Methods, compositions, and vaccines for treating a virus infection

专利摘要:
This disclosure relates to a method of treating, preventing, or reducing a symptom of a Virus infection, including a SARS—CoV—2 infection, by administering an effective amount of tdsRNA optionally With an anti—Viral agent, to a subject.
公开号:NL2027383A
申请号:NL2027383
申请日:2021-01-25
公开日:2021-09-01
发明作者:L Young Diane；K Equels Thomas；R Strayer David
申请人:Aim Immunotech Inc；
IPC主号:

专利说明:

[3] [3], like the nose and nasal pharynx [2], contains high levels of TLR3 receptors which are required for Rintatolimod to induce both innate and adaptive immune responses including antiviral responses. Thus, the well-tolerated safety profile and bio-activity of Rintatolimod administered intranasally is well documented in both animal models [4,5,6] and humans [7] and a similar bio-activity and safety profile is expected following oral administration.
[1] [1] Gowen BB, Wong M-H, Jung K-H, Sanders AB, Mitchell WM, Alexopoulou L, et al. TLR3 is essential for the induction of protective immunity against Punta Toro virus infection by the double- stranded RNA (dsRNA), Poly{E:C12U), but not Poly(1:C): differential recognition of synthetic dsRNA molecules. J Immunol 2007:178:5200-8.
[2] [2] Hewson CA, Jardine A, Edwards MR, Laza-Stanca V, Johnston SL. Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells. J Virol 2005;79(19):12273-9.
[3] [3] Uehara A, et al. Various human epithelial cells express functional Toll-like receptors, NOD} and NOD2 to produce anti-microbial peptides, but not proinflammatory cytokines, Molecular Immunology 44 (2007) 3100-3111
[4] [4] Ichinohe T, Kawaguchi A, Tamura S, Takahashi H, Sawa H, Ninomiya A, et al. Intranasal immunization with HSN1 vaccine plus Poly I: Poly C12U, a toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect 2007;9:1333—40.
[5] [5] Ichinohe T, Ainai A, Yasushi A, Nagata N, Iwata N, Kawaguchi A, et al. Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A HSN! virus. J Med Virol 2010;82:1754-61.
[6] [6] Ichinohe T, Tamura S, Kawaguchi A, Imai M, Itamura S, Odagin T, et al. Cross-protection against HSN! influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine. J Infect Dis 2007:196:1313-20.
[7] [7] Overton ET, et al. Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, increased cross-reactive IgA antibody formation against avian H5SN1 and H7N9 influenza HA in humans. Vaccine 32 (2014) 5490-5495
[8] [8] Strayer DR, Carter WA, Stouch BC, Stevens SR, Bateman L, Cimoch PJ, et al. A double-blind, placebo-controlled, randomized, clinical trial of the TLR-3agonist rintatolimod in severe cases of chronic fatigue syndrome. PLoS ONE2012;7(3}:e31334. Example 6 Intravenous Rintatolimod or tdsRNA Administration Protocol Schema Study Title: A Phase VII Study to Evaluate the Safety and Activity of Rintatolimod (Poly I:Poly CU) in Patients with Early Onset Coronavirus Disease-2019 (COVID-19) Design: Up to 4 weeks of open-label treatment followed by a follow-up phone call 30+3 days after last dose of study medication. Population: Up to 40 patients who meet the following criteria. I. Positive nasal swab RT-PCR test for COVID-19 a. If symptomatic, treatment initiated within 96 hours of COVID-19 symptom(s) onset. b. If asymptomatic, treatment initiated within 96 hours of positive SARS-COV-2 test. A concurrent control group of up to 40 patients who will not be receiving Rintatolimod, who would otherwise have qualified for the Rintatolimod infusions, will be followed using best standard of care. 92
[2] [2] Hewson CA, Jardine A, Edwards MR, Laza-Stanca V, Johnston SL. Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells. J Virol 2005;79(19):12273-9.
[3] [3] Uehara A, et al. Various human epithelial cells express functional Toll-like receptors, NOD1 and NOD 2 to produce anti-microbial peptides, but not proinflammatory cytokines, Molecular Immunology 44 (2007) 3100-3111
[4] [4] Ichinohe T, Kawaguchi A, Tamura S, Takahashi H, Sawa H, Ninomiya A, et al. Intranasal immunization with HSN 1 vaccine plus Poly I: Poly C12U, a toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect 2007,9:1333-40.
[5] [5] Ichinohe T, Amai A, Yasushi A, Nagata N, Iwata N, Kawaguchi A, et al. Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A HSN1 virus. J Med Virol 2010;82:1754-61.
[6] [6] Ichinohe T, Tamura S, Kawaguchi A, Imai M, Itamura S, Odagiri T, et al. Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine. J Infect Dis 2007;196:1313-20.
[7] [7] Overton ET, et al. Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, increased cross-reactive IgA antibody formation against avian HSN1 and H7N9 influenza HA in humans, Vaccine 32 (2014) 5490-5495
[8] [8] Strayer DR, Carter WA, Stouch BC, Stevens SR, Bateman L, Cimoch PJ, et al. A double- blind, placebo-controlled, randomized, clinical trial of the TLR-3agonist rintatolimod in severe cases of chronic fatigue syndrome. PLoS ONE2012;7(3):¢31334.
[10] [10] Day, C.W; Baric, R.; Cai, S.X.; Frieman, M.; Kumaki, Y.; Morrey, J.D.; Smee, D.F.; Barnard, D.L, A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo. Virology, 2009, 395(2), 210-222. Example 8: Universal Corona Virus Vaccine Influenza continues to present a worldwide problem even with the existing vaccines with approximately 15-20 million cases annually. These yearly epidemics results in approximately 40,000 deaths in the United States alone. 104
More than 100 national influenza centers in over 100 countries conduct year-round surveillance for influenza. This involves receiving and testing thousands of influenza virus samples from patients and sending representative viruses to five World Health Organization (WHO) Collaborating Centers for Reference and Research on Influenza. Based on the submitted data, the local or regional authorities, such as the Food and Drug Administration (FDA) in the United States, then decide on the components of the next seasonal influenzas vaccine. Because it is not feasible to vaccinate a population against every flu strain discovered, only a subset of the flu strains are selected for inclusion in the vaccine. Because of this selection process, current influenza vaccines offer only limited protection against influenza strains that are not included in the vaccine.
Rintatolimod is a double-stranded RNA (dsRNA) and a generally well tolerated selective Toll-like receptor 3 (TLR3) agonist with induction of innate and adoptive immune responses. TLR3 is expressed in high concentrations on human airway epithelial cells and serves as a recognition system for many respiratory pathogens.
The mucosal surfaces of the nose and respiratory track serves as an ideal environment for Rintatolimod to exert its pronounced ability to enhance the innate response to respiratory pathogens like influenza virus, adenovirus, and coronavirus. Indeed, the intranasal instillation of inactivated or attenuated influenza viruses contained in seasonal influenza vaccine when used in combination with Rintatolimod has been shown to be able to induce a broad anti-viral IgA response with cross-reactivity against highly pathogenic human viruses such as various H5N1 clades (A/Indonesia 5/2000, A/Hong Kong/483/97, and A/Vietnam/1194/2001/) in mice, non- human primates, and humans.
The unique ability of Rintatolimod to be able to safely enhance the mucosal IgA response to both homologous as well as heterogenous strains of influenza virus is dependent on the interaction at the mucosal environment of Rintatolimod and foreign protein epitopes present in the viruses. This interaction results in epitope spreading and the generation of secretory IgA (S-1gA) with a very broad cross-reactivity against more distantly related clades and even different strains of viruses.
This immune enhancement process is not unique to the influenzas virus, but is also adaptable to other respiratory viruses such as coronavirus. Indeed, this disclosure utilized the recently isolated highly pathogenic coronavirus (SARS-CoV-2) isolated from patients with a severe respiratory infection that originated in Wuhan, China and has spread using human to human transmission around the world.
