TREATMENT OF VIRAL INFECTION

Abstract

Disclosed are molecules useful in the treatment or prevention of viral infections in humans and animals and/or the treatment or prevention of the associated symptoms, diseases and/or conditions. The disclosure provides molecules which comprise sugar (carbohydrate/polysaccharide/sialic acid/glycan)-binding protein(s) which are useful in the treatment or prevention of Coronavirus infections and/or diseases, symptoms and/or conditions caused or contributed to by the same.

Claims

1. A method of treating or preventing a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus, said method comprising administering a glycan binding molecule to a subject in need thereof.

2. The method of claim 1, wherein the disease or condition caused or contributed by a Coronavirus is COVID-19 or SARS or MERS.

3. The method of claim 1, wherein the treatment of a Coronavirus infection and/or a disease or condition caused or contributed by a Coronavirus comprises the treatment of one or more of the symptoms associated with the Coronavirus infection, disease or condition.

4. The method of claim 3, wherein the symptom(s) is/are a continuous cough and/or a fever and/or a change/loss in/of taste/smell.

5. The method of claim 1, wherein the glycan binding molecule comprises a carbohydrate binding module (CBM).

6. The method of claim 5, wherein the CBM is selected from the group consisting of: (i) A Family 40 CBM; and (ii) A Family 32 CBM.

7. The method of claim 6, wherein the CBM is selected from the group consisting of: (i) a Clostridium perfringens CBM32 (CpCBM32); (ii) a Streptococcus pneumoniae CBM40 (SpCBM40); (iii) a Vibrio cholerae CBM40 (VcCBM40); (iv) a Vibrio cholerae NanH sialidase CBM; (v) a Vibrio cholerae NanH sialidase CBM sialic acid binding fragment thereof (vi) a Streptococcus pneumoniae nanA sialidase CBM; and (vii) a Streptococcus pneumoniae nanA sialidase CBM sialic acid binding fragment thereof.

8. The method of claim 1, wherein the Coronavirus is SARS-CoV-2.

9. The method of claim 8, wherein the Coronavirus is a SARS-CoV-2 variant.

10. The method of claim 9, wherein the SARS-CoV-2 variant comprises a mutation within the spike protein, the mutation being an amino acid change relative to the amino acid sequence of the spike protein of the Wuhan-Hu-1 isolate with accession codes: QHD43416.1/YP_009724390.1.

11. The method of claim 10, wherein the variant comprises one or more of the following spike protein mutations: (i) HV69-70 deletion; and/or (ii) N501Y; and/or (iii) E484K.

12. The method of claim 9 wherein the SARS-CoV-2 variant is the B.1.1.7 variant and/or the B.1525 variant and/or the B.1.351 variant.

13. (canceled)

14. The method of claim 1, wherein the glycan binding molecule is a CBM32, and the disease or condition caused or contributed by a Coronavirus is COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19.

15. The method of claim 14, wherein the CBM32 comprises the amino acid sequence of SEQ ID NO: 1: TABLE-US-00019 AIIETAIPQSEMTASATSEEGQDPASSAIDGNTNTMWHTKWNGSDALPQS LSVNLGSSRKVSSIAITPRTSGNNGFITKYEIHAINNGVETLVAEGTWEE NNLVKTVTFDSPIDAEEIKITAIQGVGGFASIAELNVYE or a glycan/carbohydrate binding fragment thereof.

16. A method of treating or preventing: a Coronavirus infection, a disease or condition caused or contributed by a Coronavirus; or (ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19; said method comprising administering a CBM40 to a subject in need thereof.

17. The method of claim 16, wherein the CBM40 comprises the amino acid sequence of SEQ ID NO: 4: TABLE-US-00020 ALFDYNATGDTEEDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGG RAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPI ISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFY FDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGD or SEQ ID NO: 6 VIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDAKAPAFYN LFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPG QWNSVTFTVEKPTAELPKGRVRLYVNGVLSRTSLRSGNFIKDMPDVTHV QIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRS or a glycan/sialic acid binding fragment of either.

