Patent Grant
10076567
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 1, 2016, is named 1234300_00156US2_SL.txt and is 36,232 bytes in size.
The present invention relates in general, to a formulation suitable for use in inducing anti-HIV-1 antibodies, and, in particular, to a formulation comprising Toll Like Receptor (TLR) agonists with HIV-1 gp41 membrane proximal external region (MPER) peptide-liposome conjugates for induction of broadly reactive anti-HIV-1 antibodies. The invention also relates to methods of inducing neutralizing anti-HIV-1 antibodies using such formulations.
One of the major challenges to HIV-1 vaccine development has been the inability of immunogens to induce broadly neutralizing antibodies (nAb). nAbs are generated during HIV-1 infection. However, most of the nAbs generated neutralize only the autologous viruses or closely related strains (Moog et al, J. Virol. 71:3734-3741 (1997), Gray et al, J. Virol. 81:6187-6196 (2007)). HIV envelope (Env) constantly mutates to escape from existing nAb response (Albert et al, Aids 4:107-112 (1990), Wei et al, Nature 422:307-312) (2003)). nAb responses do evolve over the course of the HIV infection. However, with the mutation capacity of HIV-1 viruses, neutralizing antibody responses always seem to “lag behind” virus evolution (Wei et al, Nature 422:307-312 (2003)), Richman et al, Proc. Natl. Acad. Sci. USA 100:4144-4149 (2003), Geffin et al, Virology 310:207-215 (2003)).
After extensive research, a handful of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1 have been identified (Buchacher et al, AIDS Res. Hum. Retroviruses 10:359-369 (1994), Zwick et al, J. Virol. 75:10892-10895 (2001), Burton et al, Proc. Natl. Acad. Sci. USA 888:10134-10137 (1991)). Two such antibodies, 2F5 and 4E10, target the conserved membrane-proximal external region (MPER) of HIV-1, have a broad spectrum of neutralization (Binley et al, J. Virol. 78:13232-13252 (2004)), and have been shown to neutralize 80% and 100% of newly transmitted viruses (Mehandru et al, J. Virol. 78:14039-14042 (2004)), respectively. When passively administered in combination with several other broadly neutralizing monoclonal antibodies, a cocktail of mAbs composed of 2G12, 2F5 and 4E10 successfully protected the host from virus infection in animal models (Baba et al, Nat. Med. 6:200-206 (2000), Ferrantelli et al, J. Infect. Dis. 189:2167-2173 (2004), Mascola et al, Nat. Med. 6:207-210 (2000), Ruprecht et al, Vaccine 21:3370-3373 (2003)), or delayed virus rebound after cessation of antiretroviral therapy (Trkola et al, Nat. Med. 11:615-622 (2005)).
The potential of using 2F5 and 4E10 to prevent HIV infection is greatly compromised by the fact that HIV infected patients rarely develop these antibodies spontaneously (Dhillon et al, J. Virol. 81:6548-6562 (2007)), and there has been no success in inducing 2F5- and 4E10-like antibodies by vaccination (Kim et al, Vaccine 25:5102-5114 (2006), Coeffier et al, Vaccine 19:684-693 (2000), Joyce et al, J. Biol. Chem. 277:45811-45820 (2002), Ho et al, Vaccine 23:1559-1573 (2005), Zhang et al, Immunobiology 210:639-645 (2005)). Identification of subjects that develop 2F5- or 4E10-like antibodies during natural HIV-1 infection, and developing an understanding of the mechanism of, or hindrance to, these broadly neutralizing antibodies is important for AIDS vaccine design.
The present invention results, at least in part, from the identification and characterization of a rare Env mutation in the HIV-1 MPER region which is associated with an increase in neutralization sensitivity to 2F5 and 4E10 mAbs. The invention also results from the development of constructs that can modulate B cell tolerance and enhance antibody responses against poorly immunogenic HIV-1gp41MPER epitopes.
In general, the present invention relates to a formulation suitable for use in inducing anti-HIV-1 antibodies. More specifically, the invention relates to a formulation comprising TLR agonists with HIV-1 gp41MPER peptide-liposome conjugates, and to methods of inducing broadly reactive neutralizing anti-HIV-1 antibodies using same.
In certain aspects the invention provides MPER peptides, including but not limited to MPER peptides with carboxy-terminal anchor, for example but not limited to a trans-membrane protein, and MPER-liposome conjugates. In certain embodiments, the MPER peptides of the invention bind 2F5 and 4E10 mAbs. In certain embodiments, compositions comprising these MPER peptides are expected to be immunogenic and provide a global population coverage in HIV-1 vaccination. In non-limiting embodiments, the immunogenic composition and vaccination methods comprise a combination of MPER peptide-liposomes as described herein.
Additional objects and advantages of the present invention will be clear from the description that follows.
In certain aspects the invention provides a composition comprising a peptide-liposome conjugate, wherein the peptide comprises a membrane proximal external region (MPER) epitope and a transmembrane anchor/linker. In certain embodiments, the linker is amphiphatic. In certain embodiments, the peptide is embedded in the liposome via the GTH linker. In certain embodiments, the composition comprises an adjuvant. In some embodiments, the adjuvant is alum. In other embodiments, the adjuvant is a TLR ligand. In some embodiments, the TLR4 adjuvant is Monophosphoryl Lipid A (MPLA) or synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA). In certain aspects the invention provides a peptide-liposome conjugate comprising a TLR ligand, wherein the peptide comprises a membrane proximal external region (MPER) epitope and a transmembrane anchor/linker. Without being bound by theory, the anchor/linker is required for the 4E10 epitope region to adopt the proper helical structure, leading to antigen presentation. See Dennison et al. Journal of Virol. 83(19): 10211-10-223 (2009). The anchor/linker provides liposome anchorage of the MPER peptide, which anchorage induces helical structure of MPER peptides, resulting in enhanced binding of mAb 4E10. In non-limiting embodiments, the anchor/linker is a transmembrane domain, or an amphiphatic alpha-helix peptide. In certain embodiments, the peptide is synthetic or recombinantly produced. In certain embodiments, the peptide comprises two MPER epitopes.
In certain embodiments, the MPEP epitope is any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 18, 19, 20, 21, 22, 23, 24, 25, or any combination thereof. In certain embodiments, the peptide comprises or has SEQ ID NO: 9 (NEQELLELDKWASLWNWFNITNWLWYIK), SEQ ID NO: 10 (NEQDLLALDKWASLWNWFDISNWLWYIK), SEQ ID NO: 11 (NEKDLLALDSWKNLWNWFSITKWLWYIK), SEQ ID NO: 12 (NEQELLALDKWNNLWSWFDITNWLWYIR), or SEQ ID NO: 26 (CAP206_0moB5_MPER656 of the sequence NEKDLLALDSWKNLWNWFDITKWLWYIK). In certain embodiments, the peptide and compositions of the invention does not comprise the MPER epitopes of SEQ ID NOs: 1, 18, 2 and/or 22.
In certain embodiments, the transmembrane anchor/linker is at the carboxy-terminal of the peptide. In certain embodiments, the transmembrane anchor/linker is alpha-helical HIV-1 p24 gag sequence GTH1 (SEQ ID NO: 17 YKRWIILGLNKIVRMYS). In certain embodiments the anchor/linker is TMD peptide.
In certain embodiments, the peptide comprises or has SEQ ID NO: 13, 14, 15, or 16.
In certain embodiments, the TLR is a TLR 9 ligand. In certain embodiments, the TLR 9 ligand is oligo CpG. In certain embodiments, the TLR ligand is a TLR 7/8 ligand. In certain embodiments, wherein the TLR 7/8 ligand is R-848. In certain embodiments, the TLR ligand is a TLR 4 ligand. In certain embodiments, the TLR 4 ligand is monophosphorylipid A.
In certain embodiments, the conjugate comprises a TLR 9 ligand and a TLR 7/8 ligand. In certain embodiments, the TLR 9 ligand is oligo CpG and the TLR 7/8 ligand is R-848. In certain embodiments, the conjugate comprises a TLR 9 ligand and a TLR 4 ligand. In certain embodiments, the TLR 9 ligand is oligo CpG and the TLR 4 ligand is R-848. In certain embodiments, the conjugate further comprises interferon-.alpha. encapsulated therewithin.
In certain aspects, the invention provides a pharmaceutical composition comprising a synthetic peptide-liposome conjugate as described herein, or any combination thereof. In certain embodiments, the compositions of the invention are immunogenic. In certain embodiments the compositions comprise an adjuvant.
In certain embodiments, the composition further comprises a synthetic peptide-liposome complex comprising a peptide of SEQ ID NO: 9 or 13. In certain embodiments, the composition comprises the peptides of SEQ ID NOs: 9, and 10. In certain embodiments, the composition comprises the peptides of SEQ ID NOs: 9, 10 and 11. In certain embodiments, the composition comprises the peptides of SEQ ID NOs: 9, 10, 11 and 12. In certain embodiments, the composition comprises the peptides of SEQ ID NOs: 13 and 14. In certain embodiments, the composition comprises the peptides of SEQ ID NOs; 13, 14, and 15. In certain embodiments, the composition comprises the peptides of SEQ ID NOs; 13, 14, 15 and 16. In certain embodiments, the compositions of the invention consist essentially of any one of or any combination of the MPER liposomes described herein. In certain embodiments, the compositions of the invention are immunogenic.
In certain aspects, the invention provides a method of inducing an immune response in a subject, the method comprising administering to the subject any one of the inventive compositions in an amount sufficient to induce the immune response, wherein in certain embodiments the response comprises induction of anti-HIV-1 antibody. In certain aspects, the invention provides a method of inducing an anti-HIV-1 antibody in a subject, the method comprising administering to the subject any one of the inventive compositions in an amount sufficient to induce the anti-HIV-1 antibody. In certain embodiments of the methods, the composition is administered as a prime or/and a boost. In certain aspects, the composition is administered as a prime followed by the same or a different composition as a boost. In certain embodiments, the composition is administered as a boost following a prime of another suitable antigen.
In certain embodiments of the methods, the antibody binds an epitope comprised in the peptides of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 26, 27 or any combination thereof. In certain embodiments, the antibody does not bind SEQ ID NO: 9 or 13. In certain embodiments, if the antibody binds SEQ ID NO: 9 and/or 13, then the antibody also binds to SEQ ID NOs: 10, 11, 12, 14, 15, and/or 16.
In certain embodiments of the methods, the antibody binds a nominal epitope of SEQ ID NOs: 1, 3, 5, 7, 18, 19, 20, 21 or any combination thereof. In certain embodiments, the antibody does not bind SEQ ID NO: 1, 18 and/or 5. In certain embodiments, the antibody binds a nominal epitope of SEQ ID NO: 1, 3, 7, 18, 19, 20, and/or 21.
In certain embodiments of the methods, the antibody binds a nominal epitope of SEQ ID NOs: 2, 4, 6, 8, 22-25 or any combination thereof. In certain embodiments, the antibody does not bind SEQ ID NO: 2 or 22. In certain embodiments, the antibody binds a nominal epitope of SEQ ID NO: 2, 4, 6, 8, 22-25 or any combination thereof. In certain embodiments, if the antibody binds SEQ ID NO: 2 or 22, it also binds to SEQ ID NO: 4, 6, 8, 23-25, or any combination thereof.
In certain aspects, the invention provides a synthetic peptide comprising SEQ ID NO: 3, 5, 7, 4, 6, 8, 18-24, or any combination thereof. In certain aspects, the invention provides a synthetic peptide comprising SEQ ID NO: 9, 11, or 12. In certain aspects, the invention provides a synthetic peptide comprising SEQ ID NO: 14, 15, or 16. Peptide length can be from 28-45, 28-50, 28-55, 28-60, 26-65, 28-70, 28-75, 28-80, 28-85, 28-90, 28-100, 28-110, 28-120, 28-130, 28-140, 28-150, or 45-50, 45-55, 45-60, 26-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-100, 45-110, 45-120, 45-130, 45-140, 45-150 amino acids long.
