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 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 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 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 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). Barry et al. components. Recombinant Systems in which the immunogen 35 (Nature 377:632–635, 1995) discloses a different approach is produced as a part of a larger chimeric molecule represent to genetic immunization referred to as “expression-library a Second approach to improving immunogenicity and deliv immunization.” In expression-library immunization an ery of immunogens. Direct delivery of DNA represents expression library of multiple DNA sequences defining a particular pathogen is prepared and used via genetic immu another non-traditional vaccine System. nization to Stimulate a protective immune response. This Liposomes and microbeads have been Suggested as 40 approach, in theory, permits exposure of a host to an array conjugate/carrier Systems. Because liposomes have the abil of pathogen antigens without the concomitant risks normally ity to fuse with biological membranes, they have been tested asSociated with the use of the pathogen itself. AS with any in the peroral delivery of a variety of other immunogens use of DNA per se, its potential integration into the host (Rouse, J. Am. Vet. Med. Assoc. 181:988–991, 1982; genome and disruption of normal gene function must be Childers et al., Regional Immunol. 3:289-296, 1991). As an 45 alternative, antigens have been incorporated into Synthetic, considered. degradable microbeads which are thought to Stimulate the SUMMARY OF THE INVENTION immune System and deliver molecules as particulate entities. This delivery System can be adapted to contain incorporated The invention features a composition that includes Sub adjuvant and/or cytokines. Moreover, microbeads can be 50 Stantially pure recombinant gas vesicles which have at least designed to provide a Sequential release of antigen through one heterologous peptide inserted into at least one gas timed biodegradation of the particle. (O'Hagan et al., Vac vesicle Structural protein. The recombinant gas vesicles, cine 9:768, 1991; O'Hagan et al., Immunology 73:239, when administered to a mammal, are capable of eliciting 1991). In addition, bacterial S-layers also have been studied antibodies which Specifically bind to the heterologous pep as conjugate/carrier vehicles. Because their natural repeat 55 tide. The heterologous peptide can be any peptide against spacing provides a defined geometric matrix, the chemical which one wishes to raise antibodies, e.g., a peptide found crosslinking of immunogen(s) to this carrier results in the in the gp120 protein of human immunodeficiency virus display of epitopes at known intervals and defined densities (HIV). Preferably the gas vesicle structural protein is a (Herzenberg et al., J. Exp. Med. 155:1730, 1982; Schneers gVpA-related orgVpc-related protein. son et al., Infect. C& Immun. 52:519, 1986; Schultze et al., J. 60 By "peptide derived from a particular organism or pro Immunol., 135:2319, 1987; Schultze et al., J. Immunol, tein is meant a peptide having a Sequence that is the same as 135:2319, 1987; Russell et al., Infect. & Immun. 59:4061, all or part of a protein found in that organism or protein. 1991). By "gVpA-related protein' is meant a protein which is Recombinant vaccines represent a Second type of non both homologous to and functionally equivalent to the gVp A traditional of vaccine System. Recombinant vaccines involve 65 protein of Halobacterium halobium. By “gvpC-related pro genetically engineering the immunogen and its associated tein' is meant a protein which is both homologous to and carrier, which can be an attenuated pathogen, as a Single functionally equivalent to the gVpc protein of Halobacte 5,824,309 3 4 rium halobium. Preferred proteins for insertion of peptides WTTNPGV. In this set of experiments BSA-TNP was used are the gVpA or the gvpc protein of Halobacterium halo as the antigen for ELISA. bium. Preferred gas vesicles are the gas vesicles of Halo FIG. 1, panel B is a graph depicting the results of ELISA bacterium halobium. used to measure the level of particular antibodies present in The heterologous peptide is inserted, in frame, into the the Serum of mice 2 weeks after a first immunization with gas vesicle Structural protein by preparing a chimeric gene. WTTNPGV. In this set of experiments BSA-TNP was used Those skilled in the art can prepare a nucleic acid molecule as the antigen for ELISA. encoding