Patent Grant
9555097
The present invention provides an immunogen-specific adjuvant for a vaccine or inoculum. More specifically, the invention provides a vaccine or inoculum adjuvant comprising recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein. The recombinant fusion protein contains two sequences that are peptide-linked together in which one sequence is a protein body-inducing sequence (PBIS) and the other is a T-cell stimulatory polypeptide that corresponds to a portion of a pathogenic polypeptide sequence present in or encoded by a vaccine or inoculum.
Protein bodies (PBs) are subcellular organelles (or large vesicles, about 1-3 microns in diameter, surrounded by a membrane) that specialize in protein accumulation. They are naturally formed in some specific plant tissues, like seeds, and serve as principal source of amino acids for germination and seedling growth.
The storage proteins are co-translationally inserted into the lumen of the endoplasmic reticulum (ER) via a signal peptide to be packaged either in the ER or into the vacuoles (Galili et al., 1993 Trends Cell Biol. 3:437-443) and assembled into multimeric units inside these subcellular compartments, developing specific organelles called (ER)-derived protein bodies (PBs) or protein storage vacuoles (PSV) (Okita and Rogers, 1996 Annu. Rev. Plant Physiol Mol. Biol. 47:327-350; Herman and Larkins, 1999 Plant Cell 11:601-613; Sanderfoot and Raikel, 1999 Plant Cell 11:629-642).
The storage proteins dicotiledoneous plants are primarily soluble proteins such as the 7S globulin or vicilin type, 11S globulins or legumin-type proteins and are sequestered in PSVs together with other proteins (i.e., protease inhibitors, proteolytic enzymes, lectins and the like), sugars and salts.
In contrast to PSVs, PBs (1-3 microns) sequester predominantly prolamins, which are highly hydrophobic storage proteins of cereals (such as zeins of maize and gliadins of wheat), and lack of other auxiliary proteins (Herman et al., 1999 Plant Cell 11:601-613).
At present, no PBs have been found in tissues other than plant seeds, with the exception of the ER bodies. The ER bodies are small in size (0.2-0.4 micrometers) and are formed in Arabidopsis leaves only by wounding and chewing by insects but do not develop under normal conditions (Matsushima et al., 2003 Plant J. 33:493-502).
Genetic engineering approaches have been used to study plant PBs formation, storage protein assembly and targeting. It has been shown that when recombinant proteins, predominantly plant storage proteins are expressed and packaged in Arabidopsis and tobacco, plant tissues that did not contain PBs (as vegetative tissues), develop these organelles “de novo” (Bagga et al., 1997 Plant Cell 9:1683-1696 and Bagga et al., 1995 Plant Physiol. 107:13-23, and U.S. Pat. No. 5,990,384, U.S. Pat. No. 5,215,912, and U.S. Pat. No. 5,589,616; and Geli et al., 1994 Plant Cell 6:1911-1922).
Maize beta-zein when expressed in transgenic tobacco plants was correctly targeted in new formed ER-derived PBs in leaf cells (Bagga et al., 1995 Plant Physiol. 107:13-23). Maize gamma-zein and, truncated gamma-zein cDNAs expressed in Arabidopsis plants also accumulate in a novel ER-derived PBs in leaves (Geli et al., 1994 Plant Cell 6:1911-1922). Lysine-rich gamma-zeins expressed in maize endosperms (Torrent et al. 1997 Plant Mol. Biol. 34(1):139-149) accumulate in maize PBs and co-localized with endogenous zeins. Transgenic tobacco plants expressing alpha-zein gene demonstrated that alpha-zein was not able to form PBs. However, when alpha- and gamma-zein were co-expressed, the stability of alpha-zein increased and both proteins co-localized in ER-derived protein bodies (Coleman et al., 1996 Plant Cell 8:2335-2345). Formation of novel PBs has been also described in transgenic soybean transformed with methionine-rich 10 kDa delta-zein (Bagga et al., 2000 Plant Sci. 150:21-28).
Recombinant storage proteins are also assembled in PBs-like organelles about 100 to about 400 nm in diameter in a non-plant host system such as Xenopus oocytes and in yeast. Rosenberg et al., 1993 Plant Physiol 102:61-69 reported the expression of wheat gamma-gliadin in yeast. The gene expressed correctly and the protein was accumulated in ER-derived PBs. In Xenopus oocytes, Torrent et al., 1994 Planta 192:512-518 demonstrated that gamma zein also accumulates in PB-like organelles when transcripts encoding the protein were microinjected into oocytes. Hurkman et al., 1981 J. Cell Biol. 87:292-299 with alfa-zeins and Altschuler et al., 1993 Plant Cell 5:443-450 with gamma-gliadins had similar results in Xenopus oocytes.
One of the fundamental achievements of the field of the biotechnology (genetic engineering) is the ability to genetically manipulate an organism to produce a protein for therapeutic, nutraceutical or industrial uses. Methods are provided for producing and recovering recombinant proteins from fermentation broth of bacteria, yeast, crop plants and mammalian cell cultures. Different approaches for protein expression in host cells have been described. The essential objectives of these approaches are: protein expression level, protein stability and protein recovery (Menkhaus et al., 2004 Biotechnol. Prog. 20: 1001-1014; Evangelista et al., 1998 Biotechnol. Prog. 14:607-614).
One strategy that can solve a problem with protein recovery is secretion. However, secretion involves some times poor expression levels and product instability. Another strategy is the accumulation of the recombinant protein in the most beneficial location in the cell. This strategy has been extensively used by directing recombinant proteins to the ER by engineering C-terminal extension of a tetrapeptide (HDEL/KDEL) (Conrad and Fiedler, 1998 Plant Mol. Biol. 38:101-109).
Fusion proteins containing a plant storage protein or storage protein domains fused to the heterologous protein have been an alternative approach to direct recombinant proteins to the ER (WO 2004003207). One interesting fusion strategy is the production of recombinant proteins fused to oleosins, constitutive protein of plant oil bodies. The specific characteristics of oil bodies benefit of the easy recovery of proteins using a two-phase system (van Rooijen and Moloney, 1995 Bio/Technology 13:72-77).
Heterologous proteins have been successfully expressed in plant cells (reviews Horn et al., 2004 Plant Cell Rep. 22:711-720; Twyman et al., 2003, Trends in Biotechnology 21:570-578; Ma et al., 1995, Science 268: 716-719; Richter et al., 2000 Nat. Biotechnol. 18:1167-1171), and in some, the expression of the recombinant protein has been directed to ER-derived PB or PSV (PSV). Yang et al., 2003 Planta 216:597-603, expressed human lysozyme in rice seeds using the seed-specific promoters of glutelin and globulin storage proteins. Immunocytochemistry results indicated that the recombinant protein was located in ER-PBs and accumulated with endogenous rice globulins and glutelins. The expression of glycoprotein B of the human cytomegalovirus (hCMV) in transgenic tobacco plants has been carried out using a glutelin promoter of rice. Tackaberry et al., 1999 Vaccine 17:3020-3029. Recently, Arcalis et al., 2004 Plant Physiology 136:1-10 expressed human serum albumin (HSA) with a C-terminal extension (KDEL) in rice seeds. The recombinant HSA accumulated in PSVs with the endogenous rice storage proteins.
One obstacle for the application of plants as biofactories is the need for more research regarding the downstream processing. Protein purification from plants is a difficult task due to the complexity of the plant system. Plant solids of the extract are large, dense and relative elevated (9-20 percent by weight) (see review Menkhaus et al., 2004 Biotechnol. Prog. 20:1001-1014). At present, recombinant protein purification techniques include clarification of the extracts, treatment with solvents to remove lipids and pigments and protein or peptides purification by several ion-exchange and gel-filtration chromatography columns. The existing protocols rely upon the use of specific solvents or aqueous solutions for each plant-host system and recombinant protein. There is a need in the art for efficient and general procedures for recombinant protein recovery from transformed hosts. This need is especially relevant in cases where recombinant proteins produced in plant hosts must to be isolated. The diversity of hosts and proteins and the different physical-chemical traits between them required an efficient method to concentrate and recover recombinant products.
Immunologic adjuvants are agents that enhance specific immune responses to vaccines and inocula. An immunologic adjuvant can be defined as any substance or formulation that, when incorporated into a vaccine or inoculum, acts generally or specifically to accelerate, prolong, or enhance the quality of specific immune responses to the immunogenic materials in the preparation.
The word adjuvant is derived from the Latin verb adjuvare, which means to help or aid. Adjuvant mechanisms of action include the following: (1) increasing the biological or immunologic half-life of vaccine or inoculum immunogens; (2) improving immunogen delivery to antigen (immunogen)-presenting cells (APCs), as well as antigen (immunogen) processing and presentation by the APCs; and (3) inducing the production of immunomodulatory cytokines.
Possession of biological activity that resembles an activity of a natural pathogen or other agent is particularly relevant for vaccines or inocula, which must induce a correct immune response in an immunized human or other animal to be effective. Several new vaccines and inocula are composed of synthetic, recombinant, or highly purified subunit immunogens (antigens) that are thought to be safer than whole-inactivated or live-attenuated vaccines. However, pathogen-related immunomodulatory adjuvant components that are typically associated with attenuated or killed pathogen vaccines are absent from such synthetic, recombinant, or highly purified subunit immunogens, which often results in weaker immunogenicity for such preparations.
