Virus-specific T cells and methods for treating and preventing viral infections

Abstract

The present invention relates to pharmaceutical compositions comprising viral peptide-specific cytotoxic T lymphocytes (CTLs), methods for generating viral peptide-specific CTLs, and methods for treating or preventing viral infections. In one embodiment, the present disclosure relates to a method for treating viral infections. The method may comprise administering to a human patient in need thereof an effective amount of cells comprising viral peptide-specific cytotoxic T lymphocytes (CTLs), which are specifically enriched for cells reactive to the viral peptide. The human patient may be an elderly patient or an immunocompromised patient. Administering may be performed by intravenous infusion. The infusion may be delivered to the patient via a central line or midline. The viral peptide-specific CTLs may be derived from a single donor.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 123,109, filed Dec. 9, 2020, and U.S. Provisional Patent Application No. 63 / 186,098, filed May 8, 2021. [0001.1] Reference to Sequence Listing This application includes a sequence listing in computer-readable form. The computer-readable form is incorporated herein by reference. The ASCII copy was created on Dec. 9, 2021, has the name 767095_000023_SL.txt, and has a size of 15,982 bytes.

Background Art

[0002] Infectious diseases were the leading global burden of premature death and disability until the end of the 20th century, when they ceded that ignominious position to non-infectious diseases. Among infectious diseases, viral infections are a major global health problem with the potential threat of an impending global pandemic. For example, in the United States alone, the number of cases identified between January 2020, when the first case of COVID-19 was reported, and August 2020 was at least 5,821,819, and the number of deaths attributable to COVID-19 was 179,708. (Johns Hopkins Coronavirus Resource Center, 2020). Although progress has been made in developing treatment methods for infections in the sickest patients, virus-related deaths continue to be a significant problem, especially in certain populations at high risk of infection (Recovery Collaborative Group, 06 / 22 / 2020). The elderly, immunocompromised, those with comorbidities (such as diabetes or cardiovascular disease), the unvaccinated, and minorities have a higher mortality rate due to viral infection compared to the rest of the population. (Guan et al., 2020, Stokes et al., 2020).

[0003] While several vaccine platforms are approved for human use against viral infections, several factors remain that restrict post-infection treatment options. For example, the lack of vaccination options, vaccine refusal, breakthrough infections in vaccinated populations, and immunity evasion by viral pathogens can lead to viral populations that no longer respond to existing antiviral strategies (such as small molecules and vaccines). Furthermore, the persistence of immunity after viral infection and vaccination remains questionable (To et al., 2020). There is still a need for treatment options that can rapidly adapt to the evolutionary changes that occur throughout the viral infection cycle, and this applies to COVID-19 as well. [Overview of the Initiative] [Means for solving the problem]

[0004] In one embodiment, the present disclosure relates to a method for treating a viral infection. The method may include administering an effective dose to a human patient in need of it, cells containing viral peptide-specific cytotoxic T lymphocytes (CTLs), which are cells specifically enriched to be reactive to viral peptides. The human patient may be an elderly patient or an immunocompromised patient. The administration may be carried out by intravenous infusion. The infusion may be delivered to the patient via a central line or midline. The viral peptide-specific CTLs may originate from a single donor.

[0005] CTLs can be sensitized in vitro to multiple peptides constrained to a single HLA allele. At least 20% of such CTLs may be reactive to viral peptides, and / or such cells may contain less than 2.5% naive T cells, monocytes, NK cells, or any combination thereof.

[0006] Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to the HLA-A1 allele (such as one or more peptides that can be selected from Table 1). Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to the HLA-A2 allele (such as one or more peptides that can be selected from Table 2 or Table 9). Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to the HLA-B7 allele (such as one or more peptides that can be selected from Table 3). Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to the HLA-B40 allele (such as one or more peptides that can be selected from Table 4). Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to the HLA-Cw7 allele (such as one or more peptides that can be selected from Table 5). Viral peptide-specific CTLs can be sensitized to combinations of viral peptides that bind to any one or a combination of the following HLA-A1 alleles: HLA-A2 allele, HLA-B7 allele, HLA-B40 allele, and HLA-Cw7 allele.

[0007] Viral peptides may originate from the severe acute respiratory syndrome (SARS) virus, the SARS-coronavirus 2 (COVID-19) virus, or the influenza virus.

[0008] In one embodiment, the disclosure relates to a method for preparing viral peptide-specific cytotoxic T cells (CTLs), which are cells specifically enriched to be reactive to viral peptides. The method may include a first stimulation step in which a subset of monocytes may be treated to induce maturation into dendritic cells, the dendritic cells may be pulsed with one or more virus-specific peptides, and the dendritic cells may be co-cultured with lymphocytes for at least 6 days. Induction of maturation into dendritic cells may include a first treatment of monocytes for at least about 24 hours using GM-CSF, IL-4, or a combination of the two, and a second treatment of monocytes for at least about 24 hours after the first treatment using TNF-alpha, IL-1 beta, IL-6, prostaglandin E2, or any combination thereof. Pulsing of dendritic cells with viral peptides may include incubating dendritic cells with at least about 2 μg / mL of each viral peptide.

[0009] The method may include a second stimulation step in which monocytes are used to present a virus-specific peptide, stimulated lymphocytes are cultured for at least 7 days, and peptide-specific CTLs can be selected due to the selective adhesion of T cells that recognize the pulsed peptide to the adherent monocyte layer. Stimulated lymphocytes may be further selected by treating the co-culture with human interleukin-1 (IL-1).

[0010] The method may include a third stimulation step in which a subset of monocytes is pulsed with multiple virus-specific peptides to constrain them to a single HLA allele, thereby generating virus peptide-specific CTLs, of which at least 20% may be reactive to the virus peptides. The virus-reactive CTLs may be allogeneic mononuclear leukocytes collected from a single donor.

[0011] Viral peptide-specific CTLs can be sensitized to one or more peptides constrained to one or more of the following HLA-A1, HLA-A2, HLA-B7, HLA-B40, or HLA-Cw7 alleles. Examples of peptides are shown in Tables 1-5 and 9.

[0012] Viral peptides may originate from the severe acute respiratory syndrome (SARS) virus, the SARS-coronavirus 2 (COVID-19) virus, or the influenza virus.

[0013] In one embodiment, the disclosure relates to a pharmaceutical composition that may comprise cells comprising viral peptide-specific cytotoxic T lymphocytes (CTLs), which may be specifically enriched with cells that are reactive to viral peptides. The CTLs are derived from multiple donors. The CTLs may be sensitized to multiple peptides constrained to a single HLA allele by in vitro stimulation. At least 20% of the CTLs may be reactive to viral peptides. The cells may comprise less than 2.5% naive T cells, monocytes, NK cells, or any combination thereof.

[0014] CTLs may be sensitized to one or more viral peptides that bind to one or more specific HLA-A1, HLA-A2, HLA-B7, HLA-B40, or HLA-Cw7 alleles. Examples of peptides are shown in Tables 1-5 and 9.

[0015] Viral peptide-specific CTLs may be specific to viruses selected from the group consisting of SARS-CoV-2 (COVID-19), influenza, parainfluenza, respiratory syncytial virus (RSV), metapneumovirus, hepatitis B virus (HBV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), human herpesvirus (HHV), and adenovirus. In one embodiment, the virus may be severe acute respiratory syndrome (SARS) virus, SARS-coronavirus 2 (COVID-19) virus, or influenza virus.

[0016] The pharmaceutical composition may further comprise CTLs cryopreserved in DMSO, RPM1-1640, albumin, or a combination thereof, and / or one or more additional antiviral agents.

[0017] The pharmaceutical composition may be in a form suitable for intravenous administration. [Brief explanation of the drawing]

[0018] [Figure 1] A–C are a series of graphs showing the proliferation of viral (COVID-19) CTLs by applying the proposed laboratory process. A shows the background level of CD8+ CTLs. B shows the cell content of CD8+ CTLs one week after in vitro culture. C shows that CD8+ CTLs were enriched to 15% after selection and further proliferation through approximately 3–4 weeks of in vitro culture.

[0019] [Figure 2A] This is a series of graphs showing the results of flow cytometry (FITC) tetramer analysis, which measured the CD8+ T cell content after the first peptide stimulation. [Figure 2B] This is a series of graphs showing the results of flow cytometry (FITC) tetramer analysis, which measured the CD8+ T cell content after the final stimulation (B).

[0020] [Figure 3] A and B are a series of graphs showing the results of cytotoxicity analysis, measuring virus-specific T cell-mediated lysis as a function of (A) effector cell-to-T cell ratio (E:T) and (B) peptide concentration. In the presence of the sensitized viral peptide, T cells robustly kill the target with an 80% lysis rate until the effector (cytotoxic T cell)-to-target ratio (E:T ratio) drops to 3:1. In the absence of the peptide, cell death is at a background level.

[0021] [Figure 4]This is a graph showing the results of flow cytometry (FITC) tetramer analysis which measured the CD8+ T cell content after stimulation twice with influenza peptides.

Mode for Carrying Out the Invention

[0022] The applicant is developing a method for treating infected patients by administering a novel preparation of virus peptide-specific cytotoxic T lymphocytes (CTL) products that can be used for the immunological treatment of patients severely affected by viral infections (e.g., influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), human herpesvirus (HHV), adenovirus, or coronavirus). The role of T cells in eradicating viral infections provides the scientific and clinical rationale for this effort.

