Respiratory synthetic viral antigen compositions and methods

Abstract

Described herein are compositions comprising particles, the particles comprising a multilayer film, the multilayer film comprising two or more layers of polyelectrolytes, adjacent layers comprising oppositely charged polyelectrolytes, one of the polyelectrolytes comprising a designed polypeptide represented by the structure: (Pam3Cys or Pam2Cys)-(surface adsorption region 1)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region 2).

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 63 / 324,824, filed March 29, 2022, which is incorporated by reference in its entirety. The present disclosure relates to compositions and methods for preventing infection with respiratory syncytial virus, and in particular to multi-layer film compositions containing antigenic epitopes. [Background technology]

[0002] Respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease in infants and young children worldwide and is also a threat to older and immunocompromised patients. In the United States, RSV infections result in up to 126,000 infant hospitalizations and up to 60,000 older adult hospitalizations annually. Natural RSV infections do not induce persistent long-term immunity, so patients are susceptible to reinfection with the same and different virus strains throughout their lives. RSV has been associated with secondary infections, such as otitis media, which may predispose young children to asthma-related illnesses later in life.

[0003] After more than 40 years of effort, there is no safe and effective RSV vaccine. The earliest attempt to develop a formalin-inactivated, alum-precipitated RSV (FI-RSV) vaccine around 1960 actually found that vaccinated children were more susceptible to more severe disease and even death during subsequent natural infections. The exact mechanism of this response has not been fully characterized but appears to depend on skewing of the immune response toward a proinflammatory Th2-dominant phenotype characterized by inappropriate activation of cytokine and chemokine pathways.

[0004] Given the economic impact of RSV disease, estimated at approximately $700 million annually in the United States in 2004, and the life-threatening complications that can result from RSV infection in infants, the elderly, and immunocompromised patients, the development of a safe and effective RSV vaccine is a high priority.

[0005] No. 9,487,593 describes antigenic RSV compositions and methods. However, there is a need for improved antigenic compositions suitable for stimulating an immune response to RSV. Summary of the Invention

[0006] In an embodiment, the composition comprises a particle, the particle comprises a multilayer film, the multilayer film comprises two or more layers of polyelectrolytes, adjacent layers comprise oppositely charged polyelectrolytes, one of the polyelectrolytes having the structure (Pam3Cys or Pam2Cys)-(Surface Adsorption Region 1)-(RSV-G Peptide Epitope)-L1-(RSV-M2 Peptide Epitope)-L2-(Surface Adsorption Region 2) and a designed polypeptide having the formula: The surface adsorption regions 1 and 2 each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, and the net charge per residue of the designed polypeptide is 0.1 or more; the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA) or (ESYIGSINNITKQSASVA); the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT); and L1 or L2 is a linker comprising 0 to 100 uncharged amino acid residues; Designed non-polypeptide polyelectrolytes include polycationic or polyanionic materials having a molecular weight greater than 1,000 and at least 5 charges per molecule; The multilayer film is deposited onto a core particle or forms a hollow particle to provide the composition. [Brief description of the drawings]

[0007] [Figure 1A] FIG. 1 shows antibody responses in mice immunized with ACT-1190. [Figure 1B]Figure 1 shows antibody responses of mice immunized with ACT-1190. BALB / c mice were immunized with the indicated doses of ACT-1190 on days 0 and 21. Mice were bled one week after the boost and serum was tested by ELISA against RSV-G protein. (1A) Results show individual mice (open circles) and group means (bars) at a 1:50 dilution of serum. (1B) Results show the average response of 16 animals per group.

[0008] [Diagram 2] Figure 2 shows T cell responses of mice immunized with ACT-1190. On day 28, 3 mice / group from Figure 1 were sacrificed, spleen cells were harvested, and restimulated in vitro with RSV-M2 and RSV-G peptides in IFNγ and IL-5 ELISPOT plates. Data show mean ± SD spots / 106 cells of 3 mice / group.

[0009] [Diagram 3] Figure 1 shows pulmonary RSV burden after challenge with strain A2. On day 35 (14 days after boost), the mice from Figure 1 were challenged with RSVA2. Five days after challenge, lungs were harvested and viral load was measured by standard plaque assay. Results are shown for individual mice (circles) and group means (bars); inset shows number of mice fully protected from challenge (>90% reduction in viral load), group mean percent reduction in plaque count compared to naive group and P value by Student's t-test compared to naive group; n=12 mice per group.

[0010] [Figure 4A] FIG. 1 shows antibody responses elicited by immunization with RSV microparticles. [Figure 4B]Figure 4. Antibody responses induced by immunization with RSV microparticles. BALB / c mice were immunized with the indicated treatments on days 0 and 21. Sera collected on day 28 were tested by ELISA against RSV-G protein. (4A) Results show the mean ± SEM of 10 mice per group. (4B) Sera were tested at 1:50 and plates were probed with isotype-specific detection antibodies. Results show the mean ± SEM of 10 mice per group.

[0011] [Diagram 5] Figure 1 shows T cell responses to RSV microparticles. On day 28, splenocytes were harvested from 3 mice per group from Figure 4 and restimulated with RSV-G+M2 peptide in IFNγ and IL-5 ELISPOT plates. Data show the mean ± SD of 3 mice per group.

[0012] [Figure 6A] FIG. 1 shows efficacy of RSV vaccines containing PAM3Cys. [Figure 6B] Figure 6: Efficacy of RSV vaccine containing PAM3Cys. Mice from Figure 4 were challenged with RSV on day 35 (14 days post-boost). Lungs were harvested 5 days after challenge and viral load was measured. (6A) Plaque assay results show individual mice (circles) and group means (bars). (6B) qPCR results show mean ± SEM of 10 mice per group. In both panels, insets show the number of mice protected (>90% reduction in viral load), mean group percent reduction and P-values ​​(all compared to PBS group).

[0013] [Figure 7A] FIG. 1 shows antibody responses of mice immunized with ACT-1190, -1193, -1213 and -1214. [Figure 7B] FIG. 1 shows antibody responses of mice immunized with ACT-1190, -1193, -1213 and -1214. [Figure 7C]Figure 7 shows antibody responses of mice immunized with ACT-1190, -1193, -1213 and -1214. BALB / c mice were immunized on days 0 and 21 and control mice were included as described in the text. Mice were bled one week after the boost and sera were tested by ELISA against RSV-G peptide. (7A) Results show the mean response of 10 animals per group. (7B) Results show individual mice (open circles) and group means (bars) at a 1:50 dilution of sera. (7C) Results show the mean ± SD of 10 animals per group at a 1:50 dilution of sera.

[0014] [Figure 8A] FIG. 1 shows T cell responses in mice immunized with ACT-1190, -1193, 1-1213 and -1214. [Figure 8B] FIG. 1 shows T cell responses in mice immunized with ACT-1190, -1193, 1-1213 and -1214. [Figure 8C] Figure 8 shows T cell responses of mice immunized with ACT-1190, -1193, 1-1213 and -1214. On day 28, 3 mice / group from Figure 7 were sacrificed, splenocytes were harvested and restimulated in vitro with RSV-M2 (8A), RSV-G (8B) or both peptides (8C) in IFNγ and IL-5 ELISPOT plates. Data show mean ± SD spots / 106 cells of 3 mice / group.

[0015] [Figure 9] Figure 1 shows pulmonary RSV burden after challenge with A2 strain. On day 35 (14 days post boost), mice from Figure 7 were challenged with live RSV A2. Five days after challenge, mice were sacrificed and pulmonary viral load was measured by standard plaque assay on Vero cells. Results are shown for individual mice (circles) and group means (bars). Inset shows the number of fully protected individual mice, % reduction in mean viral titer per group and P value for group 1214 compared to naive group mean. For all other groups, 10 / 10 were protected, group mean was 100% protected, P<0.0000001.

[0016] [Figure 10A] FIG. 1 shows the numbers of EOS and MΦ cells in bronchoalveolar lavage (BAL) fluid. [Figure 10B] FIG. 1 shows the numbers of EOS and MΦ cells in bronchoalveolar lavage (BAL) fluid. [Figure 10C] Figure 1 shows the number of EOS and MΦ cells in bronchoalveolar lavage (BAL) fluid. Mice from Figure 7 were challenged with RSV on day 35, and BAL cells were collected and analyzed by flow cytometry as described in the text. Data represent the mean ± SD cells / 50,000 events collected from 3 mice / group at each time point.

[0017] [Figure 11A] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11B] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11C] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11D] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11E] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11F] FIG. 1 shows cytokine and chemokine content in the lungs following RSV challenge of immunized mice. [Figure 11G] Figure 1 shows cytokine and chemokine content in lungs after RSV challenge of immunized mice. Mice from Figure 7 were challenged with live RSV on day 35 and BAL fluid was collected from lungs 6 days later. After removal of cells by centrifugation, BAL fluid was analyzed by 16plex cytokine and chemokine ELISA. Results show the mean ± SD of 3 mice per group per specimen.

[0018] [Figure 12A] FIG. 1 shows antibody responses in BAL fluid. [Figure 12B] FIG. 1 shows antibody responses in BAL fluid. [Figure 12C] FIG. 1 shows antibody responses in BAL fluid. [Figure 12D] FIG. 1 shows antibody responses in BAL fluid. [Figure 12E] Figure 12: Antibody responses in BAL fluid. BAL fluid was collected from mice from Figure 7 (3 mice per group per sacrifice day) before challenge (day 0) and on days 6 and 10 after challenge and tested for total IgG and against RSV-G peptide. (12A) Mean ± SD peptide-specific responses. (12B) Mean ± SD total IgG levels. (12C), (12D) and (12E) Mean ± SD isotype levels on the indicated days. n=3 per group.

[0019] [Figure 13A] FIG. 1 shows antibody responses induced by immunization with RSV-GM2 microparticles. [Figure 13B] FIG. 1 shows antibody responses induced by immunization with RSV-GM2 microparticles. [Figure 13C] Figure 13 shows antibody responses induced by immunization with RSV-GM2 microparticles. Mice from Figure 12 were bled on day 28 (day 7 after boost) and sera were tested by ELISA against RSV-G peptide. (13A) Results show the mean ± SD of 12 mice per group. (13B) Results show individual mice (circles) and group means (bars) at 1:50 serum dilution. (13C) Sera were tested at 1:50 and plates were probed with isotype-specific detection antibodies. Results show the mean ± SD of 12 mice per group.