Phagocytosis involves the entry of large particles, such us apoptotic cells or whole microbes, into another cell. The capacity of the cells to engulf large particles likely appeared as a nutritional function in unicellular organisms; however complex organisms have taken advantage of the phagocytic machinery to fulfill additional functions. For instance, the phagocytosis of immunogens undertaken by the macrophages, the B-cells or the dendritic cells represents a key process in innate and adaptive immunity. Indeed, phagocytosis and the subsequent killing of microbes in phagosomes form the basis of an organism's innate defense against intracellular pathogens. Furthermore, the degradation of pathogens in the phagosome lumen and the production of antigenic peptides, which are presented by phagocytic cells to activate specific lymphocytes, also link phagocytosis to adaptive immunity (Jutras et al., 2005 Annual Review in Cell Development Biology. 21:511-527).
The proteins present on and in engulfed particles encounter an array of degrading proteases in phagosomes. Yet, this destructive environment generates peptides that are capable of binding to MHC class II molecules. Newly formed immunogen-MHC class II complexes are delivered to the cell surface for presentation to CD4+ T cells (Boes et al., 2002 Nature 418:983-988). The activation of these cells induces the Th2 subset of cytokines such as IL-4 and IL-5 that help B cells to proliferate and differentiate, and is associated with humoral-type immune response.
A large body of evidence indicates that, in addition to the clear involvement of the MHC class II pathway in the immune response against phagocytosed pathogens, immunogens from pathogens, including mycobacteria, Salmonella, Brucella, and Leishmania, can elicit an immunogen cross-presentation. That is to say, the presentation of an engulfed immunogen by phagocytosis by the MHC class I-dependent response promotes the proliferation of CD8+ cytotoxic T cells (Ackerman et al., 2004 Nature Immunology 5(7):678-684; Kaufmann et al., 2005 Current Opinions in Immunology 17(1):79-87).
Dendritic cells play a central immunogen presentation role to induce the immune system (Blander et al., Nature Immunology 2006 10:1029-1035). Although rare, dendritic cells are the most highly specialized APC, with ability both to instigate and regulate immune reactivity (Lau et al. 2003 Gut 52:307-314). Although dendritic cells are important in presenting immunogens, particularly to initiate primary immune responses, macrophages are the APC type most prominent in inflammatory sites and specialized for clearing necrotic and apoptotic material. Macrophages can act not only as APC, but can also perform either pro- or anti-inflammatory roles, dependent on the means by which they are activated.
Considering that APCs play a central role in the induction and regulation of the adaptive immunity (humoral and cellular), the recognition and phagocytosis of the immunogen by those cells can be considered a key step in the immunization process. A wide variety of techniques based on the uptake of fluorescent particles have been developed to study phagocytosis by the macrophages (Vergne et al., 1998 Analytical Biochemistry 255:127-132).
An important aspect in veterinary vaccines is the genetic diversity of the species being considered and the requirement for generic systems that work across different species. To a large degree, this diversity limits the use of molecular targeting techniques to cell surface markers and immune modulators such as cytokines, because for many species including wildlife, only minimal knowledge of these molecules is available. Thus, adjuvants that rely on universal activation signals of the innate immune response (i.e. that are identical in different species) are to be preferred. Taking these requirements into consideration, particulate vaccine delivery systems are well suited for veterinary and wildlife vaccine strategies (Scheerlinck et al., 2004 Methods 40:118-124).
In Third World countries, cervical cancer (cc) is one of the major causes of cancer-related deaths. About 80% of women dying from this disease originate from low-budget countries where screening programs for early detection and the medical infrastructure for treatment are not available. In contrast, in the more developed world the mortality was reduced (by 70% in the US) during the last 50 years as a consequence of cytological screening programs [American Cancer Society, Cancer facts and figures 2004. Atlanta, Ga.] Treatment of cc patients by surgery, radiotherapy or chemotherapy results in a significant loss of quality of life. Even when optimal treatment is available about 40% of all cc patients die of this disease [Gatta et al., 1998 Eur J Cancer 34(14 Spec. No.):2218-2225]. Therefore, the development of an effective and save therapeutic vaccine is needed.
A necessary event for the development of premalignancies like cervical intraepithelial neoplasia (CIN) and cc is infection by hr-HPVs [Walboomers et al., 1999 J Pathol 189(1):12-19]. So far over 120 HPV types are identified [de Villiers et al., 2004 Virology 324(1):17-27], 18 of which were found to be associated with cc [Munoz et al., 2003 N Engl J Med 348(6):518-527].
HPV-16 is responsible for about 50% of the cases [Bosch et al., 1995 J Natl Cancer Inst 87(11):796-802]. Due to the fact that the oncoprotein E7 of the hr-HPVs is exclusively and consistently expressed by HPV-infected tumor cells [von Knebel Doeberitz et al., 1994 J Virol 68(5):2811-2821], that protein represents a specific target for an immune therapy directed against cc and its premalignant dysplasia. The E7 protein, however, is an oncoprotein with transforming activity that operates by interfering with the cell cycle control. The E7 alters cell growth regulation by inactivating the pRB (retinoblastoma) tumor suppressor protein [Dyson et al., 1989 Science 243(4893):934-937; Munger et al., 1992 Cancer Surv 12:197-217] and contains two metal-binding motifs (C-XX-C) [Edmonds et al., 1989 J Virol 63(6):2650-2656; Watanabe et al., 1990 J Virol 64(1):207-214].
For safety reasons a functional oncogene cannot be applied to humans. Therefore, efforts were made to inactivate the oncogenic properties of the HPV-16 E7. Some investigators have introduced point mutations into the sites of the E7-oncogene that are associated with transforming potential [Shi et al., 1999 J Virol 73(9):7877-7881; Smahel et al., 2001 Virology 281(2):231-238], whereas others have used HLA- (human leukocyte antigen) restricted singular epitopes [Doan et al., 2000 Cancer Res 60(11):2810-2815; Velders et al., 2001 J Immunol 166(9):5366-5373]. These approaches, however, can lead to an unwanted loss of a naturally occurring epitope that is potentially associated with a decrease in vaccine efficacy.
An aim of the present inventors was to supply several to all potential naturally occurring T cell epitopes, covering the broad range of MHC restriction. In consequence, prior knowledge of the patient's HLA-haplotype is not required. This is especially important in the outbred human population.
In addition, a more potent immune response may be induced, involving all occurring HLA-restriction elements in the vaccine. A “proof-of-principle” was generated in a study using an artificial HPV-16 E7 gene (HPV-16 E7SH) of the first generation [Osen et al., 2001 Vaccine 19(30):4276-4286]. It was shown in that study that an oncoprotein with a rearranged primary sequence still induces E7WT-specific CTLs in mice but is devoid of transforming properties. That study took advantage of the earlier finding that fusion with the VP22 gene of Herpes Simplex Virus Type 1 strongly enhances the CTL response in mice [Michel et al., 2002 Virology 294(1):47-59].
The HIV-1 virus is comprised of several layers of proteins and glycoproteins that surround its RNA, and its associated proteins integrase and reverse transcriptase. The RNA is encapsidated by a capsid protein (CA), p24. The capsid environment also contains other viral proteins such as integrase and reverse transcriptase. The capsid is in turn encapsidated by a layer of matrix protein (MA), p17. This matrix protein is associated with a lipid bilayer or envelope.
The great diversity among human immunodeficiency virus type 1 (HIV-1) subtypes, which are prevalent in various regions of the world, is a major impediment to the development of broad-based prophylactic HIV-1 vaccines. Thus, it may be necessary to develop vaccines that match local epidemics more closely (Morris et al., 2001). In southern Africa, subtype C infections predominate (UNAIDS, 2006), and isolates of this subtype have been selected for the development of a DNA vaccine in South Africa (Williamson et al., 2003). This candidate vaccine has been constructed and characterized (van Harmelen et al., 2003) and is scheduled to be evaluated in clinical trials shortly.
DNA vaccines encoding HIV or simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) antigens have been studied extensively and shown to induce both humoral and cellular immune responses in animal models as well as in humans [Boyer et al., 1997 J Infect Dis. 176(6):1501-1509; Calarota et al., 1998 Lancet 351(9112):1320-1325; Estcourt et al., Immunol. Rev. 2004 199:144-155; Letvin et al., 1997 Proc Natl Acad Sci USA. 94(17):9378-9383; Yasutomi et al., 1996 J Virol. 70(1):678-681]. However, although DNA vaccines have been shown to be safe, immunization generates low and transient levels of immune responses. Various approaches to augment DNA vaccines have been tested [Barouch et al., 2000 Intervirology 43(4-6):282-287; Hemmi et al., 2003 J Immunol 170(6):3059-3064; Raviprakash et al., 2003 Virology. 315(2):345-352], including their use in heterologous prime-boost immunization regimens [Casimiro et al., 2003 J. Virol. 77(13):7663-7668; Cherpelis et al., 2001 Immunol Lett. 79(1-2):47-55; Leung et al., 2004 AIDS 18(7):991-1001; Pal et al., 2006 Virology 348(2):341-353; Robinson et al., 1999 Int J Mol. Med. 4(5):549-555; Suh et al., 2006 Vaccine 24(11):1811-1820. Epub 2005 Oct. 25].