18. (canceled)

19. The method of claim 5, wherein the CBM comprises a modified CBM comprising a wild type CBM sequence which has been modified to include one or more mutations.

20. The method of claim 19, wherein the modified CBM comprises the sequence of SEQ ID NO: 17: TABLE-US-00021 GAMVIEKEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPA FYNLFSVSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKV KPGQWNSVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDV THVQIGATKRANNTVWGSNLQIRNLTVYNRALTPEEVOKRSGGGSGVIE KEDVETNASNGQRVDLSSELDKLKKLENATVHMEFKPDPKAPAFYNLFS VSSATKKDEYFTMAVYNNTATLEGRGSDGKQFYNNYNDAPLKVKPGQWN SVTFTVEKPTAELPKGRARLYVNGGLSRTSLRSGNFIKDMPDVTHVQIG ATKRANNTVWGSNLQIRNLTVYNRALTPEEVQKRSGGSLGVPDFESDWF DVSSNSLYTLSHGLQRSPRRVVVEFARSSSPSTWNIVMPSYFNDGGHKG SGAQVEVGSLNIKLGTGAAVWGTGYFGGIDNSATTRFATGYYRVRAWI or a glycan acid binding fragment thereof.

21. A method of treating or preventing: a Coronavirus infection, a disease or condition caused or contributed by a Coronavirus and/or a symptom of a Coronavirus infection/disease; or (ii) COVID-19, a disease or condition caused or contributed by SARS-CoV-2 and/or a symptom of COVID-19; said method comprising administering a glycan acid binding molecule to a subject in need thereof, wherein the glycan binding molecule is selected from the group consisting of: Cp2CBM32TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM32s) from Clostridium perfringens fused to a trimerisation domain); (ii) Sp2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Streptococcus pneumoniae fused to a trimerisation domain; (iii) Vc2CBM40TD (comprising, consisting essentially of or consisting of: 2 CBMs (CBM40s) from Vibrio cholerae fused to a trimerisation domain; and (iv) Vc4CBM (comprising, consisting essentially of or consisting of: 4 CBMs (CBM40s) from Vibrio cholerae).

Description

[0277] The present disclosure will now be described with reference to the following figures which show:

[0278] FIG. 1: Building blocks of the multivalent CBM forms and their affinities for sialic acid. a, VcCBM, residues 25-216 of the V. cholerae sialidase (PDB:1w0p) with α-2,3-sialyllactose drawn as spheres. b, SpCBM, residues 121-305 of S. pneumoniae NanA sialidase with α-2,3-sialyllactose (PDB:4c1w). c, TD, the trimerisation domain, residues 333-438, of the P. aeruginosa pseudaminidase (PDB:2w38) in rainbow colours; the other two monomers in single colours. d, Multivalent forms: their molecular weights, valencies and binding affinities for α2,3-sialyllactose as determined by surface plasmon resonance (SPR) at 25° C. (KD values for VcCBM, Vc2CBM and Vc3CBM had been reported previously (Connaris et al, 2009)). Tandem repeat CBMs, and oligomeric CBMs fused to TD are linked by a 5-amino linker (details in Connaris, H. et al., (2014). PNAS 111:6401-6406).

[0279] FIG. 2: Graph showing the results of the condition 1 assay. There is an observable anti-viral effect with all CBM compounds especially when tested at 3 mg/mL.

[0280] FIG. 3: Plaque assay showing the results of the condition 1 assay; again, there is an anti-viral effect shown for all 3 CBMs especially when used at the higher concentration.

