CN114025792A

CN114025792A - Vaccines against pathogenic immune-activating cells during infection

Info

Publication number: CN114025792A
Application number: CN202080037286.4A
Authority: CN
Inventors: D·扎古里
Original assignee: 21C Bio SAS
Current assignee: 21C Bio SAS
Priority date: 2019-03-21
Filing date: 2020-03-20
Publication date: 2022-02-08
Also published as: AU2020243095A1; EP3941517A1; JP2022526628A; WO2020188111A1; CA3134209A1

Abstract

In the present invention, Applicants provide a novel method of preventing or treating infectious diseases in a subject in need thereof. In particular, the method comprises administering a combination, pharmaceutical combination, medicament or kit comprising a first portion of a CD8 vaccine comprising a CD8 vaccine specific for at least one infectious disease associated antigen, optionally comprising an interferon alpha blocker A second part, and a third part comprising Type III interferon and/or an agent that stimulates the production of Type III interferon.

Description

Vaccine against pathogenic immune-activated cells during infection

Technical Field

In the present invention, applicants provide a novel method of preventing or treating an infectious disease in a subject in need thereof. In particular, the methods comprise administering a combination, pharmaceutical combination, medicament, or kit (kit-of-parts) comprising a first portion comprising a CD8 vaccine specific for at least one infectious disease associated antigen, optionally a second portion comprising an interferon alpha blocker, and a third portion comprising a type III interferon and/or an agent that stimulates production of type III interferon.

Background

Shortly after the initial discovery that Human Immunodeficiency Virus (HIV) is responsible for Acquired Immune Deficiency Syndrome (AIDS), it has been determined that very few HIV-infected patients remain free of AIDS for decades. These so-called HIV Elite Controllers (EC) generally have a relatively high CD4⁺T cell count and ability to maintain clinically undetectable plasma HIV-1RNA levels (HIV RNA) over extended periods of time in the absence of any antiretroviral therapy (ART)<50 copies/mL). In recent years, extensive research has been conducted to define the mechanisms by which these rare individuals control HIVAnd (5) preparing.

Interestingly, the EC status indicates a low activation profile of T cells and a unique MHC-1b/E restricted CD8⁺The presence of T cell population can inhibit pathogenic HIV antigen presentation CD4⁺Early activation of T cells (Lu et al (2016) front. Immunol.7: 134). Furthermore, recent advances in the field of SIV vaccinology have also emphasized the MHC-1b/E restricted CD8⁺Role of T Cell responses in controlling SIV infection in rhesus monkeys (rhesus macaque) (Hansen et al (2013) Science 24; 340(6135): 1237874; Hansen et al (2016) Science; 351(6274), 714-20; Lu et al (2012) Cell Rep.2(6), 1736-46; Andrieu et al (2014) Front immunol.5: 297). These observations suggest alternative strategies for the development of HIV vaccines.

In fact, due to CD4⁺The activation state of T cells is also a prerequisite for proliferative HIV infection in vivo, and is therefore dormant (quiescent) CD4⁺Replication in T cells is essentially non-productive and is often inefficient, presumably by interfering with CD4⁺It is possible that T cells activate to inhibit viral replication. Thus, several groups have attempted to use induced MHC-1b/E restricted CD8⁺Vaccine for cells to inhibit virus-specific CD4⁺T cell activation.

For example, Andrieu et al have developed a method capable of inducing MHC-1b/E restricted CD8 in cynomolgus monkeys⁺T cell vaccine. This vaccine consists of inactivated Simian Immunodeficiency Virus (SIV) particles with a tolerogenic adjuvant, such as Lactobacillus plantarum. Although this vaccine strategy was effective in immunizing and inducing the inhibitory MHC-1b/E restricted CD8 in macaca mulatta⁺T cells, but indian-derived macaques immunized with the same adjuvanted vaccine were not protected.

Hansen et al, modified Cytomegalovirus (CMV) vector determinants controlling unconventional T cell priming (priming), demonstrated unique modulation of CD8⁺It is possible that T cell responses could maximize prophylactic or therapeutic protection. In particular, it was found that the use of such SIV protein-expressing rhesus cytomegalovirus vectors in Rhesus Monkeys (RM) inducesAiming at the aseptic protection of SIV after the toxic attack. However, this protection was only effective in 50% of vaccinated RMs.

These results have extended the current paradigm, on a global scale, from one focused on prophylactic HIV vaccines to one in which immunotherapy of HIV/AIDS can become an important part of combating this epidemic. Thus, there remains a need for an effective therapy to treat individuals co-existing with HIV-1, in addition to prophylactic vaccines.

Based on the discovery of novel biological properties of type III interferons (IFN-III), the present inventors propose to improve existing vaccines for the prevention or treatment of HIV and other infectious diseases (i.e., induction of inhibitory MHC-1b/E restricted CD8⁺T cell population).

In particular, applicants have demonstrated that type III interferons can greatly enhance existing CD 8-inhibitory vaccines. Indeed, in contrast to type I interferons, type III interferons appear to have a more specific role in innate antiviral defense and do not inhibit the initiation of adaptive immune responses following HIV infection. In particular, IFN-III is capable of preventing or limiting viral replication without inhibiting CD4 responsive to HIV infection⁺Proliferation of T cells.

Applicants further demonstrated that blockers of interferon alpha (IFN- α) as a supplement to CD 8-suppressive vaccines can potentiate type III interferons and thus greatly improve vaccine efficacy. In fact, IFN- α is an aberrant type I interferon cytokine that is capable of preventing or limiting viral replication, while promoting unwanted chronic immune activation. Since chronic immune activation is essential for HIV replication, IFN- α may play a detrimental role during HIV infection. Furthermore, as demonstrated by the inventors, IFN- α has the effect of inhibiting CD4⁺Antiproliferative activity of T cell proliferation and thus immune decline, viral replication and AIDS in infected patients. Thus, by blocking IFN- α, applicants aimed to enhance type III interferon activity, thereby promoting lower chronic immune activation and CD4⁺Better response of T cells.

Thus, in the present invention, applicants provide a novel method of preventing or treating an infectious disease in a subject in need thereof, comprising administering to the subject:

1) a CD8 vaccine specific for at least one infectious disease associated antigen,

2) optionally, an interferon alpha blocker, and

3) type III interferon and/or agents that stimulate the production of type III interferon.

Disclosure of Invention

The present invention relates to a combination comprising:

2) optionally, an interferon- α blocker, and

In one embodiment, the interferon- α blocker is selected from the group consisting of: an agent that neutralizes circulating interferon-alpha, an agent that blocks interferon-alpha signaling, an agent that depletes cells that produce IFN-alpha, and/or an agent that blocks IFN-alpha production, wherein the agent that neutralizes circulating interferon-alpha is selected from the group comprising: an active anti-IFN- α vaccine comprising an anti-IFN- α antibody or a passive anti-IFN- α vaccine comprising an anti-IFN- α antibody or an anti-IFN- α hyperimmune serum (anti-IFN- α hyper-immune serum), wherein the blocking agent of interferon- α signaling is selected from the group of anti-type I interferon R1 or R2 antibodies or from an endogenous modulator of interferon- α comprising SOSC1 or an arene receptor, wherein the agent depleting the IFN- α producing cells is an agent depleting plasmacytoid dendritic cells (pdcs), and wherein the agent blocking IFN- α production is an agent blocking the production of IFN- α by pdcs.

In one embodiment, the type III interferon comprises at least one IFN- λ selected from the group of IFN- λ 1, IFN- λ 2, IFN- λ 3, and IFN- λ 4, wherein the agent that stimulates production of the type III interferon comprises a TLR ligand, a RIG-I ligand, and/or an MDA5 ligand; and wherein the agent that stimulates production of type III interferon may also stimulate production of type I interferon.

The present invention further relates to a combination for use in the prevention or treatment of an infectious disease in a subject in need thereof, wherein the combination comprises:

1) a CD8 vaccine specific for at least one pathogen-specific antigen,

2) optionally, an interferon- α blocker, and

In one embodiment, the CD8 vaccine elicits or comprises the inhibitor MHC-1b/E restricted CD8⁺T cells.

In one embodiment, the CD8 vaccine is an active vaccine (active vaccine), wherein the CD8 vaccine is a live viral vector comprising at least one infectious disease associated antigen, and wherein the live viral vector is selected from the group of cytomegalovirus, lentivirus, vaccinia virus, adenovirus or a plasmid.

In one embodiment, the CD8 vaccine is an active vaccine, wherein the CD8 vaccine is a Cytomegalovirus (CMV) vector comprising:

-a first nucleic acid sequence encoding at least one infectious disease associated antigen,

optionally, a second nucleic acid sequence comprising a first MicroRNA Recognition Element (MRE) operably linked to a CMV gene that is essential or enhanced for CMV growth, wherein the MRE is expressed by default in the presence of microRNAs expressed by the endothelial cell lineage,

and wherein the CMV vector does not express an active UL128 protein or ortholog (ortholog) thereof; does not express active UL130 protein or ortholog thereof; does not express active UL146 or an ortholog thereof; does not express active UL147 protein or ortholog thereof,

and wherein the CMV vector expresses at least one active UL40 protein or ortholog thereof; expressing at least one active US27 protein or ortholog thereof and/or expressing at least one active US28 protein or ortholog thereof.

In another embodiment, the CD8 vaccine is an active vaccine, wherein the CD8 vaccine comprises at least one infectious disease associated antigen and a non-pathogenic bacterium.

In one embodiment, the infectious disease associated antigen is selected from the group of viruses, viral particles, virus-like particles, recombinant viruses, recombinant viral particles, conjugated viral proteins (conjugated viral proteins) and concatemer viral proteins (concatemer viral proteins), wherein the virus, viral particle or recombinant viral particle is attenuated (attenuated) or inactivated (inactivated).

In one embodiment, the non-pathogenic bacteria are live.

In one embodiment, the non-pathogenic bacteria are selected from attenuated or inactivated pathogenic bacteria, and preferably wherein the non-pathogenic bacteria are Lactobacillus bacteria (Lactobacillus plantarum), more preferably Lactobacillus plantarum.

In another embodiment, the CD8 vaccine is an active vaccine, wherein the CD8 vaccine is an ex vivo generated dendritic, natural killer, or B cell population that presents MHC-1B/E restricted and MHC-II restricted antigens, wherein the MHC-1B/E restricted antigen is an infectious disease associated antigen.

In another embodiment, the CD8 vaccine is a passive vaccine (passive vaccine), and wherein the CD8 vaccine is autologous MHC-1b/E restricted CD8 produced ex vivo⁺T cell population in which MHC-lb/E restricted CD8⁺The T cell population recognizes MHC-1b/E restricted infectious disease associated antigens.

In one embodiment, the infectious disease-associated antigen is a pathogen-specific antigen, preferably a viral pathogen-specific antigen.

In one embodiment, the infectious disease associated antigen is derived from a pathogen selected from the group consisting of: human immunodeficiency virus, simian immunodeficiency virus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, papilloma virus, Plasmodium paradoxum, and Mycobacterium tuberculosis.

In one embodiment, the infectious disease-associated antigen is a viral pathogen-specific antigen, wherein the viral pathogen-specific antigen is derived from an HIV or SIV pathogen, such as gag, env, rev, tat, nef, pol, and/or vif antigens.

The invention further relates to an ex vivo method for generating an autologous dendritic, natural killer or B-cell population presenting MHC-1B/E restricted peptides comprising the steps of:

1) optionally, reducing MHC-1a expression in immature dendritic cells, natural killer cells or B cells using an agent that inhibits TAP expression or activity,

2) loading HLA-DR and/or MHC-lb/E restricted peptides onto immature dendritic cells, natural killer cells or B cells, and

3) maturing the loaded immature dendritic cells, natural killer cells or B cells.

The invention further relates to a method for generating autologous MHC-1b/E restricted CD8⁺An ex vivo method of T cells comprising the steps of:

1) culturing naive CD8 in the presence of dendritic, natural killer, or B cell populations presenting MHC-1B/E restricted peptides⁺T cells (

CD8⁺T cell), thereby generating MHC-1b/E restricted CD8⁺A T cell; and

2) amplification of MHC-lb/E restricted CD8⁺T cells.

Definition of

In the present invention, the following terms have the following meanings:

-a digit preceded by "about" encompasses plus or minus 10% or less of the value of the digit. It is to be understood that the value to which the term "about" refers is also itself specifically and preferably disclosed.

As used herein, the term "adjuvant" refers to agents that aid and enhance the pharmacological action of a drug or vaccine, or increase the immunogenic response, including CD8⁺Immune responses (e.g., CD8 restricted by MHC-Ib/E used in the treatment of infectious diseases at high percentages⁺T cell response is an immune response characterized by).

The term "administering" means either directly administering a compound or composition of the invention, or administering a prodrug (produgs), derivative or analogue which will form an equivalent amount of the active compound or substance in vivo. For example, according to one embodiment, the fact that the agent is administered to the subject by any effective route, such as a composition comprising an effective amount of a HCMV vector containing the foreign antigen. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

The term "antigen" refers to a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions that are injected or absorbed into the animal. The antigen reacts with the products of a particular humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all relevant epitopes. An "epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to an epitope when presented with an MHC molecule. Epitopes can be formed of contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed from tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3, and more typically, at least 5, about 9, or about 8-10 amino acids with a unique spatial conformation. Methods for determining spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. In some embodiments, the antigen is a pathogen-specific antigen. In the context of the present disclosure, a pathogen-specific antigen is an antigen that elicits an immune response against a pathogen and/or is unique to the pathogen (e.g., a virus, bacterium, fungus, or protozoan).

The term "attenuated" in the context of a live virus or bacterium refers to a virus or bacterium that has a reduced ability to infect a cell or subject (e.g., eliminated) and/or a reduced ability to induce or cause disease (e.g., eliminated) as compared to a wild-type virus or wild-type bacterium. Typically, the attenuated virus or bacterium retains at least some ability to elicit an immune response after administration to an immunocompetent subject. In some cases, the attenuated virus or bacteria are capable of eliciting a protective immune response without causing any signs or symptoms of infection. In some embodiments, the ability of an attenuated virus or bacterium to cause a disease in a subject is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% relative to a wild-type virus or wild-type bacterium.

The term "CMV" (cytomegalovirus) refers to a member of the beta subclass of the herpes virus family. CMV is a large (-230 kB genome) double-stranded DNA virus with host-range specific variants, such as MCMV (murine CMV), RhCMV (rhesus CMV), and HCMV (human CMV). In the context of the present invention, "RhCMV" refers to any strain, isolate or variant of rhesus CMV. In the context of the present invention, "HCMV" refers to any strain, isolate or variant of human CMV.

The term "reducing" means reducing the mass, quantity or intensity of something. For example, a therapy (e.g., a method provided herein) reduces the infectious load or titer of a pathogen, or one or more symptoms associated with an infection.

The term "deletion" refers to the removal of a DNA sequence which joins the regions flanking the removed sequence.

The term "expression" refers to the translation of a nucleic acid into a protein, for example the translation of mRNA encoding a tumor-specific or pathogen-specific antigen into a protein.

The term "expression control sequence" refers to a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence operably linked thereto, such as the expression of a heterologous polynucleotide spliced into the CMV genome and encoding an antigenic protein operably linked to the expression control sequence. The expression control sequence is operably linked to the nucleic acid sequence when the expression control sequence controls and regulates the transcription and translation of the nucleic acid sequence, as the case may be. Thus, expression control sequences may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) in front of the protein-encoding gene, splicing signals for introns, and maintaining the correct reading frame for the gene to allow for proper translation of mRNA, and a stop codon. The term "control sequences" is intended to include at least components whose presence can affect expression, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences. The expression control sequence may include a promoter. A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements sufficient for promoter-dependent gene expression to be cell-type specific, tissue-specifically controllable, or inducible by an external signal or agent; such elements may be located in the 5 'or 3' regions of the gene. Including constitutive and inducible promoters (see, e.g., Bitter et al, Methods in Enzymology 153:516-544, 1987). For example, when cloning is performed in a bacterial system, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of phage lambda, and the like can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or from a mammalian virus (e.g., the retroviral long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of a nucleic acid sequence. The polynucleotides may be inserted into expression vectors, including viral vectors, containing promoter sequences that facilitate efficient transcription of the inserted gene sequences in a host. Expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection of transformed cells.

The term "fragment" refers to the part of the polypeptide that displays at least one useful epitope. The phrase "functional fragment of a polypeptide" refers to all polypeptide fragments that retain the activity or a measurable portion of the activity of the polypeptide from which the fragment is derived. The size of the fragments may vary, for example, being as small as being able to bind an epitope of an antibody molecule, as large as being able to participate in the characteristic induction or programming of a phenotypic change in a cell. An epitope is a region of a polypeptide that is capable of binding to an immunoglobulin produced in response to contact with an antigen.

The term "heterologous" as used herein refers to a heterologous polypeptide or polynucleotide (e.g., antigen or protein) derived from a different source or species. In some embodiments of the invention, the heterologous sequence is from a different genetic source than the second sequence, e.g., a virus or other organism. In a specific example, the heterologous antigen is not derived from CMV.

The term "immunogenic peptide" (or "antigenic peptide") refers to a peptide comprising an allele-specific motif or other sequence (e.g., an N-terminal repeat) such that the peptide will bind to MHC molecules and induce a cytotoxic T lymphocyte ("CTL") response or a B cell response (e.g., antibody production) against the antigen from which the immunogenic peptide is derived. In one embodiment, the immunogenic peptides are identified using sequence motifs or other methods, such as neural networks (neural nets) or polynomial determinations (polymodal determination) as known in the art. Typically, an algorithm is used to determine the "binding threshold" of peptides to select those peptides that have a score that indicates that they are likely to bind with a particular affinity and that are immunogenic. The algorithm is based on the effect of a particular amino acid at a particular position on MHC binding, the effect of a particular amino acid at a particular position on antibody binding, or the effect of a particular substitution in a motif-containing peptide on binding. In the context of immunogenic peptides, a "conserved residue" is a residue that occurs at a particular position in the peptide with a significantly higher frequency than would be expected from a random distribution. In one embodiment, a conserved residue is a residue that the MHC structure may provide a point of contact with the immunogenic peptide.

The term "immune" refers to a condition capable of producing a protective response upon exposure to an immunogenic agent. The protective response may be antibody-mediated or immune cell-mediated, and may be directed against a particular pathogen or tumor antigen. Immunization may be achieved actively (e.g., by exposure to an immunogenic agent, either naturally or in a pharmaceutical composition) or passively (e.g., by administration of antibodies or T cells stimulated and expanded in vitro).

For biological components (e.g., nucleic acid molecules, proteins, organelles, or cells), the terms "isolated" or "non-naturally occurring" refer to a biological component that is altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" from the coexisting materials in its natural state, either partially or completely. An isolated nucleic acid or peptide may be present in a substantially purified form, or may be present in a non-natural environment such as a host cell. Typically, an isolated preparation of nucleic acids or peptides contains a nucleic acid or peptide that is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure. Nucleic acids and proteins that are "non-naturally occurring" or that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An "isolated polypeptide" is a polypeptide that has been identified and isolated and/or recovered from a component of its natural environment.

The terms "subject" and "patient" are used interchangeably herein and refer to an animal, such as a human, that is provided for treatment, including prophylactic treatment, using the pharmaceutical compositions of the invention.

The term "subject" as used herein refers to mammals, primates and/or humans, and includes all mammals, e.g. non-human primates (especially higher primates), sheep, dogs, rodents (e.g. mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, horses.

The term "mutation" refers to any difference of a nucleic acid or polypeptide sequence from a normal, consensus or "wild-type" sequence. A mutant is any protein or nucleic acid sequence that comprises a mutation. Furthermore, a cell or organism having a mutation may also be referred to as a mutant. Some types of coding sequence mutations include point mutations (single nucleotide or amino acid differences); silent mutations (nucleotide differences that do not result in amino acid changes); deletions (deletion of one or more nucleotide or amino acid differences up to and including deletion of the entire coding sequence of the gene); frame shift mutations (where the amino acid sequence is altered by a difference in the deletion of a number of nucleotides not divisible by 3). Mutations that result in differences in amino acids may also be referred to as amino acid substitution mutations. Amino acid substitution mutations can be described by amino acid changes at a particular position in the amino acid sequence relative to the wild type.

As used herein, an "inactivating mutation" is any mutation in a viral gene that ultimately results in a reduction or complete loss of function of a viral protein.

The term "operably linked" means that a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, join two protein coding regions, in the same reading frame.

The term "open reading frame" (ORF) refers to a series of nucleotide triplets (codons) encoding amino acids without any internal stop codon. These sequences are generally translatable into peptides.

As used herein, the term "prevention" refers to a prophylactic measure, wherein the objective is to reduce the chance of a subject developing a pathological condition or disorder within a given period of time. Such a reduction may be reflected, for example, in a delayed onset of at least one symptom of the pathological condition or disorder in the subject.

The term "prophylactic" refers to a treatment administered to a subject showing no signs of disease or showing only early signs, with the aim of reducing the risk of developing pathology. In particular, prophylactic treatment of HIV or SIV infection in a subject refers to treatment that allows the subject to become an Elite Controller (EC), i.e., one with relatively high CD4⁺T cell count (e.g., better than 500 CD4 per microliter)⁺T cells) and/or maintain clinically undetectable plasma HIV-1RNA levels (e.g., HIV RNA) for extended periods of time in the absence of any antiretroviral therapy (ART)<50 copies/mL).

The term "therapeutic" refers to a treatment administered to a subject exhibiting early or established signs of disease.

The term "curative" refers to a treatment administered to a subject suffering from a disease for the purpose of curing the disease, i.e. to make any sign of the disease disappear or become undetectable.

The term "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). A polynucleotide consists of four bases; adenine, cytosine, guanine and thymine/uracil (uracil is used in RNA). The coding sequence from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogues, and it may be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that are modified by nature or intervention; such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or bioactive component.

The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within the cell or production reaction chamber (as the case may be).

The term "recombinant" refers to a nucleic acid having a sequence that is not naturally occurring or having a sequence that is prepared by an artificial combination of two additional separate sequence segments. Such artificial combination may be achieved by chemical synthesis, or more commonly by artificial manipulation of isolated nucleic acid segments, for example by genetic engineering techniques.

