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 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 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, , 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 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 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. cell, progeny Virion particles are not assembled, thus limit 0008 Alphaviruses share replication strategies and ing the replication to a single cycle within the infected target genomic organization. The complete replicative cycle of cell. Experimental VRP vaccines have been successful in alphaviruses occurs in the cytoplasm of infected cells. vaccinating rodents against virus, Lassa fever Expression from the alphavirus genome is Segregated into virus and Marburg virus (Pushko et al., 1997; Hevey et al., two regions. The four enzymatic nonstructural proteins 1998). In nonhuman primates, VRP vaccines have demon (nsP1-nsP4) are synthesized from the 5' two-thirds of the Strated complete efficacy against lethal Marburg virus chal genome-length RNA and are required for RNA replication. lenge (Hevey et al., 1998), shown partial but significant US 2002/014 1975 A1 Oct. 3, 2002 protection against SIV infection and disease (Davis et al., 0017. After these inoculations, three of four test 2000) and induced an anti-HA response at a level consistent macaques had measurable CTL-specific killing directed with protection of humans against influenza virus infection. against both SIV gag and env, all four had gp160 IgG 0013 The alphavirus based replicon vector systems, and antibody by ELISA, and the three animals which harbored in particular the VEE-based Systems, present Several advan SIV-specific CTL also showed neutralizing antibody to tages in vaccination, including Safety and high immunoge SIVsmH-4. nicity/efficacy. VEE is unique among the alphaviruses in that 0018 Four of four vaccinated animals were protected a live attenuated IND VEE vaccine, TC-83, (Kinney et al., against disease for at least 16 months following intravenous 1989; Kinney et al., 1993) has been inoculated into approxi challenge with the pathogenic SIV Swarm, while the two mately 8,000 humans. This allows direct safety and efficacy vehicle controls required euthanasia at week 10 and week comparisons between human, nonhuman primate and rodent 11, post challenge. In two of the vaccinees, plasma virus responses to the same VEE derivative. A large body of levels were below the limit of detection by branched chain experience Strongly Suggests that the animal models gener DNA assay. At 64 weeks post challenge, all four vaccinated ally reflect the human Susceptibility and disease course, animals showed no clinical Signs of disease. One animal except that mice are far more susceptible to lethal VEE remained VDNA negative at 64 weeks. disease than humans or nonhuman primates. Furthermore, the VEE replicon vectors express high levels of the gene of 0019. The results of this highly pathogenic challenge interest in cell culture, and in Vivo expression is targeted to demonstrated that the immune response induced by vacci lymphoid tissues, reflecting the natural tropism mediated by nation with SIV-VRP was effective in preventing early the YEE glycoproteins. Cells in the draining lymph node of mortality and increasing the ability to Suppress challenge VRP-inoculated mice contain detectable amounts of the virus replication. The ability to control SIV replication and reduce viral load to undetectable levels was closely corre desired gene product within hours of inoculation. This lated with the Strongest measurable antibody and cellular expression continues for up to five dayS. immune responses. 0.014) To date, VRP vector vaccines have been used in over 2000 rodents and in 94 macaques at doses up to 5x10 0020 While these results are encouraging, the level of i.u., with no indication of any clinical manifestations. protection obtained would not be acceptable for a human vaccine against HIV infection. Thus, there remains a need 0015. In work reported by Pushko et al. (1997), indi for a robust, effective and safe vaccine against HIV infection vidual mice were immunized Sequentially with Lassa virus in humans. Development of a HIV Vaccine comprising the N-VRP and influenza virus HA-VRP Groups of mice, which complete, or immunogenic fragments of the, gag gene received two inoculations of 3x10" or 3x10° iu. of Lassa (Gag-VRP), an immunogenic portion of the pol gene (Pol N-VRP followed by two inoculations of 2x10 iu. of HA VRP), and the complete, or immunogenic fragments of the, VRP, all responded with serum antibodies to both antigens. env gene (Env-VRP), would increase the diversity of avail The level of anti-influenza antibody induced in these able CTL epitopes substantially and thus address this need. Sequentially inoculated mice was equivalent to a control group, which received two inoculations of buffer followed SUMMARY OF THE INVENTION by two inoculations of 2x10 iu. of HA-VRP. All HA-VRP immunized mice were completely protected against influ 0021. The present invention provides a composition com enza virus challenge. Furthermore, Sequential immunization prising two or more isolated nucleic acids Selected from the of mice with two inoculations of N-VRP prior to two group consisting of an isolated nucleic acid encoding an env inoculations of HA-VRP induced an immune response to gene product or an immunogenic fragment thereof of a both HA and N equivalent to immunization with either VRP human immunodeficiency virus, an isolated nucleic acid construct alone. Primary and booster immunization with a encoding a gag gene product or an immunogenic fragment VRP preparation expressing an immunogen from one patho thereof of a human immunodeficiency virus, wherein the gen did not interfere with the development of a protective gag gene product or immunogenic fragment thereof is response to Subsequent primary immunization and boosting modified to inhibit formation of virus-like particles by the with VRP expressing an immunogen from a Second patho gag gene product or the immunogenic fragment thereof and gen, thus showing that the VRP-based system can be used to their release from a cell, and an isolated nucleic acid induce immunity to a variety of pathogens in the same encoding a pol gene product or an immunogenic fragment individual over time. thereof of a human immunodeficiency virus, wherein the pol 0016 Four macaques were inoculated subcutaneously at gene product or immunogenic fragment thereof is modified week 0 with 10 i.u. each of SIV-gp160-VRP (env) and SIV to inhibit activity. MA/CA-VRP (gag), boosted by the same route at week 7 0022. Also provided is a composition comprising a popu with 107 iu. of each VRP vaccine, and intravenously at lation of alphavirus replicon particles comprising two or weeks 12 and 20 with 5x10 i.u. of each VRP. Two control more isolated nucleic acids Selected from the group consist animals were inoculated with equivalent doses of HA-VRP ing of 1) an isolated nucleic acid encoding an env gene (haemagglutinin, a glycoprotein from influenza virus), and product or an immunogenic fragment thereof of a human two with the vehicle only. The four SIV-VRPs immunized immunodeficiency virus, 2) an isolated nucleic acid encod monkeys received subcutaneously an additional dose of ing a gag gene product or an immunogenic fragment thereof 2x10 i.u. of gp140-VRP at week 41, followed by a final of a human immunodeficiency virus, wherein the gag gene boost of 2x107 i.u. each of gp140-VRP and MA/CA-VRPat product or immunogenic fragment thereof is modified to week 49. Four weeks after the final immunization, all eight inhibit formation of virus-like particles by the gag gene macaques were challenged intravenously with the patho product or the immunogenic fragment thereof and their genic virus, SIVsmE660. release from a cell, and 3) an isolated nucleic acid encoding US 2002/014 1975 A1 Oct. 3, 2002 a pol gene product or an immunogenic fragment thereof of alphavirus particles as determined by passage on a human immunodeficiency virus, wherein the poll gene permissive cells in culture; product or immunogenic fragment thereof is modified to remove protease, integrase and RNase H regions and to 0031 (b) producing the alphavirus particles in the inhibit reverse transcriptase activity, and wherein the nucleic helper cell; and acids are each contained within a separate alphavirus repli 0032 (c) collecting the alphavirus particles from the con particle. helper cells, 0023. In addition, the present invention provides a com 0033 B) (a) providing a second helper cell for pro position comprising a population of alphavirus replicon ducing a Second population of infectious, replication particles comprising two or more isolated nucleic acids defective alphavirus particles, comprising in an Selected from the group consisting of 1) an isolated nucleic alphavirus-permissive cell: acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an 0034 (i) an alphavirus replicon RNA, wherein the isolated nucleic acid encoding a gag gene product or an replicon RNA comprises an alphavirus packaging immunogenic fragment thereof of a human immunodefi Signal and a nucleic acid encoding a gag gene ciency virus, wherein the gag gene product or immunogenic product or an immunogenic fragment thereof of a fragment thereof is modified to inhibit formation of virus human immunodeficiency virus, wherein the gag like particles containing the gag gene product or the immu gene product or immunogenic fragment thereof is nogenic fragment thereof and their release from a cell, and modified to inhibit formation of virus-like particles 3) an isolated nucleic acid encoding a pol gene product or an containing the gag gene product or the immunogenic immunogenic fragment thereof of a human immunodefi fragment thereof and their release from a cell, and ciency virus, wherein the poll gene product or immunogenic wherein the replicon RNA lackS Sequences encoding fragment thereof is modified to inhibit reverse transcriptase alphavirus Structural proteins, activity, and wherein the nucleic acids are each contained 0035 (ii) a first helper RNA separate from said within a separate alphavirus replicon particle, and further replicon RNA, said first helper RNA encoding at wherein the alphavirus replicon particles comprise a repli least one alphavirus Structural protein and further con RNA or at least one structural protein which comprises more not encoding at least one other alphavirus one or more attenuating mutations. Structural protein; and 0024 10. A method of making a population of alphavirus 0.036 (iii) one or more additional helper RNA(s) replicon particles of this invention is provided herein, com Separate from Said replicon RNA and Separate from prising; said first helper RNA, said additional helper RNA(s) 0025 A) (a) providing a first helper cell for producing encoding at least one other alphavirus Structural a first population of infectious, replication defective protein not encoded by said first helper RNA; alphavirus particles, comprising in an alphavirus-per 0037 and with at least one of said helper RNAs missive cell: lacking an alphavirus packaging Signal; 0026 (i) an alphavirus replicon RNA, wherein the 0038 wherein the combined expression of the replicon RNA comprises an alphavirus packaging alphavirus replicon RNA and the helper RNAS pro Signal and a nucleic acid encoding an enV gene duces an assembled alphavirus particle which is able product or an immunogenic fragment thereof of a to infect a cell, and is unable to complete viral human immunodeficiency virus, and wherein the replication, and further wherein the Second popula replicon RNA lackS Sequences encoding alphavirus tion contains no detectable replication-competent Structural proteins, alphavirus particles as determined by passage on 0027 (ii) a first helper RNA separate from said permissive cells in culture; replicon RNA, said first helper RNA encoding at 0039 (b) producing the alphavirus particles in the least one alphavirus Structural protein and further helper cell; and more not encoding at least one other alphavirus Structural protein; and 0040 (c) collecting the alphavirus particles from the helper cells, 0028 (iii) one or more additional helper RNA(s) Separate from Said replicon RNA and Separate from 0041 C) (a) providing a third helper cell for producing said first helper RNA, said additional helper RNA(s) a third population of infectious, replication defective encoding at least one other alphavirus Structural alphavirus particles, comprising in an alphavirus-per protein not encoded by said first helper RNA; missive cell: 0029) and with at least one of said helper RNAs 0042 (i) an alphavirus replicon RNA, wherein the lacking an alphavirus packaging Signal; replicon RNA comprises an alphavirus packaging Signal and a nucleic acid encoding a pol gene product 0030 wherein the combined expression of the or an immunogenic fragment thereof of a human alphavirus replicon RNA and the helper RNAS pro immunodeficiency virus, wherein the pol gene prod duces an assembled alphavirus particle which is able uct or immunogenic fragment thereof is modified to to infect a cell, and is unable to complete viral inhibit reverse transcriptase activity, and wherein the replication, and further wherein the first population replicon RNA lackS Sequences encoding alphavirus contains no detectable replication-competent Structural proteins, US 2002/014 1975 A1 Oct. 3, 2002

0043 (ii) a first helper RNA separate from said duces an assembled alphavirus particle which is able replicon RNA, said first helper RNA encoding at to infect a cell, and is unable to complete viral least one alphavirus Structural protein and further replication, and further wherein the first population more not encoding at least one other alphavirus contains no detectable replication-competent Structural protein; and alphavirus particles as determined by passage on 0044) (iii) one or more additional helper RNA(s) permissive cells in culture, and further wherein at Separate from Said replicon RNA and Separate from least one of said replicon RNA, said first helper said first helper RNA, said additional helper RNA(s) RNA, and said one or more additional helper encoding at least one other alphavirus Structural RNA(s) comprises one or more attenuating muta protein not encoded by said first helper RNA; tions, 0057 (b) producing the alphavirus particles in the 0045 and with at least one of said helper RNAS lacking an alphavirus packaging Signal; helper cell; and 0.058 (c) collecting the alphavirus particles from the 0046 wherein the combined expression of the helper cells, alphavirus replicon RNA and the helper RNAS pro duces an assembled alphavirus particle which is able 0059 B) (a) providing a second helper cell for pro to infect a cell, and is unable to complete viral ducing a Second population of infectious, replication replication, and further wherein the third population defective alphavirus particle, comprising in an alphavi contains no detectable replication-competent rus-permissive cell: alphavirus particles as determined by passage on 0060 (i) an alphavirus replicon RNA, wherein the permissive cells in culture; replicon RNA comprises an alphavirus packaging Signal and a nucleic acid encoding a gag gene 0047 (b) producing the alphavirus particles in the product or an immunogenic fragment thereof of a helper cell; and human immunodeficiency virus, wherein the gag 0048 (c) collecting the alphavirus particles from the gene product or immunogenic fragment thereof is helper cells, and modified to inhibit formation of virus-like particles 0049 D) combining the first population of alphavirus containing the gag gene product or the immunogenic particles produced from the first helper cell, the Second fragment thereof and their release from a cell, and population of alphavirus particles produced from the wherein the replicon RNA lackS Sequences encoding Second helper cell and the third population of alphavi alphavirus structural proteins, rus particles produced from the third helper cell, 0061 (ii) a first helper RNA separate from said thereby producing the population of alphavirus repli replicon RNA, said first helper RNA encoding at con particles. least one alphavirus Structural protein and further more not encoding at least one other alphavirus 0050 Also provided is a method of making a population Structural protein; and of alphavirus replicon particles, comprising: 0062 (iii) one or more additional helper RNA(g) 0051 A) (a) providing a first helper cell for producing Separate from Said replicon RNA and Separate from a first population of infectious, replication defective said first helper RNA, said additional helper RNA(s) alphavirus particles, comprising in an alphavirus-per encoding at least one other alphavirus Structural missive cell: protein not encoded by said first helper RNA; 0.052 (i) an alphavirus replicon RNA, wherein the 0063 and with at least one of said helper RNAS replicon RNA comprises an alphavirus packaging lacking an alphavirus packaging Signal; Signal and a nucleic acid encoding an enV gene 0064 wherein the combined expression of the product or an immunogenic fragment thereof of a alphavirus replicon RNA and the helper RNAS pro human immunodeficiency virus, and wherein the duces an assembled alphavirus particle which is able replicon RNA lackS Sequences encoding alphavirus to infect a cell, and is unable to complete viral Structural proteins, replication, and further wherein the Second popula 0053 (ii) a first helper RNA separate from said tion contains no detectable replication-competent replicon RNA, said first helper RNA encoding at alphavirus particles as determined by passage on least one alphavirus Structural protein and further permissive cells in culture, and further wherein at more not encoding at least one other alphavirus least one of said replicon RNA, said first helper Structural protein; and RNA, and said one or more additional helper RNA(s) comprises one or more attenuating muta 0054 (iii) one or more additional helper RNA(s) tions, Separate from Said replicon RNA and Separate from said first helper RNA, said additional helper RNA(s) 0065 (b) producing the alphavirus particles in the encoding at least one other alphavirus Structural helper cell; and protein not encoded by said first helper RNA; 0066 (c) collecting the alphavirus particles from the helper cells, 0055 and with at least one of said helper RNAs lacking an alphavirus packaging Signal; 0067 C) (a) providing a third helper cell for producing a third population of infectious, replication defective 0056 wherein the combined expression of the alphavirus particles, comprising in an alphavirus-per alphavirus replicon RNA and the helper RNAS pro missive cell: US 2002/014 1975 A1 Oct. 3, 2002

0068 (i) an alphavirus replicon RNA, wherein the reverse transcriptase functions in the poll gene product or replicon RNA comprises an alphavirus packaging immunogenic fragment thereof. Signal and a nucleic acid encoding a poll gene product 0077. In addition, the present invention provides a com or an immunogenic fragment thereof of a human position comprising a population of alphavirus replicon immunodeficiency virus, wherein the pol gene prod particles comprising two or more isolated nucleic acids uct or immunogenic fragment thereof is modified to Selected from the group consisting of 1) an isolated nucleic inhibit reverse transcriptase activity, and wherein the acid encoding an env gene product or an immunogenic replicon RNA lackS Sequences encoding alphavirus fragment thereof of a human immunodeficiency virus, 2) an Structural proteins, isolated nucleic acid encoding a gag gene product or an 0069 (ii) a first helper RNA separate from said immunogenic fragment thereof of a human immunodefi replicon RNA, said first helper RNA encoding at ciency virus, wherein the gag gene product or immunogenic least one alphavirus Structural protein and further fragment thereof is modified to inhibit formation of virus more not encoding at least one other alphavirus like particles containing the gag gene product or the immu Structural protein; and nogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an 0070 (iii) one or more additional helper RNA(s) immunogenic fragment thereof of a human immunodefi Separate from Said replicon RNA and Separate from ciency virus, wherein the poll gene product or immunogenic said first helper RNA, said additional helper RNA(s) fragment thereof comprises a modification resulting in dele encoding at least one other alphavirus Structural tion or inactivation of protease, integrase, RNase H and protein not encoded by said first helper RNA; reverse transcriptase functions in the poll gene product or 0071 and with at least one of said helper RNAS immunogenic fragment thereof, and wherein the nucleic lacking an alphavirus packaging Signal; acids are each contained within a separate alphavirus repli con particle. 0072 wherein the combined expression of the 0078. Also provided herein is a composition comprising alphavirus replicon RNA and the helper RNAS pro a population of alphavirus replicon particles comprising two duces an assembled alphavirus particle which is able or more isolated nucleic acids Selected from the group to infect a cell, and is unable to complete viral consisting of 1) an isolated nucleic acid encoding an env replication, and further wherein the third population gene product or an immunogenic fragment thereof of a contains no detectable replication-competent human immunodeficiency virus, 2) an isolated nucleic acid alphavirus particles as determined by passage on encoding a gag gene product or an immunogenic fragment permissive cells in culture, and further wherein at thereof of a human immunodeficiency virus, wherein the least one of said replicon RNA, said first helper gag gene product or immunogenic fragment thereof is RNA, and said one or more additional helper modified to inhibit formation of virus-like particles contain RNA(s) comprises one or more attenuating muta ing the gag gene product or the immunogenic fragment tions, thereof and their release from a cell, and 3) an isolated 0073 (b) producing the alphavirus particles in the nucleic acid encoding a poll gene product or an immunogenic helper cell; and fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment 0074 (c) collecting the alphavirus particles from the thereof comprises a modification resulting in deletion or helper cells, and inactivation of protease, integrase, RNase H and reverse 0075 D) combining the first population of alphavirus transcriptase functions in the poll gene product or immuno particles produced from the first helper cell, the Second genic fragment thereof, and wherein the nucleic acids are population of alphavirus particles produced from the each contained within a separate alphavirus replicon par Second helper cell and the third population of alphavi ticle, and further wherein the alphavirus replicon particles rus particles produced from the third helper cell, comprise a replicon RNA or at least one Structural protein thereby producing the population of alphavirus repli which comprises one or more attenuating mutations. con particles. 0079. In these embodiments, the gag gene product or immunogenic fragment thereof can be modified by mutation 0.076 Furthermore, the present invention provides a com of the Second codon, whereby a glycine is changed to an position comprising two or more isolated nucleic acids alanine and the poll gene product or immunogenic fragment Selected from the group consisting of an isolated nucleic thereof can be modified by mutation of the nucleotide acid encoding an env gene product or an immunogenic sequence encoding the active site motif, whereby YMDD is fragment thereof of a human immunodeficiency virus, an changed to YMAA or HMAA. In addition, the pol gene isolated nucleic acid encoding a gag gene product or an product or immunogenic fragment thereof is modified to immunogenic fragment thereof of a human immunodefi remove protease, integrase and RNase H regions and to ciency virus, wherein the gag gene product or immunogenic produce only p51 of the poll gene product or immunogenic fragment thereof is modified to inhibit formation of virus like particles containing the gag gene product or the immu fragment thereof. nogenic fragment thereof and their release from a cell, and 0080. The present invention provides a method of making an isolated nucleic acid encoding a poll gene product or an a population of alphavirus replicon particles, comprising: immunogenic fragment thereof of a human immunodefi 0081 A) (a) providing a first helper cell for producing ciency virus, wherein the poll gene product or immunogenic a first population of infectious, replication defective fragment thereof comprises a modification resulting in dele alphavirus particles, comprising in an alphavirus-per tion or inactivation of protease, integrase, RNase H and missive cell: US 2002/014 1975 A1 Oct. 3, 2002

0082 (i) an alphavirus replicon RNA, wherein the 0094 wherein the combined expression of the replicon RNA comprises an alphavirus packaging alphavirus replicon RNA and the helper RNAS pro Signal and a nucleic acid encoding an enV gene duces an assembled alphavirus particle which is able product or an immunogenic fragment thereof of a to infect a cell, and is unable to complete viral human immunodeficiency virus, and wherein the replication, and further wherein the Second popula replicon RNA lackS Sequences encoding alphavirus tion contains no detectable replication-competent Structural proteins, alphavirus particles as determined by passage on 0083 (ii) a first helper RNA separate from said permissive cells in culture; replicon RNA, said first helper RNA encoding at 0.095 (b) producing the alphavirus particles in the least one alphavirus Structural protein and further helper cell; and more not encoding at least one other alphavirus 0096 (c) collecting the alphavirus particles from the Structural protein; and helper cells, 0084 (iii) one or more additional helper RNA(s) 0097 C) (a) providing a third helper cell for producing Separate from Said replicon RNA and Separate from a third population of infectious, replication defective said first helper RNA, said additional helper RNA(s) alphavirus particles, comprising in an alphavirus-per encoding at least one other alphavirus Structural missive cell: protein not encoded by said first helper RNA: 0098 (i) an alphavirus replicon RNA, wherein the 0085 and with at least one of said helper RNAs replicon RNA comprises an alphavirus packaging lacking an alphavirus packaging Signal; Signal and a nucleic acid encoding a pol gene product 0086 wherein the combined expression of the or an immunogenic fragment thereof of a human alphavirus replicon RNA and the helper RNAS pro immunodeficiency virus, wherein the pol gene prod duces an assembled alphavirus particle which is able uct or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of to infect a cell, and is unable to complete viral protease, integrase, RNase H and reverse tran replication, and further wherein the first population Scriptase functions in the poll gene product or immu contains no detectable replication-competent nogenic fragment thereof, and wherein the replicon alphavirus particles as determined by passage on RNA lackS Sequences encoding alphavirus Structural permissive cells in culture; proteins, 0087 (b) producing the alphavirus particles in the 0099 (ii) a first helper RNA separate from said helper cell; and replicon RNA, said first helper RNA encoding at 0088 (c) collecting the alphavirus particles from the least one alphavirus Structural protein and further helper cells, more not encoding at least one other alphavirus Structural protein; and 0089 B) (a) providing a second helper cell for pro ducing a Second population of infectious, replication 0100 (iii) one or more additional helper RNA(s) defective alphavirus particles, comprising in an Separate from Said replicon RNA and Separate from alphavirus-permissive cell: said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus Structural 0090 (i) an alphavirus replicon RNA, wherein the protein not encoded by said first helper RNA; replicon RNA comprises an alphavirus packaging Signal and a nucleic acid encoding a gag gene 0101 and with at least one of said helper RNAS product or an immunogenic fragment thereof of a lacking an alphavirus packaging Signal; human immunodeficiency virus, wherein the gag 0102 wherein the combined expression of the gene product or immunogenic fragment thereof is alphavirus replicon RNA and the helper RNAS pro modified to inhibit formation of virus-like particles duces an assembled alphavirus particle which is able containing the gag gene product or the immunogenic to infect a cell, and is unable to complete viral fragment thereof and their release from a cell, and replication, and further wherein the third population wherein the replicon RNA lackS Sequences encoding contains no detectable replication-competent alphavirus Structural proteins, alphavirus particles as determined by passage on 0091 (ii) a first helper RNA separate from said permissive cells in culture; replicon RNA, said first helper RNA encoding at 0103) (b) producing the alphavirus particles in the least one alphavirus Structural protein and further helper cell; and more not encoding at least one other alphavirus Structural protein; and 0104 (c) collecting the alphavirus particles from the helper cells, and 0092 (iii) one or more additional helper RNA(s) 0105 D) combining the first population of alphavirus Separate from Said replicon RNA and Separate from particles produced from the first helper cell, the Second said first helper RNA, said additional helper RNA(s) population of alphavirus particles produced from the encoding at least one other alphavirus Structural Second helper cell and the third population of alphavi protein not encoded by said first helper RNA; rus particles produced from the third helper cell, 0093) and with at least one of said helper RNAs thereby producing the population of alphavirus repli lacking an alphavirus packaging Signal; con particles. US 2002/014 1975 A1 Oct. 3, 2002

0106 An additional method of making a population of least one alphavirus Structural protein and further alphavirus replicon particles is provided, comprising: more not encoding at least one other alphavirus 0107 A) (a) providing a first helper cell for producing Structuraltructural protein;tein; andan a first population of infectious, replication defective 0118 (iii) one or more additional helper RNA(s) alphavirus particles, comprising in an alphavirus-per Separate from Said replicon RNA and Separate from missive cell: said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus Structural 0108 (i) an alphavirus replicon RNA, wherein the protein not encoded by said first helper RNA, replicon RNA comprises an alphavirus packaging Signal and a nucleic acid encoding an enV gene 0119) and with at least one of said helper RNAS product or an immunogenic fragment thereof of a lacking an alphavirus packaging Signal; human immunodeficiency virus, and wherein the 0120 wherein the combined expression of the replicon RNA lackS Sequences encoding alphavirus alphavirus replicon RNA and the helper RNAS pro Structural proteins, duces an assembled alphavirus particle which is able 0109 (ii) a first helper RNA separate from said to infect a cell, and is unable to complete viral replicon RNA, said first helper RNA encoding at replication, and further wherein the Second popula least one alphavirus Structural protein and further tion contains no detectable replication-competent more not encoding at least one other alphavirus alphavirus particles as determined by passage on Structural protein; and permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper 0110 (iii) one or more additional helper RNA(s) RNA, and said one or more additional helper Separate from Said replicon RNA and Separate from RNA(s) comprises one or more attenuating muta said first helper RNA, said additional helper RNA(s) tions, encoding at least one other alphavirus Structural 0121 (b) producing the alphavirus particles in the protein not encoded by said first helper RNA; helper cell; and 0111 and with at least one of said helper RNAS 0122 (c) collecting the alphavirus particles from the lacking an alphavirus packaging Signal; helper cells, 0112 wherein the combined expression of the 0123 C) (a) providing a third helper cell for producing alphavirus replicon RNA and the helper RNAS pro a third population of infectious, replication defective duces an assembled alphavirus particle which is able alphavirus particles, comprising in an alphavirus-per to infect a cell, and is unable to complete viral replication, and further wherein the first population missive cell: contains no detectable replication-competent 0124 (i) an alphavirus replicon RNA, wherein the alphavirus particles as determined by passage on replicon RNA comprises an alphavirus packaging permissive cells in culture, and further wherein at Signal and a nucleic acid encoding a pol gene product least one of said replicon RNA, said first helper or an immunogenic fragment thereof of a human RNA, and said one or more additional helper immunodeficiency virus, wherein the pol gene prod RNA(s) comprises one or more attenuating muta uct or immunogenic fragment thereof comprises a tions, modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse tran 0113) (b) producing the alphavirus particles in the Scriptase functions in the poll gene product or immu helper cell; and nogenic fragment thereof, and wherein the replicon 0114 (c) collecting the alphavirus particles from the RNA lackS Sequences encoding alphavirus Structural helper cells, proteins, 0115 B)(a) providing a second helper cell for produc 0125 (ii) a first helper RNA separate from said ing a Second population of infectious, replication defec replicon RNA, said first helper RNA encoding at tive alphavirus particle, comprising in an alphavirus least one alphavirus Structural protein and further permissive cell: more not encoding at least one other alphavirus Structural protein; and 0116 (i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging 0126 (iii) one or more additional helper RNA(s) Signal and a nucleic acid encoding a gag gene Separate from Said replicon RNA and Separate from product or an immunogenic fragment thereof of a said first helper RNA, said additional helper RNA(s) human immunodeficiency virus, wherein the gag encoding at least one other alphavirus Structural gene product or immunogenic fragment thereof is protein not encoded by said first helper RNA; modified to inhibit formation of virus-like particles 0127 and with at least one of said helper RNAS containing the gag gene product or the immunogenic lacking an alphavirus packaging Signal; fragment thereof and their release from a cell, and wherein the replicon RNA lackS Sequences encoding 0128 wherein the combined expression of the alphavirus Structural proteins, alphavirus replicon RNA and the helper RNAS pro duces an assembled alphavirus particle which is able 0117 (ii) a first helper RNA separate from said to infect a cell, and is unable to complete viral replicon RNA, said first helper RNA encoding at replication, and further wherein the third population US 2002/014 1975 A1 Oct. 3, 2002