[0281] FIG. 4: Graph showing the results of the condition 2 assay. In this case, cells were exposed to CBM before SARS-CoV-2 infection; thus, the condition 2 assay represents a prophylaxis model. An anti-viral effect is shown for at least CBM2 & CBM3

[0282] FIG. 5: Graph showing the results of the condition 3 assay. In this case, infected cells (with SARS-CoV-2) were treated with the various CBMs. An anti-viral effect is shown for all 3 CBMs—particularly CBM3.

[0283] FIG. 6: Plaque assay showing the results of the condition 3 assay. Again, this shows the anti-viral effect of all 3 CBMs—particularly CBM3.

[0284] FIG. 7: the mean and SEM of the total clinical observations for group 2 (Control) and 3 (Neumifil) from 0 DPC until day of cull (7 DPC). FIG. 7 also shows the mean (with SEM) percentage weight change for the same period (right y-axis).

[0285] FIG. 8: Detection of Neumifil (HEX17) binding to SARS-CoV-2 Spike S1 variants. The dotted lines represent 4PL curve fits of the data. Inset: EC50 values for each variant.

[0286] FIG. 9: Detection of Neumifil (HEX17) binding to recombinant human ACE2. The dotted line represents a 4PL curve fitting of the data. Inset: EC50 value.

METHODS

[0287] Plaque Reduction Assay

[0288] Thaw vial of titrated SARS CoV-2 on ice.

[0289] Prepare 2 working stocks of SARS-CoV-2, diluting the virus in serum free (SF) DMEM to 500 pfu/mL (working stock for condition 1) and 250 pfu/mL (working stock for conditions 2 and 3). Store the prepared stocks on ice.

[0290] For test agent preparation, thaw an aliquot of each CBM (100 μL per vial at 10 mg/mL) on ice and transfer contents into a new sterile 1.5 mL Eppendorf tube an centrifuge at 13,000 rpm for 5 min to pellet any precipitates that may have formed. Transfer the supernatant into new sterile 1.5 mL Eppendorf tubes.

[0291] From the CBM supernatants, prepare 2 working stock concentrations (3 mg/mL and 1 mg/mL, from a master stock of 10 mg/mL) of the 3 CBMs to be tested, diluting the CBMs in a 50:50 mix of serum-free DMEM:PBS (see Table 1)

[0292] The final concentration of CBMs will vary between conditions. Condition 1: final CBM concentrations 1.5 mg/mL and 0.5 mg/mL, and for conditions 2 and 3, final CBM concentrations 3 mg/mL and 1 mg/mL.

TABLE-US-00014 TABLE 1 Diluent* Vials [Working] Final CBM volume required/ Condition (mg/mL) volume (μl) volume (μL) (μL) CBM 1 3 400 120 280 1 400 40 360 Total CBM 160 2 needed (μL) Diluent* Vials [Working] Final CBM volume required/ (mg/mL) volume (μl) volume (μL) (μL) CBM 2 3 500 150 350 1 500 50 450 Total CBM 200 2 needed (μL) 3 3 500 150 350 1 500 50 450 Total CBM 200 2 needed (μL) *50-50 mix of serum free DMEM:PBS

[0293] Condition 1: virus and CBM mixed prior to adding to cells [0294] Mix equal volumes of SARS CoV-2 and CBM and incubate on ice for 1 hour. For the positive control mix SARS-CoV-2 with SF diluent (50-50 mix of SF DMEM and PBS) in place of the CBM. For the negative control mix CBM 1 at 3 mg/mL with SF DMEM in place of SARS-CoV-2. [0295] Tube 1: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM1 at 3 mg/mL [0296] Tube 2: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM2 at 3 mg/mL [0297] Tube 3: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM3 at 3 mg/mL [0298] Tube 4: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM1 at 1 mg/mL [0299] Tube 5: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM1 at 1 mg/mL [0300] Tube 6: 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM1 at 1 mg/mL [0301] Tube NC: negative control: 300 μL SF DMEM plus 300 μL CBM diluent [0302] Tube PC: positive control, 300 μL SARS-CoV-2 at 500 pfu/mL plus 300 μL CBM diluent [0303] After 1 hr, remove serum free media from the cells and wash with sterile PBS (<0.5 mL/well) [0304] After the PBS wash, add 200 μL of the prepared inoculums to the appropriate wells, with tubes 1-3 (and controls) on plate 1 and tubes 4-6 (and controls) on plate 2. [0305] Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate [0306] at 37° C. 5% CO.sub.2 for 1 hr. [0307] After 1 hr, remove the inoculum and add 1 mL overlay to each well. [0308] Transfer plates to a flat-bottomed sealable container [0309] Incubate for 5 days at 37° C. 5% CO.sub.2.