The term "sample" or "biological sample" refers to a biological sample, e.g. a liquid of a cell, tissue sample, obtained from a subject. In some cases, the biological sample contains genomic DNA, RNA (including mRNA and microRNA), proteins, or a combination thereof. Examples of samples include, but are not limited to, saliva, blood, serum, urine, spinal fluid, tissue biopsies, surgical specimens, cells (e.g., PBMCs, leukocytes, lymphocytes, or other cells of the immune system), and autopsy material.

The term "sequence identity" refers to the similarity between two nucleic acid sequences, or between two amino acid sequences, expressed in terms of similarity between the sequences, and is also referred to as sequence identity. Sequence identity is typically measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences.

Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: smith and Waterman (adv. Appl. Math.2:482,1981); needleman and Wunsch (J.mol.biol.48:443,1970); pearson and Lipman (PNAS USA 85:2444,1988); higgins and Sharp (Gene,73: 237-; higgins and Sharp (CABIOS 5: 151-; corpet et al (Nuc. acids Res.16:10881-10890, 1988); huang et al (Comp. appls biosci.8:155-165, 1992); and Pearson et al (meth.mol.biol.24:307-31, 1994). Sequence alignment methods and homology calculations are described in detail by Altschul et al (Nature Genet.,6:119-129, 1994). Alignment tools ALIGN (Myers and Miller, CABIOS4:11-17,1989) or LFASTA (Pearson and Lipman,1988) can be used to perform sequence comparisons (Internet)

1996, w.r. pearson and the University of Virginia, fasta20u63 version 2.0u63, release date 12 months 1996). ALIGN compares entire sequences to each other, while LFASTA compares locally similar regions. These alignment tools and their respective courses can be found on the NCSA website. Alternatively, for comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function can be used using the default BLOSUM62 matrix set as default parameters (gap existence cost of 11, and gap cost per residue of 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for alignment using the PAM30 matrix set as default parameters (open gap 9, extension gap 1 penalty). BLAST sequence comparison systems are available, for example, from the NCBI website; see also Altschul et al, J.mol.Biol.215:403-410,1990；Gish.&States, Nature Genet.3: 266-; madden et al meth.Enzymol.266:131-141, 1996; altschul et al, Nucleic Acids Res.25:3389-3402, 1997; and Zhang&Madden,Genome Res.7:649-656,1997。

Orthologs of proteins are generally characterized by having greater than 75% sequence identity, when counted in full-length alignment with the amino acid sequence of a particular protein, using ALIGN set as a default parameter. Proteins having even greater similarity to a reference sequence will exhibit an increased percentage of identity, e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity, when assessed by this method. In addition, sequence identity can be compared over the entire length of a particular domain of the disclosed peptides.

When comparing sequence identities that are significantly less than the entire sequence, homologous sequences will typically have at least 80% sequence identity, and possibly at least 85%, at least 90%, at least 95%, or at least 99% sequence identity, over a short window of 10-20 amino acids, depending on their similarity to the reference sequence. Sequence identity over such short windows can be determined using LFASTA; the method is described at the NCSA website. Those skilled in the art will appreciate that these ranges of sequence identity are provided for guidance only; it is entirely possible to obtain very important homologues outside the ranges provided.

However, due to the degeneracy of the genetic code, nucleic acid sequences that do not show a high degree of identity may also encode similar amino acid sequences. It will be appreciated that such degeneracy can be used to alter a nucleic acid sequence to produce a plurality of nucleic acid sequences each encoding a substantially identical protein.

The term "treatment" as used herein refers to an intervention that improves a sign or symptom of a disease or pathological condition. For example, in the case of HIV infection, HIV RNA (viral load) and CD 4T lymphocyte (CD4) cell count are two alternative markers of anti-retroviral therapy (ART) response and HIV disease progression, and have been used for decades to manage and monitor HIV infection. Thus, plasma diseases in humans can be treated before and after treatmentToxic RNA load, and treatment is considered effective if it is reduced by at least about 10%, 20%, 30%, 40%, 50%, more preferably by at least about 70%, still more preferably by at least about 75% or 80% or 85% or 90% or 95% or 98% or 99% or even more (99.5%, 99.8%, 99.9%, 100%), and/or treatment effect can be assessed by monitoring CD4 cell count before and after treatment, and treatment is considered effective if the absolute count of CD4 cells is increased by at least about 5%, 10%, 15%, 20%, 25%, more preferably by at least about 30%, still more preferably by at least about 35% or 40% or 45% or 50% or 55% or 60% or 65% or even more. As used herein, the terms "treatment" and "treating" with respect to a disease, pathological condition, or symptom also refer to any observable beneficial effect of the treatment. The beneficial effects can be demonstrated, for example, by: delayed onset of clinical symptoms of the disease in a susceptible subject, reduced severity of some or all clinical symptoms of the disease, slower progression of the disease, reduced number of disease relapses, improvement in the overall health or well-being of the subject, or by other parameters known in the art to be specific for a particular disease. Therapeutic treatment is treatment administered to a subject after signs and symptoms of the disease have developed. Prophylactic treatment is treatment administered to a subject who shows no signs of disease or only early signs, with the aim of reducing the risk of pathological development. In particular, prophylactic treatment of HIV or SIV infection in a subject refers to treatment that allows the subject to become an Elite Controller (EC), i.e. one with a relatively high CD4 for a long period of time in the absence of any antiretroviral therapy (ART)⁺T cell count (e.g., greater than 500 CD4 per microliter)⁺T cells) and/or maintain clinically undetectable plasma HIV-1RNA levels (e.g., HIV RNA)<50 copies/mL). A prophylactic treatment is a treatment administered to a subject suffering from a disease with the aim of curing the disease, i.e. causing any signs of the disease to disappear or become undetectable.

The term "vector" may include nucleic acid sequences which allow it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct expression of the nucleic acid. The vector may be a viral vector, such as a CMV vector. Viral vectors can be constructed from wild-type or attenuated viruses, including replication-defective viruses. The vector may also be a non-viral vector, including any plasmid known in the art.

The term "virus" refers to microscopic infectious organisms that multiply within living cells. Viruses essentially consist of a nucleic acid core (viral genome) surrounded by a protein coat (capsid) and have the ability to replicate only in living cells. "viral replication" is the generation of additional viral particles by the occurrence of at least one viral life cycle. Viruses can disrupt the normal function of a host cell, causing the cell to behave in a virus-determined manner. For example, viral infection may cause cells to produce or respond to cytokines, while uninfected cells do not typically do so. The term "lytic" or "acute" viral infection refers to a viral infection in which the viral genome is replicated and expressed to produce polypeptides necessary for the production of the viral capsid. The mature viral particles leave the host cell, causing cell lysis. Alternatively, a particular virus species may enter into a "lysogenic" or "latent" infection. In the latent establishment, the viral genome is replicated, but the capsid proteins are not produced and assembled into viral particles.

As used herein, the term "microRNA" or "miRNA" refers to a class of major biological molecules involved in the control of gene expression. For example, in human heart, liver or brain, mirnas play a role in tissue specification or cell lineage determination. In addition, mirnas affect multiple processes, including early development, cell proliferation and cell death, as well as apoptosis and fat metabolism. The large number of miRNA genes, diverse expression patterns, and abundant potential miRNA targets suggest that mirnas can be an important source of genetic diversity.

Mature mirnas are typically 18-25 nucleotide non-coding RNAs that regulate expression of mrnas that include sequences complementary to mirnas. These small RNA molecules are known to control gene expression by modulating mRNA stability and/or translation. For example, mirnas bind to the 3' UTR of target mrnas and inhibit translation. mirnas can also bind to target mrnas and mediate gene silencing via the RNAi pathway. mirnas may also regulate gene expression by causing chromatin condensation.

mirnas silence the translation of one or more specific mRNA molecules by binding to a MiRNA Recognition Element (MRE), defined as any sequence that directly base pairs with and interacts with a miRNA somewhere on the mRNA transcript. Typically, MRE is present in the 3 'untranslated region (UTR) of an mRNA, but it can also be present in the coding sequence or in the 5' UTR. MREs need not be perfectly complementary to mirnas, they typically have only a few bases complementary to mirnas, and typically contain one or more mismatches in these complementary bases. The MRE may be any sequence that is sufficiently bound by the miRNA such that translation of a gene operably linked to the MRE (e.g., a CMV gene essential or enhanced for in vivo growth) is inhibited by a miRNA silencing mechanism, such as RISC.

Detailed Description

The present invention relates to a method of preventing or treating an infectious disease in a subject in need thereof, comprising administering to the subject:

2) an interferon alpha blocker, and/or

In one embodiment, the method comprises administering to the subject 1) a CD8 vaccine specific for at least one infectious disease associated antigen, 2) an interferon alpha blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

In another embodiment, the method comprises administering to the subject 1) a CD8 vaccine specific for at least one infectious disease associated antigen and 2) an interferon alpha blocker.

In another embodiment, the method comprises administering to the subject 1) a CD8 vaccine specific for at least one infectious disease associated antigen and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

In some embodiments, the method is a method of prophylactic treatment or a method of curative treatment.

In a particular embodiment, the method is a prophylactic method and comprises administering to the subject 1) a CD8 vaccine specific for at least one infectious disease-associated antigen, 2) an interferon alpha blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

The present invention further relates to a combination for use as a medicament, wherein the combination comprises:

2) an interferon alpha blocker, and/or

The present invention also relates to a combination for use in the prevention or treatment of an infectious disease in a subject in need thereof, wherein the combination comprises:

2) an interferon alpha blocker, and/or

In one embodiment, the combination used comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, 2) an interferon alpha blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

In one embodiment, the combination used comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen and 2) an interferon alpha blocker.

In one embodiment, the combination used comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

In a specific embodiment, the combination is to be used prophylactically and comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, 2) an interferon alpha blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon.

The present invention further relates to a kit for preventing or treating an infectious disease in a subject in need thereof, wherein the kit comprises at least 2 parts and comprises:

1) comprising a first portion of a CD8 vaccine specific for at least one infectious disease associated antigen,

2) optionally, a second moiety comprising an interferon- α blocker, and

3) a third portion comprising a type III interferon and/or an agent that stimulates production of a type III interferon.

As used herein, the term "vaccine" refers to an immunogenic product or composition that can be administered to a mammal, such as a human, to confer immunity to a disease or other pathological condition, e.g., passive or active immunity. Vaccines can be used prophylactically or therapeutically (prophylactically or curatively). Thus, vaccines can be used to reduce the likelihood of developing a disease (e.g., infection) or reduce the severity of symptoms of a disease or condition, limit the progression of a disease or condition (e.g., infection), or limit the recurrence of a disease or condition.

In one embodiment, the CD8 vaccine is a prophylactic (preventative) vaccine. In another embodiment, the CD8 vaccine is a therapeutic (therpeutic) vaccine. As used herein, a therapeutic vaccine may be a prophylactic (therapeutic) vaccine or a curative (curative) vaccine. In some embodiments, the CD8 vaccine is a prophylactic vaccine. In other embodiments, the CD8 vaccine is a curative vaccine.

In one embodiment, the CD8 vaccine induces immune tolerance to at least one infectious disease-associated antigen. In one embodiment, the CD8 vaccine is therefore specific for at least one infectious disease-associated antigen.

As used herein, the terms "immune tolerance" and "Ts" are synonymous. Immune tolerance is the physiological ability of the immune system to recognize an antigen and produce anergy (anergy) that is commonly associated with other immune modifications when the same antigen is subsequently encountered. In the present invention, the main feature of immune tolerance is CD8⁺Activity of T cells inhibiting CD4 presenting at least one antigen associated with an infectious disease⁺Activation of T cells. Overall, whenever one or several infectious disease-associated antigens are involved in CD4⁺MHC-1 b/E-restricted CD8 produced by the CD8 vaccine of the invention upon specific activation of T cells presenting epitopes derived from antigens associated with infectious diseases⁺T cells can cause CD4⁺Specific inhibition/prevention of T cell activation.

In one embodiment, the CD8 vaccine of the invention elicits the inhibitor MHC-1b/E restricted CD8⁺T cells. In another embodiment, the CD8 vaccine of the invention comprises the inhibitor MHC-1b/E restricted CD8⁺T cells consist essentially of or consist of T cells.

As used herein, with respect to a population of cells, "consisting essentially of … …" means the inhibitor MHC-1b/E restricted CD8⁺The T cell population is the only one biologically active therapeutic or pharmaceutical agent in the composition.

In one embodiment, the inhibitor is MHC-1b/E restricted CD8⁺T cells are generated by ex vivo or in vivo induction of HLA-1a deprived dendritic cells, natural killer cells or B cells.

In one embodiment, the inhibitor is MHC-1b/E restricted CD8⁺The T cell is cytolytic CD8⁺T cells. In one embodiment, the inhibitor is MHC-1b/E restricted CD8⁺T cells are non-cytolytic CD8⁺T cells.

In one embodiment, the CD8 vaccine is an active vaccine. In another embodiment, the CD8 vaccine is a passive vaccine.

As used herein, the term "active vaccine" refers to a vaccine that induces active immunity, and refers to the process of exposing the body to antigens to generate an adaptive immune response: the reaction takes days/weeks to develop, but can last for a long time-even for life. "passive vaccine" induces passive immunity and refers to a process of providing, for example, antibodies or cells to prevent infection; it provides immediate but transient protection-weeks to months.

In some embodiments, the CD8 vaccine comprises the inhibitor MHC-1b/E restricted CD8⁺T cells.

In some embodiments, the CD8 vaccine is a priming inhibitor MHC-1 b/E-restricted CD8⁺A vaccine for T cells, said vaccine selected from the group consisting of:

-an active vaccine which is a live viral vector comprising at least one pathogen-specific antigen, wherein the live viral vector is selected from the group consisting of cytomegalovirus, lentiviruses, vaccinia virus, adenoviruses and plasmids;

-an active vaccine comprising at least one pathogen-specific antigen and at least one non-pathogenic bacterium, preferably at least one attenuated or inactivated pathogenic bacterium;

-an active vaccine which is an ex vivo generated dendritic cell, natural killer cell or B cell population presenting at least one MHC-1B/E restricted antigen and at least one MHC-II restricted antigen, and wherein the MHC-1B/E restricted antigen is a pathogen specific antigen;

passive vaccines, autologous MHC-1b/E restricted CD8 produced in vitro⁺T cell population recognizing MHC-1b/E restricted pathogen specific antigens.

According to one embodiment of the invention, the CD8 vaccine is a live viral vector comprising at least one infectious disease associated antigen.

In one embodiment, the live viral vector described above is an active vaccine.

In one embodiment, the live viral vector described above is selected from the group of cytomegalovirus, lentiviruses, vaccinia virus, adenoviruses and plasmids.

In one embodiment, the live virus vector described above is a recombinant vector selected from the group of recombinant cytomegalovirus, recombinant lentivirus, recombinant vaccinia virus, recombinant adenovirus and recombinant plasmid.

According to one embodiment, the live viral vector is a recombinant vaccinia virus. Recombinant vaccinia viruses have been produced from different strains of vaccinia virus. For example, a variety of highly attenuated, host-restricted, non-replicating or poorly replicating poxvirus strains have been developed for use as substrates in recombinant vaccine development, including orthopoxviruses (Orthopoxvirus), Modified Vaccinia Ankara (MVA), NYVAC, avipoxviruses (Avipoxvirus), ALVAC and TROVAC.

According to one embodiment of the invention, the CD8 vaccine is a CMV vector.

In one embodiment, the CMV vector is an active vaccine.

In one embodiment, the CMV vector comprises a nucleic acid sequence encoding at least one infectious disease-associated antigen. In a specific embodiment, the CMV vector comprises a nucleic acid sequence encoding at least one Human Immunodeficiency Virus (HIV) antigen.

In one embodiment, the CD8 vaccine is a recombinant CMV that expresses at least one infectious disease-associated antigen, wherein the antigen is a heterologous antigen. Thus, in one embodiment, the infectious disease associated antigen may be derived from any protein that is not naturally expressed in CMV.

In one embodiment, the CMV vector does not express active UL128 and UL130 proteins or orthologs thereof.

As used herein, the term "ortholog" refers to a homologous gene of CMV that infects other species.

In one embodiment, the CMV vector does not express active UL146 and UL147 proteins or orthologs thereof.

In one embodiment, the CMV vector expresses at least one active UL40 protein, and/or at least one active US27 protein, and/or at least one active US28 protein. In one embodiment, the at least one active UL40 protein, the at least one active US27, and the at least one active US28 protein may be orthologs or homologs of UL40, US27, and US 28.

In some embodiments, the CMV vector does not express an active UL128, UL130, UL146, or UL147 protein due to a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147, or an ortholog thereof.

As used herein, the term "mutation" may refer to any mutation that results in the lack of expression of an active UL128, UL130, UL146, or UL147 protein. Such mutations may include point mutations, frameshift mutations, deletions of less than all of the sequences encoding the protein (truncation mutations), or deletions of all of the nucleic acid sequences encoding the protein, or any other mutation. CMV comprising such mutations are described, for example, in WO2014138209, which is incorporated herein by reference in its entirety.

In a further embodiment, the vector does not express an active UL128, UL130, UL146 or UL147 protein or ortholog thereof, because a nucleic acid sequence comprising an antisense or RNAi sequence (siRNA or miRNA) is present in the vector, which inhibits expression of the UL128, UL130, UL146 or UL147 protein or ortholog thereof.

In one embodiment, mutations and/or antisense and/or RNAi can be used in any combination to generate a CMV vector that lacks active UL128, UL130, UL146, or UL147, or orthologs thereof.

In one embodiment, the CMV vector comprises all of the above modifications and further comprises a nucleic acid sequence that functions as a MiRNA Response Element (MRE), which is expressed recombinantly in the presence of miRNA expressed by endothelial cells.

As used herein, the term "miRNA response element" or "MRE" refers to any sequence that directly base pairs with and interacts with a miRNA somewhere on an mRNA transcript. Thus, mirnas can silence the translation of one or more specific mRNA molecules by binding to MiRNA Recognition Elements (MREs). Typically, MRE is present in the 3 'untranslated region (UTR) of an mRNA, but it can also be present in the coding sequence or in the 5' UTR. MREs are not necessarily perfectly complementary to mirnas, usually only have a few bases complementary to mirnas, and usually contain one or more mismatches in these complementary bases. The MRE may be any sequence that is capable of being sufficiently bound by the miRNA to translate a gene operably linked to the MRE. Examples of such genes include, but are not limited to, the IE2 and UL79 genes, or orthologs thereof, or any CMV gene that is essential for or enhances growth in vivo. CMV comprising such MREs are described, for example, in WO201875591, which is incorporated herein by reference in its entirety.

In one embodiment, the MRE may be any miRNA recognition element that is expressed by default in the presence of mirnas expressed by endothelial cells. In one embodiment, the MRE of the vector is expressed silently in the presence of one or more of miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR-296, and miR-328.

In one embodiment, MRE is expressed silently in the presence of miR-126-3 p.

In one embodiment, MRE disrupts the expression of UL122(IE2) and UL79 in the presence of miR-126-3 p.

One skilled in the art can select from the literature validated, putative or mutated MRE sequences that will be predicted to induce silencing in the presence of mirnas expressed in endothelial cells or bone marrow cells such as macrophages. One skilled in the art can then obtain an expression construct in which a reporter gene (e.g., a fluorescent protein, enzyme, or other reporter gene) has expression driven by a promoter, such as a constitutively active promoter or a cell-specific promoter. The MRE sequence may then be introduced into the expression construct. The expression construct may be transfected into a suitable cell and the cell transfected with the miRNA of interest. The absence of expression of the reporter gene indicates that MRE silences gene expression in the presence of miRNA.

In one embodiment, the CMV vector comprises a first nucleic acid sequence encoding at least one infectious disease-associated antigen and does not express active UL128, UL130, UL146, and UL147 proteins or orthologs thereof and expresses at least one active UL40, US27, and/or US28 protein or ortholog thereof.

In another embodiment, the CMV vector comprises a first nucleic acid sequence encoding at least one infectious disease-associated antigen, optionally a second nucleic acid sequence comprising a first MicroRNA Recognition Element (MRE) operably linked to a CMV gene essential for CMV growth or enhancing CMV growth, wherein the MRE is constitutively expressed in the presence of micrornas expressed by cells of endothelial lineage and does not express active UL128, UL130, UL146, and UL147 proteins or orthologs thereof, and expresses at least one active UL40, US27, and/or US28 protein or ortholog thereof.

In one embodiment, the CMV vector may comprise additional inactivating mutations known in the art to provide a different immune response, such as an inactivating US11 mutation or an inactivating UL82(pp71) mutation, or any other inactivating mutation.

In one embodiment, the CMV vector may further comprise at least one inactivating mutation in one or more viral genes encoding viral proteins known in the art to be essential for or enhance viral transmission (i.e., transmission from cell to cell) in vivo. Such inactivating mutations may be from point mutations, frame shift mutations, truncation mutations, or deletions of all nucleic acid sequences encoding viral proteins. Inactivating mutations include any mutation in the viral gene that ultimately results in a reduction in the function or complete loss of function of the viral protein.

In one embodiment, the CMV vectors described herein may comprise mutations that prevent transmission between hosts, thereby rendering the virus incapable of infecting immunocompromised or other subjects that may be exposed to complications from CMV infection. In another embodiment, the CMV vectors described herein can further comprise mutations that result in the presentation of immunodominant and non-immunodominant epitopes and non-classical MHC restriction. Such CMV mutations are described, for example, in U.S. patent publication 2013-0136768; 2014-0141038; and PCT application publication WO 2014/138209, which is incorporated herein by reference in its entirety.

In one embodiment, a mutation in a CMV vector described herein does not affect the ability of the vector to re-infect a subject that has been previously infected with CMV. Thus, in one embodiment, the CMV vector is capable of re-infecting an organism.

In one embodiment, the CMV vector is a human CMV (hcmv) or rhesus CMV (rhcmv) vector.

In one embodiment, the CMV vectors disclosed herein can be prepared by inserting DNA comprising a sequence encoding an infectious disease-associated antigen into an essential or non-essential region of the CMV genome.

