Complexes of cytomegalovirus proteins

Abstract

An isolated human cytomegalovirus (HCMV) membrane protein complex that comprises gH, gL and at least one more HCMV glycoprotein is provided. In some embodiments the complex consists of gH, gL and gO. In other embodiments the complex consists of gH, gL, pUL128, pUL130 and pUL131A. Processes for expressing and purifying such complexes, and subsequent uses of such complexes in immunogenic compositions and vaccines, are also provided.

1 . A process for producing an isolated HCMV membrane protein complex comprising gH, gL and at least one additional HCMV glycoprotein, wherein said process comprises recombinant expression of said gH, gL and at least one more HCMV glycoprotein. 
     
     
         2 . A process for expressing an HCMV membrane protein complex comprising gH, gL and at least one more HCMV glycoprotein by:
 introducing one or more recombinant nucleic acid molecules which encode gH, gL and at least one more HCMV glycoprotein into an expression system,   expressing said one or more nucleic acids in said expression system; and   purifying said membrane protein complex.   
     
     
         3 . The process of  claim 2 , which comprises the step o f transfecting mammalian cells with a fast nucleic acid construct which encodes a fragment of gH that lacks the transmembrane domain, a second nucleic acid construct which encodes the gL protein; and a third nucleic acid construct which encodes at feast one more HCMV glycoprotein. 
     
     
         4 . The process of any preceding claim, wherein the HCMV membrane protein complex consists of gH, gL and gO. 
     
     
         5 . The process of any preceding claim, wherein the HCMV membrane protein complex consists of gH, gL, pUL128, pUL130 and pUL131A. 
     
     
         6 . The process of  claim 5 , wherein said:
 pUL128 comprises or consists of any one of the sequences recited in SEQ ID NOs: 13, 14, 15 or 33;   pUL130 comprises or consists of any one of the sequences recited in SEQ ID NOs: 16, 17 or 34, and/or   pUL131A comprises or consists of any one of the sequences recited in SEQ ID NOs: 18, 19, 20 or 35.   
     
     
         7 . The process of any preceding claim, wherein the HCMV membrane protein complex has a purity of >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94% of >95% by mass. 
     
     
         8 . The process of any preceding claim, wherein one or more of gH, gL, gO, pUL128, pUL130 and pUL131A in said HCMV membrane protein complex:
 have a mammalian glycosylation pattern; and/or   do not contain an insect cell pattern of glycosylation.   
     
     
         9 . A purified HCMV membrane protein complex comprising gH, gL and at least one more HCMV glycoprotein. 
     
     
         10 . An HCMV membrane protein complex comprising gH, gL and at least one more HCMV glycoprotein, w herein said complex is produced by the process of any preceding claim. 
     
     
         11 . An immunogenic composition comprising die isolated HCMV membrane complex of  claim 9  or  claim 10 . 
     
     
         12 . The immunogenic composition of  claims 11 , wherein said composition is a vaccine. 
     
     
         13 . The immunogenic composition of  claim 11 , wherein said composition comprises an adjuvant. 
     
     
         14 . The immunogenic composition of  claim 12 , wherein said adjuvant is an oil-in-water emulsion or an aluminium salt. 
     
     
         15 . An immunogenic composition comprising:
 a self-replicating RNA molecule that encodes an HCMV membrane protein complex; and   the HCMV membrane protein complex of  claim 9  or  claim 10 .   
     
     
         16 . A kit comprising:
 a priming composition comprising a self-replicating RNA molecule that encodes an HCMV membrane protein complex; and   a boosting composition comprising the HCMV membrane protein complex of  claim 9  or  claim 10 .   
     
     
         17 . A recombinant nucleic acid molecule which encodes gL, gH that lacks a transmembrane domain, and at least one additional HCMV glycoprotein, wherein said recombinant nucleic acid:
 (a) is not a self-replicating RNA molecule;   (b) is not an alphavirus replicon;   (c) does not encode any alphavirus nonstructural proteins, such as NSP1, NSP2, NSP3 and NSP4;   (d) does not contain; an Internal Ribosomal Entry Site (IRES), such as EMCV or EV71; and/or   (e) does not contain a viral 2A site, such as FMDV.   
     
     
         18 . The recombinant nucleic acid molecule of  claim 17 , wherein said recombinant nucleic acid molecule encodes:
 gL, gH that lacks a transmembrane domain, pUL128, pUL130 and pUL131A; or   gL, gH that lacks a transmembrane domain and gO.   
     
     
         19 . A plurality of recombinant nucleic acids, wherein said plurality of recombinant nucleic acids encode gL, gH that lacks a transmembrane domain, and at least one additional HCMV glycoprotein, wherein one or more or all of said plurality of recombinant nucleic acids:
 (a) is not a self-replicating RNA molecule;   (b) is not an alphavirus replicon;   (c) does not encode any alphavirus nonstructural proteins, such as NSP1, NSP2, NSP3 and NSP4;   (d) does not contain: an Internal Ribosomal Entry Site (IRES), such as EMCV or BV71; and/or   (c) does not contain a viral 2A site, such as FMDV.   
     
     
         20 . The plurality of recombinant nucleic acids of  claim 19  comprising:
 a first construct encoding gH that lacks a transmembrane domain and gL; and 
 a second construct encoding one additional HCMV glycoprotein. 
 
     
     
         21 . The plurality of recombinant nucleic acids of  claim 20 , wherein said second construct encodes:
 pUL128, pUL130 and pUL131A; or   g .   
     
     
         22 . The plurality of recombinant nucleic acids of  claim 21  comprising:
 a first recombinant nucleic acid molecule which encodes gL; 
 a second recombinant nucleic acid molecule which encodes a fragment of gH that lacks a transmembrane domain; and 
 one or more third recombinant nucleic acid molecules which encode one or more additional HCMV proteins. 
 
     
     
         23 . A cell comprising gH, gL and al least, one additional HCMV glycoprotein, wherein said cell docs not:
 (a) contain the HCMV genome;   (b) produce HCMV virions;   (c) contain self-replicating RNA molecules encoding said gH, gL and at least one additional HCMV glycoprotein; and/or   (d) contain alphavirus replicons.   
     
     
         24 . A process for producing an isolated or a purified HCMV membrane protein complex comprising gH, gL and at least one additional HCMV glycoprotein, wherein said process involves growing the cell of  claim 23  in growth medium. 
     
     
         25 . The process of  claim 24 , whereto said HCMV membrane protein complex is secreted into said growth medium. 
     
     
         26 . The process of  claim 25 , wherein sad HCMV membrane protein complex accumulates to a concern ration of >0.8 mg, >0.85 mg, >0.88 mg, >0.9 mg, >0.95 mg, >1 mg, >1.5 mg, >2 mg, >2.5 mg, >3 mg, >3.5 mg, >4 mg, >4.5 mg, >5 mg of complex per lure of growth medium. 
     
     
         27 . An RNA prime-protein boost regimen comprising:
 performing one or more priming immunization(s) with RNA that encodes one or more of the protein components of an HCMV membrane protein complex, wherein said HCMV membrane protein complex comprises gH, gL and at least one additional HCMV glycoprotein,   performing one or more boosting immunization(s) later with a purified HCMV membrane protein complex, wherein sad purified HCMV membrane protein complex comprises gH, gL and at least one additional HCMV glycoprotein.