[0023] In addition to the treatment of acute viral infections, virus-specific CTLs can also be used for the prevention and / or treatment of virus infection-related malignancies. For example, EBV is associated with various B cell cancers including post-transplant lymphoproliferative disorders. Targeted CTLs can also be used for the treatment of other EBV-related malignancies of the B cell lineage as well as nasopharyngeal carcinoma.

[0024] Viral infections can also be the basis for future cancers. Thus, there is a strong association between chronic hepatitis B infection and the development of hepatocellular carcinoma. Eradicating the virus via targeted CTLs early after infection is promising in avoiding the subsequent occurrence of malignancies. The methods described herein are not limited to merely the treatment of acute viral infections, and antiviral CTLs can be used to treat and / or prevent various types of cancers.

[0025] Existing production methods and ready-made products (e.g., AlloVir) yield T cell mixtures with far lower enrichment compared to the compositions and methods disclosed herein. Such products are marketed for the same or similar methods as those disclosed herein, using specialized terminology (e.g., “virus-specific T cells” (i.e., VST)). In contrast to commercially available products, the cells produced herein are known to be HLA class I-restricted CD8+ cytotoxic T cells. Thus, the methods disclosed herein are superior to those known in the art, as demonstrated by the high-purification. While not wishing to be constrained by theory, the high-purification of the cells disclosed herein is expected to result from the claimed method steps, which include, for example, restorative and prolonged culture between production steps, and which ultimately enable scalability to clinical grade.

[0026] At a higher level, this disclosure relates to a method for treating viral infections by administering virus-specific T cells to patients in need, a method for generating virus-specific CTLs, and pharmaceutical compositions of virus-specific CTLs. Such virus-reactive CTLs may be used in diagnostic methods for detecting the presence of viruses or viral products in biological samples using virus-specific T cells primed against human viruses (e.g., among many viruses, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus, and coronavirus). The method described herein substantially improves existing methodologies used for generating virus peptide-specific T cells by enriching CD8+ T cells against CD4+ T cells, identifying specific HLA allele-viral peptide associations, and increasing the cytotoxicity efficiency of virus-specific CTLs by about 40 times compared to conventional methods.

[0027] By using the compositions and methods described herein, responses can be concentrated on diverse targets, thereby providing a safety net to prevent viral evasion due to mutations. For example, many viral vaccines target surface-exposed glycoproteins (such as "S proteins"). If viral mutations occur in the S protein, the immune response may be impaired. Existing T cell-based therapeutic and prophylactic treatments are not rationally designed and generally contain mixtures of CD4 and CD8, making it difficult, if not impossible, to identify specific viral genome regions that induce a response. In contrast, the present invention allows those skilled in the art to identify not only epitopes specific to acquiring immunity, but also which part of the viral genome originates from the epitope. Thus, it is possible to individually evaluate responses to various viral peptides within the entire peptide repertoire and attack viruses resulting from the diversity of antigenic targets. As a result of the diversity included in the present invention, the likelihood of the virus evading immunological detection is lower compared to when a single antigen (such as the S protein) is focused on.

[0028] Currently, infectious diseases are one of the leading causes of death worldwide, with coronaviruses alone accounting for over 2 million deaths in 2020-2021. Viral genomes boast a remarkably high mutation rate compared to other genetic material, primarily due to their relatively small size, rapid spread, large number of offspring, and strong selective pressures. Conventional viral treatments, for example, in cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), focus on the surface-exposed spike glycoprotein. In COVID-19 variants, mutations frequently occur in these glycoproteins, significantly altering their folding and theoretically altering antibody binding. This could undermine the effectiveness of current treatments and preventative measures against new COVID-19 variants. Similarly, the duration of vaccine-induced immunity (particularly against a single mutable viral element) is uncertain. According to several reports, innate immunity to newly emerging viruses (such as coronaviruses) is short-lived (Eldridge, AWD et al. Nature Medicine 2020, 26:1691-1693).

[0029] This specification describes a method for treating patients with viral infection using novel viral peptide-specific CTLs. Viral peptide-specific CTLs, produced using a three-step (optionally four-step) process including an in vitro stimulation-proliferation cycle to generate the final CTL product described herein, can be injected into infected patients. Viral peptide-specific CTLs may originate from allogeneic mononuclear leukocytes collected from one or more donors using standard leukocyte apheresis techniques.

[0030] definition

[0031] As used herein, the term “pharmaceutical composition” refers to a preparation containing an effective amount of viral peptide-specific cytotoxic T lymphocytes (CTLs). The pharmaceutical composition may further contain at least one pharmaceutically acceptable pharmaceutical excipient.

[0032] As used herein, the terms “administer” or “to administer” refer to introducing virus peptide-specific T cells into the bloodstream of a patient. Pharmacopoeia of virus-specific CTLs may be administered to a patient, for example, via intravenous administration.

[0033] As used herein, the term “effective dose” refers to the amount of drug required to achieve the desired effect. The actual effective dose for a particular use may vary depending on the mode of administration and the patient’s physiological parameters (including, in particular, the patient’s age, weight, overall health, and the severity of the symptoms or condition being treated). The appropriate dose and administration schedule of viral peptide-specific CTLs for a particular patient may be determined by a clinician of usual skill, based on these and other considerations.

[0034] As used herein, the term “pharmaceutically acceptable pharmaceutical additive” refers to a pharmaceutical additive that can be administered without significant adverse toxicity. Such pharmaceutical additives are generally considered safe (GRAS) by the U.S. Food and Drug Administration.

[0035] As used herein, the terms “viral-like illness” or “viral infection” refer to any disease or illness that presents with symptoms that resemble those of a virus (meaning symptoms that appear in an infection caused by a virus).

[0036] As used herein, the terms “CD8+ T cells” or “CD8+ cytotoxic T lymphocytes (CTLs)” refer to CTLs that possess a CD8 coreceptor that binds to MHC class I molecules. CD8+ T cells and CD4+ T cells have different roles: CD8+ T cells or cytotoxic T cells mediate the death of cells presenting non-self epitopes bound to MHC class I molecules, while CD4+ T cells regulate the immune response by recognizing a different set of non-self epitopes bound to MHC class II molecules. A notable difference between CD8+ T cells and CD4+ T cells is that CD8+ T cells are more efficient at antigen presentation than CD4+ T cells. CD8+ T cells can be detected from samples containing heterogeneous T cell populations using tetramer assays or tetramer staining.

[0037] As used herein, the terms “peptide” or “viral peptide” refer to at least two consecutive amino acids covalently linked via peptide bonds. Viral peptides described herein may include short amino acid sequences (on the order of approximately 5 to 20 amino acids) that serve as high-affinity ligands for a given HLA allele. The term “viral peptide” means that the peptide is identical to a naturally occurring peptide sequence in a virus. Viral peptides do not need to be isolated from a virus and can be produced by peptide synthesis, recombinant DNA-based peptide expression systems, or other peptide production methods known to those skilled in the art. Hereinafter, a viral peptide identical to a naturally occurring peptide sequence in a virus may be referred to as “derived” from that virus, even if such viral peptide was not isolated from that virus.

[0038] As used herein, the term “human leukocyte antigen (HLA)” refers to the human leukocyte antigen (HLA) complex encoded by the major histocompatibility complex (MHC) genes. HLA complexes are cell surface-displayed receptors that function to bind to and display short peptides. HLA molecules are highly specific with respect to the peptide sequences they can present. HLA class I molecules typically bind to peptides of 8–12 amino acids (aa). HLA class I (HLA-I) and HLA class II (HLA-II) molecules present peptides that are typically recognized as complexes by CD8+ T cells and CD4+ T cells, respectively.

[0039] Treatment methods, prevention methods, and methods for reducing transmission

[0040] In one embodiment, the present invention relates to a method for treating and reducing the transmission of viruses (e.g., influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), human herpesvirus (HHV), adenovirus, coronavirus, and the like). The method comprises administering an effective amount of viral peptide-specific T cells to a patient in need, preferably by intravenous infusion. The viral peptide-specific T cells can be administered by intravenous delivery to the patient, which may be done, for example, by administration via a central line, midline, or peripheral IV.

[0041] Most viral infections are eradicated by T cells, with a particular emphasis on cytotoxic T cells. The majority of treatments for viral infections rely on measures to eradicate the virus by delaying viral replication and / or sufficiently alleviating symptoms to buy time for endogenous immunity to develop. In contrast, virus-specific CTLs are effective in specific response percentages, which vary depending on the virus, post-transplant characteristics, the persistence of immunosuppressive therapy, and other clinically variable factors. The CTL panel disclosed herein includes some of the most common HLA alleles, thereby providing a therapeutic opportunity to the majority of the population.

[0042] In some embodiments, patients undergo complete HLA typing before being treated with the appropriate CTLs, prior to administration of viral peptide-specific CTLs.

[0043] In one embodiment, prior to administration of viral peptide-specific CTLs, the patient undergoes a blood test for rapid, low-resolution HLA typing using high-resolution PCR sequence-specific primers (SSPs) as supplementation to determine whether the patient possesses potentially therapeutically suitable HLA antigens (HLA-A1, HLA-A2, HLA-B7, HLA-B40, HLA-Cw7). The use of high-resolution supplementation confirms that the patient is compatible with HLA-A*01:01, HLA-A*02:01, HLA-B*07:02, HLA-B*40:01, and HLA-C*07:02, and therefore matches the CTL for one or more alleles. Matching the CTL with the HLA allele(s) requires matching / selection of the correct viral antigenic peptide(s) (see Tables 1-5 and 14) to ensure that the injected cells recognize any potentially viral targets present on the patient's infected cells.

