PARTICLE COMPRISING AN RSV-F PROTEIN FOR USE IN RSV VACCINATION

Abstract

The present invention relates to the epicutaneous vaccination against RSV vaccination with a skin patch device loaded with a particle exposing an RSV-F protein, a variant or a fragment thereof.

Claims

1. A particle comprising an RSV-F protein, a variant or a fragment thereof for use in a method of prevention of a disease caused by RSV, by epicutaneous vaccination with said particle.

2. A particle comprising an RSV-F protein, a variant or a fragment thereof for use in a method for vaccinating an infant against RSV by maternal epicutaneous vaccination with said particle.

3. The particle for use according to claim 1 or 2, wherein said vaccination leads to the generation of neutralizing antibodies directed against RSV-F protein in a subject treated with said particle.

4. The particle for use according to any one of claim 1 to 3, wherein said fragment of said RSV-F protein is a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, and wherein said variant of said RSV-F protein is a cyclic peptide comprising an amino acid sequence (I), wherein said amino acid sequence (I) comprises, preferably consists of, the amino acid sequence: TABLE-US-00007 (SEQ ID NO: 44) X1-X2-X3-C4-X5-X6-X7-C8-X9-X10-X11-P12-I13-T14- N15-D16-Q17-K18-K19-L20-C21-X22-X23-X24-C25-X26- X27-X28-X29-X30, wherein X1, X2, X3, X5, X6, X7, X9, X10, X11, X22, X23, X24, X26, X27, X28 and X29 are independently of each other an amino acid; C4, C8, C21 and C25 are independently of each other cysteine; P12 is proline; 113 is isoleucine; T14 is threonine; N15 is asparagine; D16 is aspartic acid; Q17 is glutamine; K18 and K19 are independently of each other lysine; L20 is leucine; and X30 is an amino acid or a deletion, wherein said cysteines C4 and C25 form a first disulfide bond and said cysteines C8 and C21 form a second disulfide bond.

5. The particle for use according to claim 4, wherein said amino acid sequence (I) comprises or preferably consists of an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 45-88.

6. The particle for use according to claims 1 to 5, wherein said particle is applied epicutaneously to an infant of less than 6 months.

7. The particle for use according to any one of claims 1 to 6, wherein said particle is applied epicutaneously to a pregnant female during the second and third quarters of the pregnancy, preferably during the second quarter or a mother during lactation.

8. The particle for use according to any one of claims 1 to 7, wherein said particle is a synthetic virus-like-particle (SVLP).

9. The particle for use according to claim 8, wherein said SVLP consists of conjugates, wherein each conjugate comprises, preferably consists of: a peptide chain comprising a coiled coil-domain and optionally a T-helper epitope, a lipid moiety comprising two or three, preferably two, hydrocarbyl chains, and said RSV-F protein, said variant or said fragment thereof, wherein the peptide chain is linked to said RSV-F protein, said variant or said fragment thereof and to the lipid moiety.

10. The particle for use according to claim 8 or 9, wherein: said peptide chain comprises a coiled coil peptide chain segment comprising 3 to 8 repeat units, preferably 4 repeat units, wherein said repeat unit consists of the sequence IEKKIE-X0 (SEQ ID NO: 115), wherein X0 represents an amino acid, preferably said repeat unit consists of the sequence selected from IEKKIEG (SEQ ID NO: 116), IEKKIEA (SEQ ID NO: 117) or IEKKIES (SEQ ID NO:118), more preferably said repeat unit consists of the sequence IEKKIES (SEQ ID NO:118); said lipid moiety comprises the formula LM-II, ##STR00038## wherein R.sup.1 and R.sup.2 are independently C.sub.11-15alkyl, preferably R.sup.1 and R.sup.2 are independently —C.sub.11H.sub.23, —C.sub.13H.sub.27 or —C.sub.15H.sub.31, and further preferably R.sup.1 and R.sup.2 are —C.sub.15H.sub.31; R.sup.3 is hydrogen or —C(O)C.sub.11-15alkyl, preferably R.sup.3 is H or —C(O)C.sub.15H.sub.31; and wherein the wavy line in formula LM-II indicates the linkage site of said lipid moiety to said peptide chain; and said RSV-F protein, said variant or said fragment thereof is a sequence selected from group consisting of SEQ ID NO: 2-5 and 45-88, preferably SEQ ID NO: 45-88, more preferably SEQ ID NO: 45 or 85.

11. The particle for use according to claim 10, wherein said conjugate is selected from any one of the formulae (38), (39), (40), (41) or (42), wherein preferably said conjugate is of formula (38) ##STR00039## ##STR00040## ##STR00041##

12. The particle for use according to anyone of claims 1 to 11, wherein said particle is applied using a skin patch device, preferably an electrostatic skin patch device.

13. The particle for use according to claim 12, wherein said epicutaneous vaccination is performed by the application of said skin patch device on a pretreated skin.

14. A method for preparing a skin patch device comprising depositing, preferably by electrospraying, at least one particle comprising an RSV F protein, a variant or a fragment thereof on a surface of a skin patch device.

15. A skin patch device comprising an application surface, wherein the application surface contains an SVLP comprising an RSV-F protein, a variant or a fragment thereof.

16. Use of a particle comprising an RSV-F protein, a variant or a fragment thereof in the manufacture of a drug for the prevention of a disease caused by RSV, wherein the drug is delivered by means of a skin patch by epicutaneous route to provide vaccination against RSV.

17. Use of a particle comprising an RSV-F protein, a variant or a fragment thereof in the manufacture of a drug for passively vaccinating an infant against RSV, wherein the drug is administered by means of a skin patch by epicutaneous route to the infant's mother.

18. The use according to claim 16, wherein the drug is epicutaneously administered to an infant of less than 6 months.

19. The use according to claim 17, wherein the drug is epicutaneously administered to the mother during the second or third quarters of the pregnancy, preferably during the second quarter or during breastfeeding period.

20. The use according to any one of claims 16 to 19, wherein the particle is as defined in any one of claims 4, 5 and 8-11.

