Patent Application
20120102582
The present invention relates, in general, to animal models suitable for testing candidate immunogens and, in particular, to knock-in mice expressing heavy and light chains of membrane proximal external region (MPER) HIV-1 broadly neutralizing antibodies and to methods of screening candidate immunogens using same.
The first antibodies that are made in acute HIV-1 infection are against the CD4 binding site (Moore et al, J. Virol. 68(8) 5142 (1994)), the CCR5 co-receptor binding site (Choe et al, Cell 114(2):161-170 (2003)), and the V3 loop (Moore et al, J. Acquit. Immun. Def. Syn. 7(4):332 (1994)). However, these antibodies do not control HIV-1 and are easily escaped (Burton et al, Nature Immun. 5:233-236 (2004), Wei et al, Nature 422(6929):307-312 (2003)). Neutralizing antibodies against autologous virus develop fifty to sixty days after infection, but antibodies capable of neutralizing heterologous HIV-1 strains do not arise until after the first year of infection (Richman et al, Proc. Natl. Acad. Sci. USA 100(7):4144-4149 (2003), Wei et al, Nature 422(6929):307-312 (2003)).
The four epitopes on HIV-1 envelope to which rare broadly reactive neutralizing antibodies bind are the CD4 binding site (CD4BS) (nab (monoclonal antibody) IgG1b12) (Zwick et al, J. Virol. 77(10):5863-5876 (2003)), the membrane proximal external region (MPER) epitopes defined by human mabs 2F5 and 4E10 (Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004), Stiegler and Katinger, J. Antimicrob. Chemother. 51:757-759 (2003), Zwick et al, Journal of Virology 79:1252-1261 (2005), Purtscher et al, AIDS 10:587 (1996)) (
A number of epitopes of the HIV-1 envelope have been shown to cross-react with host tissues (Pinto et al, AIDS Res. Hum. Retrov. 10:823-828 (1994), Douvas et al, AIDS Res, Hum. Retrov. 10:253-262 (1994), Douvas et al, AIDS Res. Hum. Retrov. 12:1509-1517 (1996)), and autoimmune patients have been shown to make antibodies that cross-react with HIV proteins (Pinto et al, AIDS Res. Hum. Retrov. 10:823-828 (1994), Douvas et al, AIDS Res, Hum. Retrov.
10:253-262 (1994), Douvas et al, AIDS Res, Hum. Retrov, 12:1509-1517 (1996), Barthel et al, Semin. Arthr. Rheum. 23:1-7 (1993)). Similarly, induction of immune responses to self-epitopes has been suggested to be a cause of the autoimmune abnormalities and T cell depletion in AIDS (Douvas et al, AIDS Res. Hum. Retrov. 12:1509-1517 (1996), Ziegler et al, Clin. Immunol. Immunopath. 41:305-313 (1986)).
The present invention results from studies designed to directly examine the role of B cell tolerance in regulating MPER-specific B cells and to determine the mechanisms involved/B cell subsets affected. The knock-in mouse models described herein can be used to yield genetic information on the spectrum of heavy and light chains within the MPER-specific B cell repertoire capable of conferring autoreactivity and/or neutralization activity. The disclosed mouse models can also be used to facilitate examination of lead candidate immunogens in eliciting MPER bnAbs, regardless of whether tolerance is involved or not.
In general, the present invention relates to animal models suitable for testing candidate immunogens. More specifically, the invention relates to knock-in mice expressing heavy and light chains of MPER HIV-1 broadly neutralizing antibodies. The invention further relates to methods of screening candidate immunogens using such mice.
Objects and advantages of the present invention will be clear from the description that follows.
