T cells specific for multiple respiratory viral antigens and methods for producing and using same in therapy

Abstract

Embodiments of the present disclosure relate to T cell lines specific for multiple respiratory viruses and methods of using same to treat and prevent viral infections.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 823,446, filed March 25, 2019, which is incorporated by reference in its entirety herein. [Technical field]

[0002] Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, immunology, and medicine. [Background technology]

[0003] Viral infections are a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), a treatment option for a variety of disorders. However, after transplantation, graft-versus-host disease (GVHD), recurrence of primary disease, and viral infections remain the major causes of morbidity and mortality. Airway infections with community-acquired respiratory viruses, including respiratory syncytial virus, influenza, parainfluenza virus, and human metapneumovirus, can cause severe symptoms, including pneumonia and bronchiolitis, are detected in up to 40% of allogeneic hematopoietic stem cell transplant recipients, and can be fatal. Other respiratory viruses, including adenoviruses (AdVs), rhinoviruses, and coronavirus strains, including SARS-CoV, SARS-CoV-2, MERS-CoV, as well as epidemic CoVs that commonly affect immunocompromised patients, can also cause severe symptoms, especially in immunocompromised individuals, and the recent SARS-CoV2 pandemic has revealed how ill-prepared we are to treat and prevent such infections. Given the lack of effective antiviral drugs and data from our group demonstrating that adoptively transferred ex vivo expanded virus-specific T cells could be clinically beneficial in the treatment of both latent (Epstein-Barr virus, cytomegalovirus, BK virus, human herpesvirus 6) and lytic (adenovirus) viruses, we investigated the possibility of extending this immunotherapy approach to respiratory viruses. Antiviral drugs are available for some viruses but are not always effective, highlighting the need for novel therapies. One strategy to treat these viral infections is by adoptive T cell transfer, whereby virus-specific T cells (VSTs) are expanded ex vivo from the peripheral blood of healthy donors and then infused into individuals with viral infections, e.g., stem cell transplant recipients.

[0004] In vitro expanded donor-derived and third-party virus-specific T cells targeting Adv, EBV, CMV, BK, and HHV6 have been shown to be safe when adoptively transferred into stem cell transplant patients with ongoing viral infections. The virus-specific T cells reconstituted antiviral immunity against Adv, EBV, CMV, BK, and HHV6, were effective in eliminating disease, and showed substantial proliferation in vivo. Adoptively transferred in vitro expanded virus-specific T cells have also been shown to be safe and associated with clinical benefit when adoptively transferred into patients.

[0005] Embodiments of the present disclosure fulfill a long-standing need in the art by providing treatment against specific viruses by administering ex vivo expanded, non-genetically modified, virus-specific T cells to control viral infection and ameliorate / eliminate one or more disease symptoms. Summary of the Invention

[0006] In some embodiments, the present disclosure provides a composition comprising a polyclonal population of virus-specific T lymphocytes (VSTs) that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one first antigen from PIV and at least one second antigen from one or more second viruses. In some embodiments, the VSTs are generated by contacting peripheral blood mononuclear cells (PBMCs) with a plurality of pepmix libraries, each pepmix library containing a plurality of overlapping peptides spanning at least a portion of the viral antigens, at least one of the plurality of pepmix libraries spanning a first antigen from PIV-3, and at least one additional pepmix library of the plurality of pepmix libraries spanning each second antigen. In some embodiments, the VST is generated by contacting a T cell with a dendritic cell (DC) stimulated with a plurality of pepmix libraries, each pepmix library containing a plurality of overlapping peptides spanning at least a portion of a viral antigen, at least one of the plurality of pepmix libraries spanning a first antigen from PIV-3, and at least one additional pepmix library of the plurality of pepmix libraries spanning each second antigen. In some embodiments, the VST is generated by contacting a T cell with a dendritic cell (DC) nucleofected with at least one DNA plasmid encoding a PIV-3 antigen and at least one DNA plasmid encoding each second antigen. In some embodiments, the plasmids encode at least one PIV-3 antigen and at least one second antigen. In some embodiments, the VST comprises CD4+ T lymphocytes and CD8+ T lymphocytes. In some embodiments, the VST expresses an αβ T cell receptor. In some embodiments, the VST comprises an MHC-restricted T lymphocyte. In some embodiments, the one or more second viruses are selected from the group consisting of respiratory syncytial virus (RSV), influenza, human metapneumovirus (hMPV), and combinations thereof. In some embodiments, the one or more second viruses include respiratory syncytial virus (RSV), influenza, human metapneumovirus, and combinations thereof.In some embodiments, the one or more second viruses comprise respiratory syncytial virus (RSV), influenza, human metapneumovirus, and combinations thereof. In some embodiments, the composition comprises one, two, three, or four first antigens. In some embodiments, the first antigen is selected from the group consisting of PIV-3 antigen M, PIV-3 antigen HN, PIV-3 antigen N, PIV-3 antigen F, and combinations thereof. In some embodiments, the four first antigens are as follows: PIV-3 antigen M, PIV-3 antigen HN, PIV-3 antigen N, and PIV-3 antigen F. In some embodiments, the composition comprises two or three second viruses. In some embodiments, the composition comprises three second viruses. In some embodiments, the three second viruses are influenza, RSV, and hMPV. In some embodiments, the composition comprises at least two second antigens per each second virus. In some embodiments, the composition comprises one, two, three, four, five, six, seven, or eight second antigens. In some embodiments, the second antigen is selected from the group consisting of influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, hMPV antigen N, and combinations thereof. In some embodiments, the second antigen comprises influenza antigen NP1, influenza antigen MP1, or both. In some embodiments, the second antigen comprises RSV antigen N, RSV antigen F, or both. In some embodiments, the second antigen comprises hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, hMPV antigen N, and combinations thereof. In some embodiments, the second antigen comprises each of influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the multiple antigens include PIV-3 antigen M, PIV-3 antigen HN, PIV-3 antigen N, PIV-3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N.In some embodiments, the plurality of antigens consists of or consists essentially of PIV-3 antigen M, PIV-3 antigen HN, PIV-3 antigen N, PIV-3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the VSTs are cultured ex vivo in the presence of both IL-7 and IL-4. In some embodiments, the multi-virus VSTs have grown sufficiently within 9-18 days of culture to be ready for administration to a subject. In some embodiments, the VSTs exhibit one or more properties selected from: (a) negligible alloreactivity; (b) less activation-induced cell death of antigen-specific T cells recovered from a subject than corresponding antigen-specific T cells recovered from the same subject but not cultured in the presence of both IL-7 and IL-4; and (c) greater than 70% viability. In some embodiments, the composition is negative for bacteria and fungi for at least 7 days in culture; exhibits less than 5 EU / ml endotoxin, and is negative for mycoplasma. In some embodiments, the pepmix is ​​chemically synthesized, and is optionally >90% pure. In some embodiments, the VST is Th1 polarized. In some embodiments, the VST is capable of lysing viral antigen-expressing target cells. In some embodiments, the VST does not significantly lyse uninfected autologous or allogeneic target cells.

[0007] The present disclosure also provides a pharmaceutical composition comprising any one of the compositions disclosed herein, formulated for intravenous delivery, which is negative for bacteria and fungi in culture for at least 7 days; exhibits endotoxin less than 5 EU / ml, and is negative for mycoplasma. For example, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a polyclonal population of virus-specific T lymphocytes (VSTs) that recognize multiple viral antigens, the multiple viral antigens comprising at least one first antigen from PIV and at least one second antigen from one or more second viruses. In some embodiments, the second viruses comprise respiratory syncytial virus (RSV), influenza, and human metapneumovirus. In some embodiments, the VST is generated by contacting peripheral blood mononuclear cells (PBMCs) with a plurality of pepmix libraries, each pepmix library containing a plurality of overlapping peptides spanning at least a portion of a viral antigen, at least one of the plurality of pepmix libraries spanning a first antigen from PIV-3, and at least one further pepmix library of the plurality of pepmix libraries spanning a respective second antigen, and the pharmaceutical composition is formulated for intravenous delivery, the composition is negative for bacteria and fungi in culture for at least 7 days; exhibits less than 5 EU / ml endotoxin, and is negative for mycoplasma.

[0008] The present disclosure also provides a method of lysing a target cell with any one or more of the compositions or pharmaceutical compositions disclosed herein. For example, in some embodiments, the present disclosure provides a method of lysing a target cell, comprising contacting the target cell with a polyclonal population of virus-specific T lymphocytes (VSTs) that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one first antigen from PIV and at least one second antigen from one or more second viruses. In some embodiments, the second viruses comprise respiratory syncytial virus (RSV), influenza, and human metapneumovirus. In some embodiments, the VSTs are generated by contacting peripheral blood mononuclear cells (PBMCs) with a plurality of pepmix libraries, each pepmix library containing a plurality of overlapping peptides that span at least a portion of the viral antigen, at least one of the plurality of pepmix libraries spanning a first antigen from PIV-3, and at least one additional pepmix library of the plurality of pepmix libraries spanning each second antigen. In some embodiments, the contacting occurs in vivo in a subject. In some embodiments, the contacting occurs in vivo via administration of a VST to a subject.

[0009] The present disclosure also provides a method of treating or preventing a viral infection comprising administering to a subject in need of such treatment or prevention any one or more of the compositions or pharmaceutical compositions disclosed herein. For example, in some embodiments, the present disclosure provides a method of treating or preventing a viral infection comprising administering to a subject in need of such treatment or prevention a polyclonal population of virus-specific T lymphocytes (VSTs) that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one first antigen from PIV and at least one second antigen from one or more second viruses. In some embodiments, the second viruses comprise respiratory syncytial virus (RSV), influenza, and human metapneumovirus. In some embodiments, the VST is generated by contacting peripheral blood mononuclear cells (PBMCs) with a plurality of pepmix libraries, each pepmix library containing a plurality of overlapping peptides spanning at least a portion of a viral antigen, at least one of the plurality of pepmix libraries spanning a first antigen from PIV-3, and at least one additional pepmix library of the plurality of pepmix libraries spanning a respective second antigen. In some embodiments, the subject is administered VST 5x10 6 ~5x10 7 pieces / m 2In some embodiments, the subject is administered multiple doses of VST. In one embodiment, the subject is administered VST, then the subject's viral load is monitored, and if the viral load increases, the subject is administered a second dose of VST. In some embodiments, the subject is immunocompromised. In some embodiments, the subject has acute myeloid leukemia, acute lymphoblastic leukemia, or chronic granulomatous disease. In some embodiments, the subject has undergone (a) a matched related donor transplant with reduced intensity conditioning; (b) a matched unrelated donor transplant with myeloablative conditioning; (c) a haploidentical transplant with reduced intensity conditioning; or (d) a matched related donor transplant with myeloablative conditioning prior to receiving VST. In some embodiments, the subject has (a) undergone a solid organ transplant; (b) undergone chemotherapy; (c) has HIV infection; (d) has a genetic immunodeficiency; and / or (e) has undergone an allogeneic stem cell transplant. In some embodiments, the composition is administered multiple times to the subject. In some embodiments, administration of the composition effectively treats or prevents a viral infection in a subject, the viral infection being selected from the group consisting of parainfluenza virus type 3, respiratory syncytial virus, influenza, human metapneumovirus, and combinations thereof. In some embodiments, the subject is a human.

