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United States Patent (19) 11 Patent Number: 5,824,309 Dassarma Et Al

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United States Patent (19) 11 Patent Number: 5,824,309 Dassarma Et Al USOO5824309A United States Patent (19) 11 Patent Number: 5,824,309 DasSarma et al. (45) Date of Patent: Oct. 20, 1998 54 RECOMBINANT GAS VESICLES AND USES Muster et al., “A Conserved Neutralizing Epitope on gp41 of THEREOF Human Immunodeficiency Virus Type 1 J. of Virology 67(11):6642–6647, 1993. 75 Inventors: Shiladitya DasSarma; Fazeela Blaseio et al., “Transformation of Halobacterium halobium: Morshed; Elizabeth Stuart, all of Development of vectors and investigation of gas vesicle Amherst; Samuel Black, Leverett, all of synthesis” Proc. Natl., Acad. Sci. USA87:6772-6776, 1990. Mass. DasSarma et al., “Wild-Type Gas Vesicle Formation Requires at Least Ten Genes in the gVp Gene Cluster . ' 73 Assignee: University of Massachusetts, Boston, J. of Bacteriology 176(24):7646–7652, 1994. Mass. DasSarma et al., “Gas Vesicle Proteins and Genes' Mol. Biol. 12:93-98, 1993. 2 1. Appl. No.: 759,444 Halladay et al., “The Rightward Gas Vesicle Operon in Halobacterium Plasmid pNRC100: . J. of Bacteriology Filed: Dec. 5, 1996 175(3):684-692, 1993. | Int. Cl." .......................... A61K 39/21; A61K 39/02; Halladay et al., Genetic transformation of a halophilic A61K 39/295; CO7K 14/195 archaebacterium with a gas vesicle gene cluster restores. 52 U.S. Cl. ..................................... 424/188.1; 424/190.1; Jones et al., “Structure and organization of the gas vesicle 424/2011; 424/204.1; 424/207.1; 424/208.1; gene cluster on the Halobacterium Halobium plasmid 424/234.1; 514/2; 530/350 pNRC100” Gene 102:117–122, 1991. 58 Field of Search ................................ 435/69.1, 320.1, Primary Examiner David Guzo 435/172.1, 69.3, 172.3; 530/350; 514/2; Attorney, Agent, or Firm Fish & Richardson, P.C. 424/185.1, 234.1, 186.1, 187.1, 188.1, 190.1, 57 ABSTRACT 2011, 204.1, 207.1, 208.1 The invention features a composition that includes a Sub 56) References Cited Stantially pure recombinant gas vesicles which have at least PUBLICATIONS one heterologous peptide inserted into at one of their struc tural proteins. The recombinant gas vesicle, when adminis Aldovini et al., “Synthesis of the complete trans-activation tered to a mammal, is capable of eliciting antibodies which gene product of human T-lymphotropic virus type III in Specifically bind to the heterologous peptide. The heterolo Escherichia Coli: . Proc. Natl. Acad. Sci. USA gous peptide can be any peptide against which one Wishes to 83:6672–6676, 1986. raise antibodies, e.g., a peptide found in the gp120 protein J.S. Allan, “Major Glycoprotein Antigens Taht Induce Anti of human immunodeficiency virus (HIV). bodies in AIDS Patients Are Encoded by HTLV-III" Science 228:1091–1094, 1985. 10 Claims, 12 Drawing Sheets U.S. Patent Oct. 20, 1998 Sheet 1 of 12 5,824.309 F.G. 1A F.G. 1B 1 O 16 O 2560 1 O 16 O 256 O Reciprocal serum dilution Reciprocal serum dilution OD 405 F.G. 1 C OD 405 F.G. 1D 2.. O 18 1.6 .. 