Cellular vaccine and method of inducing an immune response in a subject

Abstract

The present 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.

Claims

1. A method of inducing or enhancing an immune response against an immunogen in a subject, the method comprising: a) administering to the subject a first nucleic acid sequence encoding an immunogenic polypeptide linked to a first promoter and a second nucleic acid sequence encoding a cell death inducing polypeptide linked to a second promoter, wherein the cell death inducing polypeptide is perforin and wherein the first promoter has a greater relative efficacy than the second promoter, and b) expressing in a target cell in the subject the immunogenic polypeptide from the first nucleic acid sequence and the cell death inducing polypeptide from the second nucleic acid sequence, thereby inducing necrosis of the target cell and a pro-inflammatory immune response.

2. The method of claim 1, wherein the target cell is a somatic cell.

3. The method of claim 1, wherein the target cell is an antigen presenting cell.

4. The method of claim 1, wherein step b) further includes the step of presenting the expressed immunogenic polypeptide on the surface of the target cell.

5. The method of claim 1, wherein the immunogenic polypeptide is a viral peptide.

6. The method of claim 5, wherein the immunogenic polypeptide is a human immunodeficiency virus (HIV) or a hepatitis C virus (HCV) protein.

7. The method of claim 1, wherein the first promoter is a CMV promoter; and the second promoter is a SV40 promoter.

8. The method of claim 1, wherein the expression level of perforin is lower relative to the expression level of the immunogenic polypeptide.

9. The method of claim 8, wherein the expression level of perforin is at least 10-fold lower than the expression level of the immunogenic polypeptide.

10. The method of claim 3, wherein the step of administering the cell first and second nucleic acid sequence to the subject, thereby inducing a pro-inflammatory immune response, is carried out by processing of the immunogenic polypeptide by the antigen presenting cell via the class II pathway, such that the processed immunogenic polypeptide is detectable by nave CD4.sup.+ T cells.

11. The method of claim 3, wherein the step of administering the first and second nucleic acid sequence to the subject, thereby inducing a pro-inflammatory immune response, is carried out by processing of the immunogenic polypeptide by the antigen presenting cell via the class I pathway, such that the processed immunogenic polypeptide is detectable by nave CD8.sup.+ T cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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:

(2) FIG. 1 shows a schematic of the design of DNA vaccines encoding PRF or DTa that induce cell death in vitro according to Example 1. (A) Schematic diagrams of the bicistronic DNA vaccine constructs. All vaccines use the pVAX backbone with a gene encoding an antigen (HIV-1 GAG or firefly luciferase (LUC)) under the control of the CMV promoter and PRF or DTa under the control of the SV40 promoter. An additional construct encodes the antigen and PRF as a polyprotein controlled by the CMV promoter that self-cleaves at the FMDV 2A protease sequence. (B) Phase microscopy images (100?) reveal rounded, detached HEK293T cells at 48 hours post transfection with DNA vaccines encoding DTa or PRF. (C) Flow cytometry to identify dead cells, and Annexin V and PI staining to identify apoptosis (Annexin V+) and necrosis (Annexin V+ PI+) 48 hours post transfection.

(3) FIG. 2 shows a table of the proportion of apoptotic and necrotic cells within the dead cell population gate. Apoptotic cells were defined as Annexin V+ PI?, secondary necrotic cells were defined as Annexin V+PI+. A third dead cell population that was Annexin V? PI+ was identified as cells that had undergone non-apoptotic cell death, likely primary necrosis.

(4) FIG. 3 shows in vivo imagining of luciferase for tracking of antigen expression from DNA vaccines in vivo, according to Example 1. (A) Representative IVIS images of luminescence in the mouse dermis on day 7, 14 and 28 after ID vaccination with 50 ?g CMV-LUC+/?PRF or DTa. (B) The longitudinal expression of LUC after ID vaccination as determined by quantification of luminescence in vaccinated mice. (C) Detection of LUC-specific antibody by ELISA. Mice received three vaccinations of 50 ?g CMV-LUC+/?DTa or PRF, and serum samples were taken on day 10 after the final dose. (D) IFN ELISPOT to detect LUC-specific T cell responses in splenocytes re-stimulated with an immunodominant LUC peptide. Spleens were harvested at day 10 after the final vaccination. Vaccinations were performed on groups of 5 female C57BL/6 mice. Graphs show mean+/?SEM.

(5) FIG. 4 shows IFN ELISPOT to detect GAG-specific T cell responses, according to Example 1. Mice were vaccinated ID with 50 ?g DNA on 3 occasions, 4 weeks apart and spleens harvested on day 10 after the final vaccination. Splenocytes were restimulated with 4 different pools of overlapping peptides covering the complete GAG protein (pools 1-4 shown in A-D respectively), or a final pool of C57BU6 MHCI and II restricted immunodominant peptides (E). Vaccinations were performed on groups of 8 female C57BL/6 mice. Graphs show mean SFU per 10^6 splenocytes (+/?SEM).

