Patent Application
20230159597
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 including or with SEQ ID:NO2 or SEQ ID NO:3. In certain aspects, the invention provides a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
In certain aspects, a SARS lipid-peptide fusion includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and a lipid tag.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a SARS lipid-peptide fusion inhibitor includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, and a spacer.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the SARS lipid-peptide fusion inhibitor further includes a cell penetrating peptide sequence (CPP). In some embodiments, the CPP is HIV-TAT.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and a pharmaceutically acceptable excipient.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, 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. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the coronavirus lipid-peptide fusion inhibitor further includes a cell penetrating peptide sequence (CPP). In some embodiments, the CPP is HIV-TAT.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor and a pharmaceutically acceptable excipient. The SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor further includes a peptide selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, a lipid tag, a spacer, and a CPP.
In some embodiments, the peptide is SEQ ID NO:2 or SEQ ID NO:3.
In certain aspects, the invention provides a method of treating COVID-19 that includes administering to a patient an antiviral pharmaceutical composition. The antiviral pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, a spacer, a CPP, and pharmaceutically acceptable excipients.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
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.
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 inventors discovered that that this type of lipid-peptide molecule is highly effective at preventing and even treating lethal infections of other viruses, like measles, lethal Nipah virus, influenza, and others. The designed peptides are highly effective at inhibiting live SARS-CoV-2 (COVID) virus infection in cultured cells and ex vivo.
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 designed specific peptides, linked to lipids, that inhibit viral fusion and infection by binding to transitional stages of the spike protein, preventing its function. Importantly, based on evidence from the other viruses that the inventors targeted, these antivirals can be given by the airway, by nasal drops, 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 feasible for widespread use.
The inventors designed several assays for assessing potency and mechanism in BSL2 laboratory conditions, which thus far precisely predict efficacy vs. live SARS-CoV-2 in cell culture. The prototype peptides are highly effective in blocking SARS-CoV-2 spike protein fusion and viral entry assays in cultured cells, and at inhibiting live SARS-CoV-2 (COVID) virus infection in vitro and ex vivo. Improvements to these antivirals will make them even more effective, more resistant to being broken down in the lungs or blood, and better at interacting with the spike protein to block its transitional states. 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 including or with SEQ ID:NO2 or SEQ ID NO:3. In certain aspects, the invention provides a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
In certain aspects, a SARS lipid-peptide fusion includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and a lipid tag.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a SARS lipid-peptide fusion inhibitor includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, and a spacer.
In some embodiments, the spacer is a polyethylene glycol (PEG). In some embodiments, the spacer is PEG4. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the SARS lipid-peptide fusion inhibitor further includes a cell penetrating peptide sequence (CPP). In some embodiments, the CPP is HIV-TAT.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and a pharmaceutically acceptable excipient.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, a pharmaceutically acceptable excipient.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In certain aspects, a pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, 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. In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
In some embodiments, the coronavirus lipid-peptide fusion inhibitor further includes a cell penetrating peptide sequence (CPP). In some embodiments, the CPP is HIV-TAT.
In certain aspects, a SARS-COV-2 (COVID-19) antiviral composition includes a SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor and a pharmaceutically acceptable excipient. The SARS-COV-2 (COVID-19) lipid-peptide fusion inhibitor further includes a peptide selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, a lipid tag, a spacer, and a CPP.
In some embodiments, the peptide is SEQ ID NO:2 or SEQ ID NO:3.
In certain aspects, the invention provides a method of treating COVID-19 that includes administering to a patient an antiviral pharmaceutical composition. The antiviral pharmaceutical composition includes a peptide including or with SEQ ID:NO2 or SEQ ID NO:3, or a peptide including or with a sequence with more than 80%, 85%, 90%, 95%, but less than 100% homology with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, a lipid tag, a spacer, a CPP, and pharmaceutically acceptable excipients.
In some embodiments, the lipid tag is Cholesterol, Tocopherol, or Palmitate.
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.
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.
Coronavirus Infection
Coronaviruses (CoVs) can cause life-threatening diseases. The latest disease was recently 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. No vaccines or treatments for COVID-19 are yet available. Antivirals that target viral entry into the host cell have been proven effective against a wide range of viral diseases.
