BACKGROUND Yersinia pestis (Y. pestis) is the etiological agent of plague, which causes virulent and lethal infections in humans, and which is one of the agents of major concern for use as a biological weapon against civilian or military communities. Although antibiotics can be used to treat the disease, successful vaccination against Y. pestis infection would be a more effective approach to prevent plague. Currently, there is no plague vaccine available, and vaccines made in the past had problems regarding safety and efficacy. The experimental Y pestis vaccine candidates under development by US and UK military biomedical researchers have not shown efficient protection against a pneumonic form of plague, a rapidly fatal disease induced by aerosol infection, and the form most likely to result from a biological attack.

SUMMARY The invention features nucleic acid and polypeptide compositions that can elicit a immune responses against Yersinia pestis. In particular, these nucleic acids and polypeptides can induce protective responses against pneumonic and bubonic forms of Yersinia pestis infection. The nucleic acid compositions encoding Yersiniapestis antigens can include modifications to enhance expression in a subject, such as sequences encoding signal peptides that direct secretion of the Yersiraia pestis antigens in a host (e. g.

a mammalian host). The invention also includes methods of using the nucleic acid and polypeptide compositions.

In one aspect, the invention features isolated polynucleotides including a first nucleic acid that encodes a signal sequence or a biologically active fragment thereof, and a second nucleic acid that encodes a Yersinia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid and the second nucleic acid are linked such that the signal sequence and the Yersinia pestis antigen polypeptide are expressed as a fusion, and wherein the signal sequence or biologically active fragment thereof is <BR> <BR> sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell). The<BR> Yersinia pestis antigen polypeptide can be selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, and a YscF antigen polypeptide.

The signal sequence can be a heterologous signal sequence, e. g. , a mammalian signal sequence, or a bacterial signal sequence. The signal sequence can be a tissue plasminogen activator (tPA) signal sequence. In various embodiments, the polynucleotide, when expressed in a cell, produces a Yersinia pestis antigen polypeptide that is free of the signal sequence, e. g. , when the polynucleotide is expressed in a mammalian cell, the signal sequence is cleaved in the cell.

In various embodiments, the second nucleic acid encodes a V antigen <BR> <BR> polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID<BR> NO : 13, or an antigenic fragment thereof ; an Fl antigen polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO : 15, or an antigenic fragment <BR> <BR> thereof; a YopB antigen polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO : 16, or an antigenic fragment thereof; a YopD antigen <BR> <BR> polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID<BR> NO : 17, or an antigenic fragment thereof; a YopO antigen polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO : 18, or an antigenic fragment thereof; a Pla antigen polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO : 20, or an antigenic fragment thereof; or a YscF antigen

polypeptide, e. g. , that is at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO : 21, or an antigenic fragment thereof.

For example, the second nucleotide can include a sequence at least 85% identical to SEQ ID NO: 2, or a portion thereof; a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 4, or a portion thereof; a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 5, or a portion thereof; a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 6, or a portion thereof; a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 7, or a portion thereof; a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 9, or a portion thereof; or a sequence at least 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 10, or a portion thereof.

The invention also features expression vectors including the polynucleotides described herein. In various embodiments, the expression vector includes a promoter that is constituitively active in human, cells (e. g. , a CMV promoter).

The invention also features cells including the polynucleotides described herein, e. g., eukaryotic cells, such as a mammalian (e. g. , human) cells.

In another aspect, the invention features a process for producing a Ye7si71ia pestis antigen polypeptide by culturing a cell under conditions sufficient to produce the Yersinia pestis antigen polypeptide, and isolating the Yersinia pestis antigen polypeptide, wherein the cell includes a polynucleotide including a first nucleic acid that encodes a signal sequence or a biologically active fragment thereof, and a second nucleic acid that encodes a Yersinia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid and the second nucleic acid are linked such that the signal sequence and the Yersiniapestis antigen polypeptide are expressed as a fusion, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell).

The invention also features compositions that include polynucleotides, wherein the polynucleotides include a first nucleic acid that encodes a signal sequence or a biologically active fragment thereof, and a second nucleic acid that encodes a Yersinia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid

and the second nucleic acid are linked such that the signal sequence and the Yersinia pestis antigen polypeptide are expressed as a fusion, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell). The Yersinia pestis antigen polypeptide can be selected from a V antigen polypeptide, an Fl antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, and a YscF antigen polypeptide. The polynucleotides can include other features described herein.

The compositions induce an immune response in the subject to which they are <BR> <BR> administered, e. g. , a mammalian subject, e. g. , a human subject. In various embodiments,<BR> the response is a protective response, e. g. , partially or fully protective against a subsequent challenge by Yersinia pestis after immunization.

The compositions can further include one or more polynucleotides encoding a Y. pestis antigen polypeptide selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a YopB polypeptide, a YopD polypeptide, a YopO polypeptide, a YscF polypeptide, and antigenic fragments thereof.

The compositions can further include one or more polypeptides selected from a V antigen polypeptide, an F 1 antigen polypeptide, a YopB polypeptide, a YopD polypeptide, a YopO polypeptide, a YscF polypeptide, and antigenic fragments thereof.

In one embodiment, the composition includes a V antigen, e. g. , a multimeric form of the V antigen.

In another aspect, the invention features kits that include a polynucleotide and one or more Yersinia pestis antigen polypeptides selected from, e. g. , the following: a V antigen polypeptide, an F1 antigen polypeptide, a YopB polypeptide, a YopD polypeptide, a YopO polypeptide, a YscF polypeptide, and antigenic fragments thereof.

The polynucleotide can include a first nucleic acid that encodes a signal sequence or a biologically active fragment thereof, and a second nucleic acid that encodes a Yersinia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid and the second nucleic acid are linked such that the signal sequence and the Yersinia pestis antigen polypeptide are expressed as a fusion, and wherein the signal sequence or

biologically active fragment thereof is sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell). The Yersinia pestis antigen polypeptide encoded by the polynucleotide can be selected from a V antigen polypeptide, an Fl antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, and a YscF antigen polypeptide. The polynucleotides can include other features described herein. The kits can include instructions for administering the polynucleotides to a subject in an amount sufficient to induce an immune response, e. g. , a protective immune response.

In another aspect, the invention features modified Yersinia pestis antigen polypeptides that include a Yersinia pestis antigen polypeptide sequence or antigenic fragment thereof, linked to a signal sequence or biologically active fragment of the signal sequence. The Yersinia pestis antigen polypeptide is selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, a YscF antigen polypeptide, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion from a cell.

The signal sequence can be a signal sequence that increases secretion and/or expression of the polypeptide or antigenic fragment thereof from a cell, e. g. , a mammalian cell, relative to a polypeptide that does not contain the signal sequence.

For example, the signal sequence can be a mammalian signal sequence, e. g. , a tissue plasminogen activator (tPA) signal sequence.

In another aspect, the invention features compositions including a Yersinia pestis antigen polypeptide that includes a Yersiiiia pestis antigen polypeptide sequence or antigenic fragment thereof, linked to a signal sequence or biologically active fragment of the signal sequence. The Yersinia pestis antigen polypeptide can be selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, a YscF antigen polypeptide, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion from a cell.

The invention also features compositions that include Yersinia pestis antigen polypeptides or antigenic fragments thereof, which are substantially free of bacterial antigens other than the Yef°sifxia pestis antigen polypeptide. The Yersinia pestis antigen polypeptide can be selected from a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, a YscF antigen polypeptide. For example, the Yersinia pestis <BR> <BR> antigen polypeptides are produced in a mammalian cell and, e. g. , the compositions<BR> include less than 10%, 5%, 2.5%, 1%, 0.5% bacterial antigens (e. g. , proteins, lipids, carbohydrates) other than the Yersinia pestis polypeptide. The Yersinia pestis polypeptides of the composition can include features characteristic of polypeptides produced in mammalian cells, such as glycosylations, signal peptide cleavage, and other post-translational modifications.

In one embodiment, the composition includes a V antigen or antigenic fragment thereof. The composition can further include a YopB polypeptide, a YopD polypeptide, a YopO polypeptide, a YscF polypeptide, and antigenic fragments thereof.

In another aspect, the invention features cells that include a modified Yersinia pestis antigen polypeptide, e. g. , a Yersinia pestis antigen polypeptide that includes a Yersina pestis antigen polypeptide sequence or antigenic fragment thereof, linked to a signal sequence or biologically active fragment of the signal sequence. The Yersinia pestis antigen polypeptide can be selected from, e. g. , a V antigen polypeptide, an Fl antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, a YscF antigen polypeptide, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion from a cell. The cells can be eukaryotic cells, e. g. , mammalian cells.

In another aspect, the invention features methods of inducing an immune response in a mammal. The methods include, for example: administering to a mammal susceptible to or having a Yersinia pestis infection an expression vector that includes a polynucleotide encoding a Yersinia pestis polypeptide antigen in an amount sufficient to produce an immune response, e. g. , a protective immune response, in the mammal.