The methods of this disclosure include the use of a vaccine combined with Rintatolimod and administering intranasally (IN), with the generation of a mucosal S-IgA response and 105 having a broad cross-reactivity against other coronavirus including SARS, MERS, and human coronaviruses 229E, NL63, and OC43. The vaccine may contain inactivated (dead) SARS-CoV- 2, attenuated SARS-CoV-2, an antigen of SARS-CoV-2, an RNA encoding an antigen of SARS-CoV-2, or a similarly isolated virus from patients infected with SARS-CoV-2 This universal coronavirus vaccine would have antiviral activity not only against currently identified. coronaviruses but also against newly emerging coronavirus that currently are in wild animal populations such as bats and are likely to emerge in the future to infect human populations similarly to SARS, MERS, and SARS-CoV-2.
Example 9: Collecting and detecting SARS-CoV-2 virus Nasopharyngeal and oropharyngeal swab specimens are collected with synthetic fiber swabs; each swab was inserted into a separate sterile tube containing 2 to 3 ml of viral transport medium using established techniques. See, e.g., Holshue et al., N Engl J Med 2020; 382:929-
936.
Detection of SARS-CoV-2 may be made by polymerase chain reaction. Sequences for PCT are available through GenBank, for example, in accession number MN985325. Methods for detecting SARS-CoV-2, for example, by real-time reverse-transcriptase-polymerase-chain- reaction (rRT-PCR) assay, are known and published. See, ¢.g., Holshue et al., N Engl J Med 2020; 382:929-936.
Example 10: Growing SARS-CoV-2 Virus in Vitro: Method 1 Methods for culturing cells that can host SARS-CoV-2 has been published in Journals. See, e.g., Harcourt et al., Emerging Infectious Diseases, Vol. 26, No. 6, June 2020, pages 1266-
1273.
Human airway epithelial cell culture has been known for over 20 years (see, e.g., Lechner, J. F., Haugen, A., McLendon, L A, and Pettis, E. W. (1982) Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18, 633--642.). Human airway epithelial cells are harvested directly from humans according to established protocols (Jonsdottir HR, Dijkman R. Coronaviruses and the human airway: a universal system for virus- host interaction studies. Virol J 2016;13:24-24). Human airway epithelial cell cultures maintained at an air-liquid interface (ALI) is known and well described (Fulcher, M.L., Gabriel, S.; Burns, K.A., Yankaskas, J.R., Randell, S.H., Well-Differentiated Human Airway Epithelial Cell Culture, in Methods in Molecular Medicine, Vol. 107: Human cell Culture Protocols, Second Edition, Edited by: J. Picot, Humana Press Inc. Totowa, NJ). Primary cells such as “Normal Human Bronchial Epithelial Cells-P1” (catalog number: NhBE-P1) are also available commercially for example, by Novabiosis (North Carolina, U.S.A.). These cells are suitable for 106 growing SARS-CoV-2 cells in vitro (Zhu, N., et al, “A Novel Coronavirus from Patients with Pneumonia in China, 2019”; published on the web from the New England Journal of Medicine January 24, 2020). Bronchoalveolar-lavage fluid are collect from infected subjects and the collected samples are centrifuged to remove cellular debris. The supernatants containing coronavirus (e.g., SARS-CoV-2) are propagated on human airway epithelial cells as described herein.
To prepare cells for virus propagation, human airway epithelial cells are expanded on plastic substrate to generate passage-1 cells and are subsequently plated at a density of 2.5x10° cells per well on permeable Transwell-COL (12-mm diameter) supports. Human airway epithelial cell cultures are generated in an air-liquid interface for 4 to 6 weeks to form well- differentiated, polarized cultures resembling in vivo pseudostratified mucociliary epithelium.
Prior to infection, the apical surfaces of the human airway epithelial cells are washed three times with phosphate-buffered saline. Infection is initiated by adding 150 ul of supernatant from bronchoalveolar-lavage fluid samples (as described above) or from a previous SARS- CoV-2 preparation onto the apical surface of the cell cultures. After a 2-hour incubation at 37°C, unbound virus is removed by washing with 500 ul of phosphate-buffered saline for 10 minutes. The human airway epithelial cells are maintained in an air-liquid interface incubated at 37°C with 5% carbon dioxide. Every 48 hours, 150 ul of phosphate-buffered saline is applied to the apical surfaces of the human airway epithelial cells, and after 10 minutes of incubation at 37°C the samples are harvested as new SARS-CoV-2 virus harvests. The viral title may be monitored by RT-PCR and by infecting new cultures and observing cytopathic effects (CPE).
Example 11: Example of Growing Host Cells Susceptable to SARS-CoV-2, Infecting the Cells with SARS-CoV-2. and Testing Rintatolimod To test the efficacy of rintatolimod made by the methods of this disclosure, experiments were performed an in vitro study to determine the antiviral efficacy of rintatolimod against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human-derived tracheal/bronchial epithelial cells. All rintatolimod in this Example were made by us following the procedures and methods of this disclosure.
The antiviral activity of AIM ImmunoTech's compound rintatolimod made according to the methods disclosed in this disclosure was evaluated against SARS-CoV-2 (strain USA- WA1/2020) in a highly differentiated, three-dimensional (3-D), in vitro model of normal, human-derived tracheal/bronchial epithelial (TBE) cells. The compounds were tested at 5 concentrations in triplicate inserts of the 3D tissue models of the human airway (MatTek Life 107
Sciences) as indicated in Table 1. Antiviral activity was measured by virus yield reduction assays 5 days after infection.
Compounds: Rintatolimod was provided as 25 frozen | mL aliquots at 2.5 mg/mL and stored at -20°C upon arrival.
A fresh vial of the compound was diluted to the test dilutions (100, 50, 25, 12.5, and 6.25 ug/ml.) in the MatTek culture medium (AIR-100-MM) just prior to each drug addition using RNase-free tubes, pipettes and pipet tips.
Remdesivir (MedChemExpress, cat# HY-104077) was tested in singlet wells at 1, 0.1, 0.01, and 0.001 ng/mL as the positive control.
Cell Culture: The EpiAirway™ Model consists of normal, human-derived tracheal/bronchial epithelial (TBE) cells which have been cultured to form a multi layered, highly differentiated model which closely resembles the epithelial tissue of the respiratory tract.
The cell cultures were made to order by MatTek Life Sciences (https://www.mattek.com) (Ashland, MA) and arrived in kits with either 12- or 24-well inserts each.
The TBE cells were grown on 6mm mesh disks in transwell inserts.
During transportation the tissues were stabilized on a sheet of agarose, which was removed upon receipt.
One insert was estimated to consist of approximately 1.2 x 10° cells.
Kits of cell inserts (EpiAirway™ AIR-100) originated from a single donor, # 9831, a 23-year old, healthy, non-smoking, Caucasian male.
The cells have unique properties in forming layers, the apical side of which is exposed only to air and that creates a mucin layer.
Upon arrival (Tuesday, 4Aug2020), the cell transwell inserts were immediately transferred to individual wells of a 6-well plate according to manufacturer’s instructions, and 1 mL of MatTek’s proprietary culture medium (AIR-100-MM) was added to the basolateral side, whereas the apical side was exposed to a humidified 5% CO, environment.
The TBE cells were cultured at 37°C for 5 h.
Viruses: SARS-CoV-2 strain USA-WA 1/2020 was passaged three times in Vero 76 cells to create the virus stock.
Virus was diluted in AIR-100-MM medium before infection, yielding a multiplicity of infection (MOI) of approximately 0.005 CCID per cell.
Experimental design: After the 5 h equilibration period, the cells were treated with drug on the basal side of the transwells and cultured at 37°C for 18 h.
The mucin layer, secreted from the apical side of the cells, was removed by washing with 400 uL pre-warmed 30 mM HEPES buffered saline solution 3X.