[0044] Premedication may be administered before administration of viral peptide-specific CTLs. In some embodiments, patients may be given premedication (such as diphenhydramine and acetaminophen). The diphenhydramine dose may be approximately 15 mg, 20 mg, 25 mg, or 30 mg. The acetaminophen dose may be approximately 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or 750 mg.

[0045] Patients may also be treated with antiviral drugs (e.g., remdesivir) or other standard therapeutic drug formulations before, concurrently with, or after administration of viral peptide-specific CTLs.

[0046] The effective dose of viral peptide-specific CTLs is based on body weight and can range from 1 × 10⁵ total cells / kg to 3 × 10⁶ total cells / kg. Doses of 1 × 10⁵ total cells / kg, 2 × 10⁵ total cells / kg, 3 × 10⁵ total cells / kg, 4 × 10⁵ total cells / kg, 5 × 10⁵ total cells / kg, 6 × 10⁵ total cells / kg, 7 × 10⁵ total cells / kg, 8 × 10⁵ total cells / kg, 9 × 10⁵ total cells / kg, 1 × 10⁶ total cells / kg, 2 × 10⁶ total cells / kg, 3 × 10⁶ total cells / kg, 4 × 10⁶ total cells / kg, 5 × 10⁶ total cells / kg, 6 × 10⁶ total cells / kg, 7 × 10⁶ total cells / kg, 8 × 10⁶ total cells / kg, or 9 × 10⁶ total cells / kg may be administered. In some embodiments, the dose is measured by the number of virus-reactive cells instead of the total amount. For example, the effective dose may range from 1 × 10⁵ virus-reactive cells / kg to 3 × 10⁶ virus-reactive cells / kg. Doses of 1 × 10⁵ virus-reactive cells / kg, 2 × 10⁵ virus-reactive cells / kg, 3 × 10⁵ virus-reactive cells / kg, 4 × 10⁵ virus-reactive cells / kg, 5 × 10⁵ virus-reactive cells / kg, 6 × 10⁵ virus-reactive cells / kg, 7 × 10⁵ virus-reactive cells / kg, 8 × 10⁵ virus-reactive cells / kg, 9 × 10⁵ virus-reactive cells / kg, 1 × 10⁶ virus-reactive cells / kg, 2 × 10⁶ virus-reactive cells / kg, 3 × 10⁶ virus-reactive cells / kg, 4 × 10⁶ virus-reactive cells / kg, 5 × 10⁶ virus-reactive cells / kg, 6 × 10⁶ virus-reactive cells / kg, 7 × 10⁶ virus-reactive cells / kg, 8 × 10⁶ virus-reactive cells / kg, or 9 × 10⁶ virus-reactive cells / kg may be administered. In some embodiments, the effective dose will be based on actual body weight. In certain embodiments, if the actual weight is heavier than the ideal weight, the dosage will be based on a corrected weight (ideal weight + 40% of the difference between the actual weight and the ideal weight). The ideal weight for each height is calculated using the formula by BJ Devine (1974): Men: 50.0 kg + 2.3 kg for each inch beyond 5 feet, Women: 45.5 kg + 2.3 kg for each inch beyond 5 feet.

[0047] The “effective dose” of a pharmaceutical composition containing viral peptide-specific CTLs is administered to an individual in need (such as an individual with a viral infection, an individual with a viral-like illness, an individual with viral-like symptoms, or an individual at risk of viral infection). The effective dose is sufficient to achieve the desired therapeutic or preventive effect, and such doses include those sufficient to reduce the virus, viral-like illness, or viral-like symptoms; those sufficient to reduce the duration of the disease; those sufficient to reduce the viral titer in an individual; those sufficient to reduce the number of days an infected individual develops viral-like symptoms and / or requires oxygen by any means; those sufficient to reduce the number of patients who develop virus-associated cytokine release syndrome; and / or those sufficient to reduce the incidence or rate of viral infection. A clinician with normal skills can determine the appropriate dose and optional antiviral agent, which is based, for example, on the individual’s age, susceptibility, tolerance, and overall health. Viral peptide-specific CTLs may be administered in single or multiple doses, as indicated.

[0048] The time required for intravenous delivery of CTLs (e.g., via peripheral, central, or midline infusion) should be less than 10 minutes. In some embodiments, the infusion time of CTLs is approximately 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute.

[0049] In one embodiment, the method includes administering an effective amount of a pharmaceutical composition to an individual suspected of having a virus, an individual in which a virus has been confirmed, or an individual at risk of infection with a virus (e.g., coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus, and similar). The method also includes administering an effective amount of a pharmaceutical composition to an individual having a viral-like disease.

[0050] The pharmaceutical composition may be intended for administration into the patient's bloodstream and may be administered in any appropriate form (such as intravenous).

[0051] In some embodiments, the treatment method involves administering an effective amount of the pharmaceutical composition of the present invention to an individual suspected of having a virus or an individual at risk of having a virus. For example, in some embodiments, an individual is suspected of having a virus (e.g., coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus, and similar) and may have one or more symptoms of the virus. Symptoms of the virus are well known and may include, depending on the specific virus, fever, cough, and shortness of breath, diarrhea, cystitis / hematuria, and hepatitis. Additional symptoms of some viral infections may include difficulty breathing, persistent chest pain or pressure, confusion, coma, and bluing of the lips or face.

[0052] In some embodiments, the method is for treating a viral infection and includes administering an effective amount of the pharmaceutical composition of the present invention to an individual in need. In other embodiments, the method is for preventing a viral infection and includes administering an effective amount of the pharmaceutical composition of the present invention to an individual at risk of infection by a virus (e.g., coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus). In other embodiments, the method is for reducing the spread of a viral infection and includes administering an effective amount of the pharmaceutical composition of the present invention to an individual infected with a virus (e.g., coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus) or an individual at risk of infection by a virus (e.g., coronavirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, hepatitis B virus, EBV, CMV, BKV, JCV, HHV, adenovirus).

[0053] The appropriate interval between doses to achieve the desired therapeutic effect may be determined based on the severity of the condition (e.g., infection), the patient's overall health, the patient's tolerance to the pharmaceutical composition, and other considerations. Based on these and other considerations, a clinician can determine the appropriate interval between doses. Generally, the pharmaceutical composition is administered at once, but may be administered every 1 to 4 days or once a week as needed.

[0054] The therapeutic methods and uses of the present invention offer particular benefits when an individual is suspected of having a virus, when an individual is confirmed to have a virus, when an individual is at risk of viral infection (e.g., individuals over 60 years of age, individuals with severe chronic conditions (heart disease, diabetes, lung disease, etc.), immunocompromised individuals, patients who have recently received cancer treatment), or when an individual has a lung disease (e.g., asthma (e.g., allergic / atopic, juvenile, late-onset, cough-type, or chronic obstructive), airway hypersensitivity, allergic rhinitis (seasonal or non-seasonal), bronchiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, cystic fibrosis, juvenile wheezing, and similar conditions) in addition to a viral-like illness. These patient populations are particularly susceptible to viral and other respiratory infections, and such infections often cause acute exacerbations of underlying lung disease. Therefore, the methods and therapeutic uses described herein may provide additional benefits to these patient populations by reducing the occurrence, duration, and / or severity of acute exacerbations of underlying lung disease.

[0055] Following administration, the success of the treatment will be determined by examining patient blood, urine, or stool samples and / or nasal or nasopharyngeal swab samples for viral diagnosis, CTL persistence, endogenous CTL formation, and antibody response to the virus. The response to the treatment may be examined, for example, 4, 7, 14, 28, 2, 3, and 6 months after infusion.

[0056] Method for preparing viral peptide-specific CTLs

[0057] The viral peptide-specific CTLs used in the therapeutic methods provided herein can be prepared using any suitable method, for example, by collecting allogeneic mononuclear leukocytes from a donor using standard leukocyte apheresis techniques and then sensitizing them with viral peptides. Lymphocytes can be exposed to a limited number of peptides known to bind to the HLA-restricting element of interest. For example, lymphocytes can be stimulated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-5, 1-10, 1-15, 1-20, 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 10-20, or 15-20 peptides known to bind to the HLA-restricting element of interest. The viral peptide-specific CTLs may originate from peripheral blood lymphocytes.

[0058] For example, the HLA-restrictive element of interest may be selected from the three classical HLA-I genes (HLA-A, HLA-B, and HLA-C) expressed in all human nucleated cells. HLA-I molecules present peptides derived from intracellular proteins. The intracellular antigen presentation pathway may include cleavage of viral proteins in the cytosol by proteasomes, transposition to the endoplasmic reticulum (ER) lumen, trimming by ER-endogenous aminopeptidases, loading onto HLA, and presentation on the cell surface. HLA-II genes (HLA-DR, HLA-DP, and HLA-DQ) are constitutively expressed only in cell subsets specialized for antigen presentation (such as dendritic cells, B cells, and macrophages), although expression may be induced in additional cell types, for example, in response to cytokine stimulation. HLA-II molecules present peptides derived from extracellular proteins taken into cells via endocytosis and phagocytosis, and peptides derived from intracellular proteins that access the autophagy-mediated HLA-II processing pathway.