In one embodiment, the infectious disease associated antigen is a heterologous antigen of CMV.

In one embodiment, the method may further comprise deleting one or more regions from the CMV genome. In one embodiment, the method may comprise in vivo recombination. Thus, the method may comprise transfecting a cell with CMV DNA in a cytocompatible medium in the presence of a donor DNA comprising heterologous DNA flanked by DNA sequences that are partially homologous to the CMV genome, thereby introducing the heterologous DNA into the CMV genome, and then optionally recovering the CMV modified by in vivo recombination.

In one embodiment, the method may further comprise cleaving the CMV DNA to obtain cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMV DNA to obtain hybrid CMV-heterologous DNA, transfecting the cell with the hybrid CMV-heterologous DNA, and then optionally recovering the CMV modified by the presence of the heterologous DNA. Because in vivo recombination is involved, the method also provides a plasmid comprising a donor DNA encoding a non-CMV-native polypeptide that is not naturally present in CMV, the donor DNA being located within a CMV DNA segment that would otherwise be collinear (co-linear) with an essential or non-essential region of the CMV genome such that DNA from the CMV essential or non-essential region flanks the donor DNA. When desired, heterologous DNA can be inserted into the CMV to produce a recombinant CMV in any orientation that results in stable integration of this DNA and its expression.

In one embodiment, the DNA encoding the infectious disease associated antigen in the recombinant CMV vector may further comprise a promoter. The promoter may be from any source, such as herpes virus, including endogenous CMV promoters, such as HCMV, RhCMV, murine CMV (mcmv), or other CMV promoters. The promoter may also be a non-viral promoter, such as the EF1a promoter. The promoter may be a truncated transcriptionally active promoter comprising a region transactivated by a virally provided transactivator and the minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter may comprise a combination of a DNA sequence corresponding to the minimal promoter and an upstream regulatory sequence. The minimal promoter comprises a CAP site and a TATA box (minimal sequence for basal transcription level; unregulated transcription level); an "upstream regulatory sequence" comprises one or more upstream elements and one or more enhancer sequences. Furthermore, the term "truncated" means that the full-length promoter is not completely present, i.e., some portion of the full-length promoter has been removed. Also, the truncated promoter may be derived from a herpes virus, such as MCMV or HCMV, such as HCMV-IE or MCMV-IE. The size of the full-length promoter can be reduced by up to 40%, or even up to 90%, based on base pairs. The promoter may also be a modified non-viral promoter. For the HCMV promoter, reference is made to U.S. Pat. nos. 5,168,062 and 5,385,839, which are incorporated herein by reference. For expression by transfecting cells with plasmid DNA, reference is made to Feigner et al (1994), J.biol.chem.269,2550-2561, which is incorporated herein by reference. Also, with respect to direct injection of plasmid DNA as a simple and effective vaccination against a variety of infectious diseases, reference is made to Ulmer et al (1993) science 259:1745-49, which is incorporated herein by reference. Therefore, it is within the scope of the present invention that the vector may be used by direct injection of vector DNA.

Also disclosed are expression cassettes that can be inserted into recombinant viruses or plasmids containing truncated transcriptionally active promoters. The expression cassette may further comprise a functionally truncated polyadenylation signal; for example, a SV40 polyadenylation signal that is truncated but still functional. It is indeed surprising that the truncated polyadenylation signal is functional, given that nature provides a larger signal. The truncated polyadenylation signal solves the problem of insert size limitation of recombinant viruses (e.g., CMV). The expression cassette may also include DNA heterologous to the virus or system in which it is inserted; and the DNA may be heterologous DNA as described herein.

In order to express the disclosed infectious disease associated antigens in a vector, the protein coding sequence of the infectious disease associated antigen should be "operably linked" to regulatory or nucleic acid control sequences that direct the transcription and translation of the protein.

According to another embodiment of the invention, the CD8 vaccine comprises at least one infectious disease associated antigen and a non-pathogenic bacterium.

In one embodiment, the CD8 vaccine comprises at least one infectious disease associated antigen and at least one non-pathogenic bacterium.

In one embodiment, the CD8 vaccine comprising at least one infectious disease associated antigen and a non-pathogenic bacterium is an active vaccine.

As used herein, the term "non-pathogenic bacteria" refers to bacteria that do not normally induce any pathology in a mammal, preferably a human.

In one embodiment, the non-pathogenic bacteria are live.

In one embodiment, the non-pathogenic bacteria described herein are commensal bacteria.

As used herein, the term "commensal bacterium" or "commensal bacterium" refers to a microorganism that is present on the surface of the body covered by epithelial cells and exposed to the external environment (e.g., the gastrointestinal and respiratory tracts, vagina, skin, etc.). Among the many proposed health benefits attributed to gut commensal bacteria, their ability to interact with the host immune system is now well documented. Commensal bacteria are well known to the skilled person. Non-limiting examples include Bacillus (Bacillus sp.) (e.g., Bacillus coagulans), Lactobacillus (Lactobacillus sp.), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium lactis (Bifidobacterium lactis), Escherichia coli (Escherichia coli), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus johnsonii (Lactobacillus sanobacter), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus salivarius, Lactobacillus paracasei (Lactobacillus salivarius), Lactobacillus salivarius, Lactobacillus sporogenes (Lactobacillus salivarius), Lactobacillus salivarius, Lactobacillus sporogenes (Bacillus salivarius), Lactobacillus salivarius, Lactobacillus sporogenes (Bacillus salivarius), Lactobacillus salivarius, Lactobacillus sporogenes (Lactobacillus salivarius), Lactobacillus sporogenes (Lactobacillus sporogenes, Lactobacillus sporogenes, Lactobacillus, Lactobacillus delurekii, Lactobacillus bulgaricus (Lactobacillus delurekii), Lactobacillus delurekii, Lactobacillus lactis (Lactobacillus delurekii lactis), Lactococcus lactis (Lactobacillus lactis), Streptococcus thermophilus (Streptococcus thermophilus), and the like.

In one embodiment, the commensal bacterium is selected from the group consisting of: lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium bifidum, Bifidobacterium breve, lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus reuteri.

In one embodiment, the commensal bacterium is a lactobacillus, preferably lactobacillus plantarum.

In another embodiment, the commensal bacterium is a lactobacillus, preferably lactobacillus rhamnosus.

In one embodiment, non-pathogenic bacteria may be used, such as a combination of two or more commensal bacteria.

In another embodiment, the non-pathogenic bacteria described herein are selected from attenuated or inactivated pathogenic bacteria.

As used herein, the term "pathogenic bacteria" refers to bacteria that induce pathology in humans. Such bacteria are well known to the skilled person and include Listeria species (Listeria species) (e.g. Listeria monocytogenes), Corynebacterium species (Corynebacterium species), Mycobacterium species (Mycobacterium species), rhodococcus species (Rhococcus species), eubacterial species (eubacterium species), bordetella species (Bortadella species) and Nocardia species (Nocardia species), among others. Preferably, the pathogenic bacteria are selected from mycobacterial species, and more preferably Mycobacterium bovis (Mycobacterium bovis).

As used herein, the term "attenuated pathogenic bacteria" refers to bacteria that are less virulent than their wild-type counterparts as a result of one or more mutations or one or more attenuation treatments (e.g., chemical treatments and/or serial passages on a particular medium). Such attenuated pathogenic bacteria are well known to those skilled in the art. Non-limiting examples of attenuated pathogenic bacteria include attenuated Salmonella typhimurium (Salmonella typhimurium) and mycobacteria. Methods for preparing such inactivated pathogenic bacteria are part of the common general knowledge in the art. As examples of such methods, phage-mediated lysis, chemical inactivation such as formalin treatment, heat inactivation, physical inactivation such as lyophilization (e.g., Extended Freeze Drying), or u.v. or gamma irradiation or microwave exposure, or any combination thereof, may be cited.

In one embodiment, the non-pathogenic bacteria described herein can be recombinant or non-recombinant.

In one embodiment, the attenuated pathogenic bacteria described herein are attenuated derivatives of pathogenic bacteria, such as BCG. In one embodiment, the attenuated derivative of the pathogenic bacterium corresponds to a recombinant salmonella typhimurium or a recombinant mycobacterium (e.g., BCG) that expresses or produces at least one HIV protein. In another embodiment, the derivative of the pathogenic bacterium does not express any HIV protein.

In one embodiment, the non-pathogenic bacteria described herein are used as tolerogenic adjuvants for CD8 vaccines. Thus, in one embodiment, the non-pathogenic bacteria is a tolerogenic adjuvant.

As used herein, the term "tolerogenic adjuvant" is an entity which, when administered by mucosal or intradermal or intraepithelial route together with a suitable infectious disease associated antigen as defined below, will induce and will preferably maintain an immune-tolerized state to the antigen, thereby enabling the treatment of infectious disease infections in humans.

In one embodiment, the tolerogenic adjuvant, when combined with an infectious disease-associated antigen, induces or maintains immune tolerance to the viral antigen, thereby treating the associated infectious disease.

In one embodiment, nonpathogenic bacteria, especially probiotics (probiotics) and commensal bacteria, may be used as tolerogenic adjuvants in the context of the present invention.

In a particular embodiment, lactobacillus, preferably lactobacillus plantarum and/or lactobacillus rhamnosus, may be used as tolerogenic adjuvant in the context of the present invention.

In one embodiment, a non-pathogenic bacterium, such as a combination of two or more commensal bacteria, may be used as a tolerogenic adjuvant in the context of the present invention.

In another embodiment, instead of or in addition to attenuation, the pathogenic bacteria described herein may be inactivated for use as tolerogenic adjuvants in the context of the present invention, but attenuated pathogenic bacteria may also be used after inactivation.

In some embodiments, the tolerogenic adjuvant comprising a non-pathogenic bacterium or an attenuated pathogenic bacterium further comprises a prebiotic (prebiotic).

As used herein, a "prebiotic" is a substance that induces the growth or activity of certain bacteria. Prebiotics have different properties, including for example carbohydrates, such as oligosaccharides and polysaccharides.

Any prebiotic may be used in combination with the non-pathogenic bacteria or attenuated pathogenic bacteria described herein.

In some embodiments, the tolerogenic adjuvant comprises at least one prebiotic selected from the group consisting of: fructooligosaccharides (FOS), Galactooligosaccharides (GOS), inulin (inulin), trans-galactooligosaccharides (TOS), beneo Synergy 1(SYN1), oligofructose-inulin (oligofructose-inulin), lactulose (lactulose), oat fiber (oat fiber), malted barley (germinited barley), hydrolyzed guar gum (hydrolyzed gum), resistant starch (resistant starch), plantago asiatica (planta ovata), beta-glucan (beta glucana), and pectin (pectin).

According to another embodiment of the invention, the CD8 vaccine is an ex vivo generated dendritic, natural killer or B cell population presenting MHC-II and MHC-1B/E restricted antigens.

In one embodiment, the ex vivo generated dendritic, natural killer or B cell population presenting MHC-II and MHC-1B/E restricted antigens is an active vaccine.

In one embodiment, the MHC-1b/E restricted antigen is an infectious disease associated pathogen specific antigen.

In one embodiment, the infectious disease-associated antigen or pathogen-specific antigen is an HIV or SIV-derived MHCIb/E binding antigen.

In one embodiment, the HIV-derived MHCIb/E binding antigen described herein is selected from the group of SEQ ID NO 1 to SEQ ID NO 4.

In one embodiment, the HIV-derived mhc ib/E binding antigen has an amino acid sequence selected from the group consisting of seq id no: sequence RMYSPVSIL (SEQ ID NO:1), sequence PEIVIYDYM (SEQ ID NO:2), sequence TALSEGATP (SEQ ID NO:3) and sequence RIRTWKSLV (SEQ ID NO: 4).

In some embodiments, the MHC-II restricted peptide or antigen is an HLA-DR restricted peptide or antigen. Examples of HLA-DR-restricted peptides include, for example, HLA-DR-binding antigens having one of the following sequences: QGQMVHQAISPRTLN (SEQ ID NO:7) (Gag p24), GEIYKRWIILGLNKI (SEQ ID NO:8) (Gag p24), KRWIILGLNKIVRMY (SEQ ID NO:9) (Gag p24) or FRKYTAFTIPSINNE (SEQ ID NO:10) (Pol RT).

In one embodiment, the HLA-DR-restricted peptide is derived from HIV, preferably from HIV-1.

In one embodiment, the HLA-DR-restricted peptide is an HIV-derived HLA-DR binding antigen having an amino acid sequence selected from the group consisting of: sequence QGQMVHQAISPRTLN (SEQ ID NO:7) (Gag p24), sequence GEIYKRWIILGLNKI (SEQ ID NO:8) (Gag p24), sequence KRWIILGLNKIVRMY (SEQ ID NO:9) (Gag p24) and sequence FRKYTAFTIPSINNE (SEQ ID NO:10) (Pol RT).

In one embodiment, the dendritic, natural killer, or B cell population presenting MHC-II and MHC-1B/E restricted peptides is an allogeneic cell population. In a preferred embodiment, the dendritic, natural killer or B cell population presenting MHC-II and MHC-1B/E restricted peptides is an autologous cell population.

As used herein, "allogeneic cell" refers to a cell that is isolated from one subject (donor) and injected into another subject (recipient or host).

As used herein, "autologous cell" refers to a cell that is isolated and injected back into the same subject (recipient or host).

The invention thus also relates to ex vivo methods for generating dendritic, natural killer or B cell populations that present MHC-II and MHC-1B/E restricted peptides.

In one embodiment, an ex vivo method for generating a dendritic, natural killer, or B cell population that presents MHC-II and MHC-1B/E restricted peptides comprises:

a. optionally, reducing MHC-1a expression in immature dendritic cells, natural killer cells or B cells using an agent that inhibits TAP expression or activity,

b. loading HLA-DR and/or MHC-1b/E restricted peptides onto immature dendritic cells, and

c. maturing the loaded immature dendritic cells, natural killer cells or B cells.

In one embodiment, the immature dendritic cells are produced from monocytic dendritic cell precursor (MO-DC) precursors.

As used herein, the term "monocytic dendritic cell precursors" refers to monocytes and other bone marrow precursors (e.g., myeloid precursors). These cells can be isolated from any tissue in which they are located, especially lymphoid tissues such as spleen, bone marrow, lymph nodes and thymus. Monocytic dendritic cell precursors can be isolated from cord blood. Monocytic dendritic cell precursors can also be isolated from peripheral blood mononuclear cells or bone marrow samples by any technique known in the art. Monocytic dendritic cell precursors can also be isolated from frozen samples. Methods for isolating MO-DC precursors and immature dendritic cells from the various sources provided above, including blood and bone marrow, can be accomplished in a variety of ways. Typically, a population of cells is collected from an individual and enriched for MO-DC precursors. For example, mixed cell populations comprising MO-DC precursors can be obtained from peripheral blood by leukapheresis (leukapheresis), apheresis (apheresis), density centrifugation, differential lysis (differential lysis), filtration, antibody panning (e.g., flow cytometry, positive or negative selection), or preparation of buffy coat (buffy coat). In one embodiment, the MO-DC precursor is inactive, and thus, in one embodiment, the method selected must not activate the MO-DC precursor. For example, if antibody panning is selected to enrich the precursors of a cell population, the selected antibody must not activate the cells (e.g., by inducing influx of calcium ions, which may be the result of cross-linking of molecules on the surface to which the antibody binds). In general, in antibody panning, an antibody that eliminates macrophages, B cells, natural killer cells, T cells, and the like is used. The antibodies can also be used for positive selection of monocyte-like cells expressing CD 14.

In one embodiment, the MO-DC precursors and immature dendritic cells can be obtained from autologous PBMCs (peripheral blood mononuclear cells). In one embodiment, the MO-DC precursors and immature dendritic cells can be obtained from autologous tissue. In one embodiment, the MO-DC precursors and immature dendritic cells can be obtained from an HLA-matched healthy individual.

In one embodiment, the immature dendritic cells can be obtained from induced pluripotent stem cells (iPS). In one embodiment, the immature dendritic cells can be derived from CD34⁺Obtaining dendritic cell precursor. In one embodiment, the immature dendritic cells can be obtained from a human dendritic cell line. In one embodiment, the immature dendritic cells can be derived from CD34⁺Obtaining a dendritic cell precursor cell line. A non-limiting example of a cell line that can be used to generate immature dendritic cells is CD34⁺Human acute myeloid leukemia cell line (MUTZ-3), see, e.g., Masterson et al, (2002) Blood,100: 701-703.

In another embodiment, MO-DC precursors and immature dendritic cells can be obtained from an HLA-matched healthy individual for conversion to immature dendritic cells, maturation, activation, and administration to an HLA-matched subject in need thereof.

In one embodiment, the MO-DC precursor or CD34 will never be activated⁺The dendritic cell precursor-enriched cell population is cultured ex vivo or in vitro for differentiation, maturation and/or expansion.

Briefly, ex vivo differentiation generally involves culturing MO-DC precursors or CD34 in the presence of one or more differentiating agents⁺Dendritic cell precursors, orComprising unactivated MO-DC precursor or CD34⁺A cell population of dendritic cell precursors. Such agents typically comprise granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 4(IL-4), interleukin 6(IL-6), interleukin 3(IL-3), Stem Cell Factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3-L), or a combination thereof. Such agents may be used alone or in combination. For example, GM-CSF may be used alone or in combination with one or more cytokines, such as IL-4, IL-6, IL-3, SC, and/or Flt 3-L. In one embodiment, the unactivated MO-DC precursor or CD34⁺Dendritic cell precursors are differentiated to form immature dendritic cells capable of inducing activation and proliferation of a large number of T cells.

Suitable culture conditions for the production and maintenance of immature dendritic cell precursors are well known in the art. Such media include, but are not limited to

RPMI 1640、DMEM、X-VIVO

And similar products supplemented with cytokines. The culture medium may be supplemented with serum, amino acids, vitamins, divalent cations, etc., to promote differentiation of the cells into dendritic cells. In one embodiment, the dendritic cell precursors can be cultured in serum-free media. Such culture conditions may optionally exclude any animal-derived products. Typically, GM-CSF is added to the culture medium at a concentration of GM-CSF of about 2 to about 200ng/ml, or typically 20ng/ml, and IL-4 is added to the culture medium at a concentration of IL-4 of about 2 to about 200ng/ml, or typically 20 ng/ml. IL-6 is added to the medium at a concentration of about 2 to about 200ng/ml, or typically 20ng/ml, of IL-6. IL-3 is added to the culture medium at a concentration of about 2 to about 200ng/ml, or typically 20ng/ml, SCF is added to the culture medium at a concentration of about 10 to about 1000ng/ml, or typically 100ng/ml, SCF, and Flt3-L is added to the culture medium at a concentration of about 10 to about 1000ng/ml, or typically 100ng/ml, Flt 3-L. When differentiated to form immature dendritic cells, the precursors are typically epitopicallyShow a typical expression pattern of cell surface proteins seen in immature dendritic cells, e.g., the cells are usually CD14^-、HLA-DR⁺、CD11c⁺、CD83^-And express low levels of CD 86. Non-limiting examples of the production of immature dendritic cell precursors are described in example 2. At this stage, immature dendritic cells are able to capture soluble antigens through a specialized uptake mechanism.

In one embodiment, MHC-1a expression in immature dendritic cells or in dendritic cells is reduced by an agent that inhibits TAP expression or activity.

According to one embodiment, the immature dendritic cells or dendritic cells express a reduced level of major histocompatibility class 1a (MHC-1a) molecules on their surface. According to one embodiment, the immature dendritic cells or dendritic cells do not express major histocompatibility class 1a (MHC-1a) on their surface.

By "MHC class 1a presentation" is meant "classical" presentation by HLA-A, HLA-B and/or HLA-C molecules, whereas by MHC class Ib presentation is meant "non-classical" antigen presentation by HLA-E, HLA-F, HLA-G and/or HLA-H molecules.

Methods for inhibiting the expression of MHC-1a molecules are well known. For example, inhibition of the TAP transporter (transporter associated with antigen processing) results in decreased expression of MHC-1a molecules, thereby promoting expression of HLA-E molecules on the surface of dendritic cells.

Exemplary methods of inhibiting TAP transporters in the endoplasmic reticulum include, but are not limited to, CRISPR-CAS-9 technology, silencing RNA, DCs transfected with UL-10 viral proteins from CMV (cytomegalovirus), or using viral proteins.

Examples of viral genes or proteins that silence TAP expression include, but are not limited to, HSV-1ICP47 protein, varicella UL49.5 protein, cytomegalovirus US6 protein, or gamma herpes virus EBV BNLF2a protein, HIV nef protein.

Another approach is to use chemicals to inhibit the expression of MHC class 1a molecules without altering HLA-E expression on the surface of tolerogenic DCs. Examples of chemical products include, but are not limited to, 5 '-methyl-5' -thioadenosine (5 '-methyl-5' -thioadenosine) or leptomycin b (leptomycin b).

In one embodiment, the TAP inhibitor is RNA synthesized from pGem4Z vector containing the UL49.5 gene from BHV-1.

In one embodiment, the TAP inhibitor can be efficiently introduced into immature dendritic cells by electroporation. In another embodiment, the TAP inhibitor can be efficiently introduced into immature dendritic cells by transfection.

Depletion (depletion) of MHC-1a can be monitored by methods known in the art. For example, the antibodies can also be used to monitor immature dendritic cells or whether dendritic cells are MHC-1a^-/low。

In one embodiment, immature dendritic cells or dendritic cells can be loaded (or pulsed) in the presence of at least one predetermined antigen. In one embodiment, MHC-1a expression in immature dendritic cells or dendritic cells has been previously reduced. In another embodiment, MHC-1a expression in immature dendritic cells has not been previously reduced.

In one embodiment, the immature dendritic cells or dendritic cells present peptides or antigens that specifically bind to HLA-DR and/or MHC-1b/E molecules. Thus, in one embodiment, immature dendrites can be loaded (or pulsed) by contacting the immature dendritic cells or dendritic cells with a predetermined peptide or antigen before, after, or during maturation. In one embodiment, MHC-1a depleted immature dendritic cells or dendritic cells present peptides or antigens that specifically bind to HLA-DR and/or MHC-1b/E molecules. Thus, in one embodiment, MHC-1a depleted immature dendritic cells can be loaded (or pulsed) by contacting the immature dendritic cells or dendritic cells with a predetermined peptide or antigen before, after or during maturation.