In certain embodiments, the liposome comprises a viral membrane lipid or an anionic lipid. In certain embodiments, the anionic lipid is phosphatidylserine. In certain embodiments, the anionic lipid is phosphatidic acid. In certain embodiments, the anionic lipid is cardiolipin.
In certain embodiments, the viral membrane lipid is sphingolipid or cholesterol.
In certain embodiments, the liposome comprises POPC (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine), POPE (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine DMPA (1,2-Dimyristoyl-sn-Glycero-3-Phosphate), Cholesterol, or any combination thereof.
In certain embodiments, the liposome includes POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS). In certain embodiments, the liposome includes POPC and POPS and lysophosphorylcholine (lysoPC). In certain embodiments, the liposome includes POPC and POPE and Sphingomyelin and Cholesterol. In certain embodiments, the liposome includes POPC, POPE, DMPA, Cholesterol, or any combination thereof. In certain embodiments, the liposome includes DOPC and cholesterol. In certain embodiments, the liposome includes DPPC, DPPG and cholesterol.
In certain embodiments, the peptide embedded in the liposome binds an MPER neutralizing antibody or a Fab fragment thereof in a two-step binding model. In certain embodiments, the MPER neutralizing antibody is 2F5 or 410E. In certain embodiments, the liposome presents the peptide comprising the MPER epitope in a native HIV-1 membrane environment.
In certain embodiments, the liposome comprises POPC. In certain embodiments, the liposome comprises POPE. In certain embodiments, the liposome comprises DMPE. In certain embodiments, the liposome comprises DMPE. In certain embodiments, the liposome comprises cholesterol. In certain embodiments, the liposome comprises POPC (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine), POPE (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine), DMPA (1,2-Dimyristoyl-sn-Glycero-3-Phosphate), and Cholesterol.
The present invention relates to a liposome-based adjuvant conjugate that presents TLR ligands and HIV-1 gp41 neutralizing antigens, and to a method of inducing neutralizing anti-HIV-1 antibodies in a subject (e.g., a human subject) using same. Suitable neutralizing antigens include gp41MPER epitope peptides (Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004), Stiegler and Katinger, J. Antimicrob. Chemother. 512:757-759 (2003), Zwick et al, Journal of Virology 79:1252-1261 (2005), Purtscher et al, AIDS10:587 (1996)) and variants thereof, for example, variants that confer higher neutralization sensitivity to MPER Mabs 2F5 and 4E10. In certain embodiments, the variant is a MPER epitope peptide with an L669S mutation that confers higher neutralization sensitivity to MPER mAbs 2F5 and 4E10 (Shen et al, J. Virology 83: 3617-25 (2009)).
Liposomes suitable for use in the invention include, but are not limited to, those comprising POPC, POPE, DMPA (or sphingomyelin (SM)), DMPE, lysophosphorylcholine, phosphatidylserine, cholesterol (Ch), or any combination thereof. While optimum ratios can be determined by one skilled in the art, examples include POPC:POPE (or POPS):SM:Ch or POPC:POPE (or POPS):DMPA:Ch at ratios of 45:25:20:10. Alternative formulations of liposomes that can be used include DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) (or lysophosphorylcholine), cholesterol (Ch) and DMPG (1,2-dimyristoyl-sn-glycero-3-phoshpho-rac-(1-glycerol) formulated at a molar ratio of 9:7.5:1 (Wassef et al, ImmunoMethods 4:217-222 (1994); Alving et al, G. Gregoriadis (ed.), Liposome technology 2nd ed., vol. III CRC Press, Inc., Boca Raton, Fla. (1993); Richards et al, Infect. Immun. 66(6):285902865 (1998)). The above-described lipid compositions can be complexed with lipid A and used as an immunogen to induce antibody responses against phospholipids (Schuster et al, J. Immunol. 122:900-905 (1979)). In certain embodiments, a formulation comprises POPC:POPS:Ch at ratios of 60:30:10 complexed with lipid A according to Schuster et al, J. Immunol. 122:900-905 (1979). In certain embodiments, the liposomes can be pegylated. In these embodiments the liposomes comprise DMPE.
The liposomes can comprise the following phospholipids, POPC (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine), POPE (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine), DMPA (1,2-Dimyristoyl-sn-Glycero-3-Phosphate), DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphatidylethanolamine) Cholesterol, or any combination thereof. For liposome preparation these lipids can be dissolved in chloroform (purchased from Avanti Polar Lipids (Alabaster, Ala.).).
Synthetic liposomes can be prepared by dispensing appropriate molar amounts of phospholipids in one embodiment the ratio is (POPC:POPE:DMPA:Ch=45:25:20:10)
Peptide-lipid conjugates. Each of these peptides can be incorporated into synthetic liposomes of varying composition which include:
i) POPC:POPE:DMPA:Cholesterol
ii) POPC:POPS
iii) POPC:POPS:lysoPC
iv) POPC:POPE:Sphingomyelin:Cholesterol
v) POPC:Cholesterol:POPE:DMPA
vi) POPC:Cholesterol:POPE:DMPE
The liposome composition could comprise an adjuvant. In non-limiting embodiments the adjuvant is alum or monophosphoryl Lipid A (Avanti Polar Lipids).
The peptide-liposome immunogens of the invention can be formulated with, and/or administered with, adjuvants such as lipid A, oCpGs, TLR4 agonists or TLR 7 agonists that facilitate robust antibody responses (Rao et al, Immunobiol. Cell Biol. 82(5):523 (2004)). Non-limiting examples of other adjuvants that can be used include alum and Q521 (which do not break existing B cell tolerance). In certain embodiments, formulations comprise an adjuvant that is designed to break forms of B cell tolerance, such as oCpGs in an oil emulsion such as Emulsigen (an oil in water emulsion) (Tran et al, Clin. Immunol. 109(3):278-287 (2003)).
In accordance with the invention, immune response enhancing TLR ligands, for example, monophosphorylipid A (MPL-A, TLR4 ligand), oligo CpG (TLR 9 ligand) and R-848 (TLR 7/8 ligand), are formulated either individually or in combination into liposomes conjugated with an HIV-1 gp41MPER peptide immunogen. In certain embodiments, the combination of TLR agonists comprises oCpG (TLR9) (Hemni et al, Nature 408:740-745 (2004)) and R848 (TLR7/8) (Hemni et al, Nat. Immunol. 3:196-200 (2002)).
Additional designs of constructs of the invention include MPER peptide-liposome encapsulated with the cytokine interferon (IFN)-α and either encapsulated or membrane bound CD40 ligand. Two broadly neutralizing gp41 MPER antibodies (2F5, 4E10) bind with high affinity to such TLR ligand adjuvant-associated liposome constructs. These constructs can be used to modulate B cell tolerance, direct liposomes to certain B cell populations capable of making broadly reactive neutralizing antibodies, and in enhance antibody responses against poorly immunogenic HIV-1 gp41MPER epitopes.
The peptide-liposome immunogens can be administered, for example, IV, intranasally, subcutaneously, intraperitoneally, intravaginally, or intrarectally. The route of administration can vary, for example, with the patient, the conjugate and/or the effect sought, likewise the dosing regimen.
Autoreactive B cells can be activated by TLR ligands through a mechanism dependent on dual engagement of the B cell receptor (BCR) and TLR (Leadbetter et al, Nature 416:603 (2002); Marshak-Rothstein et al, Annu. Rev. Immunol. 25: 419-41 (2007), Herlands et al, Immunity 29:249-260 (2008), Schlomchik, Immunity 28:18-28 (2008)). In certain embodiments ofimmunogen design of the instant invention, soluble IFN-α is encapsulated into liposomes conjugated to MPER peptides such as MPER656 or MPER656-L669S peptides. IFN-α has been reported to modulate and relax the selectivity for autoreactive B cells by lowering the BCR activation threshold (Uccellini et al, J. Immunol. 181:5875-5884 (2008)). The design of the immunogens results from the observation that lipid reactivity of gp41MPER antibodies is required for both binding to membrane bound MPER epitopes and in the neutralization of HIV-1.
The B cell subsets that the liposomes can target include any B cell subset capable of making polyreactive antibodies that react with both lipids and the gp41 epitopes of the MPER. These B cell subsets include, but are not limited to, the marginal zone IgM+ CD27+ B cell subset (Weill et al, Annu. Rev. Immunol. 27:267-85 (2009), Li et al, J. Exp. Med 195: 181-188 (2002)), the transitional populations of human B cells (Sims et al, Blood 105:4390-4398 (2005)), and the human equivalent of the B cells that express the human equivalent of the mouse Immunoglobulin (Ig) light chain lambda X (Li et al, Proc. Natl. Acad. Sci. 103:11264-11269 (2006), Witsch et al, J. Exp. Med. 203:1761-1772 (2006)). All of these B cell subsets have the capacity to make multireactive antibodies and, therefore, to make antibodies that have the characteristic of reacting with both lipids and HIV-1 gp41. That the liposomes have the characteristic of having both lipids and gp41 in them, should result in the selective targeting of these immunogens to the B cells of interest. Because these liposomes can be used to transiently break tolerance of B cells or to target rare B cell subsets, it can be seen that other HIV-1 envelope immunogens, such as deglycosylated envelope preparations, such as described below, can be formulated in the liposomes containing TLR 4 agonists, TLR 7/8 agonists and IFNα.
The deglycosylated JRFL gp 140 Env protein and the CD4-binding site mutant gp140 (JRFL APA) have been described in a previous application (see, for example, WO 2008/033500). Deglycosylated env and Env mutated to not bind CD4 so as not to be immunosuppressive can be anchored in the liposomes by incorporating a transmembrane domain and, after solubilizing in detergent, can be reconstituted into synthetic liposomes. Alternatively, His-tagged (c-terminus end) versions of the Env gp140 can be anchored into liposomes as described for an intermediate form of HIV-1 gp41 (gp41-inter).
Given that many B cell subsets capable of making polyreactive antibodies also bind mammalian DNA, addition of DNA to liposomes can be used to target the immunogens to the responsive B cells.
The liposome-containing formulations of the invention can be administered, for example, by intramuscular, intravenous, intraperitoneal or subcutaneous injection. Additionally, the formulations can be administered via the intranasal route, or intrarectally or vaginally as a suppository-like vehicle. Generally, the liposomes are suspended in an aqueous liquid such as normal saline or phosphate buffered saline pH 7.0. Optimum dosing regimens can be readily determined by one skilled in the art.
Certain aspects of the invention can be described in greater detail in the non-limiting Examples that follows. See also Published PCT Application Nos. WO 2006/110831 and WO 2008/127651, U.S. Published Application Nos. 2008/0031890 and 2008/0057075, U.S. Provisional Application No. 60/960,413 and U.S. application Ser. No. 11/918,219.).
Experimental Details
Subjects
Trinidad Seroconverter Cohort was described preciously (Blattner et al, J. Infect. Dis. 189:1793-1801 (2004)). Briefly, patients from a sexually transmitted disease (STD) clinic were monitored for HIV infection and enrolled upon seroconvertion. Infections occurred through heterosexual contact and subtype B viruses accounted for all the infections. The patient of interest in this study, SC42, was naïve for antiviral therapy until 5 yr into infection.
Molecular Cloning of Full-Length Envelopes
Cloning strategy of full-length gp160 has been described previously (Wei et al, Nature 422:307-312 (2003), Li et al, J. Virol. 79:10108-10125 (2005)). Briefly, viral RNA was extracted from patient plasma samples using QIAmp Viral RNA Mini Kit (Qiagen, Valencia, Calif.) and subsequently reverse-transcribed into cDNA using SuperScript II ((Invitrogen Corp., Carlsbad, Calif.) and random hexamer primers. Full length envelope sequences were generated by nested PCR with the following primers: 1st round primers 5′OUT 5′-TAGAGCCCTGGAAGCATCCAGGAAG-3′ (SEQ ID NO: 33), nt 5852-5876 and 3′ OUT 5′-TTGCTACTTGTGATTGCTCCATG T-3′ (SEQ ID NO: 34), nt 8912-8935); and 2nd round primers 5′Intopo 5′-CACCTAGGCATCTCCTATGGCAGGAAGAAG-3′ (SEQ ID NO: 35), nt 5957-5982 and 3′IN 5′-GTCTCGAGATACTGCTCCCACCC-3′ (SEQ ID NO: 36), nt 8881-8903). The PCR products were purified and then directly ligated into the directional cloning vector pcDNA 3.1DN5-His-TOPO (Invitrogen) following the manufacturer's directions. This pcDNA 3.1DN5-His-TOPO vector contains a cytomegalovirus promoter that allows the expression of envelope proteins for subsequent pseudovirus production.