(6) FIG. 5 shows multifunctional T cells detected by flow cytometry, according to Example 1. Intracellular cytokine staining for GAG-specific T cell responses after DNA vaccination, as described in the legend for FIG. 4. Splenocytes were re-stimulated for 12 hours with the immunodominant GAG peptide pool in the presence of Brefeldin A. (A) The gating strategy to identify IFN, TNF and IL-2 positive CD4 and CD8 CD44+ T cells. (B) The percentage of IFN and TNF-secreting CD8 T cells and (C) the percentage of IFN, TNF and IL-2-secreting CD8 T cells in response to GAG peptide stimulation. (D) Percentage of CD4 T cells secreting IFN and TNF, and (E) IFN, TNF and IL-2 in response to GAG peptides. Vaccinations were performed on groups of 8 female C57BL/6 mice. Graphs show mean percentage of cytokine secreting cells (+/?SEM).

(7) FIG. 6 shows detection of activated dendritic cells. Dendritic cells in the draining lymph node are activated after a single 50 ?g DNA vaccination, according to Example 1. Auricular lymph nodes were harvested on days 3, 7, 10 and 14 post ID vaccination. (A) The gating strategy used to identify CD11c+ CD8a+ Clec9A+/? activated DC, showing representative plots from each group. pVAX empty and CMV-GAG upper middle and right panels, SV40-DTa and SV40-PRF lower middle and right panels. (B) Percentage of CD11c+ CD8a+ DC in the draining lymph node on day 14 post vaccination. (C) Percentage of Clec9A+ DC in the draining lymph nodes. (D) Histogram plots of the activation status of CD11c+ CD8+ DC as measured by MHCII and CD80 expression. DNA vaccinations were performed on groups of 4 female C57BL/6 mice. Graphs show mean percentage of cells (+/?SEM).

(8) FIG. 7 shows CMV-GAG-SV40-PRF is an effective immunogen against EcoHIV challenge, according to Example 1. EcoHIV challenge after DNA vaccination with 2 doses of 50 ?g CMV-GAG, CMV-GAG-SV40-PRF or pVAX empty, 3 weeks apart. The mice were challenged 10 days later with EcoHIV (1.5 ?g p24/mouse) and the viral load determined 7 days later. EcoHIV mRNA levels in (A) peritoneal exudate cells, (B) spleen and (C) blood. Vaccinations were performed on groups of 8 female C57BU6 mice. The graphs show mean expression normalised to RPL13a (+/?SEM).

(9) FIG. 8 shows a plasmid map of the pVAX luciferase-TK plasmid prepared for the bicistronic expression of luciferase and thymidine kinase (TK), according to Example 2.

(10) FIG. 9 shows a plasmid map of the pVAX luciferase-NSP4 plasmid prepared for the bicistronic expression of luciferase and NSP4, according to Example 2.

(11) FIG. 10 shows a plasmid map of the pVAX luciferase-PRF plasmid prepared for the bicistronic expression of luciferase and PRF, according to Example 2.

(12) FIG. 11 shows photographs of luciferase expression in mice transfected with the plasmids of FIG. 9 or pVAX (not shown), the plasmids with which the mice were transfected are indicated below the photographs, according to Example 2.

(13) FIG. 12 shows a graph representing luminescence expression over time after intra-dermal DNA vaccination of mice, according to Example 2.

(14) FIG. 13 shows a graph representing luciferase specific antibody detected in mice after vaccination with the plasmids of FIG. 8, 9 or 10, according to Example 2.

(15) FIG. 14 shows a graph representing anti-luciferase cell mediated immunity detected in mice after vaccination with the plasmids of FIG. 9, according to Example 2.

(16) FIG. 15 shows a graph representing anti-luciferase cell mediated immunity detected in mice after vaccination with the plasmids of FIGS. 8 and 10, according to Example 2.

(17) FIG. 16 shows a diagrammatic representation of the processes of necrosis of a HCV/HIV immunogen expressing cell ex vivo, the APC phagocytosing the antigen in vivo, presentation of the antigen by the APC to a T cell, and recognition of an infected cell by the activated T cell, according to Example 3.

(18) FIG. 17 shows ELIspot analysis of peptide-stimulated splenocytes representing the poorly immunogenic HCV NS3 protein harvested from mice vaccinated with the necrotic HCV NS3-positive cell line, compared to vaccination with the same live cell line. The HCV NS3-positive, necrotic cells illicit cell mediated immunity more effectively than viable cells. DC 2.4 cells were transfected with RNA encoding the HCV NS3 protein. A proportion was made necrotic. C57BI/6 mice were vaccinated by the subcutaneous route as follows; G1, 10.sup.6 untransfected, live cells G2, 10.sup.6 NS3 untransfected, necrotic cells; G3; 10.sup.6 NS# transfected, live cells; G4, 10.sup.6 NS3 transfected, necrotic cells. Splenocytes were stimulated with NS3 peptides in an ELIspot assay, according to Example 3.

