Patent Application
20240226304
Infection by coronaviruses, including the Severe acute respiratory syndrome virus SARS-COV-2 (COVID) virus, requires membrane fusion between the viral envelope and the lung cell membrane. The fusion process is mediated by the virus's envelope glycoprotein, also called spike protein or S. No therapeutic options are currently available for the prophylaxis or treatment of infected individuals. The newly emerged pathogenic virus SARS-CoV-2 (the cause of COVID-19 respiratory disease) represents a worldwide threat to human health and social order. Therefore, given the current pandemic of COVID-19, the development of an effective antiviral therapy against these coronaviruses, especially SARS-CoV-2, is of highest priority not only nationally but also worldwide.
In certain aspects, the invention provides a peptide; the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2. In certain aspects, the invention provides a peptide; the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO: 1 and SEQ ID NO:2.
In certain aspects, a SARS lipid-peptide fusion includes a lipid tag, a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate. In some embodiments, the lipid tag is Cholesterol.
In certain aspects, a SARS lipid-peptide fusion inhibitor includes a lipid tag, a spacer, a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4, PEG11, or PEG24. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate. In some embodiments, the lipid tag is Cholesterol.
In some embodiments, the SARS lipid-peptide fusion inhibitor has one peptide moiety, one spacer moiety, and one lipid tag. In some embodiments, the inhibitor has two peptide moieties, two spacer moieties, and one lipid tag. The terms “linker” and “spacer” are used interchangeably in the instant application.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and a pharmaceutically acceptable excipient.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, a lipid tag, and a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, a lipid tag, a spacer, and a pharmaceutically acceptable excipient.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4, PEG11, or PEG24. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the SARS lipid-peptide fusion inhibitor in the pharmaceutical composition has one peptide moiety, one spacer moiety, and one lipid tag. In some embodiments, the inhibitor has two peptide moieties, two spacer moieties, and one lipid tag.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor. The inhibitor further includes two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. In some embodiments, each PEG4 is flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor. The inhibitor further includes one moiety of SEQ ID NO:1, one PEG4 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. In some embodiments, the PEG4 is flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO: 1 and SEQ ID NO:2, a lipid tag, a spacer, and a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor, which further includes two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient, wherein each PEG4 is flanked by SEQ ID NO: 1 on one end and cholesterol on the other end.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor, which further includes one moiety of SEQ ID NO:1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient, wherein the PEG24 is flanked by SEQ ID NO: 1 on one end and cholesterol on the other end.
In some embodiments, the antiviral pharmaceutical composition is administered per airway or subcutaneously. In some embodiments, the antiviral pharmaceutical composition is administered intranasally. In some embodiments, the antiviral pharmaceutical composition is administered as nasal drops or a spray. In some embodiments, the antiviral pharmaceutical composition is administered as nasal powder.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject at least two times. In some embodiments, at least one administration occurs before the subject is exposed to SARS-COV-2. In some embodiments, all administrations occur before the subject is exposed to SARS-COV-2. In some embodiments, the antiviral pharmaceutical composition is administered daily.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject once. In some embodiments, the administration occurs before the subject is exposed to SARS-COV-2.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject in need thereof with one or more additional antiviral substances. In some embodiments, at least one additional antiviral substance targets a different aspect of SARS-CoV-2 life cycle than SARSHRC peptides.
In some embodiments, the peptide reaches biologically effective concentrations both in upper and lower respiratory tract of the subject. In some embodiments, the peptide reaches biologically effective concentrations in the lungs of the subject. In some embodiments, the peptide reaches biologically effective concentrations in the blood of the subject.
In some embodiments, the method prevents COVID-19 that would have been caused by SARS-COV-2 virions that comprise a Spike protein, wherein the sequence of the Spike protein differs from SEQ ID No:3. In some embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 S247R, SARS-COV-2 D614G, SARS-COV-2 S943P, and SARS-COV-2 D839Y. In some other embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 alpha beta, gamma, delta, and lambda variants.
In certain aspects, the invention provides a method of reducing the risk of a SARS-COV-2 infecting a cell in a subject. The method includes administering an effective amount of a SARS-COV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-CoV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO:1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In certain aspects wherein the SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient, intranasal administration thereof results in an equivalent level of SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor in the turbinate and in the lungs of the subject by 1 hour after administration. Two levels are equivalent if both levels, e.g., of concentration of lipid-peptide fusion inhibitor, are of the same order of magnitude, or wherein one level is within 25% of the level of the other, or wherein one level is within 50% of the level of the other. In some embodiments, equivalent levels of the SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor are maintained in both the lungs and the turbinate of the subject up to 8 hours after intranasal administration. In some embodiments, equivalent levels of the SARS-CoV-2 (COVID-19) lipid-peptide fusion inhibitor are maintained in both the lungs and the turbinates of the subject up to 24 hours after intranasal administration. In some embodiments, equivalent levels of the SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor are maintained in both the lungs and the turbinate of the subject up to 48 hours after intranasal administration.
In certain aspects, the invention provides a method of reducing the risk of COVID-19 in a subject. The method includes administering an effective amount of a SARS-CoV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO: 1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO:1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In certain aspects, the invention provides a method of reducing the risk of death from COVID-19 in a subject. The method includes administering an effective amount of a SARS-COV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO: 1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In some embodiments, the method prevents COVID-19 that would have been caused by SARS-COV-2 virions that comprise a Spike protein, wherein the sequence of the Spike protein differs from SEQ ID NO:3. In some embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 S247R, SARS-COV-2 D614G, SARS-CoV-2 S943P, and SARS-COV-2 D839Y. In some other embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 B alpha, beta, gamma, delta, and lambda variants.
This application contains at least one drawing executed in color.
The invention covers lipid-peptide molecules for the prevention and treatment of COVID-19. The invention uses designed peptides that block SARS-COV-2 entry into cells and will likely prevent and/or abrogate infection in vivo and prevent transmission. The designed lipid-peptide molecules are highly effective at inhibiting live SARS-COV-2 (COVID) virus infection in cultured cells and animal models.
