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(12) Patent Application Publication (10) Pub. No.: US 2012/0121650 A1 Johnston Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2012/0121650 A1 Johnston Et Al US 201201 21 650A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0121650 A1 Johnston et al. (43) Pub. Date: May 17, 2012 (54) CHMERIC VIRUS VACCINES Publication Classification (76) Inventors: Robert E. Johnston, Chapel Hill, (51) Int. Cl. NC (US); Christy Jurgens, A63L/7088 (2006.01) Rahway, NJ (US); Kelly Young CI2N 5/86 (2006.01) A6IP37/02 (2006.01) Poe, Durham, NC (US) CI2N 7/01 (2006.01) (21) Appl. No.: 12/377,927 (52) U.S. Cl. ................. 424/278.1; 435/235.1:435/320.1 PCT Fled: Aug. 16, 2007 (22) (57) ABSTRACT (86) PCT NO.: PCT/USO7/18046 The present invention provides novel self-replicating and self-propagating chimeric viral vectors and chimeric virus S371 (c)(1), particles comprising a modified genome of a carrier RNA (2), (4) Date: Jul. 23, 2010 virus packaged within structural proteins of a second virus. Also provided are pharmaceutical formulations comprising Related U.S. Application Data the chimeric viral vectors and virus particles and methods of (60) Provisional application No. 60/838,604, filed on Aug. inducing an immune response by administration of the chi 18, 2006, provisional application No. 60/840,645, meric viral vectors and virus particles or nucleic acids (e.g., filed on Aug. 28, 2006. DNA and/or RNA) encoding the same to the subject. Patent Application Publication May 17, 2012 Sheet 1 of 22 US 2012/O121650 A1 26S PARENTAL nSP 1-4 GENOME C E2 E1 (-) STRAND ----------------------------------- PROGENY GENOME SUBGENOMIC MRNA C E2 E1 FIG. 1 26S GAG HA) FIG. 2A 26S GENOME - a REPLICASE C E2 E1 26S REPLICON - - REPLICASE CAPSID HELPER GLYCOPROTEIN HELPER ZN." E2 E1 ( ; : FIG. 2B Patent Application Publication May 17, 2012 Sheet 2 of 22 US 2012/O121650 A1 FIG. 2E Patent Application Publication May 17, 2012 Sheet 3 of 22 US 2012/O121650 A1 pVR21 SHIV89.6P Gag 26S NSP'S GAG An -- RT primer -> 1949 bp - PCR primers pVR21 SHIV89.6P Gag 26S NSP'S GAG A(n) - NSP's - GAGRT PRIMER- -> 2139 bp - PCR PRIMERS pVR21 SHIV89.6P GagansP4 26S NSP'S A540 bp GAG HA - RT PRIMER -> 1409 BP - PCR PRIMERS FIG 3A -R H C - 29 GAG GAG NGAG a.a.S. VRP PARTICLE PARTICLE O - 5 O 5 as a < < < 5,2 2it 3 22 3g 4.g.,g g g1 <g G.g g14 g g.is 3,555 , , , sh -- H n Oits CDS GZis n Oits Oto 5 H. H. 'H. 'H. H H" - "c- H. H." 'He 'H CD CD CD CD > Z Z Do Z Z did ZZ did T. CN CO S L CO in OO O. C. va Patent Application Publication May 17, 2012 Sheet 4 of 22 US 2012/0121650 A1 26S GENOME H - . REPLICASE C E2 E1 REPLICON 26s REPLICASE gp160 CAPSID HELPER nae GLYCOPROTEIN HELPER Zagrg ENV-VRP() { Patent Application Publication May 17, 2012 Sheet 5 of 22 US 2012/O121650 A1 !=>#[].