[0310] Condition 2: cells exposed to CBM before virus infection [0311] Remove serum free DMEM and wash with Sterile PBS (<0.5 mL/well). [0312] After the PBS wash, add 200 μL CBM, with CBMs at 3 mg/mL on plate 3 and 1 mg/mL on plate 4. Incubate at 37° C. 5% CO.sub.2 for 1 hr. [0313] After 1 hr, remove CBM and wash with sterile PBS (<0.5 mL/well). [0314] After the PBS wash, add 200μL SARS-CoV-2 diluted to 250 pfu/mL to each well except the negative control wells. To the negative control wells, add 200 μL SF DMEM. [0315] Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate at 37° C. 5% CO.sub.2 for 1 hr. [0316] After 1 hr, remove the inoculum and add 1 mL overlay to each well. [0317] Transfer plates to a flat-bottomed sealable container [0318] Incubate for 5 days at 37° C. 5% CO.sub.2.

[0319] Condition 3: cells infected with SARS-CoV-2 and then treated with CBM. [0320] Remove serum free DMEM and wash with sterile PBS (0.5 mL/well) [0321] After the PBS wash, add 200 μL SARS-CoV-2 diluted to 250 pfu/mL to each well except the negative control. To the negative control, add 200 μL SF DMEM. [0322] Place plates in a flat-bottomed, sealable container and transfer to incubator. Incubate at 37° C. 5% CO.sub.2 for 1 hr. [0323] After 1 hr, remove the inoculum and add 200 μL CBM to each well, except the negative control (CBMs at 3 mg/mL on plate 5 and 1 mg/mL on plate 6). To the negative control, add 200 μL CBM diluent (SF DMEM-PBS). [0324] Place plates in a flat-bottomed sealable container and transfer to incubator. Incubate at 37° C. 5% CO.sub.2 for 1 hr. [0325] After 1 hr, remove the CBMs and add 1 mL overlay to each well. [0326] Transfer plates to a flat-bottomed sealable container [0327] Incubate for 4 days at 37° C. 5% CO2.

[0328] Specific details:

[0329] CBMs: [0330] CBM1: Vc2CBM40TD (trimeric (hexavalent) form based on V. cholerae CBM40) [0331] CBM2: Neumifil (HEX17: trimeric (hexavalent) form based on S. pneumoniae CBM40) [0332] CBM3: Cp2CBM32TD (trimeric (hexavalentform based on C. perfringens CBM32) CBMs added at two concentrations (1 mg/mL & 3 mg/mL), and (as stated) tested in three conditions: [0333] Condition 1: SARS-CoV-2 and CBM mixed prior to adding to cells [0334] Condition 2: Cells exposed to CBM before SARS-CoV-2 infection (prophylaxis model) [0335] Condition 3: Cells infected with SARS-CoV-2 and then CBM added (treatment model)

[0336] For making up of CBM: [0337] 3 mg/mL: 450 μL stock +1050 μL DMEM:PBS. [0338] 1 mg/mL: 150 μL stock +1350 μL DMEM:PBS

