Meningococcus adhesins nada, app and orf 40

Abstract

NadA, App and ORF40 function as adhesins in N. meningitidis . Adhesion can be modulated by targeting these three proteins. NadA allelic variants are disclosed. Autoproteolytic cleavage of App is disclosed, as is removal of the activity by mutagenesis. App is processed and secreted into culture medium when expressed in E. coli . Mature App proteins are disclosed. Knockout mutants are disclosed. Vesicles from non-Neisserial hosts with heterologous adhesin expression are disclosed.

1 . A protein comprising the amino acid sequence of one or more of SEQ IDs 1 to 14.  
     
     
         2 . A protein comprising an amino acid sequence having at least 50% sequence identity to one or more of SEQ IDs 1 to 14.  
     
     
         3 . A protein comprising a fragment of one or more of SEQ IDs 1 to 14.  
     
     
         4 . The protein of  claim 1 ,  claim 2  or  claim 3 , including a heptad sequence (AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 AA 7 ) r , wherein: AA 1  is Leu, Ile, Val or Met; each of AA 2  AA 3  AA 4  AA 5  AA 6  and AA 7  may independently be any amino acid; and r is an integer of 1 or more.  
     
     
         5 . The protein of  claim 3 , comprising amino acids 24 to 351 of SEQ ID 3.  
     
     
         6 . A nucleic acid encoding the protein of any one of  claims 1  to  3 .  
     
     
         7 . An immunogenic composition comprising (a) a Neisserial NadA protein and/or (b) nucleic acid encoding a Neisserial NadA protein.  
     
     
         8 . A method for raising an antibody response in a mammal, comprising administering the immunogenic composition of  claim 7  to the mammal.  
     
     
         9 . A method for protecting a mammal against a Neisserial infection, comprising administering to the mammal the immunogenic composition of  claim 7 .  
     
     
         10 . (canceled)  
     
     
         11 . (canceled)  
     
     
         12 . The method of  claim 8  or  claim 9 , wherein the NadA protein comprises the amino acid sequence of one or more of SEQ IDs 1 to 12.  
     
     
         13 . The method of  claim 8  or  claim 9 , wherein the Neisserial infection is an infection with  N. meningitidis  from hypervirulent lineages ET-5, EY-37 and cluster A4.  
     
     
         14 . The composition of  claim 7 , additionally comprising an antigen which, when administered to a mammal, elicits an immune response which is protective against a lineage III strain of  N. meningitdis.    
     
     
         15 . A method for purifying processed App protein, comprising the steps of: 
 expressing a gene encoding App protein in a non-Neisserial host cell; and purifying processed App protein from the culture medium.    
     
     
         16 . The method of  claim 15 , wherein the non-Neisserial host cell is  E. coli.    
     
     
         17 . Purified protein obtainable by the process of  claim 15  or  claim 16 .  
     
     
         18 . A method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein the ability of one or more App, ORF40 and/or NadA to bind to the epithelial cell is blocked.  
     
     
         19 . The method of  claim 18 , wherein the binding is blocked using an antibody specific for App, ORF40 and/or NadA.  
     
     
         20 . A method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein protein expression from one or more of App, ORF40 and/or NadA is inhibited.  
     
     
         21 . The method of  claim 20 , wherein expression is inhibited using antisense techniques.  
     
     
         22 . Nucleic acid comprising a fragment of x or more nucleotides from nucleic acid which encodes App, ORF40 or NadA, wherein x is at least 8.  
     
     
         23 . The nucleic acid of  claim 22  having formula 5′-(N) a -(X)-(N) b -3′, wherein 0>a>15, 0>b>15, N is any nucleotide, and X is a fragment of at least 8 contiguous nucleotides from a nucleic acid which encodes App, ORF40 or NadA.  
     
     
         24 . A  Neisseria  bacterium in which one or more of App, ORF40 and/or NadA has been knocked out.  
     
     
         25 . A method for preventing the attachment of a Neisserial cell to an epithelial cell, wherein one or more of App, ORF40 and/or NadA has a mutation which inhibits its activity.  
     
     
         26 . A mutant protein, wherein the mutant protein comprises the amino acid sequence of App, ORF40 and/or NadA, or a fragment thereof, but wherein one or more amino acids of said amino acid sequence is/are mutated.  
     
     
         27 . Nucleic acid encoding the mutant protein of  claim 26 .  
     
     
         28 . A method for producing the nucleic acid of  claim 27 , comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis on said source nucleic acid to provide nucleic acid encoding a mutant protein.  
     
     
         29 . A method for screening for compounds which inhibit the binding of a Neisserial cell to an epithelial cell, comprising the steps of (a) incubating App, ORF40 and/or NadA protein with an epithelial cell and a test compound; (b) testing the mixture to determine if the interaction between the protein and the epithelial cell has been inhibited.  
     
     
         30 . A compound identified using the method of  claim 29 .  
     
     
         31 . A composition comprising (a) an  E. coli  bacterium which expresses App and/or ORF40 (and, optionally, NadA) and (b) an epithelial cell.  
     