[0020] [Figure 14]Figure 13 shows pulmonary RSV burden after challenge with the A2 strain. Mice from Figure 12 were challenged with live RSV on day 35 (14 days post-boost) and pulmonary viral load was measured by standard plaque assay on Vero cells 5 days after challenge. Results are shown for individual mice (circles) and group means (bars); insets show % reduction in mean viral titers per group, number of individual mice with complete protection and P-values, all compared to the naive group mean.

[0021] [Figure 15A] FIG. 1 shows antibody responses and CCL2 levels in BAL fluid. [Figure 15B] FIG. 1 shows antibody responses and CCL2 levels in BAL fluid. [Figure 15C] FIG. 1 shows antibody responses and CCL2 levels in BAL fluid. [Figure 15D] Antibody responses and CCL2 levels in BAL fluid. BALB / c mice were immunized on days 0 and 21, challenged on day 35, and BAL fluid was collected on day 41. (15A) Total IgG, mean ± SD of 6 mice per group. (15B) RSV-G peptide-specific IgG (circles) and group mean (bars) in individual mice. (15C) RSV-G peptide-specific IgG1 and IgG2a, mean ± SD of 6 mice per group. (15D) CCL2 levels in individual mice; red circles indicate values ​​above the limit of detection; open circles represent values ​​below the limit of detection.

[0022] [Figure 16A] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16B] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16C] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16D]FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16E] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16F] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16G] FIG. 1 shows cytokine and chemokine content in the lungs of immunized mice following RSV challenge. [Figure 16H] Figure 14 shows cytokine and chemokine content in lungs after RSV challenge of immunized mice. Mice from Figure 14 were challenged with live RSV on day 35 and BAL fluid was collected from lungs 6 days later. After removal of cells by centrifugation, BAL fluid was analyzed by 16plex cytokine and chemokine ELISA. Results show the mean ± SD of 3 mice per group per specimen. Red columns indicate Th2 cytokines.

[0023] [Figure 17A] FIG. 1 shows inhibition of the chemotactic activity of RSV-G protein by antibodies induced by LbL-MP vaccination. [Figure 17B] Figure 17 shows the inhibition of the chemotactic activity of RSV-G protein by antibodies induced by LbL-MP vaccination. Antibodies purified from serum from mice from Figure 7 (Figure 17A) and Figure 12 (Figure 17B) were added to the lower chamber containing RSV-G protein, and human PBMCs were added to the upper chamber. After overnight incubation, the number of cells that migrated to the lower chamber was counted and the % inhibition was calculated by comparing with the control wells containing only RSV-G protein in the lower chamber. The assay was performed three times with duplicate wells each time; the results show the mean ± SD of six replicates per sample. 131-2G is a monoclonal antibody specific for the RSV-G CX3C epitope and was included as a positive control for inhibition of chemotactic activity.

[0024] [Figure 18A] FIG. 1 shows antibody responses and body weight following RSV challenge. [Figure 18B] Figure 18. Antibody responses and body weight after RSV challenge. RSV naive mice were immunized on days 0 and 21 (1211, 1216, FI-RSV) or infected with 106 pfu of live RSV on day 0. (18A) Mice were bled on day 28 and antibody levels were measured by ELISA on RSV-G coated plates. Results show the mean ± SD of individual mice (open circles) and 10 mice per group (red bars) at 1:50 serum dilution. (18B) Mice were challenged on day 35 (day 0 post-challenge on the x-axis) and weighed daily starting on the day of challenge (day 0). Results show the mean percent of starting body weight for 10 mice / group through day 5 when 7 mice / group were sacrificed for plaque assays and for the remaining 3 mice / group through day 8 when sacrificed for BAL fluid and cells.

[0025] [Figure 19A] FIG. 1 shows viral load in the lungs of BALB / c mice immunized with RSV microparticles or FI-RSV, or recovered from RSV infection. [Figure 19B] 19A-19B show viral loads in the lungs of BALB / c mice immunized with RSV microparticles or FI-RSV or recovered from RSV infection. Mice from FIG. 18 were challenged with 106 pfu 2 weeks after boost and sacrificed 5 days later to measure lung viral loads. [N]=naive; [+]=challenged; [-]=unchallenged. (19A) Plaque assay results. (19B) qPCR results. Both panels show individual mice (red=group mean). Insets show group mean % reduction vs. naive group mean, number of animals with >90% reduction in viral load and P-values ​​vs. naive-challenged group by Student's t-test, [NS]=not significant, and [*]<0.005.

[0026] [Figure 20A] FIG. 1 shows fluorescent staining of lung cells collected by lavage 8 days after viral challenge. [Figure 20B] Fluorescent staining of lung cells collected by lavage 8 days after viral challenge. Mice from FIG. 18 were sacrificed 8 days after challenge and BAL cells were harvested. Cells were stained with a cocktail of antibodies against CD3 / CD4 / CD8 (20A) or CD45 / CD11c / SiglecF (20B) and analyzed as described in the text. Results are shown as fold increase compared to the naive group mean for 3 individual mice per group.

[0027] [Figure 21] Scatter plots of lung cells collected by lavage 8 days after viral challenge of naive (left) and FI-RSV immunized (right) mice from Figure 18. Triangular region R1 contains T cells, while polygonal region R4 contains the larger, more granular CD45+ eosinophils.

[0028] [Figure 22A] FIG. 19 shows Th1 (22A) cytokine levels in lung homogenates and BAL fluid of mice from FIG. [Figure 22B] Th2 (22B) cytokine levels in lung homogenates and BAL fluid of mice from FIG. 18. Lungs were harvested, homogenized, and clarified 5 days after viral challenge (7 mice / group). Cytokines were measured using a fluorescent bead-based multi-analyte flow assay kit. Data are shown as the mean fold change for each group versus naive control. The group designated "naive, nc" was naive animals that were anesthetized but not challenged.

[0029] These and other features will be understood and appreciated by those skilled in the art from the following detailed description, drawings and appended claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Disclosed herein are multilayer films and particles that contain polypeptide epitopes derived from RSV, which can elicit an immune response in a host upon administration to the host. Specifically described herein are specifically designed TLR targeting peptides that provide multilayer films that advantageously support a more balanced immune response (much lower levels of Th2 cytokines) compared to the same peptides without TLR ligands. The immune response of the constructs described herein is comparable to mice that have recovered from previous infection with low dose live RSV prior to challenge. The TLR targeting peptides and multilayer films are advantageous because the problems of formalin inactivated vaccine trials in the 1960s, where children became ill or even died after natural exposure, compared to unvaccinated children, are widely believed to have been caused by a Th2 inflammatory response resulting from formalin inactivation, which disrupts virus:TLR interactions during vaccination. The immunogenic multilayer film constructs described herein advantageously avoid these problems of conventional RSV vaccines.

[0031] In an embodiment, the composition comprises a particle, the particle comprises a multilayer film, the multilayer film comprises two or more layers of polyelectrolytes, adjacent layers comprise oppositely charged polyelectrolytes, one of the polyelectrolytes having the structure (Pam3Cys or Pam2Cys)-(Surface Adsorption Region 1)-(RSV-G Peptide Epitope)-L1-(RSV-M2 Peptide Epitope)-L2-(Surface Adsorption Region 2) and a designed polypeptide having the formula: The surface adsorption regions 1 and 2 each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, the net charge per residue of the designed polypeptide is 0.1 or more, the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA; SEQ ID NO:1) or (ESYIGSINNITKQSASVA; SEQ ID NO:2), the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT; SEQ ID NO:3), L1 or L2 is a linker comprising 0 to 100 uncharged amino acid residues; the polyelectrolyte that is not a designed polypeptide comprises a polycationic or polyanionic material having a molecular weight of greater than 1,000 and at least 5 charges per molecule, and the multilayer film is deposited on the core particle or forms a hollow particle to provide the composition.

[0032] As described herein, the multilayer film comprises alternating layers of oppositely charged polyelectrolytes, with one layer, such as the outermost layer, comprising a designed polypeptide represented by (Pam3Cys or Pam2Cys)-(surface adsorption region 1)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region 2). The polyelectrolyte in the multilayer film that is not a designed polypeptide comprises a polycationic or polyanionic material having a molecular weight of greater than 1,000 and at least 5 charges per molecule, and the multilayer film is deposited on a core particle or forms a hollow particle to provide the composition.

[0033] More specifically, polyelectrolyte multilayer films are thin films (e.g., a few nanometers to micrometers thick) composed of alternating layers of oppositely charged polyelectrolytes. Such films can be formed by layer-by-layer assembly on a substrate. In electrostatic layer-by-layer self-assembly ("ELBL"), the physical basis for polyelectrolyte association is electrostatic attraction. Film stacking is possible because the sign of the surface charge density of the film is reversed upon deposition of successive layers. The generality and relative simplicity of the ELBL film process allows for the deposition of many different types of polyelectrolytes on many different types of surfaces. Polypeptide multilayer films are a subset of polyelectrolyte multilayer films that include at least one layer that includes a charged polypeptide, referred to herein as a designed polypeptide. An important advantage of polypeptide multilayer films over films made from other polymers is their biocompatibility. ELBL films can also be used for encapsulation. Applications of polypeptide films and microcapsules include, for example, nanoreactors, biosensors, artificial cells, and drug delivery vehicles.

[0034] The term "polyelectrolyte" includes polycationic and polyanionic materials having a molecular weight greater than 1,000 and at least 5 charges per molecule. Suitable polycationic materials include, for example, polypeptides and polyamines. Polyamines include, for example, polypeptides such as poly-L-lysine (PLL) or poly-L-omitin, polyvinylamine, poly(aminostyrene), poly(aminoacrylate), poly(N-methylaminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethylammoacrylate), poly(N,N-diethylaminoacrylate), poly(aminomethacrylate), poly(N-methylaminomethacrylate), poly(N-ethylaminomethacrylate), poly(N,N-dimethylaminomethacrylate), poly(N,N-diethylaminomethacrylate), poly(ethyleneimine), poly(diallyldimethylammonium chloride), poly(N,N,N-trimethylaminoacrylate chloride), poly(methylacrylamidopropyltrimethylammonium chloride), chitosan, and combinations comprising one or more of the foregoing polycationic materials. Suitable polyanionic materials include, for example, polypeptides such as poly-L-glutamic acid (PGA) and poly-L-aspartic acid, nucleic acids such as DNA and RNA, alginate, carrageenan, furcellaran, pectin, xanthan, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, poly(meth)acrylic acid, oxidized cellulose, carboxymethylcellulose, acid polysaccharides and croscarmelose, synthetic polymers and copolymers containing pendant carboxyl groups, and combinations comprising one or more of the above polyanionic materials. In one embodiment, the RSV epitope and the polyelectrolyte have the same sign of charge.