[0010] The disclosure also provides compositions comprising a polyclonal population of VSTs that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one antigen selected from parainfluenza virus type 3 (PIV-3), respiratory syncytial virus, influenza, human metapneumovirus, and combinations thereof. In some embodiments, the VSTs recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one antigen from each of parainfluenza virus type 3, respiratory syncytial virus, influenza, and human metapneumovirus. In some embodiments, the VSTs recognize a plurality of viral antigens, the plurality of viral antigens comprising at least two antigens from each of parainfluenza virus type 3, respiratory syncytial virus, influenza, and human metapneumovirus. In some embodiments, the plurality of antigens comprises, consists of, or consists essentially of PIV-3 antigen M, PIV-3 antigen HN, PIV-3 antigen N, PIV-3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the composition is a pharmaceutical composition formulated for intravenous delivery. In some embodiments, the composition is negative for bacteria and fungi in culture for at least 7 days; exhibits less than 5 EU / ml endotoxin, and is negative for mycoplasma.

[0011] The present disclosure also provides a method of lysing a target antigen comprising contacting a target cell with a composition comprising a polyclonal population of VSTs that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one antigen selected from parainfluenza virus type 3 (PIV-3), respiratory syncytial virus, influenza, and human metapneumovirus. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the contacting occurs in vivo in a subject. In some embodiments, the contacting occurs in vivo via administration of VSTs to a subject.

[0012] The present disclosure also provides a method of treating or preventing a viral infection comprising administering to cells of a subject in need of such treatment or prevention a composition comprising a polyclonal population of VSTs that recognize a plurality of viral antigens, the plurality of viral antigens comprising at least one antigen selected from parainfluenza virus type 3 (PIV-3), respiratory syncytial virus, influenza, and human metapneumovirus. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is administered to the subject multiple times. In some embodiments, administration of the composition effectively treats or prevents a viral infection in the subject, the viral infection being selected from the group consisting of parainfluenza virus type 3, respiratory syncytial virus, influenza, and human metapneumovirus. In some embodiments, the subject is a human. [Brief description of the drawings]

[0013] [Figure 1] Generation of polyclonal multiple respiratory virus specific T cells (Multi-R-VST) from healthy donors. Figure 1A shows a schematic of the Multi-R-VST generation protocol. Figure 1B shows the expansion fold achieved over 10 days based on cell counting using trypan blue exclusion (n=12). The manufacturing run can range from approximately 10 to 18 days. Figure 1C and Figure 1D show the phenotype of the expanded cells (mean ± SEM, n=12). Figure 1E shows minimal detection of Tregs (CD4+CD25+FoxP3+) within the expanded CD4+ T cell population (mean ± SEM, n=8).

[0014] [Diagram 2]Specificity and enrichment of multi-R-VST. Figure 2A shows the specificity of virus-reactive T cells within expanded T cell lines after exposure to individual stimulating antigens from each target virus. Data are presented as mean ± SEM SFC / 2x105 (n=12). Figure 2B shows the fold enrichment of specificity (PBMC vs. multi-R-VST; n=12). Figure 2C shows IFNγ production as assessed by ICS from CD4 helper (top) and CD8 cytotoxic T cells (bottom) after viral stimulation in one representative donor (dot plots gated on CD3+ cells), while Figure 2D shows a summary of the results for the nine donors screened (mean ± SEM). Figure 2E shows the number of donor-derived VST lines responding to individual stimulating antigens. Figure 2F shows the specificity of virus-reactive T cells within expanded T cell lines after exposure to titrating concentrations of pooled stimulating antigens from each target virus. Data are presented as mean ± SEM SFC / 2x105 (n=7). Figure 2G shows the frequency of CARV-specific T cells in peripheral blood of healthy donors after exposure to individual stimulating antigens from each target virus. Data are presented as mean ± SEM SFC / 5x105 (n=12).

[0015] [Diagram 3] Multi-R-VSTs are polyclonal and multifunctional. Figure 3A shows dual IFNγ and TNFα production from CD3+ T cells as assessed by ICS in one representative donor, while Figure 3B shows a summary of results from nine donors screened (mean ± SEM). Figure 3C shows the cytokine profile of multi-R-VSTs as measured by multiplex bead array, while Figure 3D evaluates Granzyme B production by ELIspot assay. Results are reported as SFC / 2x105 dose VST (mean ± SEM, n=9).

[0016] [Figure 4]Multi-R-VST is reactive against virus-infected targets. Figure 4A shows the cytolytic potential of Multi-R-VST assessed by a standard 4-hour Cr51 release assay using unloaded PHA blasts as control and autologous pepmix-pulsed PHA blasts as targets (E:T 40:1; n=8). Results are shown as percentage of specific lysis (mean ± SEM). Figure 4B reveals that Multi-R-VST shows negligible activity against either uninfected autologous or allogeneic PHA blasts as assessed by Cr51 release assay. Figure 4C shows the cytotoxic activity of Multi-R-VST assessed by a standard 4-hour Cr51 release assay using unloaded PHA blasts as control and autologous pepmix-pulsed PHA blasts as targets (E:T 40:1, 20:1, 10:1, 5:1). Results are presented as the percentage of specific lysis (mean±SEM, n=8).

[0017] [Diagram 5] Detection of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) specific T cells in peripheral blood of HSCT recipients. PBMCs isolated from two thrice infected HSCT recipients were tested for specificity against the infectious viruses using IFNγ ELIspot as readout. Figures 5A and 5B show results from two patients with controlled RSV-associated URTIs that coincided with a detectable increase in endogenous RSV-specific T cells, while Figure 5C shows clearance of hMPV-LRTIs along with an expansion of endogenous hMPV-specific T cells. ALC: absolute lymphocyte count.

[0018] [Figure 6]Detection of RSV and parainfluenza (PIV-3) specific T cells in peripheral blood of HSCT recipients. PBMCs isolated from three triplicate infected HSCT recipients were tested for specificity against the infecting viruses using IFNγ ELIspot as readout. Figures 6A and 6B show results from two patients with controlled RSV and PIV associated URTI and LRTI that were consistent with a detectable increase in endogenous virus-specific T cells. Figure 6C shows results from a patient with ongoing PIV associated severe URTI who was unable to mount a T cell response against the virus. ALC: absolute lymphocyte count.

[0019] [Figure 7] Structure and morphology of the RSV genome.

[0020] [Figure 8] Schematic diagram of the RSV-VST production protocol.

[0021] [Figure 9] Characterization of RSV-VST. Figure 9A shows the fold expansion achieved over a 10 day period based on cell counting using trypan blue exclusion. Figures 9B and 9C show the phenotype of the expanded cells.

[0022] [Figure 10] RSV-VST is multifunctional. Figure 10A shows IFNγ production from CD3+ T cells as assessed by EliSpot assay. Figure 10B shows Granzyme B production by ELIspot assay. Results are reported as SFC / 2x105 dose VST (mean±SEM, n=9).

[0023] [Figure 11] Cytokine profile of RSV-VST as measured by multiplex bead array. Detailed Description of the Invention

[0024] (definition) As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more." Some embodiments of the invention may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0025] The term "about" immediately preceding a numerical value means ±0%-10%, ±0%-10%, ±0%-9%, ±0%-8%, ±0%-7%, ±0%-6%, ±0%-5%, ±0%-4%, ±0%-3%, ±0%-2%, ±0%-1%, less than ±0%-1% of the numerical value, or any other value or range of values ​​therein. For example, "about 40" means ±0%-10% of 40 (i.e., 36-44).

[0026] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternative situations only or the options are not mutually exclusive, however, the present disclosure supports a definition that refers to alternative situations only and "and / or."

[0027] As used herein, the term "viral antigen" refers to an antigen that is proteinaceous in nature. In certain embodiments, the viral antigen is a coat protein. Specific examples of viral antigens include antigens from at least one virus selected from EBV, CMV, AdV, BK, JC virus, HHV6, RSV, influenza, parainfluenza, bocavirus, coronavirus, rhinovirus, LCMV, mumps, measles, hMPV, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HIV, HTLV1, HHV8, hepatitis C, hepatitis B, HTLV 1, herpes simplex virus, West Nile virus, Zika virus, and Ebola.

[0028] The terms "antigen-specific T cell line" or "virus-specific T cell" or "virus-specific T cell line" are used interchangeably herein to refer to a polyclonal T cell line with specificity and potency against one or more viruses of interest. As described herein, one or more viral antigens are presented to native T cells in peripheral blood mononuclear cells, and native CD4+ and CD8+ T cell populations proliferate in response to said viral antigens. For example, an antigen-specific T cell line or virus-specific T cells for EBV can recognize EBV, thereby proliferating T cells specific for EBV. In another example, an antigen-specific T cell line or virus-specific T cells for adenovirus and BK can recognize both AdV and BK, thereby proliferating T cells specific for adenovirus and BK.

[0029] As used herein, the terms "patient" or "subject" or "individual" are used interchangeably herein to refer to any mammal, including humans, farm animals, livestock, and zoo, sport and pet animals, such as dogs, horses, cats, and agricultural animals, including cows, sheep, pigs and goats. One particular mammal is a human, including adults, children and the elderly. The subject may also be a pet animal, including dogs, cats and horses. Examples of livestock animals include pigs, cows, sheep and goats.

[0030] As used herein, the terms "treat," "treating," "treatment," and the like, unless otherwise specified, refer to reversing, alleviating, inhibiting the process of, or preventing the disease, disorder, or condition to which such term applies, or one or more symptoms of such disease, disorder, or condition, and include administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein to prevent the onset of symptoms or complications, or to alleviate symptoms or complications, or to eliminate the disease, condition, or disorder. In some instances, treatment is curative or ameliorative.

[0031] As used herein, the terms "administering," "administer," "administration," and the like refer to any manner of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such a therapeutic agent. Such manners include, but are not limited to, intraocular, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0032] As used herein, the words "comprise," "comprising," "includes," "including," "has," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to encompass a non-exclusive inclusion of the recited elements, subject to any limitation expressly indicated to the contrary. For example, a composition and / or method "comprising" a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or properties or steps), but may include other elements (or components or properties or steps) that are not expressly recited or that are inherent to the composition and / or method.

[0033] As used herein, the phrases "consist of" and "consisting of" exclude any element, step, or component not recited. For example, "consist of" or "consisting of" used in a claim limits the claim to the components, materials, or steps specifically recited in the claim, excluding impurities normally associated therewith (i.e., impurities within a given component). When the phrase "consist of" or "consisting of" appears in a section in the body of a claim rather than immediately following the preamble, the phrase "consist of" or "consisting of" limits only the elements (or components or steps) recited in that section; other elements (or components) are not excluded from the claim as a whole.

[0034] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0035] The following discussion relates to various embodiments of the present invention. The term "invention" is not intended to refer to any particular embodiment or otherwise limit the scope of the present disclosure. One or more of these embodiments may be used, but the disclosed embodiments should not be interpreted or otherwise used as limiting the scope of the present disclosure, including the claims. Furthermore, those skilled in the art will understand that the following description has broad applicability, and the discussion of any embodiment is intended only as an example of that embodiment, and is not intended to clarify that the scope of the disclosure, including the claims, is limited to that embodiment. overview

[0036] In various embodiments, the present disclosure provides compositions and methods for treating or preventing viral infections (e.g., respiratory viral infections) and associated diseases. The present disclosure relates to the prevention or treatment of such infections by administration of ex vivo expanded, genetically unmodified, virus-specific T cells (VSTs) to control viral infections and eliminate symptoms. Without wishing to be bound by any theory, VSTs recognize and kill virus-infected cells via their native T cell receptors (TCRs) that bind to major histocompatibility complex (MHC) molecules expressed on target cells that present virus-derived peptides.

[0037] Respiratory viral infections due to community-acquired respiratory viruses (CARVs), including respiratory syncytial virus (RSV), influenza, parainfluenza virus (PIV), and human metapneumovirus (hMPV), have been detected in up to 40% of allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, who can develop severe illnesses such as bronchiolitis and pneumonia, which can be fatal. RSV-induced bronchiolitis is the most common reason for hospitalization in children under 1 year of age, while the Centers for Disease Control (CDC) estimate that influenza causes up to 35.6 million cases and 140,000-710,000 hospitalizations worldwide each year, with disease management costs of approximately $87.1 billion annually and 12,000-56,000 deaths in the United States alone.