(19) FIG. 18 shows the frequency of murine splenocytes, stimulated ex vivo with immunodominant gag peptides after vaccination with pVAXgag, pVAXgag +NSP4 or pVAXgag +PRF as determined by ELIspot, according to Example 4.

(20) FIG. 19 shows the frequency of murine CD44.sup.+ memory, CD8.sup.+ T cells stimulated ex vivo with immunodominant gag peptides after vaccination with pVAXgag, pVAXgag +NSP4 or pVAXgag +PRF as determined by multifunctional cytokine staining followed by flow cytometry, according to Example 4.

(21) FIG. 20 shows the frequency of murine CD44.sup.+ memory, CD8.sup.? T cells stimulated ex vivo with immunodominant gag peptides after vaccination with pVAXgag, pVAXgag +NSP4 or pVAXgag +PRF as determined by multifunctional cytokine staining followed by flow cytometry, according to Example 4.

(22) FIG. 21 shows flow cytometry scatter plots of the stable DC2.4 cell line expressing codon optimised NS3 g1b used in necrotic cell vaccinations according to Example 5.

(23) FIG. 22 shows the percentage of GFP positive cells in each of the single clones of the stable DC2.4 cell line expressing codon optimised NS3 g1b used in necrotic cell vaccinations according to Example 5.

(24) FIG. 23 shows the GFP expression in DLN obtained from vaccinated mice after vaccination according to Example 5.

(25) FIG. 24 shows the frequency of GFP positive cells, in DLN obtained from vaccinated mice 2, 3, 5 and 7 days after vaccination with NS3 positive, live or necrotic DC2.4 cells according to Example 5.

(26) FIG. 25 shows the total numbers of lymphocytes in DLN obtained from vaccinated mice 2, 3, 5 and 7 days after vaccination according to Example 5.

(27) FIG. 26 shows the frequency of CD11c.sup.high DCs in DLN, relative to frequency and total cell number, obtained from vaccinated mice 2, 3, 5 and 7 days after vaccination according to Example 5.

(28) FIG. 27 shows the number of CD11c.sup.high DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5.

(29) FIG. 28 shows the frequency of CD11c.sup.high CD8 DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5.

(30) FIG. 29 shows the cell number of CD11c.sup.high CD8 DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5.

(31) FIG. 30 shows the frequency of Clec9A.sup.+ CD11c.sup.high CD8 DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5.

(32) FIG. 31 shows the cell number of Clec9A+ CD11c.sup.high CD8 DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5. Grey bars represent mice vaccinated with live cells while black bars represent mice vaccinated with necrotic cells.

(33) FIG. 32 shows the frequency of Clec9A CD11c.sup.high CD8 DCs in DLN obtained from mice vaccinated with live or necrotic cells on 2, 3, 5 and 7 days after vaccination according to Example 5.

(34) FIG. 33 shows an ELISPOT of mouse splenocytes stimulated ex vivo with NS3 peptide pools after prime and boost according to Example 5.

(35) FIG. 34 shows ELISPOT data of splenocytes from individual mice after prime and boost according to Example 5.

(36) FIG. 35 shows the frequency of CD8+ CD44+ DCs in DLN obtained from vaccinated mice after vaccination according to Example 5.

(37) FIG. 36 shows the frequency of IL-2 secreting CD8+ CD44+ T cells (upper panel), and the mean fluorescent intensity of cells induced to express IL-2 (lower panel) after prime and boost according to Example 5, after stimulation or no stimulation in vitro with HCV NS3 peptides.

(38) FIG. 37 shows the frequency of TNF secreting CD8+ CD44+ T cells (upper panel), and the mean fluorescent intensity of cells induced to express TNF (lower panel) after prime and boost according to Example 5, after stimulation or no stimulation in vitro with HCV NS3 peptides.

(39) FIG. 38 shows the frequency of IFNg secreting CD8+ CD44+ T cells (upper panel) and the mean fluorescent intensity of cells induced to express IFNg (lower panel) after prime and boost according to Example 5, after stimulation or no stimulation in vitro with HCV NS3 peptides.

(40) FIG. 39 shows the design of the trial according to Example 6.

(41) FIG. 40 shows graphs depicting the IFN-g ELISPOT response of patient 1 to (A) HCV peptide pools representing the complete HCV polyprotein and (B) peptides representing proteins in the vaccine according to Example 6.

(42) FIG. 41 shows graphs depicting the IFN-g ELISPOT response of patient 2 to (A) HCV peptide pools representing the complete HCV polyprotein and (B) peptides representing proteins in the vaccine according to Example 6.