The methods can induce protective (e. g. , fully or partially protective) immune<BR> responses (e. g. , responses that reduce or inhibit infection upon challenge mitla Y pestis).

The responses can protect from pneumonic or bubonic forms of Y. pestis infection (e. g., infection by aerosolized droplets).

The expression vector can include a polynucleotide that includes a first nucleic acid encoding a signal sequence or a biologically active fragment thereof, and a second nucleic acid encoding a Yersinia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid and the second nucleic acid are linked such that the signal sequence and the Yersinia pestis antigen polypeptide are expressed as a fusion, and wherein the signal sequence or biologically active fragment thereof is sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell). The polynucleotide can include other features described herein.

The methods can further include administering a Yersinia pestis antigen polypeptide, e. g. , a Yersinia pestis antigen described herein, e. g. , a modified Yersinia<BR> pestis antigen polypeptide, e. g. , a Yersinia pestis antigen polypeptide that includes a Yersinia pestis antigen polypeptide sequence or antigenic fragment thereof, linked to a signal sequence or biologically active fragment of the signal sequence. The methods can further include administering one or more additional Y. pestis antigen polypeptides (e. g., F1, YscF, YopB, YopD, or YopO).

In another aspect, the invention features methods of inducing an immune response in a mammal. The methods can include, for example: administering to a mammal susceptible to or having a Yersinia pestis infection a composition including a modified Yersinia pestis antigen polypeptide or antigenic fragment thereof in an amount sufficient to produce an immune response, e. g. , a protective immune response, in the mammal. The<BR> modified Yersinia pestis antigen polypeptide is selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, a YscF antigen polypeptide. In one embodiment, the Yersinia pestis antigen polypeptide is administered to the mammal via an expression vector including a nucleic acid encoding the modified Yersi77ia pestis antigen polypeptide.

The methods can induce protective (e. g. , fully protective, or partially protective)<BR> immune responses (e. g. , responses that reduce or inhibit infection upon challenge with Y. pestis). The responses can protect from pneumonic or bubonic forms of Y. pestis infection (e. g. , infection by aerosolized droplets).

The invention also features kits that include compositions of isolated polynucleotides, wherein the polynucleotides include a first nucleic acid that encodes a signal sequence or a biologically active fragment thereof, and a second nucleic acid that encodes a Yersitzia pestis antigen polypeptide or antigenic fragment thereof, wherein the first nucleic acid and the second nucleic acid are linked such that the signal sequence and the Yersinia pestis antigen polypeptide are expressed as a fusion, and wherein the signal <BR> <BR> sequence or biologically active fragment thereof is sufficient for secretion (e. g. , sufficient for secretion from a mammalian cell). The Yersinia pestis antigen polypeptide can be selected from, e. g. , a V antigen polypeptide, an F1 antigen polypeptide, a Pla antigen polypeptide, a YopB antigen polypeptide, a YopD antigen polypeptide, a YopO antigen polypeptide, and a YscF antigen polypeptide.

The kits can include instructions for administration to a subject in an amount sufficient for inducing an immune response (e. g. , a protective immune response).

In another aspect, the invention features an isolated immunoglobulin which recognizes and specifically binds to a modified Yersinia pestis antigen polypeptide or <BR> <BR> antigenic fragment thereof (e. g. , a modified Yersinia pestis antigen polypeptide or<BR> antigenic fragment described herein). The immunoglobulin can be, e. g. , a monoclonal antibody, a chimeric antibody, a human antibody, a bispecific antibody, a humanized <BR> <BR> antibody, a primatized antibody, or an antibody fragment (e. g. , a Fab, Fab', F (ab') 2, F (v), or scFv).

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, immunology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e. g., <BR> <BR> Sambrook et al, "Molecular Cloning: A Laboratory Manual" (3rd edition, 2001);"Current<BR> Protocols in Molecular Biology"Volumes I-IV [Ausubel, R. M. , ed. (2002 and updated bimonthly)] ;"Cell Biology: A Laboratory Handbook"Volumes I-III [J. E. Celis, ed.

(1994) ];"Current Protocols in hnmunology"Volumes I-IV [Coligan, J. E. , ed. (2002 and<BR> updated bimonthly)] ; "Oligonucleotide Synthesis" (M. J. Gait ed. 1984);"Nucleic Acid<BR> Hybridization" [B. D. Hames & S. J. Higgins eds. (1985) ] ; "Transcription And<BR> Translation" [B. D. Hames & S. J. Higgins, eds. (1984)] ; "Culture of Animal Cells, 4th edition" [R. I. Freshney, ed. (2000)] ;"Immobilized Cells And Enzymes" [IRL Press, (1986) ]; B. Perbal, "A Practical Guide To Molecular Cloning" (1988); Using Antibodies : A Laboratory Manual : Portable Protocol No. I, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1998) ; Using Antibodies : A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999).

Unless otherwise stated, the following terms used in the specification and claims have the meanings given below: "YPVA"means Yersinia pestis V antigen.

The phrase"modified Y. pestis antigens"includes the modified YPVAs, modified Y. pestis F1 antigens, modified Y. pestis YopB antigens, modified Y. pestis YopD antigens, modified Y. pestis YopO antigens, and modified Y. pestis Pla antigens of the present invention.

An"immunoglobulin"includes antibodies and antibody fragments with immunogenic activity. The term"antibody"refers to immunoglobulins, including whole antibodies as well as fragments thereof that recognize or bind to specific epitopes. The term antibody encompasses polyclonal, monoclonal, bispecific, human, humanized, primatized, and chimeric antibodies, the last mentioned described in further detail in U. S.

Patent Nos. 4,816, 397 and 4,816, 567. The term"epitope"is used to identify one or more portions of an antigen or an immunogen which is recognized or recognizable by antibodies or other immune system components.

Exemplary immunoglobulins are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope. Antibody fragments include but are not limited to those portions known in the art as Fab, Fab', F (ab') 2, F (v), and scFv which portions are preferred for use in the therapeutic methods described herein.

Fab and F (ab') 2 portions of antibody fragments are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well-known. See, for example, U. S. Patent No. 4,342, 566 to Theofilopolous et al. Fab'antibody portions are also well-known and are produced from F (ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation with a reagent such as iodoacetamide. An antibody containing intact antibody portions is preferred herein.

An"antibody combining site"is that structural portion of an antibody comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase"monoclonal antibody"in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular epitope on an antigen. A monoclonal antibody may therefore contain a <BR> <BR> plurality of antibody combining sites, each immunospecific for a different antigen, e. g. , a bispecific monoclonal antibody.

By"animal"is meant any member of the animal kingdom including vertebrates (e. g., frogs, salamanders, chickens, fish, or horses) and invertebrates (e. g., worms, etc. ).<BR> <P>"Animal"is also meant to include"mammals. "Preferred mammals include livestock animals (e. g., ungulates, such as cattle, buffalo, horses, sheep, pigs and goats), as well as rodents (e. g., mice, hamsters, rats and guinea pigs), canines, felines, primates, lupine, camelid, cervidae, and rodent.

A DNA sequence is"operatively linked"to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term"operatively linked"includes having an appropriate start signal (e. g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

"Biological sample"is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject; wherein said sample can be blood, serum, an urine sample, a fecal sample, a tumor sample, a cellular wash, an oral sample, sputum, biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture.

A DNA"coding sequence"is a double stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e. g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3'to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

"Concurrent administration,""administration in combination,""simultaneous administration, "or"administered simultaneously"mean that the compounds are administered at the same point in time or sufficiently close in time that the results observed are essentially the same as if the two or more compounds were administered at the same point in time.

A"DNA molecule"refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double stranded DNA found, inter alia, in linear DNA molecules (e. g. , restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5'to 3'direction along the

nontranscribed strand of DNA (i. e. , the strand having a sequence homologous to the<BR> mRNA).

An"expression control sequence"is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.

By"isolated"or"purified"is meant a Y. pestis antigen nucleic acid molecule or protein, or a antigenic portion thereof, or an antibody substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an"isolated"nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i. e. , sequences located at the 5'and 3'ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the invention, "isolated"when used to refer to nucleic acid molecules, excludes isolated chromosomes. For example, in various embodiments, the isolated Y. pestis antigen nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 lcb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. An Y. pestis antigen protein that is substantially free of cellular material includes preparations of Y. pestis antigen protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non Y. pestis antigen protein. When the Y. pestis antigen protein or antigenic portion thereof is recombinantly produced, preferably, culture medium represents less than about 30%, 20%, 10%, or 5% of the volume of the protein preparation. When Y. pestis antigen protein is produced by chemical synthesis, preferably the protein preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non Y. pestis antigen chemicals.

The term"oligonucleotide, "as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The phrase"phannaceutically acceptable"refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or

similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The term"pharmaceutically acceptable carrier, "as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

"Primatized antibody"means a recombinant antibody containing primate variable sequences or antigen binding portions and human constant domain sequences.

The term"primer"as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i. e. , in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single stranded or double stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be"substantially"complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non- complementary nucleotide fragment may be attached to the 5'end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

A"promoter sequence"is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3'terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA"boxes and"CAT"boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the-10 and-35 consensus sequences.

