WO2005042564A1

WO2005042564A1 - Flagellin fusion proteins as adjuvants or vaccines and methods of use

Info

Publication number: WO2005042564A1
Application number: PCT/US2004/034910
Authority: WO
Inventors: Joseph Lustgarten
Original assignee: Sidney Kimmel Cancer Center
Priority date: 2003-10-20
Filing date: 2004-10-20
Publication date: 2005-05-12

Abstract

The invention provides a polypeptide comprising a flagellin polypeptide fused to an antigen. The invention also provides a method of stimulating an immune response by administering to an individual a composition comprising a polypeptide comprising a flagellin polypeptide fused to an antigen.

Description

FLAGELLIN FUSION PROTEINS AS ADJUVANTS OR VACCINES AND METHODS OF USE

This application relates generally to the field of immunology and more specifically to vaccines.

Vaccination remains the most effective valuable tool in the prevention of infectious disease (Hunter RL. Overview of vaccine adjuvants: present and future. Vaccine 2002; 20 Suppl 3:S7-12; Cohen J , Marshall E. Bioterrorism. Vaccines for biodefense: a system in distress. Science 2001; 294:498-501; Curtiss R, 3rd. Bacterial infectious disease control by vaccine development. J Clin Invest 2002; 110:1061- 1066). However, new vaccination strategies that induce the cellular and humoral compartments of the immune response are needed for the development of effective prophylactic vaccines against a number of infectious diseases. For the development of effective vaccines, a great need exists for the identification of safe and effective adjuvants. A number of novel adjuvants have been reported during the past few years including: bacterial toxins such as cholera (Lee SF, Halperin SA, Salloum DF, MacMillan A, Morris A. Mucosal immunization with a genetically engineered pertussis toxin SI fragment-cholera toxin subunit B chimeric protein. Infect Immun 2003; 71:2272-2275); endogenous immunomodulators like cytokines (Murtaugh MP, Foss DL. Inflammatory cytokines and antigen presenting cell activation. Vet Immunol Immunopathol 2002; 87: 109-121); bacterial wall components (Verthelyi D, Kenney RT, Seder RA, Gam AA, Friedag B , Klinman DM. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J Immunol 2002; 168:1659-1663) and others (Morse MA, Lyerly HK. Clinical applications of dendritic cell vaccines. Curr Opin Mol Ther 2000; 2:20-28). Recent progress in understanding innate immune responses is beginning to yield insight into the elucidation of receptors and pathways involved in the immune responses, leading to the characterization of immunostimulatory molecules that enhance this process (Janeway C A, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197- 216; Medzhitov R, Janeway C, Jr. Innate immunity. N Engl J Med 2000; 343 :338-344). The immune system can be divided into innate and adaptive components. The innate immune response is the first line of defense against infectious diseases (Imler JL, Hoffmann JA. Toll receptors in innate immunity. Trends Cell Biol 2001; 11 :304-311), while adaptive immune responses represent specific resistance, weak at first but develops into long-term memory responses (Heine H, Lien E. Toll-like receptors and their function in innate and adaptive immunity. Int Arch Allergy Immunol 2003; 130:180- 192). More importantly, adaptive responses are initiated when T and B cells recognize foreign molecules expressed on antigen presenting cells (APC) (Schijns VE. Induction and direction of immune responses by vaccine-adjuvants. Crit Rev Immunol 2001 ; 21:75-85). The major difference between the innate and adaptive immune systems lies in the mechanism of recognition of the antigens. In the adaptive immune response, T and B cell responses recognize the antigen through the T and B cell receptors, respectively, which has the capacity to recognize almost any antigen structure. In addition, each T or B cell expresses a unique receptor that can bind any antigen regardless its origin. The innate response is largely mediated by white blood cells such as neutrophils, monocytes, macrophages (MΦ) and dendritic cells (DCs). In contrast to the adaptive immune response, the innate immune response relies on the recognition of the antigen by receptors that recognize specific structures found exclusively in microbial pathogens termed pathogen-associated molecular patterns (PAMPs) (Barton GM, Medzhitov R. Control of adaptive immune responses by Toll-like receptors. Curr Opin Immunol 2002; 14:380-383). The recognition of PAMPs by the innate immune system can regulate the induction of adaptive immune responses (Huang Q, Liu D, Majewski P, Schulte LC, Korn JM, Young RA, Lander ES, Hacohen N. The plasticity of dendritic cell responses to pathogens and their components. Science 2001 ; 294:870-875). For example, DCs respond to some microbial product by taking up the antigen.

Concurrently, DCs synthesize a wide variety of inflammatory mediators and cytokines amplifying the immune response and, in addition, DCs can process and present antigens resulting in the activation of T and B cell responses and the establishment of protective immunity. As such, a number of microbial products are thought to function as effective adjuvants due to effects on APCs, which in turn, can influence the activation of an adaptive immune response. More than a decade ago, Janeway postulated that regulation of recognition of PAMPs must be controlled by receptors with a specificity for microbial products, thereby linking innate recognition of non-self with induction of adaptive immunity (Janeway CA, Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 1989; 54 Pt 1:1-13). Recent studies have demonstrated that recognition of PAMPs by APCs is mediated by a Toll-like receptor (TLRs) family (Means TK, Golenbock DT, Fenton MJ. Structure and function of Toll-like receptor proteins. Life Sci 2000; 68:241-258; Kaisho T, Akira S. Toll-like receptors as adjuvant receptors. Biochim Biophys Acta 2002; 1589:1-13). There are currently 10 known TLR family members capable of sensing bacterial wall components, such as LPS (TLR-2/4), lipoteichoic acids (TLR-2/4), CpG DNA (TLR-9), flagellin (TLR-5), as well as other microbial products (Takeda K, Akira S. Toll receptors and pathogen resistance. Cell Microbiol 2003; 5:143-153). A wide variety of TLRs are expressed in immature or mature DCs, MΦ and monocytes and these receptors control the activation of these APCs (Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000; 406:782-787). Recognition of PAMPs by TLRs initiates a signaling pathway that leads to activation of NF-kB transcription factors and members of the MAP kinase family (Means TK, Golenbock DT, Fenton MJ. The biology of Tolllike receptors. Cytokine Growth Factor Rev 2000; 11:219-232). All TLRs share a common intracellular domain that is similar to the IL-1 receptors and the signal is mediated through the adaptor protein MyD88 (Takeuchi O, Akira S. MyD88 as a bottle neck in Toll/IL-1 signaling. Curr Top Microbiol Immunol 2002; 270:155-167). The TLRs signaling triggers maturation and activation of APCs that includes upregulation of MHC and co-stimulatory molecules, secretion of pro-inflammatory cytokines and chemokines (Gewirtz AT, Navas TA, Lyons S , Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial pro inflammatory gene expression. J Immunol 2001; 167:1882-1885). This maturation of APCs significantly increases their ability to prime naive T cells. In this way, TLRs link the recognition of pathogens with induction of adaptive immune responses.