Each compound treatment (120 gL) and virus (120 ul) was applied to the apical side, and compound treatment only was applied to the basal side (1 mL), fora 2 h incubation, As a virus control, 4 of the cell wells were treated with placebo (cell culfare medium only). Following the 2 h infection, the apical medium was removed, and the basal side was replaced with fresh compound or medium.
The cells were maintained at the air-liquid interface. 108
The basal side compound was replaced again at 6 h, 36 h, 48 h, and 72 h after the infection. On day 5, the medium was removed and discarded from the basal side. Virus released into the apical compartment of the tissues was harvested by the addition of 400 uL of culture medium that was pre-warmed at 37°C. The contents were incubated for 30 min, mixed well, collected, thoroughly vortexed and plated on Vero 76 cells for VYR titration. Triplicate and singlet wells were used for virus control and cell controls, respectively.
Determination of virus titers from each treated cell culture: Vero 76 cells were seeded in 96-well plates and grown overnight (37°C) to 90% confluence. Samples containing virus were diluted in 10-fold increments in infection medium and 200 uL of each dilution transferred into respective wells of a 96-well microtiter plate. Four microwells were used for each dilution to determine 50% viral endpoints. After 5 days of incubation, each well was scored positive for virus if any cytopathic effect (CPE) was observed as compared with the uninfected control, and counts were confirmed for endpoint on day 7. The virus dose that was able to infect 50% of the cell cultures (CCIDs, per 0.1 mL) was calculated by the Reed- Muench method (1948). The day 5 values are reported. Untreated, uninfected cells were used as the cell controls.
MTT cytotoxicity assay: The MTT assay is used as an indicator of cell viability. The colorimetric assay is based on the reduction of a yellow tetrazoliom salt to purple formazan crystals by live cells. The formazan crystals are then dissolved using a solubilization solution (10% SDS prepared in PBS) and the resulting colored solution is quantified by measuring absorbance at 550 nanometers using a multi-well spectrophotometer.
The test compound was provided as 3 frozen 6 mL aliquots at 10.3 mg/mL prepared in MatTek’s proprietary culture medium (AIR-100-MM and stored at -20 OC upon arrival. A fresh vial of the compound was diluted to the test dilutions (10, 4.5, 1.5, and 0.5 mg/ml.) in the MatTek culture medium just prior to each drug addition using RNase-free tubes, pipettes and pipet tips. Two tissues treated with mediam only were used as the cell controls.
After the 24 h equilibration period, the mucin layer, secreted from the apical side of the cells, was removed by washing with 400 uL pre-warmed 30 mM HEPES buffered saline solution 3X. Each compound treatment (240 pl) was applied to the apical side and to the basal side (1 mL), for a 2 h incubation at 37°C. As a cell control, 2 cell wells were treated with placebo (cell culture medium only). Following the 2 h incubation (to mimic the conditions of a virus infection), the apical medium was removed, and the cells were maintained at the air-liquid interface. The basal side compound was replaced again at 48 and 96 h after the mock infection. On day 5, the apical side was washed 1X with PBS, 0.1 mL of MTT was applied to the apical side and incubated at 37°C overnight for the cytotoxicity assay. Any remaining liquid was then 109 removed from the apical and basal sides and 0.2 mL of solubilization solution added to the cell inserts and incubated at 37°C overnight. The solution was then transferred to a 96-well flat- bottom plate and read by a spectrophotometer. Triplicate and singlet wells were used for virus control and cell controls, respectively.
RESULTS The virus yield results and EC90 values are summarized in Table 1.
Rintatolimod tested at 10 mg/mL was 47% cytotoxic, 4.5 mg/mL was 12% cytotoxic, and the lower concentrations had no measurable toxicity. The data indicate that the cell cytotoxicity concentration of compound that would cause 50% cell death (CC) is >10 mg/mL in the tested tissue model of normal, human-derived tracheal/bronchial epithelial cells.
Reference: Reed, L.J., Muench, H., 1938, A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 27, 493-497. 110
Table 6. Antiviral efficacy: EC9 for AIM ImmunoTech, Inc. compound rintatolimod against SARS-CoV-2.
est Compounds oncentration *Log10 CCID50 PEC90 (ug/mL) (ng/mL) virus per 0.2 mL Rintatolimod 100 3.00 9 50 3.67 25 50
12.5 67
6.25 4.50 Rintatolimod 100 3.00 55 50 00 25 30
12.5 4.30
6.25 4.50 Rintatolimod 100 3.50 39.1 0 3.50 25 4.00
12.5 4.30
6.25 30 Remdesivir 1 3.00 0.01
0.1 3.00
0.01 3.67 ).001 30 irus Control 5.00
4.00 67
5.00 Each well was scored positive for virus if any CPE was observed as compared with the uninfected control. Vero 76 cells were scored on day 5 and confirmed on day 7. Titer results from the virus yield reduction (V YR) assay. PEC90 = 90% effective concentration (concentration to reduce virus yield by 1 log10) determined by regression analysis. Our results indicate that the SARS-CoV-2 virus count can be reduced by one order of magnitude, to 10 fold less, when tdsRNA is applied at a concentration of 55 pg/mL. To confirm 111 that this is a safe dosage for application to humans in a clinical setting, this concentration was compared to clinically achievable concentration based on the intranasal safety profile of rintatolimod as shown below.
Table 7: The EC of Rintatolimod Against SARS-CoV-2 in a 3-D In Vitro Model of Normal, Human-derived Tracheal/Bronchial Epithelial Cells was 39.1-55 pg/ml, a Clinically Achievable Concentration Based on the Intranaal Safety Profile of Rintatolimod. Intranasal Dose Volume! | Dose Rintatolimod Fold Dose Rintatolimod (ul) Concentration EC” (pg/ml) Conc. Increase dose (ug) (ug/ml) Over ECqy "Dose volume is split equally between each nostril.
“Highest EC90 value obtained.
For the well tolerated dose concentration of 2500 pg/ml, it represents a 45.5 fold increase relative to the EC90 at 55 pg/ml. As can be seen, rintatolimod made by the methods of this disclosure has high antiviral activity against SARS-CoV-2 as shown by EC at rintatolimod concentrations that are well tolerated in humans. In fact, as shown in Table 7 above, a dosage that is 45 fold higher (i.e, 45 x EC dose) is well tolerated in humans. Thus, tdsRNA and especially tdsRNA made by the process of this disclosure is effective and well tolerated for the treatment or prevention of SARS-CoV-2 infection or for the reduction of SARS-CoV-2 titer on nasal tissue.
Rintatolimod has been tested in vitro in a SARS-CoV-2 infection model in human- derived tracheal/bronchial epithelial cells. Rintatolimod decreased SARS-CoV-2 infectious viral yields by 90% (EC90) at clinically achievable intranasal dosage levels (Table 6). In the same human-derived tracheal/bronchial epithelial cell system, the cell cytotoxicity 50% (CC50) of Rintatolimod was >10 mg/mL. Rintatolimod concentrations of 1.5 mg/ml and 0.5 mg/ml induced no cellular toxicity (0%).
As a further test of the ability of tdsRNA to enhance protection against SARS-CoV-2, the following experiments were performed as on animal hosts as follows: 112
“0 day” and “0 Week” is defined as the day and week of SARS-CoV-2 infection. Therefore, -1 week refers to 1 week before infection or -7 days before infection, 10 days refers to 10 days after infection, Blood sampling was performed throughout the period of experiments.
Mice were immunized at -35 days and -21 days. The dosage of SARS-CoV-2 S protein ectodomain (referred to in this Example only as S protein) is at 100 ng per mouse when administered. The dosage of tdsRNA, in the form of rintatolimod (rler{(C12U);), was at 10 Hg per mouse when administered. All immunizations were performed by subcutaneous injection.
