Post-Exposure Vaccination Against Viral Respiratory Infections

Abstract

Pharmaceutical compositions, in particular vaccine compositions, for preventing or at least reducing the severity of, respectively, viral respiratory infections through application of said composition to a human subject post-exposure or at least presumed post-exposure of said subject to a virus causing said viral respiratory infections or pre-exposure of said subject to said virus. More particularly, in specific embodiments, the invention provides pharmaceutical compositions as such comprising at least one antigenic component of the infectious virus and a TLR-3 agonist. The invention also relates to methods of treatment and/or prevention of said viral respiratory infections through administration of the composition to the human subject post exposure or at least presumed post-exposure of said subject to the infectious virus or pre-exposure of said subject to said virus

Claims

1. A method for the prevention and/or treatment of a viral respiratory infection in a human subject comprising the step of administering, preferably by intra-nasal administration, an effective amount of a pharmaceutical composition pre-exposure or post-exposure of said subject to a virus causing said viral respiratory infection, or in a subject at least suspected to have been exposed to a virus causing said viral respiratory infection, respectively, wherein said composition comprises (i) at least one antigenic component of said virus and/or a nucleic acid encoding at least one antigenic peptide component of said virus and (ii) one or more adjuvants eliciting an innate immune response in the subject against said virus.

2. (canceled)

3. The method of claim 1 wherein the composition is self-administered by said subject.

4. The method of claim 1 wherein the composition is administered 30 seconds to 72 hours, preferably 3 min to 24 hours, more preferably 5 min to 12 hours post-exposure or post-suspected exposure to said subject, and optionally, the composition is further administered to the subject at least once daily for one week, or further administered once every 48 hours for one week, most preferably once every 72 hours for one week.

5. (canceled)

6. The method of claim 4 wherein the composition is administered at least once to the subject after completion of the optional administration as defined in claim 4, preferably at least once one week after completion of the optional administration as defined in claim 4.

7. (canceled)

8. The method of claim 1 wherein the incubation time of said viral respiratory infection is 8 days+/−2 days (95% confidence interval), preferably 5+/−1 days (95% confidence interval), most preferably 3+/−2 days (95% confidence interval).

9. (canceled)

10. The method of claim 1 wherein the at least one antigenic component of the virus is selected from the group consisting of inactivated virus, virus subunits, viral proteins, preferably viral structural proteins and/or viral non-structural proteins and/or viral enzymes, peptides comprising an epitope of a protein of said virus, and/or the nucleic acid encoding at least one antigenic peptide component of said virus is selected from the group consisting of viral proteins, preferably viral structural proteins and/or viral non-structural proteins and/or viral enzymes, and peptides comprising an epitope of a protein of said virus.

11. (canceled)

12. The method of claim 1 wherein the composition contains at least one protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family and/or at least one nucleic acid encoding a protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family.

13. The method of claim 12 wherein the SARS-like coronavirus is selected from the group consisting of SARS-coronavirus, MERS and SARS-coronavirus-2.

14. (canceled)

15. The method of claim 12 wherein the composition comprises a peptide comprising an epitope of the spike protein, preferably of the receptor binding domain (RBD), of said SARS-like coronavirus and/or a nucleic acid encoding an epitope of the spike protein preferably of the receptor binding domain (RBD), of said SARS-like coronavirus.

16. (canceled)

17. (canceled)

18. The method of claim 7 wherein the influenza virus is selected from the group consisting of H5N1, H1N1, H2N2, H3N2 and H7N1 viruses, preferably the genotype 4 (G4) Eurasian avian-like (EA) H1N1 influenza virus.

19. (canceled)

20. The method of claim 18 wherein the protein or at least a peptide comprising an epitope of said protein is selected from hemagglutinin subunits HA1 and HA2, preferably the protein or at least a peptide of said protein has an amino acid consensus sequence based on the G4 EA virus.

21. (canceled)

22. The method of claim 12 wherein the virus of the Paramyxoviridae is selected from the group consisting of Respiratory syncytial virus, Parainfluenza virus 1 to 3, Hendra virus and Nipah Virus; or wherein the virus of the Pneumoviridae family is Metapneumovirus; or wherein the virus of the Poxviridae family is selected from the group consisting of Variola major (smallpox) and monkeypox.

