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(12) Patent Application Publication (10) Pub. No.: US 2002/014 1975 A1 Olmsted Et Al US 2002O141975A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/014 1975 A1 Olmsted et al. (43) Pub. Date: Oct. 3, 2002 (54) ALPHAVIRUS VECTORS AND WIROSOMES Publication Classification WITH MODIFIED HIV GENES FOR USE IN WACCINES (51) Int. Cl." ......................... A61K 48/00; A61K 39/21; A61K 39/12; CO7H 21/04; (75) Inventors: Robert Olmsted, Chapel Hill, NC AO1N 63/00; CO7K 1/00; (US); Paula Keith, Holly Springs, NC C07K 14/00; CO7K 17/00 (US); Sergey Dryga, Chapel Hill, NC (52) U.S. Cl. ................. 424/93.2; 424/188.1; 424/186.1; (US); Ian Caley, Durham, NC (US); 530/350; 530/826; 536/23.72 Maureen Maughan, Durham, NC (US); Robert Johnston, Chapel Hill, NC (US); Nancy Davis, Chapel Hill, (57) ABSTRACT NC (US); Ronald Swanstrom, Chapel The present invention provides methods and compositions Hill, NC (US) comprising a population of alphavirus replicon particles Correspondence Address: comprising two or more isolated nucleic acids Selected from NEEDLE & ROSENBERG PC 1) an isolated nucleic acid encoding an env gene product or 127 PEACHTREE STREET N E an immunogenic fragment thereof of a human immunode ATLANTA, GA 30303-1811 (US) ficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a (73) ASSignee: Alpha Vax, Inc. human immunodeficiency virus, wherein the gag gene prod (21) Appl. No.: 09/991,258 uct or immunogenic fragment thereof is modified to inhibit formation of Virus-like particles containing the gag gene (22) Filed: Nov. 16, 2001 product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding Related U.S. Application Data a pol gene product or an immunogenic fragment thereof of (63) Continuation-in-part of application No. 09/902,537, a human immunodeficiency virus, wherein the poll gene filed on Jul. 9, 2001, now abandoned. product or immunogenic fragment thereof is modified to inhibit protease, integrase, RNase H and/or reverse tran (60) Provisional application No. 60/216,995, filed on Jul. Scriptase activity, and wherein the nucleic acids are each 7, 2000. contained within a separate alphavirus replicon particle. Aba I (12497) EcoRI (12354), KN(R) COLE1 OR EcoRI (2137) nSP2 gag 26S promoter M nsP3 Apa I (7506) nsP4 Patent Application Publication Oct. 3, 2002. Sheet 1 of 15 US 2002/0141975 A1 Xba I (12497) EcoRI (12354). T7P KN(R) COLE1 OR EcoRI (2137) nSP2 of I (9232) gag 26S promoter es ' ' ' ...? nsP3 Apa I (7506) f nsP4 FIG. 1 Patent Application Publication Oct. 3, 2002. Sheet 2 of 15 US 2002/0141975 A1 Xba I (5047) TTP EcoRI (4904) , KN(R) ^ 26S promoter COLE1 ORI Not I (1782) FIG. 2 Patent Application Publication Oct. 3, 2002. Sheet 3 of 15 US 2002/0141975 A1 Xba I (6960) EcoRI (6817) T7P KNR) \ M 26S promoter E2 COLE1 ORI Xba I (1930 Not I (3695) FIG. 3 Patent Application Publication Oct. 3, 2002. Sheet 4 of 15 US 2002/0141975 A1 COLE1 OR nSP2 Not I (9085) p51 26S promoter - InsP3 FIG. 4 Patent Application Publication Oct. 3, 2002. Sheet 5 of 15 US 2002/0141975 A1 Xba I (13549) EcoRI (13406), KNR) lalla nsP coLE1 OR - EcoRI (2131) nsP2 pERK-DU151env Noti (10284) 13584 bp DU15 env V nsP3 Xbai (8642) 26S promoter / Apa I (7500) nsP4 FIG. 5 Patent Application Publication Oct. 3, 2002. Sheet 6 of 15 US 2002/0141975 A1 . * * * . x' \* * :::::::3% . ; ; , s: ' ' ' . 8.3: vs. x - 8: ... ::::::. ::::::: ... : : FIG. 6 Patent Application Publication Oct. 3, 2002. Sheet 8 of 15 US 2002/0141975 A1 Patent Application Publication Oct. 3, 2002 Sheet 9 of 15 US 2002/0141975 A1 1 OO 75 2 > 50 - S. | 25 O 1 OO 50 25 12 6 3 E/T Ratio FIG. 9A 1 OO 50 25 12 6 3 E/T Ratio FIG. 9B 1 OO 75 92 >9. 50 SS 25 O 1 OO 50 25 12 6 3 E IT Ratio FIG. 9C Patent Application Publication Oct. 3, 2002. Sheet 10 of 15 US 2002/0141975 A1 Patent Application Publication Oct. 3, 2002 Sheet 14 of 15 US 2002/0141975A1 Patent Application Publication Oct. 3, 2002 Sheet 15 of 15 US 2002/0141975 A1 US 2002/014 1975 A1 Oct. 3, 2002 ALPHAVIRUS VECTORS AND WIROSOMES WITH Immediately following infection, the nsPs are produced by MODIFIED HIV GENES FOR USE IN WACCNES translation of parental genomes and catalyze the Synthesis of 0001. This application is a continuation-in-part of and a full-length negative-Sense copy of the genome. This Serves claims priority to, U.S. application Ser. No. 09/902,537, as a template for the Synthesis of progeny plus-Stranded filed Jul. 9, 2001 (abandoned), which claims priority to genomeS. provisional application Serial No. 60/216,995, filed Jul. 7, 0009. The negative-sense copy of the genome also serves 2000, which applications are incorporated by reference as the template for the synthesis of subgenomic mRNA at herein in their entirety. approximately 10-fold molar exceSS relative to genomic RNA in infected cells (Schlesinger and Schlesinger, 1990). BACKGROUND OF THE