The HIV-1 Gag gene encodes the precursor protein Pr55 Gag, which is the major protein that makes up the structure of the HIV viral particle. On maturation of the viral particle, Gag is cleaved by the viral protease into several smaller proteins that include the capsid (CA) protein p24, the matrix protein p17, as well as proteins p7 and p6.
HIV-1 Pr55gag precursor protein possesses an ability to self-assemble into non-replicating and non-infectious virus-like particles (VLPs) [Deml et al., 1997 Virology 235(1):26-39; Mergener et al., 1992 Virology 186(1):25-39; Sakuragi et al., 2002 Proc Natl Acad Sci USA 99(12):7956-7961; Wagner et al., 1994 Behring Inst Mitt (95):23-34; Wagner et al., 1996 Virology 220(1):128-140], and elicits strong humoral and cellular immune responses in animals [Deml et al., 1997 Virology 235(1):26-39; Deml et al., 2004 Methods Mol Med 94:133-157; Jaffray et al., 2004 J Gen Virol. 85(Pt 2):409-413], including non-human primates (NHPs) (Montefiori et al., 2001 J. Virol. 75(21):10200-10207; Paliard et al., 2000 AIDS Res Hum Retroviruses 16(3):273-282]. Recently, Chege et al., J Gen Virol 2008 89:2214-2227 have shown that subtype C Pr55gag VLPs can very efficiently boost baboons primed with a matched DNA vaccine.
In addition, HIV-1 Pr55gag VLPs are safe, easy to produce and have the potential of including chimeric immunogens (Doan et al., 2005; Halsey et al., 2008). Their particulate nature and size, which approximates that of HIV-1, make HIV-1 Pr55gag VLPs more likely to stimulate the immune system better than non-particulate immunogens.
As above, the p24 protein forms the outer capsid layer of the viral particle. This protein has a high density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168), which make it more effective in inducing a broad immune response when used as a vaccine candidate. It has also been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies. This suggests that high anti-p24 antibody titres might be necessary to maintain a disease-free state.
In addition, HIV-1 Pr55gag VLPs are safe, easy to produce and have the potential of including chimeric immunogens [Doan et al., 2005 Rev Med. Virol. 15(2):75-88; Halsey et al., 2008 Virus Res. 2008 133(2):259-268. Epub 2008 Mar. 10]. Their particulate nature and size, which approximates that of HIV-1, make HIV-1 Pr55gag VLPs more likely to stimulate the immune system better than non-particulate immunogens.
As above, the p24 protein forms the outer capsid layer of the viral particle. This protein has a high density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168), which make it more effective in inducing a broad immune response when used as a vaccine candidate. It has also been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies. This suggests that high anti-p24 antibody titres might be necessary to maintain a disease-free state.
The matrix protein, p17, facilitates the intra-membrane associations that are required for viral assembly and release (Dorfman et al., 1994 J Virol 68(12):8180-8187]. Protein p17 is also involved in the transport of the viral pre-integration complex into the nucleus (Burkinsky et al., 1993). Fused together with p24, this p17/p24 (p41) complex contains the highest density of CTL epitopes in the HIV-1 genome (Novitsky et al., J. Virol. 2002 76(20):10155-10168).
HIV-1 reverse transcriptase (RT) is an RNA-dependent DNA polymerase that makes DNA templates and synthesises DNA from RNA. It is essential for viral replication. HIV-1 RT is cleaved from the Pr160gag-pol polyprotein by the HIV-1 protease (PR). Several CTL epitopes against HIV-1 have been identified in RT, although they appear to be subdominant to Gag-specific epitopes [Dela Cruz et al., 2000 Int Immunol 12(9):1293-1302].
Several studies have indicated that enhanced immune responses can be achieved by heterologous prime-boost inoculation regimens. It has been shown that a HIV-1 DNA vaccine denominated pTHGagC used as a prime inoculation of mice is boosted effectively by Pr55Gag virus-like particles (VLPs) (Chege et al., J. Gen. Virol. 2008 89:2214-2227). Because p24 has the highest density of cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of the HIV proteome it was thought that the particulate nature of protein bodies containing p24 may have a similar boosting effect to expand immune responses after the immune system has been primed. It has also been thought that the use of combinations of protein bodies containing different HIV-1 antigens such as p41 and RT, may broaden the immune response such as has been shown in the use of the multigene DNA vaccine “grttn” (Burgers et al., AIDS Research and Human Retroviruses 2008 24(2):195-206) that contains five AIDS genes, the gag, reverse transcriptase, tat and nef genes that are expressed as a polyprotein and a truncated env gene (gp150).
The present invention contemplates an immunogen-specific adjuvant for a vaccine or inoculum. The adjuvant is comprised of particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein. The recombinant fusion protein contains two sequences peptide-linked together in which one sequence is a protein body-inducing sequence (PBIS) such as a prolamin sequence and the other is a T-cell stimulating immunogenic polypeptide whose sequence is that of a pathogenic polypeptide sequence present in or induced by a vaccine or inoculum. The adjuvant when used in an inoculum without a prior priming vaccination or inoculation does not induce production of antibodies or T cell activation to the pathogenic sequence. A contemplated adjuvant is typically used in an adjuvant-effective amount dissolved or dispersed in a pharmaceutically acceptable diluent.
In the drawings forming a part of this disclosure,
The word “antigen” has been used historically to designate an entity that is bound by an antibody or receptor, and also to designate the entity that induces the production of the antibody or cellular response such as that of a CD4+ T cell. More current usage limits the meaning of antigen to that entity bound by an antibody or receptor, whereas the word “immunogen” is used for the entity that induces antibody production or cellular response. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen is typically made according to its intended utility.
“Antigenic determinant” refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site or T-cell receptor. The term is also used interchangeably with “epitope”.
As used herein, the term “fusion protein” designates a polypeptide that contains at least two amino acid residue sequences not normally found linked together in nature that are operatively linked together end-to-end (head-to-tail) by a peptide bond between their respective carboxy- and amino-terminal amino acid residues. A fusion protein of the present invention is a chimer of a protein body-inducing sequence (PBIS) linked to a second sequence that is a T-cell stimulating polypeptide (e.g., peptide or protein) that is present in the pathogen (target) at which the vaccine or inoculum is directed.
The term “immunogen-specific” is used herein to distinguish the adjuvanticity of a contemplated recombinant adjuvant and a more general adjuvant. More particularly, a contemplated immunogen-specific adjuvant enhances the cellular (T-cell) immune response toward an immunogen that includes an amino acid residue sequence of the adjuvant and does not generally activate the immune system. Thus, the vaccine or inoculum shares an amino acid residue sequence or encodes a shared sequence with the adjuvant.
An “inoculum” is a composition that comprises an immunogenically effective amount of immunogenic chimer particles dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. When administered to a host animal in need of immunization or in which antibodies or activated T cells are desired to be induced such as a mammal (e.g., a mouse, dog, goat, sheep, horse, bovine, monkey, ape, or human) or bird (e.g., a chicken, turkey, duck or goose), an inoculum induces a B cell and/or T cell response (stimulation) in an inoculated host animal such as production of antibodies that immunoreact with the immunogen of the chimer and/or induces T cells that respond to the immunogen. A “vaccine” is a type of inoculum in which the vaccine-induced antibodies not only immunoreact with the immunogen or activated T cells respond to that immunogen, but also immunoreact with the pathogen from which the immunogen is derived in vivo, and provide protection from that disease state.
The expression “T-Cell-mediated immunity” refers to an immune response that does not involve antibodies or complement but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune system was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens. T-Cell-mediated immunity is an immune response produced when T cells, especially cytotoxic T cells, that are sensitized to foreign antigens attack and lyse target cells. In addition to direct cytotoxicity, T cells can stimulate the production of lymphokines that activate macrophages. Cell-mediated immune responses are important in defense against pathogens, autoimmune diseases, some acquired allergies, viral infection, some tumors and other immune reactions.
The present invention contemplates an immunogen-specific adjuvant for a vaccine or inoculum. The adjuvant is comprised of particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein. The recombinant fusion protein contains two sequences peptide-linked together in which one sequence is a protein body-inducing sequence (PBIS) such as a preferred prolamin sequence and the other is a T-cell stimulating immunogenic polypeptide whose sequence (a) is present in a pathogenic polypeptide sequence present in a polypeptide-containing vaccine or inoculum or (b) is encoded by a nucleic acid vaccine or inoculum. The adjuvant, at the concentration used in an inoculum in a host animal without a prior priming by vaccination or inoculation of a host animal or additional immunogen, does not induce production of antibodies in that host animal that immunoreact with or induce T cell activation to the pathogenic sequence.
The invention also contemplates a method for inducing an T-cell mediated immune response in a subject in need thereof against an immunogenic peptide which comprises the administration to a subject in need thereof of a vaccine selected from the group of
In a preferred embodiment, the method of the invention is carried out using a RPBLA wherein the PBIS forming part of the first portion includes a prolamin sequence. In a still more preferred embodiment, the prolamin sequence is present in a prolamin selected from the group consisting of gamma-zein, alpha-zein, delta-zein, beta-zein, rice prolamin and gamma-gliadin.