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 as a large homotrimer, each monomer having several domains (
Like the influenza HA, this S 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 may 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
Pathways of Viral Entry and Strategies for Inhibition
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 (F) 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 broad spectrum activity and potency of the conjugates. For the purpose of this application, the words “linker” and “spacer” are used interchangeably. 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 led to effective antiviral C-peptides for Ebola infection. In
Proof of Principle: Fusion Lipid Peptides
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.
HRC peptides inhibit viral fusion and entry in a dominant-negative manner by binding to the pre-hairpin intermediate, preventing formation of the 6HB. For strains that fuse at the cell membrane (early entry), HRC peptides without additional components can prevent viral entry, but these peptides are ineffective on strains that fuse in the endosome (late entry). The intracellular sequestration of S could make it challenging to develop HRC peptide fusion inhibitors against endosomal fusing coronavirus strains. To target endosomal fusing coronaviruses including SARS-CoV-2, in addition to the proven lipidation and pegylation strategies, we incorporated a cell penetrating peptide sequence (CPP in
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 (
For viruses that fuse at the target cell membrane, lipid conjugation to HRC peptides markedly increases antiviral potency and in vivo half-life. Lipid conjugation also enables activity against viruses that do not fuse until they have been taken up via endocytosis. For example, we showed that lipid-conjugated HRC peptides derived from MERS (see below) inhibit MERS infection, suggesting that the lipid-conjugation-based strategy generates inhibitors of fusion with endosomal membranes. A similar strategy led to effective antiviral peptides for Ebola infection, which fuses between the late endosome and the lysosome. These lipid-peptides “follow” the virus into intracellular compartments.
We designed and produced MERS-CoV-specific lipid-conjugated peptides based on a peptide sequence shown to be effective in vivo after intra-lung administration. In 2014, these peptides made by our design were tested against MERS-CoV in vitro (
Proof of Principle: Inhibition of Live Ebola Infectivity In Vitro.
We compared the efficacy of the above C-peptides vs. live ZEBOV infection in vitro in collaboration with UTMB's BSL4 facility (Table 1). Control Ebola C-peptides derived from the same HR domain but without the TAT (CPP motif) sequence were ineffective even when lipid conjugated (100 μM was the highest concentration tested). Thus, the inhibitory activity for Ebola virus in particular requires both the TAT sequence and the lipid conjugation.
indicates data missing or illegible when filed
Proof of Principle: Inhibition of Mouse Adapted (MA)-ZEBOV In Vivo:
Three C-peptide inhibitors in Table 1 (highlighted in) were first tested for acute toxicity in mice at 20 mg/ml for 14 days by intraperitoneal (i.p.) delivery, without any tolerability issues. Mouse pharmacokinetic studies confirmed the presence of the lipid conjugated C-peptide inhibitors in the plasma for at least 24 hrs. For the in vivo study presented in
Proof of Principle: Lipid-Conjugated Inhibitory Peptides Undergo Cellular Internalization.
Since the TATEBOLA-dPEG4-Toc peptide is effective in vivo (
In summary, we showed that TAT sequence and the lipid moiety both promote efficient intracellular localization and in vivo efficacy for intracellular fusing viruses, and both in various combinations may be useful for coronaviruses. Scientific premise: the coronavirus entry pathway into target cells is promiscuous.
Design, Generate and Characterize Improved Inhibitory SARS-CoV-2 S Specific C-Peptides
We identified lipid-derivatized MERS-CoV-S-derived entry inhibitors that effectively block MERS (see
Sequence of the HRC Domain of the SARSCoV-2 S Protein
The SARS-CoV-2 6HB assembly (
Peptide D-1 (
A common approach will be used to assess HRC-based peptides generated in this program. Circular dichroism (CD) measurements will indicate whether HRC derivatives coassemble with the HRN peptide, and if so, to assess assembly stability. For promising HRC derivatives, cocrystallization with the HRN peptide will provide structures analogous to that in
Use Structure Guided Mutagenesis
indicates data missing or illegible when filed
Using structure-guided mutagenesis and protein engineering to optimize the antiviral potency and bioavailability of EBOV C-peptide fusion inhibitors, as an example to support the work for SARS-CoV-2 that we discuss here. We assessed the IC50 of several peptides with modifications in the lipid moiety (either at the C or N terminal) and/or in the polyethylene glycol (PEG) spacer (size and origin). The results are shown in Table 2, above.