Flagella is expressed in Gram negative and positive bacteria and is a surface structure that confers motility to the bacteria (Chilcott GS, Hughes KT. Coupling of fiagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev 2000; 64:694-708). Flagella are composed of a basal body that serves as a rotatory motor and a filament that extends into the space around the bacterium to provide motive force (DeRosier DJ. The turn of the screw: the bacterial flagellar motor. Cell 1998; 93 : 17-20; Homma M, Fujita H, Yamaguchi S, lino T. Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion. J Bacteriol 1987; 169:291-296). The filament consists of a long homopolymer of a single protein, flagellin, with a small cap at the end. Polymerization of flagellin occurs as a result of relatively conserved structures at the N and C termini, although the intervening regions of the protein are highly diverse (Homma M, Fujita H, Yamaguchi S, lino T. Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion. J Bacteriol 1987; 169:291-296).

Recent evidence suggest that flagella enhance the pathogenicity of the bacterium by promoting adherance to host tissues and by directly activating host inflammatory signal pathways (Gewirtz AT, Simon PO Jr., Schmitt CK, Taylor LJ, Hagedorn CH, O'Brien AD, Neish AS, Madara JL. Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response. J Clin Invest 2001 ; 107:99-109). It has been demonstrated that the component of the flagella responsible for eliciting host immune responses is the filament protein flagellin (Lee LH, Burg E, 3rd, Baqar S, Bourgeois AL, Burr DH, Ewing CP, Trust TJ, Guerry P. Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni. Infect Immun 1999; 67:5799-5805). Purified or recombinant flagellin causes inflammatory responses on treated cells in vitro (Jeon SH, Arnon R. hnmunization with influenza virus hemagglutinin globular region containing the receptor-binding pocket. Viral Immunol 2002; 15:165-176) or systemically administered in vivo (Wyant TL, Tanner MK, Sztein MB. Potent immunoregulatory effects of Salmonella typhi flagella on antigenic stimulation of human peripheral blood mononuclear cells. Infect Immun 1999; 67:1338-1346). This effect is mediated by activation of TLR-5 (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S , Underhill DM Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001; 410:1099-1103). Although the use of other PAMPs like CpG DNA to boost T cell responses is well established (Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20:709-760), the use of flagellin as an adjuvant is also becoming very attractive (Reed KA, Hobert ME, Kolenda CE, Sands KA, Rathman M, O'Connor M, Lyons S, Gewirtz AT Sansonetti PJ, Madara JL. The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells. J Biol Chem 2002; 277:13346- 13353). It has been reported that flagellin is an effective adjuvant capable of enhancing T cell responses in vivo (McSorley SJ, Ehst BD, Yu Y, Gewirtz AT. Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo. J Immunol 2002; 169:3914-3919; Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, Szabo C, Salzman AL. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol 2001;

166:1248-1260). The advantage of using flagellin over CpG DNA is that CpG DNA are synthetic oligodeoxynuleotides containing unmethylated CpG motifs used as soluble adjuvants, while the flagellin can be used as a soluble adjuvant (McSorley S J, Ehst BD, Yu Y, Gewirtz AT. Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo. J Immunol 2002; 169:3914-3919) and to generate recombinant proteins (Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, Szabo C, Salzman AL. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol 2001; 166:1248-1260). There is evidence that epitopes inserted into the flagellin of Salmonella and used as live vaccine induces specific T and B cell responses against these epitopes (Newton SM, Joys TM, Anderson SA, Kennedy RC, Hovi ME, Stocker BA. Expression and immunogenicity of an 18-residue epitope of HIV 1 gp41 inserted in the flagellar protein of a Salmonella live vaccine. Res Microbiol 1995; 146:203-216; Newton SM, Jacob CO, Stocker BA. Immune response to cholera toxin epitope inserted in

Salmonella flagellin. Science 1989; 244:70-72; Verma NK Ziegler HK, Stocker BA, Schoolnik GK. Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella. Vaccine 1995; 13:235-244). If the antigen incorporated into the flagellin will interfere with folding or transport or assembly of the flagella, the bacteria cannot present the flagella on the surface because of alterations on its structure (Stocker BA, Newton SM. Immune responses to epitopes inserted in Salmonella flagellin. Int Rev Immunol 1994; 11 : 167- 178), which is the major limitation of this approach.

Thus, there exists a need to develop adjuvants that can be used to enhance the immune response to a variety of antigens. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic representation of EGFP and flagellin-EGFP fusion protein. EGFP and flagellin-EGFP were synthesized as described above. Figure 1 A shows a diagram of EGFP and flagellin-EGFP proteins. a.a., amino acids. Figure IB shows a gel in which an aliquot of each protein was loaded onto a 10% polyacrylamide gel and subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Molecular weight markers(M.W.M) are indicated in thousands.

Figure 2 shows that APCs are bound by flagellin-EGFP but not by EGFP. Bone marrow-derived APCs were incubated for 1 h with flagellin-EGFP or EGFP (thick lines). Thin lines represent APCs left untreated, as controls. Cells were analyzed by flow cytometry for fluorescence in the FLH-1 channel. Numbers within the plots are the percentage of APCs that showed increased fluorescence. Experiments were repeated three times with similar results. One representative experiment is shown.

Figure 3 shows that maturation of APCs is induced by LPS and by flagellin- EGFP, but not by EGFP. Freshly isolated APCs were incubated for 24 h with LPS (10 μg/ml), flagellin-EGFP, or EGFP (each at 10 μg/ml). APCs were stained to detect B7.1 (thick line) and class II MHC (broken line) molecules, or were left unstained as control (solid line), and analyzed by flow cytometry. Experiments were repeated three times with similar results. One representative experiment is shown.

Figure 4 shows that a flagellin-EGFP fusion protein.induces a proinflammatory response. Cultured APCs were incubated with a 10-μg/ml concentration of LPS, flagellin-EGFP, or EGFP or were left unstimulated (control). After 24 hours, the cell supernatants were collected and TNF-α and NO secretion were measured. In Figure 4A, TNF-α production in each culture was measured by ELIS A. In Figure 4B, the same supernatants were used to analyze NO production, as measured by the Griess reaction. Data are the means of three independent experiments (performed with duplicates) (error bars, standard deviations). A significant (P 0.001 (Student's t test)) difference was found between the control group and the flagellin-EGFP group.