23. (canceled)

24. (canceled)

25. The method of claim 1 wherein at least one antigenic component is linked to a mucosa-targeting moiety, preferably C-CPE; and/or wherein the nucleic acid encodes at least one antigenic peptide component of said virus linked to a mucosa-targeting moiety, preferably C-CPE.

26. (canceled)

27. (canceled)

28. The method of claim 1 wherein the nucleic acid is an mRNA, optionally containing one or more nucleotide analogues, preferably one or more pseud-uridine nucleotides.

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. The method of claim 28 wherein the composition comprises the mRNA formulated as a lipid nanoparticle.

34. The method of claim 1 wherein one of the adjuvants or the adjuvant is a TLR-3 and/or RIG-I agonist; or wherein one of the adjuvants or the adjuvant is selected from the group consisting of TLR4, TLR7/8, TLR9 and STING agonists.

35. (canceled)

36. The method of claim 34 wherein the adjuvant is a dsRNA of at least 45 bp preferably a perfectly annealed dsRNA of at least 45 bp having two blunt ends or a perfectly annealed dsRNA having one blunt end and having a single-stranded overhang of 1 to 5 nt, preferably 1 to 3 nt, at the other end of the dsRNA.

37. (canceled)

38. The method of claim 36 wherein the dsRNA comprises a free 5′-triphophate group at least on one double-stranded end of said dsRNA.

39. The method of claim 38 wherein the dsRNA has a length of 45 to 200 bp, preferably 45 to 100 bp.

40. The method of claim 1 wherein the composition comprises at least one carrier selected from the group consisting of polyethyleneimine, PGLA, cationic liposomes, sucrose, trehalose, arginine, mannitol, histidine, sodium succinate, and mixtures of two or more thereof.

41. A pharmaceutical composition comprising (a) at least one antigenic component of a virus causing a respiratory infection, said at least one antigenic component being coupled to a mucosa-targeting moiety and/or a nucleic acid encoding at least one antigenic peptide component of a virus causing a respiratory infection linked to a mucosa-targeting moiety, and (b) one or more TLR-3 and/or RIG-I agonists.

42. The composition of claim 41 wherein the composition contains at least one protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family and/or at least one nucleic acid encoding a protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family; or wherein the composition contains a nucleic acid encoding a protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family and/or at least one nucleic acid encoding a protein or at least a peptide comprising an epitope of a protein of a virus selected from the group consisting of SARS-like coronaviruses, influenza viruses, viruses of the Paramyxoviridae family, viruses of the Pneumoviridae family and viruses of the Poxviridae family.

43. The composition of claim 42 wherein the SARS-like coronavirus is selected from the group consisting of SARS-coronavirus, MERS and SARS-coronavirus-2.

44. The composition of claim 41 wherein the at least one antigenic component is linked to a mucosa-targeting moiety, preferably C-CPE; or wherein the nucleic acid encodes at least one antigenic peptide component of said virus linked to a mucosa-targeting moiety, preferably C-CPE.

45. (canceled)

46. (canceled)

47. The composition of claim 41 wherein the nucleic acid is an mRNA, optionally containing one or more nucleotide analogues, preferably one or more pseud-uridine nucleotides.

48. (canceled)

49. The composition of claim 47 wherein the composition comprises the mRNA formulated as a lipid nanoparticle.

50. The composition of claim 41 wherein the TLR-3 agonist is a dsRNA of at least 45 bp, preferably a perfectly annealed dsRNA of at least 45 bp having two blunt ends or a perfectly annealed dsRNA having one blunt end and having a single-stranded overhang of 1 to 5 nt, preferably 1 to 3 nt, at the other end of the dsRNA, optionally comprising a free 5′-triphophate group at least on one double-stranded end of said dsRNA.

51. The composition of claim 21 comprising a carrier selected from the group consisting of polyethyleneimine, PGLA, cationic liposomes, sucrose, trehalose, arginine, mannitol, histidine, sodium succinate, and mixtures of two or more thereof.

52. The composition of claim 21 being in liquid or lyophilized form.

53. A pharmaceutical kit comprising an intra-nasal delivery device and a unit dose of the pharmaceutical composition of claim 21.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 shows a schematic diagram illustrating a vaccination scheme according to the prior art leaving a protection gap in humans against respiratory viral infections such as infections by SARS-CoV.

[0084] FIG. 2 shows an illustration of the vaccination scheme according to Example 2.