A"replicon"is any genetic element (e. g., plasmid, chromosome, virus) that <BR> <BR> functions as an autonomous unit of DNA replication in vivo ; i. e. , capable of replication under its own control.

"Therapeutically effective amount"is meant an amount sufficient to prevent or reduce some feature of pathology such as for example, elevated blood pressure, respiratory output, etc.

A cell has been"transformed"by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.

This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clones a population of cells derived from a single cell or common ancestor by mitosis.

A"cell line"is a clone of a primary cell that is capable of stable growth in vitro for many generations.

As used herein, the term"substantially identical" (or"substantially homologous") refers to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e. g. , with a similar side chain, e. g. , conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have at least 80% sequence identity. In the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.

Calculations of"identity"between two sequences are performed as follows. The <BR> <BR> sequences are aligned for optimal comparison purposes (e. g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).

The length of a reference sequence aligned for comparison purposes is at least 50% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid"identity"is equivalent to amino acid or nucleic acid"homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent identity between two sequences is determined using the Needleman and Wunsch, J. Mol. Biol., 48: 444-453,1970, algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

As used herein, the term"hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions"describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be

found in Current Protocols in Molecular Biology. John Wiley & Sons, N. Y. 6.3. 1-6.3. 6, 1989, which is incorporated herein by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions: 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0. 1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions: 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C ; 3) high stringency hybridization conditions: 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0. 1 % SDS at 65°C ; and 4) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. <BR> <BR> <P>"Treating"or"treatment"refer to prophylactic treatment (i. e. , pre-infection) and<BR> therapeutic (i. e. , post-infection) methods. A protective immune response is an immune response that provides inhibition of infection by a pathogen (e. g., Yersinia pestis) after challenge with the pathogen.

A"protective immune response"is a humoral and/or cell-mediated immune response in a mammal to an immunization with a composition described herein, that interferes with or inhibits the activity, spread, and/or growth of Y. pestis following a subsequent challenge after immunization.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of plague antigen inserts used in DNA vaccine constructs. The wild-type genes and the genes with tPA leader sequence were cloned into the pJW4303 expression vector downstream of the cytomegalovirus promoter and intron A sequence.

Figure 2 is a schematic diagram depicting the schedule of DNA immunization of Balb/c mice. Each mouse received three immunizations at one month intervals, followed by a five-month resting period. Animals received the fourth immunization at week 28 and were challenged with Y pestis fourteen days later.

Figures 3A-3C are a set of graphs depicting results of ELISA assays to compare antigen-specific antibody responses to wild-type and tPA signal-sequence-encoding plasmids. Balb/c mice were immunized with V (Fig. 3A), F1 (Fig. 3B), and Pla (Fig. 3C) DNA as indicated. Sera were collected, pooled, and tested after the third vaccination.

Results shown were average titers from several ELISA assays.

Figures 4A and 4B are a set of graphs illustrating anti-V specific humoral responses in mice induced by wild-type V DNA, tPA-V DNA, or vector DNA. Arrows represent the time of immunization. Fig. 4A depicts the results of a direct ELISA assay to evaluate the quantity of anti-V antibody produced in response to tPA-V, wild-type-V, or vector DNA immunization. Fig. 4A depicts antibody responses over time and shows that tPA-V is more effective than wt-V in inducing antigen specific antibody responses.

Figures 5A-5C are graphs depicting the percent survival of immunized animals following a lethal dose of Y. pestis. Balb/c mice received DNA immunization via gene gun with DNA containing vector or wild-type/tPA Y. pestis antigen as described.

Cumulative survival curves were plotted to show the protection for groups immunized with Pla DNA (Fig. 5A), F1 DNA (Fig. 5B), and V DNA (Fig. 5C), or vector DNA as indicated in each figure.

Figure 6 is a Western blot showing wt-V and tPA-V antigen expression in vitro.

V antigen was transiently transfected into 293T cell using and detected using an antibody against the V protein. Antigens can be detected both in supernatant (S) and in cellular lysates (L).

Figure 7 is a graph depicting the results of measurements to determine the IgG isotype of mouse anti-V antibody responses from animal receiving wt-V or tPA-V DNA immunization.

Figure 8 is a schematic diagram of Y pestis antigen inserts used in DNA vaccine constructs. The wild-type genes and the fusion tPA genes with tPA leader sequence were cloned into the pJW4303 expression vector downstream of the cytomegalovirus promoter and intron A sequence as described. A subset of constructs (YopD-dTM and YobB- dTM) contained deletions in hydrophobic regions of the proteins. YopD-dTM has a deletion of amino acids 129-149. YopB-dTM has a deletion of amino acids 166-258.

Figure 9 is a Western blot of YopB antigens in transiently transfected 293T cell supernatants (S) and lysates (L) using sera from YopB DNA-immunized mice as the detecting antibody. YopB-wt: wild-type YopB DNA vaccine; YopB-dTM: modified YopB DNA vaccine with the fusion of tPA at N-terminus and deletion of the hydrophobic domain; Vector: pJW4303 vector control.

Figure 10 is a Western blot of YopD antigen in transiently transfected 293T cell supernatants (S) and lysates (L) using sera from YopD DNA-immunized mice as the detecting antibody. YopD-wt: wild-type YopD DNA vaccine; Vector: pJW4303 vector control.

Figure 11 is a Western blot of YopO antigens in transiently transfected 293T cell supernatant (S) and lysate (L) using sera from YopO DNA-immunized mice as the detecting antibody. YopO-wt: wild-type YopO DNA vaccine; YopO-tPA: DNA vaccine with the fusion of tPA at N-terminus; Vector: pJW4303 vector control.

Figure 12 is a schematic diagram of YscF plague antigen inserts used in DNA vaccine constructs. The wild-type genes and the fusion tPA genes with tPA leader sequence were cloned into the pJW4303 expression vector downstream of the cytomegalovirus promoter and intron A sequence as described.

Figures 13A-K are representations of the following exemplary nucleic acid sequences useful in compositions and methods described herein: Figure 13A. SEQ ID NO : 1 is the nucleic acid sequence of a modified Y. pestis V antigen.

Figure 13B. SEQ ID NO : 2 is the nucleic acid sequence of the wild-type Y. pestis V antigen.

Figure 13C. SEQ ID NO : 3 is the nucleic acid sequence of a modified Y. pestis F1 antigen.

Figure 13D. SEQ ID NO : 4 is the nucleic acid sequence of the wild-type Y. pestis F1 antigen.

Figure 13E. SEQ ID NO : 5 is the nucleic acid sequence of the wild-type Y. pestis YopB antigen.

Figure 13F. SEQ ID NO : 6 is the nucleic acid sequence of the wild-type Y. pestis YopD antigen.

Figure 13G. SEQ ID NO : 7 is the nucleic acid sequence of the wild-type Y. pestis YopO antigen.

Figure 13H. SEQ ID NO : 8 is the nucleic acid sequence of a modified Y. pestis Pla antigen, Figure 13I. SEQ ID NO : 9 is the nucleic acid sequence of the wild-type Pla antigen.

Figure 13J. SEQ ID NO : 10 is the nucleic acid sequence of the wild-type Y. pestis YscF antigen.

Figure 13K. SEQ ID NO : 11 is the nucleic acid sequence of a modified YscF antigen.

Figures 14A-K are representations of the following exemplary amino acid sequences useful in compositions and methods described herein: Figure 14A. SEQ ID NO : 12 is the amino acid sequence of a modified Y. pestis V antigen.

Figure 14B. SEQ ID NO : 13 is the amino acid sequence of the wild-type Y. pestis V antigen.

Figure 14C. SEQ ID NO : 14 is the amino acid sequence of a modified Y. pestis F1 antigen.

Figure 14D. SEQ ID NO : 15 is the amino acid sequence of the wild-type Y. pestis F1 antigen.

Figure 14E. SEQ ID NO : 16 is the amino acid sequence of the wild-type Y. pestis YopB antigen.

Figure 14F. SEQ ID NO : 17 is the amino acid sequence of the wild-type Y. pestis YopD antigen.

Figure 14G. SEQ ID NO : 18 is the amino acid sequence of the wild-type Y. pestis YopO antigen.

Figure 14H SEQ ID NO : 19 is the amino acid sequence of a modified Y. pestis Pla antigen.

Figure 14I. SEQ ID NO : 20 is the amino acid sequence of the wild-type Pla antigen, Figure 14J. SEQ ID NO : 21 is the amino acid sequence of the wild-type Y. pestis YscF antigen.

Figure 14K. SEQ ID NO : 22 is the amino acid sequence of a modified YscF antigen.

DETAILED DESCRIPTION There is an urgent need for a safe and effective vaccine to protect against lethal infections of Yersinia pestis. Because the bacteria is highly pathogenic and can be transmitted in aerosol form, it is feared as a potential bioterrorist agent. Currently, there is no safe and effective vaccine available to inhibit or protect from Y. pestis infection.

