HCMV ENTRY INHIBITORS
20210380664 · 2021-12-09
Assignee
Inventors
- Christian SINZGER (Reutlingen, DE)
- Cora STEGMANN (Ulm, DE)
- Kerstin LAIB SAMPAIO (Dettingen, DE)
- Barbara ADLER (Munich, DE)
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
C07K2319/74
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Subject matter of the present invention is a soluble PDGFR-alpha-Fc chimera or a PDGFR-alpha derived peptide or an anti-PDGFR-alpha antibody or a PDGFR-alpha antibody fragment or anti-PDGFR-alpha non-Ig scaffold for inhibiting HCMV entry for use in a method of treatment in a subject that has been infected by HCMV or for use in a method of prophylaxis of HCMV infection in a subject that has not yet been infected by HCMV.
Claims
1. A method for treatment of a subject that has been infected by HCMV, comprising: administering to said subject a soluble PDGFR-alpha-Fc chimera, wherein said soluble PDGFR-alpha-Fc chimera comprises a PDGFR-alpha sequence selected from the following group: I. SEQ ID No. 2 consisting of amino acides 24 to amino acids 524 of SEQ ID No. 1: TABLE-US-00011 QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRN EENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPD PDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYD SRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKT VYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVY TLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGFIEIKPTFSQ LEAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENLTEITTDVEKIQEI RYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPSSILDLVDD HHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANNVSNII TEIHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRS E, II. a sequence having 90% or more identity to SEQ ID No. 2, III. a sequence that is a truncated sequence of SEQ ID No. 2 with at least 45 amino acids or a sequence having 90% or more identity to said truncated SEQ ID No. 2, and IV. variants of sequences according to items I., II., III. having substitutions atone or more of the following positions (numbering adhered to SEQ ID. No. 1): Ile-30, Glu-52, Ser-66, Ser-67, Asp-68, Leu-80, Ser-89, His-162, Pro-169, Asp-173, Ile-188, Val-193, Lys-194, Glu-213, Lys-304, Thr-320, His-334, Arg-340, Ile-373, Lys-378, Ala-396, Ala-401, Thr-436, Thr-440, Ile-453, Val-469, Ile-476, Ser-478, Asp-480, Ser-482, Arg-487.
2. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera has at least one of the following mutations or deletions within SEQ ID No. 2 (numbering adhered to SEQ ID. No. 1): i. Deletion of aa 150-189, ii. SEQ ID No. 2 having at least one point mutation in the protein region as specified in the above item i.
3. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera has at least one of the following mutations or deletions within SEQ ID No. 2 (numbering is adhered to SEQ ID No. 1): i) Deletion of amino acids M133-I139; ii) Deletion of amino acids V184-G185; iii) Deletion of amino acids N204-Y206; iv) Deletion of amino acids T259-E262; v) Deletion of amino acids Q294-E298; vi) SEQ ID No. 2 having at least one point mutation in at least one of the protein regions as specified above under items i., ii., iii., iv., or v.
4. The method according to claim 1, wherein said subject is: (a) a pregnant woman who is infected by HCMV, (b) a congenitally HCMV-infected child, (c) a bone marrow transplant recipient infected with HCMV, or (d) a solid organ transplant recipients infected with HCMV.
5. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera comprises a sequence of SEQ ID No. 3.
6. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera comprises a sequence of human Fc as depicted in SEQ ID No. 8.
7. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera is suitable for binding specifically to HCMV.
8. A method for treatment of a subject that has been infected by HCMV comprising: administering to said subject a PDGFR-alpha peptide fragment, wherein said peptide fragment is selected from: I. SEQ ID No. 9; II. SEQ ID No. 10; M. SEQ ID No. 11; IV. SEQ ID No. 12; V. SEQ ID No. 13; VI. a peptide fragment of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13, any of them having at least 10 amino acids, and VII. a variant of the above items I to VI that exhibits at least 80% sequence identity to the peptide having the sequence of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 that exhibits at least 80% sequence identity to the peptide fragment of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13, said variant having a length of at least 10 amino acids.
9. The method according to claim 8, wherein said subject is: (a) a pregnant woman that is infected by HCMV, (b) a congenitally HCMV-infected child, (c) a bone marrow transplant recipient infected with HCMV or at risk of HCMV infection, or (d) a solid organ transplant recipient infected with HCMV or at risk with HCMV infection.
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. A method for prophylaxis of HCMV infection in a subject that has not yet been infected by HCMV, comprising: administering to said subject a soluble PDGFR-alpha-Fc chimera, wherein said soluble PDGFR-alpha-Fc chimera comprises a PDGFR-alpha sequence selected from the following group: I. SEQ ID No. 2 consisting of amino acids 24 to amino acids 524 of SEQ ID No. 1: TABLE-US-00012 QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYV PDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPA SYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEME ALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPS IKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGFIEI KPTFSQLEAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENLTEITTD VEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPS SILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTI LANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRE LKLVAPTLRSE, II. a sequence having 90% or more identity to SEQ ID No. 2, III. a sequence that is a truncated sequence of SEQ ID No. 2 with at least 45 amino acids or a sequence having 90% or more identity to said truncated SEQ ID No. 2, and IV. variants of sequences according to items I., II., III. having substitutions atone or more of the following positions (numbering adhered to SEQ ID. No. 1): Ile-30, Glu-52, Ser-66, Ser-67, Asp-68, Leu-80, Ser-89, His-162, Pro-169, Asp-173, Ile-188, Val-193, Lys-194, Glu-213, Lys-304, Thr-320, His-334, Arg-340, Ile-373, Lys-378, Ala-396, Ala-401, Thr-436, Thr-440, Ile-453, Val-469, Ile-476, Ser-478, Asp-480, Ser-482, Arg-487.
16. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera, has an EC50 value lower than 100 ng/ml.
17. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera comprises a PDGFR-alpha sequence having at least one of the following mutations or deletions within SEQ ID No. 2 (numbering is adhered to SEQ ID No. 1): i. Deletion of amino acids M133-I139 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids; further it is possible also to delete one or two or three or four or five amino acids less than amino acids M133-I139 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids M133-I139, ii. Deletion of amino acids V184-G185 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids, wherein each of the respective combinations of additional deletions; Furthermore, it is possible also to delete one amino acid less than amino acids V184-G185 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids V184-G185, iii. Deletion of amino acids N204-Y206 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids; furthermore, it is possible also to delete one or two amino acid less than amino acids N204-Y206 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids N204-Y206; iv. Deletion of amino acids N240-L245 (optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids; further it is possible also to delete one or two or three or four or five amino acids less than amino acids N240-L245 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids N240-L245, v. Deletion of amino acids T259-E262 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids, wherein each of the respective combinations of additional deletions; further it is possible also to delete one or two or three or four amino acids less than amino acids T259-E262 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids T259-E262; vi. Deletion of amino acids K270-T273 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids, wherein each of the respective combinations of additional deletions; further it is possible also to delete one or two or three amino acids less than amino acids K270-T273 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids K270-T273; vii. Deletion of amino acids Q294-E298 optionally having additional deletions at the N- and/or C-termini of at least one or at least two or at least three N-terminal amino acids and/or at least one or at least two or at least three or at least four or at least five C-terminal amino acids; further it is possible also to delete one or two or three or four amino acids less than amino acids Q294-E298 at the N-terminus and/or the C-terminus, wherein all possible combinations of fewer deleted amino acids are possible, provided that at least one amino acid remains deleted in the stretch of amino acids Q294-E298; and viii. SEQ ID No. 2 having at least one point mutation in at least one of the protein regions as specified above under items i., ii., iii., iv., v., vi, or vii.
18. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera comprises a PDGFR-alpha sequence selected from the following group: I. a sequence having 90% or more identity to SEQ ID No. 2, II. a sequence that is a truncated sequence of SEQ ID No. 2 or a sequence having 90% or more identity to said truncated SEQ ID No. 2 said sequence having at least 45 amino acids, and III. variants of sequences according to the yet aforementioned items I. and II., with substitutions at one or more of the following positions (numbering is adhered to SEQ ID. No. 1): Ile-30, Glu-52, Ser-66, Ser-67, Asp-68, Lu-80, Ser-89, His-162, Pro-169, Asp-173, Ile-188, Val-193, Lys-194, Glu-213, Lys-304, Thr-320, His-334, Arg-340, Ile-373, Lys-378, Ala-396, Ala-401, Thr-436, Thr-440, Ile-453, Val-469, Ile-476, Ser-478, Asp-480, Ser-482, Arg-487.
19. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera comprises a sequence selected from the following group: TABLE-US-00013 SEQ ID No. 3: QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYV PDPDVAFVPLGMTDYLVIVEDDDSAIIPEATVKGKKFQTIPFNVYALKA TSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGIT MLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTIS VHEKGFIEIKPTFSQLEAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLI ENLTEITTDVEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYT FELLTQVPSSILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKK CNNETSWTILANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAK NLLGAENRELKLVAPTLRSE, SEQ ID No. 4: QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYV PDPDVAFVPLGMTDYLVIVEDDDSAIIPCAVFNNEVVDLQWTYPGEVKG KGITMLEEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKK VTISVHEKGFIEIKPTFSQLEAVNLHEVKHFVVEVRAYPPPRISWLKNN LTLIENLTEITTDVEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAV KSYTFELLTQVPSSILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMICK DIKKCNNETSWTILANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVR CLAKNLLGAENRELKLVAPTLRSE, SEQ ID No. 5: QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYV PDPDVAFVPLGMTDYLVIVEDDDSAIIPAARQATREVKEMKKVTISVHE KGFIEIKPTFSQLEAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENL TEITTDVEKIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFEL LTQVPSSILDLVDDHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNN ETSWTILANNVSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAKNLL GAENRELKLVAPTLRSE, SEQ ID No. 6: QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYV PDPDVAFVPLGMTDYLVIVEDDDSAIIP, SEQ ID No. 7: YYNHTQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIP.