(43) FIG. 42 shows graphs depicting the IFN-g ELISPOT response of patient 3 to (A) HCV peptide pools representing the complete HCV polyprotein and (B) peptides representing proteins in the vaccine according to Example 6.

(44) FIG. 43 shows graphs depicting the IFN-g ELISPOT response of patient 4 to (A) HCV peptide pools representing the complete HCV polyprotein and (B) peptides representing proteins in the vaccine according to Example 6.

(45) FIG. 44 shows graphs depicting the IFN-g ELISPOT response of patients 1 to 4 (A-D) to CEF peptide pools according to Example 6.

(46) FIG. 45 shows a table of the raw HCV viral load data for patients vaccinated in the clinical trial according to Example 6.

(47) FIG. 46 shows a table of the HCV viral load as a percentage of the baseline level for the patients vaccinated in the clinical trial according to Example 6.

(48) FIG. 47 shows a graph of the change in viral load in the patients vaccinated in the clinical trial according to Example 6.

(49) FIG. 48 shows a table of the percentage viral load data for the patients vaccinated in the clinical trial according to Example 6.

(50) FIG. 49 shows a graph of the percentage change in viral load for the patients vaccinated in the clinical trial according to Example 6.

(51) SEQ ID NO: 1 is the DNA sequence of pVAX luciferase-TK plasmid.

(52) SEQ ID NO: 2 is the DNA sequence of pVAX luciferase-NSP4.

(53) SEQ ID NO: 3 is the DNA sequence of thymidine kinase (TK).

(54) SEQ ID NO: 4 is the DNA sequence of NSP4.

(55) SEQ ID NO: 5 is the DNA sequence of luciferase.

(56) SEQ ID NO: 6 is the RNA sequence of NS3.

(57) SEQ ID NO: 7 is the DNA sequence of mouse mutant perforin (PRF).

(58) SEQ ID NO: 8 is the amino acid sequence of the 2A protease.

(59) SEQ ID NO: 9 is the DNA sequence of the 2A protease.

(60) 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.

DESCRIPTION OF EMBODIMENTS

(61) Introduction

(62) DNA Vaccines

(63) 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.

(64) 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.

(65) 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.

(66) 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.

(67) 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.

(68) Innate Immunity Activates Adaptive Immune Responses

(69) 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 na?ve 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.

(70) Cell Death and Priming Immune Responses

(71) 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.

(72) 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.

(73) Although CD8.sup.+ 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.sup.+ T cell help to prime and expand CD8.sup.+ 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.

(74) Dendritic cells (DC) are the major APC able to prime na?ve T cells, the primary aim of vaccination. DC phagocytose viral antigens (released from infected or vaccine-targeted necrotic cells), process and present these to na?ve CD4.sup.+ T cells via the class II pathway (to generate Th) and to na?ve CD8.sup.+ 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.

(75) 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.

(76) Suicide Gene Therapy

(77) 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.

(78) 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.

(79) 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.

EXAMPLE 1

(80) Materials and Methods

(81) DNA Vaccines

(82) 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.

(83) Cell Culture

(84) 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.

(85) Mice

(86) 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.

(87) Immunization

(88) 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.

(89) ELISPOT

(90) IFN ELISPOT was performed on red blood cell-depleted splenocytes that were re-stimulated with 2 ?g/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 ?g/mL of the immunodominant peptide, LMYRFEEEL.

(91) Flow Cytometry

(92) 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.

(93) ELISA

(94) 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).

(95) EcoHIV Challenge

(96) 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.

(97) Statistical Analysis

(98) 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.

(99) Results

(100) DTa and PRF Cause Cell Death In Vitro

(101) 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 (FIG. 1A). The SV40 promoter resulted in 10-fold lower expression of luciferase compared to the CMV promoter, permitting lower levels of PRF or DTa expression relative to the antigen expressed from the CMV promoter. An additional construct encoded a LUC-PRF or GAG-PRF polyprotein under the control of the CMV promoter; the two proteins were separated by the FMDV 2A protease to enable autocleavage of the polyprotein. This latter construct allowed investigation of the effects of the cytolytic gene, PRF, when expressed at 10-fold higher levels from the CMV promoter.

(102) 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 (FIG. 1B-C). At 48 hours post transfection with CMV-LUC-SV40-PRF or CMV-LUC-SV40-DTa, a significant proportion of cells were shown to be dead as determined by forward and side scatter characteristics (38.2% and 43.2% respectively), compared to cells transfected with CMV-LUC DNA alone (15.4%). When PRF was encoded under the control of the stronger CMV promoter (CMV-LUC-2A-PRF) cell death was more rapid compared with CMV-LUC-SV40-PRF, with 48.9% of cells dead by 48 hours. As the transfection efficiency was generally ?70%, this indicates that a majority of the cells that expressed PRF or DTa were killed. Within these dead cell populations, the proportion of apoptotic (AnnexinV+ PI?) and necrotic (AnnexinV+ PI+) cells varied, as summarised in FIG. 2, reflecting the different mechanisms by which DTa and PRF induced cell death. Transfection with CMV-LUC-SV40-DTa resulted in cells that were primarily apoptotic or secondary necrotic, whereas CMV-LUC-SV40-PRF transfection resulted in a higher proportion of primary and secondary necrotic cells. Cells transfected with CMV-LUC-2A PRF showed the highest proportion of primary necrotic cells.

