (Tischer, B. K. et al., 2010. En passant mutagenesis: a two step markerless red recombination system. Methods Mol. Biol. 634:421-430.). (a) Red-recombination was induced by electro-transformation of the synthetic DNA fragment LIFdel94; SEQ.ID.No:15 according to the standard protocol (Tischer, B. K. et al., supra) resulting in pTB40E-BAC4-delUL94-SZeo. Recombinants were selected by picking single clones after plating the transformants on LB agar plates in the presence of 25 g/ml chloramphenicol and 30 g/ml zeocin. The correct replacement of the BAC sequences from nt122630 to 123668 reffering to SEQ.ID.No:20 with LIFdelUL94, SEQ.ID.No:15 was confirmed by restrictions pattern analysis and sequencing. (b) To remove the zeocin cassette from the UL94 locus, a second round of Red recombination was induced in liquid culture of pTB40E-BAC4-delUL94-Szeo according to the standard protocol (Tischer, B. K. et al., supra) in presence of 25 g/ml chloramphenichol and 2% of L-arabinose. Recombinants, which were coined pTB40E-BAC4-del94, were selected by picking single clones after plating of the recombinants on LB agar plates in the presence of 25 g/ml chloramphenicol 1% of L-arabinose. The correct removal of the operational sequences were confirmed by restrictions pattern analysis and sequencing. (c) A next red-recombination was induced by electro-transformation of the synthetic mutagenesis fragment LIFdel99, SEQ.ID.No:16, as described above (see a) herein) resulting in pTB40E-BAC4-delUL94-del99-SZeo. Recombinants were selected by picking single clones after plating the transformants on LB agar plates in the presence of 25 m/ml chloramphenicol and 30 g/ml zeocin. The correct replacement of the sequences from nt 130670 to 1:31243 (according to the numbering of the BAC referred to herein as SEQ.ID.No:20) was confirmed by restrictions pattern analysis and sequencing. (d) To remove the zeocin cassette from the UL99 locus, a final round of red-recombination was induced in liquid culture of pTB40E-BAC4-delUL94-delUL99-Szeo as above (see b) herein). Recombinants, which were coined pTB40E-BAC4-del94-del99, were selected by picking single clones after plating of the recombinants on LB agar plates in the presence of 25 g/ml chloramphenicol 1% of L-arabinose. The correct removal of the operational sequences from the UL99 locus were confirmed by restrictions pattern analysis and sequencing. 1. The description of the BAC modifications in the new way are the following:
[0242] M1) To generate a non-functional UL94 (or inactivate the UL94 gene) the nt sequence of pTB40E-BAC4-FRT (SEQ.ID.No:20) between nt 122630 and nt 123668 is replaced by the synthetic fragment delUL94S (SEQ.ID.No:34).
[0243] M2) To generate a non-functional UL99 (or inactivate the UL99 gene) the nt sequence of pTB40E-BAC4-FRT (SEQ.ID.No:20) between nt 130670 and nt 131243 is replaced by the synthetic fragment delUL99S (SEQ.ID.No:35). For the double mutant of UL94-UL99 this has to be done in addition to modification M1.
[0244] M3) To generate a non-functional UL50 (or inactivate the UL50 gene) the nt sequence of pTB40E-BAC4-FRT (SEQ.ID.No:20) between nt 58442 and nt 59622 is replaced by the synthetic fragment delUL50S (SEQ.ID.No:32).
[0245] M4) To generate a non-functional UL53 (or inactivate the UL53 gene) the nt sequence of pTB40E-BAC4-FRT (SEQ.ID.No.:20) between nt 62129 and at 63261 is replaced by the synthetic fragment delUL53S (SEQ.ID.No:33). For the double mutant of UL50-UL53 this has to be done in addition to modification M3.
[0246] Reconstitution of Spread-Deficient Human CMV.
[0247] 0.710.sup.7 frozen TCL94/99-BP cells were plated on a 10 cm dish in OPTI-MEM 5% FCS containing 0.2 g/ml puromycin and two days later the adherent cell were split and plated on 6 cm dishes at densities of 210.sup.6 cells per dish. On the next day two 6 cm cultures were transfected with 2 g of purified pTB40E-BAC4-FRT-del94-del99-DNA each by Lipofectamin 2000 (Invitrogen) according to the manufacturer's protocol. 24 h later the two culture were combined and plated on a 10 cm dish in OPTI-MEM 5% ECS. After 10 days the reconstitution of the recombinant TB40E-BAC4-FRT-del194-del99 virus was evident by plaque formation. After 14-16 days the most of the cells in the transfected cultures showed CPE the entire culture was harvested. The amounts of the viable viruses was determined by limiting dilution on sub-confluent TCL94/99-BP cell in 96 well plates using TCID50 (median tissue culture infectious dose) method as described in Mohr et al (Mohr, C. A. et al., 2010. A spread-deficient cytomegalovirus for assessment of first-target cells in vaccination. Virol. 2010 August; 84(15):7730-42. Epub 2010 May 12.). The spread-deficient human CMV reconstituted from TB40E-BAC4-FRT-del94-99, can be propagated using TCL94/99-BP cells after infection with 0.1 MOI per cell using standard protocols for propagation of human CMV as described by Scrivano et al. (Scrivano et al., supra),
[0248] HCMV lacking secondary envelopment complex, i.e. UL99 and UL94, is spread-deficient.
[0249] The phenotype of the UL94-UL99 double deletion CMV reconstituted from TB40E-BAC4-FRTdel94-99 was tested in cell-to-cell spread. This was investigated by infection of MRC5 and TCL94/99-BP cells as essentially described in Example 1 herein, with CMVs reconstituted from TB40E-BAC4-FRT-de194-del99 and TB40E-BAC4-FRT, respectively, followed by removal of excess virus by extensive washing after infection. Next, CFSE stained MRC5 cells were added and virus replication was permitted. After additional 72 h the culture was fixed and stained for immediate-early 1 expression as described in Example 1 herein. This resulted in cells which were either immediate-early 1-positive, CFSE-positive or positive for both stains. These cells were counted in each preparation. The missing increase of double positive cells in MRC5 after infection with TB40E-BAC4-FRT-del94-del99 is conclusive to a deficiency in cell-to-cell spread.
EXAMPLE 15
Immunization with Spread-Deficient Human CMV
[0250] After primary immunization with an additional boost with spread-deficient human CMV the human sera exhibit at least 64-fold higher neutralizing potency against endotheliotropic a human CMV strains such as TB40E or VR1814 assayed on endothelial-or epithelial cells (such as HUVEC [ATCC CRL 1730] or ARPE-19 [ATCC CRL2302], respectively, than against the same virus assayed on human fibroblasts cell line (such as MRC5, ATCC CLL-171). In addition, specific antibody response is detectable against the gene products of UL130, UL128, or UL131A by Western blot (whereby it is sufficient that at least one specificity is seen).
[0251] The following deletions of the indicated genes result in recombinant human beta-herpesviruses which are spread-deficient:
TABLE-US-00002 Effector complex UL50 gene UL53 gene UL94 gene UL99 gene NEC + NEC + NEC + + SEC + SEC + +
[0252] The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof. It has to be acknowledged that the sequence listing is part of the instant specification.