We also expanded the data in Table 3 using several strains of Ebola virus. The preliminary data show potency in the nanomolar range against several Ebola virus strains (see Table 3).
The newly identified sequence that we designed was tested against live virus (see Table 4, below). Based on biophysical data the sequence IEP (shown in highlight in Table 2) was modified to IAAILP highlight in Table 4). Contrary to our hypothesis, the TAT-EBO-IAAILP-PEG4-Chol (see table 4) had an IC50 of 0.6 uM, around 10 times higher than both the TAT-EBO-PEG4-Chol and TAT-EBO-dPEG4-Chol (see Table 2) that have similar structures (PEG4 and cholesterol). We concluded that the sequence modification was detrimental to antiviral activity. However, we found that the TAT-EBO-IAAILP-Chol without PEG4 linker had an IC50 of 3 nM, around 20 times better than the most potent peptide identified so far. Additionally, the IC90 was 27 nM—around half of the IC50 of our best peptide up to this point.
indicates data missing or illegible when filed
We prepared the TAT-EBO-Chol (the original sequence in Table 2 but without PEG). We tested the original sequence and compared it to the newly modified sequence in
Addition of Lipid and Cell-Penetrating Peptide Sequences to Improve Efficacy and Intracellular Targeting
We designed SARS-CoV-2 S specific C-peptides (see
These data will provide information regarding the most effective HRC S derived aa sequence among 7 sequences (the 5 in
Assessment of peptide toxicity in monolayer cell culture: 5 peptides will be assessed for toxicity as in previous work5. Toxicity will be evaluated by Vybrant® MTT cell proliferation assay (Invitrogen).
SARS-CoV-2 infections will be performed first in Vero cells with confirmation in Calu-3 cells, and peptides that show efficacy against live virus in these cells will move to experiments in HAE (commercially acquired). Serial dilutions of peptide inhibitors will be added either before or after infection to evaluate the effect of the peptides in preventing viral entry and whether the peptides block viral spread within the tissue after infection. In addition, we will study the HAE tissue for evidence of toxicity of the peptide using established protocols. We will use no more than ˜5 peptide inhibitors to study the ex vivo activity. We have shown that HAE are an ideal model to assess fusion inhibitory peptides activity. We have also recently shown that the human developmental lung organoid model represents the developing lung and can model several aspects of respiratory infections and we may use this model for SARS-CoV-2 in the future. These two models will be used as in our published work to assess peptides effectiveness against SARS-CoV-2. Viruses that emerge from growth in HAE (or organoids in future work) will be sequenced to assess evolution as we have done previously and to evaluate any peptide-resistant variants that emerge.
Middle East Respiratory Syndrome (MERS, caused by MERS-CoV) is a respiratory illness that was new to humans when it was first reported in 2012. We designed and produced several MERS-CoV specific lipid conjugated peptides based on a peptide sequence shown to be effective in vivo after intra-lung administration.
In 2014, these peptides designed by us were tested against MERS-CoV in fusion assays (
We have recently tested these MERS-S derived peptides in fusion assays using the SARS-CoV-2 S protein. Even without the cell penetrating sequence, the addition of lipid moieties increased the peptides' potency in fusion assays (
100% reduction in SARS-CoV-2 infection was observed using live virus and our MERS lipid-peptide in cell culture (
Lipid-peptide based on SARS-COV-2 was even more effective than the MERS lipid-peptides.
Inhibition of SARS CoV-2 glycoprotein fusion with the indicated peptides. The cell-to-cell fusion of 293T cells expressing SARS-CoV-2 glycoprotein bearing the indicated mutations and α-subunit of β-galactosidase with 293T cells transfected with ω-subunit of α-galactosidase and A) transfected hACE2 receptor or B) without transfected hACE2 receptor was assessed by a β-Gal complementation assay, in the presence of increasing concentrations of the indicated peptides. Resulting luminescence from β-galactosidase was quantified using a Tecan Infinite M1000 Pro. The percent inhibition of fusion (compared to results for control cells not treated with peptide) is shown as a function of the concentration of peptide. The values are means (±SD) of results from one experiment. The sequences of the peptides are in the diagram below (
SARS lipid-peptides were effective against SARS live virus. IC50 is estimated at around 5-10 nM, indicating that the level needed is achievable in people. Notably,
In order to understand the determinants of infection in the natural host, we will use the HAE model that has been used to characterize the polarity and cell specificity. We used this model for parainfluenza infection, confirming that it reflects virus-HAE interactions in the human lung. We and others have documented that results in immortalized monolayer cells may not be applicable when translated in vivo, and thus it is important to test our hypotheses in models that more closely represents the natural host. The HAE is ideal for assessing field isolates in experiments that replicate the clinical scenario.