Suitable predetermined antigens for use in the present invention may include any infectious disease associated antigen. Infectious disease associated antigens are described below and include, for example, HIV or SIV MHC-Ib/E peptides or antigens.

Methods for contacting dendritic cells with antigen are generally known in the art (see Steel and Nutman, J.Immunol.160:351-60 (1998); Tao et al, J.Immunol.158:4237-44 (1997); Dozmorov and Miller, Cell Immunol.178:187-96 (1997); Inaba et al, JExp Med.166:182-94 (1987); Macatonia et al, J Exp Med.169:1255-64 (1989); De Bruijn et al, Eur.J.Immunol.22:3013-20 (1992); the disclosure of which is incorporated herein by reference). In general, immature dendritic immature cells obtained by the method of the present invention can be cultured in the presence of a predetermined antigen under suitable culture conditions, as described above. Optionally, the immature dendritic cells can be mixed with a predetermined antigen in a typical dendritic cell culture medium with or without GM-CSF and/or a maturation agent. After at least about 10 minutes to about 2 days of incubation with the antigen, the antigen can be removed and the medium supplemented with a maturation agent. GM-CSF and other cytokines (e.g., IL-4) may also be added to the culture medium.

In one embodiment, immature dendritic cells or dendritic cells can be transfected with a plasmid encoding an MHC-1b/E molecule. In another embodiment, immature dendritic cells or dendritic cells can be transfected with a plasmid encoding a peptide-MHC-lb/E complex.

In one embodiment, immature dendritic cells (optionally, MHC-1a depleted and previously loaded) can be matured with a maturation agent.

In one embodiment, the immature dendritic cells can be matured to form mature dendritic cells. Mature dendritic cells lose the ability to take up antigen and the cells show up-regulation of expression of costimulatory cell surface molecules and secretion of various cytokines. For example, mature dendritic cells can express higher levels of HLA-DR and/or MHC-1b/E antigens and are generally identified as MHC-1a^-/low、CD80⁺、CD83⁺And CD86⁺. More MHC expression leads to increased antigen density on the DC surface, while upregulation of the co-stimulatory molecules CD80 and CD86 enhances the T cell activation signal by the counterpart of the co-stimulatory molecule (e.g., CD28 on T cells).

Methods for preparing mature dendritic cells are well known in the art. For example, immature dendritic cells can be matured by contacting the immature dendritic cells with an effective amount or concentration of a dendritic cell maturation agent. Dendritic cell maturation agents can include, for example, BCG, IFN γ, LPS, TNF α, IL-1 β, IL-6, PGE2, Poly I: C, TLR 7/8-ligand, or a combination thereof.

For example, immature DCs are typically contacted with an effective amount of LPS for about 1 hour to about 48 hours, preferably 24 hours. Immature dendritic cells can be cultured and matured under suitable maturation culture conditions. Suitable tissue culture media include

RPMI 1640、DMEM、X-VIVO

And the like. The tissue culture medium may be supplemented with amino acids, vitamins, cytokines such as GM-CSF, divalent cations, and the like, to promote cell maturation.

For exemplary purposes, dendritic cells can be matured in the presence of IL-1 β, IL-6, PGE2, TNF- α, LPS, Poly I: C. Typically, about 2ng/ml IL-1. beta. is used, 30ng/ml IL-6, 1. mu.g/ml PGE2, 10ng/ml TNF-. alpha., 250ng/ml LPS, 150ng/ml Poly I: C.

Maturation of dendritic cells can be monitored by methods known in the art for dendritic cells. Cell surface markers can be detected in assays well known in the art, such as flow cytometry, immunohistochemistry, and the like. The cells may also be monitored for cytokine production (e.g., by ELISA, another immunoassay, or by using an oligonucleotide array). Mature DCs of the invention also lose the ability to take up antigen, which can be analyzed by uptake assays well known to those of ordinary skill in the art.

Thus, the invention also relates to a population of mature dendritic cells presenting MHC-II and MHC-1b/E restricted peptides obtainable or obtained by the ex vivo method as described above.

In addition to dendritic cells, other immune cell types can be used to obtain mature immune cell populations that present MHC-II and MHC-1b/E restricted peptides or antigens.

In one embodiment, the CD8 vaccine is an active vaccine that is an ex vivo generated population of natural killer cells presenting at least one MHC-1b/E restricted antigen and at least one MHC-II restricted antigen.

In one embodiment, the natural killer cell is the K562 cell line.

In some embodiments, natural killer cells are modified to express MHC-1 b/E.

In another embodiment, the CD8 vaccine is an active vaccine that is an ex vivo generated natural B population that presents at least one MHC-1B/E restricted antigen and at least one MHC-II restricted antigen.

In one embodiment, the B cell is a cell line.

In some embodiments, B cells are modified to express MHC-1B/E.

Thus, the invention also relates to a population of mature natural killer cells presenting MHC-II and MHC-1b/E restricted peptides obtainable or obtained by the ex vivo method as described above.

The invention also relates to a population of mature B cells presenting MHC-II and MHC-1B/E restricted peptides obtainable or obtained by the ex vivo method as described above.

According to another embodiment of the invention, the CD8 vaccine is an MHC-1b/E restricted CD8 produced ex vivo⁺A population of T cells.

In one embodiment, the MHC-1b/E restricted CD8 is generated ex vivo⁺The T cell population is a passive vaccine.

In one embodiment, MHC-1b/E restricted CD8⁺The T cell population recognizes MHC-1b/E restricted infectious disease associated antigens.

The invention therefore also relates to a method for generating MHC-1b/E restricted CD8⁺Ex vivo methods for T cell populations.

In one embodiment, MHC-1b/E restricted CD8 is generated⁺An ex vivo method of T cell population comprising:

a. presentation of MHC-1 b/E-restricted peptides to enable production of MHC-1 b/E-restricted CD8⁺Culturing naive CD8 in the presence of dendritic, natural killer, or B cell populations of T cells⁺T cells, and

b. amplification of MHC-lb/E restricted CD8⁺T cells.

In one embodiment, CD8 is incorporated into a CD-shaped CD⁺T cells, preferably primary CD8⁺T cells, isolated from a blood sample by any technique known in the art. In one embodiment, CD8 is incorporated into a CD-shaped CD⁺T cells, preferably primary CD8⁺T cells, isolated from PBMCs (peripheral blood mononuclear cells) by flow cytometry. In one embodiment, CD8⁺T cells, preferably primary CD8⁺T cells, which can be isolated from frozen PBMCs. In one embodiment, CD8⁺The T cells are allogeneic T cells, preferably allogeneic naive T cells. In another embodiment, CD8⁺The T cells are autologous T cells, preferably autologous naive T cells. T cell isolation or purification can be achieved by positive or negative selection, including but not limited to the use of antibodies against CD8, CD56, CD57, CD45RO, CD45RA, CCR7, and the like. For example, the original CD8⁺T cell isolation can be performed in a one-step or two-step procedure. The two-step procedure may include a first step in which naive T cells are enriched by depletion of non-naive T cells, and a second step in which the enriched naive T cells are labeled with a CD8 antibody for subsequent positive selection.

In one embodiment, CD8 is incorporated into a CD-shaped CD⁺T cells, preferably primary CD8⁺T cells, more preferably autologous naive CD8⁺T cells are stimulated with dendritic cells pulsed with peptides or antigens (e.g., tolerogenic dendritic cells pulsed with MHC-Ib/E antigens) in the presence of a stimulating agent. After stimulation, the cells may be washed, for example with PBS, and may be stained with anti-CD 8 antibody and sorted using MHC peptide pentamers. Purifying CD8⁺T cells are enriched and can be used for the following activation steps.

In one embodiment, CD8⁺T cells, preferably primary CD8⁺T cells, more preferably autologous naive CD8⁺T cells and MHC-lb/E-restricted peptide-presenting dendritic cellsThe cells were incubated together. In one embodiment, the MHC-lb/E-restricted peptide-presenting dendritic cells also present an MHC-II restricted peptide.

In one embodiment, the dendritic cells do not express MHC-1a molecules on their surface. In one embodiment, the dendritic cell expresses less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of MHC-1a molecules on its surface (i.e., relative to all MHC molecules expressed on the surface of the dendritic cell). In one embodiment, the dendritic cell expresses at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of MHC-1b molecules on its surface. In one embodiment, the dendritic cells express only MHC-1b molecules on their surface.

In one embodiment, the dendritic cells express MHC-II molecules on their surface. In one embodiment, the dendritic cells express MHC-II molecules and MHC-Ib molecules on their surface.

In one embodiment, CD8 is incorporated into a CD-shaped CD⁺T cells, preferably primary CD8⁺T cells, more preferably autologous naive CD8⁺T cells contacted with tolerogenic dendritic cells as described above. Thus, at the end of the culture, the T cells are the inhibitor MHC-1b/E restricted CD8⁺T cells and can induce immune tolerance.

In one embodiment, for generating the MHC-1b/E restricted CD8 of the invention⁺The culturing of the T cells is performed for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or more. In one embodiment, for generating the MHC-1b/E restricted CD8 of the invention⁺The culturing of the T cells is performed for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or more. In one embodiment, for generating the MHC-1b/E restricted CD8 of the invention⁺The culturing of the T cells is performed for at least 1 month, at least 2 months, at least 3 months, or more.

In one embodiment, the methods described herein for generating autologous MHC-1b/E restricted CD8⁺Methods of using MHC-Ib/E natural killer cells or MHC-Ib/E B generations in T cell populationsFor MHC-Ib/E dendritic cells.

In one embodiment, the MHC-1b/E restricted CD8 is generated ex vivo⁺T cell populations are isolated by flow cytometry based on their ability to bind to a particular HLA-E antigen or peptide (e.g., a particular tetramer).

In one embodiment, the isolated MHC-1b/E restricted CD8 thus obtained is then subjected to⁺The T cell population is expanded ex vivo by culturing the cells in the presence of at least one T cell activator. Examples of T cell activators include, but are not limited to, activators that are subsequently completed. Alternatively, other examples of T cell activators that may be used during expansion include, but are not limited to, mitogens (mitogens), such as PMA/ionomycin (ionomycin), superantigens (super-antigen), anti-CD 3 antibodies, and the like. Preferably, the anti-CD 3 monoclonal antibody is coated. In one embodiment, the T cell activator may be used in the presence of feeder cells (feeder cells).

Feeder cells include, but are not limited to, Δ CD3 cells (T cell depleted helper cells), irradiated PBMCs, irradiated DCs, artificial APCs (antigen presenting cells), Sf9 cells, insect cells, PBMC or B cell pools from different subjects, KCD40L cells, EBV transformed B cell lines, and EBV transformed Lymphoblast Cells (LCLs).

In another embodiment, the isolated MHC-1b/E restricted CD8 so obtained⁺The T cell population is then expanded ex vivo by culturing these cells in the presence of antigen-specific T cell activators (e.g., anti-CD 3/CD28 antibodies, PMA/iono, cytokines, etc.). In one embodiment, the antigen-specific T cell activator may be used in the presence of feeder cells as described above.

In one embodiment, the ex vivo MHC-1 b/E-restricted CD8 for amplifying the invention⁺The culturing of the T cells is performed during at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, or more. In one embodiment, the ex vivo MHC-1 b/E-restricted CD8 for amplifying the invention⁺T cell culture at least 1Weekly, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or more weeks. In one embodiment, the invention is used to amplify the ex vivo MHC-1b/E restricted CD8⁺The culturing of the T cells is performed during at least 1 month, at least 2 months, at least 3 months, or more.

Thus, the invention also relates to MHC-1b/E restricted CD8+ T cells obtainable or obtained by the ex vivo method as described above.

In one embodiment, the infectious disease associated antigen is a pathogen specific antigen.

The "pathogen-specific antigen" may be selected from any organism known to be pathogenic or against which an immune response is to be elicited. Such pathogen-specific antigens are well known in the art and thus a person of ordinary skill in the art can select an appropriate antigen. The antigen is selected according to the type of infectious disease to be treated. For example, when the disease to be prevented or treated is acquired immunodeficiency syndrome (AIDS) or Simian Immunodeficiency Virus (SIV) infection, the CD8 vaccine contains or encodes an antigen from HIV or SIV, respectively.

In one embodiment, the pathogen-specific antigens described herein may be derived from any human or animal pathogen. In one embodiment, the pathogen-specific antigen is a viral pathogen, a bacterial pathogen, or a parasite, and the antigen may be a protein derived from a viral pathogen, a bacterial pathogen, or a parasite. In one embodiment, the parasite may be an organism or a disease caused by an organism. For example, the parasite may be a protozoan organism, a disease causing protozoan organism, a helminth organism (worm) or a worm (work), a disease caused by a helminth organism, an ectoparasite (ectoparasite) or a disease caused by an ectoparasite.

In one embodiment, the pathogen-specific antigen described herein can be an antigen from a viral pathogen. In one embodiment, the pathogen-specific antigen may be an antigen from a bacterial pathogen. In one embodiment, the pathogen-specific antigen may be an antigen from a parasitic organism. In another embodiment, the pathogen-specific antigen may be an antigen from a helminth organism.

In one embodiment, the pathogen-specific antigen described herein is non-infectious.

In one embodiment, when recombinant viruses or bacteria are used to express the antigen, these are preferably inactivated microorganisms.

In one embodiment, the pathogen-specific antigen described herein is a particulate antigen (particulate antigen).

In one embodiment, the pathogen-specific antigens described herein may result from the expression of viral nucleic acid sequences advantageously comprised in a suitable recombinant microorganism. In one embodiment, the recombinant microorganism is CMV, preferably a CMV vector as described above. In another embodiment, the recombinant microorganism is a bacterium, preferably a bacterium different from the non-pathogenic bacterium described above.

In one embodiment, the pathogen-specific antigens described herein can be codon optimized. Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and enhanced expression of antigens can be achieved by altering these codons to correspond to codons commonly used in the subject of interest (e.g., a human). For example, rare codons used in HIV proteins can be mutated to those frequently found in highly expressed human genes (Andre et al (1998) J Virol 72, 1497-1503).

In one embodiment, the pathogen-specific antigen described herein can be a consensus or chimeric antigen (mosaic antigen) containing sequence fragments from different pathogen strains.

In one embodiment, the particulate antigen described herein is a viral antigen.

In one embodiment, the particle antigen is selected from the group consisting of a viral particle, a recombinant viral particle, a virus-like particle, a recombinant viral particle, a polymeric microparticle presenting one or more viral peptides or epitopes on its surface, a conjugated viral protein, and a concatemeric viral protein.

In one embodiment, the particulate antigen described herein may be one or more viral proteins or peptides, recombinant or non-recombinant, and present in the form of a conjugate or concatemer.

In one embodiment, the pathogen-specific antigens described herein may be derived from Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), herpes simplex virus, hepatitis b virus, hepatitis c virus, papilloma virus, Plasmodium (Plasmodium paranite), and mycobacterium tuberculosis.

In one embodiment, the pathogen-specific antigen described herein is an HIV or SIV antigen. In one embodiment, the HIV or SIV antigen is selected from the group consisting of any HIV or SIV strain.

In one embodiment, the HIV or SIV antigen is selected from the group consisting of gag, tat, pol, vif, nef and env antigens.

In one embodiment, the pathogen-specific antigen described herein is derived from an immunogenic apoptotic body (apoptotic body) from an infected cell or from a tissue lysate.

The infected cells may be derived from tissue biopsy or from expansion of circulating infected cells. The infected cell may be infected by a virus, a bacterium, a parasitic organism, or a helminth organism.

Immunogenic apoptotic bodies from infected cells can be produced, for example, by anthracyclines (anthracyclines) including doxorubicin (doxorubicin), daunorubicin (daunorubicin), idarubicin (idarubicin), and mitoxantrone (mitoxantrone); infected cells that release apoptotic bodies that have been treated with oxaliplatin (oxaliplatin), UVC, UVB, or radiation.

Examples of tissue lysates include, but are not limited to, lymph nodes, synovial fluid, or inflammatory tissue lysates.

In one embodiment, the pathogen-specific antigen described herein is an antigen from an HIV or SIV source.

In one embodiment, the immunogenic body is obtained from HIVInfected CD4⁺T cells.

In one embodiment, the pathogen-specific antigen described herein is an antigen from an HIV source.

In one embodiment, the pathogen-specific antigen described herein is an HIV antigen.

Due to the great variability in the HIV genome caused by mutations, recombinations, insertions and/or deletions, HIV has been classified into groups, subgroups, types, subtypes and genotypes. There are two major HIV groups (HIV-1 and HIV-2) and many subgroups because of the continuous variation in the HIV genome. The major differences between each group and each subgroup are related to the viral envelope. HIV-1 is classified into a major group (M), which is divided into at least nine genetically distinct subtypes. These are subtypes A, B, C, D, F, G, H, J and K. There are also many other subtypes (e.g., CRF) that result from in vivo recombination of previous subtypes. In one embodiment, the HIV antigen is associated with a particular HIV group, subgroup, type, subtype, or combination of subtypes.

In one embodiment, the HIV virus is HIV-1 or HIV-2, preferably HIV-1. In another embodiment, the HIV-1 virus is from group M and subtype B (HXB 2).

In one embodiment, the HIV antigen is an inactivated whole HIV virus.

As used herein, "inactivated whole HIV" means a whole HIV particle that has been inactivated and no longer infectious.

In one embodiment, the HIV antigen is an autologous HIV antigen. In another embodiment, the HIV antigen is not an autologous HIV antigen. In one embodiment, the HIV antigen is made from an inactivated autologous HIV virus.

As used herein, "antigen made from inactivated autologous HIV virus" refers to an antigen comprising or consisting of an HIV virus that infects a human to be treated and is suitably inactivated for safe therapeutic administration to the human. Thus, in practice, for the preparation of the vaccine composition of the invention, the CD4 of the human to be treated, more particularly of said human, is taken from⁺HIV virus was isolated from T cells. Will thus separateThe HIV virus of (1) is cultured and inactivated.

In one embodiment, the HIV antigen is selected from the group consisting of HIV gag, HIV env, HIV rev, HIV tat, HIV nef, HIV pol, and HIV vif.

In one embodiment, the HIV antigen comprises one or more epitopes of HIV gag, HIV env, HIV rev, HIV tat, HIV nef, HIV pol, and HIV vif proteins.

In one embodiment, the HIV antigen comprises at least HIV gag and/or HIV pol proteins. Alternatively or additionally, the HIV virus-derived antigen may comprise one or more proteins encoded by gag, such as the capsid protein (p24) and the matrix protein (p1), and/or one or more proteins encoded by pol, such as integrase (integrase), reverse transcriptase and protease.

In one embodiment, the pathogen-specific antigen described herein is an MHCIb/E binding antigen. In one embodiment, the pathogen-specific antigen is an MHCIb/E binding peptide.

In one embodiment, the pathogen-specific antigen described herein is an HIV or SIV-derived MHCIb/E binding peptide. In one embodiment, the pathogen-specific antigen is an HIV or SIV-derived MHCIb/E binding antigen.

In one embodiment, the HIV-derived MHCIb/E binding antigen described herein is selected from the group of SEQ ID NO 1 to SEQ ID NO 4. In one embodiment, the HIV-derived MHCIb/E binding peptide is selected from the group of SEQ ID NO 1 to SEQ ID NO 4.

In one embodiment, the HIV-derived MHCIb/E binding antigen has the amino acid sequence RMYSPVSIL (SEQ ID NO: 1). In one embodiment, the HIV-derived MHCIb/E binding antigen has the amino acid sequence PEIVIYDYM (SEQ ID NO: 2). In one embodiment, the HIV-derived MHCIb/E binding antigen has the amino acid sequence TALSEGATP (SEQ ID NO: 3). In one embodiment, the HIV-derived MHCIb/E binding antigen has the amino acid sequence RIRTWKSLV (SEQ ID NO: 4).

As used herein, the term "interferon-alpha" (IFN- α) or "interferon- α" refers to a family of more than 20 related but distinct members encoded by a cluster on chromosome 9 and all binding to the same IFN receptor. Of these, IFN-. alpha.2 has 3 recombinant variants (. alpha.2a,. alpha.2b,. alpha.2c), depending on the cell source, whereas IFN-. alpha.2b is the major variant in the human genome. There is evidence that each subtype has a different binding capacity for IFNAR, regulating signal transduction events and biological effects in target cells.

In one embodiment, the interferon- α blockers described herein are agents that neutralize circulating IFN- α and/or block IFN- α signaling, and/or deplete cells producing IFN- α, and/or block IFN- α production.

In one embodiment, the interferon- α blockers described herein comprise at least one agent selected from the group consisting of: an agent that neutralizes circulating IFN- α and/or blocks IFN- α signaling, and/or an agent that depletes cells producing IFN- α, and/or an agent that blocks IFN- α production.

In one embodiment, the agent that neutralizes circulating IFN- α and/or blocks IFN- α signaling, and/or the agent that depletes IFN- α producing cells, and/or the agent that blocks IFN- α production is an IFN- α antagonist.

In some embodiments, wherein the interferon- α blocker is selected from the group consisting of: an agent that neutralizes circulating interferon-alpha, an agent that blocks interferon-alpha signaling, an agent that depletes cells that produce IFN-alpha, and/or an agent that blocks IFN-alpha production, wherein the agent neutralizing circulating interferon alpha is selected from the group comprising an active anti-IFN-alpha vaccine (including an antimeron) or a passive anti-IFN-alpha vaccine (including an anti-IFN-alpha antibody or an anti-IFN-alpha hyperimmune serum), wherein the blocker of interferon-alpha signalling is selected from the group of anti-type I interferon R1 or R2 antibodies or from endogenous modulators of interferon-alpha (including SOSC1 or arene receptors), wherein the agent that depletes IFN- α producing cells is an agent that depletes plasmacytoid dendritic cells (pDCs), and wherein the agent that blocks IFN- α production is an agent that blocks IFN- α production by pDC.