4 1.2 O.81. O s 0.4O. 6 issN O.2 C-3-O-Filee O 2 O 92 4.22 1940 891 4 256 O 20 92 4.22 1940 '894' 2560 Reciprocal End Titer Reciprocal End Titer o 1 mg GV-TNP 1gM 1 mg GV 1gM o 1 mg GV-TNP 1gg C 1mg GV 1gG a 0.1 mg GV-TNP 1gM Buffer 1gM a 0.1 mg GV-TNP 1gg D Buffer 1gg U.S. Patent Oct. 20, 1998 Sheet 2 of 12 5,824.309 OD 405 FIG. 2A O ---------- 2O 32O 5 12 O 2 O 32O 5 12 O Reciprocal serum dilution OD 405 FIG. 2C 2 O 8 O 320 1 280 5 120 20480 20 92 4.22 1940 891 4 40960 Reciprocal Serum Dilution Reciprocal Serum Dilution e GV-TNP 1gM o GV-TNP 1gG Buffer gM o Buffer igG U.S. Patent Oct. 20, 1998 Sheet 3 of 12 5,824.309 FIG. 3A 1 ... O O. 8 OD 405 O. 6 O. 4- . O2 O 40 161 649 26 O8 10480 Reciprocal Serum Dilution o GV-TNP 1gM Buffer 1gM FIG. 3B OD 405 1 O 40 16 649 2608 10480 Reciprocal Serum Dilution o GV-TNP 1gg D Buffer 1 gC U.S. Patent Oct. 20, 1998 Sheet 4 of 12 5,824.309 FG 4A 1.4 -OH 9/22 - L. 12 s K)... 9/22 - R ". -O . 9 / 22 - O Absorbance 0: 405 nm. 0.6 0.4 02a------a- -- O 1 OO 1 OOO 1 OOOO Reciprocal of Sera Dilutions FIG. 4B Absorbance 405 nm. - 2.71 O50543.12 13761 1 E-2O 1 OO 1 OOO OOOO Reciprocal of Serum Dilutions U.S. Patent Oct. 20, 1998 Sheet 5 of 12 5,824.309 F.G. 5A 8 16 -OH L - 17d *... N. KX R - 17d 1.4 N. - O O- 17d Absorbance 1-2 3. ''A'. 2nd boost repeat 405 n 1 ... B. L17-d PBS O.8- O6 O.4 O2 O 1 OO 1 OOO 1 OOOO Reciprocal of Serum Dilutions F.G. 5B 2 1.8 -H 17d repeat 1.6 '. N. KX a R-17 d - 3 Wk 14- V O. PBS Control Absorbance at 1.2 405 nm 1 - O.8 O6 0.4 O.2 O 1 O 1 OO 1 OOO 1 OOOO Reciprocal of Serum Dilutions U.S. Patent Oct. 20, 1998 Sheet 10 of 12 5,824,309 CCCAGCTATC GAATGCGATT GTGAGTTGCG TGGTCGCGGC GTTGATCTCT TGGATCCATT 6360 CGAGGAGCGA ATCCAGATCG AGTTTCTCGA ATGGCACGTC TACGTCCAGT GGGAGTTCGA 6420 ATGGGTCTTG GAAGAGGTCA AGAACGCGG TTGTGTCGAG TGAGGAGTGA CGGCGAAGT 64.80 ACATCTCGG AACCGTCTCC TGATCAACAC GTGTGGACAC GTAGAGGACA TCACTGTCTC 6540 GGCCAACAC ATCGAGGCCG CGGACGTGA ACAAGGTCTT ACCCGTGCCT GGTGCACCGT 6600 TAATGAGGAG CGTTTCCCCG GCGTCACCCA GAAAAACTG GCTGAGCTCG CGGGGGAAA 6660 ACACGATTCC GGTGTAGTCT GTGGGCGGGT GAGCTAGA GGGTGAACTC ATTACTTCTC 6720 TCCAGTCGAT GGCGGTAGAG CACTCCCGAC AGTAGGTGA GGCTTTCTTC GCTTCACGAC 6780 -- PD TGTCTAAGAA GCTITACACT CTCCGTACTT AGAAGACGA CTCATTACAG GAGACATAAC 6840 PA -- GACTGGTGAA ACCATACACA TCCTTATGTG ATGCCCGAGT ATAGTAGAG ATGGGTTAAT 69 OO CCCAGATCAC CAAGGCGCA ACCAGATTCT TCAGGCTTGG CAGAAGCCT TGATCGTGTA 6960 CTAGACAAAG GTGTCGTTGT GGACGTGGG GCTCGTGTGT CGCTTGTCGG CATCGAAATC 7020 CTGACCGTCG AGGCGCGGGT CGTCGCCGCC TCGGGGACA CCTTCCTCCA CTACGCAGAA 7080 GAAATCGCCA AGATCGAACA AGCCGAACTT ACCGCCGGCG CCGAGGCGGC ACCCGAGGCC 71.40 TGACGCACAG GCCTCCCTTC GGCCGGCGTA AGGGAGGGA ATCGCTTGCA AACCATACA 7200 *k kgwpA TTAACACCTT CTCGGGTACA CACTAATCCC ATGAGTGTCA CAGACAAACG CGACGAGATG 7260 AGTACTGCCC GCGATAAGT CGCAGAATCA CAGCAGGAG TCGAATCATA CGCTGACGAG 732O TTTGCAGCCG ATATCACGGC AAAGCAAGAC GATGICAGCG ACCTTGTCGA TGCGATCACC 7380 GACTTCCAGG CGGAGATGAC CAACACGACG GATGCATTTC ACACAATGG TGACGAGTTC 7440 GCCGCTGAGG TTGACCACCT CCGTGCCGAT ATTGACGCCC AGCGGGACG GACCGGAG 7500 ATGCAGGATG CGTTCGAGGC ATATGCTGAC ATCTTCGCTA CAGAATCGC AGACAAACAA 7560 GATATCGGCA ATCTTCTGGC TGCGATTGAG GCGCTCCGAA CAGAGATGAA CTCAACCCAC 762O GGGGCATCG AAGCATATGC GGACGACTTC GCAGCCGATG TCGCTGCGCT CCGTGAATA 768O TCTGATCTGG TTGCAGCAAT CGACGACTTC CAAGAGGAA TCATCGCCGT GCAGGACGCA 7740 TTTGACAACT ACGCGGGA CTTCGATGCG GAGATCGACC AGCTCCACGC TGCCATCGCT 7800 GACCAGCACG ACAGCTTCGA CGCTACCGCG GACGCCTTCG CAGAGTACCG AGATGAGC 7860 TATCGCATAG AGGGGAAGC ACTGCTTGAG GCGATCAACG ACTTCCAGCA GGACATCGGT 7920 FG. 5,824,309 1 2 RECOMBINANT GAS VESICLES AND USES chimeric unit. Using this broad approach, Viral capsid and THEREOF recombinant bacteriophage have been engineered to display peptides on their surface (Notkins et al., Science 228.737, CROSS REFERENCE TO RELATED 1985; Smith, Science 228:1315, 1985; Clarke et al., Nature APPLICATION 330:381, 1987; Dedieu et al., J. Virol. 