Respiratory syncytial virus antigenic compositions and methods

Abstract

Described herein is a composition comprising a particle comprising a multilayer film comprising two or more layers of polyelectrolytes wherein adjacent layers comprise oppositely charged polyelectrolytes, wherein one of the polyelectrolytes is a designed polypeptide with the following structure: (Pam3Cys or Pam2Cys)-(surface adsorption region I)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region II).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 324,824, filed on March 29, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to compositions and methods for preventing respiratory syncytial virus infection, particularly multilayer films comprising antigenic epitopes. Background Art

[0004] Respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract illness in infants and young children worldwide and is a threat to elderly and immunocompromised patients. In the United States, RSV infection leads to the hospitalization of up to 126,000 infants and up to 60,000 elderly individuals each year. Because natural RSV infection does not induce durable long-term immunity, patients are susceptible to reinfection with the same and different viral strains throughout their lives. RSV is associated with secondary infections such as otitis media, and it may predispose young children to asthma-related illnesses later in life.

[0005] After more than 40 years of effort, there is currently no safe and effective RSV vaccine. In the 1960s, the earliest attempts to develop a formalin-inactivated, alum-precipitated RSV (FI-RSV) vaccine actually appeared to cause vaccinated children to develop more severe disease and even die during subsequent natural infection. The exact mechanism of this reaction has not been fully determined, but it appears to rely on the skewing of the immune response toward an inflammatory Th2-dominant phenotype characterized by inappropriate activation of cytokine and chemokine pathways.

[0006] Given the economic impact of RSV disease (estimated at nearly $700 million per year 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.

[0007] US Patent 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

[0008] In one aspect, a composition comprises a particle comprising a multilayer film comprising two or more layers of polyelectrolytes, wherein adjacent layers comprise oppositely charged polyelectrolytes, wherein one of the polyelectrolytes comprises a designed polypeptide having the structure

[0009] (Pam3Cys or Pam2Cys)-(surface adsorption region 1)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region 2)

[0010] wherein the surface adsorption regions one and two each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, wherein the net charge of each residue of the designed polypeptide is greater than or equal to 0.1, wherein the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA) or (ESYIGSINNITKQSASVA), wherein the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT), and wherein L1 or L2 is a linker comprising 0-100 uncharged amino acid residues;

[0011] wherein the polyelectrolyte that is not the designer polypeptide comprises a polycationic material or a polyanionic material having a molecular weight greater than 1,000 and at least 5 charges per molecule, and

[0012] Wherein the multilayer film is deposited on core particles or forms hollow particles to provide the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A A and B show 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 booster immunization and the serum was tested in an ELISA for responses to RSV-G protein. (1A) Results are shown for individual mice (open circles) and group averages (bars) at 1:50 serum dilution. (1B) Results show the average responses of 16 animals per group.

[0014] Figure 2 T cell responses of mice immunized with ACT-1190 are shown. On day 28, three mice / group in Figure 1 were sacrificed, splenocytes were harvested, and restimulated in vitro with RSV-M2 and RSV-G peptides in IFNγ and IL-5 ELISPOT plates. Data depict mean ± SD of 3 mice / group Spot / 10 6 cells.

[0015] Figure 3 Lung RSV burden after challenge with strain A2 is shown. On day 35 (14 days after booster immunization), mice from Figure 1 were challenged with RSV A2. Five days after challenge, lungs were harvested and viral burden was measured by standard plaque assay. Results show individual mice (circles) and group averages (bars); annotations show the number of mice fully protected from challenge (>90% reduction in viral burden), compared to naive mice. Group mean percentage reduction in plaque number compared with the naive group and P value by Student's t test; n = 12 mice per group.

[0016] Figure 4A and 4B Antibody responses elicited by RSV microparticle immunization are shown. BALB / c mice were immunized with the indicated treatments at day 0 and day 21. Serum collected at day 28 was tested in ELISA for the response to RSV-G protein. (4A) Results show 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 mean ± SEM of 10 mice per group.

[0017] Figure 5 T cell responses to RSV particles are shown. At day 28, splenocytes were harvested from 3 mice in each group of Figure 4 and restimulated with RSV-G+M2 peptide in IFNγ and IL-5 ELISPOT plates. Data depict mean ± SD of 3 mice in each group.

[0018] Fig. 6A A and B show the efficacy of RSV vaccines containing PAM3Cys. The mice of Figure 4 were challenged with RSV on day 35 (14 days after booster immunization). Lungs were collected 5 days after challenge and viral burden was measured. (6A) Plaque assay results show individual mice (circles) and group averages (bars). (6B) qPCR results show mean ± SEM of 10 mice per group. In both figures, the number of mice protected (>90% reduction in viral burden), the average group reduction percentage, and the P value (all compared to the PBS group) are indicated.

[0019] Fig. 7A -C shows the antibody response of mice immunized with ACT-1190, ACT-1193, ACT-1213 and ACT-1214. BALB / c mice were immunized at day 0 and day 21, and control mice were included as described herein. Mice were bled 1 week after booster immunization, and the serum was tested for RSV-G peptide response in ELISA. (7A) Results show the average response of 10 animals in each group. (7B) Results show individual mice (open circles) and group averages (bars) at 1:50 serum dilution. (7C) Results show the mean ± SD of 10 animals in each group at 1:50 serum dilution.

[0020] Fig. 8A-C shows T cell responses of mice immunized with ACT-1190, -1193, 1-1213, and -1214. On day 28, three mice / group in Figure 7 were sacrificed, spleen cells were harvested, and in vitro restimulation was performed with RSV-M2 (8A) and RSV-G (8B) or both peptides (8C) in IFNγ and IL-5 ELISPOT plates. Data depicts the mean ± SD of 3 mice / group. Spots / 10 6 cells.

[0021] Fig. 9 Shown is the lung RSV burden after attacking with A2 strain.At the 35th day (14 days after booster immunization), the mice of Figure 7 were attacked with live RSV A2.Mice were killed 5 days after the attack, and the lung virus burden was measured by standard plaque assay on Vero cells.The results show individual mice (circles) and group mean values ​​(bars).The annotation shows that compared with the naive group mean value, 1214 groups have the number of individual mice with complete protection, the percentage of average virus titer reduction in each group and the P value.For all other groups, 10 / 10 were protected, the group mean value was 100% protection, and P<0.0000001.

[0022] Fig. 10A -C shows the number of EOS and MΦ cells in bronchoalveolar lavage (BAL) fluid. The mice in Figure 7 were challenged with RSV on day 35, and BAL cells were collected and analyzed by flow cytometry as described herein. The data depict the mean ± SD cells / 50,000 events of 3 mice / group collected at each time point.

[0023] Fig.11A -G shows the content of cytokines and chemokines in the lungs of immune mice after RSV challenge. The mice of Figure 7 were challenged with live RSV on day 35, and BAL fluid was harvested from the lungs 6 days later. After removing cells by centrifugation, BAL fluid was analyzed using 16-plex cytokine and chemokine ELISA. The results show the mean ± SD of 3 mice per group for each analyte.

[0024] Fig. 12A -E shows antibody responses in BAL fluid. BAL fluid was collected from mice of Figure 7 (3 mice per group per sacrifice day) before challenge (day 0) and on days 6 and 10 after challenge, and total IgG and responses to RSV-G peptide were tested. (12A) Mean ± SD of peptide-specific responses. (12B) Mean ± SD of total IgG levels. (12C), (12D) and (12E) represent the mean ± SD of isotype levels on the indicated dates. n = 3 per group.

[0025] Fig.13A-C shows the antibody response induced by RSV-GM2 microparticle immunization. The mice of Figure 12 were bled on the 28th day (7 days after booster immunization) and the serum was tested in ELISA for the reaction of RSV-G peptide. (13A) The results show the mean ± SD of 12 mice in each group. (13B) The results show the individual mice (open circles) and group mean values ​​(bars) of 1:50 serum dilution. (13C) The serum was tested at 1:50 and the plate was probed with isotype-specific detection antibodies. The results show the mean ± SD of 12 mice in each group.

[0026] Fig.14 Shown is the lung RSV burden after attack with A2 strain. The mice of Figure 12 were attacked with live RSV at day 35 (14 days after booster immunization), and lung virus burden was measured on Vero cells by standard plaque assay 5 days after attack. The results show individual mice (circles) and group averages (bars); the annotations show the percentage reduction of the average viral titer in each group, the number of individual mice fully protected, and the P-value, all values ​​compared to the average of the naive group.

[0027] Fig.15A -D shows 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 harvested on day 41. (15A) Total IgG, mean ± SD of 6 mice per group. (15B) RSV-G peptide-specific IgG, individual mice (circles) and group mean (bars). (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 detection limit; open circles indicate values ​​not above the detection limit.

[0028] Fig.16A -H shows the levels of cytokines and chemokines in the lungs of immunized mice challenged with RSV. Fig.14 6 days after the mice were treated with 10% paraformaldehyde (P < 0.05) and BAL fluid was harvested from the lungs. After removing cells by centrifugation, BAL fluid was analyzed by 16-plex cytokine and chemokine ELISA. Results show the mean ± SD of 3 mice per group for each analyte. Red bars represent Th2 cytokines.

