Allogeneic T cell-based HIV vaccines for inducing cellular and humoral immunity

Abstract

Provided herein are methods for treating patients with human immunodeficiency virus (HIV) comprising administering a cell composition comprising recombinant allogeneic cells, such as CD4+ T cells. The invention further relates to compositions and methods for generating an allogeneic T cell-based protective HIV vaccine that induces both cellular and humoral immunity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 862,432, filed June 17, 2019, which is incorporated by reference in its entirety.

[0002] Provided herein are methods for treating a patient with Human Immunodeficiency Virus (HIV) comprising administering a cell composition further comprising recombinant allogeneic cells, such as CD4+ T cells. The invention further relates to compositions and methods for generating an allogeneic T cell-based protective HIV vaccine that induces both cellular and humoral immunity. [Background technology]

[0003] Great strides have been made against the HIV epidemic; however, there are still 1.7 million new infections and 770,000 deaths worldwide annually. The most successful treatment, antiretroviral therapy (ART), can cost between $18,000 and $40,000 per year. The total amount spent on HIV from 2000 to 2016 was $562 billion, or nearly $50 billion per year. Thus, a vaccine and a cure are badly needed. Summary of the Invention

[0004] In certain embodiments, provided herein is a cell comprising a heterologous nucleic acid molecule comprising a nucleotide sequence encoding CD40L and CXCL13.

[0005] In additional embodiments, CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

[0006] In a further embodiment, the cell is a T cell.

[0007] In a further embodiment, the cells are transduced or transfected with a second and / or a third nucleic acid encoding a heterologous protein.

[0008] In a further embodiment, the second nucleic acid comprises a human immunodeficiency virus (HIV) genome, wherein the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further wherein the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0009] In certain embodiments, provided herein are CD4+ cells comprising one or more heterologous nucleic acid molecules encoding CD40L and CXCL13.

[0010] In a further embodiment, CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

[0011] In certain embodiments, provided herein are CD4+ cells comprising a heterologous CD40L protein and a heterologous CXCL13 protein.

[0012] In a further embodiment, the CD4+ cell further comprises a heterologous nucleic acid molecule comprising a human immunodeficiency virus (HIV) genome, wherein the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further, wherein the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0013] In certain embodiments, provided herein is a method of treating HIV in a subject, comprising administering a composition to the subject, comprising administering an effective amount of any of the cells described herein above.

[0014] In certain embodiments, provided herein is a method for increasing an immune response in a subject in need thereof, comprising administering an effective amount of any of the cells described herein above.

[0015] In additional embodiments, the cells are allogeneic to the subject.

[0016] In additional embodiments, the cells are HLA-mismatched with the patient.

[0017] In additional embodiments, the dosage of cells is about 1-5×10 6 The range is.

[0018] In additional embodiments, the viral infection is caused by human immunodeficiency virus (HIV).

[0019] In a further embodiment, graft-versus-host disease (GVHD) is reduced or eliminated while graft-versus-virus (GVV) is increased in a subject.

[0020] In additional embodiments, the treatment or augmentation of the immune response is repeated periodically as a maintenance treatment over a time frame of once every week to once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, once every six months, or once every seven months, or once every eight months, or once every nine months, or once every ten months, or once every eleven months, or once every year, so long as the subject shows improvement, reduced or undetectable viral titers, or shows stable / non-progressive disease.

[0021] In a further embodiment, both cellular and humoral immunity is induced in the subject. [Brief description of the drawings]

[0022] [Figure 1A] 1 shows non-limiting vector maps for generating human and macaque constructs. [Figure 1B] 1 shows non-limiting vector maps for generating human and macaque constructs. [Diagram 2] 1 shows a scheme for using the macaque embodiment provided herein. [Diagram 3] 1 shows a scheme for using the embodiments provided herein. [Figure 4] 1 shows a scheme for using the embodiments provided herein. [Diagram 5] 1 presents data demonstrating the enhanced cytotoxicity of the immune cell compositions provided herein. [Figure 6] 1 shows the expansion of NK cells following vaccination of PBMCs with cells provided herein. [Figure 7] 1 shows the expansion of NKT cells following vaccination of PBMCs with cells provided herein. [Figure 8] 1 shows increased NK cell activation following vaccination of PBMCs with cells provided herein. [Figure 9] 1 shows an increase in NK cell activation humoral responses following vaccination of PBMCs with cells provided herein. [Figure 10] 1 shows increased B cell activation following vaccination of PBMCs with cells provided herein. [Figure 11] 1 shows increased T cell activation following vaccination of PBMCs with cells provided herein. [Figure 12] 1 shows increased T cell activation following vaccination of PBMCs with cells provided herein. [Figure 13] 1 shows increased CD8 T cell activation following vaccination of PBMCs with cells provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Abbreviation: Antibody-dependent cytotoxicity: ADCC

[0024] Differentiation antigen group 3: CD3

[0025] Graft-versus-tumor effect: GVT

[0026] Graft-versus-host disease: GVHD

[0027] Graft-versus-virus: GVV

[0028] Gamma delta T cells: GDT cells, also γδ T cells.

[0029] Human immunodeficiency virus (HIV): A lentivirus that causes acquired immune deficiency syndrome.

[0030] Invariant natural killer T cells: iNK T cells, also known as type I or classical NKT cells, are a distinct population of T cells that express the invariant aβ T cell receptor (TCR) and many cell surface molecules in common with natural killer (NK) cells.

[0031] Natural killer cells: NK cells

[0032] Effective efforts to induce sufficient immune responses to protect against HIV have been limited. Although significant titers of neutralizing antibodies can be achieved and non-human primates can prevent infection, successful translation of humoral vaccines to humans has been difficult so far. Vaccines that promote cellular responses have shown promise in animals, with several currently in human trials. However, data to date indicate limited efficacy.

[0033] A vaccine that could effectively combine strong humoral and cellular responses would be a powerful approach: even if such a vaccine fails as a protective vaccine, it may still have efficacy as a therapeutic vaccine.

[0034] The present invention relates to compositions and methods for generating allogeneic (or in certain embodiments autologous) T cell-based protective HIV vaccines that induce both cellular and humoral immunity. A general overview of these methods and compositions is as follows:

[0035] T cells from a donor or cell line are infected with replication-incompetent HIV or with a live attenuated strain of HIV.

[0036] Once injected into the host, the host immune system will easily reject the entire live attenuated HIV-infected T cell population only by cytotoxic immune response, without giving the humoral immune system a chance to engage allo-T cells in the rejection process, unless the injected cell population is very large. Thus, humoral immunity is not elicited. To ensure the recruitment of host B cells to the rejection process, the infected allo-T cells are genetically modified to express human CD40L and human CXCL13 by viral or non-viral genetic modification tools. (See Figure 1A, an example of hCD40L and hCXCL13 expressing vector map).

[0037] Expression of hCD40L and hCXCL13 molecules promotes B cell-specific rejection of allo-T cells and ensures activation of the humoral immune system against allo-T cells and the HIV genome carried by them.

[0038] Thus, these constructs serve to induce humoral and cellular immunity against HIV by piggybacking the HIV genome from allogeneic T cells, ensuring their rejection when injected into a mismatched host.

[0039] The final allo-T cell product will be used as a prophylactic vaccine against HIV. The cells will be infused several times, the first acting as a primer and the next one or more infusions acting as booster shots. Optimization of dosing options and the safe dose / number of cells infused, as well as the number of infusions and their timing, are expected to be determined by further safety / efficacy studies.

[0040] So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined in this document, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used herein, and unless otherwise specified, the term "about" is intended to mean ±5% of the value it modifies. Thus, about 100 means 95 to 105. Additionally, the term "about" modifies a term in a series of terms, such as "about 1, 2, 3, 4, or 5." It should be understood that the term "about" modifies each member of the list, such that "about 1, 2, 3, 4, or 5" can be understood to mean "about 1, about 2, about 3, about 4, or about 5." The same is true for lists modified by the term "at least" or other quantifiers, such as, but not limited to, "smaller than," "greater than," and the like.

[0042] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise.

[0043] As used herein, the terms "comprise," "have," "has," and "include," and conjugations thereof, as used herein, mean "including, but not limited to." Although various compositions and methods are described in terms of "comprising" various components or steps (which should be interpreted to mean "including, but not limited to"), the compositions, methods, and devices may also "consist essentially of" or "consist of" various components and steps, and such terms should be interpreted as defining groups whose members are essentially limited.

[0044] Chemokine (CXC motif) ligand 13 (CXCL13), also known as B lymphocyte chemoattractant (BLC) or B cell-attracting chemokine 1 (BCA-1), is a protein ligand encoded by the CXCL13 gene in humans. CXCR5 is the receptor for CXCL13. Expression of the chemokine initiates a positive loop of lymphocyte recruitment and stimulation. Overexpression of CXCL13 in intestinal epithelial cells promoted a significant increase in the number of B cells in the lamina propria, as well as an increase in the size and number of lymphoid follicles in the small intestine. These results suggest that overexpression of CXCL13 in the intestine during inflammatory conditions favors the recruitment of B cells, as well as LTi and NK cells, which have immune regulatory and repair functions. In some embodiments, CXCL13 is encoded by a nucleic acid molecule comprising the following sequence: ATGAAGTTCATCTCGACATCTCTGCTTCTCATGCTGCTGGTCAGCAGCCTCTCTCCAGTCCAAGGTGTTCTGGAGGTCTATTACACAAGCTTGAGGTGTAGATGTGTCCAAGAGAGCTCAGTCTTTATCCCTAGACGCTTCATTGATCGAATTCAAATCTTGCCCCG TGGGAATGGTTGTCCAAGAAAAGAAATCATAGTCTGGAAGAAGAACAAGTCAATTGTGTGTGTGGACCCTCAAGCTGAATGGATACAAAGAATGATGGAAGTATTGAGAAAAAGAAGTTCTTCAACTCTACCAGTTCCAGTGTTTAAGAGAAAGATTCCC (SEQ ID NO: 1).