Infection by coronaviruses, including the SARS-COV-2 (COVID) virus, requires membrane fusion between the viral envelope and the lung cell membrane. The fusion process is mediated by the virus's envelope glycoprotein, also called spike protein or S. The inventors engineered specific lipid-peptide constructs, that inhibit viral fusion and infection by binding to transitional stages of the spike protein, therefore preventing its function. Importantly, these antivirals can be given by the airway, by nasal drops or other method of nasal administration including powder, are not toxic, and have good half-life in the lungs. The fact that they can be given via the nose and inhalation makes them convenient and feasible for widespread use. Testing the lead antivirals in animal models will show utility for preventing and treating infection and preventing contagion from an infected animal to a healthy animal, including treatment as nasal drops or spray to prevent infection of healthcare workers.
In certain aspects, the invention provides a peptide; the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2. In certain aspects, the invention provides a peptide; the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2.
In certain aspects, a SARS lipid-peptide fusion includes a lipid tag, a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate. In some embodiments, the lipid tag is Cholesterol.
In certain aspects, a SARS lipid-peptide fusion inhibitor includes a lipid tag, a spacer, a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO: 1 and SEQ ID NO:2.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4, PEG11, or PEG24. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate. In some embodiments, the lipid tag is Cholesterol.
In some embodiments, the SARS lipid-peptide fusion inhibitor has one peptide moiety, one spacer moiety, and one lipid tag. In some embodiments, the inhibitor has two peptide moieties, two spacer moieties, and one lipid tag. The terms “linker” and “spacer” are used interchangeably in the instant application.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and a pharmaceutically acceptable excipient.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, a lipid tag, and a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO: 1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, a lipid tag, a spacer, and a pharmaceutically acceptable excipient.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4, PEG11, or PEG24. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the SARS lipid-peptide fusion inhibitor in the pharmaceutical composition has one peptide moiety, one spacer moiety, and one lipid tag. In some embodiments, the inhibitor has two peptide moieties, two spacer moieties, and one lipid tag.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor. The inhibitor further includes two moieties of SEQ ID NO:1, two PEG4 moieties, once cholesterol tag, and a pharmaceutically acceptable excipient. In some embodiments, each PEG4 is flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor. The inhibitor further includes one moiety of SEQ ID NO:1, one PEG4 moiety, once cholesterol tag, and a pharmaceutically acceptable excipient. In some embodiments, the PEG4 is flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide is selected from SEQ ID NO:1 and SEQ ID NO:2, or a peptide where the C-terminal part of the peptide is “Gly-Ser-Gly-Ser-Cys,” and the N-terminal part of the peptide has more than 80%, 85%, 90%, 95%, but less than 100% homology with a sequence selected from SEQ ID NO:1 and SEQ ID NO:2, a lipid tag, a spacer, and a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor, which further includes two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient, wherein each PEG4 is flanked by SEQ ID NO: 1 on one end and cholesterol on the other end.
In certain aspects, the invention provides a method of preventing COVID-19 that includes administering to a subject in need an antiviral pharmaceutical composition. The pharmaceutical composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor, which further includes one moiety of SEQ ID NO: 1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient, wherein the PEG24 is flanked by SEQ ID NO: 1 on one end and cholesterol on the other end.
In some embodiments, the antiviral pharmaceutical composition is administered per airway or subcutaneously. In some embodiments, the antiviral pharmaceutical composition is administered intranasally. In some embodiments, the antiviral pharmaceutical composition is administered as nasal drops or a spray. In some embodiments, the antiviral pharmaceutical composition is administered as nasal powder.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject at least two times. In some embodiments, at least one administration occurs before the subject is exposed to SARS-COV-2. In some embodiments, all administrations occur before the subject is exposed to SARS-COV-2. In some embodiments, the antiviral pharmaceutical composition is administered daily.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject once. In some embodiments, the administration occurs before the subject is exposed to SARS-COV-2.
In some embodiments, the antiviral pharmaceutical composition is administered to the subject in need thereof with one or more additional antiviral substances. In some embodiments, at least one additional antiviral substance targets a different aspect of SARS-CoV-2 life cycle than SARSHRC peptides.
In some embodiments, the peptide reaches biologically effective concentrations both in upper and lower respiratory tract of the subject. In some embodiments, the peptide reaches biologically effective concentrations in the lungs of the subject. In some embodiments, the peptide reaches biologically effective concentrations in the blood of the subject.
In some embodiments, the method prevents COVID-19 caused by SARS-COV-2 virions that comprise a Spike protein, wherein the sequence of the Spike protein differs from SEQ ID NO:3. In some embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 S247R, SARS-COV-2 D614G, SARS-COV-2 S943P, and SARS-CoV-2 D839Y. In some other embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 alpha, beta, gamma, delta, and lambda variants.
In certain aspects, the invention provides a method of reducing the risk of a SARS-COV-2 infecting a cell in a subject. The method includes administering an effective amount of a SARS-COV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-CoV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO:1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In certain aspects, the invention provides a method of reducing the risk of COVID-19 in a subject. The method includes administering an effective amount of a SARS-CoV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO: 1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In certain aspects, the invention provides a method of reducing the risk of death from COVID-19 in a subject. The method includes administering an effective amount of a SARS-COV-2 (COVID-19) antiviral composition to inhibit SARS-COV-2 infection of a cell. The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising two moieties of SEQ ID NO:1, two PEG4 moieties, one cholesterol tag, and a pharmaceutically acceptable excipient. Each PEG4 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol tag on the other end. Alternatively, The SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor comprising one moiety of SEQ ID NO: 1, one PEG24 moiety, one cholesterol tag, and a pharmaceutically acceptable excipient. PEG24 can be flanked by a SEQ ID NO: 1 on one end and the cholesterol on the other end.
In some embodiments, the method prevents COVID-19 caused by SARS-COV-2 virions that comprise a Spike protein, wherein the sequence of the Spike protein differs from SEQ ID NO:3. In some embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 S247R, SARS-COV-2 D614G, SARS-COV-2 S943P, and SARS-CoV-2 D839Y. In some other embodiments, the SARS-COV-2 is selected from the group consisting of SARS-COV-2 alpha, beta, gamma, delta, and lambda variants.