[0059] HLA-I molecules typically bind to peptides of 8–12 amino acids (aa) in length. The peptide binding clefts of HLA-I are closed at both the N-terminus and C-terminus, and the optimal length selectivity is often biased to bind to peptides of approximately 9 units. In most HLA-I alleles, length selectivity differs between alleles. High-affinity ligands for a given HLA allele usually share common amino acid motifs with relatively strict selectivity at anchor sites (typically the second (P2) and last (PΩ) for HLA-I, and P1, P4, P6, and P9 for HLA-II), where specific interactions with the corresponding HLA-binding pocket residues are formed. HLA loci are the most polymorphic areas in the human genome, with tens of thousands of alleles reported to date. HLA variants with different peptide contact residues offer a different repertoire of peptides. HLA allele diversity in a population is an important evolutionary defense mechanism against a variety of pathogens (e.g., rapidly mutating viruses, newly emerging viruses, and similar ones). HLA alleles may be associated with the severity and prognosis of viral infections. For example, the HLA-C*15:02 allele is associated with protection from SARS-CoV-1, and HLA-B57 is highly associated with efficient HIV-1 control and long-term non-progressive infection even without antiretroviral therapy.

[0060] The specific peptides used in this method vary from virus to virus and will change over time as new peptides are discovered and the CTL results induced by existing peptides are experimentally demonstrated to be insufficient. The list of peptides provided in this application is not exhaustive and is a dynamic list that continues to evolve, and is provided as a non-exclusive illustrative list.

[0061] The methods for generating viral peptide-specific CTLs, methods for treating and preventing viral infections using viral peptide-specific CTLs, and the peptides used in the pharmaceutical compositions of viral peptide-specific CTLs described herein may originate from any virus. For example, T cell epitopes have been identified, collected, and reported for a wide range of viruses in the Immune Epitope Database and Analysis Resource (IEDB).

[0062] In one embodiment, lymphocytes are subjected to three in vitro stimulation-proliferation cycles to become the final CTL product used in the therapeutic method described herein. Each of these three stimulation-proliferation cycles has a different purpose in the overall production process and therefore follows a different procedure. Optionally, a fourth restimulation may be performed. The fourth restimulation may be performed (1) on products that are slightly below the release criteria and are expected to meet these criteria by performing an additional series of stimulation and proliferation, or (2) on products where additional cell proliferation is desired and it is thought that performing an additional series of stimulation and proliferation could significantly increase the number of therapeutic doses obtainable from that batch. Any such optional fourth restimulation may be performed according to the same process as the third stimulation.

[0063] In one embodiment, mononuclear cells derived from a healthy volunteer donor are separated into lymphocyte and monocyte fractions by elutriation. The lymphocytes are stimulated with peptides derived from known sequences of the viral genome and predicted / demonstrated to bind to specific HLA alleles. No virus-derived products are utilized. In the first stimulation, a subset of collected monocytes is processed to induce their maturation into dendritic cells. The dendritic cells are then pulsed with one or more virus-specific peptides and co-cultured with lymphocytes for 7 days. In the second and third stimulations, monocytes are used to present peptides, and the stimulated lymphocytes are grown / proliferated again for 7–12 days. The second stimulation may also include an enrichment step that helps in the selection of peptide-specific CTLs (by selective adhesion of T cells that recognize the pulsed peptide to the adherent monocyte layer) and the reduction of the content of other nonspecific "bystander" lymphocytes or other immune cells derived from the donor. In the third stimulation, monocytes and peptides are used again, but this selection step is not repeated.

[0064] Most of the procedures described herein are performed in RPMI-1640 medium containing 10% heat-inactivated AB serum. The amount of AB serum in the medium may be reduced to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Serum-free mediums and autologous plasma may be used as alternatives. These are referred to as “complete medium” or CM. Any suitable medium determined by those skilled in the art may be used, such as AIM V medium or other serum-free medium preparations (alone, containing 10% or low concentrations of pooled serum or autologous serum or plasma), or RPMI-1640 containing serum substitutes (containing or low concentrations of pooled serum or autologous serum or plasma).

[0065] In certain embodiments, lymphocytes are stimulated in a first stimulation (referred to as initial in vitro sensitization) using a pool of peptides for a given HLA allele, rather than as individual peptides. Examples 1-20, described below, show an initial list of peptides for five HLA alleles (see Tables 1-5). This list includes peptides suitable for coronaviruses and is expected to expand as new information becomes continuously available, and those skilled in the art will be able to identify additional peptides for use in the methods described herein. It is also possible to remove some peptides from the initial list.

[0066] Examples 13-14 and Table 9 show another example list of peptides for the HLA-A2 allele. Table 9 is a list of peptides suitable for influenza. This list is expected to expand as new information becomes continuously available. Those skilled in the art will be able to identify additional peptides for use in the methods described herein. Some of the peptides shown in Table 9 may turn out to be ineffective.

[0067] For initial in vitro sensitization, dendritic cells are used as antigen-presenting cells. These cells are prepared from monocytes subjected to elutriation. Fresh or freshly thawed monocytes can be concentrated by adhering them to plastic. After a suitable number of monocytes are resuspended in culture medium, the cells are transferred to tissue culture plates. Approximately half of all monocytes used initially are expected to mature into antigen-presenting dendritic cells (DCs) usable for the method. The cells are then incubated for an appropriate time (e.g., at least 60 minutes, at least 90 minutes, at least 120 minutes) until the monocytes adhere to the culture plate. After incubation, the supernatant is aspirated from the culture plate. The adherent cells can then be cultured for an appropriate time (e.g., 24 hours) in a medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, at which point mature cytokines (e.g., TNF-alpha, IL-1 beta, IL-6, and / or prostaglandin E2) may be added. This process forces monocytes to differentiate into antigen-presenting DCs. After 24 hours of incubation, the dendritic cells can be detached and prepared for collection by aspiration and centrifugation. The culture time in mature cytokines can be extended beyond 24 hours to approximately 30, 36, 42, or 48 hours, if necessary.

[0068] After collection, dendritic cells are pulsed with peptides (e.g., at concentrations of 2 micrograms / mL [μg / mL]) for an appropriate length of time (e.g., approximately 60, 75, 90, or 120 minutes). These dendritic cells then present viral peptides and are subsequently co-cultured with lymphocytes in culture medium (e.g., CM) in tissue culture flasks (e.g., 75 cm²). The ratio of lymphocytes to dendritic cells in the culture can be 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1. Lymphocytes (e.g., 80 × 10⁶ total cells, 100 × 10⁶ total cells, or 120 × 10⁶ total cells) are then added to each flask. This is considered day 0 of the CTL culture process. By not interfering with the culture during this 7-day stimulation period, intercellular interactions will begin to form.

[0069] Approximately 7 days later, a second stimulation enrichment and subsequent proliferation of virus-specific CTLs takes place. Seven days after initial sensitization, CTLs are restimulated as part of an enrichment step that helps in the selection of peptide-specific CTLs and the reduction of the content of other nonspecific "bystander" lymphocytes or other immune cells from the donor. The enrichment is based on the selective adhesion of peptide-specific CTLs to the monocyte layer that is pulsed with the specific viral peptide used in the initial sensitization. In contrast to CTLs that recognize any of the peptides presented by the appropriate HLA allele, which selectively adhere to the peptide-pulsed monocyte layer via the creation of immune synapses, "bystander" lymphocytes can be gently washed away as such interactions do not occur. Nonspecific adhesion may occur by some "bystander" lymphocytes, but this typically allows for a enrichment of peptide-specific CTLs by about 10 times or more compared to the starting material. To carry out this enrichment step, monocytes (e.g., 10 × 10⁶ cells / mL) are added to the tissue culture plate. Peptides (e.g., those with a final concentration of 2 μg / mL) are added and incubated with monocytes (e.g., for about 90 minutes). Lymphocytes (e.g., 60 × 10⁶ cells / mL, 70 × 10⁶ cells / mL, 80 × 10⁶ cells / mL, 90 × 10⁶ cells / mL, 100 × 10⁶ cells / mL, 110 × 10⁶ cells / mL, or 120 × 10⁶ cells / mL, typically the content of one 75 cm² tissue culture flask) are then added to the wells. "Bystander" lymphocytes are removed from the wells by gently washing with PBS after an appropriate length of time (e.g., about 5, 7.5, 10, or 12 minutes). The activation / restimulation process is completed by leaving the adherent lymphocytes in contact with the peptide-pulsed monocytes overnight.

[0070] The following day, lymphocytes are removed from the monocyte layer. Adherent lymphocytes obtained from the previous step are removed and transferred to a tissue culture flask containing culture medium supplemented with recombinant human IL-2 (for example, to a concentration of 50 U / ml). The addition of IL-2 over time (for example, 50 U / mL every 48 hours) promotes optimal proliferation of stimulated CD8+ T cells. The medium is replaced if the acidity of the flask increases due to the accumulation of metabolic waste products (the culture color changes from phenol red to a stronger orange / yellow). The medium may also be replaced if the glucose level falls below 60 mg / dl or if the lactate concentration rises above 11.5 mmol / L. Subsequently, the cells are cultured for a total of 7 days from the second stimulation. The enrichment on the monocyte layer as part of this second stimulation is crucial and provides true benefits that surpass conventional CTL culture methods. The enrichment on the monocyte layer is thought to be responsible for significantly increasing the purity level among total T cells, a level of purity that had not been achieved prior to the present invention. This purification step leads to increased selection of virus peptide-specific CD8+ CTLs compared to CD4+ lymphocytes and other nonspecific bystander lymphocytes, resulting in a final product containing a homogeneous sample with a purity of over 90%.