the peptide of interest. This nucleic acid molecule FIG. 1, panel C is a graph depicting the results of ELISA can be inserted into a gene encoding a gas vesicle Structural used to measure the level of particular antibodies present in protein to prepare a recombinant gas vesicle gene which the Serum of mice after a Second immunization with encodes gas vesicle protein with the peptide inserted in WTTNPGV. In this set of experiments BSA-TNP was used frame. The Sequence of the peptide can be based on the as the antigen for ELISA. Sequence of all or part of any desired protein present in the FIG. 1, panel D is a graph depicting the results of ELISA molecule or pathogen or organism to which antibodies are to used to measure the level of particular antibodies present in be raised. 15 the Serum of mice after a Second immunization with By "peptide' is meant any chain of amino acids, regard WTTNPGV. In this set of experiments BSA-TNP was used less of length or post-translational modification, e.g., gly as the antigen for ELISA. cosylation or phosphorylation, and includes polypeptides In FIG. 1, panels A-D the following symbols were used: and proteins. Preferred peptides for insertion into gas vesicle filled circles: 1 mg WTTNP GV IgM; filled triangles: 0.1 proteins are at least 50 amino acids long, more preferably at mg WTTNPGV IgM; filled diamonds: 1 mg. WTGV IgM; least 20 amino acids long, even more preferably at least 10 open circles: 1 mg WTTNPGV IgG; open triangles: 0.1 mg amino acids long. They may also be at least 7, 6, or 5 amino WTTNPGV IgG; open triangles: 0.1 mg WTTNPGV IgG; acids long or even at least. Peptides can also be 5 kD or even open diamonds: 1 mg. WTGV IgG, and open squares: buffer larger. IgG. By "Substantially pure' is meant a preparation, e.g., of 25 FIG. 2, panel A is a graph depicting the results of ELISA recombinant gas vesicles, which is at least 60% by weight used to measure the level of particular antibodies present in (dry weight) the material of interest, e.g., recombinant gas the Serum of mice 4 weeks after immunization with mutant vesicle. Preferably the preparation is at least 75%, more TNP GV. In this set of experiments BSA-TNP was used as preferably at least 90%, and most preferably at least 99%, by an antigen for ELISA. weight the compound of interest. Purity can be measured by FIG. 2, panel B is a graph depicting the results of ELISA any appropriate method, e.g., column chromatography, poly used to measure the level of particular antibodies present in acrylamide gel electrophoresis, or HPLC analysis. Amino the Serum of mice 4 weeks after immunization with mutant acid analysis is the preferred method for assessing the purity TNPGV. In this set of experiments BSA-TNP was used as of gas vesicles. an antigen for ELISA. An antibody that “specifically binds' as peptide is an 35 FIG. 2, panel C is a graph depicting the results of ELISA antibody that recognizes and binds the Selected peptide but used to measure the level of particular antibodies present in which does not Substantially recognize and bind other mol the Serum of mice after a Second immunization with mutant ecules in a Sample, e.g., a biological Sample, which naturally TNP GV. In this set of experiments BSA-TNP was used as includes the Selected peptide. Specific e binding can be an antigen for ELISA. measured using an ELISA assay in which the Selected 40 FIG. 2, panel D is a graph depicting the results of ELISA peptide is used as an antigen. used to measure the level of particular antibodies present in By "structural protein' is meant a protein present in the Serum of mice after a Second immunization with mutant mature complete gas vesicles. By "Surface protein' is meant TNP GV. In this set of experiments BSA-TNP was used as a protein present on the external Surface of mature complete 45 an antigen for ELISA. gas Vesicles. In FIG. 2, panels A-D, the following symbols were used: Unless otherwise defined, all technical and Scientific filled circles: mutant-TNP GV IgM, filled squares: buffer terms used herein have the same meaning as commonly IgM; open circles: mutant-TNP GV; and open squares: understood by one of ordinary skill in the art to which this buffer. invention belongs. Although methods and materials similar 50 FIG. 3, panel A is a graph depicting the results of ELISA or equivalent to those described herein can be used in the used to measure the level of particular antibodies present in practice or testing of the present invention, the preferred the Serum of mice after a Second immunization with mutant methods and materials are