[0035] In embodiments, a stable multilayer film is one that, once formed, retains more than half of its components after incubation in PBS at 37° C. for 24 hours.

[0036] A designed polypeptide refers to a polypeptide that has sufficient charge for stable binding to an oppositely charged surface, i.e., a polypeptide that can be deposited into a layer of a multi-layer film where the driving force for film formation is electrostatic. In another embodiment, the solubility of the designed polypeptide at pH 4-10 is about 0.1 mg / mL or more. In another embodiment, the solubility of the designed polypeptide at pH 4-10 is about 1 mg / mL or more. Solubility is a practical limitation for facilitating deposition of the polypeptide from aqueous solution. The practical upper limit for the degree of polymerization of an antigenic polypeptide is about 1,000 residues. However, it is believed that longer complex polypeptides can be achieved by appropriate synthetic methods.

[0037] In certain embodiments, the magnitude of the net charge per residue of the designed polypeptide is 0.1, 0.2, 0.3, 0.4 or 0.5 or more at pH 7.0. In one embodiment, the ratio of the number of charged residues of the same polarity minus the number of residues of the opposite polarity to the total number of residues in the polypeptide is 0.5 or more at pH 7.0. In other words, the magnitude of the net charge per residue of the polypeptide is 0.5 or more. There is no absolute upper limit to the length of the polypeptide, but generally, designed polypeptides suitable for ELBL deposition have a practical upper limit of 1,000 residues. The designed polypeptide can include sequences found in nature, such as RSV epitopes, as well as regions that provide functionality to the peptide, such as charged regions, also referred to herein as surface adsorption regions, which allow the designed polypeptide to be deposited on a polypeptide multilayer film.

[0038] The positively charged (basic) naturally occurring amino acids at pH 7.0 are arginine (Arg), histidine (His), ornithine (Orn) and lysine (Lys). The negatively charged (acidic) naturally occurring amino acid residues at pH 7.0 are glutamic acid (Glu) and aspartic acid (Asp). Mixtures of amino acid residues of opposite charge can be used as long as the overall net charge ratio meets the specified criteria. In one embodiment, the designed polypeptide is not homopolymeric. In another embodiment, the designed polypeptide is unbranched.

[0039] The designed polypeptides contain Pam3Cys or Pam3Cys at the N-terminus. Pam3Cys and Pam2Cys are TLR ligands. Pam3Cys ([N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine]) is a triacyl bacterial lipoprotein TLR1 ligand. Pam2Cys (Pam2Cys[S-[2,3-bis(palmitoyloxy)propyl]cysteine]) is a diacyl bacterial lipoprotein TLR2 ligand. The experiments described herein use Pam3Cys, but similar results are expected with Pam2Cys.

[0040] Pam3Cys and Pam2Cys can be covalently coupled to a polypeptide chain by standard polypeptide synthesis chemistry reactions. For example, Pam3Cys can be covalently linked to an antigenic polypeptide via a direct covalent linkage via an amide bond formed between the carboxylic acid of Pam3Cys-OH (commercially available from Bachem, Inc.) and the N-terminus of the peptide. A convenient way to achieve this reaction is to couple Pam3Cys-OH to a synthetic peptide on solid-phase synthesis resin beads in the presence of an amide bond forming reagent such as HBTU (O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate), HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methaneaminium) or DIPCDI (N,N'-diisopropylcarbodiimide). The progress of the coupling reaction can be monitored colorimetrically by ninhydrin assay, and after completion, excess Pam3Cys-OH and other reagents can be washed away. The synthetic Pam3Cys peptide conjugate is cleaved from the resin and purified by chromatography. For example, Pam3Cys peptide can be purified by reversed-phase HPLC using a C4 column and a water / isopropanol gradient. The advantage of this approach is that the Pam3Cys / antigenic polypeptide ratio is tightly controlled at 1:1.

[0041] In another embodiment, Pam3Cys-OH is either specifically conjugated to the side chain ε-amine of a lysine residue, specifically conjugated to a resin-bound peptide as described above, or non-specifically conjugated to an unprotected peptide or protein using a water-soluble coupling reagent such as EDC / sulfo-NHS. The product of the reaction is purified, for example, by gel permeation chromatography or dialysis, and then incorporated into particles by LBL or other methods.

[0042] The designed peptides contain two RSV epitopes, the RSV-M2 epitope and the RSV-G epitope. The RSV-M2 peptide epitope contains (ESYIGSINNITKQSACVA; SEQ ID NO: 1) or (ESYIGSINNITKQSASVA; SEQ ID NO: 2), and the RSV-G peptide epitope contains (NFVPCSICSNNPTCWAICKRIPNKKPGKKT; SEQ ID NO: 3). The full sequences of RSV-M2 and RSV-G are provided below. RSV-G-SEQ ID NO:4 1 MSKNKDQRTA KTLERTWDTL NHLLFISSCL YKLNLKSVAQ ITLSILAMII STSLIIAAII 60 61 FIASANHKVT PTTATTQDAT SQIKNTTPTY LTQNPQLGTS PSNPSEITSQ ITTILASTTP 120 121 GVKSTLQSTT VKTKNTTTTQ TQPSKPTTKQ RQNKPPSKPN NDFHFEVFNF VPCSICSNNP 180 181 TCWAICKRIP NKKPGKKTTT KPTKKPTLKT TKKDPKPQTT KSKEVPTTKP TEEPTINTTK 240 241 TNIITTLLTS NTTGNPELTS QMETFHSTSS EGNPSPSQVS TTSEYPSQPS SPPNTPRQ 298 RSV-M2-SEQ ID NO:5 1 MSRRNPCKFE IRGHCLNGKR CHFSHNYFEW PPHALLVRQN FMLNRILKSM DKSIDTLSEI 60 61 SGAAELDRTE EYALGVVGVL ESYIGSINNI TKQSACVAMS KLLTELNSDD IKKLRDNEEL 120 121 NSPKIRVYNT VISYIESNRK NNKQTIHLLK RLPADVLKKT IKNTLDIHKS ITINNPKEST 180 DTNDHAKN NDTT

[0043] The designed peptide comprises two surface adsorption regions that provide sufficient charge to assemble the peptide into a multilayer film. In an embodiment, each of the first and second surface adsorption regions independently comprises at least 2, 3, 4, 5, 6, 7 or 8 amino acid residues and has the same sign of charge as the designed polypeptide. The first and second surface adsorption regions may be the same or different.

[0044] As used herein, a surface adsorption region is a charged region of an engineered polypeptide that advantageously provides sufficient charge to allow the deposition of a peptide, for example, containing an epitope from RSV, onto a multilayer film.

[0045] The designed polypeptide also comprises amino acid linkers L1 and L2 comprising 0 to 100 uncharged amino acid residues. In an embodiment, L1 and L2 are SGS.

[0046] Specifically designed polypeptides include the following: Pam3CSKKKKNFVPCSICSNNPTCWAICKRIPNKKPGKKTSGSESYIGSINNITK QSASVASGSKKKKKKKKKKKKKKKKKKKK; SEQ ID NO:6 Pam3CSKKKKNFVPCSICSNNPTCWAICKRIPNKKPGKKTSGSESYIGSINNITK QSACVASGSKKKKKKKKKKKKKKKKKKKK; SEQ ID NO: 7

[0047] In one embodiment, the multilayer film is deposited on core particles such as CaCO3 nanoparticles, latex particles or iron particles. Particle sizes on the order of 5 nanometers (nm) to 50 micrometers (μm) in diameter are particularly useful. Particles made of other materials can also be used as cores, as long as they are biocompatible, have a controllable size distribution and have sufficient surface charge (either positive or negative) to bind to the polyelectrolyte peptide. Examples include nanoparticles and microparticles made of materials such as polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polyethylene glycol (PEG), chitosan, hyaluronic acid, gelatin or combinations thereof. Core particles can also be made from materials that are considered unsuitable for human use, as long as they can be dissolved and separated from the multilayer film after film manufacture. Examples of template core materials include organic polymers such as latex, or inorganic materials such as silica.

[0048] One design concern is the control of the stability of polypeptide ELBL films. Ionic bonds, hydrogen bonds, van der Waals interactions and hydrophobic interactions contribute to the stability of multilayer films. In addition, covalent disulfide bonds formed between sulfhydryl-containing amino acids in polypeptides within the same layer or adjacent layers can enhance structural strength. Sulfhydryl-containing amino acids include cysteine ​​and homocysteine, and these residues can be easily incorporated into synthetic designed peptides. In addition, sulfhydryl groups can be incorporated into polyelectrolyte homopolymers such as poly-L-lysine or poly-L-glutamic acid by methods well described in the literature. Sulfhydryl-containing amino acids can be used to "lock" (bond together) and "unlock" layers of multilayer polypeptide films by changes in oxidation potential. Incorporation of sulfhydryl-containing amino acids into designed polypeptides also allows the use of relatively short peptides in thin film fabrication due to intermolecular disulfide bond formation.

[0049] In one embodiment, the designed sulfhydryl-containing polypeptides, whether chemically synthesized or produced in a host organism, are assembled by ELBL in the presence of a reducing agent to prevent premature disulfide bond formation. After film assembly, the reducing agent is removed and an oxidizing agent is added. In the presence of the oxidizing agent, disulfide bonds form between the sulfhydryl groups, thereby "locking" the polypeptides together within and between layers where thiol groups reside. Suitable reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (BME), reduced glutathione, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and combinations of two or more of these chemicals. Suitable oxidizing agents include oxidized glutathione, tert-butyl hydroperoxide (t-BHP), thimerosal, diamide, 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB), 4,4'-dithiodipyridine, sodium bromate, hydrogen peroxide, sodium tetrathionate, porphyrins, sodium orthoiodobenzoate, and combinations of two or more of these chemicals.

[0050] As an alternative to disulfide bonds, chemical reactions that generate other covalent bonds can be used to stabilize ELBL films. For films composed of polypeptides, chemical reactions that generate amide bonds are particularly useful. In the presence of a suitable coupling reagent, acidic amino acids (those with side chains containing carboxylic acid groups, such as aspartic acid and glutamic acid) react with amino acids whose side chains contain amine groups (such as lysine and ornithine) to form amide bonds. Amide bonds are more stable than disulfide bonds under biological conditions, and amide bonds do not undergo exchange reactions. Many reagents can be used to activate polypeptide side chains for amide bonds. Carbodiimide reagents, such as water-soluble l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), react with aspartic acid or glutamic acid at slightly acidic pH to form intermediate products that react irreversibly with amines to generate amide bonds. To accelerate the rate and efficiency of amide formation, additives such as N-hydroxysuccinimide are often added to the reaction. After the reaction, the soluble reagents are removed from the nanoparticles or microparticles by centrifugation and aspiration. Examples of other coupling reagents include diisopropylcarbodiimide, HBTU, HATU, HCTU, TBTU and PyBOP. Other additives include sulfo-N-hydroxysuccinimide, 1-hydroxybenzotriazole and 1-hydroxy-7-aza-benzotriazole. The degree of amide crosslinking can be controlled by adjusting the stoichiometry of the coupling reagent, reaction time or reaction temperature, and can be monitored by techniques such as Fourier transform-infrared spectroscopy (FT-IR).

