Patent Application
20100275278
The present invention relates to a composition and method for producing an animal model for HIV.
HIV is a viral infection. Therefore, by definition, HIV is an intra cellular parasite. The virus must assimilate a variety of host cellular proteins, lipids, carbohydrates and nucleic acids into its own structure and reproductive cycle. Attempts at inoculating animals with HIV have all failed. Animals such as mice lack one or more cellular proteins or other cellular derived molecules necessary for viral replication, immune evasion and immune suppression. The purpose of this invention is to produce an animal that possesses the full complement of HIV immune mediated molecules in a manner that the animal can assimilate in trans the human derived proteins necessary for an HIV infection to proliferate. The animal will not recognize these foreign molecules as being foreign, and therefore, will not develop an immune response towards them. Furthermore, these human derived molecules will be directed towards Peyer's patches, the very site of HIV replication. The animal will be susceptible to HIV disease.
Rationale Behind an HIV Animal Model
A retroviral life cycle can be divided into an afferent and efferent limb. The afferent limb starts with viral attachment and ends with viral DNA integration into the host genome. The efferent limb commences with the production of viral messenger RNA and culminates with viral fission releasing immature virions.
The afferent lifecycle of the virus will be arbitrarily divided into the following steps:
The efferent lifecycle of the virus will be arbitrarily divided into the following steps:
Each step delineated above relies on host derived proteins, lipids, carbohydrates and/or nucleic acids. Animals do not support the HIV lifecycle because they lack one or more necessary host derived molecules.
HIV, as with all significant viral pathogens, is able to evade the host immune response. Furthermore, HIV down regulates or deregulates the host immunologic response.
For an animal model to be successful for HIV disease, three correlates of the disease must be expressed:
Many proteins necessary for viral replication of the host immune response are human host derived proteins that are not found in animals. These include, but are not limited to, tRNA synthetase, tRNAlys, Tsg101, Tal, Staufen, LEDGF/p75, Cyclin T, CDK9 and RNA polymerase II. To create an animal model capable of not only supporting HIV replication, but also reproducing HIV disease in the animal requires the assimilation of these proteins into the animal without the animal recognizing these proteins as foreign. Success of such an animal model would rely on the lack of an immunologic response to these human proteins. Furthermore, assimilation or targeting of these proteins into the proper target tissues, predominantly Peyer's patches, the principal site of HIV replication, is necessary to reproduce an HIV infection in an alternate host.
Viral evasion of the host's immune response requires the active participation of host derived cellular proteins such as the complement control proteins CD55, CD46 and Factor H. These proteins are necessary to prevent the host's immune cells from reacting to and destroying normal tissue. By incorporating these molecules into an intact HIV virion, the virus is able to fool the immune system in a “cloak-and-dagger” method that avoids virolysis.
Immune disregulation is accomplished by the virus skewing the host towards a Th2 immune response. This is accomplished by the virus hijacking the endosomal pathway by incorporating molecules such as Tsg101, Tal and Ubiquitin. Furthermore, the viral envelope incorporates MHC-11 and CD86 molecules which are consistent with a Th2 response.
As a corollary to the above paragraph, any given protein may exhibit different and at times divergent and conflicting functions, complicating the challenge to an animal model for HIV.
The present invention provides compositions and a method for producing an animal model for HIV induced disease. The present invention is an animal adapted to simulate a human-like immune response to HIV, which is accomplished by activation and inactivation of complement of proteins within the animal. Accordingly, the present invention stages certain human proteins within an animal by way of its gut associated lymphoid tissue followed by infection of live HIV.
The present invention is directed to an animal model for HIV and the method of producing the same. Preferably, the present invention is a mouse adapted to simulate a human-like immune response to HIV, which is generated by appropriate protein behavior within the mouse. The mouse genome has been published.1 Extensive linkage conservation/synteny between mouse and human DNA has been established.2 The present invention stages certain human proteins within a mouse by way of its gut associated lymphoid tissue (GALT).