20. The method according to claim 1, wherein said soluble PDGFR-alpha-Fc chimera further comprises the sequence of human Fc that is SEQ ID No. 8: TABLE-US-00014 LTVAGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
21. A method for inhibiting HCMV entry into a cell, comprising: bringing said cell into contact with a soluble PDGFR-alpha-Fc chimera, wherein said soluble PDGFR-alpha-Fc chimera comprises a PDGFR-alpha sequence selected from the following group: I. SEQ ID No. 2 consisting of amino acids 24 to amino acids 524 of SEQ ID No. 1: TABLE-US-00015 QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNE ENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDP DVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDS RQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTV YKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYT LTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGFIEIKPTFSQL EAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENLTEITTDVEKIQEIRY RSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPSSILDLVDDHH GSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANNVSNIITEI HSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSE, II. a sequence having 90% or more identity to SEQ ID No. 2, III. a sequence that is a truncated sequence of SEQ ID No. 2 with at least 45 amino acids or a sequence having 90% or more identity to said truncated SEQ ID No. 2, and IV. variants of sequences according to items I., II., III. having substitutions atone or more of the following positions (numbering adhered to SEQ ID. No. 1): Ile-30, Glu-52, Ser-66, Ser-67, Asp-68, Leu-80, Ser-89, His-162, Pro-169, Asp-173, Ile-188, Val-193, Lys-194, Glu-213, Lys-304, Thr-320, His-334, Arg-340, Ile-373, Lys-378, Ala-396, Ala-401, Thr-436, Thr-440, Ile-453, Val-469, Ile-476, Ser-478, Asp-480, Ser-482, Arg-487.
22. A method for inhibiting HCMV entry into a cell, comprising: bringing said cell into contact with a PDGFR-alpha peptide fragment, wherein said peptide fragment is selected from: I. SEQ ID No. 9; II. SEQ ID No. 10; III. SEQ ID No. 11; IV. SEQ ID No. 12; V. SEQ ID No. 13, VI. a peptide fragment of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13, any of them having at least 10 amino acids, and VII. a variant of the above items I to VI that exhibits at least 80% sequence identity to the peptide having the sequence of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 that exhibits at least 80% sequence identity to the peptide fragment of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13, said variant having a length of at least 10 amino acids.
23. A method for inhibiting HCMV entry into a cell, comprising: bringing said cell into contact with a delivery vector for transferring a nucleic acid sequence encoding a PDGFR-alpha derived peptide or fragment thereof suitable for inhibiting HCMV entry, wherein the peptide or fragment is releasable from the delivery vector to bind to HCMV and inhibit infection of said cell.
24. The method according to claim 23, wherein said nucleic acid sequence comprises a PDGFR-alpha sequence selected from the following group: I. SEQ ID No. 2 consisting of amino acids 24 to amino acids 524 of SEQ ID No. 1: TABLE-US-00016 QLSLPSILPNENEKVVQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIRNE ENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTEENELEGRHIYIYVPDP DVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPETPVTLHNSEGVVPASYDS RQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALKATSELDLEMEALKTV YKSGETIVVTCAVFNNEVVDLQWTYPGEVKGKGITMLEEIKVPSIKLVYT LTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGFIEIKPTFSQL EAVNLHEVKHFVVEVRAYPPPRISWLKNNLTLIENLTEITTDVEKIQEIRY RSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPSSILDLVDDHH GSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANNVSNIITEI HSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSE, II. a sequence having 90% or more identity to SEQ ID No. 2, III. a sequence that is a truncated sequence of SEQ ID No. 2 with at least 45 amino acids or a sequence having 90% or more identity to said truncated SEQ ID No. 2, and IV. variants of sequences according to items I., II., III. having substitutions atone or more of the following positions (numbering adhered to SEQ ID. No. 1): Ile-30, Glu-52, Ser-66, Ser-67, Asp-68, Leu-80, Ser-89, His-162, Pro-169, Asp-173, Ile-188, Val-193, Lys-194, Glu-213, Lys-304, Thr-320, His-334, Arg-340, Ile-373, Lys-378, Ala-396, Ala-401, Thr-436, Thr-440, Ile-453, Val-469, Ile-476, Ser-478, Asp-480, Ser-482, Arg-487.
25. The method according to claim 23, wherein said nucleic acid sequence comprises a PDGFR-alpha sequence selected from the following group: I. SEQ ID No. 9; II. SEQ ID No. 10; III. SEQ ID No. 11; IV. SEQ ID No. 12; V. SEQ ID No. 13, VI. a peptide fragment of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13, any of them having at least 10 amino acids, and VII. a variant of the above items I to VI that exhibits at least 80% sequence identity to the peptide having the sequence of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13 that exhibits at least 80% sequence identity to the peptide fragment of SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 12 or SEQ ID No. 13, said variant having a length of at least 10 amino acids.