[0051] Covalently crosslinked ELBL films have desirable properties such as increased stability. Higher stability allows for the use of more stringent conditions during the production of nanoparticles, microparticles, nanocapsules or microcapsules. Examples of stringent conditions include high temperatures, low temperatures, cryogenic temperatures, high centrifugation speeds, high salt buffers, high pH buffers, low pH buffers, filtration and long-term storage.

[0052] A method for making polyelectrolyte multilayer films involves depositing multiple layers of oppositely charged species onto a substrate, with at least one layer, preferably the outermost layer, comprising a designed polypeptide as described herein. The successively deposited polyelectrolytes have opposite net charges. In one embodiment, deposition of the polyelectrolyte comprises exposing the substrate to an aqueous solution comprising a polyelectrolyte at a pH that has a suitable net charge for ELBL. In another embodiment, deposition of the polyelectrolyte onto the substrate is achieved by sequential spraying of solutions of oppositely charged polypeptides. In yet another embodiment, deposition onto the substrate is by simultaneous spraying of solutions of oppositely charged polyelectrolytes.

[0053] In the ELBL process of forming multilayer films, the opposing charges of adjacent layers provide the driving force for assembly. It is not important that the polyelectrolytes of the opposing layers have the same net linear charge density, only that the opposing layers have opposite charges. One standard film assembly procedure by deposition involves forming an aqueous solution of polyions at a pH at which the polyions are ionized (i.e., pH 4-10), providing a substrate with a surface charge, and alternately immersing the substrate in the charged polyelectrolyte solution. The substrate is optionally washed between deposition of alternating layers.

[0054] The polyelectrolyte concentration suitable for polyelectrolyte deposition can be readily determined by one of skill in the art. Exemplary concentrations are 0.1-10 mg / mL. For typical non-polypeptide polyelectrolytes such as poly(acrylic acid) and poly(allylamine hydrochloride), typical layer thicknesses are about 3 to about 5 A depending on the ionic strength of the solution. Short polyelectrolytes typically form thinner layers than long polyelectrolytes. With regard to film thickness, polyelectrolyte film thickness depends on humidity as well as the number and composition of layers of the film. For example, a 50 nm thick PLL / PGA film shrinks to 1.6 nm when dried with nitrogen. In general, films with thicknesses of 1 nm to 100 nm or more can be formed depending on the hydration state of the film and the molecular weight of the polyelectrolyte used in the assembly.

[0055] In addition, the number of layers required to form a stable polyelectrolyte multilayer film depends on the polyelectrolytes in the film. For films containing only low molecular weight polypeptide layers, the film typically has four or more bilayers of oppositely charged polypeptides. For films containing high molecular weight polyelectrolytes such as poly(acrylic acid) and poly(allylamine hydrochloride), films containing a single bilayer of oppositely charged polyelectrolytes can be stable. Studies have shown that polyelectrolyte films are dynamic. Polyelectrolytes contained within the film can migrate between layers and can be exchanged with soluble polyelectrolytes of sticky charge when suspended in polyelectrolyte solution. In addition, polyelectrolyte films can degrade or dissolve in response to changes in the environment such as temperature, pH, ionic strength or oxidation potential of the suspension buffer. Thus, some polyelectrolytes, especially peptide polyelectrolytes, exhibit transient stability. The stability of peptide polyelectrolyte films can be monitored by suspending the film in a suitable buffer under controlled conditions for a period of time and then measuring the amount of peptide in the film with a suitable assay such as amino acid analysis, HPLC assay or fluorescence assay. Peptide polyelectrolyte films are most stable under conditions relevant for their storage and use as vaccines, such as in neutral buffers and at ambient temperatures, e.g., 4° C. to 37° C. Under these conditions, stable peptide polyelectrolyte films retain most of their component peptides for at least 24 hours, and often for up to 14 days and beyond.

[0056] For the synthesis of a designed polypeptide, each of the independent regions of the designed polypeptide (e.g., the RSV epitope and the surface adsorption region) can be synthesized separately by solution-phase peptide synthesis, solid-phase peptide synthesis, or genetic engineering of a suitable host organism. Solution-phase peptide synthesis is the method used to generate most of the approved peptide drugs on the market today. A combination of solution-phase and solid-phase methods can be used to synthesize relatively long peptides and even small proteins. Peptide synthesis companies have the expertise and experience to synthesize difficult peptides on a fee-for-service basis. Synthesis is performed under Good Manufacturing Practice (GMP) conditions and on a scale suitable for clinical trials and commercial drug launches.

[0057] Alternatively, the various independent regions can be synthesized together as a single polypeptide chain by solution phase peptide synthesis, solid phase peptide synthesis, or by genetic engineering of a suitable host organism. The choice of approach in any particular case is a matter of convenience or economy.

[0058] When the various RSV epitopes and surface adsorption regions are synthesized separately, for example, purified by ion exchange chromatography or high performance liquid chromatography, they are joined by peptide bond synthesis. That is, the N-terminus of the surface adsorption region is covalently joined to the C-terminus of the RSV epitope to produce a designed polypeptide. Alternatively, the C-terminus of the surface adsorption region is covalently joined to the N-terminus of the RSV epitope to produce a designed polypeptide. Individual fragments can be synthesized by solid-phase methods and obtained as fully protected, fully unprotected or partially protected segments. The segments can be covalently joined in solution or solid-phase reactions. When one polypeptide fragment contains a cysteine ​​as its N-terminal residue and the other polypeptide fragment contains a thioester or thioester precursor at its C-terminal residue, the two fragments spontaneously join in solution by a specific reaction commonly known (to those skilled in the art) as native ligation. Native ligation is a particularly attractive option for designed peptide synthesis, since it can be carried out with fully deprotected or partially protected peptide fragments in aqueous solution and at dilute concentrations.

[0059] In one embodiment, the RSV epitopes and / or surface adsorption regions are linked by peptidic or non-peptidic bridges, as described in U.S. Patent No. 7,723,294, which is incorporated herein by reference for its teaching of the use of non-peptidic bridges to link segments of polypeptides for use in multilayer films. Suitable non-peptidic linkers include, for example, alkyl linkers such as -NH-(CH2)sC(O)- (wherein s=2-20). The alkyl linkers are optionally substituted with non-sterically hindering groups such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. Another exemplary non-peptidic linker is a polyethylene glycol linker such as -NH-(CH2-CH2-O)n, -C(O)- (wherein n is such that the linker has a molecular weight of 100-5000 Da, particularly 100-500 Da). Many of the linkers described herein are available from commercial suppliers in a form suitable for use in solid phase peptide synthesis.

[0060] Further disclosed herein is an immunogenic composition, comprising a multilayer film comprising two or more layers of polyelectrolyte, adjacent layers comprising oppositely charged polyelectrolytes, one layer comprising an RSV epitope. The immunogenic composition optionally further comprises one or more layers comprising a designed polypeptide.

[0061] Also included are methods where the compositions described herein are administered to an individual in need of immunization against RSV.

[0062] In an embodiment, the particles described herein induce an IFNγ T cell phenotype and induce fewer IL-5 producing T cells compared to designed peptides lacking (Pam3Cys or Pam2Cys). In another embodiment, the composition induces a broader isotype distribution in RSV-G protein specific antibody responses compared to designed peptides lacking (Pam3Cys or Pam2Cys). In a further embodiment, the composition induces IgG1, IgG2a and IgG2b isotypes.

[0063] The immunogenicity of the immunogenic composition can be enhanced in several ways. In one embodiment, the multilayer film optionally comprises one or more additional immunogenic bioactive molecules. Although not required, the one or more additional immunogenic bioactive molecules typically comprise one or more additional antigenic determinants. Suitable additional immunogenic bioactive molecules include, for example, drugs, proteins, oligonucleotides, nucleic acids, lipids, phospholipids, carbohydrates, polysaccharides, lipopolysaccharides, low molecular weight immunostimulatory molecules, or combinations comprising one or more of the aforementioned bioactive molecules. Other types of additional immune enhancers include functional membrane fragments, membrane structures, viruses, pathogens, cells, cell aggregates, organelles, or combinations comprising one or more of the aforementioned bioactive structures.

[0064] In one embodiment, the multilayer film optionally includes one or more additional bioactive molecules. The one or more additional bioactive molecules can be drugs. Alternatively, the immunogenic composition is in the form of a coating surrounding a hollow shell or core. The core includes one or more additional bioactive molecules, including a variety of different encapsulating agents, e.g., drugs. Thus, the immunogenic composition designed as described herein can also be used for combination therapy, e.g., eliciting an immune response and targeted drug delivery. A micron-sized "core" of a suitable therapeutic material in "crystalline" form can be encapsulated by an immunogenic composition that includes an antigenic polypeptide, and the resulting microcapsules can be used for drug delivery. The core can be insoluble under some conditions, e.g., at high pH or low temperature, and can be soluble under conditions under which controlled release occurs. The surface charge on the crystals can be determined by zeta potential measurements (used to determine the charge of electrostatic units on colloidal particles in liquid media). The rate at which the microcapsule contents are released from the interior of the microcapsule into the surrounding environment depends on many factors, including the thickness of the encapsulation shell, the antigenic polypeptide used in the shell, the presence of disulfide bonds, the degree of cross-linking of the peptides, temperature, ionic strength and the method used to assemble the peptides. In general, the thicker the capsule, the longer the release time.

[0065] In another embodiment, the additional immunogenic biomolecule is a nucleic acid sequence that can direct the synthesis of a desired immunogen in the host organism or can prevent the expression of genetic information from a pathogen. In the former case, such a nucleic acid sequence is inserted into a suitable expression vector, for example, by methods known to those skilled in the art. Suitable expression vectors for generating highly efficient gene transfer in vivo include retrovirus, adenovirus and vaccinia virus vectors. The operational elements of such expression vectors include at least one promoter, at least one operator, at least one leader sequence, at least one terminator codon and any other DNA sequence necessary or preferred for proper transcription and subsequent translation of the vector nucleic acid. In particular, it is contemplated that such vectors contain at least one origin of replication recognized by the host organism, together with at least one selectable marker and at least one promoter sequence that can initiate transcription of the nucleic acid sequence. In the latter case, multiple copies of such a nucleic acid sequence are prepared for delivery, for example, by encapsulating the nucleic acid in a polypeptide multilayer film in the form of a capsule for intravenous delivery.