The invention is based, in part, on the discovery that DNA compositions encoding Y. pestis antigens can inhibit infection in mammals in vivo. Y. pestis antigens expressed in mammalian host cells, optionally in a form that includes a signal peptide, can generate beneficial immune responses to these antigens. Results presented herein indicate that DNA immunization is effective in inducing high quality, conformation sensitive antibody responses against bacterial infections when the antigen gene is properly engineered.

Y. Pestis V Antigen The V antigen of Y. pestis is a secreted protein acting as a regulator of the low calcium response and may also act as a virulence factor (Skrzypek and Straley, J.

Bacteriol., 177: 2530-2542,1995) suppressing innate immune responses by reducing the expression of tumor necrosis factor a and 7 interferon, in response to a Yersinia infection (Nakajima et al., Infect. Immun., 63: 3021-3029,1995). There is no putative signal peptide sequence at the N-terminus of V protein and it does not use the mechanism used by most secreted cellular proteins.

Data provided herein shows that DNA constructs encoding modified Y. pestis V gene inserts can be used to induce immune responses against Y. pestis. The data also illustrates that the same modified V gene insert design can be used to produce recombinant V proteins with mammalian expression systems. For example, it was discovered that a DNA encoding a modified V antigen from Y. pestis with a tissue plasminogen activator signal peptide sequence was highly immunogenic in inducing V- specific antibody responses in Balb/C mice. These mice were completely protected from the challenge of a lethal dose of 5000 cfu of Y. pestis (Kim Strain) by intranasal inoculation. DNA constructs expressing the wild type V antigen and another Y. pestis antigen, Fl, induced partial protection. 293T cells transiently transfected with the <BR> <BR> modified V DNA vaccine (i. e. , modified to include the mammalian tPA signal sequence) secreted much more V antigen than cells transfected with a construct encoding a wild type V antigen. The modified V antigen, but not the wild-type V antigen, was able to form oligomers, which can stimulate protective antibody responses.

Mice immunized with modified V DNA construct had higher total anti-V IgG, and, more importantly, higher IgG2a responses than those immunized with their wild- type counterpart. This indicates that the modified V antigen induced strong Thl-type responses in the animals. Therefore, addition of the tPA signal sequence to the Y. pestis V antigen produced a qualitatively different antigen which is more immunogenic and induces better immune protection against a lethal mucosal challenge of Y. pestis. This new Y. pestis V DNA construct is an attractive candidate for inducing immunity in

humans, either alone or in combination with other immunogenic antigens, or in combination with other therapies.

Y. Pestis Fl Antigen Y. pestis F1 is believed to confer resistance to phagocytosis, possibly by forming aqueous pores in the membranes of phagocytic cells (Rodrigues et al., Braz. J. Med. Biol.

Res., 25: 75-79,1992 ; Spiers etal., J. P/larm. Pharmacol., 51: 991-997,1999) or by interfering with complement-mediated opsonization (Spiers et al., J. Pharm. Pharmacol., 51: 991-997,1999 ; Williams et al., J. Infect. Dis., 126: 235-241,1972). TheFl protein forms a capsular structure at 37°C (Perry and Fetherston, Clin. Microbiol. Rev., 10: 35- 66,1997). The mature F1 protein has 149 amino acids after removal of a 21-aa leader sequence. Recombinant F1 has been expressed in Escherichia coli, harvested by ammonium sulfate precipitation, and purified by chromatography (Titball et al., Infect.

Immun., 65: 1926-1930,1997). It is readily soluble during in vitro cultivation from the bacterial expression system.

Immunogenicity of DNA constructs expressing two other Y. pestis antigens, F1 and Pla, is described herein. Two F1 DNA inserts were constructed : wt-Fl, with the original wild type sequences and tPA-Fl, with a tPA leader added to the N-terminus of the wild type F1 gene. It was discovered that the role of tPA in promoting expression of F1 was limited may not significantly enhance immune protection as compared to use of wild-type Fl. This is consistent with the fact that the Fl has an endogenous signal sequence.

Y. Pestis Pla Antigen The Pla protease is very important in bacterial pathogenesis of Y. pestis. Mapping of loci involved in expression and regulation of the Yop regulon (Goguen et al., J.

Bacteriol., 160: 842-848,1984 ; Yother and Goguen, J Bacteriol., 164: 704-711,1985) and Yop-mediated cytotoxicity (Goguen et al., Iiifect. Immun., 51: 788-794, 1986; Yother et al., J. Bacteriol., 165: 443-447,1986 ; Hoe and Goguen, J. Bacteriol., 175: 7901-7909, 1993; Hoe et al., J ; Bacteriol., 174: 4275-4286,1992) demonstrated that the Pla protease

was a true plasminogen activator, but not, as was widely believed, a coagulase (Sodeinde and Goguen, Infect. I7nmun., 57: 1517-1523,1989 ; Sodeinde et al., Infect. Imntun., 56: 2749-2752, 1988). Therefore, Pla plays a major role in determining the invasive nature of Y. pestis. The most recent studies on the suppression of neutrophil gene expression by Y. pestis showed that the activation of plasminogen by Pla was indeed a critical activity of this protease in vivo, required for the suppression of neutrophil accumulation at the foci of infection.

The importance of Pla in plague pathogenesis prompted an investigation into the efficacy of this antigen in stimulating immune responses. Pla DNA constructs were generated and tested for efficacy in blocking infection by Y. pestis. Similar to the F1 and V, a version with a tPA leader was constructed in addition to a wild type version. A prominent hydrophobic region at the N-terminus of the protein, and the addition of a tPA leader sequence alone was insufficient to lead to a detectable amount of soluble Pla antigen. These specific Pla constructs did not induce significant immune protection.

Other Y. Pestis Antigens Other Y. pestis antigens in addition to the V antigen, F1, and Pla can be expressed as described herein. Yop proteins of Y. pestis such as YopB, YopD, and YopO are virulence determinants. These proteins are secreted by Y. pestis bacteria by a complex type III secretion system (Perry and Fetherston, Clin. Microbiol. Rev., 10: 35-66, 1997).

It has been discovered that these can be modified and expressed via DNA constructs as described for the other Y. pestis antigens, above. Yet another Y. pestis antigen which can be used in inducing immune responses is YscF. Exemplary YscF constructs are described herein.

Modified Yersiiiiapestis antigens Provided herein are Y. pestis V antigens that are modified such that they include a signal sequence. Naturally occurring Yersinia pestis V antigens do not include a signal sequence. The signal sequence may be cleaved off of the modified Yersinia pestis V antigen during or after synthesis of the polypeptide. A"signal sequence"encodes a

signal peptide that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media. Signal sequences are typically located at the extreme N terminus of a polypeptide, and can be clipped off by enzymes within the host cell prior to the final steps of trafficking and secretion from the cell. Signal sequences typically have an N-terminal region of approximately 2-15 amino acids which has a net postive charge, followed by a hydrophobic region of 8 amino acids or more, and a neutral but polar C-terminal region. Residues at positions 23 and 21, relative to the signal peptidase cleavage site, must be small and neutral for cleavage to occur correctly (von Heijne, Eur. J. Bioehern., 133: 17-21,1983 ; von Heijne, J : Mol. Biol., 184 : 99-105, 1985). Numerous signal sequences are known to those of skill in the art. The use of any of these signal sequences is contemplated and those described herein are not limiting.

Certain signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes, such as tissue plasminogen activator (tPA) signal sequence, <BR> <BR> alpha factor leader sequence, and the like. "Leader sequence, signal sequence, and signal peptide"are used interchangeably herein.

In various embodiments, regions of a Y. pestis antigen may be deleted in constructing a modified antigen. For example, Yop constructs were generated in which <BR> <BR> highly hydrophobic (i. e. , putative transmembrane) regions were deleted. This strategy can allow efficient expression of the antigen.

In certain embodiments, the signal sequence is a tissue plasminogen activator signal sequence or a signal sequence that has the same function as tPA. An exemplary tPA signal sequence has the following amino acid sequence: MDAMKRGLCCVLLLCGAVFVSAS (SEQ ID NO : 29).

A modified Y. pestis V antigen is described in Example 2. This particular modified Y. pestis V antigen includes a tPA signal sequence.

Also provided herein are isolated soluble YPVA multimers. These YPVA multimers may or may not include a signal sequence. The YPVA multimers can be produced by host cells expressing modified Y. pestis V antigens. YPVA multimers can be produced by other means. The YPVA multimers can include multiple YPVA polypeptides. A multimer can be, e. g. , a dimer or a trimer.

Also provided herein are modified Fl, YopB, YopD, YopO, YscF, and Pla antigen polypeptides. Also included are compositions including any of the wild-type or modified Y. pestis antigens. A composition may include multiple Y. pestis antigens, which may be wild-type or modified (e. g. , modified to include a signal sequence). The composition may also include a polynucleotide described herein. The polynucleotides may be encode wild-type or modified polypeptides. The composition may include a vaccine, which may be a DNA vaccine.