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Coronaviruses (CoVs) can cause life-threatening diseases. The latest disease was named coronavirus disease 2019 (abbreviated “COVID-19”) by the World Health Organization. COVID-19 is caused by the coronavirus strain SARS-COV-2. Like its predecessors SARS-COV-1 and middle eastern respiratory syndrome virus MERS-COV, SARS-COV-2 is a betacoronavirus. However, SARS-COV-2 and COVID-19 differ from the other CoVs (such as MERS) and their respective diseases in striking manners, as witnessed by the entire world in 2020.
Coronavirus Entry Pathway into Target Cells
Coronaviruses employ a type I fusion mechanism to gain access to the cytoplasm of host cells. Other pathogenic viruses that employ the type I fusion mechanism include HIV, paramyxoviruses and pneumoviruses. Merger of the viral envelope and host cell membrane is driven by profound structural rearrangements of trimeric viral fusion proteins; infection can be arrested by inhibiting the rearrangement process.
Infection by coronavirus requires membrane fusion between the viral envelope and the cell membrane. Depending on the cell type and the coronavirus strain, fusion can occur at either the cell surface membrane or in the endosomal membrane. The fusion process is mediated by the viral envelope glycoprotein (S), a ˜1200 residue, heavily glycosylated type-I integral membrane protein presented as a large homotrimer, each monomer having several domains (
Like the influenza HA, the S protein exists as a trimer on the virion surface and mediates attachment, receptor binding and membrane fusion. The betacoronaviruses S proteins' host cell receptors identified thus far include angiotensin-converting enzyme 2 (ACE2) for SARS-COV-1 and dipeptidyl peptidase-4 (DPP4) for MERS-COV. SARS-COV-2 was found to use the human angiotensin-converting enzyme 2 (hACE2) for entry (and most likely uses or can use other receptors as yet unknown). S undergoes cleavage by a host protease to generate S1 and S2. Priming with the receptor and cleavage are both necessary for membrane merger
The activation step that initiates a series of conformational changes in the fusion protein leading to membrane merger differs depending on the pathway that the virus uses to enter the cell. For many paramyxoviruses, upon receptor binding, the attachment glycoprotein activates the fusion protein to assume its fusion-ready conformation at the cell surface at neutral pH. We and others have shown that for these viruses (that fuse at the cell membrane), C-peptides derived from the HRC region of the fusion protein ectodomain inhibit viral entry with varying activity and that lipid conjugation markedly enhances their antiviral potency and simultaneously increases their in vivo half-life. By targeting lipid-conjugated fusion inhibitory peptides to the plasma membrane, and by engineering increased HRN-peptide binding affinity, we have increased antiviral potency by several logs. The lipid-conjugated inhibitory peptides on the cell surface directly target the membrane site of viral fusion. By adding poly-ethylene glycol (PEG) linkers (such as PEG4) to the compounds between the lipid moiety and the peptide, we further increased the activity and potency of the conjugates. We demonstrated in vivo efficacy of lipid-conjugated fusion inhibitory peptides against lethal Nipah virus infection in golden hamsters and non-human primates, measles virus infection in mice and cotton rats, and human parainfluenza virus type 3 infection in cotton rats.
For viruses that do not fuse at the cell membrane the target for C-peptides is generally thought to be inaccessible. Example of these viruses are influenza and Ebola viruses. The fusion proteins of influenza (hemagglutinin protein; HA) and of Ebola (GP) are activated to fuse only after intracellular internalization. We showed that our lipid-conjugated peptides derived from influenza HA inhibit infection by influenza, suggesting that the lipid-conjugation-based strategy permits the use of fusion-inhibitory peptides for viruses that fuse in the cell interior. A second strategy that we adopted for influenza is the addition of HIV-TAT (a well known cell-penetrating peptide, CPP) to enhance inhibition of intracellular targets. With the combination of these two strategies, HA derived peptides are effective in vivo against human strains of influenza virus. A similar strategy also led to effective antiviral C-peptides for Ebola infection.
A major challenge in developing C-peptide fusion inhibitors for coronavirus may be that coronavirus viral entry can follow several entry pathways (
For this reason, design of entry inhibitors for coronavirus is a challenge. We explored whether adding cell penetrating peptides and lipid moieties that promote endosomal localization would increase the antiviral potency.
Earlier research on lipid-conjugated inhibitory peptides demonstrated that the lipid directs the peptide to cell membranes and increases antiviral efficacy. These conjugated peptides were shown, in published work, to inhibit both early and late entry strains of coronavirus (
The SARS-COV-2 6HB assembly (
Peptide SARS (
Infection by SARS-COV-2 requires membrane fusion between the viral envelope and the host cell membrane, at either the cell surface or the endosomal membrane. The fusion process is mediated by the viral envelope spike glycoprotein, S. Upon viral attachment or uptake, host factors trigger large-scale conformational rearrangements in S, including a refolding step that leads directly to membrane fusion and viral entry. Peptides corresponding to the highly conserved heptad repeat (HR) domain at the C-terminus of the S protein (HRC peptides) may prevent this refolding and inhibit fusion, thereby preventing infection.
We recently described a monomeric SARS-COV-2 HRC-lipopeptide fusion inhibitor against SARS-COV-2 with in vitro and ex vivo efficacy superior to previously described HRC-derived fusion inhibitory peptides. We designed a number of constructs based on the SARSHRC (also named SARS) and SARSMod peptide sequences. Basically, the SARS and SARSMod peptides were modified by attaching a glycine-serine 4-mer, GSGS, and a cysteine at their C-terminals. A PEG linker (PEG4, PEG24, or PEG11) and a cholesterol tag were further added to the constructs. The HRC peptides form six-helix bundle (6HB)-like assemblies with the extended intermediate form of the S protein trimer, thereby disrupting the structural rearrangement of S that drives membrane fusion.