The human airway epithelium (HAE) mostly consists of large airway tissue grown at air-liquid interface (
We also may utilize Human lung organoids as a model (
The clinical use of Fuzeon© for HIV-1 resulted in the emergence of drug resistant HIV-1 variants. Escape variant viruses also emerged upon in vitro passaging of HIV-1 in the presence of Fuzeon©. The resistant viral population acquired mutations within a highly conserved stretch of three HRN amino acids, glycine-isoleucinevaline (GIV). Resistance mutations in this GIV motif also exist within the viral quasi-species of patients on Fuzeon© therapy. The resistance was due to either decreased interaction between the viral HRN and Fuzeon©, or increased interaction between viral HRN and HRC. Increased kinetics of fusion led to resistance, but also to viruses whose growth depended on the drug. While anti-SARS CoV-2 therapy will be of shorter duration than that for HIV (acute vs. chronic treatment), resistance may be important clinically, as it is for influenza. Based on the results in HIV and influenza, the in vitro data on emergence of resistance will apply directly to in vivo behavior of the viruses under selective pressure of treatment, and can be used to predict resistance and preemptively improve C-peptide fusion inhibitor design.
Strategy: SARS-CoV-2 infections will be performed in HAE. At recombinant SARS-CoV-2 virus bearing the EGFP gene (EGFP-SARS-CoV-2) has been recently produced. This virus will be used to monitor viral evolution under C-peptides' selective pressure in real time. Serial dilutions of peptide inhibitors will be added either before or after infection to evaluate (i) the effect of the peptides in preventing viral entry; (ii) whether the peptides block viral spread within the tissue after infection. In addition, we will study the HAE and organoid tissue for evidence of toxicity of the peptide using established protocols. Following assessment of antiviral activity in HAE, infections will be performed under the selective pressure of optimized C-peptide fusion inhibitors to analyze the molecular basis of potential resistance; to predict the possibility of evolution of C-peptide-resistant viruses; and to provide information that will be used to identify the C-peptide fusion inhibitors least likely to select for resistance.
Ex vivo antiviral activity: We have shown that HAE are an ideal model to assess fusion inhibitory peptides activity. This model is used to assess C-peptides effectiveness against SARSCoV-2, as in
Generation of resistant variants: We will attempt to elicit SARS-CoV-2 viruses resistant to the inhibitory effect of small molecules using protocols routinely performed in our laboratory. Briefly, several dilutions of SARSCoV-2 will be passaged in HAE in the presence of several concentrations of C-peptides (ranging between 5x and 40x the IC50) for three to four days in HAE. Note C-peptides will be added after the initial infection to allow the viral polymerase complex to replicate and produce phenotypic variants for selection. Resistant viruses would spread even in the presence of C-peptides. Yields of virus will be determined by plaque assays and/or by qRTPCR. As the virus spreads in the presence and absence of inhibitor, the concentration of the inhibitor will be gradually increased to obtain a population of resistant viruses. Passaged virus will be sequenced as well as tested for inhibitor sensitivity in a plaque reduction assay. This strategy of applying selective pressure for viral evolution is similar to the informative experiments performed in our lab for neuraminidase-resistant variants and small molecule inhibitor-resistant variants.