described below. All publications, TNPGV. In this set of experiments the 7 peptide patent applications, patents, and other references mentioned (ESSGTFE) (SEQID NO:1) present in mutant GV was used herein are incorporated by reference in their entirety. In case 55 as an antigen for ELISA. of conflict, the present Specification, including definitions, FIG. 3, panel B is a graph depicting the results of ELISA will control. In addition, the materials, methods, and used to measure the level of particular antibodies present in examples are illustrative only and not intended to be limit the Serum of mice after a Second immunization with mutant ing. TNPGV. In this set of experiments the 7 amino acid peptide Other features and advantages of the invention will be 60 (ESSGTFE) (SEQID NO:1) present in mutant GV was used apparent from the following detailed description, and from as an antigen for ELISA. the claims. In FIG. 3, panels A and B, the following symbols were used: filled circles: mutant-TNP GV IgM, filled squares: BRIEF DESCRIPTION OF THE DRAWINGS buffer IgM, open circles: mutant-TNP GV; and open FIG. 1, panel A is a graph depicting the results of ELISA 65 Squares: buffer. used to measure the level of particular antibodies present in FIG. 4, panel A is a graph depicting the results of ELISA the Serum of mice 2 weeks after a first immunization with used to measure the level of particular antibodies present in 5,824,309 S 6 the serum of mice 10d after a reinoculation 8 months after peptide may also be place at the carboxy terminus of the the original immunization. protein, e.g., at the carboxy terminus of gvpc or gVpA. FIG.4, panel B is a graph depicting the results of ELISA Preferred locations for insertion are within the repeats of the used to measure the level of particular antibodies present in gvpc protein or a gvpC-related protein. the serum of mice 7d after a reinoculation 8 months after the 5 Under Some circumstances it may be desirable to insert original immunization. two or more peptides into a particular structural protein. The FIG. 5, panel A is a graph depicting the results of ELISA presence of multiple antigens can often elicit a stronger or used to measure the level of particular antibodies present in more protective immune response. When two or more the serum of mice 17d after a reinoculation 8 months after peptides are inserted into a single gas vesicle Structural the original immunization. protein, they can be derived from the same or different FIG. 5, panel B is a graph depicting the results of ELISA pathogenic organism. When multiple peptides are inserted used to measure the level of particular antibodies present in they can be inserted at different locations within the gas the serum of mice 17d and 39d after a reinoculation 8 vesicle Structurallprotein or adjacent to each other at the months after the original immunization. Same location. It is not necessary that the peptides be 15 different. Thus, one can insert multiple copies of the same FIG. 6 is the nucleotide Sequence of the gVp gene cluster peptide into a gas Vesicle Structural protein. of Halobacterium halobium (SEQ ID NO:2). The recombinant gas vesicles can be used to create DETAILED DESCRIPTION compositions Suitable for expression library immunization. In this technique a library of peptide-encoding random DNA Described below are methods which can be used to fragments is prepared and inserted into one or more Selected prepare recombinant gas Vesicles having a heterologous locations in a DNA molecule encoding a gas vesicle Struc peptide inserted into protein component of the vesicle. tural protein, e.g., a plasmid bearing the gvpMLKJIHGFE Preparation of recombinant vesicles for use in eliciting DACN gene cluster of Halobacterium halobium. This pro antibodies includes four basic steps: (1) insertion of DNA ceSS results in the creation of a population of DNA encoding the peptide of interest into a coding Sequence of a 25 molecules which can be used to transform bacteria. The halobacterium gas vesicle protein to create a chimeric gas resulting clones can be used for the production of recombi vesicle protein; (2) transformation of halobacterium or Some nant gas vesicles. By purifying gas vesicles from a mixture other Suitable bacteria capable of producing gas vesicles of gas Vesicle-producing clones, it is possible to create a with the DNA encoding the chimeric protein; (3) growing “cocktail” of recombinant gas vesicles which can be injected the bacteria under conditions which permit the expression of into a patient in order to elicit an immune