contains no detectable replication-competent removing detergent, whereby alphavirus replicon ViroSomes alphavirus particles as determined by passage on are produced. Also provided is a viroSome produced by this permissive cells in culture, and further wherein at method. least one of said replicon RNA, said first helper 0.139. Furthermore, the present invention provides a RNA, and said one or more additional helper method of eliciting an immune response in a Subject, com RNA(s) comprises one or more attenuating muta prising administering to the Subject an immunogenic amount tions, of the alphavirus replicon ViroSome of this invention in a 0129 (b) producing the alphavirus particles in the pharmaceutically acceptable carrier. helper cell; and 0140. The present invention additionally provides a 0130 (c) collecting the alphavirus particles from the method of treating or preventing infection by human immu helper cells, and nodeficiency virus in a Subject, comprising administering to the Subject an immunogenic amount of the alphavirus rep 0131 D) combining the first population of alphavirus licon viroSome of this invention, wherein the viroSome particles produced from the first helper cell, the Second comprises alphavirus replicon RNA encoding one or more population of alphavirus particles produced from the HIV immunogens. Second helper cell and the third population of alphavi rus particles produced from the third helper cell, 0.141. In further embodiments, the present invention pro thereby producing the population of alphavirus repli vides a composition comprising a population of alphavirus con particles. replicon ViroSomes comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated 0.132. In each of the methods above, the alphavirus rep nucleic acid encoding an env gene product or an immuno licon RNA of at least one of the first helper cell, the second genic fragment thereof of a human immunodeficiency virus, helper cell and the third helper cell can comprise Sequence 2) an isolated nucleic acid encoding a gag gene product or encoding at least one alphavirus Structural protein and the an immunogenic fragment thereof of a human immunode first helper RNA and the one or more additional helper ficiency virus, wherein the gag gene product or immuno RNA(s) in the at least one of the first helper cell, the second genic fragment thereof is modified to inhibit formation of helper cell and the third helper cell, can encode at least one Virus-like particles containing the gag gene product or the other alphavirus Structural protein not encoded by the rep immunogenic fragment thereof and their release from a cell, licon RNA. and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immuno 0133. Furthermore, in the methods above which recite deficiency virus, wherein the poll gene product or immuno attenuating mutations, only at least one of the first popula genic fragment thereof comprises a modification resulting in tion of alphavirus particles, the Second population of deletion or inactivation of protease, integrase, RNase H and alphavirus particles and the third population of alphavirus reverse transcriptase functions in the poll gene product or particles can comprise particles wherein at least one of the immunogenic fragment thereof, and wherein the nucleic replicon RNA, the first helper RNA, and the one or more acids are each contained within a separate alphavirus repli additional helper RNA(s) comprises one or more attenuating con ViroSome. mutations. 0142. Additionally provided herein is a composition 0134) The present invention further provides alphavirus comprising a population of alphavirus replicon ViroSomes particles produced by any of the methods of this invention. comprising two or more isolated nucleic acids Selected from 0135 The present invention further provides a method of the group consisting of 1) an isolated nucleic acid encoding inducing an immune response to human immunodeficiency an env gene product or an immunogenic fragment thereof of Virus in a Subject, comprising administering to the Subject an a human immunodeficiency virus, 2) an isolated nucleic acid immunogenic amount of the populations and/or composi encoding a gag gene product or an immunogenic fragment tions of this invention, in a pharmaceutically acceptable thereof of a human immunodeficiency virus, wherein the carrier. gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles contain 0.136 Also provided herein is a method of treating or ing the gag gene product or the immunogenic fragment preventing infection by human immunodeficiency virus in a thereof and their release from a cell, and 3) an isolated Subject, comprising administering to the Subject an immu nucleic acid encoding a poll gene product or an immunogenic nogenic amount of the populations and/or compositions of fragment thereof of a human immunodeficiency virus, this invention, in a pharmaceutically acceptable carrier. wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in inactivation of 0.137 Also provided by the present invention is an reverse transcriptase activity in the poll gene product or alphavirus replicon ViroSome comprising an alphavirus rep immunogenic fragment thereof, and wherein the nucleic licon RNA encapsidated by a lipid bilayer comprising acids are each contained within a separate alphavirus repli alphavirus glycoproteins, E1 and E2, which in one embodi con ViroSome. ment, can be Venezuelan Equine Encephalitis glycoproteins E1 and E2. 0.143 A method of producing a population of alphavirus replicon ViroSomes is provided herein, comprising: 0138 A method of producing an alphavirus replicon ViroSome is further provided, comprising: a) combining 0144 A) (a) producing a first population of alphavirus alphavirus replicon RNA, alphavirus glycoproteins E1 and replicon ViroSomes by combining alphavirus replicon E2, non-cationic lipids and detergent; and b) gradually RNA comprising nucleic acid encoding an env gene US 2002/014 1975 A1 Oct. 3, 2002

product or immunogenic fragment thereof, alphavirus 0156 C) (a) producing a third population of alphavirus glycoproteins E1 and E2, non-cationic lipids and deter replicon ViroSomes by combining alphavirus replicon gent, and RNA comprising nucleic acid encoding the pol gene product or immunogenic fragment thereof, wherein the 0145 b) gradually removing detergent, whereby pol gene product or immunogenic fragment thereof alphavirus replicon ViroSomes are produced; comprises a modification resulting in inactivation of 0146 B)(a) producing a second population of alphavi reverse transcriptase activity in the poll gene product or rus replicon ViroSomes by combining alphavirus repli immunogenic fragment thereof, alphavirus glycopro con RNA comprising nucleic acid encoding a gag gene teins E1 and E2, non-cationic lipids and detergent; and product or immunogenic fragment thereof, wherein the 0157 b) gradually removing detergent, whereby gag gene product or immunogenic fragment thereof is alphavirus replicon ViroSomes are produced; and modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic 0158 D) combining the first population of alphavirus fragment thereof and their release from a cell, alphavi replicon ViroSomes, the Second population of alphavi rus glycoproteins E1 and E2, non-cationic lipids and rus replicon ViroSomes and the third population of detergent, and alphavirus replicon ViroSomes to produce the popula 0147 b) gradually removing detergent, whereby tion of alphavirus replicon Virosomes of claim 48. alphavirus replicon ViroSomes are produced; 0159 Furthermore, the present invention provides a method of inducing an immune response in a Subject, 0148 C)(a) producing a third population of alphavirus comprising administering to the Subject an immunogenic replicon ViroSomes by combining alphavirus replicon amount of the ViroSomes of this invention, in a pharmaceu RNA comprising nucleic acid encoding the pol gene tically acceptable carrier. product or immunogenic fragment thereof, wherein the pol gene product or immunogenic fragment thereof 0160 Also provided is a method of treating or preventing comprises a modification resulting in deletion or inac infection by human immunodeficiency virus in a Subject, tivation of protease, integrase, RNase H and reverse comprising administering to the Subject an immunogenic transcriptase functions in the poll gene product or amount of the ViroSomes of this invention, in a pharmaceu immunogenic fragment thereof, alphavirus glycopro tically acceptable carrier. teins E1 and E2, non-cationic lipids and detergent; and 0.161 Additionally provided by this invention is a com 0149 b) gradually removing detergent, whereby position comprising heparin affinity-purified alphavirus rep alphavirus replicon ViroSomes are produced; and licon particles, wherein the alphavirus replicon particles comprise at least one Structural protein which comprises one 0150 D) combining the first population of alphavirus or more attenuating mutations, as well as a method of replicon ViroSomes, the Second population of alphavi preparing heparin affinity-purified alphavirus particles, com rus replicon ViroSomes and the third population of prising: alphavirus replicon ViroSomes to produce the popula tion of alphavirus replicon ViroSomes. 0162 a) producing alphavirus replicon particles, wherein the alphavirus replicon particles comprise at 0151. In addition, a method of producing a popula least one structural protein which comprises one or tion of alphavirus replicon ViroSomes is provided, more attenuating mutations, comprising: 0163 b) loading the alphavirus replicon particles of 0152 A) (a) producing a first population of alphavirus Step (a) in a heparin affinity chromatography colunn; replicon ViroSomes by combining alphavirus replicon RNA comprising nucleic acid encoding and env gene 0164 c) eluting the particles from the column of product or immunogenic fragment thereof, alphavirus Step (b) with a Salt gradient (e.g., NaCl gradient); and glycoproteins E1 and E2, non-cationic lipids and deter 0165 d) collecting the fraction from the column gent, and which contains the heparin affinity-purified alphavi 0153 b) gradually removing detergent, whereby ruS replicon particles. alphavirus replicon ViroSomes are produced; 0166 In further embodiments, the present invention pro 0154 B)(a) producing a second population of alphavi vides a method of producing VRP for use in a vaccine rus replicon ViroSomes by combining alphavirus repli comprising: con RNA comprising nucleic acid encoding and gag gene product or immunogenic fragment thereof, 0.167 a) producing a plasmid encoding the nucle wherein the gag gene product or immunogenic frag otide Sequence of an alphavirus replicon RNA, ment thereof is modified to inhibit formation of virus 0168 b) producing a plasmid encoding the nucle like particles containing the gag gene product or the otide Sequence of one or more helper RNAS, immunogenic fragment thereof and their release from a cell, alphavirus glycoproteins E1 and E2, non-cationic 0169 c) transcribing the plasmids of steps (a) and lipids and detergent; and (b) into RNA in vitro; 0155 b) gradually removing detergent, whereby 0170 d) electroporating the RNA of step (c) into a alphavirus replicon ViroSomes are produced; Vero cell line; and US 2002/014 1975 A1 Oct. 3, 2002

0171 e) purifying VRP from the Vero cell line of 0180 (b) producing the alphavirus particles in the Step (d) by heparin affinity chromatography. By this helper cell; and method, VRPS can be produced in large-scale. 0181 (c) collecting the alphavirus particles from the 0172 In additional embodiments, the present invention helper cell. provides an isolated nucleic acid encoding a pol gene product or immunogenic fragment thereof of a human 0182. In the method described above, at least one of the immunodeficiency virus, wherein the poll gene product or replicon RNA, the first helper RNA, and the one or more immunogenic fragment thereof comprises a modification additional helper RNA(s) can comprise one or more attenu resulting in deletion or inactivation of protease, integrase, ating mutations. The present invention additionally provides RNase H and reverse transcriptase functions in the pol gene alphavirus replicon particle produced according to the above product or immunogenic fragment thereof. This nucleic acid methods. can be present in a composition and in a vector. Such a 0183. Further provided is a method of inducing an vector can be present in a cell. This nucleic acid can also be immune response in a Subject, comprising administering to present in an alphavirus replicon particle. the Subject an immunogenic amount of a composition com 0173 The present invention further provides a method of prising alphavirus replicon particles encoding a poll gene making an alphavirus replicon particle comprising nucleic product or an immunogenic fragment thereof of a human acid encoding a pol gene product or immunogenic fragment immunodeficiency virus, wherein the poll gene product or thereof of a human immunodeficiency virus, wherein the pol immunogenic fragment thereof comprises a modification gene product or immunogenic fragment thereof comprises a resulting in deletion or inactivation of protease, integrase, modification resulting in deletion or inactivation of protease, RNase H and reverse transcriptase functions in the pol gene integrase, RNase H and reverse transcriptase functions in the product or immunogenic fragment thereof in a pharmaceu pol gene product or immunogenic fragment thereof, com tically acceptable carrier. prising BRIEF DESCRIPTION OF THE DRAWINGS 0174) a) providing a helper cell for producing an 0184 FIG. 1. DNA plasmid map of VEE replicon RNA infectious, defective alphavirus particle, comprising encoding the HIV gag gene (p3-40.1.6). The plasmid is in an alphavirus-permissive cell: 12523 base pairs in length and encodes a single polyprotein 0175 (i) an alphavirus replicon RNA, wherein the encoding the four non-Structural genes nsP1-4, the Clade C replicon RNA comprises an alphavirus packaging gag gene and antibiotic resistance marker, Kanamycin Signal and a nucleic acid encoding a poll gene KN(R). The plasmid contains two promoter regions, the T7 product or an immunogenic fragment thereof of a polymerase promoter and the 26S RNA promoter. The human immunodeficiency virus, wherein the pol unique Not restriction enzyme site used to linearize prior to gene product or immunogenic fragment thereof in vitro transcription is also noted. comprises a modification resulting in deletion or 0185 FIG. 2. DNA plasmid map of the capsid helper inactivation of protease, integrase, RNase H and construct (p3-13.2.2). The plasmid is 5076 base pairs in reverse transcriptase functions in the pol gene length and encodes the VEE capsid gene (C) and antibiotic product or immunogenic fragment thereof, and resistance marker, Kanamycin KNCR). The plasmid contains wherein the replicon RNA lackS Sequences encod two promoter regions, the T7 polymerase promoter and the ing alphavirus Structural proteins, 26S RNA promoter. The unique Not restriction enzyme site 0176 (ii) a first helper RNA separate from said used to linearize DNA prior to in vitro transcription is also replicon RNA, said first helper RNA encoding at noted. least one alphavirus Structural protein and further 0186 FIG. 3. DNA plasmid map of the glycoprotein more not encoding at least one other alphavirus helper construct (p3-13.4.6). The plasmid is 6989 base pairs Structural protein; and in length and encodes the VEE glycoprotein genes (E3, E2, 6K and E1) and antibiotic resistance marker, Kanamycin 0177 (iii) one or more additional helper RNA(s) KN(R). The plasmid contains two promoter regions, the T7 Separate from Said replicon RNA and Separate polymerase promoter and the 26S RNA promoter. The from said first helper RNA, said additional helper unique Not restriction enzyme site used to linearize DNA RNA(s) encoding at least one other alphavirus prior to in vitro transcription is also noted. Structural protein not encoded by Said first helper RNA; 0187 FIG. 4. DNA plasmid map of VEE replicon RNA encoding HIV pol (p51) gene (p13-60.2.14). The plasmid is 0.178 and with at least one of said helper RNAS 12379 base pairs in length and encodes a single polyprotein lacking an alphavirus packaging Signal; encoding the four non-structural genes, nsP1-4, the Clade C 0179 wherein the combined expression of the pol (p51) gene and antibiotic resistance marker, Kanamycin alphavirus replicon RNA and the helper RNAS pro KN(R). The plasmid contains two promoter regions, the T7 duces an assembled alphavirus particle which is able polymerase promoter and the 26S RNA promoter. The to infect a cell, and is unable to complete viral unique Not restriction enzyme site used to linearize prior to replication, and further wherein the population con in vitro transcription is also noted. tains no detectable replication-competent alphavirus 0188 FIG. 5. DNA plasmid map of VEE replicon RNA particles as determined by passage on permissive encoding HIV env gene (pERK-Du151env). The plasmid is cells in culture; 13584 base pairs in length and encodes a single polyprotein US 2002/014 1975 A1 Oct. 3, 2002

encoding the four non-structural genes, nsP1-4, the Clade C 0194 FIG. 11. Phylogenetic comparison of Du422 Clade env gene and antibiotic resistance marker, Kanamycin C Gag isolate with referenced Clade C Strains. Consensus KN(R). The plasmid contains two promoter regions, the T7 lade A, B, D, Mal and SA strains are also shown. Du422 the polymerase promoter and the 26S RNA promoter. The vaccine Strain had 95% amino acid Sequence homology to unique Not restriction enzyme site used to linearize prior to the South African consensus Clade C Sequence. in vitro transcription is also noted. 0189 FIG. 6. Western immunoblot, demonstrating the 0195 FIG. 12. Phylogenetic comparison of Du151 Clade expression of HIV proteins in baby hamster kidney (BHK) C isolate Env isolate with referenced Clade C strains. Du151 cells infected with VRPs. The outer lanes of the panel are the vaccine Strain had 93% amino acid Sequence homology Standard molecular weight markers. Lane 1 is the expression to the South African consensus Clade C Sequence. from VRPs encoding the p51 (pol) gene. Lane 2 is the expression from VRPs encoding the gp160 (env) gene. Lane 0196. FIG. 13. Phylogenetic comparison of Du151 Clade 3 is the expression from VRPS encoding the p55 (gag) gene. C isolate Pol isolate with referenced Clade C strains. Du151 Arrows indicate proteins migrating with the apparent the vaccine Strain had 99% amino acid Sequence homology molecular weight of each respective protein. to the South African consensus Clade C Sequence. 0190 FIG. 7. Western immunoblot of cells infected with 0197 FIG. 14. Du422HIV Gag expression as detected by the Du151env VRP. At 18 hr post infection, the cells were immunofluorescence following electroporation with Gag lysed and the lysate run in a denaturing polyacrylamide gel. replicon RNA. BHK cells were electroporated and subjected Proteins were transferred out of the gel onto a filter and the to imunofluorescence Staining with an anti-Gag monoclonal filter was probed with serum from Subject Du151 using antibody at 24 hours post-electroporation, to demonstrate Western immunoblot methods. Lane 1, uninfected expression of the Clade C protein. U87.CD4-CXCR4 cells. Lane 2, uninfected U87.CD4 0198 FIG. 15. Immunofluorescence detection of Du422 CCR5 cells. Lane 3, infection of a mixed culture of Gag protein expression in BHK cells. BHK cells were U87.CD4-CXCR4 cells and BHK cells (mixtures were used infected with VRP-Gag particles and subjected to immun as a positive control in case the U87 cells were refractory to fluorescence Staining with an anti-Gag monoclonal antibody infection by the VRP, which did not turn out to be the case). at 24 hours post-infection, to demonstrate expression of the Lane 4, infected U87.CD4-CXCR4 cells. Lane 5, infected Clade C protein. BHK cells. Lane 6, infection of a mixture of BHK cells and U87.CD4-CCR5 cells. Lane 7, infected U87.CD4-CCR5 cells. The positions of molecular weight of markers run in DETAILED DESCRIPTION OF THE the same gel are shown on the right, and the inferred INVENTION positions of gp160, gp120 and are shown on the left. 0199 AS used in the specification and the appended 0191 FIG.8. Micrographs of U87.CD4-CCR and BHK claims, the singular forms “a,”“an,” and “the” include plural cells used to examine expression and Syncytium formation referents unless the context clearly dictates otherwise. Thus, of Du151 envelope expressed from the VEE replicon. for example, reference to “a pharmaceutical carrier can U87.CD4-CCR5 cells alone (Panel 1), or a mixture of mean a single pharmaceutical carrier or mixtures of two or U87.CD4-CCR5 and BHK cells (Panel 2), BHK cells alone more Such carriers. (Panel 3) and U87.CD4-CXCR4 cells (Panel 4) were 0200. The present invention is based on the discovery of infected with Du151 env VRP at a multiplicity of infection a vaccine for the treatment and/or prevention of infection by of 3 i.u. per cell. At 18 hours post infection, the cells were HIV, comprising novel combinations of isolated nucleic examined using light microScopy for the presence of Syn acids encoding two or more distinct antigens which elicit an cytia. The U87.CD4-CCR5 in Panel 1 and 2 show clear immune response in a Subject which is effective in treating Syncytia, which was absent in the control cell types in the and/or preventing infection by HIV. In a particular embodi lower panels. In addition, no Syntycia were Seen in unin ment, the nucleic acids encoding the antigens of the vaccine fected control cells or VRP-GFP infected cells (data not are modified to enhance the immunogenicity of the antigen, shown). improve the Safety of the vaccine, or both. 0.192 FIGS. 9A-C. Antigen-specific CTL response in 0201 AS used herein, the term “isolated nucleic acid” mice to the HIV-1 Clade C VRP-Gag vaccine. Eight means a nucleic acid Separated or Substantially free from at BALB/c mice were immunized twice, first at day 0 and least Some of the other components of the naturally occur again at day 28 with 103 iu. (Panel A) or 10 i.u. (Panels B ring organism, for example, the cell Structural components and C) VRP-Gag. Eight days (Panels A and B) or 49 days commonly found associated with nucleic acids in a cellular (Panel C) post-boost, Spleen cells were isolated and stimu environment and/or other nucleic acids. The isolation of lated in vitro with virus expressing HIV Gag for 1 nucleic acids can be accomplished by well known tech week. Chromium release assays were performed using vac niques Such as cell lysis or disruption of virus particles, cinia-Gag infected target cells (diamond Symbols) or control followed by phenol plus chloroform extraction, followed by vaccinia alone-infected Sc11 target cells (Square symbols). ethanol precipitation of the nucleic acids (Sambrook et al., Clear HIV Gag-specific lysis was detected in animals vac latest edition). The nucleic acids of this invention can be cinated with the VRP-Gag vaccine. isolated according to methods well known in the art for 0193 FIG. 10. Diagrammatic representation of the isolating nucleic acids. Alternatively, the nucleic acids of the HIV-1 genome. Black bars indicate relative regions of the present invention can be Synthesized according to Standard genome Sequenced to generate phylogenetic Sequence com protocols well described in the literature for Synthesis, parative data for Clade C gag, pol and env gene isolates. cloning and amplification of nucleic acids. US 2002/014 1975 A1 Oct. 3, 2002

0202 HIV-VRP Vaccines their release from a cell, and an isolated nucleic acid 0203 The antigens of this invention can be gene products encoding a pol gene product or an immunogenic fragment which are complete proteins or any fragment of a protein thereof of a human immunodeficiency virus, wherein the pol determined to be immunogenic by methods well known in gene product or immunogenic fragment thereof is modified the art. Modifications are made to the antigens of this to inhibit reverse transcriptase activity. invention to enhance immunogenicity and/or improve the 0208. In a preferred embodiment, the invention provides Safety of administration of a vaccine containing the antigen. alphavirus replicon particles (VRPs) that can be adminis Examples of Such modifications are described in the tered as an HIV vaccine. These HIV-VRPs are propagation Examples Section herein. Furthermore, it is understood that, defective, Single cycle vector constructs that contain a where desired, other modifications and changes (e.g., Sub self-amplifying RNA (replicon RNA), e.g., from VEE, in Stitutions, deletions, additions) may be made in the amino which the Structural protein genes of the virus are replaced acid Sequence of the antigen of the present invention, which by a HIV-1 Clade C gag gene or any other HIV antigen to may not specifically impart enhanced immunogenicity or be expressed. Following introduction into packaging (or improved Safety, yet still result in a protein or fragment helper) cells in vitro, the replicon RNA is packaged into which retains all of the functional characteristics by which VRPs by Supplying the viral structural proteins in trans the protein or fragment is defined. Such changes may occur (helper RNAs). in natural isolates, may be introduced by Synthesis of the protein or fragment, or may be introduced into the amino 0209 The present invention further provides a population acid Sequence of the protein or fragment using Site-specific of alphavirus replicon particles comprising two or more mutagenesis of nucleic acid encoding the protein or frag isolated nucleic acids Selected from the group consisting of ment, the procedures for which, Such as mis-match poly 1) an isolated nucleic acid encoding an env gene product or merase chain reaction (PCR), are well known in the art. an immunogenic fragment thereof of a human immunode ficiency virus, 2) an isolated nucleic acid encoding a gag 0204. The nucleic acids of this invention can be present gene product or an immunogenic fragment thereof of a in a vector and the vector of this invention can be present in human immunodeficiency virus, wherein the gag gene prod a cell. The vectors and cells of this invention can be in a uct or immunogenic fragment thereof is modified to inhibit composition comprising the cell or vector and a pharma formation of particles, Such as virus-like particles, contain ceutically acceptable carrier. ing the gag gene product or the immunogenic fragment 0205 The vector of this invention can be an expression thereof, from a cell, and 3) an isolated nucleic acid encoding vector which contains all of the genetic components required a pol gene product or an immunogenic fragment thereof of for expression of the nucleic acids of this invention in cells a human immunodeficiency virus, wherein the poll gene into which the vector has been introduced, as are well known product or immunogenic fragment thereof is modified to in the art. For example, the expression vector of this inven inhibit reverse transcriptase activity, and wherein the nucleic tion can be a vector comprising the helper RNAS of this acids are each contained within a separate alphavirus repli invention. Such an expression vector can be a commercial con particle. expression vector or it can be constructed in the laboratory 0210. It is also contemplated that the compositions of this according to Standard molecular biology protocols. The invention comprise alphavirus replicon particles in which expression vector can comprise viral nucleic acid including, either the replicon RNA or at least one structural protein but not limited to, alphavirus, flavivirus, adenovirus, retro comprises one or more attenuating mutations. Thus, the Virus and/or adeno-associated virus nucleic acid. The present invention additionally provides a population of nucleic acid or vector of this invention can also be in a alphavirus replicon particles comprising two or more dis lipoSome or a delivery vehicle which can be taken up by a tinct types of Such particles Selected from the group con cell via receptor-mediated or other type of endocytosis. sisting of 1) particles expressing a nucleic acid encoding an 0206. In another embodiment, the nucleic acids of this env gene product or an immunogenic fragment thereof of a invention can be present in a composition comprising a human immunodeficiency virus, 2) particles expressing a population of alphavirus replicon particles which comprise nucleic acid encoding a gag gene product or an immuno two or more distinct isolated nucleic acids of this invention genic fragment thereof of a human immunodeficiency virus, and wherein the nucleic acids are each contained within a wherein the gag gene product or immunogenic fragment Separate alphavirus replicon particle (herein referred to as a thereof is modified to inhibit release of particles, Such as “VRP”). Thus, the expression vector of the present invention Virus-like particles, containing the gag gene product or the can be an alphavirus replicon particle comprising a nucleic immunogenic fragment thereof, from a cell, and 3) particles acid encoding an antigen of this invention. expressing a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodefi 0207. In a particular embodiment, the present invention ciency virus, wherein the poll gene product or immunogenic provides a composition comprising two or more isolated fragment thereof is modified to inhibit reverse transcriptase nucleic acids Selected from the group consisting of an activity; and wherein the nucleic acids are each contained isolated nucleic acid encoding an env gene product or an within a separate alphavirus replicon particle and further immunogenic fragment thereof of a human immunodefi wherein the alphavirus replicon particles comprise a repli ciency Virus, an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human con RNA or at least one structural protein which comprises immunodeficiency virus, wherein the gag gene product or one or more attenuating mutations. immunogenic fragment thereof is modified to inhibit forma 0211. In a preferred embodiment, the population of tion of particles, e.g., Virus-like particles, containing the gag alphavirus replicon particles comprises particles expressing gene product or the immunogenic fragment thereof, and the nucleic acids encoding pol, env, and gag gene products. US 2002/014 1975 A1 Oct. 3, 2002

In this embodiment, Vigorous antigen-specific cellular (e.g., Natal as described herein (see FIGS. 11-13 for isolate CTL, NK cell and T-helper) and/or humoral (e.g., antibody) names). In a further embodiment, the gag gene or gene responses can be obtained when Such particle populations fragment is from a gag Sequence having 95% or greater are administered to a Subject. amino acid identity with the South African consensus 0212. In the compositions described above, the gag gene Sequence for the gag gene. In a Specific embodiment, the gag product or immunogenic fragment thereof can be modified gene or fragment thereof is derived from HIV Subtype Clade by mutation of the Second codon, whereby a glycine is C isolate Du422 and the env andpol genes or fragments changed to an alanine. Alternatively, the gag gene product or thereof are derived from HIV isolate Du151. immunogenic fragment thereof can be modified by any other 0217. The term “alphavirus' has its conventional mean means known in the art for inhibiting the release of particles ing in the art and includes the various Species of the containing the gag gene product or immunogenic fragment alphavirus genus, Such as Eastern Equine Encephalitis virus thereof from a cell. (EEE), Venezuelan Equine Encephalitis virus (VEE), West ern Equine Encephalitis virus (WEE), Everglades virus, 0213 Furthermore, in the compositions of this invention, Mucambo virus, Pixuna virus, Sindbis virus, Semliki Forest the pol gene product or immunogenic fragment thereof can virus, South African Arbovirus No. 86, Middleburg virus, be modified by mutation of the nucleotide Sequence encod Chikungunya virus, O-Nyong-Nyong virus, Ross River ing the active site motif, whereby YMDD is changed to Virus, Barmah Forest Virus, Getah Virus, Sagiyama virus, YMAA or HMAA (the latter providing a convenient site for Bebaru virus, Mayaro virus, Una virus, Aura virus, Wha cloning, see SEQ ID NO:16). The pol gene product or taroa virus, Babanki Virus, Kyzylagach virus, Highlands J immunogenic fragment thereof can also be modified by any Virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus, means known in the art for inhibiting reverse transcriptase as well as any specific strains of these alphaviruses (e.g., activity. TR339; Girdwood) and any other virus classified by the 0214) The pol gene product or immunogenic fragment International Committee on Taxonomy of Viruses (ICTV) as thereof of this invention may be further modified such that an alphavirus. the coding Sequences for protease, integrase and RNase H 0218. An “alphavirus replicon particle” as used herein is are removed, inactivated and/or modified, e.g., by producing an infectious, replication defective, alphavirus particle only the p51 region of the poll gene product. This modifi which comprises alphavirus Structural proteins and further cation has been shown in Some Studies to reduce the possi comprises a replicon RNA. The replicon RNA comprises bility of formation of replication competent alphavirus par nucleic acid encoding the alphavirus packaging Segment, ticles during production of alphavirus replicon particles nucleic acid encoding alphavirus non-structural proteins and comprising the poll gene product or immunogenic fragment a heterologous nucleic acid Sequence encoding an antigen of thereof. This modification can be of the nucleic acid encod this invention. The non-Structural proteins encoded by the ing the poll gene product or immunogenic fragment thereof replicon RNA may be Such proteins as are required for according to methods known in the art. Thus, the particles replication and transcription. In a Specific embodiment of and compositions of this invention can comprise nucleic this invention, the Structure of the replicon RNA, Starting at acid encoding a pol gene product or an immunogenic the 5' end, comprises the 5' untranslated region of the fragment thereof of a human immunodeficiency virus, alphavirus RNA, the non-structural proteins (e.g., nsPS1-4) wherein the pol gene product or immunogenic fragment of the alphavirus, the 26S promoter (also known as the thereof comprises a modification resulting in deletion or “Subgenomic promoter”), the heterologous nucleic acid inactivation of protease, integrase, RNase H and reverse encoding an HIV antigen, and the 3' untranslated region of transcriptase functions in the poll gene product or immuno the alphavirus RNA. An example of a nucleic acid encoding genic fragment thereof. alphavirus nonstructural proteins that can be incorporated 0215. In the compositions of this invention, the gag, env into the embodiments of this invention is SEQ ID NO:2, or pol gene products or immunogenic fragments thereof can which encodes the amino acid sequence of SEQ ID NO:3. be from any HIV isolate or consensus sequence derived from 0219. Although the alphavirus replicon RNA can com HIV primary isolates now known or later identified, the prise nucleic acid encoding one or two alphavirus Structural isolation and characterization of which are well known in proteins, the replicon RNA does not contain nucleic acid the art. Also, in the compositions of this invention, the gag, encoding all of the alphavirus Structural proteins. The rep env or poll gene products or immunogenic fragments thereof licon RNA can lack nucleic acid encoding any alphavirus can be produced from the same HIV isolate or HIV con Structural protein(s). Thus, the resulting alphavirus replicon Sensus Sequence or from any combination of HIV isolates or particles of this invention are replication defective inasmuch HIV consensus Sequences. In the Examples provided herein, as the replicon RNA does not encode all of the structural the nucleic acid Sequences encoding the enV, gag and pol proteins required for encapsidation of the replicon RNA and gene products of this invention were Selected based on a consensus Sequence generated from primary isolates assembly of an infectious virion. obtained from recent Seroconvertors in KwaZulu/Natal 0220 AS used herein, “alphavirus structural protein' or Province in South Africa. Sequence analysis of these isolates "structural protein' means the alphavirus proteins required identified them as Subtype (or lade) C, and in preferred for encapsidation of alphavirus replicon RNA and packaging embodiments of the invention, the env, gag and pol genes are of the encapsidated RNA into a virus particle. The alphavi from Clade C isolates of HIV. rus structural proteins include PE2, E2, E3, 6K and E1. 0216) In preferred embodiments, each of the three HIV 0221) The alphavirus replicon particles of this invention genes are derived from one or more of the South African can comprise replicon RNA from any of the alphaviruses of isolates obtained from recent Seroconverters in KwaZulu/ this invention. Furthermore, the alphavirus replicon particles US 2002/014 1975 A1 Oct. 3, 2002

