Patent Grant
10052377
The invention relates to methods of inducing or enhancing an immune response against an immunogen in a subject. The invention further includes isolated nucleic acid vaccines, cellular vaccines, fusion proteins, expression vectors, vaccines, and immunogenic compositions for use therein.
There are approximately 33 million individuals with human immunodeficiency virus (HIV) infection worldwide. Many will die as a result of the infection which has already claimed more than 23 million deaths and 2.7 million people are newly infected each year (International AIDS vaccine initiative (IAVI), AIDS vaccine blueprint, A challenge to the field, A road map for progress, 2008, ISBN #0-9792432-8-9). Over 80% of new HIV-1 infections arise in the developing world.
There are approximately 10,000 new cases of hepatitis C virus (HCV) infections in Australia each year, 220,000 HCV-positive individuals in the country (Razali K et al., 2007, Drug Alcohol Depend 91: 228-235), approximately 200 million infected individuals in the world and an estimated 3-4 million new infections each year (Bonner, J. E., Esserman, D., Evon, D. M., 2012, Reliability and validity of a self-efficacy instrument for hepatitis C antiviral treatment regimens, J Viral Hep 19: 316-26). In developing countries, many infections result from poor medical practice (Hauri, A. M, Armstrong, G. L, Hutin Y. J, 2004). The global burden of disease attributable to contaminated injections given in health care settings. Int. J. STD AIDS, 15 (2004), pp. 7-16).
Effective vaccines are necessary to control the spread of these agents. However, existing licensed vaccines often depend on neutralising antibody to induce protection, but neutralising antibody may not effectively control challenge by HCV and HIV. These viruses mutate rapidly and generate neutralising antibody (NAb)-escape mutants. As conventional vaccines elicit NAb, an alternative approach to design vaccines for these viruses is necessary.
There exists a need for alternative vaccines for HIV and HCV. The present invention seeks to overcome, or at least ameliorate, one or more of the deficiencies of the prior art mentioned above, or to provide the consumer with a useful or commercial choice.
The above discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.
According to the invention there is provided a method of inducing or enhancing an immune response against an immunogen in a subject. The method includes the steps of:
The cell may be a somatic cell.
The method may include the further step of inducing cell death (necrosis) in the cell. The step of inducing cell death (necrosis) may be carried out before or after the step of administering the cell to the subject. The step of inducing cell death may result in lysis of the cell after vaccination. Lysis of the cell may occur in a subject after administration, thereby resulting in induction of a pro-inflammatory immune response upon processing of the lysed cell by the immune system of the subject. Typically, when the method includes the further step of inducing cell death in the cell, the cell is provided as a non-antigen presenting cell, such as a somatic cell. The cell may be selected from the group comprising allogeneic cells, HEK 293T cells and recombinantly expressing Human Leucocyte Antigen (HLA) class I and II molecules and Huh7 cells. Preferably, the somatic cell is either allogeneic or autologous. The cell may also be selected from cells which cannot express HLA proteins such as, but not limited to, CIR cells, K562 and LCL721.221 cells.
Alternatively the cell may be an antigen-presenting cell (APC).
The method may include the further step of the step of expressing the immunogen in the cell may further include the step of presenting the expressed immunogen on the surface of the cell.
When the cell is an APC, the step of expressing the immunogen in the cell may further include the step of presenting the expressed immunogen on the surface of the cell. The step of presenting the expressed immunogen on the surface of the cell may result in processing of the expressed immunogen-presenting APC by the subjects immune system, thereby inducing a pro-inflammatory immune response in the subject. The APC may be selected from the group comprising: allogeneic cells, dendritic cells (DC), macrophages, B cells, Langerhans cells, Kupffer cells. Preferably, the APC is either allogeneic or autologous.
The cell may co-express HLA. The HLA expression by the cell may be the result of a natural process or due to recombinant expression of HLA. That is, the HLA expression may be as a result of HLA expression by the cell by endogenous processes. Alternatively, the HLA expression may occur as a result of recombinant engineering and protein production.
The immunogen may be derived from HCV. When the immunogen is derived from HCV, the cell typically does not co-express HLA.
The immunogen may be derived from HIV. When the immunogen is derived from HIV, the cell may or may not co-express HLA.
In one preferred embodiment, the immune response is against an endogenous antigen so that after cell lyse the antigen is treated as exogenous antigen by the immune system resulting in cross presentation of the antigen. In a further aspect, the invention is a method of inducing or enhancing an immune response against an immunogen in a subject including the steps of:
Further features of the invention provide for the step of administering the cell to the subject, thereby inducing a pro-inflammatory immune response, to be carried out by processing of the immunogen by an APC of the subject via the class II pathway, the presented immunogen detectable by naïve CD4+ T cells.
Still further features of the invention provide for the step of administering the cell to the subject, thereby inducing a pro-inflammatory immune response, to be carried out by processing of the immunogen by the APC via the class I pathway, the presented immunogen detectable by naïve CD8+ T cells.
In a further aspect, the invention is a method of inducing or enhancing an immune response against an immunogen in a subject including the steps of:
In one example the cell is an APC.
Further features of the invention provide for the step of expressing an immunogen in an APC, thereby presenting the immunogen, to be carried out by processing of the immunogen by the APC via the class II pathway, the presented immunogen detectable by naïve CD4+ T cells.
Still further features of the invention provide for the step of expressing an immunogen in an APC, thereby presenting the immunogen, to be carried out by processing of the immunogen by the APC via the class I pathway, the presented immunogen detectable by naïve CD8+ T cells.
The cell of the method of the invention may be selected from the group comprising allogeneic or autologous cells, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), macrophages, B cells, Langerhans cells, Kupffer cells, HEK 293T cells, HEK293T cells recombinantly expressing HLA class I and II molecules and Huh7 cells. Preferably, the APC is allogeneic or autologous.
The immunogen may be of viral origin. As discussed above, the immunogen may be of HIV origin. The immunogen of HIV origin may be presented together with a Human Leucocyte Antigen (HLA) such as, but not limited to, HLA A*0201, HLA B*0702, HLA Cw7, DRB1, DRB3, DRB4 and DRB5. The immunogen, when derived from HIV, may be selected from, but not limited to, the HIV gag and/or env proteins. Alternatively, the immunogen may be derived from SIV, such as the SIV gag and/or env proteins.
In one embodiment, the method of inducing or enhancing an immune response against an HIV protein in a subject includes the steps of:
Preferably, the HLA is selected from the group consisting of: HLA-A*0101, HLA-A*0301 and HLA-A*2901 and HLA DP, DQ and DR.
The HIV antigen may be any HIV-derived protein, such as, but not limited to, gp120 or gag.
In a further embodiment, the method of inducing or enhancing an immune response against an HIV protein in a subject includes the steps of:
Further features of the invention provide for the step of inducing cell death in the cell to be carried out by inducing necrosis. The step of inducing necrosis in the cell may be carried out by exposing the cell to necrosis-inducing conditions, such as, but not limited to, heating the immunogen-presenting cell to 63° C. for at least 30 min.
Alternatively, the step of inducing cell death in the cell may be carried out by exposing the cell to a necrosis-inducing agent or expressing the necrosis agent in the cell (internal expression in the cell, rather than external exposure to the necrosis agent). Examples of the necrosis-inducing agent may include, but are not limited to, thymidine kinase (TK), perforin, granzyme, caspase, RIP proteins 1&3, adenovirus penton fibre, NSP4, streptolysin O, anthrolysin, PB-F2, and functional fragments thereof.
In another embodiment, the method of inducing or enhancing an immune response against an HIV protein in a subject includes the steps of:
Preferably, the cell is an APC.