Nucleic Acids Encoding Modified Y. pestis Antigens A modified Y. pestis antigen can include a polynucleotide including a first nucleotide that encodes a signal sequence or a biologically active fragment thereof, and a second nucleotide that encodes a Y, pestis antigen, wherein the signal sequence or biologically active fragment thereof is sufficient for secretion, e. g. , by mammalian cells.

The Y. pestis antigen can encode Y. pestis V antigen, F1 antigen, YopB antigen, YopD antigen, YopO antigen, YscF antigen, or Pla antigen, as described herein.

The modified Y. pestis antigens can be generated by molecular biological techniques standard in the genetic engineering art, including but not limited to, polymerase chain reaction (PCR), restriction enzymes, expression vectors, plasmids, and the like. By way of example, vectors may be designed which contain a signal sequence.

PCR amplified DNA fragments of a Y. pestis antigen to be modified may be digested by appropriate restriction enzymes and subcloned into the vector. Modifications of the DNA may be introduced by standard molecular biological techniques as described above.

The present invention further includes polynucleotides encoding modified F1, YopB, YopD, YopO, YscF, and Pla antigens. Also included are compositions including any of the polynucleotides encoding wild-type or modified Y. pestis antigens. A composition may include multiple polynucleotides encoding Y. pestis antigens, which may be wild-type or modified. The composition may also include a polypeptides described herein. The polypeptides may be wild-type or modified. The composition may include a vaccine, which may be a DNA vaccine.

Expression of Modified Y. pestis Antigens DNA sequences can be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate host. Such operative linking of a DNA sequence to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed in expressing wild-type and modified Y. pestis antigens. Useful expression vectors, for example, can include of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Such vectors may be plasmids, linear DNA, and the like, and can be introduced into hosts via standard methods such as transformation, transfection, electroporation, gene guns, and others. Suitable vectors include derivatives of SV40 and known bacterial plasmids (e. g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives), viral vectors, plasmids such as RP4; phage DNA (e. g. , the numerous derivatives of phage) s, e. g., NM989), and other phage DNA (e. g., M13 and filamentous single stranded phage DNA); yeast plasmids such as the 2 ; u. plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences (i. e. , sequences that control the expression of a DNA sequence operatively linked to it) can be used in these vectors to express Y. pestis antigens. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia virus, polyoma virus or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3 phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e. g., Pho5), the promoters of the yeast a-mating factors,

and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of host cells are also useful in expressing the DNA sequences of this invention. These hosts can include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts (e. g., Saccharomyces), plant cells, nematode cells, and animal cells, such as HEK-293, CHO, Rl. l, B W and L M cells, African Green Monkey kidney cells (e. g., COS-1, COS-7, BSC1, BSC40, and BMT10 cells), insect cells (e. g., Sf9 cells), and human cells and plant cells.

Proper vectors and expression control sequences will include those that are operable in the host (e. g., for expression in mammalian hosts, an appropriate vector will include promoters and other regulatory sequences used for mammalian expression). The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered in selecting a vector.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e. g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

Methods of Inducing an Immune Response in a Mammal Methods of inducing immune responses in a mammal can include administering to a mammal a nucleic acid (e. g. , a DNA expression vector or plasmid) including a polynucleotide encoding a wild-type Y pestis antigen, and the like. For example, the polynucleotide encodes a modified Y. pestis V antigen, a wild-type or modified F1

antigen, YopB, YopD, YopO, or Pla antigen. The polynucleotide can be administered in combination with an immunogen. The immunogen may be a modified Y. pestis antigen (e. g. , a modified Y. pestis antigen described herein). The method can include the administration of a wild-type or modified V, F1, YopB, YopD, YopO, YscF, or Pla antigen (or antigenic fragment thereof).

In one aspect, the method includes administering to a mammal a wild-type or modified Y. pestis antigen, e. g. , a Y. pestis V antigen or a antigenic fragment thereof. The Y. pestis antigen may be delivered in addition to other Y. pestis antigens as a mixed formulation. hi an additional aspect, both a nucleic acid and a modified Y. pestis antigen are administered to the mammal.

Also provided are processes for producing a Y. pestis V antigen, a modified Y. pestis V antigen, and other modified Y. pestis antigens. Host cells described herein may be cultured and the antigens may be obtained therefrom.

Also provided are methods of inducing an immune response in a mammal. A method can include administering to a mammal a wild-type or modified Y. pestis antigen, e. g. , a wild-type or modified Y. pestis V antigen. In a particular embodiment, the method of administering the wild-type or modified Y. pestis V antigen does not include the administration of a Y. pestis Fl antigen. The antigen may be a wild-type or modified Fl, YopB, YopD, YopO, YscF, or Pla antigen. The method may include the administration of an additional polypeptide. The method may include the administration of a polynucleotide encoding a wild-type or modified V, F1, YopB, YopD, YopO, or Pla antigen.

Nucleic acid compositions can be administered, or inoculated, to an individual as naked nucleic acid molecules (e. g. , naked DNA plasmids) in physiologically compatible solutions such as water, saline, Tris-EDTA (TE) buffer, or in phosphate buffered saline (PBS). They can also be administered in the presence of substances (e. g. , facilitating agents and adjuvants) that have the capability of promoting nucleic acid uptake or recruiting immune system cells to the site of inoculation. Nucleic acid compositions for inducing immune responses have many modes and routes of administration. They can be

administered intradermally (il), intramuscularly (IM), and by either route, they can be administered by needle injection, gene gun, or needle-less jet injection (e. g., Bioj ectofm (Bioject Inc. , Portland, OR). Other modes of administration include oral, intravenous, intraperitoneal, intrapulmonary, intravitreal, and subcutaneous inoculation. Modes of mucosal vaccination may also be employed. These include delivery, for example, via intranasal, ocular, oral, vaginal, or rectal topical routes. Delivery by these topical routes can be by nose drops, eye drops, inhalants, suppositories, or microspheres. Also possible are delivery methods which use an electroporation device, or which rely on skin surface absorption.

DNA uptake can sometimes be improved by the use of the appropriate adjuvants.

Synthetic polymers (e. g. , polyamino acids, co-polymers of amino acids, saponin, paraffin oil, muramyl dipeptide, Regressin (Vetrepharm, Athens GA), and Avridine) and liposomal formulations can be added as adjuvants to the vaccine formulation to improve DNA stability and DNA uptake by the host cells, and may decrease the dosage required to induce an effective immune response. Regardless of route, adjuvants can be administered before, during, or after administration of the nucleic acid. Not only can the adjuvant increase the uptake of nucleic acid into host cells, it can increase the expression of the antigen from the nucleic acid within the cell, induce antigen presenting cells to infiltrate the region of tissue where the antigen is being expressed, or increase the antigen-specific response provided by lymphocytes.

Nucleic acid uptake can be improved in other ways as well. For example, DNA uptake via IM delivery of vaccine can be improved by the addition of sodium phosphate to the formulation. Increased DNA uptake via IM delivery can also be accomplished by electrotransfer (e. g. , applying a series of electrical impulses to muscle immediately after DNA immunization). Adjuvants which can also be added to the vaccine to improve DNA stability and uptake as well as improve immune induction include water emulsions (e. g., complete and incomplete Freund's adjuvant), oil, Corynebacterium parvum, Bacillus Calmette Guerin, iron oxide, sodium alginate, aluminum hydroxide, aluminum and calcium salts (i. e., alum), unmethylated CpG motifs, glucan, and dextran sulfate.

Coinjection of cytokines, ubiquitin or costimulatory molecules can also help improve

immune induction. Fusions of the antigen with cytokine genes, helper epitopes, ubiquitin, or signal sequences have been successful and can also induce immune response. Fusions that aid in targeting to certain cell types can also be done. For example, antigen fused to L-selectin was successful in targeting the antigen to high endothelial venules of peripheral lymph nodes.

The medium in which the DNA vector is introduced should be physiologically acceptable for safety reasons. Suitable pharmaceutical carriers include sterile water, <BR> <BR> saline, dextrose, glucose, or other buffered solutions (e. g. , TE or PBS). Included in the medium can be physiologically acceptable preservatives, stabilizers, diluents, emulsifying agents, pH buffering agents, viscosity enhancing agents, colors, etc.

Once the DNA vaccine is delivered, the nucleic acid molecules (e. g. , DNA plasmids) are taken up into host cells, which then express the plasmid DNA as protein.

Once expressed, the protein is processed and presented in the context of self-major histocompatibility complex (MHC) class I and class II molecules. An immune response is then generated against the DNA-encoded immunogen. To improve the effectiveness of the vaccine, multiple injections can be used for therapy or prophylaxis over extended periods of time. To improve immune induction, a prime-boost strategy can be employed.

Priming vaccination with DNA and a different modality for boosting (e. g. , live viral vector or protein antigen) has been used successfully in inducing cell-mediated immunity.

The timing between priming and boosting varies and is adjusted for each vaccine.