of this invention can comprise alphavirus Structural proteins ment, either one or both of the first helper RNA and the from any of the alphaviruses of this invention. Thus, the Second helper RNA include at least one attenuating muta replicon particles can be made up of replicon RNA and tion. The attenuating mutations provide the advantage that in Structural proteins from the Same alphavirus or from differ the event of RNA-RNA recombination the resulting recom ent alphaviruses, the latter of which would be chimeric binant RNA molecules encoding the alphavirus Structural alphavirus replicon particles (e.g., a particle comprising and non-Structural genes will yield or produce virus of Sindbis virus replicon RNA and VEE structural proteins). decreased virulence. 0222. The alphavirus replicon particles of this invention 0227. The alphavirus replicon particles of this invention can be made by employing a helper cell for expressing an can be made by a) transfecting a helper cell as given above infectious, replication defective, alphavirus particle in an with a replication defective replicon RNA, b) producing the alphavirus-permissive cell. The helper cell includes (a) a alphavirus particles in the transfected cell, and c) collecting first helper RNA encoding (i) at least one alphavirus Struc the alphavirus particles from the cell. The replicon RNA tural protein, and (ii) not encoding at least one alphavirus encodes the alphavirus packaging Segment and a heterolo Structural protein; and (b) a Second helper RNA separate gous RNA. The transfected helper cell further includes the from the first helper RNA, the second helper RNA (i) not first helper RNA and second helper RNA as described above. encoding the at least one alphavirus Structural protein 0228. As described hereinabove, the structural proteins encoded by the first helper RNA, and (ii) encoding at least used to assemble the alphavirus replicon particles of this one alphavirus Structural protein not encoded by the first invention are distributed among one or more helper RNAS helper RNA, such that all of the alphavirus structural pro (i.e., a first helper RNA and a second helper RNA). As noted teins assemble together into alphavirus particles in the cell. herein, one or more Structural protein genes may be located 0223) The alphavirus structural protein genes can be on the replicon RNA, provided that at least one structural present on the helper RNAS of this invention in any com protein gene is deleted from the replicon RNA such that the bination. For example, the helper RNA of this invention can replicon RNA and resulting alphavirus particle are replica encode the alphavirus capsid and E1, capsid and E2, E1 and tion defective. As used herein, the terms “deleted” or “dele E2, capsid only, E1 only, E2 only, etc. It is also contemplated tion” mean either total deletion of the Specified nucleic acid that the alphavirus Structural proteins are provided in trans or the deletion of a Sufficient portion of the Specified nucleic from genes located on three Separate RNA molecules within acid to render the nucleic acid and/or its resultant gene the helper cell. product inoperative or nonfunctional, in accordance with 0224. In a preferred embodiment, the helper cell also standard usage. (See, e.g., U.S. Pat. No. 4,650,764 to Temin includes a replicon RNA, which encodes the alphavirus et al.) The term “replication defective” as used herein means packaging Segment and an inserted heterologous RNA. In that the replicon RNA cannot replicate in the host cell (i.e., the embodiment wherein the helper cell also includes a produce progeny infectious viral particles) in the absence of replicon RNA, the alphavirus packaging Segment may be, the helper RNA. The replicon RNA is replication defective and preferably is, deleted from both the first helper RNA and inasmuch as the replicon RNA does not include all of the the second helper RNA. For example, in an embodiment alphavirus Structural protein genes required for replication, wherein the helper cell includes a replicon RNA encoding at least one of the required Structural protein genes being the alphavirus packaging Segment and an inserted heterolo deleted therefrom. gous RNA, the first helper RNA encodes the alphavirus E1 0229. In one embodiment, the packaging segment or glycoprotein and the alphavirus E2 glycoprotein, and the “encapsidation Sequence' is deleted from at least the first Second helper RNA encodes the alphavirus capsid protein. helper RNA. In a preferred embodiment, the packaging In a preferred embodiment, the first helper RNA encodes the segment is deleted from both the first helper RNA and the E3-E2-6k-E1 cassette from an alphavirus. In an alternative Second helper RNA. In a Specific embodiment, the Second embodiment, the cassette encoded on the first helper RNA is helper RNA is constructed from a VEE cDNA clone, delet referred to as the E3-E2-E1 cassette. A specific embodiment ing all of the non-structural proteins (i.e., nsPS1-4) except of this aspect of the invention is diagrammed in FIG. 3, and approximately 500 nucleotides at the 5' end of nsP1, the an exemplary nucleotide sequence is SEQ ID NO:11. The packaging signal, and the glycoprotein cassette (E3-E2-E1). replicon RNA, first helper RNA, and second helper RNA are An example of a plasmid encoding Such a Second helper all on Separate molecules and are cotransfected, e.g., by RNA is provided in FIG. 2, and an exemplary nucleotide electroporation, into the helper cell, which can be any sequence for such a second helper RNA is SEQ ID NO:8. alphavirus permissive cell, as is well known in the art. 0230. In the preferred embodiment wherein the packag 0225. In an alternative embodiment, the helper cell ing segment is deleted from both the first helper RNA and includes a replicon RNA encoding the alphavirus packaging the second helper RNA, preferably the helper cell contains Segment and an inserted heterologous RNA and also a replicon RNA in addition to the first helper RNA and the includes the alphavirus capsid protein otherwise encoded by second helper RNA. The replicon RNA encodes the pack the second helper RNA. The first helper RNA encodes the aging Segment and an inserted heterologous RNA encoding alphavirus E1 glycoprotein and the alphavirus E2 glycopro an HIV antigen or a fragment thereof. Typically, the inserted tein. Thus, the replicon RNA and the first helper RNA are on heterologous RNA encodes a gene product which is Separate molecules, and the replicon RNA and the Second expressed in the target cell, and includes the promoter and helper RNA are on a Single molecule. regulatory Segments necessary for the expression of that 0226. The RNA encoding the structural proteins, i.e., the gene product in that cell. first helper RNA and the second helper RNA, can include 0231. In another preferred embodiment, the replicon one or more attenuating mutations. In a preferred embodi RNA, the first helper RNA and the second helper RNA are US 2002/014 1975 A1 Oct. 3, 2002 provided on Separate molecules Such that a first molecule, ogy in the art, See, e.g., Davis et el. (1980). The mutation i.e., the replicon RNA, encodes the packaging Segment and can be, for example, a Substitution mutation or an in-frame the inserted heterologous RNA, a Second molecule, i.e., the deletion mutation. The phrase “attenuating mutation' first helper RNA, encodes at least one but not all of the excludes mutations which would be lethal to the virus. Thus, required alphavirus Structural proteins, and a third molecule, according to this embodiment, the E1 RNA and/or the E2 i.e., the Second helper RNA, encodes at least one but not all RNA and/or the capsid RNA can include at least one of the required alphavirus Structural proteins. For example, attenuating mutation. In a more preferred embodiment, the in one preferred embodiment of the present invention, the E1 RNA and/or the E2 RNA and/or the capsid RNA includes helper cell includes a set of RNAS which include (a) a at least two, or multiple, attenuating mutations. The multiple replicon RNA encoding an alphavirus packaging Sequence attenuating mutations may be positioned in either the first and an inserted heterologous RNA, (b) a first helper RNA helper RNA or in the second helper RNA, or they may be encoding the alphavirus E1 glycoprotein and the alphavirus distributed randomly with one or more attenuating mutations E2 glycoprotein, and (c) a second helper RNA encoding the being positioned in the first helper RNA and one or more alphavirus capsid protein, So that the alphavirus E1 glyco attenuating mutations positioned in the Second helper RNA. protein, the alphavirus E2 glycoprotein and the capsid Appropriate attenuating mutations will be dependent upon protein assemble together into alphavirus particles contain the alphavirus used, as is well known in the art. ing the replicon RNA in the helper cell. 0235 For example, when the alphavirus is VEE, suitable 0232. In an alternate embodiment, the replicon RNA and attenuating mutations can be in codons at E2 amino acid the first helper RNA are on Separate molecules, and the position 76 which specify an attenuating amino acid, pref replicon RNA and the second helper RNA are on a single erably lysine, arginine, or histidine as E2 amino acid 76, molecule together, thereby providing a first molecule, i.e., codons at E2 amino acid position 120 which Specify an the first helper RNA, encoding at least one but not all of the attenuating amino acid, preferably lysine as E2 amino acid required alphavirus Structural proteins, and a Second mol 120; codons at E2 amino acid position 209 which specify an ecule, i.e., the replicon RNA and second helper RNA, attenuating amino acid, preferably lysine, arginine, or his encoding the packaging Segment, the inserted heterologous tidine as E2 amino acid 209; codons at E1 amino acid 272 gene product and the structural protein(s) not encoded by the which Specify an attenuating mutation, preferably threonine first helper. Thus, one or more structural protein(s) is or Serine as E1 amino acid 272, codons at E1 amino acid 81 encoded by the second helper RNA, but the second helper which Specify an attenuating mutation, preferably isoleucine RNA is located on the second molecule together with the or leucine as E1 amino acid 81; and codons at E1 amino acid replicon RNA. For example, in one preferred embodiment of 253 which specify an attenuating mutation, preferably Serine the present invention, the helper cell includes a set of RNAS or threoinine as E1 amino acid 253; and the combination including (a) a replicon RNA encoding an alphavirus pack mutation of the deletion of E3 codons 56-59 together with aging Sequence, an inserted heterologous RNA, and an codons at E1 amino acid 253 which Specify an attenuating alphavirus capsid protein, and (b) a first helper RNA encod mutation, as provided herein. Other Suitable attenuating ing the alphavirus E1 glycoprotein and the alphavirus E2 mutations within the VEE genome will be known to those glycoprotein So that the alphavirus E1 glycoprotein, the skilled in the art. alphavirus E2 glycoprotein and the capsid protein assemble 0236. In an alternate embodiment, wherein the alphavirus together into alphavirus particles in the helper cell. is the South African Arbovirus No. 86 (S.A.A.R.86), Suitable 0233. The present invention also contemplates alphavirus attenuating mutations can be, for example, in codons at nsP1 replicon particles which comprise replicon RNA encoding amino acid position 538 which Specify an attenuating amino more than one heterologous gene product. For expression of acid, preferably isoleucine as nsP1 amino acid 538; codons more than one heterologous nucleic acid from a single at E2 amino acid position 304 which Specify an attenuating replicon RNA, a promoter can be inserted upstream of each amino acid, preferably threonine as E2 amino acid 304, heterologous nucleic acid on the replicon RNA, Such that the codons at E2 amino acid position 314 which Specify an promoter regulates expression of the heterologous nucleic attenuating amino acid, preferably lysine as E2 amino acid acid, resulting in the production of more than one antigen 314, codons at E2 amino acid position 376 which specify an from a single replicon RNA Another embodiment contem attenuating amino acid, preferably alanine as E2 amino acid plates the insertion of an IRES Sequence, Such as the one 376; codons at E2 amino acid position 372 which specify an from the picomavirus, EMC virus, between the heterologous attenuating amino acid, preferably leucine as E2 amino acid genes downstream ftom a 26S promoter of the replicon, thus 372; codons at nsP2 amino acid position 96 which specify an leading to translation of multiple antigens from a single attenuating amino acid, preferably glycine as nsP2 amino replicon. acid 96; codons at nsP2 amino acid position 372 which Specify an attenuating amino acid, preferably valine as nsP2 0234. In one preferred embodiment of the present inven amino acid 372; in combination, codons at E2 amino acid tion, the RNA encoding the alphavirus Structural proteins, residues 304, 314, 372 and 376; codons at E2 amino acid i.e., the capsid, E1 glycoprotein and/or E2 glycoprotein, position 378 which specify an attenuating amino acid, contains at least one attenuating mutation. It is further preferably leucine as E2 amino acid 378; codons at nsP2 contemplated that the RNA encoding the non-structural amino acid residue 372 which Specify an attenuating muta proteins can contain at least one attenuating mutation. The tion, preferably valine as nsP2 amino acid 372; in combi phrases “attenuating mutation' and "attenuating amino nation, codons at nsP2 amino acid residues 96 and 372 acid, as used herein, mean a nucleotide mutation or an attenuating Substitution mutations at nsP2 amino acid resi amino acid coded for in View of Such a mutation which result dues 96 and 372; codons at nsP2 amino acid residue 529 in a decreased probability of causing disease in its host (i.e., which Specify an attenuating mutation, preferably leucine, at a loss of virulence), in accordance with Standard terminol nsP2 amino acid residue 529; codons at nsP2 amino acid US 2002/014 1975 A1 Oct. 3, 2002 residue 571 which Specify an attenuating mutation, prefer packaging Segment and an inserted heterologous RNA, a ably asparagine, at nsP2 amino acid residue 571; codons at first helper RNA encoding at least one alphavirus Structural nSP2 amino acid residue 682 which Specify an attenuating protein, and a Second helper RNA encoding at least one mutation, preferably arginine, at nsP2 amino acid residue alphavirus structural protein which is different from that 682; codons at nsP2 amino acid residue 804 which specify encoded by the first helper RNA; producing the alphavirus an attenuating mutation, preferably arginine, at nsP2 amino particles in the transfected cell; and collecting the alphavirus acid residue 804, codons at nsP3 amino acid residue 22 particles from the cell. which Specify an attenuating mutation, preferably arginine, 0242 Methods for transfecting the alphavirus-permissive at nsP3 amino acid residue 22, and in combination, codons cell with the replicon RNA and helper RNAS can be at nsP2 amino acid residues 529, 571, 682 and 804, and at achieved, for example, by (i) treating the cells with DEAE nsP3 amino acid residue 22, Specifying attenuating amino dextran, (ii) by lipofection, by treating the cells with, for acids at nsP2 amino acid residues 529, 571, 682 and 804 and example, LIPOFECTIN, and (iii) by electroporation, with at nsP3 amino acid residue 22. Other suitable attenuating electroporation being a preferred means of achieving RNA mutations within the S.A.A.R.86 genome will be known to uptake into the alphavirus-permissive cells. Examples of those skilled in the art. these techniques are well known in the art, See e.g., U.S. Pat. 0237) The alphavirus capsid gene used to make alphavi No. 5,185,440 to Davis et al., and PCT Publication No. WO ruS replicon particles can also be Subjected to site-directed 92/10578 to Bioption AB, the disclosures of which are mutagenesis. The altered capsid protein provides additional incorporated herein by reference in their entirety. assurance that recombination to produce the virulent virus will not occur. The altered capsid protein gene which 0243 The steps of producing the infectious viral particles functions in particle assembly but not in autoproteolysis in the cells may also be carried out using conventional provides helper function for production of replicon particles, techniques. See e.g., U.S. Pat. No. 5,185,440 to Davis et al., PCT Publication No. WO 92/10578 to Bioption AB, and but does not allow for production of a viable recombinant. U.S. Pat. No. 4,650,764 to Temin et al. (although Teminet The capsid residues required for proteolytic function are al., relates to rather than alphaviruses). The known (Strauss et al., 1990). infectious viral particles may be produced by Standard cell 0238 Suitable attenuating mutations useful in embodi culture growth techniques. ments wherein any of the alphaviruses of this invention are 0244. The steps of collecting the infectious alphavirus employed are known to or can be identified by those skilled particles may also be carried out using conventional tech in the art using routine protocols. Attenuating mutations may niques. For example, the infectious particles may be col be introduced into the RNA by performing site-directed lected by cell lysis, or collection of the Supernatant of the mutagenesis on the cDNA which encodes the RNA, in cell culture, as is known in the art. See e.g., U.S. Pat. No. accordance with known procedures. See Kunkel (1985), the 5,185,440 to Davis et al., PCT Publication No. WO disclosure of which is incorporated herein by reference in its 92/10578 to Bioption AB, and U.S. Pat. No. 4,650,764 to entirety. Alternatively, mutations may be introduced into the Temin et al. (although Temin et al. relates to retroviruses RNA by replacement of homologous restriction fragments in rather than alphaviruses). Other suitable techniques will be the cDNA which encodes for the RNA, in accordance with known to those skilled in the art. Optionally, the collected known procedures. The identification of a particular muta infectious alphavirus particles may be purified, if desired. tion in an alphavirus as attenuating is done using routine Purification techniques for viruses are well known to those experimentation according to methods well known in the art. skilled in the art, and these are Suitable for the purification 0239) Preferably, the helper RNA of this invention of Small batches of infectious alphavirus particles. includes a promoter. It is also preferred that the replicon 0245 Thus, the present invention provides a method of RNA includes a promoter. Suitable promoters for inclusion making the populations of alphavirus replicon particles of in the helper RNA and replicon RNA are well known in the art. One preferred promoter is the alphavirus 26S promoter, this invention comprising: although many Suitable promoters are available, as is well 0246 A) (a) providing a first helper cell for producing known in the art. a first population of infectious, defective alphavirus 0240. In the system wherein a first helper RNA, a second particles, comprising in an alphavirus-permissive cell; helper RNA, and a replicon RNA are all on separate mol 0247 (i) an alphavirus replicon RNA, wherein the ecules, if the same promoter is used for all three RNAS, then replicon RNA comprises an alphavirus packaging a homologous Sequence between the three molecules is Signal and a nucleic acid encoding an enV gene provided. Thus, it is advantageous to employ different product or an immunogenic fragment thereof of a promoters on the first and second helper RNAS to provide human immunodeficiency virus, and wherein the further impediment to RNA recombination that might pro replicon RNA lackS Sequences encoding alphavirus duce virulent virus. It is preferred that the selected promoter Structural proteins, is operative with the non-Structural proteins encoded by the replicon RNA molecule. 0248 (ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at 0241 The infectious, replication defective, alphavirus least one alphavirus Structural protein and further particles of this invention are prepared according to the more not encoding at least one other alphavirus methods disclosed herein in combination with techniques Structural protein; and known to those skilled in the art. The methods include, for example, transfecting an alphavirus-permissive cell with a 0249 (iii) one or more additional helper RNA(s) replication defective replicon RNA including the alphavirus Separate from Said replicon RNA and Separate from US 2002/014 1975 A1 Oct. 3, 2002

said first helper RNA, said additional helper RNA(s) 0263 (i) an alphavirus replicon RNA, wherein the encoding at least one other alphavirus Structural replicon RNA comprises an alphavirus packaging protein not encoded by said first helper RNA; Signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human 0250) and with at least one of said helper RNAS immunodeficiency virus, wherein the pol gene prod lacking an alphavirus packaging Signal; uct or immunogenic fragment thereof is modified to 0251 wherein the combined expression of the inhibit reverse transcriptase activity or is modified to alphavirus replicon RNA and the helper RNAS pro inactivate or delete protease, integrase, RNase H and duces an assembled alphavirus particle which is able reverse transcriptase functions in the pol gene prod to infect a cell, and is unable to complete viral uct or immunogenic fragment thereof, and wherein replication, and further wherein the first population the replicon RNA lackS Sequences encoding alphavi contains no detectable replication-competent rus Structural proteins, alphavirus particles as determined by passage on permissive cells in culture; 0264 (ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at 0252 (b) producing the alphavirus particles in the least one alphavirus Structural protein and further helper cell; and more not encoding at least one other alphavirus 0253) (c) collecting the alphavirus particles from the Structural protein; and helper cells, 0265 (iii) one or more additional helper RNA(s) 0254 B) (a) providing a second helper cell for pro Separate from Said replicon RNA and Separate from ducing a Second population of infectious, defective said first helper RNA, said additional helper RNA(s) alphavirus particles, comprising in an alphavirus-per encoding at least one other alphavirus Structural missive cell: protein not encoded by said first helper RNA; 0255 (i) an alphavirus replicon RNA, wherein the 0266 and with at least one of said helper RNAS replicon RNA comprises an alphavirus packaging lacking an alphavirus packaging Signal; Signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a 0267 wherein the combined expression of the human immunodeficiency virus, wherein the gag alphavirus replicon RNA and the helper RNAS pro gene product or immunogenic fragment thereof is duces an assembled alphavirus particle which is able modified to inhibit formation of virus-like particles to infect a cell, and unable to complete Viral repli containing the gag gene product or the immunogenic cation, and further wherein the third population fragment thereof and their release from a cell, and contains no detectable replication-competent wherein the replicon RNA lackS Sequences encoding alphavirus particles as determined by passage on alphavirus Structural proteins, permissive cells in culture; 0256 (ii) a first helper RNA separate from said 0268 (b) producing the alphavirus particles in the replicon RNA, said first helper RNA encoding at helper cell; and least one alphavirus Structural protein and further 0269 (c) collecting the alphavirus particles from the more not encoding at least one other alphavirus helper cells, and Structural protein; and 0270 D) combining the first population of alphavirus 0257 (iii) one or more additional helper RNA(s) particles produced from the first helper cell, the Second Separate from Said replicon RNA and Separate from population of alphavirus particles produced from the said first helper RNA, said additional helper RNA(s) Second helper cell and the third population of alphavi encoding at least one other alphavirus Structural rus particles produced from the third helper cell, protein not encoded by said first helper RNA; thereby producing the populations of alphavirus repli 0258 and with at least one of said helper RNAS con particles. lacking an alphavirus packaging Signal; 0271 In a preferred embodiment, as noted above, the 0259 wherein the combined expression of the method provided also includes a mutation in the pot gene alphavirus replicon RNA and the helper RNAS pro product or immunogenic fragment thereof resulting in inac duces an assembled alphavirus particle which is able tivation or deletion of protease, integrase and RNase H to infect a cell, and is unable to complete viral functions of the pol gene product or immunogenic fragment replication, and further wherein the Second popula thereof. In a specific embodiment of this method, the region tion contains no detectable replication-competent of the pot gene encoding the protease, RNase H and inte alphavirus particles as determined by passage on grase function of the pot gene product or immunogenic permissive cells in culture; fragment thereof has been deleted. 0260 (b) producing the alphavirus particles in the helper 0272. A method of making the populations of alphavirus cell; and replicon particles of this invention, wherein the particles 0261 (c) collecting the alphavirus particles from the comprise at least one attenuating mutation, is also provided, helper cells, comprising: 0262 C) providing a third helper cell for producing a 0273 A) (a) providing a first helper cell for producing third population of infectious, defective alphavirus a first population of infectious, defective alphavirus particles, comprising in an alphavirus-permissive cell: particles, comprising in an alphavirus-permissive cell: US 2002/014 1975 A1 Oct. 3, 2002

0274 (i) an alphavirus replicon RNA, wherein the 0285) and with at least one of said helper RNAS replicon RNA comprises an alphavirus packaging lacking an alphavirus packaging Signal; Signal and a nucleic acid encoding an enV gene 0286 wherein the combined expression of the product or an immunogenic fragment thereof of a alphavirus replicon RNA and the helper RNAS pro human immunodeficiency virus, and wherein the duces an assembled alphavirus particle which is able replicon RNA lackS Sequences encoding alphavirus to infect a cell, and is unable to complete viral Structural proteins, replication, and further wherein the Second popula tion contains no detectable replication-competent 0275 (ii) a first helper RNA separate from said alphavirus particles as determined by passage on replicon RNA, said first helper RNA encoding at permissive cells in culture, and further wherein at least one alphavirus Structural protein and further least one of said replicon RNA, said first helper more not encoding at least one other alphavirus RNA, and said one or more additional helper Structural protein; and RNA(s) comprises one or more attenuating muta 0276 (iii) one or more additional helper RNA(s) tions, Separate from Said replicon RNA and Separate from 0287 (b) producing the alphavirus particles in the said first helper RNA, said additional helper RNA(s) helper cell; and encoding at least one other alphavirus Structural 0288 (c) collecting the alphavirus particles from the protein not encoded by said first helper RNA; helper cells, 0277 and with at least one of said helper RNAS 0289 C) providing a third helper cell for producing a lacking an alphavirus packaging Signal; third population of infectious, defective alphavirus 0278 wherein the combined expression of the particles, comprising in an alphavirus-permissive cell: alphavirus replicon RNA and the helper RNAS pro 0290 (i) an alphavirus replicon RNA, wherein the duces an assembled alphavirus particle which is able replicon RNA comprises an alphavirus packaging to infect a cell, and is unable to complete viral Signal and a nucleic acid encoding a pol gene product replication, and further wherein the first population or an immunogenic fragment thereof of a human contains no detectable replication-competent immunodeficiency virus, wherein the pol gene prod alphavirus particles as determined by passage on uct or immunogenic fragment thereof is modified to permissive cells in culture, and further wherein at inhibit reverse transcriptase activity or is modified to least one of said replicon RNA, said first helper inactivate or delete protease, integrase, RNase H and RNA, and said one or more additional helper reverse transcriptase functions in the pol gene prod RNA(s) comprises one or more attenuating muta uct or immunogenic fragment thereof, and wherein tions, the replicon RNA lackS Sequences encoding alphavi 0279 (b) producing the alphavirus particles in the rus Structural proteins, helper cell; and 0291 (ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at 0280 (c) collecting the alphavirus particles from the least one alphavirus Structural protein and further helper cells, more not encoding at least one other alphavirus 0281 B) providing a second helper cell for producing Structural protein; and a Second population of infectious, defective alphavirus 0292 (iii) one or more additional helper RNA(s) particles, comprising in an alphavirus-permissive cell: Separate from Said replicon RNA and Separate from 0282 (i) an alphavirus replicon RNA, wherein the said first helper RNA, said additional helper RNA(s) replicon RNA comprises an alphavirus packaging encoding at least one other alphavirus Structural Signal and a nucleic acid encoding a gag gene protein not encoded by said first helper RNA; product or an immunogenic fragment thereof of a 0293 and with at least one of said helper RNAS human immunodeficiency virus, wherein the gag lacking an alphavirus packaging Signal; gene product or immunogenic fragment thereof is modified to inhibit release of particles, Such as 0294 wherein the combined expression of the Virus-like particles, containing the gag gene product alphavirus replicon RNA and the helper RNAS pro or the immunogenic fragment thereof from a cell, duces an assembled alphavirus particle which is able and wherein the replicon RNA lackS Sequences to infect a cell, and is unable to complete viral encoding alphavirus Structural proteins, replication, and further wherein the third population contains no detectable replication-competent 0283 (ii) a first helper RNA separate from said alphavirus particles as determined by passage on replicon RNA, said first helper RNA encoding at permissive cells in culture, and further wherein at least one alphavirus Structural protein and further least one of said replicon RNA, said first helper more not encoding at least one other alphavirus RNA, and said one or more additional helper Structural protein; and RNA(s) comprises one or more attenuating muta tions, 0284 (iii) one or more additional helper RNA(s) Separate from Said replicon RNA and Separate from 0295) (b) producing the alphavirus particles in the said first helper RNA, said additional helper RNA(s) helper cell; and encoding at least one other alphavirus Structural 0296 (c) collecting the alphavirus particles from the protein not encoded by said first helper RNA; helper cells, and US 2002/014 1975 A1 Oct. 3, 2002 19