(103) Live Imaging of Luciferase Allows Tracking of Antigen Expression after Intradermal Vaccination

(104) 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 ?g 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 (FIG. 3A-B). Mice that received CMV-LUC DNA demonstrated detectable luminescence to day 35 post-vaccination, although the level of luminescence dropped 100-fold over this period. In contrast, mice that received CMV-LUC-SV40-PRF DNA showed a more rapid decrease in luminescence, which was undetectable by day 35. The CMV-LUC-SV40-DTa and CMV-LUC-2A-PRF groups showed the most rapid decrease in luminescence, and no detectable luminescence was seen in these mice after day 14, indicating luciferase-positive cell clearance (FIG. 3B). In addition, the CMV-LUC-SV40-DTa and CMV-LUC-2A-PRF vaccinated groups demonstrated a 10-fold lower luminescence from day 1 after vaccination, indicating that there was reduced antigen expression in the dermis even at the earliest time-point. Thus the live imaging demonstrated that co-expression of DTa or PRF with luciferase from the DNA vaccine facilitated removal of luciferase-positive cells. The timing of cell death differed between DTa and PRF, and also between different levels of PRF expression controlled by the CMV or SV40 promoters. This indicates that the intrinsic properties of DTa and PRF, as well as the overall level of expression controlled by the promoter, influence the timing of cell death.

(105) 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.

(106) The Luciferase-Specific Immune Response Following Intradermal DNA Vaccination

(107) To examine the immune response to luciferase, vaccinated mice received booster vaccinations of 50 ?g DNA at day 35 (at a point when luminescence expression was 100-fold lower in the CMV-LUC control mice (FIG. 3B)). To assess the humoral immune response, serum samples were examined by ELISA to detect luciferase-specific IgG. Mice vaccinated with CMV-LUC, CMV-LUC-SV40-PRF or CMV-LUC-2A-PRF all showed end-point titers of anti-luciferase IgG 1/10,000 (FIG. 3C). Thus the inclusion of PRF in the vaccine did not significantly enhance the luciferase-specific antibody response. Interestingly, CMV-LUC-SV40-DTa vaccination induced lower levels of luciferase-specific IgG (approximately 100-fold) compared to the CMV-LUC control, suggesting that, rather than enhancing immunity, it had an inhibitory effect on the immune response, probably due to a reduction in antigen levels caused by the ability of DTa to inhibit de novo protein synthesis.

(108) An IFN ELISPOT was performed, using a single immunodominant luciferase peptide to restimulate splenocytes (FIG. 3D). The ELISPOT assay revealed low-level IFN responses to this peptide. CMV-LUC-SV40-PRF vaccination showed a clear trend towards an increased response to this immunodominant peptide compared to CMV-LUC, while CMV-LUC-SV40-DTa decreased the response.

(109) PRF Enhances the T Cell Mediated Immune Responses to HIV-1 GAG DNA Vaccines

(110) 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 ?g 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 (FIG. 4A-D). Mice vaccinated with CMV-GAG-SV40-PRF DNA showed a similar range of responses to pools 1-3 in control CMV-GAG mice, but presented a 2.5-fold higher response to peptide pool 4, compared to CMV-GAG mice (mean SFU 155 vs. 62, p=0.03) (FIG. 4D). As this pool contains none of the immunodominant GAG peptides, this represents a broadening of the immune response to non-dominant epitopes. In contrast, the CMV-GAG-SV40-DTa DNA vaccinated mice showed 5-fold lower IFN responses to peptide pool 3 (mean SFU 142 vs. 696, p=0.003) and to the immunodominant peptides in pool 5 (mean SFU 272 vs. 1270, p=0.001), compared to CMV-GAG mice (FIGS. 4C and E). These results are consistent with the results from CMV-LUC-SV40-DTa vaccination shown in FIG. 3 and confirm that co-expression of DTa with an antigen actually reduces the subsequent immune response. Surprisingly, mice vaccinated with the CMV-GAG-2A-PRF DNA vaccine showed no significant changes in IFN responses compared to mice vaccinated with CMV-GAG DNA.