In a preferred embodiment, the PBIS sequence further includes a signal peptide sequence that directs a protein towards the endoplasmic reticulum (ER) of the RPBLA-expressing cell.
In a preferred embodiment, the immunogenic peptide used in fusion protein forming the RPBLA is a peptide capable of stimulating the T-cell immune response.
In another preferred embodiment, the method of the invention is carried out using a RPBLA comprising a second portion wherein the immunogenic polypeptide sequence is selected from the group of
In a preferred embodiment, the method of the invention is carried out using a particulate recombinant protein body-like assemblies (RPBLAs) are assembled in vitro from the purified recombinant fusion protein.
In a more preferred embodiment, the administration step of the method of the invention is preceded by a priming vaccination or inoculation step using a composition comprising immunogenic polypeptide or a nucleic acid encoding said immunogenic polypeptide.
In a still more preferred embodiment, the composition comprising the immunogenic polypeptide used in the priming vaccination or stimulation step is selected from the group of
In a preferred embodiment, the immunogenic peptide used in fusion protein forming the RPBLA is a peptide capable of stimulating the T-cell immune response.
In a more preferred embodiment, the vaccine is administered intramuscularly.
In another aspect, the invention relates to vaccine for use in a method for inducing an T-cell mediated immune response in a subject in need thereof against an immunogenic peptide wherein the vaccine is selected from the group of
A contemplated adjuvant can be administered along with or separately as a boost to an anti-pathogen vaccine or inoculum. Such a vaccine or inoculum can contain an attenuated live or killed pathogen such as a bacterium or virus, a subunit vaccine or inoculum that contains only a protein portion of a pathogen, or a vaccine or inoculum that contains an immunogen that is comprised of a polypeptide linked to a carrier, wherein the immunogenic portion of the vaccine or inoculum contains a polypeptide sequence that is also present in the adjuvant. Where the vaccine or inoculum is a nucleic acid preparation such as a DNA or RNA vaccine, the nucleic acid encodes an immunogenic amino acid sequence that is also present in the adjuvant. Nucleic acid vaccines and inocula are themselves well known.
A contemplated adjuvant can be administered as a preparation of expressed RPBLAs, or as a nucleic acid preparation, such as a single or double stranded DNA sequence, that encodes the RPBLAs. In the latter circumstance, the RPBLAs are expressed in vivo in the host animal. In either situation, media in which the expressed RPBLAs or nucleic acids are dissolved or dispersed to form adjuvant compositions are also well known.
Illustrative nucleic acid sequences are provided hereinafter that encode specific portions of the RPBLAs. As is well known in the art, particular codons are preferred for encoding amino acid residues in different animals, and as a consequence the skilled worker can revise specific nucleic acid sequences to provide desired degrees of expression. In addition, several vectors are well known for expressing foreign nucleic acids and their encoded proteins in animal hosts, including humans. On expression, the polypeptides encoded self-assemble in vivo to form RPBLAs.
T-cell stimulating immunogenic polypeptide portions of a number of illustrative adjuvants are discussed hereinafter that relate to the HIV-1 virus. In those adjuvants, a DNA vaccine that comprises all of parts of the gag gene is utilized. The HIV-1 gag gene encodes four proteins: the P24 capsid (CA), P17 matrix (MA), and two nucleocapsid proteins (NC) P6 and P9. Illustrative adjuvants' fusion proteins contain the gag-encoded P24 sequence or the P41 sequence that results from fusion of the P24 and P17 sequences, and the reverse transcriptase (RT) that is encoded by the HIV-1 pol gene.
Analogously, a vaccine or inoculum against hepatitis B virus (HBV) the utilizes one or more of the surface (HBsAg) proteins as immunogen can utilize an adjuvant whose T-cell stimulating immunogenic polypeptide portion includes a sequence illustrated of a surface protein that includes the PreS1 and/or PreS2 portions of the surface protein in the table of T Cell Epitopes that follows. One such vaccine is that sold under the name RECOMBIVAX HB® hepatitis B vaccine that is a non-infectious subunit viral vaccine derived from the hepatitis B surface antigen (HBsAg) produced in yeast cells and developed in the Merck Research Laboratories. Similarly, the HBV core-based vaccine of U.S. Pat. No. 7,351,413, can be provided an adjuvant by utilization of one or more core sequences set out in the table of T Cell Epitopes that follows. Additional adjuvants can be prepared as discussed herein using the sequences in that following table or other T cell epitopes obtained from the literature.
A contemplated adjuvant is typically used in an adjuvant-effective amount dissolved or dispersed in a pharmaceutically acceptable diluent as an adjuvant composition. The amount utilized can vary widely in different host animals, with the T-cell stimulating immunogenic polypeptide portion used, and the construct used. Typical amounts are about 1 microgram (μg) of RPBLAs per kilogram (kg) of host body weight (μg/kg) to about 1 milligram (mg) of RPBLAs per kilogram of host body weight (mg/kg). More usual amounts are about 5 μg/kg of host body weight to about 0.5 mg/kg host body weight.
The diluent is typically aqueous-based and can include one or more additional adjuvants, buffers, salts and viscosity enhancing agents. The ingredients of the diluent are those materials that are often present in a vaccine or inoculum as are discussed hereinafter.
Protein Bodies and Protein Body-Inducing Sequences
Inasmuch as protein bodies (PBs) are appropriately so-named only in seeds, similar structures produced in other plant organs and in non-higher plants are referred to generally as synthetic PBs or “recombinant protein body-like assemblies” (RPBLAs). Such RPBLAs are membrane-enclosed fusion proteins that are found associated with the endoplasmic reticulum (ER) of a cell.
“Purified RPBLAs” are membrane free preparations of RPBLAs in which the membrane has been removed, usually by chemical reduction as with a mercaptan-containing reagent, and the fusion protein purified as by chromatographic means to free the fusion protein from the membrane and other expression-associated impurities. The resulting purified protein is then reassembled in vitro to form purified RPBLA particles. That reformation of particles typically takes place in an aqueous composition in the presence of salts and an oxidizing environment. The formation of such purified RPBLAs is illustrated hereinafter.
A contemplated RPBLA is a recombinantly prepared fusion protein (polypeptide) that is expressed in a cell foreign to the nucleic acids used to transform the cell. The cell(s) in which the polypeptide is expressed is a host cell(s), and can be a cell preparation or cells of an intact organism. The intact organism can itself be a group of single celled organisms such as bacteria or fungi, or multi-celled plants or animals, including humans. When a human is the host, the person is the recipient of a nucleic acid-encoded form of the adjuvant and the adjuvant is administered as part of a treatment regimen.
In living organisms, the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the gene that codes for the protein. Thus, through the well-known degeneracy of the genetic code additional DNAs and corresponding RNA sequences (nucleic acids) can be prepared as desired that encode the same fusion protein amino acid residue sequences, but are sufficiently different from a before-discussed gene sequence that the two sequences do not hybridize at high stringency, but do hybridize at moderate stringency.
High stringency conditions can be defined as comprising hybridization at a temperature of about 50°-55° C. in 6×SSC and a final wash at a temperature of 68° C. in 1-3×SSC. Moderate stringency conditions comprise hybridization at a temperature of about 50° C. to about 65° C. in 0.2 to 0.3 M NaCl, followed by washing at about 50° C. to about 55° C. in 0.2×SSC, 0.1% SDS (sodium dodecyl sulfate).
A nucleic sequence (DNA sequence or an RNA sequence) that (1) itself encodes, or its complement encodes, a fusion protein containing a protein body-inducing sequence (PBIS) and a polypeptide of interest is also contemplated herein. As is well-known, a nucleic acid sequence such as a contemplated nucleic acid sequence is expressed when operatively linked to an appropriate promoter in an appropriate expression system as discussed elsewhere herein.
Different hosts often have preferences for a particular codon to be used for encoding a particular amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired fusion protein sequence can be altered, using in vitro mutagenesis for example, so that host-preferred codons are utilized for a particular host in which the fusion protein is to be expressed.
The RPBLAs are usually present in a generally spherical form having a diameter of about 0.5 to about 3 microns (μ) and usually about 1μ. In some instances, RPBLAs are amorphous in shape and can vary widely in dimensions, but are still found associated with the ER.
The density of RPBLAs is typically greater than that of substantially all of the endogenous host cell proteins, and is typically about 1.1 to about 1.35 g/ml. The high density of contemplated RPBLAs is due to the general ability of the recombinant fusion proteins to assemble as multimers and accumulate.
A contemplated RPBLA used as an adjuvant need not be expressed in a plant. Rather, as disclosed in published US application 20060121573, RPBLAs can be expressed in other transformed eukaryotes, particularly in transformed mammalian cells.
A fusion protein of the adjuvant RPBLAs contains two proteinaceous sequences linked together by a peptide bond as is found in a naturally occurring protein or in a polypeptide expressed by a genetically engineered nucleic acid. In a contemplated fusion protein, one sequence is a protein body-inducing sequence (PBIS) such as that of a prolamin, and the other is a biologically active immunogenic polypeptide. Either of the two portions can be at the N-terminus of the fusion protein. However, it is preferred to have the PBIS at the N-terminus.