Group 1 mice were administered S protein only. Group 2 mice were administered S protein and tdsRNA. Group 3 mice were sham administered phosphate buffered saline. As discussed, infection was at O day.
Neutralization antibody in the serum was measured at -7 days which is 2 weeks after the second immunization but before infection. Group 3 mice (sham immunized) has a relative titer of 2 (logs) representing a baseline of the measuring methods. Group 1 mice (S protein only) has a relative titer of 4 (logz). Group 2 mice (S protein and tdsRNA) has a relative titer of 16 (logo).
Viral titers after infection were measured. Group 3 mice (sham immunized) has a viral titer (Log10/g) of 8.7. Group 1 mice (S protein only) has a viral titer {Log10/g) of 8. Group 2 mice (S protein and tdsRNA) have a viral titer (Logl0/g) of 6, 1/10 of the Group 1 mice because the scale is Logio/g.
Group 3 mice (sham immunized) lost weight linearly until they reached 70% of their initial weight 5 days after infection at which point they died from the infection. Group 1 mice (S protein only) had a weight reduction to 83% by day 3 and about 95% by day 10. Group 2 mice (S protein and tdsRNA) had a weight reduction to 87% by day 3 and gained weight by day 10 to reach a level of 105%. All weight percent were measured as a percentage of initial weight at the moment of infection which was set as 100%.
Survival data were most dramatic. Group 3 mice (sham immunized) 1/3 of the mice died at day 5 and all mice died by day 6. Group 1 mice (5S protein only) had 1/9 death by day 6 and survival was 8/9 by day 10. In contrast, Group 2 mice (S protein and tdsRNA) had 100% survival by day 10.
Example 12: Growing SARS-CoV-2 Virus in Vitro: Method 2 SARS-CoV has been shown to replicate in BGM, CV-1, FRhK, LLC-Mk2, MA-104, pCMK, RK-13, and Vero cell lines. These cell lines produced a cytopathic effect (CPE) (also termed cell death) as early as day 4 after inoculation (Kaye, M., Druce, J., Tran, T., Kostecki, R., Chibo, D., Morris, J., Catton, M., and Birch, C. Emerg Infect Dis. 2006 Jan; 12(1): 128- 133).
113
Vero E6 cells are grown in Vero E6 cell growth media which is formulated as follows: minimal essential media (MEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 1% L-glutamme and 1% penicillin/streptomycin. Vero E6 cells may be grown in T flasks such as, for example, NUNC T Flask (e.g., from suppliers such as ThermoFisher Scientific) in T25, T75, T175, and T2235 sizes. Passage and growth may be performed using standard tissue culture techniques.
For SARS-CoV-2 virus growth, seed 1x10” Vero E6 cells into a T175 flask and culture at 37°C in 5% CO: to achieve a 90% confluent layer. Remove growth media and wash cells with serum free media leaving about 5 ml serum free media in a T175 flask. Add 1 ml of SARS- CoV-2 virus to the flask. Distribute virus over cells and incubate for 1 hour at 37°C in 5% CO2. Replenish with growth media up to a total volume of 20m! in a T175 flask. Incubate flask for 48-72 hours at 37°C in 5% CO», or until significant cytopathic effect (CPE) (also termed cell death) is observed. Collect supernatant from the infected flask and centrifuge at 500g for 5 minutes to remove any cellular debris. Aliquot appropriate volumes (100ul-1ml) of the clarified supematant into 1.5ml screw cap tubes and store at —80°C until needed.
The same procedure scaled down can be used to titer a sample of SARS-CoV-2 virus. For example, Vero E6 cells can be grown in 96 well plates with each well seeded with 1x10* Vero E6 cells. Each well can be infected with serial dilutions of virus and cytopathic effect (CPE) (also termed cell death) for each well is observed. The titer of the SARS-CoV-2 preparation is then determined by statistical methods. Alternatively, SARS-CoV-2 titer may be determined by a plaque assay.
The produced virus may be inactivated using standard industrial techniques such as formaldehyde inactivation (24 hr at 2-7°C) is performed at a final concentration of 0.02% formalin. Another standard inactivation technique is beta-propiolactone (BPL) based inactivation (24 hr at 18-22°C) with a final BPL concentration of 0.1%.
Example 13: Growing SARS-CoV-2 Virus in Vitro: Method 3 Vero CCL-81 cells can be used for in vitro growth and amplification, isolation, and initial passage of SARS-CoV-2. Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.com).
Virus isolation is started using both nasopharyngeal (NP) and oropharyngeal (OP) swabs. For isolation, limiting dilution, and passage 1 of the virus the following procedures may be used. 50 pl of serum-free DMEM is added to columns 2-12 of a 96-well tissue culture plate, 114 then 100 u of clinical specimens are pipetted into column 1 and serially diluted 2-fold across the plate. Vero cells are trypsinized and resuspended in DMEM containing 10% fetal bovine serum, 2x penicillin/streptomycin, 2x antibiotics/antimycotics, and 2x amphotericin B at a concentration of 2.5 x 10° cells/ml. 100 ul of cell suspension is added directly to the clinical specimen dilutions and mixed gently by pipetting. The inoculated cultures are grown in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPEs) daily. Standard plague assays for SARS-CoV-2 can be used to monitor virus growth. This protocol is based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) which are known and published.
When CPEs are observed, cell monolayers are scraped with the back of a pipette tip. 50 uL of viral lysate are used for total nucleic acid extraction for confirmatory testing and sequencing. 50 pL of virus lvsate are used to inoculate a well of a 90% confluent 24-well plate.
Confirmatory testing, to determine that we are growing SARS-CoV -2 are performed by using real-time reverse transcription PCR (CDC) and full-genome sequencing. Cells in which CPE is observed are used for testing and confirmation. The CDC molecular diagnostic assay targets 3 portions of the nucleocapsid gene, and results for all 3 portions should be positive for a sample to be considered positive (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-per- detection-instructions.html and https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-per-panel- primer-probes.html). To, Fast Track Respiratory Pathogens 33 Testing (FTD Diagnostics, http://www.fast-trackdiagnostics.com) is used to confirm that no other respiratory viruses were present.
Example 14: Generating anti- SARS-CoV-2 antibodies Methods for generating SARS-CoV-2 antibodies are published in Journals. See, e.g., Harcourt et al., Emerging Infectious Diseases, Vol. 26, No. 6, June 2020, pages 1266-1273. Plasmid pBM302 (Das D, Suresh MR. Copious production of SARS-CoV spike protein employing codon optimized synthetic gene is used to express a SARS-CoV-2 nucleocapsid protein, with a C-terminal His6 tag, to high levels within the inclusion bodies of Escherichia coli. The recombinant protein is purified from the inclusion bodies by using nickel-affinity column chromatography under denaturing conditions. Stepwise dialysis against Tris/phosphate buffer is performed to refold the recombinant SARS-CoV-2 nucleocapsid protein with decreasing concentrations of urea to renature the protein. Rabbits are immunized with the renatured, full-length, SARS-CoV -2 nucleocapsid protein to generate an affinity-purified rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody.
115
Example 15: Nasal Immunization using SARS-CoV-2, tdsRNA, and a combination thereof.
SARS-CoV-2 virus is grown in vitro using published techniques as discussed in this disclosure or using protocols as disclosed in the Examples section. The collected virus supernatant from cell cultures is purified at 1500 x g for 20 min to remove cell debris. For inactivation, the purified virus is treated with 1:4000 (v/v) formalin and incubated for 3 days at 37°C and then dialyzed against PBS. Inactivation of virus is confirmed by inoculation of the virus into cells permissible for virus growth as disclosed in the other Examples — preferably Example 5.
Antigen: Three types of nasal administration composition are prepared with 3 types of antigens.