[0066] It should be further noted that in constructing recombinant expression vectors, multiple copies of the nucleic acid sequence of interest and its associated operational elements can be inserted into each vector.In such an embodiment, the host organism produces a greater amount of the desired protein per vector.The number of multiple copies of the nucleic acid sequence that can be inserted into a vector is limited only by the ability of the resulting vector to be transferred, replicated and transcribed into a suitable host microorganism due to the size of the resulting vector.

[0067] In one embodiment, the multilayer film / immunogenic composition induces a response from the immune system against a pathogen. In one embodiment, the vaccine composition comprises the immunogenic composition in combination with a pharma- ceutically acceptable carrier. Thus, a method of vaccination against a pathogenic disease comprises administering an effective amount of the immunogenic composition to a subject in need of vaccination.

[0068] Pharmaceutically acceptable carriers include, but are not limited to, large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, inactive virus particles, etc. Pharmaceutically acceptable salts, such as inorganic salts such as hydrochloride, hydrobromide, phosphate or sulfate, and salts of organic acids such as acetate, proline, malonate or benzoate, can also be used in the composition. The composition can also contain liquids such as water, saline, glycerol and ethanol, as well as substances such as wetting agents, emulsifiers or pH buffers. Liposomes can also be used as carriers.

[0069] A method for inducing an immune response against a disease or pathogen in a vertebrate (e.g., vaccination) includes administering an immunogenic composition comprising a multi-layer film containing an RSV epitope. In one embodiment, the polyelectrolyte containing the RSV epitope is in the outermost layer or solvent-exposed layer of the multi-layer film. The immunogenic composition can be administered orally, intranasally, intravenously, intramuscularly, subcutaneously, intraperitoneally, sublingually, intradermally, pulmonary or transdermally, with or without a booster dose. In general, the composition is administered in a manner compatible with the dosage formulation and in such an amount as will be prophylactically and / or therapeutically effective. The exact amount of the immunogenic composition administered will depend on the judgment of the practitioner and may be specific to each subject. It will be apparent to one skilled in the art that a therapeutically effective amount of the immunogenic composition will depend, among other things, on the administration schedule, the unit dose of the antigen administered, whether the composition is administered in combination with other therapeutic agents, and the immune and health status of the recipient. Therapeutically effective dosages can be determined by those of skill in the art based on the patient's characteristics (age, weight, sex, condition, comorbidities, other diseases, etc.), as is well known in the art. Moreover, as further routine research is conducted, more specific information will become available regarding appropriate dosage levels for the treatment of various conditions in various patients, and the skilled artisan will be able to ascertain proper dosing, taking into account the treatment situation, age, and general health of the recipient.

[0070] The immunogenic composition optionally includes an adjuvant. Adjuvants generally include substances that enhance the immune response of the host in a non-specific manner. The choice of adjuvant depends on the subject to be vaccinated. Preferably, a pharma-ceutically acceptable adjuvant is used. For example, vaccines for humans should avoid oil or hydrocarbon emulsion adjuvants, including complete and incomplete Freund's adjuvants. One example of an adjuvant suitable for human use is alum (alumina gel). However, vaccines for animals may contain adjuvants that are not suitable for human use.

[0071] It is contemplated that immune response can be induced through the presentation of any protein or peptide that can induce such a response.In one embodiment, the antigen is an important epitope that generates a strong immune response against a particular agent of infectious disease, i.e., an immunodominant epitope.If desired, two or more antigens or epitopes can be included in the immunogenic composition to increase the possibility of immune response.

[0072] As used herein, "layer" refers to a thickness increase on a template, for example for film formation, after an adsorption step. "Multilayer" refers to multiple (i.e., two or more) thickness increases. A "polyelectrolyte multilayer film" is a film that includes one or more thickness increases of polyelectrolytes. After deposition, the layers of the multilayer film may not remain as distinct layers. In fact, there may be significant intermixing of species, especially at the interfaces of the thickness increases. Entanglement or its absence can be monitored by analytical techniques such as zeta potential measurements, X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry.

[0073] "Amino acid" refers to a building block of a polypeptide. As used herein, "amino acid" includes the 20 common naturally occurring L-amino acids, all other natural amino acids, all unnatural amino acids, and all amino acid mimetics, such as peptoids.

[0074] "Naturally occurring amino acid" means glycine plus the 20 common naturally occurring L-amino acids, namely, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, ornithine, tyrosine, tryptophan, and proline.

[0075] "Unnatural amino acid" means an amino acid other than any of the 20 common naturally occurring L-amino acids. Unnatural amino acids can have either L- or D-stereochemistry.

[0076] By "amino acid sequence" and "sequence" is meant a contiguous stretch of a polypeptide chain that is at least two amino acid residues long.

[0077] "Residue" means an amino acid in a polymer or oligomer; it is the residue of the amino acid monomer from which the polymer was formed. Polypeptide synthesis involves dehydration, i.e., a single water molecule is "lost" upon addition of an amino acid to a polypeptide chain.

[0078] As used herein, "peptide" and "polypeptide" all refer to a series of amino acids connected to one another by peptide bonds between the alpha-amino and alpha-carboxy groups of adjacent amino acids, which may or may not contain modifications such as glycosylation, side chain oxidation or phosphorylation, provided that such modifications, or lack thereof, do not destroy immunogenicity. As used herein, the term "peptide" is meant to refer to both peptides and polypeptides or proteins.

[0079] "Substrate" refers to a solid material having a surface suitable for adsorbing polyelectrolytes from an aqueous solution. The surface of the substrate can have essentially any shape, e.g., planar, spherical, rod-like, etc. The substrate surface can be regular or irregular. The substrate can be crystalline. The substrate can be a bioactive molecule. The size of the substrate ranges from nanoscale to macroscale. Furthermore, the substrate optionally includes some small subparticles. The substrate can be made of organic materials, inorganic materials, bioactive materials, or combinations thereof. Non-limiting examples of substrates include silicon wafers; charged colloidal particles, e.g., CaCO3 or melamine formaldehyde microparticles; biological cells, such as red blood cells, hepatocytes, bacterial cells, yeast cells; organic polymer lattices, e.g., polystyrene or styrene copolymer lattices; liposomes; organelles; and viruses. In one embodiment, the substrate is a medical device, such as an artificial pacemaker, cochlear implant, or stent.

[0080] If the substrate is disintegrated or otherwise removed during or after film formation, it is called a "template" (for film formation). The template particles can be dissolved in a suitable solvent or removed by heat treatment. For example, when partially crosslinked melamine-formaldehyde template particles are used, the template can be disintegrated by mild chemical methods, for example, in DMSO, or by changing the pH value. After dissolution of the template particles, a hollow multilayer shell composed of alternating polyelectrolyte layers remains.

[0081] A "capsule" is a polyelectrolyte film in the form of a coating surrounding a hollow shell or core. The core contains a variety of different encapsulants, such as proteins, drugs, or combinations thereof. Capsules with a diameter of less than about 1 μm are called nanocapsules. Capsules with a diameter of more than about 1 μm are called microcapsules.

[0082] "Crosslinking" means the formation of a covalent bond, or several bonds, or many bonds, between two or more molecules.

[0083] "Bioactive molecule" refers to a molecule, macromolecule or macromolecular assembly that has a biological effect. The specific biological effect can be measured in a suitable assay and normalized per unit weight or molecule of the bioactive molecule. The bioactive molecule can be encapsulated, retained behind the polyelectrolyte film or encapsulated within the polyelectrolyte film. Non-limiting examples of bioactive molecules are drugs, drug crystals, proteins, functional fragments of proteins, protein complexes, lipoproteins, oligopeptides, oligonucleotides, nucleic acids, ribosomes, active therapeutic agents, phospholipids, polysaccharides, lipopolysaccharides. As used herein, "bioactive molecule" further encompasses biologically active structures such as, for example, functional membrane fragments, membrane structures, viruses, pathogens, cells, cell aggregates and organelles. Examples of proteins that can be encapsulated or retained behind the polypeptide film are hemoglobin; enzymes such as glucose oxidase, urease, lysozyme, etc.; extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen; and antibodies. Examples of cells that can be encapsulated or retained behind the polyelectrolyte film are transplanted pancreatic islet cells, eukaryotic cells, bacterial cells, plant cells and yeast cells.

[0084] "Biocompatible" means not causing substantial adverse health effects when ingested orally, applied topically, applied transdermally, injected subcutaneously, intramuscularly, inhaled, implanted, or injected intravenously. For example, biocompatible films include those that do not cause a substantial immune response when contacted with, for example, the human immune system.

[0085] "Immune response" means a response of the cellular or humoral immune system to the presence of a substance anywhere in the body. An immune response can be characterized in several ways, for example, by an increase in the bloodstream in the number of antibodies that recognize a particular antigen. Antibodies are proteins secreted by B cells, and immunogens are the entities that elicit an immune response. The body fights infection and inhibits reinfection by increasing the number of antibodies in the bloodstream and elsewhere.

[0086] "Antigen" means a foreign substance that induces an immune response (e.g., the production of specific antibody molecules) when introduced into the tissues of a susceptible vertebrate. An antigen contains one or more epitopes. An antigen can be a pure substance, a mixture of substances (including cells or cell fragments). The term antigen includes suitable antigenic determinants, autoantigens, self-antigens, cross-reactive antigens, alloantigens, tolerogens, allergens, haptens and immunogens, or portions thereof, as well as combinations thereof, and these terms are used interchangeably. Antigens are generally of high molecular weight and are generally polypeptides. Antigens that induce a strong immune response are said to be strongly immunogenic. The site on an antigen to which a complementary antibody can specifically bind is called an epitope or antigenic determinant.

[0087] "Antigenicity" refers to the ability of a composition to elicit an antibody or cell-mediated immune response specific to the composition.

[0088] As used herein, a "vaccine composition" is a composition that induces an immune response in a mammal to which it is administered, protecting the immunized organism from subsequent challenge with an immunizing agent or an immunological cross-reactive agent. Prevention may be complete or partial in terms of reduced symptoms or infection compared to an unvaccinated organism. The immunological cross-reactive agent may be, for example, a whole protein (e.g., glucosyltransferase) from which a subunit peptide is derived for use as an immunogen. Alternatively, the immunological cross-reactive agent may be a different protein that is recognized in whole or in part by the antibodies elicited by the immunizing agent.

