Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof

Abstract

The present invention relates to polynucleotides encoding immunogenic HIV type C polypeptides. Uses of the polynucleotides in applications including DNA immunization, generation of packaging cell lines, and production of HIV Type C proteins are also described.

1 . An expression cassette comprising a polynucleotide sequence encoding a polypeptide comprising:
 (a) an HIV Pol polypeptide, wherein the polynucleotide sequence encoding said Pol polypeptide comprises a sequence having at least 90% sequence identity to the sequence presented of  FIG. 8  (SEQ ID NO:30);  FIG. 9  (SEQ ID NO:31) or  FIG. 10  (SEQ ID NO:32);   (b) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:46, (ii) X equals Y, and (iii) Y is at least 97:   (c) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:51 (ii) X equals Y, and (iii) Y is 1494:   (d) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:99, (ii) X equals Y, and (iii) Y is 1491:   (e) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:101; SEQ ID NO:96; SEQ ID NO:134 or SEQ ID NO:135, (ii) X equals Y, and (iii) Y is at least 60:   (f) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:58; (ii) X equals Y, and (iii) Y is 354:   (g) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:60; (ii) X equals Y, and (iii) Y is 876.   (h) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:62; (ii) X equals Y, and (iii) Y is 3015:   (i) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:103; (ii) X equals Y, and (iii) Y is 3009:   (j) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:64 or SEQ ID NO:66; (ii) X equals Y, and (iii) Y is 297;   (k) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:68, (ii) X equals Y, and (iii) Y is 1965:   (l) An expression cassette comprising a polynucleotide comprising X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:70; (ii) X equals Y, and (iii) Y is 1977;   (m) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:72 or SEQ ID NO:105, (ii) X equals Y, and (iii) Y is at least 30;   (n) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:74 or SEQ ID NO:107, (ii) X equals Y, and (iii) Y is at least 30;   (o) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:76; (ii) X equals Y, and (iii) Y is 1680:   (p) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:78; (ii) X equals Y, and (iii) Y is 1668:   (q) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:109; (ii) X equals Y, and (iii) Y is 216;   (r) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:83; (ii) X equals Y, and (iii) Y is 93;   (s) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:111; (ii) X equals Y, and (iii) Y is 90;   (t) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:85, or SEQ ID NO:113; (ii) X equals Y, and (iii) Y is 579.   (u) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:87; (ii) X equals Y, and (iii) Y is 288.   (v) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:115; (ii) X equals Y, and (iii) Y is 287:   (w) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:89 or SEQ ID NO:117; (ii) X equals Y, and (iii) Y is at least 30;   (x) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:91; (ii) X equals Y, and (iii) Y is at least 30:   (y) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:93 or SEQ ID NO:94; (ii) X equals Y, and (iii) Y is 309;   (z) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:96; (ii) X equals Y, and (iii) Y is at least 60; or   (aa) X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:119, SEQ ID NO:120; SEQ ID NO:121; SEQ ID NO:122; SEQ ID NO:123; SEQ ID NO:124; SEQ ID NO:125; SEQ ID NO:126; SEQ ID NO:127; SEQ ID NO:131; SEQ ID NO:132 or SEQ ID NO:133, (ii) X equals Y, and (iii) Y is at least 60.   
     
     
         2 . (canceled) 
     
     
         3 . The expression cassette of  claim 1 , wherein the polynucleotide comprises (b) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:47, (ii) X equals Y, and (iii) Y is at least 144. 
     
     
         4 . The expression cassette of  claim 3 , comprising a polynucleotide comprising X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:49 or SEQ ID NO:97, (ii) X equals Y, and (iii) Y is at least 300. 
     
     
         5 . The expression cassette of  claim 4 , comprising a polynucleotide comprising X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:49, (ii) X equals Y, and (iii) Y is 2610. 
     
     
         6 . The expression cassette of  claim 4 , comprising a polynucleotide comprising X contiguous nucleotides, wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:97, (ii) X equals Y, and (iii) Y is 2565. 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The expression cassette of  claim 1 , wherein the polynucleotide, comprises (e) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:101; SEQ ID NO:96; SEQ ID NO:134 or SEQ ID NO:135, (ii) X equals Y, and (iii) Y is 624. 
     
     
         11 - 18 . (canceled) 
     
     
         19 . The expression cassette of  claim 1 , wherein the polynucleotide comprises (m) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:72 or SEQ ID NO:105; (ii) X equals Y, and (iii) Y is 75. 
     