[SARSHRC-PEG4]-Chol (Also Named “SARS Monomer”):
We also designed additional constructs as variations from the constructs above. The design of the peptides is demonstrated in
We have previously demonstrated that lipid conjugation of HRC-derived inhibitory peptides markedly increases antiviral potency and in vivo half-life, and successfully used this strategy to create entry inhibitors for prophylaxis and/or treatment of human parainfluenza virus type 3, measles virus, influenza virus, and Nipah virus infection. Both dimerization and peptide integration into cell membranes proved key to ensure respiratory tract protection and prevent systemic lipopeptide dissemination. The lipid-conjugated peptides administered intranasally to animals reached high, and biologically effective (in vivo) concentrations both in the upper and lower respiratory tract, and the specific nature of the lipid can be designed to modulate the extent of transit from the lung to the systemic circulation and organs. Lipid conjugation also enabled activity against viruses that do not fuse until they have been taken up via endocytosis. Here, we show that a SARS-CoV-2 S-specific lipopeptide is a potent inhibitor of fusion, prevents viral entry, and, when administered intranasally, completely prevents direct-contact transmission of SARS-COV-2 in ferrets. We propose this compound as a candidate antiviral, for pre-exposure or early post-exposure prophylaxis for SARS-COV-2 transmission in humans.
To improve the antiviral potency of the previously assessed SARS-COV-2 HRC-lipopeptide fusion inhibitor, we compared monomeric and dimeric derivatives of the SARS-CoV-2 S-derived HRC-peptide (
Despite the overall stability of the SARS-COV-2 genome, variants with mutations in S have spread globally. These mutations in S altered infectivity of cells (e.g., D614G) or were located in the putative target domain of the HRC peptide (e.g., S943P). To determine the potency of the [SARSHRC-PEG4]2-chol peptide for a range of variant SARS-COV-2 viruses, we examined fusion inhibition mediated by each of these emerging S protein mutants. In addition, to assess the potential for broad-spectrum activity, we assessed potency against the S of SARS-COV and MERS-COV (using dipeptidyl peptidase 4 (DPP4) receptor-bearing cells as the target for the latter). The dimeric cholesterol-conjugated peptide ([SARSHRC-PEG4]2-chol) also robustly inhibited fusion mediated by the S proteins of several emerging SARS-COV-2 variants (including D614G) and the S protein of SARS-COV and MERS-COV (
HIV-TAT is a well-known cell-penetrating peptide (CPP) to enhance inhibition of intracellular targets. We have previously shown that the addition of cell penetrating peptide sequence can increase the antiviral activity for both peptides targeting Ebola and Influenza viruses. For influenza, only the TAT conjugated peptides were shown to be effective in vivo. Surprisingly, addition of cell penetrating peptide sequence does not increase the antiviral activity of [SARSHRC-PEG4]2-chol, as illustrated in
Proposed anchoring of the dimeric lipopeptide in the host cell membrane and interactions with the viral S protein are shown in
For other enveloped respiratory viruses, we previously showed that both ex vivo and in vivo dimeric lipopeptides administered intranasally displayed different retentions in the respiratory tract dependent on the attached moiety of chol versus toc (Figueira, T. N. et al. J Virol 91 (2017)). Here, we compared local and systemic biodistribution of our most potent monomeric and dimeric lipopeptides (SARSHRC-PEG24-chol and [SARSHRC-PEG4]2-chol) at 1, 8, and 24 hours after intranasal inoculation or subcutaneous injection in humanized K18 hACE2 mice (
An ex vivo toxicity (MTT) assay in primary HAE cells was conducted for the fusions. The assay showed minimal toxicity even after 6 days at the highest concentrations tested (<20% at 100 μM), and no toxicity at its IC90 entry inhibitory concentrations (˜35 nM) (
Next, the lead peptide, [SARSHRC-PEG4]2-chol, was assessed for its ability to block entry of live SARS-COV-2 in VeroE6 cells or VeroE6 cells overexpressing the protease TMPRSS2, one of the host factors thought to facilitate viral entry at the cell membrane. Whereas viral fusion in VeroE6 cells predominantly occurs after endocytosis, the virus enters TMPRSS2-overexpressing cells by fusion at the cell surface, reflecting the entry route in airway cells. This difference is highlighted by chloroquine's effectiveness against SARS-CoV-2 infection in Vero cells but failure in TMPRSS2-expressing Vero cells and human lung. The [SARSHRC-PEG4]2-chol peptide dissolved in an aqueous buffer containing 2% dimethylsulfoxide (DMSO) inhibited virus entry after 8 hours with an IC50˜300 nM in VeroE6 and ˜5 nM in VeroE6-TMPRSS2 cells (
Here, we determined and characterize the potency of fusion inhibitory lipopeptides against two VOC, the alpha (B.1.1.7) variant and the South African or beta (B.1.35) variant. For both VOC, immune escape from monoclonal antibodies has been described and convalescent sera are reported as less potent against B.1.351 in neutralization assays.
We have previously described that wt SARS-COV-2 infection can be efficiently inhibited by fusion inhibitory lipopeptides in vitro and in vivo. Here, we evaluated the efficacy of two lipopeptides ([SARSHRC-PEG4]2-chol and SARSHRC-PEG24-chol) against wild type, the alpha variant (B.1.1.7) and the beta variant (B.1.351) SARS-COV-2 in an infectious virus entry assay. We directly compared the lipopeptides to a set of eleven previously described monoclonal antibodies (mAb) and eight post-vaccination sera (BNT162b2, two shots).