Analysis of resistant variants in vitro: Mutant resistant viruses before and expansion (by growth in HAE), will be sequenced. We will analyze resistant virus mutants by high depth, whole viral genome sequencing. Sequences of the HAE-grown viruses will be compared to population-derived sequences generated during the duration of the selection experiments using custom bioinformatics software specifically made for longitudinal analysis of viral evolution. This approach will prevent us from neglecting potentially important viral subpopulations or alleles present across the genome that may co-exist during or after the selection process. We will determine whether the fitness of each variant is similar to that of the parent virus, or whether the variants require the presence of inhibitor for viability. We have extensive experience and previously validated both approaches, showing that the allele frequencies match for the two approaches with Paramyxoviridae such as canine distemper virus, human parainfluenza virus 3 (HPIV3), and respiratory syncytial virus. Shotgun sequencing enables a simple, one-workflow protocol for all RNA viruses, while tiling RT-PCR enables specific selection of viral sequences from complex sample types. We will sequence these viruses to a minimum average depth of 200X and call all variants with an allele frequency >4%. Sequence reads will be analyzed using our custom bioinformatic pipeline for longitudinal analysis of viral alleles in which reads for each sample are aligned to a de novo assembly consensus reference of the day/passage 0 viral genome.
If S contains mutations, we will introduce the mutated genes into our expression vectors and evaluate the glycoprotein functions in our functional assays. If multiple mutations are found, site-specific mutagenesis will be used to introduce the mutations into the S background, and singly-mutated genes will be analyzed for their phenotypes using the same in vitro assays. Location and conservation of the mutations will tell us the extent to which the resistance mechanism(s) for different peptides are similar. If the mutants derived from different peptides are markedly different, we will analyze the contributions of the specific mutations to dissect each contribution.
Analysis of resistant variants in vivo: If we identify resistant variants that grow well in vitro and ex vivo, we will assess their in vivo fitness. Resistant variants' pathogenicity will be compared in vivo to the parent virus. The total number of animals will depend on the number of resistant variants. One mouse model we will use to assess the resistant variants here and the peptides' efficacy is a human angiotensin-converting enzyme 2 (ACE2) transgenic mouse (hACE2 mouse). This model has been shown to be a lethal model for SARS-CoV-1. A recent report shows that for SARS-CoV-2 the model is not lethal, but weight loss and pathological signs are observed. Both gross pathology and histopathology can be easily observed at both day 3 and day 5 post infection. Viral titers of 106-107 pfu/ml were obtained after 1-3 days post infection. We have pre-ordered these mice from Jax laboratory and we expect to get mice in June 2020. This animal model of infection will be used to assess whether peptide-resistant variants cause altered pathology with respect to wt and if their fitness decreases or increases as a consequence of resistance mutations. The hACE2 mice will be used for assessment of antiviral efficacy. We anticipate testing ˜4 variant viruses plus 1 wt for a total of 5 viruses (10 animals; 5 males and 5 female-per group, for a total n=50 mice).
Sample collection and analysis: Tissue samples of all major organs will be collected from each mouse for histopathology assessment and viral load (by qRT-PCR). Virus isolation will be done only from specimens positive for EGFP-SARS-CoV-2 by qRT-PCR. Virus titration will be performed by plaque assay. Samples will also be sequenced to assess viral evolution in vivo.
The sequence alterations in S will be linked with the functional alterations, and this information will be used to understand resistance mechanisms. For parainfluenza, enhanced fusion kinetics led to partial resistance to peptide inhibitors in vitro, but we showed that mutations that increase fusion kinetics have a negative impact on growth in natural host tissue; these resistance mutations are likely to significantly reduce fitness in vivo. We expect the resistant CoV variants to be less pathogenic in vivo. We propose that by assessing these mechanisms early on in antiviral development we will avoid advancing antiviral strategies that could lead to more pathogenic viruses. Determining the ease of generation of variants and the fitness of SARS-CoV-2 containing resistance mutations will permit us to predict the likelihood of evolution of clinically relevant resistant variants. If resistance is acquired within four to five passages, we will consider combining our C-peptides with the protease inhibitors discussed elsewhere, to test the hypothesis that the combination treatment will provide a higher barrier to resistance. We also consider the possibility that no resistance will be elicited —although this is unlikely—, it would suggest that the fitness cost is too high to generate a viable variant resistant to that specific C-peptide. Such a C-peptide would be an ideal candidate to move forward. The information gained here will be used to select C-peptides with the lowest likelihood of eliciting resistance. The C-peptides that are effective in ex vivo, and are the least likely to elicit resistance in vivo, will be tested for in vivo efficacy.