[0089] As used herein, "immunogenic composition" is intended to encompass a composition that elicits an immune response in an organism to which it is administered and may or may not protect the immunized mammal from subsequent challenge with the immunizing agent. In one embodiment, the immunogenic composition is a vaccine composition.

[0090] The invention is further illustrated by the following non-limiting examples. Working Example material and method

[0091] Cores and Microparticles: Substrate CaCO3 cores were formed in a controlled co-precipitation reaction with the sodium salt of poly-L-glutamic acid (PGA-Na). Solutions of sodium carbonate and calcium chloride (both containing PGA-Na) were pumped through tubing and rapidly mixed in a 1:1 ratio in a flow reaction. The PGA-Na provided a more stable particle and also served as an initial layering step on the CaCO3 microparticles.

[0092] The precipitated CaCO3 cores were subjected to electrostatic layer-by-layer (LbL) assembly to assemble charged polymers with high net positive or net negative charges on the surface of the CaCO3 microparticles. Assembly is driven by electrostatic attraction between the soluble polymer and the oppositely charged surface. Alternating layering of poly-l-lysine (PLL, positive charge) and poly-l-glutamic acid (PGA, negative charge) homopolymers was performed to assemble a total of seven layers on the CaCO3 microparticles, with the seventh layer being PGA, resulting in a net negative surface charge. The eighth layer is a designed peptide containing a C-terminal polylysine tail (K20 (SEQ ID NO: 8) or K20Y (SEQ ID NO: 9)) that is net positively charged and electrostatically stacks on the negatively charged surface of the microparticles.

[0093] Homopolymers: Both PLL and PGA are supplied by Sigma. They are synthetically made amino acid chains that are either positively charged (PLL) or negatively charged (PGA).

[0094] Designed peptides: The designed peptides were linearly synthesized by solid-phase peptide synthesis (SPPS), a process that involves alternating cycles of N-terminal deprotection and coupling (amino acid addition from the C-terminus to the N-terminus). SPPS uses an N-terminal FMOC protecting group for coupling and onium-based chemistry with microwave-assisted synthesis. The synthesis method for the peptides used in ACT-1216 was modified to utilize carbodiimide-based chemistry for higher microwave-temperature assisted synthesis resulting in faster peptide coupling times. Once the peptides were synthesized, they were subjected to trifluoroacetic acid cleavage to remove any remaining protecting groups on the peptide chain, such as FMOC, and to remove the peptide from the solid support resin on the C-terminus. After cleavage, the peptides were purified on either a C4 or C18 column and lyophilized for storage. The peptides in constructs ACT-1190 / 1211 and ACT-1193 / 1216 underwent an additional oxidation step to help fold the peptide over the region containing the four free cysteines. After this oxidation step, the peptides were purified before lyophilization.

[0095] ELISA PROTOCOL: ELISA plates were coated with RSVA2 G protein (kindly provided by Ralph Tripp, Univ, GA) or RSV-G peptide (SEQ ID NO:3) for 2 hours at room temperature, blocked with ELISA buffer (PBS+1% BSA) for 1 hour, then washed 3 times with PBS-T. Mouse serum samples serially diluted in buffer were added and incubated for 2 hours at room temperature or refrigerated overnight. Plates were washed 3 times with PBS-T, then HRP-conjugated mouse IgG secondary antibody was added for 1 hour. Plates were washed again 3 times with PBS-T. TMB solution was added and color was developed for 3 minutes and then stopped by the addition of 2M H2SO4. Plates were read immediately at OD of 450 nm.

[0096] ELISPOT PROTOCOL: ELISPOT plates were coated with mouse IL-5 or IFNγ capture antibodies overnight at 4°C. Wells were blocked with RPMI Complete+10% FBS for 2 hours at room temperature. Spleens were processed into single cell suspensions in RPMI medium and 5x104 cells / well were added. RSV-M2 (ACT-2019) or RSV-G (ACT-2183) peptides were diluted in RPMI medium and added at final concentrations of 5 and 2.5 μg / ml, respectively. ConA (final concentration of 2 μg / ml) was added to some wells as a positive control. After overnight incubation (16-20 hours) at 37°C / 5% CO2, the cell suspension was discarded and wells were washed twice with deionized water and then three times with PBS-T. IL-5 or IFNγ biotinylated detection antibodies in PBS / 1% BSA were added and plates were incubated for 2 hours at room temperature. The detection antibody was discarded and the wells were washed three times with PBS-T. Streptavidin-AP solution was added and the plates were incubated at room temperature for 45 min to 1 h, then washed three times with PBS-T and twice with PBS. BCIP / NBT solution was added and the plates were incubated in the dark for 5 to 10 min. The plates were rinsed with distilled water to stop the color development and then dried at room temperature. Spots were quantified using an automated Viruspot reader and the results were expressed as the number of spots per 106 splenocytes. Example 1: Dose response of RSV particles Table 1: Sequence of RSV particulate composition [Table 1]

[0097] This study investigated the potency, immunogenicity and efficacy of ACT-1190 particles over a 5 log dose range. BALB / c mice were immunized on days 0 and 21 with ACT-1190 concentrations ranging from 10 μg to 1 ng DP. Post-boost sera were examined by RSV-G protein ELISA and post-boost T cell responses were examined in IL-5 and IFNγ ELISPOT. Results in Figure 1A and Figure 1B show dose-dependent antibody responses over the entire dose range with only 1-2 outliers in the low dose group.

[0098] The ELISPOT results are shown in Figure 2. The IFNγ response increases in a dose-dependent manner from 1 ng to 100 ng and then remains the same up to the 10 μg dose. The IL-5 response peaks at 1 μg.

[0099] Two weeks after the booster immunization, the remaining mice were challenged with the RSVA2 strain. Lungs were harvested 5 days later to measure viral titers by qPCR and plaque assay on Vero cells. The results of the plaque assays are shown in Figure 3. A significant reduction in viral load was achieved at all doses compared to naive mice, ranging from 59.4% at the 1 ng dose to complete protection at 10 μg. All immunization groups were statistically different from the naive group, but there was no statistical difference between the 10 μg group and either the 1 μg or 100 ng groups (P>0.05 for each comparison). A similar pattern was observed when samples were analyzed by qPCR (data not shown), although the magnitude of the differences is routinely greater for plaque assays than for qPCR. Plaque assays are generally more sensitive, as they measure actual replicating virus, whereas qPCR measures total viral M2 gene expression, which does not necessarily correlate with viral replication. Example 2: PAM3CYS improves the potency of RSV particles

[0100] ACT-1193-01 contains a Pam3Cys-modified designed peptide (DP) similar to the unmodified DP in ACT-1190. Groups of BALB / c mice were immunized with 1 μg or 31.6 ng of ACT-1190(GM2) or ACT-1193(Pam3Cys.GM2) via fp on days 0 and 21. Serum was collected on day 28 for determination of RSV-G protein-specific antibody titers by ELISA. Figure 4A shows that ACT-1190 and ACT-1193 constructs elicited RSV-G protein-specific antibody responses in a dose-dependent manner, with both constructs eliciting primarily IgG1 (Th2-associated), while ACT-1193 elicited some IgG2a (Th1-associated) and IgG2b isotypes (Figure 4B). The results show that RSV particles carrying a DP modified with Pam3Cys (ACT-1193) elicit higher antibody titers and a broader isotype distribution than the control particle ACT-1190 carrying an unmodified DP.

[0101] T cell responses were measured by ELISPOT on day 28. Mice immunized with either construct mounted comparable IFNγ T cell responses, but Pam3Cys-modified ACT-1193 induced fewer IL-5 T cells than unmodified ACT-1190 (Figure 5).

[0102] The remaining 10 mice / group were challenged with RSV strain A2 on day 37 and viral load in the lungs was measured by qPCR. The results in Figures 6A and B show that all immunized groups were equally protected from viral challenge as measured by plaque assay (Figure 6A) or qPCR (Figure 6B). Because qPCR measures total RSVM2 gene expression and not actual viral replication, this assay may yield false positive values. However, the same protective trends are seen in plaque assays that measure actual virus replication.These results show that potency is potent and detectable at submicrogram doses with or without Pam3Cys, but the modification confers 100% protection in challenged mice even at the lowest dose of 31.6 ng.

[0103] This example shows that inclusion of Pam3Cys on the designed peptides improved the potency (Figure 4A and Figure 6) and phenotype of the immune response (favoring IgG2a and IgG2b in Figure 4B and favoring IFNγ over IL-5 in Figure 5). Example 3: RSV Disease Enhancement Studies

[0104] BALB / c mice were immunized with 1 μg ACT-1190 (GM2), 1 μg ACT-1193 (Pam3Cys.GM2), 0.67 μg ACT-1213 (G) or 0.33 μg ACT-1214 (M2) via fp on days 0 and 21. Doses of ACT-1193, ACT-1213 and ACT-1214 were the molar equivalent of 1 μg ACT-1190. Three control groups were included: FI-RSV (10 6 pfu equivalents); Day 0, 10 6 pfu of live virus (positive); and naive mice (negative). Post-boost sera were examined by RSV-G peptide ELISA, and post-boost T cell responses were examined in IL-5 and IFNγ ELISPOT. Figures 7A and 7B show that antibody responses in the FI-RSV and live RSV groups were very low in the RSV-G peptide ELISA. Inclusion of M2 (1190) increased antibody titers (Figures 7A and B) and induced a shift in the isotype distribution to include IgG2a (Figure 7C) compared to immunization with the G-only construct (1213), even though the amount of G epitope was the same in both groups. These results confirm that M2 provides both qualitative and quantitative improvements to the antibody response to G. The antibody response was further improved by Pam3Cys modification of the GM2 DP; the ACT-1193(Pam3Cys.GM2) group had the highest IgG titers (Figure 7A) and the highest levels of IgG2a (Figure 7C).

[0105] The ELISPOT results are shown in Figure 8A-C. The IFNγ response to RSV-M2 is dominated by the live RSV group, but positive responses are also seen in the ACT-1193, -1214 and FI-RSV groups. The response to RSV-G is highest in the ACT-1213 group (G only), followed by the ACT-1190 group.

[0106] The remaining mice were challenged with RSV strain A2, and lungs from 10 mice per group were harvested 5 days later to measure viral titers by qPCR and plaque assay on Vero cells. The plaque assay results in Figure 9 show that all mice were fully protected from challenge, except for the ACT-1214 group (RSV-M2 alone). The % reduction in viral load in this group ranged from 41.1% to 90.9% compared to naive animals, with an average reduction of 67.4%. A similar pattern of protection was observed by qPCR (data not shown).