0297 D) combining the first population of alphavirus 0302) The alphavirus replicon particles of this invention particles produced from the first helper cell, the Second can also be made in a cell free System. Such replicon population of alphavirus particles produced from the particles are herein referred to as ViroSomes. In a specific Second helper cell and the third population of alphavi embodiment of the method, Such particles are constructed rus particles produced from the third helper cell, from a mixture containing replicon RNA that does not thereby producing the populations of alphavirus repli encode all of the alphavirus Structural proteins, purified con particles of the present invention comprising at glycoproteins E1 and E2, one or more non-cationic lipids, least one attenuating mutation. Such as lecithin, and detergent. Detergent is slowly removed 0298. In a preferred embodiment, as noted above, the from the mixture to allow formation of lipid bilayers with method provided above can include a further mutation in the incorporated RNA and glycoproteins. pol gene product or immunogenic fragment thereof resulting 0303. In preferred embodiments of the methods of this in inactivation or deletion of protease, integrase and RNase invention, the glycoproteins E1 and E2 could be expressed H functions of the poll gene product or immunogenic frag in any recombinant protein expression System capable of ment thereof. In a specific embodiment of this method, the glycosylation of mammalian proteins, Such as Stably trans region of the pol gene encoding the protease, RNase H and formed cell lines, for example CHO cells, or viral vector integrase function of the poll gene product or immunogenic expression Systems. Such as vaccinia, baculovirus, herpes fragment thereof has been deleted. Virus, alphavirus or adenovirus. In a preferred embodiment, 0299. It is also contemplated regarding the method following expression of the proteins, the E1 and E2 glyco described above, that not all of the first, second and third proteins are purified from contaminating cellular proteins in populations of alphavirus particles do not all have to com the expression Supernatant. The purification of these glyco prise an attenuating mutation. For example, the first popu proteins can be achieved by affinity chromatographic col lation may comprise attenuating mutations, but the Second umn purification, for example using lectin-, heparin-, or and third populations may not, etc. antibody-affinity columns. This affinity purification step may be preceded by Selective precipitation or Selective 0300. The present invention further provides the compo extraction from the expression System Supernatant by meth Sitions of the present invention which are produced by the ods including, but not limited to, ammonium Sulfate pre methods of this invention. cipitation or detergent extraction respectively. Final polish 0301 The compositions and methods of this invention ing Steps of purification may include ion-exchange which incorporate attenuating mutations into the alphavirus chromatography or buffer exchange, for example, and tan replicon particles forming the composition and/or produced gential flow methods to generate purified glycoproteins by the methods include purified compositions and methods suitable for virosome assembly. of purification based on the presence of the attenuating 0304 Thus, the present invention provides a method of mutations. In particular, certain attenuating mutations in the producing alphavirus replicon ViroSomes, comprising: a) alphavirus Structural proteins introduce heparin binding Sites combining alphavirus replicon RNA, alphavirus glycopro into these proteins which are present on the Surface of the teins E1 and E2, non-cationic lipids and detergent; and b) alphavirus replicon particles. As an example, the V3014 E2 gradually removing detergent, whereby alphavirus replicon glycoprotein (SEQ ID NO:12 and SEQ ID NO:13) has a ViroSomes are produced. This method is described in more mutation in which a lysine is Substituted for the glutamic detail in the Examples Section herein. acid at amino acid position 209. This mutation, which creates a more positively charged glycoprotein, increases the 0305 The present invention also provides alphavirus affinity of this protein for heparin. Thus, it is possible to replicon ViroSomes comprising an alphavirus replicon RNA purify Such particles using heparin affinity chromatography. encapsidated by a lipid bilayer in which alphavirus glyco Such chromatography can be performed using any of Several proteins are embedded. The replicon RNA can be from any commercially available resins to which heparin has been alphavirus and the glycoproteins can be from any alphavi bound. The Source of heparin is variable; the commercially rus. In a specific embodiment, the alphavirus glycoproteins available resins currently use porcine heparin. The choice of are VEE E1 and E2. The advantage of the alphavirus resin will be based on its relative ease of use in a Scaled-up, replicon ViroSomes is the ease of preparation, their Stability, GMP-compliant process, e.g., price, column packing limi and their purity, Since they are devoid of any cellular tations, and potential for easy Sanitization. The use of components being made in a cell free System. heparin affinity chromatography results in a Substantial 0306 The helper cells, RNAS and methods of the present purification of the VRPs with very little loss of material, and invention are useful in in Vitro expression Systems, wherein it is a Scalable purification Step. In a preferred embodiment, the inserted heterologous RNA located on the replicon RNA a heparin affinity chromatography Step results in between an encodes a protein or peptide which is desirably produced in 8- to 27-fold reduction in total protein per ml, or from a 300 to 1000-fold reduction in total protein per VRP. Thus, the vitro. The helper cells, RNAS, methods, compositions and present invention provides heparin affinity-purified alphavi pharmaceutical formulations of the present invention are ruS replicon particles containing attenuating mutations additionally useful in a method of administering a protein or which are useful as clinical trial material and commercial peptide to a Subject in need of the desired protein or peptide, product. The present invention also provides methods for as a method of treatment or otherwise. preparing purified alphavirus replicon particles containing 0307. It is contemplated that the proteins, peptides, attenuating mutations comprising the use of heparin affinity nucleic acids, vectors and alphavirus replicon particles of chromatography, as described in the Examples provided this invention can be administered to a Subject to impart a herein. These particles can also be present in a composition therapeutic or beneficial effect. Therefore, the proteins, of this invention. peptides, nucleic acids, vectors and particles of this inven US 2002/014 1975 A1 Oct. 3, 2002 20 tion can be present in a pharmaceutically acceptable carrier. 0313 Amethod of treating and/or preventing infection by By “pharmaceutically acceptable' is meant a material that is HIV in a Subject is also provided herein, comprising admin not biologically or otherwise undesirable, i.e., the material istering to the Subject an effective amount of the particles, may be administered to a Subject, along with the nucleic acid ViroSomes and/or compositions of this invention, in a phar or vector of this invention, without causing any undesirable maceutically acceptable carrier. biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical compo 0314. The subject of this invention can be any animal in sition in which it is contained. The carrier would naturally be which an immune response can be induced or in which an Selected to minimize any degradation of the active ingredi infection by HIV can be treated and/or prevented. In a ent and to minimize any adverse Side effects in the Subject, preferred embodiment, the Subject of this invention is a as would be well known to one of skill in the art (see, e.g., mammal and most preferably is a human. Remington's Pharmaceutical Science; latest edition). 0315 Protocols and data regarding the testing of the 0308) Pharmaceutical formulations of this invention, compositions of this invention in animals and protocols for Such as Vaccines, of the present invention can comprise an administration to humans are provided in the Examples immunogenic amount of the alphavirus replicon particles as herein. disclosed herein in combination with a pharmaceutically 0316. In a particular embodiment, the present invention acceptable carrier. An "immunogenic amount' is an amount provides an isolated nucleic acid encoding a pol gene of the infectious alphavirus particles which is Sufficient to product or immunogenic fragment thereof of a human evoke an immune response (humoral and/or cellular immune immunodeficiency virus, wherein the protease, integrase, response) in the Subject to which the pharmaceutical for RNase H and reverse transcriptase functions of the pol gene mulation is administered. An amount of from about 10 to product or immunogenic fragment thereof have been inac about 10" replicon-containing particles, and preferably, tivated or deleted. Such a modification has been shown in about 10" to about 10 replicon-containing particles per dose Some studies to facilitate inhibition of the formation of is believed Suitable, depending upon the age and Species of replication competent alphavirus particles during production the Subject being treated. Exemplary pharmaceutically of alphavirus replicon particles comprising the poll gene acceptable carriers include, but are not limited to, Sterile product or immunogenic fragment thereof. pyrogen-free water and Sterile pyrogen-free physiological Saline Solution. 0317. Also provided herein is a composition comprising the pol-expressing nucleic acid described above, a vector 0309 Subjects which may be administered immunogenic comprising the nucleic acid and a cell comprising the vector. amounts of the infectious, replication defective alphavirus The pol-expressing nucleic acid can also be present in an particles of the present invention include, but are not limited alphavirus replicon particle comprising the nucleic acid. to, human and animal (e.g., horse, donkey, mouse, hamster, monkey) Subjects. Administration may be by any Suitable 0318. As noted above, the nucleic acid encoding the pol means, Such as intraperitoneal or intramuscular injection. gene product or immunogenic fragment thereof comprises a 0310) Pharmaceutical formulations for the present inven modification resulting in the inhibition of reverse tran tion can include those Suitable for parenteral (e.g., Subcuta Scriptase activity. In a preferred embodiment, a mutation is neous, intradermal, intramuscular, intravenous and intraar introduced at the active site motif that results in inhibition of ticular) administration. Alternatively, pharmaceutical reverse transcriptase activity. Such a mutation may remove formulations of the present invention may be suitable for the DNA binding domain of the enzyme, for example. A administration to the mucous membranes of a Subject (e.g., mutation from YMDD to YMAA or HMAA at this motif is intranasal administration). The formulations may be conve an example of Such a mutation. niently prepared in unit dosage form and may be prepared by 03.19. The present invention additionally provides a any of the methods well known in the art. method of making an alphavirus replicon particle compris 0311. Thus, the present invention provides a method for ing nucleic acid encoding a poll gene product or immuno delivering nucleic acids and vectors (e.g., alphavirus repli genic fragment thereof of a human immunodeficiency virus, con particles, ViroSomes) encoding the antigens of this wherein the pol gene product or immunogenic fragment invention to a cell, comprising administering the nucleic thereof comprises a modification resulting in deletion or acids or vectors to a cell under conditions whereby the inactivation of protease, integrase, RNase H and reverse nucleic acids are expressed, thereby delivering the antigens transcriptase functions from the poll gene product or immu of this invention to the cell. The nucleic acids can be nogenic fragment thereof, comprising delivered as naked DNA or in a vector (which can be a viral vector) or other delivery vehicles and can be delivered to 0320 A) providing a helper cell for producing an cells in vivo and/or ex vivo by a variety of mechanisms well infectious, defective alphavirus particle, comprising known in the art (e.g., uptake of naked DNA, Viral infection, in an alphavirus-permissive cell: liposome fusion, endocytosis and the like). The cell can be 0321 (i) an alphavirus replicon RNA, wherein the any cell which can take up and express exogenous nucleic replicon RNA comprises an alphavirus packaging acids. Signal and a nucleic acid encoding a poll gene 0312 Further provided herein is a method of inducing an product or an immunogenic fragment thereof of a immune response to an HIV antigen of this invention in a human immunodeficiency virus, wherein the pol Subject, comprising administering to the Subject an immu gene product or immunogenic fragment thereof is nogenic amount of the particles, ViroSomes and/or compo modified to delete or inactivate protease, RNase Sition of this invention, in a pharmaceutically acceptable H, integrase and reverse transcriptase functions in carrier. the poll gene product or immunogenic fragment US 2002/014 1975 A1 Oct. 3, 2002 21

thereof, and wherein the replicon RNA lacks 0332 Furthermore, the present invention provides a Sequences encoding alphavirus Structural proteins, method of treating or preventing infection by human immu nodeficiency virus in a Subject, comprising administering to 0322 (ii) a first helper RNA separate from said the Subject an effective amount of a composition comprising replicon RNA, said first helper RNA encoding at an alphavirus particle comprising nucleic acid encoding a least one alphavirus Structural protein and further pol gene product or immunogenic fragment thereof of a more not encoding at least one other alphavirus human immunodeficiency virus, wherein the poll gene prod Structural protein; and uct or immunogenic fragment thereof comprises a modifi 0323 (iii) one or more additional helper RNA(s) cation resulting in deletion or inactivation of protease, Separate from Said replicon RNA and Separate integrase, RNase H and reverse transcriptase functions in the from said first helper RNA, said additional helper pol gene product or immunogenic fragment thereof, in a RNA(s) encoding at least one other alphavirus pharmaceutically acceptable carrier. Structural protein not encoded by Said first helper 0333. In preferred embodiments of the methods of this RNA; invention, the Subject is administered an effective amount of a population of alphavirus particles comprising particles 0324 and with at least one of said helper RNAS expressing (1) nucleic acid encoding a pol gene product or lacking an alphavirus packaging Signal; immunogenic fragment thereof of a human immunodefi 0325 wherein the combined expression of the ciency virus, wherein the poll gene product or immunogenic alphavirus replicon RNA and the helper RNAS pro fragment thereof comprises a modification resulting in inac duces an assembled alphavirus particle which is able tivation or deletion of protease, integrase, RNase H and to infect a cell, and is unable to complete viral reverse transcriptase functions in the poll gene product or replication, and further wherein the population con immunogenic fragment thereof, (2) nucleic acid encoding a tains no detectable replication-competent alphavirus gag gene product or immunogenic fragment thereof of a particles as determined by passage on permissive human immunodeficiency virus, wherein the gag gene prod uct or immunogenic fragment thereof is modified to inhibit cells in culture; release of gag gene product or the immunogenic fragment 0326 (B) producing the alphavirus particles in the thereof from a cell, and (3) nucleic acid encoding an env helper cell; and gene product or an immunogenic fragment thereof of a human immunodeficiency virus in a pharmaceutically 0327 (C) collecting the alphavirus particles from acceptable carrier. the helper cell. 0334. In further preferred embodiments, the population 0328. In the method provided above, at least one of the of alphavirus particles comprises particles expressing (1) replicon RNA, the first helper RNA, and the one or more nucleic acid encoding a gag gene Sequence that has at least additional helper RNA(s) can comprise one or more attenu 95% identity with SEQ ID NO:4; (2) nucleic acid encoding ating mutations, as described herein. a pol gene sequence that has at least 99% identity with SEQ 0329. In a specific embodiment of this method, a muta ID NO:15; and (3) nucleic acid encoding an env gene tion is introduced at the active site motif in the poll gene sequence with at least 92% identity with SEQ ID NO:18. In product or immunogenic fragment thereof that results in a specific embodiment, the population of alphavirus par inhibition of reverse transcriptase activity. Such a mutation ticles comprises particles expressing (1) nucleic acid of SEQ may remove the DNA binding domain of the enzyme, for ID NO:4, (2) nucleic acid of SEQ ID NO:15, and (3) nucleic example. A mutation from YMDD to YMAA or HMAA at acid of SEO ID NO:18. this motif is an example of Such a mutation. EXAMPLES 0330. Also provided herein is an alphavirus replicon 0335 The following examples are provided to illustrate particle expressing the pol gene product or immunogenic the present invention, and should not be construed as lim fragment thereof, wherein the pol gene product or immu iting thereof. In these examples, nm means nanometer, mL nogenic fragment thereof comprises a modification resulting means milliliter, pfu/mL means plaque forming units/milli in deletion or inactivation of protease, integrase, RNase H liter, VEE means Venezuelan Equine Encephalitis virus, and reverse transcriptase functions in the pol gene product or EMC means encephalomyocarditis virus, BHK means baby immunogenic fragment thereof, produced according to any hamster kidney cells, HA means hemagglutinin gene, N of the above methods. means nucleocapsid, FACS means fluorescence activated 0331 In a further embodiment, the present invention cell sorter, and IRES means internal ribosome entry site. The provides a method of inducing an immune response in a expression “E2 amino acid (e.g., lys, thr, etc.) number Subject, comprising administering to the Subject an immu indicates the designated amino acid at the designated residue nogenic amount of a composition comprising an alphavirus of the E2 gene, and is also used to refer to amino acids at particle comprising nucleic acid encoding a pol gene product Specific residues in the E1 protein and in the E3 protein, or immunogenic fragment thereof of a human immunode respectively. ficiency virus, wherein the pol gene product or immuno genic fragment thereof comprises a modification resulting in Example 1 deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the poll gene product or VEE Replicon Particles as Vaccines immunogenic fragment thereof, in a pharmaceutically 0336 Replicon particles for use as a vaccine are pro acceptable carrier. duced using the VEE-based vector System, originally devel US 2002/014 1975 A1 Oct. 3, 2002 22 oped from a full-length, infectious clNA clone of the RNA ments, one containing the VEE nonstructural genes (e.g., genome of VEE (FIG. 1 in Davis et al., 1989). In this SEQ ID NO:2) and a single copy of the 26S subgenomic Example, one or more attenuating mutations (Johnston and RNA promoter followed by a multiple cloning site, and a Smith, 1988; Davis et al., 1990) have been inserted into the Second Smaller fragment containing a 26S Subgenomic RNA clone to generate attenuated VEE vaccine vectors (Davis et promoter followed by the VEE structural genes. The large al., 1991; 1995; Grieder et al., 1995). fragment is isolated and religated to produce the replicon, 0337 AS described herein, these constructs are geneti pVR2. A multiple cloning site (MCS) was inserted into cally modified to create an RNA replicon (i.e., an RNA that pVR2 to generate pVERV. In this example, as well as in the Self-amplifies and expresses), and one or more helper RNAS construction of the helper plasmids (Example 3), the ampi to allow packaging. The replicon RNA expresses an HIV cillin resistance gene in each plasmid was replaced with a gene, e.g., the Clade C HIV-1 gag gene. The replicon RNA kanamycin resistance gene (SEQ ID NO:6; encoding amino is packaged into virus-like particles (herein referred to as acid sequence as in SEQ ID NO:7). The kanamycin resis “virus replicon particles” or “VRPs') that are infectious for tance gene was obtained from the peT-9a plasmid, and was only one cycle. During this cycle, the characteristics of the used to aid in the cloning manipulations and for regulatory alphavirus-based vector result in very high levels of expres compliance. sion of the replicon RNA in cells to which the VRP is Example 3 targeted, e.g., cells of the lymph node. 0338. In the cytoplasm of the target cell, the replicon Construction of Helper Plasmids RNA is first translated to produce the viral replicase proteins 0342. The starting materials for the helper plasmids are necessary to initiate Self-amplification and expression. In four full-length cDNA clones: V3000, the virulent Trinidad this Example, the HIV-1 Clade C gag gene is encoded by a donkey Strain of VEE, three clones with attenuating muta Subgenomic mRNA, abundantly transcribed from a nega tions, pV3014 (E2 lys 209, E1 thr 272), V3519 (E2 lys 76, tive-Sense replicon RNA intermediate, leading to high-level E2 lys 209, E1 thr 272) and V3526 (deletion of E356-59, E1 expression of the HIV-1 Clade C gag gene product. Since the ser 253), which are in the genetic background of Trinidad VEE Structural protein genes are not encoded by the replicon donkey strain VEE. Several different helper plasmids have RNA, progeny Virion particles are not assembled, thus been made by using unique or rare restriction sites in the limiting the replication to a Single cycle within the infected full-length cDNA clone to delete portions of the nonstruc target cell. tural protein region. The full-length clone is digested with 0339) Importantly, only the replicon RNA is packaged one or two restriction enzymes, the larger DNA fragment is into VRPS, as the helper RNAS lack the cis-acting packaging isolated and then religated to form a functional plasmid. In Sequence required for encapsidation. The “split helper' or Vitro RNA transcripts from these plasmids upon transfection bipartite System (see Example 4) greatly reduces the chance of tissue culture cells would not encode a functional RNA for an intact genome being assembled by recombination, and replication complex, and also would not include an encapsi as a back-up Safety feature, one or more highly attenuating dation Signal. The helper constructs differ in the size of the mutations, Such as those contained in the glycoprotein genes nonstructural gene deletion. The helper constructs are des in V3014 (Grieder et al., 1995), are incorporated. ignated by the attenuated mutant clone used in their con Struction, and by the percentage of the nonstructural region 0340 Overall, the design of the VRPs incorporates sev deleted. The following helper constructs were generated: eral layered and redundant Safety features. In addition to the above-described split helper System and attenuating muta 0343) V3014A520-7507(93%) tions, over one-third of the genome of the virus has been removed, creating a defective genome which prevents 0344) V3519A520-7507(93%) Spread from the initially infected target cell. Nonetheless, if 0345) V3526A520-7505(93%) a Statistically rare recombination event occurs to yield replication competent virus (RCV), the resulting virus 0346) V3014A520-6965(87%) would be a highly attenuated VEE strain. 0347 V3519A1687-7507(78%) Example 2 0348) V3014A2311-7505(70%) Construction of VEE Replicon 0349) V3519A3958-7507(47%) 0341) The VEE structural protein genes (C-PE2-6K-E 1) 0350) V3526A520-7505(93%) are removed from a cDNA clone pV3014 which contained two attenuating mutations (E2 lys 209, E1 thr 272), and a 0351) V3014A3958-7505(47%) duplication of the 26S Subgenomic RNA promoter Sequence immediately downstream from the 3'-end of the E1 glyco O352) V3519A1955-3359(19%) protein gene, followed by a multiple cloning Site as 0353) V3014A520-3954(46%) described in U.S. Pat. No. 5,505,947 to Johnston et al. The pV3014 plasmid DNA is digested to completion with Apal 0354) V3014A1955-3359(19%) restriction enzyme, which cuts the VEE genomic Sequence at nucleotide 7505 (numbered from the 5'-end of the genome 0355) V3014A1951-3359(19%) Sequence). A second recognition site for this enzyme is found in the duplicate 26S Subgenomic promoter. Therefore, 0356) V3014A2311-3055(10%) digestion of pV3014 with Apal produces two DNA frag 0357 V3014A2307-3055(10%) US 2002/014 1975 A1 Oct. 3, 2002

Example 4 Sequences of each isolate were compared to the derived consensus. All isolates were found to be Subtype C of HIV, Construction of Bipartite RNA Helper Plasmids thus confirming the predominance of this Subtype in South Africa. 0358. A bipartite helper system is constructed as described herein. The V3014A520-7505(93%) helper is used 0362 A. Construction of the Gag-VRP Vaccine to construct an additional deletion of the E2 and E1 glyco 0363. Described herein is the design and manufacture of protein genes by digestion with Hpal restriction enzyme and VEE replicon particles (VRPs) engineered to express the ligation, resulting in deletion of the Sequence between gag gene from a Subtype C isolate of HIV-1. The main nucleotide 8494 (in the E3 gene) and nucleotide 11,299 (near purpose of this single antigen vaccine is to establish a Safety the 3'-end of the E1 gene). In vitro RNA transcripts of this profile for VRPs in healthy human subjects. Optimally, the glycoprotein helper plasmid (presented graphically in FIG. HIV-Gag-VRPs will be formulated as a component of a 2, an exemplary nucleotide Sequence for Such a plasmid is trivalent vaccine, also containing HIV-Pol-VRP and HIV SEQ ID NO:8, including the nucleotide sequence (SEQ ID gp160-VRP (env) made in analogous procedures to the one NO:9 and the amino acid sequence (SEQ ID NO:10 of the described herein for HIV-Gag-VRPs. VEE capsid), when electroporated into BHK cells with a replicon RNA, are replicated and transcribed to give a 0364. In this Example, the VEE particles are based on the mRNA encoding only the capsid protein of VEE. V3014 glycoprotein helper plasmid (FIG.3, SEQID NO:12 and SEQ ID NO:13), which harbors two highly attenuating 0359 The second member of the bipartite helper is mutations, one in E2 and the other in E1 (Grieder et al., constructed from the same original helper plasmid 1995). The V3014 glycoprotein helper RNA is able to 3014A5207505(93%) by cleavage with Tth111I restriction package VRPS with Significantly greater efficiency than the enzyme (at nucleotide 7544) and Spel restriction enzyme (at glycoprotein helper RNA derived from V3526 (Pushko et nucleotide 8389), resulting in deletion of the capsid gene, al., 1997). Nonetheless, safety of the VRP vector system has followed by insertion of a synthetic double-stranded oligo not been compromised since detailed pathogenesis Studies nucleotide with Tth111 I and Spel termini. The inserted clearly have shown V3014 to be avirulent in adult mice by Sequence restored the downstream portion of the 26S pro subcutaneous inoculation (Grieder et al., 1995). V3014 was moter and an ATG initiation codon followed by a Ser codon, found to be Significantly impaired in its ability to reach and such that the first amino acid residue of E3 (Ser) is the first Spread beyond the draining lymph node following Subcuta codon following the inserted AUG. The resulting glycopro neous inoculation. Unlike wild-type V3000, V3014 does not tein helper plasmid is presented graphically in FIG. 3, and establish a Viremia and does not reach the brain. In addition, an exemplary nucleic acid Sequence for Such a plasmid is on rare occasions when found, histopathological lesions in SEQ ID NO:11, encoding the VEE glycoproteins (E3-E2-6 the periphery were much leSS Severe than those induced by kD-E1), SEQ ID NO:12. The in vitro transcript of this wild-type V3000 (Grieder et al., 1995). Following inocula plasmid, when transfected into a cell with replicon RNA, tion with V3014, adult mice are protected against lethal will produce the VEE glycoproteins (SEQ ID NO:13). wild-type VEE infection. Co-electroporation of both of these helper RNAS into a cell with replicon RNA results in production of infectious par 0365. The attenuated phenotype of V3014 also was ticles containing only replicon RNA. observed in VEE challenge studies in horses. Animals inoculated subcutaneously with V3014 showed no signifi 0360. Other than the 5' and 3' ends and the 26S promoters cant leukopenia or febrile response compared to mock (40 nucleotides) of these helper RNAS, the only sequence in vaccinated controls. In addition, results indicated that these common between the capsid and glycoprotein helperS is the animals were completely protected against Virulent VEE sequence from 8389 to 8494 (106 nucleotides) (V3000) challenge. Example 5 0366 Taken together, these data indicate that if the rare recombination event did occur during VRP assembly to VEE Replicon Particles Expressing HIV Genes yield RCV, the worst case scenario would be the generation of a highly attenuated strain of VEE. 0361 The vaccines of this invention are exemplified by 0367 B. Selection and Cloning of the Heterologous the use of a propagation defective, replicon particle vector Antigen System derived from an attenuated Strain of Venezuelan equine encephalitis virus (VEE) to create a mixture of VEE 0368. The exemplary HIV genes used in this invention, replicon particles individually expressing HIV-1 gag, pol, or gag, pol and env, are derived from Subtype C (Clade C) env genes. The three genes used in this Example were viruses isolated from likely Phase III clinical trial sites in Selected based on homology to consensus Sequences gener South Africa. The HIV infection rate in South Africa and its ated from primary isolates obtained from recent Serocon long established Virology and public health infrastructure verters in KwaZulu/Natal Province, South Africa. Plasma make this country an attractive choice for clinical testing of Samples from approximately 20 recent Seroconverters in the HIV Vaccines. Focused Sequencing and phylogenetic analy Durban/Hlabisa cohort and a similar number of HIV-posi sis of the gag, pol, and env genes of these isolates has tive, asymptomatic individuals were collected. HIV viral allowed the Selection of genes representative of the Clade C RNA was isolated from the plasma, and the Sequences of the isolates circulating in this region of Africa. gag, pol and env genes were analyzed. Two regions from each gene were amplified, and the resulting PCR products 0369) 1. HIV-1 Clade C gag Gene were sequenced (see FIG. 10 for regions analyzed). A 0370. Two 400 bp regions of the gag gene were consensus Sequence was derived for each gene, and the Sequenced from approximately 30 plasma Samples collected US 2002/014 1975 A1 Oct. 3, 2002 24 from HIV seropositive individuals in South Africa. A South showing that the protein expressed in the cells is of the African consensus Sequence was then determined for the gag correct size and is immunoreactive. In FIG. 7, expression of gene as well as a consensus Sequence from the Los Alamos this ENV protein in U87.CD4. CCR5 cells is shown. These database for Subtype C virus. In addition, approximately 20 cells process the ENV protein into two components, gp120, comparable Sequences from Malawi were used, generated as gp41 and gp160. In these cells, the expressed gp160 is part of another Study, to confirm conclusions about Sequence fusogenic (see FIG. 8). variation. Several isolates that were close to the South African consensus Sequence were compared to other isolates 0377 3. HIV-1 Clade C pol Gene in distance measurements. Among these 30 isolates, one was 0378 AClade C pol gene from isolate Du151 was chosen chosen as the source for the gag gene (SEQ ID NO:4; based on its 99% amino acid identity with the South African corresponding to the amino acid sequence in SEQID NO:5) consensus Sequence. This gene was modified at the active for the following reasons. Site of the reverse transcriptase encoding Sequence to inhibit its activity, and the p51 fragment of this modified gene (SEQ 0371 This isolate had greater than 95% amino acid ID NO:15) was engineered into a VEE RNA replicon identity to the South African consensus Sequence, represent plasmid. The map of this poll plasmid is shown in FIG. 4, ing the approximate middle of the Sequence diversity of all and the nucleotide Sequence of the plasmid is provided as isolates. This isolate, known as Du422, came from a recent SEQ ID NO:14. In FIG. 6, expression of this POL p51 Seroconvertor, reflecting currently circulating Strains and the fragment (SEQ ID NO:16) in BHK cells is demonstrated transmitted phenotype. The phenotype of Du422 is NS1, (Western blot, lane 1), showing that the protein expressed in CCR5(+), and CXCR4(-). these cells is both the correct size and immunoreactive. 0372 Prior to the insertion of the gag gene into the VEE 0379 C. Immunological Response to VRP-Gag Vaccine replicon plasmid vector, the amino terminal myristylation (“myr) site of gag was removed to prevent the formation of 0380 Mice were injected subcutaneously in two doses, Gag-containing virus-like particles. Restriction enzyme with 8-9 mice in each group. The mice were immunized digests of the gag gene plasmid, the capsid helper plasmid, once, then immunized a Second time, with the same dose, 28 and the glycoprotein helper plasmid were performed to days later. Serum was collected the day prior to the first confirm the identity of the three vectors when compared to immunization, then at day 27 (“after 1 immunization) and published maps of the parental plasmid pBR322, with the at day 35 (after 2" immunization). kanamycin resistance gene Substituted for the amplicillin 0381. The vigorous, antigen-specific humoral response of resistance gene. The confirmed plasmid maps of the VEE mice to the HIV-1 Clade CVRP-Gag vaccine described in replicon plasmid containing the Du422 gag gene (p3 Example 5.A. 1. is presented in Table 1. Details of this assay 40.1.6), the capsid helper plasmid (p3-13.2.2), and the glycoprotein helper plasmid (p3-13.4.6) are presented in are described in Example 7A.1. FIGS. 1, 2, and 3, respectively. The full nucleotide sequence of each of these plasmids is presented herein as SEQ ID TABLE 1. NO:1, SEQ ID NO:8, and SEQ ID NO:11, respectively. Humoral Response to VRP-Gag Vaccine 0373) In FIGS. 6 and 15, expression of this HIV-1 Gag Total Ab Titer protein in BHK cells infected with VRPs expressing such a Dose: (log10) gag construct is demonstrated (FIG. 6: Western blot, lane 3; 10 i.u. dose: FIG. 15, immunofluorescence detection). The cells were infected at a multiplicity of infection (m.o.i.) of 3.5 infec after 1 immunization 1.3 +f- 0.1 tious units (i.u.) per cell, and expression was measured 18 after 2" immunization 2.8 +f- 1.1 hours post-infection (p. i.). Cell lysates (from approximately 10 i.u. dose 2x10 cells) were collected and fractionated either by a after 1 immunization 2.1 +f- 0.5 4-12% gradient SDS-PAGE or by 10% SDS-PAGE. The after 2" immunization 4.1 +f- 0.6 fractionated polypeptides were transferred to PVDF mem branes and probed with human HIV-1 positive serum. 0382 A robust, Gag-specific response in mice was 0374) 2. HIV-1 Clade C env Gene induced by the HIV-1 dade C VRP-Gag vaccine and is 0375 A Clade C env gene (aka “gp160) from another presented in FIG. 9. Details of this assay are described in HIV isolate, Du151, from a recent Seroconverter was chosen Example 7A.3. based on its 92% amino acid identity to the South African consensus Sequence for this gene, determined in an analo Example 6 gous method to the one described for the gag gene in Example 5.A.1. The phenotype of the Du151 isolate is NS Manufacturing Process for HIV VRP Vaccines 1, CCR5(+), CXCR4(-). This gene was engineered into a 0383 A. Manufacturing Process VEE RNA replicon plasmid as shown in FIG. 5, and the entire sequence of the plasmid is given at SEQ ID NO:17. 0384 Disclosed herein is a manufacturing process for The env gene construct used in this Example is SEQ ID VRP vaccines that is suitable for large-scale preparation of NO:18. GMP-compliant (GMP=Good Manufacturing Practices) material for use in human clinical trials or for commercial 0376) In FIG. 6, expression of this ENV protein (SEQ. manufacture. The process includes Several StepS and after ID. NO:19) in BHK cells infected with VRPs expressing this each step (as appropriate), a set of “in process control' (IPC) HIV env construct is demonstrated (Western blot, lane 2), assays or Release Tests (RT) is performed to confirm the US 2002/014 1975 A1 Oct. 3, 2002