A contemplated protein body-inducing sequence (PBIS) is preferably in whole or part from a higher plant. Illustrative, non-limiting examples of PBIS include storage proteins or modified storage proteins, as for instance, prolamins or modified prolamins, prolamin domains or modified prolamin domains. Prolamins are reviewed in Shewry et al., 2002 J. Exp. Bot. 53(370):947-958. A preferred PBIS sequence is present in a prolamin compound sequence such as gamma-zein, alpha-zein, delta-zein, beta-zein, rice prolamin and gamma-gliadin that are discussed hereinafter.
A PBIS includes a sequence that directs a protein towards the endoplasmic reticulum (ER) of the RPBLA-expressing cell. That sequence often referred to as a leader sequence or signal peptide can be from the same plant as the remainder of the PBIS or from a different plant or an animal or fungus. Illustrative signal peptides are the 19 residue gamma-zein signal peptide sequence shown in WO 2004003207 (US 20040005660), the 19 residue signal peptide sequence of alpha-gliadin or 21 residue gamma-gliadin signal peptide sequence (see, Altschuler et al., 1993 Plant Cell 5:443-450; Sugiyama et al., 1986 Plant Sci. 44:205-209; and Rafalski et al., 1984 EMBO J. 3(6):1409-11415 and the citations therein). The pathogenesis-related protein of PR10 class includes a 25 residue signal peptide sequence that is also useful herein. Similarly functioning signal peptides from other plants and animals are also reported in the literature.
The characteristics of the signal peptides responsible for directing the protein to the ER have been extensively studied (von Heijne et al., 2001 Biochim. Biophys. Acta December 12 1541(1-2):114-119). The signal peptides do not share homology at a primary structure, but have a common tripartite structure: a central hydrophobic h-region and hydrophilic N- and C-terminal flanking regions. These similarities, and the fact that proteins are translocated through the ER membrane using apparently common pathways, permits interchange of the signal peptides between different proteins or even from different organisms belonging to different phyla (See, Martoglio et al., 1998 Trends Cell Biol. October; 8(10):410-415). Thus, a PBIS can include a signal peptide of a protein from a phylum different from higher plants.
It is to be understood that an entire prolamin sequence is not required to be used. Rather, as is discussed hereinafter, only portions are needed although an entire prolamin sequence can be used.
Gamma-Zein, a maize storage protein whose DNA and amino acid residue sequences are shown hereinafter, is one of the four maize prolamins and represents 10-15 percent of the total protein in the maize endosperm. As other cereal prolamins, alpha- and gamma-zeins are biosynthesized in membrane-bound polysomes at the cytoplasmic side of the rough ER, assembled within the lumen and then sequestered into ER-derived protein bodies (Herman et al., 1999 Plant Cell 11:601-613; Ludevid et al., 1984 Plant Mol. Biol. 3:277-234; Torrent et al., 1986 Plant Mol. Biol. 7:93-403).
Gamma-Zein is composed of four characteristic domains: i) a peptide signal of 19 amino acids, ii) the repeat domain containing eight units of the hexapeptide PPPVHL (SEQ ID NO:2) [(53 amino acid residues (aa)], iii) the ProX domain where proline residues alternate with other amino acids (29 aa) and iv) the hydrophobic cysteine rich C-terminal domain (lll aa).
The ability of gamma-zein to assemble in ER-derived RPBLAs is not restricted to seeds. In fact, when gamma-zein-gene was constitutively expressed in transgenic Arabidopsis plants, the storage protein accumulated within ER-derived PBLS in leaf mesophyl cells (Geli et al., 1994 Plant Cell 6:1911-1922). Looking for a signal responsible for the gamma-zein deposition into the ER-derived protein bodies (prolamins do not have KDEL signal for ER-retention), it has been demonstrated that the proline-rich N-terminal domain including the tandem repeat domain was necessary for ER retention. In this work, it was also suggested that the C-terminal domain could be involved in protein body formation, however, recent data (WO2004003207A1) demonstrate that the proline-rich N-terminal domain is necessary and sufficient to retain in the ER and to induce the protein body formation. However, the mechanisms by which these domains promote the protein body assembly are still unknown, but evidence from in vitro studies suggests that the N-terminal portion of gamma-zein is able to self-assemble into ordered structures.
It is preferred that a gamma-zein-based PBIS include at least one repeat and the amino-terminal nine residues of the ProX domain, and more preferably the entire Pro-X domain. The C-terminal portion of gamma-zein is not needed, but can be present. Those sequences are shown in US 20040005660 and designated as RX3 and P4, respectively, and are noted hereinafter.
Zeins are of four distinct types: alpha, beta, delta, and gamma. They accumulate in a sequential manner in the ER-derived protein bodies during endosperm development. Beta-zein and delta-zein do not accumulate in large amount in maize PBs, but they were stable in the vegetative tissues and were deposited in ER-derived protein body-like structures when expressed in tobacco plants (Bagga et al., 1997 Plant Cell September 9(9):1683-1696). This result indicates that beta-zein, as well as delta-zein, can induce ER retention and PB formation.
The wheat prolamin storage proteins, gliadins, are a group of K/HDEL-less proteins whose transport via the ER appears to be complex. These proteins sequester in to the ER where they are either retained and packaged into dense protein bodies, or are transported from the ER via the Golgi into vacuoles. (Altschuler et al., 1993 Plant Cell 5:443-450.)
The gliadins appear to be natural chimeras, containing two separately folded autonomous regions. The N-terminus is composed of about 7 to about 16 tandem repeats rich in glutamine and proline. The sequence of the tandem repeats varies among the different gliadins, but are based on one or the other consensus sequences PQQPFPQ (SEQ ID NO:3), PQQQPPFS (SEQ ID NO:4) and PQQPQ (SEQ ID NO:5). The C-terminal region of the protein contains six to eight cysteines that form intramolecular disulfide bonds. The work of the Altschuler et al. indicates that the N-terminal region and consensus sequences are responsible for PB formation in the ER from gamma-gliadin. (Altschuler et al., 1993 Plant Cell 5:443-450.)
Illustrative useful prolamin-type sequences are shown in the Table below along with their GenBank identifiers.
Further useful sequences are obtained by carrying out a BLAST search in the all non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF (excluding environmental samples) data base as described in Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402 using a query such as those shown below:
RX3 Query
Alpha-Zein
Rice Prolamin Query
An illustrative modified prolamin includes (a) a signal peptide sequence, (b) a sequence of one or more copies of the repeat domain hexapeptide PPPVHL (SEQ ID NO: 2) of the protein gamma-zein, the entire domain containing eight hexapeptide units; and (c) a sequence of all or part of the ProX domain of gamma-zein. Illustrative specific modified prolamins include the polypeptides identified below as R3, RX3 and P4 whose DNA and amino acid residue sequences are also shown below.
Particularly preferred prolamins include gamma-zein and its component portions as disclosed in published application WO2004003207, the rice rP13 protein and the 22 kDa maize alpha-zein and its N-terminal fragment. The DNA and amino acid residue sequences of the gamma-zein, rice and alpha-zein proteins are shown below.
Gamma-Zein of 27 kD
DNA Sequence:
Protein Sequence:
RX3
DNA Sequence:
Protein Sequence:
R3
DNA Sequence:
Protein Sequence:
P4
DNA Sequence:
Protein Sequence:
X10
DNA Sequence:
Protein Sequence:
rP13—rice prolamin of 13 kD homologous to the clone—AB016504 Sha et al., 1996 Biosci. Biotechnol. Biochem. 60(2):335-337; Wen et al., 1993 Plant Physiol. 101(3):1115-1116; Kawagoe et al., 2005 Plant Cell 17(4):1141-1153; Mullins et al., 2004 J. Agric. Food Chem. 52(8):2242-2246; Mitsukawa et al., 1999 Biosci. Biotechnol. Biochem. 63(11):1851-1858
Protein Sequence:
DNA Sequence:
22aZt N-terminal fragment of the maize alpha-zein of 22 kD—V01475 Kim et al., 2002 Plant Cell 14(3):655-672; Woo et al., 2001 Plant Cell 13(10):2297-2317; Matsushima et al., 1997 Biochim. Biophys. Acta 1339(1):14-22; Thompson et al., 1992 Plant Mol. Biol. 18(4):827-833.
Protein Sequence (Full Length):
DNA Sequence (Full Length):
Gamma-Gliadin precursor—AAA34272—Scheets et al., 1988 Plant Sci. 57:141-150.
Protein Sequence:
DNA Sequence (M36999)
Beta zein—AF371264—Woo et al., (2001) Plant Cell 13 (10), 2297-2317.
DNA
Protein
Delta zein 10 kD—AF371266—Woo et al., (2001) Plant Cell 13 (10), 2297-2317, and Kirihara et al., (1988) Gene. November 30; 71(2):359-70.
DNA
Protein
Signal Peptides
Gamma-Zein
Alpha-Gliadin
Gamma-Gliadin
PR10
T-Cell Stimulating Immunogenic Polypeptides
A large number of T-cell-stimulating immunogenic polypeptide sequences have been identified in the literature. A partial list is provided below in the table below using the single letter code.