A key to protein variability lies in the primary, secondary, tertiary and quaternary structure of the protein itself. The protein may assume different secondary, tertiary and quaternary structures in various environmental conditions. Changes in ph, temperature, as well as the presence, absence, or concentration of cellular cofactors, such as calcium and magnesium, alter the structure and function of the protein. Most importantly however, proteins can be divided into basic building blocks or subunits known as motifs, each which possesses a specific function which is independent of the rest of the molecule. In some instances only a portion of the protein is directly involved in a certain metabolic process. The whole protein may or may not be needed to produce the desired effect. The subunits not directly involved in the cellular activity may affect the overall structure, stability, intracellular location and often function as a scaffold.
However it has also been demonstrated in other circumstances that a subunit of a protein that carries a significant function maintains that function when physically separated from the rest of the molecule. In such circumstances one may envision that only a portion of the protein is needed to perform the desired effect and is necessary to be encoded by recombinant DNA technology to develop an animal model for HIV. Invariant amino acids in each protein are always noted. For example, the cystine residue occupying the position of amino acid 261 of Cyclin T is absolutely required for interaction with Tat.3
The above conclusion has been demonstrated with in vitro models of human CyclinT1 (hCycT1) as it interacts with the Tat protein. A heterodimer of human CyclinT1 and Tat protein is a prerequisite to the binding of the heterodimer to the TAR sequence that initiates HIV RNA replication. The first 272 amino acids of the 726-aa hCycT1 protein are sufficient to support Tat function, TAR recognition and binding and ultimately viral replication. Even more specifically a critically defined region of hCycT1 located between residues 250 and 262 is critical for Tat and TAR binding and has been termed the Tat-TAR recognition motif (TRM).
All proteins have a characteristic half life usually measured in minutes or hours. Therefore, these proteins that support HIV replication and immune evasion need to be produced within the animal in a continuous pattern with a steady state level. The tissue concentration of the proteins supplied in trans should mirror that found in the normal human immunologic milieu.
All proteins administered to the animal model are encoded within the DNA. Recombinant technology allows introduction of human DNA into bacteria, fungi, yeast or viruses. Utilizing commensal organisms, found normally in the gut of an animal such as a mouse, rat or rabbit for this recombination the proteins of human origin necessary for HIV replication and immune evasion and immune disregulation can be introduced into the animal without the animal rejecting the proteins as foreign. The mechanisms of suppressor cells and regulatory cells found within the gut associated lymphoid tissue (GALT) prevent immunologic response to ingested food, commensal organisms and the products of the commensal organisms. Commensal organisms often produce vitamins necessary for the host to survive. Vitamins are protein based structures. By reasonable inference other proteins produced by the commensals would be assimilated into the host without an ensuing immunologic response. To replicate and survive the commensal bacteria continually produce protein and other components of its structure in excess of what is needed or incorporated into the replicating bacteria. These excess proteins do not elicit an immunologic response from the host animal.
GALT constitutes nearly 80% of the total body's immune cell population. GALT is the most comprehensive lymphoid organ system in humans. The function of GALT is a paradox and at times is in conflict with the systemic immune system. The systemic immune apparatus, under normal conditions, functions in a sterile environment devoid of pathogens and pathogen associated toxins. Therefore, any foreign matter encountered by the systemic immune system is regarded as a potentially harmful invader and the appropriate immunologic response follows. GALT, however, stands as a barrier between the human organism and an external environment replete with foreign tissue. The foreign matter includes a variety of commensal organisms, commensal derived products, pathogens, and pathogen derived products and ingested food. The entire GI tract from the mouth to the anus is functionally external to the human body. Unlike the systemic immune system, which responds vigorously to any foreign matter, GALT must differentiate between commensal organisms and their products, as well as ingested food to which an immunologic response would have adverse consequences and invading pathogens potentially lethal to the host.4
To affect this diversity of function, GALT is compartmentalized and, in contrast to the systemic and peripheral immune system (spleen & lymph nodes), is characterized by non-homogeneously distributed B and T cells. The phenotypic behavior, cell surface markers, developmental origins, secretory products, and hence function of the T and B cells of GALT, is markedly different from the T and B cells of the systemic system. Furthermore, GALT contains certain subsets of non-conventional lymphocytes such as γ/δ T cells. Overall GALT is characterized by afferent and efferent conduits not found in the systemic system.5