[0038] Thus, CARV is a major cause of morbidity and mortality worldwide, with individuals with naive immune systems (e.g., young children) or who are immunocompromised being the most vulnerable. For example, in allogeneic hematopoietic stem cell transplant (HSCT) recipients, the incidence of CARV-associated respiratory viral disease is as high as 40% (5). While most patients initially present with rhinorrhea, cough, and fever, approximately 50% of cases progress to the lower respiratory tract, where severe symptoms including pneumonia and bronchiolitis are seen, and the disease is characterized by a mortality rate of 23–50% (6–9). There are no approved prophylactic vaccines or antivirals for hMPV (10) and PIV (11), and for influenza, prophylactic vaccination is only indicated if patients are at least 6 months post-HSCT (12). Aerosolized ribavirin (RBV) is FDA approved for the treatment of RSV, but is very expensive (5-day course = $149,756) and logistically difficult to administer, requiring specialized nebulizer devices that connect to an aerosol tent that surrounds the patient. (13-16) Thus, the lack of approved antivirals for clinically problematic CARV and the high cost and complexity of administering aerosolized RBV highlight the need for alternative treatment strategies.

[0039] Adenoviruses (AdV), rhinoviruses and other respiratory viruses including coronavirus strains, SARS-CoV, SARS-CoV-2, MERS-CoV and epidemic CoVs affecting both immunocompetent and immunocompromised patients. These can cause severe symptoms especially in immunocompromised individuals and the 2020 SARS-CoV2 pandemic has revealed how unprepared humans should treat and prevent this infection and associated diseases. This terrible pandemic has already resulted in thousands of deaths worldwide, healthcare collapse and a global economic collapse not seen in decades. It is therefore clear that new therapies to treat these viruses are urgently needed. The present disclosure provides such a treatment.

[0040] In some embodiments, the present disclosure provides VSTs made from peripheral blood mononuclear cells (PBMCs) obtained from healthy, pre-screened, seropositive donors, which are available as partially HLA-matched "off the shelf" products. Thus, the present disclosure provides VST products that include VSTs with specificity for one or more viruses, and methods of using such VSTs to treat or prevent viral infections.

[0041] In some embodiments, the VSTs described herein are responsive to (or "specific for") one or more viruses (e.g., one or more respiratory viruses) or, more specifically, one or more antigens expressed by a virus. In some embodiments, the VSTs described herein are responsive to only one virus. For example, in one embodiment, the present disclosure provides a polyclonal population of VSTs with specificity for one or more RSV antigens. In some examples, such RSV-specific VSTs include T cells with specificity for multiple RSV antigens. In some embodiments, the present disclosure also provides a method of treating a RSV infection in a subject by administering such a RSV-specific VST. In some embodiments, the present disclosure also provides a method of preventing a RSV infection in a subject by administering such a RSV-specific VST. Such practices may be applied to any single virus other than RSV.

[0042] In some embodiments, the VSTs described herein are responsive to multiple viruses (e.g., any one or more of the viruses disclosed herein). In certain embodiments, the present disclosure provides multiple respiratory virus-specific T cells (multi-R-VSTs) that are responsive to multiple respiratory viruses (e.g., any one or more of the respiratory viruses disclosed herein). In certain aspects, the multi-R-VSTs have specificity for one or more respiratory virus antigens expressed by a virus selected from influenza, RSV, hMPV, PIV, and combinations thereof. In certain embodiments, the multi-R-VSTs have specificity for an antigen expressed by each of influenza, RSV, hMPV, and PIV. In certain aspects, the multi-R-VSTs have specificity for one or more respiratory virus antigens expressed by a virus selected from influenza, RSV, hMPV, PIV3, and combinations thereof. In certain embodiments, the multi-R-VSTs have specificity for an antigen expressed by each of influenza, RSV, hMPV, and PIV3. In some embodiments, the influenza antigen is influenza A antigen NP1. In some embodiments, the influenza antigen is influenza A antigen MP1. In some embodiments, the influenza antigen is a combination of NP1 and MP1. In some embodiments, the RSV antigen is RSV N. In some embodiments, the RSV antigen is RSV F. In some embodiments, the RSV antigen is a combination of RSV N and F. In some embodiments, the hMPV antigen is F. In some embodiments, the hMPV antigen is N. In some embodiments, the hMPV antigen is M2-1. In some embodiments, the hMPV antigen is M. In some embodiments, the hMPV antigen is a combination of F, N, M2-1 and M. In some embodiments, the PIV antigen is M. In some embodiments, the PIV antigen is HN. In some embodiments, the PIV antigen is N. In some embodiments, the PIV antigen is F. In some embodiments, the PIV antigen is a combination of M, HN, N and F.In some embodiments, the disclosure also provides a method of treating a PIV, influenza, RSV and / or hMPV infection in a subject by administering such a multi-R-VST to the subject. In some embodiments, the disclosure also provides a method of preventing a PIV, influenza, RSV and / or hMPV infection in a subject by administering such a multi-R-VST to the subject. In some embodiments, the PIV3 antigen is M. In some embodiments, the PIV3 antigen is HN. In some embodiments, the PIV3 antigen is N. In some embodiments, the PIV3 antigen is F. In some embodiments, the PIV3 antigen is a combination of M, HN, N and F. In some embodiments, the disclosure also provides a method of treating a PIV3, influenza, RSV and / or hMPV infection in a subject by administering such a multi-R-VST to the subject. In some embodiments, the disclosure also provides a method of preventing a PIV3, influenza, RSV and / or hMPV infection in a subject by administering such a multi-R-VST to the subject. Such practices may be applied to any number of viruses.

[0043] In a particular embodiment, the disclosure provides a composition comprising a polyclonal population of multi-R-VSTs having specificity for each of PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F and hMPV antigen N. The polyclonal population may comprise both CD4+ and CD8+ VSTs. The polyclonal population may be administered to a subject. The subject may have a PIV, influenza, RSV and / or hMPV infection. A method of treating a PIV, influenza, RSV and / or hMPV infection in a subject may comprise administering a polyclonal population of multi-R-VSTs to the subject. A method of preventing a PIV, influenza, RSV and / or hMPV infection in a subject may comprise administering a polyclonal population of multi-R-VSTs to the subject.

[0044] In a particular embodiment, the disclosure provides a composition comprising a polyclonal population of multi-R-VSTs having specificity for each of PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, PIV3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F and hMPV antigen N. The polyclonal population may comprise both CD4+ and CD8+ VSTs. The polyclonal population may be administered to a subject. The subject may have a PIV3, influenza, RSV and / or hMPV infection. A method of treating a PIV3, influenza, RSV and / or hMPV infection in a subject may comprise administering a polyclonal population of multi-R-VSTs to the subject. A method of preventing a PIV3, influenza, RSV and / or hMPV infection in a subject may comprise administering a polyclonal population of multi-R-VSTs to the subject.

[0045] In some embodiments, the disclosure provides compositions comprising a polyclonal population of VSTs that recognize multiple viral antigens, the multiple viral antigens including at least one first antigen from PIV and at least one second antigen from one or more additional viruses. In some specific embodiments, the disclosure provides compositions comprising a polyclonal population of VSTs that recognize multiple viral antigens, the multiple viral antigens including at least one first antigen from PIV3 and at least one second antigen from one or more additional viruses. The additional viruses can include influenza, RSV, hMPV, AdV, coronavirus, or combinations thereof. The VST may recognize additional antigens expressed by one or more additional viruses, which may include one or more or all of the group consisting of PIV antigen M (e.g., PIV3 antigen M), PIV antigen HN (e.g., PIV3 antigen HN), PIV antigen N (e.g., PIV3 antigen N), PIV antigen F (e.g., PIV3 antigen F), influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, hMPV antigen N, and AdV antigen Hexon, AdV antigen Penton, and combinations thereof. The additional antigens may, in some embodiments, include one or more coronavirus antigens. For example, the additional antigens may include one or more coronavirus (e.g., SARS-CoV or SARS-CoV2) antigens. In some embodiments, the coronavirus antigens comprise one or more SARS-CoV2 antigens selected from the group consisting of: nsp1; nsp3; nsp4; nsp5; nsp6; nsp10; nsp12; nsp13; nsp14; nsp15; nsp16; spike (S); envelope protein (E); matrix protein (M); nucleocapsid protein (N). In some embodiments, the SARS-CoV2 antigens further comprise one or more antigens selected from the group consisting of: SARS-CoV-2(AP3A); SARS-CoV-2(NS7); SARS-CoV-2(NS8); SARS-CoV-2(ORF10); SARS-CoV-2(ORF9B); and SARS-CoV-2(Y14).

[0046] The additional antigens may in some embodiments also or alternatively be from a virus selected from EBV, CMV, AdV, BK, JC virus, HHV6, RSV, influenza, parainfluenza, bocavirus, rhinovirus, coronavirus, LCMV, mumps, measles, human metapneumovirus, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HIV, HTLV1, HHV8, hepatitis C, hepatitis B, HTLV1, and West Nile virus, Zika virus, Ebola. In some embodiments, the EBV antigens are from LMP2, EBNA1, BZLF1, and combinations thereof. In some embodiments, the CMV antigens are from IE1, pp65, and combinations thereof. In some embodiments, the adenovirus antigens are from Hexon, Penton, and combinations thereof. In some embodiments, the BK virus antigens are from VP1, large T, and combinations thereof. In some embodiments, the HHV6 antigens are from U90, U11, U14, and combinations thereof.

[0047] In some embodiments, at least one pepmix covers antigens (or portions of antigens) from RSV, influenza, PIV, or hMPV. In some embodiments, at least one pepmix covers antigens (or portions of antigens) from RSV, influenza, PIV, hMPV, coronavirus (e.g., SARS-CoV or SARS-CoV2), or a combination thereof. In some embodiments, at least one pepmix covers antigens (or portions of antigens) from RSV, influenza, PIV3, hMPV, or a combination thereof. In some embodiments, at least one pepmix covers antigens (or portions of antigens) from RSV, influenza, PIV3, hMPV, coronavirus (e.g., SARS-CoV or SARS-CoV2), or a combination thereof.

[0048] In some embodiments, the first antigen is a PIV antigen. For example, in some embodiments, the first antigen can be PIV antigen M. In some embodiments, the first antigen can be PIV antigen HN. In some embodiments, the first antigen can be PIV antigen N. In some embodiments, the first antigen can be PIV antigen F. In some embodiments, the first antigen can be any combination of PIV antigen M, PIV antigen HN, PIV antigen N, and PIV antigen F. In some embodiments, the composition can include one first antigen. In some embodiments, the composition can include two first antigens. In some embodiments, the composition can include three first antigens. In some embodiments, the composition can include four first antigens. In some embodiments, the four first antigens can include PIV antigen M, PIV antigen HN, PIV antigen N, and PIV antigen F.

[0049] In some embodiments, the first antigen is a PIV3 antigen. For example, in some embodiments, the first antigen can be PIV3 antigen M. In some embodiments, the first antigen can be PIV3 antigen HN. In some embodiments, the first antigen can be PIV3 antigen N. In some embodiments, the first antigen can be PIV3 antigen F. In some embodiments, the first antigen can be any combination of PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, and PIV3 antigen F. In some embodiments, the composition can include one first antigen. In some embodiments, the composition can include two first antigens. In some embodiments, the composition can include three first antigens. In some embodiments, the composition can include four first antigens. In some embodiments, the four first antigens can include PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, and PIV3 antigen F.