[0085] FIG. 3 shows an illustration of the experimental schedule of Example 3.

[0086] FIG. 4 is a graphic representation of results of Example 3 showing the Matrix effect of naïve mouse sera (n=5) in a Luminex assay.

[0087] FIGS. 5a to 5f are graphic representations of results of endpoint titrations of SARS-CoV-2 spike binding IgG antibodies.

[0088] FIGS. 6a to 6f are graphic representations of results of endpoint titrations of SARS-CoV-2 spike binding IgA antibodies.

[0089] FIGS. 7a to 7d are graphic representations of the results of wildtype SARS-CoV-2 pseudovirus (Wuhan-Hu-1 strain) neutralization with XPOVAX-SARS-CoV-2 immunized mouse sera.

[0090] FIG. 8 is a graphic representation of wildtype SARS-CoV2 pseudovirus neutralization with XPOVAX-SARS-CoV-2 immunized mouse bronchioalveolar lavage (BAL) samples of mice from group #7 of Example 4.

[0091] FIGS. 9a to 9c are graphic representations of SARS-CoV-2 Variants of Concern (UK and SA VOC) pseudovirus neutralization titers (% neutralization) with XPOVAX-SARS-CoV-2 immunized mouse sera or BAL from group #7 of Example 4.

[0092] FIG. 9d is a graphic representation of SARS-CoV-2 Variants of Concern (UK VoC, SA VoC) pseudovirus neutralization titers (IC50 values titres) with XPOVAX-SARS-CoV-2 immunized mouse sera from group #7 of Example 4.

[0093] FIG. 10 is a graphic representation of T-cell responses (IFNγ ELISPOT with splenocytes, bulk all T-cells & CD4, CD8 T-cells separated) in mice immunized with XPOVAX SARS-CoV-2 vaccine.

[0094] FIG. 11 shows an illustration of the experimental schedule of Example 4.

[0095] FIGS. 12a to 12e show graphic representations of the reactivity of XPOVAX Influenza/SARS combination vaccine immunized mouse sera (IgG) with recombinant SARS-CoV-2 spike protein (wildtype and VoC variants) in the Luminex assay. Individual sera of group #5 mice (n=5). S-Trimer alpha=UK VoC, S-Trimer beta=SA VoC.

[0096] FIGS. 13a to 13e show graphic representations of the reactivity of XPOVAX Influenza/SARS combination vaccine immunized mouse sera (IgG) with SARS-CoV-2 recombinant receptor binding domain (RBD, wildtype, and RBD variants) in the Luminex assay. Individual sera of group #5 mice (n=5).

[0097] FIGS. 14a to 14e show graphic representations of the reactivity of XPOVAX Influenza/SARS combination vaccine immunized mouse sera (IgG) with recombinant influenza virus H1-HA1 in the Luminex assay. Individual sera of group #5 mice (n=5).

[0098] FIGS. 15a to 15e show graphic representations of the reactivity of XPOVAX Influenza/SARS combination vaccine immunized mouse nasal lavage (IgG) with recombinant SARS-RBD and H1-HA1 in the Luminex assay. Individual sera of group #5 mice (n=5).

[0099] FIGS. 16a to 16e show graphic representations of the reactivity of XPOVAX SARS/CoV-2 combination vaccine immunized mouse bronchio-alveolar lavage (IgG) with recombinant SARS-RBD and H1-HA1 in the Luminex assay. Individual sera of group #5 mice (n=5).

[0100] FIGS. 17a to 17e show graphic representations of the reactivity of XPOVAX Influenza/SARS combination vaccine immunized mouse bronchio-alveolar lavage (IgA) with recombinant SARS-RBD and H1-HA1 in the Luminex assay. Individual sera of group #5 mice (n=5).

[0101] FIGS. 18a to 18c show graphic representations of the results of pseudovirus (PV) neutralization assays (pseudotyped with spike protein of SARS-SoV-2 strain Wuhan-Hu-1) with sera and BAL from XPOVXAX Influenza/SARS immunized mice.

[0102] FIGS. 19a and 19b show graphic representations of T-cell responses (IFNγ ELISPOT, bulk all T-cells, and CD4, CD8 T-cells separated) in mice immunized with XPOVAX Influenza or XPOVAX Influenza-SARS combination vaccine, as single or prime-boost application.