[0107] BAL fluid was collected before challenge, then on days 6 and 10 after challenge (three mice per group at each time point). BAL cells were identified as CD8 + / M2-pentamer + Cells were stained for at day 6 and 10 post-challenge, as well as eosinophils (EOS) and macrophages (MΦ) at all three time points. EOS express CD45 + / SiglecF + / CD11c - and MΦ are CD45 + / SiglecF + / CD11c + For each sample, 50,000 events were collected and then CD45 +The cells were gated, allowing us to select for white blood cells and exclude any red blood cells that escaped lysis. Two-color analysis was then performed on the gated cells. Figure 10A shows that there is little difference in EOS and MΦ numbers between the groups before challenge. After challenge, the EOS and MΦ profiles of the ACT-1190 and ACT-1213 groups resemble those of the FI-RSV group (Figures 10B and 10C). The EOS numbers of the ACT-1193 group are somewhat elevated in one out of three animals at both time points after challenge, but are overall lower than either ACT-1190 or -1213. The EOS profile of the ACT-1214 group resembles that of convalescent mice, but is the only group that is not completely protected from challenge. The results of this study demonstrate that although constructs containing RSV-GDP induce eosinophil infiltration into the lungs, these constructs provide complete protection from RSV infection in the lungs and do not apparently induce any overt adverse events, and inclusion of the TLR2 ligand Pam3Cys (ACT-1193) results in a modest reduction in eosinophil infiltration in the lungs following challenge. Example 4: Lung cytokine content after challenge in RSV disease enhancement studies

[0108] In Example 3, ACT-1193(Pam3Cys.GM2) was more potent than ACT-1190(GM2) in switching T cell phenotype from IL-5 to IFNγ, yet both constructs provided 100% protection from live RSV challenge. Immunization with ACT-1190 primed mice for pulmonary eosinophil infiltration after challenge is comparable to that seen in FI-RSV immunized mice, but it is particularly interesting to see immunization with ACT-1193 primed mice for lower levels of eosinophil infiltration after challenge. 1193-immunized mice also developed higher titers and a broader isotype distribution of G-specific antibody responses in the lungs after live RSV challenge. Taken together, these results demonstrate that Pam3Cys-modified LbL-MP induces immune responses that are quantitatively (antibody titers) and qualitatively (antibody isotype, T cell phenotype and eosinophil infiltration after antigen challenge) improved compared to responses induced by unmodified LbL-MP. This observation was further strengthened when we examined the cytokine profile of lung (BAL) fluid after antigen challenge. Cell-free BAL fluid was analyzed for cytokine and chemokine levels by 16plex Luminex assay. Results from pre-antigen challenge (day 0) mice were similar to those from naïve mice 6 days after antigen challenge and are not shown for clarity. In all of the groups 10 days after antigen challenge, the majority of cytokines and chemokines were reduced below pre-antigen challenge levels and are also not shown for clarity. Results for all groups 6 days after antigen challenge are shown in Figure 11A-G. Columns highlighted in yellow are analytes expressed at higher levels in ACT-1190-immunized mice than in FI-RSV-immunized mice following challenge. These include IL-12p40, IL-15 and IL-17 (pro-inflammatory cytokines), as well as RANTES (leukocyte chemotaxis), suggesting that immunization with RSV-GM2 LbL-MP primes the host for leukocyte infiltration and activation upon subsequent challenge. All of these analytes are also detectable in ACT-1193-immunized mice, although at slightly lower levels than in ACT-1190-immunized mice. Highlighted in red are the Th2-associated cytokines IL-4, IL-5, IL-6 and IL-13, all of which are similarly elevated in the FI-RSV, ACT-1190 (GM2) and ACT-1213 (G) groups, but expressed at low or undetectable levels in the live RSV, ACT-1193 (Pam3Cys.GM2) and ACT-1214 (M2) groups. Thus, the Th2 cytokine fingerprint in the BAL fluid after challenge differentiates the immunogen into two distinct groups, as shown by the vertical alignment of the graphs in Figures 11A-G. It is particularly interesting to see that IL-4 and IL-13 are elevated in the FI-RSV and ACT-1190 groups, but are reduced or completely absent in the live RSV and ACT-1193 groups. Both of these Th2 cytokines are associated with eosinophil recruitment in pulmonary inflammatory diseases such as asthma, and therefore may be important surrogate markers of RSV disease enhancement, which is normally characterized by Th2-associated inflammation. These results demonstrate that the Pam3Cys modification of the GM2 construct favors a Th1 non-inflammatory response that may correlate with lower levels of disease enhancement following antigen challenge. Example 5: Pulmonary antibody responses in RSV disease enhancement studies

[0109] BAL fluid samples were tested for RSV-G peptide-specific IgG and isotype in ELISA. Total (non-specific) IgG levels were also measured by ELISA for normalization purposes. BAL was measured at 1:5 dilution in all ELISAs. Figure 12A shows that the amount of RSV-G specific antibodies increases dramatically over 10 days after challenge in the ACT-1193 group, but remains fairly constant in the ACT-1190 group. This pattern is also reflected in the isotype ELISA. In Figures 12C-E, we found that the ACT-1190 group has the highest level of IgG1 on the day of challenge, but the levels do not increase over time after challenge; the amount of IgG2a is low initially and increases only slightly. In the ACT-1193 group, both IgG1 and IgG2a increase over time. Figure 12B shows that total IgG levels increase in all groups except the ACT-1190 group, where total IgG was already elevated before challenge. These results are consistent with previous findings showing RSV-G-specific serum IgG2a in the ACT-1193 group but not in the ACT-1190 group. Example 6: Serum antibody responses and efficacy of a second RSV disease enhancement study

[0110] BALB / c mice were immunized with ACT-1190 (GM2) or ACT-1193 (Pam3Cys.GM2) via fp on days 0 and 21. Control groups were FI-RSV, live RSV and naive mice. Mice were bled one week after boost and RSV-G specific antibody titers in serum were measured by ELISA. The results in Figures 13A and 13B show that both constructs induced RSV-G specific antibody responses in a dose-dependent manner. As previously reported, the addition of Pam3Cys to DP (ACT-1193) resulted in both higher antibody responses and a broader isotype distribution.

[0111] Mice were bled without the use of anticoagulants so that the resulting sera could be tested in the RSV neutralization assay. Serum was pooled, heat inactivated, and serial dilutions were mixed with equal volumes of diluted RSV / A2, incubated for 1 h at 37°C, and then added to Vero cell monolayers. After 6 days at 37°C, the monolayers were fixed and immunostained. An anti-RSV-F monoclonal control antibody showed a dose-dependent reduction in viral plaque numbers, whereas only FI-RSV and live RSV sera showed neutralization (data not shown). Without wishing to be bound by theory, it is believed that higher doses of immunogen must be given before neutralizing antibodies can be detected in vitro.

[0112] The remaining mice were challenged with RSV / A2 on day 35, and six mice per group were sacrificed 5 days after challenge to assess viral load in the lungs by plaque assay and qPCR. The remaining six mice per group were sacrificed 6 days after challenge to assess lung histology as well as BAL cellularity and cytokine content. Figure 14 shows that the infection levels in the naive group were fairly consistent, but somewhat lower than desired due to overestimation of the viral titer of the infectious stock. Nevertheless, protection was 100%, except for the 10ng ACT-1193 group, where infection was detected in 2 of 6 mice. qPCR analysis showed a similar pattern in that only the ACT-1193 10ng group was not statistically different from the naive group (data not shown). Example 7: Pulmonary antibody responses in a second RSV disease enhancement study

[0113] Mice immunized with ACT-1193 had higher serum antibody titers, broader antibody isotype distribution and lower IL-5 responses than mice immunized with ACT-1190, but comparable CTL responses and efficacy. There was also a trend toward lower lung eosinophil counts after challenge. In a second study, BALB / c mice were immunized with ACT-1190 (GM2) or ACT-1193 (Pam3Cys.GM2) via fp on days 0 and 21; control groups were FI-RSV, live RSV and naive mice. Mice were challenged with live RSV after the second immunization, and BAL fluid collected from six mice per group 6 days after viral challenge was tested for RSV-G peptide-specific antibodies and total IgG by ELISA. We also measured the chemokine CCL2 / MCP-1, a Th2-associated chemokine known to be involved in pulmonary inflammation, using a sandwich ELISA and following the manufacturer's instructions. The results are shown in Figure 15. Although total IgG levels were similar in all BAL samples (FIG. 15A), RSV-G-specific IgG titers in BAL fluid from mice immunized with ACT-1193(Pam3Cys.GM2) were much higher than those immunized with ACT-1190(GM2) (FIG. 15B). There was also a shift in the RSV-G-specific isotype distribution, as BAL from 1193-immunized mice contained both IgG1 and IgG2a, whereas BAL from 1190-immunized mice contained only IgG1 (FIG. 15C). FIG. 15D shows that CCL2 was detectable in some mice in all groups except the group immunized with 1 μg ACT-1193, the group immunized with live RSV, and the naive group. The detection limit in the CCL-2 ELISA was 5 pg / ml; all samples above this level are shown as red circles.

[0114] BAL fluid was analyzed by 16plex ELISA. The results in Figures 16A-H are very similar to those obtained with the BAL samples of Example 4. Th2 cytokines are highlighted in red. Both dose levels of 1190 induced an identical Th2 fingerprint to that seen in the FI-RSV group. In contrast, the 1193 group showed a dose-dependent decrease in Th2 cytokines, with barely detectable IL-4, IL-5, IL-6 or IL-13 in the 1 μg dose group and increasing amounts in the lower dose groups. The IL-13 response appears to be particularly sensitive to vaccine dose, as this cytokine was not detectable in either the 1 or 0.1 μg 1193 dose groups, and was lower in the 0.01 μg 1193 group compared to both 1190 dose levels.