GALT (armed with a variety of immunologic cells not found in the systemic circulation, and patterned or clustered into characteristic vehicles not found elsewhere in the body) is capable of immunologic suppression as well as classically based Th-1 and Th-2 immune responses. Antigen uptake in GALT occurs through specialized epithelial cells known as “M” cells or “membranous” cells. Antigen uptake in GALT can also occur directly by epithelial cells in close proximity to underlying T and B cells. The uptake or assimilation of antigens through the “M” cells or epithelial cells may result in localized immune response, disseminated immune response and/or tolerance or immunosuppression. The vast majority of antigens interacting with GALT results in specific suppression of immunity for that antigenic structure. This is necessary because the primary function of GALT is to prevent an immunologic reaction to innocuous, and at times beneficial, foreign material.6
The final determination in GALT of immunity versus tolerance rests on many variables. These include but are not limited to the chemical structure of the antigen, the dose of the antigen administered, and the cytokine environment. Whether this phenomenon is termed suppression, anergy, deletion, ignorance, and/or immunologic deviation is irrelevant. Importantly, immunologic tolerance within GALT depends on an intact epithelial barrier.7
Many mechanisms have been described in the literature detailing the immune suppression observed with antigens derived from the large and small intestine. In classic immunology dendritic cells exposed to peripherally derived antigen assimilate the antigen (by a variety of mechanisms including but not limited to endocytosis, macrocytosis, pinocytosis, and cross presentation). Dendritic cells (DCs) lining the tissue have been described. The DCs then undergo a process of maturation and migrate to the most proximal lymph nodes. Expressing a “danger signal” the cells of the lymph node respond and eliminate the antigen expressed by the DCs. Recently however, DCs lining the GALT with an opposite function, one of tolerance have been described in the literature. These cells stimulate a protective immune response when stimulated by pathogens whose tropism (i.e., the phenomena observed in living organisms of moving towards each other) is confined to pathogens that infect or are confined to epithelial cells.8
The incorporation of the DNA encoding these human derived proteins into the commensals, herein referred to as incorporated DNA, can be done through recombinant technology with the following seven methodologies commonly used and known by those in the art.
If incorporated into a plasmid, a promoter/regulatory region controlling the plasmid activity would need to be included. The assimilation of the protein produced by the commensal into the animal may occur by passive (ATP independent) or active (ATP dependent) means. The DNA encoding a cell penetrating peptide (CPP) may be fused with the DNA encoding the human protein(s) prior to the recombinant process incorporating the DNA into the bacteria. Many cell penetrating peptides have been defined in the literature and have been used to carry cargos (attached protein, carbohydrate or lipid molecules) into cells which would normally be impermeable to these attached structures. Cell penetrating peptides can pass through cell walls, nuclear membranes, as well as the membranes enclosing other intracellular organelles with ease.15
Alternatively, the DNA encoding the below mentioned human proteins necessary for HIV viral replication, immune evasion and immune disregulation can be spliced into the DNA of an animal. Intuitively this may seem to be the most logical answer. For some proteins such as the CD4 receptor and the CCR5 and CXCR4 co-receptor, this would be workable and perhaps preferable, since the proteins would be a component of the host cell plasma membrane. Many potential problems arise using that conceptualized framework for all the proteins. Most difficult would be the targeting of the needed proteins to the sites of HIV replication (i.e., Peyer's patches). Furthermore, encoding a protein into the DNA of an organism does not equate to transcription and translation of the DNA and protein production. 70% of the DNA in a mammal is not transcribed and has been termed “junk DNA”. Production of a transgenic or chimeric animal does not equate to tissue targeting. External control of animals genetically modified at the level of embryonic cells is problematic.
These issues may be addressed as the science relating to models progresses. However, the present invention, as a first conceptualized model, involves splicing the DNA for the needed human proteins into commensal organisms.
The host proteins necessary for HIV to attach to a target cell, penetrate the target cell and replicate within the target cell, include and are not limited to the following list. The following proteins, or the nucleotide sequences encoding these proteins, preferably should be included in a working animal model for HIV:
1. Transcription factors.
2. Cellular cofactors.
g. Ran Binding Protein (RanBP1)
3. Cellular receptors.
4. Cellular coreceptors.
5. Cellular proteases.
6. Cellular proteins involved in the ubiquitin-proteasome pathway.
7. Cellular adaptor protein.
8. Human ribosomal RNA.
The host derived proteins necessary for HIV to evade the immune response include but are not limited to the following, and preferably should be included in a workable animal model for HIV. (See Table in Appendix A for a complete list of “Host Proteins Incorporated into the Intact Virus and for Pre-Integration Complex (PIC)”.