[0029] Fig.17A A and B show the inhibition of RSV-G protein chemotactic activity by antibodies induced by LbL-MP vaccination. Fig.17A ) and Figure 12( Fig. 17B) was added to the lower chamber containing RSV-G protein, and human PBMC was 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. Three determinations were performed in repeated wells each time; the results show the mean ± SD of 6 replicates for each sample. 131-2G is a monoclonal antibody specific for RSV-G CX3C epitope and is included as a positive control for inhibiting chemotactic activity.

[0030] Fig.18A A and B show antibody responses and weights after RSV challenge. Mice were immunized with naive RSV (1211, 1216, FI-RSV) on days 0 and 21 or with 10 6 pfu of live RSV infection. (18A) Mice were bled on day 28 and antibody levels were measured by ELISA on RSV-G coated plates. Results show the mean ± SD (red bars) of individual mice (open circles) and 10 mice per group at 1:50 serum dilution. (18B) Mice were attacked on day 35 (day 0 after attack on the x-axis) and weighed daily from the day of attack (day 0). Results show the average starting weight percentage of 10 mice per group on day 5, 7 mice / group were killed on this day for plaque determination, and the remaining 3 mice / group were killed on day 8 for BAL fluid and cells.

[0031] Fig.19A A and B show the viral burden in the lungs of BALB / c mice immunized with RSV particles or FI-RSV or recovered from RSV infection. 6 pfu challenged mice of Figure 18, sacrificed 5 days later and lung viral burden measured. [N] = naive; [+] = challenged; [-] = unchallenged. (19A) Plaque assay results. (19B) qPCR results. Both graphs show individual mice (red = group mean). Annotations show the group mean percent reduction compared to the naive group mean, the number of animals with >90% reduction in viral burden, and the P value compared to the naive group by Student's t-test, where [NS] = not significant and [*] <0.005.

[0032] Fig. 20A A and B show fluorescent staining of lung cells collected by lavage 8 days after viral challenge. The mice of Figure 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 herein. Results are shown as fold increase relative to the naive group averaged over 3 individual mice per group.

[0033] Fig.21 Shown are scatter plots of lung cells collected by lavage from naive (left) and FI-RSV-immunized (right) mice of Figure 18 8 days after challenge. The triangular region, R1, contains T cells, while the polygonal region, R4, contains larger and more granular CD45+ eosinophils.

[0034] Fig.22A A and B show Th1 (22A) and Th2 (22B) cytokine levels in lung homogenates and BAL fluid from mice of Figure 18. Lungs were harvested 5 days after viral challenge, homogenized, and clarified (7 / group). Cytokines were measured using a fluorescent bead-based multi-analyte flow assay kit. Data are presented as the mean fold change per group relative to a naive control group. The group named "naive, nc" is naive animals that were anesthetized but not challenged.

[0035] The above described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings and appended claims. DETAILED DESCRIPTION

[0036] Disclosed herein are multilayer films and particles comprising polypeptide epitopes from RSV, wherein the multilayer films can induce an immune response in a host after being applied to the host. Specifically, a TLR targeting peptide of a specific design is described herein, and it provides a multilayer film, which advantageously contributes to a more balanced immune response (Th2 cytokine levels are much lower) compared with the same peptides without TLR ligands. The immune response of constructs described herein is suitable for mice recovered from the infection previously carried out with low-dose live RSV before attack. TLR targeting peptides and multilayer films are advantageous, because it is generally believed that the problem of formalin inactivated vaccine trials in the 1960s (wherein children are more seriously ill than unvaccinated children after natural exposure or even die) is caused by Th2 inflammatory response, and this is due to formalin inactivation destroying virus during vaccination: the interaction of TLR. The immunogenic multilayer film constructs described herein advantageously avoid these problems of existing RSV vaccines.

[0037] In one aspect, a composition comprises a particle comprising a multilayer film comprising two or more layers of polyelectrolytes, wherein adjacent layers comprise oppositely charged polyelectrolytes, wherein one of the polyelectrolytes comprises a designed polypeptide having the structure

[0038] (Pam3Cys or Pam2Cys)-(surface adsorption region 1)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region 2)

[0039] Wherein the surface adsorption regions one and two each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, wherein the net charge of each residue of the designed polypeptide is greater than or equal to 0.1, wherein the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA; SEQ ID NO: 1) or (ESYIGSINNITKQSASVA; SEQ ID NO: 2), wherein the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT; SEQ; ID NO: 3), wherein L1 or L2 is a linker comprising 0-100 uncharged amino acid residues; wherein the polyelectrolyte that is not the designed polypeptide comprises a polycationic material or a polyanionic material having a molecular weight greater than 1,000 and at least 5 charges per molecule, and wherein the multilayer film is deposited on a core particle or forms a hollow particle to provide the composition.

[0040] As described herein, the multilayer film comprises alternating layers of oppositely charged polyelectrolytes, wherein one layer, such as the outermost layer, comprises a designed polypeptide represented by: (Pam3Cys or Pam2Cys)-(surface adsorption region one)-(RSV-G peptide epitope)-L1-(RSV-M2 peptide epitope)-L2-(surface adsorption region two). The polyelectrolyte in the multilayer film that is not the designed polypeptide comprises a polycationic material or a polyanionic material having a molecular weight greater than 1,000 and at least 5 charges per molecule, and wherein the multilayer film is deposited on a core particle or forms a hollow particle to provide the composition.

[0041] More specifically, a polyelectrolyte multilayer is a film (e.g., a few nanometers to a few micrometers thick) composed of alternating layers of oppositely charged polyelectrolytes. Such a film can be formed by assembling layer by layer on a substrate. In electrostatic layer-by-layer self-assembly ("ELBL"), the physical basis of polyelectrolyte association is electrostatic attraction. Film stacking is possible because the sign of the surface charge density of the film is reversed in the deposition of successive layers. The versatility and relative simplicity of the ELBL film process allow many different types of polyelectrolytes to be deposited on many different types of surfaces. Polypeptide multilayers are a subset of polyelectrolyte multilayers, comprising at least one layer comprising a charged polypeptide, referred to herein as a designed polypeptide. A key advantage of polypeptide multilayers over films made of 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.

[0042] 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-ornithine), polyvinylamine, poly(aminostyrene), poly(aminoacrylate), poly(N-methylaminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethylaminoacrylate), 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-trimethylaminoacrylic acid chloride), poly(methacrylamidepropyltrimethylammonium 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 gum, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, poly (methyl) acrylic acid, oxidized cellulose, carboxymethyl cellulose, acidic polysaccharides and cross-linked carboxymethyl cellulose, synthetic polymers and copolymers containing side carboxyl groups, and combinations comprising one or more of the aforementioned polyanionic materials. In one embodiment, the RSV epitope and polyelectrolyte have the same charge sign.

[0043] In one aspect, a stable multilayer film is one that, once formed, retains more than half of its composition after incubation in PBS at 37°C for 24 hours.

[0044] A designed polypeptide is one that has sufficient charge to stably bind to an oppositely charged surface, i.e., one that can be deposited into a layer of a multilayer film, wherein the driving force for film formation is electrostatic. In another embodiment, the solubility of the designed polypeptide at pH 4 to 10 is greater than or equal to about 0.1 mg / mL. In another embodiment, the solubility of the designed polypeptide at pH 4 to 10 is greater than or equal to about 1 mg / mL. The solubility is a practical limit for promoting deposition of polypeptides from aqueous solutions. The practical upper limit of the degree of polymerization of antigenic polypeptides is about 1,000 residues. However, it is conceivable that longer composite polypeptides can be achieved by appropriate synthetic methods.

[0045] In a specific embodiment, at pH 7.0, the magnitude of the net charge of each residue of the designed polypeptide is greater than or equal to 0.1, 0.2, 0.3, 0.4 or 0.5. In one embodiment, at pH 7.0, the ratio of the number of charged residues of the same polarity minus the number of residues of opposite polarity to the total number of residues in the polypeptide is greater than or equal to 0.5. In other words, the magnitude of the net charge of each residue of the polypeptide is greater than or equal to 0.5. Although there is no absolute upper limit to the length of the polypeptide, generally, the actual length upper limit of the designed polypeptide suitable for ELBL deposition is 1,000 residues. The designed polypeptide can include sequences found in nature, such as RSV epitopes, and 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 into the polypeptide multilayer film.

[0046] The naturally occurring amino acids that are positively charged (basic) at pH 7.0 are arginine (Arg), histidine (His), ornithine (Orn), and lysine (Lys). The naturally occurring amino acid residues that are negatively charged (acidic) at pH 7.0 are glutamic acid (Glu) and aspartic acid (Asp). Mixtures of oppositely charged amino acid residues may be used as long as the overall net charge ratio meets the specified criteria. In one embodiment, the designed polypeptide is not a homopolymer. In another embodiment, the designed polypeptide is unbranched.

[0047] 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. Although the experiments described herein used Pam3Cys, it is expected that similar results would be obtained using Pam2Cys.