[0045] The Genbank accession number for the nucleic acid sequence is NM_006419.2, which is incorporated herein by reference in its entirety. In some embodiments, the amino acid sequence of CXCL13 is: MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP (sequence number 3).

[0046] Due to the degeneracy of the genetic code, the sequence of SEQ ID NO:1 is merely a non-limiting example, and other nucleic acid sequences may be used to express CXCL13. In some embodiments, the nucleic acid sequence encoding SEQ ID NO:3 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. In some embodiments, the protein of CXCL13 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. This sequence may contain conservative substitutions (mutations) that do not affect the activity of CXCL13.

[0047] CD40 ligand (CD40L), also called CD154, is a protein that is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells (APCs) and has many effects depending on the type of target cell. In total, CD40L has three binding partners: CD40, α5β1 integrin and αIIbβ3. CD154 functions as a costimulatory molecule and is particularly important in a subset of T cells called follicular helper T cells (TFH cells). In TFH cells, CD40L promotes B cell maturation and function by binding to CD40 on the B cell surface, thereby facilitating cell-cell communication. Stable expression of CD40L allows recombinant allogeneic CD4+ to produce IL-12 to overcome immune suppression and to induce differentiation of memory T cells. In some embodiments, CXCL13 is encoded by a nucleic acid molecule comprising the following sequence: (Sequence number 2).

[0048] The Genbank accession number for the nucleic acid sequence is NM_000074.2, which is incorporated herein by reference in its entirety. In some embodiments, the amino acid sequence of CD40L is MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (sequence number 4).

[0049] Due to the degeneracy of the genetic code, the sequence of SEQ ID NO:2 is merely a non-limiting example and other nucleic acid sequences may be used to express CD40L. In some embodiments, the nucleic acid sequence encoding SEQ ID NO:4 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the protein of CD40L is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4. This sequence may contain conservative substitutions (mutations) that do not affect the activity of CD40L.

[0050] As used herein, the term "heterologous" when referring to a nucleic acid molecule in a cell means that the nucleic acid molecule is not native to the genome of the naturally occurring cell, even if the cell has a similar sequence or gene sequence encoding the same protein or sequence. For example, a cell containing a heterologous nucleic acid molecule encoding CD40L and / or CXCL13 refers to a cell that has been modified to contain a nucleic acid molecule(s) encoding CD40L and / or CXCL13. This may be done by transfection or transduction, or other genome editing techniques such as, but not limited to, CRISPR. When the term "heterologous" is used in reference to a protein in a cell, it refers to a protein encoded by a heterologous nucleic acid molecule. Thus, a cell may contain the same protein, a protein that is native to the cell (a protein that is not encoded by a heterologous nucleic acid molecule), and a protein that is heterologous to the cell (a protein that is encoded by a heterologous nucleic acid molecule). A heterologous nucleic acid molecule may be present in the cell transiently, by insertion into the genome of the cell, or by the presence of an episomal plasmid in the cell, or may be present stably in the cell. The heterologous nucleic acid molecule may also be introduced into the cell via viral transduction, which may also be stably integrated into the genome of the cell.

[0051] The recombinant cells expressing the heterologous protein may also be modified to express a target antigen against which an immune response is desired to be generated. For example, the target antigen may be an HIV protein or an antigenic fragment thereof. In some embodiments, cells such as T cells or CD4+ T cells that heterologously express CD40L and / or CXCL13 also heterologously express a target antigen. In some embodiments, the additional nucleic acid molecule comprises a human immunodeficiency virus (HIV) genome. In some embodiments, the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further, the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genome construct. In some embodiments, the target antigen is one or more of HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, Vif, etc., or any combination thereof. In some embodiments, the target antigen is any HIV protein. In some embodiments, the cells heterologously expressing CD40L and / or CXCL13 express each HIV protein. In some embodiments, the cells heterologously express at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HIV proteins, or fragments thereof. In some embodiments, the target antigen is HIV Tat (full length or isoforms of 72 and 101 amino acids in length). In some embodiments, the target antigen is HIV Rev. In some embodiments, the target antigen is HIV Pol. In some embodiments, the target antigen is HIV GP120. In some embodiments, the target antigen is HIV GP160. In some embodiments, the target antigen is HIV GP41. In some embodiments, the target antigen is HIV env. In some embodiments, the target antigen is HIV Gag. In some embodiments, the target antigen is HIV Gag-Pol. In some embodiments, the target antigen is HIV Nef. In some embodiments, the target antigen is HIV Vpr. In some embodiments, the target antigen is HIV Vpu.In some embodiments, the target antigen is HIV Vif. Any of these antigens may be heterologously expressed in cells heterologously expressing CD40L and / or CXCL13. In some embodiments, SHIV antigens may be used in place of HIV antigens, and equivalent antigens may be used in place of the antigens provided herein.

[0052] When the HIV genome is used, HIV genome plasmids are commercially available and exemplary mutations and systems are described, for example, in Mol Ther. 2017 Aug 2; 25(8): 1790-1804. Similarly, a commercially available plasmid backbone pVAX1 (plasmids mixed in a 1:1 ratio) is also available that carries the Chinese HIV-1 subtype C / B = env and gag genes in one plasmid and pol and nef / tat constructs designed to express fusion proteins in a second plasmid (see Advax, San Diego, CA and Clinical and Vaccine Immunology, Volume 20 Number 3, March 2013, p. 397-408.) This is a non-limiting example and other constructs may be used.

[0053] Vector maps of exemplary constructs used to generate recombinant allogeneic CD4+ cells are shown in Figures 1 and 1B, and an overview and schematic for testing these constructs is shown in Figures 2, 3, and 4.

[0054] Terms such as "co-administration" are meant to encompass the administration of selected therapeutic agents to a single patient, and are intended to include therapeutic regimens in which agents are administered by the same or different routes of administration or at the same or different times.

[0055] As used herein, the term "agonist" refers to a compound whose presence produces a biological activity of a protein that is the same as the biological activity that results from the presence of the protein's naturally occurring ligand.

[0056] As used herein, the term "partial agonist" refers to a compound whose presence produces the same type of biological activity of a protein, but to a lesser extent, that results from the presence of the protein's naturally occurring ligand.

[0057] As used herein, the term "antagonist" refers to a compound whose presence results in a decrease in the degree of biological activity of a protein. In certain embodiments, the presence of an antagonist results in complete inhibition of the biological activity of a protein. In certain embodiments, an antagonist is an inhibitor.

[0058] When used in conjunction with therapeutic compositions (e.g., protective allogeneic T cell-based HIV vaccines, recombinant allogeneic CD4+-based vaccines, and compositions comprising these products), "administering" means administering a therapeutic agent directly to a target tissue or administering a therapeutic agent to a patient, thereby having a positive therapeutic effect on a targeted tissue.

[0059] The term "subject" or "patient" as used herein includes, but is not limited to, humans and non-human vertebrates, such as wild animals, domestic animals, and farm animals. In some embodiments, the subject or patient, which may be used interchangeably, may be a non-human primate. In certain embodiments, the subject or patient described herein is an animal. In certain embodiments, the subject or patient is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject or patient is a non-human animal. In certain embodiments, the subject or patient is a non-human mammal. In certain embodiments, the subject or patient is a domestic animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject or patient is a companion animal, such as a dog or cat. In certain embodiments, the subject or patient is a domestic animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject or patient is a zoo animal. In another embodiment, the subject or patient is a research animal, such as a rodent, dog, or non-human primate. In certain embodiments, the subject or patient is a non-human transgenic animal, such as a transgenic mouse or a transgenic pig.

[0060] The term "inhibit" includes the administration of a therapeutic agent of the embodiments herein to prevent the onset of symptoms, alleviate symptoms, or eliminate a disease, condition, or disorder.

[0061] By "pharmacologically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the therapeutic agent and not deleterious to the recipient thereof.

[0062] The terms "treat", "treated" or "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to inhibit, prevent or slow (alleviate) an undesirable physiological condition, disorder or disease, or to improve, inhibit or otherwise obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, amelioration or alleviation of symptoms, reduction in the extent of a condition, disorder or disease, stabilization (i.e., not worsening) of a condition, disorder or disease state, delay in onset or slowing of progression of a condition, disorder or disease, improvement of a condition, disorder or disease state, and remission (partial or complete), or improvement or amelioration of a condition, disorder or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. "Treatment" also includes prolonging survival compared to expected survival in the absence of treatment.

[0063] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources or an immunoreactive portion of an intact immunoglobulin. Antibodies may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

[0064] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0065] The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may include either antibody production or activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will therefore encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that this embodiment includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen may be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tissue samples suspected of containing a virus, cells, or biological fluids.

[0066] The term "autoantigen" refers to any self-antigen that is mistakenly recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphorylated proteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, and examples of cell surface receptors.