1. Plasma proteins.
2. Cell membrane bound proteins.
Finally and in addition to the proteins listed above the table located in Appendix A lists the host proteins incorporated into the intact virus, the pre-integration complex (PIC) and those involved in the HIV lifecycle. It is not exhaustive as new viral protein/host protein interactions are reported in the literature with regularity. The genetic loci of the human proteins have been described in the literature and allow for restriction enzyme splicing into yeast, bacteria or plasmid DNA.
In an alternative embodiment, the activity of Human Factor H in an animal can be limited by administration of soluble complement-receptor 1 (sCR1) by adding sCR1 exogenously or by splicing the genomic sequence for sCR1 into a commensal organism. This protein binds to C3b and C4b and facilitates the breakdown of these proteins by Factor 1. By binding to C3b, sCR1 prevents complement activation by the C3 convertase. The activity of Human Factor H in thwarting the complement cascade is mimicked by sCR1.
The administration of soluble CR1 is a controlled element or variable in the animal model. sCR1 allows control of tissue levels of C3b thereby limiting the activity of the C3 and C5 convertases which mirrors the function of Factor H.
In some animal models (e.g., old world primates), and particularly cell cultures derived thereof, TRIM-α confers a potent post entry (i.e., meaning after entry into the cell) block to HIV-1 infection. Cyclophilin A (CypA) binding to viral capsid proteins results in a similar response observed in vitro for certain human cell lines. Among new world primates, only owl monkeys exhibit post-entry restriction of HIV-1 replication. More specifically, monkey kidney cells of the Aotus trivirgatus owl restrict HIV infection, but are permissive for SIV infection. HIV restriction in these cells is completely abrogated when the interaction of the HIV-1 capsid and the cellular protein CypA is disrupted. Paradoxically, the opposite is seen in human cells where capsid-CypA interaction is required for efficient intracellular HIV-1 replication. Therefore if such an animal model is used the viral capsid interaction with the host CypA protein must be severed. The use of the CypA-binding drug cyclosporine A (CsA) would be necessary if these animal models were used. Similar findings may exist in other animals but have not yet been delineated.16
The most effective weapon for immune perturbation within the HIV arsenal is the Tat protein. The Tat protein is necessary for viral replication as well. A multiplicity of immune down modulating effects of the Tat protein has been well documented in human studies. An accurate model of HIV must include Tat mediated immune suppression. This will involve the Tat protein and the host cell receptors for the Tat protein.
Expression of MHC class II genes is inhibited by the Tat protein resulting in profound immunosuppression. A central protein in class II expression is the class II trans-activator (CIITA) protein. CIITA is responsible for integrating several proteins at the promoters of MHC class II genes enhancing MHC II gene transcription and ultimately MHC II gene expression.
In human models, the Tat protein inhibits CIITA function down regulating the expression of MHC II genes. Human cyclin T1 (hCycT1) is involved in this Tat mediated immunosuppression.
In mice however, the Tat protein does not interact with the human counterpart of hCycT1, mouse cyclin T1 (mCycT1). However, the Tat protein in mice does inhibit the activity of CIITA in a mechanism that is not dependent on mCycT1. The results are the same: the down regulation of the CIITA protein, decreased MHC II production, and immunosuppression.
Co-expression of transfected human CD4, CCR5 and CXCR4 molecules into murine cell cultures allows entry of HIV-1 but replication is blocked. Murine cyclin T1 binds Tat but does not bind TAR. Transfection with human cyclin T1 restored Tat function.17
Murine cyclin T2 can bind HIV-1 Tat and facilitate TAR binding if a single residue, asparagine 260 is replaced with a cysteine residue. Interestingly, Tat from HIV-2 does bind murine cyclin T1 and murine cyclin T2. However, neither complex binds effectively the TAR residue. With both HIV-1 and HIV-2 Tat effective binding and activity of Tat on HIV replication is rescued in murine cells by the above-mentioned mutation of Cyclin T2 at amino acid number 260. Therefore, if a murine model is anticipated, mutation of Cyclin T2 at residue 260 would equate to human Cyclin T1 supplied in trans. In an alternate murine animal model, another single amino acid difference between human Cyclin T1 and murine Cyclin T1 determines species restriction of HIV-1 Tat function. In this model, replacing the tyrosine residue at amino acid 261 in the murine Cyclin T1 with a cysteine conferred effective Cyclin T1 function with Tat and TAR.18
A competent Cyclin T1 is necessary but not sufficient for HIV viral replication. This can be provided to a murine model by either one of the above-mentioned mutations in the mouse genome or by providing human Cyclin T1 in trans.