The first type comprises inactivated virus only. For nasal immunization, 50, 100, 200, 500, or 1 mg of inactivated virus may be used. In a preferred embodiment. the amount of inactivated viruses may be 5 pg to 10 pg; 10 ug to 20 ug; 20 pg to 50 pg; 50 pg to 100 ug; 100 pgto 200 pg; 200 pg to 500 pg: 500 pg to 1000 pg; 1000 ug to 1500 ug; 1500 pg to 2000 pg; or any combination thereof.
The second type comprises inactivated virus and tdsRNA. As stated above, for nasal immunization, 50, 100, 200, 500, or 1 mg of inactivated virus is used. In a preferred embodiment, the amount of inactivated viruses may be 5 ug to 10 pg; 10 ug to 20 pg; 20 ug to 50 pg; 50 ug to 100 pg; 100 gg to 200 pg; 200 pg to 500 ug; 500 ug to 1000 ug; 1000 pg to 1500 pg; 1500 ug to 2000 ug; or any combination thereof. In addition, about an equal weight amount of tdsRNA is also added. That is, any of the above listed dosage for inactivated virus may also apply to the tdsRNA.
The third type comprises tdsRNA without any inactivated virus. 50, 100, 200, 500, or 1 mg of tdsRNA may be used. In a preferred embodiment, the amount of tdsRNA may be 5 pg to 10 ug; 10 pg to 20 ug; 20 pg to 50 ug; 50 pg to 100 ug; 100 pg to 200 ug; 200 ug to 500 pg; 500 pg to 1000 pg; 1000 ug to 1500 pg; 1500 pg to 2000 pg; or any combination thereof.
For any nasal administration composition, including type 1, type 2 and type 3 listed above, the composition may optionally include Cholera Toxin. Cholera toxin B Subunit is optionally added in equal weight amounts (e.g., 50, 100, 200, 500, or 1 mg) for its synergistic effect to stimulate nasal immunity. Cholera toxin (CT) may be purchased from Sigma-Aldrich (St. Louis, Mo.). Therefore, optionally, 6 different types (3 types without cholera toxin, and 3 types with cholera toxin are prepared.
Immunization 116
Humans and animals, such as ferrets, susceptible to SARS-CoV-2 are used for intranasal immunizations. Nasal immunization may comprise the above listed dosages for nasal immunization in PBS in a total volume of between 25 uL to 100 pL.
To determine the titers of SARS-CoV-2 in the lungs of infected naive and immunized animals (such as ferrets and not humans) will be challenged by intranasal instillation of 25 pL of SARS-CoV-2 (e.g., about 600 to 6000 pfu) 11 weeks after the last immunization. The challenged animals is monitored for signs of morbidity (body weight changes, fever and hunched posture) and mortality. Animals are weighed immediately before and daily after challenge. Half of the animals from each group will be analyzed on day 4 and day 8 post- challenge. Lung homogenates will be prepared in DMEM serum-free medium to assess the viral titers, determined per g of lung tissue. See, e.g., Sha et al., Induction of CD4(+) T-cell- independent immunoglobulin responses by inactivated influenza virus. J Virol, 2000; 74(11):4999-5005. For plaque assays, we prepare serial dilutions of lung supernatants, incubate them with cells permissive for growth of SARS-CoV-2 as shown in the Examples.
Evaluation of Humoral Immune Responses The concentrations of virus-specific IgG, IgGl, 1gG2a and IgA will be determined in all sera and mucosal secretions using standard assay procedures such as ELISA plates coated with purified inactivated SARS-CoV-2. See, e.g., Sha et al, Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus. J Virol. 2000; 74(1 1):4999-5005.
Kang et al. Enhancement of mucosal immunization with virus-like particles of simian immunodeficiency virus. J Virol. 2003; 77(6):3615-23. Kang et al. Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens. J Virol. 2004; 78(18):9624-32.
We determine the hemagglutination inhibition (HI) and neutralizing antibody titers, which are both used as indicators of protective immune responses {0 viruses, as previously described. Sha et al., Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus. J Virol. 2000; 74(11):4999-5005. Novak et al., Murine model for evaluation of protective immunity to influenza virus. Vaccine 1993; 11(1):55-60.
Cellular Immune Responses is determined by Cytokine ELISA. Briefly, spleen or inguinal lymph nodes cells will be prepared from immunized mice at 2 weeks after the last immunization, and stimulated in vitro with inactivated SARS-CoV-2 virus at a final concentration of 1 ug/ml in complete RPMI medium. Sha et al., Induction of CD4(+) T-cell- independent immunoglobulin responses by inactivated influenza virus. J Virol. 2000; 74¢11):4999-5005. Kang et al. Enhancement of mucosal immunization with virus-like particles of simian immunodeficiency virus. J. Virol. 2003; 77(6):3615-23. Kang et al. Intranasal 117 immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens.J.
Virol. 2004; 78(18):9624-32. After 72 h the cells are centrifuged and the supernatant will be collected and stored at -80 °C. until assayed.
Cytokine production (TNF-alpha, IFN-gamma, IL-4, IL-6 and IL-10) will be determined according to the manufacturer's instructions. 118
P129459NLOO sequence list ST25
SEQUENCE LISTING <110> AIM IMMUNOTECH INC. <120> METHODS, COMPOSITIONS, AND VACCINCES FOR TREATING A VIRUS
INFECTION <130> P129459NL60 <140> NL 2027383 <141> 2021-01-25 <150> US 62/965,713 <151> 2020-01-24 <150> US 62/967,493 <151> 2020-01-29 <150> US 62/976,994 <151> 2020-02-14 <150> US 62/982,641 <151> 2020-02-27 <150> US 62/993,514 <151> 2020-03-23 <150> US 62/994,777 <151> 2020-03-25 <150> US 63/003,197 <151> 2020-03-31 <150> US 63/016,960 <151> 2020-04-28 <150> US 63/026,712 <151> 2020-05-18 <150> US 63/029,395 <151> 2020-05-22 <150> US 63/092,432 <151> 2020-10-15 <150> US 62/969,572 <151> 2020-02-03 <150> US 62/971,199 <151> 2020-02-06 <160> 22 Pagina 1
P129459NLOO sequence list ST25 <170> PatentIn version 3.5 <21e> 1 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome-related coronavirus <400> 1 ucucuaaacg aacuuuaaaa ucugug 26 <2105 2 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome coronavirus 2 <400> 2 ucucuaaacg aacuuuaaaa ucugug 26 <2105 3 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome-related coronavirus <400> 3 caacuaaacg aac 13 <2105 4 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome coronavirus 2 <400> 4 caacuaaacg aac 13 <210>5 5
Pagina 2
P129459NLOO sequence list ST25 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome-related coronavirus <400> 5 cacauaaacg aacuu 15 <210> 6 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome coronavirus 2 <400> 6 cacauaaacg aacuu 15 <210> 7 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome-related coronavirus <400> 7 ugaguacgaa cuu 13 <2105 8 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome coronavirus 2 <400> 8 ugaguacgaa cuu 13 <216> 9 <211> 18 <212> RNA <213> Artificial Sequence
Pagina 3
P129459NLOO sequence list ST25 <220> <223> Severe acute respiratory syndrome-related coronavirus <400> 9 ggucuaaacg aacuaacu 18 <210> 10 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> Severe acute respiratory syndrome coronavirus 2 <400> 10 ggucuaaacg aacuaaau 18 <21e> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tggggyttta crggtaacct 20 <21e> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aacrcgctta acaaagcact c 21 <2105 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 13
Pagina 4
P129459NLOO sequence list ST25 tagttgtgat gcwatcatga ctag 24 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 taatcagaca aggaactgat ta 22 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgaaggtgtg acttccatg 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 16 gcaaattgtg caatttgcgg 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccctgtgggt tttacactta a 21 <210> 18
Pagina 5
P129459NLOO sequence list ST25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 acgattgtgc atcagctga 19 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 19 ccgtctgcgg tatgtggaaa ggttatgg 28 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ggggaacttc tcctgctaga at 22 <21e> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cagacatttt gctctcaagc tg 22 <2105 22 <211> 20 <212> DNA <213> Artificial Sequence
Pagina 6
P129459NLOO sequence list ST25 <220> <223> probe <400> 22 ttgctgctgc ttgacagatt 20 Pagina 7