Alternatively, the immunogen may be of HCV origin. The immunogen when of HCV origin may be selected from, but is not limited to, the following HCV proteins: core, E1/E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B
In a further embodiment the method of inducing or enhancing an immune response against an HCV protein in a subject includes the steps of:
Further features of the invention provide for the step of inducing cell death in the cell to be carried out by inducing necrosis. The step of inducing necrosis in the cell may be carried out by exposing the cell to necrosis-inducing conditions, such as, but not limited to, heating the immunogen-presenting cell to 63° C. for at least 30 min.
Alternatively, the step of inducing necrosis in the cell may be carried out by exposing the cell to a necrosis-inducing agent or expressing the necrosis agent in the cell (internal expression in the cell, rather than external exposure to the necrosis agent). Examples of the necrosis inducing agent may include, but are not limited to, thymidine kinase (TK), perforin, granzyme, caspase, RIP proteins 1&3, adenovirus penton fibre, NSP4, streptolysin O, anthrolysin, PB-F2, and functional fragments thereof.
The cell may undergo lysis after the step of administering the cell to the subject.
In another embodiment, the method of inducing or enhancing an immune response against an HCV protein in a subject includes the steps of:
In a further embodiment, the method of inducing or enhancing an immune response against an HCV protein in a subject includes the steps of:
The invention also provides for a nucleic acid vaccine comprising a nucleic acid sequence having:
The nucleic acid vaccine may be a DNA vaccine.
The first sequence and the second sequence may be bicistronic.
The nucleic acid vaccine may further include one or more promoters, such as, but not limited to, a CMV promoter or a SV40 promoter. The nucleic acid vaccine may also include a viral internal ribosome entry site (IRES), such as but not limited to, the encephalomyocarditis virus (EMCV) IRES.
In one embodiment, the nucleic acid vaccine includes:
The nucleic acid vaccine may include the first nucleic acid sequence and the second nucleic acid sequence operably linked for monocistronic expression by way of, for example, by the encephalomyocarditis virus internal ribosome entry site (EMCV IRES) and optionally includes an intervening nucleic acid sequence between the first nucleic acid sequence and the second nucleic acid sequence, the intervening nucleic acid sequence encoding the FMDV 2A protease or other autoprotease sequence.
Thus, in another embodiment, the nucleic acid vaccine includes:
Alternatively, nucleic acids having a DNA sequence encoding a protease cleavage site may be inserted between the first and second nucleic acid sequence such as, but not limited to, the foot and mouth disease virus 2A protease sequence. It will be appreciated that a vector may be designed to insert the foot and mouth disease virus 2A protease nucleic acid sequence between the upstream first nucleic acid sequence which encodes the immunogen and the downstream second nucleic acid sequence which encodes the polypeptide having necrosis inducing activity. The resultant fusion protein produced would be expected to include the immunogen fused to the protease and the polypeptide having cell death inducing activity. The 2A protease has self-cleaving activity which would be expected to result in the production of the upstream and downstream proteins as independent proteins by co- or post-translational cleavage. The amino acid sequence of the 2A protease as described herein as SEQ ID NO: 8, and the DNA sequence of the 2A protease as described herein as SEQ ID NO: 9.
The invention also provides for a nucleic acid vaccine comprising:
Further features of the invention provide for the intervening nucleic acid molecule to have the nucleic acid sequence of the foot and mouth disease virus 2A protease, or a functional fragment thereof.
Still further features of the invention provide for the polypeptide having necrosis inducing activity to be selected from, but not limited to, the group comprising thymidine kinase (TK), perforin, granzyme, caspase, RIP proteins 1&3, adenovirus penton fibre, NSP4, streptolysin O, anthrolysin, PB-F2, and functional fragments thereof. The NSP4 protein may be from rotavirus. Alternatively, the cell death-inducing protein may be selected from the group comprising, but not limited to, Anthrolysin O; human or mouse perforin, including wild type and mutant forms (such as, but not limited to, SEQ ID NO 7); Adenovirus penton fibre; Granzyme A or B; Caspase 1; Streptolysin O; the influenza A virus protein PB-F2 and Cholera toxin.
The invention further provides for a nucleic acid vaccine comprising for use in the method of the invention, wherein:
In another embodiment of the invention, the nucleic acid vaccine further comprises:
In another embodiment of the invention, the nucleic acid vaccine further comprises:
Due to the degeneracy of the DNA code, it will be well understood to one of ordinary skill in the art that substitution of nucleotides may be made without changing the amino acid sequence of the encoded polypeptide. Therefore, the invention includes any nucleic acid sequence for a polypeptide having necrosis inducing activity comprising a nucleic acid sequence encoding a toxic protein (which is capable of inducing cell necrosis) and a nucleic acid sequence encoding an immunogenic polypeptide according to the invention. Moreover, it is understood in the art that for a given protein's amino acid sequence, substitution of certain amino acids in the sequence can be made without significant effect on the function of the peptide. Such substitutions are known in the art as “conservative substitutions.” The invention encompasses a nucleic acid sequence according to the invention comprising a first nucleic acid sequence encoding a polypeptide having necrosis inducing activity and a second nucleic acid sequence encoding an immunogenic polypeptide according to the invention that contains conservative substitutions, wherein the function of the cell necrosis inducing polypeptide and immunogenic polypeptide according to the invention is not altered.
The invention extends to a fusion protein comprising:
The invention also provides a fusion protein comprising:
Further features of the invention provide for the protease to be the foot and mouth disease virus 2A protease, or a functional fragment thereof.
The invention also provides for a recombinant virus vaccine comprising
Further features of the invention provide for the recombinant virus vaccine to be selected from, but not limited to, the group comprising a replication-competent vector and a replication-defective vector, wherein the vector does not induce cell necrosis.
The recombinant virus vaccine may be selected from the group including a baculovirus vector; a replication-defective virus including a virus selected from the group comprising a replication-defective virus derived from human and animal adenoviruses, lentivirus, recVV, MVA, AAV, PAV; a replication-competent virus including a virus selected from the group comprising replication competent viruses derived from human and animal adenoviruses, lentivirus, recVV, MVA, AAV, PAV; and a liposome/lipoplex and an virosome generated from influenza virus or similar viruses.
The invention also provides for an expression vector comprising
Further features of the invention provide for the expression vector to be selected from, but not limited to, the group comprising a replication-competent vector and a replication-defective vector, wherein the vector does not induce cell death.
The expression vector may be selected from the group including a baculovirus vector; a replication-defective virus including a virus selected from the group comprising a replication-defective virus derived from human and animal adenoviruses, lentivirus, recVV, MVA, AAV, PAV; a replication-competent virus including a virus selected from the group comprising replication viruses derived from human and animal adenoviruses, lentivirus, recVV, MVA, AAV, PAV; and a liposome/lipoplex and a virosome generated from influenza virus or similar viruses.
The invention encompasses an expression vector of the invention containing DNA that is at least 40% identical; at least 45%; at least 50%; at least 55%; at least 60%; at least 65%; at least 70%; at least 75%; at least 80%; at least 85%; at least 90%; at least 95%; or at least 97% identical; to either SEQ ID NO: 1 or SEQ ID NO: 2. Most preferably, the expression vector will be at least 99% identical to of either SEQ ID NO: 1 or SEQ ID NO: 2.
The invention yet further provides for a pro-inflammatory cellular vaccine comprising a cell; and
The invention also provides for an immunogenic composition comprising:
The invention further provides a method of inducing or enhancing an antigen-specific immune response to an antigen in a subject, the method including the step of exposing the subject to the immunogenic composition according to the invention.
The invention also provides for a method of increasing CD4+ T cell levels and/or activity and/or CD8+ T cell levels and/or activity in a subject, the method including the step of exposing the subject to the immunogenic composition according to the invention.
The invention further provides for a method of vaccinating a subject with an immunogen, the method including the step of exposing the subject to the immunogenic composition according to the invention.
The invention also provides for a method of generating a dendritic cell (DC) immune response profile in a subject, the method including the step of exposing the subject to the immunogenic composition according to the invention.
The invention further provides for a method of generating a CD4+ T cell and/or CD8+ T cell immune response profile in a subject, the method including the step of exposing the subject to the immunogenic composition according to the invention.