Description
EXAMPLES
Example 1
[0150] Cells and Viruses: Primary human foreskin fibroblast (HFFs) were propagated in MEM (plus GlutaMaxx; Gibco) supplemented with 5% fetal calf serum (FCS), 100 μg/ml gentamycin and 0.5 ng/ml basic fibroblast growth factor. During experiments the cells were kept in maintenance medium without growth factor. Conditionally immortalized human endothelial cells (HEC-LTT, short HEC) (25, 28), were proliferated on vessels coated with 0.1% gelatin in endothelial cells growth medium (bullet kit; Lonza) with 2 μg/ml doxycycline. For experiments, the HECs were withdrawn from doxycycline for 24 hours to control the cell numbers of this otherwise fast dividing cell line. The efficiently transfectable hybrid endothelial cell line EA.hy926 (ATCC: CRL-2922; Edgell 1983) was expanded in DMEM (life technologies) plus 10% FCS.
[0151] The HCMV strains TB40/E and TB40/F were isolated from the same patient. TB40/E was propagated on endothelial cells and is highly endotheliotropic, whereas TB40/F was kept on fibroblast and is non-endotheliotropic (Sinzger 1999). AD169 (Rowe 1956 Plotkin 1975) and Towne (Plotkin 1975) are widely used HCMV strains, but lack the pentameric complex and are therefore non-endotheliotropic. VR1814 (Revello 2001), VHL/E (Waldman 1991) and Merlin (Davison 2003) represent endotheliotropic HCMV strains. TB40-BAC.sub.KL7-UL32EGFP-UL100mCherry is an endotheliotropic descendant of TB40/E that was labelled to allow differentiation between enveloped and non-enveloped virus capsids (Sampaio 2013).
[0152] TB40-BAC4 is a highly endotheliotropic BACmid based on TB40/E (Sinzger 2008) and BAC4UL74stop is an (yet unpublished) BAC4 mutant in which M7 and K12 of pUL74 were changed to stop codons, resulting in loss of expression of pUL74 (gO). Virus stocks of TB40 variants, AD169, Towne, VHL/E and VR1814 were harvested from infected HFFs day 5 to 7 post infection (p.i.). Supernatants were cleared from cells and large cell debris by centrifugation at 2,700 g for 10 min before storage at −80° C. Cleared UL74stop supernatants were 50 fold concentrated by ultracentrifugation at 70,000 g for 70 min. The luciferase reporter virus contains a Gaussia expression cassette under control of the major immediate early promoter, therefore the luciferase is expressed with the same kinetics as the immediate early proteins of HCMV (10). Virus stocks of the Gaussia luciferase reporter virus were first cleared and then twice ultracentrifuged at 23000 g for 70 min to remove Luciferase that is secreted along with the virus particles.
Example 2
[0153] Antiviral drugs, chimeric receptor mokcules and PDGFRα-derived peptides: All recombinant Fc-fusion proteins used in these studies were obtained from R&D: PDGFR-alpha-Fc (6765-PR-050), PDGFRβ-Fc (385-PR-100), EGFR-Fc (344-ER-050). The 40 amino acid long peptides based on human PDGFRα isotype 1 extracellular domain were obtained from Phtdpeptides, Shanghai, China. All peptides were dissolved to a final concentration of 1 mmol/A. Depending on their physiochemical properties either water, ammonium carbonate, dimethyl sulfoxide or acetic acid were used as solvents.
Example 3
[0154] Knockdown of protein expression by siRNA: For reverse transfection of siRNAs, cells were seeded at a density of 10,000 per well. As a negative control the inventors used siGenome non-targeting pool #2 (Dharmacon), as a positive control served a highly efficient IE siRNA (Hochdorfer 2016). Targets were knocked down with pools of four different siRNAs (siGenome Dharmacon). For each transfection using Lipofectamin RNAiMAX (Life Technologies) a final concentration of 50 nM was applied. 48 h post transfection HCMV TB40/E was added to the cells at a multiplicity of 0.5 to 1. Infection was allowed for 1 day before cells were fixed and stained for viral immediate early antigens.
Example 4
[0155] Inhibition of infection: For testing the inhibitory effect of antivirals or fusion proteins on HCMV, the respective inhibitors were diluted in MEM and mixed with infectious supernatants, the mixtures were incubated for 2 h at 37° C. before addition to the cells. For fibroblast infection the virus-inhibitor mixture was incubated on the cells for about 24 h. If endothelial cells were included in the experiment, all cells were supplied with their respective maintenance media after 2 h, and further incubated for 22 h.
Example 5
[0156] Determination of infection efficiencies: Infection efficiencies were determined by immunofluorescence staining for viral proteins. For fixation and permeabilization the cells were incubated with 80% acetone for 5 min. For HCMV infected cells the immediate early proteins pUL122/123 were detected with a mouse monoclonal (clone E13, Argene) and visualized using a Cy3 conjugated goat anti-mouse secondary antibody (Jackson Immuno Research). HSV infected cultures were stained 6 h post infection for ICP0 using a mouse monoclonal (clone 11060, Santa Cruz) and goat anti-mouse AF488 (life technologies). DAPI was used to locate nuclei. Infection rates were determined by counting the number of cell nuclei positive for the respective viral protein, as well as the total number of nuclei per image. For each condition three images were evaluated.