权利要求:
Claims (1)
[1]
CONCLUSIONS
A composition for treating or preventing a viral infection in an individual caused by a virus, the composition comprising a therapeutic double-stranded RNA (tdsRNA), wherein the tdsRNA is at least one selected from the group consisting of rly ( Cx U)n (formula 1); TIn (CG) (Formula 2); TAn *TUk (formula 3); rl, TCn (Formula 4); and robust dsRNA (Formula 5); wherein x is one or more selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 4-29, 4-30, 14-30, 15-30, 11-14, and 30 -35.
The composition of claim 1, wherein the composition further comprises a vaccine against the virus.
The composition of claim 1, or any one of the preceding claims, wherein the virus is a virus of Table 2 1s.
The composition of claim 1, or any one of the preceding claims, wherein the virus is a coronavirus, preferably a SARS-CoV-2 virus.
The composition of claim 1, or any one of the preceding claims, wherein the virus is at least one selected from the group consisting of Human coronavirus 229E (HCoV-229E); Human coronavirus NL63 (HCoV-NL63, “New Haven coronavirus”); Human coronavirus OC43 (HCoV-OC43); Human coronavirus HKU; 119
“Middle East respiratory syndrome-related coronavirus” (MERS-CoV); new coronavirus 2012 (HCoV-EMC); “Severe acute respiratory syndrome-related coronavirus” (SARS-CoV); “Severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2); Ebola virus; H5 influenza; H7 influenza; H5N1 influenza; Influenza A; Influenza B; HIN1 influenza; H3N2 influenza; H7N9 influenza; H5N6 influenza; H10N8 influenza; H9N2 influenza; H6N1 influenza; West Nile virus; and Zika virus.
The composition of claim 1, or any one of the preceding claims, wherein n is a number having a value selected from the group consisting of: 40 to 50,000; 40 through 40,000; 50 to 10,000; 60 to 9,000; 70 through 8,000; 120
80 to 7,000; and 380 through 450.
The composition of claim 1, or any one of the preceding claims, wherein at least 90% by weight of the tdsRNA is greater than a size selected from the group consisting of: 40 base pairs; 50 base pairs,; 60 base pairs; 70 base pairs; 80 base pairs; and 380 base pairs.
The composition of claim 1, or any one of the preceding claims, wherein at least 90% by weight of the tdsRNA is less than a size selected from the group consisting of:
50,000 base pairs;
10,000 base pairs;
9,000 base pairs;
8,000 base pairs,;
7,000 base pairs; and 450 base pairs.
The composition of claim 1, or any one of the preceding claims, wherein n is 40 to 40,000; wherein the tdsRNA has about 4 to about 4000 helix twists of duplexed RNA strands; or wherein the tdsRNA has a molecular weight selected from the group consisting of: 2 kDa to 30,000 kDa; 25 kDa to 2,500 kDa; and 250 kDa to 320 kDa. 121
The composition of claim 1, or any one of the preceding claims, wherein the tdsRNA comprises r1, * ribo(C11.14U)s; and robust dsRNA.
The composition of claim 1, or any one of the preceding claims, wherein the robust dsRNA has a single strand consisting of r(C4-29U)n, T(C1114U)a, or r(C12U)n; and has an opposite strand consisting of r(I); wherein the single strand and the opposite strand do not base pair at the position of the uracil base, and wherein the single strand and the opposite strand are partially hybridized.
The composition of claim 1, or any one of the preceding claims, wherein the robust dsRNA has a molecular weight of about 250 kDa to 500 kDa; each strand of the robust dsRNA is about 400 to 800 base pairs in length; or the robust tdsRNA has about 30 to 100 or 30-60 twists of duplexed RNA.
The composition of claim 1, or any of the preceding claims, wherein the tdsRNA is robust dsRNA that is resistant to denaturation under conditions that allow hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands (r1, *rCn) to form. to separate.
The composition of claim 1, or any one of the preceding claims, wherein the robust dsRNA is an isolated double-stranded ribonucleic acid (dsRNA) that is enzymatically active under thermal stress comprising: 122 each strand having a molecular weight of about 250 kDa to about 500 kDa, 400-800 base pairs, or 30 to 60 spiral turns of duplex RNA, a single strand consisting of poly(ribocytosine4-29 uracilic acid) and an opposite strand of poly(riboinosin acid), with the two strands not forming a base pair at the position of the uracil base, the two strands base pairing at the position of the cytosine base, and the strands are partially hybridized.
The composition of claim 1, or any one of the preceding claims, wherein the tdsRNA is produced by a method comprising: a) synthesizing a first single-stranded RNA (first ssRNA) in a first synthesis reaction with PNPase as the sole RNA polymerase, and purifying said first ssRNA after the first synthesis reaction; b) synthesizing a second single-stranded RNA (second ssRNA) in a second synthesis reaction with PNPase as the sole RNA polymerase, and purifying said ssRNA after the second synthesis reaction; and c) hybridizing the first ssRNA to the second ssRNA to form the tdsRNA; wherein step a) and step b) are performed in random order; wherein the first synthesis reaction comprises inosine diphosphate (rIDP) as the only free ribonucleotide; wherein the second synthesis reaction comprises cytidine diphosphate (rCDP) and uridine diphosphate (UDP) as the only two free ribonucleotides 123 and a molar ratio of (free rCDP): (free rUDP) in the second synthesis reaction approximately (11 to 14): (1 ) 1s.
The composition of claim 1, or any one of the preceding claims, wherein the tdsRNA comprises 0.1-12 mol% robust dsRNA, preferably the tdsRNA comprises 0.1-5 mol% robust dsRNA.
The composition of claim 1, or any one of the preceding claims, wherein the composition comprises at least one pharmaceutically acceptable carrier.
The composition of claim 1, or any one of the preceding claims, wherein the tdsRNA is complexed with a stabilizing polymer.
The composition of claim 18, or any one of the preceding claims, wherein the stabilizing polymer is at least one selected from the group consisting of: polylysine; polylysine and carboxymethylcellulose; polyarginine; polyarginine and carboxymethylcellulose; and any combination thereof.
The composition of claim 1, or any one of the preceding claims, wherein the composition further comprises an antiviral agent that is not a tdsRNA.
The composition of claim 20, or any one of the preceding claims, wherein the antiviral agent is at least one selected from the group consisting of: an antibody to an S protein of SARS-CoV-2; an antibody to an NTD region of an S protein of SARS-CoV-2; an antibody to an HR1 region of an S protein of SARS-CoV-2; 124 an antibody to an RBD region of an S protein of SARS-CoV-2; a SARS-CoV monoclonal antibody; a MERS-CoV monoclonal antibody; a SARS-CoV-2 monoclonal antibody; a peptide; a protease inhibitor; a PIKfyve inhibitor; a TMPRSS2 inhibitor; a cathepsin inhibitor; a furin inhibitor; an antiviral peptide; an antiviral protein; an antiviral chemical compound; and an antiviral agent.