In some embodiments, the interferon-alpha blocker is an agent that neutralizes circulating alpha interferon selected from the group consisting of an active anti-IFN-alpha vaccine (including anti-interferon) or a passive anti-IFN-alpha vaccine (including anti-IFN-alpha antibodies or anti-IFN-alpha hyperimmune serum), and wherein the blocker of interferon-alpha signaling is selected from the group consisting of anti-type I interferon R1 or R2 antibodies, SOSC1, and arene receptors.

As used herein, the term "interferon-alpha antagonist" refers to a substance that interferes with or inhibits the biological activity of IFN-alpha. As used herein, "IFN-. alpha.0 biological activity" refers to any activity resulting from the binding of IFN-. alpha.to its receptor IFNAR (IFNAR1/IFNAR2 heterodimer). For example, such binding can activate the JAK-STAT signaling cascade and trigger tyrosine phosphorylation of many proteins including JAK, TYK2, STAT proteins. Thus, a blocker of interferon signaling may neutralize the immobilization of INF- α to its receptor and/or block the signaling cascade induced by IFN- α binding to its receptor. In some embodiments, the IFN- α antagonist is selected from the group of an active anti-IFN- α vaccine (e.g., antimferon) or a passive anti-IFN- α vaccine (e.g., anti-IFN- α antibody or anti-IFN- α hyperimmune serum). See, e.g.

Et al (2018). Cytokine Growth Factor Rev 40: 99-112.

In one embodiment, the agents described herein that neutralize circulating IFN- α are IFN- α ligand inhibitors.

In one embodiment, the agent that neutralizes circulating IFN- α is an anti-IFN- α antibody. Examples of anti-IFN- α antibodies include, but are not limited to, monoclonal antibody (Sifalimumab), monoclonal antibody (Rontalizumab), MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-11 clone, MMHA-13 clone, and MMHA-17 clone.

In one embodiment, the agent that neutralizes circulating IFN- α is an anti-IFN- α hyperimmune serum.

In one embodiment, the agent that neutralizes circulating IFN- α described herein is an antidiferon, such as IFN- α -Kinoid.

In one embodiment, the agent that neutralizes circulating interferon alpha is a soluble receptor that binds IFN-alpha.

In one embodiment, the agent that neutralizes circulating IFN- α does not neutralize circulating type III interferon.

In one embodiment, the agent that blocks IFN- α signaling described herein is an IFNAR antagonist.

In one embodiment, the agent that blocks IFN- α signaling is an IFNAR1 antagonist. In another embodiment, the agent that blocks IFN- α signaling is an IFNAR2 antagonist.

In one embodiment, the agent that blocks IFN- α signaling is an antibody that binds to IFNAR1 or IFNAR 2.

In one embodiment, the agent that blocks IFN- α signaling is an agent that antagonizes the type I IFN signaling pathway.

In one embodiment, the agent that blocks IFN- α signaling can be an inhibitor of the type I IFN signaling pathway. Inhibitors of the type I IFN signaling pathway are well known in the art and include, without limitation, JAK1/2 inhibitors and STAT inhibitors. Thus, in one embodiment, the agent that blocks IFN- α signaling is selected from a JAK1/2 inhibitor and a STAT inhibitor. Non-limiting examples of JAK1/2 inhibitors include Ruxolitinib (Ruxolitinib), Tofacitinib (Tofacitinib), and Baricitinib (Baricitinib).

In one embodiment, the agent that blocks IFN- α signaling can be an endogenous negative modulator of the type I IFN signaling pathway. Endogenous negative modulators are well known in the art and include, without limitation, SOCS1/3, FOXO3, arene receptor (AhR), or other negative modulators. Thus, in one embodiment, the agent that blocks interferon signaling is selected from SOCS1/3, FOXO3, or an arene receptor (AhR).

In one embodiment, the agent that blocks IFN- α signaling is a PAS (pasylilated) antagonist. Antagonists of the PASy of type I IFNs are known in the art, see e.g. nganu-Makamdop et al (2018).

In one embodiment, an IFN- α antagonist described herein is an agent that depletes IFN- α producing cells.

As used herein, the term "IFN- α producing cell" refers to any cell that produces IFN- α. In particular, plasmacytoid dendritic cells (pDCs) are well known in the art as the primary producers of IFN- α. Thus, in one embodiment, an agent that depletes IFN- α producing cells depletes pDC.

In one embodiment, the agent that depletes IFN- α producing cells is an antibody. In one embodiment, the antibody depletes pDC, such as an anti-CD 123 antibody (i.e., anti-IL-3 RA).

In one embodiment, an IFN- α antagonist described herein is an agent that blocks IFN- α production.

In one embodiment, the agent that blocks IFN- α production is an antibody. In one embodiment, the antibody blocks the production of IFN- α by pDC. The antibody may be, for example, an anti-BDCA 2(Blood DC Antigen 2) antibody.

In one embodiment, the agent that blocks interferon signaling does not block type III interferon signaling.

In some embodiments, the interferon- α blocker is selected from the group consisting of:

an anti-IFN-alpha antibody, preferably a Cefamumab, a monoclonal antibody of Longilimumab, a clone MMHA-1, a clone MMHA-2, a clone MMHA-6, a clone MMHA-8, a clone MMHA-9, a clone MMHA-11, a clone MMHA-13 or a clone MMHA-17,

an anti-IFN-alpha hyperimmune serum,

-antimferon, preferably IFN-. alpha. -Kinoid,

a soluble receptor that binds IFN-alpha,

-an IFNARl or IFNAR2 antagonist, preferably an antibody that binds to IFNARl or IFNAR2,

an inhibitor of the type I IFN signaling pathway selected from STAT inhibitors and JAK1/2 inhibitors, such as ruxotinib, tofacitinib or barrettinib,

-an endogenous negative modulator of the type I IFN signaling pathway selected from SOCS1/3, FOXO3, an arene receptor (AhR) or another negative modulator,

-a PASylated antagonist,

an antibody that depletes pDC, preferably an anti-CD 123 (i.e. anti-IL-3 RA) antibody,

antibodies blocking the production of IFN- α by pDC, preferably anti-BDCA 2(Blood DC Antigen 2) antibodies.

As used herein, the term "type III interferon," also known as interferon- λ (IFN- λ), refers to a naturally occurring and/or recombinant type III interferon- λ family of cytokines. There are four IFN-lambda members in humans, IFN-lambda 1/IL-29, IFN-lambda 2/IL-28A, IFN-lambda 3/IL-28B, IFN-lambda 4.

In one embodiment, the type III interferon is IFN- λ.

In one embodiment, the IFN- λ comprises at least one IFN- λ subtype (e.g., IFN- λ 1, IFN- λ 2, IFN- λ 3, IFN- λ 4).

In one embodiment, the IFN- λ is selected from the group of IFN- λ 1, IFN- λ 2, IFN- λ 3, IFN- λ 4, or combinations thereof.

In one embodiment, human IFN-. lambda.1 has the following accession number NP-742152.1. In one embodiment, human IFN- λ 2 has the following accession number NP _ 742150.1. In one embodiment, human IFN- λ 3 has the following accession No. NP _001333866.1 (isoform 1) or NP _742151.2 (isoform 2). In one embodiment, human IFN- λ 4 has the following accession number NP _ 001263183.2.

In one embodiment, the interferon- λ is IFN- λ 1. In one embodiment, the interferon- λ is IFN- λ 2. In one embodiment, the interferon- λ is IFN- λ 3. In one embodiment, the interferon- λ is IFN- λ 4.

In one embodiment, interferon- λ is chemically modified to improve certain properties, such as serum half-life. In one embodiment, the interferon- λ of the present invention is pegylated (i.e., the interferon- λ is covalently linked to poly (ethylene glycol), etc.). Methods for producing pegylated proteins are well known in the art, see, e.g., Chapman A et al, 2002, Advanced Drug Delivery Reviews 54: 531-545.

In one embodiment, interferon- λ is a functional mimetic of IFN- λ 1. In one embodiment, interferon- λ is a functional mimetic of IFN- λ 2. In one embodiment, interferon- λ is a functional mimetic of IFN- λ 3. In one embodiment, interferon- λ is a functional mimetic of IFN- λ 4.

As used herein, the term "functional mimetic" means a molecule that has the same or similar biological effect as a naturally occurring protein. For example, an interferon- λ functional mimetic can activate the interferon- λ receptor and drive transcription of an IFN-stimulating gene.

In one embodiment, the interferon- λ functional mimetic is a fragment of IFN- λ 1. In one embodiment, the interferon- λ functional mimetic is a fragment of IFN- λ 2. In one embodiment, the interferon- λ functional mimetic is a fragment of IFN- λ 3. In one embodiment, the interferon- λ functional mimetic is a fragment of IFN- λ 4.

In one embodiment, the interferon- λ functional mimetic is an antibody. Such interferon- λ functionally mimetic antibodies can elicit the same or similar biological effects as naturally occurring proteins. For example, antibodies can bind to an epitope on the interferon-lambda receptor, activate receptor signaling and drive transcription of IFN-stimulating genes. The heterodimeric receptor complex of interferon- λ (IFNLR) comprises IFNLR1(IFNLRA, IL-28RA) and IL10R2(IL-10 RB). IFNLR1 confers ligand specificity and enables receptor assembly, whereas IL10R2 is common to IL-10 family members and is essential for signaling.

In one embodiment, the interferon- λ derivative is a small molecule chemical entity (e.g., a chemical entity having a molecular weight of less than 900 daltons). Methods of screening chemical libraries to identify small molecule chemical entities that are potential drug candidates are known in the art. For example, chemical libraries can be tested in ligand-receptor binding assays.

In one embodiment, the agent that stimulates type III interferon production is an agent that stimulates a pattern-recognition receptor (PRR).

In one embodiment, the agent that stimulates production of type III interferon is an agent that stimulates a Pattern Recognition Receptor (PRR).

"Pattern-recognition receptors" mainly include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-1-like receptors (RLRs), and C-type lectin receptors (CLRs). They recognize different microbial signatures or host-derived danger signals and trigger immune responses, such as the production of interferons.

In one embodiment, the agent that stimulates type III interferon production comprises a toll-like receptor (TLR) ligand (e.g., TLR3, TLR5, TLR7/8, and TLR9), RIG-1 ligand, and MDA-5 ligand.

In one embodiment, the agent that stimulates production of type III interferon comprises poly I: C, CpG and/or Tat protein.

In one embodiment, the agent that stimulates production of type III interferon may also induce production of type I interferon.

The invention further relates to a combination comprising:

1) comprising a first portion of a CD8 vaccine specific for at least one infectious disease associated antigen as described herein,

2) optionally, a second moiety comprising an interferon- α blocker as described herein, and

3) a third portion comprising a type III interferon described herein and/or an agent that stimulates production of a type III interferon.

The invention also relates to a kit comprising at least 2 parts:

In one embodiment, a CD8 vaccine specific for at least one infectious disease associated antigen is included in the composition.

In one embodiment, the composition consists essentially of a CD8 vaccine specific for at least one infectious disease associated antigen.

As used herein, with respect to a composition, "consisting essentially of … …" means that the CD8 vaccine is the only therapeutic or biologically active agent in the composition.

In one embodiment, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient.

As used herein, the term "excipient" refers to any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In general, the nature of the excipient will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as vehicles. For solid compositions (e.g., in powder, pill, tablet or capsule form), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the 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 and the like, for example, sodium acetate or sorbitan monolaurate. For human administration, the formulations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory agencies such as the FDA office or EMA. In one embodiment, the excipient is an adjuvant, stabilizer, emulsifier, thickener, preservative, antibiotic, organic or inorganic acid or salt thereof, saccharide, alcohol, antioxidant, diluent, solvent, filler, binder, adsorbent, buffer, chelating agent, lubricant, colorant, or any other component.

By "pharmaceutically acceptable" is meant that the ingredients of the pharmaceutical composition are compatible with each other and not deleterious to the subject to which they are administered. Examples of pharmaceutically acceptable excipients include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof.

Pharmaceutically acceptable excipients that may be used in the pharmaceutical combination of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

In one embodiment, the composition is a vaccine composition. In one embodiment, the vaccine composition further comprises at least one adjuvant.

In one embodiment, a CD8 vaccine specific for at least one infectious disease associated antigen is included in the medicament.

In one embodiment, when the CD8 vaccine according to the invention comprises an infectious disease associated antigen and a non-pathogenic bacterium, the infectious disease associated antigen and the non-pathogenic bacterium are two separate and distinct components comprised as a mixture in a pharmaceutical composition. In another embodiment, when the CD8 vaccine according to the invention comprises an infectious disease associated antigen and a non-pathogenic bacterium, the infectious disease associated antigen and the non-pathogenic bacterium are the same component comprised in the pharmaceutical composition.

In one embodiment, the CD8 vaccine is a composition, a pharmaceutical composition, or a medicament, wherein the CD8 vaccine is conjugated to a delivery vehicle.

In one embodiment, the CD8 vaccine comprises at least one infectious disease associated antigen and a non-pathogenic bacterium, wherein the at least one infectious disease associated antigen and/or the non-pathogenic bacterium is conjugated to a delivery vehicle. In one embodiment, at least one infectious disease associated antigen is conjugated to a delivery vehicle. In one embodiment, the non-pathogenic bacteria are conjugated to a delivery vehicle. In one embodiment, the at least one infectious disease associated antigen and the non-pathogenic bacteria are conjugated to a delivery vehicle.

The term "conjugated" means that the infectious disease associated antigen and/or the non-pathogenic bacteria are physically or chemically coupled, adhered, absorbed or encapsulated onto the delivery vehicle. Examples of conjugation include, but are not limited to, covalent attachment and electrostatic complexation (electrostatic complexation). The terms "complexed", "complexed with … …" and "conjugated" are used interchangeably herein. In one embodiment, more than one copy or type of infectious disease associated antigen is conjugated to the delivery vehicle. In one embodiment, more than one copy or type of non-pathogenic bacteria is conjugated to the delivery vehicle.

Delivery vehicles are well known in the art. For example, the delivery vehicle may be selected from the group consisting of cationic lipids, liposomes, cochleates, virosomes, immunostimulatory complexes

Microparticles, microspheres, nanospheres, unilamellar vesicles (LUVs), multilamellar vesicles, creamy bodies (emulsomes) and polycationic peptides, lipoplexe, polyplexe, lipopleplexe, water-in-oil (W/O) emulsions, oil-in-water (O/W) emulsions, water-in-oil-in-water (W/O/W) multiple emulsions, microemulsions, nanoemulsions, micelles, dendrimers (dendrimers), virosomes, virus-like particles, polymeric nanoparticles (e.g., nanobeads, nanospheres or nanocapsules), polymeric microparticles (e.g., microspheres or microcapsules), chitosan, poly (lactic acid) (PLA) polymers, poly (lactic acid-co-glycolide) (PLGA) polymers, cyclodextrins, vesicles (niosomes), or polycations

And optionally, a pharmaceutically acceptable carrier. In one embodiment, the delivery vehicle is in a form suitable for oral administration, injection, topical administration, or rectal administration.

In one embodiment, the CD8 vaccine is conjugated to a nanoparticle, such as a nanobead, nanosphere, or nanocapsule. Preferably, the diameter of the nanoparticles is between 50 and 300nm, more preferably between 70 and 200nm, even more preferably between 100 and 150 nm.

Microfold cells (or M-cells) are found in gut-associated lymphoid tissue (GALT) of Peyer's patches (Peyer's disease) in the small intestine and mucosa-associated lymphoid tissue (MALT) in other parts of the gastrointestinal tract. These cells are known to initiate mucosal immune responses.

In some embodiments, the delivery vehicle is coated with or conjugated to a molecule that enhances delivery to M cells (e.g., a lectin or peptide).

M cells express a specific carbohydrate moiety (. alpha. -L-fucose) on the apical surface (apical surface). Lectin subtypes, such as Jingdou lectin 1(Ulex europaeus agglutinin 1, UEA-1) and Trichosporon aurantiaca (Aleuria aurantia), have been shown to be highly specific for alpha-L-fucose on M cells. Thus, in some embodiments, the delivery vehicle is coated with or conjugated to at least one lectin selected from the group consisting of vitellin 1(UEA-1) and dictyostelium aurantiacum.

M cells also express claudin 4 and TM4SF 3. Delivery systems using surface conjugated peptides with high affinity for sealin 4, such as CTGKSC (SEQ ID NO:11), LRVG (SEQ ID NO:12) or CKSTHPLSC (CKS9) (SEQ ID NO:13) can also be used. Thus, in some embodiments, the delivery vehicle is coated with or conjugated to at least one peptide selected from CTGKSC (SEQ ID NO:11), LRVG (SEQ ID NO:12), and CKSTHPLSC (CKS9) (SEQ ID NO: 13).

In one embodiment, an interferon- α blocker is included in the composition. In one embodiment, the composition comprises at least one interferon- α blocker selected from the group consisting of: an agent that neutralizes circulating interferon-alpha, and/or an agent that blocks interferon-alpha signaling, and/or an agent that depletes cells that produce IFN-alpha, and/or an agent that blocks IFN-alpha production.

In one embodiment, the composition consists essentially of an agent that neutralizes circulating interferon-alpha. In one embodiment, the composition consists essentially of an agent that blocks IFN- α signaling. In one embodiment, the composition consists essentially of an agent that depletes cells that produce IFN- α. In one embodiment, the composition consists essentially of an agent that blocks IFN- α production.

In one embodiment, the interferon- α blocker is comprised in a medicament.

In one embodiment, the medicament comprises at least one interferon- α blocker selected from the group consisting of: an agent that neutralizes circulating interferon-alpha, and/or an agent that blocks interferon-alpha signaling, and/or an agent that depletes cells that produce IFN-alpha, and/or an agent that blocks IFN-alpha production.

In one embodiment, the agent that neutralizes circulating interferon-alpha is included in a medicament. In one embodiment, the agent that blocks interferon-alpha signaling is included in a medicament. In one embodiment, the agent that depletes IFN- α producing cells is comprised in a medicament. In one embodiment, the agent that blocks IFN- α production is included in a medicament.

In one embodiment, the type III interferon and/or the agent that stimulates the production of the type III interferon are included in a composition.

In one embodiment, the composition consists essentially of a type III interferon. In one embodiment, the composition consists essentially of an agent that stimulates production of type III interferon. In one embodiment, the composition consists essentially of a type III interferon and an agent that stimulates the production of the type III interferon.

In one embodiment, the type III interferon and/or the agent that stimulates the production of type III interferon are contained in a medicament.

In one embodiment, the type III interferon is comprised in a medicament. In one embodiment, the agent that stimulates production of type III interferon is included in a medicament. In one embodiment, the type III interferon and the agent that stimulates the production of the type III interferon are comprised in a medicament.

Another object of the invention is a pharmaceutical composition comprising a combination of: 1) the CD8 vaccine specific for at least one infectious disease associated antigen as described above, 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon, and which further comprises at least one pharmaceutically acceptable excipient, or a kit as described above, for use in treating an infectious disease in a subject in need thereof.

Another object of the invention is a medicament comprising a combination of: 1) the CD8 vaccine specific for at least one infectious disease associated antigen as described above, 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent that stimulates the production of type III interferon, or a pharmaceutical combination as described above, or a kit as described above, for use in treating an infectious disease in a subject in need thereof.

As mentioned above, the combination, pharmaceutical combination, medicament or kit of the invention according to the invention will be administered simultaneously, separately or sequentially with respect to each other.

In one embodiment, according to the invention, 1) the CD8 vaccine specific for at least one infectious disease associated antigen, 2) the interferon-alpha blocker, and/or 3) the type III interferon and/or the agent stimulating the production of type III interferon in a combination of the invention will be administered simultaneously, separately or sequentially with respect to each other.

According to one embodiment, a 1) CD8 vaccine specific for at least one infectious disease associated antigen, 2) an interferon-alpha blocker, and/or 3) a type III interferon and/or an agent that stimulates the production of type III interferon, a combination or pharmaceutical combination thereof, a medicament or kit according to the invention will be formulated for administration to a subject.

In one embodiment, the CD8 vaccine according to the invention 1) specific for at least one infectious disease associated antigen, 2) interferon-alpha blocker, and/or 3) type III interferon and/or agent stimulating the production of type III interferon, combinations or pharmaceutical combinations thereof or medicaments may be administered orally, intragastrically, parenterally, topically, by inhalation spray, rectally, nasally, buccally, preputially, vaginally or by implanted depot (implanted reservoir).

In one embodiment, oral administration includes mucosal administration. "mucosal administration" is the delivery to mucosal surfaces, such as the sublingual, tracheal, bronchial, pharyngeal, esophageal, gastric and duodenal, small and large intestinal mucosa, including the rectal mucosa. Also preferably, the mucosal surface is the digestive mucosa (digestive mucosa).

In one embodiment, the administration of each part of the combination, pharmaceutical combination, medicament or kit according to the invention may be performed by the same route of administration or by different routes of administration.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for oral or intragastric administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is administered orally or intragastrically to a subject, e.g. as a powder, tablet, capsule or the like or as a tablet formulated for extended release or sustained release.

Examples of forms suitable for oral or intragastric administration include, but are not limited to, liquid, paste, or solid compositions, and more specifically, tablets formulated for extended or sustained release, capsules, pills, dragees, liquids, gels, syrups, slurries, suspensions and the like.

In one embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above is in a form suitable for oral or intragastric administration. Thus, in one embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above will be administered orally or intragastrically to a subject, e.g., as a capsule or as a tablet.

In another embodiment, the interferon- α blocker as described above is in a form suitable for oral or intragastric administration. Thus, in one embodiment, an interferon- α blocker as described above will be administered orally or intragastrically to a subject, e.g., as a capsule or as a tablet.

In another embodiment, the type III interferon and/or the agent that stimulates the production of type III interferon as described above is in a form suitable for oral or intragastric administration. Thus, in one embodiment, a type III interferon as described above and/or an agent that stimulates the production of a type III interferon will be administered orally or intragastrically to a subject, for example as a capsule or as a tablet.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for parenteral administration.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for injection, e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, transdermal injection or infusion. Thus, the combination, pharmaceutical combination, medicament or kit according to the invention will be injected into a subject by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for injection, e.g. intravenous, intramuscular, intraperitoneal injection or infusion. Thus, the combination, pharmaceutical combination, medicament or kit according to the invention will be injected into a subject by intravenous, intramuscular, intraperitoneal injection or infusion.