The invention also provides for a method of inducing an antibody immune response in a subject, the method including the step of exposing the subject to the immunogenic composition according to the invention.
Further features of the invention provide for the step of exposing the subject to the immunogenic composition according to the invention to comprise the following steps:
Still further features of the invention provide for a method of preparing an immunogenic composition, the method including the steps of:
The invention extends to an immunogenic composition prepared according to the method of the invention.
The invention also provides for a method of presenting an immunogenic polypeptide to a subject, the method including the steps of:
The invention further provides for a method of generating a cytotoxic T cell response against HIV in a subject, the method comprising:
The HLA may include a plurality of HLA proteins and/or other cell proteins that are in the HIV lipid envelope.
The invention also provides for a pro-inflammatory cellular vaccine comprising an allogeneic and/or autologous cell comprising HIV antigen and human leukocyte antigen (HLA) and wherein the allogeneic and/or autologous cell does not comprise live HIV.
The invention further provides for a method of generating a cytotoxic T cell response against HCV in a subject, the method comprising:
The invention also provides for a pro-inflammatory cellular vaccine comprising an allogeneic and/or autologous cell comprising HCV antigen wherein the cell does not comprise live HCV.
The invention further provides for an isolated antibody capable of binding to the immunogen as produced by one or more of the following:
The invention further provides a method of prophylactically or therapeutically treating an infection in a patient, said method comprising administering to the patient an effective amount of the immunogenic composition according to the invention. Preferably, when the immunogenic composition comprises cells, the dose is selected from the list comprising: between 1×102 to 1×109 cells per dose; between 1×103 to 1×108 cells per dose; between 1×104 to 1×107 cells per dose; and between 1×105 to 1×106 cells per dose. Preferably, when the immunogenic composition comprises a nucleic acid vaccine, fusion protein, recombinant virus vaccine or expression vector, the dose of nucleic acid vaccine, fusion protein, recombinant virus vaccine or expression vector is selected from the list comprising: between 10 μg to 100 mg per dose; between 100 μg to 10 mg per dose; between 300 μg to 5 mg per dose; between 300 μg to 3 mg per dose; and between 500 μg to 1 mg per dose. Preferably the immunogenic composition is administered intramuscularly, intra-dermally or intravenously. Preferably, the immunogenic composition is administered on a routine selected from the list comprising; three times daily, twice daily, twice weekly, once monthly, twice monthly, every two months, every three months, every sixth months, yearly twice yearly every two years and every five years.
The invention further provides a use of the immunogenic composition of the invention in the manufacture of a medicament for the prophylactically or therapeutically treatment of an infection in a patient.
The invention further provides a pharmaceutical composition comprising the immunogenic composition of the invention, together with an acceptable diluent or carrier.
The invention further provides a therapeutic vaccine comprising the immunogenic composition of the invention.
The invention further provides a prophylactic vaccine comprising the immunogenic composition of the invention.
The invention further provides a dosage form comprising the immunogenic composition of the invention.
The invention further provides a kit comprising the dosage form of the invention sealed in a vial or container. Preferably, the sealed vial or container is labelled with instructions that the kit is used for the prophylactic or therapeutic treatment of an infection in a patient.
The term “immunogenic composition” as used herein comprises the substances of the present invention and optionally one or more pharmaceutically acceptable carriers. The immunogenic compositions can be conveniently administered by any of the routes conventionally used for vaccine administration, for instance, parenterally or by inhalation. The substances may be administered in conventional dosage forms prepared by combining the immunogenic composition with standard pharmaceutical carriers according to conventional procedures. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the methods described above. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. Dosage can be varied and delivered as one or multiple doses. The DNA can be delivered as naked DNA or complexed with lipids, liposomes, lipoplexes, virosomes, immunoliposomes, or other commercial or non-commercially available compounds.
The dose administered to the patient, in the context of the present invention, should be sufficient to induce an immune response as detected by conventional assays measuring CD4+ and/or CD8+ response, conventional assays measuring antibody response, and/or effect a beneficial therapeutic response in a subject over time such as, but not limited to, a decrease in viral load or a decrease in other viral associated symptoms.
Some of the vaccines of the invention described above may have application as therapeutic vaccines. That is, some of the vaccines of the invention may be applied as injected therapy for administering to patients already infected with HIV or HCV. In such instances, the use of some of the vaccines of the invention may have application in substantially increasing the immune response to HIV or HCV, and substantially decreasing disease progression.
Some of the vaccine of the invention described above may have application as prophylactic vaccines. That is, some of the vaccines of the invention may be applied as preventative therapy for administering to patients not already infected with HIV or HCV. In such instances, the use of some of the vaccines of the invention may have application in substantially generating an immune response to HIV or HCV in a previously uninfected subject.
The vaccines of the invention described herein may be delivered by various routes of delivery eg. Intranasal, oral, subcutaneous, intradermal, intramuscular, intrathecal, pulmonary, intrarectal, topical, intravaginal, by scarification, intralesionally, intrabuccal, particle bombardment, nanoparticles, ID pulse followed by IN sublingual boost or any other combination.
Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:
SEQ ID NO: 1 is the DNA sequence of pVAX luciferase-TK plasmid.
SEQ ID NO: 2 is the DNA sequence of pVAX luciferase-NSP4.
SEQ ID NO: 3 is the DNA sequence of thymidine kinase (TK).
SEQ ID NO: 4 is the DNA sequence of NSP4.
SEQ ID NO: 5 is the DNA sequence of luciferase.
SEQ ID NO: 6 is the RNA sequence of NS3.
SEQ ID NO: 7 is the DNA sequence of mouse mutant perforin (PRF).
SEQ ID NO: 8 is the amino acid sequence of the 2A protease.
SEQ ID NO: 9 is the DNA sequence of the 2A protease.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Introduction
DNA Vaccines
DNA vaccines induce cell-mediated (CMI) and humoral immune responses, and result in antigen expression and processing that mimics viral protein production. DNA vaccines also have practical advantages, as they are highly stable and more easily and rapidly manufactured than protein or recombinant-virus vaccines.
Attempts to improve DNA vaccine immunogenicity have largely focused on the co-expression of immunostimulatory proteins such as cytokines, co-stimulatory molecules CD80 and CD86, and Toll-like receptor ligands. Previously, the improvement of immune responses to vaccines has involved targeting antigen directly to dendritic cells (DC). Protein vaccines have been conjugated to antibody that binds DC surface receptors such as DEC205 and Clec9A. This strategy increased DC antigen presentation and T cell responses following protein vaccination, which has traditionally induced mainly humoral responses. However, such approaches require additional adjuvants such as Poly I:C to ensure DC activation.
In Example 1, the inventors have developed a DNA vaccination strategy for DC targeting of antigen and for providing adjuvant signals for activation via induction of necrosis. Necrosis provides danger signals to the immune system via the release of intracellular factors that act as ‘damage-associated molecular-patterns’, (DAMPs). DAMPs such as uric acid and HMGB-1 are capable of signaling to DC via diverse receptors. More recently the receptor Clec9A and its ligand F-actin have been identified as a mechanism by which DC sense necrosis and target antigen for cross-presentation. Furthermore, necrosis also leads to NLRP3 inflammasome activation within cells and release of pro-inflammatory cytokines. In addition, tissue damage resulting in release of double-stranded DNA into the extracellular space has been identified as one of the underlying mechanisms of Alum adjuvants. The DNA vaccine described herein encodes an antigen and a cytolytic gene that results in targeted necrosis of antigen-positive cells. As such, this DNA vaccine substantially mimics the effect of a lytic virus, and lytic viruses such as vaccinia are known to be highly immunogenic.
Suicide genes, that generally induce apoptosis of target cells, have been proposed for use as cancer therapies. The inventors have surprisingly found that suicide genes or cytolytic genes which induce necrosis may be used as adjuvants for prophylactic vaccines.
