HYBRID INTERFERONS FOR TREATING VIRAL INFECTIONS

Abstract

Compositions and methods for preventing or treating a coronavirus infection and viral infections more generally are provided. In particular, compositions for preventing or treating infection from Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Human Coronavirus NL63, Human Coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2 or Covid19) are provided.

Claims

1. (canceled)

2. (canceled)

3. A method for the treatment and/or prophylaxis of a coronavirus infection, said method comprising the step of: administering to a subject in need thereof a therapeutically effective amount of an interferon alpha subtype, wherein the interferon alpha subtype is HYBRID 2 or a functionally active fragment or variant thereof.

4. The method of claim 1, wherein HYBRID 2 comprises an amino acid sequence of SEQ ID NO: 3: TABLE-US-00005 CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQF QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDL EACVIQEVGVEETPLMNEDSILAVKKYFORITLYLIERKYSPCAWEVV RAEIMRSLSFSTNLQKRLRRKD.

5. The method of claim 3, wherein the coronavirus is Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Human Coronavirus NL63, Human Coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2 or Covid19).

6. The method of claim 4, wherein the interferon alpha subtype HYBRID 2 consists of the amino acid sequence of SEQ ID NO:3: TABLE-US-00006 CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQF QKAQAISVLHEMMQQTFNLESTKNSSAAWDETLLEKFYIELFQQMNDL EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEVV RAEIMRSLSFSTNLQKRLRRKD.

7. The method of claim 3, wherein the method of administration is sublingual, oral or injection administration.

8. The method of claim 3, wherein the therapeutically effective amount of the interferon alpha subtype is a low dose.

9. An interferon alpha subtype, wherein the interferon alpha subtype is HYBRID 2 or a functionally active fragment or variant thereof for use in the treatment and/or prophylaxis of a viral infection, wherein the viral infection is coronavirus infection.

10. The interferon alpha subtype for use in the treatment and/or prophylaxis of a viral infection of claim 9, wherein the interferon alpha subtype is HYBRID 2 wherein HYBRID 2 comprises an amino acid sequence of SEQ ID NO: 3: TABLE-US-00007 CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQF QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDL EACVIQEVGVEETPLMNEDSILAVKKYFORITLYLIERKYSPCAWEVV RAEIMRSLSFSTNLQKRLRRKD.

11. (canceled)

12. The interferon alpha subtype for use in the treatment and/or prophylaxis of a viral infection of claim 11, wherein the coronavirus is Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Human Coronavirus NL63, Human Coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2 or Covid19).

13. The interferon alpha subtype for use in the treatment and/or prophylaxis of a viral infection of claim 9, wherein the interferon alpha subtype is administered by sublingual, oral or injection administration.

14. The interferon subtype of claim 13, wherein the interferon alpha subtype is administered at a low dose.

15. (canceled)

16. (canceled)

17. (canceled)

18. A composition comprising an interferon alpha subtype, wherein the interferon alpha subtype is HYBRID 2 or a functionally active fragment or variant thereof, for use in the treatment and/or prophylaxis of a coronavirus infection.

19. (canceled)

20. (canceled)

21. The interferon alpha subtype for use in the treatment and/or prophylaxis of a viral infection of claim 9, wherein the interferon alpha subtype is HYBRID 2 wherein HYBRID 2 consists of the amino acid sequence of SEQ ID NO: 3: TABLE-US-00008 CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQF QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDL EACVIQEVGVEETPLMNEDSILAVKKYFORITLYLIERKYSPCAWEVV RAEIMRSLSFSTNLQKRLRRKD.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0149] FIG. 1 shows the effect of IFNα-14 on the cytopathic effect following infection from SARS-CoV-1 Coronavirus.

[0150] FIG. 2 shows a graph demonstrating the effect of IFNα-14 on plaque formation following infection from SARS-CoV-1 Coronavirus.

[0151] FIG. 3 shows results of Plaque Inhibition Assays using Vero Cells of a SARS-CoV-2 Inhibition plaque assay in which IFNalpha 2a (the commercially available subtype) is compared with IFNalpha 14 and shows the IC50s—this indicates Alpha-14 is 20× more potent against the Covid 19 virus than Alpha-2a

[0152] FIG. 4 shows results of Plaque Inhibition Assays using Vero Cells of studies comparing 5 interferons—INTERFERON-ALPHA 14, HYBRID 2, HYBRID 1, INTERFERON-BETA 1a, INTERFERON-ALPHA 2a—this gives a comparison based again on Alpha-2a of the 4 other ‘INTERFERONS’ of 20×, 10×, 5× more potent than Alpha-2a.