21. A method for providing vaccination against RSV to a subject which comprises administering the subject with a particle comprising an RSV-F protein, a variant or a fragment thereof by epicutaneous route, preferably by means of a skin patch.

22. A method for providing passive vaccination to an infant against RSV, which comprises administering the infant's mother with a particle comprising an RSV-F protein, a variant or a fragment thereof by epicutaneous route, preferably by means of a skin patch.

23. The method according to claim 21, wherein the drug is epicutaneously administered to an infant of less than 6 months.

24. The method according to claim 22, wherein the drug is epicutaneously administered to the mother during the second and third quarters of the pregnancy, preferably during the second quarter or during breastfeeding period.

25. The method according to any one of claims 21 to 24 wherein the particle is as defined in any one of claims 4, 5 and 8-11.

Description

LEGEND TO THE FIGURES

[0277] FIG. 1: Evaluation of the efficacy of Viaskin-SVLP-FsII as a boost epicutaneous vaccine. Mice were primed with a subcutaneous injection of 150 μg of SVLP-FsII, (also called herein V-306 SVLP (groups 2 to 5). Three weeks later, mice were boosted with a single application of Viaskin patch loaded with 100 or 200 μg of SVLP-FsII, for 48 hours (groups 3 and 4). As a negative control for boost immunization, mice received a single application of Viaskin patch loaded with excipient alone (PBS 1×), for 48 hours (group 2). As a positive control for boost immunization, mice received an injection of 150 μg of V-306 SVLP-FsII, namely V-306 SVLP, by subcutaneous route (group 5). As a negative control for prime and boost immunizations, mice received two application of Viaskin patch loaded with excipient alone (PBS 1×), for 48 hours, at day 0 and day 21 (group 1).

[0278] FIG. 2: Measurement of anti-FsII antibodies in mouse sera and BAL by FsII-specific ELISA. Mice were immunized as described in FIG. 1. Blood samples were collected three weeks after the prime immunization (day 21) and two weeks after the boost immunization (day 35) to prepare sera. At day 35, mice were sacrificed and bronchoalveolar lavages (BAL) were collected. Anti-FsII antibody titers were measured by ELISA from sera (A) and BAL (B) using FsII peptide as a coating antigen and mouse anti IgG (H+L) secondary antibody. Data are median±interquartile range of individual IgG titers (n=10 per experimental group). P values were determined using the Man-Whitney non-parametric test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n.s., non-significant).

[0279] FIG. 3: Measurement of neutralizing and palivizumab-competitive antibody (PCA) titers in sera. Mice were immunized as described in FIG. 1. (A) Neutralizing antibody titers were measured from heat inactivated sera (30 min at 56° C.) collected at day 35. Briefly, neutralization assay was performed using Hep2 cells on microplates and using RSV A Memphis strain. Neutralizing antibody titers were determined by the Reed and Muench method as the reciprocal of the highest dilution of each serum, which suppressed cyto-pathogen effect. (B1) PCA titers were measured from sera collected at day 35 by competitive ELISA. Briefly, serial dilutions of sera were mixed 1:1 with a fixed concentration of Palivizumab. This mixture was then added to an ELISA plate coated with FsII peptide. Residual biding of Palivizumab was revealed using a peroxidase-conjugated anti-human IgG and TMB colorimetric substrate. Optical density at 450 nm (B1) was plotted against mouse serum dilution. Non-linear curve fit was performed using Boltzmann sigmoidal equation. PCA titers (B2) were defined as the reciprocal dilution of sera that inhibit 50% of the optical density measured at 450 nm. The limit of detection was indicated with a dotted line. Data are median±interquartile range of individual titers (n=6-10 per experimental group). P values were determined using the Man-Whitney non-parametric test (**, P<0.01; * P<0.001; ****, P<0.0001; n.s., non-significant).

[0280] FIG. 4: Evaluation of the capacity of Viaskin-SVLP-FsII boost epicutaneous vaccine to enhance protection against RSV infection. Mice were primed with a subcutaneous injection of 150 μg of SVLP-FsII (also called herein V-306 SVLP) (groups 2 to 4). Three weeks later, mice were boosted with a single application of Viaskin patch loaded with 150 μg of SVLP-FsII, preferably V-306 SVLP, for 48 hours (group 3). As a negative control for boost immunization, mice received a single application of Viaskin patch loaded with excipient alone (PBS 1×), for 48 hours (group 2). As a positive control for boost immunization, mice received an injection of 150 μg of SVLP-FsII, by subcutaneous route (group 4). As a negative control for prime and boost immunizations, mice received an injection of excipient (PBS 1×) by subcutaneous route at day 0 and an application of Viaskin patch loaded with excipient alone (PBS 1×), for 48 hours, at day 21 (group 1). As a positive control for viral protection, mice were infected intranasally at day 0 with RSV A2. As a control for vaccine-induced immunopathology, mice were primed and boosted at day 0 and day 21 by an injection of formalin-inactivated RSV by intramuscular route (group 6). A blood sample was collected three weeks after the boost immunization (day 42) and mice were challenged intranasally with 1×10.sup.6 plaque-forming units (pfu) of RSV A2 strain. Mice were sacrificed at 5 days post-infection and lungs were collected to measure pulmonary viral load, perform histological analysis and to extract mRNA.

[0281] FIG. 5: Measurement of F-specific I2G1/I2G2a and RSV-neutralizing antibodies in vaccinated mice and pulmonary viral load following RSV challenge. Mice were immunized as described in FIG. 4. Blood samples were collected three weeks after the boost immunization (day 42) to prepare sera. (A) Anti-F antibody titers were measured by ELISA from sera using F protein as a coating antigen and mouse anti-IgG1 (left panel) or anti-IgG2a (right panel) secondary antibodies. Data are median±interquartile range of individual IgG titers (n=10 per experimental group). (B) Neutralizing antibody titers were measured by plate reduction assay from heat inactivated sera (30 min at 56° C.) collected at day 35. (C) Palivizumab-competitive antibody titers were evaluated as described in FIG. 3. (D) Mice were infected at day 42 and were sacrificed at day 5 post-infection. Lungs were collected and homogenized to measure viral load by plate titration. Viral titers were normalized to the weight of individual lungs to obtain a viral concentration expressed as the number of plaque-forming units (pfu) per gram of lung. Data are mean+SEM of individual data (n=4-10 per experimental group). P values were determined using the Man-Whitney non-parametric test (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n.s., non-significant). For D panel, the significance measured between the group 1 (non-vaccinated group) and each of other groups is indicated above each histogram.