The inventors believe that for optimum protection, vaccines against HIV and HCV require cell mediated immunity (CMI) to be induced in addition to neutralising antibody (NAb) or antibody with other characteristics eg. ADCC activity. CMI is most readily generated by natural infection, live- or recombinant-virus vaccines, or DNA vaccines, resulting in expression of the immunogens.
Innate Immunity Activates Adaptive Immune Responses
Innate immunity is activated by pathogen associated molecular patterns (PAMPs) or DAMPs as a prelude to adaptive immune responses. The most common PAMPs are LPS and dsRNA which bind to recognised pattern recognition receptors (PRR), including toll-like receptors (TLRs) but these PAMPs are not expressed after vaccination with naked DNA or recombinant, replication-defective vaccine vectors eg. RecAd. Consequently, although these vaccines generate viral antigens for uptake by DC, this will not result in mature DC because an additional stimulation signal or DAMP is necessary to “licence” mature, antigen-loaded DC with the capacity to migrate to draining lymph nodes and activate naïve T cells. Necrotic cells in particular are a rich source of DAMPs which include the heat shock proteins (HSP), uric acid, high mobility group box 1 protein (HMGB1) and extracellular ATP, chromatin, nucleosomes, DNA, galectins, thioredoxin, S100 proteins, cathelicidins, and defensins, and act as natural adjuvants. Apoptotic cells, although not generally inflammatory in nature, may develop secondary necrosis. HSP70 and HMGB1 have been used successfully to increase the immunogenicity of viral proteins. One study used a mixture of plasmids which encoded HIV gag and HMGB1, whereas the results described herein used DNA vaccines which encode both proteins in a single vector.
Cell Death and Priming Immune Responses
B lymphocytes express immunoglobulin and class II MHC molecules on their cell surface and are able to produce antibody which recognises the same antigen as that of their immunoglobulin receptor, the B cell receptor. B cells can also act as an APC. The antigen is recognized by the immunoglobulin receptor, phagocytosed, processed and the epitopes presented to a helper T cell in a MHC class II-restricted manner. This results in activation of the T cell and in the production of cytokines which in turn induce the B cell to divide and secrete specific antibodies. In this context, the B cell acts as an APC that can phagocytose antigen, analogous to a Class II APC.
However, in a similar manner to that described above for DC, additional stimuli, provided by PAMPs or DAMPS, are required to activate the B cell. Thus, many antigens are dependent on T cell help in order to elicit an effective antibody response.
Although CD8+ T cells are recognised as the effector cells that eliminate virus-infected cells or secrete cytokines that inhibit virus replication, there is also a need for CD4+ T cell help to prime and expand CD8+ T cells. By mimicking virus-related cell death the invention described herein facilitates the cross presentation of the immunogen and the induction of CD8 T cell responses and also facilitates the induction of CD4 T cell responses by uptake of exogenous immunogen that results from cell lysis.
Dendritic cells (DC) are the major APC able to prime naïve T cells, the primary aim of vaccination. DC phagocytose viral antigens (released from infected or vaccine-targeted necrotic cells), process and present these to naïve CD4+ T cells via the class II pathway (to generate Th) and to naïve CD8+ T cells via the class I pathway (cross presentation) to generate CTL. Cross presentation is proposed to be required for CD8 T cell immunity to viruses, because immunity would otherwise only occur if the virus or a vaccine infected DC to express antigen. If vaccine delivery vehicles are to be successful, they must target DC or otherwise direct the immunogen to DC. It is possible, even likely, that cross presentation is the major mechanism to induce cell mediated immune responses.
Dead cells are highly immunogenic due to release of DAMPs, pro-inflammatory “danger signals”. The uptake of viral antigen-positive dead or dying cells by DC and other APC represents a fundamental mechanism to elicit immunity against pathogens or the payload of vaccine delivery vehicles that do not intrinsically infect DC.
Suicide Gene Therapy
Suicide gene therapy has been used to treat various tumours by inducing cell death. The prototype suicide gene is thymidine kinase (TK), usually derived from herpes simplex virus (HSV). In the first step, the gene is delivered for protein expression in the target cell. The second step is to administer a prodrug, ganciclovir (GCV), which is activated by the expressed protein. The HSV TK gene, used in combination with GCV, is one of several approaches to kill cells and can induce necrosis, depending on cell type. GCV, commonly used to treat patients with herpesvirus infections, is a nucleoside analog which is converted to the monophosphate form by the HSV TK, then into the triphosphate form by cellular kinases, prior to incorporation into DNA. This results in DNA chain termination and cell death. Non apoptotic cell death (necrosis) results in expression of HSP70, and DC express receptors for HSP. Thus TK/GCV-induced cell death is likely to result in cross presentation of a protein immunogen in DC and phagocytosis by macrophages. The immune response to tumour cells was more effective after necrosis, characterized by increased levels of HSP70, than after apoptosis, although apoptotic cells also induced anti-tumour immunity. In that study, apoptosis or necrosis was induced in different cell lines by TK/GCV treatment and immunogenicity was increased after co-transfection of the tumour cells with HSP70 cDNA.
Importantly, work in this field has been limited to the use of suicide gene therapy in treating cancers, for the direct destruction of the cancer cell. The invention relates to methods of duplicating the effect of live attenuated viruses which result in the production of endogenous immunogen and thereby during expression of the viral antigen the cell will lyse to result in exogenous protein that will be phagocytosed by antigen presenting cells (including dendritic cells), which will then cross present the phagocytosed antigen.
Other prodrug/activator combinations can be used to convert non-toxic prodrugs into toxic metabolites eg. cytosine deaminase activated by 5-fluorocytosine and purine nucleoside phosphorylase activated by fludarabine. More recently, the use of a suicide protein which is directly cytotoxic and does not require activation was reported. In the study, a protein immunogen and the cytotoxic protein M from the infectious hematopoietic necrosis virus I (IHNV) were co-expressed from DNA after vaccination of fish. Protein M expression was controlled by an inducible promoter viz. the metallothionein promoter by the addition of zinc to the water in which fish were kept. This resulted in elimination of residual DNA after DNA vaccination, but not increased levels of protection against challenge with IHNV. Although these approaches are generally effective in vitro and may have some effect in vivo, they are dependent on the administration of an activation- or inducing-reagent, after vaccination. This adds a degree of complexity to the vaccination process and it is likely that a significant proportion of human vaccines will be non-compliant if this procedure was necessary for a human vaccine to be effective.
Materials and Methods
DNA Vaccines
All DNA vaccine constructs are based on the pVAX plasmid backbone (Invitrogen). Genes for Firefly luciferase2 (Promega) or codon optimised HIV-1 gag (clade B) were inserted downstream of the CMV promoter. An additional promoter, the SV40 promoter, and a poly-adenylation sequence were inserted into the pVAX backbone and all cytolytic/suicide gene candidates were inserted downstream of this second promoter. All DNA vaccines were purified using the Qiagen Endotoxin-free Mega kit.
Cell Culture
HEK293T cells (human embryonic kidney cell line) were grown in DMEM supplemented with 10% FCS and 1% Pen/Strep, and transfected with DNA using Fugene 6 reagent (Promega). Cell viability was determined by trypan blue staining. Cell death was tracked over time by luciferase assay using firefly luciferase as a reporter and D-Luciferin (Perkin Elmer) substrate. Markers of apoptosis and secondary necrosis were assayed by flow cytometry using Annexin-V (Invitrogen) and PI (BDBiosciences) staining.
Mice
All experiments were approved by the University of Adelaide and the Women's and Children's Health Network animal ethics committees. C57BL/6 mice from the University of Adelaide Laboratory Animal Services were maintained under specific-pathogen-free conditions and vaccinated at 6-8 weeks. All interventions were performed under Domitor/Ketamine anesthetic, injected i.p. The anesthetic was reversed with Antisedan i.p. Blood samples were obtained by retro-orbital bleed with haematocrit tubes. Live imaging was performed on isofluorane sedated animals using an IVIS live imager (PerkinElmer) after injection of D-luciferin K+ 3 mg/20 g mouse.