successful completion of the step. The IPC/RT tests and 0388 To produce HIV-VRP vaccine for clinical use, both process Steps and the accompanying IPC assay(s) or RTS replicon and helper plasmids are linearized by digestion at (described in more detail in Example 6D.1 and 6D.2) are as the unique Not I site and used as templates for Synthesis of follows: run-off transcripts. The quality of the transcription products (i.e., the replicon and the two helper RNAS) is evaluated by agarose gel electrophoresis. Process Step IPC/RT Tests and Process Steps 0389 C. Characterization of the Vero cells Linearize 3 DNA plasmids IPC: Check for linearity 0390 Vero cells are used in the production of HIV-VRPs In vitro RNA transcription IPC: Size, integrity and (WHO Vero MCBP139, BioPeliance Inc., Rockville, Md.). concentration Vials contained approximately 1x10 cells/mL in a cryopro Electroporation of certified Vero tectant Solution of 90% fetal bovine serum and 10% dim cell line Harvest culture fluids IPC: ethyl sulfoxide. A Cell Certification Summary is provided Titration/Identity with each lot. BioPeliance Inc. has filed a Master File with Test for replication-competent the FDA regarding the WHO Vero MCB P139. virus (RCV) Pool the culture fluid RT: 0391) Vials of WHO Vero MCBP139 cells are expanded Mycoplasma into flaskS. Each of the flaskS is then expanded again in order Adventitious virus to prepare the Master Cell Bank (MCB). The Working Cell PERT assay Bank (WCB) is prepared from the MCB. The MCB is tested Purification of bulk VRP by heparin IPCs: affinity chromatography Heparin residual assay for purity and identity. The WCB is tested for adventitious BSA assay agents (detection of mycoplasma and viruses). Viability tests Bovine IgG assay are performed on both the MCB and the WCB. Filtration of bulk VRP RT: Test for RCW 0392 Tumorigenicity tests are performed once at the end Titration/Identity of the production period. Contaminating protein/DNA Sterility 0393 D. Electroporation Endotoxin Formulate, Fill, Release RT: 0394 Vero cells are cotransfected by electroporation with Titration/Identity RNA mixtures comprising replicon RNA transcripts encod Sterility ing HIV-gag, VEE capsid helper RNA transcripts, and VEE General Safety glycoprotein helper RNA transcripts. The transfected cells are transferred to tissue culture vessels and incubated in well-defined culture medium. Following harvest, the HIV 0385 B. Preparation of plasmid DNAS Gag-VRP is purified from pooled culture fluid Supernatants by affinity column chromatography. Prior to formulation and 0386 Stock solutions of replicon plasmid DNA, capsid filling, purified, bulk HIV-Gag-VRP is tested for the pres helper plasmid DNA and glycoprotein helper plasmid DNA ence of RCV. are produced in Eschericia coli XL2 Blue cells (Stratagene, cati 200150). All plasmids harbor the kanamycin resistance 0395 E. Final formulated product gene marker. The three plasmid DNAS were manufactured 0396 The HIV-Gag-VRP vaccine is vialed at four dif and purified by PureSyn, Inc. (Malvern, PA) under appro ferent doses. The material is filtered (0.22 um) and added to priate GLP/GMP procedures, with a complete Batch Record Vials at the appropriate concentration and Volume, Stop with full traceability. Following fermentation and cell har pered, quick-frozen and Stored at -20° C. vest, cell paste was lysed with base and plasmid DNAS were purified by ion pair chromatography on PolyFlo'TM separa 0397) F. Control tests of the Gag-VRP vaccine tion media. 0398 1. In-Process Controls 0387 Prior to release by appropriate quality assurance/ 0399 Table 3 below summarizes the In-Process Controls quality control oversight, each lot of each plasmid DNA is performed during the manufacturing process of the HIV analyzed to confirm identity, purity and quality (Table 2). An Gag-VRP Vaccine. approved certificate of analysis for each DNA is then established for each plasmid DNA lot. TABLE 3 TABLE 2 IPCs during the manufacture of HIV-Gag-VRP Vaccine Plasmid DNA Release Tests Test Method Target Test Method Specification Check for linearity Agarose Gel electrophoresis Report Size, integrity and Agarose Gel electrophoresis Report DNA homogeneity Agarose gel electrophoresis >90% supercoiled concentration of RNAs E. coli genomic DNA Southern Blot <50 tug/mg plasmid Titration/Identity Indirect immunofluorescence Report E. coli RNA Agarose gel electrophoresis No detectable bands assay(IFA), using standardized Endotoxin Limulus Amoebocyte <0.1 EU/mg Gag-specific antibody preparation Lysate (LAL) Test for RCW CPE Assay Report Total protein Abs 260/28O 1.8-1.9 Heparin Residual Chromogenic Inhibitory Assay Report Sterility Bioburden assay, USP23 1 CFU BSA residual ELISA Report Identity Restriction enzyme analysis Matches map Bovine IgG Residual ELISA Report US 2002/014 1975 A1 Oct. 3, 2002 26

0400 2. Release tests mammalian cells. Antigen Specificity also is confirmed by 04.01 Tables 4 and 5 below summarize the release tests immunoblot analysis. Anti-VEE responses are monitored by performed on the HIV-Gag-VRP Vaccine. ELISA (Johnston and Smith, 1988). 0407 A.2Humoral Immune Response in Rabbits TABLE 4 0408. Three groups of five female New Zealand white Pool of the Culture Fluids rabbits are inoculated subcutaneously with 10, 10°, or 107 i.u. of the HIV-Gag-VRP. The fourth group, Control Group, Test Method Target receives the vehicle only. Immediately prior to inoculation, Adventitious Virus (in vivo) European guidelines Negative and at weeks 3, 5, 8 and 10 post-inoculation, blood Samples Adventitious Virus (in vitro) 5 cell lines No growth are collected for humoral immune response evaluations. Mycoplasma 21CFR 610.30 No Growth Reverse Transcriptase PERT Assay Negative 04.09 Humoral immune responses are evaluated as described in Section A.1. 0410 A.3 Cell-Mediated Immune Response in Mice 0402 0411 Three groups of five female BALB/c mice are TABLE 5 inoculated subcutaneously with 10, 10°, or 107 iu. of the HIV-Gag-VRP at day 0 and day 28. The fourth group, Bulk VRP and Final Vial testing Control Group, receives the vehicle only. Blood Samples are collected at week 3 post-inoculation. Spleens are harvested Test Method Target Result for Splenocyte collection on day 7 following the Second Replication Cytopathic effect (CPE) Absence (in VERO cells, inoculation for evaluation of cell-mediated immune competent assay sensitivity is 1-10 pfu V3.014) responses. virus (RCV) VRP identity, Indirect 10° to 10 i.u. per mL potency immunofluorescence 0412. The cell-mediated immune response is evaluated assay (IFA) by determining the ability of splenic T cells from immunized Cellular Protein Pierce BCA protein assay Total protein content per dose mice to proliferate eX Vivo in the presence of either Gag Contaminant protein or Gag peptide(s). The ability of splenic T and CD4+ Cellular DNA Southern Blot or PCR <10 ng per dose T cells to produce interferon-Y and interleukin-4 respec Contaminant Sterility 21 CFR S 610.12 Pass tively, is determined. Finally, the ability of cytotoxic T Endotoxin LAL <5 EU/dose lymphocytes to lyse target cells that present murine major General Safety 21 CFR s 610.11 Pass histocompatibility complex class-I restricted epitopes for Particulates USP Pass HIV-1 Clade C Gag protein is measured (see Betts et al., Stability IFA 10° to 10 i.u. per mL 1997 for methods) 0413 B. Safety Study Example 7 0414. Three groups of six male and six female New Zealand white rabbits are inoculated subcutaneously with Preclinical Studies 10, 10°, or 3x107 i.u. of the HIV-Gag-VRP. The fourth group, Control Group, receives the vehicle only. Animals 0403 Pilot lots are manufactured following written pro receive four injections at week 0, week 3, week 6 and Week cedures (SOPs and STMs) and according to the manufac 9. Half of the animals are sacrificed two days after the last turing Scheme described in Example 6. These pilot lots are injection (week 9) and the other half at three weeks after the prepared and used for two major tasks. The first one is a last injection (week 12). Similar studies are performed in preclinical immunogenicity evaluation, which includes Stud mice with a high dose at 10 iu. This level is 10-100 times ies to assess the immune response and the cell-mediated the likely primate dose, based on efficacy Studies in rhesus immune response in vaccinated animals. The Second major macaque.S. task is a preclinical Safety evaluation, which includes evalu 0415. In addition to system toxicity (record of mortality/ ations of System toxicity, hematopoietic and immune System morbidity, body temperature, body weight, food consump toxicity, and local reactogenicity. tion and ophthalmic examinations), hematopoietic toxicity is 0404 A. Immunogenicity Studies evaluated by quantitating cellular components of peripheral blood, and immune System toxicity is assessed by histo 04.05 A.1 Humoral Immune Response in Mice pathologic evaluation of the lymphoid organs. Local reac 0406 Three groups of five female BALB/c mice (4-6 togenicity is evaluated by examining the injection Sites weeks of age) are inoculated subcutaneously with 10, 10, grossly and microscopically to determine irritation potential. or 107 iu. of the HIV-Gag-VRP at three time points: on day Serum Samples are also tested for the presence of replication 0, and at weeks 4 and 8. The fourth group, Control Group, competent virus by blind passage in cell culture. receives the vehicle only. Immediately prior to inoculation, and at weeks 3, 5, 8 and 10 post-inoculation, blood Samples 0416) C. In Situ Hybridization Study in Mice are collected for humoral immune response evaluations. Gag 0417. Three groups of five female BALB/c mice are protein-Specific Serum antibody titers and Seroconversion inoculated subcutaneously with 10, 10°, or 107 iu. of the rates are measured by ELISA (Caley et al., 1997) against HIV-Gag-VRP. The fourth group, Control Group, receives purified, recombinant Gag protein. The Source of the antigen the vehicle only. A Single injection is performed in each is the homologous Clade C gag gene expressed in insect or grOup. US 2002/014 1975 A1 Oct. 3, 2002 27

0418. To verify expression of HIV-GAG-VRP in lym 0427 Cell Culture phoid tissue, the draining lymph nodes, Spleen, and thymus 0428 BHK cells were used as a cell Substrate and were of the mice are examined by in situ hybridization at 24 hours maintained in growth medium (alpha-MEM (Life Technolo and 48 hours after the Single inoculation. gies), supplemented with 10% Fetal Bovine Serum (HyClone), 1x Glutamine (Life-Technologies)), in an atmo Example 8 sphere of 5% CO at 37° C. Prior to electroporation, cells 0419 Heparin Affinity Chromatography of VRPs were detached from the cell culture vessel using 0.05% trypsin-0.53 mM EDTA solution (Life Technologies). 0420 Generally, the majority of contaminating protein is Trypsin was neutralized with growth medium, and cells non-VEE protein from the conditioned media. Heparin were washed twice with cold Phosphate-Buffered Saline column capacity requirements for GMP manufacturing runs (PBS, BioWhittaker) and resuspended at a concentration of are therefore based on the Volume of conditioned media, 1.5x10" cells/ml. RNA Transcription, Electroporation and rather than the concentration of VRPs. Column parameters Virosome Harvest Plasmid DNA pVR-GFP (green fluores are optimized at room temperature, but variations in tem cent protein) was linearized using restriction endonuclease perature do not greatly affect performance. The expected NotI (New England Biolabs) as recommended by the manu yields of VRPs can range from 50% to >90%. facturer. DNA was extracted with phenol:chloroform:iso 0421) While only minimal leaching of heparin from the amyl alcohol (25:24:1, Gibco BRL) and precipitated with columns has been detected, GMP requirements stipulate that ethanol, following the addition of NHAc to 2.5 M final a residual heparin assay be performed as an IPC test fol concentration. RNA was Synthesized in an in vitro transcrip lowing the chromatography Step. tion reaction using an Message mMachine(E) kit (Ambion) as recommended by the manufacturer. This RNA, without 0422 A. Pharmacia HiTrap(R) Heparin further purification, was used to transfect BHK cells. Helper 0423 Five mL columns of Pharmacia HiTrap(R) Heparin RNA was prepared in a similar fashion. A BHK cell sus (cat no. 17-0407-01, Amersham Pharmacia Biotech), pre pension in PBS (0.8 ML, 1.2x107 cells) was mixed with 10 equilibrated with 25 mM HEPES/0.25 M NaCl, pH 7.5, tug of each RNA, and the mixture was electroporated. were loaded with HIV-Gag-VRPS produced in Vero cells. Electroporation settings for Gene-Pulser(R) (Bio-Rad Labo After column washing with the equilibration buffer, VRPs ratories) were: 850 V, 25 uF, 3 pulses. Culture Supernatant were eluted with a 15 column volume gradient from 0.25-1.0 was collected at 18-24 hr post-electroporation and clarified M. NaCl gradient in 25 mM HEPES, pH 7.5. The HIV-Gag by centrifugation for 10 min at 1000 rpm. VRPs eluted at a conductivity of approximately 48 mS/cm. The wash step was optimized (based on the Aso peak) at a 0429 Titration of Virosomes NaCl concentration between 0.25 M and 0.3 M. 0430. The presence of infectious virosome particles was demonstrated using an immunofluorescence assay to titer the 0424 B. Heparin Sepharose 6 Fast Flow(R) Resin virosomes by detecting the fluorescence of the GFP encoded 0425 Heparin Sepharose 6 Fast Flow(R) resin (catalog no. by the replicon RNA in the virosomes. Serial dilutions of the 90-1000-2; Amersham Pharmacia Biotech) is supplied as a cell culture Supernatant were added to 12-well plates of bulk resin which allows various size columns to be packed BHK cells. Following an 18-24 hour incubation in an as needed. Fast Flow(R) resins have the advantages of excel atmosphere of 5% CO at 37 C., the medium was removed lent flow characteristics and ability to be sanitized with from each plate. ViroSome infectious titer was then deter Sodium hydroxide Solutions, which are particularly useful in mined by counting the number of green-fluorescent Single a GMP manufacturing process. A6 mL column was prepared cells at a particular dilution, followed by a back-calculation by packing the Heparin Sepharose 6 Fast Flow(R) bulk resin to determine total infectious units (i.u.) per mL. A final titer in a BioPad(R) Econo-Column chromatography column, of 440 i.u./mL was collected. which was then pre-equilibrated with 25 mM HEPES/0.12 0431 Confirmation of Virosome Identity M. NaCl, pH 7.5. VRPs were loaded onto the column, which was then washed with the equilibration buffer. Initial experi 0432 Three independent experimental methods were ments indicated that the VRPs eluted at a lower conductivity used to determine that the infectious particles were in fact (36 mS/cm) with this resin as compared to the HiTrap(R) ViroSomes, rather than replication competent Viral particles Heparin, So the wash conditions were modified accordingly. or naked RNA being carried over from the electroporated The VRPS were eluted from the Fast Flow(E) resin with a 15 cells. column volume gradient from 0.12 M to 1M NaCl in 25 mM 0433) i) The virosome-containing supernatant was HEPES, pH 7.5. passaged a Second time by removing the cell Super natant from the 12-well plate used for titration and Example 9 placing this Supernatant onto a fresh monolayer of BHK cells. At 18-24 hours post-passage, the mono ViroSome Formation layer was examined under U/V fluorescence and 0426. The feasibility of virosome formation is demon found to contain 0 (zero) GFP-positive cells, indi Strated in a Series of experiments in which replicon RNA and cating the infectious particles produced using this RNA encoding the glycoprotein E1 and E2 genes (glyco method can undergo only a single round of replica protein helper) were first transfected into BHK cells by tion, a critical characteristic of a ViroSome. electroporation. After 18-24 hours, cell Supernatants were 0434) ii) To establish that the infectious titer harvested and tested for the presence of ViroSomeS as detected following ViroSome packaging was not due described briefly below. to carry-over of RNA used in the electroporation, the US 2002/014 1975 A1 Oct. 3, 2002 28

Supernatant was treated with RNase A (Invitrogen) at 0442. Subjects: Healthy adult volunteers without a a concentration of 100 lug/mL for 15 minutes at 37 history of identifiable high-risk behavior for HIV-1 C. The treated and untreated control Supernatants infection as determined by a comprehensive Screening were titered according to the methods outlined questionnaire. above. The RNase-treated sample contained 400 i.u./mL and the control group had 440 i.u./mL, 0443) No. Subjects: 40 indicating that the RNASe treatment had no signifi 0444 Route: Subcutaneous injection cant effect on ViroSome titer. 0445 Scheme: The volunteers are arranged in four 0435 iii) To establish that the infectious particles groups, ten Subjects per group. In each group, two were enveloped in the E1 and E2 glycoproteins, Subjects receive a placebo, while the other eight Sub anti-VEE mouse serum was used to treat the cell jects receive either 10, 10, 107, or 10 i.u. of HIV Supernatant in a neutralization assay. As a control, Gag-VRPs. Subjects are vaccinated on day 0, day 30, normal mouse Serum was used to treat the ViroSome and day 120. Supernatant. In addition, VEE replicon particles 0446 Estimated Duration: Forty weeks expressing GFP were used in the assay, the infectiv ity of which is known to be inhibited by this serum. 0447 A. Selection of Subjects 0448 Subjects are healthy HIV-1 seronegative adults who fully comprehend the purpose and details of the Study Particle Titer (i.u./mL) as described in the informed consent. Subjects whom either Anti-VEE Normal Mouse themselves or whose Sexual partners have identifiable higher Sel Serum No serum risk behavior for HIV-1 infection are not eligible. Higher Virosome Supernatant 2O 440 530 risk behavior is determined by a prescreen Series of ques VRP-GFP O 530 890 tions designed to identify risk factors for HIV-1 infection. An assessment of absolute exclusion criteria using the Self-administered and interview questions is conducted. 0436 The infectivity of the virosomes was inhibited Subsequently, investigators proceed with phlebotomy, his similar to that of VRP-GFP, indicating that the virosome tory and physical examination, and final questions regarding particles were enveloped by the E1 and E2 glycoproteins. Sexual behavior and other practices. Eligibility determina tions for the trial depend on results of laboratory tests and 0437. These examples clearly demonstrate the ability to answers to these Self-administered and interview questions. produce infectious viroSome particles comprising replicon RNA enveloped with only the alphavirus E1 and E2 glyco 0449 The criteria used to define low risk behavior are as proteins. Testing confirmed that these viroSomes are infec follows: tious agents, but that they undergo only a Single round of 04.50 Either All of the Following: replication, as indicated by the inability to passage the agent. In addition, the agents contained the E1 and E2 glycopro 0451 1. No newly acquired higher risk associated STD teins, as evidenced by the ability to block infection with only in the last Six months VEE specific serum. Finally, the infectious RNA is protected 0452 2. No possibly safe or unsafe sex with a known from RNase enzymatic digestion, indicating an enveloped HIV+individual or an active injection drug user in the particle. past Six months 0438. The natural lipid content in BHK cells is primarily 0453 3. No unsafe sexual activity non-cationic. ViroSomes made in a completely cell free System can be made by using one or more non-cationic 0454 4. Possiblvy Safe sexual activitvy with two or fewer lipids, Such as lecithin (phosphatidycholine). partners within the last six months 0455 5. No injection drug use Example 10 0456 Or Both of the Following: Phase I Clinical Protocol 0457 1. Mutually monogamous relationship with a known or presumed HIV seronegative partner for the 0439) Phase I Safety and Immunogenicity Trial of an HIV last Six months Subtype C Gag-VEE Replicon Particle Vaccine in HIV-1 Seronegative Human Subjects 0458 2. No injection drug use 0440 A Phase I trial is conducted to evaluate the safety 0459 A.1 Inclusion Criteria and immunogenicity of the HIV Gag-VRP prototype vaccine 0460 Age: 18-60 component in healthy Seronegative adult Volunteers. The doses are Selected based on preclinical Studies in rodents and 0461 Sex: Male or Female For females, negative nonhuman primates. The Schedule mimics previous preclini pregnancy test at time of entry and assurance that cal efficacy studies with the SIV model that demonstrated adequate birth control measures will be used for one the capacity of SIV-VRP to induce SIV specific neutralizing month prior to immunization and the duration of the antibodies and CTL. Study 0441) PurprooSe: TO eValuate1 theh candidatedid Vaccine COm 0462 Normal history and physical examination ponent in an open-labeled, placebo-controlled Study. 0463 Lower risk sexual behavior as defined above. US 2002/014 1975 A1 Oct. 3, 2002 29

0464) Normal complete blood count and differential 0490 Active syphilis NOTE: If the serology is defined as: documented to be a false positive or due to a remote (>Six months) treated infection, the Volunteer is 0465 Hematocrit 34% for women; 38% for men eligible 0466 White count 3500 cells/mm with normal differ 0491) Active tuberculosis NOTE: Volunteers with a ential positive PPD and a normal chest X-ray showing no 0467 Total lymphocyte count 800 cells/mm evidence of TB and not requiring INH therapy are eligible. 0468 Absolute CD4 count 400 cells/mm 0492 History of anaphylaxis or other serious 0469 Platelets (150,000-550,000) adverse reactions to vaccines 0470 Normal ALT (~1.5x institutional upper normal 0493 Prior receipt of HIV vaccines or a placebo limit) and creatinine recipient in an HIV vaccine trial 0471 (1.6 mg/dl) 0494 Pregnant or lactating women 0472. Normal urine dipstick with esterase and nitrite 0495 B. Safety and Immunogenicity Monitoring 0473] Negative for hepatitis B surface antigen 0496 Safety is evaluated by monitoring volunteers for 0474 Negative ELISA for HIV within eight weeks of adverse reactions during the course of the trial. Volunteers immunization are followed for a total of 26 weeks post-final inoculation. The main toxicity associated with the Subcutaneous injection 0475 Availability for follow-up for planned duration in this study is that associated with Subcutaneous injection of of the study (68 weeks) any immunogen, i.e., pain, redness and Swelling at the injection Site, as well as the possibility of fever, chills, aches 0476 Aviable EBV transformed autologous B cell line and pains and perhaps fatigue. 0477 A.2 Exclusion Criteria 0497 Safety monitoring includes periodic review of data 0478. History of immunodeficiency, chronic illness, from the trial with particular emphasis on monitoring for malignancy, autoimmune disease, or use of immu adverse reactions including the following evaluations: noSuppressive medications 0498 Hematologic: CBC, differential, platelets 0479 Medical or psychiatric condition or occupa 0499 Hepatic/renal: ALT, creatinine, urinalysis tional responsibilities which preclude Subject com pliance with the protocol 0500 Neurologic: headache, paralysis, anxiety, con fusion, weakness, tremors. 0480 Subjects with identifiable higher risk behavior for HIV infection as determined by Screening ques 0501 Systemic symptoms: fever, gastrointestinal tionnaire designed to identify risk factors for HIV complaints, myalgia, malaise, fatigue, headache, infection; Specific exclusions include: anaphylaxis, immune complex disease, and other hyperSensitivity reactions 0481. History of injection drug use within the last 12 months prior to enrollment. 0502. Local toxicity at the site of injection: e.g., pain, tenderness, erythema, regional lymphadenopa 0482 Higher risk sexual behavior defined as one or thy, limitation of limb movement more of the following behaviors: 0503) The immunogenicity monitoring includes the fol 0483 1. A newly acquired higher risk associated lowing immunological assays, all utilizing HIV Subtype C STD within the past six months based reagents: 0484 2. Possibly safe or unsafe sex with a known 0504 Humoral Responses: HIV+individual in the past six months 0505 HIV Subtype C Gag-specific ELISA 0485 3. Possibly safe sexual activity with twelve or more partners in the past Six months 0506 Anti-VEE ELISA 0486 4. Unsafe sexual activity with four or more 0507 Cellular Immune Responses: partners within the past Six months. 0508 Standard cell-killing assay (i.e., chromium 0487 Live attenuated vaccines within 60 days of release) to measure CD8+Gag-specific CTL activity study NOTE: Medically indicated subunit or killed Vaccines (e.g., influenza, pneumococcal) are not 0509 ELISPOT assay to measure IFN-? exclusionary, but should be given at least two weeks 0510 Mucosal Immune Responses: away from test article immunizations. 0511 Standardized assay for assessment of Gag-spe 0488 Use of experimental agents within 30 days cific IgA prior to Study 0512 Throughout this application, various publications 0489 Receipt of blood products or immunoglobulin are referenced. The disclosures of these publications in their in the past Six months entireties are hereby incorporated by reference into this US 2002/014 1975 A1 Oct. 3, 2002 30 application in order to more fully describe the State of the art ciency virus with Venezuelan equine encephalitis virus to which this invention pertains. replicon particles. J. Virol. 74:371-378 0524) Grieder, F. B., N. L. Davis, J. F. Aronson, P. C. REFERENCES Charles, D. C. Sellon, K. Suzuki and R. E. Johnston. 0513 Barany F. 1985. Single-stranded hexameric link 1995. Specific restrictions in the progression of Ven ers: a System for in-phase insertion mutagenesis and eZuelan equine encephalitis virus induced disease protein engineering. Gene 37(1-3):111-23. resulting from Single amino acid changes in the glyco proteins. Virology 206:994-1006. 0514 Betts, M. R., J. Krowka, C. Santamaria, K. Balsamo, F. Gao, G. Mulundu, C. Luo, N. N'Gandu, H. 0525 Hevey, M., D. Negley, P. Pushko, J. Smith and A. Sheppard, B. H. Hahn, S. Allen and J. A. Frelinger. Schmaljohn. 1998. Marburg virus vaccines based upon 1997. Cross-clade human immunodeficiency virus alphavirus replicons protect guinea pigs and nonhuman (HIV)-specific cytotoxic T-lymphocyte responses in primates. Virology 251:28-37. HIV-infected Zambians. J. Virol. 71:8908-8911. 0526 Hirsch, V., T. R. Fuerst, G. Sutter, M. W. Carroll, L. C. Yang, S. Goldstein et al. 1996. Patterns of viral 0515 Caley, I.J., M. R. Betts, D. M. Irlbeck, N. L. replication correlate with outcome in SIV-infected Davis, R. Swanstrom, J. A. Frelinger and R. E. macaques: effect of prior immunization with a trivalent Johnston. 1997. Humoral, mucosal and cellular immu SIV vaccine in modified vaccinia virus Ankara. J Virol. nity in response to an HIV-1 vaccine candidate. J Virol. 70:3741-3752. 71:3031-3O38. 0527 Johnston, Robert E. and Jonathan F. Smith. 0516 Davis et al. 1980. In: Microbiology, 3d ed., p. 1988. Selection for accelerated penetration in cell cul 132. ture co-Selects for attenuated mutants of Venezuelan equine encephalitis virus. Virology 162:437-443. 0517 Davis, N. L., L. V. Willis, J. F. Smith and R. E. Johnston. 1989. In vitro synthesis of infectious Ven 0528 Johnston, R. E. and C.J. Peters. 1996. Alphavi ezuelan equine encephalitis virus RNA from a cDNA ruses. In: Virology, Third Edition, B. N. Fields, D. M. clone: Analysis of a viable deletion mutant. Virology Knipe and P. M. Howley, eds., Raven Press, New York. 171189-204. pp. 843-898. 0518) Davis, N. L., L. V. Willis, J. F. Smith, G. 0529) Kinney, R. M., B.J. B. Johnson, J. B. Welch, K. Greenwald and R. E. Johnston. 1990. In vitro synthesis R. Tsuchiya and D. W. Trent. 1989. The full-length of infectious VEE virus RNA from a cDNA clone: nucleotide Sequences of the virulent Trinidad donkey Analysis of a viable deletion mutant and mutations Strain of Venezuelan equine encephalitis virus and its affecting virulence. In: Vaccines 90, Cold Spring Har attenuated vaccine derivative, strain TC-83. Virology bor Press, Cold Spring Harbor, N.Y. pp. 109-113. 170:19-30. 0519) Davis, N. L., N. Powell, G. F. Greenwald, L. V. 0530 Kinney, R. M., G-J. Chang, K. R. Tsuchiya, J. M. Willis, B. J. Johnson, J. F. Smith and R. E. Johnston. Sneider, J. T. Roehrig, T. M. Woodward and D. W. 1991. Attenuating mutations in the E2 glycoprotein Trent. 1993. Attenuation of Venezuelan equine gene of Venezuelan equine encephalitis virus: Con encephalitis virus strain TC-83 is encoded by the Struction of Single and multiple mutants in a full-length 5'-noncoding region and the E2 envelope glycoprotein. cDNA clone. Virology 183:20-31. J. Viro. 67: 1269-1277. 0520) Davis, N. L., K. W. Brown, G. F. Greenwald, A. 0531 Kunkel. 1985. Proc. Natl. Acad. Sci. USA J. Zajac, V. L. Zacny, J. F. Smith and R. E. Johnston. 82:488. 1995. Attenuated mutants of Venezuelan equine 0532 Paredes, A. M., D. T. Brown, R. Rothnagel, W. encephalitis virus containing lethal mutations in the Chiu, R. J. Schoepp, R. E. Johnston and B. V. Prasad. PE2 cleavage Signal combined with a Second-Site Sup 1993. Three-dimensional structure of a membrane pressor mutation in E1. Virology 212:102-110. containing virus. Proc. Natl. Acad. Sci., USA90:9095 0521. Davis, N. L., K. W. Brown and R. E. Johnston. 9099. 1996a. A viral vaccine vector that expresses foreign 0533) Pushko, P., M. Parker, G. V. Ludwig, N. L. genes in lymph nodes and protects against mucosal Davis, R. E. Johnston and J. F. Smith. 1997. Replicon challenge. J. Virol. 70:3781-3787. helper Systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in 0522 Davis, N. L., P. Pushko, K. W. Brown, P. C. Charles, I.J. Caley, M. Parker, G. Ludwig, J. F. Smith Vitro and immunization against heterologous pathogens and R. E. Johnston. 1996b. Immunization against influ in vivo. Virology 239:389-401. enza with attenuated Venezuelan equine encephalitis 0534 Rosenberg, A. H., et al 1987. Vectors for selec virus vectors. In: Options for the Control of Influenza tive expression of cloned DNAS by T7 RNA poly III, L. E. Brown and A. W. Hampson, eds. Elsevier, Amsterdam. pp.803-809. merase. Gene. 56(1): p. 125-35. 0535 Schlesinger, S. and M. J. Schlesinger. 1990. 0523 Davis, N. L., I. J. Caley, K. W. Brown, M. R. Replication of Togaviridae and Flaviviridae. In: Virol Betts, D. L. Irlbeck, K. M. McGrath, M. J. Connell, D. C. Montefiori, J. A. Frelinger, R. Swanstrom, P. R. ogy, Fields, B. N. and Knipe, D. M. (eds.) Raven Press. Johnson and R. E. Johnston. 2000. Vaccination of pp. 697-711. macaques against pathogenic Simian immunodefi 0536 Strauss et al. 1990. Seminars in Virology 1:347. US 2002/014 1975 A1 Oct. 3, 2002 31