[0071] Subsequently, a third stimulation is performed to further increase the number of virus peptide-specific CTLs. The enrichment step performed as part of the second stimulation is typically not repeated as part of the third stimulation. However, if the evaluation of the percentage of virus-reactive lymphocytes (measured by intracellular cytokine assay or tetramer assay) falls below a certain threshold (e.g., approximately 12-18%, 12%, 13%, 14%, 15%, 16%, 17%, or 18%) on the day of the planned third stimulation, the procedure for the second stimulation may be repeated instead of the usual procedure for the third stimulation. Since the enrichment step contains most of the operations in the entire CTL generation process and is the most vulnerable point to contamination, it is desirable to avoid repeating this step multiple times whenever possible. Even without repeating the enrichment step, a further enrichment of the percentage of virus peptide-specific CTLs is expected after the third stimulation. This takes into account the fact that stimulated cells will proliferate in IL-2-containing medium, while unstimulated "bystander" cells will not. Unstimulated "bystander" cells die over time during culture, leading to increased enrichment of virus-specific CTL products. By setting the above threshold for when the enrichment step resulting from the second stimulation can be repeated, it is expected that this enrichment step will be repeated infrequently, only when essential to the manufacturing process.

[0072] After approximately 7 days of concentrated culture in tissue culture flasks (e.g., on day 22, 23, or 24 of the entire CTL stimulation / culture), the number of cells is counted, cell viability is tested, and the cells are re-stimulated in cell culture flasks (such as G-Rex® (Wilson Wolf Manufacturing Corporation, St. Paul, MN) or T75 (Thermo Fisher Scientific, Waltham, MA) brand cell culture flasks) with monocytes and peptides. The lymphocyte:monocyte ratio may be 4:1-5:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1. Each peptide is added again at a concentration of 2 μg / mL. Lymphocytes (1.5 × 10⁷ cells) are added to each cell culture flask for this second re-stimulation. Sensitization is carried out in a culture medium (e.g., 40 mL of complete medium) supplemented with IL-2 (e.g., 50 U / mL).

[0073] After the third stimulation, the CTLs are cultured again (e.g., for 7 days). The culture medium and IL-2 may be changed every 3-4 days, depending on when a color change in the medium is observed.

[0074] After a full three and a half weeks of stimulation and proliferation, starting from day 0, the CTLs are evaluated to determine if they meet the required criteria. If they do, they are collected for cryopreservation (e.g., within the next 24 hours). Optionally, and depending on the circumstances, a fourth stimulation may be performed after the third stimulation. This is typically done when further proliferation of the cells is deemed desirable to increase the number of CTLs being produced, or when the product slightly falls short of the release criteria and an additional series of stimulation / proliferation is thought to allow the product to meet all criteria. Restimulation steps may be performed at 6-10 day intervals (e.g., 6-day, 7-day, 8-day, 9-day, 10-day intervals), but these steps may be shortened or extended by one day from this typical 7-day interval as needed.

[0075] After completing three in vitro stimulation-growth cycles, the product is screened for adequate cell content, function, viability, and sterility. Cell content can be screened using tetramer analysis (e.g., using fluorescein isothiocyanate (FITC)) to determine the concentration of CD8+ and / or CD3+CD8+ CTLs in the sample, as well as the overall heterogeneity of the sample.

[0076] Adequate cell content is determined based on intracellular cytokine (ICC) staining or tetramer binding, where at least 20% of cells (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) are virus-free. This means that the cells react to the peptide, and that the content of naive T cells, monocytes, and NK cells in the product is less than 2.5% (e.g., about 2.4%, 2.3%, 2.2%, 2.1%, 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%). Preferably, at least 70% of the cells will react to the viral peptide based on tetramer bonds.

[0077] The CTLs described herein for use in the methods for generating virus-specific CTLs, methods for treating and preventing viral infections using virus-specific CTLs, and pharmaceutical compositions of virus-specific CTLs are substantially CD8+ T cells based on their cell content. The CTLs described herein contain, based on total cell content, at least about 60% CD8+ T cells, at least about 70% CD8+ T cells, at least about 80% CD8+ T cells, at least about 85% CD8+ T cells, at least about 90% CD8+ T cells, at least about 95% CD8+ T cells, or at least about 99% CD8+ T cells. The CTLs may begin as a CD8+ enriched cell composition in which all other lymphocyte types are depleted. The CD8+ T cell content may be determined using flow cytometry with an anti-CD8 antibody.

[0078] Of the CD8+ T cell population constituting CTLs, based on cell content, at least approximately 60% of CD8+ T cells may be peptide-specific CD8+ T cells, at least approximately 70% may be peptide-specific CD8+ T cells, at least approximately 80% may be peptide-specific CD8+ T cells, at least approximately 85% may be peptide-specific CD8+ T cells, at least approximately 90% may be peptide-specific CD8+ T cells, at least approximately 95% may be peptide-specific CD8+ T cells, and at least approximately 99% may be peptide-specific CD8+ T cells.

[0079] The methods for generating virus-specific CTLs, methods for treating and preventing viral infections using virus-specific CTLs, and CTLs for use in pharmaceutical compositions of virus-specific CTLs described herein are substantially depleted of CD4+ T cells based on their cell content. The CTLs described herein contain, based on total cell content, at least about 30% or less CD4+ T cells, at least about 20% or less CD4+ T cells, at least about 15% or less CD4+ T cells, at least about 10% or less CD4+ T cells, at least about 5% or less CD4+ T cells, at least about 2.5% or less CD4+ T cells, at least about 2.0% or less CD4+ T cells, at least about 1.5% or less CD4+ T cells, and at least about 1.0% or less CD4+ T cells.

[0080] The CTLs used in the methods for generating virus-specific CTLs, methods for treating and preventing viral infections using virus-specific CTLs, and pharmaceutical compositions of virus-specific CTLs described herein are substantially depleted of naive T cells, NK cells, monocytes, dendritic cells (DCs), and / or B cells. The total cell content of naive T cells, NK cells, monocytes, DCs, and / or B cells in CTLs for use in the therapeutic methods described herein is at least about 2.5% or less of the total cell content, either individually or collectively (e.g., about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%). These cell populations were considered to pose particular concern among the various complications of cell therapy, including the induction of graft-versus-host disease (e.g., naive T cells), HLA alloimmunity, transfer of leukocyte pathogens, or induction of immune exhaustion.

[0081] The function is based on the fact that when the effector:target ratio is 40:1, the CTL exhibits at least approximately 40% cell-lytic activity against the peptide pulse target. In some embodiments, the function is based on the fact that when the effector-to-target ratio is 30:1, 10:1, and up to 3:1, the CTL exhibits approximately 80% cell-lytic activity against the peptide pulse target.

[0082] The survival rate should exceed 70%, and for example, survival rates of 75%, 80%, 85%, 90%, 95%, or 99% may be appropriate. Virus-specific CTLs may be cryopreservable.

[0083] The method can be scaled to obtain sufficient cells for experiments, treatments, or clinical trials. For example, the Phase I-II trials planned by the inventors required approximately 3.5 × 10⁹ CTLs (total cell count) to support the trials. The method described herein began with 2.9 × 10⁹ lymphocytes isolated from a donor, yielding 9.2 × 10⁹ lymphocytes, of which at least 76% were detectable as CD8+ by tetramer analysis, which was sufficient material for clinical trials.

[0084] Sterility can be assessed through predetermined bacterial and fungal cultures, as well as mycoplasma and endotoxin assays. CTLs can be cryopreserved in cryobags at any desired concentration (e.g., a concentration of approximately 2 × 10⁶ viable cells / mL) and stored for later use in the methods disclosed herein.

[0085] Pharmaceutical composition containing viral peptide-specific cytotoxic T cells (CTLs)

[0086] In one embodiment, the present invention relates to a pharmaceutical composition for intravenous delivery comprising a viral peptide-specific CTL. The pharmaceutical composition is intended for intravenous delivery to an individual requiring it, and this intravenous delivery is, for example, by infusion via a peripheral IV, central line, or midline catheter. The pharmaceutical composition typically comprises one or more carriers or pharmaceutical excipients (such as DMSO) suitable for delivery of cryopreserved CTLs, and may also include the like.

[0087] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a SARS-CoV-2 (COVID-19) peptide that binds to a specific HLA-A2 allele (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or a combination thereof).

[0088] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a COVID-19 peptide that binds to a specific HLA-A1 allele (e.g., SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or a combination thereof).

[0089] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a COVID-19 peptide that binds to a specific HLA-B7 allele (e.g., SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, or a combination thereof).

[0090] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a COVID-19 peptide that binds to a specific HLA-B40 allele (e.g., SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, or a combination thereof).

[0091] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a COVID-19 peptide that binds to a specific HLA-Cw7 allele (e.g., SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a combination thereof).

[0092] In one embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a COVID-19 peptide that binds to a specific HLA-Cw7 allele (e.g., SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, or a combination thereof).