Immunization
C57BL/6 mice received a 50 L dose of DNA vaccine (50 g in saline) injected into the dermal layer of the ear (25 l/ear). Unless otherwise stated, mice received a total of 3 doses of vaccine at 4-week intervals, and peripheral blood samples were taken prior to each vaccination. At 10 days post-final vaccination, the mice were anesthetised and killed, and spleen, peripheral blood and draining lymph nodes collected.
ELISPOT
IFN ELISPOT was performed on red blood cell-depleted splenocytes that were re-stimulated with 2 μg/mL 15-mer, 11a.a overlapping gag peptide pools (NIH AIDS reagent bank #8117) or MHC I and II restricted immunodominant peptides (listed in the HIV molecular immunology database, http://www.hiv.lanl.gov/). Multiscreen-IP HTS plates (Millipore) were coated with anti-mouse IFN (clone AN18, MabTech) and secreted IFN was detected with anti-mouse IFN-biotin (clone R4-6A2, MabTech), streptavidin-AP (Sigma) and SigmaFast BCIP/NBT. Cells for the luciferase ELISPOT were re-stimulated with 2 μg/mL of the immunodominant peptide, LMYRFEEEL.
Flow Cytometry
Multi-colour intracellular cytokine staining was performed on splenocytes re-stimulated with immuno-dominant gag peptides for 12 hours in the presence of Brefeldin A. Staining was performed with BD FACS Cytofix/Cytoperm and BD anti-mouse antibodies (CD3-PercP-Cy5.5, CD8-APC-Cy7, CD44-APC, IL-2-FITC, IFN-Pecy7, TNF-PE, BDbiosciences). Dendritic cell staining was performed on cell suspensions obtained from the auricular draining lymph nodes using BD antibodies (CD8a-APC-Cy7, CD11c-PeCy7, CD80-APC, CD86-PE, MHCII-FITC, BDbiosciences). Cells were analysed on a BD FACS Canto and results analysed with FlowJo software.
ELISA
Corning 96 well EIA/RIA plates were coated with 500 ng/well recombinant luciferase (Promega). Serum samples were incubated for 2 hours at 37° C. and detected with anti-mouse IgG-HRP (GE healthcare) and SigmaFast OPD (Sigma).
EcoHIV Challenge
EcoHIV/NL4-3 challenge and qRT PCR monitoring were performed as described previously (Potash M J, et al. (2005) Proc Natl Acad Sci USA 102(10):3760-3765; Roshorm Y, et al. (2009) Eur J Immunol 39(7):1831-1840; Suhrbier A, et al. (2013) PLoS One In Press). Briefly, EcoHIV stocks were prepared by transfection of pEcoHIV into HEK293T cells. Purified virus supernatant (or conditioned media control) was administered to mice via i.p. injection at a concentration of 1.5 ug p24 (Zeptometrix p24 antigen ELISA). Seven days post EcoHIV challenge, mice were culled, and spleen, peritoneal exudate cells (PEC) and peripheral blood samples were collected. RNA was isolated using the Trizol method and used to generate cDNA (Qiagen Quantitect RT-PCR kit). 50 ng of cDNA was used as template for quantitative real-time PCR using the Quantifast Sybr Green kit (Qiagen) to determine levels of MLV mRNA (5′-GAGGTCGGGTGGAAGTACCA-3′ 5′-TGCA TCTTGGCCTTTTCCTT-3′). Results were normalised to RPL13a expression (5′-TAGGGCCAAACCCCGTTCTG-3′ 5′-GCCGGTGGAAGTTGGGTAGG-3′) after validation of primer amplification efficiency, using the CT method of quantification.
Statistical Analysis
Data are presented as the mean+/−SEM. Data analysis and generation of graphs was performed using Graphpad Prism 5.0b and SAS Version 9.3. Nonparametric Kruskal-Wallis test was used in comparing the difference between the multiple vaccine groups (pVAX GAG Standard Vaccine, pVAX GAG DTa (A), pVAX GAG PRF (B) and pVAX GAG 2A PRF(C). If the global test showed significant difference between the groups then Wilcoxon tests were performed to compare the post-hoc difference between Standard Vaccine group vs. A, Standard Vaccine group vs. B and Standard Vaccine group vs. C separately.
Results
DTa and PRF Cause Cell Death In Vitro
DNA vaccines were constructed using the plasmid pVAX, (Invitrogen), which contains either a luciferase (LUC) reporter gene or the HIV-1 GAG gene under the control of the constitutive CMV promoter. To investigate the effect of cell death following DNA vaccination, bi-cistronic constructs were designed to also encode either (i) perforin (PRF), an endogenous mediator that disrupts cellular membranes and causes cell lysis, or (ii) Diphtheria Toxin subunit A (DTa), which inhibits protein synthesis and causes apoptosis. These genes were inserted under the control of the weaker SV40 promoter (
The DNA vaccines were first tested for their ability to induce cell death in vitro. After transfection with the bi-cistronic plasmids, HEK293T cells showed visible signs of cell death 48 hours later, and cell death was confirmed by flow cytometry using Annexin V and propidium iodide (PI) staining (
Live Imaging of Luciferase Allows Tracking of Antigen Expression after Intradermal Vaccination
To investigate the effects of cell death after DNA vaccination in vivo, luciferase was used as a model antigen. This allowed live imaging of mice and tracking of antigen expression after vaccination. Female C57BL/6 mice were vaccinated with a single dose of 50 μg DNA via the intradermal (ID) route (ear pinnae dermis) and imaged periodically for luminescence. The inclusion of the PRF or DTa gene changed the kinetics of luciferase expression after vaccination (
All mice that received the ID vaccination showed some irritation (redness and swelling) around the site of injection, however this was observed even for mice that received an injection of saline, and rapidly subsided by days 2-3 post vaccination. No tissue damage was observed macroscopically, or microscopically by histological analysis in any vaccinated group. Thus co-expression of DTa or PRF led to specific killing of luciferase-positive cells without excessive inflammation or tissue damage.
The Luciferase-Specific Immune Response Following Intradermal DNA Vaccination
To examine the immune response to luciferase, vaccinated mice received booster vaccinations of 50 μg DNA at day 35 (at a point when luminescence expression was 100-fold lower in the CMV-LUC control mice (
An IFN ELISPOT was performed, using a single immunodominant luciferase peptide to restimulate splenocytes (
PRF Enhances the T Cell Mediated Immune Responses to HIV-1 GAG DNA Vaccines
To investigate the effect of PRF and DTa on the immune response to a viral antigen that contains multiple T cell epitopes, the luciferase reporter gene was replaced with a codon-optimised Clade B HIV-1 GAG gene. Mice received 3 doses of 50 μg CMV-GAG, CMV-GAG-SV40-PRF, CMV-GAG-SV40-DTA or CMV-GAG-2A-PRF DNA as noted above, and the immune responses were assayed 10 days post final vaccination. IFN ELISPOTs were performed using four overlapping peptide pools (30 15-mer peptides per pool) representing the complete GAG sequence, and a final pool containing only immunodominant peptides (which are otherwise present in pools 2 and 3). Mice vaccinated with CMV-GAG DNA showed responses ranging from 50-1200 mean SFU to all peptide pools (
Intracellular cytokine staining demonstrated that individual CD4 and CD8 T cells responded to vaccination by producing IFN, TNF and IL-2 (
Thus, only PRF increased the immune response to the GAG peptide pools, and only when it was expressed from the weaker SV40 promoter (CMV-GAG-SV40-PRF). An increased level of expression of PRF from the CMV promoter (CMV-GAG-2A-PRF) had no effect on the immune response, and CMV-GAG-SV40-DTa DNA actively decreased the immune response, when compared to the control CMV-GAG DNA. Therefore more rapid killing of GAG antigen-positive cells over 7-14 days did not improve immune responses compared to slower killing over 28-35 days.