The composition of claim 20, or any one of the preceding claims, wherein the antiviral agent is at least one selected from the group consisting of: 1A9; 201; 311mab-31B5; 311mab-32D4; 47D11; A4A8; 4C2; 80R; Apilimod; B38; camostat mesylate; Casirivimab; 125
CR3014; CR3022; D12; E-64D; EK1;
EK1C4; H4; HR2P; IBP02;
Imdevimab; m336; MERS-27; MERS-4; MI-701;
n3088; n3130; P2B-2F6; P2C-1F11; piss;
S230; S309; SARS-CoV-2 S HR2P fragment (aa1168-1203); tetrandrin; Viracept (nelfinavir mesylate);
YM201636; -1-PDX; favipiravir; IFN-g; IFN-alb;
IFN-a2a;
126 lopinavir-ritonavir; Q-Griffithsin (Q-GRFT); and Griffithsin;
oseltamivir;
zanamivir; abacavir; zidovudine; zalcitabine; didanosine;
stavudine; efavirenz; indinavir; ritonavir; nelfinavir; amprenavir; ribavirin; Remdesivir; chloroquine; hydroxychloroquine; rIFN-alpha-2a; rIFN-beta-1b; rIFN-gamma; nIFN-alpha; nIFN beta; nIFN-gamma; IL-2; PD-L1; Anti-PD-L1; a checkpoint inhibitor; an interferon; 127 interferon mixture; recombinant or natural interferon; alferon; alpha interferon species; recombinant or natural interferon alpha; recombinant or natural interferon alpha 2a; recombinant or natural interferon beta; recombinant or natural interferon beta 1b; and recombinant or natural interferon gamma.
The composition of claim 22, or any one of the preceding claims, wherein the alpha interferon species is a mixture of at least seven alpha interferon species produced by human white blood cells, the seven species being: interferon alpha 2; interferon alpha 4; interferon alpha 7; interferon alpha 8; interferon alpha 10; interferon alpha 16; and interferon alpha 17.
The composition of claim 1, or any one of the preceding claims, wherein the composition is at least one selected from the group consisting of an aqueous solution, a powder, a dry particle, a liquid particle, a gel particle, a semi-dry particle, an isotonic formulation, and 128 a composition for nasal administration.
The composition of claim 2, or any one of the preceding claims, wherein the vaccine comprises at least one of the group consisting of: an inactivated virus, an attenuated virus, a virus antigen, a messenger RNA encoding a protein containing a virus antigen.
The composition of claim 25, or any one of the preceding claims, wherein the virus antigen is an antigen from the S, E, M, or N structural proteins of SARS-CoV-2.
A method of treating a viral infection in a subject caused by a virus comprising: determining that the subject is infected with the virus; and administering an effective amount of a composition of claim 1, or any of the preceding claims, to the virus-infected individual.
The method of claim 27, or any one of the preceding claims, wherein the individual has been infected by the virus for no more than two to seven days, or up to 14 days.
29. A method of preventing a viral infection in an individual caused by a virus comprising: determining that the individual is not infected by the virus; and administering an effective amount of a composition of claim 1, or any one of the preceding claims, to the individual not infected by the virus.
A method of treating a viral infection in an individual caused by a virus: 129 administering a composition comprising an effective amount of a composition of claim 1, or any one of the preceding claims, to the individual infected with the virus, at risk of being infected by the virus through exposure to a second individual infected with the virus, or at risk of being infected by the virus through presence in an area where cases of viral infection have been reported.
A method of immunizing an individual against a viral infection caused by a virus, the method comprising: administering at least a first compound and a second compound in random order to the individual, together or separately, the first compound comprises an effective amount of a vaccine, and wherein the second compound is an effective amount of a composition of claim 1, or any of the preceding claims.
The method of claim 31, or any one of the preceding claims, wherein the vaccine comprises at least one selected from the group consisting of: an inactivated virus, an attenuated virus, a virus antigen, and a messenger RNA encoding a virus antigen.
The method of claim 31, or any one of the preceding claims, wherein the method produces an immune response in the individual. 130
The method of claim 33, or any one of the preceding claims, wherein the immune response is at least one selected from the group consisting of virus-specific immunoglobulin production; virus specific IgG production; virus specific IgG1 production; virus specific IgG2a production; virus specific IgA production; and virus specific IgM production.
The method of claim 31, or any one of the preceding claims, wherein the virus antigen is an antigen from the S, E, M, or N structural proteins of SARS-CoV-2.
The method of claim 31, or any one of the preceding claims, wherein the method induces an enhanced cross-reactive immune response and cross-protection against a second different virus in an individual.
The method of claim 34, or any one of the preceding claims, wherein the second virus is a variant, a different strain, or a mutation of the virus.
The method of claim 31, or any one of the preceding claims, wherein the method provides a vaccine effect superior to a coronavirus antigen administered alone.
The method of claim 31, or any one of the preceding claims, wherein the first compound and second compound are administered together as a mixture; or wherein the first compound and second compound are administered simultaneously or separately.
The method of claim 31, or any one of the preceding claims, wherein the first compound and second compound are administered separately, but within a time period selected from the group consisting of: 2 months; 1 month; 3 weeks; 2 weeks; 1 week; 3 days; 1 day; 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, and 30 minutes.
131
The method of claim 27, or any one of the preceding claims, wherein the virus or second other virus is a virus from Table 2.
The method of claim 27, or any one of the preceding claims, wherein the virus is a coronavirus, preferably a SARS-CoV-2 virus.
The method of claim 27, or any one of the preceding claims, wherein the virus is at least one virus selected from the group consisting of Human coronavirus 229E (HCoV-229E); Human coronavirus NL63 (HCoV-NL63, “New Haven coronavirus”); Human coronavirus OC43 (HCoV-0OC43); Human coronavirus HKU; “Middle East respiratory syndrome-related coronavirus” (MERS-CoV); new coronavirus 2012 (HCoV-EMC); “Severe acute respiratory syndrome-related coronavirus” (SARS-CoV); “Severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2); Ebola virus; H5 influenza; H7 influenza; H5N1 influenza; Influenza A; Influenza B; HIN1 influenza; H3N2 influenza; H7N9 influenza; H5N6 influenza; 132
H10N8 influenza; H9N2 influenza; H6N1 influenza; West Nile virus; and Zika virus.
The method of claim 27, or any one of the preceding claims, wherein the effective amount is a therapeutically effective amount or a prophylactically effective amount of the tdsRNA.