[0093] In another embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to an influenza virus peptide (e.g., SEQ ID NOs. 78-87 or a combination thereof) that binds to a specific HLA-A2 allele.

[0094] In another embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to one or more viral peptides bound to any one or a combination thereof of the HLA-A1 allele, HLA-A2 allele, HLA-B7 allele, HLA-B40 allele, and HLA-Cw7 allele. In yet another embodiment, the pharmaceutical composition comprises a virus-specific CTL sensitized to a combination of viral peptides bound to a combination of alleles (for example, a combination of half-dose A2 CTL and half-dose B7 CTL).

[0095] In some embodiments, the pharmaceutical composition comprises CTLs cryopreserved in DMSO, RPMI-1640, albumin, or a combination thereof.

[0096] If desired, the pharmaceutical compositions described herein may also include one or more additional antiviral agents (such as remdesivir). Antiviral agents suitable for intravenous injection may include acyclovir, peramivir, zanamivir, oseltamivir, ganciclovir, foscarnet, and similar agents, or antiviral agents suitable for intravenous injection may also be included in the pharmaceutical composition of CTL for patient use, or antiviral agents suitable for intravenous injection may be provided as a combination therapy.

[0097] The pharmaceutical composition may be in any form suitable for intravenous administration. [Examples]

[0098] Example 1: Pre-treatment period / screening For patients with a diagnosis of viral infection, a blood test for rapid, low-resolution human leukocyte antigen (HLA) typing is performed using high-resolution PCR SSP as a supplement to determine if the patient has HLA antigens (HLA-A1, HLA-A2, HLA-B7, HLA-B40, HLA-Cw7) that are potentially suitable for testing. The use of high-resolution supplementation confirms that the patient is HLA-A*01:01, HLA-A*02:01, HLA-B*07:02, HLA-B*40:01, HLA-C*07:02, and therefore matches CTLs for one or more alleles.

[0099] If a patient meets the eligibility criteria for the study based on age and comorbidities (multiple comorbidities are possible), and the patient has at least one HLA allele common to the cryopreserved CTL product, then such patient will be administered a virus-specific CTL. Blood samples will be collected and analyzed for confirmation by high-resolution typing. Based on the severity of the viral infection, CTL treatment will not be withheld while awaiting the results of high-resolution confirmation HLA typing in the central laboratory. Patients who do not have an HLA allele common to the cryopreserved CTL will be placed in the co-virus comparison group.

[0100] Example 2: Preparation of peptides Select COVID-19 peptides that bind to specific HLA alleles from the literature and add them to a peptide table. Add peptides sequentially to this table, assigning a laboratory notation to each peptide that reflects its binding HLA allele, with a dash followed by two extended digits. For example, A*02:01-03 refers to the third peptide in the table that binds to HLA-A*02:01. Peptides used to stimulate CTLs for clinical use should be purchased from CS Bio or similar vendors capable of preparing in vivo-grade materials. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0101] Example 3: Preparation of dendritic cells from monocytes Prepare dendritic cells to facilitate the generation of specific cytotoxic T lymphocytes (CTLs). Typically, start with 1 × 10⁷ monocytes or a multiple thereof. After centrifugation, decant the supernatant and resuspend in complete medium (RPMI1640 supplemented with 10% heat-inactivated normal AB serum) with DNase. Examine and record cell count and viability using trypan blue. Transfer to a 6-well plate, incubate at 37°C, and then wash. Add complete medium supplemented with GM-CSF and IL-4. The day on which monocyte culture is started in GM-CSF and IL-4 is referred to as day +1 of the procedure. On the following day (day +2), add four mature cytokines (IL-6, IL-1 beta, TNF-alpha, PGE2). The final concentrations of mature cytokines vary, but one example is as follows: [Table 6]

[0102] Return the 6-well plate to the incubator at 37°C. On day 3, transfer the dendritic cells to a conical tube, centrifuge, decant the supernatant, and resuspend them in complete medium to which peptides have been added to a concentration of 2 micrograms / mL, then incubate. At this point, the dendritic cells are ready for co-culture with lymphocytes. As monocytes mature into dendritic cells, changes occur in their shape and desorbency. Approximately half the number of dendritic cells compared to the starting monocytes are recovered.

[0103] Example 4: Co-culture of dendritic cells and lymphocytes The dendritic cells prepared in Example 3 are resuspended at a cell concentration of 1 × 10⁶ cells / mL in peptide-containing complete medium containing peptides at a concentration of 2 micrograms / mL each. Lymphocytes are prepared while the dendritic cells are pulsed with peptides. 100 million lymphocytes are thawed. Cell count and viability are tested using trypan blue. The target is a lymphocyte:monocyte (DC) ratio of 20:1 (100 million:5 million). Lymphocyte viability should be greater than 95%. After centrifugation, the thawed lymphocytes are resuspended in complete medium containing penicillin / streptomycin, transferred to a culture flask, and incubated. After incubation of dendritic cells with peptides, they are removed from the incubator, centrifuged again, and then resuspended in complete medium containing penicillin / streptomycin. The dendritic cell suspension is added to the flask containing the lymphocytes. After incubation of this mixture, additional complete medium containing penicillin / streptomycin is added. Seven days (in the range of 6-8 days) after co-culture, the cells are ready for analysis and re-stimulation.

[0104] Example 5: Stimulation and concentration on a monocyte monolayer This procedure is for enriching T cells that respond to specific peptides on a monocyte monolayer. These T cells can be enriched, activated, and proliferated in culture. Start with 10 × 10⁶ monocytes, verify the cell count, and assess viability. Viability should be 92% or higher. Resuspend the monocytes in complete medium with DNase. Incubate, then carefully remove the medium. Rinse the monocyte membrane with PBS, then remove the PBS from the well. After removing the PBS, add complete medium containing the peptides (each peptide at a concentration of 2 micrograms / mL) and incubate (prepare lymphocytes during this incubation). After incubation, remove the unbound peptides. Peptide removal is performed as follows: after removing the medium as described, wash with PBS.

[0105] Collect the cultured lymphocytes (carefully stir the flask gently to collect all lymphocytes from the bottom), centrifuge, decant the supernatant, resuspend in complete medium, and perform cell count and viability tests. Return to the incubator.

[0106] After washing the monocytes, the activated lymphocytes, which have been transferred to complete culture medium, are gently added to the wells containing the monocytes, and the plate is placed in the incubator. The moment the plate is placed in the incubator, T cell selection begins. Once the plate is in the incubator, the cells must not be disturbed at all during the selection process.

[0107] The selection time to be used depends on the number of lymphocytes being subjected to the selection process. If the lymphocyte count is less than 90 million, the selection time should be 10 minutes. If the lymphocyte count is 90 million or more, the selection time should be 7.5 minutes. The selection time should end when the timer sounds, and the cells should be removed from the incubator.

[0108] Remove the culture medium containing non-adherent cells. After removing the medium, wash with PBS as described above. As before, it is very important to be gentle at this stage, because the lymphocytes are adhering to the monocytes, and furthermore, the lymphocytes will loosen as the monocytes begin to die. After the third wash, you should notice a white film at the bottom of the plate. This is a good sign (evidence that the lymphocytes are adhering to the monocytes) and may be so opaque that it is impossible to see through to the bottom. Take care not to let the cells dry out.

[0109] Add complete medium containing penicillin / streptomycin and place in an incubator. Remove the plate from the incubator and examine the wells under a microscope. If excessive suspension cells are observed, remove the medium and wash again with warm PBS. If few or no suspension cells are observed, incubate overnight. After a fourth wash (if necessary), add complete medium containing penicillin / streptomycin and incubate overnight.

[0110] T cell proliferation

[0111] The following day, use a transfer pipette to transfer all remaining cells from the plate to a tissue culture flask containing IL-2-containing complete medium with penicillin / streptomycin. IL-2 should be added to the culture every two days.

[0112] Example 6: Stimulation of monocytes in a flask This procedure involves stimulating lymphocytes with monocytes pulsed with peptides, without performing a T cell enrichment / selection step (such as the one used in Example 5).

[0113] Typically, 10 million monocytes are required. The lymphocyte-to-monocyte ratio may vary somewhat depending on the available monocytes and the rate of lymphocyte proliferation after stimulation, but this ratio is typically close to 5:1. DNase should be added to the culture medium.

[0114] After counting and confirming viability, resuspend the monocytes in DNase-containing complete medium to which peptides are added at a concentration of 2 micrograms / mL. The cells should be resuspended in a conical tube. Briefly vortex the tube and place it in the incubator. Incubate, centrifuge, and then decant the supernatant. Add complete medium and vortex to resuspend. While the monocytes are pulsed with peptides, count the lymphocytes to be stimulated by the monocytes and confirm their viability. Resuspend the lymphocytes in complete medium and place them in a tissue culture flask. Add the monocytes to the lymphocytes and then incubate.

[0115] After incubating monocytes and lymphocytes as described above, add IL-2-containing complete medium with penicillin / streptomycin. Return the flask to the incubator. IL-2 should be added to the culture every two days.

[0116] Example 7: Intracellular cytokine (ICC) assay This procedure is for preparing ICC cells prior to staining and flow cytometry. Prepare monocytes before co-culture with lymphocytes. Thaw monocytes as described in the cell thawing SOP. Count cells using trypan blue and confirm viability. Monocytes must be primed with peptide(s) before adding lymphocytes (except for negative controls). Negative controls, which are monocytes not primed with any peptide, should always be included in parallel.