INVENTION Synthesis of Subgenomic 26S mRNA is initiated from the highly active internal 26S mRNA promoter, which is func 0002) 1. Field of the Invention tional only on the negative-Sense RNA. The Subgenomic 0003. The present invention relates to vaccines using mRNA corresponds to the 3' one-third of the genome and Viral antigens, and in particular, to vaccines for the treatment encodes the alphavirus Structural proteins. and prevention of human immunodeficiency virus (HIV) 0010 Full-length, infectious cDNA clones of the RNA infection. The vaccines of this invention comprise alphavi genome of VEE Davis et al., 1989) have been constructed, ruS RNA replicon Systems which contain nucleic acid a panel of mutations which Strongly attenuate the virus have Sequence encoding antigens for eliciting an immune been identified (Johnston and Smith, 1988; Davis et al., response to HIV. 1990), and various constellations of these attenuating muta 0004 2. Background tions have been inserted into the clones to generate Several live attenuated VEE vaccine candidates (Davis et al., 1991; 0005. The successful control of the AIDS epidemic will 1995b, Grieder et al., 1995). The resulting vaccine candi require an effective vaccine for human immunodeficiency dates are avirulent and provide complete protection against virus type 1 (HIV) that significantly reduces or prevents the lethal virus challenge in rodents, horses and nonhuman Spread of infection. Currently, Several viral vector Systems primates. as well as naked DNA are at various Stages of pre-clinical and clinical evaluation as candidate HIV Vaccines. Recom 0011. The alphavirus VRPs are propagation defective, binant poxyiruses are the most widely Studied virus vectors Single cycle vectors that contain a Self-amplifying alphavirus and are furthest along in clinical development (e.g., RNA (replicon RNA) in which the structural protein genes ALVAC). of the virus are replaced by a heterologous antigen gene to be expressed. Alphavirus VRPs are typically made in cul 0006 The alphavirus-based replicon particle systems, tured cells, referred to as packaging cells. Following intro Such as the ones described in U.S. Pat. No. 5,792,462 and duction into mammalian cells, the replicon RNA ig pack herein referred to as “VRPs,” have multiple distinct prop aged into VRP by Supplying the Structural proteins in erties that make them attractive as an HIV vaccine delivery “trans, i.e., the cells are co-transfected with both replicon technology. These properties include: natural targeting to RNA and one or more separate helper RNAS which together and expression in lymphoid tissues (an optimal site for encode the full complement of alphavirus Structural pro induction of an immune response); high antigen expression teins. Importantly, only the replicon RNA is packaged into levels, e.g., up to 20% of total cell protein; induction of VRP, as the helper RNA(s) lack the cis-acting packaging balanced humoral, cellular, and mucosal immune responses, Sequence required for encapsidation. Thus, the VRPs are Sustained efficacy over multiple Simultaneous or Sequential defective, in that they can only infect target cells in culture inoculations of the vector; and a high margin of Safety. or in Vivo, where they express the heterologous antigen gene 0007 Venezuelan equine encephalitis virus (VEE) is a to high level, but they lack critical portions of the VEE member of the Alphaviruses group, which also includes the genome (i.e., the VEE Structural protein genes) necessary to prototype Sindbis virus (SIN) and Semliki Forest virus produce virus particles which could spread to other cells. (SFV), and is comprised of enveloped viruses containing 0012 Delivery of the replicon RNA into target cells (for plus-Stranded RNA genomes within icosahedral capsids vaccination) is facilitated by the VRP following infection of (Strauss, 1994). Alphavirus genomes are: approximately the target cells. In the cytoplasm of the target cell, the 11.5 kb long, capped, polyadenylated, and infectious under replicon RNA is first translated to produce the viral replicase appropriate transfection conditions. The nucleocapsid is proteins necessary to initiate Self-amplification and expres composed of 240 molecules of the capsid protein arranged Sion. The heterologous antigen gene is encoded by a Sub as a T=4 icosahedron, and is Surrounded by a lipoprotein genomic mRNA, abundantly transcribed from the replicon envelope (Paredes et al., 1993). Protruding from the virion RNA, leading to high level expression of the heterologous Surface are 80 glycoprotein Spikes, each of which is a trimer antigen gene product. Since the VEE Structural protein genes of Virally encoded E1 and E2 glycoprotein heterodimers. are not encoded by the replicon RNA delivered to the target The Virions contain no host proteins.
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