[0050] In some embodiments, the one or more second viruses can be RSV. In some embodiments, the one or more second viruses can be influenza. In some embodiments, the one or more second viruses can be hMPV. In some embodiments, the one or more second viruses can include RSV, influenza, and hMPV. In some embodiments, the one or more second viruses can consist of RSV, influenza, and hMPV. In some embodiments, the one or more second viruses can be selected from any suitable virus as described herein.

[0051] In some embodiments, the composition may include two or three second viruses. In some embodiments, the composition may include three second viruses. In some embodiments, the three second viruses may include influenza, RSV, and hMPV. In some embodiments, the composition includes at least two second antigens for each second virus. In some embodiments, the composition includes one second antigen. In some embodiments, the composition includes two second antigens. In some embodiments, the composition includes three second antigens. In some embodiments, the composition includes four second antigens. In some embodiments, the composition includes five second antigens. In some embodiments, the composition includes six second antigens. In some embodiments, the composition includes seven second antigens. In some embodiments, the composition includes eight second antigens. In some embodiments, the composition includes nine second antigens. In some embodiments, the composition includes ten second antigens. In some embodiments, the composition includes eleven second antigens. In some embodiments, the composition includes twelve second antigens. In some embodiments, the composition comprises any number of second antigens suitable for the composition as described herein.

[0052] In some embodiments, the second antigen can be influenza antigen NP1. In some embodiments, the second antigen can be influenza antigen MP1. In some embodiments, the second antigen can be RSV antigen N. In some embodiments, the second antigen can be RSV antigen F. In some embodiments, the second antigen can be hMPV antigen M. In some embodiments, the second antigen can be hMPV antigen M2-1. In some embodiments, the second antigen can be hMPV antigen F. In some embodiments, the second antigen can be hMPV antigen N. In some embodiments, the second antigen can be any combination of influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N.

[0053] In some embodiments, the second antigen comprises influenza antigen NP1. In some embodiments, the second antigen comprises influenza antigen MP1. In some embodiments, the second antigen comprises both influenza antigen NP1 and influenza antigen MP1. In some embodiments, the second antigen comprises RSV antigen N. In some embodiments, the second antigen comprises RSV antigen F. In some embodiments, the second antigen comprises both RSV antigen N and RSV antigen F.

[0054] In some embodiments, the second antigen comprises hMPV antigen M. In some embodiments, the second antigen comprises hMPV antigen M2-1. In some embodiments, the second antigen comprises hMPV antigen F. In some embodiments, the second antigen comprises hMPV antigen N. In some embodiments, the second antigen comprises a combination of hMPV antigen M, hMPV antigen M2-1, hMPV antigen F and hMPV antigen N.

[0055] In some embodiments, the second antigen comprises each of influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens comprises PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens consists of PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens consists essentially of PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the second antigen may comprise any suitable antigen for the composition as described herein.

[0056] In some embodiments, the second antigen comprises each of influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens comprises PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, PIV3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens consists of PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, PIV3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens consists essentially of PIV3 antigen M, PIV3 antigen HN, PIV3 antigen N, PIV3 antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the second antigen may comprise any suitable antigen for the composition as described herein.

[0057] In some embodiments, the VST in the composition disclosed herein is produced by contacting PBMCs with a plurality of pepmix libraries. In some embodiments, each pepmix library contains a plurality of overlapping peptides that span at least a portion of a viral antigen. In some embodiments, at least one of the plurality of pepmix libraries spans a first antigen from PIV. In some embodiments, at least one of the plurality of pepmix libraries spans a first antigen from PIV3. In some embodiments, at least one additional pepmix library of the plurality of pepmix libraries spans each second antigen.

[0058] In some embodiments, the VSTs disclosed herein are generated by contacting T cells with antigen-presenting cells (APCs), such as dendritic cells (DCs), nucleofected with at least one DNA plasmid. In some embodiments, the DNA plasmid may encode at least a portion of one antigen. In some embodiments, the DNA plasmid may encode a PIV antigen (e.g., a PIV3 antigen). In some embodiments, at least one DNA plasmid encodes each second antigen. In some embodiments, the plasmid encodes at least one PIV antigen and at least one second antigen. In some embodiments, the compositions described herein include CD4+ T lymphocytes and CD8+ T lymphocytes. In some embodiments, the compositions include VSTs that express the αβ T cell receptor. In some embodiments, the compositions include MHC-restricted VSTs.

[0059] In some embodiments, the present disclosure provides multiple respiratory virus specific T cells (multi-R-VST) with specificity for one or more respiratory viruses selected from influenza, RSV, hMPV, PIV, and one or more additional viruses. The PIV antigen can be from PIV3. For example, in some instances, the additional virus comprises a coronavirus. The coronavirus can be an alphacoronavirus. For example, in certain embodiments, the alphacoronavirus is selected from HCoV-E229, HCoV-NL63, and combinations thereof. In certain embodiments, the alphacoronavirus comprises each of HCoV-E229 and HCoV-NL63. The coronavirus can be a betacoronavirus. For example, in certain embodiments, the betacoronavirus is selected from SARS-CoV, SARS-CoV2, MERS-CoV, HCoV-HKU1, HCoV-OC43, and combinations thereof. In certain embodiments, the betacoronavirus includes each of SARS-CoV, SARS-CoV2, MERS-CoV, HCoV-HKU1, HCoV-OC43, and combinations thereof. In some examples, the additional virus includes an adenovirus. In some examples, the additional virus is selected from the group consisting of EBV, CMV, AdV, BK, JC virus, HHV6, bocavirus, rhinovirus, coronavirus, LCMV, mumps, measles, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HIV, HTLV1, HHV8, hepatitis C, hepatitis B, HTLV1, West Nile virus, Zika virus, Ebola, and combinations thereof.

[0060] In one embodiment, the disclosure provides a multi-R-VST with specificity for influenza, RSV, hMPV, PIV, and a coronavirus (e.g., SARS-CoV2). In one embodiment, the disclosure provides a multi-R-VST with specificity for influenza, RSV, hMPV, PIV, one or more AdVs, and a coronavirus (e.g., SARS-CoV or SARS-CoV2). In one embodiment, the disclosure provides a multi-R-VST with specificity for influenza, RSV, hMPV, PIV3, and a coronavirus (e.g., SARS-CoV2). In one embodiment, the disclosure provides a multi-R-VST with specificity for influenza, RSV, hMPV, PIV3, one or more AdVs, and a coronavirus (e.g., SARS-CoV or SARS-CoV2).

[0061] In some embodiments, the VST may be cultured ex vivo in the presence of both IL-7 and IL-4. In some embodiments, the multiviral VST has grown sufficiently within 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days of culture (including all ranges and subranges therebetween) to be ready for administration to a patient. A typical manufacturing run (culture / growth under the above conditions) is 10-18 days, more typically 14-16 days. In some embodiments, the multiviral VST has grown sufficiently within any number of days appropriate for the compositions as described herein.

[0062] The present disclosure provides compositions comprising VST that exhibit negligible alloreactivity. In some embodiments, the compositions comprise VST such that antigen-specific T cells recovered from a patient have less activation-induced cell death than corresponding antigen-specific T cells recovered from the same patient. In some embodiments, the compositions are not cultured in the presence of both IL-7 and IL-4. In some embodiments, the compositions comprising VST exhibit greater than 70% viability.

[0063] In some embodiments, the composition is negative for bacteria and fungi in culture for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days. In some embodiments, the composition is negative for bacteria and fungi in culture for at least 7 days. In some embodiments, the composition exhibits endotoxins less than 1 EU / ml, less than 2 EU / ml, less than 3 EU / ml, less than 4 EU / ml, less than 5 EU / ml, less than 6 EU / ml, less than 7 EU / ml, less than 8 EU / ml, less than 9 EU / ml, less than 10 EU / ml. In some embodiments, the composition exhibits endotoxins less than 5 EU / ml. In some embodiments, the composition is negative for mycoplasma.

[0064] In some embodiments, the pepmix used to construct the VST polyclonal is chemically synthesized. In some embodiments, the pepmix is ​​optionally >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, or >90% pure (including all ranges and subranges therebetween). In some embodiments, the pepmix is ​​optionally >90% pure.

[0065] In some embodiments, the VST is Th1 polarized. In some embodiments, the VST is capable of lysing viral antigen-expressing target cells. In some embodiments, the VST is capable of lysing other suitable types of antigen-expressing target cells. In some embodiments, the VST in the composition does not significantly lyse non-infected autologous target cells. In some embodiments, the VST in the composition does not significantly lyse non-infected autologous allogeneic target cells.

[0066] The present disclosure provides pharmaceutical compositions, including any compositions formulated for intravenous delivery. In some embodiments, the compositions are negative for bacteria during at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days of culture. In some embodiments, the compositions are negative for bacteria during at least 7 days of culture. In some embodiments, the compositions are negative for fungi during at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days of culture. In some embodiments, the compositions are negative for fungi during at least 7 days of culture.

[0067] In some embodiments, the pharmaceutical composition exhibits endotoxins of less than 1 EU / ml, less than 2 EU / ml, less than 3 EU / ml, less than 4 EU / ml, less than 5 EU / ml, less than 6 EU / ml, less than 7 EU / ml, less than 8 EU / ml, less than 9 EU / ml, less than 10 EU / ml. In some embodiments, the pharmaceutical composition is negative for mycoplasma.

[0068] The disclosure also provides methods of treating or preventing a viral infection comprising administering to a subject an effective amount of one or more of the VSTs disclosed herein (e.g., multi-R-VSTs disclosed herein with specificity for PIV, influenza, RSV, and hMPV, etc.). The disclosure also provides compositions (e.g., pharmaceutical compositions) comprising any of the VSTs disclosed herein (e.g., multi-R-VSTs disclosed herein with specificity for PIV, influenza, RSV, and hMPV, etc.) and methods of treating or preventing a viral infection comprising administering to a subject an effective dose of one or more of such pharmaceutical compositions comprising a VST disclosed herein. Preparation of pepmix library

[0069] In some embodiments of the present disclosure, a library of peptides is provided to PBMCs to ultimately generate VSTs. The library, in certain cases, comprises a mixture of peptides ("pepmix") that span part or all of the same antigen. The pepmixes utilized in the present disclosure may be from commercially available peptide libraries that, in certain aspects, comprise peptides that are 15 amino acids long and overlap each other by 11 amino acids. In some cases, they may be synthetically generated. Examples include those from JPT Technologies (Springfield, VA) or Miltenyi Biotec (Auburn, CA). In certain embodiments, the peptides are, for example, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or more amino acids in length, and in certain embodiments, the overlap is, for example, at least 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, 28, 29, 30, 31, 32, 33, or 34 amino acids in length.

[0070] In some embodiments, the amino acids used in the pepmix have a purity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99, at least 99.9% (including all ranges and subranges therebetween). In some embodiments, the amino acids used in the pepmix herein have a purity of at least 70%.

[0071] A mixture of different peptides can include any ratio of the different peptides, but in some embodiments, each particular peptide is present in the mixture in substantially the same number as another particular peptide. Methods for preparing and producing pepmixes for multi-viral cytotoxic T cell proliferation with broad specificity are described in U.S. Patent Application Publication No. 2018 / 0187152, which is incorporated by reference in its entirety. Preparation of VST

[0072] In some embodiments, the method of making VST includes isolating mononuclear cells (MNC) or having isolated MNC from blood obtained from a donor. In some embodiments, the MNC is PBMC. MNC and PBMC are isolated by using methods known to those skilled in the art. As an example, density centrifugation (gradient) (Ficoll-Paque) may be used to isolate PBMC. In another example, cell preparation tubes (CPT) and SepMate tubes containing freshly collected blood may be used to isolate PBMC.

