Antigenic mimics of discontinuous epitopes of pathogen recognized by broadly neutralizing antibodies
The present invention relates to an anti-idiotypic polypeptide scaffold that includes two or more peptide sequences that mimic a discontinuous epitope of a pathogen that is recognized by or induces formation of a broadly neutralizing antibody. Using a fibronectin FNfn10 scaffold bearing two or more modified discontinuous loops, scaffolds that recognize broadly neutralizing antibodies in vitro and from patient serum have been identified. These scaffolds should induce an immune response or mobilize germline specificities to initiate their affinity maturation.
1. A modified polypeptide comprising:
β-strand domain sequences A, B, C, D, E, F, and G of a wild-type mammalian Fn3 domain of fibronectin with loop region sequences AB, BC, CD, DE, EF, and FG linked between adjacent β-strand domain sequences; and
optionally, an N-terminal tail of at least 2 amino acids, a C-terminal tail of at least 2 amino acids, or both;
wherein at least one loop region sequence is selected from the BC and/or FG loop regions and comprises a modified amino acid sequence which varies from the wild-type sequence by deletion, insertion, or replacement of at least two amino acids from a different loop region in the wild-type mammalian Fn3 domain of fibronectin, resulting in a recombinant, modified Fn3 domain, wherein the modified amino acid sequence of the at least one recombinant, modified BC and/or FG loop region sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:117, SEQ ID NO:118, 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:128, or SEQ ID NO:129.
2. The modified polypeptide according to claim 1 , wherein the modified polypeptide binds specifically to a neutralizing monoclonal antibody (Mab) selected from the group consisting of: HIV Mab b12, HIV F105 Mab, HIV PG9 Mab, HIV PG16 Mab, HIV 2G12 Mab, HIV 2F5 Mab, HIV 4E10 Mab, HIV VRC01 Mab, Dengue 1 F1 Mab, Influenza CR6261 Mab, Influenza F10 Mab, Influenza D8 Mab, and combinations thereof.
3. The modified polypeptide according to claim 1 , wherein the modified polypeptide is capable of inducing an immune response against a viral pathogen, wherein the viral pathogen is selected from the group consisting of HIV, dengue virus, and/or influenza virus.
4. The modified polypeptide according to claim 1 , wherein said at least one recombinant, modified loop region sequence is a BC loop region sequence, wherein the modified amino acid sequence comprises SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, or SEQ ID NO:128.
5. The modified polypeptide according to claim 1 , wherein said at least one recombinant, modified loop region sequence is a FG loop region sequence, wherein the modified amino acid sequence comprises SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, or SEQ ID NO:129.
6. The modified polypeptide according to claim 1 , wherein the at least one recombinant, modified loop region sequence is a combination of the BC loop region sequence and the FG loop region sequence, wherein the modified BC and FG loop region sequences comprise, respectively, SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:22 and SEQ ID NO:23; SEQ ID NO:13 and SEQ ID NO:24; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:145 and SEQ ID NO:146; SEQ ID NO:147 and SEQ ID NO:148; SEQ ID NO:149 and SEQ ID NO:150; SEQ ID NO:151 and SEQ ID NO:152; SEQ ID NO:153 and SEQ ID NO:154; SEQ ID NO:155 and SEQ ID NO:156; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:159 and SEQ ID NO:160; SEQ ID NO:161 and SEQ ID NO:162; SEQ ID NO:163 and SEQ ID NO:164; SEQ ID NO:165 and SEQ ID NO:167; SEQ ID NO:166 and SEQ ID NO:168; SEQ ID NO:117 and SEQ ID NO:118; SEQ ID NO:119 and SEQ ID NO:120; SEQ ID NO:117 and SEQ ID NO:121; SEQ ID NO:122 and SEQ ID NO:123; SEQ ID NO:124 and SEQ ID NO:125; SEQ ID NO:126 and SEQ ID NO:127; or SEQ ID NO:128 and SEQ ID NO:129.
7. The modified polypeptide according to claim 1 , wherein the wild-type Fn3 domain of fibronectin is a wild-type tenth Fn3 domain of fibronectin.
8. An isolated nucleic acid molecule encoding the modified polypeptide of claim 1 .
9. The isolated nucleic acid molecule according to claim 8 , wherein the isolated nucleic acid is DNA.
10. A DNA construct comprising the DNA molecule of claim 9 , further comprising a promoter operably coupled to the 5′ of the DNA molecule and a transcription termination DNA molecule operably coupled to the 3′ of the DNA molecule.
11. An expression vector comprising the DNA construct of claim 10 .
12. An isolated host cell transformed with a DNA construct according to claim 10 .
13. A composition comprising a pharmaceutically acceptable carrier and the modified polypeptide according to claim 1 .
14. An immunogenic composition comprising a pharmaceutically acceptable carrier and either the modified polypeptide according to claim 1 , a DNA molecule encoding the modified polypeptide, or a combination thereof.
15. A method of administering an immunogenic composition according to claim 14 to a mammal under conditions effective to induce an immune response against the modified polypeptide.
16. An isolated antibody or binding fragment thereof specific for the modified polypeptide according to claim 1 .
17. A method for detecting a neutralizing antibody in serum comprising:
providing the modified polypeptide of claim 1 conjugated to a label;
contacting the labeled, modified polypeptide with serum from an individual; and
detecting whether the labeled, modified polypeptide binds to an antibody present in the serum, wherein said detecting is carried out using the label.