(111) Intracellular cytokine staining demonstrated that individual CD4 and CD8 T cells responded to vaccination by producing IFN, TNF and IL-2 (FIG. 5). Mice vaccinated with CMV-GAG-SV40-PRF DNA showed 2-fold higher levels of TNF and IFN-secreting CD8 T cells, compared to mice vaccinated with CMV-GAG DNA (mean percentage 0.21 vs. 0.1, p=0.03) (FIG. 5B). There was a trend showing an increase in rare, multifunctional CD8 T cells that secreted TNF, IFN and IL-2 simultaneously (mean percentage 0.078 vs. 0.04) in mice vaccinated with CMV-GAG-SV40-PRF when compared to CMV-GAG (FIG. 5C). All vaccinated groups (except mock-vaccinated) produced CD4 T cells that secreted multiple cytokines in response to GAG immunodominant peptides with no differences between groups (FIG. 5 D-E). Similarly, the CMV-GAG-2A-PRF DNA vaccine induced no significant changes in CD8 and/or CD4 T cell cytokine production compared to CMV-GAG DNA.

(112) 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.

(113) PRF Increases the Frequency of Cross-Presenting CD8a+ and Necrosis-Sensing Clec9A+ DC

(114) 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+/? (FIG. 6). Mice vaccinated with CMV-GAG, CMV-GAG-SV40-PRF or CMV-GAG-SV40-DTa showed an increase in the frequency of CD11c+ DC in the lymph node and also in the expression of markers of DC activation, compared with the control group vaccinated with pVAX only. At day 14 post vaccination, a significant increase in the frequency of cross-presenting CD11c+ CD8a+ DCs was detected in the draining lymph node of mice vaccinated with CMV-GAG-SV40-PRF compared to mice vaccinated with CMV-GAG (mean percentage 1.3 vs. 1.0, p=0.02) (FIG. 6B).

(115) 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) (FIG. 6C). This DC subset is capable of sensing necrotic cells and targeting antigen for cross-presentation. Interestingly, there was a decrease in the frequency of Clec9A+ DCs in mice vaccinated with CMV-GAG-SV40-DTa compared to CMV-GAG (mean percentage 1.8 vs. 2.2, p=0.002), suggesting that, although DTa led to antigen-positive cell death, the mechanism did not affect CD8a+ DC, and reduced the frequency of Clec9A+ DC.

(116) The activation status of the DC from the draining lymph node, as measured by MHCII and CD80 expression, was also determined (FIG. 6D). Vaccination with CMV-GAG-SV40-PRF, but not with CMV-GAG-SV40-DTa, induced the up-regulation of both MHCII and CD80 on CD11c+ CD8a+ DC above that observed after vaccination with CMV-GAG (90.5% vs. 84%, p=0.002, and 24.4% vs. 13.4%, p=0.002). The Clec9A+ DCs were already highly activated in all groups (including mock-vaccinated controls), as MHCII and CD80 were expressed on 90-100% and 60-70% of cells respectively, and further increases in activation after vaccination were not detected. Thus, DTa and PRF induced different effects on DC subpopulations after vaccination, and only PRF led to an increase in the frequency of cross-presenting and necrosis-sensing DC, as well as an increase in activation markers, consistent with necrosis-enhanced cross-presentation of antigen.

(117) PRF DNA Vaccination Reduces the EcoHIV Viral Load after Challenge

(118) 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 ?g 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 ?g p24 by the IP route, as described previously. Mice were culled 7 days after challenge and tissues were collected to determine the viral load (FIG. 7). Vaccination with CMV-GAG DNA reduced the viral load in the peritoneal exudate cells (PEC) below that detected in mock-vaccinated mice (2-fold reduction in normalised mRNA levels, p=0.02) while vaccination with CMV-GAG-SV40-PRF DNA resulted in a further decrease (5-fold reduction, p=0.001) (FIG. 7A). In the spleen, the CMV-GAG-SV40-PRF DNA, but not the CMV-GAG DNA, vaccination reduced the viral load (2.5-fold, p=0.02) compared to that observed in mock-vaccinated mice (FIG. 7B). Likewise, in the peripheral blood, the viral load was significantly reduced by CMV-GAG-SV40-PRF (2.5-fold, p=0.01) but not by CMV-GAG DNA vaccination (FIG. 7C). Thus, the immune response induced by DNA vaccination containing the cytolytic gene, PRF, resulted in improved control of viral replication at the site of infection, and systemically, in EcoHIV-challenged mice.

(119) 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.

(120) 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 (FIGS. 3 and 4). This fact could only be determined by using the technology of live imaging to track antigen expression. Other cytolytic proteins may also be useful in this system, particularly if their expression results in lytic cell death at a time-point after the threshold of the immunogenic antigen expression is achieved.

(121) 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 na?ve CD8 T cells (FIG. 6). The T cell-mediated immune response following vaccination was also enhanced by the co-expression of perforin, as determined by increased numbers of IFN-secretion by memory T cells in response to stimulation by GAG peptides (FIG. 4). The CMV-GAG vaccine induced strong immune responses when administered via the ID route, but the addition of perforin broadened the immune response by enhancing T cell responses to non-dominant epitopes. Perforin also increased the frequency of CD8 T cells capable of secreting multiple cytokines, a T cell subset shown to be important in control of HIV-1 infection (FIG. 5).