[0073] In some embodiments, the MNCs are PBMCs. By way of example, PBMCs may include lymphocytes, monocytes, and dendritic cells. By way of example, lymphocytes may include T cells, B cells, and NK cells. In some embodiments, MNCs as used herein are cultured or cryopreserved. In some embodiments, the step of culturing or cryopreserving cells may include contacting the cells in culture with one (or a portion thereof) or more antigens under suitable culture conditions to stimulate and proliferate antigen-specific T cells. In some embodiments, the one or more antigens may include one or more viral antigens.

[0074] In some embodiments, culturing or cryopreserving the cells may include contacting the cells in culture under suitable culture conditions with one or more epitopes from one or more antigens. In some embodiments, contacting the MNCs or PBMCs with one or more antigens or one or more epitopes from one or more antigens stimulates and expands a polyclonal population of antigen-specific T cells derived from each of the MNCs or PBMCs of an individual donor. In some embodiments, the antigen-specific T cell lines may be cryopreserved.

[0075] In some embodiments, one or more antigens may be in the form of a whole protein. In some embodiments, one or more antigens may be a pepmix that includes a series of overlapping peptides that span part or all of the sequence of each antigen. In some embodiments, one or more antigens may be a combination of a whole protein and a pepmix that includes a series of overlapping peptides that span part or all of the sequence of each antigen.

[0076] In some embodiments, the culture of PBMCs or MNCs is performed in a vessel comprising a gas permeable culture surface. In one embodiment, the vessel is an infusion bag or rigid vessel having a gas permeable portion. In one embodiment, the vessel is a G-Rex® bioreactor. In one embodiment, the vessel can be any vessel, bioreactor, etc. suitable for culturing PBMCs or MNCs as described herein.

[0077] In some embodiments, the PBMCs or MNCs are cultured in the presence of one or more cytokines. In some embodiments, the cytokine is IL4. In some embodiments, the cytokine is IL7. In some embodiments, the cytokine is IL4 and IL7. In some embodiments, the cytokine includes IL4 and IL7, but does not include IL2. In some embodiments, the cytokines can be any combination of cytokines suitable for culturing PBMCs or MNCs as described herein.

[0078] In some embodiments, the culture of MNC or PBMC can be in the presence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different pepmixes. A pepmix, which is a plurality of peptides, comprises a series of overlapping peptides that span a portion or the entire sequence of an antigen. In some embodiments, MNC or PBMC can be cultured in the presence of a plurality of pepmixes, where each pepmix covers at least one antigen that is different from the antigens covered by each of the other pepmixes in the plurality of pepmixes. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more antigens are covered by the plurality of pepmixes. In some embodiments, at least one antigen from at least two different viruses is covered by the plurality of pepmixes.

[0079] In some embodiments, the pepmix comprises 15-mer peptides. In some embodiments, the pepmix comprises peptides suitable for the methods described herein. In some embodiments, the peptides in the pepmix that span the antigen overlap in sequence by 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids. In some embodiments, the peptides in the pepmix that span the antigen overlap in sequence by 11 amino acids.

[0080] In some embodiments, PBMCs or MNCs are cultured in the presence of a pepmix spanning influenza A antigens NP1 and MP1, RSV antigens N and F, hMPV antigens F, N, M2-1 and M, and PIV antigens M, HN, N and F. In some embodiments, PBMCs or MNCs are cultured in the presence of a pepmix spanning influenza A antigens NP1 and MP1, RSV antigens N and F, hMPV antigens F, N, M2-1 and M, and PIV antigens M, HN, N and F, and one or more coronavirus (e.g., SARS-CoV or SARS-CoV2) antigens disclosed herein. In some embodiments, PBMCs or MNCs are cultured in the presence of a pepmix spanning EBV antigens LMP2, EBNA1, and BZLF1, CMV antigens IE1 and pp65, adenovirus antigens Hexon and Penton, BK virus antigens VP1 and large T, and HHV6 antigens U90, U11, and U14. In some embodiments, antigen-specific T cells are tested for antigen-specific cytotoxicity.

[0081] The present disclosure provides a method of lysing a target cell, comprising contacting the target cell with a composition or pharmaceutical composition described herein. In some embodiments, the contact between the target cell and the composition or pharmaceutical composition is performed in vivo in a subject. In some embodiments, the contact between the target cell and the composition or pharmaceutical composition is performed in vivo via administration of VST to the subject. In some embodiments, the subject is a human.

[0082] The present disclosure provides a method of treating or preventing a viral infection, comprising administering to a subject in need thereof a composition or pharmaceutical composition described herein. In some embodiments, the VST is at least 5x10 3 ~5x10 9 VST / m 2 , 5x10 4 ~5x10 8 VST / m 2 , 5x10 5 ~5x10 7 VST / m 2, 5x10 4 ~5x10 8 VST / m 2 , 5x10 6 ~5x10 9 VST / m 2 (including all ranges and subranges therebetween). In some embodiments, a VST is administered to the subject. In some embodiments, the subject is immunocompromised. In some embodiments, a subject with a PIV infection is administered a multi-R-VST disclosed herein specific for PIV, RSV, hMPV, and influenza. In some embodiments, the multi-R-VSTs have cross-specificity such that they are effective against a different viral infection than the virus for which they are made. For example, and not by way of limitation, in some embodiments, the PIV-specific VST in the multi-R-VST is made against a PIV3 antigen. In some embodiments, the PIV infection being treated is PIV3. In some embodiments, the PIV infection being treated is a serotype other than PIV3. In some embodiments, a subject with a RSV infection is administered a multi-R-VST disclosed herein specific for PIV, RSV, hMPV, and influenza. In some embodiments, a subject with a hMPV infection is administered a multi-R-VST disclosed herein specific for PIV, RSV, hMPV, and influenza. In some embodiments, a subject with an influenza infection is administered a multi-R-VST disclosed herein specific for PIV, RSV, hMPV, and influenza. In some embodiments, a subject with a coronavirus (e.g., SARS-CoV or SARS-CoV2) infection is administered a multi-R-VST disclosed herein specific for PIV, RSV, hMPV, influenza, and coronavirus (e.g., SARS-CoV or SARS-CoV2).

[0083] In some embodiments, the subject may have one or more medical conditions. In some embodiments, the subject undergoes reduced intensity conditioning prior to undergoing VST and undergoes a matched related donor transplant. In some embodiments, the subject undergoes myeloablative conditioning prior to undergoing VST and undergoes a matched unrelated donor transplant. In some embodiments, the subject undergoes reduced intensity conditioning prior to undergoing VST and undergoes a haploidentical transplant. In some embodiments, the subject undergoes myeloablative conditioning prior to undergoing VST and undergoes a matched related donor transplant. In some embodiments, the subject has undergone a solid organ transplant. In some embodiments, the subject has undergone chemotherapy. In some embodiments, the subject has HIV infection. In some embodiments, the subject has a genetic immune deficiency. In some embodiments, the subject has undergone an allogeneic stem cell transplant. In some embodiments, the subject has a pre-existing condition that makes them more susceptible to viral infection and / or has a significant adverse outcome following viral infection. For example, in some embodiments, the subject has cardiovascular disease. In some embodiments, the subject has diabetes. In some embodiments, the subject has chronic respiratory disease. In some embodiments, the subject has hypertension. In some embodiments, the subject has cancer. In some embodiments, the subject is obese. In some embodiments, the subject is elderly. In some embodiments, the subject has more than one medical condition as described in this paragraph. In some embodiments, the subject has all of the medical conditions as described in this paragraph. In some embodiments, the patient is infected with a coronavirus (e.g., SARS-CoV or SARS-CoV2). In some embodiments, the patient has been diagnosed with COVID-19. In some embodiments, the patient is immunocompromised. As used herein, immunocompromised means that the immune system is weakened. For example, an immunocompromised patient has a reduced ability to fight infections and other diseases. In some embodiments, the patient is immunocompromised due to a treatment the patient has received to treat the disease or condition or another disease or condition. In some embodiments, the cause of the immunocompromise is age.In one embodiment, the cause of the immunocompromised state is due to young age. In one embodiment, the cause of the immunocompromised state is due to advanced age. In some embodiments, the patient is in need of transplant therapy. In some embodiments, the subject has no other medical conditions other than infection with a coronavirus (e.g., SARS-CoV or SARS-CoV2). In some embodiments, the subject has acute myeloid leukemia, acute lymphoblastic leukemia, or chronic granulomatous disease.

[0084] In some embodiments, the therapeutic efficacy is measured after administration of the VST cell line. In other embodiments, the therapeutic efficacy is measured based on viremic resolution of the infection. In other embodiments, the therapeutic efficacy is measured based on viruric resolution of the infection. In other embodiments, the therapeutic efficacy is measured based on the resolution of viral load in a sample from the patient. In other embodiments, the therapeutic efficacy is measured via chest imaging to track disease resolution in the lungs. In some embodiments, the sample is from a nasal swab. In other embodiments, the therapeutic efficacy is measured based on viremic resolution of the infection, viruric resolution of the infection, and the resolution of viral load in a sample from the patient. In some embodiments, the therapeutic efficacy is measured by monitoring detectable viral load in the patient's peripheral blood. In some embodiments, the therapeutic efficacy includes resolution of gross hematuria. In some embodiments, the therapeutic efficacy includes reduction in symptoms of hemorrhagic cystitis as measured by CTCAE-PRO or similar assessment tools that examine outcomes reported by the patient and / or clinician.

[0085] In some embodiments, the sample is selected from a tissue sample from the patient. In some embodiments, the sample is selected from a body fluid sample from the patient. In some embodiments, the sample is selected from cerebrospinal fluid (CSF) from the patient. In some embodiments, the sample is selected from BAL from the patient. In some embodiments, the sample is selected from stool from the patient.

[0086] In some embodiments, a composition as described herein is administered to a subject multiple times. In some embodiments, a composition as described herein is administered to a subject more than once. In some embodiments, a composition as described herein is administered to a subject more than two times. In some embodiments, a composition as described herein is administered to a subject more than three times. In some embodiments, a composition as described herein is administered to a subject more than four times. In some embodiments, a composition as described herein is administered to a subject more than five times. In some embodiments, a composition as described herein is administered to a subject more than six times. In some embodiments, a composition as described herein is administered to a subject more than seven times. In some embodiments, a composition as described herein is administered to a subject more than eight times. In some embodiments, a composition as described herein is administered to a subject more than nine times. In some embodiments, a composition as described herein is administered to a subject more than ten times. In some embodiments, a composition as described herein is administered to a subject as many times as is appropriate for the subject. When multiple doses of the composition are provided to an individual, the period of time between doses can be any suitable length of time, such as 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or longer, including all ranges and subranges therebetween.

[0087] In some embodiments, two or more compositions described herein comprising a polyclonal population of VSTs (e.g., multi-R-VST) are administered to a subject in combination. Two or more compositions may be administered to a subject sequentially or simultaneously. Two or more compositions may be pooled and administered as a single composition. Two or more compositions may be administered as separate compositions at different times. In one embodiment, a first multi-R-VST composition comprising a polyclonal population of VSTs with specificity for PIV, influenza, RSV, and hMPV is administered to a subject, and a second separate VST composition comprising a polyclonal population of VSTs with specificity for another virus is also administered to the subject. In certain embodiments, the other virus is a coronavirus (e.g., SARS-CoV2). In some embodiments, a single composition comprising a pool of a first multi-R-VST composition comprising a polyclonal population of VSTs with specificity for PIV, influenza, RSV, and hMPV and a second VST composition comprising a polyclonal population of VSTs with specificity for another virus is administered to a subject. In certain embodiments, the other virus is a coronavirus (e.g., SARS-CoV2). In some embodiments, the other virus is selected from BV, CMV, AdV, BK, JC virus, HHV6, RSV, influenza, parainfluenza, bocavirus, coronavirus, rhinovirus, LCMV, mumps, measles, hMPV, parvovirus B, rotavirus, Merkel cell virus, herpes simplex virus, HPV, HIV, HTLV1, HHV8, hepatitis C, hepatitis B, HTLV 1, herpes simplex virus, West Nile virus, Zika virus, and Ebola.