[0117] Resuspend 500,000 monocytes in 2 mL of complete medium at 37°C in a 5 or 10 mL tube. Add peptides (do not add peptides to negative controls). The final concentration of each peptide should be 2 micrograms / mL. If multiple peptides are added to the tube, each peptide should be present at a concentration of 2 micrograms / mL. Perform "monocyte priming" by incubating at 37°C for 90 minutes. This is done at 37°C using 5 cc or 10 cc tubes that have been loosened at the cap, placed on their side, and balanced on a 6-well plate. After 90 minutes, centrifuge at room temperature and 482 g for 10 minutes. Decant the supernatant, taking care not to decant the monocytes and not to let the monocytes dry out after decanting. Add 2 million lymphocytes to each tube (see below for lymphocyte preparation). In this example, the lymphocyte-to-monocyte ratio is 4:1, which is suitable when the frequency of peptide-specific lymphocytes is low (less than 20%). This is expected to apply after the initial peptide priming. As the frequency of peptide-reactive cells in the lymphocyte population increases with successive priming, this ratio should be reduced. For example, when the purity of peptide-specific T cells approaches over 80%, the lymphocyte:monocyte ratio should approach 1:1.

[0118] Lymphocyte preparation

[0119] Typically, this is started after pulsed peptide into monocytes and initiating a 90-minute incubation with the peptide. Count each “lymphocyte culture”. Each ICC experiment (“reaction”) requires 2 million lymphocytes. Take an appropriate volume of 2 million lymphocytes and centrifuge at room temperature, 482 g for 10 minutes. Decant and resuspend in 2 mL of CM at 37°C. After decanting the supernatant from the monocytes (above), add these 2 mL to the monocyte tubes. Ensure all cells are mixed by gently vortexing. Loosen the caps, lay the tubes on their sides, and incubate them in 5 cc or 10 cc tubes balanced on a 6-well plate for 2 hours at 37°C. Add brefelzin A at the end of the 2-hour incubation and incubate for 4 hours at 37°C.

[0120] Stain the ICC. The number of cells that react with the peptide will be very small (less than 1% after initial sensitization in some cases), but it should increase to a much higher level with repeated stimulation.

[0121] Example 8: Production of third-party virus-specific CTLs Lymphocytes are exposed to a limited number (up to 20) of peptides known to bind to the target HLA-restricting element. Lymphocytes are initially stimulated with peptide-pulsed dendritic cells and then stimulated twice more with peptide-pulsed monocytes. As part of the first monocyte-peptide restimulation, the target CTLs are enriched due to their selective binding to the monocyte-peptide monolayer. While the highest frequency of virus-reactive lymphocytes reported in the literature to the inventors is less than 2% (Leen et al., 2006), the inventors' shipping standard requires at least 20% virus-reactive CTLs. The product will be produced by the Jefferson Cell Processing Laboratory in Philadelphia. The shipping standard is as follows: (1) The product must demonstrate that at least 20% of the CTLs react to the viral peptide in an ICC assay (note that tetramer assays are a more stringent measure, as reactivity to peptides is always higher in tetramer assays than in ICC assays). (2) Effector: When the target ratio is 20:1, 40% of the appropriate target cells are lysed. (3) Flow cytometry must reveal that the monocyte content of the cell product is 2.5% or less, the NK cell content is 2.5% or less, and the naive T cell content is 2.5% or less. These latter populations have been considered a concern in BMT studies for inducing GVHD. The inventors do not consider these latter populations to be important to the viral population, but until more data on safety becomes available, such populations will be included in the inventors' shipping criteria.

[0122] Figures 1A–1C show the proliferation of viral CTLs by applying the proposed laboratory process. These graphs show background levels (left panel; Figure 1A), detection of a small population of CD8+ T cells after one week of in vitro culture (Figure 1B), and enrichment up to 15% after selection and further proliferation (right panel; Figure 1C). The x-axis reflects staining with a CD8 marker to identify a cytotoxic subset of T cells. The y-axis reflects interferon-gamma production in response to stimulation with a viral peptide capable of binding to HLA-A*02:01. Further enrichment is expected to consistently occur with subsequent stimulation / proliferation steps in our process.

[0123] Example 9: Tetramer Assay The purity and concentration of CTLs were quantified using a tetramer assay. By first staining the CTL samples to identify anti-CD8 pMHC tetramers, we identified which CTLs had been converted into antigen-specific T cell populations (viral peptide-specific CD8+ T cells). This allows for simultaneous staining of antigen-specific T cells and the diverse phenotypic populations that resulted, without the distortion often associated with function-based profiling. CTLs have been generated from all convalescent donors tested to date. Only 7 of the 20 test peptides derived from the COVID-19 genome have been demonstrated to stimulate CTLs. As an alternative to standard interferon production assays (used in other viral models, but with insufficient readout for SARS-CoV-2 (COVID-19) (Habel, J. Ret al. PNAS 2020, 117:24384-24391., Keller, M. Ret al. Blood 2020, 136:2905-2917.)), a tetramer assay was used to quantify the purity and enrichment of CTLs. Over 90% of the generated CTLs were CD3+CD8+, and over 60% were positive in the tetramer assay. Tetramer analysis (Figure 2A) measured CD8+ T cells after the first peptide stimulation, showing that at least approximately 2.3% of the total cells were virus peptide-specific CD8+ CTLs. Tetramer analysis (Figure 2B) further demonstrated that over 75% of the cells in the sample were CD8+ CTLs after the final stimulation (third stimulation). Furthermore, after the final stimulation, more than 90% of the cell products were CD8+ and / or CD3+CD8+.

[0124] Example 10: Cytotoxic Assay Functional analysis of virus-specific CTLs was performed as a function of CTL cytotoxicity against viral peptide pulse targets. Effector cells were pulsed with 2 μg / mL viral peptide and incubated at 37°C for an appropriate time. After incubation with the viral peptide, the medium was aspirated, the effector cells were rinsed with warm PBS, and replaced with fresh complete medium. CTLs were added to wells seeded with effector cells at ratios of 30:1, 10:1, 3:1, and 1:1 and titrated (non-viral peptide pulse effector cell conditions were maintained as a control). Cytotoxicity was measured as a function of the release of the radioactive tracer (51Cr) from the lysed cells (compared to a control in which all cells were chemically lysed). CTL-mediated cytotoxicity observed at E:T ratios of 30:1 to 3:1 was typically over 80% (Figure 3A). When the effector-to-target ratio was 1:1, the cytotoxicity was approximately 60%.

[0125] Example 11: Scalability The method can be extended to obtain enough cells for clinical trials. For example, a planned Phase I-II trial requires approximately 3.5 × 10⁹ CTLs to support the trial. As shown in Table 7, 2.9 × 10⁹ lymphocytes were isolated from the donor, yielding 9.2 × 10⁹ lymphocytes. At least 76% of these were detectable as CD8+ by tetramer analysis, which was sufficient material for a clinical trial. [Table 7]

[0126] Example 12: Comparative Data We compared the use of a limited number of peptides to increase the concentration of stimulation with the use of virus-infected cells, cells transfected with full-length viral protein genes, or a large peptide library. Adding a selection step using stimulating viral peptides improved uniformity compared to existing methods and eliminated unrelated lymphocytes present in the donor sample.

[0127] In contrast to commercially available virus-specific CTL products, which have been found to be primarily CD4+ populations, the method disclosed herein statistically significantly enriched the CD8+ T cell population. To date, CTL generation has been successfully achieved from all convalescent donors. The cell products were typically over 90% CD3+CD8+ T cells, with over 60% positive in tetramer assays (Table 8). Cytotoxicity to peptide pulse targets was over 60% at an E:T ratio of 3:1. This method can be extended for clinical trials.

[0128] As a result, the cytotoxic and lytic responses of cells pulsed with the peptide were far stronger compared to existing methods. Nearly four times more T cell death (a nearly 40-fold increase in efficiency) was observed even with less than one-tenth the number of T cells used compared to other CTL products. While other commercially available virus-specific CTL products can induce cytotoxicity, it is often unclear which cells in the mixture are therapeutically important. The method disclosed herein demonstrates that the dominant virus-specific CTLs are entirely CD8+ subsets, cytotoxic, and specifically targeted to HLA-class I peptides. Existing products, on the other hand, differ significantly in T cell composition, purity, and target identification. [Table 8]

[0129] Example 13: Preparation of influenza peptide Influenza peptides that bind to specific HLA-A2 alleles are selected from the literature and added to a peptide table. Peptides are sequentially added to this table, and each peptide is assigned a laboratory notation reflecting its binding HLA allele, with a dash followed by an extended two-digit number. For example, A*02:01-03 refers to the third peptide in the table that binds to HLA-A*02:01. Peptides used to stimulate CTLs for clinical use are purchased from CS Bio or similar vendors capable of preparing in vivo-grade materials.