(122) 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.

(123) 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.

(124) 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.

EXAMPLE 2

(125) Materials and Methods

(126) 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).

(127) Construction of the Luciferase/TK DNA Vaccine

(128) 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.

(129) 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 (FIG. 8).

(130) Construction of the Luciferase/NSP4 DNA Vaccine

(131) 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.

(132) 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.

(133) 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 (FIG. 9).

(134) Construction of the Luciferase/PRF DNA Vaccine

(135) 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.

(136) 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.

(137) 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 (FIG. 10).

(138) Vaccination Schedule

(139) Eight week old C57BI/6 mice were pre-bled and vaccinated with 50 ?g 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 ?g 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 ?g DNA at 4 week intervals via the intradermal route and the immune response also examined by ELIspot or intracellular cytokine staining 10 days later.

(140) Temporal Expression of Luciferase

(141) Luminescence was detected from day 1 using a Xenogen IVIS live imager. Mice were sedated and then injected with 150 ?L 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 (FIG. 11). Luminescence is a direct measure of recent luciferase expression as luciferase has a half-life of only 3 hours. Thus, luciferase expression (FIG. 12) continued for at least 4 weeks and slowly decreased until on day 35 it was 1-2 log fold lower than on day 3.

(142) 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.

(143) 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. (FIG. 12) Additional experiments showed that luminescence disappeared completely between days 35 and 49, depending on the initial level of expression.

(144) Anti-Luciferase Antibody Titres in Vaccinated Mice

(145) 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 ?g 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 (FIG. 13). Importantly, the antibody titre rose much more rapidly in the pVAXLUC +NSP4 and pVAXLUC +PRF vaccinated mice (FIG. 13).

(146) Cell Mediated Responses to Luciferase in Vaccinated Mice

(147) 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 (FIGS. 14, 15, 18-20).

(148) 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.

(149) 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.

(150) An effective vaccine will, by necessity, need to induce an inflammatory response that is unlikely to be achieved by suicide genes which induce apoptosis.

EXAMPLE 3

(151) 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.

(152) Necrotic, Viral Antigen Positive Cells are Highly Immunogenic

(153) Preclinical studies of a necrotic whole cell vaccine were performed. A schematic diagram of the strategy is shown in FIG. 16. A mouse cell line, DC2.4, was transfected with RNA encoding the HCV NS3 protein (This RNA sequence was published by R Trowbridge and E J Gowans (Trowbridge R and Gowans E J (1998). Molecular cloning of an Australian isolate of hepatitis C virus. Arch. Virol 143:501-511) and the sequence deposited in Genebank under accession number AJ000009) and 48 hours later these cells were induced to become necrotic by heating the cells to 63? C. for 30 minutes. Subcutaneous vaccination with these cells in groups of 6 mice induced far higher CMI responses (80-100 fold in different experiments, as determined by ELIspot analysis of peptide-stimulated splenocytes representing the poorly immunogenic HCV NS3 protein), compared to vaccination with the same live cell line (FIG. 17). In these experiments, the mice received approximately 100 ng of the NS3 protein by comparison of the intensity of the band detected in Western blot with that of commercially available purified NS3.

(154) 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/10.sup.6 cells.

EXAMPLE 4

(155) In a similar manner described in FIGS. 3 and 4, the gag sequence from the HIV genome was inserted into pVAX upstream of the NSP4 and PRF cistrons respectively, to result in DNA vaccines pVAXgag +NSP4 and pVAXgag +PRF. C57BI/6 mice were vaccinated with 50 ?g of these DNA vaccines by the intradermal route described above, on 3 occasions, 4 weeks apart. These results showed that the pVAXgag +NSP4 induced approximately a 1.5-fold increase in IFN secreting T splenocytes while pVAXgag +PRF induced a 1.3-fold increase over pVAXgag alone when the splenocytes were stimulated with recognised gag immunodominant peptides in an ELIspot assay (FIG. 18).

(156) To analyse these responses in greater detail, CD8.sup.+ and CD8.sup.? memory cells (CD44.sup.+) 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.sup.+ 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 (FIG. 19).

(157) Likewise, although CD8.sup.+ T cells are often recognized as the cells which kill virus-infected cells, CD4.sup.+ cells which express multiple cytokines are necessary for T cell help. Consequently, CD8.sup.?, CD44.sup.+ 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 (FIG. 20).

EXAMPLE 5

(158) Materials and Methods

(159) Preparation of Stable DC2.4 Cell Line Expressing Codon Optimised NS3 g1B

(160) 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?10.sup.6 cells in 18 ml of medium in a 75-cm.sup.2 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.)

(161) For the transduction of the murine DC2.4 cell line, cells were seeded at a density of 2?10.sup.5 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%.