Suitable doses of nucleic acid compositions for humans can range from 1 pg/kg to 1 mg/kg of total nucleic acid in a composition, e. g. , from 5 pg/kg-500 mg/kg of a nucleic acid composition, 10 llg/kg-250 pg/kg of a nucleic acid composition, or 10 pg/kg-170 llg/kg of a nucleic acid composition. In one embodiment, a human subject (18-50 years of age, 45-75 kg) is administered 1.2 mg-7.2 mg of a nucleic acid composition. The nucleic acid composition can include compositions containing a pool <BR> <BR> multiple nucleic acids encoding two or more distinct antigens, e. g. , V antigen, F1, YopB,<BR> YopD, YopO, and YscF. DNA vaccines can be administered multiple times, e. g. , between

two-six times, e. g. , three times. In an exemplary method, 100 llg of a DNA composition is administered to a human subject at 0,4, and 12 weeks (100 pg per administration).

Pharmaceutical compositions A modified Y. pestis antigen, polynucleotide, vaccine, antibody, composition and the like can be used in pharmaceutical compositions. The preparation of therapeutic compositions that contain polypeptides, analogs, or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. Solid forms can also be injected directly. The preparation can also be emulsified. The compositions can be delivered by oral or perinteral routes. DNA compositions can be delivered by gene gun and other means. The active ingredient in the composition is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical compositions can include a modified Y. pestis antigen or polynucleotide to induce immune responses in a host, or an antibody, or combinations thereof.

A modified Y. pestis antigen, polynucleotide, vaccine, or antibody can be formulated into a therapeutic composition as a neutralized pharmaceutically acceptable salt forms. The term"pharmaceutically acceptable salts"refers to physiologically and pharmaceutically acceptable salts of the compounds, i. e. , salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Pharmaceutically acceptable salts include the acid addition salts (i. e., formed with the free amino groups of the polypeptide or antibody), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium,

potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e. g., Berge et al. ,"Pharmaceutical Salts, "J. Pharma. Sci. , 1977,66 : 1-19).

Certain factors may influence the dosage required to effectively treat a subject, include the severity of the disease or condition, disorder, or disease, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the composition (s) can include a single treatment or, preferably, can include a series of treatments.

For nucleic acid compositions, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e. g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e. g. , lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e. g., magnesium stearate, talc or silica); disintegrants (e. g., potato starch or sodium starch glycolate); or

wetting agents (e. g. , sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e. g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e. g., lecithin or acacia); non-aqueous vehicles (e. g. , almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e. g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffers, salts, flavorings, colorings, and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active composition.

The compositions may be formulated for parenteral administration by injection, e. g., by bolus injection or continuous infusion. Formulations for injection may be <BR> <BR> presented in unit dosage form, e. g. , in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e. g. , sterile pyrogen-free water, before use.

For administration by inhalation, the compositions can be delivered in the form of an aerosol spray, presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e. g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e. g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the composition and a suitable powder base such as lactose or starch.

Pharmaceutical compositions (e. g., gene, gene transcript or protein product modulatory agents as described herein) of the present invention include, but are not

limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

In one embodiment, compositions of nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol, to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in Controlled Release of Drugs: Polymers and Aggregate Systems, 185-215 (Rosoff, M. , Ed. , 1989, VCH Publishers, New York).

Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, 271 (Mack Publishing Co. , Easton, Pa. , 1985).

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with co-surfactants. The co-surfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use of co-surfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and ki-glycerides » polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including <BR> <BR> peptides (Constantinides et al., Pharm. Res. , 1994,11 : 1385-90 ; Ritschel, Meth. Find.<BR> <P>Exp. Clin. Pharmacol. , 1993,13 : 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, <BR> <BR> improved clinical potency, and decreased toxicity (Constantinides et al. , 1994; Ho et al.,<BR> J. Pharm. Sci. , 1996,85 : 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides.

Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids and other active agents from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids and other active agents within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

Microemulsions can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and

nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants <BR> <BR> (Lee et al. , Crit. Rev. Therap. Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers, and vesicles. Vesicles, such as liposomes, are useful because of their specificity and the duration of action. As used in the present invention, the term"liposome"means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. Selection of the appropriate liposome depending on the agent to be encapsulated would be evident given what is known in the art.

To cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include: (a) liposomes obtained from natural phospholipids are biocompatible and biodegradable; (b) liposomes can incorporate a wide range of water and lipid soluble drugs; (c) liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the

cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. , Biochem. Biophys. Res. Comm. , 1987,147 : 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. , J. Controlled Release, 1992,19 : 269-74).

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids and other agents, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i. e. , surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

For pharmaceutical compositions comprising oligonucleotides, agents that enhance uptake of oligonucleotides at the cellular level may also be added to the

pharmaceutical and other compositions of the present invention. For example, cationic <BR> <BR> lipids, such as lipofectin (Junichi et al. , U. S. Pat. No. 5,705, 188), cationic glycerol<BR> derivatives, and polycationic molecules, such as polylysine (Lollo et al. , PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes (e. g., limonene and menthone).

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein,"carrier compound"or"carrier"can refer to a nucleic <BR> <BR> acid, or analog thereof, which is inert (i. e. , does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or <BR> <BR> 4-acetamido-4'-isothiocyano-stilbene-2, 2'-disulfonic acid (Miyao et al. , Antisense Res.<BR> <P>Dev. , 1995,5 : 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev. , 1996,6 : 177-183).

The pharmaceutical compositions disclosed herein may also comprise a excipients. In contrast to carrier compounds described above, these excipients include a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids or other active agents to an animal.

The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc. , when combined with a nucleic acid or other active agent and the other components of a given pharmaceutical composition.

Typical pharmaceutical carriers include, but are not limited to, binding agents (e. g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl <BR> <BR> methylcellulose, etc. ) ; fillers (e. g. , lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc. ) ; lubricants (e. g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, <BR> <BR> polyethylene glycols, sodium benzoate, sodium acetate, etc. ) ; disintegrants (e. g., starch,<BR> sodium starch glycolate, etc. ) ; and wetting agents (e. g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids and other contemplated active agents may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids or other contemplated active agents can be used.

Antibodies Antibodies, including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the modified Y. pestis antigens and/or their biologically active fragments or subunits may possess certain diagnostic applications and may, for example, be utilized for the purpose of detecting and/or measuring conditions such as viral infection or the like. For example, the modified Y. pestis antigen or fragments or subunits thereof may be used to produce both polyclonal and monoclonal antibodies, to the modified Y. pestis antigen or fragments or subunits thereof, in a variety

of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity (ies) of the modified Y. pestis antigens of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.

The present invention likewise extends to the development of antibodies against the modified Y. pestis antigens, including naturally raised and recombinantly prepared antibodies. For example, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the modified Y. pestis antigens.

The general methodology for making monoclonal antibodies by hybridomas is well known. Methods for producing monoclonal antibodies are also well-known in the art. See, e. g., Niman et al., Proc. Natl. Acad. Sci. UNA, 80: 4949-4953,1983 ; and Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999). Methods for producing polyclonal anti-polypeptide antibodies are also well-known in the art. See, e. g. , U. S. Patent No. 4,493, 795.

Antibodies raised against Y. pestis antigens can be used for various diagnostic techniques, including immunoassays. Assay systems can be prepared in the form of test kits for the analysis of the extent of the presence of a Y. pestis antigen. The system or test kit may comprise a labeled component and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner (s).

Proteins Encoded by Y. pestis DNA Sequences Proteins encoded by Y. pestis DNA sequences are useful in vaccines, in the treatment or prevention of disease, in the diagnosis or detection of a disease, and the like.

These proteins may be delivered alone, in combination with other proteins, in combination with DNA, and the like.

The present invention relates to the production of proteins encoded by Y. pestis DNA sequences described herein. These proteins may be produced in eukaryotic and

prokaryotic cells, including, but not limited to, yeast cells, bacterial cells, fungal cells, insect cells, nematode cells, plant cells, and animal cells. Suitable animal cells include, but are not limited to, HEK-293 cells, HeLa cells, COS cells, and various primary mammalian cells.

In a particular embodiment, the wild-type or modified protein may be expressed in a eukaryotic expression system. In a further embodiment, the modified protein may be produced in a bacterial system. Additional non-limiting embodiments are described herein.

Kits The invention also includes kits including the nucleic acid compositions described herein. The kits can include one or more other elements including: instructions for use; other reagents, e. g. , a diluent, devices or other materials for preparing the composition for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject. Instructions for use can include instructions for therapeutic application (e. g. , DNA vaccination and boosting) including suggested dosages and/or<BR> modes of administration, e. g. , in a human subject, as described herein. Instructions can<BR> also provide directions for prophylactic treatment, e. g. , in patients who are susceptible to<BR> Y. pestis infection, e. g. , as described herein.

The kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e. g. , a diagnostic agent to monitor a response to immune response to the compositions in the subject, or an additional therapeutic agent.

In one embodiment, the kit includes a vial (or other suitable container) containing nucleic acids encoding one or more distinct Y. pesais antigens (e. g. , a modified V antigen, YopB, YopD, YscF, or antigenic fragments thereof).