Corynebacterium
diptheriae
Borrelia
burgdorferi
Trypanosoma
cruzi
Plasmodium
falciparum
P. vivax
P. yoelii
Streptococcus
sobrinus
Clostridium
tetani
Neisseria
meningitidis
M. tuberculosis
Citations:
A group of preferred T-cell stimulating immunogenic sequences are present in HPV-16, in the E7 gene. In order to translate the previously discussed therapeutic DNA vaccine candidate [Osen et al., 2001 Vaccine 19(30):4276-4286] into a vaccine for use in a clinical trial, the safety features were further enhanced. For this reason, no heterologous genes were fused. Rather, immunogenicity was enhanced by placing a Kozak-sequence [Kozak et al., 1987 Nucleic Acids Res 15(20):8125-8148] in front of the gene [Steinberg et al., 2005 Vaccine 23(9):1149-1157]. A plasmid-vector pTHamp [Hanke et al., (1998) Vaccine 16(4):426-435] applicable to humans [Hanke et al., 2000 Nat Med 6(9):951-955] was selected. Many expression vectors are known and available for use for DNA vaccines. See for example, U.S. Pat. No. 7,351,813 B2 and EP 1 026 253 B1 and the citations therein.
More importantly E7 itself was redesigned. The sequence was taken apart exactly at the positions that are critical for transforming properties of the protein (pRB-binding site, C-X-X-C motifs) and reassembled in a “shuffled” order as “core” gene. This sequence was codon optimized to humans (almost identical to mice). The original junctions destroyed by the dissection were added as an “appendix” with a non-codon optimized sequence to minimize recombination events reconstituting the wild-type sequences (see also
Tumor protection and regression studies provide a first impression on immunogenicity and effectivity of tumor vaccines. Those studies do not fully reflect, however, the responses induced in humans. “In vitro immunization” of human lymphocytes by antigen-loaded dendritic cells (DCs) can be used as a model of human responses [Norm et al., 2003 J Cancer Res Clin Oncol 129(9):511-520]. Loading of DCs by DNA transfection is a suitable technique [Lohmann et al., 2000 Cancer Gene Ther 7(4):605-614] and specific T cell priming verifies the potential immunogenicity of the DNA vaccine candidate.
The results shown hereinafter illustrate that the HPV-16 E7SH DNA vaccine candidate of the second generation induces specific immunity in vivo in mice and after in vitro immunization of human lymphocytes and, therefore, can provide for a safe therapeutic HPV-vaccine.
The sequence of the gene that expresses the HPV-16 E7SH protein is as follows from 5′ to 3′:
Another group of preferred T-cell stimulating immunogenic sequences are present in HIV-1. In particularly preferred practice, a polypeptide sequence present in HIV-1 is encoded by the HIV-1 gag gene. These sequences are thus present in the P24, P17, P6 or P9 proteins encoded by the gag gene, or a polypeptide such as the P41 polypeptide.
A particular T-cell stimulating immunogenic sequence need not itself be present as a distinct polypeptide in HIV-1 or any other pathogen. Rather, such a sequence is present as a portion of a distinct polypeptide or proteinaceous material encoded by an open reading frame of a pathogenic genome.
Specific T-cell stimulating immunogenic sequences useful herein are provided below.
p41 DNA Sequence 5′ to 3′
p41 Amino Acid Sequence
Translation of P41 (1-1083)
Universal code
Total amino acid number: 360, MW=40309
Max ORF starts at AA pos 1 (may be DNA pos 1) for 360 AA (1080 bases),
MW=40309
Origin
p24 DNA Sequence 5′ to 3′
p24 Amino Acid Sequence
Translation of p24 (1-699)
Universal code
Total amino acid number: 232, MW=25660
Max ORF starts at AA pos 1 (may be DNA pos 1) for 232 AA (696 bases),
MW=25660
Origin
RT DNA Sequence 5′ to 3′
RT Amino Acid Sequence
Translation of RT (1-1731)
Universal code
Total amino acid number: 576, MW=65360
Max ORF starts at AA pos 1 (may be DNA pos 1) for 576 AA (1728 bases),
MW=65360
Origin
NSs DNA Sequence 5′ to 3′
NSs is a silencing suppressor used in the agroinfiltration of tobacco plants.
Translation of NSs (1-1404)
Universal code
Total amino acid number: 467, MW=52121
Max ORF starts at AA pos 1 (may be DNA pos 1) for 467 AA (1401 bases),
MW=52121
Origin
Gag CD8 Peptide Amino Acid Sequence
Gag CD4 (13) Peptide Amino Acid Sequence
Gag CD4 (17) Peptide Amino Acid Sequence
RT CD4 Peptide Amino Acid Sequence
RT CD8 Peptide Amino Acid Sequence
T-cell stimulating immunogenicity of a contemplated adjuvant can be measured by a variety of well known techniques. In usual practice, a host animal is inoculated with a contemplated RPBLA vaccine or inoculum, and peripheral mononuclear blood cells (PMBC) are thereafter collected. Those PMBC are then cultured in vitro in the presence of the biologically active polypeptide (T cell immunogen) for a period of about three to five days. The cultured PMBC are then assayed for proliferation or secretion of a cytokine such as IL-2, GM-CSF of IFN-γ. Assays for T cell activation are well known in the art. See, for example, U.S. Pat. No. 5,478,726 and the art cited therein.
A contemplated adjuvant is typically prepared from a recovered RPBLA particles by dispersing the RPBLAs in a physiologically tolerable (acceptable) diluent vehicle such as water, saline, phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, or the like to form an aqueous composition. The diluent vehicle can also include oleaginous materials such as peanut oil, squalane, squalene and the like as are well known.
The preparation of adjuvants that contain proteinaceous materials as active ingredients is also well understood in the art. Typically, such adjuvants are prepared as parenterals, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified, which is particularly preferred.
The immunogenically active RPBLAs are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, an adjuvant can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the immunogenic effectiveness of the composition.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the detailed examples below, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever.
DNA encoding HIV-1 p24, p41 and RT from a cloned South African HIV isolate Du422 (GenBank accession no. AF544010) was fused to Zera® using PCR and subsequently cloned into an A. tumefaciens binary expression vector pTRAc (Meyers, BMC Biotechnology 2008 8:53) in E. coli to yield the recombinant clone pTRAcRX3p24, pTRAcRX3p41 and pTRAcRX3RT.
The HPV-16 E7SH gene was engineered by three consecutive PCR reactions as described in [Steinberg et al., 2995 Vaccine 23(9):1149-1157]. The resulting HPV16 E7SH gene was amplified by PCR and an enterokinase site was added to the 5′ end of the protein. This construct was then fused to RX3 by replacing GFP in an A. tumefaciens binary vector pTRA C [Mclean et al., 2007 J Gen Virol 88:1460-1469] that contained a RX3 GFP fusion gene.
The recombinant clone was purified from E. coli, and electroporated into competent host A. tumefaciens GV3101::pMP90RK cells. Recombinant A. tumefaciens cultures containing pTRAcRX3p24, pTRAcRX3p41, pTRAcRX3RT and pRX3E7SH were injection-infiltrated into the leaves of 6-week old N. benthamiana plants using a needle and syringe. The leaves were co-infiltrated with A. tumefaciens LBA4404 containing a silencing suppressor pBIN-NS to enhance transient protein expression. The infiltrated plants were grown at 22° C. under a 16 hour: 8 hour light:dark cycle.
Harvesting and Purification of Transiently-Expressed RX3 Fusion Proteins
Approximately 10 g of infiltrated leaf tissue was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl2, 10 mM EDTA and 0.4M NaCl). This suspension was homogenized for 3 minutes and then filtered through miracloth (a quick filtration material for gelatinous homogenates and for protoplast isolation that is composed of rayon polyester with a pore size of 22-25 mm and an acrylic binder that is available from Calbiochem®, San Diego, Calif.). The filtrate was loaded on top of a density step gradient. The gradient comprised of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep® density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80 000Xg in a Beckman SW28 rotor at 4° C. The pellet was resuspended in 500 μl of buffer PBP3 and an aliquot stored for analysis. The remainder was stored at −70° C.
HIV Group Study
Female BALB/c mice (8 to 10 weeks old) were divided into the appropriate number of groups (5 mice per group):
Group 1—vDNA prime
Group 2—vDNA prime+vDNA boost
Group 3—vDNA prime+RPBLAs boost
Group 4—RPBLAs prime
The DNA vaccines (vDNA) used in the prime and boost inoculations correspond to: (i) pTHGagC that expresses the Du422 HIV-1 subtype C Gag (van Harmelen, 2003), was manufactured by Aldevron, Fargo, N. Dak., USA and resuspended at 1 mg DNA/ml saline and (ii) pVRCgrttn, that express five HIV-1 subtype C genes gag, reverse transcriptase (RT), tat, and nef (Burgers et al., AIDS Research and Human Retroviruses 2008 24(2):195-206). The vDNA (100 μg DNA/100 μl saline) was administered by injecting 50 μl into each tibialis anterior muscle.