The present invention relates to knock-in animal (e.g., mouse) models expressing MPER HIV-1 broadly neutralizing antibodies and to methods of screening lead candidate immunogens using same. The invention results from the construction of a series of knock-in mouse lines expressing the heavy and light chains of two broadly neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies (2F5 and 4E10) at their endogenous immunoglobulin loci (see
Thus, in one embodiment, the present invention relates to a targeted transgenic mouse, the genome of which comprises a nucleic acid sequence encoding a heavy and/or a light chain variable region of a human HIV-1 broadly neutralizing antibody. In accordance with the invention, the nucleic acid sequence can be present in the genome operably linked to a promoter so that the nucleic acid sequence is expressed and the heavy and/or light chain variable region of the human HIV-1 broadly neutralizing antibody (e.g., 2F5 or 4E10) is produced. Advantageously, the nucleic acid sequence is present in the genome operably linked to an endogenous enhancer element
In a preferred embodiment, the nucleic acid sequence encoding the heavy chain variable region of the human HIV-1 broadly neutralizing antibody is operably liked to a J558 1110 family VH promoter (Love et al, Mol. Immunol. 37:29-39 (2000)). In another preferred embodiment, the nucleic acid sequence encoding the light chain variable region of the human HIV-1 broadly neutralizing antibody is operably liked to a VkOx-1 family Vkappa promoter (Sharpe et al, EMBO 10(8):2139-2145 (1991)).
The invention further relates to chimeric HIV-1 broadly neutralizing antibodies isolatable from the above-described targeted transgenic mouse, particularly, a mouse that contains in its genome both a nucleic acid sequence encoding a heavy chain variable region of a human HIV-1 broadly neutralizing antibody and a nucleic acid sequence encoding a light chain variable region of a human HIV-1 broadly neutralizing antibody. The invention also relates to hybridomas derived by fusing antibody-producing B cells of the above-described mouse with myeloma cells using, for example, standard techniques. The invention includes monoclonal antibodies produced by such hybridomas.
In yet another embodiment, the invention relates to a method of identifying a candidate agent capable of inducing the production of HIV-1 broadly neutralizing antibodies. The method can comprise: i) administering to the above-described mouse a test compound (e.g., a compound comprising a protein or peptide) under conditions such that antibodies can be produced or such that B cells can be induced to express antibodies, ii) obtaining an antibody-containing sample or an antibody expressing, B cell-containing sample from the mouse, and iii) assaying the sample for the presence or absence of antibodies specific for the HIV-1 membrane proximal external region (MPER), or MPER-specific B cells (e.g., using an ELISA, ELISPOT, Surface Plasmon Resonance, Luminex or flow cytometry-based assay). The presence of the MPER-specific antibodies or B cells in the sample, relative to a control sample (e.g., a non-treated mouse), indicates that the test compound is a candidate agent. The antibody-containing sample or the antibody expressing, B cell-containing sample can be a serum sample or a sample of a mucosal extract (e.g., a saliva, stool or vaginal wash sample). The B-cell containing sample can be a sample obtained from a systemic or mucosal immune tissue of the mouse. For example, the sample can be a bone marrow, spleen or peripheral blood lymphocyte sample. The sample can also be an enteric lymph node or Peyer's patch sample or a female reproductive tract sample or a lung sample. A candidate agent identified using the method of the invention can be assayed for HIV-1 neutralizing activity, for example, using TZM-bl assay (see, for example, Polonis et al, virol. 375(2) 315 (2008)).
The invention further relates to a method of identifying an agent capable of inducing the production of HIV-1 broadly neutralizing antibodies that comprises: i) administering to the above-described mouse a test compound (e.g., a proteinaceous compound) under conditions such that antibodies can be produced or such that B cells can be induced to express antibodies, ii) obtaining an antibody-containing sample or an antibody expressing, B cell-containing sample from the mouse, and iii) assaying the sample for HIV-1 neutralizing activity, relative to a control sample (e.g., from a non-treated mouse). As above step (iii) can be effected using, for example, a TZM-bl assay.
The data provided in the Examples below demonstrate that both heterozygous and homozygous versions of 2F5 VH knock-in mice have been characterized. Both exhibit a major blockade in bone marrow B cell development at the pre-B to mature B cell transition, indicating that the developmental block cannot be due to a defect in transgene expression but, rather, must involve an active process involving encounter of the 2F5 HC with self-antigens. The ability of the 2F5 VH-containing heavy chain to pair with multiple endogenous mouse light chain partners in vivo is demonstrated in Example 2 (see hybridoma analysis in Table 6), and strongly argues against the developmental block being due to improper association with mouse light chains. Despite diminished total numbers of mature splenic B cells with lower surface density, there are normal ratios of mature B cells in these mice, indicating that a small percentage of cells escape initial deletion in the bone marrow. Importantly, this shows that the developmental block is profound, but not complete, and residual cells have the potential to be rescued by a variety of strategies. In serum from heterozygous and homozygous 2F5 VH knock-in mice, there are substantial levels of total serum Igs, but which lack reactivity to the MPER epitope or to human/murine self antigens. The ability of these mice to produce normal Ig levels shows that the lack of reactivity is not due to improper expression of the transgene but rather must be due to elimination of this reactivity by various tolerance mechanisms. In vitro, the 2F5 VH insertion can produce chimeric human/mouse 2F5 antibodies that are functionally similar to the original human 2F5 antibody, including comparable reactivity to the MPER epitope+human/murine self-antigens, and the ability to neutralize HIV-1. This demonstrates that the lack of serum Ig reactivity in these mice is also not due to the chimeric nature of the 2F5 HC.