We performed a previously established infectious virus entry assay. Briefly, FIP, mAb and sera were serially diluted (10-fold, 5-fold or 2-fold, respectively) and incubated with a fixed amount of virus particles for one hour at 37 C. Virus-inhibitor mix was subsequently added to VeroE6 cells overexpressing TMPRSS2 (VeroE6-TMPRSS2) or Calu3 cells and incubated for eight hours at 37 C. Cells were washed, fixed and stained with a primary mouse-anti-SARS-COV nucleocapsid (Biorad) and a secondary goat-anti-mouse IgG/FITC antibody (Invitrogen). Fluorescent spots were recorded, counted and inhibition was calculated in percentage of infection control. We calculated IC50 values using a four-parameter dose response model and defined potencies within each class based on log-transformed IC50 values (shown in
We detected comparable potency of both lipopeptides against wt, the alpha variant (B.1.1.7) and the beta variant (B.1.351), independent of the cell line used. On VeroE6-TMPRSS2 cells 2 out of 11 mAb inhibited viral entry for all three viruses efficiently (2-15, 1-16); 4 out of 11 mAb did not (or only at high concentrations) inhibit viral entry; 2 out of 11 mAb efficiently inhibited wt SARS-COV-2 entry but not the alpha variant (B.1.1.7) or the beta variant (B.1.351) entry (1-21, 1-22); 2 out of 11 mAb blocked wt and the alpha variant (B.1.1.7) entry at comparable levels but did not block the beta variant (B.1.351) entry (1-18, 217); and 1 out of 11 mAb had slightly increased efficacy to the alpha variant (B.1.1.7) (2-02). Although IC50 values were in general lower on Calu3 cells, we observed a similar trend.
Polyclonal post-vaccination sera showed a broad spectrum of reactivity against all variants. However, compared to wt SARS-COV-2, we measured overall lower titers to the beta variant (B.1.351) and comparable or higher titers to the alpha variant (B.1.1.7).
In conclusion, we show equal efficacy of the evaluated SARS-COV-2 specific lipopeptides against wt SARS-COV-2 and the VOC the alpha variant (B.1.1.7) and the beta variant (B.1.351). Furthermore, we confirmed immune escape from multiple well-characterized mAb by at least one of the tested VOC, as well as lower serum titers against the beta variant (B.1.351).
Ferrets are an ideal model for assessing respiratory virus transmission, either by direct contact or by aerosol transmission. Mustelids are highly susceptible to infection with SARS-COV-2, as also illustrated by frequent COVID-19 outbreaks at mink farms. Direct contact transmission of SARS-COV in ferrets was demonstrated in 2003, and both direct contact and airborne transmission have been shown in ferrets for SARS-COV-22. Direct contact transmission in the ferret model is highly reproducible (100% transmission from donor to acceptor animals), but ferrets display limited clinical signs. After infection via direct inoculation or transmission, SARS-COV-2 can readily be detected in and isolated from the throat and nose, and viral replication leads to seroconversion.
To assess the efficacy of [SARSHRC-PEG4]2-chol in preventing SARS-COV-2 transmission, naive ferrets were dosed prophylactically with the lipopeptide before being co-housed with SARS-COV-2 infected ferrets. In this setup, transmission via multiple routes can theoretically occur (aerosol, orofecal, and scratching or biting), and ferrets are continuously exposed to infectious virus during the period of co-housing, providing a stringent test for antiviral efficacy. The study design is shown in
The viral loads (detection of viral genomes via RT-qPCR) for directly inoculated donor animals (grey), mock-treated recipient animals (red) and lipopeptide-treated recipient animals (green) are shown in
Seroconversion occurred in donor ferrets and 6/6 mock-treated animals by 21 DPI, but in none of the peptide-treated recipient animals, as shown by S- and N-specific IgG enzyme-linked immunosorbent assay (ELISA) and virus neutralization (
In light of the persistence of the dimeric lipopeptide in the murine lung (
The intranasal [SARSHRC-PEG4]2-chol peptide presented in this study is the first successful prophylaxis that prevents SARS-COV-2 transmission in a relevant animal model, providing complete protection during a 24-hour period of intense direct contact. Parallel approaches to prevent transmission that target the interaction between S and ACE2 have shown promise in vitro (e.g., the “miniprotein” approach). The lipopeptide described here acts on the S2 domain after shedding of S1 (
Ethics statement. Influenza virus, SARS-COV-2 and Aleutian Disease Virus seronegative female (weighing 900-1200 g) and male (weighing 1000-1500 g) ferrets (Mustela putorius furo) were obtained from a commercial breeder (Triple F Farms, PA, USA). Animals were housed and experiments were performed in compliance with the Dutch legislation for the protection of animals used for scientific purposes (2014, implementing EU Directive 2010/63). Research was conducted under a project license from the Dutch competent authority (license number AVD1010020174312) and the study protocol was approved by the institutional Animal Welfare Body (Erasmus MC permit number 17-4312-07, -08 and -09). Animal welfare was monitored on a daily basis. K18-hACE2 mice [B6. Cg-Tg(K18-hACE2)2Prlmn/J] (4-6 weeks old) were purchased from the Jackson Laboratory and bred in house (at CUIMC, NY, USA). All mouse experiments were conducted in accordance with protocols approved by the Columbia University Institutional Animal Care and Use Committee (AC-AABG9559). The Institute of Comparative Medicine (ICM) at Columbia University is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and complies with the regulations under the Animal Welfare Act (AWA), the Health Research Extension Act of 1985, and the National Research Council (NRC).
SARS-COV-2 S protein mediated fusion modeling. Molecular Maya (https://clarafi.com/tools/mmaya/) was used to model and simulate the inhibitory lipopeptide, the full-length SARS-COV 2 Spike (S) pre-fusion, pre-hairpin and post-fusion structures, using a combination of molecular mechanics force fields. The pegylated cholesterol, inhibitory peptides, and S protein respectively were parametrized using the MMFF94 (1), CHARMM C36 (2), and Martini (3) force fields. Simulations were run using Autodesk Maya's nucleus solver and additional restraints native to the nucleus solver (nConstraints) were used to stabilize the molecules during interactive steering.
To model the initial full-length pre-fusion S protein, we used the SARS-COV 2 S protein sequence from UniProt entry PODTC2, PDB 6XR8 (4) from the wild-type SARS-CoV 2 S protein and 2FXP (5) from the SARS-COV S protein. Remaining structural gaps were modeled using Molecular Maya's Modeling kit.
To model the S pre-hairpin intermediate, we aligned the post-fusion structure from PDB 6XRA (+) to our full-length pre-fusion model, considering only the central helix (CH) region in the alignment (residues 968-1035). Simulations were then run to progressively steer the HRN region (residues 910-985) towards the aligned post-fusion structure, leading to the extension of the CH coiled coil by HRN. The remaining regions of the model were restrained with elastic networks or position restraints to preserve local secondary structure. The fusion peptide region was then released from the elastic network and steered towards the host-cell membrane, resulting in the pre-hairpin model.