[0282] FIG. 6: Evaluation of lung pathology in vaccinated mice following RSV challenge. Mice were immunized and challenged as described in FIG. 4. Lungs were collected at day 5 post infection and fixed using formalin. Fixed lungs were embedded in paraffin and histological sections were performed. Lung sections were colored using Hematoxylin Eosin Safran (HES) staining and analyzed. Four pathology criteria were evaluated (alveolitis, interstitial pneumonia, perivasculitis, peribronchiolitis) and a pathology score ranging from 0 to 100 was given to each sample. (A) Data are median+interquartile range of individual data (n=4-10 per experimental group). (B) A representative photograph of lung sections was shown for groups 2, 3, 4, 5, as indicated.

[0283] FIG. 7: Measurement of pulmonary cytokine secretion in vaccinated mice following RSV challenge. Mice were immunized and challenged as described in FIG. 4. Lungs were collected at day 5 post infection and messenger RNA (mRNA) was extracted from lung tissues. The quantity of IL-5, IL-13, IFN-γ and IL-2 transcripts was measured by quantitative RT-PCR using specific primers. Data are mean+SEM of individual data and are expressed as relative mRNA level (n=4-10 per experimental group). The significance measured between the non-infected group and each of other groups is indicated above each histogram.

[0284] FIG. 8. Structural characterization. A, The sequences of peptides FsIIm (SEQ ID NO: 130), V-306p (Nle=L-norleucine, Dab=L-diaminobutyric acid, D-Ala=D-alanine), the synthetic lipopeptide (shown in single amino acid letter code), with Pam2Cys, the coiled coil heptad repeat IEKKIES that forms a trimeric helical bundle, with T helper epitope underlined, and C-terminal KKKCa, (a=D-alanine) and, V-306. Pam2Cys is S—[R-2,3-bis(palmitoyloxy)propyl]-R-cysteine. B, Left, Solution NMR structures of FsIIm. A superimposition of the final 20 structures is shown. Right, Superimposition of one typical NMR structure of FsIIm and the Motavizumab antigen from PDB file 3IXT. C, Schematic representation of the components and assembly of a SVLP: SVLPs, such as V-306 SVLP, are formed by aggregation of multimeric conjugate bundles, here trimeric conjugate bundles comprising three conjugates, such as V-306 (ball=epitope mimetic, such as V-306p, cylinder=helical coiled-coil domain, wavy line=lipid moiety, such as Pam2Cys lipid) resulting in ca. 25-30 nm diameter micelle-like nanoparticles.

[0285] FIG. 9. Synthesis of V-306. The synthetic route to conjugate V-306.

[0286] FIG. 10. EM and DLS data for V-306. A, Negative staining transmission electron micrograph of SVLPs formed by the V-306 lipopeptide dissolved in tris(hydroxymethyl)amino-methane (Tris) buffer containing 0.9% NaCl, pH 7.4. Scale bar 5×100 nm. B, V-306 lipopeptide was dissolved in PBS (0.5 mg/mL) at pH 7.4, and analyzed in a Wyatt DynaPro DLS instrument at 25° C. Size distributions are shown by regularization as intensity distributions. The average radius, % polydispersity and polydispersity index (PDI) are indicated.

EXAMPLES

Example 1: Evaluation of the Efficacy of Viaskin-SVLP-FsII as an Epicutaneous Boost Vaccine for the Induction of Specific RSV-F Humoral Response and the Protection Against RSV Infection

[0287] The inventors aim to evaluate the capacity of Viaskin patch loaded with SVLP-FsII to induce an RSV-F specific humoral immunity when administered epicutaneously as a boost vaccine. Two experiments were designed requiring 50 and 60 BALB/c mice respectively. The first experiment was performed at DBV Technologies (Montrouge, France) and the second experiment was performed at Sigmovir Biosystems Inc. (Rockville, USA).

[0288] Study Design

[0289] SVLP-FsII used in Example 1 and FIGS. 1-7 is V-306 SVLP (cf. FIG. 8 and description thereof), which is an SVLP comprising trimeric bundles of conjugates of formula (38) and which is formed by self-aggregation of conjugates of formula (38) into trimeric bundles that self-assemble further into SVLPs. Conjugate of formula (38) comprises the mimetic of SEQ ID NO: 85 (i.e. SEQ ID NO: 45 with an N terminal AOAc residue), herein also called V-306p. Each of said trimeric bundles of conjugates included in V-306 SVLP consists of 3 conjugates of formula (38).

[0290] For the first experiment (FIG. 1), mice were primed at day 0 with SVLP-FsII, namely V-306 SVLP (150 μg) by subcutaneous route (groups 2 to 5). Three weeks later (day 21), mice were boosted with a single application of Viaskin-SVLP-FsII patch (100 or 200 μg of SVLP-FsII, namely V-306 SVLP, per patch groups 3 and 4, respectively). As a negative control for boost immunization, mice received a Viaskin patch loaded with excipient alone (PBS 1×) at day 21 (group 2). As a positive control, mice received a boost injection of SVLP-FsII (150 μg SVLP V-306) by subcutaneous route at day 21 (group 5). As a negative control for prime and boost immunizations, mice received two Viaskin patches loaded with excipient alone (PBS 1×) at day 0 and day 21 (group 1). A blood sample was collected at day 21, before boost immunization. Two weeks after the boost immunization, mice were sacrificed and a final blood sample and bronchoalveolar lavages were collected for the evaluation of FsII specific humoral responses (FsII-specific ELISA and palivizumab competition assay) and for the measurement of RSV-neutralizing antibodies.