[0153] FIG. 5 shows the effect of INTERFERON-ALPHA 14, on CXCL-10 synthesis by Human Endothelial cells. CXCL-10 is the attractant for anti-viral NK cells.

[0154] FIG. 6 shows rapid induction of Granzyme B with INTERFERON ALPHA 14. Granzyme B causes release to kill cells with virus.

[0155] FIG. 7 shows suppression of IL-17A synthesis by Hybrid 2. INTERFERON ALPHA 14 inhibits both 17 A and F very strongly. IL17 inflammation is considered to be a significant issue in ARDS.

[0156] FIG. 8 shows suppression of IL 17F with Hybrid 2.

[0157] FIG. 9 shows the effect of Hybrid 2 on the production of CXCL 10 from human leucocytes.

[0158] FIG. 10 shows interferon gamma production.

[0159] FIG. 11 shows TNF alpha secretion.

[0160] FIG. 12 shows images from immune cells and Hybrid 2.

[0161] FIG. 13 shows Hybrid 2 can induce apoptosis.

[0162] FIG. 14 shows NK cell clustering.

[0163] FIG. 15 shows direct activation of NK cells by Hybrid 2.

[0164] FIG. 16 shows HYBRID 2's inhibition of a plaque assay with human RSV.

EXPERIMENTAL DATA

[0165] The present inventor has examined the ability of the synthetic alpha-interferon Hybrid 2 to inhibit viral infection using a model system based on the cytopathic effect and plaque formation caused as a result of infection with SARS-CoV-1 and SARS-CoV-2. The inventor has also examined the inhibition of the cytopathic effect in comparison with the anti-viral compound Ribavirin and the multi-subtype interferon Multiferon™. Additionally the inventor has utilised a RSV to demonstrate the anti-viral effect of HYBRID 2. RSV infection is the most important cause of hospitalisation in infants and one of the leading causes of infant mortality. As one of the respiratory viruses, along with influenza, rhinoviruses and coronaviruses, it is both interesting in its own right and provides an indicative model.

Experiment 1: The Effect of IFNα-14 on the Cytopathic Effect Following Infection from SARS-CoV-1 Coronavirus

[0166] The effectiveness of the interferon alpha subtype IFN-α14 to inhibit the cytopathic effect following SARS-CoV-1 infection was tested in a cytopathic endpoint assay. All endpoint assays were carried out using the multi-subtype Multiferon and IFN-α 14 as well as the anti-viral Ribavirin for comparison.

Preparation of Anti-Viral Treatments.

[0167] A broad range of concentrations (obtained by ten-fold dilutions) encompassing the inhibitory dosages commonly used for other viral-host combinations was tested. Compounds were dissolved Hank's buffered-saline solution.

[0168] For plaque assays, 5-fold drug dilutions were prepared using growth media as specified below. SARS-CoV-1 production and infection African Green Monkey (Vero E6) cells (American Type Culture Collection, Manassas, Va., USA) were propagated in 75 cm cell culture flasks containing growth medium consisting of Medium 199 (Sigma, St Louis, USA) supplemented with 10% foetal calf serum (FCS; Biological Industries, Israel). SARS-HCoV2003VA2774 (an isolate from a SARS patient in Singapore) was propagated in Vero E6 cells. Briefly, 2 ml of stock virus was added to a confluent monolayer of Vero E6 cells and incubated at 37° C. in 5% CO.sub.2 for one hour. 13 ml Medium 199, supplemented with 5% FCS, was then added. The cultures were incubated at 37° C. in 5% CO.sub.2 and the inhibition of the cytopathic effect gauged by observing each well through an inverted microscope. Where 75% or greater inhibition was observed after 48 hours, the supernatant was harvested. The supernatant was clarified at 2500 rpm and then aliquoted into cryovials and stored at −80° C. until use.

Virus Handling and Titration

[0169] Virus titre in the frozen culture supernatant was determined using a plaque assay carried out in duplicate. Briefly, 100 microlitres of virus in a 10-fold serial dilution was added to a monolayer of Vero E6 cells in a 24 well-plate. After incubation for an hour at 37° C. in 5% CO.sub.2, the viral Medium 199 supplemented with 5% FCS was added. Cells were fixed with 10%(v/v) formalin and stained with 2% (w/v) crystal violet. The plaques were counted visually and the virus titre in plaque forming units per ml (pfu/ml) calculated.