Mutagenesis for Introduction of Single Mutation
A QuikChange XL Site-directed Mutagenesis Kit (Invitrogen Corp) was used to introduce S669L mutation into HS-MPER to generate HS-MPER/S669L, and K665N mutation into HS-MPER to generate HS-MPER/K665N following the manufactures instructions. The primers for introducing S669L mutation into HS-MPER were: fN-MPER_S669L (5′-GGATAAGTGGGCAAGTTTGTGGAATTGGTTTGAC-3′ (SEQ ID NO: 37)) and r7534.5_S669L (5′-GTCAAACCAATTCCACAAACTTGCCCACTTATCC-3′ (SEQ ID NO: 38)); the primers for introducing K665N into HS-MPER were: fHS-MPER_K665N (5′-gaattattagaattggataaCtgggcaagttcgtgg-3′ (SEQ ID NO: 39)) and r7534.5_K665N (5′-CCACGAACTTGCCCAGTTATCCAATTCTAATAATTC-3′ (SEQ ID NO: 40)).
Production and Titration of Env-Pseudoviruses
Production and titration of the env-pseudoviruses was conducted following procedures modified from methods previously described (Li et al, J. Virol. 79:10108-10125 (2005)) with minor modifications. Full-length env clones in pcDNA3.1DN5-His-TOPO vector were co-transfected into 293T cells with an env-deficient HIV-1 backbone (pSG3Aenv) using FuGENE® HD transfection reagent (Roche Applied Science, Basel, Switzerland). Tissue culture fluid was harvested after 24-36 h of incubation and fresh fetal bovine serum was added to the virus stock to make a final concentration of 20%.
The 50% tissue culture infectious dose (TCID50) of each virus preparation was determined on JC53-BL cells as previously described (Li et al, J. Virol. 79:10108-10125 (2005)). Briefly, serial diluted virus stocks were used to infect JC53-BL cells on 96-well-flat-bottom-plates for 48 h. The cells were then lysed with and the relative luminescence units (RLU) determined by BriteLite™ assay system (PerkinElmer, Inc., Waltham, Mass.). Wells with luciferase luminescence 2.5-fold over that of the cells only control were considered positive for virus infection. TCID50 was calculated using the Reed-Muench formula.
Neutralization Assay
Neutralization assays for the pseudoviruses were performed on JC53-BL cells on 96-well-flat-bottom-plates as previously described (Li et al, J. Virol. 79:10108-10125 (2005)). Briefly, serially diluted serum samples or purified Abs were incubated with testing viruses, followed by addition of JC53-BL cells. The relative luminescence unit (RLU) of each well was measured with BriteLite™ assay system and the IC50 was determined as the highest dilution of serum (in cases of serum samples) or the lowest concentration of Ab (in cases of purified Abs) that was able to inhibit virus infection by 50% compared to the virus control.
Peptide Absorption Neutralization Assay
Peptide absorption neutralization assay was modified from neutralization assay. Serially diluted serum samples or purified Abs were pre-incubated with properly diluted peptide for 1 h before addition of virus, followed by regular neutralization assays.
Surface Plasmon Resonance (SPR) Assays
SPR binding assays were performed on a BIAcore 3000 (BIAcore Inc, Piscattaway, N.J.) maintained at 20° C. as previously described (Alam et al, J. Immunol. 178:4424-4435 (2007)). Biotinylated versions of SP62 peptides-gp4 652-671 (QQEKNEQELLELDKWASLWN (SEQ ID NO: 41)) and SP62-L669S (gp41 652-671) (QQEKNEQELLELDKWASSWN (SEQ ID NO: 55)), and control peptides with scrambled sequences (2F5656-670 Scrambled and 2F5656-670/L669S Scrambled), were individually anchored on a BIAcore SA sensor chip as described (Alam et al, J. Immunol. 178:4424-4435 (2007), Alam et al, AIDS Res. Hum. Retroviruses 20:836-845 (2004)). Each peptide was injected until 100 to 150 response unit (RU) of binding to streptavidin was observed. Specific binding responses of mAb binding were obtained following subtraction of non-specific binding on the scrambled 2F5 peptide surface. Rate constants were measured using the bivalent analyte model (to account for the avidity of bivalent Ig molecules) and global curve fitting to binding curves obtained from 2F5 titrations, which ranged from 0.01 to 119 nM for mAb 2F5. mAb 2F5 were injected at 30 uL/min for 2-6 min and Glycine-HCl pH 2.0 and surfactant P20 (0.01%) were used as the regeneration buffer.
SPR assay with liposome-anchored peptides were done in a similar fashion as described above. The peptides used are SP62 (gp41 652-671)-GTH1 (QQEKNEQELLELDKWASLWNYKRWIILGLNKIVRMYS-biotin (SEQ ID NO: 42), containing the consensus 2F5 epitope) and SP62-L669S (gp41 652-671)-GTH1 (QQEKNEQELLELDKWASSWNYKRWIILGLNKIVRMYS-biotin (SEQ ID NO: 43), containing the 2F5 epitope with the L669S substitution).
Fitness Assay
The dual infection fitness assay was performed as previously described (Lu et al, J. Virol. 78:4628-4637 (2004)) with minor modifications. HIV-1 infectious chimeric viruses containing TND-669S or TND-669L env and a marker sequence (either Salmonella enterica serovar Typhimurium histidinol dehydrogenase [hisD] gene or the human placental heat-stable alkaline phosphatase [PLAP] gene) were generated by cotranfecting env PCR product and NL4-3 background vector with a reporter gene. In a dual infection fitness assay, two chimeric viruses with specific input ratio (as determined by real-time PCR of the reporter genes) were used to co-infect PBMC (MOI=0.001). Relative production of the viruses with the two Env species in the culture were measured by the corresponding marker (hisD or PLAP) using real-time RT-PCR. Production of an individual virus in a dual infection was determined by calculating the percentage of the individual virus in the total virus population at specific time points (Day 4, 7, and 10). The relative fitness value (1+S) of the individual virus was determined by following equation as previously described (Wu et al, J. Virol. 80:2380-2389 (2006)):
(1+S=exp(d)=exp{ln [(TM(t2)×TL(t1))/(TL(t2)×TM(t1))]/Δt}
1+S=exp, where S is the selection coefficient; Mt, M0, Lt, and L0 are the proportion of more fit variant or less fit variant at time point t and the initial proportion (0) in the inoculum respectively.
Results
Identification of TND-669S Envelope
Multiple longitudinal Env clones were obtained from plasma samples of SC42, NL4-3 Env-pseudotyped viruses were made from the Env clones, and neutralizing sensitivity of selected Env clones against autologous as well as heterologous sera was tested. An envelope strain that was highly sensitive to neutralization by autologous sera was identified. TND-669S, an envelope clone obtained from a chronically infected HIV+ subject showed unexpectedly high sensitivity to neutralization by both autologous and heterologous sera. TND-669S was neutralized by contemporaneous and 27 month (post enrollment) autologous sera with titers of 845 and 1,353 respectively, while TND-669L, another isolate the neutralization sensitivity of which was typical of envelope clones obtained from the same time point (15 month post enrollment) and was retrospectively selected for comparison based on its envelope sequence, was not sensitive to contemporaneous autologous serum neutralization and was neutralized by 27 months post enrollment autologous serum with a titer of only 26 (
Identification of the L669S Mutation
The protein and DNA sequences for TND-669S and TND 669L gp 160 were examined for genetic variations responsible for the increased neutralizing sensitivity of TND-669S envelope. There are 6 nucleotide differences between the two env DNA sequences. However, 5 of those are synonymous mutations, resulting in a single amino acid difference between TND-669S and TND-669L Env. The single amino acid difference is located at position 669, near the C-terminus of the 2F5 epitope and 2 aa upstream of the 4E10 epitope in the MPER (
Sensitivity of the L669s Mutant to Monoclonal Antibodies
Based on the location of the L669S mutation, sensitivity of the TND-669S and TND-669L to 2F5 and 4E10 mAbs was tested. Not surprisingly, TND-669S was highly sensitive to 2F5 mAb while TND-669L was only moderately sensitive (
Sensitivity of TND-669S and TND-669L pseudoviruses to several other neutralizing agents, including the glycan dependent mAb 52D and the entry inhibitor T20 was also tested (
Single L669S Mutation Accounts for the Phenotypic Change
To confirm that the L669S mutation alone is responsible for the phenotypic change, a S669L mutation was introduced into the TND-669S envelope by site-directed mutagenesis. The resulting TND-669S/S669L showed only moderate sensitivity to 2F5 comparable to that of TND-669L, confirming the sole contribution of the L669S mutation in the TND 669S to the increased sensitivity to neutralization. Next, the role of the virus backbone in the phenotypic change associated with the L669S mutation was investigated. A L669S mutation was introduced into the envelope of another primary isolate, QZ4734. The L669S mutation rendered the QZ4734 Env-pseudovirus more than two logarithmic magnitudes more sensitive to neutralization by the 2F5 mAb (
Neutralizing of TND-669S Envelope is Mediated by 2F5 Binding to its Conventional Epitope
Characterization of a 2F5-resistant Env variant has shown that a K665N mutation in the DKW core region abrogates 2F5 binding and results in 2F5 resistance (Purtscher et al, Aids 10:587-593 (1996)). This suggests that the DKW in the core region of the 2F5 epitope EQELLELDKWASLWN (SEQ ID NO: 44) is essential for 2F5 binding. To test whether the potent neutralization of the TND-669S envelope by 2F5 is also mediated though binding of the 2F5 mAb to the core amino acids of the conventional 2F5 epitope, a TND-669S/K665N mutant was made and its sensitivity to 2F5 and 4E10 mAbs was tested. Introduction of the K665N mutation into the TND 669S envelope resulted in a fully 2F5-resistant phenotype while the sensitivity of the envelope against 4E10 was not affected.
Ability of the 2F5 Peptides to Absorb the Neutralizing Activity of the 2F5 mAb
To investigate the possible mechanisms involved in the ability of the L669S substitution to increase the MPER neutralizing sensitivity, peptides containing either the consensus 2F5 epitope (2F5656-670) or the 2F5 epitope with the L669S substitution (2F5656-670/L669S) were synthesized and subsequently tested for their ability to absorb 2F5 mAb neutralizing activity. The 2F5 mAb was pre-absorbed with either the F5con or the F5mut peptide prior to the neutralization assay. Surprisingly, F5mut did not inhibit 2F5 mAb neutralization more potently than F5con. As show in
L669S Mutation Did not Increase the Binding Avidity of the 2F5 mAb for its Epitope
To investigate the possibility that the L669S mutation enhances the avidity of the 2F5 epitope to the 2F5 mAb, peptides containing either the consensus 2F5 epitope (2F5656-670) or the 2F5 epitope with the L669S mutation (2F5656-6701L669S), along with the scrambled version for each peptide, were tested in a BIAcore SPR (surface plasmon resonance) assay for 2F5-binding thermodynamics. The equilibrium dissociation constants (KD) for the F5con and F5mut peptides were 11.0 and 28.1 nM, respectively (
Binding of the Peptides to 2F5 mAb in Lipid Environment
In HIV-1 virus, MPER is in close proximity to the envelope lipid bilayer. Direct binding SPR assay has shown that 2F5 mAb binds to F5con and F5con peptides with comparable avidity. To further examine the possible influence of the L669S substitution on binding of the 2F5 mAb to its epitope in a lipid environment, a SPR binding assay was performed using peptides anchored to phospholipid-containing liposomes. As shown in
Fitness of TND-669S Virus is Greatly Impaired
To determine if the alteration in MPER structure resulted in a fitness defect, the relative fitness of TND 669S and TND-669L viruses was examined by a dual infection competition assay in peripheral blood lymphocytes, using replication competent recombinant viruses containing the NL4-3 backbone and the TND-669S and TND-669L envelope sequences, respectively. With an input ratio of 9:1 (TND-669S:TND-669L), the TND-669S virus was outgrown by the TND-669L virus at 4 days post infection (
In summary, a mutation in the HIV-1 envelope, L669S, has been identified that significantly increases the neutralization sensitivity of the envelope to both 2F5 and 4E10 mAb neutralization. The mean IC50 of the TND-669S and TND-669L Env-pseudoviruses against mAbs 2F5 and 4E10 are 0.014 and 0.031 μg/ml, respectively. In a study by Binley et al (J. Virol. 78:13232-13252 (2004)), where a panel of 93 HIV-1 strains were examined for neutralizing sensitivity to various mAbs, most isolates were neutralized by 2F5 and 4E10 with IC50 of 1-10 μg/ml, while only 9 strains were neutralized at IC50<1 μg/ml by 2F5 mAb, and 9 neutralized by 4E10 mAb at IC50 of <1.0 μg/ml. The IC50 of TND-669S against 2F5 and 4E10 mAbs was even lower than the most 2F5/4E10 mAb sensitive strain (BUSxxxMNc), which was neutralized by 2F5 and 4E10 mAb with IC50 values of 0.05 and 0.17 μg/ml, respectively. In comparison, the L669S mutation renders the envelope 4- and 5-fold more sensitive to 2F5 and 4E10 mAb neutralization, respectively, than the most sensitive virus previously reported.