Discussion: The primary focus is to obtain an effective C-peptide for the SARS-CoV-2 virus, but at the same time from these experiments we will know whether the coronavirus family can be inhibited by one C-peptide. We have already shown (
We will conduct pharmacokinetic and safety studies in mice. We will determine whether the in vitro improved peptides identified have the desired serum half-life and tissue biodistribution profiles, and whether they are safe and well tolerated in vivo. We will use the human angiotensin-converting enzyme 2 (ACE2) transgenic mouse50-52 (hACE2 mouse) to assess in vivo anti-SARS-CoV-2 efficacy. A recent report shows that for SARS-CoV-2 the model is not lethal, but weight loss and pathological signs are observed.
(https://www.biorxiv.org/content/10.1101/2020.02.07.939389v3). Both gross pathology and histopathology can be easily observed at both day 3 and day 5 post infection. Viral titers of 106-107 pfu/ml are obtained after 1-3 days post infection.
We showed that lipid modification not only increases the antiviral efficacy of the lead SARS-Cov-2 fusion-inhibitory peptide but also overcomes the typically poor pharmacokinetics of peptide drugs, prolonging the peptides' circulatory half-life to clinically useful levels. We will determine the extent to which the mutations and backbone modifications designed to increase anti-SARS-Cov-2 potency and protease resistance affect the pharmacokinetic properties of select improved SARS-Cov-2 peptides. Our goal is to ensure that the improved peptides reach an effective concentration in vivo, and to evaluate (i) the minimal dosage and (ii) frequency of administration required to maintain it. Here we also assess potential side effects and the kinetics of drug clearance. It is encouraging that in our in vivo paramyxovirus experiments and in preliminary pharmacokinetic studies no toxic effects were observed in mice and hamsters treated for up to 21 days at 20 mg/kg.
Pharmacokinetics (PK) of select improved peptides in mice (or in any other animal model that we find advantageous, as discussed below) will be assessed as we have previously done for similar peptide inhibitors. We will assess 4 peptides. Mice (6 per group, 3 males+3 females to capture sex as a variable) will be injected subcutaneously (s.q.), intraperitoneally (i.p.), intranasally (i.n), and (i.t) (we will initially test all four routes). Our preliminary data indicate that i.p. delivery is effective for MERS treatment (see
Our published data show both prophylactic and therapeutic efficacy of lipidated peptides in vivo for Nipah virus, measles virus (MV), and influenza, and preliminary data for Ebola (not shown here) and MERS (
In vivo efficacy vs. Nipah (lethal virus) infection in golden hamsters: 2 mg/kg/d subcutaneous delivery of the lipid-peptide was effective (
In vivo efficacy vs. Nipah (lethal virus) infection in golden hamsters: the lipid-peptide was administered intranasally. An administration at 1 day before, day of, 1 day after can provide 60% protection from lethal infection (
In vivo efficacy vs. influenza infection. Peptides given intranasally three times:1 day before, day of, 1 day after 1000x lower viral titer in cotton rats (
In vivo efficacy for preventing measles infection (fatal encephalitis) in mice with measles peptides. Both subcutaneous and intranasal administration were explored (
Comparison of quantitative fusion assay, with different expression level of hACE2 receptor. These data (shown above in
The most effective two peptide fusion inhibitors in vitro with favorable toxicity/biodistribution profiles will be tested in SARS-CoV-2 infections in hACE2 mice, and/or in the other relevant animal models as these become necessary and advantageous. We will first determine whether protection is afforded by i.n. peptide administration, prior to, concomitant with, or up to 10 days after the infection, and will establish the optimal dosage. Based on these data from we will undertake prophylactic and therapeutic studies using alternative delivery routes (s.q.).
Dosing: For the initial screening of two optimized peptide inhibitors, and for determining the optimal dose, groups of 10 animals will be treated with 3 different doses of the peptide i.n. and s.q. one day prior to challenge and then daily for up to 2 days. Infection will be performed with 105 TCID50 of SARS-CoV-2 i.n.
Efficacy: Once we determine an effective dose, we will focus on determining the therapeutic window for postexposure treatment. This is important, as this is an important likely use of the product to manage an outbreak. We will determine how many days after infection a peptide treatment can provide protection. See VA section.