[0088] In some embodiments, administration of the composition effectively treats or prevents a viral infection in a subject. In some embodiments, the viral infection is PIV. In some embodiments, the viral infection is PIV3. In some embodiments, the viral infection is RSV. In some embodiments, the viral infection is influenza. In some embodiments, the viral infection is hMPV. In some embodiments, the viral infection is a coronavirus (e.g., SARS-CoV or SARS-CoV2). In some embodiments, the viral infection is SARS-CoV. In some embodiments, the viral infection is MERS-CoV. In some embodiments, the viral infection is HCoV-HKU1. In some embodiments, the viral infection is HCoV-OC43. In some embodiments, the viral infection is HCoV-E229. In some embodiments, the viral infection is HCoV-NL63.

[0089] The present disclosure provides a composition comprising a polyclonal population of VSTs that recognize multiple viral antigens. The present disclosure provides that the multiple viral antigens comprise at least one antigen. In some embodiments, the at least one antigen can be a coronavirus (e.g., SARS-CoV or SARS-CoV2). In some embodiments, the at least one antigen can be from PIV. In some embodiments, the at least one antigen can be from RSV antigen. In some embodiments, the at least one antigen can be from influenza. In some embodiments, the at least one antigen can be from hMPV.

[0090] In some embodiments, the disclosure provides a polyclonal population of VSTs that recognize a plurality of viral antigens, including at least one antigen from each of PIV, RSV, influenza, and hMPV. In some embodiments, the disclosure provides a polyclonal population of VSTs that recognize a plurality of viral antigens, including at least two antigens from each of PIV, RSV, influenza, and hMPV. In some embodiments, the plurality of antigens includes PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, the plurality of antigens can be selected from any of PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N. In some embodiments, a polyclonal population of VSTs is administered to a patient infected with influenza, RSV, PIV and / or hMPV.

[0091] In at least some methods of the disclosure, the generated VST is administered to an individual, for example, an immunocompromised individual. In some cases, the individual has undergone or will have an allogeneic stem cell transplant. In certain embodiments, the cells are administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal injection. In some embodiments, the individual has lymphoma or leukemia. In some embodiments, the VST is further defined as polyclonal CD4+ and CD8+ VST. The PBMCs may be allogeneic to the individual or autologous to the individual. In some embodiments, the method of the present invention further comprises exposing the VST to one or more compositions that stimulate cell division, such as phytohemagglutinin; in some aspects, the compound is a mitogen.

[0092] In some embodiments, the disclosure provides a pharmaceutical composition comprising a composition as described herein formulated for intravenous delivery. In some embodiments, the disclosure provides a pharmaceutical composition comprising a population of VSTs as disclosed herein and one or more carriers, excipients, diluents, buffers and / or delivery vehicles. In some particular embodiments, the disclosure provides a pharmaceutical composition comprising one or more VST compositions as described herein formulated for intravenous delivery. In certain embodiments, the composition formulated for intravenous delivery may comprise one or more propagated VSTs as disclosed herein suspended or resuspended in their culture medium. The composition formulated for intravenous delivery may additionally or alternatively comprise one or more propagated VSTs resuspended in a suitable carrier, excipient, diluent, buffer and / or delivery vehicle. In certain embodiments, the composition formulated for intravenous delivery may comprise one or more propagated VSTs as disclosed herein resuspended in saline. In some embodiments, the composition as described herein is negative for bacteria. In some embodiments, the composition as described herein is negative for fungi. In some embodiments, the compositions as described herein are negative for bacteria or fungi in culture for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, In some embodiments, the compositions as described herein are negative for bacteria or fungi in culture for at least 7 days.

[0093] In some embodiments, the pharmaceutical composition formulated for intravenous delivery exhibits endotoxins of less than 1 EU / ml, less than 2 EU / ml, less than 3 EU / ml, less than 4 EU / ml, less than 5 EU / ml, less than 6 EU / ml, less than 7 EU / ml, less than 8 EU / ml, less than 9 EU / ml, less than 10 EU / ml. In some embodiments, the pharmaceutical composition formulated for intravenous delivery is negative for mycoplasma. EXAMPLES

[0094] Example 1 method Unless otherwise indicated, the examples provided below utilized the following materials and methods. Flow cytometry Immunophenotyping

[0095] Multi-R-VST were surface stained with monoclonal antibodies against CD3, CD25, CD28, CD45RO, CD279 (PD-1) [Becton Dickinson (BD), Franklin Lakes, NJ], CD4, CD8, CD16, CD62L, CD69 (Beckman Coulter, Brea, CA) and CD366 (TIM-3) (BioLegend, San Diego, CA). Cells were pelleted in phosphate buffered saline (PBS) (Sigma-Aldrich) and antibodies were then added in saturating volumes (5 μl) followed by incubation for 15 min at 4° C. Cells were then washed and resuspended in 300 μl PBS and at least 20,000 viable cells were acquired on a Gallios™ Flow Cytometer and analyzed with Kaluza® Flow Analysis Software (Beckman Coulter). Intracellular cytokine staining (ICS)

[0096] Multi-R-VST were harvested and resuspended in VST medium (2x106 / ml) and 200μl was added per well of a 96-well plate. Cells were incubated overnight with 200ng of each test or control (irrelevant non-viral, e.g. SURVIVIN, WT1) pepmix together with Brefeldin A (1μg / ml), Monensin (1μg / ml), CD28 and CD49d (1μg / ml) (BD). VST were then washed with PBS, pelleted and surface stained with CD8 and CD3 (5μl / antibody / tube) for 15 minutes at 4°C, then washed, pelleted, fixed and permeabilized with Cytofix / Cytoperm solution (BD) for 20 minutes at 4°C in the dark. After washing with Perm / Wash Buffer (BD), cells were incubated with 10μl of IFNγ and TNFα antibodies (BD) for 30 minutes at 4°C in the dark. Cells were then washed twice with Perm / Wash Buffer and at least 50,000 viable cells were acquired on a Gallios™ Flow Cytometer and analyzed with Kaluza® Flow Analysis Software. FoxP3 staining

[0097] FoxP3 staining was performed using the eBioscience FoxP3 kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Briefly, 1x106 cells were surface stained with CD3, CD4 and CD25 antibodies, then washed, resuspended in 1 ml of fixation / permeabilization buffer and incubated in the dark at 4°C for 1 hour. After washing with PBS, cells were resuspended in permeabilization buffer and incubated with 5 μl of isotype or FoxP3 antibody (clone PCH101) for 30 minutes at 4°C, then washed and acquired on a Gallios™ flow cytometer followed by analysis with Kaluza® Flow Analysis Software. Functional studies Enzyme-linked immunospot (ELIspot)

[0098] ELIspot analysis was used to quantify the frequency of IFNγ and granzyme B secreting cells. Briefly, PBMC and multiR-VST were resuspended in VST medium at 5x106 and 2x106 cells / ml, respectively, and 100 μl of cells were added to each ELIspot well. Antigen-specific activity was measured after individual stimulation [NP1, MP1 (influenza); N, F (RSV); F, N, M2-1, M (hMPV); M, HN, N, F (PIV)] or direct stimulation (500 ng / peptide / ml) with a control pepmix (Survivin, WT1). Staphylococcal enterotoxin B (SEB) (1 μg / ml) and PHA (1 μg / ml) were used as positive controls for PBMC and VST, respectively. After 20 h of incubation, plates were developed as described above, dried overnight at room temperature, and then sent to Zellnet Consulting (New York) for quantification. Spot forming cells (SFC) and input cell number were plotted and the specificity threshold for VST was defined as ≧30 SFC / 2×10 5 input cells. Multiplex

[0099] Multi-R-VST cytokine profiles were evaluated using the MILLIPLEX High Sensitivity Human Cytokine Panel (Millipore, Billerica, MA). 2 × 105 VSTs were mixed with pepmix (NP1, MP1,

[0100] The cells were stimulated with 1 μg / ml of 10 ... Chromium release assay

[0101] A standard 4-hour chromium (Cr51) release assay was used to measure the specific cytolytic activity of multi-R-VST using autoantigen-loaded PHA blasts (20 ng / pepmix / 1x106 target cells) as targets. Effector:target (E:T) ratios of 40:1, 20:1, 10:1 and 5:1 were used to analyze specific lysis. The percentage of specific lysis was calculated as [(experimental release-spontaneous release) / (maximum release-spontaneous release)]x100. To measure the auto- and alloreactive potential of multi-R-VST strains, only autologous and allogeneic PHA blasts were used as targets. Example 2 Generation of polyclonal multi-R-VST from healthy donors

[0102] In this study, we investigated the feasibility of using ex vivo expanded T cells to target multiple clinically problematic respiratory viruses. Specifically, we generated VSTs with specificity for influenza, RSV, hMPV and PIV and demonstrated clinical efficacy in transplant recipients that successfully controlled active infection. background

[0103] CARV-associated acute upper and lower RTIs are a major public health problem, with young children, the elderly, and those with suppressed or compromised immune systems being most vulnerable (1-3). These infections are associated with symptoms including cough, dyspnea, and wheezing, with dual / multiple co-infections being common, with a frequency that may exceed 40% in children under 5 years of age, and with an increased risk of morbidity and hospitalization (22-26). Among immunocompromised allogeneic HSCT recipients, up to 40% experience CARV infections that may range from mild (rhinorrhea, cough, and associated symptoms including fever) to severe (bronchiolitis and pneumonia), with associated mortality rates as high as 50% in LRTI patients (5-9). Treatment options are limited. For hMPV and PIV, there are currently no approved prophylactic vaccines or therapeutic antivirals, although off-label use of the nucleoside analog RBV and investigational use of DAS-181 (a recombinant sialidase fusion protein) have had limited clinical benefit (10, 11, 27, 28). Annual prophylactic influenza vaccines are not recommended for allogeneic HSCT recipients until at least 6 months after transplant (and are excluded in recipients of intensive chemotherapy or anti-B cell antibodies), while neuraminidase inhibitors are not always effective in treating active infection (12). In the case of RSV, aerosolized RBV is FDA approved for the treatment of severe bronchiolitis in infants and children and is also used off-label for the prevention of upper or lower RTIs and treatment of RSV pneumonia in HSCT recipients (13, 15, 16). However, its widespread use is limited by the need for cumbersome nebulizers and ventilation systems for drug delivery and the high associated costs. For example, in 2015, the cost of aerosolized RBV was $29,953 per day, with a typical treatment course of 5 days. (14) Thus, the lack of approved treatments, coupled with the high cost of antivirals, prompted the inventors to explore the feasibility of using adoptively transferred T cells to prevent and / or treat CARV infection in this patient population.