[0157] For screening the PDGFRα peptides for their neutralizing capacity a recently developed Gaussia-Luciferase-Assay was utilized (10). The Gaussia luciferase is secreted into the cell culture supernatants, therefore it is not necessary to fix the cells and instead a sample of the Gaussia containing supernatant is taken and mixed with the substrate coelenterazine (PjK) at a final concentration of 0.2 μg/ml. The resulting light emission was detected at 495 nm. For all obtained values background signals were subtracted and neutralization efficiency was determined relative to the control samples which contained only virus, no peptide.
Example 6
[0158] Quantification of adsorption and penetration: To distinguish between adsorption and penetration of viral particles the inventors made use of dual fluorescent HCMV TB40-BAC.sub.K0.7-UL32EGFP-UL100mCherry (Sampaio 2013). HFFs and HECs were seeded at a density of 40,000 cells per well on gelatin-coated IBIDI plates. Overnight produced cell-free infectious supernatant of the fluorescent virus was pre-incubated with PDGFR-Fcs for 2 h at 37° C. Before addition of the mixture the cells were pre-incubated with MEM for 30 min. Penetration of virus particles which had been pre-incubated for 2 h with 500 ng/ml of Fc-fusion protein, was allowed for 2 h at 37° C. After fixation with acetone, the EGFP signal of pUL32-EGFP was enhanced using mouse-anti-GFP (clone 3E6, Invitrogen) and Alexa488-anti-mouse (Life Technologies). DAPI (4′,6-diamidino-2-phenylindole) was added to mark the cell nuclei. The number of mCherry and EGFP positive particles which are enveloped was compared to the number of particles which were only green per cell. These EGFP-mCherry double positive particles have lost their UL100mCherry containing envelope, presumably by fusion with cellular membranes.
Example 7
[0159] Analysis of post adsorption inhibitory effects: For the analysis of post adsorption inhibition, HFFs were seeded at a density of 40,000 per well on IBIDI plates and incubated for 1 day before infection. Infectious supernatants of TB40E which were produced within 24 hours were cleared by centrifugation. Two identical plates were treated as follows: Cells and virus dilutions were precooled on ice for 15 min before attachment of the virus was allowed on ice for 1 h. The virus containing medium was exchanged by pre-cooled MEM with or without 200 ng/ml PDGFR-alpha-Fc. After 2 h incubation of the inhibitor with the cells on ice, one plate was directly shifted to 37° C., whereas the other cells were treated with pre-warmed 50% PEG (Roche) for 30 sec. The PEG was washed off by five times washing with pre-warmed PBS. Supplied with pre-warmed MEM containing again 200 ng/ml PDGFR-alpha-Fc, the cells were then incubated at 37° C. After 2 h incubation the medium was exchanged on PEG-treated and untreated cells and infection was allowed to proceed for 24 h before infection efficiencies were assessed by immediate early staining. Efficient PEG fusion was controlled visually by detection of syncytia.
Example 8
Binding of Chimeric Receptors to HCMV Particles:
[0160] To assess the binding of Fc-Proteins to virus particles, HFFs were seeded at a density of 40,000 cells per well on IBIDI plates 1 day prior to infection. Virus preparations were pre-incubated with Fc-fusion proteins at a final concentration of 500 ng/ml for 2 h at 37° C. The virus/Fc-protein mixtures were incubated with the cells for 1.5 h on ice. Before fixation with 80% acetone, the cells were washed once with MEM. For staining of viral particles mouse hybridoma recognizing the abundant viral protein pp150 (generously provided by W. Britt, Sanchez 2000) used. As a secondary antibody goat anti-mouse Cy3 (Jackson Immuno Research was used. Visualization of bound Fc-proteins was achieved by applying anti-human Alexa488 (Invitrogen). For better orientation, cell nuclei were stained with DAPI. For quantification of PDGFR-alpha-Fc binding to HCMV particles, the grey values of 100 particles per condition were quantified using AxioVision Software (Zeiss).
Example 9
Knockdown of PDGFR-Alpha Prevents HCMV Infection of Fibroblasts but not of Endothelial Cells
[0161] Two cellular growth factor receptor molecules, PDGFR-alpha and EGFR have been reported to promote HCMV infection in fibroblasts (33, 39). However, only fibroblast-restricted virus strains lacking the pentameric complex were used in those analyses, and in subsequent studies their relevance for HCMV infection was questioned (21, 35). As we aimed at exploring the potential of these molecules to serve as a basis for the development of HCMV entry inhibitors, the first step was to confirm their contribution to HCMV infection. To address the diverse entry pathways of HCMV the inventors applied a virus strain expressing both gH/gL complexes on two model cell types representing the restricted tropism (fibroblasts) or the extended tropism (endothelial cells).