An effective block of HIV replication in a murine model is the inability of the virion to assimilate murine Factor H. HIV directly activates the classical complement pathway in rabbit, mouse and guinea pig serum. This activation results in viral neutralization by lysis.19 Factor H is bound at multiple sites to gp120 and gp41 in the intact virus.20 Factor His the main contributor to HIV evasion of complement mediated lysis.21 Murine and human Factor His composed of twenty repetitive units and each unit is approximately sixty amino acids long.22 Neither murine Factor H nor human Factor H is characterized by an alpha helix or a beta pleated sheet. Both human and murine Factor H exists in two different confomiational states (φ1 and φ2) that can be separated by hydrophobic chromatography. Both have equal function.23 Although murine Factor H possesses a high degree of homology to human Factor H, it does not bind to the HIV virus. Establishing an effective HIV infection in a murine model would require the assimilation of human Factor H.
A variety of sialic acids (characterized by a 9 carbon backbone) and/or a glycan chain (composed of mostly 5 and 6 carbon sugars) are expressed on the surfaces of animals, fungi, plants, protozoa, bacteria and viruses. Mammals possess a variety of sialic acid recognizing proteins known as Siglecs. To date, eleven functional Siglecs and one Siglecs like molecule (Siglec L1) have been characterized. Macrophages express Siglec 1 (sialoadhesin), B cells express Siglec 2 (CD22) and monocytes express Siglec 3 (CD33). Cells involved in the innate immune response including natural killer cells and granulocytes are characterized by Siglecs 1, 3, 5, 7 and 10. The function of a protein and its potential immunogenicity are in part related to its surface glycan or sialic acid residues. Therefore, a potential rejection and function issue exists if proteins from animals expressing different surface sugar molecules co-exist in the same animal. Interestingly, the CMP-Neu5Ac synthetase genes that encode the enzymatic machinery necessary for sialic acids are found with one exception only in fruit flies, rainbow trout, mice and humans. Surprisingly, one bacteria Streptomyces coelicolor also expresses this genetic machinery. Lateral gene transfer between this bacterium and a eukaryotic host best explains this anomaly.24 Therefore, a murine model obviates this overwhelming concern.
The mucosa of the murine GI tract has been well described. The surface of Peyer's patches is covered by epithelium associated with a variety of lymphoid cells known as the follicle-associated epithelium (FAE). The FAE is composed of a variety of cells including cells known as M cells. These cells exhibit slender cytoplasmic extensions around lymphoid cells. The basolateral surface of the M cell is deeply invaginated forming a pocket that shortens the distance from the apical to the basolateral surface. The pocket is rich in B cells, T cells, macrophages and dendritic cells. Antigen uptake by M cells does not result in intracellular degradation but rather delivery of the intact molecule to the underlying lymphoid tissue. The apical surface of the M cell lacks the brush border of typical gut lining enterocytes. Furthermore, the M cells are not coated with the thick glycocalyx found on enterocytes. Finally, the distribution of actin-associated protein villin in M cells differs from enterocytes. These characteristics make M cells ideal targets for absorption of proteins produced by recombinant commensal organisms needed for HIV replication.25
A variety of methods will target the M cells for absorption of defined proteins. These include, but are not limited to: (1) cholera toxin-B subunit, (2) carbohydrate lectins, (3) genetically engineered IgA or the secretory component of IgA. Splicing the genetic DNA sequence for a defined protein needed for HIV replication and linking that protein to 1, 2 or 3, above, will target the protein to the M cells and ultimately to the underlying immune tissue.26
Alternatively, attenuated viruses particularly the mouse reovirus, attenuated Poliovirus type 1 and the attenuated Sabin strain selectively adhere to M cells. These viruses can be exploited for transporting a defined protein into Peyer's patches.27
Certain attenuated bacteria also target the M cell apical membrane. These include Vibrio Cholerae, Salmonella, Shigella, Yersinia and BCG. Attenuation of these organisms renders them non-virulent. They can be exploited in targeting recombinant proteins to the M cells and the underlying immune tissue.28
As a final step, the described proteins are administered to the animal by way of its GALT followed by infection of live HIV. Infection with live HIV will result in Tat protein transcription and translation with the resulting Tat mediated immune suppression. Alternatively, Tat protein or the incorporation of the DNA encoding the Tat protein can be administered directly in combination with other proteins or incorporated into the commensal through recombinant technology described above.