The post-fusion model was obtained by progressively steering the HRC region from the pre-hairpin model towards the post-fusion structure to obtain the HRC-HRN 6-helix bundle. During the transition, the position restraints on the C-terminal transmembrane domains of the S were translated to allow HRC reaching the steering targets, and the entire post-fusion structure target was reoriented to avoid overlaps with the viral membrane proxy.
Finally, to model the inhibitory lipopeptide's interaction with the S protein, its amino-acids were steered towards the matching HRC residues in the target post-fusion structure, while the cholesterol moiety was positioned on the host-cell membrane plane. The S's pre-hairpin to post-fusion transition was interrupted before sterical clashes between HRC and the inhibitory peptide were detected.
Lipopeptide synthesis. The peptide (SARSHRC) corresponding to residues 1168-1203 of SARS-COV-2 S with a C-terminal-GSGSGC spacer sequence was prepared by solid phase peptide synthesis (SPPS). The SARSHRC peptide was acetylated at the N-terminus and amidated at the C-terminus. The crude peptide was purified by reverse-phase high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). SARSHRC-chol,SARSHRC-PEG4-chol, SARSHRC-PEG24-chol, [SARSHRC]2-PEG11, and [SARSHRC-PEG4]2-chol were synthesized via chemoselective Thiol-Michael addition reactions between the terminal thiol group on the peptide cysteine residue and either bromoacetyl chol (with and without the indicated PEG linkers), maleimide functional PEG linkers or PEG-cholesterol linkers as previously described (6). Purification by HPLC and lyophilization yielded the peptide-lipid conjugates as white powders. The identity of the conjugates was verified by MALDI-TOF MS (
Dissolving lipopeptides for use in experiments. [SARSHRC-PEG4]2-chol was supplied as a white powder in aliquots of 10 mg. For in vivo experiments in ferrets, 10 mg of [SARSHRC-PEG4]2-chol was dissolved in 33.3 μl DMSO, which was subsequently added to 1632.7 μl de-ionized H2O. This yielded a final aqueous solution of lipopeptide dissolved at a concentration of 6 mg/mL containing 2% DMSO. To obtain peptide dissolved in aqueous solution without DMSO, 100 mg/ml of [SARSHRC-PEG4]2-chol or [HPIV3HRC-PEG4]2-chol in DMSO (10 mg of peptide in 100 μl of DMSO) and 1 mg/ml of sucrose in sterile water were prepared. 10 μl of the peptide solution (1 mg) was added to 100 μl of sucrose (0.1 mg). Lyophilization of the peptide solution (DMSO+sucrose) was performed over-night and dry powder was resuspended in 50 μl of DI water to a final concentration of 20 mg/ml in water without any DMSO.
Plasmids. The cDNAs coding for hACE2 fused to the fluorescent protein Venus, dipeptidyl peptidase 4 (DPP4) fused to the fluorescent protein Venus, SARS-COV-2 S and the indicated S variants, SARS-COV S, and MERS-S(codon optimized for mammalian expression) were cloned in a modified version of the pCAGGS (with puromycin resistance for selection).
Virus. SARS-COV-2 (isolate BetaCoV/Munich/BavPat1/2020; kindly provided by Prof. Dr. C. Drosten) was propagated to passage 3 on VeroE6 cells in OptiMEM I (1×)+GlutaMAX (Gibco), supplemented with penicillin (10,000 IU/mL, Lonza) and streptomycin (10,000 IU/mL, Lonza) at 37° C. VeroE6 cells were inoculated at a multiplicity of infection (MOI) of 0.01. Supernatant fluid was harvested 72 hours post inoculation (HPI), cleared by centrifugation and stored at −80° C. All live virus work was performed in a Class II Biosafety Cabinet under BSL-3 conditions at Erasmus MC. HPIV3-GFP was commercially obtained from Viratree, propagated to passage 3 on Vero cells in DMEM supplemented with 10% foetal bovine serum (FBS), penicillin (10,000 IU/mL, Lonza) and streptomycin (10,000 IU/mL, Lonza) at 37° C.
Cells. Human embryonic kidney (HEK) 293T and Vero (African green monkey kidney) cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific) supplemented with 10% FBS and antibiotics in 5% CO2 at 37ºC. VeroE6 (ATCC CRL-1586) and VeroE6-TMPRSS2 cells were grown in DMEM (Gibco) with 10% FBS, 2 mM L-glutamine (Gibco), 10 mM Hepes (Lonza), 1.5 mg/ml sodium bicarbonate (NaHCO3, Lonza), penicillin (10,000 IU/mL) and streptomycin (10,000 IU/mL) (7).
β-Gal complementation-based fusion assay. We previously adapted a fusion assay based on alpha complementation of β-galactosidase (β-Gal) (8). In this assay, hACE2 receptor-bearing cells (or dipeptidyl peptidase 4 (DPP4) receptor-bearing cells for MERS-CoV-2 experiments) expressing the omega peptide of β-Gal are mixed with cells co-expressing SARS-COV or SARS-COV-2 glycoprotein S and the alpha peptide of β-Gal, and cell fusion leads to alpha-omega complementation. Fusion is stopped by lysing the cells and, after addition of the substrate (® The Tropix Galacto-Star™ chemiluminescent reporter assay system, Applied Biosystem), luminescence is quantified on a Tecan M1000PRO microplate reader.
HAE cultures & toxicity assay. The EpiAirway AIR-100 system (MatTek Corporation) consists of normal human-derived tracheal/bronchial epithelial cells that have been cultured to form a pseudostratified, highly differentiated mucociliary epithelium closely resembling that of epithelial tissue in vivo (9). HAE cultures were incubated at 37° C. in the presence or absence of 1, 10, or 100 μM concentrations of the different peptides, which were added to the feeding medium every 2 days for 7 days. Cell viability was determined on day 7. Cycloheximide (CHE, a protein synthesis inhibitor in eukaryotes) was used as positive control for toxicity. Cell viability was determined after 24 h using the Vybrant MTT Cell proliferation Assay Kit according to the manufacturer's guidelines. The absorbance was read at 540 nm using Tecan M1000PRO microplate reader.