[0103] FIG. 20 shows a table of results of pseudovirus neutralization titers (IC.sub.50) of XPOVAX-SARS-CoV-2 immunized mouse sera and BAL.

DETAILED DESCRIPTION OF THE INVENTION

[0104] The following non-limiting examples further illustrate the present invention.

EXAMPLES

Example 1: Components for Pharmaceutical Composition

[0105] Component 1: Antigenic Component

[0106] The following constructs were provided by trenzyme GmbH, Konstanz, Germany.

[0107] Component 1.1: RBD of SARS-CoV-2

[0108] A his-tagged RBD of SARS-CoV-2 was prepared by recombinant expression in HEK293 cells. The construct was purified by one-step Ni-column chromatography. High-affinity binding was confirmed by SPR analysis (binding to human ACE2) (Kd of about 1 nM).

[0109] Component 1.2: Fusion protein construct of SARS-CoV-2 with C-CPE A fusion construct of his-tagged RBD of SARS-CoV with C-CPE was prepared by recombinant expression in HEK293 cells and purified as described for component 1.1.

[0110] Component 2: Adjuvant

[0111] A double-stranded TLR-3 agonist according to WO 2015/091578 A1 (dsRNA 100 bp, wherein one strand is polyC and the complementary strand is poly(G:I) and comprising a 5′-triphosphate at the polyC strand) was provided by RiboxX GmbH, Radebeul, Germany), hereinafter also denoted as “RIBOXXIM”.

[0112] Components 1 and 2 were combined and solubilized in water for injection (WFI) followed by lyophilization. The freeze-dried composition was reconstituted by addition of WFI.

[0113] The following compositions were prepared:

TABLE-US-00004 TABLE 1 Amount of Amount of Volume of liquid component component for reconstitution Formulation 1.1 [μg] 2 [μg] [μl] 1 100 1000 100 2 100  500 100 3 100  250 100 4 100  125 100 5 100   61 100

Example 2 Testing the Immune Response Elicited by Vaccination of Mice with RIBOXXIM and RBD-Fragment of SARS-CoV2 Spike Protein (Designated RBD) as Well as Modified RBD-Fragment (Designated RBD-PF), Both Provided by Trenzyme GmbH, Konstanz, Germany

[0114] Immunization of C57/BL6 mice with a combination of a TLR3 agonist (RIBOXXIM) and a viral structural protein of SARS-CoV-2 generates a local (IFN Type I, IgA) and systemic immune response (IgM, IgG)

[0115] Material and Methods

[0116] Reagents

[0117] Vaccine compositions containing component 2 of Example 1 (RIBOXXIM) and/or RBD-protein (component 1.1 of Example 1) or RBD-PF-Protein (component 1.2 of Example 1) and/or Chitosan are supplied in a volume of 250 μl as listed in the following Table 2:

TABLE-US-00005 TABLE 2 Group RIBOXXIM Chitosan RBD RBD-PF Total volume # (μg) (μg) (μg) (μg) (μl) 1 50 — 10 — 300 2 50 — 10 — 300 3 50 10 10 — 300 4 — — 10 — 300 5 50 — — — 300 6 50 10 — — 300 7 50 — — 10 300 8 — — — 10 300 9 (PBS) — — — — 300

[0118] Collection of Samples

[0119] For collection of nasal swabs, nasosorption (Mucosal Diagnostics, Midhurst, UK) is used according to the instructions of the manufacturer.

[0120] Collection of blood samples is performed in the tail vein. Bronchoalveolar lavage and collection of splenocytes are performed after sacrifice of the animals.

[0121] Animals

[0122] C57/BL6 mice (n=5 per Group) are used.

[0123] Application of the Vaccine and Schedule

[0124] Each animal from Groups 1, 3, 4, 5, 6, 7, 8 and 9 receives 25 μl of the vaccine or the Vehicle (PBS, Group #8) in each nostril, corresponding to a total application of 50 μl per animal.

[0125] Animals from Group 2 receive 25 μl of the vaccine sub-cutaneous at day 0 and 25 μl of the vaccine intra-nasal at day 21.

[0126] Application occurs at day 0 and day 21.

[0127] For Groups 1, 3, 4, 5, 6, 7 and 9: nasal swabs are collected at days 0, 1, 3 and 5 and frozen at −20° C.

[0128] For Groups 1 to 9: blood is drawn at days 0 and day 21 and frozen at −20° C.