For intramuscular inoculation with the RPBLAs containing the corresponding RX3 fusion proteins (RX3-p24, RX3-p41 and RX3-RT), 100 μl of the corresponding RPBLAs fraction isolated as described above and resuspended in saline buffer The following amounts of RPBLAs were injected into the tibialis anterior muscle: 3.6 μg for RX3-p24; 3.1 μg for RX3-p41; and 4 μg for RX3-RT.
HPV Group Study
DNA Vaccination
Six- to eight-week-old female C57BL/6 mice were injected with 50 μl of 10 μM cardiotoxin into each tibialis anterior muscle 5-6 days prior to DNA injection. For vaccination, 50 μl of plasmid DNA (1 μg/μl in PBS) was injected into each pretreated muscle. Ten days later, mice were sacrificed and splenocytes were isolated from the spleen.
Tumor Protection and Regression Studies
Tumor protection and regression studies were performed essentially as described in (Öhlschlager et al., Vaccine 2006 24(2):2880-2893). The specific modifications of the protocol are indicated in the corresponding experiments.
HIV Antigens
A single cell suspension of splenocytes was prepared from spleens harvested on day 12 or 40 and pooled from 5 mice per group. IFN-γ ELISPOT responses were measured using a mouse IL-2 or IFN-γ ELISPOT set (BD Pharmingen). Splenocytes were plated in triplicate at 5×105/well in a final volume of 200 μl R10 culture medium (RPMI with 10% heat inactivated FCS, Gibco, containing 15 mM β-mercaptoethanol, 100 U penicillin per ml, and 100 μg streptomycin).
The peptides (>95% pure, Bachem, Switzerland) GagCD8 AMQMLKDTI (SEQ ID NO:116) gag CD4(13) NPPIPVGDIYKRWIIGLNK (SEQ ID NO:117) gag CD4(17) FRDYVDRFFKTLRAEQATQE (SEQ ID NO:118), RT(CD8) VYYDPSKDLIA (SEQ ID NO:120), or RT(CD4) PKVKQWPLTEVKIKALTAI (SEQ ID NO:119), were used as stimuli in the assay at a final concentration of 4 μg/ml. Reactions containing an irrelevant H-2Kd binding peptide TYSTVASSL (SEQ ID NO:1), (obtained from Elizabeth Reap, AlphaVax) or without peptide served as background controls. Spots were detected with the detection antibody at 22 hours, developed with Nova Red then counted using a CTL Analyzer (Cellular Technology, Ohio, USA) with Immunospot Version 3.0 software. The average number of spots in triplicate wells was calculated and results are expressed as the average number of spot-forming units (SFU) per 106 splenocytes±the standard deviation (SD). For each group of mice, the average background spots obtained in the absence of peptide and in the presence of the irrelevant peptide plus one standard deviation of this average was considered as the cut-off for a positive response.
HPV Antigens
In all studies, ELISPOTs were performed ex vivo essentially as described in (Steinberg et al., 2005 Vaccine 23:1149-1157). Murine IFN-gamma Elispot assays were performed ex vivo and 5 or 6 days after each in vitro restimulation as described earlier (Ohlschlager et al., 2006). The granzyme B Elispot assay was performed similarly to the IFN-gamma Elispot Assay. For this assay, the anti-mouse granzyme capture antibody (100 ng/well, clone R4-6A2; PharMingen, San Diego, USA) and the biotinylated anti-mouse granzyme detection antibody (50 ng/well, clone XMG1.2; PharMingen, San Diego, USA) were used.
Western blots were carried out using a LAV Blot I commercial kit (Biorad). Mouse serum from inoculated mice was used to detect antibodies with goat anti-mouse IgG conjugated to alkaline phosphatase.
Approximately 10 g of leaf tissue agroinfiltrated with the corresponding construct (pRX3-p24, pRX3-p41 or pRX3-RT) was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl2, 10 mM EDTA and 0.4M NaCl). This was homogenized for 3 minutes on ice using a Polytron homogenizer and then filtered through miracloth. The corresponding filtrate was loaded on top of a density step gradient, comprising of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep® density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80,000×g in a Beckman SW28 rotor at 4° C. The pellet was resuspended in 500 μl buffer PBP3 to check for the presence of RPBLAs by optic microscopy, and an aliquot stored for analysis. The remainder was stored at −70° C.
To verify that the RPBLAs fraction contained the corresponding RX3 fusion protein, an aliquot of it was analyzed by western blot using anti-RX3 and anti-p24 antibodies to verify the integrity of the fusion protein (
To determine the cellular immune response induced by the administration of RX3-p24 containing RPBLAs, four groups of mice were inoculated as follows: (i) mice inoculated with the DNA vaccine (pTHGagx1), (ii) mice inoculated with the DNA vaccine and boosted with another dose of the same DNA vaccine (pTHGagx2), (iii) mice inoculated with the DNA vaccine and boosted with RPBLAs containing RX3-p24 and no further DNA (pTHGag+RX3-p24), and (iv) mice inoculated exclusively with RPBLAs containing RX3-p24 (RX3-p24).
IFN-γ and IL-2 ELISPOT assays indicated that mice inoculated with the DNA vaccine alone (pTHGagx1) induced a cellular response. As shown in
When the same assays were performed with the mouse group inoculated exclusively with the RPBLAs containing RX3-p24 (RX3-p24), no significant response was observed. This result suggested that the immunogen aggregated inside RPBLAs is not able to trigger the cellular response. Nevertheless, when IFN-γ and IL-2 ELISPOT assays were performed on T-cells from mice inoculated with the DNA vaccine and boosted with a second inoculation consisting of RPBLAs containing RX3-p24 and no further DNA (pTHGag+RX3-p24), a surprising 3-fold higher cellular response was observed compared to the pTHGagx1 group. The lack of cellular response observed in the p24 mouse group probably indicates that a higher amount of RPBLAs should be inoculated.
These data indicate that RPBLAs are a suitable immunogen presentation vehicle able to induce a cellular response.
It has been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies, suggesting that high anti-p24 antibody titres might be necessary to maintain a disease-free state.
To determine the presence of antibodies against the p24 antigen, strips containing a representation of the HIV virus proteins [LAV Blot I commercial kit (Biorad)] were incubated with mouse serum from the four inoculation groups (pTHGagx1, pTHGagx2, pTHGag+RX3-p24 and RX3-p24). Antibodies against the p24 protein were detected only in mice inoculated with the DNA vaccine and boosted with a second round of the DNA inoculation or with RPBLAs containing the RX3-p24 and no further DNA (
As indicated previously, p41 which results from the fusion of p17 and p24 fragments (p17/24) of the HIV Gag protein, contains the highest density of CTL epitopes in the HIV-1 genome (Novitsky et al., J. Virol. 2002 76(20):10155-10168). In this context the efficiency of RPBLAs containing the RX3-p41 fusion protein to trigger the cellular response of the immune system was examined.
As occurred previously in Example 6, IFN-γ and IL-2 ELISPOT assays indicated that mice inoculated with a single dose of the DNA vaccine induced a small cellular response, which was significantly increased when those mice were boosted with a second inoculation with the DNA vaccine (compare pTHGagx1 versus pTHGagx2 in
As an effective multivalent vaccine against HIV includes several antigens, similar studies were performed with HIV viral protein RT.
IFNγ ELISPOT assays indicated that mice inoculated with a single dose of the DNA vaccine (pVRCgrttnx1) induced a very poor cellular response. Exclusively CD8 T-cells incubated with the stimulating peptides RT(CD8) secreted a larger amount of IFN-γ than the same cells incubated with un irrelevant peptide TYSTVASSL (SEQ ID NO:1;
DNA vaccines encoding HIV antigens have been studied extensively and shown to induce both humoral and cellular immune responses in animal models as well as in humans (Estcourt et al., Immunol. Rev. 2004 199:144-155). However, although DNA vaccines have been shown to be safe, immunizations have generated low and transient levels of immune responses.
pTHGag was shown in the mouse model to induce a potent cytotoxic lymphocyte response. Pr55Gag expressed in a variety of cell systems can assemble and bud through the plasma membrane to form highly immunogenic virus-like particles (VLPs). RPBLAs can not been considered as classical VLPs, because their assembly is induced by the aggregation capacity of RX3, which is not a viral protein involved in the formation of the virus particles. However, the suitability of a DNA vaccine expressing RPBLAs containing the RX3-24, RX3-41, RX3-RT and RX3E7SH was examined. Interestingly, once the corresponding pTH-derived vectors (pTHRX3-p24, pTHRX3-p41, pTHRX3-RT and pTHRX3-E7SH) were administrated as the pTHGag in previous studies, significant humoral and cellular immune responses were observed. This unexpected result indicates that RPBLAs can be administered by DNA vaccination; in spite of this organelles are stored in the ER and are not supposed to bud through the plasma membrane to form highly immunogenic virus-like particles (VLPs).