Antibodies against S-derived HRC peptides (HRCSARS). Polyclonal antibodies against linear epitopes of HRCSARS were generated (Genscript) in rabbits and validated in our western blot and ELISA assays. Genscript provided a full report confirming epitope recognition by the antibodies in an ELISA. The purified sera were aliquoted and lyophilized (10-20 mg in sealed bottles). Several aliquots of the purified sera were conjugated to biotin. Lyophilized aliquots were kept at −80° ° C. Once an aliquot was re-suspended, multiple liquid aliquots (50-100 μl) were made and re-frozen)(−80° ° C.
Mouse biodistribution experiments. Intranasal inoculation (
ELISA for semi-quantitative peptide assessment. 96 well plates Maxisorp (Nunc) were coated overnight with purified rabbit anti-HRCSARS antibodies in carbonate/bicarbonate buffer (pH=7.4, 20 μg/ml). Plates were washed twice in 1×PBS and blocked in 3% BSA/1×PBS for 30 min. The blocking buffer was replaced by 2 dilutions of each sample in 3% BSA/1×PBS in duplicate and incubated for 1.5 h at room temperature (RT). Wells were washed 3× in 1×PBS, and developed with purified rabbit anti-HRCSARS antibodies conjugated to biotin for 1 h at RT. Wells were washed 3 times in 1×PBS, developed using streptavidin conjugated to peroxidase in 3% BSA/1×PBS for 30′ at RT followed by 5 washes and incubation with Ultra TMB substrate solution (Sigma-Aldrich), and stopped with sulfuric acid (12%). Absorbance was read at 450 nm.
Immunohistochemical detection. Lung sections were de-paraffinized and blocked with 10% donkey serum in PBS for 1 h at RT. Rabbit anti-HRCSARS antibody was added and incubated for 12 h at 4° C. Sections were stained with donkey anti-rabbit secondary antibody (Invitrogen, #A31572) for 1 hr at RT. Sections were treated with DAPI, mounted in Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, CA), covered, and imaged with DMi8 (Leica Microsystems, Buffalo Grove, IL).
In vitro potency of fusion inhibitory lipopeptides. Potency of [SARSHRC-PEG4]2-chol and [HPIV3HRC-PEG4]2-chol was determined in an in vitro infectious virus fusion assay. Original stocks and working dilutions for animal experiments were tested in triplicate in VeroE6 and VeroE6-TMPRSS2 cells at concentrations of 0.06 nM to 5 μM (5-fold dilution series). Peptides were pre-incubated with the cells for 1 h at 37° C. After pre-incubation, SARS-COV-2 (600 TCID50/well) was added. After 8 h at 37° C., cells were washed and fixed with 4% PFA for 20 min at RT. Plates were submerged in 70% ethanol and stained in a BSL-2 laboratory. In short, cells were washed with PBS and blocked with 10% normal goat serum (NGS) for 30 min at RT. Primary mouse-anti-SARS-COV nucleocapsid antibody (Biorad) was incubated for 1 h at RT in 10% NGS. After washing, secondary goat-anti-mouse IgG/FITC antibody (Invitrogen) was incubated for 45 min at RT in 10% NGS. Fluorescent spots were visualized with an Amersham Typhoon Biomolecular Imager (GE Healthcare) and counted with ImageQuant TL 7.0 software (GE Healthcare). The activity of [HPIV3HRC-PEG4]2-chol against HPIV3 was determined using a similar assay. Original stocks were tested in triplicate in Vero cells at the same concentrations. After pre-incubation, rHPIV3-GFP (300 TCID50/well) was added. Cells were washed and fixed with 2% PFA after 37 h and fluorescent spots were visualized and counted.
Ferret transmission experiment. For the experiment with DMSO-formulated lipopeptide: Three donor ferrets were inoculated intranasally with 5×105 TCID50/ml of SARS-COV-2 in 450 μl (225 μl instilled dropwise in each nostril) and were housed together in a negatively pressurized HEPA-filtered ABSL-3 isolator. This was considered the start of the experiment (0 days post inoculation, DPI). At the same time, twelve direct contact ferrets were divided over three other isolators. Ferrets were either mock-treated (vehicle, 2% DMSO in DI water) or treated with [SARSHRC-PEG4]2-chol on 1-4 DPI. The peptide was inoculated intranasally in 450 μl (225 μl instilled dropwise in each nostril), HRC dimer-chol treated ferrets received a peptide dose of ˜2.7 mg/kg. Leftover batches were stored at −80° C. for later use in in vitro potency assays (
To assess susceptibility of ferrets to SARS-COV-2 post-treatment, previously mock-treated or [SARSHRC-PEG4]2-chol-treated ferrets were re-housed in pairs of the same treatment schedule into six isolators. Ferrets were challenged in a titration fashion with 5×103, 5×104 or 5×105 TCID50/ml (in 450 μl) of SARS-COV-2. For each dose, two mock-treated and two peptide-treated ferrets were inoculated intranasally. Daily throat and nose swabs were collected from the animals until day 7. All susceptible animals were productively infected with SARS-COV-2 in a dose-dependent manner (
A second ferret transmission experiment (
All animal handling was performed under anesthesia with a mixture of ketamine/medetomidine (10 mg/kg and 0.05 mg/kg, respectively) antagonized by atipamezole (0.25 mg/kg). All animal experiments were performed in class III isolators in a negatively pressurized ABSL3 facility. Ferrets were weighed on a daily basis. Body weights of all ferrets remained stable over time in both the experiment with DMSO-formulated peptides and sucrose-formulated peptides (
RNA isolation and RT-qPCR on throat and nose swabs. Sixty μl of sample (virus transport medium in which swabs are stored) was added to 90 μl of MagNA Pure 96 External Lysis Buffer (Roche). A known concentration of phocine distemper virus (PDV) was added to the sample as internal control for the RNA extraction. The 150 μl of sample/lysis buffer was added to a well of a 96-well plate containing 50 μl of magnetic beads (AMPure XP, Beckman Coulter). After thorough mixing, the plate was incubated for 15 min at room temperature. The plate was then placed on a magnetic block (DynaMag™-96 Side Skirted Magnet (ThermoFisher Scientific)) and incubated for 3 min to allow the displacement of the beads towards the side of the magnet. Supernatants were carefully removed and beads were washed three times for 30 sec at room temperature with 200 μl/well of 70% ethanol. After the last wash, a 20 μl multi-channel pipet was used to remove residual ethanol. Plates were air-dried for 2 min at room temperature. Plates were removed from the magnetic block and 50 μl of PCR grade water was added to each well and mixed. Plates were incubated for 5 min at room temperature and then placed back on the magnetic block for 2 min to allow separation of the beads. Supernatants were pipetted in a new plate and RNA was stored at −80° C. The RNA was directly used for RT-qPCR using primers and probes targeting the E gene of SARS-COV-2 as previously described.