[0067] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0068] The term "allogeneic" as used herein refers to antigenically distinct HLA or MHC loci. Thus, cells or tissues transferred from the same species can be antigenically distinct. Syngeneic mice can differ at one or more loci (congenic) and allogeneic mice can have the same background.

[0069] The term "antigen" as used herein is defined as a molecule that elicits an immune response, which may include either antibody production or activation of specific immunologically competent cells, or both.

[0070] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0071] The term "donor" refers to a mammal, e.g., a human, who is not the recipient of the patient. The donor may, for example, have HLA identity with the recipient, or may have partial or greater HLA disparity with the recipient.

[0072] The term "haploidentical" as used herein with respect to cells, cell types and / or cell lines refers to cells that share a haplotype, or have substantially the same alleles in a set of closely related genes on one chromosome. A haploidentical donor does not have complete HLA identity with the recipient, but rather has partial HLA disparity.

[0073] T cells and activated T cells (including CD3+ cells): T cells (also called T lymphocytes) belong to a group of white blood cells called lymphocytes. Lymphocytes are generally involved in cell-mediated immunity. The "T" in "T cell" refers to cells that originate from the thymus or mature under the influence of the thymus. T cells can be distinguished from other lymphocyte types, such as B cells and natural killer (NK) cells, by the presence of a cell surface protein known as the T cell receptor. The term "activated T cell" as used herein refers to a T cell that has been stimulated to generate an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant presented in the context of a class II major histocompatibility complex (MHC) marker. T cells are activated by the presence of antigenic determinants, cytokines and / or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, etc. and combinations thereof). Cells that express a cluster of differentiation protein are often said to be "positive" for expression of that protein on the surface of T cells (e.g., cells positive for expression of CD3 or CD4 are called CD3+ or CD4+). The CD3 and CD4 proteins are cell surface receptors or co-receptors and may be directly and / or indirectly involved in T cell signaling.

[0074] As used herein, the term "peripheral blood" refers to the cellular components of blood (e.g., red blood cells, white blood cells, and platelets) obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow.

[0075] "Peripheral blood mononuclear cells", "PBMCs" or "mononuclear cells" refer to mononuclear cells isolated from peripheral blood that are commonly used in anti-cancer immunotherapy. Peripheral blood mononuclear cells can be obtained from collected human blood using known methods such as Ficoll-Hypaque density gradients.

[0076] According to one exemplary embodiment of the present invention, "peripheral blood mononuclear cells" may be obtained from any suitable person. The source of donor lymphocyte cells, including sources such as peripheral blood mononuclear cells as used herein, may be allogeneic or autologous to the recipient patient for isolation of desired lymphocyte cells including: CD4+ cells, NK cells, γδ T cells, iNKT cells, CD3 T cells, or other combinations for use in anti-HIV, protective and / or therapeutic methods according to the present invention. In certain embodiments, the recombinant cells are allogeneic, while in other embodiments, they may be autologous.

[0077] As used herein, the term "ex vivo" refers to "outside" the body.

[0078] "Disease" refers to the health condition of a subject in which the subject is unable to maintain homeostasis and the health of the animal continues to deteriorate if the disease is not improved.In contrast, a "disorder" in a subject refers to a health condition in which the subject can maintain homeostasis, but the health of the subject is less favorable than if the disorder did not exist.If left untreated, the disorder does not necessarily cause the health of the subject to further deteriorate.

[0079] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0080] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.

[0081] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue or system.

[0082] As used herein, the term "exogenous" refers to any material introduced into or produced outside of an organism, cell, tissue or system. A heterologous nucleic acid molecule within a cell is an exogenous nucleic acid molecule.

[0083] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0084] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), that incorporate a recombinant polynucleotide.

[0085] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, and multiplied by 100. For example, if 6 out of 10 positions of two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed when the two sequences are aligned to give maximum homology.

[0086] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE.

[0087] "Isolated" means changed or removed from a natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may be present in a substantially purified form or may be present in a non-native environment, such as, for example, a host cell. An "isolated" biological component (such as a nucleic acid, protein, or cell) has been substantially separated or purified away from other biological components (such as cell debris, other proteins, nucleic acids, or cell types). An "isolated" biological component includes a component that has been purified by standard purification methods.

[0088] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of the disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0089] As used herein, recombinant generally refers to: a recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or that is made by the artificial combination of two otherwise isolated segments of sequence, which is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques.

[0090] As used herein, the following abbreviations for commonly occurring nucleobases are used: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0091] As used herein, the term "leukocyte" or "white blood cell" refers to any immune cell, including monocytes, neutrophils, eosinophils, basophils, and lymphocytes. As used herein, the term "lymphocyte" refers to cells commonly found in lymphocytes, including natural killer cells (NK cells), T cells, and B cells. It will be understood by those skilled in the art that the above immune cell types can be divided into further subsets.

[0092] As used herein, the term "tumor-infiltrating leukocytes" refers to leukocytes present in solid tumors.

[0093] As used herein, the term "blood sample" refers to any sample prepared from blood, such as plasma, blood cells isolated from blood, etc.

[0094] As used herein, the term "purified sample" refers to any sample in which one or more cell subsets are enriched. A sample can be purified by removing or isolating cells based on characteristics such as size, protein expression, etc.

[0095] The pharma- ceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional agents.

[0096] The compositions and cells provided herein may be administered by any suitable method. The compositions and cells of the embodiments provided herein may be in a variety of forms. These include, for example, liquids and semi-solids, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The form depends on the intended mode of administration and therapeutic application. In some embodiments, the composition is in the form of an injectable or infusible solution. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the therapeutic composition (pharmaceutical composition) is administered by intravenous infusion or injection. In some embodiments, the therapeutic molecule is administered by intramuscular or subcutaneous injection. In some embodiments, the therapeutic composition is administered locally, for example, by injection at the target site. The phrases "parenteral administration" and "administered parenterally", as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and inhalation.

[0097] Generally, the nature of the carrier or vehicle suitable for delivery depends on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids, including pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicles. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or monolaurate.

[0098] In some embodiments, the composition, either in solution, suspension or other form, may contain one or more of the following: DMSO, a sterile diluent such as water for injection, a physiological solution, preferably saline, Ringer's solution, isotonic sodium chloride, a fixed oil such as synthetic mono- or diglycerides (which may serve as a solvent or suspending medium), polyethylene glycol, glycerin, propylene glycol, or other solvents, etc., an antibacterial agent such as benzyl alcohol or methylparaben, an antioxidant such as ascorbic acid or sodium bisulfite, a chelating agent such as ethylenediaminetetraacetic acid, a buffer such as acetate, citrate, or phosphate, and an agent for adjusting isotonicity such as sodium chloride or dextrose.

[0099] Diseases that the compositions and methods described herein may treat include microbial infections, such as viral infections.

[0100] As used herein, "viral infection" refers to an infection caused by the presence of a virus in the body. Viral infections include chronic or persistent viral infections, which are viral infections that can infect a host and grow in the host's cells for extended periods of time, usually weeks, months, or years, before becoming fatal. Viruses that cause chronic infections include, for example, human papilloma virus (HPV), herpes simplex and other herpes viruses, hepatitis B and C viruses, and other hepatitis viruses, human immunodeficiency virus, and measles virus, all of which can cause significant clinical disease. Prolonged infections can eventually lead to the induction of disease, which can be fatal to the patient, for example, in the case of hepatitis C virus liver cancer. Other chronic viral infections that can be treated according to the present invention include Epstein-Barr virus (EBV), as well as other viruses such as viruses that can be associated with tumors.

[0101] Examples of viral infections that may be treated or prevented by recombinant allogeneic CD4+ HIV vaccine cells or compositions and methods comprising such cells described herein include, but are not limited to, viral infections caused by retroviruses (e.g., human T-cell lymphotropic virus (HTLV)), particularly types I and II, and human immunodeficiency virus (HIV). Treating and / or preventing a viral infection includes, but is not limited to, alleviating one or more symptoms associated with the infection, inhibiting, reducing or suppressing viral replication and / or viral load, and / or enhancing the immune response.

[0102] As used herein, "immunodeficiency" means lacking at least one essential function of the immune system. As used herein, an "immunodeficient subject" (such as a human) is a subject that lacks a particular component of the immune system or lacks the function of a particular component of the immune system (such as, for example, B cells, T cells, NK cells, or macrophages). In some cases, an immunodeficient subject contains one or more genetic alterations that prevent or inhibit the development of functional immune cells (such as B cells, T cells, or NK cells). In some examples, the genetic alterations are in IL17 or the IL17 receptor.

[0103] As used herein, "immunosuppressed" refers to the reduced activity or function of the immune system.A subject may be immunosuppressed, for example, due to treatment with an immunosuppressant compound, or as a result of a disease or disorder (e.g., immunosuppression due to HIV infection or genetic defects).In some cases, immunosuppression occurs as a result of genetic mutations that prevent or inhibit the development of functional immune cells, such as T cells.