66:3161, 1992). Some This application claims the benefit of provisional appli recombinant vaccines use live, attenuated bacteria to deliver cation Ser. No. 60/008,200, filed Dec. 5, 1995. an exogenous antigen (Schodel et al., Infect. Immun. 62:1669, 1994, Fairweather et al., Infect. C& Immunol. BACKGROUND OF THE INVENTION 58:1323, 1990; Sutter et al., Proc. Natl Acad. Sci.89:10847, The invention relates to recombinant gas vesicles and uses 1992; Scheiflinger et al. J. Bacteriol. 174:595, 1992; Andino thereof. et al., Science 265:1448, 1995). Traditional vaccines consist of killed or attenuated patho Recombinant vaccines may be able to Serve as multivalent gens or their isolated toxins and normally include a variety antigen delivery vehicles capable of repeated use. However, of epitopes. However, it has long been known that portions there may be Significant drawbacks associated with the use of molecules can be recognized by the immune System and 15 of these recombinant vaccines. First, it is possible that that antibodies developed against the appropriate epitope(s) Subsequent exposure of vaccinated hosts to Such carriers can result in protective immunity. As a result, there has been could result in Severe immunological reactions because of considerable interest in developing vaccines composed of Sensitization to the carrier. Second, where attenuated patho less than the entire pathogen. Subunit vaccines, which gens are used, genetic recombination may pose a long term include all or a part of a protein Subunit of the infectious threat of reactivating the attenuated Strain. Finally, the organism, and peptide vaccines are examples of Such vac expense of cell culture and the need for large quantities cines. The immunogens of Subunit and peptide vaccines are viable recombinants, may make large Scale vaccination typically not Sufficiently immunogenic unless they are programs prohibitively expensive. administered in conjunction with an adjuvant, or are croSS DNA-mediated immunization represents a third approach linked to a large molecular weight protein Such as keyhole 25 non-traditional approach to vaccination. In this approach, limpet hemocyanin (KLH). DNA encoding the relevant antigen is introduced directly or The desire to use vaccines which do not involve admin indirectly into the individual being immunized. For istration of killed or attenuated pathogens has led to the example, the relevant DNA sequences can be inserted into a development of Specialized vaccine Systems. Among these plasmid which is carried by bacteria. The plasmid DNA is newer Systems are conjugate/carrier Systems that are taken up by the eukaryotic cells and this host's biosynthetic designed to improve the immunogenicity and delivery of machinery then is co-opted to produce the encoded protein Smaller antigens by physically associating, often by chemi (s) (see, e.g., Ulmer et al., Science 259:1745, 1993; cal means, Separately produced epitope and carrier/adjuvant Sizemore et al., Science 270:299-302, 1995).
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