[0339] SARS-CoV-2 [0340] Vial labelled: SARS CoV2, England 2, P2 HCM/V/52, 05.03.20 [0341] Stock at 2.4×10.sup.5 pfu/mL [0342] .fwdarw.100 μL stock +900 μL media=2.4×10.sup.4 pfu/mL [0343] .fwdarw.200 μL above +1800 μL media=2.4×10.sup.3pfu/mL [0344] .fwdarw.1.5 mL above +6 mL media =480 pfu/mL ←Used for studies

[0345] VeroE6 cells [0346] P15, split 1:2 25/3/20 (obtained from PHE HCM group)

[0347] Reagent Details [0348] DMEM Sigma D5796, Lot RNBH6732, Exp Jun. 2020 [0349] PBS Gibco 10010-023, Lot 2098597, Exp 30 Jun. 2021 [0350] Overlay (aliquots made, each sufficient for 2 μlates): [0351] 7.5 mL 4% CMC PHE media, MR/19/1044, Prod date 4 Dec. 19 [0352] 7.5 mL 2% CMC PHE media, MR/19/1043, Prod date 4 Dec. 19 [0353] 15 mL 2xMEM Gibco 21935-028, Lot 2150451, Exp 31 Dec. 2020 [0354] 800 μL FCS Gibco 10100-139, Lot 216386RP, Exp May 2024 [0355] 400 μL anti-anti Sigma A5955, Lot 035M4800V

[0356] RESULTS

[0357] Condition 1: Virus and CBM mixed prior to adding to cells

[0358] In this phase of testing, SARS-CoV-2 virus and the CBM compounds were incubated for 1 hour prior to addition to cells. Plate 1 were compounds tested at a working concentration of 3 mg/mL and plate 2 at 1 mg/mL.

[0359] Negative control wells were all intact and the positive control gave counts between 119-164 plaques per well. The results are shown in FIGS. 2 (graph) and 3 (plaque assay). The results show that there is an observable anti-viral effect with all CBM compounds.

[0360] 3.2. Condition 2: Cells exposed to CBM before virus infection

[0361] In this phase of testing, VeroE6 cells were exposed to CBM compounds prior to addition of SARS-CoV-2 virus. Plate 3 were compounds tested at a working concentration of 3 mg/mL and plate 4 at 1 mg/mL. Plaques were counted after staining.

[0362] As shown in FIG. 4, there was an observable anti-viral effect with the CBM2 and CBM3 compounds when tested at a working concentration of 1 mg/mL.

[0363] 3.3. Condition 3: Cells infected with SARS-CoV-2 and then treated with CBM

[0364] In this phase of testing, VeroE6 cells were infected with SARS-CoV-2 virus before addition of CBM compounds. Plate 5 were compounds tested at a working concentration of 3 mg/mL and plate 6 at 1 mg/mL. Plaques were counted after staining. Negative control wells were all intact and the positive control gave counts between 117-132 plaques per well.

[0365] When plotted on a graph (see FIG. 5), all 3 CBM compounds are shown to have an anti-viral effect, particularly CBM3 which showed the greatest reduction. Results from both working concentrations of CBM1 and CBM2 were similar. The results of the plaque assay are also shown in FIG. 6.

[0366] Conclusion

[0367] All CBMs showed some anti-viral activity against SARS-CoV-2 virus in vitro CBM2 (Neumifil: Hex17) and CBM3 (CBM32-based) are very promising. The results demonstrated here provide the basis for urgent in vivo studies in a suitable animal model.

EXAMPLE 2

Testing CBM vs 0C43

[0368] OC43 is a human coronavirus OC43-(HCoV-OC43); it is a Betacoronavirus and is associated with occurrences of a ‘common cold’ type illness. Its S protein binds to sugar-based receptor-determinants, specifically to 9-O-acetylated sialic acids (9-O-Ac-Sias) attached as terminal residues to glycan chains on glycoproteins and lipids.

[0369] A series of experiments were completed to determine whether or not CBMs could be used to treat or prevent OC43 infections.