[0291] For the second experiment (FIG. 4), mice were primed at day 0 with SVLP-FsII (SVLP V-306 150 μg) by subcutaneous route (groups 2 to 4). Three weeks later (day 21), mice were boosted with a single application of Viaskin-SVLP-FsII patch (150 μg of SVLP-FsII, namely SVLP V-306 per patch, group 3). As a negative control for boost immunization, mice received a Viaskin patch loaded with excipient alone (PBS 1×) at day 21 (group 2). As a positive control, mice received a boost injection of SVLP-FsII (150 μg SVLP V-306) by subcutaneous route at day 21 (group 4). As a negative control for prime and boost immunizations, mice received an injection of excipient (PBS 1×) by subcutaneous route at day 0 and a Viaskin patch loaded with excipient alone (PBS 1×) at day 21 (group 1). As a model of vaccine-induced immunopathology, mice were primed at day 0 and boosted at day 21 with formalin-inactivated RSV (group 5) and, as a positive control for protection, mice were infected intranasally at day 0 with RSV A2 (group 6). A single blood sample was collected three weeks after the boost immunization (day 42) for the evaluation of F-specific humoral responses (F-specific ELISA [IgG1 and IgG2a] and palivizumab competition assay) and for the measurement of RSV-neutralizing antibodies. Following blood sample, all mice were challenged intranasally with RSV A2 strain (1×10.sup.6 pfu per mouse). At day 5 post-infection, mice were sacrificed, and lungs were collected for the measurement of viral load, histological analysis and mRNA extraction for qPCR on IL-5, IL-13, IFN-γ and IL-2 transcripts.

[0292] Results

[0293] 1. Viaskin-SVLP-FsII is Able to Systemically and Locally Boost Anti-F Antibody Responses Mice were immunized as described in FIG. 1. Sera samples were collected from individual mice three weeks after le prime immunization (day 21) and two weeks after the boost immunization (day 35). From these sera, FsII-specific antibody titers were measured by ELISA (FIG. 2).

[0294] In mice previously primed subcutaneously with SVLP-FsII, epicutaneous boost immunization with Viaskin-SVLP-FsII induced a strong and a significant increase of FsII specific antibody titers compared to the mice that received Viaskin-excipient patch (FIG. 2A) (increase of 0.6 to 0.7 Log 10 between day 21 and day 35, based on median values; mean antibody titer of 5.8±0.2 log 10 for Viaskin-SVLP-FsII 100 μg at day 35, p<0.001 compared to day 21; mean antibody titer of 5.9±0.2 for Viaskin-SVLP-FsII 200 μg at day 35, p<0.0001 compared to day 21). This boost effect was in the same range than that observed after a subcutaneous boost immunization with SVLP-FsII (mean antibody titer of 6.1±0.2 log 10 at day 35, p<0.0001 compared to day 21, p<0.01 compared to Viaskin-SVLP-FsII 100 μg at day 35 and non-significant compared to Viaskin-SVLP-FsII 200 μg at day 35). The dose effect observed between Viaskin-SVLP-FsII 100 μg and Viaskin-SVLP-FsII 200 μg was in favor of the highest dose.

[0295] To evaluate the capacity of Viaskin-SVLP-FsII to boost local antibody response in lungs, bronchoalveolar lavages (BAL) were collected at day 35. Anti-FsII antibody response was measured by ELISA (FIG. 2B). Epicutaneous boost immunization with Viaskin-SVLP-FsII induced a significant increase of FsII specific IgG titers in BAL compared to the mice that received Viaskin-excipient patch (mean antibody titer of 3.1±0.2 log 10 for Viaskin-SVLP-FsII 100 μg, p<0.001 compared to the Viaskin-excipient group; mean antibody titer of 3.4±0.4 for Viaskin-SVLP-FsII 200 μg, p<0.0001 compared to the Viaskin-excipient group). The boost effect observed with Viaskin-SVLP-FsII 200 μg was in the same range than that observed after a subcutaneous boost immunization with SVLP-FsII (mean antibody titer of 3.6±0.2 log 10, p<0.0001 compared to the Viaskin-excipient group, p<0.0001 compared to Viaskin-SVLP-FsII 100 μg and non-significant compared to Viaskin-SVLP-FsII 200 μg). Again, the dose effect observed between Viaskin-SVLP-FsII 100 μg and Viaskin-SVLP-FsII 200 μg was in favor of the highest dose but not significant.

[0296] To conclude, these results demonstrate that epicutaneous boost immunization with Viaskin-SVLP-FsII is able to significantly increase FsII-specific antibody response in mice primed by subcutaneous route.

[0297] 2. Epicutaneous Boost Immunization with Viaskin-SVLP-FsII Increases the Level of RSV-Neutralizing Antibodies and the Level of Palivizumab-Competitive Antibodies (PCA) in Mice

[0298] To evaluate the capacity of antibodies induced by epicutaneous boost immunization with Viaskin-SVLP-FsII to neutralize RSV infectivity in vitro, sera samples were sent to hVIVO services LTD (London, UK) for microneutralization assays. RSV microneutralization assay were performed on Hep2 cells using RSV A Memphis strain.

[0299] In mice previously primed subcutaneously with SVLP-FsII, epicutaneous boost immunization with Viaskin-SVLP-FsII induced a significant increase of RSV-neutralizing antibody titers compared to the mice that received Viaskin-excipient patch (FIG. 3A) (mean neutralizing antibody titer of 3.1±0.7 log 10 for Viaskin-SVLP-FsII 100 μg, p<0.01 compared to the Viaskin-excipient group; mean antibody titer of 3.4±0.4 for Viaskin-SVLP-FsII 200 μg, p<0.001 compared to the Viaskin-excipient group). The boost effect observed with Viaskin-SVLP-FsII was similar to that observed after the subcutaneous boost immunization with SVLP-FsII (mean antibody titer of 3.5±0.3 log 10, p<0.0001 compared to the Viaskin-excipient group, non-significant compared to Viaskin-SVLP-FsII (100 or 200 μg). As it was assessed by ELISA, the dose effect observed between Viaskin-SVLP-FsII 100 μg and Viaskin-SVLP-FsII 200 μg was in the favour of the highest.