A single amino acid mutation L669S is responsible for this specific phenotype, as supported by site directed mutagenesis of the L669S mutation into another primary isolate, QZ4734, which rendered the QZ4734/L669S Env-pseudovirus more than 2 logs more sensitive to 2F5 mAb neutralization. To further confirm this, the serine at position 669 of the TND-669S was also mutated back to leucine resulting in the loss of the ultra sensitivity observed in TND-669S envelope.
Both TND-669S and TND 669L envelopes were obtained through bulk PCR. Single genome amplification (SGA) was performed later but the envelope sequences were not identified indicating that the L669S mutation was not circulating in vivo. Additionally, the L669S mutation results in a significant loss of fitness indicating that even if present in natural infection, it would not have circulated long because of its poor fitness level.
In an elegant alkaline-scanning mutagenesis study by Zwick et al, J. Virol. 79:1252-1261 (2005), 13 out of 21 MPER Ala mutants were more sensitive to 2F5 or 4E10 mAb, or both, than the parental MPER. An L669A mutation in HIV-1 JR2 was 50- and 45-fold more sensitive to neutralization by 2F5 and 4E10 mAbs, respectively, and was among the most sensitivity-enhancing mutations. These findings, together with present data, suggest that there may be some common mechanisms shared by the 2F5 and 4E10 epitopes, such as the structure or the accessibility of the MPER, that greatly affects Env sensitivity to MPER neutralizing antibodies.
The mechanisms of the L669S substitution-associated increase in HIV-1 envelope sensitivity to MPER neutralization warrants in depth study because it sheds light on the neutralizing mechanisms of 2F5 and 4E10, and provides important information regarding immunogen design to elicit these types of antibodies.
There are multiple ways through which this mutation may increase neutralizing sensitivity. First, the mutation could have caused dramatic changes in Env and affected the expression level of functional Env spikes on viral particles. Neutralizing assays with multiple other neutralizing agents showed that the increase in neutralizing sensitivity of the TND-669S envelope is not a global effect, making it unlikely that L669S mutation enhances neutralizing sensitivity through changes in Env expression levels. Secondly, this mutation could have changed the fusion kinetics of gp41, resulting in a slower fusion process. Env with reduced fusion kinetics have been shown to be more sensitive to 2F5 and 4E10 neutralization (Reeves et al, J. Virol. 79:4991-4999 (2005)). This is unlikely since the sensitivity of the TND-669S envelope to T20 was only 3-fold that of the TND-669L envelope, suggesting the fusion kinetics is not changed considerably by L669S mutation. Thirdly, it is possible that the L669S mutation itself renders higher avidity binding of the 2F5 mAb to the 2F5 epitope. This hypothesis, however, is not supported by the surface plasmon resonance (SPR) assay results for peptide binding to 2F5, where the 2F5 consensus peptide (containing the consensus 2F5 epitope sequence) bound with slightly higher avidity than did the 2F5 mutant peptide (containing the L669S mutation). Moreover, this hypothesis cannot explain the similar fold of increase in the sensitivity of the TND-669S envelope to both 2F5 and 4E10 mAbs. Fourthly, the L669S mutation could have caused dramatic conformational change of Env, resulting in a more “open” MPER structure, and thus allowing for easier access of antibodies targeting 2F5 and 4E10. This hypothesis can very well explain the similar magnitude of increase in sensitivity of the TND-669S envelope to both 2F5 and 4E10 mAbs. The 447-52D sensitivity changes associated with the L669S mutation (>161×) suggests that the conformational change may have caused changes in the V3 loop as well. Steric constraints for neutralizing antibodies targeting MPER have been suspected by many groups. Several studies have observed possible antagonism between 2F5 and 4E10 (Zwick et al, J. Virol. 79:1252-1261 (2005), Nelson et al, J. Virol. 81:4033-4043 (2007)), suggesting that space limitation may be a factor affecting 2F5 and 4E10 neutralization of HIV virus. Interestingly, when 2F5 epitope was inserted to MLV Env (Ou et al, J. Virol. 80:2539-2547 (2006)), the Env with 2F5 epitope in surface unit is more than 10 times more sensitive to 2F5 neutralization than the Env with 2F5 epitope in the transmembrane unit. In addition, grafting 2F5 epitope into V1, V2, V4 regions of HIV Env also was shown to increase the binding of gp140 to 2F5 (Joyce et al, J. Biol. Chem. 277:45811-45820 (2002), and grafting 2F5 and 4E10 epitopes to the MPER of HIV-2 has been shown to be associated with substantial increase in 2F5-/4E10-neutralization sensitivity (Decker et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, Colo., 2006), presumably through improved epitope accessibility. These data reflected the influence of epitope accessibility on 2F5 sensitivity. The characteristic of TND-669S is in concordance with a likely more “open” MPER structure. The TND-669S isolate can be used to detect the presence of 2F5 and 4E10-like antibodies elicited by vaccination or natural infection (studies to date have failed to detect 2F5 or 4E10 in HIV-1 infected patients and vaccines). An ultra-sensitive isolate can provide crucial information as to whether or not 2F5/4E10 is generated at extremely low levels during natural infection or vaccination. Furthermore, the demonstration that a more exposed MPER, as TND-669S envelope appears to have, has significant applications for vaccine immunogen design.
Description of some embodiments of gp41MPER Peptide-Liposome Conjugates
Description of gp41 MPER Peptide-Adjuvant Conjugates
Toll-like receptor ligands, shown in
The construction of Lipid A and R-848 containing MPER peptide liposomes utilized the method of co-solubilization of MPER peptide having a membrane anchoring amino acid sequence and synthetic lipids 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE), 1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA) and Cholesterol at mole fractions 0.216, 45.00, 25.00, 20.00 and 1.33 respectively (Alam et al, J. Immunol. 178:4424-4435 (2007)). Appropriate amount of MPER peptide dissolved in chloroform-methanol mixture (7:3 v/v), Lipid A dissolved in Chloroform or R-848 dissolved in methanol, appropriate amounts of chloroform stocks of phospholipids were dried in a stream of nitrogen followed by overnight vacuum drying. Liposomes were made from the dried peptide-lipid film in phosphate buffered saline (pH 7.4) using extrusion technology.
Construction of oligo-CpG complexed MPER peptide liposomes used the cationic lipid
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-ethylphospho choline (POEPC) instead of POPC. Conjugation of oCpG was done by mixing of cationic liposomes containing the peptide immunogen with appropriate amounts of oCpG stock solution (1 mg/ml) for the desired dose.
Surface Plasmon Resonance (SPR) assay for the binding of 2F5 mAb to its epitope in the peptide-liposome constructs revealed that incorporation or conjugation of TLR adjuvants does not affect binding of HIV neutralizing antibody 2F5. Strong binding of both mAbs 2F5 and 4E10 were observed.
The long CDR H3 loops of MPER neutralizing mAbs 4E10 and 2F5 have a hydrophobic face, postulated to interact with virion membrane lipids (Ofek et al, J. Virol. 78:10724 (2004); Cardoso et al, Immunity 22:163-173 (2005)). CDRH3 mutants of 4E10 (scFv) and 2F5 (IgG) have been constructed (see
Thus, this strategy has the potential to modulate B cell tolerance, target immunogens to responsive B cell subsets, and allow the induction of polyreactive B cells that bind to phospholipids and gp41MPER epitopes. When used in combination with TLR ligands, the delivery of IFN-α in liposomes has the potential to allow TLR-dependent activation of B cells from the autoreactive pool and with the desired specificity for gp41MPER epitopes.
Description of Constructs
The HIV-1 gp41MPER peptides (
The assessment of the presentation of MPER epitopes on the adjuvanted liposome constructs can be done by SPR analysis of 2F5 and 4E10 mAb binding as described in
Experimental Details
Representative data from two immunized animals show the application of a prime/boost strategy for the induction of MPER specific antibody responses following repeated immunizations with MPER peptide liposomes (see
Results
The presented experimental data shows the application of the designed MPER liposomal immunogens in the induction of antibodies that are targeted to the neutralizing epitopes on gp41 of HIV-1 Envelope protein. The data shows that the constructed MPER peptide liposomes are immunogenic in small animals like guinea pigs and non-human primates (NHP) and that the induced antibody responses are specific for the core neutralizing epitope on gp41MPER. These studies also demonstrate the application of prime-boost strategy in enhancement of the MPER specific responses and in focusing of the antibody responses to the core neutralizing epitopes that include the 2F5 core residues DKW. In the presented immunization scheme, the data shows a shift in the binding epitope in initial responses from residues that are N-terminus to the core DKW to responses that include all three residues of the core neutralizing epitope (DKW) that are induced in later time points. Final immunizations with the MPER liposomes resulted in focusing of the antibody responses to the core DKW residues of the broad neutralizing mAb 2F5. These data represents application of the design of MPER immunogens in liposomal form for the induction of MPER specific antibodies in experimental animals like guinea pigs (
As shown in
As shown in
Binding data from four NHP immunized sera are shown. Binding response measurements and epitope mapping experiments were done as described in
The original MPER vaccine sequence matches HXB2—it has a single rare amino acid in it, otherwise it resembles the B clade consensus. That substitution doesn't impact 4E10 or 10E8 but it is in the contact region, other antibodies against this epitope might be weakened. There is extensive variation in the epitope, but it doesn't affect 4E10 or 10E8. To extend coverage of multiple clades, the invention provides three additional MPER peptides (MPER656.1-3). In certain aspects the invention provides that much of that extensive epitope variation can be covered with a four-valent vaccine, for example but not-limited to the peptide design described in
mAb 2F5 does have a lot of sequence and clade specificity. The original MPER peptide matches the B clade in that epitope. A polyvalent vaccine, for example but not limited to a four-valent vaccine, provides a candidate for a global vaccine, as it is believed that adding the variants could impact breadth. There are 368 variants of the 2F5 epitope “ELDKWA” (SEQ ID NO: 45) found in 4036 sequences from different individuals in the database, and there were 104 different forms found in just the 1010 B clade sequences. Non-limiting example are provided herein which are “central”—they each are common regionally, and the more diverged forms of the epitope tend to build off of these basic forms.
In certain embodiments the anchor is TMD peptide (US Pub. 20100047331 A1).