Cytopathic Endpoint Assay

[0170] The effect of each anti-viral treatment was tested in quadruplicate. Briefly, 100 microlitres of serial 10-fold dilutions of each treatment were incubated with 100 microlitres of Vero E6 cells giving a final cell count of 20,000 cells per well in a 96-well plate. Incubation was at 37° C. in 5% CO.sub.2 overnight for the interferon preparations and for one hour for the of infection (MOI) (virus particles per cell) of 0.5. The plates were incubated at 37° C. in 5% CO.sub.2 for three days and the plates were observed daily for cytopathic effects. The end point was the diluted concentration that inhibited the cytopathic effect in all four set-ups.

[0171] To determine cytotoxicity, 100 microlitres of serial 10-fold dilutions of each of the treatments were incubated with 100 microlitres of Vero E6 cells giving a final cell count of 20,000 cells per well in a 96-well plate, without viral challenge. The plates were then incubated at 37° C. in 5% CO.sub.2 for three days and toxicity effects were observed for using an inverted microscope.

[0172] 10 microlitres of virus at a concentration of 10,000 pfu/well were then added to each test well. This equates to a multiplicity of infection (MOI) (virus particles per cell) of 0.5. The plates were incubated at 37° C. in 5% CO.sub.2 for three days and the plates were observed daily for cytopathic effects. The end point was the diluted concentration that inhibited the cytopathic effect in all four set-ups (CIA100).

[0173] To determine cytotoxicity, 100 microlitres of serial 10-fold dilutions of each treatment were incubated with 100 microlitres of Vero E6 cells giving a final cell count of 20,000 cells per well in a 96-well plate, without viral challenge. The plates were then incubated at 37° C. in 5% CO.sub.2 for three days and toxicity effects were observed for using an inverted microscope. Interferons which showed complete inhibition were tested further at the lower viral titres of 10.sup.3 and 10.sup.2 pfu/well.

Experiment 2: The Effect of IFNα-14 on Plaque Formation Following Infection from SARS-CoV-1 Coronavirus

[0174] The effectiveness of the interferon alpha subtype IFN-α14 to inhibit plaque formation following SARS-CoV-1 infection was tested in a plaque reduction assay.

[0175] The plaque assay was performed using 10-fold dilutions of a virus stock, and 0.1 ml aliquots were inoculated onto susceptible cell monolayers. After an incubation period, which allowed virus to attach to cells, the monolayers were covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. After plate incubation, the original infected cells released viral progeny. The spread of the new viruses is restricted to neighbouring cells by the gel. Consequently, each infectious particle produced a circular zone of infected cells called a plaque. Eventually the plaque became large enough to be visible to the naked eye. Dyes to stain living cells were used to enhance the contrast between the living cells and the plaques. Only viruses that caused visible damage to cells were assayed in this way i.e. SARS-CoV-1.

[0176] These results clearly demonstrate the ability of the synthetic alpha-interferon, Interferon-alpha-14 (SEQ ID NO:1) to inhibit the cytopathic effect and plaque formation caused as a result of infection with SARS-CoV-1. The results demonstrate the inhibition of the cytopathic effect in comparison with the anti-viral compound Ribavirin and the multi-subtype interferon Multiferon™. Surprisingly Interferon-alpha14 (SEQ ID NO:1) is active against SARS-CoV-1 while Ribavirin and the multi-subtype interferon Multiferon™ show much less inhibitory activity. HYBRID 1 and HYBRID 2 show similar functional effects. These results are a clear indication of the potential superiority of IFNα-14, in particular SEQ ID NO:1, HYBRID 1, in particular SEQ ID NO:2 or HYBRID 2, in particular SEQ ID NO:3 or a variant or fragment thereof, for the treatment of a coronavirus infection over existing anti-viral treatments.

Experiment 3: The Effect of IFNα-14 on Plaque Formation Following Infection from SARS-CoV-2 Coronavirus

[0177] The effectiveness of the interferon alpha subtype IFN-α14 to inhibit plaque formation following SARS-CoV-2 infection was tested in a plaque reduction assay.