Figure 5 shows that APCs pulsed with flagellin-EGFP process and present EGFP. APCs cells were pulsed for 18 h with Ad-EGFP, flagellin-EGFP, or EGFP or were unpulsed. The cytotoxic activity of an anti-EGFP-specific CD8-T-cell line was analyzed against the pulsed APCs in a 6-h ⁵ Cr release assay at the indicated ratios of effector to target cells (E:T). Data are the means of two independent experiments (performed with triplicates) (error bars, standard deviations). A significant (P 0.001 (Student's t test)) difference was found between the control group and the flagellin-EGFP group.

Figure 6 shows the induction of specific CD8-T-cell responses by flagellin-EGFP fusion protein. BALB/c mice were immunized with adenovirus-EGFP (Figure 6A), flagellin-EGFP (Figure 6B) and EGFP (Figure 6C). After 2 weeks, spleens were removed, and T cells were stimulated in vitro for 5 days. Cytotoxic activity was tested against BM-185-w.t, BM-185-w.t. pulsed with the H2-K^d-EGFP-peptide, and BM-185- EGFP cells in a 6-h ⁵¹Cr release assay at the indicated ratio of effector cells to target cells (E:T). Data represent the means of results for three individually analyzed mice per group in two independent experiments (error bars, standard deviations). A significant (P 0.001 (Student's t test)) difference was found between the control group and mice immunized with flagellin-EGFP.

Figure 7 shows induction of anti-EGFP antibody production after immunization with flagellin-EGFP fusion protein. Balb/c mice wee immunized multiple times with the flagUein-EGFP and EGFP proteins and anti-EGFP (Figure 7A) and anti-flagellin (Figure 7B) antibody production was measured.

Figure 8 shows activation of specific CD4 T cells by flagellin-EGFP fusion protein. BALB/c mice were immunized with flagellin-EGFP or EGFP. After 2 weeks, spleens were removed, and CD4 T cells were enriched. CD4 T cells were incubated for 4 days with APCs pulsed with adenovirus-EGFP or empty adenovirus. After 3 days of incubation, tritiated thymidine (1 μCi/well for the last 18 h) was added, and thymidine incorporation was measured. Data show the means of results for three individually analyzed mice per group in two independent experiments (error bars, standard deviations). A significant (P 0.001 (Student's t test)) difference was found between the control group and mice immunized with flagellin-EGFP.

Figure 9 shows the amino acid sequence (SEQ ID NO:l) of the phase II flagellin encoded by the fljB gene of Salmonella enterica serovar Typhimurium (GenBank accession No. AB 108532).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a polypeptide comprising a flagellin polypeptide fused to an antigen. As disclosed herein, the use of a flagellin polypeptide fused to an antigen can advantageously be used as an adjuvant to enhance an immune response against the antigen. The use of attenuated bacteria in vaccination represents an unknown risk. The present invention is advantageous in that a recombinant fusion protein incorporating both the antigen of interest and the adjuvant (flagellin) can eliminate problems associated with vaccine risk. In addition, the fusion of both elements in one single molecule have the advantage of targeting the antigen to the APCs for uptake, processing and presentation. As disclosed herein, a flagellin-EGFP fusion protein was generated. The flagellin-EGFP fusion protein activates APCs and is capable of stimulating specific T-cell responses. These data indicate that flagellin fusion proteins are suitable carriers for the development of vaccines to induce strong and specific immune responses against antigens of interest.

In the present invention, to be able to use flagellin as an adjuvant/vaccine and overcome the limitation of incorporating into the flagellin primary sequence any antigen of any size, we produced a tandem recombinant flagellin fusion protein. In particular, to test the efficacy of the tandem recombinant flagellin-fusion proteins as an adjuvant/vaccine and as a proof of concept, we generated a tandem flagellin-EGFP (enhanced green fluorescent protein) fusion protein.

We have characterized the immune-effect of this fusion protein, and as described in the Preliminary Results Section, tandem flagellin-fusion proteins induce the activation and maturation of APCs, and more importantly induce specific T and B cell responses. Moreover, multiple exposures to the tandem flagellin-fusion protein did not induce a neutralization response against the flagellin. Taken together, the preliminary data presented herein demonstrate that tandem flagellin-fusion proteins are suitable carriers for the development of adjuvant/vaccines to induce and boost immune responses against infectious diseases. We believe that a tandem recombinant fusion protein could be superior compared to live attenuated vaccines for safety reasons.

These studies demonstrate the effects that tandem flagellin-fusion proteins have on the immune response and the use of these fusion proteins as adjuvants/vaccines against infectious disease. The tandem flagellin-EGFP fusion protein is practical for performing experiments because it is possible to track the protein once it is phagocytosed due to its fluorescent nature and serves as a proof of concept to demonstrate its ability to stimulate immune responses.

In the design of vaccines, one of the major considerations is to overcome the low immunogenicity of the antigen. The common approaches to this issue are the coupling of the antigen to an immunogenic carrier or adjuvant. The critical issue concerning adjuvants is their toxic effects, whether the adjuvant is immunogenically capable of inducing a neutralizing response against itself and whether it will be approved for human use.

Our findings demonstrate a genus of vaccines which comprise tandem recombinant flagellin fusion proteins. We have found that tandem recombinant flagellin- fusion proteins effectively activate the innate and adaptive immune responses, and, as such, provide a method for developing recombinant proteins for use as vaccines, and use of tandem recombinant vaccines in vaccination strategies.

The invention provides a polypeptide comprising a flagellin polypeptide fused to an antigen. As disclosed herein, the use of a flagellin polypeptide fused to an antigen can advantageously be used as an adjuvant to enhance an immune response against the antigen. The invention also provides a nucleic acid encoding the polypeptide a polypeptide comprising a flagellin polypeptide fused to an antigen.

The invention also provides composition comprising a polypeptide comprising a flagellin polypeptide fused to an antigen. The composition can further comprise a physiologically acceptable carrier and can be used as a vaccine to stimulate an immune response.

The invention additionally provides a method of stimulating an immune response by administering to an individual a composition comprising a polypeptide comprising a flagellin polypeptide fused to an antigen. As exemplified herein, a flagellin-EGFP fusion was effective at stimulating an immune response to EGFP. It is understood that EGFP is merely an exemplary antigen and that other desired antigens can be fused to a flagellin polypeptide as an adjuvant to stimulate an immune response.