[0370] In each experiment the following CBMs were used: [0371] 1) Vc2CBM40TD (VC2) [0372] 2) Cp2CBM32TD (CBM32) [0373] 3) HEX17

[0374] Each CBM was used at 1 mg/ml and at 3 mg/ml.

[0375] Condition 1: Prophylactic CBM treatment

[0376] Cells were treated with CBMs for 1 hour before incubation with hCoV-OC43 for 1 hour. The assay showed that treatment with any of the CBMs tested was able to prevent a subsequent hCoV-OC43 infection as compared to cells infected but not pre-treated with any CBM. The effect was most pronounced with VC2 (at 3 mg/ml and at 1 mg/ml), HEX17 (at 1 mg/ml) and CBM32 (at 3 mg/ml and 1 mg/ml).

[0377] Condition 2: Simultaneous CBM treatment

[0378] Cells treated with CBMs and hCoV-OC43 for 1 hour simultaneously. The assay showed that simultaneous treatment with any of the CBMs and at any concentration (1 mg/ml or 3 mg/ml) was able to reduce hCoV-OC43 infection as compared to cells infected but not treated with any CBM.

EXAMPLE 3

[0379] Animal (Hamster) Studies Testing Neumifil Against SARS-CoV-2

[0380] Protocol/Study Design

TABLE-US-00015 TABLE 2 detail study protocol Action/Procedure/Sample (to include all animals related activity other than routine husbandry i.e. Study Day Licenced procedure, clinical (DPC- scoring, weight/temperature days post Group/Number of measurement, challenge) animals scheduled kill/necropsy) Number of samples −5 (−12 DPC) 27 Hamsters Delivery of animals n/a 0 (−7 DPC) 27 Hamsters, Chip all 27 animals (4 n/a groups 1-5 groups × 6 animals, and 1 group × 3 animals) assigned as following: Group 1 (Mock Challenge/Sentinel-PBS 5% Glycerol), n = 3 (3M) Group 2 (Challenge Control- PBS 5% Glycerol), n = 6 (3M, 3F) Group 3 (Challenge- Neumifil), n = 6 (3M, 3F) Group 4 (Challenge Control- PBS 5% Glycerol), n = 6 (3M, 3F) Group 5 (Challenge- Neumifil), n = 6 (3M, 3F) Give 1st Dose to groups 1-5: (1) PBS 5% Glycerol-100 ul intranasal (50 ul per nare) (2) PBS 5% Glycerol-100 ul intranasal (50 ul per nare) (3) Neumifil, 5000 ug/kg- 100 ul intranasal (50 ul per nare) (4) PBS 5% Glycerol-100 ul intranasal (50 ul per nare) (5) Neumifil, 5000 ug/kg- 100 ul intranasal (50 ul per nare) Temperature check-daily Clinical parameters measured (scored), then checked daily until Challenge, then twice-daily thereafter) 4 (−3 DPC) 27 Hamsters, (Clinical observations (scored) n/a groups 1-5 and temperature checked daily) Groups 1-5: Give 2nd Dose- same as previous 6 (−1 DPC) 27 Hamsters, (Clinical observations (scored) 6 × Throat swabs-‘media tube’ groups 1-5 and temperature checked daily) Groups 1-5: Give 3rd Dose- same as previous Also group 2: Throat swab 7 (0 DPC) 24 Hamsters, Challenge with SARS-CoV2 n/a groups 2-5 virus −5E+04 PFU groups 2&3 1E+02 Groups 4&5 (No challenge for group 1) (Clinical observations (scored) and temperature checked twice daily, from hereafter) 9 (2 DPC) 6 Hamsters, (Clinical observations (scored) 6 × Throat swabs-‘media tube’ Group 2 and temperature checked twice daily) Group 2: Throat swab 10 (3 DPC) 12 Hamsters, (Clinical observations (scored) 12 × 400 ul Nasal Wash −1.5 ml sarstedt groups 4 and 5 and temperature checked 12 × Throat Swab-‘media tube’ twice daily) 12 × Lung in 1.5 ml sarstedt Terminate animals from 12 × Lung in RNAlater tube groups 4 and 5 (12 animals) Histology: Nasal cavity*, Collect, per animal: trachea, lung, spleen Nasal wash *Collect whole head and Throat swab thoracic pluck Lung tissue (2 × sample) For Histology: Nasal cavity, trachea, lung, spleen *collect whole head and thoracic pluck 11 (4 DPC) 6 Hamsters, (Clinical observations (scored) 6 × Throat swabs-‘media tube’ Group 2 and temperature checked twice daily) Group 2: Throat swab 13 (6 DPC) 6 Hamsters, (Clinical observations (scored) 6 × Throat swab-‘media tube’ Group 2 and temperature checked twice daily) Group 2: Throat swab 14 (7 DPC) 15 Hamsters, (Clinical observations (scored) 15 × 400 ul Nasal Wash −1.5 ml sarstedt groups 1, 2 and temperature checked 15 × Throat Swab-‘Media tube’ and 3 twice daily) 15 × Lung in 1.5 ml sarstedt Terminate 15 animals, groups 15 × Lung in RNAlater tube 1, 2 and 3 Histology: Nasal cavity*, Collect, per animal: trachea, lung, spleen Nasal wash *Collect whole head and Throat swab thoracic pluck Lung tissue (2 samples) For Histology: Nasal cavity, trachea, lung, spleen *collect whole head and thoracic pluck