[0104] In some embodiments of the invention, a "therapeutically effective amount" is an amount of recombinant allogeneic or autologous T cells, such as CD4+, comprising a target antigen as described herein, e.g., an HIV protein, vaccine cell, or a composition comprising such cells, that results in at least a 2.5%, at least a 5%, at least a 10%, at least a 15%, at least a 25%, at least a 35%, at least a 45%, at least a 50%, at least a 75%, at least a 85%, at least a 90%, at least a 95%, or at least a 99% reduction in viral titer in a subject / patient / animal administered recombinant allogeneic CD4+ HIV vaccine cells or a composition comprising such cells and treated with the associated methods described herein, compared to the viral titer or microbial titer in an animal or group of animals (e.g., 2, 3, 5, 10 or more animals) that have not been administered the recombinant T cell (e.g., CD4+ T cell) vaccine cells, or a composition comprising such cells of the invention.

[0105] In certain embodiments, recombinant allogeneic or autologous CD4+ HIV vaccine cells or compositions comprising such cells may be administered simultaneously with antibacterial, antiviral and / or other therapeutic agents. Alternatively, recombinant allogeneic or autologous CD4+ HIV vaccine cells or compositions comprising such cells may be administered at a selected time prior to the time that antibacterial, antiviral and other therapeutic agents are administered.

[0106] As provided herein, recombinant T cells (e.g., CD4+ cells), in some embodiments, may be allogeneic relative to the patient to which the cells are administered, or they may be autologous or HLA-matched.

[0107] cell source Peripheral blood mononuclear cells (PBMCs) may be isolated by Ficoll-Hypaque density gradient centrifugation of samples obtained from discarded de-identified leukocyte reduction filters (American Red Cross), or by blood donations from healthy volunteers with informed consent. Descriptions of cell populations, sources, and methods for selecting or enriching for desired cell types may be found, for example, in U.S. Pat. No. 9,347,044. A population of cells for use in the methods described herein for treating a mammal must be of a compatible species, such as human cells. The cells may be obtained from an animal, e.g., a human patient. If the cells are obtained from an animal, they may have first been established in culture, e.g., by transformation, or, more preferably, they may have been subjected to a preliminary purification method. For example, the cell population may be manipulated by positive or negative selection based on expression of cell surface markers, may be stimulated with one or more antigens in vitro or in vivo, may be treated with one or more biological modifiers in vitro or in vivo, or may be a combination of any or all of these. In an exemplary embodiment, the cell population is subjected to negative selection for depletion of non-T cells and / or specific T cell subsets. Negative selection can be performed based on cell surface expression of various molecules, including B cell markers such as CD19 and CD20, monocyte marker CD14, NK cell marker CD56, etc. Alternatively, the cell population may be subjected to negative selection for depletion of non-CD34+ hematopoietic cells and / or specific hematopoietic cell subsets. Negative selection can be performed based on cell surface expression of various molecules, such as a cocktail of antibodies (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a), which can be used for separation of other cell types, such as through MACS or column separation.

[0108] Populations of cells include peripheral blood mononuclear cells (PBMCs), whole blood or fractions thereof, including mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a tumor. Suitable donors include immunized donors, non-immunized (naive) donors, treated or untreated donors. A "treated" donor is one that has been exposed to one or more biological modifiers. An "untreated" donor is one that has not been exposed to one or more biological modifiers.

[0109] The method of obtaining a population of cells that includes T cells is well known in the art.For example, peripheral blood mononuclear cells (PBMCs) can be obtained as described according to the method known in the art.Examples of such methods are described in Examples and discussed by Kim et al. (1992), Biswas et al. (1990), Biswas et al. (1991).

[0110] It is also possible to obtain a cell sample from a subject and enrich it for the desired cell type. For example, PBMCs can be isolated from blood as described herein. Counterflow centrifugation (elutriation) can be used to enrich T cells from PBMCs. Cells can be separated from other cells using various techniques, such as separation and / or activation with antibodies that bind to cell surface epitopes of the desired cell type; for example, some T cell separation kits use antibody-bound beads to activate cells, followed by column separation with the same beads. Another method that can be used includes negative selection, which uses antibodies against cell surface markers to selectively enrich for a particular cell type without activating the cells by receptor engagement.

[0111] Bone marrow cells may be obtained from the iliac crest, femur, tibia, spine, rib, or other bone marrow cavities. Bone marrow may be removed from a patient and separated by various separation and washing procedures. Known procedures for separating bone marrow cells include the steps of: a) centrifuging the bone marrow suspension into three fractions and collecting the intermediate fraction, i.e., the buffy coat, b) centrifuging the buffy coat fraction from step (a) again in a separation fluid, usually Ficoll (trademark of Pharmacia Fine Chemicals AB), and collecting the intermediate fraction containing the bone marrow cells, c) washing the fraction collected from step (b) for recovery of retransfusion-capable bone marrow cells.

[0112] If one wishes to use a population of cells enriched for T cells, such a population of cells may be obtained from a mixed population of cells by leukapheresis and mechanical apheresis using a continuous flow cell separator. For example, T cells may be separated from buffy coats by any known method, such as separation on a Ficoll-Hypaque™ gradient, separation on a Percoll gradient, or by elutriation.

[0113] Methods of viral vector-mediated gene transfer In certain embodiments, the transgene is incorporated into a viral particle to mediate gene transfer into cells. Usually, the virus is simply exposed to a suitable host cell under physiological conditions to allow viral uptake. (See U.S. Patent No. 9,089,520.) The method of the present invention can be advantageously used with various viral vectors, including lentiviral vectors, as discussed below.

[0114] 1. Adenovirus Adenoviruses are particularly suitable for use as gene transfer vectors thanks to their medium-sized DNA genome, ease of manipulation, high titer, wide target cell range, and high infectivity. The approximately 36 kb viral genome is surrounded by 100-200 base pair (bp) inverted terminal repeats (ITRs) that contain the cis-acting elements required for viral DNA replication and packaging. The early (E) and late (L) regions of the genome, which contain different transcription units, are divided by the onset of viral DNA replication.

[0115] The E1 region (E1A and E1B) codes for proteins involved in the transcriptional regulation of the viral genome and some cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff (Renan, MJ (1990) Radiother Oncol., 19, 197-218). The products of the late genes (L1, L2, L3, L4, and L5), including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 map units) is particularly efficient at the late stage of infection, and all mRNAs arising from this promoter have a 5' tripartite leader (TL) sequence, making them available for translation.

[0116] In order to optimize adenovirus for gene therapy, it is necessary to maximize the payload capacity so that it can include large segments of DNA. It is also highly desirable to reduce the toxicity and immune response associated with certain adenovirus products. The two goals are somewhat on the same level, in that the removal of adenovirus genes serves both purposes. By practicing the method of the present invention, it is possible to achieve both of these goals while retaining the ability to relatively easily engineer therapeutic constructs.

[0117] All cis elements required for viral DNA replication are localized in the inverted terminal repeats (ITRs) (100-200 bp) at either end of the linear viral genome, allowing large displacements of DNA. Plasmids containing ITRs can replicate in the presence of non-defective adenovirus (Hay, RT, et al., J Mol. Biol. 1984 Jun. 5; 175(4): 493-510). Thus, inclusion of these elements in adenoviral vectors may allow replication.

[0118] Furthermore, a packaging signal for viral encapsidation is localized between 194 and 385 bp (0.5-1.1 map units) at the left end of the viral genome (Hearing et al., J. (1987) Virol., 67, 2555-2558). This signal mimics a protein recognition site in bacteriophage lambda DNA, where a specific sequence close to the left end but outside the sticky end sequence mediates binding to proteins required for insertion of DNA into the head structure. It has been shown by Ad E1 replacement vectors that a 450 bp (0-1.25 map units) fragment at the left end of the viral genome can direct packaging in 293 cells (Levrero et al., Gene, 101:195-202, 1991).

[0119] Previously, it has been shown that certain regions of adenovirus genome can be integrated into the genome of mammalian cells, and the genes encoded thereby can be expressed.These cell lines can support the replication of adenovirus vectors that lack the adenovirus functions encoded by these cell lines.There have also been reports of the complementation of replication-deficient adenovirus vectors by "helping" vectors such as wild-type virus or conditionally defective mutants.

[0120] Replication-defective adenoviral vectors can be complemented in trans by helper viruses. This observation alone does not allow the isolation of replication-defective vectors, but the presence of helper viruses necessary to provide replication functions will contaminate any preparation. Therefore, an additional element was required that would add specificity to the replication and / or packaging of replication-defective vectors. This element is derived from the packaging function of adenovirus.

[0121] The adenovirus packaging signal has been shown to reside at the left end of the conventional adenovirus map (Tibbetts et.al. (1977) Cell, 12, 243-249). Later studies showed that mutants with deletions in the E1A (194-358 bp) region of the genome grew poorly even in cell lines that complemented early (E1A) functions (Hearing and Shenk, (1983) J. Mol. Biol. 167, 809-822). When compensatory adenovirus DNA (0-353 bp) was recombined to the right end of the mutant, the virus was packaged normally. Further mutational analysis identified a short, repeated, position-dependent element at the left end of the Ad5 genome. A single copy of the repeat was found to be sufficient for efficient packaging when present at either end of the genome, but not when moved toward the interior of the Ad5 DNA molecule (Hearing et al., J. (1987) Virol., 67, 2555-2558).

[0122] By using mutated versions of the packaging signal, it is possible to create helper viruses that are packaged with different efficiencies. The mutations are usually point mutations or deletions. When helper viruses with low efficiency packaging are grown in helper cells, the virus is packaged, albeit at a slower rate compared to the wild-type virus, thereby allowing the helper to grow. However, when these helper viruses are grown in cells with viruses containing wild-type packaging signals, the wild-type packaging signal is recognized preferentially over the mutated version. Given the limited amount of packaging factors, viruses containing wild-type signals are preferentially packaged when compared to the helper. If the preference is large enough, near-homogeneous stocks can be achieved.