[0300] To go further and to evaluate the capacity of the antibodies induced by epicutaneous boost immunization with Viaskin-SVLP-FsII to compete with Palivizumab binding to FsII peptide, a Palivizumab-competitive ELISA was set up (FIG. 3B). In agreement with neutralization results, epicutaneous boost immunization Viaskin-SVLP-FsII induced an increase of Palivizumab-competitive antibodies (PCA) as compared to the mice that received Viaskin-excipient patch (mean PCA titer of 2.2±0.2 log 10 for Viaskin-SVLP-FsII 100 μg, non-significant compared to the Viaskin-excipient group; mean PCA titer of 2.3±0.1 log 10 for Viaskin-SVLP-FsII 200 μg, p<0.001 compared to the Viaskin-excipient group).

[0301] To conclude, these results demonstrated that epicutaneous boost immunization with Viaskin-SVLP-FsII is able to significantly raise the level of high-quality and functional antibodies in mice.

[0302] 3. Epicutaneous Boost Immunization with Viaskin-SVLP-FsII Increases the Level of F-Specific Neutralizing Antibodies and Protects Animal Against RSV Infection

[0303] To confirm previous results and to evaluate the capacity of epicutaneous boost immunization with Viaskin-SVLP-FsII to increase protection against RSV infection, a new experiment was performed at Sigmovir Biosystems, Inc, following the study design presented in FIG. 4. A unique dose of 150 μg was chosen for Viaskin-SVLP-FsII boost immunization since it constitutes a pertinent compromise between 100 and 200 μg doses used in the first study. Moreover, it permits a more rigorous comparison with subcutaneous immunization that was performed at the same dose. As a control for immunopathology, mice were immunized with formalin-inactivated RSV (group 5) that corresponds to the formulation used for the very first clinical trial in the 60.sup.ies. As a positive control for protection, mice were infected at day 0 with RSV (group 6). All mice were challenged intranasally with RSV A2 at day 42.

[0304] A blood sample was collected at day 42, before RSV challenge, and F-specific IgG1 and IgG2a antibody titers were measured by ELISA (FIG. 5A). A significant increase of F-specific IgG1 was measured for mice boosted epicutaneously with Viaskin-SVLP-FsII compared to mice boosted with Viaskin-excipient patch (mean IgG1 titer of 6.7±0.3 log 10 for Viaskin-SVLP-FsII versus 6.0±0.2 log 10 for Viaskin-excipient, p<0.01; mean IgG2a titer of 4.7±0.5 log 10 for Viaskin-SVLP-FsII versus 4.2±0.3 log 10 for Viaskin-excipient, non-significant). Of note, IgG1 and IgG2a titers obtained for mice boosted epicutaneously with Viaskin-SVLP-FsII were significantly lower than those obtained for mice boosted subcutaneously with SVLP-FsII. However, IgG1/IgG2a ratio was identical between the two groups (mean ratio of 1.4±0.1 for both group) suggesting that the orientation of the immune response was not affected by the route of immunization. As expected, formalin-inactivated vaccine and RSV infection induced poor anti-F antibody titers.

[0305] To validate the functionality and the quality of these antibodies, a neutralization assay and a PCA were performed from sera collected at day 42 (FIGS. 5B and 5C). In line with our previous set of data, a significant increase of RSV-neutralizing and PCA titers was measured from mice boosted epicutaneously with Viaskin-SVLP-FsII compared to mice boosted with Viaskin-excipient (mean neutralization titer of 7.4±0.6 log 10 for Viaskin-SVLP-FsII versus 5.2±1.5 log 10 for Viaskin-excipient, p<0.05; mean PCA titer of 2.1±0.3 log 10 for Viaskin-SVLP-FsII versus 1.7±0.3 log 10 for Viaskin-excipient, p<0.05). Of note, PCA titers obtained from mice boosted epicutaneously with Viaskin-SVLP-FsII were significantly lower than those obtained from mice boosted subcutaneously with SVLP-FsII. However, RSV neutralizing titres were found similar. As expected, and in agreement with the low F-specific antibody titers measured by ELISA, formalin-inactivated vaccine and RSV infection did not induce any RSV-neutralizing and PCA antibodies.

[0306] In order to evaluate the capacity of epicutaneous boost immunization with Viaskin-SVLP-FsII to give an advantage for protection against RSV infection, mice were challenged 3 weeks after the boost immunization. Five days later, mice were sacrificed, and lungs were collected. A part of each lung was homogenized, and RSV viral load was measured by plate titration (FIG. 5D). A significant decrease of viral load was observed from mice boosted epicutaneously with Viaskin-SVLP-FsII compared to mice boosted with Viaskin-excipient or non-vaccinated mice (mean pfu per gram of lung of 3.0±0.4 log 10 for Viaskin-SVLP-FsII versus 4.0±1.0 log 10 for Viaskin-excipient, p<0.05, versus 4.7±0.1 log 10 for non-vaccinated mice). This decrease was similar to that observed from mice boosted subcutaneously with SVLP-FsII (mean pfu per gram of lung of 2.8±0.2 log 10). As expected, a strong protection was observed from mice vaccinated with formalin-inactivated virus or infected at day 0, probably through the induction of cellular response.

[0307] To conclude, these data demonstrate that epicutaneous boost immunization with Viaskin-SVLP-FsII is able to induce neutralizing antibodies, leading to efficient protection against RSV replication in mouse lungs.

[0308] 4. Epicutaneous Boost Immunization with Viaskin-SVLP-FsII is Safe and does not Exacerbate Lung Inflammation Following RSV Challenge

[0309] The main issue related to the first RSV vaccine tested in human (formalin-inactivated virus) was the exacerbation of lung inflammation following RSV infection. This aberrant reaction was due to the poor quality of the immunity induced by the vaccine that was mainly of Th2 orientation.