[0048] Pam3Cys and Pam2Cys can be covalently coupled to a polypeptide chain by standard peptide synthesis chemistry. For example, Pam3Cys can be covalently linked directly to an antigenic polypeptide 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 method for accomplishing this reaction is to couple Pam3Cys-OH to a synthetic peptide on a solid phase synthetic resin bead in the presence of an amide bond forming agent such as HBTU (o-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate), HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanoammonium) or DIPCDI (N,N'-diisopropylcarbodiimide). The progress of the coupling reaction can be monitored colorimetrically by a ninhydrin assay, and upon completion, excess Pam3Cys-OH and other reagents can be washed away. The synthesized Pam3Cys peptide conjugate is cleaved from the resin and purified by chromatography. For example, the Pam3Cys peptide can be purified by reverse phase HPLC using a C4 column and a water / isopropanol gradient. The advantage of this method is that the Pam3Cys / antigenic polypeptide ratio is strictly controlled at 1:1.

[0049] In another embodiment, Pam3Cys-OH is conjugated specifically to the side chain ε-amine of lysine residues, or specifically to a resin-bound peptide as described above, or non-specifically to an unprotected peptide or protein using a water-soluble coupling agent 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.

[0050] The designed peptide comprises two RSV epitopes, RSV-M2 epitope and RSV-G epitope. The RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA; SEQ ID NO: 1) or (ESYIGSINNITKQSASVA; SEQ ID NO: 2), wherein the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT; SEQ ID NO: 3). The full sequences of RSV-M2 and RSV-G are provided as follows.

[0051] RSV-G - SEQ ID NO:4

[0052]

[0053] RSV-M2-SEQ ID NO:5

[0054] 1MSRRNPCKFE IRGHCLNGKR CHFSHNYFEW PPHALLVRQN FMLNRILKSM DKSIDTLSEI 60

[0055] 61SGAAELDRTE EYALGVVGVL ESYIGSINNI TKQSACVAMS KLLTELNSDDIKKLRDNEEL120

[0056] 121NSPKIRVYNT VISYIESNRK NNKQTIHLLK RLPADVLKKT IKNTLDIHKS ITINNPKEST180

[0057] DTNDHAKN NDTT

[0058] The designed peptide includes two surface adsorption regions, which provide sufficient charge for the peptide to assemble into a multilayer film. In one aspect, the first and second surface adsorption regions each independently comprise at least 2, 3, 4, 5, 6, 7 or 8 amino acid residues and have the same charge sign as the designed polypeptide. The first and second surface adsorption regions can be the same or different.

[0059] As used herein, a surface adsorption region is a charged region of a designed polypeptide that advantageously provides sufficient charge so that a peptide containing, for example, an epitope from RSV can be deposited into a multilayer film.

[0060] The designed polypeptide further comprises amino acid linkers L1 and L2, which comprise 0-100 uncharged amino acid residues. In one aspect, L1 and L2 are SGS.

[0061] Specific designed peptides include:

[0062] SEQ ID NO:6

[0063] SEQ ID NO:7

[0064] In one embodiment, the multilayer film is deposited on a core particle, such as CaCO nanoparticles, latex particles or iron particles. Particle sizes of 5 nanometers (nm) to 50 microns (μm) in diameter are particularly useful. Particles made of other materials can also be used as cores, provided that they are biocompatible, have a controlled size distribution, and have enough surface charge (positive or negative charge) to combine polyelectrolyte peptides. 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 a combination thereof. Core particles can also be made of materials that are considered unsuitable for human use, provided that they can be dissolved and separated from the multilayer film after film manufacturing. Examples of template core materials include organic polymers such as latex or inorganic materials such as silicon dioxide.

[0065] One design consideration is to control the stability of the polypeptide ELBL membrane. Ionic bonds, hydrogen bonds, van der Waals interactions, and hydrophobic interactions contribute to the stability of the multilayer membrane. In addition, covalent disulfide bonds formed between thiol-containing amino acids in polypeptides in the same layer or adjacent layers can increase structural strength. Thiol-containing amino acids include cysteine ​​and homocysteine, and these residues can be easily incorporated into synthetically designed peptides. In addition, thiol groups can be incorporated into polyelectrolyte homopolymers such as poly-L-lysine or poly-L-glutamate by methods well described in the literature. Thiol-containing amino acids can be used to "lock" (bind together) and "unlock" the layers of a multilayer polypeptide membrane through changes in oxidation potential. In addition, the incorporation of thiol-containing amino acids in designed polypeptides enables the use of relatively short peptides in membrane fabrication due to the formation of intermolecular disulfide bonds.

[0066] In one embodiment, the designed thiol-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 the membrane is assembled, the reducing agent is removed and an oxidant is added. In the presence of an oxidant, disulfide bonds are formed between thiols, thereby "locking" the polypeptides in and between the layers where thiol groups exist together. Suitable reducing agents include dithiothreitol (DTT), 2-mercaptoethanol (BME), reduced glutathione, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and a combination of more than one of these chemicals. Suitable oxidants 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, porphyrin, sodium o-iodobenzoate, and a combination of more than one of these chemicals.

[0067] As an alternative to disulfide bonds, chemicals that produce other covalent bonds can be used to stabilize ELBL membranes. For membranes containing polypeptides, chemicals that produce amide bonds are particularly useful. In the presence of an appropriate coupling agent, acidic amino acids (amino acids with carboxylic acid groups on the side chains, such as aspartic acid and glutamic acid) react with amino acids with amine groups on the side chains (such as lysine and ornithine) to form amide bonds. Under biological conditions, amide bonds are more stable than disulfide bonds, and amide bonds do not undergo exchange reactions. Many reagents can be used to activate polypeptide side chains to form amide bonds. Carbodiimide reagents, such as water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), will react with aspartic acid or glutamic acid at a slightly acidic pH to form an intermediate that will react irreversibly with amines to produce amide bonds. Additives such as N-hydroxysuccinimide are often added to the reaction to accelerate the rate and efficiency of amide formation. After the reaction, the soluble reagents are removed from the nanoparticles or microparticles by centrifugation and aspiration. Examples of other coupling agents include diisopropylcarbodiimide, HBTU, HATU, HCTU, TBTU, and PyBOP. Examples of other additives include sulfo-N-hydroxysuccinimide, 1-hydroxybenzotriazole, and 1-hydroxy-7-aza-benzotriazole. The extent of amide crosslinking can be controlled by adjusting the stoichiometry of the coupling agent, the reaction time, or the reaction temperature, and can be monitored by techniques such as Fourier transform-infrared spectroscopy (FT-IR).

[0068] The covalently cross-linked ELBL membrane has desirable properties, such as increased stability. Higher stability allows the use of more stringent conditions in the nanoparticle, microparticle, nanocapsule or microcapsule manufacturing process. Examples of stringent conditions include high temperature, low temperature, cryogenic temperature, high centrifugal speed, high salt buffer, high pH buffer, low pH buffer, filtration and long-term storage.

[0069] A method for preparing a polyelectrolyte multilayer film comprises depositing multiple layers of oppositely charged chemical substances on a substrate. At least one layer, preferably the outermost layer, comprises a designed polypeptide as described herein. The successively deposited polyelectrolytes will have opposite net charges. In one embodiment, the deposition of the polyelectrolyte comprises exposing the substrate to an aqueous solution containing the polyelectrolyte at a certain pH value, at which the polyelectrolyte has a net charge suitable for ELBL. In other embodiments, the deposition of the polyelectrolyte on the substrate is achieved by sequentially spraying oppositely charged polypeptide solutions. In other embodiments, the deposition is carried out on the substrate by spraying oppositely charged polyelectrolyte solutions simultaneously.

[0070] In the ELBL method of forming a multilayer film, the opposite charges of adjacent layers provide the driving force for assembly. It is not critical that the polyelectrolytes in the opposite layers have the same net linear charge density, the key is that the opposite layers have opposite charges. A standard membrane assembly method by deposition includes forming an aqueous solution of polyions at a pH value (i.e., pH 4-10) at which the polyions are ionized, providing a substrate carrying a surface charge, and alternately immersing the substrate in a charged polyelectrolyte solution. Optionally, the substrate is cleaned between the deposition of alternating layers.

[0071] One of ordinary skill in the art can readily determine the polyelectrolyte concentration suitable for depositing the polyelectrolyte. Exemplary concentrations are 0.1 to 10 mg / mL. For typical non-polypeptide polyelectrolytes, such as poly(acrylic acid) and poly(allylamine hydrochloride), typical layer thicknesses are from about 3 to about 100 μm. This depends on the ionic strength of the solution. Short polyelectrolytes generally form thinner layers than long polyelectrolytes. Regarding film thickness, polyelectrolyte film thickness depends on humidity as well as the number of layers and composition of the film. For example, a 50nm thick PLL / PGA film shrinks to 1.6nm after drying with nitrogen. In general, films with a thickness of 1nm to 100nm or more can be formed, depending on the hydration state of the film and the molecular weight of the polyelectrolyte used in the assembly.