[0123] To improve the tropism of ADV constructs for specific tissues or species, receptor-binding fiber sequences can often be substituted between adenovirus isolates. For example, the Coxsackie-adenovirus receptor (CAR) ligand found in adenovirus 5 can be substituted for the CD46-binding fiber sequence of adenovirus 35, resulting in a virus with greatly improved binding affinity for human hematopoietic cells. The resulting "pseudotyped" virus, Ad5f35, has become the basis of several clinically developed virus isolates. In addition, a variety of biochemical methods exist to modify the fiber to allow retargeting of the virus to target cells. Methods include the use of bifunctional antibodies (one end binds the CAR ligand and one end binds the target sequence) and metabolic biotinylation of the fiber to allow association with customized avidin-based chimeric ligands. Alternatively, ligands (e.g., anti-CD205 (e.g., PEG-containing) by heterobifunctional linkers may be attached to the adenovirus particle.

[0124] 2. Retroviruses Retroviruses are a group of single-stranded RNA viruses characterized by their ability to convert RNA to double-stranded DNA in infected cells by a process of reverse transcription (Coffin, (1990) In: Virology, ed., New York: Raven Press, pp. 1437-1500). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs the synthesis of viral proteins. The integration results in the retention of viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes (gag, pol, and env) that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream of the gag gene, called psi, functions as a signal for packaging the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also necessary for integration into the host cell genome (Coffin, 1990).

[0125] To construct retroviral vectors, a nucleic acid encoding a promoter is inserted into the viral genome in place of certain viral sequences to generate a virus that is replication-defective. To generate virions, a packaging cell line is constructed that contains the gag, pol, and env genes but does not contain the LTR and psi components (Mann et al., (1983) Cell, 33, 153-159). When a recombinant plasmid containing human cDNA is introduced into this cell line along with the retroviral LTR and psi sequences (e.g., by calcium phosphate precipitation), the psi sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles that are then secreted into the culture medium (Nicolas, JF, and Rubenstein, JLR, (1988) In: Vectors: a Survey of Molecular Cloning Vectors and Their Uses, Rodriquez and Denhardt, Eds.). Nicolas and Rubenstein; Temin et al., (1986) Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188; Mann et al., 1983). The medium containing the recombinant retrovirus is collected, concentrated if necessary, and used for gene transfer. Retroviral vectors can infect a wide variety of cell types. However, the integration and stable expression of many types of retroviruses requires the division of host cells (Paskind et al., (1975) Virology, 67, 242-248). An approach designed to allow specific targeting of retroviral vectors has recently been developed based on the chemical modification of retroviruses by chemical addition of galactose residues to the viral envelope. This modification may allow specific infection of cells such as hepatocytes via the asialoglycoprotein receptor, which may be desirable.

[0126] Another approach to targeting recombinant retroviruses was designed and used biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. Streptavidin was used to bind the antibodies via the biotin moiety (Roux et al., (1989) Proc. Nat'l Acad. Sci. USA, 86, 9079-9083). Antibodies against major histocompatibility complex class I and class II antigens were used to demonstrate infection of various human cells bearing these surface antigens with ecotropic viruses in vitro (Roux et al., 1989).

[0127] 3. Adeno-associated virus AAV utilizes a linear, single-stranded DNA of approximately 4700 base pairs. Inverted terminal repeats flank the genome. There are two genes within the genome that give rise to many different gene products. The first, the cap gene, produces three different virion proteins (VPs), called VP-1, VP-2, and VP-3. The second, the rep gene, encodes four nonstructural proteins (NS). One or more of these rep gene products are responsible for transactivation of AAV transcription.

[0128] The three promoters of AAV are designated by their location in map units within the genome. From left to right, these are p5, p19, and p40. Transcription gives rise to six transcripts, two initiated at each of the three promoters, and one of each pair is spliced. The splice sites, derived from map units 42-46, are the same in each transcript. The four nonstructural proteins are apparently derived from the longer transcript, and all three virion proteins arise from the smallest transcript.

[0129] AAV is not associated with human pathology. Interestingly, for efficient replication, AAV requires "help" functions from viruses such as herpes simplex virus I and II, cytomegalovirus, pseudorabies virus, and of course adenovirus. The best characterized helper is adenovirus, and many of the "early" functions of this virus have been shown to support AAV replication. Low levels of expression of the AAV rep protein are thought to suppress constitutive expression of AAV, and helper virus infection is thought to remove this block.

[0130] The terminal repeats of the AAV vector may be obtained by restriction endonuclease digestion of a plasmid such as p201 containing an AAV or modified AAV genome (Samulski et al., J. Virol., 61:3096-3101 (1987)), or by other methods including, but not limited to, chemical or enzymatic synthesis of the terminal repeats based on the published sequence of AAV. The terminal repeats can be determined, for example, by deletion analysis to determine the minimal sequence or part of the AAV ITRs required to enable function, i.e., stable site-specific integration. It is also possible to determine which small changes in sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable site-specific integration.

[0131] AAV-based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are being developed and tested at preclinical and clinical stages for a wide range of potential applications in gene therapy, both ex vivo and in vivo. (Carter and Flotte,(1995)Ann.NYAcad.Sci.,770;79-90;Chatteijee,et al.,(1995)Ann.NYAcad.Sci.,770,79-90;Ferrari et al.,(1996)J.Virol.,70,3227-3234;Fisher et al. al.,(1996)J.Virol.,70,520-532;Flotte et al.,Proc.Nat'l Acad.Sci.USA,90,10613-10617,(1993);Goodman et al.(1994),Blood,84,1492-1500;Kaplitt et al.,(1994)Nat'l Genet.,8,148-153;Kaplitt,MG,et al.,Ann Thorac Surg.1996 December;62(6):1669-76;Kessler et al.,(1996)Proc.Nat'l Acad.Sci.USA,93,14082-14087;Koeberl et al.,(1997)Proc.Nat'l Acad.Sci.USA,94,1426-1431;Mizukami et al. al., (1996) Virology, 217, 124-130).

[0132] Efficient AAV-mediated gene transfer and expression in the lung has led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1995; Flotte et al., Proc. Nat'l Acad. Sci. USA, 90, 10613-10617, (1993)). Similarly, the prospects for treating muscular dystrophy by AAV-mediated gene delivery of the dystrophin gene to skeletal muscle, treating Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, treating hemophilia B by factor IX gene delivery to the liver, and potentially treating myocardial infarction with the vascular endothelial growth factor gene to the heart appear promising, since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al., (1996) J. Virol., 70, 520-532; Flotte et al., 1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al., (1996) Brain Res., 713, 99-107; Ping et al., (1996) Microcirculation, 3, 225-228; Xiao et al., (1997) J. Virol ... al., (1996) J. Virol., 70, 8098-8108).

[0133] 4. Lentiviral Vectors In certain embodiments, CXCL13 and CD40L are transduced into CD4 T cells, T cell subsets and / or T cell precursors using lentivirus, gammaretrovirus, alpharetrovirus or adenovirus by electroporation or by transfection of nucleic acid, protein, site-specific nuclease, self-replicating RNA virus or integration-defective lentiviral vector (see U.S. Pat. No. 10,131,876 for such vectors).

[0134] In certain embodiments, recombinant modification of CD4 T cells, T cell subsets and / or T cell progenitors may be performed by transduction, transfection, or electroporation.

[0135] Preferably, transduction is carried out using lentiviruses, gamma, alpha retroviruses or adenoviruses, or by electroporation or transfection with nucleic acids (DNA, mRNA, miRNA, antagonists, ODN), proteins, site-specific nucleases (zinc finger nucleases, TALEN, CRISP / R), self-replicating RNA viruses (e.g., equine encephalopathy virus) or integration-deficient lentiviral vectors.

[0136] More preferentially, recombinant modification of CD4 T cells, T cell subsets and / or T cell precursors may be performed by transducing such cells with lentiviral vectors (see Cockrell Adam S et al., "Gene delivery by lentivirus vectors", Molecular Biotechnology, vol. 36, No. 3, July 2007).

[0137] Lentiviral vectors pseudotyped with VSVG allow efficient transduction under automated manufacturing methods. However, the method of the present invention is fully suitable for use with any type of lentiviral vector (e.g., measles virus (ML-LV), gibbon ape leukemia virus (GALV), feline endogenous retrovirus (RD114), baboon endogenous retrovirus (BaEV) derived pseudotype envelope). Other viral vectors such as gamma or alpha retroviral vectors may also be used. Transduction enhancer reagents may be added as necessary using the automated manufacturing described in the present invention.

[0138] 5. Other viral vectors Other viral vectors may be used as expression constructs in the methods and compositions of the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, (1988) In: Vectors: A survey of molecular cloning vectors and their uses, pp. 467-492; Baichwal and Sugden, (1986) In, Gene Transfer, pp. 117-148; Coupar et al., Gene, 68:1-10, 1988), canarypox virus, and herpes virus have been employed. These viruses offer several features that make them useful for gene transfer into a variety of mammalian cells.