Virus isolation from throat and nose swabs. SARS-COV-2 was isolated in VeroE6 using an infectious center assay determining the tissue culture infectious dose-50 (TCID50/ml). Cells were inoculated with 50 μl of sample (virus transport medium in which swabs are stored, first dilution 1:3), which was diluted in a 3-fold dilution series in quadruplicate. VeroE6 were screened for cytopathic effect (CPE) after 6 days of culture, the infectious titer in TCID50/ml was calculated.
Detection of SARS-COV-2-specific antibodies in ferret sera. Seroconversion of ferrets was tested with ferret sera obtained at 0, 7, 14 and 21 DPI. A S and nucleocapsid (N) ELISA were performed. High-binding ELISA plates were coated with 20 ng recombinant His-tagged S protein (SinoBiological) or 100 ng recombinant His-tagged N protein (SinoBiological) in PBS overnight at 4° C. Subsequently, plates were washed with PBS-Tween followed by a blocking step with Blocker blotto in TBS (Life technologies) containing 0.01% Tween-20 (37° ° C., 1 hr). Sera were tested in duplicate at a concentration of 1:100 diluted in blocking buffer. After 1 hr incubation at 37° C., plates were washed and incubated with goat-anti-ferret IgG H&L/HRP (Abcam) for 1 h at 37° C. After washing, TMB substrate (Seracare) was incubated for 5 minutes in the dark. The reaction was stopped using sulfuric acid and absorbance was measured at 450 nm in a Tecan M200 plate reader. Virus neutralizing antibodies were detected by endpoint titration assay. Briefly, duplicates of ferret sera were incubated with 100 TCID50 of SARS-COV-2 in a 2-fold dilution series starting at a concentration of 1:8 for 1 hr at 37° C. Virus-sera mix was added to VeroE6 cells and incubated for 5 days at 37° C. CPE was used as readout to determine the minimal serum concentration required to inhibit CPE.
Statistics. Inhibitory concentrations 50% and 90% (IC50 and IC90, respectively) in fusion assays were calculated by performing three parameter nonlinear regression. The difference between IC50s were compared by Two-way ANOVA. IC50 and IC90 in infectious virus assays were calculated by performing four parameter nonlinear regression with variable slope on normalized and transformed data. All line graphs in in vivo experiments were compared by 2-way ANOVA repeated measures. Areas under the curve were calculated with GraphPad Prism on basis of curves per animal, and were compared by Mann-Whitney test. All statistics were performed with GraphPad Prism V9.
SARS-COV-2 is a Betacoronavirus that emerged from China in 2019. It is responsible for the COVID-19 (Coronavirus disease 2019) pandemic that has already caused millions of deaths worldwide. Although vaccines are available, it is important to have alternative and complementary prophylaxis, especially for people who are vulnerable or refractory to vaccination.
The entry of SARS-COV-2 occurs through the attachment and fusion of the viral envelope with the plasma membrane of the host cell and is mediated by the viral glycoprotein S. This trimeric class I protein has N- and C-terminal repeated heptades (HR) organized in 6 anti-parallel helixes. We generated peptides from the HR region at the C-terminal position of the S protein of SARS-COV-2, coupled to a lipid. These peptides inhibit viral entry by binding the N-terminal heptad-repeat (HR) regions of the surface proteins. We tested their ability to inhibit viral entry into the cell both in vitro and ex vivo and to prevent viral transmission. The ability of these peptides to protect an animal model sensitive to SARS-CoV-2 was also investigated through an in vivo study.
Certain peptides inhibit viral fusion with a 90% inhibitory concentration (IC90) in the nanomolar range, and subsequently infection and viral dissemination in mice. These peptides were then administered to transgenic mice expressing the human ACE2 receptor, under the control of cytokeratin K18 (B6.Cg-Tg (K18-ACE2) 2Prlmn/J, Jackson) promoter intranasally, prior to infection with SARS-COV-2. Although infection was generally 100% lethal within 10 days post-infection in K18-hACE2 mice, 80-100% of animals treated with these 2 peptides survived respectively, with significantly reduced viral loads in the lungs 2 days post-infection, compared to untreated animals.
In conclusion, these results demonstrate that the peptides that inhibit fusion between the virus and its host cell also block the respiratory infection of SARS-COV-2 in vivo in a mouse model, thus constituting a new antiviral approach to be developed through administration of the inhibitory peptides, in order to fight the current covid-19 pandemic.
This application claims priority to United States Provisional Application Nos. 63/091,915 filed on Oct. 14, 2020; 63/107,429 filed on Oct. 29, 2020; 63/139,302 and 63/139,306 filed on Jan. 19, 2021; 63/144,606 filed on Feb. 2, 2021; and 63/145,453 filed on Feb. 3, 2021, all of which are herein incorporated by reference in their entirety. All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application. This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
This invention was made with government support under grants AI114736 and AI121349 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/54789 | 10/13/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63091915 | Oct 2020 | US | |
| 63107429 | Oct 2020 | US | |
| 63139302 | Jan 2021 | US | |
| 63139306 | Jan 2021 | US | |
| 63144606 | Feb 2021 | US | |
| 63145453 | Feb 2021 | US |