[0129] For Groups 1 to 9: at day 28, blood, BAL, nasal wash is collected and frozen at −20° C.

[0130] For Groups 1, 2, 3, 4, 7, 8, 9: at day 28, splenocytes are collected and frozen at −20° C.

[0131] FIG. 2 shows an illustration of the vaccination scheme.

Example 3 Immune Responses of Mice Immunized with RIBOXXIM and RBD-Fragment of SARS-CoV2 Spike Protein (Designated RBD), as Well as Claudin-4 Targeted RBD-Fragment (Designated RBD-C-CPE)

[0132] Materials and Methods

[0133] XPOVAX SARS-CoV-2 Vaccine Composition

[0134] The vaccine contained SARS-CoV-2 RBD-protein or RBD-C-CPE-Protein, adjuvanted with RIBOXXIM or unadjuvanted, with or without Chitosan (see Table 3).

TABLE-US-00006 TABLE 3 Amount of antigen (SARS-COV-2 RBD or SARS-COV-2 RBD-C- CPE), adjuvant (Riboxxim), and Chitosan, in different formulations of XPOVAX-SARS-COV-2 vaccine, per one dose in 50 μl volume (divided over two nostrils) in different treatment groups (1-9) Total volume Group RIBOXXIM Chitosan RBD RBD-C- administered # (μg) (μg) (μg) CPE (μg) per dose (μl) 1 20 — 20 — 50 (2 × 25) 2 20 — 20 — 50 (2 × 25) 3 20 20 20 — 50 (2 × 25) 4 — — 20 — 50 (2 × 25) 5 20 — — — 50 (2 × 25) 6 20 20 — — 50 (2 × 25) 7 20 — — 20 50 (2 × 25) 8 — — — 20 50 (2 × 25) 9 — — — 50 (2 × 25)

[0135] Animals

[0136] Female Balb/c mice (n=5 per group) were immunized at 6-8 weeks of age

[0137] Route and Schedule of Immunizations

[0138] Animals from groups 1, 3, 4, 5, 6, 7, 8 and 9 received 25 μl of the vaccine or the vehicle control (PBS, Group #9) in each nostril at days 0 and 21, resulting in a total application of 50 μl per animal per immunization. Animals from group 2 received 50 μl of the vaccine subcutaneously at day 0, and 50 μl of the vaccine intranasally at day 21.

[0139] The first immunization was performed on day 0 (prime), the second immunization on day 21 (boost).

[0140] FIG. 4 shows a graphic representation of the experimental schedule.

[0141] Biological Sample Collection

[0142] Collection of blood samples was performed via the tail vein. Bronchoalveolar-lavage and collection of splenocytes was performed after sacrificing of the animals. Nasal lavages were performed at the end of the experiment. A small incision was made into the trachea using a surgical knife. A blunted 20-gauge needle was inserted through the incision and a lavage was performed by flushing 500 μl of sterilized PBS through the trachea. The nasal lavage was collected through the nostrils in a 1.5-ml micro tube. For Groups #1 to #9: Blood, BAL and nasal fluid were collected on day 0 and 28, and frozen at −20° C.

[0143] For Groups #1, 2, 3, 4, 7, 8, 9 splenocytes were collected on day 28, frozen and stored at −80°

[0144] Luminex Antibody Assays

[0145] Polyclonal antibody binding to immobilized analyte protein (recombinant SARS-CoV-2 spike, RBD, or recombinant Influenza H1-HA.sub.1) was done in a Luminex bead-based assay according to the manufacturer's instructions (Bio-Plex, Bio-Rad Laboratories, Hercules, Calif., USA). Recombinant analyte proteins were conjugated to beads via carbodiimide chemistry. Biotinylated analytes were bound to streptavidin coated plates. The following SARS-CoV-2 spike or RBD variants were used: Wuhan-Hu-1 strain, GenBank Acc #MN908947.3. Point mutations in Wuhan RBD: Y453F, N439K, N501Y, E484K. Recombinant proteins with different labels were obtained from trenzyme GmbH (Konstanz, Germany), Sino Biological (Beiing, China) and ACROBiosystems (Newark, Del., USA). Sequence of H1-HA.sub.1 Influenza A virus: A consensus sequence was constructed based on the sequence of A/swine/Guangxi/2499/2011(H1N1), see the following Tab. 4:

[0146] Table. 4: Consensus Protein Sequence H1N1-G4-EA HA1 [0147] 1) HA1 with C-terminal His-tag (secretion signal cleaved-off in final protein) (SEQ ID NO: 1) [0148] 2) HA1-C-CPE with N-terminal His-tag (secretion signal cleaved-off in final protein) (SEQ ID NO: 2)

[0149] Tissue Culture

[0150] HEK293T cells were cultured using DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

[0151] Pseudotype Virus Production

[0152] Production of SARS CoV 2 pseudotypes (PV) was carried out as described Di Genova et al. (2020). Briefly, HEK 293T cells were seeded for next day transfection at 50% confluence. On the day of transfection, media was replaced with complete DMEM. Plasmids used for transfection were 1000 ng pCAGGS-SARS CoV 2 Spike (Wuhan, B.1.1.7 or B.1.351), 1500 ng lentiviral vector expressing firefly luciferase pCSFLW, and 1000 ng second generation lentiviral packaging plasmid p8.91 expressing gag, pol and rev, were mixed in 200 μL optimem for 5 minutes, followed by addition of FuGENE HD at a ratio of 1:3 (DNA:FuGENE HD), and 15 min incubation at RT before adding the transfection mix to the cells. PVs were harvested after 48 hours by filtration using a 0.45 μm cellulose acetate filter.

[0153] PV representing two “variants of concern (VoC)” were generated with the spike sequences for SARS-CoV2 strains B.1.1.7 (South African variant, SA) and B.1.351 (British variant, UK), according to Di Genova et a. (2020).

[0154] Pseudotype Titration and Neutralization

[0155] The day prior to titration or neutralization, target HEK293T cells were transfected using plasmids expressing ACE2 (pcDNA3.1+) and TRSSMP2 (pCAGGS). For titration, 100 μL of harvested pseudotyped virus was added in the first row of a 96 white well plate, followed by 50 μL of DMEM to all other wells. A 2-fold serial dilution was carried out. Target cells were then added at a density of 10,000 cells per well, and plates were returned to the incubator. 48 hours later, media was aspirated and Bright-Glo (Promega) was added to the wells, incubated at room temperature for 5 minutes, and then luciferase expression levels were quantified using a GloMax Navigator microplate luminometer.

[0156] For neutralization, target cells were transfected as described above. Patient sera was serially diluted 2-fold in complete DMEM starting at a 1:10 dilution in a 96 white well plate and mixed with SARS CoV 2 pseudotypes, for 1 hour at 37° C., 5% CO2. Target cells were then added at a density of 10,000 cells per well, and plates were returned to the incubator for 48 hours prior to assaying luciferase expression levels. Data analysis to derive half-maximal inhibitory concentrations (IC50s) was carried out using GraphPad Prism 8 software. FIG. 4 shows neutralization of PV with sera and BAL from XPOVAX-immunized mice.

[0157] ELISpot Assay

[0158] IFNγ production of OVA-specific cells was analyzed using a commercially available mouse ELISPOT antibody pair according to the manufacturer's protocol (#551881, BD Biosciences). Briefly, splenocytes of immunized mice were seeded at 5×105 cells per well onto pre-coated MultiScreen®HTS Filter Plates (Merck Millipore, Burlington, Mass., USA) and re-stimulated with 10 μM of the respective peptide for 20 h. After incubation with the biotinylated secondary antibody specific for IFNγ, a streptavidin-alkaline phosphatase enzyme conjugate was added. After the addition of the BCIP®/NBT substrate solution (#1911, Merck KGaA, Darmstadt, Germany), a purple precipitate is formed as spots at the sites of captured IFNγ. Automated spot analysis and quantification were performed using the ImmunoScan® analyzer and Immunospot software v.6.0.0.2 (CTL Europe GmbH, Bonn Germany)

[0159] Results

[0160] Antibody Responses Induced by the XPOVAX-SARS-CoV-2 Vaccine

[0161] Binding antibody responses (IgG, IgA) to spike protein were measured in nasal lavage (NAL), bronchioalveolar lavage (BAL), and serum, at day 28 after immunization, using the SARS-CoV-2 spike protein (Wuhan-Hu-1) as analyte. FIG. 5 shows background reactivity of sera from control mice (group #9) immunized with PBS in the Luminex assay, and calculation of cut-off value. The cut-off value was used to determine antibody titers. No binding antibodies were detected in sera of mice immunized with vaccine containing only the RBD without adjuvant or chitosan (group 4), or animals receiving the RBD and chitosan (group 3) (data not shown).