Methods of Inducing Antibodies with the Composition of the Invention
Various immunization schedules are contemplated by the methods of the invention, either in a sequential prime and boost schedule using individual MPER-liposome conjugates, for example but not limited to the ones described in
In a non-limiting embodiment, the vaccination schedule contemplates sequential vaccination using compositions comprising individual MPER-liposome conjugates, for example each one of the four MPER-liposome conjugates as described in
In another non-limiting embodiment, the vaccination schedule contemplates swarm vaccination using compositions comprising various combinations of the MPER-liposome conjugates. In a non-limiting example a prime is given with all four MPER-liposome conjugates as described in
In another non-limiting embodiment, the vaccination schedule contemplates priming with the original MPER656-liposome conjugate, followed by immunization with combinations of other liposomes, for example a combination of the original MPER656-liposome conjugate and the MPER656.1-liposome conjugate, followed by a combination of the original MPER656-liposome conjugate, the MPER656.1-liposome conjugate and MPER656.2-liposome conjugate, followed by a combination of the original MPER656-liposome conjugate, the MPER656.1-liposome conjugate, MPER656.2-liposome conjugate and MPER656.3-liposome conjugate.
Immunogenicity of the inventive compositions and antibody induction in response to immunization by various schedules as contemplated by the methods of the invention can be measured by various methods known in the art.
NHP #22-JRFL gp140 natively deglycosylated Env prime (that binds UCAs of 2F5) (Ma, B et al. PLoS Pathogens 9: e10022000, 2011), and MPER peptide-liposome boost (J. Virol. 85: 11725, 2011). Goal: induce MPER BnAb lineages such as 2F5 or 4E10. Criteria: induced antibodies bind gp41 inter, map to nAb epitope in MPER, bind by two step mechanism.
Analyses showed that of 2F5 BnAb have the following characteristics: Long hydrophobic loop with LF at loop tip—binds to viral membrane; RR at base of the HCDR3—binds to “D664” of DKW of gp41 MPER; Aspartic acid triplet (DDD) in HCDR2—binds to “K665” of DKW of gp41 MPER.
Data presented in example 7,
Additional MPER-liposomes combinations as contemplated herein can be analyzed by the methods and assays described in Example 7.
This example provides results of non-human primate (NHP) immunization studies with peptide liposome immunogens formulated with different adjuvants. The immunization strategy is shown in
Further studies will be carried to out to determine plasma neutralization properties. Samples from various time points post immunization could be analyzed by any suitable method to determine whether specific antibodies or antibody lineages are induced. Such methods include but are not limited to deep sequencing, single cell sorting of B-cells to isolate and characterize antibodies induced by the MPER peptide immunizations.
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
This application is the National Stage of International Application No. PCT/US2014/058032, filed Sep. 29, 2014, which claims the benefit of and priority to U.S. Provisional Patent Application No. 61/883,306 filed Sep. 27, 2013, the entire contents of which application is herein incorporated by reference.
This invention was made with government support under Grant No. AI 067854 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/058032 | 9/29/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/048635 | 4/2/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4994440 | Creaven | Feb 1991 | A |
| 5565332 | Hoogenboom et al. | Oct 1996 | A |
| 5693752 | Katinger et al. | Dec 1997 | A |
| 5707626 | Douvas et al. | Jan 1998 | A |
| 5733743 | Johnson et al. | Mar 1998 | A |
| 5756674 | Katinger et al. | May 1998 | A |
| 5831034 | Katinger et al. | Nov 1998 | A |
| 5837242 | Holliger et al. | Nov 1998 | A |
| 5858657 | Winter et al. | Jan 1999 | A |
| 5866694 | Katinger et al. | Feb 1999 | A |
| 5871907 | Winter et al. | Feb 1999 | A |
| 5877218 | Herzig et al. | Mar 1999 | A |
| 5910306 | Alving et al. | Jun 1999 | A |
| 5916588 | Popescu et al. | Jun 1999 | A |
| 5919459 | Nacy et al. | Jul 1999 | A |
| 6156337 | Barenholz et al. | Dec 2000 | A |
| 6300308 | Schroit | Oct 2001 | B1 |
| 6761888 | Schenk | Jul 2004 | B1 |
| 6806354 | Schroit | Oct 2004 | B2 |
| 7195768 | Haynes et al. | Mar 2007 | B2 |
| 7459165 | Charland et al. | Dec 2008 | B2 |
| 8956627 | Haynes | Feb 2015 | B2 |
| 9402893 | Haynes | Aug 2016 | B2 |
| 9402917 | Alam | Aug 2016 | B2 |
| 20010036461 | Haynes et al. | Nov 2001 | A1 |
| 20030181406 | Schetter et al. | Sep 2003 | A1 |
| 20040006032 | Lopez | Jan 2004 | A1 |
| 20040006242 | Hawkins et al. | Jan 2004 | A1 |
| 20040053880 | Krieg | Mar 2004 | A1 |
| 20040067905 | Krieg | Apr 2004 | A1 |
| 20040092472 | Krieg | May 2004 | A1 |
| 20040131610 | Thorpe et al. | Jul 2004 | A1 |
| 20040131621 | Thorpe et al. | Jul 2004 | A1 |
| 20040131622 | Thorpe et al. | Jul 2004 | A1 |
| 20040152649 | Krieg | Aug 2004 | A1 |
| 20040161429 | Thorpe et al. | Aug 2004 | A1 |
| 20040171086 | Fink et al. | Sep 2004 | A1 |
| 20040198680 | Krieg | Oct 2004 | A1 |
| 20040213779 | Thorpe et al. | Oct 2004 | A1 |
| 20040241641 | Stiegler et al. | Dec 2004 | A1 |
| 20040265367 | Thorpe et al. | Dec 2004 | A1 |
| 20050025761 | Thorpe et al. | Feb 2005 | A1 |
| 20050059619 | Krieg et al. | Mar 2005 | A1 |
| 20050080240 | Kunert et al. | Apr 2005 | A1 |
| 20050095282 | Schroit | May 2005 | A1 |
| 20060089326 | Krieg et al. | Apr 2006 | A1 |
| 20080031890 | Haynes et al. | Feb 2008 | A1 |
| 20080057075 | Haynes | Mar 2008 | A1 |
| 20090035360 | Lemoine | Feb 2009 | A1 |
| 20090220536 | Ofek et al. | Sep 2009 | A1 |
| 20100028415 | Haynes et al. | Feb 2010 | A1 |
| 20100047331 | Haynes et al. | Feb 2010 | A1 |
| 20110142869 | Berkower | Jun 2011 | A1 |
| 20120070488 | Haynes et al. | Mar 2012 | A1 |
| 20120128758 | Alam et al. | May 2012 | A1 |
| 20120183597 | Haynes et al. | Jul 2012 | A1 |
| 20130323299 | Haynes et al. | Dec 2013 | A1 |
| 20140322262 | Spicer et al. | Oct 2014 | A1 |
| 20150147387 | Haynes et al. | May 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 102485273 | Jun 2012 | CN |
| 1250933 | Oct 2002 | EP |
| 2006-512391 | Apr 2006 | JP |
| WO-8201047 | Apr 1982 | WO |
| WO-9117271 | Nov 1991 | WO |
| WO-19950004524 | Feb 1995 | WO |
| WO-19950007354 | Mar 1995 | WO |
| WO-19950025124 | Sep 1995 | WO |
| WO-19960033219 | Oct 1996 | WO |
| WO-19960040243 | Dec 1996 | WO |
| WO-19990033522 | Jul 1999 | WO |
| WO-20000016746 | Mar 2000 | WO |
| WO-20030022879 | Mar 2003 | WO |
| WO-20030059953 | Jul 2003 | WO |
| WO-20040006847 | Jan 2004 | WO |
| WO-20040087738 | Oct 2004 | WO |
| WO-2005021574 | Mar 2005 | WO |
| WO-20060110831 | Oct 2006 | WO |
| WO-20080033500 | Mar 2008 | WO |
| WO-20080127651 | Oct 2008 | WO |
| WO-20090111304 | Sep 2009 | WO |
| WO-20100042942 | Apr 2010 | WO |
| WO-2010045613 | Apr 2010 | WO |
| WO-2010114628 | Oct 2010 | WO |
| WO-20100114629 | Oct 2010 | WO |
| WO-20120139097 | Oct 2012 | WO |
| Entry |
|---|
| Verkoczy et al., ‘Induction of ffIV 1 broad neutralizing antibodies in 2F5 knock in mice: selection against membrane proximal external region associated autoreactivity limits T dependent responses’ The Journal of Irmlunology, Vo1.191, No. 5, pp. 2538 2550 (e pub. Aug. 5, 2018). |
| Verkoczy, L., et al., 2013, Induction of HIV-1 broad neutralizing antibodies in 2F5 knock-in mice: selection against membrane proximal external region-associated autoreactivity limits T-dependent responses, J. Immunol. 191:2538-2550. |
| Zwick, M. B., 2005, The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring, AIDS 19:1725-1737. |
| Kuiken, C., et al., eds., 2012, HIV Sequence Compendium, published by Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, NM, LA-UR 12-24653, pp. 358-371. |
| Aguilar, L., et al., “Phospholipid Membranes Form Specific Nonbilayer Molecular Arrangements That Are Antigenic”, The Journal of Biological Chemistry, vol. 274, No. 36, pp. 25193-25196 (Sep. 3, 1999). |
| Alam, S. M. et al., “An Inducible HIV Type 1 gp41 HR-2 Peptide-Binding Site on HIV Type 1 Envelope gp120,” AIDS Research and Human Retroviruses, vol. 20, No. 8, pp. 836-845 (Aug. 2004). |
| Alam, S. M. et al., “Human Immunodeficiency Virus Type 1 gp41 Antibodies That Mask Membrane Proximal Region Epitopes: Antibody Binding Kinetics, Induction, and Potential for Regulation in Acute Infection,” Journal of Virology, vol. 82, No. 1, pp. 115-125 (Jan. 2008). |
| Alam, S. M., et al., “Role of HIV membrane in neutralization by two broadly neutralizing antibodies”, PNAS, vol. 106, No. 48, pp. 20234-20239 (Dec. 1, 2009). |
| Alam, S. M., et al., “The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes”, The Journal of Immunology, vol. 178, No. 7, pp. 4424-4435, Author Manuscript—25 pages (Apr. 1, 2007). |
| Alam, S. M., et al., “Differential reactivity of germ line allelic variants of a broadly neutralizing HIV-1 antibody to a gp41 fusion intermediate conformation,” J. Virol., vol. 85, No. 22, pp. 11725-11731 (Nov. 2011). |
| Alving, C. R. et al., “HIV-1, Lipid Rafts, and Antibodies to Liposomes: Implications for Anti-Viral-Neutralizing Antibodies (Review),” Molecular Membrane Biology, vol. 23, No. 6, pp. 453-466 (Nov.-Dec. 2006). |
| Alving, Carl R., “Immunologic aspects of liposomes: presentation and processing of liposomal protein and phospholipid antigens,” Biochimica et Biophysica Acta, vol. 1113, No. 3-4, pp. 307-322 (Dec. 11, 1992). |
| Alving, C. R., et al., “Chapter 21: Preparation and Use of Liposomes in Immunological Studies,” Liposome Technology, 2nd Edition, vol. III, Interactions of Liposomes with the Biological Milieu, pp. 317-343, 29 pages (1993). |
| Armbruster, C., et al., “Passive immunization with the anti-HIV-1 human monoclonal antibody (hMab) 4E10 and the hMab combination 4E10/2F5/2G12”, Journal of Antimicrobial Chemotherapy, vol. 54, pp. 915-920 (Sep. 29, 2004). |
| Barbato, G., et al., “Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion,” J. Mol. Biol., vol. 330, pp. 1101-1115 (2003). |
| Barthel, H. R. and Wallace, D. J., “False-Positive Human Immunodeficiency Virus Testing in Patients With Lupus Erythematosus”, Seminars in Arthritis and Rheumatism, vol. 23, No. 1, pp. 1-7 (Aug. 1993). |
| Bate, et al., “Phospholipids coupled to a carrier induce IgG antibody that blocks tumour necrosis factor induction by toxic malaria antigens”, Immunology, vol. 79, pp. 138-145 (1993). |