[0139] Once the construct is delivered to the cell, the nucleic acid encoding the transgene is positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the transgene is stably integrated into the genome of the cell. This integration is in the cognate location and orientation via homologous recombination (gene replacement) or in a random, non-specific location (gene augmentation). In yet another embodiment, the nucleic acid is stably maintained in the cell as a separate episomal segment of DNA. Such a nucleic acid segment or "episome" encodes sufficient sequences to allow maintenance and replication independent of or in synchrony with the host cell cycle. How the expression construct is delivered to the cell and where the nucleic acid remains in the cell depends on the type of expression construct used.

[0140] These methods of introducing heterologous nucleic acid molecules into cells are non-limiting and any method may be used to heterologously express CD40L, CXCL13, and / or target antigens (which may be HIV proteins as provided herein).

[0141] Methods for Treating Disease The methods of the invention also encompass methods of treatment or prevention of diseases in which administration of cells, for example by injection, may be beneficial. In some embodiments, the disease is a viral infection. In some embodiments, the infection is an HIV infection. In some embodiments, the method comprises administering a composition or cells as provided herein to a subject with a viral infection. In some embodiments, the method increases an immune response, such as a humoral immune response and / or a cellular immune response. In some embodiments, the immune response is against HIV infection.

[0142] In some embodiments, a method of treating HIV in a subject is provided. In some embodiments, the method includes administering a composition to the subject, comprising administering an effective amount of any of the cells provided herein. In some embodiments, the composition may be referred to as a pharmaceutical composition. As provided herein, the composition may be administered by any suitable route. In some embodiments, the composition is administered intravenously or by infusion. In some embodiments, the cells are allogeneic or not HLA-matched to the subject. In some embodiments, the cells are autologous to the subject. In some embodiments, the dosage of cells in the composition is about 1×10 6 ~Approx. 5×10 6 It is.

[0143] In some embodiments, a method of increasing an immune response in a subject in need thereof is provided. In some embodiments, the method comprises administering an effective amount of any of the cells provided herein. In some embodiments, the increased immune response is against a target antigen. In some embodiments, the increased immune response is a humoral immune response and / or a cellular immune response. In some embodiments, the increased immune response is an increase in NK cells. In some embodiments, the increased immune response is an increase in NKT cells. In some embodiments, the increased immune response is an increase in activated NK cells. In some embodiments, the increased immune response is an increase in activated B cells. In some embodiments, the increased immune response is an increase in activated CD8 T cells. In some embodiments, the increased immune response is an increase in activated T cells as measured by the percentage of CD3+ and CD38+ cells. In some embodiments, the increased immune response is an increase in activated T cells as measured by the percentage of CD3+ and CD25+ cells. In some embodiments, the target antigen is an HIV protein. In some embodiments, the HIV protein is one or more of HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof. In some embodiments, the target antigen is expressed as the entire HIV genome, such as from a heterologous nucleic acid molecule. In some embodiments, the HIV genomic nucleic acid comprises a mutation in the retroviral reverse transcriptase, and further, the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genome construct. In some embodiments, the HIV genome does not generate replication-competent HIV viral particles. In some embodiments, the HIV genome does not produce HIV viral particles capable of infecting T cells. In some embodiments, the cells are allogeneic to the subject. In some embodiments, the cells are not HLA-matched to the patient.In some embodiments, the dosage of cells is about 1 x 10 6 ~Approx. 1×10 6 In some embodiments, the immune response is against a viral infection, and the viral infection may be a human immunodeficiency virus (HIV) infection.

[0144] In some embodiments, the method comprises administering the composition two or more times.In some embodiments, the composition is administered as a maintenance therapy once every week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every year.The composition may be administered, for example, as long as the subject shows improvement, reduction, or undetectable viral titer, or stable / non-progressive disease being treated.

[0145] For example, cells, such as recombinant allogeneic or autologous CD4+ HIV vaccine cells, or compositions comprising such cells may be used in cell therapy. The cells may be donor-derived or patient-derived. The cells may be used, for example, in regeneration, to replace the function of diseased cells. The cells may also be modified to express heterologous genes, so that biological factors can be delivered to specific microenvironments, such as diseased bone marrow or metastatic deposits. Mesenchymal stromal cells have also been used, for example, to provide immunosuppressive activity and may be used in the treatment of graft-versus-host disease and autoimmune disorders.

[0146] In other embodiments, recombinant allogeneic or autologous CD4+ HIV vaccine cells or compositions comprising such cells are used to treat a variety of diseases and conditions.

[0147] The term "unit dosage form", when referring to an inoculum, refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined amount of a pharmaceutical composition calculated to produce a desired immunogenic effect in association with the required diluent. The specifications for the unit dose of an inoculum are dictated by and dependent upon the unique characteristics of the pharmaceutical composition and the particular immunogenic effect to be achieved.

[0148] An effective amount of a pharmaceutical composition comprising recombinant allogeneic or autologous CD4+ HIV vaccine cells or a composition comprising such cells is an amount such that greater than 60%, 70%, 80%, 85%, 90%, 95%, or 97% of HIV infected cells are killed. This term is also synonymous with "sufficient amount."

[0149] The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. Effective amounts of the particular compositions presented herein may be empirically determined without necessitating undue experimentation.

[0150] The terms "contacted" and "exposed", when applied to a cell, tissue or organism, are used herein to describe the process by which a pharmaceutical composition and / or another agent, e.g., a chemotherapeutic or radiotherapeutic agent, is delivered to or directly juxtaposed with a target cell, tissue or organism. To achieve cell killing or stasis, the pharmaceutical composition and / or additional agent(s) are delivered to one or more cells in a combined amount effective to kill the cell(s) or prevent them from dividing. Administration of the pharmaceutical composition may precede, be simultaneous with, and / or follow the other agent(s) by an interval ranging from minutes to weeks. In embodiments in which the pharmaceutical composition and the other agent(s) are applied separately to a cell, tissue or organism, it is generally ensured that no significant period of time expires between the time of each delivery, so that the pharmaceutical composition and agent(s) may still exert an advantageously combined effect on the cell, tissue, or organism. For example, in such cases, it is contemplated that a cell, tissue, or organism may be contacted with the pharmaceutical composition in two, three, four, or more ways substantially simultaneously (i.e., within about one minute). In other embodiments, the one or more agents may be administered substantially simultaneously, from about one minute, from about 24 hours, from about seven days, from about one to about eight weeks or more, and any range derivable therein, prior to and / or following administration of the expression vector. Additionally, various combination regimens of the pharmaceutical compositions and one or more agents presented herein may be used.

[0151] Formulation and route of administration to patients Where clinical applications are contemplated, it is necessary to prepare the pharmaceutical compositions (expression constructs, expression vectors, fusion proteins, transfected or transduced cells) in a form appropriate for the intended application. Generally, this involves preparing compositions that are essentially free of pyrogens and other impurities that could be harmful to humans or animals.

[0152] Recombinant allogeneic or autologous CD4+ HIV vaccine cells or compositions comprising such cells may be delivered in doses of, for example, about 1 to 5 million cells per dose. For example, vials or other containers containing the recombinant cells may be provided in a volume per vial of about 0.25 ml to about 10 ml, e.g., about 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ml, e.g., about 2 ml.

[0153] When recombinant cells are introduced into a patient, it may generally be desirable to use appropriate salts and buffers. The phrase "pharmacologically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to animals or humans. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known. Any conventional media or agent is contemplated for use in the therapeutic compositions unless incompatible with the vector or cells. Supplementary active ingredients may also be incorporated into the compositions.

[0154] Once formulated, the solution is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent may first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, a sterile aqueous medium may be used. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations will meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0155] Moreover, in certain patients, this treatment is repeated periodically, hopefully to boost the immune system's response to the remaining virus / virions. Such periodic treatment may vary from once every week as a maintenance therapy, to once every two weeks, once every three weeks, once a month, up to once every two months, up to once every three months, up to once every four months, up to once every five months, up to once every six months, or once every seven months, or once every eight months, or once every nine months, or once every ten months, or once every eleven months, or once every year, as long as necessary to achieve stable or undetectable disease in the patient.

[0156] In some embodiments, provided herein is an isolated cell transfected or transduced with a nucleic acid comprising a nucleotide sequence encoding CD40L and CXCL13.

[0157] In some embodiments, CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 and CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1.

[0158] In some embodiments, the cell is a T cell.

[0159] In some embodiments, the cells are transduced or transfected with a second and / or a third nucleic acid encoding a heterologous protein.

[0160] In some embodiments, the second nucleic acid comprises a human immunodeficiency virus (HIV) genome, wherein the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further wherein the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0161] In some embodiments, provided herein are CD4+ cells comprising one or more heterologous nucleic acid molecules encoding CD40L and CXCL13.

[0162] In some embodiments, CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 and CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1.

[0163] In some embodiments, provided herein are CD4+ cells comprising a heterologous CD40L protein and a heterologous CXCL13 protein.

[0164] In some embodiments, the CD4+ cells further comprise a heterologous nucleic acid molecule comprising a human immunodeficiency virus (HIV) genome, wherein the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further, wherein the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0165] In some embodiments, provided herein is a method of treating HIV in a subject, comprising administering a composition to the subject, comprising administering an effective amount of any of the cells described herein above.

[0166] In some embodiments, provided herein is a method for increasing an immune response in a subject in need thereof, comprising administering an effective amount of any of the cells described herein above.

[0167] In some embodiments, the cells are allogeneic to the subject.

[0168] In some embodiments, the cells are not HLA-matched to the patient.

[0169] In some embodiments, the dose of cells is about 1-5 x 10 6 The range is.