The sterile injectable form of the combination, pharmaceutical combination, medicament or kit according to the invention may be a solution or an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span and other emulsifiers or bioavailability enhancers (which are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms), may also be used for formulation purposes.

In one embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above is in a form suitable for parenteral administration and/or injection. Thus, in another embodiment, a CD8 vaccine specific for at least one infectious disease associated antigen as described above will be administered and/or injected parenterally by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion (preferably by intravenous injection) to a subject.

In another embodiment, the interferon- α blocker described above is in a form suitable for parenteral administration and/or injection. Thus, in another embodiment, the interferon- α blockers described above are administered and/or injected parenterally by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion (preferably by intravenous injection).

In another embodiment, the type III interferon described above and/or the agent that stimulates the production of type III interferon are in a form suitable for parenteral administration and/or injection. Thus, in another embodiment, the type III interferon described above and/or the agent that stimulates production of the type III interferon will be administered and/or injected parenterally by intravenous, intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermal injection or infusion (preferably by intravenous injection).

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for topical administration. Thus, the combination, pharmaceutical combination, medicament or kit according to the invention will be administered topically.

Examples of forms suitable for topical administration include, but are not limited to, liquids, pastes, or solid compositions, and more specifically, aqueous solutions, drops, dispersions, sprays, microcapsules, microparticles or nanoparticles, polymeric patches or controlled release patches, and the like.

In another embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above is in a form suitable for topical administration. Thus, a CD8 vaccine according to the invention, as described above, specific for at least one infectious disease associated antigen will be administered topically.

In another embodiment, the interferon- α blocker described above is in a form suitable for topical administration. Thus, the agent interferon- α blocker described above will be administered topically.

In another embodiment, the type III interferon described above and/or the agent that stimulates the production of type III interferon are in a form suitable for topical administration. Thus, a CD8 vaccine according to the invention, as described above, specific for at least one infectious disease associated antigen will be administered topically.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is in a form suitable for rectal administration. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention is to be administered rectally.

Examples of forms suitable for rectal administration include, but are not limited to, suppositories, microaclysters, enemas, gels, rectal foams, creams, ointments and the like.

In another embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above is in a form suitable for rectal administration. Thus, in one embodiment, the CD8 vaccine specific for at least one infectious disease associated antigen described above will be administered rectally.

In another embodiment, the interferon- α blocker described above is in a form suitable for rectal administration. Thus, in one embodiment, the interferon- α blockers described above will be administered rectally.

In another embodiment, the type III interferon described above and/or the agent that stimulates the production of type III interferon are in a form suitable for rectal administration. Thus, in one embodiment, the type III interferon and/or the agent that stimulates the production of type III interferon described above will be administered rectally.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, all in a form suitable for parenteral administration and/or injection.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, all in a form suitable for oral administration.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, in a form suitable for oral administration, and 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, in a form suitable for parenteral administration and/or injection.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, in a form suitable for rectal administration, and 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, in a form suitable for parenteral administration and/or injection.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises 1) a CD8 vaccine specific for at least one infectious disease associated antigen, in a form suitable for vaginal administration, and 2) optionally, an interferon- α blocker, and 3) a type III interferon and/or an agent stimulating the production of type III interferon, in a form suitable for parenteral administration and/or injection.

As mentioned above, the administration of each part of the combination, pharmaceutical combination, medicament or kit according to the invention may be simultaneous, separate or sequential.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a first part comprising a CD8 vaccine specific for at least one infectious disease associated antigen, optionally a second part comprising an interferon- α blocker, and a third part comprising a type III interferon and/or an agent stimulating the production of a type III interferon, all administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more at the same time.

In one embodiment of the invention, the first part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the second part and the third part of the combination, pharmaceutical combination or kit.

In another embodiment of the present invention, the second part of the combination, pharmaceutical combination or kit of parts is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part and the third part of the combination, pharmaceutical combination or kit of parts.

In another embodiment of the present invention, the third part of the combination, pharmaceutical combination or kit is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part and the second part of the combination, pharmaceutical combination or kit.

In one embodiment of the invention, the first part and the second part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the third part of the combination, pharmaceutical combination or kit.

In another embodiment of the present invention, the first part and the third part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the second part of the combination, pharmaceutical combination or kit.

In another embodiment of the present invention, the second and third parts of the combination, pharmaceutical combination or kit will be administered one, two, three, four, five, six, seven, eight, nine, ten or more times before the first and second parts of the combination, pharmaceutical combination or kit.

In one embodiment of the invention, the first part and the second part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more at the same time.

In one embodiment of the invention, the first part and the third part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more at the same time.

In one embodiment of the invention, the second and third parts of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more at the same time.

In one embodiment of the invention, the first part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the second part of the combination, pharmaceutical combination or kit and the second part will be administered once, twice, three times or more before the third part of the combination, pharmaceutical combination or kit.

In another embodiment of the present invention, the second part of the combination, pharmaceutical combination or kit will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part of the combination, pharmaceutical combination or kit, and the first part will be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the third part of the combination, pharmaceutical combination or kit.

In one embodiment of the invention, the second part of the combination, pharmaceutical combination or kit is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the third part of the combination, pharmaceutical combination or kit, and the third part is to be administered once, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part of the combination, pharmaceutical combination or kit.

In one embodiment of the invention, the third part of the combination, pharmaceutical combination or kit is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part of the combination, pharmaceutical combination or kit and the first part is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the second part of the combination, pharmaceutical combination or kit.

In one embodiment of the invention, the third part of the combination, pharmaceutical combination or kit is to be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the second part of the combination, pharmaceutical combination or kit and the second part is to be administered once, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more before the first part of the combination, pharmaceutical combination or kit.

In another embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a first part comprising a CD8 vaccine specific for at least one infectious disease associated antigen, optionally a second part comprising an interferon- α blocker, and a third part comprising a type III interferon and/or an agent stimulating the production of a type III interferon, all of which are administered once daily for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more at the same time.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a first part comprising a CD8 vaccine specific for at least one infectious disease associated antigen, optionally a second part comprising an interferon- α blocker, and a third part comprising a type III interferon and/or an agent stimulating the production of a type III interferon, all administered once a month for the same time period for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months or more.

In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a first part comprising a CD8 vaccine specific for at least one infectious disease-associated antigen, optionally a second part comprising an interferon- α blocker, and a third part comprising a type III interferon and/or an agent stimulating the production of type III interferon, all administered once a year at the same time for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more.

In one embodiment, the administration of each part of the combination, pharmaceutical combination, medicament or kit according to the invention may be performed at the same time or at different times.

Another object of the invention is a method of preventing or treating an infectious disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of:

2) optionally, an interferon- α blocker, and

According to one embodiment, the therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above is to be administered in combination with a therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above and a therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above for the treatment of an infectious disease in a subject in need thereof. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above and a therapeutically effective dose of the third part as described above.

According to one embodiment, the therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above is to be administered in combination with a therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above for the treatment of an infectious disease in a subject in need thereof. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above.

According to one embodiment, the therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above is to be administered in combination with a therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit of parts described above for the treatment of an infectious disease in a subject in need thereof. Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the third part as described above.

In one embodiment, the administration of each part of the combination, pharmaceutical combination, medicament or kit according to the invention may be performed according to a prime/boost mode. Thus, the present invention also encompasses a variety of prime-boost regimens.

In one embodiment, the prime/boost mode comprises the following administration steps:

-one or more primary immunizations, wherein the booster immunization comprises: a therapeutically effective dose of the first part, a therapeutically effective dose of the second part and/or a therapeutically effective dose of the third part, and

one or more boosters.

In a prime/boost regimen, the composition of each part of the combination according to the invention may be the same or different for each immunization and the type of composition, route and formulation of each part of the combination, pharmaceutical combination, medicament or kit according to the invention may also vary. For example, if an expression vector is used for the priming and boosting steps, it may be of the same or different type (e.g., DNA or bacterial or viral expression vector). For example, one useful prime-boost regimen provides at least two prime immunizations, separated by two weeks, followed by at least one boost immunization after the last prime immunization (e.g., at 4-5 weeks and/or 8-9 weeks). It should also be apparent to one of skill in the art that the various permutations and combinations encompassed by the use of the disclosed DNA, bacterial and viral expression vectors or bacteria to provide priming and boosting protocols. For example, CMV vectors can be reused when expressing different antigens derived from the same or different pathogens.

In one embodiment, the boosting step comprises administering a non-infectious dose of SIV or HIV, or an attenuated SIV or HIV (e.g., HIV or SIV depleted in the protein nef). In one embodiment, the boosting is in a form suitable for oral, rectal, or vaginal administration.

Attenuated SIV or HIV viruses are well known in the art. A non-limiting example of such an attenuated virus is HIV or SIV with a depletion of the protein nef (see, e.g., Giorgi et al, J Med Primatol.1996 Jun; 25(3): 186-91).

In some embodiments, the second part and/or the third part of a combination, pharmaceutical combination, medicament or kit according to the invention will be administered at a time and by a route of administration that is separate from the first part.

In one embodiment, the first part of a combination, pharmaceutical combination, medicament or kit according to the invention is to be administered at least 2 times (e.g. on days 0 and 14). In another embodiment, the second part and/or the third part of the combination, pharmaceutical combination, medicament or kit according to the invention will be administered at least 2 times (e.g. on days-7 and-3) before the first part and at least 9 times (e.g. on

days

3, 11, 38, 45, 52, 59, 66, 73 and 80) after the administration.

In one embodiment, the first part of a combination, pharmaceutical combination, medicament or kit according to the invention is to be administered at least 7 times (e.g. on

days

0,1, 3, 7, 28 and 29). In another embodiment, the second part and/or the third part of the combination, pharmaceutical combination, medicament or kit according to the invention will be administered before the administration of the first part (e.g. on days-3, 0, 28 and 29) and at least 1 more time (e.g. on day 57).

In one embodiment, the first part of a combination, pharmaceutical combination, medicament or kit according to the invention will be administered at least 7 times during the priming and boosting steps (e.g. priming on

days

0,1, 2, 3, 5, first boosting on days 28 and 29). The second part of the combination, pharmaceutical combination, medicament or kit according to the invention will be administered at least once during the priming and first boosting steps (e.g. from day 0 to day 40) and at least once after the last administration of the first part of the combination, pharmaceutical combination, medicament or kit according to the invention. The third part of the combination, pharmaceutical combination, medicament or kit according to the invention will be administered 0,1, 2 or 3 days before each administration of the first part of the combination, pharmaceutical combination, medicament or kit according to the invention.

In one embodiment, the prime/boost mode comprises the step of administering a non-infectious dose (e.g., on day 60) of SIV or HIV or attenuated SIV or HIV.

It will be appreciated that the total daily amount of the first part, the total daily amount of the second part and the total daily amount of the third part of the combination, pharmaceutical combination, medicament or kit according to the invention will be decided by the attending physician within the scope of sound medical judgment. The specific dose for any particular subject will depend upon a variety of factors, such as the infectious disease to be treated; the age, weight, general health, sex, and diet of the subject, and similar factors well known in the medical arts. Thus, the combination, pharmaceutical combination, medicament or kit according to the invention may be administered to a subject one or more times. Preferably, there is a set time interval between the individual administrations of the combination, pharmaceutical combination, medicament or kit according to the invention. Although this interval varies for each subject, it is typically 1 day to several weeks, and typically 1 day, 2 days, 4 days, 6 days, or 8 days, or 1 week, 2 weeks, 4 weeks, 6 weeks, or 8 weeks. In one embodiment of the invention, the interval is typically 1 to 6 weeks. In one embodiment of the invention, the intervals are longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks, 70 weeks or 80 weeks. In one embodiment, the administration regimen typically has 1 to 20 administrations of 3 different fractions according to the invention, but may be as few as one or two or four or eight or ten. In another embodiment, the administration regimen is once a year, once two years, or other long interval (5-10 years).

In one embodiment, the subject is a mammal, primate, preferably a human.

As an example, when the first part of the pharmaceutical combination, medicament or kit according to the invention comprises a CMV vector as described above, and when the subject to be treated is a mammal, primate or human, the therapeutically effective dose of said CMV vector may range from a few micrograms to a few hundred micrograms (e.g. 5 to 500 μ g per administration). The CMV vector may beAny suitable amount is administered to achieve expression at these dosage levels. In a non-limiting example, the CMV vector can be at least 10 per administration¹、10²、10³、10⁴、10⁵、10⁶、10⁷Or 10⁸The amount of pfu is administered. Thus, a CMV vector can be administered at least 10 per administration¹pfu, or about 10¹pfu to about 10⁸Range of pfu administration. CMV vectors can be lyophilized (resuspended at the time of administration) or can be in solution.

In one embodiment, the amount of CMV vector as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷Or 10⁸pfu. In one embodiment, the amount of CMV vector administered per administration ranges from about 10, as described above¹To about 10⁸Preferably about 10²To about 10⁷More preferably about 10³To about 10⁶And even more preferably about 10⁴To about 10⁵Including all integer values within these ranges. In one embodiment, the daily amount of CMV vector as described above administered to a subject daily is at least 10 days ¹10 daily²10 daily³10 daily⁴10 daily⁵10 daily⁶10 daily⁷Or 10 days⁸pfu. In one embodiment, the daily amount of CMV vector, as described above, administered daily ranges from about 10 days¹To about 10⁸Preferably about 10 days²To about 10⁷More preferably about 10 days³To about 10⁶And even more preferably about 10 days⁴To about 10⁵Including all integer values within these ranges. In one embodiment, the amount of CMV vector as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷Or 10⁸Individual virus/kg body weight.

As an example, when the pharmaceutical combination according to the invention,When the first part of the medicament or kit comprises at least one infectious disease associated antigen and a non-pathogenic bacterium as described above, and when the subject to be treated is a human, the therapeutically effective dose of the non-pathogenic bacterium (i.e. lactobacillus or lactobacillus plantarum) may range from about 10 per administration¹To about 10¹⁸cfu, and the therapeutically effective dose of the infectious disease associated antigen (i.e., inactivated SIV or HIV virus) may range from about 10 per administration¹To about 10¹⁴And (4) viruses.

In one embodiment, the amount of non-pathogenic bacteria as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴cfu. In one embodiment, the amount of non-pathogenic bacteria as described above administered per administration ranges from about 10¹To about 10¹⁸Preferably about 10²To about 10¹⁶More preferably about 10⁴To about 10¹⁴And even more preferably about 10⁶To about 10¹²Including all integer values within these ranges. In one embodiment, the daily amount of non-pathogenic bacteria as described above administered to a subject daily is at least 10 days ¹10 daily²10 daily³10 daily⁴10 daily⁵10 daily⁶10 daily⁷10 daily⁸10 daily⁹10 daily¹⁰10 daily¹¹10 daily¹²10 daily¹³10 daily¹⁴10 daily¹⁵10 daily¹⁶10 daily¹⁷10 daily¹⁸cfu. In one embodiment, the daily amount of non-pathogenic bacteria as described above administered daily ranges from about 10 daily¹To about 10¹⁸Preferably about 10 days²To about 10¹⁶More preferably about 10 days⁴To about 10¹⁴And even more preferably about 10 days⁶To about 10¹²Including all integer values within these ranges. In one embodiment, the amount of non-pathogenic bacteria as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴Individual bacteria/kg body weight.

In one embodiment, the amount of inactivated SIV or HIV virus as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴And (4) viruses. In one embodiment, the amount of inactivated SIV or HIV virus as described above administered per administration ranges from about 10¹To about 10¹⁸Preferably about 10²To about 10¹⁶More preferably about 10⁴To about 10¹⁴And even more preferably about 10⁶To about 10¹²Including all integer values within these ranges. In one embodiment, the daily amount of inactivated SIV or HIV virus as described above administered to a subject daily is at least 10 days ¹10 daily²10 daily³10 daily⁴10 daily⁵10 daily⁶10 daily⁷10 daily⁸10 daily⁹10 daily¹⁰10 daily¹¹10 daily¹²10 daily¹³10 daily¹⁴10 daily¹⁵10 daily¹⁶10 daily¹⁷10 daily¹⁸And (4) viruses. In one embodiment, the daily amount of inactivated SIV or HIV virus as described above administered daily ranges from about 10 daily¹To about 10¹⁸Preferably about 10 days²To about 10¹⁶More preferably about 10 days⁴To about 10¹⁴And even more preferably about 10 days⁶To about 10¹²Bag (bag)Including all integer values within these ranges. In one embodiment, the amount of inactivated SIV or HIV virus as described above administered to a subject is at least 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³Or 10¹⁴Individual virus/kg body weight.

In one embodiment the subject is a mammal, a primate, preferably a human, and said therapeutically effective dose of the combination, pharmaceutical combination, medicament or kit according to the invention in the first part is a daily dose to be administered in one, two, three or more administrations or one, two, three or more injections.

In one embodiment the subject is a mammal, a primate, preferably a human, and said therapeutically effective dose of the combination, pharmaceutical combination, medicament or second part of the kit according to the invention is a daily dose to be administered in one, two, three or more administrations or one, two, three or more injections.

In one embodiment the subject is a mammal, a primate, preferably a human, and said therapeutically effective dose of the combination, pharmaceutical combination, medicament or third part of the kit according to the invention is a daily dose to be administered in one, two, three or more administrations or one, two, three or more injections.

According to the present invention, a combination, a pharmaceutical combination, a medicament or a kit as described above is used alone.

Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention is used alone and comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above and a therapeutically effective dose of the third part as described above. In one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention is for use alone and comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above. In another embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention is used alone and comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the third part as described above.

In one embodiment, the combination, pharmaceutical combination, medicament or kit of the invention as described above is used in combination with at least one other therapeutic agent.

Such administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may be administered as one composition or as separate compositions as appropriate. Other therapeutic agents are often associated with the condition to be treated.

Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above and a therapeutically effective dose of the third part as described above and is used in combination with at least one further therapeutic agent. In one embodiment, the combination, pharmaceutical combination, medicament or kit according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above, and is used in combination with at least one further therapeutic agent. In another embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the third part as described above and is used in combination with at least one further therapeutic agent.

In one embodiment, the other therapeutic agent is anti-retroviral therapy (ART).

As used herein, the term "antiretroviral therapy" or "highly active antiretroviral therapy" refers to any combination of Antiretroviral (ARV) drugs to maximize inhibition of the HIV virus (e.g., reduce viral load, reduce HIV proliferation), and prevent progression of HIV disease. There are multiple classes of HIV drugs, for example, non-nucleoside reverse transcriptase inhibitors (NNRTIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), post-attachment inhibitors (post-attachment inhibitors), protease inhibitors (PIs ), CCR5 antagonists, integrase strand transfer inhibitors (intis), fusion inhibitors (fusion inhibitors). Typically, the initial treatment regimen will typically include two NTRIs in combination with a third active antiretroviral drug (which may belong to the INSTI, NNRTI or PI classes). They may sometimes include boosters (boster), which may be cobicistat (Tybost) or ritonavir (ritonavir, Norvir).

In one embodiment, the combination, pharmaceutical combination, medicament or kit as described above is used in combination with antiretroviral therapy (ART).

Thus, in one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above and a therapeutically effective dose of the third part as described above and is used in combination with antiretroviral therapy. In one embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the second part as described above, for use in combination with antiretroviral therapy. In another embodiment, the combination, pharmaceutical combination, medicament or kit of parts according to the invention comprises a therapeutically effective dose of the first part as described above and a therapeutically effective dose of the third part as described above and is used in combination with antiretroviral therapy.

According to the present invention, the combination, pharmaceutical combination, medicament or kit as described above is for use in the prevention or treatment of an infectious disease in a subject in need thereof.

As used herein, the term "infectious disease" refers to a disease caused by a pathogen, such as a fungus, parasite, protozoa, bacteria or virus. Examples of "infectious diseases" include, but are not limited to, influenza virus infection, monkeypox virus infection, west nile virus infection, Chikungunya virus infection, ebola virus infection, hepatitis c virus infection, poliovirus infection, dengue fever, acquired immunodeficiency syndrome (AIDS) or Simian Immunodeficiency Virus (SIV) infection and recombinant RhCMV or HCMV vectors encoding antigens from HIV or SIV, cutaneous warts (skin warts), genital warts (genetic warts), respiratory papillomatosis (respiratory papillomatosis), malaria, ebola hemorrhagic fever, tuberculosis, herpetic diseases (e.g., genital herpes, chicken pox or shingles, infectious mononucleosis), tuberculosis infection (caused by mycobacterium tuberculosis), typhoid infection or fever (caused by salmonella typhi).

In one embodiment, the infectious disease to be prevented or treated is preferably acquired immunodeficiency syndrome (AIDS), Human Immunodeficiency Virus (HIV) infection, or Simian Immunodeficiency Virus (SIV) infection.

In one embodiment, the infectious disease to be prevented or treated is Acquired Immune Deficiency Syndrome (AIDS).

In some embodiments, the combination, pharmaceutical combination, medicament or kit as described above is for use in the prophylactic or curative treatment of an infectious disease in a subject in need thereof.

Brief description of the drawings

FIG. 1: antiviral activity of type I and type III interferons. (A) ISG expression in HepG 2. HepG2 cells were treated with IFN α 2a or IFN λ 1-4(10 ng/ml). After 4h stimulation, mRNA levels of interferon-induced genes IFIT1, MX1 and OASL were detected using qRT-PCR and 2 by comparison to untreated cell controls^-ΔΔCtMethods and normalization using endogenous S14 mRNA levels were used to calculate fold changes. (B) Antiviral activity of type I and type III IFNs on EMCV. IFN alpha 2a or IFN lambda 1/2/3/4(10ng/ml) was added to HepG2 cells 24h prior to challenge with EMCV. Cell viability was determined by bioassay 48 after infection with EMCV. The A570 value is proportional to cell viability and therefore to the antiviral activity of the corresponding IFN. IFN-alpha therapy without viral challengeTherapy was used as a baseline for cell viability.

FIG. 2: type I and type III interferon pair CD4⁺Antiproliferative activity of T cells. Stimulation of CFSE-stained CD4 with allogeneic poly I: C mature DCs in 96 round-bottomed microwells in the absence (control) or presence of 10ng/ml of IFN-. alpha.2a or IFN-. lambda.1 or IFN-. lambda.2 or IFN-. lambda.3 or IFN-. lambda.4⁺T cells (10X 10)⁴/well) for 5 days. Anti-interferon type I receptor antibodies were added as indicated. The percentage of CFSE dilution was assessed by flow cytometry.