| Binley, J. M., et al., “Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies,” J. Virol., vol. 78, No. 23, pp. 13232-13252 (Dec. 2004). |
| Blattner, W. A., et al., “Rapid Clearance of Virus after Acute HIV-1 Infection: Correlates of Risk of AIDS,” J. Infect. Dis., vol. 189, pp. 1793-1801 (May 15, 2004). |
| Brown, B. K., et al, “Monoclonal Antibodies to Phosphatidylinositol Phosphate Neutralize Human Immunodeficiency Virus Type 1: Role of Phosphate-Binding Subsites”, Journal of Virology, vol. 81, No. 4, pp. 2087-2091 (Feb. 2007). |
| Burton, D. R., et al., “A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals,” Proc. Natl. Acad. Sci. USA, vol. 88, pp. 10134-10137 (Nov. 1991). |
| Callahan, M. K., et al., “Phosphatidylserine on HIV Envelope Is a Cofactor for Infection of Monocytic Cells”, The Journal of Immunology, vol. 170, pp. 4840-4845 (2003). |
| Cardoso, R. M., et al., “Broadly Neutralizing Anti-HIV Antibody 4E10 Recognizes a Helical Conformation of a Highly Conserved Fusion-Associated Motif in gp41,” Immunity, vol. 22, No. 2, pp. 163-173 (Feb. 2005). |
| Chang, J.-S., et al., “Immunogenicity of synthetic HIV-1 V3 loop peptides by MPL adjuvanted pH-sensitive liposomes”, Vaccine, vol. 17, pp. 1540-1548 (1999). |
| Chen, X. et al., “Novel recombinant engineered gp41 N-terminal heptad repeat trimers and their potential as anti-HIV-1 therapeutics or microbicides,” The Journal of Biological Chemistry, vol. 285, No. 33, pp. 25506-25515 (Aug. 13, 2010) (23 pages). |
| Chukwuocha, R. U., et al., “Molecular and genetic characterizations of five pathogenic and two non-pathogenic monoclonal antiphospholipid antibodies”, Molecular Immunology, vol. 39, pp. 299-311 (2002). |
| Coeffier, E. et al., “Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MaIE protein,” Vaccine, vol. 19, No. 7-8, pp. 684-693 (Nov. 22, 2000). |
| Cornet, B., et al., “Virosomes reconstituted from human immunodeficiency virus proteins and lipids”, Biochem. Biophys. Res. Comm., vol. 167, No. 1, pp. 222-231 (Feb. 28, 1990). |
| Creaven, P. J., et al, “Initial Clinical Trial of Muramyl Tripeptide Derivative (MTP-PE) Encapsulated in Liposomes: An Interim Report”, Abstract, Accession No. 90657770 CancerLit (Jan. 1990) (1 page). |
| Darland-Ransom et al, “Role of C. elegans TAT-1 Protein in Maintaining Plasma Membrane Phosphatidylserine Asymmetry”, Science, vol. 320, pp. 528-531 (Apr. 25, 2008). |
| Del Papa et al., “Human beta2-Glycoprotein I Binds to Endothelial Cells Through a Cluster of Lysine Residues That Are Critical for anionic Phospholipid Binding and Offers Epitopes for anti-beta2-Glycoprotein I Antibodies”, The Journal of Immunology, vol. 160, pp. 5572-5578 (1998). |
| Dennison et al., “Stable docking of neutralizing human immunodeficiency virus type 1 gp41 membrane-proximal external region monoclonal antibodies 2F5 and 4E10 is dependent on the membrane immersion depth of their epitope regions,” Journal of Virology, vol. 83, No. 19, pp. 10211-10223 (Oct. 2009). |
| Dennison, S. M., et al., “Induction of Antibodies in Rhesus Macaques That Recognize a Fusion-Intermediate Conformation of HIV-1 gp41,” Plos One, vol. 6, Issue 11, e27824, pp. 1-14 (Nov. 2011). |
| Dhillon, A. K., et al., “Dissecting the Neutralizing Antibody Specificities of Broadly Neutralizing Sera from Huma Immunodeficiency Virus Type 1-Infected Donors,” J. Virol., vol. 81, No. 12, pp. 6548-6562 (Jun. 2007). |
| Doyle, C. M., et al., “Consequences of receptor editing at the λ locus: Multireactivity and light chain secretion,” PNAS, vol. 103, No. 30, pp. 11264-11269 (Jul. 25, 2006). |
| Extended European Search Report issued by the European Patent Office for European Application No. 10759166.1 dated Apr. 20, 2015 (6 pages). |
| Fairn, G. D. and Grinstein, S., “A One-Sided Signal”, Science, vol. 320, pp. 458-460 (Apr. 25, 2008). |
| Ferrantelli, F., et al., “Complete Protection of Neonatal Rhesus Macaques against Oral Exposure to Pathogenic Simian-Human Immunodeficiency Virus by Human Anti-HIV Monoclonal Antibodies,” J. Infect. Dis., vol. 189, pp. 2167-2173 (Jun. 15, 2004). |
| Frey, G. et al., “A fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies,” PNAS, vol. 105, No. 10, pp. 3739-3744 (Mar. 11, 2008). |
| Frisch et al., “Synthetic Peptide-Based Highly Immunogenic Liposomal Constructs”, Methods in Enzymology, vol. 373, pp. 51-73 (2003). |
| Geffin, R., et al., “A longitudinal assessment of autologous neutralizing antibodies in children perinatally infected with human immunodeficiency virus type 1,” Virology, vol. 310, pp. 207-215 (2003). |
| Gómara, M. J., et al., “Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion”, FEBS Letters, vol. 580, No. 11, pp. 2561-2566 (May 15, 2006). |
| Gray, E. S., et al., “Neutralizing Antibody Responses in Acute Human Immunodeficiency Virus Type 1 Subtype C Infection,” J. Virol., vol. 81, No. 12, pp. 6187-6196 (Jun. 2007). |
| Guenaga, J., et al., “Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant,” PLoS One, vol. 6, Issue 1, e16074, pp. 1-12 (Jan. 2011). |
| Hammel, M., et al., “Mechanism of the Interaction of beta2-Glycoprotein I with Negatively Charged Phospholipid Membranes”, Biochemistry, vol. 40, pp. 14173-14181 (2001). |
| Haynes, B. F. et al., “Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies,” Science, vol. 308, No. 5730, pp. 1906-1908 (Jun. 24, 2005). |
| Herlands, R. A., et al., “T Cell-Independent and Toll-like Receptor-Dependent Antigen-Driven Activation of Autoreactive B Cells,” Immunity, vol. 29, No. 2, pp. 249-260, Author Manuscript—26 pages (Aug. 15, 2008). |
| Hinz, A. et al., “Characterization of a trimeric MPER containing HIV-1 gp41 antigen,” Virology, vol. 390, No. 2, pp. 221-227 (Aug. 1, 2009). |
| Ho, J. et al., “Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response,” Vaccine, vol. 23, pp. 1559-1573 (2005). |
| Huang, X., et al., “A Monoclonal Antibody that Binds Anionic Phospholipids on Tumor Blood Vessels Enhances the Antitumor Effect of Docetaxel on Human Breast Tumors in Mice”, Cancer Res., vol. 65, No. 10, pp. 4408-4416 (May 15, 2005). |
| Huang, X., et al., “Anti-Tumor Effects and Lack of Side Effects in Mice of Immunotoxin Directed Against Human and Mouse Prostate-Specific Membrane Antigen”, The Prostate, vol. 61, pp. 1-11 (2004). |
| Huarte, N., et al., “Recognition of membrane-bound fusion-peptide/MPER complexes by the HIV-1 neutralizing 2F5 antibody: implications for anti-2F5 immunogenicity,” PLoS One, vol. 7, Issue 12, e52740, pp. 1-13 (Dec. 2012). |
| Huarte, N., et al., “The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5”, Journal of Virology, vol. 82, No. 18, p. 8986-8996, Sep. 2008, 4 pages of supplemental material (15 pages total). |
| Hunt, J. and Krilis, S., “The Fifth Domain of beta2-Glycoprotein I Contains a Phospholipid Binding Site (Cys281-Cys288) and a Region Recognized by Anticardiolipin antibodies”, Journal of Immunology, vol. 152, pp. 653-659 (1994). |
| Ichikawa, H. T., et al., “Activation of APCs Through CD40 or Troll-like Receptor 9 Overcomes Tolerance and Precipitates Autoimmune Disease”, The Journal of Immunology, vol. 169, pp. 2781-2787 (2002). |
| International Preliminary Report on Patentability issued by the International Bureau of WIPO for International Application No. PCT/US2006/013684 dated Apr. 28, 2009 (4 pages). |
| International Preliminary Report on Patentability issued by the International Bureau of WIPO for International Application No. PCT/US2008/004709 dated Oct. 13, 2009 (5 pages). |
| International Preliminary Report on Patentability issued by the International Bureau of WIPO for International Application No. PCT/US2012/032717 dated Oct. 8, 2013 (5 pages). |
| International Search Report and Written Opinion issued by the Korean Intellectual Property Office as International Searching Authority for International Application No. PCT/US2012/032717 dated Oct. 29, 2012 (8 pages). |
| International Search Report and Written Opinion issued by the Korean Intellectual Property Office as International Searching Authority for International Application No. PCT/US2014/058032 dated Jan. 12, 2015 (11 pages). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as International Searching Authority for International Application No. PCT/US06/13684 dated Jul. 31, 2008 (4 pages). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office for International Application No. PCT/US2008/04709 dated Sep. 17, 2008 (7 pages). |
| International Search Report and Written Opinion issued by the Korean Intellectual Property and Trademark Office as International Searching Authority for International Application No. PCT/US2010/001017 dated Jan. 18, 2011 (11 pages). |
| Japanese Office Action issued by the Japan Patent Office for Japanese Application No. 2012-503432 dated Jun. 16, 2014 (4 pages—English translation only). |
| Jiang, Z.-H. And Rao, K. R., “Synthetic vaccines: the role of adjuvants in immune targeting”, Current Medicinal Chemistry, vol. 10, No. 15, pp. 1423-1439 (Aug. 2003)—Medline, Abstract, PMID: 12871139 (1 page). |
| Li, J., et al., “The interaction between the membrane-proximal external region and the N-trimer region of HIV-1 gp41: Involvement in viral fusion,” Chinese Science Bulletin, vol. 54, No. 10, pp. 1707-1712 (May 2009). |
| Joyce, J. G. et al., “Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design,” J. Biol. Chem., vol. 277, No. 48, pp. 45811-45820, 12 pages (Nov. 29, 2002). |
| Kim, M., et al., “Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization,” Nat. Struct. Mol. Biol., vol. 18, No. 11, pp. 1235-1243, Author Manuscript—22 pages (Nov. 1, 2012). |
| Kim, M., et al., “Immunogenicity of membrane-bound HIV-1 gp41 membrane-proximal external region (MPER) segments is dominated by residue accessibility and modulated by stereochemistry,” J. Biol. Chem., vol. 288, No. 44, pp. 31888-31901 (Nov. 1, 2013). |
| Kim, M., et al., “Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state,” Vaccine, vol. 25, No. 27, pp. 5102-5114, Author's Manuscript—24 pages (Jun. 28, 2007). |
| Lenz, O., et al, “Trimeric membrane-anchored gp41 inhibits HIV membrane fusion”, J. Biol. Chem., vol. 280, No. 6, pp. 4095-4101 (Feb. 11, 2005). |
| Letvin, N. L. and Walker, B. D., “Immunopathogenesis and immunotherapy in AIDS virus infections”, Nature Medicine, vol. 9, No. 7, pp. 861-866 (Jul. 2003). |
| Li, M., et al., “Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies,” J. Virol., vol. 79, No. 16, pp. 10108-10125 (Aug. 2005). |