0537) Strauss, J. H. and E. G. Strauss. 1994. The 0538 Studier, F. W., et al. 1990. Use of T7 RNA alphaviruses: Gene expression, replication, and evolu- polymerase to direct expression of cloned genes. 1990. tion. Micro. Rey: 58:491-562. Methods Enzymol. 185:60-89.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 19 <210> SEQ ID NO 1 &2 11s LENGTH 12523 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &22O > FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 1 atggg.cggcg catgaga gaa gC cca gacca attacct acc caaaatggag aaagttcacg 60 ttgacatcga ggaag acago coatticcitca gagctttgca gcggagcttic cogcagtttg 120 aggtagaagc caagcagg to actdataatg accatgctaa toccagagcg ttitt.cgcatc 18O tggcttcaaa acto atc gala acggaggtogg acco atc.cga cacgatccitt gacattggaa 240 gtgc.gc.ccgc cc.gcagaatg tattotaagc acaagtatca ttgttatctgt cogatgagat 3OO gtgcggaaga to cqgacaga ttgtataagt atgcaactaa gCtgaagaaa aact gtaagg 360 aaataactga taaggaattig gacaagaaaa togaaggagct cqcc.gc.cgtc atgagc gacc 420 citgacctgga aacto agact atgtgccitcc acgacgacga gtcgtgtc.gc tacgaagggc 480 aagtc.gctgt ttaccaggat gtatacgcgg ttgacgg acc gacaagttct c tatcaccalag 540 ccaataaggg agittagagtic goctactgga taggctttga caccacccct tittatottta 600 agaacttggc to gag catat coatcatact citaccaact g g gcc.gacgaa accgtgttaa 660 cggctcqtaa cataggccta to cagotctg acgittatgga gC gg to acgt agagggatgt 720 ccattcttag aaagaagitat ttgaaac cat coaacaatgttctattotct gttggctoga 78O ccatctacca cq agaagagg gacittactga ggagctggca cotgcc gitot gtattt cact 840 tacgtggcaa gCaaaattac acatgtcggt gtgagacitat agittagttgc gacgggtacg 9 OO togttaaaag aatagot atc agtcCaggcc totatoggala gC ctitc aggc tatgctgcta 96.O cgatgcaccg cq agggattic ttgttgctgca aagtgacaga cacattaaac ggggagaggg O20 totcitttitcc cgtgtgcacg tatgtgc.cag ctacattgttg to accalaatg actogcatac O8O tggcaacaga totcagtgcg gacgacgc.gc aaaaactgct ggttgggctic aaccagogta 1 4 0 tag togtoaa cqgtogcacc cagagaaa.ca coaataccat gaaaaattac cittittgcc.cg 200 tagtggcc.ca gg catttgct aggtgggcaa aggaatataa goaagatcaa galagatgaaa 260 ggccactagg actacgagat agacagttag to atggggtg ttgttgggct tittagaaggc 320 acaagataac atctattitat aag.cgc.ccgg at accolaaac catcatcaaa gtgaac agcg 38O attitccactic attcgtgct g cc caggatag gCagtaacac attggagatc gggctdagaa 4 40

caagaatcag gaaaatgtta gaggagcaca aggagcc.gtc. acct ct catt accgcc gagg 5 OO acgtacaaga agctaagtgc goagc.cgatg aggctaagga ggtgcgtgaa gC cqaggagt 560

tgcgc.gcago to taccacct ttggcagot g atgttgagga gcc.cactctg. galagcc gatg 62O

togacttgat gttacaa gag gCtgggg.ccg gCtcagtgga gacaccitcgt ggcttgataa 680

US 2002/014 1975 A1 Oct. 3, 2002 40

-continued Tyr Lys Val Asin Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Val 930 935 940 aac gtc. cita citg acc cqc acg gag gac cqc atc gtg togg aaa aca cita 2880 Asn Val Lieu Lieu. Thr Arg Thr Glu Asp Arg Ile Val Trp Llys Thr Lieu 945 950 955 96.O gcc ggc gac coa togg ata aaa aca citg act gcc aag tac cot ggg aat 292.8 Ala Gly Asp Pro Trp Ile Lys Thr Lieu. Thir Ala Lys Tyr Pro Gly Asn 965 970 975 titc act gcc acg ata gag gag togg caa gCa gag cat gat goc atc atg 2976 Phe Thr Ala Thr Ile Glu Glu Trp Glin Ala Glu His Asp Ala Ile Met 98O 985 99 O agg cac atc ttg gag aga ccg gac cot acc gac gtc titc cag aat aag 3024 Arg His Ile Leu Glu Arg Pro Asp Pro Thr Asp Val Phe Glin Asn Lys 995 10 OO 1005 gca aac gtg tot togg gcc aag got tta gtg cc.g. gtg citg aag acc got 3 OF2 Ala Asn Val Cys Trp Ala Lys Ala Lieu Val Pro Val Lieu Lys Thr Ala 1010 1015 1020 ggc at a gac at g acc act gaa caa tog aac act gtg gat titt gala 312 O Gly Ile Asp Met Thr Thr Glu Gln Trp Asn Thr Val Asp Tyr Phe Glu 1025 1030 1035 1040 acg gac aaa gCt cac to a gca gag at a gta ttgaac caa cita toc gtg 31.68 Thr Asp Lys Ala His Ser Ala Glu Ile Val Lieu. Asn Glin Lieu. Cys Wal 1045 105 O 1055 agg ttc titt gga citc gat citg gac toc ggit cta ttt tot goa coc act 3216 Arg Phe Phe Gly Lieu. Asp Leu Asp Ser Gly Lieu Phe Ser Ala Pro Thr 1060 1065 1070 gtt cog tta toc att agg aat aat cac togg gat aac toc cog tog cct 3264 Val Pro Leu Ser Ile Arg Asn. Asn His Trp Asp Asn. Ser Pro Ser Pro 1075 1080 1085 aac atg tac ggg citg aat aaa gala gtg gtc. c.gt cag citc. tct cqc agg 3312 Asn Met Tyr Gly Lieu. Asn Lys Glu Val Val Arg Glin Leu Ser Arg Arg 1090 1095 1100 tac coa caa citg cct c gg gca gtt gcc act gga aga gito tat gac atg 3360 Tyr Pro Gln Leu Pro Arg Ala Val Ala Thr Gly Arg Val Tyr Asp Met 1105 1110 1115 1120 aac act ggt aca citg cqc aat tat gat cog cqc ata aac cta gta cot 34.08 Asn Thr Gly Thr Lieu Arg Asn Tyr Asp Pro Arg Ile Asn Lieu Val Pro 1125 1130 1135 gta aac aga aga citg cct cat gct tta gtc. citc. cac cat aat gaa cac 345 6 Val Asn Arg Arg Lieu Pro His Ala Lieu Val Lieu. His His Asn. Glu His 1140 1145 1150 cca cag agt gac titt tot to a titc gtc agc aaa titg aag ggc aga act 3504 Pro Glin Ser Asp Phe Ser Ser Phe Val Ser Lys Leu Lys Gly Arg Thr 1155 1160 1165 gto: ct g g to gtc. g g g gala aag ttg to c gtc. cca ggc aaa at g gtt gac 3552 Val Leu Val Val Gly Glu Lys Leu Ser Val Pro Gly Lys Met Val Asp 1170 1175 118O tgg ttg to a gac cqg cct gag gCt acc titc aga gct c gg ct g g at tta 3600 Trp Leu Ser Asp Arg Pro Glu Ala Thr Phe Arg Ala Arg Lieu. Asp Lieu 1185 11.90 11.95 1200 ggc atc cca ggit gat gtg ccc aaa tat gac ata ata titt gtt aat gtg 3648 Gly Ile Pro Gly Asp Val Pro Llys Tyr Asp Ile Ile Phe Val Asin Val 1205 1210 1215 agg acc cca tat aaa tac cat cac tat cag cag togt gaa gac cat gcc 36.96 Arg Thr Pro Tyr Lys Tyr His His Tyr Glin Gln Cys Glu Asp His Ala 1220 1225 1230 att aag citt agc atg titg acc aag aaa gCt tot citg cat citg aat coc 3744 US 2002/014 1975 A1 Oct. 3, 2002 41

-continued Ile Lys Lieu Ser Met Lieu. Thir Lys Lys Ala Cys Lieu. His Lieu. Asn Pro 1235 1240 1245 ggc gga acc tot gtc. agc ata ggit tat ggit tac got gac agg gcc agc 3792 Gly Gly. Thir Cys Val Ser Ile Gly Tyr Gly Tyr Ala Asp Arg Ala Ser 1250 1255 1260 gala agc atc att got gct ata gC g c gq cag titc aag titt toc cqg gta 384 O Glu Ser Ile Ile Gly Ala Ile Ala Arg Glin Phe Lys Phe Ser Arg Val 1265 1270 1275 1280 tgc aaa cc g aaa toc to a citt gala gag acg gaa gtt citg titt gta titc 3888 Cys Llys Pro Llys Ser Ser Leu Glu Glu Thr Glu Val Leu Phe Val Phe 1285 1290 1295 att ggg tac gat cqc aag gCd cqt acg cac aat cot tac aag citt to a 3936 Ile Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro Tyr Lys Lieu Ser 1300 1305 1310 toa acc titg acc aac att tat aca ggit to c aga citc. cac gala gCC gga 3984 Ser Thr Leu Thr Asn Ile Tyr Thr Gly Ser Arg Leu. His Glu Ala Gly 1315 1320 1325 tgt gca coc to a tat cat gtg gtg cqa ggg gat att gcc acg gCC acc 4032 Cys Ala Pro Ser Tyr His Val Val Arg Gly Asp Ile Ala Thr Ala Thr 1330 1335 1340 gala gga gtg attata aat gct gct aac agc aaa goa caa cot ggc gga 408 O Glu Gly Val Ile Ile Asn Ala Ala Asn. Ser Lys Gly Glin Pro Gly Gly 1345 1350 1355 1360 ggg gtg togc gga gcg citg tat aag aag titc cc.g. gala agc titc gat tta 4128 Gly Val Cys Gly Ala Leu Tyr Lys Llys Phe Pro Glu Ser Phe Asp Lieu 1365 1370 1375 cag cog atc gala gta gga aaa gC g c ga ct g g to aaa gqt gca gct aaa. 416 Glin Pro Ile Glu Val Gly Lys Ala Arg Lieu Val Lys Gly Ala Ala Lys 1380 1385 1390 cat atc att cat gcc gta gga cca aac titc aac aaa gtt tog gag gtt 4224 His Ile Ile His Ala Val Gly Pro Asn Phe Asn Lys Val Ser Glu Val 1395 14 OO 1405 gala ggit gac aaa cag titg gca gag gCt tat gag toc atc got aag att 4272 Glu Gly Asp Lys Glin Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile 1410 1415 1420 gto: aac gat aac aat tac aag to a gta gC g att coa citg ttg to c acc 4320 Val Asn Asp Asn. Asn Tyr Lys Ser Val Ala Ile Pro Leu Lleu Ser Thr 1425 1430 1435 1440 ggc atc titt toc gog aac aaa gat cq a cita acc caa to a ttgaac cat 4368 Gly Ile Phe Ser Gly Asn Lys Asp Arg Lieu. Thr Glin Ser Lieu. Asn His 1445 1450 1455 ttg citg aca gCt tta gac acc act gat gca gat gta gcc at a tac toc 4 416 Leu Lieu. Thir Ala Lieu. Asp Thir Thr Asp Ala Asp Wall Ala Ile Tyr Cys 1460 1465 1470 agg gac aag aaa togg gaa atg act citc aag gaa goa gtg gCt agg aga 4 464 Arg Asp Llys Lys Trp Glu Met Thr Lieu Lys Glu Ala Wall Ala Arg Arg 1475 1480 1485 gala gCa gtg gag gag ata toc at a to c gac gac tot to a gtg aca gaa 4512 Glu Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr Glu 1490 1495 15 OO cct gat gca gag citg gtg agg gtg cat cog aag agt tot ttg gct gga 45 60 Pro Asp Ala Glu Lieu Val Arg Val His Pro Lys Ser Ser Lieu Ala Gly 1505 1510 1515 1520 agg aag ggc tac agc aca agc gat ggc aaa act titc. tca tat ttg gala 4608 Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser Tyr Leu Glu 1525 1530 1535 ggg acc aag titt cac cag gCd gcc aag gat ata gca gaa att aat gcc 465.6 US 2002/014 1975 A1 Oct. 3, 2002 42

-continued Gly Thr Lys Phe His Glin Ala Ala Lys Asp Ile Ala Glu Ile Asn Ala 1540 1545 1550 atg togg ccc gtt gca acg gag gCC aat gag cag gta toc atg tat atc 4704 Met Trp Pro Val Ala Thr Glu Ala Asn Glu Glin Val Cys Met Tyr Ile 1555 15 60 1565 citc gga gaa agc atg agc agt att agg tog aaa toc ccc gtc. gala gag 4752 Leu Gly Glu Ser Met Ser Ser Ile Arg Ser Lys Cys Pro Val Glu Glu 1570 1575 1580 tog gala gC c toc aca cca cct agc acg citg cct togc titg toc atc cat 4800 Ser Glu Ala Ser Thr Pro Pro Ser Thr Leu Pro Cys Leu Cys Ile His 1585 159 O 1595 1600 gcc at g act coa gaa aga gta cag cqc cta aaa goc to a cqt coa gaa 4848 Ala Met Thr Pro Glu Arg Val Glin Arg Lieu Lys Ala Ser Arg Pro Glu 1605 1610 1615 caa att act gtg tac toa toc titt coa ttg cog aag tat aga atc act 4896 Glin Ile Thr Val Cys Ser Ser Phe Pro Leu Pro Lys Tyr Arg Ile Thr 1620 1625 1630 ggit gtg cag aag atc caa toc tocc cag cot ata ttg titc. tca cog aaa. 494 4 Gly Val Glin Lys Ile Glin Cys Ser Glin Pro Ile Leu Phe Ser Pro Lys 1635 1640 1645 gtg cct gcg tat att cat coa agg aag tat citc gtg gaa aca coa cog 4992 Val Pro Ala Tyr Ile His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro 1650 1655 1660 gta gac gag act cog gag cca to g gca gag aac caa toc aca gag ggg 5040 Val Asp Glu Thr Pro Glu Pro Ser Ala Glu Asn Gln Ser Thr Glu Gly 1665 1670 1675 1680 aca cot gaa caa cca cca citt ata acc gag gat gag acc agg act aga 5088 Thr Pro Glu Gln Pro Pro Leu Ile Thr Glu Asp Glu Thr Arg Thr Arg 1685 1690 1695 acg cct gag cog atc atc atc gala gag gala gaa gag gat agc ata agt 51.36 Thr Pro Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser 17 OO 1705 1710 ttg citg to a gat ggc ccg acc cac cag gtg citg caa gito gag gCa gac 51.84 Leu Lleu Ser Asp Gly Pro Thr His Glin Val Lieu Glin Val Glu Ala Asp 1715 1720 1725 att cac ggg ccg ccc tot gta tot agc to a toc tagg toc att cot cat 5232 Ile His Gly Pro Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro His 1730 1735 1740 gca to c gac titt gat gtg gac agt tta to cata citt gac acc citg gag 528 O Ala Ser Asp Phe Asp Wall Asp Ser Lieu Ser Ile Leu Asp Thr Lieu Glu 1745 175 O 1755 1760 gga gCt agc gtg acc agc ggg gca acg to a gCo gag act aac tot tac 5328 Gly Ala Ser Val Thr Ser Gly Ala Thr Ser Ala Glu Thr Asn Ser Tyr 1765 1770 1775 titc gca aag agt atg gag titt citg gC g cqa cog gtg cct gcg cct cqa 5376 Phe Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Val Pro Ala Pro Arg 1780 1785 1790 aca gta titc agg aac cct coa cat coc got cog cqc aca aga aca cog 5 424 Thr Val Phe Arg Asn Pro Pro His Pro Ala Pro Arg Thr Arg Thr Pro 1795 1800 1805 to a citt go a coc agc agg gcc toc to g aga acc agc cta gtt to c acc 54.72 Ser Leu Ala Pro Ser Arg Ala Cys Ser Arg Thr Ser Leu Val Ser Thr 1810 1815 1820 cc.g. cca ggc gtgaat agg gtg atc act aga gag gag citc gag gC g citt 552O Pro Pro Gly Val Asn Arg Val Ile Thr Arg Glu Glu Lieu Glu Ala Lieu 1825 1830 1835 1840 acc cc g to a cqc act cot agc agg to g g to tcg aga acc agc ct g g to 5568

US 2002/014 1975 A1 Oct. 3, 2002 45

-continued Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val Gly. Thr 2450 2455 2460 to c atc at a gtt atg gcc atg act act cita gCt agc agt gtt aaa to a 440 Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val Lys Ser 2465 2470 24.75 titc agc tac citg aga ggg gcc cct ata act citc tac ggc 7479 Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly 24.80 2485 24.90

<210> SEQ ID NO 3 &2 11s LENGTH 24.92 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 3 Met Glu Lys Wal His Val Asp Ile Glu Glu Asp Ser Pro Phe Lieu Arg 1 5 10 15 Ala Leu Glin Arg Ser Phe Pro Glin Phe Glu Val Glu Ala Lys Glin Val 2O 25 30 Thr Asp Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala Ser 35 40 45 Lys Lieu. Ile Glu Thr Glu Val Asp Pro Ser Asp Thr Ile Leu Asp Ile 50 55 60 Gly Ser Ala Pro Ala Arg Arg Met Tyr Ser Lys His Lys Tyr His Cys 65 70 75 8O Ile Cys Pro Met Arg Cys Ala Glu Asp Pro Asp Arg Lieu. Tyr Lys Tyr 85 90 95 Ala Thr Lys Lieu Lys Lys Asn. Cys Lys Glu Ile Thr Asp Lys Glu Lieu 100 105 110 Asp Llys Lys Met Lys Glu Lieu Ala Ala Wal Met Ser Asp Pro Asp Lieu 115 120 125 Glu Thr Glu Thir Met Cys Lieu. His Asp Asp Glu Ser Cys Arg Tyr Glu 130 135 1 4 0 Gly Glin Val Ala Val Tyr Glin Asp Val Tyr Ala Val Asp Gly Pro Thr 145 15 O 155 160 Ser Leu Tyr His Glin Ala Asn Lys Gly Val Arg Val Ala Tyr Trp Ile 1.65 170 175 Gly Phe Asp Thr Thr Pro Phe Met Phe Lys Asn Leu Ala Gly Ala Tyr 18O 185 190 Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Thr Val Leu Thr Ala Arg 195 200 2O5 Asn. Ile Gly Lieu. Cys Ser Ser Asp Wal Met Glu Arg Ser Arg Arg Gly 210 215 220 Met Ser Ile Leu Arg Lys Lys Tyr Lieu Lys Pro Ser Asn. Asn. Wall Leu 225 230 235 240 Phe Ser Val Gly Ser Thr Ile Tyr His Glu Lys Arg Asp Leu Lieu Arg 245 250 255 Ser Trp His Leu Pro Ser Val Phe His Leu Arg Gly Lys Glin Asn Tyr 260 265 27 O Thr Cys Arg Cys Glu Thir Ile Val Ser Cys Asp Gly Tyr Val Val Lys 275 280 285 US 2002/014 1975 A1 Oct. 3, 2002 46

-continued Arg Ile Ala Ile Ser Pro Gly Lieu. Tyr Gly Lys Pro Ser Gly Tyr Ala 29 O 295 3OO Ala Thr Met His Arg Glu Gly Phe Leu Cys Cys Lys Val Thr Asp Thr 305 310 315 320 Leu Asin Gly Glu Arg Val Ser Phe Pro Val Cys Thr Tyr Val Pro Ala 325 330 335 Thr Leu Cys Asp Gln Met Thr Gly Ile Leu Ala Thr Asp Val Ser Ala 340 345 350 Asp Asp Ala Glin Lys Lieu Lleu Val Gly Lieu. Asn Glin Arg Ile Val Val 355 360 365 Asn Gly Arg Thr Glin Arg Asn. Thir Asn. Thir Met Lys Asn Tyr Lieu Lieu 370 375 38O Pro Val Val Ala Glin Ala Phe Ala Arg Trp Ala Lys Glu Tyr Lys Glu 385 390 395 400 Asp Glin Glu Asp Glu Arg Pro Leu Gly Lieu Arg Asp Arg Glin Lieu Val 405 410 415 Met Gly Cys Cys Trp Ala Phe Arg Arg His Lys Ile Thr Ser Ile Tyr 420 425 430 Lys Arg Pro Asp Thr Glin Thir Ile Ile Llys Val Asn. Ser Asp Phe His 435 4 40 4 45 Ser Phe Val Leu Pro Arg Ile Gly Ser Asn Thr Leu Glu Ile Gly Leu 450 455 460 Arg Thr Arg Ile Arg Lys Met Leu Glu Glu His Lys Glu Pro Ser Pro 465 470 475 480 Lieu. Ile Thr Ala Glu Asp Val Glin Glu Ala Lys Cys Ala Ala Asp Glu 485 490 495 Ala Lys Glu Val Arg Glu Ala Glu Glu Lieu Arg Ala Ala Lieu Pro Pro 5 OO 505 510 Leu Ala Ala Asp Val Glu Glu Pro Thr Lieu Glu Ala Asp Wall Asp Lieu 515 52O 525 Met Leu Glin Glu Ala Gly Ala Gly Ser Val Glu Thr Pro Arg Gly Lieu 530 535 540 Ile Llys Val Thir Ser Tyr Ala Gly Glu Asp Lys Ile Gly Ser Tyr Ala 545 550 555 560 Val Lieu Ser Pro Glin Ala Val Lieu Lys Ser Glu Lys Lieu Ser Cys Ile 565 570 575 His Pro Leu Ala Glu Glin Val Ile Val Ile Thr His Ser Gly Arg Lys 58O 585 59 O Gly Arg Tyr Ala Val Glu Pro Tyr His Gly Lys Val Val Val Pro Glu 595 600 605 Gly His Ala Ile Pro Val Glin Asp Phe Glin Ala Leu Ser Glu Ser Ala 610 615 62O Thir Ile Val Tyr Asn Glu Arg Glu Phe Val Asn Arg Tyr Leu. His His 625 630 635 640 Ile Ala Thr His Gly Gly Ala Lieu. Asn. Thir Asp Glu Glu Tyr Tyr Lys 645 650 655 Thr Val Lys Pro Ser Glu His Asp Gly Glu Tyr Lieu. Tyr Asp Ile Asp 660 665 670 Arg Lys Glin Cys Wall Lys Lys Glu Lieu Val Thr Gly Lieu Gly Lieu. Thr 675 680 685 Gly Glu Leu Val Asp Pro Pro Phe His Glu Phe Ala Tyr Glu Ser Leu US 2002/014 1975 A1 Oct. 3, 2002 47

-continued

69 O. 695 7 OO Arg Thr Arg Pro Ala Ala Pro Tyr Glin Val Pro Thr Ile Gly Val Tyr 705 710 715 720 Gly Val Pro Gly Ser Gly Lys Ser Gly Ile Ile Lys Ser Ala Val Thr 725 730 735 Lys Lys Asp Leu Val Val Ser Ala Lys Lys Glu Asn. Cys Ala Glu Ile 740 745 750 Ile Arg Asp Wall Lys Lys Met Lys Gly Lieu. Asp Wall Asn Ala Arg Thr 755 760 765 Val Asp Ser Val Lieu Lieu. Asn Gly Cys Lys His Pro Val Glu Thir Lieu 770 775 78O Tyr Ile Asp Glu Ala Phe Ala Cys His Ala Gly Thr Lieu Arg Ala Lieu 785 790 795 8OO Ile Ala Ile Ile Arg Pro Lys Lys Ala Val Lieu. Cys Gly Asp Pro Lys 805 810 815 Gln Cys Gly Phe Phe Asn Met Met Cys Leu Lys Val His Phe Asn His 820 825 830 Glu Ile Cys Thr Glin Val Phe His Lys Ser Ile Ser Arg Arg Cys Thr 835 840 845 Lys Ser Val Thr Ser Val Val Ser Thr Leu Phe Tyr Asp Llys Lys Met 85 O 855 860 Arg Thr Thr Asn Pro Lys Glu Thr Lys Ile Val Ile Asp Thr Thr Gly 865 870 875 88O Ser Thr Lys Pro Lys Glin Asp Asp Lieu. Ile Lieu. Thr Cys Phe Arg Gly 885 890 895 Trp Wall Lys Glin Leu Glin Ile Asp Tyr Lys Gly Asn. Glu Ile Met Thr 9 OO 905 910 Ala Ala Ala Ser Glin Gly Lieu. Thir Arg Lys Gly Val Tyr Ala Val Arg 915 920 925 Tyr Lys Val Asin Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Val 930 935 940 Asn Val Lieu Lieu. Thr Arg Thr Glu Asp Arg Ile Val Trp Llys Thr Lieu 945 950 955 96.O Ala Gly Asp Pro Trp Ile Lys Thr Lieu. Thir Ala Lys Tyr Pro Gly Asn 965 970 975 Phe Thr Ala Thr Ile Glu Glu Trp Glin Ala Glu His Asp Ala Ile Met 98O 985 99 O Arg His Ile Leu Glu Arg Pro Asp Pro Thr Asp Val Phe Glin Asn Lys 995 10 OO 1005 Ala Asn Val Cys Trp Ala Lys Ala Lieu Val Pro Val Lieu Lys Thr Ala 1010 1015 1020 Gly Ile Asp Met Thr Thr Glu Gln Trp Asn Thr Val Asp Tyr Phe Glu 1025 1030 1035 1040 Thr Asp Lys Ala His Ser Ala Glu Ile Val Lieu. Asn Glin Lieu. Cys Wal 1045 105 O 1055 Arg Phe Phe Gly Lieu. Asp Leu Asp Ser Gly Lieu Phe Ser Ala Pro Thr 1060 1065 1070 Val Pro Leu Ser Ile Arg Asn. Asn His Trp Asp Asn. Ser Pro Ser Pro 1075 1080 1085 Asn Met Tyr Gly Lieu. Asn Lys Glu Val Val Arg Glin Leu Ser Arg Arg 1090 1095 1100 US 2002/014 1975 A1 Oct. 3, 2002 48

-continued

Tyr Pro Gln Leu Pro Arg Ala Val Ala Thr Gly Arg Val Tyr Asp Met 1105 1110 1115 1120 Asn Thr Gly Thr Lieu Arg Asn Tyr Asp Pro Arg Ile Asn Lieu Val Pro 1125 1130 1135 Val Asn Arg Arg Lieu Pro His Ala Lieu Val Lieu. His His Asn. Glu His 1140 1145 1150 Pro Glin Ser Asp Phe Ser Ser Phe Val Ser Lys Leu Lys Gly Arg Thr 1155 1160 1165 Val Leu Val Val Gly Glu Lys Leu Ser Val Pro Gly Lys Met Val Asp 1170 1175 118O Trp Leu Ser Asp Arg Pro Glu Ala Thr Phe Arg Ala Arg Lieu. Asp Lieu 1185 11.90 11.95 1200 Gly Ile Pro Gly Asp Val Pro Llys Tyr Asp Ile Ile Phe Val Asin Val 1205 1210 1215 Arg Thr Pro Tyr Lys Tyr His His Tyr Glin Gln Cys Glu Asp His Ala 1220 1225 1230 Ile Lys Lieu Ser Met Lieu. Thir Lys Lys Ala Cys Lieu. His Lieu. Asn Pro 1235 1240 1245 Gly Gly. Thir Cys Val Ser Ile Gly Tyr Gly Tyr Ala Asp Arg Ala Ser 1250 1255 1260 Glu Ser Ile Ile Gly Ala Ile Ala Arg Glin Phe Lys Phe Ser Arg Val 1265 1270 1275 1280 Cys Lys Pro Lys Ser Ser Leu Glu Glu Thr Glu Val Leu Phe Val Phe 1285 1290 1295 Ile Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro Tyr Lys Lieu Ser 1300 1305 1310 Ser Thr Leu Thr Asn Ile Tyr Thr Gly Ser Arg Leu. His Glu Ala Gly 1315 1320 1325 Cys Ala Pro Ser Tyr His Val Val Arg Gly Asp Ile Ala Thr Ala Thr 1330 1335 1340 Glu Gly Val Ile Ile Asn Ala Ala Asn. Ser Lys Gly Glin Pro Gly Gly 1345 1350 1355 1360 Gly Val Cys Gly Ala Leu Tyr Lys Llys Phe Pro Glu Ser Phe Asp Lieu 1365 1370 1375 Glin Pro Ile Glu Val Gly Lys Ala Arg Lieu Val Lys Gly Ala Ala Lys 1380 1385 1390 His Ile Ile His Ala Val Gly Pro Asn Phe Asn Lys Val Ser Glu Val 1395 14 OO 1405 Glu Gly Asp Lys Glin Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile 1410 1415 1420 Val Asn Asp Asn. Asn Tyr Lys Ser Val Ala Ile Pro Leu Lleu Ser Thr 1425 1430 1435 1440 Gly Ile Phe Ser Gly Asn Lys Asp Arg Lieu. Thr Glin Ser Lieu. Asn His 1445 1450 1455 Leu Lieu. Thir Ala Lieu. Asp Thir Thr Asp Ala Asp Wall Ala Ile Tyr Cys 1460 1465 1470 Arg Asp Llys Lys Trp Glu Met Thr Lieu Lys Glu Ala Wall Ala Arg Arg 1475 1480 1485 Glu Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr Glu 1490 1495 15 OO US 2002/014 1975 A1 Oct. 3, 2002 49