[0162] FIGS. 5a to 5e show the detection of IgG antibodies in NAL, BAL and sera of mice receiving the RBD plus Riboxxim (group #1), or RBD-C-CPE with Riboxxim (group #7). Shown are 2-fold endpoint titrations, starting at an initial serum dilution of 1:20. The RBD-C-CPE fusion protein is more immunogenic than the RBD domain alone, as shown by induction of 5.7-11.9-fold higher IgG geometric mean antibody titers in the biological fluids tested (table 3). The GMTs of IgA antibodies did not differ between groups (see FIGS. 6a to 6e and Tab. 5).

TABLE-US-00007 TABLE 5 Geometric mean titers of IgG and IgA antibodies induced in NAL, BAL, and serum of mice immunized with XPOVAX- SARS-COV-2 vaccine (groups #1 and #7 shown) Group GMT Group GMT #1 NAL-IgG 320 #1 NAL-IgA 452 #1 BAL-IgG 14481 #1 BAL-IgA 2560 #1 Serum-IgG 579261 #1 Serum-IgA 2262 #7 NAL-IgG 3800 #7 NAL-IgA 970 #7 BAL-IgG 142631 #7 BAL-IgA 2560 #7 Serum-IgG 3276800 #7 Serum-IgA 4222

[0163] FIGS. 6a to 6f show the detection of IgA antibodies in nasal lavages, BAL and sera of mice receiving the RBD plus Riboxxim, or RBD-C-CPE with Riboxxim. Shown are 2-fold endpoint titrations, starting at an initial serum dilution of 1:20.

[0164] Neutralizing antibody titers were determined in sera collected on day 28 after immunization. No neutralizing antibodies were detectable in sera of mice from groups #3, 4, 5, 6, 8 and 9. Neutralizing antibodies against SARS-CoV-2 wt and VoC PV were detectable in all sera and one BAL of mice belonging to groups #1, 2, and 7, with highest titers observed in the mice which received the RBD-C-CPE antigen together with Riboxxim (see FIGS. 7 and 8). All sera with neutralizing antibodies neutralized the wildtype (wt) and UK-VoC PV with similar IC50 titers, whereas titers were approx. 10-fold lower for the SA-VoC (see FIGS. 9a, 9b, 9c and 9d, as well as FIG. 20).

[0165] T-Cell Responses

[0166] Animals in groups 1 and 2 showed RBD-specific CD4 and CD8 T-cell responses against recombinant wildtype SARS-CoV-2 RBD, which were significantly increased in group 7 that had been immunized with the RBD-C-CPE construct (FIG. 10).

Example 4 Immune Responses of Mice Immunized with RIBOXXIM and Influenza H1-HA1 (Consensus Sequence Based on Strain H1N1 G4 EA), the Claudin-4 Targeted H1-HA1 (Designated H1-HA1-C-CPE) and the Claudin-4 Targeted RBD-Fragment of the SARS-CoV-2 Spike (Strain Wuhan-Hu-1, Designated SARS-CoV-2 RBD-C-CPE)

[0167] XPOVAX Influenza/SARS Combination Vaccine Composition

[0168] The XPOVAX Influenza vaccine contained H1-HA1 or H1-HA1-C-CPE protein, adjuvanted with Riboxxim, the Influenza/SARS-CoV-2 combination vaccine contained recombinant H1-HA1-C-CPE and SARS-CoV-2 RBD-C-CPE-Protein, adjuvanted with RIBOXXIM (Table 6).

TABLE-US-00008 TABLE 6 Amount of antigen (Influenza H1-HA.sub.1, Influenza H1-HA.sub.1-C-CPE, SARS-CoV-2 RBD-C-CPE), adjuvant (Riboxxim), in different formulations of XPOVAX SARS/Influenza vaccine, per one dose in 50 μl volume (divided over two nostrils), in different treatment groups SARS- Influenza Group CoV-2 H1 Influenza Total volume # RIBOXXIM RBD-C- HA.sub.1-C- H1 HA.sub.1 administered (n = 5) (μg) CPE (μg) CPE (μg) (μg) Dosing per dose 1 20 20 — — Single 50 (2 × 25) 2 20 — 20 — Single 50 (2 × 25) 3 20 — 20 — Prime- 50 (2 × 25) boost 4 20 20 20 — Single 50 (2 × 25) 5 20 20 20 — Prime- 50 (2 × 25) boost 6 20 — — — Single 50 (2 × 25) 7 20 — — 20 Prime- 50 (2 × 25) boost