Animal numbers: For dosing: (2 peptides+scrambled+mock treated)×10 mice X 2 inoculation routes X 3 doses=240 mice. Efficacy: 2 peptides X 10 mice X 1 inoculation route X 4 time points=80+10 untreated mice.
Viral load from lung will be determined by plaque assay and qRT-PCR. Sample tissues from treated and untreated animals will also be sent for sequencing to determine whether viral evolution occurred during treatment.
The readout of the model is clear, and statistically significant groups will be formed. Animals of both sexes will be used to ensure the capture of sex as a variable. We expect that prophylactic administration of the peptides will protect against infection. Whether the peptides are also effective in a post-exposure regimen will be determined. Intranasal delivery will likely work well for prophylaxis but it is possible that s.q. will work better after initial infection, and depending on the results we may decide to treat post-exposure via s.q. injection.
Quantitation of peptide concentration is done by ELISA, since we found that HPLC-MS has a limit of detection too high for assessment of peptides in certain organs (data not shown). Block amphiphiles such as our peptides could have surfactant properties, leading to epithelial irritation; therefore, testing toxicity is important. Only peptides that do not exhibit toxic effects will progress to the efficacy study. Therapy for coronaviruses is expected to be of short duration; however, it is possible that antibodies may be generated against peptides during treatment that may affect the treatment. We have not observed such an effect while studying Nipah-infected hamsters, measles infected mice and cotton rats. To test for the possibility of antibody antagonism to treatment, we will collect the serum from animals used for toxicity studies outlined above and assess for interference with the peptide's inhibitory activity. We consider the possibility that the explanation for the non-lethalitiy of in vivo infection with SARS-CoV-2 in hACE mice may be due to the old age of the animals (6-11 months). We will determine whether younger mice may permit a lethal model of infection. Survival curves would be a more statistically significant read out for efficacy.
From these experiments we will know whether we have an effective inhibitor for SARS-CoV-2 for the currently circulating CoV (and whether coronavirus family viruses can be inhibited by one peptide). We will obtain a good understanding of the molecular basis for the inhibitory activity of the peptides and correlation between structural and stability properties and inhibitory potency, useful to guide peptide design. As the result of the proposed work, we are confident—based on the data presented here and our published data that an effective prophylactic regimen will be achieved. Developing a post-infection treatment is more difficult; however, prophylaxis itself will be critically important. Health care workers would benefit directly from our prophylactic approach since it could be easily administered (e.g., once a day i.n.) and is effective immediately and at least for 24-hours (vs. a longer-term vaccine strategy). At risk people will also benefit from such a prophylactic treatment. At the end of this project, we will have (1) identified SARS-CoV-2 peptide fusion inhibitors; (2) optimized their antiviral activity in vitro and ex vivo; (3) tested the efficacy of these novel fusion inhibitors in a relevant animal model.
We will determine the anti-SARS-CoV-2 potency, protease resistance, and the pharmacokinetic properties of the SARS-CoV-2 C-peptides. Our goal is to ensure that the HRC peptides reach an effective concentration in vivo, and to evaluate (i) the minimal dosage and (ii) frequency of administration required to maintain it. Here we also assess potential side effects and the kinetics of drug clearance.
Pharmacokinetics (PK) of the HRC peptides in mice will be assessed as we have previously done for similar peptide inhibitors. The intratracheally (i.t.) delivery in mice provides consistent results compared to i.n. delivery and this will help the biodistribution study and be used for prophylactic studies to represent delivery via airway if needed. We will assess 4 peptides. Mice (6 per group, 3 males+3 females to capture sex as a variable) will be injected subcutaneously (s.q.), intraperitoneally (i.p.), intranasally (i.n), and (i.t) (we will initially test all four routes). Our preliminary data indicate that i.p. delivery is effective for MERS treatment (see
Immunofluorescence: Cryo-sections will be stained with specific rabbit anti-SARS-Cov-2 HRC antibody (that we will generate using a contractual company as we have done regularly). Tissue sections will be analyzed using confocal microscopy.
ELISA for biodistribution studies: Organs will be homogenized using a “BeadBug” homogenizer. Peptide concentration in tissue samples and serum will be determined as we have done before5,7,8. Standard curves will be established for the lead peptides, using the same ELISA conditions as for the test samples (note this is more sensitive than the LC/MS/MS we used previously).