[0072] In addition, the number of layers required to form a stable polyelectrolyte multilayer will depend on the polyelectrolytes in the membrane. For membranes containing only low molecular weight polypeptide layers, the membranes typically have a bilayer of 4 or more oppositely charged polypeptides. For membranes containing high molecular weight polyelectrolytes (such as poly(acrylic acid) and poly(allylamine hydrochloride)), membranes containing a bilayer of a single oppositely charged polyelectrolyte can be stable. Studies have shown that polyelectrolyte membranes are dynamic. When suspended in a polyelectrolyte solution, the polyelectrolytes contained in the membrane can migrate between layers and can be exchanged with soluble polyelectrolytes with the same charge. In addition, polyelectrolyte membranes can decompose or dissolve in response to changes in the environment, such as temperature, pH, ionic strength, or the oxidation potential of the suspension buffer. Therefore, some polyelectrolytes, especially peptide polyelectrolytes, exhibit short-term stability. The stability of peptide polyelectrolyte membranes can be monitored by suspending the membrane in a suitable buffer for a fixed period of time under controlled conditions and then measuring the amount of peptides in the membrane with a suitable assay (such as amino acid analysis, HPLC assay, or fluorescence assay). Peptide polyelectrolyte membranes are most stable under conditions relevant to their storage and use as vaccines, such as in neutral buffers and at ambient temperatures, such as 4° C. to 37° C. Under these conditions, stable peptide polyelectrolyte membranes will retain most of their component peptides for at least 24 hours, and often up to 14 days or more.

[0073] For the synthesis of the designed polypeptide, each independent region of the designed polypeptide (e.g., RSV epitope and surface adsorption region) can be synthesized separately by liquid phase peptide synthesis, solid phase peptide synthesis or genetic engineering of a suitable host organism. Liquid phase peptide synthesis is a method currently used to produce most approved peptide drugs on the market. The combination of solution phase and solid phase methods can be used to synthesize relatively long peptides and even small proteins. Peptide synthesis companies have expertise and experience and can synthesize difficult peptides on a paid service basis. These syntheses are carried out under good manufacturing practice (GMP) conditions and are scaled to be suitable for clinical trials and commercial drug listings.

[0074] Alternatively, the multiple independent regions can be synthesized together as a single polypeptide chain by liquid phase peptide synthesis, solid phase peptide synthesis, or genetic engineering of a suitable host organism. The choice of method in any particular case will be a matter of convenience or economy.

[0075] If multiple RSV epitopes and surface adsorption regions are synthesized separately, once purified, for example, by ion exchange chromatography or high performance liquid chromatography, they are synthetically linked together by peptide bonds. That is, the N-terminus of the surface adsorption region and the C-terminus of the RSV epitope are covalently linked to produce the polypeptide of the design. Alternatively, the C-terminus of the surface adsorption region and the N-terminus of the RSV epitope are covalently linked to produce the polypeptide of the design. Each fragment can be synthesized by a solid phase method and obtained as a fully protected, fully unprotected or partially protected fragment. The fragment can be covalently bound in a solution phase reaction or a solid phase reaction. If a polypeptide fragment contains cysteine ​​as its N-terminal residue and another polypeptide fragment contains a thioester or thioester precursor as its C-terminal residue, the two fragments will be spontaneously coupled in solution by a specific reaction commonly referred to as natural connection by (those skilled in the art). Natural connection is a particularly attractive option for the synthesis of designed peptides because it can be carried out in aqueous solution and at dilute concentrations with fully deprotected or partially protected peptide fragments.

[0076] In one embodiment, the RSV epitope and / or surface adsorption region is connected by a peptide bond or a non-peptide bond, as described in U.S. Patent No. 7,723,294, which is incorporated herein by reference, wherein the use of non-peptide bonds to connect polypeptide fragments for multilayer membranes is taught. Suitable non-peptide linkers include, for example, alkyl linkers, such as -NH-(CH2)sC(O)-, where s=2-20. Alkyl linkers are optionally substituted with non-steric steric groups, such as low alkyl (e.g., C1-C6), low acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. Another exemplary non-peptide linker is a polyethylene glycol linker, such as -NH-(CH2-CH2-O)nC(O)-where n makes the molecular weight of the linker be 100 to 5000Da, particularly 100 to 500Da. Many linkers described herein are available from commercial suppliers in a form suitable for solid phase peptide synthesis.

[0077] Further disclosed herein is an immunogenic composition comprising a multilayer film comprising two or more layers of polyelectrolytes, wherein adjacent layers comprise polyelectrolytes with opposite charges, wherein one layer comprises an RSV epitope. The immunogenic composition optionally further comprises one or more layers comprising a designed polypeptide.

[0078] Further included are methods of administering the compositions described herein to an individual in need of RSV immunity.

[0079] In one aspect, the particles described herein elicit an IFNγ T cell phenotype and elicit fewer IL-5 producing T cells compared to a designed peptide lacking (Pam3Cys or Pam2Cys). In another aspect, the composition elicits a broader isotype distribution in RSV-G protein specific antibody responses compared to a designed peptide lacking (Pam3Cys or Pam2Cys). In another aspect, the composition elicits IgG1, IgG2a, and IgG2b isotypes.

[0080] The immunogenicity of the immunogenic composition can be enhanced in many ways. In one embodiment, the multilayer film optionally comprises one or more additional immunogenic bioactive molecules. Although not necessary, one or more additional immunogenic bioactive molecules will 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 a combination comprising one or more aforementioned bioactive molecules. Additional immunopotentiators of other types include functional membrane fragments, membrane structures, viruses, pathogens, cells, cell aggregates, organelles or a combination comprising one or more aforementioned bioactive structures.

[0081] In one embodiment, the multilayer film optionally comprises one or more additional bioactive molecules. One or more additional bioactive molecules can be drugs. Alternatively, the immunogenic composition is in the form of a hollow shell or a coating surrounding the core. The core includes a variety of different encapsulating agents, such as one or more additional bioactive molecules, including, for example, drugs. Therefore, the immunogenic composition designed as described herein can also be used for combined therapy, such as eliciting an immune response and for targeted drug delivery. The micron-sized "core" of a suitable therapeutic material in the form of a "crystal" can be encapsulated by an immunogenic composition comprising an antigenic polypeptide, and the resulting microcapsules can be used for drug delivery. Under certain conditions, such as high pH or low temperature, the core may be insoluble, but soluble under conditions of controlled release. The surface charge on the crystal can be determined by zeta potential measurement (for determining the charge of an electrostatic unit on a colloidal particle in a liquid medium). The rate at which the contents of the microcapsule are released from the inside of the microcapsule to the surrounding environment will depend on many factors, including the thickness of the encapsulating shell, the antigenic polypeptide used in the shell, the presence of disulfide bonds, the degree of cross-linking of the peptide, temperature, ionic strength, and the method for assembling the peptide. Generally, the thicker the capsule, the longer the release time.

[0082] In another embodiment, the additional immunogenic biomolecule is a nucleic acid sequence that can direct the host organism to synthesize the desired immunogen or interfere with the expression of the genetic information of the pathogen. In the former case, this nucleic acid sequence is inserted into a suitable expression vector, for example, by methods known to those skilled in the art. Expression vectors suitable for producing efficient gene transfer in vivo include retrovirus, adenovirus and vaccinia virus vectors. The operating elements of this expression vector 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 the appropriate transcription and subsequent translation of the vector nucleic acid. In particular, it is expected that this vector will contain at least one replication origin recognized by the host organism, as well as 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 this nucleic acid sequence will be prepared for delivery, for example, by encapsulating the nucleic acid in a polypeptide multilayer film in the form of a capsule for intravenous delivery.

[0083] In the construction of recombinant expression vectors, it should also be noted that multiple copies of the target nucleic acid sequence and its accompanying operational elements can be inserted into each vector. In such an embodiment, the host organism will produce a larger amount of the desired protein in each vector. The number of multiple copies of the nucleic acid sequence that can be inserted into the vector is limited only by the ability of the resulting vector to be transferred into a suitable host microorganism due to its size and to replicate and transcribe therein.

[0084] In one embodiment, the multilayer film / immunogenic composition causes the immune system to respond to the pathogen. In one embodiment, the vaccine composition comprises an immunogenic composition and a pharmaceutically acceptable carrier. Therefore, a vaccination method for a pathogenic disease comprises administering an effective amount of the immunogenic composition to a subject in need of vaccination.

[0085] Pharmaceutically acceptable carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, inactive viral particles, etc. Pharmaceutically acceptable salts may also be used in the compositions, such as mineral salts such as hydrochlorides, hydrobromides, phosphates or sulfates, and organic acid salts such as acetates, propionates, malonates or benzoates. The compositions may also include liquids such as water, saline, glycerol and ethanol, and materials such as wetting agents, emulsifiers or pH buffers. Liposomes may also be used as carriers.

[0086] A method (for example, vaccination) for inducing an immune response to a disease or pathogen in a vertebrate, comprising administering an immunogenic composition comprising a multilayer film, the multilayer film comprising an RSV epitope. In one embodiment, the polyelectrolyte containing the RSV epitope is located in the outermost layer of the multilayer film or in a layer exposed to a solvent. The immunogenic composition can be administered orally, intranasally, intravenously, intramuscularly, subcutaneously, intraperitoneally, sublingually, intradermally, pulmonary or transdermally, with or without a booster dose. Typically, the composition is administered in a manner compatible with a dosage formulation, and is administered in an amount effective for prevention and / or treatment. The precise amount of the immunogenic composition to be administered depends on the judgment of the practitioner, and may vary from subject to subject. It is clear to those skilled in the art that the therapeutically effective amount of the immunogenic composition depends particularly on the administration regimen, the unit dose of the antigen administered, whether the composition is administered in combination with other therapeutic agents, and the immune status and health status of the recipient. As well known in the art, the therapeutically effective dose can be determined by ordinary skilled medical workers according to patient characteristics (age, weight, sex, condition, complication, other diseases, etc.). Moreover, as further routine studies are conducted, more specific information will emerge regarding appropriate dosage levels for treating various conditions in various patients, and the ordinarily skilled artisan will be able to determine appropriate dosages taking into account the treatment background, age, and general health of the recipient.

