Enhanced first generation adenovirus vaccines expressing codon optimized HIV1-Gag, Pol, Nef and modifications

Abstract

First generation adenoviral vectors and associated recombinant adenovirus-based HIV vaccines which show enhanced stability and growth properties and greater cellular-mediated immunity are described within this specification. These adenoviral vectors are utilized to generate and produce through cell culture various adenoviral-based HIV-1 vaccines which contain HIV-1 gag, HIV-1 pol and/or HIV-1 nef polynucleotide pharmaceutical products, and biologically relevant modifications thereof. These adenovirus vaccines, when directly introduced into living vertebrate tissue, preferably a mammalian host such as a human or a non-human mammal of commercial or domestic veterinary importance, express the HIV1-Gag, Pol and/or Nef protein or biologically modification thereof, inducing a cellular immune response which specifically recognizes HIV-1. The exemplified polynucleotides of the present invention are synthetic DNA molecules encoding HIV-1 Gag, encoding codon optimized HIV-1 Pol, derivatives of optimized HIV-1 Pol (including constructs wherein protease, reverse transcriptase, RNAse H and integrase activity of HIV-1 Pol is inactivated), HIV-1 Nef and derivatives of optimized HIV-1 Nef, including nef mutants which effect wild type characteristics of Nef, such as myristylation and down regulation of host CD4. The adenoviral vaccines of the present invention, when administered alone or in a combined modality regime, will offer a prophylactic advantage to previously uninfected individuals and/or provide a therapeutic effect by reducing viral load levels within an infected individual, thus prolonging the asymptomatic phase of HIV-1 infection.

1 - 19 . (canceled)  
     
     
         20 . An HIV vaccine composition comprising recombinant, replication-defective adenovirus particles harvested and purified from a cell line transfected with a recombinant adenoviral vector; said cell line which expresses adenovirus E1 protein at complementing levels; and said recombinant adenoviral vector which is at least partially deleted in E1 and devoid of E1 activity, and comprises: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to between from about base pair 400 to about base pair 458 of a wildtype adenovirus genome; and    b) at least one gene encoding an HIV protein selected from the group consisting of HIV gag nef, pol, and immunologically relevant modifications thereof.    
     
     
         21 . An HIV vaccine composition of  claim 20  which comprises a physiologically acceptable carrier.  
     
     
         22 - 23 . (canceled)  
     
     
         24 . A method of generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a vaccine composition of  claim 20 .  
     
     
         25 . A method according to  claim 24  which further comprises administration to the individual a DNA plasmid vaccine, optionally administered with a biologically effective adjuvant, protein or other agent capable of increasing the immune response.  
     
     
         26 . A method according to  claim 25  wherein the DNA plasmid vaccine is administered to the individual prior to administration of the vaccine composition.  
     
     
         27 . A method according to  claim 24  wherein the vaccine composition is preceded by a vaccine composition comprising purified recombinant, replication-defective adenovirus particles of a different serotype.  
     
     
         28 . A method according to  claim 24  which comprises administering and readministering the vaccine composition to the individual.  
     
     
         29 - 38 . (canceled)  
     
     
         39 . An HIV vaccine composition comprising recombinant, replication-defective adenovirus particles harvested and purified from a cell line transfected with a recombinant adenoviral vector; said cell line which expresses adenovirus E1 protein at complementing levels; and said recombinant adenoviral vector which is at least partially deleted in E1 and devoid of E1 activity and comprises: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to about base pair 450 of a wildtype adenovirus genome:    b) a region corresponding to from about base pair 3511 to about base pair 5798 of a wildtype adenovirus genome: and    c) a gene expression cassette comprising 
 i) SEQ ID NO: 27:  
 ii) a heterologous promoter operatively linked to i): and  
 iii) a transcription termination sequence;  
   wherein the vector has a deletion corresponding to from about base pair 451 to about base pair 3510 of a wildtype adenovirus genome.    
     
     
         40 . An HIV vaccine composition of  claim 39  which comprises a physiologically acceptable carrier.  
     
     
         41 - 42 . (canceled)  
     
     
         43 . A method of generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a vaccine composition of  claim 39 .  
     
     
         44 . A method according to  claim 43  which further comprises administration to the individual a DNA plasmid vaccine, optionally administered with a biologically effective adjuvant, protein or other agent capable of increasing the immune response.  
     