The use of a flagellin fusion polypeptide as an adjuvant is exemplified herein using flagellin encoded by the fljB gene from Salmonella enterica serovar Typhymurium LT2 (see examples). An exemplary flagellin sequence is shown in Figure 9. Although exemplified with this flagellin, it is understood that other flagellins can similarly be used as fusion polypeptides with an antigen to stimulate an immune response. For example, a number of phase 2 flagellin (flj^'B) genes from Salmonella enterica strains are known and publicly available on GenBank. Exemplary sequences include, but are not limited to, GenBank accession numbers AB108532, AY352264, AY353263, AY353262, AY353285, AY353284, AY353283, AY353282, AY353281, AY353280, AY353279, AY353278, AY353277, AY353276, AY353275, AY353274, AY353273, AY353272, AY353271, AY353271, AY353269, AY353268, AY353267, AY353266, AY353265, AY353307, AY353306, AY353305, and AY353303. In addition, other types of flagellins from Salmonella species can be used, so long as the flagellins can function as an adjuvant in a fusion polypeptide. For example, the phase 1 flagellin encoded by the fliC gene can be used. Exemplary fliC gene sequences are available on GenBank and include, but are not limited to, AY353368, AY353367, AY353366, AY353365, AY353364, AY353363, AY353362, AY353361, AY353360, AY353359, AY353358, AY353357, AY353356, AY353355, AY353354, AY353353, AY35352, AY353351, AY353350, and AY353349. Furthermore, flagellins from other Salmonella species, including flagellins having similar functions as those encoded by flj^'B and fliC genes, are well known and can be similarly be used as fusion polypeptides with an antigen to stimulate an immune response. Exemplary flagellin sequences are available on GenBank and include, but are not limited to, AY353261, AY353260, AY353259, AY353258, AE008826, D12510, AJ295701, AJ295700, AJ295699, AJ295698, AJ295697, AJ295696, AF045151, U17177, U17176, U17175, U17174, U17172, U17171, D13690, and D13689.

In addition to Salmonella, flagellins from other microorganisms can be used to enhance an immune response. It is understood by those skilled in the art that various flagellin polypeptides from various Salmonella strains or species or from other microorganisms can be used in the invention to generate flagellin fusion polypeptides with a desired antigen. A composition of the invention containing a flagellin polypeptide fused to an antien can be combined with a physiologically acceptable carrier useful in a vaccine by including any of the well known components useful for immunization. The components of the physiological carrier are intended to facilitate or enhance an immune response to an antigen administered in a vaccine. The formulations can contain buffers to maintain a preferred pH range, salts or other components that present the antigen to an individual in a composition that stimulates an immune response to the antigen. The physiologically acceptable carrier can also contain one or more adjuvants that enhance the immune response to the antigen. Formulations can be administered subcutaneously, intramuscularly, intradermally, or in any manner acceptable for immunization.

The invention provides a flagellin polypeptide fused to an antigen that can function as an adjuvant. It is understood that a composition containing a flagellin fusion polypeptide to be used to stimulate an immune response, which itself can function as an adjuvant, can additionally contain other adjuvants, if desired. An adjuvant refers to a substance which, when added to a composition of the invention, nonspecifically enhances or potentiates an immune response to the antigen in the recipient host upon exposure to the mixture. Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as, polysytrene, starch, polyphosphazene and polylactide/polyglycosides. Adjuvants can also include, for example, squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. Nature 344:873-875 (1990). For veterinary use and for production of antibodies in animals, mitogenic components of Freund's adjuvant

(both complete and incomplete) can be used. In humans, Incomplete Freund's Adjuvant (IF A) is a useful adjuvant. Various appropriate adjuvants are well known in the art (see, for example, Warren and Chedid, CRC Critical Reviews in Immunology 8:83 (1988); Allison and Byars, in Vaccines: New Approaches to Immunological Problems. Ellis, ed., Butterworth-Heinemann, Boston (1992)). Additional adjuvants include, for example, bacille Calmett-Gύerin (BCG), DETOX (containing cell wall skeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL)), and the like (see, for example, Hoover et al., J. Clin. Oncol. 11:390 (1993); Woodlock et al., Immunotherapy 22:251-259 (1999)).

A major consideration in the design of vaccines is to overcome the low immunogenicity of the antigen itself, which is commonly achieved by coadministering antigens with an immunogenic carrier or adjuvant. In the development of efficient vaccines, a great need exists for the identification of safe and effective adjuvants. A number of microbial products are thought to function as effective adjuvants due to effects on APCs, which in turn promote the activation of an immune response. In this regard, it has been demonstrated that flagellin, which signals via the TLR-5 pathway (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S , Underhill DM Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001; 410:1099-1103), induces the activation of proinflammatory signals such as the secretion of TNF-α and NO (Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, Szabo C, Salzman AL. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol 2001; 166:1248-1260). We hypothesize that flagellin fusion proteins can serve as adjuvants or vaccines for the induction of specific immune responses. As a proof of concept we generated a flagellin- EGFP fusion protein. Herein, we demonstrate that flagellin-EGFP is capable of inducing the maturation of APCs and the secretion of TNF-α and NO. These proinflammatory signals elicited by flagellin-EGFP are specific and derived from the flagellin component, since treatment of APCs with recombinant EGFP does not stimulate these cells. These data demonstrate that the flagellin component of the fusion protein retains its stimulatory effects. Moreover, we show that APCs loaded with the flagellin fusion protein take up, process, and present the antigen, because they are specifically recognized by anti-EGFP T cells. More importantly, animals immunized with flagellin-EGFP develop specific CD8⁺- and CD4⁺-T-cell responses, while no immune response is detected in animals immunized with recombinant EGFP. These results suggest that flagellin fusion proteins are suitable carriers for the development of adjuvants or vaccines to induce and boost immune responses against the antigen of interest.