Evaluation of SARS-CoV-2 C-peptide toxicity in mice: We will undertake acute systemic toxicity testing in mice (for the peptides with the best biodistribution profile) to evaluate the toxicity and dose tolerance of the improved SARS-CoV-2 peptides. Purpose-bred outbred mice (n=6 per group, 3 male and 3 female) will receive a single s.q. injection of 5, 20 or 200 mg/kg of fusion inhibitory C-peptide. Harlan isovolumetric saline will serve as the control. Animals will be closely monitored for survival and/or signs of distress. For 15-day toxicity study, animals will be inoculated s.q. with peptide (20 mg/kg) for 15 consecutive days, and monitored daily. For chronic toxicity, peptides will be administered s.q. and i.n. to mice (n=6 per group) twice weekly at a dose of 20 mg/kg for 30 (i.e., 8 inoculations) or 60 (i.e., 16 inoculations) days. On days 30 and 60, animals will be sacrificed for determination of body and organ weights and gross pathologic examination (as described; 5,7,52-54) as well as for histopathology. Statistical significance of the mean of the treated group compared with that of the control group will be analyzed by a one-way analysis of variance, followed by Dunnett's multiple comparison tests using the Prism program (Graphpad, San Diego). Differences will be considered statistically significant if p<0.05.
These experiments will determine the effective half-life for the therapeutic dose and the gross biodistribution of SARS-CoV-2 peptide inhibitors in mice, and whether (as we anticipate) the SARS-Cov-2 peptide fusion inhibitor is non-toxic at likely therapeutic doses. More potent inhibitors will allow for lower dosages. Only the peptides that do not exhibit toxicity will progress to the efficacy study. We consider the possibility that a combination of the different delivery routes (s.q., i.p., i.t., and i.n.) may yield a favorable balance between biodistribution profile and ease of use with minimal adverse side-effects. Delivery to mucosal surfaces (e.g. i.n./i.t.) would be an easy and effective way to treat prophylactically, and this strategy would be applicable in the field or in hospitals (e.g. to protect health care providers). For critically ill patients or others who cannot tolerate i.n. medication, parenteral administration will be preferable. We expect to show that i.n. with a large volume (i.e., 50 μl) will result in consistent lung delivery 10 and this will be assessed by comparison to i.t., since i.t. has been shown to mimic delivery via airway. This will be important for in vivo challenge since (especially for prophylaxis) all animals should receive consistent dosage via i.n. In case i.n does not consistently result in distribution similar to i.t. we will consider i.t., at least for single prophylactic doses. From this we will select the peptides based on the longest biodistribution in the lungs.
Peptide immunogenicity studies: Measurement of antibodies associated with administration of peptides will be performed when conducting repeated dose toxicity studies. Anti-peptide antisera will be used to assay for antibodies generated during the chronic toxicity studies described above. We will attempt to evaluate effects of antibody responses on pharmacokinetics, incidence and/or severity of adverse effects, complement activation, or pathological changes related to immune complex formation and deposition.
Beyond mouse, we will also use different animal models as these are elucidated and become available. (www.sciencemag.org/news/2020/04/mice-hamsters-ferrets-monkeys-which-lab-animals-can-help-defeat-new-coronavirus)
The first animal model we will use is the ferret (Kim et al., Infection and Rapid Transmission of SARS-CoV-2 in Ferrets, Cell Host & Microbe (2020)) for assessing whether our prototype peptide prevents direct transmission of SARS-CoV-2 from an infected animal to uninfected direct contacts. Ferrets are an ideal model for studying prophylaxis and transmission. This animal transmits SARS-CoV-2 very readily to uninfected ferrets, either by direct contact or from cage to cage. (Kim et al.) Ferrets will be treated with nose drops and assessed for protection from infection during contact with SARS-CoV-2 infected contact animals. All direct contacts become infected by 2 days. Ferrets will be treated with nose drops and assessed for protection from infection during contact with SARS-CoV-2 infected contact animals (
We look towards human safety/efficacy in health care workers and other first responders first, after permissible results can be gained from animal tests.
This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/015,479, filed Apr. 24, 2020, the contents of which are hereby incorporated by reference in its 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/US21/28667 | 4/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63015479 | Apr 2020 | US |