Advantages DNA immunization offers a unique advantage of producing antigens in their native conformation, because the antigens are produced in vivo and will not be subjected to in vitro manipulations during the protein purification process. Other studies reported

the use of recombinant V proteins as plague vaccine candidates, either alone or as part of an V-F1 fusion protein, which was produced from a bacterial expression system requiring cleavage and separation from a GST tag (Adamovicz, J. Efficacy of the Fl-V fusion protein vaccine in non-human primates. NIH Plague Vaccine Meeting Nov. 22,2002 ; Williamson, D. The F1 + V combined sub-unit vaccine. NIH Plague Vaccine Meeting Nov. 22,2002). This V-F1 fusion protein tended to aggregate. Previous attempts at DNA immunization with wild type V or F1 expressing DNA plasmids produced only partial protection (Carr et al. Kaccine, 18: 153-159,1999).

The compositions and methods described here offer additional options for the development of a clinical plague vaccine. DNA vaccines can be used not only alone, but also as a component in a DNA prime plus protein boost formulation. Such a combination can offer a number of advantages over the conventional single modality vaccines, because DNA and protein are individually effective in inducing different sets of immune responses. Using DNA components in the priming phase can significantly reduce the amount of protein boost, or decrease the numbers of protein immunizations required to induce the same level of protective immune responses. Therefore, DNA vaccine can substantially reduce the cost and improve the safety of a final subunit based vaccine against Y. pestis in a larger human population.

DNA components are also effective in inducing cell-mediated immune responses including Thl type immune responses. Because Y. pestis is believed to be a partially intracellular pathogen, a strong cell mediated immune response may be important in the final protection against a lethal challenge.

In various embodiments, the highly protective V antigen serves as a core component for developing a multi-antigen plague vaccine using additional Y. pestis antigens.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention, and would be readily known to the skilled artisan. Additionally, the invention is not to be construed to be limited by the following examples.

EXAMPLES The invention will be further explained by the following illustrative examples, which are intended to be non-limiting.

Example 1. Materials and Methods Bacterial strains Y. pestis strain KIM was employed as the sources of genes for plague antigens and the challenge strain in this study.

Constructions of DNA vaccines expressing plague antigens.

Three virulence related genes (V, Fl and Pla) were isolated in plasmids pPCPl, pCDland pMTl respectively. Genes coding for these plague antigens were first PCR amplified with Pfu polymerase (Stratagene), and then cloned into pJW4303 vector.

The gene encoding V antigen was amplified with primers Plague-V-1 (5'gtcgctcc AAGCTT GCTAGC ATG ATT AGA GCC TAC GAA CAA AAC CC 3'), (SEQ ID NO : 23), and Plague-V-2 (5'agtcac GGATCC TCA TTT ACC AGA CGT GTC ATC TAG C 3') (SEQ ID NO : 24). The following PCR primers were used to amplify the Fl gene: Plague-Fl-1 (5'gtcgctcc AAGCTT GCTAGC ATG AAA AAA ATC AGT TCC GTT ATC GCC 3') (SEQ ID NO: 25), and Plague-Fl-2 (5'agtcac GGATCC TTA TTG GTT AGA TAC GGT TAC GG 3') (SEQ ID NO: 26). Pla gene was amplified with PCR primers Plague-Pla-1 (5'gtcgctcc AAGCTT GCTAGC ATG AAG AAAAGT TCT ATT GTG GC 3'), SEQ ID NO: 27, and Plague-Pla-2 (5'gagc AGGCC TCA GAA GCG ATA TTG CAG ACC CGC 3') (SEQ ID NO: 28). Each of the plague gene inserts was subcloned into DNA vaccine vector pJW4303 (Lu, et al. 1998 paper). For each of the gene inserts, there were two versions. In one, the original wild type plague gene was inserted into the DNA vaccine vector. In the other, an additional tissue plasminogen activator (tPA) leader sequence was added at the 5'end of the inserted plague gene sequences. Once the correct DNA vaccine clones were confirmed by expected restriction enzyme analysis, large DNA preps were made using the Mega plasmid purification kit (Qiagen).

In vitro expression of Y. pestis antigens In vitro expression of the V, F1 and Pla antigens was conducted by transient transfection of 293T cells with DNA vaccine constructs. Transfection was carried out by calcium phosphate co-precipitation of 10 Ag of plasmid DNA to 293T cells at approximately 50% confluence on 60-mm dishes. The supernatants and cell-lysates were harvested 72 hours after transfection. Protein expression was confirmed by Western blot and ELISA.

DNA immunization of BalblC mice Six to eight weeks old female Balb/C mice were purchased from Taconic Farms (Germantown, NY) and housed in the animal facility managed by the Department of Animal Medicine at the University of Massachusetts Medical School in accordance with IACUC (Institutional Animal Care and Use Committees) approved protocols. Balb/C mice were first anesthetized with 50 Al of a mixture of Ketamine (26.7 mg/ml) and Xylazine (6.67 mg/ml) by IP injection before each immunization. The animals received three monthly DNA immunizations by a Bio-Rad Helios gene gun (Bio-Rad, Hercules, CA). The DNA vaccines and the pJW4303 vector plasmids were coated onto the 1.0- micron gold beads at a concentration of 2 Ag of DNA for each mg of gold. Each shot delivered 1 ttg of DNA. A total of 6 non-overlapping shots were delivered to each mouse on shaved abdominal skin. The blood samples were collected periorbitally prior to the first and 2 weeks after each immunization.

Western Blot The V, F1 or Pla antigens transiently expressed from 293T-cell supernatants and cell lysates were subjected to denaturing SDS-PAGE and blotted onto PVDF membranes (BioRad). Blocking was done with 0.1% I-Block (Tropix, Bedford, MA). Mouse serum immunized with corresponding DNA vaccine was used as the detecting antibody at 1: 200 dilution and incubated for 45 minutes. Subsequently, the membranes were washed with blocking buffer and then reacted with AP-conjugated goat anti-mouse IgG (Tropix) at 1: 5000 dilution. After final wash, Western-light substrate was applied to the membranes for 5 minutes. Once the membranes were dry, Kodak films were exposed to the membrane and developed with an X-Omat processor.

ELISA (enzyme-linked immunosorbent assay) Sera collected from each group of immunized mice were pooled to test for specific IgG antibody responses against individual Y. pestis antigens by ELISA. The 96- well microtiter plates were coated with 100 ttl of the antigens (V, F1 or Pla protein harvested from 293T) at 1 yg/ml and incubated overnight at 4°C. The plates were washed 5 times with washing buffer (PBS at pH 7.2 with 0.1% Triton X-100). Blocking was done with 200, ul/well of 4% milk-whey blocking buffer for 1 hour at room temperature. After removal of the blocking buffer and five more washes, 100 il of serially diluted, pooled mouse sera were added in duplicate and incubated for 1 hour.

The plates were washed five times and incubated with 100 jtd ofbiotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA) diluted at 1: 1000 for 1 hour followed with washes. Then, horseradish peroxidase-conjugated streptavidin (Vector Laboratories) diluted at 1: 2000 was added (100 ltvwell) and incubated for 1 hour. After the final washes, 100 Itl of fresh TMB substrate (Sigma) was added per well and incubated for 3.5 min. The reaction was stopped by adding 25/dof2M H2S04, and the optical density (OD) of the plate was measured at 450 mn.

Isotype analysis of Y. pestis V antigen specific IgG responses in mice To determine the amount of different antibody isotypes, standard curves for IgG isotype were first established by ELISA in which 100 ul of unlabeled mouse IgGl or IgG2a (Southern Technology Associates, Al) were serially diluted, starting from 1yg/ml in PBS in duplicate to coat the microtiter plates. The remaining wells were coated with 100 y1 of transiently expressed V antigen. ELISA was performed according to the <BR> <BR> protocol described above, except using horseradish peroxidase (HRP) -conjugated goat- anti-mouse IgG1 or IgG2 (Southern Technology Associates, Al) at 1: 2000 dilution for detection. The concentrations for V-specific mouse IgGl or IgG2a were then calculated from the standard curve.

Intranasal challenge of DNA vaccine immunized BalblC mice with lethal dose of virulent Y. pestis An intranasal challenge model was used in which each mouse received 5,000 cfu of Y. pestis KIM strain (in 50 Itl saline through its nostril by a small pipette tip. The

challenge dose was determined with the previous titration of the lethal doses (LD). The current challenge dose reflects about 15 LD50 (i. e. , median lethal dose) by intranasal infection. The immunized Balb/C mice were first anesthetized with 50 jU. l of a mixture of Ketamine (26.7 mg/ml) and Xylazine (6.67 mg/ml) by i. p. injection before the challenge.

Mice were observed twice daily, to monitor for sickness and survival. Dead mice were removed immediately from the cages once identified. All the studies were conducted in a Biosafety Level 3 facility at the Department of Animal Medicine, University of Massachusetts Medical School, following the procedure approved by the Institutional Biosafety Committee (IBC) at the University of Massachusetts Medical School. Survival was calculated by the method of Kaplan-Meier and comparisons made by ANOVA test.

P values less than the 0.05 were considered insignificant.