Furthermore, other knock-in models can be readily genetically manipulated to remove the above-described counterselection pressures (
Certain aspects of the invention can be described in greater detail in the non-limiting Examples that follows. (See also U.S. Provisional Application No. 61/166,625, filed Apr. 3, 2009 and U.S. Provisional Application No. 61/166,648, filed Apr. 3, 2009.)
Two strategies aimed at testing MPER lead candidate immunogens in 2F5 knock-in mice in the absence of negative selection pressure are set forth in
Expression/characterization of m2F5 and generation of 2F5 VH mice. The methods and reagents used to generate m2F5 and the binding, immunofluorescence, and neutralization assays used to characterize its functional properties are described in Example 3, as are the reagents and methods used for the site-directed targeting of 2F5 VH into the mouse Igh locus.
Mice and flow cytometry. Female C57BL/6 and C57BL/6 Igha, inbred mouse strains (8-12 wks of age) were purchased from Charles River Laboratories. 3H9 mice, originally produced in the laboratory of Dr. Martin Weigert on a BALB/c background, were backcrossed onto the C57BL/6 background for >14 generations in the laboratory of Dr. Robert Eisenberg (University of Pennsylvania, Philadelphia, Pa.).
For flow cytometric analysis, BM cells and splenocytes were isolated from 9-12 is week old female mice. Total BM B-cells (gated as singlet, live, CD19+, lin− lymphocytes; lin=Ter 119, Gr-1, CD11b, CD4, CD8) were stained with APC anti-B220 and PE anti-CD43 antibodies or FITC anti-IgD and PE anti-IgM antibodies; singlet, live, lymphocyte-gated splenocytes were stained using the combination of FITC anti-B220, PE anti-IgM, APC anti-CD93, and PE-Cy7 anti-CD23 antibodies. Data were acquired using a BD LSRII flow cytometer equipped with FACS Diva software and analyzed using FloJo software.
Allotype screening. 2F5 VH IgHb/WT IgHa and WT IgHb/WT IgHa F1 mice were generated by breeding C57BL/6 Igha congenic mice with 2F5 VH+/− mice and WT littermate controls, respectively. BM cells and splenocytes from 8-16 week old female F1 mice from each group were surface stained with PE-IgMa and FITC-IgMb antibodies, distinguishing targeted 2F5 VH μHCs bearing the allotype of the targeted IgH allele (IgMb) from endogenous μHCs bearing the IgMb allotype.
ELISA analyses. Serum samples were collected from naïve female WT, 2F5 VH+/−, and 2F5 VH+/+, and where applicable, MRL/lpr mice. Serum concentrations of total IgG and IgM were determined using quantitative mouse IgG and IgM ELISA kits, respectively (Bethyl). ELISA measurements of cardiolipin and gp41 MPER 2F5 reactivity of total (IgM+IgG-specific) Igs was determined by optical density readings, as previously described (Haynes et al, Science 308:1906-1908 (2005), Alam et al, J. Immunol. 178:4424-4435 (2007)), and serum reactivity of total Igs to nuclear auto-antigens was determined using a mouse anti-ANA quantitative ELISA kit (Alpha Diagnostics). Cardiolipin and ANA assays were done using serum from 12-32 weeks; all other assays were done using serum from 8-16 week old mice.