[0115] Although anti-RSV-G IgG responses in the serum of 1193-immunized mice are slightly higher than in 1190-immunized mice, the main differences between the two groups are the serum and BAL isotype distributions (1193 induces both IgG1 and IgG2a in both samples), the BAL cytokine profile (1193 inhibits Th2 cytokine responses), the T cell phenotype (1193 inhibits IL-5 responses [Th2] without affecting IFNγ responses [Th1]) and the BALCCL-2 response, which is present at higher levels in mice immunized with 1 μg ACT-1193 than in mice immunized with 1 μg ACT-1190. This body of evidence clearly demonstrates that LbL-MPs loaded with Pam3Cys-modified DPs favor a stronger Th1 response than LbL-MPs loaded with the same DPs minus the Pam3Cys modification. Although CTL activity and efficacy are similar between the two candidates (at the doses tested so far), the reduction in Th2 responses induced by Pam3Cys-modified particles may confer protection from disease enhancement following antigen challenge. Example 8: Induction of a chemotaxis-inhibiting antibody response

[0116] The RSV-G epitopes described herein include mimics of fractalkine, a CX3C chemokine with chemotactic activity. The particles are designed to elicit an antibody response that not only prevents viral infection, but also inhibits the inflammatory properties of RSV-G protein, including chemotaxis or migration of lymphoid cells to sites of inflammation. Serum from disease enhancement studies was analyzed for inhibition of chemotactic inhibition. Serum antibodies were immunoprecipitated using a pool of IgA and IgG conjugated Dynal beads and dialyzed against PBS using a cutoff molecular weight of 100,000. The purified and dialyzed antibodies were incubated with native RSV-G protein in the lower chamber of a modified Boyden chamber at 1 / 5 the concentration of purified RSV-G protein. A thawed resting pool of human lymphocytes from three adult donors was added to the upper chamber of the Boyden chamber and the chamber was incubated overnight. The next day (18-24 hours later), the number of viable lymphocytes that had migrated into the lower chamber was scored and the chemotaxis index (CI) was determined. From this CI the % migration inhibition was determined. Monoclonal antibody 131-2G was included as a positive control for inhibition of migration; it was incubated with a 50-fold concentration of RSV-G protein. The assay was repeated on three separate nights with different lymphocyte pools in duplicate wells per night; thus n=6 for each serum sample. The results in Figures 17A and 17B show that antisera raised against any LbL-MP containing the RSV-G epitope inhibited lymphocyte migration by at least 30%, equivalent to the activity in the 131-2G positive control wells. Antisera raised against ACT-1214 (RSV-M2) failed to inhibit chemotaxis, as expected. Inclusion of M2 or addition of Pam3Cys to the vaccine did not appear to improve antichemotactic activity beyond that seen with sera from mice immunized with LbL-MP containing only the RSV-G epitope. Example 9: Antibody Responses and Efficacy of RSV Disease Enhancement Marker Studies

[0117] The aim of this study was to continue the search for reliable markers of disease enhancement. RSV naïve BALB / c mice (10 per group) were immunized im on days 0 and 21 with either 1 μg ACT-1211 (GM2) or ACT-1216 (Pam3.GM2). FI-RSV and live RSV groups were included as positive and negative controls, respectively. As an additional control, a naïve group that was anesthetized but not challenged was also included. One mouse in the live RSV group died within a few days of the study, reducing the number of mice in this group to nine. Mice were bled one week after the boost and RSV-G specific IgG was measured by ELISA against the RSV-G peptide (Figure 18A). ACT-1211 titers were lower than expected, but this is only the second study testing the 1 μg dose via the im route. The ACT-1211 and ACT-1216 batches used in this study are both over 4 years old, so an aliquot of each was recently tested by DLS to determine particle size and dispersity. Results showed that both constructs appeared stable with minimal changes in size and dispersity (data not shown). Thus, the relatively low antibody response in the 1211 group may simply be due to between-study variability or the lower dose of 1 μg tested.

[0118] Mice were challenged on day 35 and weight loss was monitored for 8 days after challenge. Figure 18B shows the group mean % change in body weight relative to the day of viral challenge (day 0). All groups lost some weight after challenge, including the naïve group that was anesthetized but did not receive virus. This indicates that at least a portion of the weight loss can be attributed to the mice going under anesthesia. However, the FI-RSV group lost the most weight of all groups, losing 5% on day 2, in contrast to previous studies in which the same batch of FI-RSV did not result in any significant weight loss. A second weight loss event occurred between days 5 and 6. This loss could be due to the influx of cells into the lungs that occurs at this time. However, this explanation does not explain the weight loss in the naïve animals that were not challenged. Without being bound by theory, it is believed that this apparent loss could be an artifact, since the mean weights from days 6-8 are those of the remaining 3 mice / group that were kept for BAL. In addition, these remaining mice were placed in clean cages on day 5, and this decrease may simply be due to increased activity in the "new" environment.

[0119] For analysis of lung viral load and cytokine / chemokine content, mice were sacrificed 5 days after challenge (6–7 mice / group). Both plaque and qPCR were used to measure viral load in the lungs. Results in Figure 19A and Figure 19B show that the efficacy of ACT-1211 was not as high as previously observed, with only a mean reduction of 75.6% compared to the naive group, and no individual mice had a >90% reduction in viral load. This result correlates with the low serum antibody titers in this treatment group. ACT-1216 and FI-RSV each resulted in a significant reduction in viral load, whereas pre-infection with RSV completely protected mice from challenge.

[0120] The remaining 3 mice / group were sacrificed 8 days after challenge for analysis of BAL cell density and cytokine / chemokine content. Example 10: Lung cells and cytokine content in RSV disease enhancement marker studies

[0121] Three mice per group were sacrificed 8 days after challenge and BAL fluid was collected for analysis of cytokine and chemokine content and cellularity using markers for T cells, macrophages and eosinophils. T cells were stained with a cocktail of anti-CD3-FITC / CD4-APC / CD-8-PE and CD3 + Gate the cells and detect CD4 + and CD8 + A similar strategy was used to characterize eosinophils (CD45 + / CD11c - / SiglecF + ) and macrophages (CD45 + / CD11c + / SiglecF + ) were characterized. The results are shown in FIG. 20 as the fold increase in cell counts for each group relative to the naive group. FIG. 20A shows that there is an increase in CD4+ cells in the ACT-1216, FI-RSV and live RSV groups, with the greatest increase in the FI-RSV group. T cell counts are increased in all challenged mice compared to non-challenged naive mice. FIG. 20B shows, to various degrees, an increase in eosinophil counts in all immunization groups after challenge. One mouse in the ACT-1216 group had a 5-fold increase in eosinophils, while two mice in the live RSV group showed approximately 3- and 15-fold increases. The most significant increase in eosinophils was in the FI-RSV and ACT-1211 groups. The inset in FIG. 20B shows the fold increase for the three values ​​above the scale of the graph.

[0122] During analysis of BAL cells, it was noted that the presence of eosinophils could be easily identified in the SSC / FSC dot plot. Figure 21 shows the scatter plots of one naïve and one FI-RSV mouse. The triangular region (R1) contained T cells and was present in all mice in all groups, but was greatly reduced in number in the non-challenged naïve mice. The polygonal region (R4) contains larger, more granular cells, i.e. eosinophils and other CD45 +Corresponding to R4 cells. Mice with increased numbers of eosinophils (determined by fluorescent marker) had significantly more cells in R4 of the corresponding scatter plot. Thus, only the scatter plot gives an indication of the presence or absence of infiltrating eosinophils in individual animals.

[0123] Th1 / 2 cytokines and proinflammatory chemokines were measured in both clarified lung homogenates collected 5 days after challenge (7 mice / group) and BAL collected 3 days after challenge (3 mice / group) using a multi-analyte flow assay kit from BioLegend. Figure 22 shows the fold increase or decrease compared to the naive group for the day 5 samples only (most day 8 samples showed little change or decrease in all samples compared to the naive control, with no significant distinction between treatment groups); asterisks indicate cytokines that are related to both Th1 and Th2, but are primarily related to the phenotypes they are shown to represent. Figure 22A shows elevated levels of Th1-related TNFα in all challenged groups, but IFNγ was only elevated in the 1211-immunized group. Figure 22B shows an increase in Th2-related IL-4, IL-5 and IL-13 in the 1211 and FI-RSV groups (inset shows fold increase for IL-5 values ​​above the scale of the graph). This pattern is similar to that reported in Figure 11 and Figure 13, again suggesting that TLR activation (either TLR2 via Pam3Cys at 1216 or TLR4 via live RSV viral proteins) dampens the inflammatory cytokine response.

[0124] The use of the terms "a" and "an" and "the" and similar referents (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second, etc., as used herein are not meant to indicate any particular order, but are merely meant to indicate a plurality, e.g., layers, for convenience. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. The endpoints of all ranges are included within the ranges and can be independently combined. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") is intended merely to better describe the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention as used herein.

[0125] Although the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes can be made and elements thereof can be substituted with equivalents without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. Therefore, the invention is not limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but the invention is intended to include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A composition comprising particles, the particles comprising a multilayer film, the multilayer film comprising two or more layers of polyelectrolytes, adjacent layers comprising oppositely charged polyelectrolytes, one of the polyelectrolytes having the structure (Pam3Cys or Pam2Cys)-(surface adsorption region 1)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region 2) wherein surface adsorption regions 1 and 2 each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, and the designed polypeptide has a net charge per residue of 0.1 or greater; the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA) or (ESYIGSINNITKQSASVA); the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT); and L1 or L2 is a linker comprising 0-100 uncharged amino acid residues; the non-designed polyelectrolyte comprises a polycationic or polyanionic material having a molecular weight greater than 1,000 and at least 5 charges per molecule; The multilayer film is deposited on a core particle or forms a hollow particle to provide the composition.

2. The composition of claim 1, wherein the composition induces an IFNγ T cell phenotype and induces fewer IL-5 producing T cells compared to a designed peptide lacking the (Pam3Cys or Pam2Cys).

3. The composition of claim 1, wherein the composition induces a broader isotype distribution in RSV-G protein-specific antibody responses compared to designed peptides lacking the (Pam3Cys or Pam2Cys).

4. The composition of claim 3, wherein the composition induces IgG1, IgG2a and IgG2b isotypes.

5. The designed polypeptide comprises: Pam3CSKKKKNFVPCSICSNNPTCWAICKRIPNKKPGKKTSGSESYIGSINNITKQSASVASGSKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 6), or Pam3CSKKKKNFVPCSICSNNPTCWAICKRIPNKKPGKKTSGSESYIGSINNITK QSAC VASGSKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 7) The composition of claim 1 comprising:

6. The composition of claim 1 , wherein L1 and L2 are SGS.

7. The composition of claim 1 , wherein two or more of the layers of the multilayer film are covalently crosslinked.

8. The composition of claim 7 , wherein two or more of the layers of the multilayer film are covalently crosslinked by amide bonds.

9. The composition of claim 1 , wherein the multilayer film is deposited on a core particle.

10. The composition of claim 1 , wherein the multilayer film is in the form of a hollow capsule.

11. A method of administering the composition of claim 1 to an individual in need of immunization against RSV.

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