[0087] The immunogenic composition optionally comprises an adjuvant. An adjuvant typically comprises a substance that enhances the host immune response in a non-specific manner. The choice of adjuvant depends on the subject to be vaccinated. Preferably, a pharmaceutically acceptable adjuvant is used. For example, oily or hydrocarbon emulsion adjuvants, including complete and incomplete Freund's adjuvants, should be avoided for vaccines used in humans. An example of an adjuvant suitable for humans is alum (alumina gel). However, vaccines used in animals may contain adjuvants that are not suitable for humans.

[0088] It is contemplated that such an immune response can be elicited by presenting any protein or peptide capable of eliciting an immune response. In one embodiment, the antigen is a key epitope, i.e., an immunodominant epitope, which elicits a strong immune response to a specific infectious disease agent. If desired, more than one antigen or epitope may be included in the immunogenic composition to increase the likelihood of an immune response.

[0089] As used herein, "layer" refers to the thickness increment after the adsorption step, for example, on a template for film formation. "Multilayer" refers to multiple (i.e., two or more) thickness increments. A "polyelectrolyte multilayer" is a film comprising a thickness increment of one or more polyelectrolytes. After deposition, the layers of the multilayer may not remain as discrete layers. In fact, there may be significant species mixing, especially at the interfaces of the thickness increments. The presence or absence of mixing can be monitored by analytical techniques such as zeta potential measurements, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry.

[0090] "Amino acid" refers to the building blocks 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.

[0091] “Naturally occurring amino acids” refers to 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.

[0092] "Unnatural amino acids" refer to amino acids other than the 20 common, naturally occurring L-amino acids. Unnatural amino acids can have L- or D-stereochemistry.

[0093] "Amino acid sequence" and "sequence" refer to a contiguous length of a polypeptide chain of at least two amino acid residues in length.

[0094] "Residue" refers to an amino acid in a polymer or oligomer; it is the residue of an amino acid monomer from which the polymer is formed. Peptide synthesis involves dehydration, that is, the "loss" of a single water molecule as an amino acid is added to the polypeptide chain.

[0095] As used herein, "peptide" and "polypeptide" both refer to a series of amino acids linked to each other by peptide bonds between the α-amino and α-carboxyl groups of adjacent amino acids, and may or may not contain modifications, such as glycosylation, side chain oxidation or phosphorylation, provided that such modifications or lack of modifications do not destroy immunogenicity. As used herein, the term "peptide" refers to peptides and polypeptides or proteins.

[0096] "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, such as a plane, a spherical shape, a rod shape, etc. The surface of the substrate can be regular or irregular. The substrate can be a crystal. The substrate can be a bioactive molecule. The size of the substrate ranges from nanoscale to macroscale. In addition, the substrate optionally includes a plurality of small sub-particles. The substrate can be made of organic materials, inorganic materials, bioactive materials, or a combination thereof. Non-limiting examples of substrates include silicon wafers; charged colloidal particles, such as microparticles of CaCO3 or melamine formaldehyde; biological cells, such as red blood cells, hepatocytes, bacterial cells, or yeast cells; organic polymer lattices, such as polystyrene or styrene copolymer lattices; liposomes; organelles; and viruses. In one embodiment, the substrate is a medical device, such as an artificial pacemaker, a cochlear implant, or a stent.

[0097] When the substrate is decomposed or removed during or after film formation, it is called a "template" (for film formation). The template particles can be dissolved in an appropriate solvent or removed by heat treatment. For example, if partially cross-linked melamine-formaldehyde template particles are used, the template can be decomposed by mild chemical methods, such as in DMSO, or by changing the pH value. After the template particles are dissolved, a hollow multilayer shell composed of alternating polyelectrolyte layers is left.

[0098] A "capsule" is a polyelectrolyte membrane in the form of a hollow shell or coating surrounding a core. The core includes a variety of different encapsulating agents, such as proteins, drugs, or combinations thereof. Capsules with a diameter less than about 1 μm are called nanocapsules. Capsules with a diameter greater than about 1 μm are called microcapsules.

[0099] "Cross-linking" refers to the formation of a covalent bond, a few bonds, or many bonds between two or more molecules.

[0100] "Biologically active molecules" refer to molecules, macromolecules or macromolecular assemblies that have biological effects. Specific biological effects can be measured in appropriate assays and normalized according to the unit weight or molecule of the biologically active molecule. The biologically active molecule can be encapsulated, retained behind or encapsulated in a polyelectrolyte membrane. Non-limiting examples of biologically active 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, "biologically active molecules" further include biologically active structures, such as functional membrane fragments, membrane structures, viruses, pathogens, cells, cell aggregates and organelles. Examples of proteins that can be encapsulated or retained behind a polypeptide membrane 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 a polyelectrolyte membrane are transplanted pancreatic islet cells, eukaryotic cells, bacterial cells, plant cells, and yeast cells.

[0101] "Biocompatible" means that it does not cause substantial adverse health effects when taken orally, topically, transdermally, subcutaneously, intramuscularly, inhaled, implanted, or intravenously. For example, biocompatible films include films that do not cause a significant immune response when in contact with, for example, the human immune system.

[0102] An “immune response” is a reaction of the cellular or humoral immune system to a substance present anywhere in the body. An immune response can be characterized in a variety of ways, for example, by an increase in the number of antibodies in the bloodstream that recognize a certain antigen. Antibodies are proteins secreted by B cells, while immunogens are entities that trigger an immune response. The body fights infection and inhibits reinfection by increasing the number of antibodies in the blood and elsewhere.

[0103] "Antigen" refers to a foreign substance that, when introduced into the tissues of a susceptible vertebrate organism, elicits an immune response (e.g., the production of specific antibody molecules). An antigen comprises one or more epitopes. An antigen may be a pure substance or 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, and combinations thereof, which terms may be used interchangeably. Antigens are typically high molecular weight and are typically polypeptides. Antigens that elicit a strong immune response are considered to be strongly immunogenic. The site on an antigen to which a complementary antibody can specifically bind is called an epitope or antigenic determinant.

[0104] "Antigenicity" refers to the ability of a composition to generate antibodies or a cell-mediated immune response specific for the composition.

[0105] As used herein, a "vaccine composition" is a composition that elicits an immune response in a mammal to which it is administered and protects the immunized organism from subsequent attack by an immunizing agent or an immune cross-reacting agent. Protection may be complete or partial in terms of reduction of symptoms or infection compared to an unvaccinated organism. An immune cross-reacting agent may be, for example, a whole protein (e.g., a glucosyltransferase) from which a subunit peptide used as an immunogen is derived. Alternatively, an immune cross-reacting agent may be a different protein that is recognized in whole or in part by antibodies elicited by the immunizing agent.

[0106] As used herein, "immunogenic composition" is intended to include compositions that elicit an immune response in an organism to which the composition is administered, and which may or may not protect the immunized mammal from subsequent challenge with an immunizing agent. In one embodiment, the immunogenic composition is a vaccine composition.

[0107] The present invention is further illustrated by the following non-limiting examples.

[0108] Example

[0109] Materials and methods

[0110] Core and Microparticles: The base CaCO3 core is formed in a controlled coprecipitation reaction with poly-L-glutamic acid sodium salt (PGA-Na). Sodium carbonate and calcium chloride solutions (both containing PGA-Na) are pumped through tubing and rapidly mixed in a 1:1 ratio in a flow reaction. PGA-Na provides a more stable particle and also serves as an initial layering step on the CaCO3 microparticles.

[0111] The precipitated CaCO3 cores were subjected to electrostatic layer-by-layer (LbL) assembly, where charged polymers with high net positive or net negative charge were assembled on the surface of the CaCO3 microparticles. The assembly was driven by electrostatic attraction between soluble polymers and oppositely charged surfaces. Poly-l-lysine (PLL, positive charge) and poly-l-glutamic acid (PGA, negative charge) homopolymers were alternately layered to assemble a total of 7 layers on the CaCO3 microparticles, where the 7th layer was PGA to produce a net negative surface charge. The 8th layer was the designed peptide, which contained a C-terminal polylysine tail (K20 (SEQ ID NO: 8) or K20Y (SEQ ID NO: 9)), which was a net positive charge and would be electrostatically layered on the negatively charged surface of the microparticles.

[0112] Homopolymers: PLL and PGA were obtained from Sigma. They are synthetically prepared chains of amino acids with either positive (PLL) or negative charge (PGA).

[0113] Designed peptides: Designed peptides were linearly synthesized by solid phase peptide synthesis (SPPS), a process with repeated cycles of alternating N-terminal deprotection and coupling reactions (C-terminal to N-terminal amino acid addition). SPPS uses the N-terminal FMOC protecting group for coupling and microwave-assisted synthesis using onium-based chemistry. The synthesis method for the peptides used in ACT-1216 was modified to utilize carbodiimide-based chemistry for higher microwave temperature-assisted synthesis to speed up peptide coupling time. Once the peptides were synthesized, they were cleaved with trifluoroacetic acid to remove any remaining protecting groups on the peptide chain, such as FMOC, and to remove the peptide from the solid support resin at the C-terminus. After cleavage, the peptides were purified by C4 or C18 columns and stored lyophilized. The peptides in constructs ACT-1190 / 1211 and ACT-1193 / 1216 underwent an additional oxidation step to aid in the folding of the peptide in a region containing four free cysteines. After this oxidation step, the peptides were purified before lyophilization.