FIG. 3: IFN-alpha 2a but not IFN-type III induces CD4⁺ISG expression in T cells. CD4⁺T cells with IFN alpha 2a or IFN lambda 1/2/3/4(10ng/ml) treatment. After 4h stimulation, mRNA levels of interferon-induced genes IFIT1, MX1 and OASL were detected using qRT-PCR and 2 by comparison to untreated cell controls^-ΔΔCtMethods and normalization using endogenous S14 mRNA levels were used to calculate fold changes.

FIG. 4: IFN-alpha 2a but not IFN-III stimulates CD4⁺Stat1 phosphorylation in T cells. Will CD4⁺10ng ml for T cells^-1IFN- λ 1, IFN- λ 2, IFN- λ 3, IFN- λ 4, or IFN- α 2a for 20 minutes, or no stimulation (control). Increase in pSTAT1 was assessed as the proportion of induction relative to baseline levels (MFI fold change ═ MFI cytokine stimulated/MFI untreated cells).

FIG. 5: IFN-alpha 2a but not IFN-type III increases CD3/CD28 stimulated CD4⁺CD38 expression in T cells. In the presence of Δ CD 3-feeder cells (4X 10)⁴Perwell) and plate-bound anti-CD 3mAb (2. mu.g/ml), soluble anti-CD 28 mAb (2. mu.g/ml) and increasing doses of IFN-. alpha.2a or type III IFN, CFSE-stained CD4 was cultured in 96 round-bottom microwells⁺T cells (4X 10)⁴Hole/bore). At the end of the culture by flow cytometry at CD3⁺7-AAD-CFSE⁺Stimulated CD4⁺The Median Fluorescence Intensity (MFI) of CD38 was measured in T cells.

FIG. 6: production and amplification of peptide-specific CD8HLA-E restricted by peptide-loaded m-DC. TAP-inhibited mDC were pulsed with peptide (10. mu.M, duration 1h) and combined with autologous naive CD8⁺T cells andco-culture at a ratio of 1: 10. Monitoring of peptide positive CD8 by flow cytometry analysis using MHC-peptide pentamers on day 0 and one week after the last stimulation⁺T cells. Data represented as CD8⁺Percentage of tetramer positive cells in T cells.

FIG. 7: schematic of immunization protocol in macaques using RhCMV/SIV vectors.

FIG. 8: schematic of an oral immunization protocol in macaques using inactivated SIV and live lactobacillus plantarum.

FIG. 9: schematic of oral immunization protocol in BLT mice using inactivated SIV and live lactobacillus plantarum.

FIG. 10: CM CD8 in HIV-1 infected subjects⁺Comparison of T cell distribution and serum IFN- α levels and the key pathogenic role of IFN- α in HIV-1 infection in humans. (A) CM CD8 in HIV-1 infected subjects⁺Comparison of T cell distribution; (B) comparison of serum IFN- α levels in HIV-1 infected subjects; (C) CD8 in untreated HIV patients (EC and pre-cART groups)⁺Relationship between CM frequency and serum IFN- α levels.

FIG. 11: schematic of an oral immunization protocol in macaques using inactivated SIV and live lactobacillus plantarum.

FIG. 12: schematic of oral immunization protocol in BSLT mice using inactivated SIV and live lactobacillus plantarum.

Examples

The invention is further illustrated by the following examples.

Example 1: effect of type I and type III interferons on innate and adaptive immune responses

Materials and methods

Human cell line

HCC HepG2 and normal renal epithelial Vero cell lines were obtained from ATCC. Cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, 1% penicillin and streptomycin solution, 2% hypoxia (hypoxia). Cancer cell lines were grown to 70-100% confluence, followed by a maximum of 5 passages, and then thawed fresh. Cells were detached using accutase, resuspended in FBS-containing medium and harvested using centrifugation (300g, 3 min). The cell number was determined using trypan blue.

Human blood sample

From Etablessment

du Sang (EFS, Paris) blood samples from healthy individuals. Blood cells were collected using standard procedures.

Cell purification and culture

Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation on Ficoll-Hypaque (pharmacia). PBMCs can be used as fresh cells or stored frozen in liquid nitrogen. T cell subsets and T cell depleted helper cells (Δ CD3 cells) were isolated from fresh or frozen PBMCs. T cell depleted helper cells (Δ CD3 cells) were isolated from PBMC by negative selection by incubation with anti-CD 3 coated dynabeads (dynal biotech) and irradiated at 3000rad (termed Δ CD 3-feeder cells). Using CD4⁺T cell isolation kit (Miltenyi Biotec) negative selection of CD4 from PBMC⁺T cells, producing CD4 with a purity of 96-99%⁺A population of T cells. T cell subsets were cultured in 2% hypoxic IMDM supplemented with 5% SVF, 100IU/ml penicillin/streptomycin, 1mM sodium pyruvate, 1mM non-essential amino acids, glutamax and 10mM HEPES (IMDM-5 medium).

Freezing and thawing of cells

Cells were frozen in FBS containing 10% DMSO. Cryo tubes (Cryo tubes) were placed in CoolCell (Bioprecision) freezer and incubated at-80 ℃. After 2 days, the tubes were transferred to liquid nitrogen and stored until needed. Cell thawing was performed by placing the cryopreserved tubes in a 37 ℃ water bath for about 30 seconds. Next, the cell suspension was mixed with an equal volume of pre-warmed medium and then transferred to a falcon centrifuge tube containing the same medium. The cells were pelleted by centrifugation (300g, 3min) to remove DMSO. The cell pellet was resuspended in cell culture medium.

Real-time PCR for ISG detection

Refining HepG2Cells at 2X 10 per well⁵The density of individual cells was seeded in 12-well plates and incubated for 24 h. Then, fresh medium containing the indicated interferon was added. Cells were incubated for 4 hours and then lysed and RNA purified using an extraction kit (Qiagen) according to the manufacturer's instructions. The synthesis of cDNA was performed using PrimeScript RT Reagent kit (TAKARA). Quantitative PCR was performed on a LightCycler 480 instrument (Roche) using a Power SYBR Green PCR Master Mix (Applied Biosystems). Each reaction was performed in duplicate in a total volume of 100 μ L. Using Primer3 or Primer

v3.0 software (Applied Biosystems) primers were designed to be intron-spanning. To determine the cellular transcriptional response to IFN stimulation, 3 ISG targets MXI, OASL and ISG15 were selected according to published results studying transcriptional responses in IFN-stimulated PBMCs (see, e.g., Waddell et al (2010) PLoS one.5(3): e 97532). For gene induction assays, fold change values were calculated using the Δ Δ Ct method. The geometric mean of the Ct values of the reference gene S14 was used as reference value.

Virus production

The EMCV virus used (FA strain) was grown on a monolayer of Vero cells to complete the cytopathic effect or until all cells were affected by infection (as determined by microscopy) and prepared by two cycles of freezing and thawing, followed by centrifugation at 5,000xg for 30 minutes to remove cell debris.

Antiviral assays

HepG2 cells were assayed for antiviral activity at 1.5X10⁴Is seeded in DMEM supplemented with 10% FCS in 96-well plates and left to stand. Cells were incubated with the indicated doses of IFN for 24 hours prior to challenge with EMCV. Cells were incubated with virus for 48 hours. The medium was removed between each step. Analyzing the viability of the cells by a bioassay based on a dehydrogenase system; this system in intact cells converts the substrate MTT to formazan (blue), which in turn can be measured spectrophotometrically. Briefly, MTT was provided to the cells and incubated for 2 hours. Adding extract buffer to cellsWash (containing 6% to 11% sodium dodecyl sulfate and 45% N, N-dimethylformamide) and then incubate cells at 37 ℃ overnight. Subsequently, the absorbance at 570nm was measured using the extraction buffer as a blank probe. A570 is directly proportional to antiviral activity.

Flow cytometry analysis

⁺CD 3T cell staining:anti-CD 4(SK3) -APC, anti-CD 3(UCHT1) -FITC, anti-CD 8(RPA-T8) -BV421 were from Becton Dickinson. Cells were stained for surface markers using a mixture of abs diluted in PBS containing 3% FBS, 2mM EDTA (FACS buffer) (30 min in the dark at 4 ℃).

STAT1 signaling assay:the BD Phosflow technique was used according to the manufacturer's instructions (BD Bio-sciences, San Jose, Calif.) at CD4⁺STAT1 phosphorylation (pSTAT1) was analyzed by flow cytometry in T cells. Will CD4⁺T cells were stimulated or untreated by incubation with type I and type III interferons for 20min at 37 ℃. Activation was stopped by immobilization using BD Phosflow Lyse/Fix buffers (BD Biosciences) and cells were permeabilized using BD Perm Buffer III (BD Biosciences). With antibodies recognizing specific phosphorylated STAT tyrosines: p-STAT1(Y701) -PE stained the cells. In a multiparameter immunotyping experiment, cells were stained simultaneously with anti-CD 3-FITC and 7-AAD. The increase in pSTAT1 was determined as the proportion of induction relative to baseline levels (MFI fold change — MFI cytokine stimulated/MFI untreated cells).

CFSE staining:using 1. mu.M CFSE (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS at 1.10⁷Concentration of individual cells/ml CD4⁺T cells were stained at 37 ℃ for 8 min. Cells were washed 2 times with RPMI-1640 medium containing 10% FBS to stop the labeling reaction. The cells were then resuspended at the desired concentration and subsequently used in the proliferation assay.

7-AAD staining:determination of stimulated CFSE labeled CD4 Using 7-AAD assay⁺Apoptosis of T. Briefly, cultured cells were treated with the nuclear dye 7-amino-actinomycin D (7-AAD; Sigma-Aldrich, St-Q) at 20. mu.g/mLuentin Fallavier, france) were stained for 30min at 4 ℃. FSC/7-AAD dot plots live cells (FSC)^{Height of}/7-AAD^-) And apoptosis (FSC)^{Height of}/7-AAD⁺) Cell and apoptotic bodies (FSC)^{Is low in}/7-AAD⁺) And debris (FSC)^{Is low in}/7-AAD^-) Are distinguished. Identifying viable cells as CD3⁺7-AAD-FSC⁺A cell.

Appropriate isotype control abs were used for each staining combination. Samples were collected on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). The results are expressed as a percentage (%) or as a Mean Fluorescence Intensity (MFI).

Functional assay

T cell proliferation:t cell proliferation was assessed using the CFSE dilution assay. For the CFSE dilution assay, stimulated CFSE-labeled CD4 was harvested at the completion of co-cultivation⁺T cells, co-stained with anti-CD 3mAb and 7-AAD, and gated CD3 determined by flow cytometry⁺7-AAD^-Percentage of proliferating cells in the cells (defined as CFSE low fraction).

Activation of T cells:CD 38-expressed CD38 Median Fluorescence Intensity (MFI) at the end of the culture by flow cytometry at CD3⁺7-AAD-CFSE⁺Stimulated CD4⁺Measured in T cells.

⁺Polyclonal stimulation of CD 4T cells:CFSE stained CD4⁺T cells (5X 10)⁴Perwell) on Δ CD 3-feeder cells (1X 10)⁵Per well) and plate bound anti-CD 3 antibody (2 μ g/ml), soluble anti-CD 28 mAb (2 μ g/ml) were cultured in 96 round bottom microwells. CD4 pair by flow cytometry using the CFSE dilution assay as described above⁺T cell proliferation was evaluated. Cells were stimulated in the presence of varying amounts of recombinant cytokines.

Allogenic mixed lymphocyte reaction:culturing CFSE-stained CD4 in 96 round-bottom microwells in the presence of allogeneic mature DCs⁺T cells (5X 10)⁴Hole/bore). Determination of allo-activated CD4 by flow cytometry using the CFSE dilution assay as described above⁺T cells proliferate. Cells were stimulated in the presence of varying amounts of recombinant cytokines.

Stat1 phosphorylation assay:will CD4⁺T cells were stimulated with IFN-. lambda.1, IFN-. lambda.2, IFN-. lambda.3, IFN-. lambda.4 or IFN-a2a (10ng/ml) for 20min, or not (control). Levels of phosphorylated Stat1 were assessed by flow cytometry as described above.

Results

Type I interferons (IFN-. alpha./β) and the recently identified type III interferons (IFN-. lambda.) serve as the first line of defense against viral infection and regulate the development of innate and adaptive immune responses. Type III IFNs were initially identified as novel ligand-receptor systems that act in parallel with type I IFNs, but subsequent studies provide increasing evidence for the different effects of each IFN family.

The present inventors aimed to evaluate the innate (antiviral) and adaptive immune response (CD4) to type I and type III interferons⁺T cell proliferation).

Antiviral activity of type I and type III

The ability of type I and III interferons to induce the expression of Interferon Stimulated Genes (ISGs) was analyzed by qPCR.

Briefly, type I and type III antiviral activity was tested in HepG2 cells treated with IFN- α 2a, IFN λ 1, IFN λ 2, IFN λ 3, or IFN λ 4 for 4 hours. Induction of the well-known Interferon Stimulated Genes (ISG) MX1, IFIT1, and OASL was then examined by qPCR.

As shown in fig. 1A, all five interferons clearly induced all three ISGs.

Since the ISGs studied were functionally related to antiviral defenses, the inventors further evaluated the ability of both IFNs to protect HepG2 cells from EMCV-induced cytopathic effects.

Briefly, cells were seeded in 96-well microtiter plates and treated with the indicated amount of IFN for 24h, and then challenged with EMCV for 20 h. Cell survival was measured by MTT staining assay.

As shown in FIG. 1B, type III IFN and IFN-. alpha.2a have intrinsic cellular antiviral activity and are able to fully protect HepG2 cells that are challenged by EMCV.

Type I and type III interferons against CD4⁺Antiproliferative activity of T cell proliferation

Evaluation of type I and type III IFNs against CD4 in response to polyclonal or allogeneic stimulation in Mixed Lymphocyte Reaction (MLR) assays⁺Effects of T cell proliferation.

Briefly, CFSE labeled CD4 was first labeled⁺T cells were stimulated with poly I: C mature allogeneic dendritic cells in the presence of different doses of IFN. CFSE fluorescence dilutions were analyzed 5 days after activation.

As shown in figure 2, IFN-. alpha.2a inhibits stimulated CD4⁺Proliferation of T cells, whereas type III IFNs do not exhibit the ability to inhibit their proliferation. Notably, CD4 when MLR is performed in the presence of anti-interferon type I receptor antibodies⁺T cells exhibit greater proliferation. Thus, type I IFN, but not type III IFN, inhibits allo-activated CD4⁺Proliferation of T cells.

In addition, for IFN treatment of CD4⁺Analysis of the mRNA levels of the interferon inducible genes (ISG) IFIT1, MX1 and OASL in T cells confirmed that CD4⁺T cells lack or are minimally sensitive to type III interferon.

In fact, as shown in FIG. 3, ISG was only on IFN-. alpha.2a-stimulated CD4⁺Induced in T cells. Thus, IFN-. alpha.2a, but not type III IFN, induced CD4⁺ISG expression in T cells.

Since the JAK-STAT1/2 pathway is a major regulator of ISG transcription, the inventors analyzed CD4⁺The Stat1 protein in T cells responds to the phosphorylation levels of type I IFN or type III interferon.

As shown in FIG. 4, only IFN-. alpha.2a was able to stimulate CD4⁺Stat1 phosphorylation in T cells. Thus, IFN-. alpha.2a, but not type III IFN, induced CD4⁺Tyrosine phosphorylation of STAT1 in T cells.

Induction of chronic immune activation in the presence of type I and type III interferons.

Chronic immune activation has been recognized as a receptor for HIV-1 infectionImportant drivers of disease progression in the test subjects, and thus by measuring CD4⁺Expression of activation markers on the surface of T cells to monitor disease progression. Thus, the inventors evaluated two IFNs by flow cytometry to increase stimulated CD4⁺The ability of CD38 expression on T cells.

As shown in FIG. 5, IFN-. alpha.2a alone enhanced CD38 in stimulated CD4⁺Expression on T cells.

Collectively, these ex vivo experiments showed that although exhibiting antiviral activity as well as IFN- α, type III interferons were directed against CD4 as compared to immunosuppressive IFN- α⁺T cell activation and proliferation had no effect. In fact, type III interferons do not inhibit the initiation of an adaptive immune response as does IFN-. alpha.2a.

In summary, although type I and type III interferons are induced by the same viral stimulator and exhibit similar profiles (signature profiles), their biological activities do not appear to be redundant, but rather complementary. Indeed, after viral infection, type III interferons exert their antiviral action at mucosal sites during the innate phase of the immune response, whereas IFN- α acts more systemically throughout the organism. In addition, the subsequent adaptive immune response is at its initial level via IFN-alpha in activated CD4⁺Immunosuppression on T cells is inhibited.

⁺Example 2: ex vivo generation and expansion of antigen (Ag) -specific CD8HLA-E restricted T cells

Materials and methods

Human blood sample

From Etablessment

Cell purification and culture

Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation on Ficoll-Hypaque (pharmacia). PBMC can be used as fresh cells or frozenStored in liquid nitrogen. T cell subsets and T cell depleted helper cells (Δ CD3 cells) were isolated from fresh or frozen PBMCs. T cell depleted helper cells (Δ CD3 cells) were isolated by negative selection by incubating PBMCs with anti-CD 3 coated dynabeads (dynal biotech) and irradiated at 3000rad (called Δ CD 3-feeder cells). Isolation of initial CD8 from PBMCs by negative selection using the MACS System⁺T cells. CD14⁺Monocytes were isolated from PBMCs by positive selection using the MACS system. T cell subsets were cultured in IMDM supplemented with 5% SVF, 100IU/ml penicillin/streptomycin, 1mM sodium pyruvate, 1mM non-essential amino acids, glutamax and 10mM HEPES (IMDM-5 medium), hypoxic by 2%.

Freezing and thawing of cells

Cells were frozen in FBS containing 10% DMSO. The cryopreserved tubes were placed in a CoolCell (Bioprecision) freezer and incubated at-80 ℃. After 2 days, the tubes were transferred to liquid nitrogen and stored until needed. Cell thawing was performed by placing the cryopreserved tubes in a 37 ℃ water bath for about 30 seconds. Next, the cell suspension was mixed with an equal volume of pre-warmed medium and then transferred to a falcon centrifuge tube containing the same medium. The cells were pelleted by centrifugation (300g, 3min) to remove DMSO. The cell pellet was resuspended in cell culture medium.

Dendritic cell generation

Monocytes were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, 1% penicillin and streptomycin solution (RPMI medium) in the presence of IL-4(20ng/ml) and GM-CSF (20 ng/ml). On day 6, DCs were matured in different mixtures (cocktails) overnight: a (IL-1. beta. (2ng/ml) IL-6(30ng/ml), PGE2(1microg/ml) and TNF-. alpha. (10ng/ml), LPS 250ng/ml, Poly I: C (150 ng/ml).

In vitro production of TAP-inhibited stimulator cells for MLR assays

Mature DCs obtained as described above were electroporated with 20 μ g of RNA synthesized from pGem4Z vector containing the UL49.5 gene from BHV-1 (see, e.g., Lampen et al (2010) J Immunol.185(11): 6508-17).

Human Ag-specific CD 8T cell (HLA-E restricted) induction and expansion

TAP-inhibited mature DCs (TAP-mDC) were pulsed with 50. mu.g/ml synthetic peptide. The DCs were then mixed with naive CD 8T cells at a ratio of 1: 10. IL-21(30ng/ml) was added immediately after the start of the culture. After 3 days, half of the medium was replaced and 30ng/mL IL-21, 20ng/mL interleukin 15(IL-15) and 500ng/mL soluble Fc fused IL 15-receptor alpha (sIL15Ra-Fc, R & DSystems) were added. After 10 days of co-culture, T cells were re-stimulated with peptide-pulsed TAP-inhibited mature DCs in the presence of IL-21, IL-15 and Fc fused IL 15-receptor alpha. IL-2(50IU/ml) and IL-7(10ng/ml) were added 1 day after the second stimulation to further promote the expansion of activated Ag-specific T cells. Peptide-specific expansion of T cells was monitored by flow cytometry analysis using MHC peptide pentamers.

Flow cytometry analysis

T cells were transferred to each v-bottom 96-well plate, washed (300g, 2min) and stained in 100. mu.L FACS buffer (PBS, 3% FBS, 2mM EDTA) containing the corresponding peptide-MHC pentamer (1:10, ProImmune) for 1 hour at 4 ℃. Cells were washed 3 times in FACS buffer and analyzed by flow cytometry.

Results

Recent advances in the field of SIV vaccinology have highlighted MHC-1b/E restricted CD8⁺Effect of T cell response on controlling SIV infection in rhesus monkeys, thereby increasing HLA-E-restricted CD8⁺Adoptive transfer of T cells helps control the possibility of HIV-1 infection. Thus, the inventors established an experimental procedure to generate and amplify autologous CD8 against peptides presented by HLA-E (used as HLA-E peptides), CMV UL 40-derived peptides (VMAPRTLIL, SEQ ID NO:5), and mature DCs that are TAP-inhibited by stimulatory cells⁺T cell line. The use of VMAPRTLVL-HLA-E pentamer (VMAPRTLVL, SEQ ID NO:6) allows assessment of specific T cell expansion.

As shown in fig. 6, through two72% of CD8 in culture after round of stimulation⁺T cells were tetramer positive, indicating that the inventors have developed an Ag-specific CD8 promoting HLA-E restriction⁺Culture system for T cell expansion and generation.

Such ex vivo expanded cellular material itself represents an example of the principle of adoptive T cell therapeutic activity.Examples 3: prophylactic vaccine against SIV in macaques using recombinant RhCMV/SIV vector Figure 7 is a schematic of the immunization protocol in macaques.

Immunization protocol (DNA vaccination)

The CD8 vaccine composition is in a form suitable for intramuscular administration and comprises a RhCMV/SIV vector (see Hansen et al (2013) Science 24; 340(6135): 1237874; Hansen et al (2016) Science; 351 (6274)). Chinese rhesus monkeys received 2 intramuscular (i.m.) injections of the CD8 vaccine composition on

days

0 and 14.