(162) Stimulation of Necrosis Stable DC2.4 Cell Line Expressing Codon Optimised NS3 g1B

(163) 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.

(164) Flow Cytometric Detection of Necrotic Cells

(165) 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.

(166) Results

(167) Flow Cytometric Detection of Necrotic Cells

(168) 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 (FIG. 21A). This population of GFP positive transduced cells were collected 7-8 days post-transduction using standard flow cytometric sorting techniques. After the fourth sort, the purity of the GFP positive cells was improved to 93-95% (FIG. 21B-F).

(169) 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% (FIG. 22A-G), as detected using the flow cytometric techniques described above, the gating parameters for which are illustrated in FIG. 22H.

(170) Kinetics of Cell Activation Following a Single Dose of Necrotic or Live Vaccination

(171) A single dose of 10.sup.6 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.

(172) FIG. 23 shows the frequency of GFP positive cells at days 2, 3, 5, and 7 post-vaccination. FIG. 24A-D shows GFP expression in DLN at days 2, 3, 5, and 7. The frequency of necrotic GFP positive cells in DLN was found to be significantly increased after 2, 3, and 5 days. After 7 days, the frequency of live GFP positive cells was significantly increased.

(173) CD11c.sup.high Cells in DLN Post Single Dose of Necrotic or Live Vaccine.

(174) As shown in FIG. 25, the number of necrotic cells in LN was significantly increased relative to the number of live cells 5 days post vaccination. However, the number of necrotic CD11c.sup.high cells in DLN was highest 7 days post vaccination (FIG. 26). The relative frequency of necrotic CD11c.sup.high cells to live cells relative to frequency and total cell number total cells was significantly increased 5 days post vaccination (FIG. 27).

(175) The frequency of necrotic CD11c.sup.high CD8 cells in DLN did not significantly change over time (FIG. 28). However, the number of necrotic CD11c.sup.high CD8 cells in DLN was highest 7 days post vaccination (FIG. 29).

(176) The frequency of necrotic CD11c.sup.high CD8+ Clec9A+ cells in DLN did not significantly change over time (FIG. 30). However, the number of necrotic CD11c.sup.high CD8+ Clec9A+ cells in DLN was significantly higher than live cells 5 days and 7 days post vaccination (FIG. 31).

(177) The frequency of necrotic CD11c.sup.high CD8+ MHCII+ Clec9A+ cells in DLN significantly higher than live cells 2 days post vaccination (FIG. 32).

(178) NS3 Cells in DLN Post Boost Dose of Necrotic or Live Vaccine.

(179) Mice were vaccinated with 10.sup.6 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.

(180) As shown in FIGS. 33A-D and FIG. 34, the number of interferon-gamma secreting cells was significantly greater in mice vaccinated with the necrotic NS3 cells in all three pools, compared with mice vaccinated with the viable NS3 cells, indicating greater IFNg responses for all pools in mice vaccinated with necrotic cells.

(181) 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 (FIGS. 36-38).

EXAMPLE 6

(182) Materials and Methods

(183) 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.

(184) 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?10.sup.8 cells/ml. CD14 Microbeads (Miltenyi Biotec) were then added to the cells at 20 ?l per 1?10.sup.7 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?10.sup.6 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.

(185) As this was a dose escalation trial, successive patients received increasing doses of necrotic cells, ranging from 1?10.sup.4 to 1?10.sup.7 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 FIG. 39.

(186) Results

(187) 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?10.sup.4 and 2?10.sup.4 cells respectively, these patient failed to show any increase in the HCV cell mediated immunity (FIGS. 40 and 41, upper and lower panels respectively) although there was a suggestion of a NS3-specific response in patient #2. Furthermore, although patients #3 and #4 showed some responses to a broad range of peptides, there was a clear increase in the HCV cell mediated immunity to peptides contained in the vaccine (FIGS. 42 and 43). As the clinical trial is still ongoing, no additional ELIspot data are available. To confirm that the ELIspot protocol was reproducible, ELIspot responses to the CEF peptides (peptides to CMV, EBV and influenza virus) showed minimal fluctuations, although there was a substantial increase in patient #4, from baseline to week 2 that may reflect a non-specific response to the vaccination (shown in FIG. 44). The viral load data were generated by the local pathology provider and are still not complete at this stage of the clinical trial. Most patients responded to the interferon induction and the raw data are shown in FIGS. 45 and 46 while the interpretation of the data are shown in various graphs (FIGS. 46-48). The data show that after vaccination, patients #6 and #3 showed viral loads that were 57% and 64% of baseline, while patients #2 and #4 showed viral loads of 75% and 77% of baseline. Patients #1 and #5 showed no responses to the vaccination, and indeed patient #5 showed a rebound greater than baseline (FIG. 49).

(188) 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.

(189) 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.

(190) 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.

(191) 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.

(192) 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.

(193) 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.