[0310] In order to validate the absence of immunopathology in mice boosted epicutaneously with Viaskin-SVLP-FsII, histological sections were performed from lungs collected at day 5 post-infection (day 42). Then, histological slices were coloured by Haematoxylin-Eosin-Safran staining and analysed (FIGS. 6A and 6B). A significant reduction of lung pathology was observed from mice boosted epicutaneously with Viaskin-SVLP-FsII or subcutaneously with SVLP-FsII compared to mice that received formalin-inactivated RSV (p<0.0001 for both criteria) or that were boosted with Viaskin-excipient patch (p<0.01 for perivasculitis). Moreover, lung pathology was not significantly increased or even lower in mice boosted epicutaneously with Viaskin-SVLP-FsII or subcutaneously with SVLP-FsII compared to non-infected mice.

[0311] Then, in order to evaluate the orientation of the immune response recalled by RSV infection, mRNA was extracted from lung and analyzed by qPCR to measure the expression of Th1- (IFN-γ; IL-2) or Th2- (IL-5; IL-13) related cytokines (FIG. 7). A strong reduction of the expression of Th2-related cytokines was observed for mice that have been boosted epicutaneously with Viaskin-SVLP-FsII or subcutaneously with SVLP-FsII compared to mice that have been vaccinated with formalin-inactivated RSV (IL-5 and IL-13: p<0.001 and p<0.0001 for epicutaneous and subcutaneous boosts, respectively). Conversely, a strong increase of the expression of Th1-related cytokines was observed for mice that have been boosted epicutaneously with Viaskin-SVLP-FsII or subcutaneously with SVLP-FsII compared to mice that have been vaccinated with formalin-inactivated RSV (IFN-γ: p<0.001 for epicutaneous and subcutaneous boosts; IL-2: p<0.001 and p<0.01 for epicutaneous and subcutaneous boosts, respectively). Of note, this increase was more pronounced in mice that have been boosted epicutaneously with Viaskin-SVLP-FsII, compared to mice that have been boosted subcutaneously with SVLP-FsII, especially for IL-2 (IFN-γ: p<0.01: IL-2: p<0.001). This suggest that epicutaneous route is more efficient than subcutaneous route to promote Th1 local effectors that can be restimulated following infection.

[0312] To conclude, these data demonstrate that epicutaneous boost with Viaskin-SVLP-FsII gives protection against RSV replication in lung without inducing inflammation, by promoting the induction of Th1 local effectors.

CONCLUSIONS

[0313] Overall, these results indicate that Viaskin-SVLP-FsII is efficient as an epicutaneous boost vaccination strategy against RSV.

[0314] Indeed, the inventors have shown that Viaskin-SVLP-FsII V-306 patch was able to significantly boost FsII antibody titers in mice that have been previously primed subcutaneously with SVLP-FsII V-306. Importantly, these antibodies could efficiently neutralize RSV infectivity and compete with Palivizumab binding in vitro. Of note, this boost effect was almost as efficient or even more efficient as that observed after boosting with a subcutaneous injection of SVLP-FsII V-306.

[0315] Even more importantly, epicutaneous boost with Viaskin-SVLP-FsII gave a significant benefit for protection against RSV replication in lung without exacerbating local inflammation. Moreover, epicutaneous boost with Viaskin-SVLP-FsII was able to promote Th1 effectors in lung that were recalled following RSV infection. This Th1 orientation was assessed by the local increase of IFN-γ and IL-2 expression. Of note, IFN-γ and IL-2 expressions were higher in mice boosted by epicutaneous route than in mice boosted by subcutaneous route, suggesting that upper skin is a preferable route to enhance Th1 immunity.

[0316] The non-invasive epicutaneous patch would advantageously reduce the number of injections required, especially if repeated boosters are required over the years to maintain a stable level of protective immunity. One possible approach would be to propose a subcutaneous priming dose of V-306 to all women of childbearing age followed by repeated epicutaneous boosters of the same antigen, before and during pregnancy. This may lead to a pronounced increase in the level of RSV-neutralizing antibodies that likely would be transferred to the fetus through the placenta. As assessed by PCA, these antibodies would be analogous to palivizumab, for which the best correlation with protection has been established to date, and for which a low proportion of adverse events have been reported in the prior art.

[0317] Epicutaneous patches can also be used for boosting RSV-specific immunity acquired through natural infection. Whilst most adults have experienced several RSV infections during their life, specific immunity is short lived, leading to a high heterogenicity between individuals in terms of protective immunity. In this regard, a non-invasive epicutaneous booster vaccine would be a way to enhance specific humoral immunity by recalling memory B-cells naturally induced by RSV.

Example 2: An Epitope-Specific Chemically Defined Nanoparticle Vaccine for Respiratory Syncytial Virus

[0318] The inventors developed the RSV vaccine V-306 which relates to an SVLP comprising a bundle of conjugates of formula (38). V-306 elicits strong long-lasting RSV-neutralizing antibody responses in mice and rabbits that protect mice from RSV infection and disease enhancement in a validated preclinical RSV challenge model.

[0319] Results

[0320] 1. Design of Epitope Mimetic

[0321] Design of a conformationally constrained peptide mimicking the epitope recognized by Motavizumab, led to the FsII site mimetics of SEQ ID NO: 47 and SEQ ID NO: 129 (FsIIm), with stabilizing sequence modifications and cysteines for cyclization via disulfide bridges at specific antigenically non-critical positions, shown in FIG. 8A. The solution structure of this peptide was determined by homonuclear 1H NMR spectroscopy. SEQ ID NO: 47 and SEQ ID NO: 129 (FsIIm) adopt a stable helix-rich folded conformation in water (FIG. 8B). The solution structure superimposed very closely on that of the Motavizumab-bound peptide (PDB 3IXT), showing that it is an excellent structural mimetic of the epitope. Further optimization of the physical and immunological properties led to the mimetic of SEQ ID NO: 45 and V-306p (FIG. 8A, SEQ ID NOs: 85). The latter has been conjugated to a lipopeptide building block resulting in a conjugate of formula (38). SVLPs comprising bundles of three conjugates of formula (38) were assembled (FIG. 8C) and used as RSV vaccine candidates.