TABLE-US-00016 TABLE 3 study design summary Day Post Challenge Sentinel Control Treatment Control Treatment −7 Dose, PBS Dose, PBS Dose, Dose, PBS Dose, Neumifil Neumifil −6 −5 −4 −3 Dose, PBS Dose, PBS Dose, Dose, PBS Dose, Neumifil Neumifil −2 −1 Dose, PBS Dose, PBS Dose, Dose, PBS Dose, Neumifil Neumifil 0 Challenge Challenge Challenge Challenge 5E+04/PFU 5E+04/PFU 1E+02/PFU 1E+02/PFU 1 2 3 Cull Cull 4 5 6 7 Cull Cull Cull

[0381] Results

TABLE-US-00017 TABLE 4 clinical observations for group 2 (PBS) and 3 (Neumifil) following challenge with 5E+04 PFU SARS-CoV-2. Clinical Observations, Days Post Challenge Animal 0.0 0.25 1.00 1.25 1.98 2.25 2.96 3.26 3.96 4.25 4.96 5.25 5.96 6.25 6.93 2-1 H H H H H H H H H H H R, P R, P R, P R, P 2-2 H H H H H H H H H H Lb Lb, R Lb, R, Lb, R, R P P 2-3 H H H H H H H H A A A, R, A, R, Lb, R, R, P R, P P P P 2-4 H H H H H H H H H H P P H R, P, R, P D 2-5 H H H H H H H H P P Lb, P Lb, P H R, P R, P 2-6 H H H H H H H H H P Lb, R, Lb, R, Lb, R, R, P, R, P P P P D 3-1 H H H H H H H H H H Lb Lb, P, Lb, R R R R 3-2 H H H H H H H H H H H H R, P R R, P 3-3 H H H H H H H H H H H H H R H 3-4 H H H H H H H H H H H H H R H 3-5 H H H H H H H H H H H H H H H 3-6 H H H H H H H H H H P H R, P, D R, P R H = Healthy, A = Arched/Hunched, R = Ruffled fur, Lb = Laboured breathing, P = Pinched/Wasp waisted, D = Dehydrated not drinking Clinical observations were made twice-daily from 7 days prior to challenge onwards. No clinical symptoms were observed in any group prior to challenge. Note also that in the Neumifil treated group, the average number of symptoms (A, R, Lb, P and/or D) is generally lower than in the control group. Moreover there are fewer instances of Lb in the Neumifil treated group. Additional results are presented in FIG. 7. As compared to the control group(s), the Neumifil treated group (group 3) exhibited fewer total clinical observations