-continued Pro Asp Ala Glu Lieu Val Arg Val His Pro Lys Ser Ser Lieu Ala Gly 1505 1510 1515 1520 Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser Tyr Leu Glu 1525 1530 1535 Gly Thr Lys Phe His Glin Ala Ala Lys Asp Ile Ala Glu Ile Asn Ala 1540 1545 1550 Met Trp Pro Val Ala Thr Glu Ala Asn Glu Glin Val Cys Met Tyr Ile 1555 15 60 1565 Leu Gly Glu Ser Met Ser Ser Ile Arg Ser Lys Cys Pro Val Glu Glu 1570 1575 1580 Ser Glu Ala Ser Thr Pro Pro Ser Thr Leu Pro Cys Leu Cys Ile His 1585 159 O 1595 1600 Ala Met Thr Pro Glu Arg Val Glin Arg Lieu Lys Ala Ser Arg Pro Glu 1605 1610 1615 Glin Ile Thr Val Cys Ser Ser Phe Pro Leu Pro Lys Tyr Arg Ile Thr 1620 1625 1630 Gly Val Glin Lys Ile Glin Cys Ser Glin Pro Ile Leu Phe Ser Pro Lys 1635 1640 1645 Val Pro Ala Tyr Ile His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro 1650 1655 1660 Val Asp Glu Thr Pro Glu Pro Ser Ala Glu Asn Gln Ser Thr Glu Gly 1665 1670 1675 1680 Thr Pro Glu Gln Pro Pro Leu Ile Thr Glu Asp Glu Thr Arg Thr Arg 1685 1690 1695 Thr Pro Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser 17 OO 1705 1710 Leu Lleu Ser Asp Gly Pro Thr His Glin Val Lieu Glin Val Glu Ala Asp 1715 1720 1725 Ile His Gly Pro Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro His 1730 1735 1740 Ala Ser Asp Phe Asp Wall Asp Ser Lieu Ser Ile Leu Asp Thr Lieu Glu 1745 175 O 1755 1760 Gly Ala Ser Val Thr Ser Gly Ala Thr Ser Ala Glu Thr Asn Ser Tyr 1765 1770 1775 Phe Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Val Pro Ala Pro Arg 1780 1785 1790 Thr Val Phe Arg Asn Pro Pro His Pro Ala Pro Arg Thr Arg Thr Pro 1795 1800 1805 Ser Leu Ala Pro Ser Arg Ala Cys Ser Arg Thr Ser Leu Val Ser Thr 1810 1815 1820 Pro Pro Gly Val Asn Arg Val Ile Thr Arg Glu Glu Lieu Glu Ala Lieu 1825 1830 1835 1840 Thr Pro Ser Arg Thr Pro Ser Arg Ser Val Ser Arg Thr Ser Leu Val 1845 1850 1855 Ser Asn Pro Pro Gly Val Asn Arg Val Ile Thr Arg Glu Glu Phe Glu 1860 1865 1870 Ala Phe Val Ala Glin Gln Glin Arg Phe Asp Ala Gly Ala Tyr Ile Phe 1875 1880 1885 Ser Ser Asp Thr Gly Glin Gly His Leu Glin Gln Lys Ser Val Arg Glin 1890 1895 1900 Thr Val Leu Ser Glu Val Val Leu Glu Arg Thr Glu Leu Glu Ile Ser US 2002/014 1975 A1 Oct. 3, 2002 50

-continued

1905 1910 1915 1920 Tyr Ala Pro Arg Lieu. Asp Glin Glu Lys Glu Glu Lieu Lleu Arg Lys Lys 1925 1930 1935 Leu Gln Lieu. Asn. Pro Thr Pro Ala Asn Arg Ser Arg Tyr Glin Ser Arg 1940 1945 1950 Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile Leu Glin Gly 1955 1960 1965 Leu Gly. His Tyr Lieu Lys Ala Glu Gly Lys Val Glu Cys Tyr Arg Thr 1970 1975 1980 Leu. His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg Ala Phe Ser 1985 1990 1995 2OOO Ser Pro Llys Val Ala Val Glu Ala Cys Asn Ala Met Leu Lys Glu Asn 2005 2010 2015 Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro Glu Tyr Asp Ala Tyr 2020 2025 2030 Leu Asp Met Val Asp Gly Ala Ser Cys Cys Lieu. Asp Thr Ala Ser Phe 2O35 20 40 2O45 Cys Pro Ala Lys Lieu Arg Ser Phe Pro Lys Lys His Ser Tyr Lieu Glu 2O5 O 2O55 2060 Pro Thr Ile Arg Ser Ala Val Pro Ser Ala Ile Glin Asn Thr Leu Gln 2O65 2070 2O75 2080 Asn Val Lieu Ala Ala Ala Thr Lys Arg Asn. Cys Asn Val Thr Gln Met 2O85 209 O 2095 Arg Glu Lieu Pro Val Lieu. Asp Ser Ala Ala Phe Asn Val Glu Cys Phe 2100 2105 2110 Lys Lys Tyr Ala Cys Asn. Asn. Glu Tyr Trp Glu Thr Phe Lys Glu Asn 2115 2120 2125 Pro Ile Arg Leu Thr Glu Glu Asn Val Val Asn Tyr Ile Thr Lys Leu 21.30 2135 214 O Lys Gly Pro Lys Ala Ala Ala Lieu Phe Ala Lys Thr His Asn Lieu. Asn 2145 2150 215.5 216 O Met Leu Glin Asp Ile Pro Met Asp Arg Phe Wal Met Asp Leu Lys Arg 21 65 217 O 21.75 Asp Val Lys Val Thr Pro Gly. Thr Lys His Thr Glu Glu Arg Pro Lys 218O 21.85 21.90 Val Glin Val Ile Glin Ala Ala Asp Pro Leu Ala Thr Ala Tyr Lieu. Cys 21.95 22 OO 2205 Gly Ile His Arg Glu Lieu Val Arg Arg Lieu. Asn Ala Val Lieu Lleu Pro 2210 2215 2220 Asn. Ile His Thr Lieu Phe Asp Met Ser Ala Glu Asp Phe Asp Ala Ile 2225 22.30 2235 2240 Ile Ala Glu His Phe Glin Pro Gly Asp Cys Wall Leu Glu Thir Asp Ile 22 45 225 O 2255 Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met Ala Lieu. Thir Ala Lieu 2260 2265 2270 Met Ile Leu Glu Asp Lieu Gly Val Asp Ala Glu Lieu Lleu Thir Lieu. Ile 2275 228O 2285 Glu Ala Ala Phe Gly Glu Ile Ser Ser Ile His Leu Pro Thr Lys Thr 2290 2295 2300 Lys Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met Phe Leu Thr Leu 2305 2310 2315 2320 US 2002/014 1975 A1 Oct. 3, 2002 51

-continued

Phe Wall Asn Thr Wall Ile Asn. Ile Wall Ile Ala Ser Arg Wall Leu Arg 2325 2330 2335

Glu Arg Lieu. Thr Gly Ser Pro Cys Ala Ala Phe Ile Gly Asp Asp Asn 234. O 2345 2350

Ile Wall Lys Gly Val Lys Ser Asp Llys Lieu Met Ala Asp Arg Cys Ala 2355 2360 2365

Thr Trp Lieu. Asn Met Glu Wall Lys Ile Ile Asp Ala Val Val Gly Glu 2370 2375 2380

Lys Ala Pro Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp Ser Val Thr 2385 2390 2395 2400

Gly Thr Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Lieu Phe Lys Lieu 2405 2410 24.15

Gly Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp Arg Arg Arg 2420 24.25 24.30 Ala Lieu. His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly Ile Leu Ser 2435 24 40 2445 Glu Lieu. Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val Gly Thr Ser 2450 2455 2460

Ile Ile Wal Met Ala Met Thr Thr Leu Ala Ser Ser Val Lys Ser Phe 2465 2470 24.75 24.80 Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly 2485 24.90

SEQ ID NO 4 LENGTH 1476 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence; Note synthetic construct NAME/KEY: CDS LOCATION: (1) . . . (1476) <400 SEQUENCE: 4 atg gct gCg aga gcq toa alta tta aga ggg gaa a.a.a. tta gat a.a.a. tgg 48 Met Ala Ala Arg Ala Ser Ile Telu Arg Gly Glu Teu Asp Lys Trp 1 5 10 15 gaa aag att agg tta agg cca ggg gga aag a.a.a. cat tat atg tta a.a.a. 96 Glu Lys Ile Arg Teu Arg Pro Gly Gly Lys His Met Telu 2O 25 30 cac ata gta tgg gcq agc agg gag citg gaa aga titt gca citt a.a. C. cct 144 His Ile Wall Trp Ala Ser Arg Glu Telu Glu Arg Phe Ala Teu Asn Pro 35 40 45 ggC citt tta gaa a Ca toa gaa gga tgt a.a.a. Cala alta atg aaa cag cita 192 Gly Telu Telu Glu Thr Ser Glu Gly Cys Glin Ile Met Glin Telu 5 O 55 60

Cala cca gct citc. cag a Ca gga aCa gag gaa citt a.a.a. toa tta tac a.a. C. 240 Glin Pro Ala Telu Glin Thr Gly Thr Glu Glu Teu Ser Teu Tyr Asn 65 70 75 a Ca gta gca act ctic tat tgt gta cat gaa aag alta gaa gta cga gac 288 Thr Wall Ala Thr Teu Cys Wall His Glu Lys Ile Glu Wall Arg Asp 85 90 95 acc aag gaa gcc tta gat aag ata gag gaa gaa Cala aac a.a.a. tgt cag 336 Thr Lys Glu Ala Teu Asp Lys Ile Glu Glu Glu Glin Asn Tys Cys Glin 100 105 110

Cala aaa acg cag cag goa aaa gC g gct gaC ggg a.a.a. caa aat 384 US 2002/014 1975 A1 Oct. 3, 2002 52

-continued Glin Lys Thr Glin Glin Ala Lys Ala Ala Asp Gly Lys Val Ser Glin Asn 115 120 125 tat cot at a gtg cag aat citc caa ggg caa atg gta cat caa gCC at a 432 Tyr Pro Ile Val Glin Asn Leu Gln Gly Glin Met Val His Glin Ala Ile 130 135 1 4 0 to a cot aga acc ttgaat gca togg gta aaa gta ata gaa gala aag gCt 480 Ser Pro Arg Thr Lieu. Asn Ala Trp Wall Lys Val Ile Glu Glu Lys Ala 145 15 O 155 160 titt agc cca gag gta ata ccc atg titt aca gCa tta to a gaa gga gCC 528 Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala 1.65 170 175 acc cca caa gat tta aac acc atg tta aat aca gtg g g g g g a cac caa 576 Thr Pro Glin Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Glin 18O 185 190 gca gCC at g caa atg tta aaa gat act att aat gaa gag got gca gaa 624 Ala Ala Met Glin Met Lieu Lys Asp Thir Ile Asn. Glu Glu Ala Ala Glu 195 200 2O5 tgg gat aga tta cat coa gttc cat gc g g g g cct att gca coca ggc cag 672 Trp Asp Arg Lieu. His Pro Wal His Ala Gly Pro Ile Ala Pro Gly Glin 210 215 220 atg aga gaa coa agg gga agt gac at a gCa gga act act agt acc citt 720 Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly. Thir Thr Ser Thr Leu 225 230 235 240 cag gaa caa at a gca togg atg aca agt aac coa cct att coa gtg gga 768 Gln Glu Glin Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly 245 250 255 gac atc tat aaa aga tigg ata att cit g g g g tta aat aaa at a gtg aga 816 Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Lieu. Asn Lys Ile Val Arg 260 265 27 O atg tat agc cc g g to agc att ttg gac ata aga caa gqg cca aag gala 864 Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Glin Gly Pro Lys Glu 275 280 285 ccc titt cqa gac tat gta gat cqg titc titt aaa act tta aga gct gala 912 Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Lieu Arg Ala Glu 29 O 295 3OO caa gCt aca caa gaa gta aaa aat togg at g aca gac acc ttg tta gtc 96.O Glin Ala Thr Glin Glu Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val 305 310 315 320 caa aat gcg aac cca gat tdt aag acc att ttg aga gca tta gga cca OO 8 Glin Asn Ala Asn. Pro Asp Cys Lys Thir Ile Leu Arg Ala Leu Gly Pro 325 330 335 ggg gCt aca tta gaa gaa atg at g aca gCa tot caa gqg gtg gga gga O56 Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Glin Gly Val Gly Gly 340 345 350 cct ggc cac aaa goa aga gta ttg gct gag gCa atg agt caa aca aac 104 Pro Gly. His Lys Ala Arg Val Lieu Ala Glu Ala Met Ser Glin Thr Asn 355 360 365 agt gga aac ata atg atg cag aga agc aat titt aaa gqc cot aga aga 152 Ser Gly Asn. Ile Met Met Glin Arg Ser Asn. Phe Lys Gly Pro Arg Arg 370 375 38O att gtt aaa tot titt aac tot ggc aag gala ggg cac ata gCC aga aat 200 Ile Val Lys Cys Phe Asn. Cys Gly Lys Glu Gly His Ile Ala Arg Asn 385 390 395 400 tgc aga gC c cot agg aaa aaa ggc tot tog aaa tot goa aaa gaa gga 248 Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly 405 410 415 cac caa at g aaa gac toc act gag agg cag gCt aat ttt tta ggg aaa. 296 US 2002/014 1975 A1 Oct. 3, 2002 53

-continued His Gln Met Lys Asp Cys Thr Glu Arg Glin Ala Asn. Phe Leu Gly Lys 420 425 430 att togg cct tcc cac aag ggg agg cca ggg aat titc citt cag aac aga 1344 Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn. Phe Leu Glin Asn Arg 435 4 40 4 45 cca gag coa aca gcc cca cca gCa gag agc titc agg titc gala gag aca 1392 Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu Thr 450 455 460 acc ccc gct cog aaa cag gag cog at a gaa agg gaa ccc tta act tcc 1440 Thr Pro Ala Pro Lys Glin Glu Pro Ile Glu Arg Glu Pro Leu Thir Ser 465 470 475 480 citc aaa to a citc titt g g c agc gac coc titg tot caa 1476 Leu Lys Ser Lieu Phe Gly Ser Asp Pro Leu Ser Glin 485 490

<210 SEQ ID NO 5 &2 11s LENGTH 492 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 5 Met Ala Ala Arg Ala Ser Ile Leu Arg Gly Glu Lys Lieu. Asp Llys Trp 1 5 10 15 Glu Lys Ile Arg Lieu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Lieu Glu Arg Phe Ala Lieu. Asn Pro 35 40 45 Gly Lieu Lieu Glu Thir Ser Glu Gly Cys Lys Glin Ile Met Lys Glin Lieu 50 55 60 Glin Pro Ala Leu Glin Thr Gly Thr Glu Glu Lieu Lys Ser Leu Tyr Asn 65 70 75 8O Thr Val Ala Thr Leu Tyr Cys Val His Glu Lys Ile Glu Val Arg Asp 85 90 95 Thr Lys Glu Ala Lieu. Asp Lys Ile Glu Glu Glu Glin Asn Lys Cys Glin 100 105 110 Glin Lys Thr Glin Glin Ala Lys Ala Ala Asp Gly Lys Val Ser Glin Asn 115 120 125 Tyr Pro Ile Val Glin Asn Leu Gln Gly Glin Met Val His Glin Ala Ile 130 135 1 4 0 Ser Pro Arg Thr Lieu. Asn Ala Trp Wall Lys Val Ile Glu Glu Lys Ala 145 15 O 155 160 Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala 1.65 170 175 Thr Pro Glin Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Glin 18O 185 190 Ala Ala Met Glin Met Lieu Lys Asp Thir Ile Asn. Glu Glu Ala Ala Glu 195 200 2O5 Trp Asp Arg Lieu. His Pro Wal His Ala Gly Pro Ile Ala Pro Gly Glin 210 215 220 Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly. Thir Thr Ser Thr Leu 225 230 235 240 Gln Glu Glin Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly US 2002/014 1975 A1 Oct. 3, 2002 54

-continued

245 250 255 Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Lieu. Asn Lys Ile Val Arg 260 265 27 O Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Glin Gly Pro Lys Glu 275 280 285 Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Lieu Arg Ala Glu 29 O 295 3OO Glin Ala Thr Glin Glu Val Lys Asn Trp Met Thr Asp Thr Leu Leu Val 305 310 315 320 Glin Asn Ala Asn. Pro Asp Cys Lys Thir Ile Leu Arg Ala Leu Gly Pro 325 330 335 Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Glin Gly Val Gly Gly 340 345 350 Pro Gly. His Lys Ala Arg Val Lieu Ala Glu Ala Met Ser Glin Thr Asn 355 360 365 Ser Gly Asn. Ile Met Met Glin Arg Ser Asn. Phe Lys Gly Pro Arg Arg 370 375 38O Ile Val Lys Cys Phe Asn. Cys Gly Lys Glu Gly His Ile Ala Arg Asn 385 390 395 400 Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly 405 410 415 His Gln Met Lys Asp Cys Thr Glu Arg Glin Ala Asn. Phe Leu Gly Lys 420 425 430 Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn. Phe Leu Glin Asn Arg 435 4 40 4 45 Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Arg Phe Glu Glu Thr 450 455 460 Thr Pro Ala Pro Lys Glin Glu Pro Ile Glu Arg Glu Pro Leu Thir Ser 465 470 475 480 Leu Lys Ser Lieu Phe Gly Ser Asp Pro Leu Ser Glin 485 490

<210> SEQ ID NO 6 &2 11s LENGTH 813 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <221 NAME/KEY: CDS <222> LOCATION: (1) . . . (813) <400 SEQUENCE: 6 atg agc cat att caa cigg gaa acg tot toc tog agg ccg cqa tta aat 48 Met Ser His Ile Glin Arg Glu Thir Ser Cys Ser Arg Pro Arg Lieu. Asn 1 5 10 15 to c aac at g gait gct gat tta tat ggg tat aaa togg gct cqc gat aat 96 Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn 2O 25 30 gto ggg caa to a ggit gcg aca atc tat cqa ttg tat ggg aag coc gat 144 Val Gly Glin Ser Gly Ala Thr Ile Tyr Arg Lieu. Tyr Gly Lys Pro Asp 35 40 45 gcq coa gag ttg titt citg aaa cat ggc aaa ggit agc gtt goc aat gat 192 Ala Pro Glu Lieu Phe Lieu Lys His Gly Lys Gly Ser Val Ala Asn Asp 5 O 55 60 US 2002/014 1975 A1 Oct. 3, 2002 55

-continued gtt aca gat gag at g g to aga cita aac togg citg acg gaa titt at g cct 240 Val Thr Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro 65 70 75 8O citt cog acc atc aag cat titt atc cqt act cot gat gat gca tag tta 288 Leu Pro Thir Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Lieu 85 90 95 citc acc act gcg atc ccc ggg aaa aca gCa titc cag gta tta gaa gaa 336 Leu Thir Thr Ala Ile Pro Gly Lys Thr Ala Phe Glin Val Leu Glu Glu 100 105 110 tat cot gat to a ggit gala aat att gtt gat gog citg gca gtg titc citg 384 Tyr Pro Asp Ser Gly Glu Asn. Ile Val Asp Ala Leu Ala Val Phe Lieu 115 120 125 cgc cqg ttg cat tcg att cot gtt tagt aat tdt cot titt aac agc gat 432 Arg Arg Lieu. His Ser Ile Pro Val Cys Asn. Cys Pro Phe Asn. Ser Asp 130 135 1 4 0 cgc gta titt cqt citc gct cag gC g caa to a cqa atgaat aac ggt ttg 480 Arg Val Phe Arg Lieu Ala Glin Ala Glin Ser Arg Met Asn. Asn Gly Lieu 145 15 O 155 160 gtt gat gcg agt gat titt gat gac gag cqt aat ggc tigg cct gtt gala 528 Val Asp Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu 1.65 170 175 caa gtc. tog aaa gaa atg cat aag citt ttg cca titc. tca cog gat to a 576 Glin Val Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser 18O 185 190 gto gtc act cat ggit gat titc to a citt gat aac citt att titt gac gag 624 Val Val Thr His Gly Asp Phe Ser Lieu. Asp Asn Lieu. Ile Phe Asp Glu 195 2OO 2O5 ggg aaa tta at a ggit tot att gat gtt gga cqa gtc gga atc gca gac 672 Gly Lys Lieu. Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp 210 215 220 cga tac cag gat citt gcc atc cta tog aac toc citc ggit gag titt tot 720 Arg Tyr Glin Asp Leu Ala Ile Leu Trp Asn. Cys Lieu Gly Glu Phe Ser 225 230 235 240 cct to a tta cag aaa cqg citt titt caa aaa tat ggt att gat aat cot 768 Pro Ser Lieu Gln Lys Arg Lieu Phe Glin Lys Tyr Gly Ile Asp Asn Pro 245 250 255 gat atgaat aaa ttg cag titt cat ttg atg citc gat gag titt ttc 813 Asp Met Asn Lys Lieu Glin Phe His Lieu Met Lieu. Asp Glu Phe Phe 260 265 27 O

<210 SEQ ID NO 7 &2 11s LENGTH 271 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 7 Met Ser His Ile Glin Arg Glu Thir Ser Cys Ser Arg Pro Arg Lieu. Asn 1 5 10 15 Ser Asn Met Asp Ala Asp Leu Tyr Gly Tyr Lys Trp Ala Arg Asp Asn 2O 25 30 Val Gly Glin Ser Gly Ala Thr Ile Tyr Arg Lieu. Tyr Gly Lys Pro Asp 35 40 45 Ala Pro Glu Lieu Phe Lieu Lys His Gly Lys Gly Ser Val Ala Asn Asp 50 55 60 Val Thr Asp Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro US 2002/014 1975 A1 Oct. 3, 2002 56

-continued

65 70 75 8O Leu Pro Thir Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Lieu 85 90 95 Leu Thir Thr Ala Ile Pro Gly Lys Thr Ala Phe Glin Val Leu Glu Glu 100 105 110 Tyr Pro Asp Ser Gly Glu Asn. Ile Val Asp Ala Leu Ala Val Phe Lieu 115 120 125 Arg Arg Lieu. His Ser Ile Pro Val Cys Asn. Cys Pro Phe Asn. Ser Asp 130 135 1 4 0 Arg Val Phe Arg Lieu Ala Glin Ala Glin Ser Arg Met Asn. Asn Gly Lieu 145 15 O 155 160 Val Asp Ala Ser Asp Phe Asp Asp Glu Arg Asn Gly Trp Pro Val Glu 1.65 170 175 Glin Val Trp Lys Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser 18O 185 190 Val Val Thr His Gly Asp Phe Ser Lieu. Asp Asn Lieu. Ile Phe Asp Glu 195 200 2O5 Gly Lys Lieu. Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp 210 215 220 Arg Tyr Glin Asp Leu Ala Ile Leu Trp Asn. Cys Lieu Gly Glu Phe Ser 225 230 235 240 Pro Ser Lieu Gln Lys Arg Lieu Phe Glin Lys Tyr Gly Ile Asp Asn Pro 245 250 255 Asp Met Asn Lys Lieu Glin Phe His Lieu Met Lieu. Asp Glu Phe Phe 260 265 27 O

<210 SEQ ID NO 8 &2 11s LENGTH 5076 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 8 atagg.cgg.cg catgagagaa goccagacca attacct acc caaaatggag aaagttcacg 60 ttgacatcga ggalagacago coatt.cctica gagctittgca gcggagcttic cc.gcagtttg 120 aggtagaa.gc caag cagg to actgataatg accatgctaa toccagagcg tttitcgcatc 18O tggcttcaaa act gatcgaa acggaggtgg acco atc.cga cac gatcctt gacattggaa 240 gtgcgc.ccgc cc.gcagaatg tattotaagc acaagtatca ttgttatctgt cogatgagat 3OO gtgcggaaga toc ggacaga ttgtataagt atgcaactaa gotgaagaaa aactgtaagg 360 aaataact ga taagga attg gacaagaaaa taaggagct c gcc.gc.cgtc atgagcg acc 420 citgacctgga aactgagact atgtgcctico acgacgacga gtcgtgtc.gc tacga agggc 480 aagtc.gctgt ttaccaggat gtatacgcgg ttgacgg acc citata actot citacggctaa 540 cctgaatgga citacga cata gtctagtc.cg ccalagatgtt coc gttccag ccaatgitatic 600 cgatgcagoc aatgcc citat cqcaa.ccc.gt togcggc.ccc gcgcaggc.cc tdgttcc cca 660 galaccg acco ttittctgg.cg atgcaggtgc aggaattaac cc.gctcqat g g citaacct ga 720 cgttcaag.ca acgc.cgggac gogccaccitg aggggccatc cqctaagaaa cc.gaagaagg 78O aggCCtcgca aaalacaga aa gagggaggcc aagggaagaa gaagaagaac Caagggalaga 840

US 2002/014 1975 A1 Oct. 3, 2002 60

-continued titc cca tot got caa cca cca att toc tac gac aga aaa coca gca gag 912 Phe Pro Cys Ala Glin Pro Pro Ile Cys Tyr Asp Arg Lys Pro Ala Glu 29 O 295 3OO act ttg gCC at g citc agc gtt aac atc cct gct ggg agg atc agc cqt 96.O Thr Lieu Ala Met Leu Ser Val Asn. Ile Pro Ala Gly Arg Ile Ser Arg 305 310 315 320 aat tat tat aat togg citt got got ggc tac tat tdt ggc cat gta cqt 1008 Asn Tyr Tyr Asn Trp Lieu Gly Ala Gly Tyr Tyr Cys Gly His Val Arg 325 330 335 gct gac caa coa gaa aca 1026 Ala Asp Gln Pro Glu Thr 340

<210> SEQ ID NO 10 <211& LENGTH 342 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 10 Met Phe Pro Phe Gln Pro Met Tyr Pro Met Gln Pro Met Pro Tyr Arg 1 5 10 15 Asn Pro Phe Ala Ala Pro Arg Arg Pro Trp Phe Pro Arg Thr Asp Pro 2O 25 30 Phe Leu Ala Met Glin Val Glin Glu Leu Thr Arg Ser Met Ala Asn Leu 35 40 45 Thr Phe Lys Glin Arg Arg Asp Ala Pro Pro Glu Gly Pro Ser Ala Lys 50 55 60 Lys Pro Llys Lys Glu Ala Ser Glin Lys Gln Lys Gly Gly Gly Glin Gly 65 70 75 8O Lys Lys Lys Lys Asn Glin Gly Lys Lys Lys Ala Lys Thr Gly Pro Pro 85 90 95 Asn Pro Lys Ala Glin Asn Gly Asn Lys Lys Lys Thr Asn Lys Llys Pro 100 105 110 Gly Lys Arg Glin Arg Met Wal Met Lys Lieu Glu Ser Asp Llys Thr Phe 115 120 125 Pro Ile Met Leu Glu Gly Lys Ile Asn Gly Tyr Ala Cys Val Val Gly 130 135 1 4 0 Gly Lys Lieu Phe Arg Pro Met His Val Glu Gly Lys Ile Asp Asn Asp 145 15 O 155 160 Val Lieu Ala Ala Leu Lys Thr Lys Lys Ala Ser Lys Tyr Asp Leu Glu 1.65 170 175 Tyr Ala Asp Val Pro Glin Asn Met Arg Ala Asp Thr Phe Lys Tyr Thr 18O 185 190 His Glu Lys Pro Gln Gly Tyr Tyr Ser Trp His His Gly Ala Val Glin 195 200 2O5 Tyr Glu Asn Gly Arg Phe Thr Val Pro Lys Gly Val Gly Ala Lys Gly 210 215 220 Asp Ser Gly Arg Pro Ile Leu Asp Asn Glin Gly Arg Val Val Ala Ile 225 230 235 240 Val Leu Gly Gly Val Asn Glu Gly Ser Arg Thr Ala Leu Ser Val Val 245 250 255 Met Trp Asin Glu Lys Gly Val Thr Val Lys Tyr Thr Pro Glu Asn Cys US 2002/014 1975 A1 Oct. 3, 2002 61

-continued

260 265 27 O Glu Gln Trp Ser Leu Val Thir Thr Met Cys Leu Leu Ala Asn Val Thr 275 280 285 Phe Pro Cys Ala Glin Pro Pro Ile Cys Tyr Asp Arg Lys Pro Ala Glu 29 O 295 3OO Thr Lieu Ala Met Leu Ser Val Asn. Ile Pro Ala Gly Arg Ile Ser Arg 305 310 315 320 Asn Tyr Tyr Asn Trp Lieu Gly Ala Gly Tyr Tyr Cys Gly His Val Arg 325 330 335 Ala Asp Gln Pro Glu Thr 340