[0322] 2. Construction and Structural Characterization of V-306

[0323] The mimetic V-306p contains an N-terminal aminooxyacetyl group for conjugation to an engineered synthetic lipopeptide (FIG. 8A and FIG. 9) that contains a promiscuous CD4+T helper epitope, a coiled-coil motif (heptad repeat IEKKIES) that forms a very stable helical trimer, and at the N-terminus the TLR-2/6 ligand Pam2Cys. V-306p was linked to this lipopeptide via a maleimide-PEG-aldehyde linker to give the vaccine construct V-306 (FIGS. 8A and 9). The conjugate V-306 in phosphate buffered saline (PBS) was analyzed by Dynamic Light Scattering (DLS) and transmission electron microscopy (FIG. 10). DLS revealed nanoparticle formation in phosphate buffered saline (PBS) with a mean hydrodynamic radius (Rh) of ca. 13 nm and a polydispersity index of 0.05, consistent with formation of highly monodisperse nanoparticles of about 26 nm diameter. Based on computer modelling, about 60-90 copies of each V-306 lipopeptide chain should comprise each nanoparticle, with the lipid chains buried in the core of the micelle-like particle and the epitope mimetic exposed in its surface (depicted in FIG. 8C). Transmission electron microscopy also revealed the formation of nanoparticles in a similar size range 25-30 nm (see FIG. 10) which bound to Palivizumab in ELISA, indicating that the conformational epitope remained intact on the nanoparticle surface.

[0324] The V-306p mimetic was then conjugated to a synthetic lipopeptide that contains a coiled-coil domain and a universal T-helper epitope. The resulting conjugate V-306 spontaneously self-assembles into chemically defined micelle-like nanoparticles in PBS with the epitope mimetic displayed in a multivalent format over the surface of the nanoparticle.

[0325] Methods

[0326] Synthesis of V-306p, FsIIm and further peptides disclosed herein: Peptides were synthesized by solid-phase peptide synthesis using Fmoc-chemistry and Rink amide resin, using procedures known in the prior art. For the synthesis of FsIIm, the completed peptide chain was acetylated at the N-terminus prior to cleavage from the resin and removal of side chain protecting groups, by treatment with trifluoroacetic acid (TFA), thioanisole, H.sub.2O, ethanedithiol (87.5:5:5:2,5) for 2.5 h. The peptide was precipitated and washed with iPr.sub.2O. For oxidation, the reduced peptide was dissolved in 0.33 M ammonium bicarbonate buffer, pH 7.8 and stirred in air overnight. The peptide was purified by reverse phase (RP)-HPLC on a preparative C.sub.18 column and lyophilized to afford a white powder. Analytical RP-HPLC (Vydac 218TP54, 5 μm, 4.6 mm×250 mm column, 0-60% MeCN in H.sub.2O (+0.1% TFA) over 40 min): Purity: 90.4%; t.sub.R=25.07 min. ESI-MS: Mass calculated for C.sub.135H.sub.227N.sub.43O.sub.49S.sub.4: 3349.52; m/z [M+3H].sup.3+ 1117.51.

[0327] For the synthesis of V-306 (FIG. 9), Bis-Boc-aminooxyacetic acid N-hydroxysuccinimide ester (Boc.sub.2-Aoa-OSu) was coupled to the N-terminus of the peptide chain. Removal from the resin, deprotection and oxidation to give V-306p were as described above. The disulfide cross-linked peptide was then purified by reverse phase (RP)-HPLC on a preparative C18 column and lyophilized to afford a white powder. Analytical RP-HPLC (Waters BEH C.sub.8, 1.7 μm, 2.1×150 mm column, 10-50% MeCN in H.sub.2O (+0.05% TFA) over 45 min, 0.2 mL/min, 30° C.): Purity: 92.8%; t.sub.R=29.95 min. ESI-MS: Mass calculated for C.sub.135H.sub.230N.sub.44O.sub.49S.sub.4: 3379.57 Da; m/z [M+H].sup.+: 3380.60 Da (±0.3%).

[0328] Linker was prepared by first reacting N-hydroxysuccinimidyl-([N-maleimidopropionamido]-hexa-ethyleneglycol ester (SM-PEG.sub.6, Thermo Fisher Scientific) with aminoacetaldehyde dimethyl acetal (Aldrich) in H.sub.2O. SM-PEG.sub.6 (7.6 mg, 12.6 μmol) was suspended in H.sub.2O (0.3 mL) and a solution of aminoacetaldehyde dimethyl acetal in H.sub.2O (17 μl of a 1:10 (v/v)) was added. The mixture was stirred for 90 min. at room temp. The product was purified by RP-HPLC on a C8 column and lyophilized. Analytical RP-HPLC Waters BEH C.sub.8, 150×2.1 mm, 1.7 μm, 0 to 20% MeCN in H.sub.2O (+0.05% formic acid) over 20 min, 0.4 mL/min, 40° C.: Purity 94.6%, t.sub.R=14.29 min. ESI-MS: monoisotopic mass C.sub.26H.sub.45N.sub.3O.sub.12: 591.30 Da; [M+H].sup.+ found: 591.62 Da (±0.1%). Just before conjugation, hydrolysis of the dimethyl acetal was performed with 95% TFA, 5% H.sub.2O for 5 min. The TFA was removed in vacuo to give the linker. ESI-MS C.sub.24H.sub.39N.sub.3O.sub.11: 545.26 Da; [M+H].sup.+ found: 545.28 Da (±0.05%).

