Meningococcal multivalent native outer membrane vesicle vaccine, methods of making and use thereof

Abstract

The present technology provides vaccine compositions comprising native outer membrane vesicles (NOMVs) from at least one genetically modified strain of Neisseria which provides protective immunity to meningococcal disease, more preferably subtype B meningococcal disease. The present technology further provides methods of immunizing an animal or human against meningococcal disease comprising administering the vaccine composition of the present invention.

1 . A vaccine comprising native outer membrane vesicles obtained from at least two meningococcal strains that have been genetically modified to provide broad based protection, wherein the native outer membrane vesicles include three different sets of antigens based on PorA, LOS, and conserved outer membrane proteins; wherein the genetically modified strains have been modified to provide enhanced safety based on inactivation of lpxL1, synX, and lgtA genes; and wherein at least one of the genetically modified strains expresses at least two different PorA subtype proteins or subtype epitopes. 
     
     
         2 . The vaccine of  claim 1  where the LOS expressed by each strain has a different LOS core structure and has an alpha chain consisting of glucose and galactose. 
     
     
         3 . The vaccine of  claim 1  where each strain expresses at least two different PorA subtype proteins or subtype epitopes which are chosen based on the most prevalent of PorA subtypes among group B case isolates. 
     
     
         4 . The vaccine of  claim 1  where a different conserved surface protein with demonstrated capacity to induce bactericidal antibodies is over-expressed in each strain and are taken from the group consisting of FHBP (GNA1870) variants 1, FHBP variants 2, and FHBP variants 3; NadA; App; NspA; TbpA and TbpB. 
     
     
         5 . A combination of NOMVs from three genetically modified, antigenically diverse  Neisseria meningitidis  strains, wherein at least one of the strains is selected from:
 (1) H44/76 HOPS-DL which has the following genetic modifications or characteristics:   inactivation of the genes synX, lpxL1, and lgtA;   insertion of a second porA gene (subtype P1.7-1,1) in the place of opaD;   increased expression of NadA; and   stabilized high expression of Opc and PorA;   (2) 8570 HOPS-G A L which has the following genetic modifications or characteristics:   inactivation of the genes synX, lpxL1, and lgtA;   insertion of a second porA gene in place of opaD;   increased expression of factor H binding protein variant 1; and   stabilized high expression of PorA and Opc; and   (3) B16B6 HPS-G 2 A which has the following genetic modifications or characteristics:   inactivation of the genes synX, lpxL 1, and lgtA;   insertion of a second porA gene in place of opaD;   increased expression of factor H binding protein variant 2; and   stabilized high expression of PorA and Opc.   
     
     
         6 . The combination of vaccine strains of  claim 5  wherein strain H44/76 HOPS-DL was derived from the ET-5 wild type strain H44/76 (B:15: P1.7,16: L,3,7:P5.5,C). 
     
     
         7 . The combination of vaccine strains of  claim 5  wherein strain 8570 HOPS-G 1 L was derived from the ET-5 wild type strain 8570 (B:4: P 1.19,15: L3,7v: P5.5,11,C). 
     
     
         8 . The combination of vaccine strains of  claim 5  wherein strain B16B6 HPS-G 2 L is derived from the ET-37 wild type strain B16B6 (B:2a:P 1.5,2: L2:P5.1,2,5). 
     
     
         9 .- 13 . (canceled) 
     
     
         14 . A vaccine composition against meningococcal disease comprising native outer membrane vesicles (NOMVs) from one or more genetically modified strains of  Neisseria meningitidis , wherein the one or more genetically modified strains has been modified by:
 a. inactivation of the synX gene,   b. inactivation of the lpxL1 gene,   c. inactivation of the lgtA gene in each strain resulting in expression of a shortened or truncated lipooligosaccharides (LOS) that lacks lacto-N-neotetraose tetrasaccharide, and   d. insertion of at least one second antigenically different porA gene in place of the opa gene.   
     
     
         15 . (canceled) 
     
     
         16 . The vaccine composition of any one of  claim 14 , wherein the genetically modified strain further comprises stabilized expression of at least one outer membrane protein, wherein the outer membrane protein is selected from the group comprising Opc and PorA. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A genetically modified vaccine strain of  Neisseria meningitidis  subtype B derived from: H44/76 strain comprising the genetic modifications of
 i) inactivation of a synX gene,   ii) inactivation of the lpxL1 gene,   iii) inactivation of the lgtA gene,   iv) insertion of a second porA gene in the place of a opaD gene,   v) increased expression of NadA compared with the native strain, and   vi) stabilized increased expression of Opc and PorA proteins.   
     
     
         21 . The genetically modified strain of  claim 20 , wherein strain H44/76 HOPS-DL was derived from the ET-5 wild type strain H44/76 (B:15: P1.7,16: L,3,7:P5.5,C). 
     
     
         22 . (canceled) 
     
     
         23 . A genetically modified vaccine strain of  Neisseria meningitidis  subtype B strain: derived from 8570 comprising the genetic modifications of:
 i) inactivation of a synX gene,   ii) inactivation of the lpxL1 gene,   iii) inactivation of the lgtA gene,   iv) insertion of a second porA gene in place of opaD;   v) increased expression of factor H binding protein variant 1; and   vi) stabilized increased expression of PorA and Opc proteins.   
     
     
         24 . The genetically modified strain of  claim 23 , wherein the genetically modified strain was derived from the ET-5 wild type strain 85 70(B:4: P 1.19,15: L3,7v: P5.5,11,C). 
     