     
         20 . (canceled) 
     
     
         21 . The expression cassette of  claim 1 , comprising a wherein the polynucleotide comprising X contiguous nucleotides, comprises (n) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:74 or SEQ ID NO:107; (ii) X equals Y, and (iii) Y is 246. 
     
     
         22 - 30 . (canceled) 
     
     
         31 . The expression cassette of  claim 1  wherein the polynucleotide comprises (w) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:89; (ii) X equals Y, and (iii) Y is 267. 
     
     
         32 . The expression cassette of  claim 1  wherein the polynucleotide comprises (w) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:117; (ii) X equals Y, and (iii) Y is 261. 
     
     
         33 . (canceled) 
     
     
         34 . The expression cassette of  claim 1  wherein the polynucleotide comprises (x) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:91; (ii) X equals Y, and (iii) Y is 321. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The expression cassette of  claim 1  wherein the polynucleotide comprises (z) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:96; (ii) X equals Y, and (iii) Y is 624. 
     
     
         38 . (canceled) 
     
     
         39 . The expression cassette of  claim 1 , wherein the polynucleotide comprises (aa) and wherein (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:119, SEQ ID NO:120; SEQ ID NO:121; SEQ ID NO:122; SEQ ID NO:123; SEQ ID NO:124; SEQ ID NO:125; SEQ ID NO:126; SEQ ID NO:127; SEQ ID NO:131; SEQ ID NO:132 or SEQ ID NO:133, (ii) X equals Y, and (iii) Y is at least 300. 
     
     
         40 . The expression cassette of  claim 39 , wherein:
 (a) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:123 or SEQ ID NO:124, (ii) X equals Y, and (iii) Y is 2433;   (b) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:122, (ii) X equals Y, and (iii) Y is 2301;   (c) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:125; (ii) X equals Y, and (iii) Y is 2517;   (d) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:126 or SEQ ID NO:127, (ii) X equals Y, and (iii) Y is 2520;   (e) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:119, (ii) X equals Y, and (iii) Y is 1377;   (f) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:120 or SEQ ID NO:121, (ii) X equals Y, and (iii) Y is 1839; or   (g) (i) the X contiguous nucleotides have at least 90% percent identity to Y contiguous nucleotides of SEQ ID NO:132 or SEQ ID NO:133, (ii) X equals Y, and (iii) Y is 1890.   
     
     
         41 - 46 . (canceled) 
     
     
         47 . A polynucleotide comprising the sequence depicted in SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:128, or fragments derived therefrom. 
     
     
         48 . The polynucleotide of  claim 47 , wherein said fragments comprise coding sequence for the gene products selected from the group consisting of Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, Env and Nef. 
     
     
         49 . The polynucleotide of  claim 48 , wherein the fragment comprises a Gag gene product. 
     
     
         50 . The polynucleotide of  claim 48 , wherein the fragment comprises an Env gene product. 
     
     
         51 . The polynucleotide of  claim 50 , wherein the Env gene product is gp160, gp140 or gp120. 
     
     
         52 - 58 . (canceled) 
     
     
         59 . The expression cassette of  claim 1 , further comprising one or more nucleic acids encoding one or more viral polypeptides or antigens. 
     
     
         60 . The expression cassette of  claim 59 , wherein the viral polypeptide or antigen is selected from the group consisting of Gag, Env, vif, vpr, tat, rev, vpu, nef and combinations thereof. 
     
     
         61 . The expression cassette of  claim 1 , further comprising one or more nucleic acids encoding one or more cytokines. 
     
     
         62 . A recombinant expression system for use in a selected host cell, comprising, an expression cassette of  claim 1 , and wherein said polynucleotide sequence further comprises control elements capable of driving expression in the selected host cell. 
     
     
         63 . The recombinant expression system of  claim 62 , wherein said control elements are selected from the group consisting of a transcription promoter, a transcription enhancer element, a transcription termination signal, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. 
     
     
         64 . The recombinant expression system of  claim 62  wherein said transcription promoter is selected from the group consisting of CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, and metallothionein. 
     
     
         65 . A cell comprising an expression cassette of  claim 1 , and wherein said polynucleotide sequence further comprises control elements compatible with expression in the selected cell. 
     