One of the advantages of using flagellin fusion proteins is that it might be possible to effectively target APCs for the stimulation of specific immune responses. We do not yet understand the mechanism through which flagellin fusion proteins are internalized by the APCs. Sparwasser et al. (Sparwasser et al., Eur. J. Immunol. 30:3591- 3597 (2000) linked CpG-DNA to the soluble protein OVA, and under these conditions the CpG-DNA facilitates the uptake of OVA by DCs through a receptor-mediated mechanism and, at the same time, these CpG-DNA complexes retain their stimulatory potential. It is possible that not only does the binding of flagellin- EGFP to TLR-5 on APCs trigger the activation of these cells, but also the intemalization of the complex and subsequent processing and presentation of the antigen result in the activation of an immune response. Further studies are needed to understand the regulation and activation of APCs following interaction of TLR-5 with flagellin. Then it would be possible to design more efficient vaccination strategies to achieve complete protective immunity. Recently, Means et al. showed that they could not detect expression of TLR-5, by reverse transcription-PCR, on DCs isolated from C57BL/6 mice (Means et al. J. Immunol. 170:5165-5175 (2003)). In their experiments, murine DCs were not responsive to flagellin. However, Renshaw et al. (J. Immunol. 169:4697-4701 (2002)) demonstrated that murine peritoneal MΦ express TLR-5 and that treatment with flagellin induces the production of interleukin-6 and TNF-α. Moreover, McSorley et al. demonstrated that flagellin is able to boost CD4 -T-cell responses in vivo (McSorley SJ, Ehst BD, Yu Y, Gewirtz AT. Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo. J Immunol 2002; 169:3914-3919). Furthermore, Madrazo et al. found that murine osteoblasts are activated after stimulation with flagellin (Madrazo et al. Infect. Immun. 71 :5418-5421 (2003). Sebastiani et al. also showed that TLR-5 is expressed in lungs and liver (Sebastiani et al., Genomics 64:230-240 (2000). Taken together, these results indicate that TLR-5 is expressed in different tissues and cell types, which are thus capable of interacting with flagellin. It is possible that the murine strain or the ex vivo culture conditions of the DCs affect the expression of TLR-5 and the responsiveness of these cells to flagellin and are responsible for the results obtained by Means et al. We do not discard the possibility that murine DCs do not express TLR-5. In fact, in our experiments we used a pool of APCs derived from bone marrow that in addition to DCs includes MΦ and monocytes, and these cells have the capacity to respond to the stimulation with the flagellin-EGFP fusion protein. In addition, we cannot discard the possibility that flagellin also binds to a receptor different from TLR-5 and that the effect observed by the flagellin-EGFP fusion protein may not be exclusively mediated by the TLR-5. Nevertheless, our results demonstrate that the flagellin fusion protein is active and capable of activating specific immune responses. We still need to understand how this flagellin-EGFP fusion protein activates the immune responses in vivo. Further studies are necessary to elucidate the effect that flagellin and flagellin fusion proteins exert on the different APCs.

There are several reports demonstrating that certain epitopes inserted into the flagellin of Salmonella and used as live vaccines induce specific T-cell responses against these epitopes, which further demonstrates the adjuvant abilities of flagellin (34, 35, 44 Newton SM, Jacob CO, Stocker BA. Immune response to cholera toxin epitope inserted in Salmonella flagellin. Science 1989; 244:70-72; Newton SM, Joys TM, Anderson SA, Kennedy RC, Hovi ME, Stocker BA. Expression and immunogenicity of an 18-residue epitope of HIV 1 gp41 inserted in the flagellar protein of a Salmonella live vaccine. Res Microbiol 1995; 146:203-216; Verma NK Ziegler HK, Stocker BA, Schoolnik GK. Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella. Vaccine 1995; 13:235-244). The major drawback of this approach is that if the antigen incorporated into the fusion protein interferes with the folding, transport, or assembly of flagellin, bacteria cannot express this molecule in its surface (Stocker BA, Newton SM. Immune responses to epitopes inserted in Salmonella flagellin. Int Rev Immunol 1994; 11:167-178). Other advantages for the use of flagellin fusion proteins over epitopes inserted into flagellin and used as live vaccines are as follows. First, fusion proteins eliminate the limitation of using known epitopes of restricted size. Instead, it will be possible to incorporate in one molecule single or multiple antigens of larger size, which promises very interesting possibilities. Second, recombinant fusion proteins will be rid of the safety risk associated with live attenuated vaccines. Third, a very weak or no significant antiflagellin immune response appears to be developed upon administration of flagellin (Ben-Yedidia T, Amon R. Effect of pre-existing carrier immunity on the efficacy of synthetic influenza vaccine. Immunol Lett 1998; 64:9-15). A major concern for the efficacy of an adjuvant is that prior or subsequent exposures to the adjuvant may induce an immuno-neutralizing response against it, resulting in a diminished or suppressed immune response against the antigen of interest. In this regard, Ben-Yedidia and Amon (Ben-Yedidia T, Amon R. Effect of pre-existing carrier immunity on the efficacy of synthetic influenza vaccine. Immunol Lett 1998; 64:9-15) demonstrated that prior exposure to flagellin did not result in an antiflagellin response and did not suppress the immune responses against influenza antigens. We have preliminary data showing that immunization with the flagellin-EGFP induces a minimal immune response against flagellin (data not shown), further supporting the findings of Ben-Yedidia and Amon. A simple explanation as to why a strong antibody response is not induced against flagellin could be because hosts are tolerant or partially tolerant to flagellin. It is well established that a natural flora of bacteria exists in the body very early in life. As such, we might have developed some level of tolerance over time whereby T- or B-cell responses are not induced or are / minimally induced against the flagellin. However, the interaction of flagellin with TLR-5 expressed on DCs, monocytes and other cells induces the activation of these cells. Finally, another advantage of using flagellin fusion proteins is that less antigen and less adjuvant might be needed relative to other vaccination methods, because the adjuvant (flagellin) would also target the fusion protein to the APCs.

Even though only weak responses are generated against the flagellin, it is still possible that any antiflagellin response could diminish the full potential of flagellin- derived vaccines. Flagellin contains conserved structures at the N- and C-terminal ends and an intervening hypervariable region (Eaves-Pyles TD, Wong HR, Odoms K, Pyles RB. Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein. J Immunol 2001; 167:7009-7016). It has been hypothesized that the binding of flagellin to the TLR-5 is mediated by the recognition of the conserved structures in the flagellin protein. In this manner, TLR-5 expressed on APCs can guarantee the recognition of any flagellin. Recently, Eaves- Pyles et al. (Eaves-Pyles TD, Wong HR, Odoms K, Pyles RB. Salmonella flagellin- dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein. J Immunol 2001; 167:7009-7016) demonstrated that the constant regions of flagellin, but not the hypervariable region, were capable of inducing a proinflammatory response in cultured human monocytes. The results indicate that the conserved region of flagellin contains the binding domain to TLR-5. The identifi- cation of flagellin minimal binding domains will facilitate the construction of smaller effective molecules, allowing easier tissue penetration and perhaps evoking milder immune responses against flagellin. The construction of other flagellin fusion proteins containing other antigens (e.g., against infectious pathogens) will determine the capacity of these proteins to provide protective irnmunity. In conclusion, have we shown that flagellin fusion proteins are capable of stimulating APCs and, more importantly, that they have the capacity to induce specific immune responses. Therefore, flagellin fusion proteins are suitable carriers for the development of adjuvants or vaccines to induce and boost specific immune responses. It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLE I Materials and Methods

These experiments were performed essentially as described in Cuadros et al., Infect. Immun. 72:2810-2816 (2004), which is incorporated herein by reference.

Mice and cell lines. BALB/c mice were purchased from Harlan (Indianapolis, Ind.) and housed under pathogen-free conditions. The cell lines BM-185-w.t. (wild type), BM-185 expressing EGFP (BM-185-EGFP), and BM-185 expressing EGFP and CD80 (BM-185-EGFP-CD80) were kindly provided by D. Kohn at the University of Southern California, Los Angeles. All cell lines were maintained in complete RPMI medium (RPMI 1640) supplemented with 10% fetal calf serum, 2 mM glutamine, 5xl0-⁵ M2- mercaptoethanol, and gentamicin (50 μg/ml).