[0170] In some embodiments, the viral infection is caused by human immunodeficiency virus (HIV).

[0171] In some embodiments, graft-versus-host disease (GVHD) is reduced or eliminated while graft-versus-virus (GVV) is increased in a subject.

[0172] In some embodiments, the treatment or augmentation of the immune response is repeated periodically as a maintenance treatment over a time frame of once every week, up to once every 2 weeks, up to once every 3 weeks, up to once every month, up to once every 2 months, up to once every 3 months, up to once every 4 months, up to once every 5 months, up to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once every year, as long as the subject shows improvement, reduced or undetectable viral titers, or shows stable / non-progressive disease.

[0173] In some embodiments, cellular and humoral immunity is induced in a subject.

[0174] In some embodiments, the embodiments provided herein also include, but are not limited to, the following:

[0175] 1. A cell comprising a heterologous nucleic acid molecule comprising a nucleotide sequence encoding CD40L and CXCL13.

[0176] 2. The cell of embodiment 1, wherein said CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and said CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.

[0177] 3. The cell of embodiment 1, which heterologously expresses CD40L and CXCL13.

[0178] 4. The cell of embodiment 1, which is an isolated cell.

[0179] 5. The cell of embodiment 1, which is a T cell, such as a CD4+ T cell.

[0180] 6. The cell of embodiment 1, which is transduced or transfected with a second and / or third nucleic acid encoding a heterologous protein or a target antigen.

[0181] 7. The cell of embodiment 6, wherein the second nucleic acid comprises a human immunodeficiency virus (HIV) genome, the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further, the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0182] 8. The cell described in embodiment 6, wherein the target antigen is an HIV protein.

[0183] 9. The cell of embodiment 8, wherein the HIV protein is HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof.

[0184] 10. A cell according to any one of embodiments 1 to 9, which is a CD4+ T cell.

[0185] 11. A CD4+ T cell comprising one or more heterologous nucleic acid molecules encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3 and / or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4.

[0186] 12. The CD4+ T cell of embodiment 11, which expresses CD40L.

[0187] 13. A CD4+ T cell according to embodiments 11 and 12, which expresses CXCL13.

[0188] 14. The CD4+ T cell of embodiment 11, which expresses CD40L and CXCL13.

[0189] 15. The CD4+ T cell of embodiment 11, which is an isolated CD4+ T cell.

[0190] 16. A CD4+ T cell comprising a heterologous CD40L protein and a heterologous CXCL13 protein.

[0191] 17. The CD4+ T cell of embodiment 16, further comprising a heterologous nucleic acid molecule comprising a human immunodeficiency virus (HIV) genome, wherein said HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further wherein said HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

[0192] 18. The CD4+ T cell of embodiment 16, further comprising a heterologous nucleic acid molecule encoding a target antigen.

[0193] 19. The CD4+ T cell of embodiment 18, wherein the target antigen is an HIV protein.

[0194] 20. The CD4+ T cell of embodiment 19, wherein the HIV protein is HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof.

[0195] 21. A method of treating HIV in a subject, comprising administering to the subject a composition comprising administering an effective amount of any of the cells of any one of embodiments 1-20.

[0196] 22. The method of embodiment 22, wherein the composition is a pharmaceutical composition.

[0197] 23. The method of embodiment 21, wherein the composition is administered intravenously or by infusion.

[0198] 24. The method of any one of embodiments 21-23, wherein the cells are allogeneic to the subject or are HLA-mismatched to the subject.

[0199] 25. The method of any one of embodiments 21-23, wherein the cells are autologous to the subject.

[0200] 26. The method according to any one of embodiments 21 to 25, wherein the dosage of cells in the composition is about 1 × 10 6 ~Approx. 5×10 6 The method according to claim 1,

[0201] 27. A method for increasing an immune response in a subject in need thereof, comprising administering an effective amount of any of the cells according to any one of embodiments 1-20.

[0202] 28. The method of embodiment 27, wherein the increased immune response is against a target antigen.

[0203] 29. The method of embodiment 28, wherein the target antigen is an HIV protein.

[0204] 30. The method of embodiment 29, wherein the HIV protein is HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof.

[0205] 31. The method of any of embodiments 27-29, wherein the cells are allogeneic to the subject.

[0206] 32. The method of any of embodiments 27-29, wherein the cells are not HLA-matched to the patient.

[0207] 33. The dose of the cells is about 1 to 5 × 10 6 The method according to any one of embodiments 27 to 32, wherein the range is

[0208] 34. The method according to any one of embodiments 27 to 33, wherein the immune response is against a viral infection, and the viral infection may be a human immunodeficiency virus (HIV) infection.

[0209] 35. The method according to any of embodiments 21-34, wherein the treatment or augmentation of immune response is repeated periodically as a maintenance treatment in the following time frames: once every week, up to once every 2 weeks, up to once every 3 weeks, up to once every month, up to once every 2 months, up to once every 3 months, up to once every 4 months, up to once every 5 months, up to once every 6 months, or once every 7 months, or once every 8 months, or once every 9 months, or once every 10 months, or once every 11 months, or once every year, as long as the subject shows improvement, reduction or undetectable viral titers, or shows stable / non-progressive disease.

[0210] 36. The method according to any of embodiments 21 to 35, wherein cellular and humoral immunity are induced in the subject.

[0211] The following examples are illustrative, but not limiting, of the compounds and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments. EXAMPLES

[0212] Example 1

[0213] Constructing CD4+ cells transduced with CD40L and CXCL13 and further loading them with the HIV genome

[0214] Step 1: CD4+ cells (or other T cells) are transduced with lentivirus / adenovirus that overexpresses CD40L and CXCL13 (B cell attractant molecules) to produce recombinant allogeneic CD4+ T cells expressing CD40L and CXCL13. These recombinant allogeneic CD4+ cells function within the host to attract B cells to the area in front of the CD4 cells.

[0215] Step 2: Plasmid transfection and / or transposon delivery of the HIV genome into CD4+CD40L+CXCL13+ cells. CD4+CD40L+CXCL13+ cells are loaded with non-functional HIV (replication-incompetent or live attenuated genome). In a preferred embodiment, a complete HIV genome is utilized in which the reverse transcriptase (RT) contains at least one mutation (or deletion) rendering it non-functional, and in which there is a mutation (or complete deletion) in the packaging signal (creating a replication-incompetent HIV genome, but otherwise complete genome).

[0216] To generate replication-incompetent HIV genomic constructs, the following rationales and options are utilized (RT mutations and packaging signal mutations): RT disables infection Packaging signal mutation (CD4 will produce virions even in the absence of packaging signal mutation). Recombinant CD4 cells bud empty virions - the envelope glycoprotein is the part of the genome that induces neutralizing antibodies (generates a humoral response). In certain embodiments, the construct may further comprise nucleic acids encoding different strains of different envelope proteins. In certain embodiments, the construct may carry multiple variable envelope regions, creating multiple glycoprotein (gp) structures (creating glycoprotein diversity). Therefore, CD4+CD40L+CXCL13+ cells are loaded with: Generation of CD4+ cells expressing HIV plasmids, RT mutated, with no or disabled packaging signal, multiple envelope proteins, CD40L+CXCL13+, and expressing HIV envelope and glycoproteins to generate a humoral immune response by the patient. - Generate allogeneic CD4 cells as an HIV vaccine vehicle.

[0217] Example 2

[0218] Evaluation of ENOB HV-11 and ENOB HV-12 in macaques as potential HIV preventive and therapeutic vaccines

[0219] As proof of concept, both human and non-human primate (NHP) sequences of two expression cassettes (CD40L & CXCL13) are tested using lentiviral vectors (LV) to investigate the biological activity of ENOB HV-11 preventive HIV vaccine and ENOB HV-12 therapeutic HIV vaccine in non-human primates (macaques) and provide proof of concept data.

[0220] Allogeneic cells are potent stimuli of immune responses. Allogeneic T cells expressing HIV antigens genetically engineered to express high levels of the B cell promoters CD40L and CXCL13 are expected to induce strong cellular and humoral responses effective as protective or therapeutic vaccines. Such recombinant allogeneic cells should be rapidly killed by the host's immune system and not induce graft-versus-host disease if provided in sufficiently low numbers.

[0221] In the examples described herein as proof of concept (POC), non-human primates are transfected with a plasmid containing a non-replicating attenuated SHIV, and millions (e.g., 1-5×10 6 ) allogeneic T cells are injected subcutaneously in succession. The simian / human immunodeficiency viruses (SHIV) are a series of laboratory-created chimeras whose genetic material is a combination of simian immunodeficiency virus (SIV) and HIV genes. The virus can infect almost any species of non-human primate that can be infected with SIV.

[0222] Neutralizing antibody titers are measured. Once a level of protection is achieved, animals are challenged mucosally with SHIV. If animals are not protected from infection, they are challenged intravenously. Infected macaques, including a control cohort, receive therapeutic vaccination with T cells engineered with the same cassette (i.e., human vs. macaque) infected with a non-replicating attenuated SHIV. Vector constructs for HV-11 (to transduce human CXCL13 and human CD40L) are shown in FIG. 1A. Vector constructs for HV-12 (to transduce macaque CXCL13 and macaque CD40L) are shown in FIG. 1B.