response. the chimeric protein and formation of gas vesicles; and (4) A preferred site for insertion of peptides is between the V and harvesting and purifying the gas Vesicles. and E of the following gvpC sequence of Halobacterium U.S. patent application Ser. No. 08/271,270 halobium. EADADVINSERTION SITEEAEAE (SEQ ID (Recombinant Vector and Process for Cell Floatation), now NO:3). abandoned, includes useful methods related to gas vesicles 35 Preparation and Isolation of Recombinant Gas Vesicles and is incorporated by reference. Simon et al. (-A Laboratory Manual Introduction of Heterologous Peptides Halophiles, DasSarma et al., eds., Cold Spring Harbor Any Selected peptide, e.g., a peptide of a pathogen, can be Laboratory Press, 1995) describes useful techniques for introduced into a gas vesicle protein to produce recombinant working with halophilic bacteria. gas vesicles capable of eliciting an immune response to the 40 The preferred means of inducing a bacterial cell to pro Selected peptide when the vesicle is introduced into a duce gas vesicles is to transform the cell with a plasmid mammal. Recombinant gas vesicles can bear a peptide bearing the gvpMLKJIHGFEDACN gene cluster of Halo normally found in HIV, Plasmodium falciparum, Salmonella bacterium halobium. The plasmids pNRC100, p.JHGV3, and typhi, mycoplasm, or any other pathogenic organism. In pFL2 (DasSarma et al., J. Bact. 176:7646, 1994) is a suitable Some cases it may be desirable to introduce two or more 45 plasmid bearing genes required for expression of gas heterologous peptides into a single recombinant gas vesicle. vesicles. The plasmids pHGV3 and pFL2 are H. halobium The several peptides can be derived from the same or Eschericia coli shuttle plasmids that are particularly useful different pathogenic organisms and can be inserted into the for the preparation of recombinant gas vesicles. Same gas Vesicle protein or into different gas Vesicle pro It is preferable that the heterologous plasmid be inserted teins. 50 into the GvpA gene or the GvpC gene which have been The recombinant gas vesicles of the invention can be used identified in Haloferax mediterranei (Englert et al., J. Biol. to present a wide variety of antigens. Among the Suitable Chem. 268:9329, 1993), Halobacterium halobium (Halladay peptides are peptides derived from the coat protein of HIV, et al., J. Bateriol. 175:684, 1993), and other bacteria particularly the principal neutralizing domain of HIV (Walsby et al., J.Gen. Microbiol., 134:2647, 1990). Walsby (Dedieu et al., J. Virol. 66:3161, 1992); peptides derived 55 (Microbiol. Rev. 58:94, 1994) describes bacteria that express from herpes simplex virus type 1 glycoprotein D (Notkins et gas Vesicles. Under Some circumstances it is desirable to al., Science 228:737, 1985); and peptides derived from engineer Suitable restriction Sites into Gvp.A., GvpC, or other hepatitis B virus core-pre-S protein (Schodel et al., Infect. gas vesicle proteins. Suitable insertion Sites, e.g., restriction Immun. 62:1669, 1994). Other useful peptides can be Sites, can be generated by Site-directed mutagenesis. Current derived from peptide toxins produced by pathogens. 60 Protocols in Molecular Biology, (John Wiley & Sons, New Generally, many of the peptides presented by genetically York, 1994) describes the use of site-directed mutagenesis, engineered vaccines can be used. A detailed description of as well as a wide variety of other techniques that can be used peptides used in genetically engineered vaccines can be to construct recombinant gas Vesicles useful in the inven found in Ciardi et al., “Genetically Engineered Vaccines' tion. (Plenum Press, New York, 1992). 65 H. halobium can be cultured in a medium containing The Selected heterologous peptide can be inserted into a 4.3M NaCl as described by Das.Sarma et al. (Proc. Natl gas vesicle Structural protein at any Suitable location. The Acad. Sci. USA 85:6861, 1988). The nucleotide sequence of 5,824,309 7 8 the gVp gene cluster of H.halobium is disclosed in Halladay C and D present the secondary IgM and IgG response (filled (J. Bact. 175:684, 1993). A partial sequence is disclosed in circles: 1 mg WTTNP GV IgM; filled triangles: 0.1 mg Jones et al. (Gene 102: 117, 1991). FIG. 6 includes a WTTNPGV IgM; filled diamonds: 1 mg WTGV IgM; open Sequence of the gVp gene cluster of H. halobium. circles: 1 mg WTTNP GV IgG; open triangles: 0.1 mg H. halobium can be transformed using the EDTA WTTNPGV IgG; open triangles: 0.1 mg WTTNPGV IgG; polyethylene glycol procedure of Cline et al. (J. Bateriol. open diamonds: 1 mg. WTGV IgG; open