The isolation of RPBLAs by density gradient permits the recovery of a highly enriched fraction of RPBLAs, but a certain degree of contaminants are co-purified. To remove as much contaminants as possible, the RX3 fusion proteins (RX3-p24, RX3-p41, RX3-RT and RX3-E7SH) were solubilized from the corresponding RPBLA fraction in 20 mM Tris pH8, 2% DOC, 10 mM DTT incubated 1 hour at room temperature in soft agitation, and purified in RP-FPLC. The elution fractions containing the RX3 fusion proteins with more than 95% purity were pooled and lyophilized. The corresponding pellet was recovered in distilled water in the presence of 200 mM of NaCl and 50 mM of CaCl. In these conditions, the fusion proteins containing the RX3 peptide reassemble spontaneously to reform RPBLAs in vitro, outside of the plant ER.
In vitro-assembled RPBLAs containing the corresponding RX3 fusion protein were inoculated into mice and the IFNγ, IL-2 and Granzyme B ELISPOT assays showed that RPBLAs boost significantly the cellular response in equivalent studies as the those performed using in vivo-formed RPBLAs. This surprising result indicates that in vitro-assembled RPBLAs maintain the capacity of inducing the cellular response.
RX3 fusion proteins can be induced to assemble in vitro and form RPBLAs in the following conditions: (i) reducing the pH value of the solution, (ii) increasing salt content, (iii) reducing or removing the concentration of reducing agents, (iv) adding oxidizing agents, (v) decreasing the temperature, or a combination of this factors. Obviously, in vitro RPBLAs are not surrounded by a membrane. Preferred salts to induce the assembly in vitro are NaCl, CaCl and KCl and preferred pH values are below 7.
As indicated before, a double immune response (cellular plus humoral) produces a more protective effect against AIDS. The presence of antibodies against the p24, p41 and RT antigens was shown by using the HIV strips (LAV Blot I commercial kit (Biorad)) in mice primed with the DNA vaccine and boosted with the corresponding in vitro assembled RPBLAs.
Approximately 10 g of leaf tissue agroinfiltrated with the HPV-16 antigen E7SH fused to RX3 (pRX3-E7SH) was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl2, 10 mM EDTA and 0.4M NaCl). This was homogenized for 3 minutes on ice using a Polytron homogenizer and then filtered through miracloth. The corresponding filtrate was loaded on top of a density step gradient, comprising of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep® density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80,000×g in a Beckman SW28 rotor at 4° C. The pellet was resuspended in 500 μl buffer PBP3 to check for the presence of RPBLAs by optic microscopy, and an aliquot stored for analysis. The remainder was stored at −70° C.
The gene sequence for the RX2-E7SH fusion protein is shown below from 5′ to 3′:
To verify that the RPBLAs fraction contained the corresponding RX3 fusion protein, an aliquot of it was analyzed by western blot using anti-RX3 to verify the integrity of the fusion protein (
To determine the cellular immune response induced by the administration of RX3-E7SH containing RPBLAs, five groups of mice were inoculated as follows: (i) mice inoculated with the DNA vaccine expressing E7SH antigen (pTHamp-E7SH), (ii) mice inoculated with the corresponding DNA vaccine negative control (pTHamp) with the sequence:
(iii) mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH), (iv) mice co-inoculated with RPBLAs containing RX3-E7SH and incomplete Freund's adjuvant (RX3-E7SH/IFA), and finally (v) mice inoculated with RPBLAs containing RX3 fused to Gfp (RX3-Gfp) as a negative control of RPBLAs.
As expected, IFN-γ and Granzyme B ELISPOT assays indicated clearly that mice inoculated with the DNA vaccine (pTHamp-E7SH) induced a cellular response. As shown in
Surprisingly, it was also observed that splenocytes isolated from mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH) also released a large amount of IFN-γ and Granzyme B (equivalent to pTHamp-E7SH mice group) in the presence or absence of IFA co-administration
These results demonstrate clearly that E7SH antigen administered in fusion with RX3 in a RPBLAs particle is able to trigger efficiently a cellular response. The fact that no adjuvant was needed to achieve the maximum effect indicates that RX3-E7SH in RPBLAs is an efficient antigen presentation vehicle able to induce a cellular response. This conclusion was supported by the observation that it is necessary to co-administer IFA to ovalbumin (OVA) in order to induce an efficient cellular response
The amino acid sequence for ovalbumin in single letter code is shown below:
To determine, if the specifically activated splenocytes had cytolytic activity, 51Cr-release assays were performed. 51Cr release assays were performed 5-6 days after an in vitro restimulation of murine spleen cells as described elsewhere [Steinberg et al., (2005) Vaccine 23(9):1149-1157.] An animal was scored positive when the specific lysis of the specific target (RX3-E7 or pTHamp-E7SH cells) was at least 10% above the lysis of the control target (RX3-Gfp or pTHamp cells) for the protein and DNA based vaccines. After a first round of in vitro restimulation strong specific cytolytic activity against the E7WT-expressing RMA-E7 transfectants was shown (see table below.)
51C-release assay
Surprisingly, the mean of specific lysis of the RMA-E7 cells was comparable in the RX3-E7SH-group (33%) and the pTHamp-E7SH immunized animals (26%), and significantly higher than the corresponding control groups RX3-Gfp (12%) and the pTHamp (8%). This result indicates that RX3-E7SH RPBLAs was able to induce a specific cytolytic activity against E7 expressing cells as efficiently as has already been shown by using the DNA vaccine pTHamp-E7SH [Öhlschlager et al., (2006) Vaccine 24:2880-2893]. Moreover, the fact that the cytolytic activity was not increased when the RPBLAs containing RX3-E7SH fusion protein was co-administered with IFA (see RX3-E7SH/IFA-group in table) suggests that even a lower dose would be effective to trigger the cytolytic effect, supporting the idea that RPBLAs provide an efficient administration vehicle to trigger a specific cytolytic effect.
It is important to add that it has been widely demonstrated that a cytolytic response is a crucial element for controlling tumor growth [Akazawa, 2004 Cancer Res 64:757-764] and viral infection (Vine et al., 2004 J Immunol 173:5121-5129].
The aim of a therapeutic tumor vaccine is the induction of an effective immune response eradicating established tumors. Therefore, vaccination with the E7SH gene was examined to determine whether a cellular immune response could be induced that was able to control established E7-expressing tumor cells in vivo. In four tumor regression studies, a total of 80 animals were transplanted with a tumorigenic dose of syngeneic C3-tumor cells (day 0). When the tumors had reached a mean size of 4-9 mm2 at days 5-18, the animals were inoculated with: (i) 100 μg of the E7SH-encoding plasmid (pTHamp-E7SH), (ii) 100 μg of empty pTHamp vector (pTHamp), (iii) RPBLAs containing 5 μg of RX3-E7SH(RX3-E7SH), and (iv) the same amount of RX3-E7SH RPBLAs co-administered with IFA (RX3-E7SH/IFA) (day zero). Tumor size was determined every two days by measuring with a ruler until the end of the study (day 14).
As shown in
To exclude an unspecific tumor growth reduction due to some contaminants present in the RPBLAs preparation, or by the RX3 polypeptide by itself, equivalent amounts of RPBLAs containing RX3-Gfp were inoculated in an independent study, and no effect on tumor growth was observed when compared to pTHamp DNA control group (see
It must be pointed out that mice were inoculated only once with RPBLAs with RX3-E7SH. In a prime boost study it is expected to have an enhanced therapeutic effect.
Taking into consideration a goal of the application of a protective (prophylactic) vaccine based on RPBLAs in addition to a therapeutic vaccine, rechallenge studies were undertaken to determine whether the RPBLAs containing RX3-E7SH were able to protect animals from an outgrowth of E7-expressing syngeneic tumors.
Those mice that showed complete regression after the tumor regression experiment were injected again with 0.5×106 C3 cells s.c. in 100 μl PBS into the left flank 3 weeks after completion of the tumor regression study. The first C3 inoculation was given into the right flank. As a control, the same number of non-immunised mice received the same treatment. Twenty days after this injection, all control mice showed tumors with a size range of 100-400 mm2, whereas the immunized mice developed no tumors, and therefore showed clear protection from tumor growth (see
Each of the patents, patent applications and articles cited herein is incorporated by reference. The use of the article “a” or “an” is intended to include one or more.
The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.
This is a U.S. National Phase of International Appl. No. PCT/EP2009/063223, filed on Oct. 9, 2009, which claims benefit of priority of U.S. application Ser. No. 61/104,403 filed on October 2008, each of which is hereby incorporated by reference in its entirety and is a Continuation-in-Part of copending U.S. patent application Ser. No. 11/709,527 filed on Feb. 22, 2007, now U.S. Pat. No. 8,163,880 issued on Apr. 24, 2012, which claims priority to Provisional Application No. 60/776,391 filed on Feb. 23, 2006. The content of the electronically submitted sequence listing (Name: sequencelisting_ascii.txt, Size: 103,230 bytes; and Date of Creation: Jul. 5, 2011) is herein incorporated by reference in its entirety.
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| PCT/EP2009/063223 | 10/9/2009 | WO | 00 | 7/5/2011 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2010/040847 | 4/15/2010 | WO | A |
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| Number | Date | Country | |
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| 20110262478 A1 | Oct 2011 | US | |
| 20160243213 A9 | Aug 2016 | US |
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|---|---|---|---|
| 60776391 | Feb 2006 | US | |
| 61104403 | Oct 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11709527 | Feb 2007 | US |
| Child | 13123510 | US |