[0104] The crucial role of functional T cell immunity in mediating CARV viral control has only recently come to the forefront. For example, a retrospective study of 181 HSCT patients with RSV URTI reported lymphopenia (defined as ALC ≤ 100 / mm3) as a key determinant in identifying patients whose infection would progress to LRTI, whereas RSV neutralizing antibody levels were not significantly associated with disease progression (29). Moreover, in a recent retrospective analysis of 154 adult patients with hematological malignancies with or without HSCT treated for RSV LRTI, lymphopenia was significantly associated with higher mortality (30). Both of these studies suggest the importance of cell-mediated immunity in mediating protective immunity in vivo. Donors and cell lines

[0105] Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and HSCT recipients with informed consent using Baylor College of Medicine IRB-approved protocols (H-7634, H-7666) and used to generate phytohemagglutinin (PHA) blasts and multiR-VSTs. PHA blasts were generated as previously described (20) and cultured in VST medium [45% RPMI 1640 (HyClone Laboratories, Logan, Utah), 45% Click's medium (Irvine Scientific, Santa Ana, California), 2 mM GlutaMAX TM-I (Life Technologies, Grand Island, New York), and 10% human AB serum (Valley Biomedical, Winchester, Virginia)] supplemented with interleukin 2 (IL2) (100 U / mL; NIH, Bethesda, Maryland) (replenished every 2 days). VST generation Generation and phenotypic characterization of T cells specific for multiple respiratory viruses

[0106] The inventors generated virus-specific T cell (VST) T cell lines containing subpopulations of cells reactive against influenza, RSV, hMPV and PIV by the following method:

[0107] PBMCs (2.5x10 7 ) were harvested as described above and then transferred to G-Rex 10 (Wilson Wolf Manufacturing Corporation, St. Paul, MN) containing 100 ml of VST medium supplemented with IL7 (20 ng / ml), IL4 (800 U / ml) (R&D Systems, Minneapolis, MN) and pepmix (2 ng / peptide / ml) and cultured at 37°C and 5% CO for 10–13 days (Figure 1A).

[0108] The pepmix was a peptide library (15mer, 11 aa overlap) spanning influenza A antigens (NP1, MP1), RSV antigens (N, F), hMPV antigens (F, N, M2-1, M) (JPT Peptide Technologies, Berlin, Germany) and PIV antigens (M, HN, N, F) (Genemed Synthesis, San Antonio, TX). The lyophilized pepmix was reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and stored at -80 °C until use. result

[0109] Over a period of 10-13 days, we achieved an average of 8.5-fold increase in cells (Figure 1B) [PBMC 0.25x10 7 pieces / cm 2 Average cells from 1.9±0.2x10 7 pieces / cm 2 (median: 2.05x10 7 , Range: 0.6~2.82x10 cells 7 pieces / cm 2; n=12). We used flow cytometry to analyze the immunophenotype of cells expanded as described above. Expanded cells were composed almost exclusively of CD3+ T cells (96.2±0.6%; mean±SEM) with no evidence of regulatory T cell proliferation as assessed by CD4 / CD25 / FoxP3+ staining, along with a mixture of cytotoxic (CD 8+; 18.1±1.3%) and helper (CD4+; 74.4±1.7%) T cells [Figure 1C] [Figure 1E]. Furthermore, expanded cells displayed a phenotype consistent with effector function and long-term memory, as evidenced by upregulation of activation markers CD25 (50.2 ± 3.8%), CD69 (52.8 ± 6.3%), and CD28 (85.8 ± 2%) as well as expression of central (CD45RO+ / CD62L+: 61.4 ± 3%) and effector memory markers (CD45RO+ / CD62L-: 20.3 ± 2.3%), with minimal surface expression of PD1 (6.9 ± 1.4%) or Tim3 (13.5 ± 2.3%) [Figures 1C-1D].

[0110] Thus, the methods disclosed herein result in rapid expansion of polyclonal populations of activated cytotoxic and helper T cells without signs of exhaustion, suggesting the expansion of VSTs with specificity for respiratory viral antigens. Example 3 Characterization of antiviral specificity of multi-R-VSTs

[0111] Next, to determine whether the expanded populations were antigen-specific, we performed IFNγ and granzyme B secreting cell ELIspot assays using each of the individual stimulating antigens as immunogens. ELIspot analysis was performed as described above. All 12 lineages generated demonstrated reactivity against all of the target viruses [Table 1, Figure 2E]. [Table 1]

[0112] Figure 2A summarizes the magnitude of activity for each stimulating antigen, while Figure 2F shows the response of our proliferating VSTs to titrating concentrations of viral antigens. Remarkably, over 10-13 days of culture, we achieved 14.6 ± 4.3-fold (PIV-HN) to 50.4 ± 9.9-fold (RSV-N) enrichment of virus-specific T cells [Figure 2B; precursor frequency of CARV-reactive T cells within donor PBMCs is summarized in Figure 2G]. Collectively, these data suggest that respiratory virus-specific T cells exist within a memory pool and can be readily expanded ex vivo using GMP-compliant manufacturing methods.

[0113] Next, to assess whether the virus specificity was contained with CD4+ or CD8+ or both T cell subsets, we performed ICS, gating on CD4+ and CD8+ IFNγ producing cells. Figure 2C shows a representative result from one donor with activity against all four viruses detected in both T cell compartments [(CD4+: Influenza - 5.28%; RSV - 11%; hMPV - 6.57%; PIV - 3.37%), (CD8+: Influenza - 2.26%; RSV - 4.36%; hMPV - 2.69%; PIV - 2.16%)], while Figure 2D shows a summary of the results for the nine donors screened, confirming that our multiR-VST is polyclonal and multispecific.

[0114] Thus, these data confirm that these methods generate multi-R-VSTs that are polyclonal and contain both CD4+ and CD8+ T cells. Example 4 Evaluation of in vitro efficacy of multi-R-VST

[0115] The production of multiple proinflammatory cytokines and expression of effector molecules have been shown to correlate with enhanced cytolytic function and improved in vivo T cell activity. Therefore, we next investigated the cytokine profile of our multi-R-VST after antigen exposure. As shown in Figure 3, the majority of IFNγ-producing cells also produced TNFα in addition to GM-CSF as measured by Luminex arrays [Figure 3C-left panel] at baseline levels of prototypic Th2 / inhibitory cytokines [Figure 3C-right panel] [Figure 3A-detailed ICS results from one donor; summary of results for nine donors; Figure 3B]. Furthermore, upon antigen stimulation, our cells produced the effector molecule granzyme B, suggesting the cytolytic potential of these proliferating cells [Figure 3D, n=9]. Collectively, this data reveals the Th1 polarizing and multifunctional properties of our multi-R-VST.

[0116] To examine the cytolytic potential of these expanded cells in vitro, multi-R-VSTs were co-cultured with autologous Cr51-labeled PHA blasts, loaded with viral pepmix, and unloaded PHA blasts served as controls. As shown in Figure 4A and Figure 4C, targets loaded with viral antigens were specifically recognized and lysed by our expanded multi-R-VSTs (40:1 E:T-influenza: 13 ± 5%, RSV: 36 ± 8%, hMPV: 26 ± 7%, PIV: 22 ± 5%, n = 8). Finally, even though these VSTs received only a single stimulus, there was no evidence of activity against uninfected autologous targets, nor of alloreactivity (potentially graft-versus-host) when using HLA-mismatched PHA blasts as targets [Figure 4B]. This is an important consideration if these cells are to be administered to allogeneic HSCT recipients.

[0117] Thus, Multi-R-VST is effective in vitro and safe. Example 5 Evaluation of in vivo efficacy of multi-R-VST

[0118] To evaluate the potential clinical relevance of multi-R-VST, we investigated whether allogeneic HSCT recipients with active / recent CARV infections exhibited high levels of reactive T cells during / after active viral episodes. Figure 5A shows the results for patient #1, a 64-year-old male with acute myeloid leukemia (AML) who underwent matched related donor (MRD) transplant with reduced-intensity conditioning. The patient developed severe URTI 9 months after HSCT, confirmed to be RSV-related by PCR analysis. The patient was not immunosuppressed at the time of infection, but received prednisone on the day of infection diagnosis to control lung inflammation. Within 4 weeks, the patient's symptoms resolved without specific antiviral treatment. To evaluate whether T cell immunity contributed to viral clearance, we analyzed the circulating frequency of RSV-specific T cells over the course of the patient's infection. Just prior to infection, the patient had RSV antigens N and F (6.5SFC / 5x10 5 PBMC), but within 1 month of viral exposure, RSV-specific T cells expanded in vivo (527 SFC / 5x10 5 PBMC), as seen in Figure 5A, represented an 81-fold increase in reactive cells that then declined, coinciding with viral clearance. Of note, the observed RSV-specific response did not follow a global increase in lymphocyte / CD4+ counts, thus indicating that T-cell proliferation was virus-driven and not due to general immune reconstitution. Similarly, patient #2, a 23-year-old male with acute lymphoblastic leukemia (ALL) who underwent matched unrelated donor (MUD) transplant with myeloablative conditioning, developed severe RSV-associated URTI 5 months after HSCT during tapering doses of tacrolimus. The patient's infection resolved within a week, coinciding with administration of ribavirin. To investigate whether endogenous immunity also contributes to viral clearance, we monitored reactive T-cell counts over time. As seen in Figure 5B, viral clearance was associated with an increase in the circulating frequency of RSV-specific T cells (peak 93 SFC / 5x10 5hMPV was associated with PBMCs and subsequently returned to baseline levels. The same patient was hospitalized 7 months post-transplant for subsequent pneumococcal pneumonia, at the same time hMPV was detected in sputum (by PCR). The patient's pneumonia was treated with antibiotics, and subsequent recovery from disease and viral clearance coincided with a marked expansion of hMPV-specific T cells (reactive against F, N, M2-1, and M), which increased from 4 SFC to a peak of 70 SFC, then declined to baseline levels [Figure 5C]. Again, the observed RSV and hMPV-specific responses were independent of an overall increase in lymphocyte / CD4+ counts. Figure 6 shows the results of three additional HSCT recipients who developed CARV infection. Patient #3 was a 15-year-old female with AML who received a haploidentical transplant with reduced-intensity conditioning and developed RSV-induced URTI and LRTI 5 weeks after transplant while on tacrolimus. The patient was administered ribavirin, and the infection resolved within 4 weeks. The inventors monitored RSV-reactive T cells over time and, as can be seen in FIG. 6A, viral clearance was associated with a significant increase in the frequency of RSV-specific T cells (from 0 to 506 SFC / 5×10 5 PBMC). Similarly, patient #4, a 10-year-old male patient with ALL who underwent MUD transplant with myeloablative conditioning, developed PIV3-associated URTI and LRTI 1 month after HSCT while receiving tacrolimus. His infection resolved within 5 weeks, coinciding with administration of ribavirin. To examine whether endogenous immunity also contributes to viral clearance, we monitored PIV3-reactive T cell numbers over time. As seen in Figure 6B, viral clearance was associated with an increase in the circulating frequency of T cells specific for PIV3 antigens M, HN, N, and F (peak 38 SFC / 5x10 5PBMC) and then declined. Finally, we present Patient #5, a 3-year-old male with chronic granulomatous disease who underwent MRD transplantation with myeloablative conditioning and developed severe PIV3-associated URTI 4 months after HSCT while on cyclosporine. The patient received ribavirin but continued to show disease symptoms (at last evaluation) and could not demonstrate PIV3-specific T cells (Figure 6C). Taken together, these data suggest the in vivo relevance of CARV-specific T cells in controlling viral infections in immunocompromised patients. conclusion

[0119] Respiratory viral infections due to community-acquired respiratory viruses (CARVs), including respiratory syncytial virus (RSV), influenza, parainfluenza virus (PIV) and human metapneumovirus (hMPV), are detected in up to 40% of allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, causing severe disease such as bronchiolitis and pneumonia that can be fatal. Given the lack of approved antivirals for these CARVs and data demonstrating that adoptively transferred ex vivo expanded virus-specific T cells (VSTs) could be clinically beneficial for the treatment of both latent [Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), human herpesvirus 6 (HHV6)] and lytic [adenovirus (AdV)] viruses in allo-HSCT recipients, it was considered to explore the possibility of extending this approach to at least influenza, RSV, hMPV and PIV3.