PRF Increases the Frequency of Cross-Presenting CD8a+ and Necrosis-Sensing Clec9A+ DC
The data presented above show that expression of the cytolytic protein PRF and the apoptotic protein DTa resulted in opposite effects on the immune response to GAG. To determine whether PRF and DTa-induced cell death resulted in dendritic cell (DC) activation, the auricular draining lymph nodes were collected at different time points post vaccination. DC subpopulations were identified as CD3e−, CD11c+, and CD8a+/− or Clec9A+/− (
Importantly, at this time-point there was also an increase in the frequency of Clec9A+ DCs in the CMV-GAG-SV40-PRF vaccinated mice compared to CMV-GAG vaccinated mice (mean percentage 2.6 vs. 2.2, p=0.008) (
The activation status of the DC from the draining lymph node, as measured by MHCII and CD80 expression, was also determined (
PRF DNA Vaccination Reduces the EcoHIV Viral Load after Challenge
Significant changes in cytokine production by T cells and increased DC activation were observed after vaccination with DNA encoding HIV-1 GAG and the cytolytic protein, PRF. To determine whether these changes represent enhanced immunity to viral infection, vaccinated mice were challenged with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV encodes all other HIV proteins, including GAG, from the HIV-1 Clade B NL4-3 strain. Mice received 2 doses of 50 μg CMV-GAG DNA or CMV-GAG-SV40-PRF DNA by the ID route and were then challenged 10 days later with EcoHIV at a dose of 1.5 μg p24 by the IP route, as described previously. Mice were culled 7 days after challenge and tissues were collected to determine the viral load (
The embodiment of the invention described herein includes a method to target antigen for uptake by DC by utilising pathways involved in cellular necrosis, to increase the level of cross presentation of the antigen. The DNA vaccine described, which encodes an antigen of interest and the cytolytic protein PRF, induced necrosis in antigen-positive cells, enhanced DC activation and anti-viral T-cell mediated immunity, and provided increased levels of protection against viral challenge. This approach has advantages over more general mechanisms of inducing cell death at the site of vaccination, such as the use of Alum adjuvants or electroporation, as it limits tissue damage and more closely mimics the effects of lytic viruses, including live attenuated virus vaccines.
Two candidate genes are described, DTa and PRF, which showed differing effects on the kinetics of antigen expression and the subsequent immune response. The necessity for a threshold level and a minimum period of antigen expression is highlighted in this study, as reduced antigen expression and more rapid cell death resulting from increased levels of expression of perforin (CMV-GAG-2A-PRF) failed to enhance the antigen-specific immune response (
This approach induced no more tissue damage than expected from standard ID injection protocols, as cell death was restricted to cells that were previously transfected by the DNA vaccine. However, this approach resulted in an increased frequency of activated CD11c+ CD8a+ DC in the draining lymph nodes, a cell population that is believed to be essential in cross-presentation of antigen to naïve CD8 T cells (
Conversely, DTa failed to activate DCs or enhance immune responses, but instead reduced the immune response to some GAG epitopes. This highlights the importance of the mechanism of cell death in this model. DTa inhibits protein synthesis and results in apoptosis, which is known to be less inflammatory than necrotic cell death. Viral antigen-positive cells that become apoptotic and are cleared before they undergo secondary necrosis are expected to substantially limit DC activation and cross-presentation. These results indicate that necrotic cell death, in human setting, may be equally beneficial compared to, if not better than, apoptotic cell death in immune activation.
A DNA vaccine is described herein encoding an antigen and a cytolytic protein which elicits a higher level of protective immunity as determined by viral challenge than a DNA vaccine which only encodes the antigen, not only at the site of infection in the peritoneal space, but also systemically in the spleen and peripheral blood.
These results confirm that inducing necrosis in viral antigen-positive cells results in increased dendritic cell activation without excessive inflammation, and enhances the type of immune response important to control infection.
Materials and Methods
Briefly, DNA encoding luciferase was used as proof of principle. TK/GCV was used to determine if co-expression of luciferase and TK, followed by administration of ganciclovir increased the efficacy of DNA vaccination in mice. Constitutive co-expression of luciferase and a toxic protein was carried out to detect any increase in the level of the immune response against luciferase. To ensure that the expression of a toxic protein was not premature, and result in early death of cells targeted by the vaccine, the expression of the toxin was placed under the control of the SV40 promoter that is weaker than the CMV immediate early promoter (termed CMV promoter in this document) used to drive expression of the luciferase (or other immunogenic protein).
Construction of the Luciferase/TK DNA Vaccine
The modified Luciferase gene was amplified from a plasmid, and was inserted into the pVAX backbone (Invitrogen) under the control of the CMV promoter and upstream of the BGH polyadenylation sequence. The pVAX plasmid was digested with NheI and EcoR1 restriction enzymes and the modified Luciferase 2 gene ligated into the digested pVAX with T4 DNA ligase (NEB). The DNA was transformed into electrocompetent DH5alpha E. coli and positive clones were selected on kannamycin agar plates. The presence of the insert was confirmed by re-digestion and DNA sequencing.
The SV40 promoter, thymidine kinase (TK) gene and polyadenylation sequence were amplified from a plasmid. This plasmid contained the herpes simplex virus TK gene. The SV40 promoter, TK gene and SV40 poly (A)+ site were inserted into the pVAX-luciferase backbone by digestion with the restriction enzyme, PmII, ligated into pVAX with T4 DNA ligase (NEB) and positive clones were selected and tested as described above (
Construction of the Luciferase/NSP4 DNA Vaccine
The modified Luciferase gene was amplified from a plasmid and was inserted into the pVAX backbone (Invitrogen) under the control of the CMV promoter and upstream of the BGH polyadenylation sequence. The pVAX plasmid was digested with NheI and EcoR1 restriction enzymes and the modified Luciferase 2 gene ligated into the digested pVAX with T4 DNA ligase (NEB). The DNA was transformed into electrocompetent DH5alpha E. Coli and positive clones were selected on kannamycin agar plates. The presence of insert was confirmed by re-digestion and DNA sequencing.
The SV40 promoter and polyadenylation sequence were amplified from a plasmid. This plasmid was based on pcDNA3. The SV40 promoter and SV40 poly (A)+ site were inserted into the pVAX-luciferase backbone by digestion with the restriction enzyme, PmlI, ligated with T4 DNA ligase (NEB) and positive clones were selected and tested as described above.
The NSP4 gene was amplified by PCR from an expression construct, and inserted into the pVAX-luciferase plasmid under the control of the SV40 promoter and upstream of the SV40 polyadenylation sequence by digestion with SmaI restriction enzyme, the fragments ligated and selected as described above (
Construction of the Luciferase/PRF DNA Vaccine
The modified Luciferase gene was amplified from a plasmid and was inserted into the pVAX backbone (Invitrogen) under the control of the CMV promoter and upstream of the BGH polyadenylation sequence. The pVAX plasmid was digested with NheI and EcoR1 restriction enzymes and the modified Luciferase 2 gene ligated into the digested pVAX with T4 DNA ligase (NEB). The DNA was transformed into electrocompetent DH5alpha E. Coli and positive clones were selected on kannamycin agar plates. The presence of insert was confirmed by re-digestion and DNA sequencing.
The SV40 promoter and polyadenylation sequence were amplified from a plasmid. This plasmid was based on pcDNA3. The SV40 promoter and SV40 poly (A)+ site were inserted into the pVAX-luciferase backbone by digestion with the restriction enzyme, PmlI, ligated with T4 DNA ligase (NEB) and positive clones were selected and tested as described above.