[0130] [Table 9]

[0131] Example 22: Production of third-party influenza-specific CTLs Lymphocytes are exposed to a limited number (up to 20) of peptides known to bind to the target HLA-restricting element. Lymphocytes are initially stimulated with peptide-pulsed dendritic cells and then stimulated twice more with peptide-pulsed monocytes. As part of the first monocyte-peptide restimulation, the target CTLs are enriched due to their selective binding to the monocyte-peptide monolayer. While the highest frequency of virus-reactive lymphocytes reported in the literature to the inventors is less than 2% (Leen et al., 2006), the inventors' shipping standard requires at least 20% virus-reactive CTLs. The product will be produced by the Jefferson Cell Processing Laboratory in Philadelphia. The shipping standard is as follows: (1) The product must demonstrate that at least 20% of the CTLs react to the viral peptide in an ICC assay (note that tetramer assays are a more stringent measure, as reactivity to peptides is always higher in tetramer assays than in ICC assays). (2) Effector: When the target ratio is 20:1, 40% of the appropriate target cells are lysed. (3) Flow cytometry must reveal that the monocyte content of the cell product is 2.5% or less, the NK cell content is 2.5% or less, and the naive T cell content is 2.5% or less. These latter populations have been considered a concern in BMT studies for inducing GVHD. The inventors do not consider these latter populations to be important to the viral population, but until more data on safety becomes available, such populations will be included in the inventors' shipping criteria.

[0132] Figure 4 shows the proliferation of influenza CTLs by applying the proposed laboratory process. The graph shows the detection of a CD8+ T cell population after a second stimulation. The x-axis reflects staining with a CD8 marker to identify a cytotoxic subset of T cells. The y-axis reflects interferon-gamma production in response to stimulation with an influenza peptide capable of binding to HLA-A2.

[0133] All teachings of all documents cited herein are incorporated herein by reference.

Claims

1. A composition for treating a viral infection in a human patient in need thereof, the composition comprising cells including viral peptide-specific cytotoxic T lymphocytes (CTLs), which are specifically enriched for cells reactive to the viral peptide, the CTLs being sensitized by in vitro stimulation to multiple peptides restricted to a single HLA allele, at least 20% of the CTLs being reactive to the viral peptide, and the cells comprising less than 2.5% naive T cells, monocytes, NK cells, or any combination thereof.

2. The composition of claim 1 , wherein the human patient is an elderly patient or an immunocompromised patient.

3. The composition of claim 1 , wherein the composition is administered by intravenous infusion.

4. 4. The composition of claim 3, wherein the injection is delivered to the patient via a central line or midline.

5. The composition of claim 1 , wherein the viral peptide-specific CTLs are derived from a single donor.

6. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-A1 allele.

7. 7. The composition of claim 6, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and combinations thereof.

8. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-A2 allele.

9. 9. The composition of claim 8, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof.

10. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-B7 allele.

11. 11. The composition of claim 10, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and combinations thereof.

12. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-B40 allele.

13. 13. The composition of claim 12, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and combinations thereof.

14. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to the HLA-Cw7 allele.

15. 15. The composition of claim 14, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and combinations thereof.

16. The composition of any one of claims 1 to 5, wherein the viral peptide-specific CTLs are sensitized to a combination of viral peptides that bind to any one or combination of HLA-A1 alleles, HLA-A2 alleles, HLA-B7 alleles, HLA-B40 alleles, and HLA-Cw7 alleles.

17. The composition of any one of claims 1 to 16, wherein the viral peptide is derived from the Severe Acute Respiratory Syndrome (SARS) virus.

18. 18. The composition of claim 17, wherein the viral peptide is derived from the SARS-coronavirus 2 (COVID-19) virus.

19. The composition of claim 8, wherein the one or more peptides are selected from the list consisting of SEQ ID NOs: 78-87 and combinations thereof.

20. 1. An in vitro method for preparing viral peptide-specific cytotoxic T cells (CTLs), which are specifically enriched for cells reactive to a viral peptide, comprising the steps of: a. a first stimulation step in which a subset of monocytes is treated to induce their maturation into dendritic cells, said dendritic cells being pulsed with one or more virus-specific peptides and co-cultured with lymphocytes for at least 6 days; b. A second stimulation step in which monocytes are used to present the virus-specific peptides, the stimulated lymphocytes are cultured for at least 7 days, and peptide-specific CTLs are selected due to the selective adhesion of T cells that recognize the pulsed peptides to the adherent monocyte layer; c. A third stimulation step in which a subset of monocytes is pulsed with multiple virus-specific peptides and restricted to a single HLA allele, thereby generating virus-peptide-specific CTLs; wherein at least 20% of the CTLs are reactive to a viral peptide.

21. The method of claim 20, wherein the virus-reactive CTLs are allogeneic mononuclear leukocytes collected from a single donor.

22. 22. The method of claim 20 or 21, wherein the induction of maturation into dendritic cells comprises a first treatment of the monocytes with GM-CSF, IL-4, or a combination of the two, for at least about 24 hours, and a second treatment of the monocytes after the first treatment with TNF-alpha, IL-1 beta, IL-6, prostaglandin E2, or any combination thereof, for at least about 24 hours.

23. The method of any one of claims 20 to 22, wherein pulsing the dendritic cells with viral peptides comprises incubating the dendritic cells with at least about 2 μg / mL of each viral peptide.

24. The method of any one of claims 20 to 23, wherein the stimulatory lymphocytes in the second stimulation step are further selected by treating the co-culture with human interleukin-1 (IL-1).

25. The method of any one of claims 20 to 24, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-A1 allele.

26. 26. The method of claim 25, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and combinations thereof.

27. The method of any one of claims 20 to 24, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-A2 allele.

28. 28. The method of claim 27, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof.

29. The method of any one of claims 20 to 24, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-B7 allele.

30. 30. The method of claim 29, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and combinations thereof.

31. The method of any one of claims 20 to 24, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to an HLA-B40 allele.

32. 32. The method of claim 31 , wherein the one or more peptides are selected from the list consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and combinations thereof.

33. The method of any one of claims 20 to 24, wherein the viral peptide-specific CTLs are primed against one or more peptides restricted to the HLA-Cw7 allele.

34. 34. The method of claim 33, wherein the one or more peptides are selected from the list consisting of SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and combinations thereof.

35. The method according to any one of claims 20 to 24, wherein the viral peptide-specific CTLs are sensitized to a combination of viral peptides that bind to any one or more of the following combinations: HLA-A1 allele, HLA-A2 allele, HLA-B7 allele, HLA-B40 allele, and HLA-Cw7 allele.

36. The method of any one of claims 20 to 35, wherein the viral peptide is derived from the Severe Acute Respiratory Syndrome (SARS) virus.

37. 37. The method of claim 36, wherein the viral peptide is derived from the SARS-coronavirus 2 (COVID-19) virus.

38. 28. The method of claim 27, wherein the one or more peptides are selected from the list consisting of SEQ ID NOs: 78-87 and combinations thereof.

39. A pharmaceutical composition comprising cells including viral peptide-specific cytotoxic T lymphocytes (CTLs), which are specifically enriched cells reactive to a viral peptide, wherein the CTLs are sensitized to multiple peptides restricted to a single HLA allele by in vitro stimulation, at least 20% of the CTLs are reactive to the viral peptide, and the cells comprise less than 2.5% naive T cells, monocytes, NK cells, or any combination thereof.

40. The pharmaceutical composition of claim 39, wherein the CTLs are derived from multiple donors.

41. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to a specific HLA-A1 allele, and the one or more viral peptides are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and combinations thereof.

42. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to a specific HLA-A2 allele, and the one or more viral peptides are selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and combinations thereof.

43. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to a specific HLA-B7 allele, and the one or more viral peptides are selected from the group consisting of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and combinations thereof.

44. The CTLs are primed against one or more viral peptides that bind to a specific HLA-B40 allele, and the one or more viral peptides are selected from the group consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:

41. The pharmaceutical composition of claim 39 or 40, wherein the nucleic acid is selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and combinations thereof.

45. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to a specific HLA-Cw7 allele, and the one or more viral peptides are selected from the group consisting of SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and combinations thereof.

46. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to any one or combination of HLA-A1 alleles, HLA-A2 alleles, HLA-B7 alleles, HLA-B40 alleles, or HLA-Cw7 alleles.

47. The pharmaceutical composition of claim 39 or 40, wherein the pharmaceutical composition comprises CTLs cryopreserved in DMSO, in RPM1-1640, in albumin, or a combination thereof.

48. 41. The pharmaceutical composition of claim 39 or 40, wherein the pharmaceutical composition comprises one or more additional antiviral agents.

49. 41. The pharmaceutical composition of claim 39 or 40, wherein the pharmaceutical composition is in a form suitable for intravenous administration.

50. The pharmaceutical composition of claim 39 or 40, wherein the viral peptide-specific CTL is specific to a virus selected from the group consisting of SARS-CoV-2 (COVID-19), influenza, parainfluenza, respiratory syncytial virus (RSV), metapneumovirus, hepatitis B virus (HBV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), human herpes virus (HHV), and adenovirus.

51. 41. The pharmaceutical composition of claim 39 or 40, wherein the viral peptide is derived from the Severe Acute Respiratory Syndrome (SARS) virus.

52. 52. The pharmaceutical composition of claim 51, wherein the viral peptide is derived from the SARS-coronavirus 2 (COVID-19) virus.

53. The pharmaceutical composition of claim 39 or 40, wherein the CTLs are sensitized to one or more viral peptides that bind to specific HLA-A2 alleles selected from the group consisting of SEQ ID NOs: 78 to 87 and combinations thereof.