EXAMPLE 4

[0382] Angiotensin-converting enzyme 2 (ACE2) plays a major role in SARS-CoV-2 recognition, binding, fusion and entry into host cells [1]. Glycans, including sialic acid, may also be important in this interaction. There are eight glycosylation sites within the ACE2 receptor, three of which (N90, N322 and N546) may play a critical role in the interaction with SARS-CoV-2 Spike. Glycans on the SARS-CoV-2 Spike may also modulate the conformation of the Spike's receptor binding domain (RBD), which is responsible for ACE2 recognition and binding. Deletion of these glycans significantly reduces ACE2 binding.

[0383] Multiple new variants of SARS-CoV-2 have emerged and are circulating globally [1,2]. Of particular concern are the B.1.1.7 and B.1525 variants identified in the UK, the B.1.351 South African variant and the P.1 Brazil variant. The B.1.1.7 UK (Kent) virus is characterized by the HV69-70 deletion and N501Y in the Spike protein which increases transmissibility and may be associated with increased risk of death. The B.1.351 S. African variant shares some mutations with B.1.1.7 alongside additional differences, including the potential vaccine escape mutation E484K. The Brazil variant, P.1 is spreading to multiple countries and is associated with reinfections. P.1 also includes E484K, in addition to the N501Y mutation linked to increased infectivity of the UK variant. A further variant, B1525, has been identified in the UK and contains the important E484K mutation alongside the Kent B.1.1.7 mutations.

[0384] Aims: 1) To determine whether Neumifil interacts with SARS-CoV-2 Spike 51 protein and whether the affinity is affected by new variant mutations. 2) To determine whether Neumifil interacts with human ACE2.

[0385] Methods:

[0386] Table 1 summarizes the variant 51 Spike sequence information provided by the manufacturers. Each of the Spike and ACE2 proteins were recombinantly expressed in HEK293 cells.

TABLE-US-00018 TABLE 5 Spike S1 protein variants used in the study with corresponding accession codes and mutations. Spike Variant Product sequence information Wuhan Wuhan-Hu-1 (Dec 2019 isolate). Accession code: QHD43416.1 B.1.351 (S. K417N, E484K, N501Y, D614G in Wuhan- African) Hu-1 background B.1.1.7 (UK HV69-70 deletion, Y144 deletion, N501Y, A570D, (Kent)) D614G, P681H in Wuhan-Hu-1 background

[0387] EC50 (half-maximal effective concentration) values were determined by ELISA. Spike or ACE2 proteins were immobilized overnight at 4° C. on a high-binding ELISA plate at a concentration of 1 μg/mL. The wells were then incubated for 1.5 h with Neumifil (3-fold dilution series: 29160, 9720, 3240, 1080, 360, 120, 40, 0 ng/mL) in triplicate. Immunodetection of Neumifil binding was performed by incubation with rabbit anti-Neumifil (1 h), followed by HRP-labelled anti-rabbit IgG (1 h) and TMB substrate development. Binding curves were analysed using 4 parameter logistic (4PL) curve fitting to determine the inflection point (EC50).

[0388] Results:

[0389] As shown in FIG. 8, Neumifil binds to the original Spike protein (consisting of the Wuhan-Hu-1 Dec. 2019 isolate sequence) with an EC50 of 174 ng/mL. The binding profiles and EC50s for the variants indicate that the affinity is not significantly affected by the mutations present in the South African and UK (Kent) sequences. FIG. 9 shows Neumifil binding to ACE2 with an EC50 of 235 ng/ml.

REFERENCES

[0390] 1. Understanding variants of SARS-CoV-2 (2021) The Lancet World Report 397(102), P462

[0391] 2. McNally, A. (2021) BMJ 372, 504