The PRF gene was amplified by PCR from an expression construct and inserted into the pVAX-luciferase plasmid under the control of the SV40 promoter and upstream of the SV40 polyadenylation sequence by digestion with SmaI restriction enzyme, the fragments ligated and selected as described above (
Vaccination Schedule
Eight week old C57BI/6 mice were pre-bled and vaccinated with 50 μg of DNA by the intradermal route (ear) on 2 occasions, 5 weeks apart with pVAXLUC alone, pVAXLUC-TK+/−GCV, or pVAXLUC-NSP4. Luciferase expression was measured by luminescence every 7 days until luminescence decreased. Four weeks later, the mice were culled and blood collected to examine the humoral immune response to luciferase. In further experiments to analyse the antibody response, the mice were vaccinated via the intradermal route with 50 μg of pVAXLUC, pVAXLUC-TK+/−GCV, pVAXLUC-NSP4 or LUC-PRF on day 0, day 28 and day 56 and the antibody titer measured 10 days later. In experiments to analyse the cell mediated immune response, pVAXLUC, pVAXLUC-NSP4, pVAXLUC-TK+/−GCV or pVAXLUC-PRF, and pVAX, pVAXGagNSP4,or pVAXGagPRF vaccinated mice received 3×50 μg DNA at 4 week intervals via the intradermal route and the immune response also examined by ELIspot or intracellular cytokine staining 10 days later.
Temporal Expression of Luciferase
Luminescence was detected from day 1 using a Xenogen IVIS live imager. Mice were sedated and then injected with 150 μL D-luciferin by the intraperitoneal route. After 10 minutes incubation to allow the DNA vaccine-expressed luciferase to react with the luciferin, the mice were placed in the Xenogen IVIS and photographed. Luminescence was detected by Living Image software as photons per second (
No difference was seen in luminescence levels between the luciferase controls (pVAXluc, pVAXluc +GCV, pVAXluc +TK), or luciferase with adjuvant (pVAXluc +TK +GCV or +NSP4 or +PRF) groups after the initial DNA vaccine prime (day 0). All groups showed high levels of luciferase expression between days 3 and 21, with a slow reduction in expression between day 21 and day 35 at which point it was 1-2 log fold lower than on day 3.
After the DNA vaccine boost, the pVAXluc +NSP4 and pVAXluc +PRF groups showed a more rapid reduction in luciferase expression over time, compared to the other groups. (
Anti-Luciferase Antibody Titres in Vaccinated Mice
Recombinant firefly luciferase protein was used to coat ELISA plates, and serial dilutions of mouse serum were incubated at 37° C. for one hour. Anti-luciferase IgG was then detected with anti-mouse IgG conjugated with horseradish peroxidase (HRP). After incubation with orthophenylene diamine (OPD) substrate, the colour change in the substrate was measured by a spectrophotometer. Initial experiments in which mice were vaccinated with 50 μg DNA on 2 occasions showed that the titre of anti-LUC, detected by Elisa was 1/256 for the LUC and 1/4096 for the LUC+NSP4 DNA. Similar anti-LUC antibody titres were generated with TK/GCV treatment, but co-expression of TK without the later administration of GCV had no effect on anti-LUC titres. The anti-luciferase antibody titre was then measured in mice vaccinated with pVAXLUC, pVAXLUC (with the later administration of GCV, as a control) or pVAXLUC +TK (without the later administration of GCV) and was approximately 1/10,000. In mice vaccinated with pVAXLUC +TK (plus the later administration of GCV) or with pVAXLUC +NSP4 the anti-luciferase antibody titre was approximately 1/20,000 while vaccination with pVAXLUC +PRF resulted in an anti-luciferase antibody titer of approximately 1/40,000 (
Cell Mediated Responses to Luciferase in Vaccinated Mice
The cell mediated responses to luciferase were measured by ELIspot. Splenocytes were harvested from the mice, 7 days post final vaccination and stimulated with a peptide derived from luciferase. The results of this experiment showed that pVAXLuc +NSP4 vaccination resulted in a 3-fold increase in the frequency of IFN-expressing cells. Similarly, vaccination with pVAXLuc +PRF resulted in a 2-fold increase in frequency of IFN positive cells over vaccination with pVAXLuc along, and in the frequency of multifunctional T cells (
The results set out herein show that the use of suicide gene therapy can increase the immune response against a specific immunogen encoded by a DNA vaccine. This strategy can be applied equally to immunogens encoded by replication-defective recombinant vaccine vectors or even to replication-competent vaccine vectors both of which may normally be non-cytolytic.
The results set out herein also show that constitutive expression of a toxic protein simultaneously with the immunogen resulted in increased levels of immunity most likely as a result of increased cross presentation. Thus, there is no need for the subsequent administration of reagents such as a prodrug or a compound which can activate an inducible promoter, so that it is no longer necessary to administer ganciclovir to patients for example. Although the use of zinc to activate a metallothionein promoter may be effective in animals, it is unlikely to be equally effective in humans, in whom zinc is a crucial element that is continually present.
An effective vaccine will, by necessity, need to induce an inflammatory response that is unlikely to be achieved by suicide genes which induce apoptosis.
The following data illustrate another embodiment of the invention in which a mouse cell line, DC 2.4, was transfected with RNA encoding the HCV NS3 protein. The transfected cells were made necrotic by heat treatment at 63° C. for 30 min, to produce a syngeneic vaccine of the invention.
Necrotic, Viral Antigen Positive Cells are Highly Immunogenic
Preclinical studies of a necrotic whole cell vaccine were performed. A schematic diagram of the strategy is shown in
Furthermore, the responses elicited by the necrotic cells were much higher than detected with other (canonical) vaccination regimens, such as DNA vaccination, that typically generate ˜100 spot forming units/106 cells.
In a similar manner described in
To analyse these responses in greater detail, CD8+ and CD8− memory cells (CD44+) were stimulated ex vivo with the immunodominant gag peptides and the expression of IFN, TNF and IL-2 examined by intracellular cytokine staining followed by flow cytometry. The expression of multiple cytokines in individual cells was suggested to represent a prerequisite for a successful vaccine able to protect against challenge (R Seder, P A Darrah and M Roederer (2008) T-cell quality in memory and protection: implications for vaccine design. Nat Rev 8: 247-258). The results of these experiments showed that the frequency of CD8+ memory cells with the ability to synthesize all three cytokines in individual cells was increased by 1.7-fold by pVAXgag +NSP4 and by 2.7-fold by pVAXgag +PRF (
Likewise, although CD8+ T cells are often recognized as the cells which kill virus-infected cells, CD4+ cells which express multiple cytokines are necessary for T cell help. Consequently, CD8−, CD44+ memory T cells from the vaccinated mice were also examined by multicolour flow cytometry to analyse the synthesis of IFN, TNF and IL-2 in individual cells. The results showed that, although pVAX +NSP4 showed no increase in the frequency of these multifunctional T cells after peptide stimulation, T cells from mice vaccinated with pVAXgag +PRF showed a 3-fold increase in this cell population (
Materials and Methods
Preparation of Stable DC2.4 Cell Line Expressing Codon Optimised NS3 g1B
Third-generation lentiviral vectors containing the human hepatitis B virus posttranscriptional regulatory element (PRE) and the HIV central polypurine tract (cPPT) element (described in Barry et al, 2001 and Brown et al, 2010) were used for all experiments. For NS3 g1b overexpression, the backbone was reengineered to contain the elongation factor (EF)-1 promoter (plvEIG), a Gateway cloning site (att, CAT ccdB att), and an internal ribosome entry site (IRES) to drive the marker gene encoding green fluorescent protein (GFP). To generate the HCV NS3 delivery vector, this backbone was engineered to contain a Gateway cassette (att, CAT ccdB att) upstream of the Rev-responsive element into which the NS3 cassette was recombined. HCV NS3 g1b was inserted under the control of the EF-1 promoter, and the marker gene (GFP) was expressed from the IRES. To generate NS3 lentiviral stocks, HEK293T cells were seeded at a density of 6×106 cells in 18 ml of medium in a 75-cm2 tissue culture flask. Cells were transfected with 12.5 g of NS3 transfer vector (plvEIG), 7.5 g of Gag/Pol (8.2), 6.25 g of Rev (pRSV-Rev), and 3.75 g of Env (pCMV-VSV-G), using Lipofectamine LTX reagent (Invitrogen) and Opti-MEM (Invitrogen) reduced serum medium, in accordance with the standard protocol. The next day, the medium was replaced with 10 ml of fresh RPMI and 48 hr later supernatant was collected, filtered (pore size, 0.45 m), and used immediately or stored frozen (−70° C.)