[0162] Using an siRNA approach, the respective growth receptor was knocked down 2 days before infection with HCMV strain TB40/E at an MOI of 1. Cells treated with non-targeting siRNAs served as negative controls while cells in which viral IE RNAs were knocked down served as positive controls. One day after infection, cell cultures were fixed, viral IE antigens were immunostained, and the fraction of IE-antigen-positive cells was determined. In each of three experiments, the relative infection efficiency as compared to the non-targeting control was determined. As expected, knockdown of viral IE RNAs partially reduced the infection efficiency. Knockdown of PDGFR-alpha almost completed prevented HCMV infection of fibroblasts whereas it had no inhibitory effect in endothelial cells (
[0163] In line with these results PDGFR-alpha was only found on the surface of fibroblasts but not on endothelial cells in immunofluorescence stainings, and surface expression in fibroblasts was suppressed to levels below the detection limit when they were treated with the respective siRNAs (data not shown).
[0164] In conclusion, of the two growth factor receptor molecules that had previously been reported to promote HCMV entry, only the contribution of PDGFR-alpha was confirmed in the present experimental setting.
Example 10
[0165] Pretreatment of HCMV with a Soluble PDGFR-Fc Chimera Inhibits Infection of Fibroblasts and Endothelial Cells
[0166] The strong dependence of HCMV infection on expression of PDGFR-alpha suggested that viral particles interacted physically with this cellular growth factor receptor during the entry process in HFFs. The instant inventors found that pre-treatment of viral particles with soluble forms of this cellular molecule might block the respective interaction sites of the surface of HCMV virions and hence inhibit infection. To test this, the inventors pre-incubated cell free preparations of HCMV strain TB40/E with variable concentrations of soluble PDGFR-alpha-Fc chimeras for 2 h before adding them to HFFs and HECs. After 2 h the virus was removed and replaced with the appropriate cell culture medium for an overnight incubation. Cultures were then fixed, and the fraction of infected cells was determined by indirect immunofluorescence staining of viral IE antigens. Actually, PDGFR-alpha-Fc inhibited infection of HFFs in a dose dependent manner with an EC.sub.50 of about 10-20 ng/ml and a complete abrogation of infection at 200 ng/ml (
[0167] To address the possibility that the effect is rather due to the Fc part of the chimeric molecule than to the growth receptor part, the inventors compared PDGFR-alpha-Fc with EGFR-Fc and PDGFR-ß-Fc regarding their inhibitory potential on HCMV infection. Cell free preparations of TB40/E were pre-incubated with increasing concentrations of the various Fc chimeras for 2 h. HFFs were then incubated with the mixtures for 2 h followed by a medium exchange and an overnight incubation. Evaluation of the infection rates by immunofluorescence staining of viral IE antigen showed that only PDGFR-alpha-Fc blocked infection in a dose dependent fashion, whereas neither PDGFR-beta-Fc nor EGFR-Fc had an effect (
[0168] Next, it was tested whether soluble PDGFR-alpha-Fc would inhibit not only strain TB40/E but also other strains of HCMV. The inventors prepared cell free stocks of five HCMV strains (AD169, Towne, Merlin, VR1814, VHL/E) that represent the envelope glycoprotein variants described for HCMV (32), pre-incubated them with PDGFR-alpha-Fc at a concentration (250 ng/ml) that was sufficient for complete inhibition of strain TB40/E in the previous dose response experiment. In addition. TB40/F was included, a variant of TB40/E that lacks the pentameric gH/gL complex. After pre-incubation of the various HCMV preparations with PDGFR-alpha-Fc for 2 h, the mixture was added to HFFs in a 96-well format for 2 h and then replaced with medium. After an overnight incubation, the fraction of infected cells was determined by immunofluorescence staining of viral immediate early antigen. All strains were strongly inhibited by pretreatment with the soluble receptor, and with the exception of strain VR1814 (residual infection rate <2%) the reduction was complete (
[0169] Finally, to test whether this inhibitory effect was specific for HCMV the inventors repeated the experiment and included another herpes virus, HSV-1 strain F. While the inhibitory effect on HCMV was always reproduced, HSV infection was not affected by PFGFR-alpha (data not shown), indicating that the effect is specific for HCMV.