| Li, Y., et al., “Autoreactive B Cells in the Marginal Zone that Express Dual Receptors,” J. Exp. Med., vol. 195, No. 2, pp. 181-188 (Jan. 21, 2002). |
| Liao, H.-X., et al., “A Group M Consensus Envelope Glyoprotein Induces Antibodies That Neutralize Subsets of Subtype B and C HIV-1 Primary Viruses”, Virology, vol. 353, No. 2, pp. 268-282 (2006). |
| Lin, W.-S., et al., “Some antiphospholipid antibodies recognize conformational epitopes shared by β2GPI and the homologous catalytic domains of several serine proteases,” Arth. Rheum., vol. 56, No. 5, pp. 1638-1647, Author Manuscript—18 pages (May 2007). |
| Lopez, P. H. H. and Schnaar, R. L., “Determination of Glycolipid-Protein Interaction Specificity”, Methods in Enzymology, vol. 417, pp. 205-220, Author Manuscript—14 pages (2006). |
| Lorizate, M., et al., “Recognition and Blocking of HIV-1 gp41 Pre-transmembrane Sequence by Monoclonal 4E10 Antibody in a Raft-like Membrane Environment”, The Journal of Biological Chemistry, vol. 281, No. 51, pp. 39598-39606 (Dec. 22, 2006). |
| Lu et al., “Identification of Polyclonal and Monoclonal Antibodies Against Tissue Plasminogen Activator in the Antiphospholipid Syndrome”, Arthritis & Rheumatism, vol. 52, No. 12, pp. 4018-4027 (Dec. 2005). |
| Lu, J., et al., “Relative Replicative Fitness of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide (T-20),” J. Virol., vol. 78, No. 9, pp. 4628-4637 (May 2004). |
| Luo et al., “Induction of neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) by immunization with gp41 membrane-proximal external region (MPER) fused with porcine endogenous retrovirus (PERV) p15e fragment”, Vaccine (Netherlands), vol. 24, No. 4, pp. 435-442 (Jan. 23, 2006)—Medline, Abstract, PMID: 16143433 (1 page). |
| Luster, T. A., et al., “Plasma Protein beta-2-Glycoprotein 1 Mediates Interaction between the Anti-tumoro Monoclonal Antibody 3G4 and Anionic Phospholipids on Endothelial Cells”, The Journal of Biological Chemistry, vol. 281, No. 40, pp. 29863-29871 (Oct. 6, 2006). |
| Ma, B.-J., et al., “Envelope Deglycosylation Enhances Antigenicity of HIV-1 gp41 Epitopes for Both Broad Neutralizing Antibodies and Their Unmutated Ancestor Antibodies,” PLoS Pathogens, vol. 7, No. 9, e1002200, pp. 1-16 (Sep. 2011). |
| Maksyutov, A. Z., et al., “Exclusion of HIV epitopes shared with human proteins in prerequisite for designing safer AIDS vaccines”, Journal of Clinical Virology, vol. 749, pp. 1-13 (2004). |
| Marshak-Rothstein, A. and Rifkin, I. R., “Immunologically Active Autoantigens: The Role of Toll-Like Receptors in the Development of Chronic Inflammatory Disease,” Annu. Rev. Immunol., vol. 25, pp. 419-441, 26 pages (2007). |
| Mascola, J. R., et al., “Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies,” Nat. Med., vol. 6, No. 2, pp. 207-210 (Feb. 2000). |
| McGaughey, G. B., et al., “HIV-1 Vaccine Development: Constrained Peptide Immunogens Show Improved Binding to the Anti-HIV-1 gp41 MAb,” Biochemistry, vol. 42, pp. 3214-3223 (2003). |
| Mehandru, S. et al., “Neutralization Profiles of Newly Transmitted Human Immunodeficiency Virus Type 1 by Monoclonal Antibodies 2G12, 2F5, and 4E10,” Journal of Virology, vol. 78, No. 24, pp. 14039-14042 (Dec. 2004). |
| Mercer, J. and Helenius, A., “Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells”, Science, vol. 320, pp. 531-535 (Apr. 25, 2008). |
| Montefiori, David C., “Neutralizing antibodies take a swipe at HIV in vivo”, Nature Medicine, vol. 11, No. 6, pp. 593-594 (Jun. 2005). |
| Moog, C., et al., “Autologous and Heterologous Neutralizing Antibody Responses following Initial Seroconversion in Human Immunodeficiency Virus Type 1-Infected Individuals,” J. Virol., vol. 71 No. 5, pp. 3734-3741 (May 1997). |
| Muster et al., “Cross-Neutralizing Activity against Divergent Human Immunodeficiency Virus Type 1 Isolates Induced by the gp41 Sequence ELDKWAS”, Journal of Virology, vol. 68, No. 6, pp. 4031-4034 (Jun. 1994). |
| Nabel, Gary J., “Close to the Edge: Neutralizing the HIV-1 Envelope”, Science, vol. 308, pp. 1878-1879 (Jun. 24, 2005). |
| Nelson, J. D. et al., “An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10,” J. Virol., vol. 81, No. 8, pp. 4033-4043 (Apr. 2007). |
| Ofek et al., “Structure and Mechanistic Analysis of the Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5 in Complex with Its gp41 Epitope”, Journal of Virology, vol. 78, No. 19, pp. 10724-10737 (Oct. 2004). |
| Ofek, G., et al., “Elicitation of structure-specific antibodies by epitope scaffolds,” PNAS, vol. 107, No. 42, pp. 17880-17887 (Oct. 19, 2010). |
| Ou, W. et al., “Effect of epitope position on neutralization by anti-human immunodeficiency virus monoclonal antibody 2F5,” J. Virol., vol. 80, No. 5, pp. 2539-2547 (Mar. 2006). |
| Perelson, A. S., et al, “HIV-1 Dynamics in Vivo: Virion Clearance Rate, Infected Cell Life-Span, and Viral Generation Time”, Science, vol. 271, pp. 1582-1586 (Mar. 15, 1996). |
| Petrovas et al., “Anti-phospholipid Antibodies in HIV Infection and SLE With or Without Anti-phospholipid Syndrome: Comparisons of Phospholipid Specificity, Avidity and Reactivity with beta2-GPI”, Journal of Autoimmunity, vol. 13, pp. 347-355 (Nov. 1999). |
| Pinto, L. A., et al., “Panel of Anti-gp120 Monoclonal Antibodies Reacts with Same Nuclear Proteins in Uninfected Cells as Those Recognized by Autoantibodies from Patients with Systemic Lupus Erythematsous”, AIDS Research and Human Retroviruses, vol. 10, No. 7, pp. 823-828 (1994). |
| Putscher, M., et al., “Restricted antigenic variability of the epitope recognized by the neutralizing gp41 antibody 2F5,” AIDS, vol. 10, No. 6, pp. 587-593 (Jun. 1996). |
| Ran, S., et al., “Antitumor Effects of a Monoclonal Antibody that Binds Anionic Phospholipids on the Surface of Tumor Blood Vessels in Mice”, Clinical Cancer Research, vol. 11, pp. 1551-1562 (Feb. 15, 2005). |
| Ran et al., “Increased Exposure of Anionic Phospholipids on the Surface of Tumor Blood Vessels”, Cancer Research, vol. 62, pp. 6132-6140 (Nov. 1, 2002). |
| Rao, M., et al., “Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein,” Immunology and Cell Biology, vol. 82, pp. 523-530 (2004). |
| Rauch, J. and Janoff, A. S., “Phospholipid in the hexagonal II phase is immunogenic: Evidence for immunoregonition of nonbilayer lipid phases in vivo”, Proc. Natl. Acad. Sci. USA, vol. 87, pp. 4112-4114 (Jun. 1990). |
| Reardon, P. N. et al., “Structure of an HIV-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer,” PNAS, vol. 111, No. 4, pp. 1391-1396, 10 pages (Jan. 28, 2014). |
| Reeves, J. D., et al., “Enfuvirtide Resistance Mutations: Impact on Human Immunodeficiency Virus Envelope Function, Entry Inhibitor Sensitivity, and Virus Neutralization,” J. Virol., vol. 79, No. 8, pp. 4991-4999 (Apr. 2005). |
| Richards, R. L., et al., “Liposomes Containing Lipid a Serve as an Adjuvant for Induction of Antibody and Cytotoxic T-Cell Responses against RTS,S Malaria Antigen,” Infection and Immunity, vol. 66, No. 6, pp. 2859-2865 (Jun. 1998). |
| Richman, D. D., et al., “Rapid evolution of the neutralizing antibody response to HIV type 1 infection,” Proc. Natl. Acad. Sci. USA, vol. 100, No. 7, pp. 4144-4149 (Apr. 1, 2003). |
| Sakaue et al., “HIV Mucosal Vaccine: Nasal Immunization with gp 160-Encapsualted Hemagglutinating Virus of Japan-Lipsome Induces Antigen-Specific CTLs and Neutralizing Antibody Responses”, The Journal of Immunology, vol. 170, pp. 495-502 (Jan. 1, 2003). |
| Sanchez, P. J. et al., “Combined TLR/CD40 Stimulation Mediates Potent Cellular Immunity by Regulating Dendritic Cell Expression of CD70 In Vivo,” the Journal of Immunology, vol. 178, No. 3, pp. 1564-1572 (Feb. 1, 2007). |
| Scherer, E. M., et al, “Difficulties in eliciting broadly neutralizing anti-Hiv antibodies are not explained by cardiopin autoreactivity”, AIDS, vol. 21, No. 16, pp. 2131-2139 (Oct. 18, 2007)—Medline, Abstract, PMID: 18090039 (1 page). |
| Schnaar, Ronald L., “Isolation of Glycosphingolipids”, Chapter 22, Methods in Enzymology, vol. 230, pp. 348-370 (1994). |
| Serrano et al., “Structure and Immunogenicity of a Peptide Vaccine Including the Complete HIV-1 gp41 2F5 Epitope. Implications for Antibody Recognition Mechanism and Immunogen Design,” J. Biol. Chem., vol. 289, No. 10, pp. 6565-6580 (Mar. 7, 2014). |
| Shen, X. et al., “In vivo gp41 antibodies targeting the 2F5 monoclonal antibody epitope mediate human immunodeficiency virus type 1 neutralization breadth,” J. Virol., vol. 83, No. 8, pp. 3617-3625 (Apr. 2009). |
| Shen, X. et al., “Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution,” PNAS, vol. 107, No. 13, pp. 5972-5977 (Mar. 30, 2010). |
| Shlomchik, Mark J., “Sites and Stages of Autoreactive B Cell Activation and Regulation,” Immunity, vol. 28, pp. 18-28 (Jan. 2008). |
| Sims, G. P., et al., “Identification and characterization of circulating human transitional B cells,” Blood, vol. 105, No. 11, pp. 4390-4398, 23 pages in total, (Jun. 1, 2005). |
| Singh, H., et al, “Reactivity profiles of broadly neutralizing anti-HIV-1 antibodies are distinct from those of pathogenic autoantibodies”, AIDS, vol. 25, No. 10, pp. 1247-1257, Author Manuscript—19 pages (Jun. 19, 2011). |
| Stiegler, G. and Katinger, H., “Therapeutic potential of neutralizing antibodies in the treatment of HIV-1 infection”, Journal of Antimicrobial Chemotherapy, vol. 51, pp. 757-759 (2003). |
| Stults, C. L. M., et al., “Glycosphingolipids: Structure, biological Source, and Properties”, Chapter 14, Methods in Enzymology, vol. 179, pp. 167-214 (1989). |
| Sullards, M. C., et al., “Structure-Specific Quantitative Methods for Analysis of Sphingolipids by Liquid Chromatography-Tandem Mass Spectrometry: “Inside-Out” Sphingolipidomics”, Chapter Four, Methods in Enzymology, vol. 432, pp. 83-115 (2007). |
| Supplementary European Search Report issued by the European Patent Office for Application No. EP 06740904.5 dated Sep. 2, 2010 (14 pages). |
| Supplementary European Search Report issued by the European Patent Office for Application No. 08742782.9 dated May 19, 2011 (11 pages). |
| Supplementary European Search Report issued by the European Patent Office for Application No. EP 12768170.8 dated Aug. 20, 2014 (5 pages). |
| Thorpe, Philip E., “Vascular Targeting Agents as Cancer Therapeutics”, Clinical Cancer Research, vol. 10, pp. 415-427 (Jan. 15, 2004). |
| Uccellini, M. B., et al., “Autoreactive B Cells Discriminate CpG-rich and CpG-poor DNA and This Response is Modulated by IFN-α1,” J. Immunol., vol. 181, No. 9, pp. 5875-5884, Author Manuscript—21 pages (Nov. 1, 2008). |