squares: buffer 169:1341, 1987). IgG). Recombinant Gas Vesicles Can Elicit a Long-Lived Immune FIG.2 depicts the results of experiments in which mutant Response TNP GV were used to immunize mice and BSA-TNP was Wild type (WT) and peptide-inserted gas vesicles (GV) used as an antigen for ELISA. FIG.2, panels A and B present were prepared as follows. Halobacterium halobium SD109 the results for the 4 week primary IgM response and the 4 (pFL2) (WTGV) and SD109(pFL2C:K1A) (Modified GV; week primary IgG response respectively while panels C and includes the peptide ESSGTF insert into gvpc of Halobac D present the Secondary IgM and IgG response (filled terium halobium at between the V and E of the sequence circles: mutant-TNP GV IgM, filled squares: buffer IgM; EADADVEAEAE) (SEQ ID NO:3) were grown to conflu 15 open circles: mutant-TNPGV; open squares: buffer). ency on peptone-Salt plates Supplemented with 10 uM B FIG. 3 depicts the results of experiments in mutant-TNP mevinoloin. Gas vesicles were isolated by centrifugally GV were used to immunize mice and the 7 amino acid accelerated floatation (Simon et al., Supra). The yield of gas peptide (ESSGTFE) (SEQ ID NO: 1) present in mutant GV vesicles was approximately 10 mg/l of plates (plate Surface was used as an antigen for ELISA. FIG. 3, panels A and B area/liter was approximately 1134 cm). present the results for Secondary IgM and Secondary IgG A portion of the WT and peptide inserted GV were used response (filled circles: mutant-TNPGV IgM, filled squares: to prepare trinitrophenol (TNP)-modified WT and peptide buffer IgM, open circles: mutant-TNP GV IgG, open inserted GV as follows. Modification used was performed Squares: buffer IgG). according to the modification procedure described by Little These results demonstrate that GV can effectively present et al. (Methods in Immunology and Immunochemistry, Vol. 25 TNP and peptide haptens in the absence of added adjuvant. 1, pp. 128-133, 1967). Briefly, approximately 10 mg of each These experiments also demonstrate that both IgG and IgM type of GV was incubated with 2,4,6 trinitrobenzene Sul responses can be elicited. The IgG response increased after fonic acid over night at room temperature in the dark. The the Secondary immunization while the IgM response peaked GV were then purified by dialysis against PBS (150 mM prior to the boost. NaCl; 10 mM phosphate, pH 7.5). FIG. 5, panel A, FIG. 5, panel B, FIG. 6, panel A, and FIG. Groups of 8 week old mice (4 per group) were injected 6, panel B present the results of ELISA used to demonstrate intraperitoneally (approximately 0.5 mg/GV per animal) the immune response elicited by the gas vesicles lasts for with WT, mutant, WTTNP, or mutant-TNP gas vesicles. A months. group of mice were injected with PBS as a control. Serum was collected 2 weeks and 4 weeks after the primary 35 These results demonstrate that gas vesicles having a immunization. The mice were then boosted with an addi peptide inserted into a gas vesicle Structural protein can tional 0.5 mg of GV. Serum was collected again 10 days after elicit a long-lived immune response. Accordingly, Such gas the Secondary immunization. The injections did not have a vesicles can be used as a vaccine. Significant effect on weight gain. No lesions were observed Use at the Site of injection. 40 The recombinant gas vesicles of the invention may be Immune response was measured by ELISA using either used to immunize patients using Standard methods. Gener bovine serum albumin conjugated to TNP (BSA-TNP) or the ally they are mixed with a pharmaceutically acceptable Seven amino acid peptide present in mutant GV as antigen. carrier and administered by injection. Anti-mouse IgG horseradish peroxidase or anti-mouse IgM Animal models, e.g., the murine model described herein, horseradish peroxidase. The results of this analysis are 45 can be used to test the immune response elicited by a presented in FIGS. 1-3. Selected recombinant gas Vesicle. Animal models can also be FIG. 1 depicts the results of experiments in which used by those skilled in the art to estimate the dosage WTTNP GV were used to immunize mice and BSA-TNP required to provide protection against a given pathogen. In was used as an antigen for ELISA. FIG. 1, panels A and B general, methods and dosages for recombinant vaccines present the results for the 2 week primary IgM response and 50 used by those skilled in the art are applicable to the vaccines the 2 week primary IgG response respectively while panels of the present invention.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 3