     
         25 . (canceled) 
     
     
         26 . A genetically modified vaccine strain of  Neisseria meningitidis  subtype B derived from B16B6 comprising the genetic modifications of:
 i) inactivation of a synX gene,   ii) inactivation of the lpxL1 gene,   iii) inactivation of the lgtA gene,   iv) insertion of a second porA gene (subtype P1.22-1,4) in place of opaD;   v) increased expression of factor H binding protein variant 2; and   vi) stabilized increased expression of PorA and Opc proteins.   
     
     
         27 . The genetically modified strain of  claim 26 , wherein the genetically modified strain is derived from the ET-37 wild type strain B16B6 (B:2a:P 1.5,2: L2:P5.1,2,5). 
     
     
         28 .- 29 . (canceled) 
     
     
         30 . A vaccine composition comprising NOMV from one or more genetically modified strain of  Neisseria meningitidis  subtype B selected from the group consisting of:
 a) genetically modified vaccine strain of  Neisseria meningitidis  subtype B derived from: H44/76 strain comprising the genetic modifications of
 i) inactivation of a synX gene, 
 ii) inactivation of the lpxL1 gene, 
 iii) inactivation of the lgtA gene, 
 iv) insertion of a second porA gene in the place of a opaD gene, 
 v) increased expression of NadA compared with the native strain, and 
 vi) stabilized increased expression of Opc and PorA proteins; 
   b) a genetically modified vaccine strain of  Neisseria meningitidis  subtype B strain derived from 8570 comprising the genetic modifications of:
 i) inactivation of a synX gene, 
 ii) inactivation of the lpxL1 gene, 
 iii) inactivation of the lgtA gene, 
 iv) insertion of a second porA gene in place of opaD; 
 v) increased expression of factor H binding protein variant 1; and 
 vi) stabilized increased expression of PorA and Opc proteins; and 
   c) a genetically modified vaccine strain of  Neisseria meningitidis  subtype B derived from B16B6 comprising the genetic modifications of:
 i) inactivation of a synX gene, 
 ii) inactivation of the lpxL1 gene, 
 iii) inactivation of the lgtA gene, 
 iv) insertion of a second porA gene (subtype P1.22-1,4) in place of opaD; 
 v) increased expression of factor H binding protein variant 2; and 
 vi) stabilized increased expression of PorA and Opc proteins. 
   
     
     
         31 . The vaccine composition of  claim 30 , wherein the vaccine composition comprises NOMVs from two or more genetically modified strains. 
     
     
         32 . (canceled) 
     
     
         33 . The vaccine composition of  claim 30 , wherein the vaccine composition comprises NOMVs from three or more genetically modified strains. 
     
     
         34 .- 37 . (canceled) 
     
     
         38 . A vaccine against meningococcal disease comprising a variety of native outer membrane vesicles (NOMVs), wherein at least some of the NOMVs are essentially free of expression or sialylation of lipooligosaccharide (LOS), contain LOS that includes a lipid A with a penta-acyle structure and contain increased expression levels of at least one minor conserved outer membrane protein, wherein the minor conserved outer membrane protein is selected from proteins that induce bactericidal antibodies. 
     
     
         39 . The vaccine of  claim 38 , wherein the minor conserved outer membrane protein is selected from the group consisting of NadA, factor H binding protein (FHBP) variant 1, and FHBP variant 2. 
     
     
         40 . The vaccine of  claim 38 , wherein at least some of the NOMV comprise shortened or truncated LOS that are essentially free of lacto-N-neotetraose (LNnT) tetrasaccharide. 
     
     
         41 . The vaccine of  claim 38 , wherein at least some of the NOMV comprise two or more different PorA proteins. 
     
     
         42 .- 44 . (canceled) 
     
     
         45 . A method of preparing a genetically modified strain of  N. meningitidis  comprising:
 a) selecting a strain of meningococcal type B able to be genetically modified;   b) genetically modifying the strain by inactivating the synX gene,   c) genetically modifying the strain by inactivating the lpxL1 gene,   d) genetically modifying the strain by inactivating the lgtA gene, and   e) genetically modifying the strain by increasing expression of one or more minor conserved outer membrane proteins.   
     
     
         46 . The method of  claim 45 , further comprising:
 genetically modifying the strain by inserting at least one second antigenically different porA gene into the open reading frame of the opa gene.   
     
     
         47 . The method of  claim 45 , further comprising:
 genetically modifying the strain to stably express or overexpress at least one outer membrane protein by replacing the poly-C sequence within the promoter or open reading frame of the at least one outer membrane protein with a sequence containing G and C nucleotides.   
     
     
         48 . A method of preparing a vaccine against meningococcal disease comprising the steps of:
 a) culturing a genetically modified strain of  N. meningitidis  comprising one or more modification selected from the group consisting of:
 a. inactivation of the synX gene, 
 b. inactivation of the lpxL1 gene, 
 c. inactivation of the IgtA gene, 
 d. insertion of at least one second antigenically different porA gene in place of the opa gene, 
 e. increased or stable expression of at least one minor conserved outer membrane protein, and 
 f. stabilized expression of at least one outer membrane protein; 
   b) expanding the culture by fermentation using the cultured strain of a) to inoculate medium in a fermentor;   c) inactivating the fermented culture;   d) harvesting  N. meningitidis  cultured cells by continuous flow centrifugation and collecting cell paste;   e) isolating NOMVs from the cell paste; and   f) resuspending NOMVs in buffer or carrier suitable for vaccine administration.