[0169] Animals

[0170] Female Balb/c mice (n=5 per group) were immunized at 6-8 weeks of age Schedule of Immunization with XPOVAX-Influenza/SARS Combination Vaccine

[0171] Animals from groups 1-7 received 25 μl of the vaccine in each nostril at days 0 and 21, resulting in a total application of 50 μl per animal per immunization. Animals from group 3, 5, and 7 received 50 μl of the vaccine intranasally on day 0, and 50 μl of the vaccine intranasally on day 21 (prime-boost). Animals from groups 1, 2, 4 and 6 received 50 μl of the vaccine intranasally on day 0 only (single immunization). A schematic representation of the schedule of immunization of present Example 4 is shown in FIG. 11.

[0172] Biological Sample Collection

[0173] Collection of blood samples was performed via the tail vein. Bronchoalveolar-lavage and collection of splenocytes was performed after sacrificing of the animals.

[0174] For Groups 1 to 9: Blood, BAL and nasal fluid were collected on day 28, and frozen at −20° C. For Groups 1, 2, 3, 4, 7, 8, 9 splenocytes were collected on day 28, frozen stored at −80° C.

[0175] Luminex Antibody Assays

[0176] Polyclonal antibody binding to immobilized analyte protein (SARS-CoV-2 RBD, or H1-HA1) was done in a Luminex bead-based assay according to the manufacturer's instructions (Bioplex, Biorad). Recombinant analyte proteins were conjugated to beads via carbodiimide chemistry. Biotinylated analytes were bound to streptavidin coated plates. The following SARS-CoV-2 spike or RBD variants were used: wildtype Wuhan-Hu-1 strain (GenBank: MN908947.3). RBD point mutations: Y453F, N439K, N501Y, E484K. For the influenza H1-HA1 construct a consensus sequence based on the Influenza A virus strain A/swine/Guangxi/3843/2011 was constructed (table 2). Recombinant proteins with different tags were purchased from trenzyme GmbH (Konstanz, Germany), Sino Biological (Beijing, China) and ACROBiosystems (Newark, Del., USA).

[0177] Results

[0178] Binding antibodies were measured in mouse sera collected on day 28 post immunization. Animals immunized with the XPOVAX Influenza/SARS combination vaccine in a prime-boost regimen (group 5) had high IgG titers detectable against the SARS-CoV-2 spike and RBD, and against Influenza H1-HA1, in serum (FIGS. 12 to 14), nasal lavage (FIG. 15) and bronchio-alveolar lavage (FIG. 16), as measured by Luminex. Lower IgA titers were also detectable in bronchio-aveolar lavage (FIG. 17).

[0179] Pseudovirus Neutralization Assay (SARS-CoV-2 Strain Wuhan-Hu-1)

[0180] Neutralizing antibodies were detected against SARS-CoV-2 in sera, NAL and BAL of mice from group #5 (see FIG. 18).

[0181] ELISpot Assay

[0182] IFNγ production of OVA-specific cells was analyzed using a commercially available mouse ELISPOT antibody pair according to the manufacturer's protocol (#551881, BD Biosciences). Briefly, splenocytes of immunized mice were seeded at 5×105 cells per well onto pre-coated MultiScreen®HTS Filter Plates (Merck Millipore) and re-stimulated with 10 μM of the respective peptide for 20 h. After incubation with the biotinylated secondary antibody specific for IFNγ, a streptavidin-alkaline phosphatase enzyme conjugate was added. After the addition of the BCIP®/NBT substrate solution (#1911, Merck), a purple precipitate is formed as spots at the sites of captured IFNγ. Automated spot analysis and quantification were performed using the ImmunoScan® analyzer and Immunospot software v.6.0.0.2 (CTL Europe, Germany)

[0183] Animals immunized with the XPOVAX Influenza/SARS-CoV-2 combination vaccine (group 5) had robust T-cell responses against both immunization antigens (see FIG. 19).

[0184] Animal immunized with a single dose of the H1-HA1 antigen also showed robust T-cell responses against the homologous antigen (cf. FIG. 21).

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