( 2) INFORMATION FOR SEQ ID NO:1: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

5,824,309

-continued (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

i i MOLECULETYPE: peppeptide ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:3: G 1 u A la A s p A a A s p V a 1 G 1 u A a G 1 u A a G 1 u 1. 5 1 O

We claim: 7. The composition of claim 4 wherein said peptide is 1. A composition comprising Substantially pure recombi inserted into the gVpA protein. nant gas vesicles comprising a Selected peptide inserted 15 8. A method for eliciting, in a non-human mammal, in-frame into a structural protein of Said recombinant gas antibodies which specifically bind a Selected peptide, Said vesicle, Said composition, when injected into a mammal, method comprising: elicits antibodies which specifically bind Said Selected pep injecting into Said mammal Substantially pure recombi tide. nant gas vesicles comprising a Selected peptide inserted 2. The composition of claim 1 wherein Said peptide is in-frame into a Surface protein of Said recombinant gas derived from a pathogen. Vesicle, wherein Said mammal produces antibodies 3. The composition of claim 5 wherein said viral coat which Specifically bind Said Selected peptide. protein is gp120. 9. The method of claim 8 wherein said recombinant gas 4. The composition of claim 1 wherein Said gas Vesicle is vesicles comprise distinct recombinant gas vesicles each a Halobacterium halobium gas vesicle. 25 comprising a different Selected peptide. 5. The composition of claim 2 wherein said peptide is 10. The method of claim 8 wherein said gas vesicles derived from a viral coat protein. comprise at least two Selected peptides. 6. The composition of claim 4 wherein Said peptide is inserted into the gvpC protein. k k k k k

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5, 824, 309

DATED : October 20, 1998

INVENTOR(S) : Shiladitya Das Sarma, Fazeela Morshed, Elizabeth Stuart and Samuel Black

it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In column l, before "CROSS REFERENCE TO RELATED APPLICATIONS" insert the following paragraph:

--This invention was made with government support under grant #MCB-922 1144 by the National Science Foundation, and the government has certain rights to the invention. --

Signed and Sealed this Twenty-fifth Day of May, 1999

Q. TODD DICKINSON

Attesting Officer Acting Connaissioner of Patents and Trademarks