It is possible for the proteins, composition of proteins and or compositions of incorporated DNA encoding the proteins to be administered as a pharmaceutical formulation or preparation, optionally with supplements or other compositions as described above. If protein carriers are used they must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The coupling of protein carriers (e.g., complement proteins) is known within pharmacology.
Administration may be made in a variety of routes, for example orally, transbucally, transmucosally, sublingually, nasally, rectally, vaginally, intraocularly, intramuscularly, intralymphatically, intravenously, subcutaneously, transdermally, intradermally, intra tumor, topically, transpulmonarily, by inhalation, by injection, or by implantation, etc. Various forms of the composition may include, without limitation, capsule, gel cap, tablet, enteric capsule, encapsulated particle, powder, suppository, injection, ointment, cream, implant, patch, liquid, inhalant, or spray, systemic, topical, or other oral media, solutions; suspensions, infusion, etc. Because some of the first targets for infection with HIV are epithelial cells and Langerhans cells in the skin and rectal mucosa, then a preferable embodiment of delivery is dermal combined with rectal suppositories.
Those skilled in the art will recognize that for administration by injection, formulation in aqueous solutions, such as Ringer's solution or a saline buffer may be appropriate. Liposomes, emulsions, and solvents are other examples of delivery vehicles. Oral administration would require carriers suitable for capsules, tablets, liquids, pills, etc, such as sucrose, cellulose, etc.
The preferred method of administration would be via commensal organisms genetically modified to express one or more human derived proteins needed for HIV replication. The preferred area of administration would be the intestines targeting Peyer's patches. The delivery and deliberate infection of live HIV is well known in the art and includes intra vaginal, rectal and systemic portals.
In conclusion, the present invention provides compositions and a method for producing an animal model for HIV induced disease. The present invention is an animal adapted to simulate a human-like immune response to HIV, which is accomplished by activation and inactivation of complement of proteins within the animal. Accordingly, the present invention stages certain human proteins within an animal by way of its GALT followed by infection of live HIV.
The analysis and development of the animal model for HIV induced disease should incorporate a wide range of doses of the proteins necessary for viral replication and immune evasion, deregulation and/or suppression for evaluation. Animal trials should consider differences in size, species, and immunological characteristics.
The above examples should be considered to be exemplary embodiments, and are in no way limiting of the present invention. Thus, while the description above refers to particular embodiments, it will be understood that many modifications may be made without departing from the spirit thereof.
Prokaryotic organisms lack the post translational modification machinery found in eukaryotic organisms. Yeast such as Saccharomyces cerevisiae are eukaryotes often found as commensal organisms in GALT. Yeast may therefore be preferable as recombinatorial vectors.
A blend of genetic manipulations may yield the optimal animal model. A mouse with one or the other above-mentioned amino acid substitutions in the Cyclin T protein that renders it Tat and TAR processive would be a good starting point. This murine model could then assimilate the CD4 receptor and the CCR5 and CXCR4 co-receptors by transgenic technology. Other proteins the mouse is lacking to affect HIV replication, immune evasion and immune disregulation could be supplied in trans via recombinatorial GALT vectors.
The following information is generally known by those in the art and can be found in medical texts generally including by way of example, Mucosal Vaccines, Hematology Basic Principles and Practices, and Immunology, Infection and Immunity and journals such as Immunologic Reviews, Nature, Virology, and Molecular Immunology.
The present application is a Continuation-in-Part of application Ser. No. 11/702,260 filed Feb. 5, 2007 which claims priority to Provisional Application No. 60/765,315, filed on Feb. 3, 2006, which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60765315 | Feb 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11702260 | Feb 2007 | US |
| Child | 12794102 | US |