The method of claim 27, or any one of the preceding claims, wherein administering at least one method of administration is selected from the group consisting of: intravenous administration; intradermal administration; subcutaneous administration; intramuscular administration; intranasal administration (pulmonary airway administration); intranasal administration and oral administration; intraperitoneal administration; intracranial administration; intravesical administration; oral administration (by mouth, by breathing through the mouth); topical administration; inhalation administration; aerosol administration; intra-airway administration; tracheal administration; bronchial administration; instillation; bronchoscopic instillation; intratracheal administration; 133 mucosal administration; dry powder administration; spray application; contact administration; administration by means of a cotton swab; intratracheal deposition administration; intrabronchial deposition administration; bronchoscopic deposition administration; lung administration; nasal passage administration; respirable solid administration; respirable liquid administration; dry powder inhalant administration; and a combination thereof.
The method of claim 45, or any one of the preceding claims, wherein intranasal administration is at least one selected from the group consisting of: administration to nasal passages; administration to nasal epithelium; administration to lung; administration by inhalation; administration to the larynx; administration to bronchi; administration to alveoli; administration by inhalation; administration by nasal instillation; and a combination thereof.
The method of claim 27, or any one of the preceding claims, wherein administration is administration to at least one tissue or cell selected from the group consisting of: 134 an airway tissue; nasal tissue;
oral tissue; alveoli tissue;
pharyngeal tissue; trachea tissue; bronchial tissue; carina tissue; bronchial tissue;
bronchial tissue; lung tissue; lung lobe tissue; alveoli tissue; nasal passage tissue:
nasal epithelial tissue; laryngeal tissue; bronchial tissue; inhalation tissue; an epithelial cell;
an airway epithelial cell; a ciliated cell; a goblet cell; a non-ciliated cell; a basal cell;
a lung cell; a nasal cell; a tracheal cell; a bronchial cell; a bronchiolar epithelial cell;
an alveolar epithelial cell; and
135 a sinus cell.
The method of claim 27, or any one of the preceding claims, wherein administration is by at least one delivery system selected from the group consisting of: a nebulizer; a sprayer; a nasal pump; a squeeze bottle; a nasal spray; a spray nozzle or plunger nozzle (a syringe that provides pressure to a connected nozzle or nozzle); a nasal aerosol device; a controlled particle dispersion device; a nasal aerosol device; a nasal nebulizer; a pressure-driven jet nebulizer; ultrasonic nebulizer; a breath-actuated nasal delivery device; an atomized nasal medication device; an inhaler; a powder dispenser; a dry powder generator; an aerosol; an intrapulmonary aerosol; a sub-miniature aerosol; a pressurized gas based metered dose inhalers; a dry powder inhaler; an installation device; an intranasal installation device; an intravesical instillation device; 136 a cotton swab; a pipette; a nasal irrigation device; a nasal rinse; an aerosol device; a metered aerosol device; a pressurized proportioner; a powder aerosol; a spray aerosol; a sprayer; a metered sprayer; a suspension purge device; and a combination thereof.
The method of claim 27, or any one of the preceding claims, wherein the method reduces nasal virus titer at least 10-fold or 100-fold, or prevents or reduces nasal delivery of virus at least 10-fold or 10-fold
The method of claim 27, or any one of the preceding claims, wherein the tdsRNA is administered at a dose of about 25-700 milligrams, 20 mg to 200 mg, 50 mg to 150 mg, 80 mg to with 140 mg.
The method of claim 27, or any one of the preceding claims, wherein the individual is a mammal, preferably a host of the virus, and most preferably a human.
A delivery system or medical device comprising a composition of claim 1, or any one of the preceding claims.
The delivery system or medical device of claim 52, or any one of the preceding claims, wherein the delivery system or medical device is selected from the group consisting of: 137 a nebulizer; a sprayer; a nasal pump; a squeeze bottle; a nasal spray;
a spray nozzle or a suction nozzle (a syringe that provides pressure to a connected nozzle or nozzle); a nasal aerosol device; a controlled particle dispersion device;
a pressure-driven jet nebulizer; ultrasonic nebulizer a breath-actuated nasal delivery device; an atomized nasal medication device; an inhaler;
a powder dispenser; a dry powder generator; an aerosol; an intrapulmonary aerosol; a sub-miniature aerosol;
a pressurized gas based metered dose inhalers; a dry powder inhaler device; an installation device; an intranasal installation device; an intravesical instillation device;
a cotton swab; a pipette; a nasal irrigation device; a nasal rinse; an aerosol device;
a metered aerosol device;
138 a pressurized dispenser; a powder aerosol device; a spray aerosol device; a rinsing device; a metered sprayer; a suspension sprayer; and a combination thereof. 139

类似技术:

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公开号 | 公开日 | 专利标题

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Zhu et al.2015|Broad-spectrum antiviral agents

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公开号 | 公开日

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WO2021151100A1|2021-07-29|

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申请号 | 申请日 | 专利标题

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US202062965713P| true| 2020-01-24|2020-01-24|

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US202062967493P| true| 2020-01-29|2020-01-29|

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US202062969572P| true| 2020-02-03|2020-02-03|

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US202062971199P| true| 2020-02-06|2020-02-06|

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US202062976994P| true| 2020-02-14|2020-02-14|

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US202062982641P| true| 2020-02-27|2020-02-27|

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US202062993514P| true| 2020-03-23|2020-03-23|

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US202062994777P| true| 2020-03-25|2020-03-25|

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US202063003197P| true| 2020-03-31|2020-03-31|

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US202063016960P| true| 2020-04-28|2020-04-28|

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US202063026712P| true| 2020-05-18|2020-05-18|

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US202063029395P| true| 2020-05-22|2020-05-22|

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US202063092432P| true| 2020-10-15|2020-10-15|

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US202063125950P| true| 2020-12-15|2020-12-15|

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