[0329] The lipopeptide was synthesized and purified by RP-HPLC as described elsewhere (Boato, F. et al. Angew. Chem. Int. Ed. 46, 9015-9018 (2007), Ghasparian, A. et al. Chembiochem 12, 100-109 (2011), Perriman, A. W. et al. Small 6, 1191-1196 (2010), Sharma, R. et al. J. Immunol. 199, 734-749 (2017)). Analytical RP-HPLC Waters BEH C.sub.8, 150×2.1 mm, 1.7 μm, 64 to 91% MeOH in H.sub.2O (+0.05% TFA) over 37.5 min, 0.4 mL/min, 70° C.: Purity 97.0%, t.sub.R=21.80 min. ESI-MS: monoisotopic mass C.sub.312H.sub.552N.sub.74O.sub.89S.sub.3: 6856.0 Da; m/z [M+H].sup.+ found 6860.0 Da (±0.05%).

[0330] To prepare V-306, a solution of peptide V-306p (12 mg, 3.6 μmol) in 0.25 ml 0.1 M sodium acetate buffer, pH 3.5 was added to linker (3.8 mg, 7.2 μmol) in 0.25 ml 0.1 M sodium acetate buffer, pH 3.5. The mixture was stirred for 2.5 h and the product oxime (called VMX-3067) was purified by RP-HPLC on a preparative C.sub.8 column. Analytical UPLC (Waters BEH C8, 1.7 μm, 2.1×150 mm, 10 to 40% MeCN in H.sub.2O (+0.05% formic acid) over 37.5 min, 0.4 mL/min, 26° C.: Purity 95%, t.sub.R=21.5 min. ESI-MS: mass calculated for C158H263N47O59S4: 3893.32 Da; m/z [M+H].sup.+ found 3893.48 (0.3%). The oxime (4.0 mg, 1.0 μmol) was dissolved in 0.5 ml H.sub.2O and added to a solution of lipopeptide (6.2 mg, 0.9 μmol) in 2 ml 50% MeCN. The pH was adjusted to pH=6.5 with 0.1 N NaOH/0.1 N HCl and the mixture was stirred at r.t. for 2.5 h. The conjugate V-306 was purified by RP-HPLC on a C8 column. The TFA salt was converted first to an acetate salt and then to a hydrochloride salt using AG-X2 anion exchange resin. The conjugate was analyzed by analytical UPLC and MS (FIG. 9). UPLC (Waters BEH C8, 1.7 μm, 2.1×150 mm, 20 to 80% MeCN in H.sub.2O (+0.03% TFA) over 60 min, 26° C.: Purity 90%, t.sub.R=51.5 min. ESI-MS: Monoisotopic mass calc. for C470H815N121O148S7: 10746.9 Da; m/z [M+13H]13+ found 827.6875 Da (±0.1%). Conjugate V-306 was suspended in PBS, equilibrated for 30 minutes, diluted to 1.0 mg/ml and analyzed by Dynamic Light Scattering (DLS) on a DynaPro Nanostar instrument (Wyatt Technology) at 25° C. The size distribution by regularization analysis was monomodal. The mean hydrodynamic radius (Rh) was ca. 13 nm, and the polydispersity (Pd) index was 0.038.

Example 3: Preparation of Epicutaneous Patches

[0331] V-306p-conjugated lipopeptide lyophilizate was dissolved to a concentration of 2 mg/mL in sterile PBS 1× for reconstitution and incubated 30 min at room temperature (RT). During this time, solution was gently mixed by vortex for 1 min every 10 min to ensure the formation of SVLPs (V-306). Then, 50, 75 or 100 μL (100, 150 or 200 μg, respectively) of V-306 solution was deposited on Viaskin® patches (DBV Technologies). Patches were dried in a ventilated oven. One day before patch application, mice were anaesthetized with ketamine and xylazine (50 and 10 mg/kg, respectively) and hair was removed from the back using electric clippers and depilatory cream. Patches were applied on the depilated back (intact skin) and secured using a bandage for 48 h.

Example 4: V-306 SVLP Stability on Epicutaneous Patch

[0332] To initially validate the compatibility of V-306 with the epicutaneous patch, and to evaluate the stability of the combined product, 100 or 200 μg of V-306 SVLP were loaded on patches and further stored for 1 week at 4° C., 1 month at 4° C. and 1 month at RT. Then, V-306 SVLP was recovered from the patches using water and analyzed by DLS and UPLC/MS. The totality of V-306 could be recovered from patches stored 1 week at 4° C. Furthermore, this recovery leads to the formation of nanoparticles with 20-25 nm size, identical to reference V-306 SVLP. The recovery rate was slightly lower for patches stored 1 month at 4° C., and even lower for patches stored 1 month at RT, suggesting a partial degradation of V-306 over time (Table 1). However, at least half of the loaded material could be recovered, leading to the formation of well-shaped SVLPs. To investigate the capacity of trans-epidermal water loss to dissolve V-306 from patches in vivo, patches containing 100 μg of V-306 SVLP were applied to mice for 48 h and analyzed as described above by DLS and UPLC/MS (n=2). No remaining material could be retrieved from these patches, suggesting that the whole deposit was dissolved by skin humidity and the totality of antigen reached the upper layer of the skin

[0333] Characterization of V-306 nanoparticles following recovery from epicutaneous patches:

TABLE-US-00006 Hydro- dynamic Duration radius of Storage of Recovery.sup.a particles Material conditions storage [%] [nm] Identity Patch [100 μg] 4° C. 1 Week 122 10.73 Complies Patch [200 μg] 4° C. 1 Week 101 11.37 Complies Patch [100 μg] 4° C. 1 Month  71 10.43 Complies Patch [200 μg] 4° C. 1 Month  85 11.12 Complies Patch [100 μg] RT 1 Month  44 11.13 Complies Patch [200 μg] RT 1 Month  52 11.84 Complies Reference NA NA NA 10.05 NA V-306

[0334] a Recovery was defined as the percentage of protein quantity recovered from patch compared to the actual quantity loaded on patch. b Identity was assessed using UPLC/MS by comparing the molecular weight of the protein recovered from patch to the molecular weight of reference V-306 material.