     
         32 . A method for preparing an outer membrane vesicle (OMV) from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.  
     
     
         33 . A method for preparing an OMV from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of the following proteins: 
 (A) Even SEQ IDs 2-892 from WO99/24578;    (B) Even SEQ IDs 2-90 from WO99/36544;    (C) Even SEQ IDs 2-3020 from WO99/57280;    (D) Even SEQ IDs 3040-3114 from WO99/57280;    (E) SEQ IDs 3115-3241 from WO99/57280;    (F) The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [supra];    (G) A protein comprising the amino acid sequence of one or more of (A) to (F);    (H) A protein sharing sequence identity with the amino acid sequence of one or more of (A) to (F); and    (I) A protein comprising a fragment of one or more of (A) to (F).    
     
     
         34 . The method of  claim 32  or  claim 33 , wherein the non-Neisserial host cell is  E. coli.    
     
     
         35 . OMVs obtainable by the process of  claim 32  or  claim 33 .  
     
     
         36 . An outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding App, ORF40 or NadA protein.  
     
     
         37 . An outer membrane vesicle from a non-Neisserial host cell, characterised in that said cell expresses a gene encoding one or more of proteins (A) to (I) as defined in  claim 33 .  
     
     
         38 . A protein comprising the amino acid sequence of App, except that amino acid Asp-158, His-115 and/or Ser-267 is mutated.  
     
     
         39 . The protein of  claim 38 , wherein Ser-267, Asp-158 or His-115 is replaced with one of the 19 other naturally-occurring amino acids.  
     
     
         40 . A protein comprising the amino acid sequence of App, except that one or more amino acid (s) between Ser-1064 and Arg-1171 is mutated.  
     
     
         41 . The protein of  claim 40 , wherein the mutation is a deletion, an insertion, a truncation or a substitution.  
     
     
         42 . The protein of  claim 40 , wherein the residue which is mutated is S-1064, D-1065, K-1066, L-1067, G-1068, K-1069, A-1070, E-1071, A-1072, K-1073, K-1074, Q-1075, A-1076, E-1077, K-1078, D-1079, N-1080, A-1081, Q-1082, S-1083, L-1084, D-1085, A-1086, L-1087, I-1088, A-1089, A-1090, G-1091, R-1092, D-1093, A-1094, V-1095, E-1096, K-1097, T-1098, E-1099, S-1100, V-1101, A-1102, E-1103, P-1104, A-1105, R-1106, Q-1107, A-1108, G-1109, G-1110, E-1111, N-1112, V-1113, G-1114, I-1115, M-1116, Q-1117, A-1118, E-1119, E-1120, E-1121, K-1122, K-1123, R-1124, V-1125, Q-1126, A-1127, D-1128, K-1129, D-1130, T-1131, A-1132, L-1133, A-1134, K-1135, Q-1136, R-1137, E-1138, A-1139, E-1140, T-1141, R-1142, P-1143, A-1144, T-1145, T-1146, A-1147, F-1148, P-1149, R-1150, A-1151, R-1152, R-1153, A-1154, R-1155, R-1156, D-1157, L-1158, P-1159, Q-1160, L-1161, Q-1162, P-1163, Q-1164, P-1165, Q-1166, P-1167, Q-1168, P-1169, Q-1170 and/or R-1171.  
     
     
         43 . A protein comprising the amino acid sequence of App, except that one or more of amino acids Phe-956, Asn-957, Ala-1178 & Asn-1179 (numbered according to SEQ ID 32 herein) is mutated.  
     
     
         44 . The protein of  claim 43 , wherein the mutation is a deletion, an insertion, a truncation or a substitution.  
     
     
         45 . A protein comprising the amino acid sequence of a processed App, wherein said processed App does not comprise the C-terminus domain which is downstream of an autoproteloytic cleavage site in full-length App.  
     
     
         46 . The protein of  claim 45 , comprising one or more of SEQ IDs 33 to 36.  
     
     
         47 . A protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed App is Phe-956 (numbered according to SEQ ID 32 herein).  
     
     
         48 . A protein comprising the amino acid sequence of a processed App, wherein the C-terminus of said processed App is Ala-1178 (numbered according to SEQ ID 32 herein).  
     
     
         49 . A protein comprising an amino acid sequence selected from the group consisting of SEQ IDs 33, 34, 35, 36, 37, 38 & 39.  
     
     
         50 . A protein comprising an amino acid sequence with at least 50% sequence identity to one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39.  
     
     
         51 . A protein comprising a fragment of one or more of SEQ IDs 33, 34, 35, 36, 37, 38 & 39.  
     
     
         52 . Nucleic acid encoding the protein of any one of  claims 38  to  51 .  
     
     
         53 . A method for producing the nucleic acid of  claim 52 , comprising the steps of: (a) providing source nucleic acid encoding App, ORF40 or NadA, and (b) performing mutagenesis on said source nucleic acid to provide nucleic acid encoding a protein of any one of  claims 38  to  51 .