<210> SEQ ID NO 11 &2 11s LENGTH 6989 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 11 atagg.cgg.cg catgagagaa goccagacca attacct acc caaaatggag aaagttcacg 60 ttgacatcga ggalagacago coatt.cctica gagctittgca gcggagcttic cc.gcagtttg 120 aggtagaa.gc caag cagg to actgataatg accatgctaa toccagagcg tttitcgcatc 18O tggcttcaaa actgatcgaa acggaggtgg acccatcc.ga cacgatcctt gacattggaa 240 gtgcgc.ccgc cc.gcagaatg tattotaagc acaagtatca ttgttatctgt cogatgagat 3OO gtgcggaaga toc ggacaga ttgtataagt atgcaactaa gotgaagaaa aactgtaagg 360 aaataact ga taagga attg gacaagaaaa taaggagct c gcc.gc.cgtc atgagcg acc 420 citgacctgga aactgagact atgtgcctico acgacgacga gtcgtgtc.gc tacga agggc 480 aagtc.gctgt ttaccaggat gtatacgcgg ttgacgg acc citata actot citacggctaa 540 cctgaatgga citacga cata gtctagtc.cg ccalagatgtc act agtgacc accatgtgtc 600 tgctic gocaa totgacgttc ccatotgcto aaccaccaat ttgctacgac agaaaaccag 660 cagagactitt goc catgcto agc gttaacg ttgacaa.ccc gggctacgat gagctgctogg 720 aag cagctgt taagtgcc cc ggaaggaaaa gqagatccac cqaggagct g tittaaggagt 78O ataagctaac go.gc.ccittac atggccagat gcatcagatg togcagttggg agctgccata 840 gtocaatago aatcgaggca gtaaagagcg acggg cacga cqgttatgtt agacittcaga 9 OO cittcct cqca gtatggcc to gattoctocq goaacttaaa gogg cagg acc atgcgg tatg 96.O acatgcacgg gaccattaaa gagataccac tacatcaagt gtcactc.cat a catctogcc 1020 cgtgtcacat tdtggatggg cacggittatt toctogcttgc caggtgcc.cg gcagggg act 1080 ccatcaccat ggaatttaag aaagattcc.g. tca cacactc. citgctcqgtg cc.gtatgaag 1140 tgaaatttaa toc totaggc agagaactict atact catcc cccagaacac ggagtagagc 1200 aag.cgtgcca agt citacgca catgatgcac agaacagagg agctitatgtc. gagatgcacc 1260 toccaggcto agaagtggac agcagtttgg titt.ccitt gag cqg cagttca gtcaccgtga 1320 caccitcct gt toggactago gocctggtgg aatgc gagtg togcggcaca aagatctoca 1380 agac catcaa caagacaaaa cagttcagcc agtgcacaaa gaaggag cag tdcagag cat 1440

US 2002/014 1975 A1 Oct. 3, 2002 65

-continued Tyr Gly Lieu. Asp Ser Ser Gly Asn Lieu Lys Gly Arg Thr Met Arg Tyr 115 120 125 gac at g cac ggg acc att aaa gag at a coa cita cat caa gtg to a citc 432 Asp Met His Gly Thr Ile Lys Glu Ile Pro Leu. His Glin Val Ser Leu 130 135 1 4 0 cat aca tot cqc cog togt cac att gtg gat ggg cac ggit tat titc citg 480 His Thr Ser Arg Pro Cys His Ile Val Asp Gly His Gly Tyr Phe Leu 145 15 O 155 160 citt goc agg toc cog gca ggg gac toc atc acc atg gaa titt aag aaa. 528 Leu Ala Arg Cys Pro Ala Gly Asp Ser Ile Thr Met Glu Phe Lys Lys 1.65 170 175 gat to c gtc. aca cac toc toc to g g to cog tat gaa gtg aaa titt aat 576 Asp Ser Val Thr His Ser Cys Ser Val Pro Tyr Glu Val Lys Phe Asn 18O 185 190 cct gta ggc aga gaa citc tat act cat coc coa gaa cac gga gta gag 624 Pro Val Gly Arg Glu Leu Tyr Thr His Pro Pro Glu His Gly Val Glu 195 200 2O5 caa gC g togc caa gtc. tac goa cat gat gca cag aac aga gga gct tat 672 Glin Ala Cys Glin Val Tyr Ala His Asp Ala Glin Asn Arg Gly Ala Tyr 210 215 220 gto gag at g cac citc cca ggc to a gaa gtg gac agc agt ttg gtt to c 720 Val Glu Met His Leu Pro Gly Ser Glu Val Asp Ser Ser Leu Val Ser 225 230 235 240 ttg agc ggc agt to a gtc acc gtg aca cot cot gtt gog act agc goc 768 Leu Ser Gly Ser Ser Val Thr Val Thr Pro Pro Val Gly Thr Ser Ala 245 250 255 citg gtg gala toc gag tdt ggc ggc aca aag atc. tcc aag acc atc aac 816 Leu Val Glu Cys Glu Cys Gly Gly Thr Lys Ile Ser Lys Thir Ile Asn 260 265 27 O aag aca aaa cag titc agc cag toc aca aag aag gag cag toc aga gCa 864 Lys. Thir Lys Glin Phe Ser Glin Cys Thr Lys Lys Glu Glin Cys Arg Ala 275 280 285 tat cqg citg cag aac gat aag togg gtg tat aat tot gac aaa citg ccc 912 Tyr Arg Lieu Glin Asn Asp Lys Trp Val Tyr Asn. Ser Asp Llys Lieu Pro 29 O 295 3OO aaa gCa gc g g g a gcc acc tta aaa goa aaa citg cat gtc. cca titc ttg 96.O Lys Ala Ala Gly Ala Thr Lieu Lys Gly Lys Lieu. His Val Pro Phe Lieu 305 310 315 320 citg gC a gac ggc aaa toc acc gtg cct cita gCa cca gaa cot at g ata OO 8 Leu Ala Asp Gly Lys Cys Thr Val Pro Leu Ala Pro Glu Pro Met Ile 325 330 335 acc titc ggt titc aga. tca gtg to a citg aaa citg cac cct aag aat coc O56 Thr Phe Gly Phe Arg Ser Val Ser Leu Lys Leu. His Pro Lys Asn Pro 340 345 350 aca tat cita acc acc cgc caa citt got gat gag cot cac tac acg cat 104 Thr Tyr Leu Thir Thr Arg Gln Leu Ala Asp Glu Pro His Tyr Thr His 355 360 365 gag citc at a tot gaa cca gct gtt agg aat titt acc gtc acc gga aaa. 152 Glu Leu Ile Ser Glu Pro Ala Val Arg Asin Phe Thr Val Thr Gly Lys 370 375 38O ggg togg gag titt gta togg gga aac cac cog ccg aaa agg ttt togg gca 200 Gly Trp Glu Phe Val Trp Gly Asn His Pro Pro Lys Arg Phe Trp Ala 385 390 395 400 cag gala aca gCa ccc gga aat coa cat ggg cita cc.g. cac gag gtg ata 248 Gln Glu Thr Ala Pro Gly Asn Pro His Gly Leu Pro His Glu Val Ile 405 410 415 act cat tat tac cac aga tac cot atg toc acc atc citg ggit ttg to a 296

US 2002/014 1975 A1 Oct. 3, 2002 67

-continued Gly Glu Ile Tyr Asn Tyr Asp Phe Pro Glu Tyr Gly Ala Gly Glin Pro 725 730 735 gga gCa titt gga gat ata caa toc aga aca gttc. tca agc to a gat citg 2256 Gly Ala Phe Gly Asp Ile Glin Ser Arg Thr Val Ser Ser Ser Asp Lieu 740 745 750 tat gcc aat acc aac cita gtg citg cag aga coc aaa goa gga gcg atc 23O4. Tyr Ala Asn. Thir Asn Lieu Val Lieu Glin Arg Pro Lys Ala Gly Ala Ile 755 760 765 cac gtg cca tac act cag gCa cct tcg ggt titt gag caa tog aag aaa. 2352 His Val Pro Tyr Thr Glin Ala Pro Ser Gly Phe Glu Gln Trp Llys Lys 770 775 78O gat aaa gCt coa to a titg aaa titt acc goc cott titc gga toc gala ata 24 OO Asp Lys Ala Pro Ser Lieu Lys Phe Thr Ala Pro Phe Gly Cys Glu Ile 785 790 795 8OO tat aca aac coc att cqc gcc gala aac tot act gta ggg to a att coa 2448 Tyr Thr Asn Pro Ile Arg Ala Glu Asn Cys Thr Val Gly Ser Ile Pro 805 810 815 tta gcc titt gac att coc gac goc ttg titc acc agg gtg to a gala aca 2496 Leu Ala Phe Asp Ile Pro Asp Ala Leu Phe Thr Arg Val Ser Glu Thr 820 825 830 ccg aca citt to a gcg gcc gaa toc act citt aac gag togc gtg tat tot 2544 Pro Thr Leu Ser Ala Ala Glu Cys Thr Leu Asn Glu Cys Val Tyr Ser 835 840 845 to c gac titt ggt ggg atc gcc acg gtc. aag tac to g g cc agc aag to a 2592 Ser Asp Phe Gly Gly Ile Ala Thr Val Lys Tyr Ser Ala Ser Lys Ser 85 O 855 860 ggc aag togc goa gtc. cat gtg cca to a ggg act gct acc cta aaa gaa 264 O Gly Lys Cys Ala Val His Val Pro Ser Gly Thr Ala Thr Leu Lys Glu 865 870 875 88O gca gCa gtc gag cita acc gag caa gogg tog gC g act atc cat titc tog 2688 Ala Ala Val Glu Leu Thr Glu Gln Gly Ser Ala Thr Ile His Phe Ser 885 890 895 acc goa aat atc. cac ccg gag titc agg ctic caa ata toc aca to a tat 2736 Thr Ala Asn Ile His Pro Glu Phe Arg Leu Glin Ile Cys Thr Ser Tyr 9 OO 905 910 gtt acg togc aaa got gat tdt cac coc cog aaa gac cat att gtg aca 2784 Val Thr Cys Lys Gly Asp Cys His Pro Pro Lys Asp His Ile Val Thr 915 920 925 cac cot cag tat cac goc caa acattt aca gCo gcg gtg toa aaa acc 2832 His Pro Glin Tyr His Ala Gln Thr Phe Thr Ala Ala Val Ser Lys Thr 930 935 940 gcq togg acg tog tta aca toc ct g citg gga gga to a gcc gta att att 2880 Ala Trp Thir Trp Leu Thir Ser Leu Leu Gly Gly Ser Ala Val Ile Ile 945 950 955 96.O ata att ggc titg gtg citg gct act att gtg gCC atg tac gtg citg acc 292.8 Ile Ile Gly Leu Val Leu Ala Thr Ile Val Ala Met Tyr Val Leu Thr 965 970 975 aac cag aaa cat aat 2943 Asn Gln Lys His Asn 98O

<210> SEQ ID NO 13 &2 11s LENGTH 981 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct US 2002/014 1975 A1 Oct. 3, 2002 68

-continued <400 SEQUENCE: 13 Met Ser Leu Val Thr Thr Met Cys Leu Leu Ala Asn Val Thr Phe Pro 1 5 10 15 Cys Ala Glin Pro Pro Ile Cys Tyr Asp Arg Llys Pro Ala Glu Thir Lieu 2O 25 30 Ala Met Leu Ser Val Asn Val Asp Asn Pro Gly Tyr Asp Glu Lieu Lieu 35 40 45 Glu Ala Ala Wall Lys Cys Pro Gly Arg Lys Arg Arg Ser Thr Glu Glu 50 55 60 Leu Phe Lys Glu Tyr Lys Lieu. Thir Arg Pro Tyr Met Ala Arg Cys Ile 65 70 75 8O Arg Cys Ala Val Gly Ser Cys His Ser Pro Ile Ala Ile Glu Ala Val 85 90 95 Lys Ser Asp Gly His Asp Gly Tyr Val Arg Lieu Glin Thr Ser Ser Glin 100 105 110 Tyr Gly Lieu. Asp Ser Ser Gly Asn Lieu Lys Gly Arg Thr Met Arg Tyr 115 120 125 Asp Met His Gly Thr Ile Lys Glu Ile Pro Leu. His Glin Val Ser Leu 130 135 1 4 0 His Thr Ser Arg Pro Cys His Ile Val Asp Gly His Gly Tyr Phe Leu 145 15 O 155 160 Leu Ala Arg Cys Pro Ala Gly Asp Ser Ile Thr Met Glu Phe Lys Lys 1.65 170 175 Asp Ser Val Thr His Ser Cys Ser Val Pro Tyr Glu Val Lys Phe Asn 18O 185 190 Pro Val Gly Arg Glu Leu Tyr Thr His Pro Pro Glu His Gly Val Glu 195 200 2O5 Glin Ala Cys Glin Val Tyr Ala His Asp Ala Glin Asn Arg Gly Ala Tyr 210 215 220 Val Glu Met His Leu Pro Gly Ser Glu Val Asp Ser Ser Leu Val Ser 225 230 235 240 Leu Ser Gly Ser Ser Val Thr Val Thr Pro Pro Val Gly Thr Ser Ala 245 250 255 Leu Val Glu Cys Glu Cys Gly Gly Thr Lys Ile Ser Lys Thir Ile Asn 260 265 27 O Lys. Thir Lys Glin Phe Ser Glin Cys Thr Lys Lys Glu Glin Cys Arg Ala 275 280 285 Tyr Arg Lieu Glin Asn Asp Lys Trp Val Tyr Asn. Ser Asp Llys Lieu Pro 29 O 295 3OO Lys Ala Ala Gly Ala Thr Lieu Lys Gly Lys Lieu. His Val Pro Phe Lieu 305 310 315 320 Leu Ala Asp Gly Lys Cys Thr Val Pro Leu Ala Pro Glu Pro Met Ile 325 330 335 Thr Phe Gly Phe Arg Ser Val Ser Leu Lys Leu. His Pro Lys Asn Pro 340 345 350 Thr Tyr Leu Thir Thr Arg Gln Leu Ala Asp Glu Pro His Tyr Thr His 355 360 365 Glu Leu Ile Ser Glu Pro Ala Val Arg Asin Phe Thr Val Thr Gly Lys 370 375 38O Gly Trp Glu Phe Val Trp Gly Asn His Pro Pro Lys Arg Phe Trp Ala 385 390 395 400 US 2002/014 1975 A1 Oct. 3, 2002 69

-continued

Gln Glu Thr Ala Pro Gly Asn Pro His Gly Leu Pro His Glu Val Ile 405 410 415 Thr His Tyr Tyr His Arg Tyr Pro Met Ser Thr Ile Leu Gly Leu Ser 420 425 430 Ile Cys Ala Ala Ile Ala Thr Val Ser Val Ala Ala Ser Thr Trp Leu 435 4 40 4 45 Phe Cys Arg Ser Arg Val Ala Cys Leu Thr Pro Tyr Arg Leu Thr Pro 450 455 460 Asn Ala Arg Ile Pro Phe Cys Lieu Ala Val Lieu. Cys Cys Ala Arg Thr 465 470 475 480 Ala Arg Ala Glu Thir Thr Trp Glu Ser Lieu. Asp His Leu Trp Asn. Asn 485 490 495 Asn Glin Gln Met Phe Trp Ile Glin Lieu Lieu. Ile Pro Leu Ala Ala Lieu 5 OO 505 510 Ile Val Val Thr Arg Leu Leu Arg Cys Val Cys Cys Val Val Pro Phe 515 52O 525 Leu Val Met Ala Gly Ala Ala Gly Ala Gly Ala Tyr Glu His Ala Thr 530 535 540 Thr Met Pro Ser Glin Ala Gly Ile Ser Tyr Asn Thr Ile Val Asin Arg 545 550 555 560 Ala Gly Tyr Ala Pro Leu Pro Ile Ser Ile Thr Pro Thr Lys Ile Lys 565 570 575 Leu Ile Pro Thr Val Asn Leu Glu Tyr Val Thr Cys His Tyr Lys Thr 58O 585 59 O Gly Met Asp Ser Pro Ala Ile Lys Cys Cys Gly Ser Glin Glu Cys Thr 595 600 605 Pro Thr Tyr Arg Pro Asp Glu Gln Cys Llys Val Phe Thr Gly Val Tyr 610 615 62O Pro Phe Met Trp Gly Gly Ala Tyr Cys Phe Cys Asp Thr Glu Asn Thr 625 630 635 640 Glin Val Ser Lys Ala Tyr Val Met Lys Ser Asp Asp Cys Lieu Ala Asp 645 650 655 His Ala Glu Ala Tyr Lys Ala His Thr Ala Ser Val Glin Ala Phe Lieu 660 665 670 Asn Ile Thr Val Gly Glu His Ser Ile Val Thir Thr Val Tyr Val Asn 675 680 685 Gly Glu Thr Pro Val Asn Phe Asin Gly Val Lys Leu Thr Ala Gly Pro 69 O. 695 7 OO Leu Ser Thr Ala Trp Thr Pro Phe Asp Arg Lys Ile Val Glin Tyr Ala 705 710 715 720 Gly Glu Ile Tyr Asn Tyr Asp Phe Pro Glu Tyr Gly Ala Gly Glin Pro 725 730 735 Gly Ala Phe Gly Asp Ile Glin Ser Arg Thr Val Ser Ser Ser Asp Lieu 740 745 750 Tyr Ala Asn. Thir Asn Lieu Val Lieu Glin Arg Pro Lys Ala Gly Ala Ile 755 760 765 His Val Pro Tyr Thr Glin Ala Pro Ser Gly Phe Glu Gln Trp Llys Lys 770 775 78O Asp Lys Ala Pro Ser Lieu Lys Phe Thr Ala Pro Phe Gly Cys Glu Ile 785 790 795 8OO US 2002/014 1975 A1 Oct. 3, 2002 70

-continued Tyr Thr Asn Pro Ile Arg Ala Glu Asn Cys Thr Val Gly Ser Ile Pro 805 810 815 Leu Ala Phe Asp Ile Pro Asp Ala Leu Phe Thr Arg Val Ser Glu Thr 820 825 830 Pro Thr Leu Ser Ala Ala Glu Cys Thr Leu Asn Glu Cys Val Tyr Ser 835 840 845 Ser Asp Phe Gly Gly Ile Ala Thr Val Lys Tyr Ser Ala Ser Lys Ser 85 O 855 860 Gly Lys Cys Ala Val His Val Pro Ser Gly Thr Ala Thr Leu Lys Glu 865 870 875 88O Ala Ala Val Glu Leu Thr Glu Gln Gly Ser Ala Thr Ile His Phe Ser 885 890 895 Thr Ala Asn Ile His Pro Glu Phe Arg Leu Glin Ile Cys Thr Ser Tyr 9 OO 905 910 Val Thr Cys Lys Gly Asp Cys His Pro Pro Lys Asp His Ile Val Thr 915 920 925 His Pro Glin Tyr His Ala Gln Thr Phe Thr Ala Ala Val Ser Lys Thr 930 935 940 Ala Trp Thir Trp Leu Thir Ser Leu Leu Gly Gly Ser Ala Val Ile Ile 945 950 955 96.O Ile Ile Gly Leu Val Leu Ala Thr Ile Val Ala Met Tyr Val Leu Thr 965 970 975 Asn Gln Lys His Asn 98O

<210> SEQ ID NO 14 &2 11s LENGTH 12379 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 14 atggg.cgg.cg catgagagaa goccagacca attacct acc caaaatggag aaagttcacg 60 ttgacatcga ggalagacago coatt.cctica gagctittgca gcggagcttic cc.gcagtttg 120 aggtagaa.gc caag cagg to actgataatg accatgctaa toccagagcg tttitcgcatc 18O tggcttcaaa act gatcgaa acggaggtgg acco atc.cga cac gatcctt gacattggaa 240 gtgcgc.ccgc cc.gcagaatg tattotaagc acaagtatca ttgttatctgt cogatgagat 3OO gtgcggaaga toc ggacaga ttgtataagt atgcaactaa gotgaagaaa aactgtaagg 360 aaataact ga taagga attg gacaagaaaa taaggagct c gcc.gc.cgtc atgagcg acc 420 citgacctgga aactgagact atgtgcctico acgacgacga gtcgtgtc.gc tacga agggc 480 aagtc.gctgt ttaccaggat gtatacgcgg ttgacgg acc gacaagttct c tat caccaag 540 ccaataaggg agittagagtc gcc tactgga taggctttga caccaccc.ct tittatgttta 600 agaacttggc tiggagcatat coatcatact citaccalacto gg.ccgacgaa accgtgttaa 660 cggctogtaa catagg ccta to cagctotg acgittatgga gcggtoacgt agagggatgt 720 ccattcttag aaagaagitat ttgaaaccat coaacaatgttctattotot gttggctcga 78O ccatctacca cq agaagagg gacittact ga ggagctogca cct gcc.gtot gtatttcact 840 tacgtggcaa goaaaattac acatgtcggt gtgagacitat agittagttgc gacgggtacg 9 OO

US 2002/014 1975 A1 Oct. 3, 2002 76

-continued citaacaatgc gct catcg to atccitcqgca cc.gto accot goatgctgtc. tagaggatcc 12360 ctaatacgac toactatag 12379

<210 SEQ ID NO 15 &2 11s LENGTH 1323 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <221 NAME/KEY: CDS <222> LOCATION: (1) . . . (1323) <400 SEQUENCE: 15 atg cca atc agt coc att gala act gta coa gta aaa citg aag coa gga 48 Met Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly 1 5 10 15 atg gat ggc cca aag gtt aaa caa togg ccg tta aca gaa gtg aaa ata 96 Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Val Lys Ile 2O 25 30 aaa gCa tta aca gca att tot gala gaa at g gala aag gaa gga aaa att 144 Lys Ala Lieu. Thir Ala Ile Cys Glu Glu Met Glu Lys Glu Gly Lys Ile 35 40 45 aca aaa att ggg cct gala aat coa tat aac act coa ata titc gcc at a 192 Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Ile Phe Ala Ile 5 O 55 60 aaa aag gaa gac agc act aag togg aga aaa tta gta gat titc agg gaa 240 Lys Lys Glu Asp Ser Thr Lys Trp Arg Lys Lieu Val Asp Phe Arg Glu 65 70 75 8O citc aat aaa aga act caa gac titt togg gag gtt caa tta gga at a coa 288 Lieu. Asn Lys Arg Thr Glin Asp Phe Trp Glu Val Glin Lieu Gly Ile Pro 85 90 95 cac coa gca ggg tta aaa aag aaa aaa to a gtg aca gta citg gat gtg 336 His Pro Ala Gly Lieu Lys Lys Lys Lys Ser Val Thr Val Lieu. Asp Wal 100 105 110 gga gat gca tat ttt to a gtt cot tta gat gala ggc titc agg aaa tat 384 Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Gly Phe Arg Lys Tyr 115 120 125 act gca titc acc ata cct agt ata aac aat gala aca cca ggg att aga 432 Thr Ala Phe Thr Ile Pro Ser Ile Asin Asn Glu Thr Pro Gly Ile Arg 130 135 1 4 0 tat caa tat aat gtg citt coa caa gga tog aaa gog to a coca gca ata 480 Tyr Glin Tyr Asn Val Leu Pro Glin Gly Trp Lys Gly Ser Pro Ala Ile 145 15 O 155 160 titc cag gCt agc atg aca aaa atc cta gag coc titt aga gCt aaa aat 528 Phe Glin Ala Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Ala Lys Asn 1.65 170 175 cca gaa at a gtc atc tat caa cat at g gC g gCattg tat gta gga tot 576 Pro Glu Ile Val Ile Tyr Gln His Met Ala Ala Leu Tyr Val Gly Ser 18O 185 190 gac tta gaa at a ggg caa cat aga gca aaa ata gaa gag tta aga. gala 624 Asp Leu Glu Ile Gly Glin His Arg Ala Lys Ile Glu Glu Lieu Arg Glu 195 200 2O5 cat cita tta aag togg gga titt acc aca coa gac aaa aaa cat cag aaa. 672 His Lieu Lleu Lys Trp Gly Phe Thir Thr Pro Asp Lys Lys His Gln Lys 210 215 220 gaa coc coa titt citt togg at g g g g tat gaa citc cat cot gac aaa togg 720 Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu. His Pro Asp Llys Trp US 2002/014 1975 A1 Oct. 3, 2002 77

-continued

225 230 235 240 aca gta cag cot ata cag citg cca gala aaa gat agc tigg act gtc. aat 768 Thr Val Glin Pro Ile Glin Leu Pro Glu Lys Asp Ser Trp Thr Val Asn 245 250 255 gac at a cag aag tta gtg gga aaa tta aac togg aca agt cag att tac 816 Asp Ile Glin Lys Lieu Val Gly Lys Lieu. Asn Trp Thr Ser Glin Ile Tyr 260 265 27 O cca ggg att aaa gta agg caa citt tot aag citc citt agg ggg acc aaa. 864 Pro Gly Ile Llys Val Arg Glin Lieu. Cys Lys Lieu Lleu Arg Gly Thr Lys 275 280 285 gca cita aca gac ata gta cca cita act gala gaa goa gaa tta gaa ttg 912 Ala Lieu. Thir Asp Ile Val Pro Leu Thr Glu Glu Ala Glu Lieu Glu Lieu 29 O 295 3OO gca gag aac agg gaa att cita aaa gaa cca gtg cat gga gta tat tat 96.O Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Wal His Gly Val Tyr Tyr 305 310 315 320 gac coa toa aaa gac ttgata gCt gala at a cag aaa cag ggg gat gac OO 8 Asp Pro Ser Lys Asp Lieu. Ile Ala Glu Ile Glin Lys Glin Gly Asp Asp 325 330 335 caa togg aca tat caa att tac caa gaa cca ttcaaa aac citg aag aca O56 Gln Trp Thr Tyr Glin Ile Tyr Glin Glu Pro Phe Lys Asn Leu Lys Thr 340 345 350 gga aag tat gca aaa agg agg act acc cac act aat gat gta aaa cag 104 Gly Lys Tyr Ala Lys Arg Arg Thr Thr His Thr Asn Asp Wall Lys Glin 355 360 365 tta aca gag gCa gtg caa aaa ata toc ttg gaa agc ata gta aca togg 152 Leu Thr Glu Ala Val Glin Lys Ile Ser Leu Glu Ser Ile Val Thr Trp 370 375 38O gga aag act colt aaa titt aga cita coc atc caa aaa gaa aca togg gala 200 Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln Lys Glu Thir Trp Glu 385 390 395 400 ata togg togg aca gac tat tog caa gCC aca togg att cot gag togg gag 248 Ile Trp Trp Thr Asp Tyr Trp Glin Ala Thr Trp Ile Pro Glu Trp Glu 405 410 415 titt gtt aat acc cct coc cta gta aaa cita tag tac cag cta gaa aaa 296 Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys 420 425 430 gaa coc at a gCa gga gca gaa act titc 323 Glu Pro Ile Ala Gly Ala Glu Thr Phe 435 4 40

<210> SEQ ID NO 16 <211& LENGTH 441 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence; Note = synthetic construct <400 SEQUENCE: 16 Met Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro Gly 1 5 10 15 Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Val Lys Ile 2O 25 30 Lys Ala Lieu. Thir Ala Ile Cys Glu Glu Met Glu Lys Glu Gly Lys Ile 35 40 45 Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Ile Phe Ala Ile 50 55 60 US 2002/014 1975 A1 Oct. 3, 2002 78

-continued

Lys Lys Glu Asp Ser Thr Lys Trp Arg Lys Lieu Val Asp Phe Arg Glu 65 70 75 8O Lieu. Asn Lys Arg Thr Glin Asp Phe Trp Glu Val Glin Lieu Gly Ile Pro 85 90 95 His Pro Ala Gly Lieu Lys Lys Lys Lys Ser Val Thr Val Lieu. Asp Wal 100 105 110 Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Glu Gly Phe Arg Lys Tyr 115 120 125 Thr Ala Phe Thr Ile Pro Ser Ile Asin Asn Glu Thr Pro Gly Ile Arg 130 135 1 4 0 Tyr Glin Tyr Asn Val Leu Pro Glin Gly Trp Lys Gly Ser Pro Ala Ile 145 15 O 155 160 Phe Glin Ala Ser Met Thr Lys Ile Leu Glu Pro Phe Arg Ala Lys Asn 1.65 170 175 Pro Glu Ile Val Ile Tyr Gln His Met Ala Ala Leu Tyr Val Gly Ser 18O 185 190 Asp Leu Glu Ile Gly Glin His Arg Ala Lys Ile Glu Glu Lieu Arg Glu 195 200 2O5 His Lieu Lleu Lys Trp Gly Phe Thir Thr Pro Asp Lys Lys His Gln Lys 210 215 220 Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu. His Pro Asp Llys Trp 225 230 235 240 Thr Val Glin Pro Ile Gln Leu Pro Glu Lys Asp Ser Trp Thr Val Asn 245 250 255 Asp Ile Glin Lys Lieu Val Gly Lys Lieu. Asn Trp Thr Ser Glin Ile Tyr 260 265 27 O Pro Gly Ile Llys Val Arg Glin Lieu. Cys Lys Lieu Lleu Arg Gly Thr Lys 275 280 285 Ala Lieu. Thir Asp Ile Val Pro Leu Thr Glu Glu Ala Glu Lieu Glu Lieu 29 O 295 3OO Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Wal His Gly Val Tyr Tyr 305 310 315 320 Asp Pro Ser Lys Asp Lieu. Ile Ala Glu Ile Glin Lys Glin Gly Asp Asp 325 330 335 Gln Trp Thr Tyr Glin Ile Tyr Glin Glu Pro Phe Lys Asn Leu Lys Thr 340 345 350 Gly Lys Tyr Ala Lys Arg Arg Thr Thr His Thr Asn Asp Wall Lys Glin 355 360 365 Leu Thr Glu Ala Val Glin Lys Ile Ser Leu Glu Ser Ile Val Thr Trp 370 375 38O Gly Lys Thr Pro Lys Phe Arg Leu Pro Ile Gln Lys Glu Thir Trp Glu 385 390 395 400 Ile Trp Trp Thr Asp Tyr Trp Glin Ala Thr Trp Ile Pro Glu Trp Glu 405 410 415 Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys 420 425 430 Glu Pro Ile Ala Gly Ala Glu Thr Phe 435 4 40

<210 SEQ ID NO 17 &2 11s LENGTH 13584