For the transduction of the murine DC2.4 cell line, cells were seeded at a density of 2×105 cells per well in a 6-well plate. Medium was replaced with 3 ml of viral supernatant (unconcentrated) containing Polybrene at a final concentration of 8 g/ml. NS3-CFP-positive cells were sorted by flow cytometry (FACSAria; BD Biosciences, San Jose, Calif.) until purity reached 96%.
Stimulation of Necrosis Stable DC2.4 Cell Line Expressing Codon Optimised NS3 g1B
NS3 DC2.4 cells were resuspended in PBS. The cells were heated to 63° C. for 30 min to induce necrosis. Necrosis was confirmed by trypan blue staining and flow cytometry.
Flow Cytometric Detection of Necrotic Cells
Necrotic NS3 DC 2.4 cells were assayed by flow cytometry (FACSCanto, BD Biosciences, San Jose, Calif.). Necrosis was confirmed by checking FFC and SSC characteristics of DC2.4.
Results
Flow Cytometric Detection of Necrotic Cells
A stable DC2.4 cell line was prepared expressing codon optimized NS3 g1b. 97.56% of the sample of stable DC2.4 cells expressing codon optimized NS3 g1B were collected (
Seven stable DC2.4 cell line clones were prepared. The percentage of GFP positive cells of each cloned cell line varied between 52.1% to 82.8% (
Kinetics of Cell Activation Following a Single Dose of Necrotic or Live Vaccination
A single dose of 106 cells, representing live or necrotic cells of the stable NS3 g1b expressing DC2.4 cell line prepared, was administered to mice, and samples taken on days 0, 2, 3, 5 and 7. Samples from draining LN (localised) and spleen (systemic) were taken and assayed for i) T cell activation (CD3/CD4/CD8/CD25/CD69); ii) DC activation (CD11c/CD8/MHCII/CD80/CD86); iii) Clec9A DCs (Clec9A/CD11c/CD8/MHCII/CD4); iv) GFP expression in draining LN post vaccination; and v) cell numbers in draining LN.
CD11chigh Cells in DLN Post Single Dose of Necrotic or Live Vaccine.
As shown in
The frequency of necrotic CD11chigh CD8 cells in DLN did not significantly change over time (
The frequency of necrotic CD11chigh CD8+ Clec9A+ cells in DLN did not significantly change over time (
The frequency of necrotic CD11chigh CD8+ MHCII+ Clec9A+ cells in DLN significantly higher than live cells 2 days post vaccination (
NS3 Cells in DLN Post Boost Dose of Necrotic or Live Vaccine.
Mice were vaccinated with 106 cells prime, then a boost was similarly administered 7 days later. Samples were taken 14 days after the boost was administered and 3 pools of NS3 cells collected.
As shown in
The amount of IL-2, TNF, and IFNg secreted by NS3 stimulated cells was significantly increased in mice vaccinated with the NS3 positive necrotic cells compared with the NS3 positive viable cells (
Materials and Methods
To examine the potential of vaccination with HCV antigen-positive, necrotic dendritic cells on the outcome of HCV infection, a number of HCV-positive human individuals who had previously failed conventional interferon-based therapy were vaccinated. Baseline blood samples were taken. Four weeks prior to vaccination with the HCV antigen-positive, necrotic dendritic cells, each patient was treated with interferon- for a total of 4 weeks. One week prior to the vaccination with the cells, a blood sample was taken from the patient and monocyte-derived dendritic cells (Mo-DC) were prepared from each patient essentially as we described previously (Gowans E J, Roberts S, Jones K, Dinatale I, Latour P A, Chua B, Eriksson E M Y, Chin R, Li S, Wall D M, Sparrow R L, Moloney J, Loudovaris M, Ffrench R, Prince H M, Hart D, Zeng W, Torresi J, Brown L E, Jackson D C (2010). A phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals. J Hepatology. 53(4): 599-607), a process that took 5 days. Essentially, peripheral blood specimens were collected in 9 ml lithium heparin vacutainer tubes (Becton Dickinson). For every 15 ml of blood an equal volume of RPMI (R-) (Invitrogen) was mixed by swirling into a 50 ml Falcon centrifuge tube. Ficoll-paque solution was then underlaid beneath the 30 ml of diluted blood using a syringe and cannula. Tubes were placed in a centrifuge with aerosol containment, and centrifuged at 400 g for 30 min at 22° C.
The cloudy mononuclear cell interface was aspirated using a syringe/cannula. The cells were then transferred into a separate fresh sterile 50 ml tube (Falcon) and made up to a volume of 45 ml using RPMI to wash cells. The cells were then centrifuged at 400 g for 8 min at room temperature. The supernatant was discarded and the cells from each tube were pooled into a 50 ml centrifuge tube and resuspended in in MACS buffer (PBS, 2 mM EDTA, 1% BSA) at a concentration of 2.5×108 cells/ml. CD14 Microbeads (Miltenyi Biotec) were then added to the cells at 20 μl per 1×107 cells. This mixture was incubated at 4° C. for 15 min and then washed with MACS buffer. The cells were then applied to an LS+ MACS column (Miltenyi) in a magnetic field supplied by a MACS magnet. CD14 negative cells were allowed to pass through the column to waste. The column was then removed from the magnet, and CD14 positive cells eluted from the column by applying 10 ml of MACS buffer using the plunger supplied in the kit. The CD14 positive monocytes were then washed with MACS buffer and resuspended in Aim-V media (Invitrogen) at concentration of 1×106 cells per ml, with 1000 U/ml of GM-CSF (Berlex) and IL-4 (R&D Systems). The cells were then incubated for 5 days at 37° C. to obtain immature monocyte derived DC (Mo-DC).The MO-DC were then infected with a recombinant human adenovirus which encodes the HCV proteins, E1, E2 and NS3, from a genotype 1b virus (Trowbridge and Gowans, 1998) and incubated at 37° C. for 48 hours to ensure HCV antigen expression. The cells were then incubated at 63° C. for 30 minutes to induce necrosis. For patients who received multiple doses of HCV antigen-positive Mo-DC, the live antigen positive cells were frozen in liquid nitrogen, thawed on the day of vaccination and then made necrotic as described above.
As this was a dose escalation trial, successive patients received increasing doses of necrotic cells, ranging from 1×104 to 1×107 cells, in phosphate buffered saline (PBS) on 1, 2 or 3 occasions by the intradermal route. Blood samples were taken at regular intervals after vaccination, and the HCV-specific cell mediated immune response measured by ELIspot analysis of PBMC stimulated ex vivo with peptide pools which collectively represented the complete HCV polyprotein or proteins contained in the vaccine. The viral load was also measured by quantitative RT-PCR. The design of the trial and details of the dose and dosing schedule are shown schematically in
Results
Although this Phase I trial was primarily designed to test safety, ELIspot and qRT-PCR were performed to examine any changes in the HCV cell mediated immunity and viral load as a measure of efficacy. None of the patients showed any adverse events. As patients #1 and #2 only received 1×104 and 2×104 cells respectively, these patient failed to show any increase in the HCV cell mediated immunity (
The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.
The invention described herein may include one or more range of values (e.g. size, displacement and field strength etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. The term “active agent” may mean one active agent, or may encompass two or more active agents.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.
Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20170106081 A1 | Apr 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14401182 | US | |
| Child | 15397214 | US |