[0223] Dosing and schedule details of study design (see Figures 2-4): 9 macaques; n=3 per group; Potentially a two-phase study spanning ENOB HV-11 and ENOB HV-12: ⇒ 3 animals received fully MLA / HLA-mismatched (CD4+) T cells transfected with a plasmid containing an attenuated, replication-incompetent SHIV and transduced with a human vector (cohort A) (Fig. 1A). ⇒ 3 animals received fully mismatched CD4 + T cells transfected with a plasmid containing an attenuated, replication-incompetent SHIV and transduced with a macaque vector (cohort B) (Fig. 1B). ⇒3 animals will be in the control group (not receiving the product) (Cohort C).

[0224] ENOB HV-11 (Figure 3) Injection / Administration / Evaluation Schedule (Cohorts A and B) 5 million cells subcutaneously per week for 4 weeks; if there is a reaction after the first injection, reduce the dose to 2 million cells per week. After the fourth injection, neutralizing antibody titers will be measured 7 days after the fourth injection. If the desired titer is not achieved, administer a second cycle of 4 sc injections of 2 million cells every 10 days, then, if necessary, administer a third cycle of 4 sc injections of 2 million cells every 15 days. Once the desired titer is achieved, mucosal challenge with SHIV virus is performed. If mucosal challenge does not result in infection, intravenous challenge is performed. · There will be weekly monitoring, antibody titers and safety labs for each study subject.

[0225] ENOB HV-12 (Figure 4) Infected macaques in cohorts A, B, and C receive ART for 3 months until they achieve viral suppression (<50 copies / ml). After achieving suppression, they receive therapeutic vaccination with fully MLA-mismatched CD4+ T cells pulsed with a replication-incompetent attenuated SHIV and matching vector (i.e., cohort A receives human vector and cohort B receives macaque vector) as shown below and in Figures 2-4. An option is to utilize the same dosing regimen for HV-12 as used for HV-11.

[0226] For cohorts A and B, the T cells will be from a different, mismatched donor than the prophylactic vaccine. Injection / administration / evaluation schedule (Cohorts A, B and C) 5 million cells per week subcutaneously for 4 weeks, if there is a response after the first injection, reduce the dose to 2 million cells per week. An option is to start dosing at the peak of viremia (e.g. peak viremia in n-3 control animals, induced but without protective vaccine). Measure plasma viremia 7 days after the fourth injection. If not less than 1 copy per ml, begin subcutaneous injections of 2 million cells every 4 weeks. After the 4th injection (8th injection in total), measure plasma viremia. If not less than copies per ml, cycle 2 million cells per week for 4 weeks. Weekly monitoring for 6 months Plasma viremia, lymphocyte subsets and safety labs Seven days after the last subcutaneous injection of each cycle Plasma viremia, lymphocyte subsets, safety labs, GALT biopsy

[0227] Follow-up period: One year after the last injection of either HV-11 or HV-12, macaques are sacrificed for systemic evaluation of the presence of SHIV and toxicity, such as lymphoma.

[0228] Toxicity monitoring during proof-of-concept (POC) studies o Toxicity due to the ROA used. o Persistence of injected product and associated expression. o Biodistribution of the injected product. o Immune response to the injected product (humoral and cellular) 〇Tumorogenicity. 〇 Macroscopic observation of weight and behavior, etc. o Microscopic histopathology. 〇Other

[0229] Example 3: T cells transduced with CD40L and CXCL13 enhance cytotoxicity and increase immune cell activation. Vaccination in combination with engineered allogeneic effector cells expressing the target antigens CD40L and CXCL13 enhances cytotoxic activity against the antigen. An in vitro model was developed to mimic in vivo cytotoxicity. A Jurkat-GFP expressing line was created to serve as a cytotoxic target to quantitatively measure the specific killing activity of the effectors. Normal donor PBMCs were then obtained and vaccinated with the three sets of cells to generate a specific immune response against Jurkat cells. Briefly, PBMCs are "vaccinated" with the recombinant. (a) PBMCs were vaccinated with untransduced Jurkat cells ("UTD Jurkat"), (b) PBMCs were vaccinated with GFP-transduced Jurkat cells (serving as a non-specific vector-transduced control), and (c) PBMCs were vaccinated with Jurkat cells transduced with CD40L and CXCL13 (HV11). "Vaccination" in this example refers to mixing PBMCs with Jurkat cells as described above. This is similar to injecting recombinant CD4 T cells with CD40L and CXCL13 into patients with or at risk for HIV, where GFP is replaced by HIV proteins that serve as training antigens for PBMCs. Nine days after PBMC vaccination, which allows for the expansion of Jurkat-specific T cells, Jurkat-GFP expressing target cells were co-cultured with "vaccinated" PBMC cells (effector cells) for 18 hours to assay specific cytolytic activity. Cytolytic function was measured by flow cytometry (FACS) analysis to confirm the change in GFP-positive cells after co-culture. The data show that GFP expressing cells were killed more effectively by PBMCs vaccinated with T cells expressing GFP and CD40L and CXCL13 compared to both non-transduced Jurkat and GFP only transduced Jurkat T cells. The data are shown in Figure 5. The data indicate enhanced cytolytic function and were found to be dose-dependent.Thus, PBMCs vaccinated with increasing amounts of CD40L and CXCL13 cells increased the cytotoxicity of PBMCs. The effects on various types of immune cells were also measured. As shown in Figure 6, PBMCs vaccinated with T cells heterologously expressing CD40L and CXCL13 showed significant and substantial increases in NK cells (Figure 6), NKT cells (Figure 7), NK cell activation (Figure 8), NK cell humoral activation (Figure 9), B cell activation (Figure 10), T cell activation measured by percentage of CD3+ and CD38+ cells (Figure 11), T cell activation measured by percentage of CD3+ and CD25+ cells (Figure 12), and CD8 T cell activation (Figure 13). Cells and activation were measured by flow cytometry using the surface markers indicated in the figures. Thus, these embodiments and data demonstrate the surprising and unexpected consequences of the ability to generate T cells capable of enhanced cytotoxicity against a particular antigen by expressing the antigen along with CD40L and CXCL13, or active fragments thereof, in T cells.

[0230] Example 4: Standard Method Standard methods of molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also found in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY (describing cloning in bacterial cells (Vol. 1)), (describing cloning in mammalian cells and yeast (Vol. 2)), (describing complex glycosidases and protein expression (Vol. 3)), and (describing bioinformatics (Vol. 4)).

[0231] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins have been described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0232] All references described herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent was specifically and individually indicated to be incorporated by reference. This incorporation by reference statement is intended by the applicant to relate to each and every individual publication, database entry (Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identified in accordance with U.S.C. § 1.57(b)(1), even if such citation is not immediately adjacent to the specific incorporation by reference statement. The inclusion of a specific incorporation by reference statement, if any, in this specification in no way weakens this general incorporation by reference statement. The citation of a reference herein is not intended as an admission that the reference is relevant prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0233] The embodiments of the present invention are not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the embodiments shown herein and the appended claims.

Claims

1. 1. A CD4+ T cell comprising a heterologous CD40L protein and a heterologous CXCL13 protein, the CD4+ T cell further comprising at least one nucleic acid encoding at least one Human Immunodeficiency Virus (HIV) protein.

2. 2. The CD4+ T cell of claim 1, further comprising a heterologous nucleic acid molecule comprising a human immunodeficiency virus (HIV) nucleic acid, wherein the HIV genomic nucleic acid comprises a mutation in a retroviral reverse transcriptase, and further wherein the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

3. 2. The CD4+ T cell of claim 1, wherein the HIV protein is one or more of HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof.

4. 10. A pharmaceutical composition for use in a method of treating HIV in a subject, the pharmaceutical composition comprising an effective amount of the CD4+ T cells of any one of claims 1 to 3, the CD4+ T cells comprising a heterologous nucleic acid molecule comprising a nucleic acid sequence encoding CD40L and CXCL13, and the CD4+ T cells further comprising a second and / or a third nucleic acid encoding at least one Human Immunodeficiency Virus (HIV) protein.

5. The pharmaceutical composition described in claim 4, wherein the CD40L comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and the CXCL13 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

3.

6. The pharmaceutical composition of claim 4, wherein the cells heterologously express CD40L and CXCL13.

7. The pharmaceutical composition described in claim 4, wherein the cell is an isolated cell.

8. The pharmaceutical composition of claim 4, wherein the second nucleic acid comprises HIV genomic nucleic acid, the HIV genomic nucleic acid comprising a mutation in a retroviral reverse transcriptase, and further, the HIV genomic nucleic acid does not encode a retroviral packaging signal, creating a disabled HIV genomic construct.

9. The pharmaceutical composition of claim 4, wherein the HIV protein is one or more of HIV Tat (full length or isoforms of 72 and 101 amino acids in length), Rev, Pol, GP120, GP160, GP41, env, Gag, Gag-Pol, Nef, Vpr, Vpu, or Vif, or any combination thereof.

10. The pharmaceutical composition of claim 4, wherein at least one HIV protein comprises an attenuated, replication-deficient HIV.

11. The pharmaceutical composition of claim 4 , wherein the composition is administered intravenously or by infusion.

12. The pharmaceutical composition of claim 4, wherein the cells are allogeneic to the subject or are HLA-mismatched to the subject.

13. The pharmaceutical composition of claim 4 , wherein the cells are autologous to the subject.

14. The dosage of cells in the composition is about 1×10 6 ~Approx. 5×10 6 The pharmaceutical composition according to any one of claims 4 to 13,

Images