[0178] The plaque assay was performed using 10-fold dilutions of a virus stock, and 0.1 ml aliquots were inoculated onto susceptible cell monolayers. After an incubation period, which allowed virus to attach to cells, the monolayers were covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. After plate incubation, the original infected cells released viral progeny. The spread of the new viruses is restricted to neighbouring cells by the gel. Consequently, each infectious particle produced a circular zone of infected cells called a plaque. Eventually the plaque became large enough to be visible to the naked eye. Dyes to stain living cells were used to enhance the contrast between the living cells and the plaques. Only viruses that caused visible damage to cells were assayed in this way i.e. SARS-CoV-2.

[0179] These results (FIGS. 3 and 4) clearly demonstrate the ability of the synthetic alpha-interferon, Interferon-alpha-14 (SEQ ID NO:1) to inhibit the cytopathic effect and plaque formation caused as a result of infection with SARS-CoV-2. The results demonstrate the inhibition of the cytopathic effect in comparison with the other interferons. Interferon-alpha14 (SEQ ID NO:1), HYBRID 1 and HYBRID 2 are active against SARS-CoV-2. These results are a clear indication of the potential superiority of IFNα-14, in particular SEQ ID NO:1, HYBRID 1, in particular SEQ ID NO:2 or HYBRID 2, in particular SEQ ID NO:3 or a variant or fragment thereof, for the treatment of a coronavirus infection over existing anti-viral treatments.

Assays in Relation IL-17, Granzyme B. Interferon Gamma, Interferon Alpha, NK Cells

[0180] Assays utilising interferon alpha 14 and Hybrid 2 in relation to key immune modulators were undertaken as would be understood in the art.

Effect on HYBRID 2 on Immune Cell Killing

[0181] The effect of a hybrid recombinant interferon was tested in a live cell imaging assay of immune cell killing on an IncuCyte ZOOM platform.

[0182] SK-OV-3 ovarian cancer cells with red labelled nuclei (SK-OV-3 NucLight Red) were used as target cells in the study. For immune cell killing, the cells were co-cultured with natural killer (NK) cells and positive controls consisted of cells treated with IL-2 and IL-12. Apoptosis was detected by staining caspase 3/7 positive objects while cell number was determined by counting of red nuclei. The results from the co-culture model were compared to those of a mono-culture model consisting of SK-OV-3 NucLight Red cells alone.

[0183] An initial optimisation experiment was carried out that tested 4 ratios of target and effector cells. These results determined that 5,000 natural killer cells and 2,000 SK-OV-3 NucLight Red cells per well of a 96-well plate gave a suitable assay window for detecting immune cell killing.

[0184] Eight doses of hybrid recombinant interferon ranging from 10 IU/ml to 3×10.sup.6 IU/ml were tested for effects on immune cell killing. Results were compared to no treatment controls, vehicle treated controls and IL-2/IL-12 treated cells. All conditions were tested using cells in co-culture (SK-OV-3 NucLight and NK cells) and mono-culture (SK-OV-3 NucLight Red).

[0185] Cells were monitored for 4 days using an IncuCyte ZOOM, and IncuCyte software was used to measure green (apoptosis) and red (cell number) object count over time. Area under the curve (AUC) analysis was used to quantitate apoptosis and cell number over the time course.

[0186] IL-17, Il-6, CCL-5 and the immune response have been determined to be modulated following Covid-19 infection, in particular Covid-19 induced Acute Respiratory Distress Syndrome where IL-17 inflammation has been indicated as being significant.

[0187] Hybrid recombinant interferon caused a strong induction in apoptosis in the co-culture model, with an increase in AUC from 200 in vehicle controls to 1174 at the top dose of hybrid recombinant interferon. An EC.sub.50 of 1.5×10.sup.6 IU/ml was derived for apoptosis induction. In contrast, cells in mono-culture displayed only a marginal response to hybrid rIFN.

[0188] Hybrid recombinant interferon also caused a reduction in cell number. AUC values for cell number fell from 39921 in vehicle controls to 19501 at the top dose of hybrid rIFN. An IC50 value of 1.3×10.sup.6 IU/ml was determined for the reduction in cell number.

[0189] There was evidence of very strong direct activation of natural killer cells by hybrid rIFN, with cell clustering of NK cells observed in response to hybrid recombinant interferon treatment in a NK cell monoculture model.

[0190] As indicated in the FIGS. 5 to 15, IFN alpha 14 and in particular Hybrid 2 has been demonstrated to modulate Il-17 (inhibits both 17 A and F very strongly), Il-6, CCL-5, and CCl-2 and to provide an antiviral NK response.

[0191] Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.