Generation of a flagellin-EGFP fusion protein. A flagellin-EGFP chimeric protein was prepared by PCR amplification of the entire flj^'B gene from Salmonella enterica serovar Typhimurium LT2 and the EGFP gene. The products were inserted in frame into the pRSET-A expression vector (Invitrogen) to form the chimeric gene (Fig. 1). The pRSET-A-flagellin-EGFP plasmid was used to transform E. coli BL21. One selected colony was grown at 37°C in Luria broth containing ampicillin (100 μg/ml). Stationary-phase bacteria were transferred to fresh broth, grown to an optical density of approximately 0.5 at 600 nm, and supplemented with 1 mM isopropyl-β-o- thiogalactopyranoside (IPTG) to induce the fusion protein synthesis. After 4 h of induction, bacteria were harvested and resuspended in chilled STE buffer (10 mM Tris [pH 8], 50 mM NaCl, 1 mM EDTA) containing lysozyme (100 μg/ml) and 0.2 mM phenylmethylsulfonyl fluoride and incubated 30 min on ice. Cells were lysed by two freeze-thaw cycles followed by sonication, and the lysates were cleared by centrifugation (15,000 x g for 30 min). This recombinant His-tagged flagellin fusion protein was purified by affinity chromatography on Ni-nitrilotriacetic acid agarose columns (Qiagen). Potential LPS contamination of the purified protein was neutralized with a polymyxin B column, and the levels of LPS were tested by the chromogenic Limulus amebocyte test. The proteins used in the experiments described below do not contain any detectable levels of endotoxin. Purified proteins were dialyzed against PBS, and each preparation was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Fig. 1) to check the size and purity of the protein, which was quantified by the Bradford assay. For control purposes, we prepared a recombinant EGFP protein using the same protocols (Fig. 1).

Isolation of APCs. Bone marrow cells from B ALB/c mice were depleted of lymphocytes with magnetic beads conjugated with anti-CD4, anti-CD8, and anti- B220 antibodies (Dynal, Oslo, Norway). The remaining cells were cultured in complete RPMI medium containing 3% granulocyte-macrophage colony-stimulating factor (GM-CSF) (supernatant from J558L cells transfected with the murine GM-CSF gene, obtained from R Steinman, Rockefeller University, New York, N.Y.). The medium was changed every second day, each time applying fresh RPMI medium containing 3% GM-CSF. On day 8, the cells were ready to use. These cells are a pool of activated APCs, including DCs, MΦ, and monocytes (data not shown). Binding of flagellin-EGFP and EGFP proteins to APCs. We took advantage of the fluorescent properties of the EGFP (which are conserved in the chimeric protein upon fusion with flagellin) for detection and localization purposes. Bone marrow-derived APCs were recovered on day 8 and plated (10 ) on Lab-Tek II chambered coverglasses (Nalge Nunc Int., Naperville, HI.). They were incubated in complete medium at 37°C for 1 h with a 10-μg/ml concentration of purified flagellin-EGFP or EGFP proteins. Then, APCs were washed twice with fluorescence- activated cell sorter (FACS) buffer (PBS supplemented with 0.5% serum and 0.01 % NaN₃), cell fluorescence intensity was analyzed by flow cytometry (FACScan; Becton Dickinson, San Diego, Calif), and data analysis was performed using the CellQuest software.

Maturation of APCs induced by flagellin-EGFP fusion proteins. The maturation of pulsed APCs was confirmed by FACS analysis evaluating the levels of B7- 1 and class II MHC expression. For these experiments 10³ freshly isolated APCs (not cultured with GM-CSF) were incubated with 10-μg/ml concentrations of flagellin-EGFP, EGFP, and LPS (used as control, known to induce the maturation of APCs). After 24 h cells were washed and the expression of class II MHC and B7.1 was evaluated by FACS analysis. To detect the expression of surface molecules, APCs were stained with a primary murine biotinylated antibody directed against the B7.1 (PharMingen, San Diego, Calif.) or class II MHC (Pharmingen) molecules, followed by incubation with fluorescein-conjugated streptavidin. Samples were analyzed by flow cytometry and data analysis was performed using the CellQuest software.

Measurement of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) production by APCs. NO^" was measured using the Griess reaction. Culture supernatants (100 μl) from stimulated APCs were added to 100 μl of 1% sulfanilamide-0.1% naphfhylethylene diamine dihydrochloride-2% H₃PO₄ and incubated for 10 min at room temperature. The absorbance at 562 nm was measured on an ELISA plate reader. A serial dilution of NaNO₂ was used as standard. The limit of detection of this assay was 10 μM NO₂. The production of TNF- was determined with a commercial ELISA kit (R&D Systems) on the same supernatants used for NO^" measurement.

Determination of processing and presentation of antigens derived from flagellin- EGFP fusion proteins. Cultured APCs (10⁶) were incubated with a 10-μg/ml concentration of flagellin-EGFP, EGFP, or adenovirus-EGFP. After 24 h of incubation APCs were washed and the cytotoxic activity of anti-EGFP-specific T cells (previously established in our laboratory) against these pulsed cells was measured by a ⁵¹Cr release assay. Pulsed APCs were incubated with 150 μCi of [⁵¹Cr]sodium chromate for 1 h at 37°C. Cells were washed three times and resuspended in complete RPMI medium. For the cytotoxic assay, ⁵¹Cr-labeled target cells (10⁴) were incubated with various concentrations of effector cells in a final volume of 200 μl in U-bottom 96-well microtiter plates. Supernatants were recovered after 6 h of incubation at 37°C and the percent lysis was determined by the formula: percent specific lysis 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release).

Stimulation of T-cell responses by a flagellin-EGFP protein. BALB/c mice were immunized with a subcutaneous injection of flagellin-EGFP or EGFP (50 μg/injection). As a control, animals were immunized with an adenovirus-EGFP vector. Two weeks later, spleens from primed animals were removed and splenocytes were restimulated in vitro with BM- 185-EGFP-CD80 cells. After 5 days, CTLs were assayed for lytic activity. The BM-185-w.t, BM-185-w.t. pulsed with the H2-K^d-HYLSTQSAL peptide (a K^d-EGFP-derived immunodominant epitope), and BM-185-EGFP cells were incubated with 150 μCi of [⁵¹Cr] sodium chromate for 1 h at 37°C. Cells were washed three times and resuspended in complete RPMI medium. For the cytotoxic assay we followed the same protocol as described above. For the CD4 proliferation assays, CD4⁺ T cells were enriched from spleens of immunized animals and incubated for 4 days with APCs pulsed with adenovirus-EGFP. After 3 days of incubation, tritiated thymidine (1 μCi/well for the last 18 h) was added, and thymidine incorporation was measured.