The human 2F5 VDJ rearrangement forms functional chimeric antibodies with mouse CH. An in vitro test was first made to determine whether mouse C regions impacted the association and binding properties of the original human IgG1 2F5 mAb (herein referred to as h2F5). To do this, 2F5 VH/mouse Cγ1 and 2F5 VL/mouse Cκ expression constructs were generated and co-transfected into 293T cells. The 2F5 chimeric mouse/human recombinant antibody (m2F5) was assessed for its ability to bind lipid and mouse and human cell antigens. Indeed, m2F5 bound both gp41 and lipids comparably to the human IgG1 2F5 mAb (h2F5;
Also assessed was the ability of chimeric 2F5 HCs to pair with mouse K light chains (LC) in vitro by co-transfection of the 2F5VH/mouse Cγ1 expression construct with mouse κ LCs obtained from C57BL/6 splenic B cells by 5′ RACE PCR. To do this, four mouse κ LCs were arbitrarily selected to include the 4-52, 4-60, 4-70, and 9-96 V genes representing two Vκ families (Vκ4 and Vκ9) frequently-utilized in the splenic C57BL/6 LC repertoire. In each case, co-transfections of the m2F5 HC resulted in the production of secreted, functional mAbs (Table 2). Significantly, of the four chimeric recombinant antibodies generated by these transfections, three exhibited cardiolipin polyreactivity as determined by surface plasmon resonance and ELISA (
†LC containing 2F5 V-1 rearrangement fused to mouse κLC constant region.
Generation of 2F5 VH knock-in mice. To determine if the 2F5 mAb HC was sufficiently autoreactive to be regulated by immunological tolerance, the original, somatically-mutated 2F5 VHDJH rearrangement (Muster et al, J. Virol. 68:4031-4034 (1994), Muster et al, J. Virol. 67:6642-6647 (1993)) was knocked into the mouse igh locus, replacing the JH1-4 region (
The majority of B cells expressing 2F5 VH are deleted in the BM at the pre-B to immature B-cell stage. To examine the effect of the targeted 2F5 VDJ insert at one or both Igh alleles on B-cell development, a comparison was made of B-cell ontogeny in BM of heterozygous (2F5 VH+/−) and homozygous (2F5 VH+/+) knock-in mice with that of C57BL/6 controls. Fractionation of total BM B-cells from 2F5 VH+/− and 2F5 VH+/+ mice into pro-B/large pre-B (B220loCD43+), small pre-B (B220loCD43−), and immature/mature B (B220hiCD43−) fractions (Hardy et al, J. Exp. Med. 173:1213-1225 (1991)) demonstrated a profound reduction in surface immunoglobulin (sIg+) B-cell subsets (B220hiCD43−), both in frequency (˜4-fold for both 2F5 VH+/− and 2F5 VH+/+ mice;
2F5 VH+/− knock-in splenic B cells preferentially express endogenous heavy chains. It was suspected that if 2F5 HC-expressing B-cells it heterozygote 2F5 VH mice escaped BM deletion, they should be counter-selected in favor of B cells expressing endogenous Igh rearrangements, as in 3119-76R mice (Chen et al, Nature 373:252-255 (1995)). Thus, an examination was made of surface expression of endogenous (IgMa) relative to 2F5 VH-targeted (IgMb) alleles in 2F5 VH+/− IgMaIgMb F1 (F1) mice. Indeed, most IgM+ splenocytes from 2F5 F1 mice expressed surface IgMa, indicating strong selection for the endogenous HC (
2F5 VH knock-in mice have severely diminished numbers of mature splenic B-cell populations with low surface Ig density. Selection against 2F5 Ig HC+ BM B-cells should lead to diminished numbers of peripheral B-cells. Indeed, compared to littermate controls, the numbers of splenic B-cells (B220+CD19+lin−, live-gated) in 2F5 VH+/− and 2F5 VH+/+ mice were reduced by 72% and 86%, respectively (Table 5). To determine if transitional, MZ, and mature B-cell subset frequencies within this remnant splenic B cell population were altered, 2F5 VH B220+ B-cells were stained with antibodies specific for CD23, CD93, and IgM (Allman et al, J. Immunol. 167:6834-6840 (2001)). Interestingly, within this residual B cell population, the frequency of transitional IgMlo (T3) B-cells was little changed, but transitional IgMhi (T1 and T2) subset frequencies were significantly reduced relative to normal controls in both 2F5 VH+/− and 2F5 VH+/+ mice (
2F5 VH knock-in mice lack serum reactivity to cardiolipin and anti-nuclear autoantigens despite having substantial levels of serum IgG. 2F5 VH+/− and 2F5 NTH+/+ mice exhibited normal to elevated serum IgG levels relative to normal controls, respectively, but 2F5 VH+/+ mice alone expressed significantly lower levels of serum IgM (
In summary, the development of a safe and effective HIV-1 vaccine has been blocked by the inability to design HIV-1 immunogens that induce antibodies that potently neutralize diverse HIV-1 strains. While the HIV-1 Env has conserved regions to which rare, broadly neutralizing human antibodies bind, either on immunogens or in the context of natural infections, these conserved regions only rarely induce broadly neutralizing antibodies (Burton et al, Proc. Natl. Acad. Sci. USA 102:14943-14948 (2005), Haynes and Montefiori, Expert Rev. Vaccines 5:579-595 (2006), Simek et al, J. Virol. 83:7337-7348 (2009)). Moreover, even on the rare occasions that broadly neutralizing antibodies are induced by HIV-1 infection, they only arise months after infection (Shen et al, J. Virol. 83:3617-3625 (2009)).