Macaques received (i.p.) injections of interferon λ 1(50-100 μ g) and/or anti-interferon α antibody (PBL, 100 μ g/kg) on days-7, -3, 11, 38, 45, 52, 59, 66, 73, and 80.

Before attacking toxin

Optionally, the macaque receives an intrarectal injection of a non-infectious dose of SIV or an attenuated SIV (e.g., a protein nef-depleted SIV).

Counteracting toxic substances

On

days

45, 52, 59, 66, 73, and 80, macaques received a sub-optimal dose of SIVmac239 injected intrarectally.

The acquisition of SIV infection was determined as a plasma viral load >30 copies eq/mL and/or the development of an immune response to SIV Vif (i.e. antigen not contained in RhCMV/SIV vector).

Example 4: prophylactic vaccine against SIV in macaques using inactivated SIV and live Lactobacillus plantarum

Fig. 8 is a schematic of a macaque midriff immunization protocol.

Oral primary immunization protocol

The CD8 vaccine composition is in a form suitable for intragastric administration and comprises: 4x10 in maltodextrin (20%) solution⁷Inactivated SIV and 3X10 copies/mL⁹cfu/mL of viable Lactobacillus plantarum. Chinese rhesus monkeys received 30mL of the CD8 vaccine composition on

days

0,1, 3, 5, 7 and 28, 29, and then 25mL of the same composition every 30 minutes for 3 hours.

Prior to each series of immunizations at day-3 and

days

0, 28 and 29, macaques received (i.p.) injections of different combinations of agents according to the protocol described below:

scheme 1: is free of

Scheme 2: poly I C (100. mu.g/kg) and interferon lambda 1 (50-100. mu.g)

Scheme 3: polyclonal anti-IFN-alpha antibody (PBL, 100. mu.g/kg) and poly I: C (100. mu.g/kg)

Scheme 4: polyclonal anti-IFN-alpha antibody (PBL, 100. mu.g/kg), poly I: C (100. mu.g/kg) and interferon lambda 1 (50-100. mu.g)

On day 57, macaques received injections of polyclonal anti-IFN- α antibody (PBL, 100. mu.g/kg), poly I: C (100. mu.g/kg) and/or interferon lambda 1 (50-100. mu.g).

Enhanced immunity (before attacking toxin)

On day 60, macaques received intrarectal injections of non-infectious doses of SIV or attenuated SIV (e.g., SIV depleted in protein nef).

Immune response assay

anti-SIV immune responses were analyzed on

days

25, 57 and 80. Analysis of anti-SIV immune responses included monitoring of:

plasma SIV IgM/IgG/IgA antibody titers, and/or

SIV Gag-specific CD8⁺T cells and CD8⁺T cell mediated antiviral activity.

Counteracting toxic substances

On day 90, only when anti-SIV specific CD8 was observed⁺Inhibitory T cells (as described herein) macaques receive an infectious dose of SIV for intrarectal injection.

After challenge, anti-SIV immune responses and plasma viremia were monitored every two weeks.

Example 5: prophylactic vaccine against HIV in BLT mice

Figure 9 is a schematic of an oral immunization regimen in BLT mice.

BLT mice are a valuable humanized model for studying HIV infection. In fact, BLT mice recapitulate important aspects of human immunity, including T cell immunity (Marshall E.Karpel et al, Curr Opin Virol.2015Aug; 13: 75-80).

Oral immunization protocol

The CD8 vaccine composition is in a form suitable for intragastric administration and comprises: 4x10 in maltodextrin (20%) solution⁷Inactivated HIV-1 and 3x10 copies/mL⁹cfu/mL of viable Lactobacillus plantarum. BLT mice received 0.2mL of the CD8 vaccine composition on

days

0,1, 3, 5, 7 and 28, 29, and then 0.2mL of the same composition every 30 minutes for 3 hours.

Prior to each series of immunizations at day-3 and

days

0, 28 and 29, BLT mice received injections of different combinations of agents (i.p.) according to the protocol described below:

scheme 1: is free of

Scheme 2: poly I C (100. mu.g/kg) and interferon lambda 1 (50-100. mu.g)

On day 57, BLT mice received injections of polyclonal anti-IFN- α antibody (PBL, 100. mu.g/kg), poly I: C (100. mu.g/kg) and/or interferon lambda 1 (50-100. mu.g).

Enhanced immunity (before attacking toxin)

On day 60, BLT mice received intrarectal injections of a non-infectious dose of HIV-1 or attenuated HIV-1 (e.g., HIV-1 with a depletion of the protein nef).

Immune response assay

anti-HIV immune responses were analyzed on

days

25, 57 and 80. Analysis of the anti-HIV immune response included monitoring of:

plasma HIV IgM/IgG/IgA antibody titers, and/or

HIV Gag-specific CD8⁺T cells and CD8⁺T cell mediated antiviral activity.

Counteracting toxic substances

On day 90, only when anti-HIV specific CD8 was observed⁺BLT mice received intrarectal injections of infectious doses of HIV when suppressor T cells (as described herein) were administered.

After challenge, anti-HIV immune responses and plasma viremia were monitored every two weeks.

Example 6: antigen (Ag) pair of HLA-E01033 transfected derivatives using K562 cell lineSpecificity CD8+ Ex vivo generation and expansion of HLA-E restricted T cells

Materials and methods

Human blood sample

From Etablessment

Cell purification and culture

Peripheral Blood Mononuclear Cells (PBMC) were isolated by density gradient centrifugation on Ficoll-Hypaque (pharmacia). PBMC were used as fresh cells or stored frozen in liquid nitrogen. Primary CD8+ T cells were isolated from PBMCs by negative selection using the MACS system. T cell subsets were cultured in 2% hypoxic IMDM supplemented with 5% SVF, 100IU/ml penicillin/streptomycin, 1mM sodium pyruvate, 1mM non-essential amino acids, glutamax and 10mM HEPES (IMDM-5 medium).

HLA-E01033 transfected derivatives of the K562(K562/HLA-E) cell line were maintained in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 25mM HEPES and antibiotics in the presence of 0.4mg/ml G-418(Calbiochem, San Diego, Calif.). When used as antigen presenting cells, the pulsed K562/HLA-E cell line was irradiated at 80000 rad.

peptide-HLA molecule binding assay

Evaluation by HLA-E stabilization assay using HLA-E01033 transfected derivatives of K562(K562/HLA-E) cell linePeptide binding. Briefly, cells were plated at1 × 10⁶The individual cells/ml were resuspended in serum-free medium. Where appropriate, peptides were added (see table 1). After overnight incubation at 26 ℃, cells were washed with PBS to remove free peptide. Next, HLA surface expression was monitored after staining with anti-HLA-E mAb. The analysis was performed on a FACScalibur cytometer (BD Biosciences). Results are expressed as MFI of cells stained with anti-HLA-E mAb.

Peptide-specific HLA-E-restricted CD8⁺Generation and expansion of T cell lines

Initial CD8⁺T in the irradiated K562/HLA-E cell line as antigen presenting cell (0.5X 10)⁶Ml) and the K562/HLA-E cell line pulsed with the appropriate peptide (10. mu.g) in complete medium supplemented with IL-21(30 ng/ml). After 3 days, half of the medium was replaced and 30ng/mL IL-21, 20ng/mL interleukin 15(IL-15) and 500ng/mL soluble Fc fused IL 15-receptor alpha (sIL15Ra-Fc, R)&D Systems). After 10 days, the irradiated K562/HLA-E cells pulsed with the corresponding peptide were restimulated in the presence of IL-21, IL-15 and Fc fused IL 15-receptor alpha. IL-2(50IU/ml) and IL-7(10ng/ml) were added 1 day after the second stimulation to further promote the expansion of activated Ag-specific T cells. Cultures were then stimulated weekly for 4-10 weeks. Peptide-specific expansion of T cells was monitored by flow cytometry analysis using a cytotoxicity assay.

Cytotoxicity assays

Cell-based flow cytometry assays were used to measure the specific cytotoxic activity of the ex vivo generated peptide-specific HLA-E-restricted CD8+ T cell line. Briefly, CFSE-labeled targets were incubated overnight at 26 ℃ in the presence or absence of synthetic peptide (K562/HLA-E) and co-cultured with CD8+ T cell lines at different ratios (10:1 and 1:1) for 5 hours. Control tubes (target cells without the CD8+ T cell line) were also assayed to determine spontaneous cell death. After 5 hours of co-culture, the cells were stained with 7-AAD. For data analysis, CFSE-positive target cells were examined for cell death by uptake of 7-AAD. CFSE and 7-AAD double positive cells were considered dead target cells. The percentage of specific cytotoxic activity was then calculated using the following equation:

cytotoxicity (%) ═ target cell death-spontaneous death x100

Results

HLA-E expression on K562 cell line after peptide loading

Untransfected K562 cells did not show surface expression of HLA-E as assessed by flow cytometry, as did HLA-E transfected K562 in the absence of peptide loading (see Table 1). HLA-E surface expression was induced after pulsing K562/HLA-E overnight at 26 ℃ using specific HLA-E peptides from different sources (see Table 1).

Table 1: peptide-HLA molecule binding assay

Detection of antigen-specific HLA-E-restricted CD8+ T cells using a cytotoxicity assay

The inventors wanted to use a cytotoxicity assay to assess their specific functional activity, seeing long-term stimulation of cell proliferation in culture. Cells pulsed with the K562/HLA-E peptide induced activation and expansion of the antigen specific CD8+ T cell line, as the proliferated cells showed a highly cytotoxic response to the candidate antigen without significant activity against unrelated antigens (see table 2).

Table 2: specific cytotoxic Activity of expanded CD8+ T cells

Example 7: key pathogenic role of IFN-alpha in human HIV-1 infection

Materials and methods

Cryopreserved PBMCs were thawed in RPMI 1640 containing 10% Fetal Bovine Serum (FBS) and washed in FACS buffer. By comparison with viability markers (AmCyan live-dead kit from Invitrogen) and with CD3, CD4,CD8, CD45RA, CCR7 conjugated antibodies were incubated together for 10⁶Individual cells were phenotypically stained. Subsequently, the cells were washed, fixed with 4% paraformaldehyde for 5 minutes, washed, and collected with an AURORA cytometer (Cytek).

The frozen serum was thawed at 4 ℃ and centrifuged at 4000G for 10 minutes at 4 ℃. Using high sensitivity

The IFN-. alpha.serum concentration was measured by the technique (digital ELISA technique) (Quanterix).

Results

Comparison of Central Memory (CM) CD8+ T cell distribution in HIV-1 infected subjects

In the study of subjects chronically infected with HIV-1, the following groups were studied:

(i) elite Controllers (EC) for natural inhibition of HIV-1 in the absence of combination antiretroviral therapy (c-ART)

(ii) Non-controllers before (pre) and after (post) cART treatment, and

(iii) a group of age-matched Healthy Donor (HD) subjects.

The relative frequency of CM populations in the CD8+ T cell compartment (component) was evaluated in each subject group.

The gating strategy defining this subpopulation is as follows. Briefly, singlet (single) cells were defined and then gated on lymphocytes and viable cells. Among the viable cells, CD3+ T lymphocytes were identified, followed by the definition of a CD8+ sub-population. Subsequently, CD8+ T lymphocytes were analyzed for expression of CD45RA and CCR 7. Central memory T Cells (TCM) were CD45RA-CCR7 +.

Figure 10A shows that levels of CM CD8+ cells were lower in non-controller before cART than in other groups. Furthermore, combination antiretroviral therapy (cART) resulted in an increase in CM CD8+ cells.

Comparison of serum IFN-alpha levels in HIV-1 infected subjects

Serum IFN- α levels were determined in 4 groups. Figure 10B shows that non-controller patients had significantly higher serum IFN- α levels prior to treatment as compared to post-treatment.

CM CD8 in IFN-a and untreated HIV-infected patients⁺The percentage of cells is inversely related

In a population of HIV-infected patients (EC cART pre-patients), the inventors explored CM CD8⁺Potential correlation between cellular levels and serum IFN- α levels. In this study, there was a significant negative correlation between CM CD8+ cell frequency and serum IFN- α levels (spearman correlation r ═ 0,667; p<0.005). This reflects the key pathogenic role of IFN- α on T cell proliferation in secondary organs (secondary organ) (see FIG. 10C).

Example 8: prophylactic vaccine against SIV in macaques using inactivated SIV and live Lactobacillus plantarum

This scheme includes the following two steps.

FIG. 11 is a schematic diagram of the protocol plan of step 1.

Step 1: determination of the most effective protocol for inducing virus-specific CD8+ suppressor T cells in Rhesus Monkeys (RM)

The immunization protocol is based on experimental work performed on chinese rhesus monkeys, which is described in Lu et al (2012) Cell rep.2(6), 1736-46.

This scheme includes three steps:

1) oral priming using a preparation containing inactivated SIVmac239 as the activation principle and live Lactobacillus Plantarum (LP) as adjuvant,

2) oral fortification using the same preparation, and

3) intrarectal boosting with a non-infectious dose of live virus.

Including 8 male RMs, 2 were of chinese origin and 6 were of indian origin.

Primary oral immunization was performed for 5 consecutive days. Daily intragastric administration to monkeys at 4x10 in maltodextrin (20%) solution⁷Inactivated SIV and 3x10 copies/ml⁹cfu/ml of a 30ml preparation of live LP. They then received 25ml of the same preparation every 30 minutes in the stomach for 3 hours.

Oral boosting was performed on

days

28 and 29, and if necessary, on days 60 and 61.

Two intrarectal boosts were performed at 2 week intervals from day 90. Day-2, day 3 of oral priming and two days prior to each oral boost, i.e., day 26, and, if necessary, day 58 and the day prior to the IR boost, indian-derived monkeys received additional Intraperitoneal (IP) injections of poly I: C and lambda IFN.

Indian-derived 6 RMs were divided into two groups (B and C) depending on when anti-IFN α antibodies were added during immunization. For group B, on day 32, and if necessary, on day 64, the anti-IFN α antibody was administered the day before each IR boost. For group C, anti-IFN α antibody was administered only five days after the first IR boost. For all macaques, induction of virus-specific CD8+ suppressor T cells was monitored on day 50, and if necessary, on

days

80 and 126. Plasma viral load was followed weekly after IR-boost.

Step 2: vaccine efficacy was assessed by rectal challenge using SIVmac 239.

Only monkeys with virus-specific CD8+ suppressive T-cell elevation (mount) were subjected to intrarectal challenge with a high infective dose of SIVmac239(100,000TCID 50). Plasma viral load was followed weekly for 6 weeks.

Example 9: prophylactic vaccine against HIV in BSLT mice

This scheme includes the following two steps.

FIG. 12 is a schematic diagram of the protocol plan for step 1.

Step 1: determining the most effective protocol for inducing anti-HIV-1 specific CD8+ inhibitory T cells in BSLT mice

The immunization protocol is based on experimental work performed on chinese rhesus monkeys, which is described in Lu et al (2012) Cell rep.2(6), 1736-46. This scheme includes three steps:

1) oral priming by the intragastric route using a preparation containing inactivated HIV-1 as the activation principle and live Lactobacillus Plantarum (LP) as adjuvant;

2) oral boosting by intragastric route using the same preparation;

3) low dose rectal boost with live virus was used.

Four groups (A, B, C and D) of 10 mice were included. Group D is a control group to monitor the efficacy of challenge. Mice in this group D received no immunization or immunization with PBS.

Oral primary immunization by the intragastric route consists of daily oral ingestion of inactivated HIV-1 and live LP in a maltodextrin (20%) solution for more than 5 days. Mice received the same preparation daily, 3 times every 30 minutes, for 1 hour, in the stomach. Mice received 200mcl of the preparation.

Oral boosting by the intragastric route was administered on

days

28 and 29, and if necessary, on days 60 and 61.

Two intrarectal boosts were performed at 2 week intervals on

days

90 and 104. Groups B and C additionally received intraperitoneal injections of poly I: C and lambda IFN (100-.

Groups B and C differ in when anti-IFN α antibodies were added during immunization. For group B, anti-IFN α antibody was administered on day 32 during oral boosting of the intragastric route, and if necessary, on day 64 during oral boosting of the intragastric route. For group C, anti-IFN α antibody was administered only five days after IR boost. For all mice, induction of virus-specific CD8+ suppressor T cells was monitored on day 50, and if necessary, on

days

80 and 126. Plasma viral load was followed weekly after IR-boost.

Step 2: vaccine efficacy was assessed by rectal challenge using HIV.

Two months after the last anti-IFN α antibody administration, only mice with an increase in virus-specific CD8+ suppressor T cells were subjected to intrarectal challenge with a high infective dose of HIV (100,000TCID 50). Plasma viral load was followed weekly for 6 weeks.

Sequence listing

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Claims

1. A combination comprising:

1) a CD8 vaccine specific for at least one pathogen-specific antigen,

2) optionally, an interferon-alpha blocker, and

3) Type III interferon and/or an agent that stimulates the production of type III interferon.

2. The combination of claim 1, wherein the interferon-alpha blocker is selected from the group consisting of: agents that neutralize circulating alpha interferon, agents that block interferon-alpha signaling, depletion production A cellular agent for IFN-α and/or an agent for blocking IFN-α production, wherein the agent for neutralizing circulating α-interferon is selected from the group comprising: an active anti-IFN-α vaccine comprising antiferon or an active anti-IFN-α vaccine comprising antiferon Passive anti-IFN-alpha vaccine of IFN-alpha antibody or anti-IFN-alpha hyperimmune serum, wherein the agent that blocks interferon-alpha signaling is selected from anti-type I interferon R1 or R2 antibodies or selected from An endogenous modulator of interferon-alpha comprising SOSC1 or an aryl hydrocarbon receptor, wherein the agent that depletes IFN-alpha-producing cells is an agent that depletes plasmacytoid dendritic cells (pDC), wherein the blocking An IFN-α-producing agent is an agent that blocks pDC production of IFN-α.

3. The combination of claim 1 or 2, wherein the type III interferon comprises at least one IFN-λ selected from the group consisting of IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4, wherein the Agents that stimulate the production of Type III interferon include at least one TLR ligand, RIG-I ligand and/or MDA5 ligand.

4. The combination of any one of claims 1 to 3 for preventing or treating an infectious disease in a subject in need thereof, wherein the combination comprises:

1) a CD8 vaccine specific for at least one pathogen-specific antigen,

2) optionally, an interferon-alpha blocker, and

5. The combination for prophylaxis or treatment of claim 4, wherein the CD8 vaccine elicits or comprises the inhibitor MHC-1b/E-restricted CD8 ⁺ T cells.

6. The combination for prophylaxis or treatment of any one of claims 4 to 5, wherein the CD8 vaccine is an active vaccine, wherein the CD8 vaccine is a live virus comprising at least one infectious disease-associated antigen A vector, wherein the live viral vector is selected from the group of cytomegalovirus, lentivirus, vaccinia virus, adenovirus or plasmids.

7. The combination for prevention or treatment of any one of claims 4 to 6, wherein the CD8 vaccine is an active vaccine, wherein the CD8 vaccine is a cytomegalovirus (CMV) vector comprising:

- a first nucleic acid sequence encoding at least one pathogen-specific antigen,

- optionally, a second nucleic acid sequence comprising a first microRNA recognition element (MRE) operably linked to a CMV gene that is required or enhanced for CMV growth, wherein the MRE is in a microRNA expressed by endothelial cell lineages silent expression in the presence of

Wherein the CMV vector does not express active UL128 protein or its ortholog; does not express active UL130 protein or its ortholog; does not express active UL146 or its ortholog; does not express active UL147 protein or its ortholog things,

wherein the CMV vector expresses at least one active UL40 protein or its ortholog; expresses at least one active US27 protein or its ortholog and/or expresses at least one active US28 protein or its ortholog.

8. The combination for prophylaxis or treatment of claim 4, wherein the CD8 vaccine is an active vaccine, wherein the CD8 vaccine comprises at least one pathogen-specific antigen and non-pathogenic bacteria.

9. The combination for prophylaxis or treatment of claim 4 or 8, wherein the pathogen-specific antigen is selected from the group consisting of viruses, virus particles, virus-like particles, recombinant viruses, recombinant virus particles, conjugated viral proteins, and polynucleotides. A set of paraviral proteins wherein the virus, viral particle or recombinant viral particle is attenuated or inactivated.

10. The combination for prevention or treatment according to any one of claims 4, 8 to 9, wherein the non-pathogenic bacteria are live non-pathogenic bacteria, wherein the non-pathogenic bacteria is selected from attenuated or inactivated pathogenic bacteria, preferably wherein the bacteria are Lactobacillus bacterium, more preferably Lactobacillus plantarum.

11. The combination of claim 4, wherein the CD8 vaccine is an active vaccine, wherein the CD8 vaccine is ex vivo produced to present MHC-1b/E-restricted and MHC-II-restricted A dendritic, natural killer or B cell population of antigens, wherein the MHC-1b/E restricted antigen is a pathogen specific antigen.

12. The combination of claim 4, wherein the CD8 vaccine is a passive vaccine, wherein the CD8 vaccine is an autologous MHC-1b/E-restricted CD8 ⁺ T cell population produced ex vivo, wherein the MHC-lb/E-restricted CD8 ⁺ T cell population recognizes MHC-lb/E-restricted pathogen-specific antigens.

13. The combination of any one of claims 1 to 3, or the combination for prophylaxis or treatment of any one of claims 4 to 12, wherein the pathogen-specific antigen is a viral pathogen-specific A sexual antigen, wherein the pathogen-specific antigen is derived from a pathogen selected from the group consisting of human immunodeficiency virus, simian immunodeficiency virus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, hepatitis B virus, Hepatitis C virus, Papilloma virus, Plasmodium and Mycobacterium tuberculosis, more preferably wherein the viral pathogen specific antigen is derived from HIV or SIV pathogens such as gag, env, rev, tat, nef, pol and/or or vif antigen.

14. The combination of any one of claims 1 to 3, wherein the CD8 vaccine is a therapeutic vaccine, such as a prophylactic or curative vaccine.

15. The combination for treatment of any one of claims 4 to 12, wherein the treatment is prophylactic or curative.