EXAMPLE II Generation of Flagellin-fusion Proteins

Flagellin is an immunostimulatory molecule of the innate immune system. Flagellin can be used as an adjuvant to induce and boost immune responses. To test the efficacy of flagellin as an adjuvant and as a proof of concept, we generated a flagellin- EGFP protein fusion protein. Since EGFP is immunogenic, we established a model in which we measure T and B cell responses and, because of the fluorescent nature of the protein, we follow the protein, once it is phagocytosed by APCs. The flagellin-EGFP protein was prepared by amplifying by PCR the entire fljB gene from Salmonella thyphimurium and the EGFP gene. The products were inserted into pRSET-A expression vector (Invitrogen) to form the chimeric gene (Fig 1). The pRSET-A-EGFP-flagellin plasmid was used to transform E. coli BL21 and a selected colony was grown to produce and purify the fusion protein. The fusion protein was purified through an affinity chromatography Ni-NTA column (Qiagen). The LPS activity of the purified protein was neutralized with a polymyxin B column and the levels of LPS were confirmed by the chromogenic Limulus amebocyte test. The fusion proteins used in the experiments described below do not contain any detectable levels of LPS/endotoxin. Each preparation was analyzed by SDS-PAGE (Fig 1). Quantification of the fusion protein was calculated by Bradford assay. For control purposes, we prepared a recombinant EGFP protein using the same protocol as described above (Fig 1).

Functional activity of the flagellin-EGFP fusion protein. To test the activity and antigenic and immunostimulatory properties of the flagellin-EGFP fusion protein, we first analyzed the capacity of the flagellin-EGFP protein to bind dendritic cells (DCs)(antigen presenting cells; APCs). Bone marrow derived DCs were incubated with 10 μg/ml of the purified flagellin-EGFP and EGFP proteins for one hour. As demonstrated by FACS analysis (Fig 2), almost 50% of the DCs incubated with the flagellin-EGFP are fluorescent, while DCs incubated with the EGFP protein showed that only 3% of the cells are fluorescent. Moreover, after one hour, multiple molecules of the flagellin-EGFP are internalized into the cytoplasm of the DCs (data not shown), while no intemalization of the EGFP protein was observed after this time. Next, we evaluated whether the flagellin-EGFP fusion protein would induce the maturation of DCs. When maturation occurs, APCs increase the expression of surface molecules such as class II MHC and B7.1 (Gewirtz AT, Navas TA, Lyons S , Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial pro inflammatory gene expression. J Immunol 2001; 167:1882-1885). To this end, DCs were incubated overnight in the presence of 10 μg/ml of the purified flagellin-EGFP, EGFP proteins or commercial LPS (used as control, known to induce maturation of DCs) and the expression of class II and B7.1 molecules were analyzed. As shown in Fig 3, DCs cells treated with flagellin-EGFP and LPS induced the expression of these molecules, while treatment of DCs with EGFP-protein does not. We also evaluated the expression of B7.2. Interestingly, we observed that after stimulation with the flagellin- EGFP fusion protein, APCs also expressed B7.2, but to a lower extent than B7.1 (data not shown)

After confirming that flagellin-EGFP binds and induces the maturation of APCs, we examined whether this adjuvant- antigen chimeric protein is also capable of stimulating APCs to produce a proinflammatory response. To this end, the production of TNF-α and NO was measured. TNF-α and NO are known to be produced upon APC stimulation and to participate in the proinflammatory response (Banchereau et al., Annu. Rev. Immunol. 18:767-811 (2000). To evaluate whether the flagellin-EGFP protein induced a pro-inflammatory response, the production of TNF- α and Nitrite/Nitrate (NO) was determined. As shown in Fig 4, flagellin-EGFP and LPS (used as a control) treated DCs produced TNF-α and NO while no production is detected after treatment with the EGFP protein. Taken together, these results demonstrate that the flagellin-EGFP fusion protein can bind specifically to APCs and that this protein is functionally capable of inducing the maturation and stimulation of APCs. It is important to mention that we found similar results to those described above in experiments performed with purified monocytes (data not shown). EXAMPLE III Induction of Specific T cell Responses by Flagellin-EGFP Fusion Proteins

The preceding results demonstrate that flagellin-EGFP recombinant protein is bioactive and specifically activates APCs. Critical for the development of a vaccine, is the induction of specific immune responses. We next tested whether the flagellin-EGFP induced specific T cell responses. For evaluation of T cell responses against EGFP, we utilized the identified EGFP restricted H2-K^d-HYLSTQSAL peptide (Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tuting T, Robbins PD, DeLeo AB. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther 2000; 7:2036-2040) and a pre-B cell line (BM-185 w.t.) expressing the EGFP gene (BM-185-EGFP) (Stripecke R, Carmen Villacres M, Skelton D, Satake N, Halene S, Kohn D. Immune response to .green fluorescent protein: implications for gene therapy. Gene Ther 1999; 6:1305-1312). Balb/c mice were immunized s.c. with the flagellin-EGFP or the EGFP protein (50 μg/injection). As a control, animals were immunized with an adenovirus-EGFP vector. Two weeks later spleens from primed animals were removed and spleen cells were in vitro re-stimulated with BM-185 cells expressing the EGFP and CD80 genes (BM-185- EGFP-CD80). The T cell responses were measured against a BM-185 w.t, BM-185- w.t pulsed with the H2-K^d-HYLSTQS AL peptide and BM- 185-EGFP cells. As shown in Fig. 5, CD8⁺ T cells derived from mice immunized with either adenovirus-EGFP (Fig. 5A) or flagellin-EGFP (Fig. 5B) induced comparable cytotoxic activities against both BM-185-EGFP cells and H2-K^d-HYLST QSAL-pulsed BM-185-w.t. cells. No cytolytic activity was observed against the parental BM-185-w.t. cells, demonstrating that the immune response is specific. As shown in Fig 5, flagellin-EGFP immunized animals induced EGFP-specific T cell responses at almost the a level similar to animals immunized with the adenovirus-EGFP vector. In contrast, animals immunized with the soluble EGFP did not induce a T cell response. To confirm that the antigen from the flagellin-EGFP fusion protein that was phagocytosed by the APCs was processed and presented, DCs were incubated overnight with the flagellin-EGFP, EGFP proteins or adenovirus-EGFP, and cytotoxic activity of specific-EGFP T cells against these pulsed cells was measured. As demonstrated in Fig 6, specific-EGFP T cells were able to effectively kill DCs pulsed with the flagellin- EGFP and Adenovirus-EGFP vector, while a much lower cytotoxic activity was observed on DCs pulsed with the EGFP protein. No killing activity was observed on DCs that were no pulsed, indicating that the killing was specific. These experiments demonstrate that antigens from flagellin- EGFP are more effectively processed and presented by APCs than antigens from recombinant EGFP.