[0114] ELISA protocol: ELISA plates were coated with RSV A2 G protein (a generous gift from Ralph Tripp, University of Georgia) or RSV-G peptide (SEQ ID NO: 3) for 2 hours at room temperature, blocked with ELISA buffer (PBS + 1% BSA) for 1 hour, and then washed three times with PBS-T. Mouse serum samples serially diluted in buffer were added and incubated at room temperature for 2 hours or refrigerated overnight. The plates were washed three times with PBS-T, and then HRP-conjugated α mouse IgG secondary antibody was added for 1 hour. The plates were washed three times with PBS-T again. TMB solution was added to allow the color to develop for 3 minutes, and then 2M H2SO4 was added to stop the color development. The plates were immediately read at OD of 450nm.

[0115] ELISPOT protocol: ELISPOT plates were coated with mouse IL-5 or IFNγ capture antibodies at 4°C overnight. 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 5×10 4cells / well. 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 incubation overnight (16-20 hours) at 37°C / 5% CO2, the cell suspension was discarded, the wells were washed twice with deionized water, and then washed three times with PBS-T. IL-5 or IFNγ biotinylated detection antibodies in PBS / 1% BSA were added, and the plates were incubated at room temperature for 2 hours. The detection antibodies were 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 hour, 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 5-10 min. The plates were rinsed with distilled water to stop color development and then dried at room temperature. The spots were quantified using an automated virus spot reader and the results were expressed as the number of spots / 10 6 Splenocytes are represented.

[0116] Example 1: Dose response of RSV particles

[0117] Table 1: Sequences of RSV particle compositions

[0118]

[0119]

[0120] This study examined the potency, immunogenicity, and efficacy of ACT-1190 particles over a 5-log dose range. BALB / c mice were immunized with ACT-1190 at concentrations ranging from 10 μg to 1 ng DP on days 0 and 21. Serum after boosting was tested by RSV-G protein ELISA, and T cell responses after boosting were tested by IL-5 and IFNγ ELISPOT. Figure 1A The results in A and B show dose-dependent antibody responses across the entire dose range, with only 1-2 outliers in the lower dose groups.

[0121] ELISPOT results Figure 2 The IFNγ response increased in a dose-dependent manner from 1 ng to 100 ng and then remained constant until the 10 μg dose. The IL-5 response peaked at 1 μg

[0122] Two weeks after the booster, the remaining mice were challenged with RSV A2 strain. Lungs were harvested 5 days later and viral titers were measured by qPCR and plaque assay on Vero cells. The results of the plaque assay are shown in Figure 3As shown. Significant reductions in viral burden were achieved at all doses compared to naive mice, ranging from 59.4% at the 1 ng dose to complete protection at the 10 μg dose. Although all immunized groups were statistically different from the naive group, there were no statistical differences between the 10 μg group and the 1 μg group or the 100 ng group (P>0.05 for each comparison). A similar pattern was observed when the samples were analyzed by qPCR (data not shown), although the extent of differences in the plaque assay was generally greater than that in qPCR. The plaque assay is generally more sensitive because it measures actual replicating virus, whereas qPCR measures total viral M2 gene expression, which is not necessarily associated with viral replication.

[0123] Example 2: PAM3CYS improves the potency of RSV particles

[0124] ACT-1193-01 contains a Pam3Cys-modified designer peptide (DP), similar to the unmodified DP in ACT-1190. Groups of BALB / c mice were immunized by fp with 1 μg or 31.6 ng of ACT-1190 (GM2) or ACT-1193 (Pam3Cys.GM2) on days 0 and 21. Serum was collected on day 28 and RSV-G protein-specific antibody titers were determined by ELISA. Figure 4A The ACT-1190 and ACT-1193 constructs were shown to elicit RSV-G protein-specific antibody responses in a dose-dependent manner, and both constructs elicited predominantly IgG1 (Th2-associated), whereas ACT-1193 elicited some IgG2a (Th1-associated) and IgG2b isotypes ( Figure 4B ). The results showed that RSV particles carrying DP modified with Pam3Cys (ACT-1193) elicited higher antibody titers and broader isotype distribution than control particles ACT-1190 carrying unmodified DP.

[0125] T cell responses were measured by ELISPOT on day 28. Mice immunized with either construct generated equivalent IFNγ T cell responses, whereas Pam3Cys-modified ACT-1193 elicited fewer IL-5 T cells than unmodified ACT-1190 ( Figure 5 ).

[0126] The remaining 10 mice / group were challenged with RSV strain A2 on day 37, and viral burden in the lungs was measured by qPCR. Fig. 6A and 6B The results in the study showed that when the plaque assay ( Fig. 6A ) or qPCR( Figure 6B) measured by qPCR. Because qPCR measures total RSV M2 gene expression, not actual viral replication, this assay may produce false-positive values. However, the same trend of protection was observed in the plaque assay, which measures actual viral replication. These results suggest that potency was effective and detectable at sub-microgram doses with or without Pam3Cys, although the modification did result in 100% protection of challenged mice, even at the lowest dose of 31.6 ng.

[0127] This example demonstrates that inclusion of Pam3Cys on the designed peptide improves the potency of the immune response ( Figure 4A and 6) and phenotype ( Figure 4B In favor of IgG2a and IgG2b, Figure 5 IFNγ is more favorable than IL-5).

[0128] Example 3: RSV Enhanced Disease Studies

[0129] 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) by fp on days 0 and 21. The 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 equivalent); on day 0, 10 6 pfu live virus (positive); and naive mice (negative). The serum after boosting was tested by RSV-G peptide ELISA, and the T cell response after boosting was tested by IL-5 and IFNγ ELISPOT. Fig. 7A and 7B The 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 compared to immunization with the G-only construct (1213). Fig. 7A and B) and induced a shift in the isotype distribution to include IgG2a ( Figure 7C ), even though the amount of G epitope was the same in both groups. These results confirm that M2 improves the antibody response against G both qualitatively and quantitatively. Pam3Cys modification of GM2 DP further improves the antibody response; note that the ACT-1193 (Pam3Cys.GM2) group has the highest IgG titer ( Fig. 7A ) and the highest levels of IgG2a ( Figure 7C).

[0130] ELISPOT results Fig. 8A -C. The IFNγ response to RSV-M2 was predominant in the live RSV group, although positive responses were also observed in the ACT-1193, ACT-1214, and FI-RSV groups. The response to RSV-G was highest in the ACT-1213 group (G only), followed by the ACT-1190 group.

[0131] The remaining mice were challenged with RSV strain A2, and lungs of 10 mice per group were harvested 5 days later, and viral titers were measured by qPCR and plaque assay on Vero cells. Fig. 9 Plaque assay results in the 2019 study showed that all mice were completely protected from challenge except for the ACT-1214 group (RSV-M2 alone). Compared with naive animals, the viral burden in this group of animals was reduced by 41.1% to 90.9%, with an average reduction of 67.4%. Similar protection patterns were observed by qPCR (data not shown).

[0132] BAL fluid was collected before challenge and then 6 and 10 days after challenge (3 mice per group at each time point). BAL cells were stained for CD8 + / M2-pentamer + cells (days 6 & 10 after challenge) and eosinophils (EOS) and macrophages (MΦ) (at all 3 time points). EOS is CD45 + / SiglecF + / CD11c - And MΦ is CD45 + / SiglecF + / CD11c + For each sample, 50,000 events were collected and then + A gate was set on the cells, which allowed us to select for white blood cells and exclude any red blood cells that escaped lysis. The gated cells were then subjected to two-color analysis. Fig. 10A It shows that there was almost no difference in the number of EOS and MΦ between the groups before challenge. After challenge, the EOS and MΦ curves of the ACT-1190 and ACT-1213 groups were similar to those of the FI-RSV group ( Fig. 10BAnd C). In the ACT-1193 group, the number of EOS in one of the three animals was elevated at two time points after the challenge, but was generally lower than that of ACT-1190 or ACT-1213. The EOS curve of the ACT-1214 group was similar to that of the recovery mice, but the ACT-1214 group was the only group that was not completely protected from the challenge. The results of this study show that although the construct containing RSV-G DP caused eosinophils to infiltrate the lungs, the construct can completely protect the lungs from RSV infection and apparently does not cause any obvious adverse events, and the inclusion of the TLR2 ligand Pam3Cys (ACT-1193) leads to a moderate reduction in eosinophils infiltrating the lungs after the challenge.

