Adenoviral Vectors Comprising Partial Deletions of E3

Abstract

This disclosure provides replication-incompetent adenoviral vectors useful in vaccine development and gene therapy. The disclosed vectors comprise a selective deletion of E3 and are particularly useful for preparation of vaccines development and for gene therapy using toxic transgene products that result in vector instability that occurs when the entire E3 domain is deleted.

Claims

1. A method of inducing an immune response in a subject, the method comprising administering to the subject a replication-incompetent adenovirus vector comprising: a) open reading frame (ORF) ORF1 and ORF2 of E3 genomic region; b) selective deletion of E3 genomic region consisting of ORF3, ORF4, ORF5, ORF6, and ORF7; and, c) at least one ORF encoding a non-adenoviral anti-apoptotic protein.

2. The method of claim 1, wherein the anti-apoptotic protein is selected from the group consisting of a p53 inhibitor, Bcl-XL, BCL2, BCL2L1, BCL2A1, BAG1, TRAF1, BIRC3, BIRC5, BAK1, cIAP1, c-IAP2, XIAP, and API5.

3. The method of claim 1, wherein the replication-incompetent adenovirus vector is a human serotype.

4. The method of claim 1, wherein the replication-incompetent adenovirus vector has group A serotype, group B serotype, group C serotype, group D serotype, group E serotype, or group F serotype.

5. The method of claim 1, wherein the replication-incompetent adenovirus vector has serotype Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad11, Ad20, Ad21, Ad22, Ad23, Ad24, Ad25, Ad26, Ad28, Ad34, Ad35, Ad40, Ad41, Ad48, Ad49, or Ad50.

6. The method of claim 1, wherein the replication-incompetent adenovirus vector has serotype AdC1, AdC3, AdC6, AdC7, or AdC68.

7. The method of claim 1, wherein the replication-incompetent adenovirus comprises a deletion in E1 genomic region.

8. The method of claim 1, wherein the replication-incompetent adenovirus vector further comprises an ORF encoding an antigenic protein.

9. A replication-incompetent adenovirus vector comprising: a) open reading frame (ORF) ORF1 and ORF2 of E3 genomic region; b) selective deletion of E3 genomic region consisting of ORF3, ORF4, ORF5, ORF6, and ORF7; c) at least one ORF selected from the group consisting of ORF8 and ORF9; and d) at least one ORF encoding an antigenic protein.

10. The replication-incompetent adenovirus vector of claim 9, which is a human serotype.

11. The replication-incompetent adenovirus vector of claim 9, which has a serotype of a group selected from the group consisting of group A, group B, group C, group D, group E, and group F.

12. The replication-incompetent adenovirus vector of claim 9, which has a serotype selected from the group consisting of Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad11, Ad20, Ad21, Ad22, Ad23, Ad24, Ad25, Ad26, Ad28, Ad34, Ad35, Ad40, Ad41, Ad48, Ad49, and Ad50.

13. The replication-incompetent adenovirus vector of claim 9, which has a serotype selected from the group consisting of Ad C1, Ad C3, Ad C6, Ad C7, and Ad C68.

14. The replication-incompetent adenovirus vector of claim 9, comprising a deletion in E1.

15. An immunogenic composition comprising the replication-incompetent adenovirus vector of claim 9.

16. A dosage unit of the immunogenic composition of claim 15, which comprises 108 to 1011 adenoviral particles.

17. A method of inducing an immune response to the antigenic protein in a subject, comprising administering to the subject the immunogenic composition of claim 15.

18. A packaging cell line comprising the replication-incompetent adenovirus vector of claim 9.

19. The packaging cell line of claim 18 comprising HEK293 cells.

20. The replication-incompetent adenovirus vector of claim 9, which has the serotype Ad C7.

Description

EXAMPLE 1

[0021] Construction of pAdC6 020

[0022] The E3 region of AdC6 contains 5052 base pairs (bp), with a total of nine open reading frames: ORF1 is 325 bp; ORF2 is 624 bp; ORF3 is 531 bp; ORF4 is 684 bp; ORF5 is 612 bp; ORF6 is 867 bp; ORF7 is 276 bp; ORF8 is 435 bp; and ORF9 is 408 bp. ORF3, ORF4, ORF5, ORF6 and ORF7 were deleted to create the vector “AdC6 020” (the total length of the deleted sequence was 3218 bp).

EXAMPLE 2

[0023] Construction of Stable Adenoviral Vectors Comprising HIV Envelope Sequences

[0024] pAdC6 020 was used to create two vectors encoding HIV envelope sequences, “AdC6 020-HIVgp140-DU172” and “AdC6 020-HIVgp140-DU422.” The HIV strains that served as donors for the envelope sequences were isolated from clinical materials. The coding sequences were codon-optimized to permit enhanced expression in human cells. To create the pAdC6 020 plasmid, PCR-based cloning strategies were used to obtain the fragment without ORF3, ORF4, ORF5, ORF6 and ORF7 of E3 region. By using that PCR product, the pXY-E3 deleted plasmid was generated. Both pXY-E3 deleted (donor for insert) and pC6 E3 deleted (donor for backbone) plasmids were digested with PspX I and Nco I restriction enzymes to result in the pC6 E3 modified plasmid. Finally the modified E3 fragment was excised out from pC6 E3 modified plasmid by Sbf I digestion and cloned into pAdC6 000-eGFP plasmid (E1 deleted only) with E3 removed by Sbf I digestion. This resulted in pAdC6 020-eGFP. AdC6 vectors comprising the HIV coding sequences and a deletion of E1 or a deletion of E1 and of all the ORFs of E3 could not be rescued. Similar problems were encountered using adenovirus vectors of human serotype 5 (Ad5) and 26 (Ad26). Upon modifying the E3 deletion within AdC6, Ad5 and Ad26, however, all of those vectors could readily be rescued. These vectors passed quality control assays including restriction enzyme mapping, and Western blots showed that vectors expressed HIV-1 gp140 upon transfection of cells. These vectors were stable after 12 sequential passages shown by restriction enzyme mapping. Vectors rescued successfully by modified E3 region are summarized in the following table.

TABLE-US-00001 Viral Western Blot Viral Vector Name Rescued Stability for transgene Genome AdC6 020-HIVgp140-DU172 yes stable yes correct AdC6 020-HIVgp140-DU422 yes stable yes correct AdC7 010-HIVgp140-DU172 yes stable yes correct AdC7 010-HIVgp140-DU422 yes stable yes correct AdC6 020-SIVgp160 yes N.D. N.D. correct AdC6 020-HIVgag yes N.D. N.D. N.D. Ad5 060-HIVgp140-DU422 yes stable yes correct Ad5 060-SIVgp160 yes N.D. N.D. N.D. Ad26 011-HIVgp140-DU422 yes N.D. N.D. correct N.D.—Not Determined