……..,! Patent Application Publication May 17, 2012 Sheet 6 of 22 US 2012/0121 650 A1 PELLETED CELL SUPERNATANT LYSATE S.5 Sco 3à S.d5 9.c. 253 CD d CD D 25O 160 105 75 50 35 30 1600 212001400 2 1000 P 800 a 600 400 200 O O 6 10 14 18 22 TIME (HPI) FIG. 7A Patent Application Publication May 17, 2012 Sheet 7 of 22 US 2012/O121650 A1 TIME (HPI) 6 10 14 18 22 GAG 800 700 600 500 400 3OO -D- GAG 200 ...A. GAG OO O O 6 1 O 14 18 22 TIME (HPI) FIG. 7B 26S W. RIBOPROBE s - - is a Patent Application Publication May 17, 2012 Sheet 8 of 22 US 2012/0121 650 A1 N1 s 8 9 O CDCC D CD > CD FIG. 9 Patent Application Publication May 17, 2012 Sheet 9 of 22 US 2012/O121650 A1 pVR21-SHIV89.6PEnv 26S ENV “, pVR21-SHIV89.6PGag Swal Not FIG. 1 OA Patent Application Publication May 17, 2012 Sheet 10 of 22 US 2012/0121 650 A1 26S 26S GENOME REPLICASE GAG ENV O O Patent Application Publication May 17, 2012 Sheet 11 of 22 US 2012/O121650 A1 PNGase TREATED UNTREATED CELL PELLETED CELL PELLETED LYSATE SUPERNATANT LYSATE SUPERNATANT D Z CD CC (S : - ENV FIG. 1 OC s Ol 250 16O ENV 105 75 - 50 35 Patent Application Publication May 17, 2012 Sheet 12 of 22 US 2012/O121650 A1 O O CN CN Ost siCC Si. y8, S N S S Ygg55 itS 3S s 2995 d5 889999a CD CD CD CD 25O 16O 105 P55 GAG HEAVY CHAIN LIGHT CHAIN 1 2 3 4 5 6 7 8 FIG 1 OE FIG. 1 1A FIG. 1 1B Patent Application Publication May 17, 2012 Sheet 13 of 22 US 2012/0121 650 A1 FIG. 1 1 C FIG. 1 1D FIG. 11E FIG. 11 F FIG. 12A FIG. 12B Patent Application Publication May 17, 2012 Sheet 14 of 22 US 2012/O121650 A1 FIG. 12C FIG. 12D GAGENV ENVGAG PARTICLE PARTICLES O - GAG - VEE O-VEE NON-STRUCTURALS NON-STRUCTURALS FIG. 13A Patent Application Publication May 17, 2012 Sheet 15 of 22 US 2012/O121650 A1 ENV VRP SEDIMENTATION (H. FRACTION 3 4 5 6 7 8 9 10 250 160 105 75 50 35 30 GAGENV SEDIMENTATION FRACTION 3 4 5 6 7 8 9 1 O 250 160 105 75 P55 50 GAG 35 30 ENVGAG SEDIMENTATION FRACTION 3 4 5 6 7 8 9 10 250 160 105 75 P55 50 GAG 35 30 FIG. 13B Patent Application Publication May 17, 2012 Sheet 16 of 22 US 2012/O121650 A1 n CL CS 2 2 GAG 1-510 VEE CAPSID 2do N1CP (D CD Y2 CD CDOl 1-125 E O - CE O - C FIG. 14A D 2 (D 9 a 99. 2 d as C D. C O CN wS oo 9 9. 9. to toO) o s s CD CD CD 160 75 Gag-Pro (p65) 50 Gag (p55) 35 - Ma/Ca (p42) 3O 2?? Ca/NC y CN CELLLYSATE y ve w O s CD : i 60 Gag-Pro (p65) 75 Ee (p55) 35 YMa/Ca (p42) 30 N??? Ca/NC N CAPSID (p27) V ON CO Y D co N. oo o O - CN ty y - CONCENTRATED SUPERNATANT FIG. 15B Patent Application Publication May 17, 2012 Sheet 17 of 22 US 2012/O121650 A1 GAG-PRO-RT PRECURSOR (p130) PRO RT: 010 O51/O66 MATRIX CAPSID Eps *SAFETY MUTATIONS IN O17 O24 O7 POLYMERASE AND RNASeH C C 5 2 - d > 5 & St S if & cis H. H. H. H. H. H. a C. C. C. 9 9 S 9 9 9 a o3 g g g g g : CD CD CD CD CD CD CD 105 75 50 35 3O 25 CELLLYSATES o 22 = n > '? S if &: ci3 &. 9. &. 5. n n a g g : CD (D CD CD C5 CD CD 160 105 Ma/Ca CONC. SUPERNATANTS FIG. 15C Patent Application Publication May 17, 2012 Sheet 18 of 22 US 2012/0121 650 A1 VEE CAPSID - CDNA SEOUENCE Ttgttc.ccgttcCagccaatgitat CCGatgcagcCaatgCCCtatC gCaacCCott CoCggCCCC9C9 CaggCCCtggitt CCCCagaaCC9a CCCttittctggCdatgcaggtgcaggaattaa CCCdCtcgatggct aacCtgacgttcaa.gcaa.cgc.cgggacgcgCCaCCtgagggg CCat CccCtaagaaacCgaagaaggaggctt.cgcaaaaacagAAAGGGGG AGGCCAAGGGAAGAAGAAGAAGAACCAAGGGAAGAAGAAGGCTAAG ACAGGGCCGCCCAATCCGAAGGCACAGAATGGAAACAAGAAGAAGA