That the 2F5 VH knock-in mouse shows a profound block in expression of the 2F5 VH at the immature B cell stage demonstrates that the 2F5 VH is sufficiently autoreactive to invoke tolerance control of 2F5 VH expression, and supports the notion that expression of this specificity is regulated by tolerance mechanisms in vivo. In this regard, many of the broadly neutralizing antibodies such as mAbs 4E10 and 1B12 share some characteristics of the 2F5 HC, including long hydrophobic CDR3s and polyreactivity, characteristics previously associated with antibodies marked for deletion in human BM (Meffre et al, J. Clin. Invest. 108:879-886 (2001)).
It is possible that the profound block in B cells expressing 2F5 VH-containing HCs may be enhanced by incomplete and/or inefficient pairing of chimeric 2F5 VH/mouse CH HCs with endogenous mouse LCs. The data provided herein, however, do not support this possibility. First, the relatively normal pre-B compartment in 2F5 VH knock-in mice (comparable to that in the 3H9 knock-in mouse; Tables 4-7) is most consistent with the ability of the 2F μHC to associate efficiently with surrogate LC and support continued differentiation to the immature B-cell stage, although this could also be due to compensation of an earlier, partial pre-B cell defect by autoreactive, immature B cells arrested at the pre-B cell stage. Either possibility, however, is consistent with the ability of the 2F5 μHC to form signaling-competent BCR and/or pre-BCR complexes rather than pairing incompatibility. Second, the co-transfection of the m2F5 HC with four distinct mouse LCs produced functional recombinant antibodies that reacted with self-antigens (
a,b,cGroups having different letters differ at the 0.05 level, as determined by a Student's t test.
The peripheral phenotype in 2F5 VH+/+ mice is consistent with additional mechanisms for controlling autoreactivity in residual splenic 2F5 VH-bearing B cells that have escaped central tolerance. In particular, the relative enrichment for the T3 IgMlo population in 2F5 VH+/+ mice (
Mice bearing conventional or targeted autoreactive Ig transgenes have been critical in defining the developmental stages in which self-reactive B cells are eliminated (Shlomchik, Immunity 28:18-28 (2008)). The 2F5 VDJ knock-in mouse line demonstrates that the great majority of B-lineage cells that express the 2F5 VDJ rearrangement are halted in their development at the transition from small pre-B to immature B-cells (
Both the 2F5 and 4E10 mAbs bind to the gp41 membrane proximal region on HIV-1 virions as well as to the lipid bilayer (Alain et al, J. Immunol. 178:4424-4435 (2007)). Mutation of hydrophobic residues in the 2F5 HC CDR3 abrogates both lipid binding and neutralization of HIV-1 (Alam et al, Proc. Natl. Acad. Sci. USA epub (Nov. 11, 2009)). The induction of neutralizing antibodies specific for this region will likely require the targeting of B-cell populations that can make antibodies that bind both lipids and gp41 Env epitopes. This requirement may be facilitated by the activation of dendritic cells or other antigen presenting cells capable of promoting vaccine-induced B-cell responses that normally do not occur. The 2F5 mAb has been safely administered to a number of humans and 2F5 does not have characteristics of a pathogenic lipid autoantibody (i.e., it does not require (3-2-glycoprotein-1 to bind to lipids) (haynes et al, Science 308:1906-1908 (2005), de Groot and Derksen, Thromb. Haemost 3:1854-1860 (2005), Vcelar et al, AIDS 21:2161-2170 (2007)). However, if these antibodies can be induced, safety monitoring in non-human primate trials will be of paramount importance.