[0133] Example 4: Lung cytokine levels after RSV enhanced disease study challenge

[0134] Example 3 demonstrates that ACT-1193 (Pam3Cys.GM2) is more potent than ACT-1190 (GM2) and switches the T cell phenotype from IL-5 to IFNγ, with both constructs providing 100% protection against live RSV challenge. Of particular interest is that mice immunized with ACT-1190 produced post-challenge lung eosinophil infiltration comparable to that observed in mice immunized with FI-RSV, whereas mice immunized with ACT-1193 produced lower levels of post-challenge eosinophil infiltration. 1193-immunized mice also produced higher titers and a wider isotype distribution of G-specific antibody responses in the lungs after live RSV challenge. In summary, these results indicate that the immune responses elicited by Pam3Cys-modified LbL-MPs are both quantitatively (antibody titers) and qualitatively (antibody isotypes, T cell phenotypes, and post-challenge eosinophil infiltration) improved compared to responses elicited by unmodified LbL-MPs. This observation was further strengthened when we examined the cytokine profile of lung (BAL) fluid after challenge. The levels of cytokines and chemokines in cell-free BAL fluid were analyzed by 16-plex Luminex assay. The results from pre-challenge (day 0) mice were similar to those from naive mice at day 6 after challenge, which are not shown for clarity. In all groups at day 10 after challenge, most cytokines and chemokines decreased to pre-challenge levels or lower, which are also not shown for clarity. Fig.11A-G shows the results of all groups on the 6th day after the attack. The columns highlighted in yellow are analytes expressed at higher levels in mice immunized with ACT-1190 than in mice immunized with FI-RSV after the attack. These analytes include IL-12p40, IL-15 and IL-17 (proinflammatory cytokines) and RANTES (leukocyte chemotaxis), indicating that immunization with RSV-GM2 LbL-MPs produces leukocyte infiltration and activation in subsequent attacks on the host. All of these analytes can also be detected in mice immunized with ACT-1193, although their levels are slightly lower than those of mice immunized with ACT-1190. The red highlighted columns are Th2-related cytokines IL-4, IL-5, IL-6, and IL-13, which were also elevated in the FI-RSV, ACT-1190 (GM2), and ACT-1213 (G) groups, but expressed at lower or undetectable levels in the live RSV, ACT-1193 (PAM 3Cys.GM2), and ACT-1214 (M2) groups. Therefore, the Th2 cytokine fingerprint in the BAL fluid after challenge distinguished the immunogens into two different groups, such as Fig.11A -G are shown in the vertical alignment of the graphs. Of particular interest was the finding that IL-4 and IL-13 were elevated in the FI-RSV and ACT-1190 groups, but decreased or completely absent in the live RSV and ACT-1193 groups. Both of these Th2 cytokines have been implicated in eosinophil recruitment in pulmonary inflammatory diseases such as asthma, and therefore they may be important surrogate markers for RSV-enhanced disease, which is often characterized by Th2-associated inflammation. These results suggest that Pam3Cys modification of the GM2 construct favors a Th1 non-inflammatory response, which may be associated with lower levels of disease exacerbation following challenge.

[0135] Example 5: RSV Enhanced Disease Study Lung Antibody Response

[0136] 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 a dilution of 1:5 in all ELISAs. Fig. 12A The results showed that the amount of RSV-G-specific antibodies increased significantly 10 days after challenge in the ACT-1193 group but remained fairly stable in the ACT-1190 group. This pattern was also reflected in the isotype ELISA. Fig. 12C -E, we see that the ACT-1190 group had the highest levels of IgG1 on the day of challenge, but that level did not increase over time after challenge; the amount of IgG2a was initially low and increased only slightly. In the ACT-1193 group, both IgG1 and IgG2a increased over time. Fig. 12BTotal IgG levels were shown to increase in all groups except the ACT-1190 group, where total IgG was already elevated before challenge. These results are consistent with previous results showing RSV-G-specific serum IgG2a in the ACT-1193 group, but not in the ACT-1190 group.

[0137] Example 6: Second RSV Boost Disease Study Serum Antibody Response and Efficacy

[0138] BALB / c mice were immunized with ACT-1190 (GM2) or ACT-1193 (Pam3Cys.GM2) by fp at day 0 and day 21. The control group was FI-RSV, live RSV and naive mice. Blood was collected from mice 1 week after booster immunization, and RSV-G specific antibody titers in serum were measured by ELISA. Fig.13A and 13B The results in show that both constructs elicited RSV-G specific antibody responses in a dose-dependent manner. As previously reported, the addition of Pam3Cys to DP (ACT-1193) resulted in higher antibody responses and a broader isotype distribution.

[0139] Because no anticoagulant is used when mice are bled, the serum produced can be tested in RSV neutralization assay. Merge serum and heat inactivate, serial dilutions are mixed with equal-volume diluted RSV / A2, incubated at 37°C for 1 hour, and then added to Vero cell monolayer. After 6 days at 37°C, the monolayer is fixed and immunostained. Although anti-RSV-F monoclonal control antibodies show a dose-dependent reduction in the number of viral plaques, only FI-RSV and live RSV serum show any neutralization (data not shown). Not bound by theory, it is believed that it is necessary to give a higher dose of immunogen to detect neutralizing antibodies in vitro.

[0140] The remaining mice were challenged with RSV / A2 on day 35, and 6 mice per group were sacrificed 5 days after challenge to assess the viral burden in the lungs by plaque assay and qPCR. The remaining 6 mice per group were sacrificed 6 days after challenge to assess lung histology and BAL cellularity and cytokine content. Fig.14 The infection levels in the naive groups were shown to be fairly consistent, but somewhat lower than expected due to overestimation of the viral titer of the infection reservoir. However, except for the 10 ng ACT-1193 group (infection was detected in 2 of 6 animals), the protection of the other groups was 100%. qPCR analysis showed a similar pattern, with only the ACT-1193 10 ng group being statistically indistinguishable from the naive group (data not shown).

[0141] Example 7: Secondary RSV Enhanced Disease Study Lung Antibody Responses

[0142] Compared with mice immunized with ACT-1190, mice immunized with ACT-1193 had higher serum antibody titers, a wider distribution of antibody isotypes, and lower IL-5 responses, but had equivalent CTL responses and efficacy. There was also a trend of decreased lung eosinophil counts after attack. In the second study, BALB / c mice were immunized with ACT-1190 (GM2) or ACT-1193 (Pam3Cys.GM2) by fp on days 0 and 21; the control group was FI-RSV, live RSV, and naive mice. Mice were attacked with live RSV after the second immunization, and BAL fluid was collected from 6 mice in each group 6 days after the virus attack, and RSV-G peptide-specific antibodies and total IgG were tested by ELISA. We also measured the chemokine CCL2 / MCP-1, a Th2-related chemokine known to be associated with lung inflammation, using sandwich ELISA and following the manufacturer's instructions. The results are shown in Figure 15. Although the total IgG levels in all BAL samples were roughly equal ( Fig.15A ), but the RSV-G-specific IgG titers in BAL fluid of mice immunized with ACT-1193 (Pam3Cys.GM2) were much higher than those of mice immunized with ACT-1190 (GM2) ( Fig. 15B There was also a change 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 It is shown that CCL2 was detectable in some mice in each group except the group immunized with 1 μg ACT-1193, the group immunized with live RSV, and the naive group. The detection limit of the CCL-2 ELISA is 5 pg / ml; all samples above this level are indicated by red circles.

[0143] BAL fluid was analyzed by 16-plex ELISA. Fig.16A-H results are very similar to those obtained with BAL samples from Example 4. Th2 cytokines are highlighted in red. Any dose level of 1190 triggered the same Th2 fingerprint as seen in the FI-RSV group. In contrast, the 1193 group showed a dose-dependent reduction in Th2 cytokines, with almost no IL-4, IL-5, IL-6 or IL-13 detected in the 1 μg dose group, while the amount increased in the lower dose groups. The IL-13 response appears to be particularly sensitive to vaccine dose, as this cytokine was not detected in the 1 or 0.1 μg 1193 dose groups, and was lower in the 0.01 μg 1193 group compared to any dose level of 1190.

[0144] Although anti-RSV-G IgG responses in the serum of mice immunized with 1193 were slightly higher than those of mice immunized with 1190, the major differences between the two groups were in the serum isotype distribution and BAL isotype distribution (1193 elicited IgG1 and IgG2a in both samples), the BAL cytokine profile (1193 inhibited Th2 cytokine responses), the T cell phenotype (1193 inhibited IL-5 responses [Th2] without affecting IFNγ responses [Th1]), and the presence of higher levels of BAL CCL-2 responses in mice immunized with 1 μg ACT-1193 than in mice immunized with 1 μg ACT-1190. These lines of evidence clearly indicate that LbL-MPs loaded with Pam3Cys-modified DPs favor more potent Th1 responses than LbL-MPs loaded with the same DPs but without Pam3Cys modification. Although CTL activity and efficacy were similar between the two candidates (at the doses tested to date), the reduction in Th2 responses elicited by Pam3Cys-modified particles could provide protection against enhanced disease after challenge.

[0145] Example 8: Induction of antibody responses that inhibit chemotaxis

[0146] RSV-G epitope described herein includes the mimetics of fractalkine, which is a CX3C chemokine with chemotactic activity. The particle is designed to elicit an antibody response that not only interferes with viral infection, but also inhibits the inflammatory properties of RSV-G protein, including chemotaxis or lymphocyte migration to inflammatory sites. Analysis of the inhibition of chemotaxis inhibition by serum from enhanced disease research. Dynal beads pool immunoprecipitation serum antibodies conjugated with IgA and IgG were used, and PBS was dialyzed using a molecular weight cutoff of 100,000. The dialyzed antibody purified was incubated in the lower chamber of the improved Boyden chamber with natural RSV-G protein, at a concentration of 1 / 5 of the purified RSV-G protein. The thawed, static human lymphocyte pool from 3 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 live lymphocytes that migrated to the lower chamber was scored and the chemotaxis index (CI) was determined. The percentage of migration inhibition was determined based on the CI. 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 in two wells every night, using different lymphocyte pools on 3 separate nights; therefore, n=6 for each serum sample. Fig.17A The results in A and B 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. As expected, antisera raised against ACT-1214 (RSV-M2) failed to inhibit chemotaxis. Inclusion of M2 in the vaccine or addition of Pam3Cys did not appear to increase anti-chemotactic activity above the levels seen in sera from mice immunized with LbL-MP containing only the RSV-G epitope.