The experiments described above demonstrate that flagellin-EGFP induces CD8 T cell response. It is well established that CD4 T cell responses are also critical for an optimal immune response. Next, we assayed whether animals immunized with flagellin- EGFP fusion protein stimulate a CD4 T cell response. Balb/c mice were immunized s.c. with the flagellin-EGFP or EGFP proteins. Two weeks later spleens were removed and CD4⁺ T cells were enriched utilizing magnetic beads (Miltenyi Biotec). CD4 T cell responses were measured in a proliferation assay against DCs pulsed with adenovirus EGFP. Our data demonstrate that CD4⁺ T cells from flagellin-EGFP immunized mice proliferate after this in vitro stimulation while no proliferation is observed on CD4⁺ T cells from EGFP immunized mice (Figure 8). Taken together, these results demonstrate that: 1) the flagellin-EGFP fusion protein is phagocytosed with high efficiency by APCs when compared to soluble antigen (EGFP) and 2) the antigen fused to the flagellin was processed and presented correctly to induce a specific CD4⁺ and CD8⁺ T cell response.

EXAMPLE IV Induction of Humoral Responses by Flagellin-EGFP Fusion Proteins

A vaccine should not only induce cellular responses, but the activation of humoral responses are also very important for the induction of a complete protective immunity. We evaluated whether the flagellin-EGFP would induce an antibody response against the EGFP. One of our concerns was whether the flagellin-EGFP would induce an anti-flagellin response that could neutralize the immune response if subsequent immunizations with the flagellin-EGFP protein would be used. Several reports indicate that previous exposure to the flagellin alone does not neutralize the immune response against viral epitopes inserted in the flagellin of a live vaccine of Salmonella (Ben- Yedidia T, Amon R. Effect of pre-existing carrier immunity on the efficacy of synthetic influenza vaccine. Immunol Lett 1998; 64:9-15; Cattozzo EM, Stocker BA, Radaelli A, De Giuli. Morghen C, Tognon M. Expression and immunogenicity of V3 loop epitopes of HIV- 1, isolates SC and WMJ2, inserted in Salmonella flagellin. J. Biotechnol 1997; 56:191-203). We tested the effect of multiple immunizations with the flagellin-EGFP protein. To this end, Balb/c mice were immunized with flagellin-EGFP or EGFP proteins four times at intervals of three weeks between each immunization and the animals were bled ten days after each immunization. Serum was analyzed for antibodies specific against EGFP and flagellin. As shown in Fig 7A, a stronger anti-EGFP response is observed in animals immunized with the flagella-EGFP protein overtime, while a much weaker response is observed in animals immunized with the EGFP protein. We found a low response against the flagellin (Fig 7B). These results further support the findings of other laboratories (Ben-Yedidia T, Amon R. Effect of pre-existing carrier immunity on the efficacy of synthetic influenza vaccine. Immunol Lett 1998; 64:9- 15; Cattozzo EM, Stocker BA, Radaelli A, De Giuli. Morghen C, Tognon M. Expression and immunogenicity of V3 loop epitopes of HIV-1, isolates SC and WMJ2, inserted in Salmonella flagellin. J. Biotechnol 1997; 56:191-203) that prior exposure to the flagellin does not neutralize the immune response. A simple explanation as to why a strong antibody response is not induced against the flagellin protein could be because hosts are "tolerant" or "partially tolerant" to flagellin. It is well established that a natural flora of bacteria exists in the body very early in life. As such, we might have developed some level of tolerance overtime whereby T or B cell responses are not induced or are minimally induced against the flagellin. However, the innate immune cells such as, DCs, monocytes and other cells still interact with the flagellin through the TLR-5 receptor and these cells are activated.

Taken together, these results show, for the first time, that recombinant flagellin fusion proteins are functional and it might be possible to incorporate into the flagellin fusion protein any antigen of interest regardless of its size. More importantly, our data show that flagellin fusion proteins stimulate T and B cell responses indicating that they can serve as adjuvants/vaccines. Our next step is to test whether flagellin-fusion proteins carrying an infectious disease antigen will provide a prophylactic and therapeutic protective immunity. Influenza continues to pose a substantial high risk world wide. Vaccines containing recombinant antigens of the flu virus might be a conceivable option to prevent the disease. Most of the flu vaccines are directed against hamagglutinin (HA) and neuramidase (NA) that provide only antibody mediated protective immunity (Ulmer JB. Influenza DNA vaccines. Vaccine 2002; 20 Suppl 2:S74-76). However, the constant changes of the HA and NA antigenic determinants makes it more difficult to develop a universal vaccine against influenza. Alternatively, the influenza nucleoprotein (NP), a core virus of the flu virus, is a more conserved protein. Antigenic changes are rare and only to a minor extent (Gschoesser C, Almanzar G, Hainz U, Ortin J, Schonitzer D, Schild H, Saurwein-Teissl M, Grubeck-Loebenstein B. CD4(+) and CD8(+) mediated cellular immune response to recombinant influenza nucleoprotein. Vaccine 2002; 20:3731-3738). Cellular immunity against NP is valuable, as it is directed at different variants of NP epitopes. Therefore, recombinant influenza NP might be a potentially valuable vaccine due to its cross-reactivity against even distantly related influenza virus subtypes. As a proof of concept for the induction of a prophylactic therapeutic response against infectious diseases, we will test the efficacy of flagellin-NP and flagellin-HA as adjuvant/vaccine against influenza viral infection in these studies. Another consideration is that protecting the population against major outbreaks of flu encounter great difficulties, such as providing large quantities of vaccine corresponding to the newly emerged virus strain in a short period of time. Recombinant-flagellin fusion proteins can provide a solution by producing relatively quickly large quantities of a desired vaccine carrying the antigenic determinant of interest. The results of these studies will lay the foundation for the use of flagellin-fusion proteins for the development of vaccines against different infectious diseases.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

Claims

What is Claimed Is: 1. A polypeptide comprising a flagellin polypeptide fused to an antigen.

2. A nucleic acid encoding the polypeptide of claim 1.

3. A composition comprising a polypeptide comprising a flagellin polypeptide fused to an antigen.

4. The composition of claim 3, further comprising a physiologically acceptable carrier.

5. A method of stimulating an immune response comprising administering to an individual a composition comprising a polypeptide comprising a flagellin polypeptide fused to an antigen.

6. The method of claim 5, wherein the composition further comprises a physiologically acceptable carrier.