Example 11
[0170] Inhibition of HCMV Infection by PDGFR-Alpha Occurs at the Level of Viral Entry
[0171] The findings that removal of PDGFRα from the cell surface as well as pre-treatment of virus with soluble PDGFRα abrogated infection suggest interference with viral entry as the mode of action. It seemed therefore most likely that PDGFR-alpha-Fc binds directly to HCMV virus particles. The inventors tested this by staining of adsorbed virus particles with the Fc-fusion proteins. HCMV particles were pre-incubated with PDGFR-alpha-Fc, PDGFR-beta-Fc or EGFR-Fc for 90 min at 37° C. before the virus was attached to the cells for 90 min on ice. Virus particles were stained for the capsid protein pp150 and bound Fc-fusion proteins. The anti-human antibody visualized only those particles that were pre-treated with PDGFR-alpha-Fc, indicating that only this growth factor receptor-chimera binds to the virus (
[0172] The inventors analyzed the mode of inhibition by PDGFR-alpha-Fc. For this, the binding of different concentrations of the fusion protein was quantified by assessing the particle intensities after staining with anti-human antibody (
[0173] To further investigate, which of the initial steps of infection are blocked, the inventors performed a series of experiments that allowed discriminating between adsorption and penetration. They used the dual fluorescent virus TB40-BAC.sub.KL7-UL32EGFP-UL100mCherry (Sampaio 2013) as it allows to discriminate between enveloped (EGFP-positive and mCherry positive) and non-enveloped particles (only EGFP positive). They compared adsorption and penetration of untreated particles with particles pre-incubated with 100 ng/ml PDGFR-alpha or beta by counting the number of enveloped versus naked particles. On both cell types HFFs and HECs, adsorption of PDGFR-alpha-treated particles was reduced (50% on HFFs and 75% on HECs), whereas penetration was affected only in fibroblasts (
[0174] As the inhibition of penetration indicated that virions treated with PDGFR-alpha-Fc are defective for fusion of their envelope with the cellular plasma membrane, the inventors tested whether the chemical fusogen PEG was able to rescue this post-attachment inhibition by PDGFR-alpha-Fc. HCMV virus particles were adsorbed to HFFs for 1 h on ice. The virus containing medium was then exchanged by medium containing PDGFR-alpha-Fc at a concentration of 200 ng/ml. Inhibition of pre-adsorbed virus was allowed for 2 more hours on ice, before the cells were either directly shifted to 37° C. to allow entry or first treated with pre-warmed PEG for 30 sec. The PEG was washed off before the addition of PDGFR-alpha-Fc containing medium. After 2 h of incubation at 37° C. the cells were supplied with fresh medium without inhibitor and further incubated overnight. After 24 h the cells were fixed and stained for the viral immediate early antigens.
[0175] PDGFR-alpha-Fc reduced infectivity of already adsorbed viruses to 50% (
[0176] As a possible way of inactivation of HCMV, the inventors found that PDGFR-alpha-Fc binds the viral envelope glycoprotein pUL74. It was recently demonstrated that HCMV lacking pUL74 is deficient for fusion into host cells (42). To test whether pUL74 is an interaction partner of PDGFR-alpha-Fc, the inventors tested whether gO deficient particles can be stained with the soluble molecule similarly to wild type particles (shown in
[0177] To further investigate this, inhibition assays were performed with the UL74stop virus (
Example 12
[0178] Peptides Derived from the Extracellular Domain of PDGFR-Alpha Inhibit HCMV Infection
[0179] The surprising finding that only PDGFR-alpha-Fc but not EGFR-Fc or PDGFR-beta-Fc inhibits HCMV infection had indicated that the inhibitory effect is due to the PDGFR-alpha part of the chimeric molecule, which is actually only the extracellular domain of the native PDGFR-alpha transmembrane molecule. The inventors unexpectedly found that short peptides derived from this protein could also inhibit infection, and therefore tested a set of overlapping 40mer peptides covering the whole sequence of the extracellular PDGFR-alpha domain regarding the inhibitory potential of the individual peptides. Cell free preparations of strain TB40/E were pre-incubated with the individual peptides at concentrations reaching from 0.05-50 nmol/ml for 2 h and the mixtures were then incubated with HFF cultures in a 96-well format. The various peptides differed greatly regarding their inhibitory potential with a region between aa120 and aa280 being absolutely ineffective and the peptides surrounding this region having the highest anti-HCMV effect (
Example 13
[0180] Quantification of the Inhibitory Potential of PDGFR-Alpha-Fc Variants with Small Deletions within the Proposed Ligand Binding Sites on HCMV Infection
[0181] Deletion mutant PDGFR-alpha-Fc proteins set forth below and non-deleted PDGFR-alpha-Fc were expressed in 293T cells and purified using protein A. These proteins were initially diluted in cell culture medium to a concentration of 8000 ng/ml and were subsequently further diluted in a row of 2-fold dilutions to a minimum concentration of 4 ng/ml. In the inhibition assays, controls were used that contain the same amount of dilution medium and protein dilution buffer in order to rule out the occurrence of a non-specific inhibition of binding through the respective buffers. Diluted probes containing deletion mutants of PDGFR-alpha-Fc or whole PDGFR-alpha-Fc (without deletions) were mixed at a ratio of 1:1 with HCMV expressing luciferase and subsequently incubated for 2 h at 37° C. These mixtures were subsequently used in infection assays of human fibroblasts. After 2 h incubation of cells with the virus and respectively diluted deletion mutants or non-deleted PDGFR-alpha-Fc as control were removed from the cells and the cells were incubated for additional 24 h in cell culture medium. Thereafter, the activity of the luciferase was determined as a measure of the extent of infection. The background noise measured in the controls with probes containing no deletion mutants of PDGFR-alpha-Fc or without whole PDGFR-alpha-Fc were subtracted from the measurements with deletion mutants of PDGFR-alpha-Fc and whole PDGFR-alpha-Fc, respectively.
[0182] Experimental Design:
[0183] Cells: HFF at 1.5×10.sup.4/well seeded the day before on 96-well flat bottom cell culture plates coated freshly with 0.1% gelatin
[0184] Virus: BAC4 GLuc (yields 60-70% infection at 1:50 dilution; expresses Gaussia luciferase under control of the HCMV LE promotor)
[0185] Soluble Receptor:
[0186] Recombinant Human PDGFR alpha Fc Chimera and variants with small deletions within the predicted ligand binding sites. All Proteins were expressed in HEK 293T cells and purified using Protein A sepharose. The proteins were eluted in elution buffer (Thermo) with 10% 1 M Tris pH 8.