CCAACAAGAAACCAGGCAAGAGACAGCGCATGGTCATGAAATTGGA ATCTGACAAGACGTTCCCAATCATGTTGgaagggaagataaacggC tacgCttgttgttggtCGgagggaagttatt CaggCCgatgCatgttgg aaggcaagat Coacaacgacgttctggcc.gc.gcttalaga C9aagaa agcatcCaaatacgatCttgagtatgcagatgtgCCaCagaacatg CgggCCdataCattcaaataCaCCCatgagaaacCCCaaggCtatt acagctggCatCatcgagCagtC CaatatgaaaatgggCdttt CaC ggtgCCgaaaggagttgggg CCaagggaga CagCdgaCCaCCCatt CtggataaCCaggga C999 togt CCCtattgttgctgggaggtgttga atgaaggat Ctagga cagcCCttt CagtCct catgtggaacgagaa gggagitta CCG tdaagtatact CCGgagaactg.cgagCaatcg (SEQ ID NO:1) VEE CAPSID - AMINO ACID SEOUENCE AfpfgpmypmqpmpyrnpfaaprirpWifprt.dpflamdVoeltrs manlitfkgrrdappegpsakkpkkeasqkqKGGGQGKKKKNQGK KKAKTGPPNPKAQNGNKKKTNKKPGKRQRMVMKLESDKTFPIML egkingyacv voggklfrpmhvegkidndvlaalktkkaskydle yadvpcnmradtfkythekpdgyySWhhgavoyengriftvpkg V gakgdsgrpildinqqrvvai VlggVinegSrtal SV Vimwnekgvt vkytpenced (SEQID NO:2) FIG. 16 Patent Application Publication May 17, 2012 Sheet 20 of 22 US 2012/O121650 A1 INFECTIOUS CONSTRUCT DESCRIPTION OF CONSTRUCT PARTICLES FROM 25ug RNA MOCK NO RNA GAG VEE GENOME EXPRESSING GAG ONLY ENV VEE GENOME EXPRESSINGENV ONLY VEE GENOME EXPRESSING GAG+ENV GAG AND ENV VEE GENOME EXPRESSING GAGC-ENV GAG FUSED TO VEE CAPSID FRAGMENT AND ENV VEE GENOME EXPRESSING GAG(dZF)C GAG WITH ZINC FINGERS ENV DELETED FUSED TO VEE CAPSID FRAGMENT AND ENV VEE GENOME EXPRESSING GAG(dNC)C GAG WITH NUCLEOCAPSID -ENV REGION (AA 4-48) DELETED 1. E-04 FUSED TO VEE CAPSID FRAGMENT AND ENV VEE GENOME EXPRESSING GAG(dNC GAG WITH NUCLEOCAPSID +Csub)+ REGION (AA 4-48) REPLACED BY ENV VEE CAPSID FRAGMENT (AA 75-132) AND ENV VEE GENOME EXPRESSING GAG6GlyC GAGFUSED WIAA 6-GLYCINE ENV LINKER TO VEE CAPSID FRAGMENT AND ENV VEE GENOME EXPRESSING GAG WITH ZINC FINGERS GAG(dZF)- DELETED FUSED WIAA 6GlyC+ENV 6-GLYCINE LINKER TOVEE CAPSID FRAGMENT AND ENV FIG. 18 Patent Application Publication May 17, 2012 Sheet 21 of 22 US 2012/0121 650 A1 CELLLYSATES CONC. SUPERNATANTS XOOWN XOOWN OHCH-5)\/€) 75 50 35 Ma/Ca 30 Ca WrigZ+OHd-?W?OS OHd-?WE)Wri09+OS OHCH-5)\/S)ZWNS- XOOWN OHd-5)\/5) 160 105 75 5O 35 30 CONCENTRATED SUPERNATANTS FIG. 19A Patent Application Publication US 2012/O121650 A1 CELLLYSATES != CO O N.LOCYD LOO ©:- OLO CONCENTRATED SUPERNATANTS FIG. 19B US 2012/01 21 650 A1 May 17, 2012 CHMERIC VIRUS VACCINES 0016 said modified genome comprising: 0017 protein coding sequences and cis-acting FIELD OF THE INVENTION sequences sufficient for replication of the modified 0001. The present invention relates to live virus vaccines, genome; and in particular, live self-propagating virus vaccines. 0.018 structural protein coding sequences from a retro virus sufficient to form a virion; and BACKGROUND OF THE INVENTION 0019 said pseudotyped virion comprising: 0020 retrovirus structural proteins encoded by the 0002 Vaccination against disease is effected by inducing a modified genome; and protective immune response to a pathogenic organism with 0021 a pseudotyping virion structural protein that is out causing disease.
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