     
         66 . The cell of  claim 65 , wherein the cell is selected from the group consisting of a mammalian cell, an insect cell, a bacterial cell, a yeast cell, a plant, an antigen presenting cell, a primary cell, an immortalized cell, and a tumor derived cell. 
     
     
         67 . The cell of  claim 66 , wherein the cell is selected from the group consisting of BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells. 
     
     
         68 . The cell of  claim 67 , wherein said cell is a CHO cell. 
     
     
         69 . The cell of  claim 66 , wherein the cell is either  Trichoplusia ni  (Tn5) or Sf9 insect cells. 
     
     
         70 . The cell of  claim 66 , wherein the antigen presenting cell is a lymphoid cell selected from the group consisting of macrophage, monocytes, dendritic cells, B-cells, T-cells, stem cells, and progenitor cells thereof. 
     
     
         71 . A composition for generating an immunological response, comprising an expression cassette of  claim 1 . 
     
     
         72 . The composition of  claim 71 , further comprising one or more Pol polypeptides. 
     
     
         73 . The composition of  claim 72 , further comprising an adjuvant. 
     
     
         74 . A composition for generating an immunological response, comprising an expression cassette of claim  52 . 
     
     
         75 . The composition of  claim 74 , further comprising a Pol polypeptide. 
     
     
         76 . The composition of  claim 74 , further comprising one or more polypeptides encoded by the nucleic acid molecules encoding a viral polypeptide or antigen selected from the group consisting of Gag, Env, vif, vpr, tat, rev, vpu, nef and combinations thereof. 
     
     
         77 . The composition of  claim 76 , further comprising an adjuvant. 
     
     
         78 . A method of immunization of a subject, comprising, introducing a composition of  claim 71  into said subject under conditions that are compatible with expression of said expression cassette in said subject. 
     
     
         79 . The method of  claim 78 , wherein said expression cassette is introduced using a gene delivery vector. 
     
     
         80 . The method of  claim 79 , wherein the gene delivery vector is a non-viral vector. 
     
     
         81 . The method of  claim 79 , wherein said gene delivery vector is a viral vector. 
     
     
         82 . The method of  claim 79 , wherein said gene delivery vector is selected from the group consisting of an adenoviral vector, a vaccinia viral vector, an AAV vector, a retroviral vector, a lentiviral vector and an alphaviral vector. 
     
     
         83 . The method of  claim 82 , wherein said gene delivery vector is a Sindbis-virus derived vector. 
     
     
         84 . The method of  claim 82 , wherein said gene delivery vector is a cDNA vector. 
     
     
         85 . The method of  claim 82 , wherein said gene delivery vector is a eukaryotic layered viral initiation system (ELVIS). 
     
     
         86 . The method of  claim 79 , wherein said composition delivered using a particulate carrier. 
     
     
         87 . The method of  claim 79 , wherein said composition is coated on a gold or tungsten particle and said coated particle is delivered to said subject using a gene gun. 
     
     
         88 . The method of  claim 79 , wherein said composition is encapsulated in a liposome preparation. 
     
     
         89 . The method of  claim 79 , wherein said subject is a mammal. 
     
     
         90 . The method of  claim 89 , wherein said mammal is a human. 
     
     
         91 . A method of generating an immune response in a subject, comprising: providing an expression cassette of  claim 1 , expressing said polypeptide in a suitable host cell, isolating said polypeptide, and administering said polypeptide to the subject in an amount sufficient to elicit an immune response. 
     
     
         92 . A method of generating an immune response in a subject, comprising introducing into cells of said subject an expression cassette of  claim 1 , under conditions that permit the expression of said polynucleotide and production of said polypeptide, thereby eliciting an immunological response to said polypeptide. 
     
     
         93 . The method of  claim 92 , where the method further comprises co-administration of an HIV polypeptide. 
     
     
         94 . The method of  claim 93 , wherein co-administration of the polypeptide to the subject is carried out before introducing said expression cassette. 
     
     
         95 . The method of  claim 93 , wherein co-administration of the polypeptide to the subject is carried out concurrently with introducing said expression cassette. 
     
     
         96 . The method of  claim 93 , wherein co-administration of the polypeptide to the subject is carried out after introducing said expression cassette. 
     
     
         97 . The expression cassette of  claim 59 , wherein the viral polypeptide or antigen is selected from the group consisting of polypeptides derived from hepatitis B, hepatitis C and combinations thereof.