[0147] Example 9: RSV Enhanced Disease Marker Studies Antibody Response and Efficacy

[0148] The aim of this study was to continue the search for reliable markers of boosted disease. RSV-naive BALB / c mice (10 / group) were immunized im on days 0 and 21 with 1 μg ACT-1211 (GM2) or ACT-1216 (Pam3.GM2). FI-RSV and live RSV groups served as positive and negative controls, respectively. As an additional control, a naive group that would be anesthetized but not challenged was also included. One mouse in the live RSV group died a few days after the start of the study, reducing the number of mice in this group to 9. Mice were bled 1 week after the boost immunization, and RSV-G-specific IgG ( Fig.18A). ACT-1211 titers were lower than expected, although this was only the second study to test the 1 μg dose via the im route. Because both the ACT-1211 and ACT-1216 batches used in this study are older than 4 years, aliquots of each batch were recently examined by DLS to determine particle size and dispersibility. Results showed that both constructs appeared stable with minimal variation in size and dispersibility (data not shown). Therefore, the relatively lower antibody responses in the 1211 group may simply be due to differences between the different studies or the lower 1 μg dose tested.

[0149] Mice were challenged on day 35 and body weight loss was monitored for 8 days post-challenge. Fig.18B Shown are the average percent weight changes of the groups relative to the day of virus challenge (day 0). All groups lost weight after the challenge, including the naive group that was anesthetized but did not receive the virus. This suggests that at least part of the weight loss can be attributed to the mice being anesthetized. However, the FI-RSV group lost the most weight of all the groups, dropping by 5% on the second day, which contrasts with previous studies in which the same batch of FI-RSV did not cause any significant weight loss. The second weight loss occurred between the 5th and 6th days. This reduction may be caused by the influx of cells into the lungs that occurs at this time. However, this explanation cannot explain the weight loss of naive animals. Without being bound by theory, it is believed that this apparent reduction may be artificial because the average weight on days 6-8 is the average weight of the remaining 3 / groups reserved for BAL. In addition, these remaining mice were placed in clean cages on the 5th day, and the reduction may simply be due to their increased activity in the "new" environment.

[0150] Mice were sacrificed 5 days after challenge and lung viral burden and cytokine / chemokine content were analyzed (6-7 mice / group). Both plaque and qPCR were used to measure viral burden in the lung. Fig.19A and 19B Results in the study showed that the efficacy of ACT-1211 was not as high as previously observed, with an average reduction of only 75.6% compared to the naive group, and no individual mice had a reduction of >90% in viral burden. This result correlated with low serum antibody titers in this treatment group. Both ACT-1216 and FI-RSV resulted in significant reductions in viral burden, while prior infection with RSV completely protected mice from challenge.

[0151] The remaining 3 mice / group were sacrificed 8 days after challenge for analysis of BAL cellularity and cytokine / chemokine content.

[0152] Example 10: RSV Enhanced Disease Marker Studies of Lung Cell Properties and Cytokine Content

[0153] Eight days after the challenge, three mice / group were sacrificed and BAL fluid was collected to analyze the content of cytokines and chemokines and the cell properties using markers for T cells, macrophages, and eosinophils. CD3 + Cells were gated and CD4 + and CD8 + A similar strategy was used to characterize eosinophils (CD45 + / CD11c - / SiglecF + ) and macrophages (CD45 + / CD11c + / SiglecF + ). The results are shown in FIG20 as the fold increase in the number of cells in each group relative to the naive group. Fig. 20A CD4+ cells were shown to increase in the ACT-1216, FI-RSV, and live RSV groups, with the greatest increase in the FI-RSV group. All challenged mice had an increase in T cell numbers relative to unchallenged naive mice. Fig. 20B The results showed that eosinophil numbers increased to varying degrees in all immunized groups after challenge. In one mouse in the ACT-1216 group, eosinophils increased 5-fold, while in 2 mice in the live RSV group, eosinophils increased approximately 3-fold and 15-fold. By far, the FI-RSV and ACT-1211 groups had the greatest increase in eosinophils. Fig. 20B The callouts in show the multiple increases for the three values ​​above the chart range.

[0154] During analysis of BAL cells, it was noted that the presence of eosinophils could be easily seen in the SSC / FSC dot plot. Fig.21 Scatter plots for a naive mouse and a FI-RSV mouse are shown. The triangular region (R1) contains T cells and was present in all mice in all groups, although the numbers were greatly reduced in the unchallenged naive mouse. The polygonal region (R4) corresponds to larger, more granular cells, i.e., eosinophils and other CD45 + In R4 of the corresponding scatter plot, mice with increased numbers of eosinophils (determined by fluorescent labeling) have significantly more cells. Thus, the scatter plot alone gives an indication of the presence or absence of infiltrating eosinophils in individual animals.

[0155] Using a multi-analyte flow assay kit from BioLegend, Th1 / 2 cytokines and pro-inflammatory chemokines were measured in clarified lung homogenates collected 5 days after challenge (7 / group) and BAL collected 3 days later (3 / group). Figure 22 shows that only the 5th day samples increased or decreased in fold compared to the naive group (most of the 8th day samples showed little change or reduction in all analytes compared to the naive control group, and there was no significant difference between the treatment groups); asterisks indicate cytokines that are associated with both Th1 and Th2 but are primarily associated with the phenotypes they display. Fig.22A It was shown that Th1-associated TNFα levels were elevated in all challenged groups, whereas IFNγ was elevated only in the 1211-immunized group. Fig. 22B An increase in Th2-related IL-4, IL-5, and IL-13 in the 1211 and FI-RSV groups is shown (the callout shows the fold increase in IL-5 values, above the range of the graph). This pattern is similar to that reported in Figures 11 and 13, and again suggests that TLR activation (TLR2 via Pam3Cys in 1216 or TLR4 via viral proteins in live RSV) inhibits inflammatory cytokine responses.

[0156] Unless otherwise specified herein or the context is clearly contradictory, the use of the terms "one", "an" and "the" and similar indicators (especially in the context of the following claims) should be interpreted as covering the singular and plural. The terms first, second, etc. used herein are not meant to represent any particular order, but are merely for the convenience of representing multiple, such as multiple layers. Unless otherwise specified, the terms "include", "have", "include" and "contain" should be interpreted as open terms (i.e., meaning "including, but not limited to"). Unless otherwise specified herein, the description of the numerical range is intended only to be used as a shorthand method for individually referring to each individual numerical value falling within the range, and each individual numerical value is incorporated into the specification as if it is individually described herein. The endpoints of all ranges are included in the range and can be independently combined. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in a suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") is only to better illustrate the present invention, rather than to limit the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0157] Although the present invention has been described with reference to exemplary embodiments, it will be appreciated by those skilled in the art that various changes may be made and its elements may be replaced with equivalents without departing from the scope of the present invention. In addition, many modifications may be made to adapt specific situations or materials to the teachings of the present invention without departing from the basic scope of the present invention. Therefore, the present invention is not intended to be limited to the specific embodiments disclosed as the best mode for implementing the present invention, but the present invention will include all embodiments falling within the scope of the appended claims. Unless otherwise specified herein or clearly contradictory to the context, the present invention includes any combination of the above-mentioned elements in all possible variations.

Claims

1. A composition comprising a particle, the particle comprising a multilayer film, the multilayer film comprising two or more layers of polyelectrolytes, wherein adjacent layers comprise polyelectrolytes with opposite charges, wherein one of the polyelectrolytes comprises a designed polypeptide 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 the surface adsorption region one and two each independently comprise at least two amino acid residues and have the same charge sign as the designed polypeptide, wherein the net charge of each residue of the designed polypeptide is greater than or equal to 0.1, wherein the RSV-M2 peptide epitope comprises (ESYIGSINNITKQSACVA) or (ESYIGSINNITKQSASVA), wherein the RSV-G peptide epitope comprises (NFVPCSICSNNPTCWAICKRIPNKKPGKKT), and wherein L1 or L2 is a linker comprising 0-100 uncharged amino acid residues; wherein the polyelectrolyte that is not the designer polypeptide comprises a polycationic material or a polyanionic material having a molecular weight greater than 1,000 and at least 5 charges per molecule, and Wherein the multilayer film is deposited on core particles or forms hollow particles to provide the composition.

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

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

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

5. The composition of claim 1, wherein the designed polypeptide comprises or Pam3CSKKKKNFVPCSICSNNPTCWAICKRIPNKKPGKKTSGSESYIGSIN NITKQSACVASGSKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 7).

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

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

8. The composition of claim 7, wherein two or more layers of the multilayer film are covalently cross-linked via 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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