Example 2. Construction of DNA Vaccines Expressing Plague Antigens DNA vaccines expressing the V and F1 antigens of Y. pestis were constructed. F1 has a clear signal peptide sequence, presumably responsible for its secretion and extracellular functions. On the other hand, the V protein does not posses a putative leader sequence, and it is injected by the type III secretion system of Y. pestis. Two versions of DNA vaccines were designed for each of these two antigens: one with the <BR> <BR> wild type gene insert (e. g. , wt-V) and the other with a tPA leader sequence (e. g. , tPA-V).

The addition of a leader sequence with strong secretion potential can lead to higher production of the antigen (e. g. , in mammalian host cells in which the antigen is expressed) and, thus, better immunogenicity. Each of the Y. pestis antigen-encoding inserts was subcloned into the DNA vaccine vector pJW4303 (Lu et al., Antigen engineering in DNA immunization. In : Methods in Molecular Medicine. Edited by Lowrie DB, Whalen RG 29: 355-374,1998). This vector contains the CMV immediate early promoter, an intron A sequence, and the bovine growth hormone polyA tail. Figure 1 illustrates the design of these DNA vaccines expressing the V or F1 antigens. Similar designs were applied to Pla, a known Y. pestis virulence factor (Fig. 1). The Pla protein sequence has a hydrophobic region in its very N-terminal end which may be inhibitory for the secretion of Pla antigen even if a tPA leader is used. DNA vaccines expressing

YopB, YopD, YopO, and YscF antigens were also constructed in the same manner as the V, F1, and Pla vaccines. The gene inserts of the YopB, YopD, and YopO vaccines are depicted in Figure 8. For YopB and YopD, constructs were made in which a hydrophobic (e. g. , putative transmembrane) regions was deleted. The YscF vaccines are depicted in Figure 12.

Example 3. Anti-Y. Pestis Antibody Response from Immunized Animals Groups of 6-8 weeks old Balb/C mice (4-6 animals per group) received three monthly DNA immunizations delivered in the abdominal skin by a Bio-Rad gene gun (Fig. 2). A total of 6 shots (1 ug/shot) were given at each immunization. Both Fl and V DNA vaccines induced high titer IgG antibody responses against their respective plague antigens as detected by ELISA (Fig. 3). Data are presented as the end titration titers of pooled mouse sera from the same animal groups. For mice immunized with the F1 DNA vaccines, the final titers of anti-Fl IgG responses were-1 : 100,000, while V DNA vaccines induced even higher anti-V IgG responses (Figs. 3A and 3B). No significant anti-Pla IgG antibody response was detected from animals immunized with either wt-Pla or tPA-Pla DNA vaccines (Fig. 3C). Low levels of intracellular expression of Pla antigen was observed in 293T cells transfected with these two DNA plasmids and analyzed by Western blot using rabbit anti-Pla sera. Sera from mice immunized with empty vector DNA plasmids were used as negative controls and did not show specific antibody responses against any of the Y. pestis antigens (Fig. 3).

The V and F1 DNA vaccines with the tPA leader sequence appeared to be more immunogenic than their wild type counterparts (Fig. 3). An example of antibody titration for anti-V IgG is shown in Fig. 4A. The modified V gene insert with a tPA leader was able to induce an early rise of antibody responses after only one DNA immunization, while the wild type V DNA vaccine required at least two DNA immunizations to have a significant high titer antibody response (Fig. 4B). Thus, V antigens expressed with a signal sequence can generate a more robust response with fewer doses, and more rapidly, than the antigen expressed without a signal sequence.

Example 4. Protection of Immunized Mice Against Lethal Intranasal Y. pestis Challenges As illustrated in Fig. 2, the animals had a 5-month resting period after three monthly immunizations. Animals received another DNA immunization at week 28 and were challenged 14 days later with a lethal dose of the Kim strain of Yersiniapestis (5000 cfu =-15 LD50 of freshly grown challenge stock). Animals were monitored twice a day for signs of sickness and mortality. Survival of animals from each group was plotted by Kaplan-Meier curve. Animals that received the negative control vector DNA died within three to four days (Fig. 5A) with early signs of sickness within the first 24-36 hours post- challenge. Mice immunized with either the wt-Pla or tPA-Pla DNA vaccines had almost the same outcome as the vector control group mice, dying within 3-4 days post challenge (Fig. 5A). On the other hand, the tPA-V DNA vaccine achieved 100% protection (Fig.

5C). Most significantly, none of the animals in this group showed any sign of sickness.

The wt-V, wt-Fl, and tPA-Fl DNA vaccines induced partial protection, with 50-75% animal surviving (Figs. 5B-5C). Animals in these partially protected groups had variable signs of sickness. In summary, these data show that DNA administration of F1 and V antigens induces protection from Y. pestis challenge, and expression of a tPA signal sequence with these antigens enhances protection.

Example 5. Different Expression Between tPA-V and Wt-V Antigens The difference in levels of protection between the tPA-V DNA vaccine and the wt-V DNA vaccine was examined. Supernatant and cell lysate samples were collected from 293T cells transiently transfected with either the wt-V or tPA-V DNA vaccines.

Compared to the wild type construct, the tPA-V DNA construct had a much higher level of expression and more secreted antigens (Figure 6). It is interesting to note that the tPA-V antigen formed dimers and possibly tetramer in addition to the monomer form, while wt-V antigen was mainly monomeric. Because the antibody titers differ by only a few folds between these two constructs, while the protection with tPA-V was more complete than the wt-V antigen, the data suggest that the oligomer forms of the Y. pestis V antigen may be critical in inducing better neutralizing antibody responses.

In contrast, addition of a tPA leader sequence did not change the expression of the F1 antigen, suggesting that the natural F1 sequence was already able to express a significant level of the secreted antigen, but this antigen alone, though immunogenic, was not sufficient in inducing complete protection from lethal mucosal challenge.

Example 6. Improved Thl Type Immune Responses With tPA-V Antigen It was investigated whether the protective humoral immune responses induced by the tPA-V DNA vaccine were qualitatively different from those induced by wt-V DNA vaccine. Mouse sera were analyzed for their isotypes of anti-V IgG responses. Both antigens induced predominantly IgGl antibody responses as is typical of gene gun inoculation (Fig. 7). However, the tPA-V DNA vaccine group had close to 10-fold higher IgG2a responses than the wt-V DNA vaccine group (Fig. 7). Because an IgG2a response represents a Thl type immune response in mice and recent reports have suggested the wild type Y. pestis V protein may suppress Thl type immune responses (Sing et al., J. Exp. Med., 196: 1017-1024,2002 ; Sing et al., J. Immu7mol., 168 : 1315- 1321,2002), the data suggest that the modified V antigen expressed by the tPA-V DNA vaccine may overcome immune suppressive effects of this protein. This may be an important mechanism contributing to the superior protection over the wild type V antigen. The oligomer structure of tPA-V may be an important factor contributing to such favorable changes to the subtypes of T helper immune responses.

These results show that a DNA vaccine expressing a modified Y. pestis V gene insert was able to induce a complete protection in mice against lethal dose of intranasal challenges. It is a; sp shown here that DNA immunization is also effective in the induction of protective antibody responses, which are critical for inhibiting bacterial infections. Proper antigen engineering to improve the expression and secretion of protective bacterial antigenic proteins, as demonstrated in this study, offers great potential for the development of vaccines against other major bacterial pathogens in biodefense efforts.

Both wt-V and tPA-V antigens induced high levels of IgGl antibody responses when inoculated by gene gun. However, the tPA-V group led to a 10-fold higher level of

IgG2a response than the wt-V group. The tPA-V antigen, whether secreted or intracellular, is more likely to form dimers or even tetramer than wt-V (Fig. 6). Such oligomer conformations may be critical in the induction of protective immune responses.

Alternatively, the way the V protein is secreted may also contribute to the structure and immunogenicity of this antigen. The Y. pestis type III secretion apparatus secrets the natural V protein. Its secretion by mammalian cells as the result of DNA transfection may be hindered due to the lack of such secretion apparatus. Therefore, the addition of tPA leader sequences may become critical in the final secretion of the V antigen, which further affects its immunogenicity.

DNA vaccines expressing F1 antigen induced partial protection against the lethal mucosal challenge. DNA vaccines expressing Pla antigen did not induce any protection.

It is most likely due to its poor expression and poor immunogenicity in inducing anti-Pla antibody responses. The Pla gene has a very unusual hydrophobic sequence at its N- terminus which may be responsible for the poor expression of soluble Pla antigens.

Example 7. Immunogenicity of YopB, YopD, and YopO DNA Vaccines Balb/C mice were immunized with wild-type and modified YopB, YopD, and YopO DNA vaccines as described in Example 1, above. Sera from these mice was collected and assayed for reactivity with lysates and supematants of Yop-transfected cells. As shown in Figure 9, antisera from YopB-immunized mice reacted with YopB antigens by Western blotting. Similarly, sera from mice immunized with YopD reacted with YopD antigens (Fig. 10), and sera from mice immunized with YopO reacted with YopO antigens (Fig. 11).

OTHER EMBODIMENTS A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.