[0187] Treatment
[0188] Pre-Incubation of Virus with the Recombinant Proteins
[0189] For each recombinant Protein a 2-fold dilution series starting with 8 μg/ml was prepared. As a negative reference sample control dilution series containing the same volumes of Elution buffer were prepared. PDGFR-alpha-Fc serves as a positive control.
[0190] Total volume per dilution: 120 N1
TABLE-US-00009 Protein cone. Vol protein + BCA (E2) % dilution buffer Volume [μg/ml] for 8 μg/ml for 8 μg MEM5G delM1334139 21.5 37.2 89.3 151 del V184-G186 11.2 71.4 171.4 69 del N204-208 80.9 9.9 21.7 216 del242-247 20.6 38.8 93.2 147 del261-264 16.2 49.4 118.5 121 del272-275 17.5 45.7 109.7 130 del296-300 82.7 9.7 23.2 217 PDGFR-alpha-Fc 12.6 63.5 152.4 88 Conc. 8000 4000 2000 1000 500 250 125 62.5 31.25 15.63 7.81 3.9 [ng/ml] [0191] 100 μl of each inhibitor dilution were mixed with 100 N1 of HCMV BAC4Gluc (1:25 diluted) [0192] =>effective concentration of soluble receptor after addition of virus [ng/ml]:
TABLE-US-00010 Conc. 4000 2000 1000 500 250 125 [ng/ml] Conc. 62.5 31.25 15.63 7.81 3.9 195 [ng/ml] [0193] incubate for 2 h at 37° C.
[0194] Infection: [0195] Cell culture medium was replaced with the virus-receptor mixtures [0196] Cultures were incubated for 2 h at 37° C. [0197] After 2 h, virus was removed and replaced with medium. [0198] Cultures were then incubated o/n.
[0199] Measurement of Gaussia luciferase: [0200] The Luciferase containing culture media was removed from the cells. A proportion (20 μl) was mixed with Gaussia substrate Coelenterazine and light emission was measured at 492 nm. [0201] The light emission of samples treated with only buffer was subtracted from the values of the respective samples. [0202] Cells were fixed with 80% acetone for 5 min at RT to allow for IE staining at a later time point. [0203] The results of these experiments are shown in
Example 14
[0204] Focus Expansion Assays with the Repaired Strain Merlin (Initial Infection with Supernatant)
[0205] The effect of a substance of interest on viral spread is tested by a focus expansion assay essentially as previously described (Sinzger et al., 1997) with the following modifications. Instead of co-culturing infected with uninfected cells, indicator cells are directly infected with cell-free infectious preparations of the repaired strain Merlin (suitable for conditional expression of RL13 and UL128L). HFFs (or other indicator cells of choice), seeded in gelatin-coated 96-well plates at a density of 15,000 cells/well are infected with a virus dose resulting in about 50 infected cells/well, and are subsequently cultured for 7 days in the presence or absence of the substance to be tested. Plates are then fixed with 80% acetone for 5 min at ambient temperature and stained for HCMV immediate-early antigen by indirect immunofluorescence using primary antibody E13 (Argene) and secondary antibody Cy3-goat anti-mouse IgG F(ab′).sub.2 (Jackson ImmunoResearch). Nuclei of all cells are stained with DAPI. The number of infectious foci per well is counted; “infectious foci” being defined as clusters of at least three infected cells. In addition, the number of infected cells of randomly selected infectious foci is counted and “focus size” is given as infected cells/focus. The distribution of values for “focus size” is plotted for each combination of substance with one dot representing one focus, and virus and values of the central tendency (mean or median) are plotted in addition. The results are shown in
[0206] Focus Expansion Assays with Clinical Isolates or Stain Merlin (Initial Infection by Coculture):
[0207] The effect of a substance of interest on viral spread is tested by a focus expansion assay essentially as previously described (Sinzger et al., 1997). Aliquots of infected cell cultures (HFFs or HFFF-tet cells with about 10% CPE) are thawed, washed with MEM and co-cultured with an 100-fold excess of uninfected indicator cells (e.g. fibroblasts, endothelial cells or epithelial cells) for 7 days in gelatin-coated 96-well plates in the presence or absence of the substance to be tested. Plates are then fixed with 80% acetone for 5 min at ambient temperature and stained for HCMV immediate-early antigen by indirect immunofluorescence using primary antibody E13 (Argene) and secondary antibody Cy3-goat anti-mouse IgG F(ab′)2 (Jackson ImmunoResearch). Nuclei of all cells are stained with DAPI. The number of infectious foci per well is counted; “infectious foci” being defined as clusters of at least three infected cells. In addition, the number of infected cells of randomly selected infectious foci is counted and “focus size” is given as infected cells/focus. The distribution of values for “focus size” is plotted for each combination of substance with one dot representing one focus, and virus and values of the central tendency (mean or median) are plotted in addition.
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