     
         45 . A method according to  claim 44  wherein the DNA plasmid vaccine is administered to the individual prior to administration of the vaccine composition.  
     
     
         46 . A method according to  claim 43  wherein the vaccine composition is preceded by a vaccine composition comprising purified recombinant, replication-defective adenovirus particles of a different serotype.  
     
     
         47 . A method according to  claim 43  which comprises administering and readministering the vaccine composition to the individual.  
     
     
         48 - 57 . (canceled)  
     
     
         58 . An HIV vaccine composition comprising recombinant replication-defective adenovirus particles harvested and purified from a cell line transfected with a recombinant adenoviral vector; said cell line which expresses adenovirus E1 protein at complementing levels; and said recombinant adenoviral vector which is at least partially deleted in E1 and devoid of E1 activity and comprises: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to about base pair 450 of a wildtype adenovirus genome:    b) a region corresponding to from about base pair 3511 to about base pair 5798 of a wildtype adenovirus genome; and    c) a gene expression cassette comprising 
 i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7;  
 ii) a heterologous promoter operatively linked to i); and  
 iii) a transcription termination sequence;  
   wherein the vector has a deletion corresponding to from about base pair 451 to about base pair 3510 of a wildtype adenovirus genome.    
     
     
         59 . An HIV vaccine composition ot  claim 58  which comprises a physiologically acceptable carrier.  
     
     
         60 - 61 . (canceled)  
     
     
         62 . A method of generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a vaccine composition of  claim 58 .  
     
     
         63 . A method according to  claim 62  which further comprises administration to the individual a DNA plasmid vaccine, optionally administered with a biologically effective adjuvant, protein or other agent capable of increasing the immune response.  
     
     
         64 . A method according to  claim 63  wherein the DNA plasmid vaccine is administered to the individual prior to administration of the vaccine composition.  
     
     
         65 . A method according to  claim 62  wherein the vaccine composition is preceded by a vaccine composition comprising purified recombinant, replication-defective adenovirus particles of a different serotype.  
     
     
         66 . A method according to  claim 62  which comprises administering and readministering the vaccine composition to the individual.  
     
     
         67 - 76 . (canceled)  
     
     
         77 . An HIV vaccine composition comprising recombinant, replication-defective adenovirus particles harvested and purified from a cell line transfected with a recombinant adenoviral vector: said cell line which expresses adenovirus E1 protein at complementing levels, and said recombinant adenoviral vector which is at least partially deleted in E1 and devoid of E1 activity, and comprises: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to about base pair 450 of a wildtype adenovirus genome;    b) a region corresponding to from about base pair 3511 to about base pair 5798 of a wildtype adenovirus genome; and    c) a gene expression cassette comprising 
 i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15;  
 ii) a heterologous promoter operatively linked to i); and  
 iii) a transcription termination sequence;  
   wherein the vector has a deletion corresponding to from about base pair 451 to about base pair 3510 of a wildtype adenovirus genome.    
     
     
         78 . An HIV vaccine composition of  claim 77  which comprises a physiologically acceptable carrier.  
     
     
         79 - 80 . (canceled)  
     
     
         81 . A method of generating a cellular-mediated immune response against HIV in an individual comprising administering to the individual a vaccine composition of  claim 77 .  
     
     
         82 . A method according to  claim 81  which further comprises administration to the individual a DNA plasmid vaccine, optionally administered with a biologically effective adjuvant, protein or other agent capable of increasing the immune response.  
     
     
         83 . A method according to  claim 82  wherein the DNA plasmid vaccine is administered to the individual prior to administration of the vaccine composition.  
     
     
         84 . A method according to  claim 81  wherein the vaccine composition is preceded by a vaccine composition comprising purified recombinant, replication-defective adenovirus particles of a different serotype.  
     
     
         85 . A method according to  claim 81  which comprises administering and readministering the vaccine composition to the individual.  
     