[0120] Thus, we exposed PBMCs from healthy donors to a cocktail of pepmixes (overlapping peptide libraries) spanning immunogenic antigens from specific target viruses [influenza-NP1 and MP1; RSV-N and F; hMPV-F, N, M2-1 and M; PIV3-M, HN, N and F], followed by expansion in the presence of activating cytokines in G-Rex. Over a 10-13 day period, we achieved an average of 8.5-fold expansion (from 0.25x107 PBMCs / cm2 to an average of 1.9±0.2x107 cells / cm2; n=12). Cultures contained almost exclusively a mixture of CD3+ T cells (96.2±0.6%; mean±SEM), cytotoxic (CD8+) and helper (CD4+) T cells, with a phenotype consistent with immediate effector function and long-term persistence, as evidenced by upregulation of activation markers CD25, CD69 and CD28, and upregulated expression of central (CD45RO+ / CD62L+) and effector memory markers (CD45RO+ / CD62Li), with minimal PD1 or Tim3. The antiviral specificity of the multi-respiratory VST was tested in an IFNγ ELISpot assay using each of the individual stimulating antigens as immunogen. All 12 strains screened were reactive to each target virus [influenza: mean 735±75.6 SFC / 2x105, RSV: 758±69.8, hMPV: 526±100.8, PIV3: 391±93.7]. The propagated VSTs were Th1 polarized effector cells as evidenced by production of TNFα, GM-CSF and granzyme B, with only baseline levels of Th2 / inhibitory cytokines.

[0121] The cells were tested in a standard Cr51 release assay and were able to lyse autologous PHA blasts loaded with viral pepmix (40:1 E:T-influenza: 13±5%, RSV: 36±8%, hMPV: 26±7%, PIV: 22±5%, n=8) without evidence of auto- or alloreactivity, demonstrating both their selectivity and their safety for clinical use in HSCT recipients.

[0122] Finally, to evaluate the clinical relevance of these findings, we examined peripheral blood from five allogeneic HSCT recipients with active RSV, hMPV, and PIV3 infections. Four of these patients successfully controlled the virus within 1-5 weeks, consistent with the expansion of endogenous reactive T cells and their subsequent return to baseline levels upon viral clearance, whereas one patient was unable to mount an immune response to the infecting virus and was thus far unable to clear the infection as well. This data suggests that adoptive transfer of ex vivo expanded cells should be clinically beneficial in patients who lack their own cellular immunity.

[0123] In conclusion, the inventors have shown that it is feasible to rapidly generate a single preparation of polyclonal (CD4+ and CD8+) multiple respiratory (multi-R)-VSTs with specificity for 12 immunodominant antigens from four target viruses: influenza, RSV, hMPV and PIV3 using GMP-compliant manufacturing methods. The expanded cells are Th1 polarized, polyfunctional and selectively capable of responding to and killing viral antigen-expressing target cells without activity against uninfected autologous or allogeneic targets, demonstrating both their selectivity for viral targets and their safety for clinical use. In various embodiments, such multiple respiratory virus-targeted cells (multi-R-VSTs) provide a wide range of benefits to immunocompromised individuals with uncontrolled CARV infections, including in immunocompromised individuals. Example 6 Generation of polyclonal RSV-specific VSTs from healthy donors

[0124] RSV is a particularly dangerous respiratory disease. It results in over 57,000 hospitalizations of young children (<5 years old) in the United States annually, 177,000 hospitalizations of adults (>65 years old) in the United States annually, and 14,000 deaths (Centers for Disease Control and Prevention). RSV often progresses to lower respiratory tract infections causing diseases such as pneumonia, which can be fatal (Paulsen and Danziger-Isakov, Clin Chest Med 38 (2017)), and is a major cause of disease in immunocompromised individuals, including patients who have undergone allogeneic hematopoietic stem cell transplantation or solid organ transplantation. Furthermore, although ribavirin is FDA approved to treat children with severe pneumonia caused by RSV, this treatment is expensive, difficult to administer, has associated toxicity issues, and is not approved for other patient groups. Thus, there is a great need in the art for an effective RSV treatment.

[0125] As shown in Figure 7, in addition to the RSV antigens N and F that were included in the multi-R-VST described in the examples above, the RSV genome also contains other antigens: G, M2-1, M, NS1, NS2, M2-2, P, L and SH (Figure 7). Having demonstrated the efficacy of our multi-R-VST for treating RSV infection, despite being made with only the pepmix RSV antigens N and F, we sought to determine whether we could make a VST with specificity for a broader range of RSV antigens.

[0126] For that purpose, PBMCs were isolated as described in Example 1 and 2.5×10 6 pieces / cm 2 PBMCs were cultured for 10-15 days as described in Examples 1 and 2 with IL4, IL7 and a pepmix covering all of the RSV antigens listed above. result

[0127] Over a 10-day period, we achieved an average of approximately 5-fold cell expansion (Figure 9A). We used flow cytometry to analyze the immunophenotype of cells expanded as described above. Expanded cells contained almost exclusively CD3+ T cells, along with a mixture of cytotoxic (CD8+; approx. 33%) and helper (CD4+; approx. 66%) T cells [Figure 9B]. Furthermore, expanded cells displayed a phenotype consistent with effector function and long-term memory, as evidenced by upregulation of activation markers CD25, CD69, and CD28, and minimal PD1 or Tim3 surface expression [Figure 9C].

[0128] We investigated the cytokine profile of our RSV-VSTs after antigen exposure. As shown in Figure 10A, VSTs produced large amounts of IFNγ in response to RSV antigens N, F, and G, and the response induced by the addition of other RSV antigens was weak. A similar profile of granzyme B production was seen after pepmix stimulation, suggesting the cytolytic potential of these proliferating cells [Figure 10B]. Furthermore, analysis of the Th1 cytokines GM-CSF, IFNγ, and TNFα (Figure 11A) and the Th2 cytokines IL-5, IL-6, and IL-10 (Figure 11B) clearly demonstrated that RSV-specific VSTs were Th1-skewed. Taken together, these data reveal the Th1-polarizing and multifunctional properties of our multiR-VSTs.

[0129] Thus, the methods disclosed herein result in rapid expansion of polyclonal populations of activated cytotoxic T cells and helper T cells without signs of exhaustion, suggesting the expansion of VSTs with specificity for RSV antigens.

[0130] The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified, if necessary, to use concepts from the various patents, applications, and publications to provide further embodiments.

[0131] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure. References 1.Hodinka RL. Respiratory RNA Viruses. Microbiol Spectr. 2016;4(4). 2.Gill PJ, Richardson SE, Ostrow O, et al. Testing for Respiratory Viruses in Children: To Swab or Not to Swab. JAMA Pediatr. 2017;171(8):798-804. 3.Nair H, Simoes EA, Rudan I, et al. Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis. Lancet. 2013;381(9875):1380-1390. 4.Shi T, McAllister DA, O'Brien KL, et al. 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Claims

1. A composition comprising a polyclonal population of cytotoxic T lymphocytes (CTLs) that recognize multiple viral antigens, the multiple viral antigens including: (a) Parainfluenza virus (PIV) antigens: PIV antigen-M, PIV antigen-HN, PIV antigen-N, and PIV antigen-F; (b) Respiratory syncytial virus (RSV) antigens: RSV antigen N and RSV antigen F; (c) influenza antigens: influenza antigen NP1 and influenza antigen MP1; and (d) human metapneumovirus (hMPV) antigens: hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N; wherein the composition is negative for bacteria and fungi in culture for at least 7 days, exhibits less than 5 EU / ml endotoxin, and is negative for mycoplasma.

2. The composition of claim 1 , wherein the PIV is human parainfluenza virus type 3 (PIV3).

3. The composition according to claim 1 or 2, wherein the plurality of antigens consist of PIV antigen M, PIV antigen HN, PIV antigen N, PIV antigen F, influenza antigen NP1, influenza antigen MP1, RSV antigen N, RSV antigen F, hMPV antigen M, hMPV antigen M2-1, hMPV antigen F and hMPV antigen N.

4. The CTL, a. Negligible alloreactivity; b. The antigen-specific T cells recovered from the patient have less activation-induced cell death than corresponding antigen-specific T cells recovered from the same patient but that were not cultured in the presence of both IL-7 and IL-4; and c. Survival rate of over 70% The composition of claim 1 , wherein the composition exhibits one or more properties selected from the group consisting of:

5. The composition of any one of claims 1 to 4, wherein the CTLs comprise reactive T cells against one or more pepmix libraries comprising multiple overlapping peptides spanning all or part of multiple viral antigens from PIV, RSV, influenza, and hMPV.

6. 6. The composition of claim 5, wherein the pepmix is ​​chemically synthesized and optionally is >90% pure.

7. The composition of claim 5 or 6, wherein the peptide is at least 7 amino acids in length.

8. The composition of claim 5 , wherein the peptides have an overlap of three amino acids.

9. 8. The composition of claim 5, wherein the peptides are each 15 amino acids in length and overlap by 11 amino acids.

10. The composition of claim 1 , wherein the CTLs are Th1 polarized.

11. The composition of claim 1 , wherein the CTL is capable of lysing a viral antigen-expressing target cell.

12. The composition of claim 1 , wherein the CTLs do not significantly lyse uninfected autologous or allogeneic target cells.

13. 13. A pharmaceutical composition comprising the composition of any one of claims 1 to 12 formulated for intravenous delivery.

14. 14. Use of a composition according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 in the manufacture of a medicament for lysing a target cell.

15. The use of claim 14, wherein the composition is formulated for administration to a subject in vivo.

16. 14. Use of a composition according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 in the manufacture of a medicament for treating or preventing a viral infection in a subject in need thereof.

17. The drug is 5×10 ６ ~5×10 ７ CTL / m ２ 17. The use according to any one of claims 14 to 16, formulated for administration in a single dose.

18. 18. The use according to any one of claims 15 to 17, wherein the subject is immunocompromised.

19. 19. The use according to any one of claims 15 to 18, wherein the subject has acute myeloid leukemia, acute lymphoblastic leukemia or chronic granulomatous disease.

20. Prior to receiving the CTLs, the subject a. Reduced-intensity conditioning matched related donor transplants; b. Matched unrelated donor transplant with myeloablative conditioning; c. Haploidentical transplant with reduced intensity conditioning; or d. Myeloablative conditioning with matched related donor transplantation 20. The use according to any one of claims 15 to 19,

21. The object is a. Have had a solid organ transplant; b. Have had chemotherapy; c. Having HIV infection; d. have a genetic immune deficiency; and / or e. Have had an allogeneic stem cell transplant; 21. Use according to any one of claims 15 to 20.

22. 22. The use according to any one of claims 15 to 21, wherein the composition is formulated so as to be administered to a subject for multiple treatments.

23. 23. The use according to any one of claims 15 to 22, wherein the viral infection is selected from the group comprising parainfluenza virus type 3, respiratory syncytial virus, influenza and human metapneumovirus.

24. 24. The use according to any one of claims 15 to 23, wherein the subject is a human.

25. 1. A method of making a composition for use in T cell therapy comprising a polyclonal population of cytotoxic T lymphocytes (CTLs) that recognize multiple viral antigens, the multiple viral antigens comprising: (a) Parainfluenza virus (PIV) antigens: PIV antigen-M, PIV antigen-HN, PIV antigen-N, and PIV antigen-F; (b) Respiratory syncytial virus (RSV) antigens: RSV antigen N and RSV antigen F; (c) influenza antigens: influenza antigen NP1 and influenza antigen MP1; and (d) human metapneumovirus (hMPV) antigens: hMPV antigen M, hMPV antigen M2-1, hMPV antigen F, and hMPV antigen N; wherein the composition is negative for bacteria and fungi in culture for at least 7 days, exhibits less than 5 EU / ml endotoxin, and is negative for mycoplasma.

26. The method of claim 25, wherein the PIV is human parainfluenza virus type 3 (PIV3).

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