These studies demonstrate that the HIV-1 broadly neutralizing antibody 2F5-containing MC is sufficiently autoreactive to trigger immunological tolerance in the setting of a knock-in mouse. These findings have important implications for the design of strategies to induce neutralizing antibodies to the HIV-1 Env gp41 membrane proximal region. HIV-1 vaccine development should focus on vaccine regimens that might safely circumvent these tolerance controls. Moreover, efforts should concentrate on accelerating and broadening those neutralizing antibody responses that are readily made in response to HIV-1, such as autologous neutralizing antibodies that arise months after natural HIV-1 infection (Richnab et akm Uric, Batk, /acad, /scu, YSA 100:4144-4149 (2003), Wei et al, Nature 422:307-312 (2003)).
Generation and characterization of m2F5. To make m2F5, human V+mouse C constructs in which the original 2F5 VH region, ligated to mouse Cγ1 (m2F5 HC) and the original 2F5 Vκ region, fused to mouse Cκ (m2F5 LC), were cloned into the pcDNA 3.1 expression vector, co-expressed in 293T cells by transient transfection, and the resulting recombinant antibody (m2F5) was purified by standard methods. For SPR binding measurements, biotinylated MPER peptides were anchored to streptavidin sensor chip and non-specific binding to scrambled version of the MPER peptide was subtracted. Liposomes with the indicated phospholipid compositions were prepared and 500 RU of each liposomes was anchored to the L1 sensor chip as described earlier (Alam et al, J. Immunol. 178:4424-4435 (2007)). Each antibody was injected at 100 μg/mL and non-specific binding of antibodies was assessed on liposomes with phosphatidylcholine. For detection of m2F5 and h2F5 reactivity with mouse nuclear antigens, NIH-3T3 cells were grown under standard conditions on glass slides and subsequently fixed and permeabilized (Wardernann et al, Science 301:1374-1377 (2003)). Fixed cells were then incubated in medium containing 100 μg/ml m2F5 or h2F5, and bound antibodies were visualized with goat anti-mouse Igκ or goat anti-human IgG-FITC, respectively, using a Zeiss Axiovert 200M confocal immunofluorescence microscope (50 ms exposure). For detection of m2F5 and h2F5 reactivity with human nuclear antigens, HEp-2 epithelial cell slides (Zeus scientific, Raritan, N.J.) were incubated with 100 μg/ml of m2F5 and h2F5 antibodies, followed by saturating amounts of goat anti-mouse Igκ (or goat anti-human Ig, and visualized as above. Human mAb 17b, a non-autoreactive CD4i gp120 mAb, was used as a negative control for background staining. The chimeric m2F5 antibody and the h2F5 antibody were tested in the standard TZMB/L pseudovirus infection inhibition assay with the Env HIV-1 pseudoviruses B.BG1168, B.SF162, B.QH0692, A.92UG037, and C.TV-1.
Generation of 2F5 VH Mice: The targeting vector contained the rearranged 2F5 VH gene inserted within the joining (JH) region of the immunoglobulin heavy chain, disrupting all endogenous JH segments. The murine immunoglobulin JH region and the upstream and downstream regions from JH, used to generate the 3′ and 5′ homology mills, were isolated from a mouse C57BL/6 genomic library-derived BAC clone. The targeting backbone contained CAG-DTA and loxP-flanked Neo selection cassettes. Homologous recombination of ES cells was confirmed by Southern blotting using Nde I or Bam HI. Targeted ES clones were subjected to in vitro Crc recombinase-mediated deletion of the neo selection cassette and four correctly targeted, neo− clones were injected into C57BL/6J Tyrc-2J blastocysts, two of which produced chimeric mice that transmitted the 2F5 VH insertion. 2F5 VH+/− and 2F5 VH+/+ genotypes were determined in the offspring by PCR primers specific for WT or targeted alleles and a primer common to both alleles (see
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
This application claims priority from U.S. Provisional Application No. 61/202,778, filed Apr. 3, 2009, the entire content of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2010/030011 | 4/5/2010 | WO | 00 | 12/27/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61202778 | Apr 2009 | US |