     
         86 . A multivalent adenovirus vaccine composition which comprises recombinant, replication-defective adenovirus particles harvested and purified from a cell line transfected with a recombinant adenoviral vector; said cell line which expresses adenovirus E1 protein at complementing levels; said recombinant adenoviral vector which is at least partially deleted in E1 and devoid of E1 activity, and comprises: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to between from about base pair 400 to about base pair 458 of a wildtype adenovirus genome: and    b) gene expression cassette or cassettes comprising nucleotide sequences encoding HIV proteins selected from the group consisting of: 
 i) gag, pol, and nef, expressed independently from three individual vectors;  
 ii) gag, pol, and nef, expressed independently from one vector with the encoding nucleic acid sequences operatively linked to distinct promoters and transcription termination sequences;  
 iii) gag, pol, and nef, expressed via two vectors, one expressing a pol-nef fusion, and another expressing gag;  
 iv) gag, pol, and nef, expressed via two vectors, one expressing a gag-pol fusion and another expressing nef;  
 v) gag, pol and nef, expressed via two vectors, one expressing a nef-gag fusion and another expressing pol;  
 vi) gag, pol, and nef, expressed via one vector expressing a gag-pol-nef fusion;  
 vii) gag and pol, expressed independently from two individual vectors;  
 viii) h) gag and pol, expressed independently from one vector with the encoding nucleic acid sequences operatively linked to distinct promoters and transcription termination sequences;  
 ix) pol and nef, expressed independently from two individual vectors;  
 x) pol and nef, expressed independently from one vector with the encoding nucleic acid sequences operatively linked to distinct promoters and transcription termination sequences;  
 xi) nef and gag, expressed independently from two individual vectors;  
 xii) nef and gag, expressed independently from one vector with the encoding nucleic acid sequences operatively linked to distinct promoters and transcription termination sequences;  
 xiii) gag and pol, expressed via one vector expressing a gag-pol fusion;  
 xiv) pol and nef, expressed via one vector expressing a pol-nef fusion; and  
 xv) nef and gag, expressed via one vector expressing a nef-gag fusion.  
   
     
     
         87 . A multivalent adenovirus vaccine composition in accordance with  claim 86  wherein the gag-pol fusion comprises SEQ ID NO: 35.  
     
     
         88 . A multivalent adenovirus vaccine composition in accordance with  claim 86  wherein the fused sequences have the encoding nucleic acid sequences operatively linked to distinct promoters and transcription termination sequences.  
     
     
         89 . A multivalent adenovirus vaccine composition in accordance with  claim 86  wherein the fused sequences have the encoding nucleic acid sequences operatively linked to a single promoter; and the encoding nucleic acid sequences operatively linked by an internal ribosome entry sequence (“IRES”).  
     
     
         90 . A recombinant adenoviral vector at least partially deleted in E1 and devoid of E1 activity, comprising: 
 a) an adenovirus cis-acting packaging region corresponding to from about base pair 1 to about base pair 450 of a wildtype adenovirus genome;    b) a region corresponding to from about base pair 3511 to about base pair 5798 of a wildtype adenovirus genome; and    c) a gene expression cassette comprising 
 i) SEQ ID NO: 3;  
 ii) a heterologous promoter operatively linked to i); and  
 iii) a transcription termination sequence;  
   wherein the vector has a deletion corresponding to from about base pair 451 to about base pair 3510 of a wildtype adenovirus genome.    
     
     
         91 . An adenoviral vector in accordance with  claim 90  wherein the gene expression cassette is in an E1 parallel orientation.  
     
     
         92 . An adenoviral vector in accordance with  claim 90  wherein the gene expression cassette is in an E1 antiparallel orientation.  
     
     
         93 . An adenoviral vector in accordance with  claim 90  wherein the promoter is a cytomegalovirus promoter devoid of intronic sequences.  
     
     
         94 . An adenoviral vector in accordance with  claim 90  wherein the transcription termination sequence is a bovine growth hormone polyadenylation and transcription termination sequence.  
     
     
         95 . An adenoviral vector in accordance with  claim 90  which is at least partially deleted in E3.  
     
     
         96 . A cell comprising the adenoviral vector of  claim 90 .  
     
     
         97 . Recombinant, replication-defective adenovirus particles harvested and purified subsequent to transfection of the adenoviral vector of  claim 90  into a cell line which expresses adenovirus E1 protein at complementing levels.  
     
     
         98 . A method of producing recombinant, replication defective adenovirus particles containing the adenoviral genome of the adenoviral vector of  claim 90  which comprises introducing the adenoviral vector into a host cell which expresses adenoviral E1 protein, and harvesting the resultant recombinant, replication-defective adenovirus.  
     
     
         99 . A method according to  claim 98  wherein the cell expresses a transgene inclusive of nucleotides 459-3510 of adenovirus serotype 5.