| Vcelar, B., et al. “Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data”, AIDS, vol. 21, No. 16, pp. 2161-2170 (Oct. 18, 2007)—Abstract only—2 pages. |
| Vega-Ostertag, M., et al., “A human monoclonal antiprothrombin antibody is thrombogenic in vivo and upregulates expression of tissue factor and E-selectin on endothelial cells”, British Journal of Haematology, vol. 135, pp. 214-219 (2006). |
| Verkoczy, et al., “Induction of HIV-1 broad neutralizing antibodies in 2F5 knock-in mice: selection against membrane proximal external region-associated autoreactivity limits T-dependent responses,” The Journal of Immunology, vol. 191, No. 5, pp. 2538-2550 (Aug. 5, 2013). |
| Vogel, F. R., “The role of adjuvants in retroviral vaccines”, International Journal of Immunopharmacology, vol. 17, No. 2, pp. 85-90 (Feb. 1995)—Medline, Abstract, PMID: 7657411 (1 page). |
| Wang, Y. and Krieg, A. M., “Induction of autoantibody production but not autoimmune disease in HEL transgenic mice vaccinated with HEL in combination with CpG or control oligodeoxynucleotides”, Vaccine, vol. 22, pp. 2641-2650 (2004). |
| Wassef, N. M., et al., “Liposomes as Carriers for Vaccines,” Immunomethods, vol. 4, pp. 217-222 (1994). |
| Wei, X., et al., “Antibody neutralization and escape by HIV-1,” Nature, vol. 422, pp. 307-312 (Mar. 20, 2003). |
| Wei et al, “Viral dynamics in human immunodeficiency virus type 1 infection”, Nature, vol. 373, pp. 117-122 (Jan. 12, 1995). |
| White et al., “Antibody and cytotoxic T-lymphocyte responses to a single liposome-associated peptide antigen”, Vaccine, vol. 13, No. 12, pp. 1111-1122 (Aug. 1995)—Medline, Abstract, PMID: 7491819 (1 page). |
| Witsch, E. J., et al., “Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction,” J. Exp. Med., vol. 203, No. 7, pp. 1761-1772 (Jul. 10, 2006). |
| Wu, H., et al., “Modeling and Estimation of Replication Fitness of Human Immunodeficiency Virus Type 1 In Vitro Experiments by Using a Growth Competition Assay,” J. Virol., vol. 80, No. 5, pp. 2380-2389 (Mar. 2006). |
| Zhang, G. et al., “Neutralization of HIV-1 primary isolate by ELDKWA-specific murine monoclonal antibodies,” Immunobiology, vol. 210, No. 9, pp. 639-645 (2005). |
| Zhang, M. Y. et al., “Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on gp41 and Lacks Reactivity against Self-Antigens,” Journal of Virology, vol. 82, No. 14, pp. 6869-6879 (Jul. 2008). |
| Zhu, M., et al., “Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies”, British Journal of Haematology, vol. 105, pp. 102-109 (1999). |
| Ziegler, J. L. and Stites, D. P., “Hypothesis: AIDS Is an Autoimmune Disease Directed at the Immune System and Triggered by a Limphotropic Retrovrius”, Clinical Immunology and Immunopathology, vol. 41, pp. 305-313 (1986). |
| Zwick et al., “Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Antibodies 2F5 and 4E10 Require Surprisingly Few Crucial Residues in the Membrane-Proximal External Region of Glycoprotein gp41 to Neutralize HIV-1”, Journal of Virology, vol. 79, No. 2, pp. 1252-1261 (Jan. 2005). |
| Zwick et al., “The Long Third Complementarity-Determining Region of the Heavy Chain is Important in the Activity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5”, Journal of Virology, vol. 78, No. 6, pp. 3155-3161 (Mar. 2004). |
| Zwick, M. B. et al., “Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41,” J. Virol., vol. 75, No. 22, pp. 10892-10905 (Nov. 2001). |
| Zwick, Michael B., “The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring,” AIDS, vol. 19, pp. 1725-1737 (2005). |
| Williams, L.D., et al., Potent and broad HIV-neutralizing antibodies in memory B cells and plasma, Science Immunology, vol. 2, eaa12200, pp. 1-15 (Jan. 27, 2017). |
| “HIV Sequence Compendium 2011,” Published by Theoretical Biology and Biophysics, Group T-6, Mail Stop K710, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 U.S.A., 442 total pages including Table of Contents, Preface, Chapters I-VII (2011). |
| Huber, M. and Trkola, A., “Humoral immunity to HIV-1: neutralization and beyond,” Journal of Internal Medicine, vol. 262, pp. 5-25 (2007). |
| Scherer, E. M., et al, “Difficulties in eliciting broadly neutralizing anti-HIV antibodies are not explained by cardiolipin autoreactivity”, AIDS, vol. 21, No. 16, pp. 2131-2139 (Oct. 18, 2007). |
| Burton, D. R., et al., “Efficient Neutralization of Primary Isolates of HIV-1 by a Recombinant Human Monoclonal Antibody”, Science, vol. 266, pp. 1024-1027 (1994). |
| Burton, D. R., et al., “HIV vaccine design and the neutralizing antibody problem”, Nature Immunol. vol. 5, No. 3, pp. 233-236 (2004). |
| Choe, H. et al., “Tyrosine Sulfation of Human Antibodies Contributes to Recognition of the CCR5 Binding Region of HIV-1 gp120”, Cell, vol. 114, No. 2, pp. 161-170 (2003). |
| Derdeyn, C. A., et al., “Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120”, Journal of Virology, vol. 74, pp. 8358-8367 (2000). |
| DeRue, G.J., et al., “Fetal loss in systemic lupus: association with anticardiolipin antibodies”, J. Obest. Gynecol. Neonatal Nurs., vol. 5, pp. 207-209 (1985). |
| Fox, R. A. and Isenberg, D. A., “Human Immunodeficiency Virus Infection in Systemic Lupus Erythematosus,” Arth. Rheum., vol. 40, pp. 1168-1172 (1997). |
| Gregoriadis, Gregory, “Liposome Technology: vol. III, Interactions of Liposomes with the Biological Milieu,” Chapter 21, pp. 317-343 (1993). |
| Grunewald, T., et al., “Anti-phospholipid antibodies and CD5+ B cells in HIV infection”, Clin. Exp. Immunol., vol. 115, pp. 464-471 (1999). |
| Harris, E. N., et al., “Evaluation of the anti-cardiolipin antibody test: report of an international workshop held Apr. 4, 1986”, Clin. Exp. Immunol., vol. 68, p. 215-222 (1987). |
| Hemmi, H. et al., “A Toll-like receptor recognizes bacterial DNA,” Nature, vol. 408, No. 6813, pp. 740-745 (Dec. 7, 2000) (includes erratum which was published in Nature, vol. 409, p. 646 and dated Feb. 1, 2001). |
| Hohman, V. S., et al., “Genomic clone for sandbar shark λ light chain: Generation of diversity in the absence of gene rearrangement”, Proc. Natl. Acad. Sci. USA, vol. 90, pp. 9882-9886 (1993). |
| Huse, W. D., et al., “Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda,” Science, vol. 246, pp. 1275-1281, 8 total pages (1989). |
| Kunert, R. E., et al., “Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5,” AIDS, vol. 16, Issue 4, pp. 667-668, 4 pages (Mar. 8, 2002). |
| Kunert, R., et al., “Characterization of Molecular Features, Antigen-Binding, and in Vitro Properties of IgG and IgM Variants of 4E10, an Anti-HIV Type 1 Neutralizing Monoclonal Antibody,” AIDS Res. Hum. Retroviruses, vol. 20, No. 7, pp. 755-762 (2004). |
| Liao, H.-X., et al., “Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins,” Journal of Virology, vol. 78, No. 10, pp. 5270-5278 (May 2004). |
| Litman, G. W., et al., “Evolution of Antigen Binding Receptors,” Ann. Rev. Immunol., vol. 17, pp. 109-147 (1999). |
| Mascola, J. R., et al., “Recommendations for the Design and Use of Standard Virus Panels to Assess Neutralizing Antibody Responses Elicited by Candidate Human Immunodeficiency Virus Type 1 Vaccines,” Journal of Virology, vol. 79, No. 16, pp. 10103-10107 (Aug. 2005). |
| Meffre, E., et al., “Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development,” J. Clin. Invest., vol. 108, pp. 879-886 (2001). |
| Moll, S. and Ortel, T. L., “Monitoring Warfarin Therapy in Patients with Lupus Anticoagulants,” Annals of Internal Medicine, vol. 127, No. 3, pp. 177-185 (Aug. 1, 1997). |
| Moore, J. P., et al., “Development of the Anti-gp120 Antibody Response during Seroconversion to Human Immunodeficiency Virus Type 1,” Journal of Virology, vol. 68, No. 8, pp. 5142-5155 (Aug. 1994). |
| Moore, J. P., et al., “Studies with Monoclonal Antibodies to the V3 Region of HIV-1 gp120 Reveal Limitations to the Utility of Solid-Phase Peptide Binding Assays”, J. Acquir. Immun. Def. Syn., vol. 7, No. 4, pp. 332-339 (1994). |
| Nyambi, P. N., et al., “Conserved and Exposed Epitopes on Intact, Native, Primary Human Immunodeiciency Virus Type 1 Virions of Group M,” Journal of Virology, vol. 74, No. 15, pp. 7096-7107 (Aug. 2000). |
| Pancio, H. A., et al., “Interaction of Human Immunodeficiency Virus Type 2 Vpx and Invariant Chain”, Journal of Virology, vol. 74, pp. 6168-6172 (Jul. 2000). |
| Platt, E. J., et al., “Effects of CCR5 and CD4 Cell Surface Concentrations on Infections by Macrophagetropic Isolates of Human Immunodeficiency Virus Type 1,” Journal of Virology, vol. 72, No. 4, pp. 2855-2864 (Apr. 1998). |
| Ramsland, P. A., et al., “Incorporation of Long CDR3s into V Domains: Implications for the Structural Evolution of the Antibody-Combining Site,” Exp. Clin. Immunogenet, vol. 18, pp. 176-198 (2001). |
| Robinson, J. E., et al., “Identification of Conserved and Variant Epitopes of Human Immunodeficiency Virus Type 1 (HIV-1) gp120 by Human Monoclonal Antibodies Produced by EBV-Transformed Cell Lines,” AIDS Res. Hum. Retrovirol., vol. 6, p. 567-579 (1990). |
| Ruprecht, R. M. et al., “Antibody protection: passive immunization of neonates against oral AIDS virus challenge,” Vaccine, vol. 21, No. 24, pp. 3370-3373 (Jul. 28, 2003). |
| Scanlan, C. N., et al., “The Carboydrate Epitope of the Neutralizing Anti-HIV-1 Antibody 2G12,” Glycobiology and Medicine,Kluwer Academic/Plenum Publishers, New York, pp. 205-218 (2003). |
| Stiegler, G., et al., “A Potent Cross-Glade Neutralizing Human Monoclonal Antibody against a Novel Epitope on gp41 of Human Immunodeficiency Virus Type 1,”AIDS Res. Hum. Retroviruses, vol. 17, No. 18, pp. 1757-1765 (2001). |
| Trkola, A., et al., “Human Monoclonal Antibody 2G12 Defines a Distinctive Neutralization Epitope on the gp120 Glycoprotein of Human Immunodeficiency Virus Type 1,” Journal of Virology, vol. 70, No. 2, pp. 1100-1108 (Feb. 1996). |
| Wei, X., et al., “Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy,” Antimicrobial Agents and Chemotherapy, vol. 46, No. 6, pp. 1896-1905 (Jun. 2002). |
| Weiss, L., et al., “Anti-Cardiolipin Antibodies Are Associated with Anti-Endothelial Cell Antibodies but Not with Anti-beta2 Glycoprotein I Antibodies in HIV Infection,” Clin. Immunol. Immunopathol., vol. 77, No. 1, pp. 69-74 (1995). |
| Yuste, E. et al., “Simian Immunodeficiency Virus Engrafted with Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Epitopes: Replication, Neutralization, and Survey of HIV-1-Positive Plasma,” Journal of Virology, vol. 80, No. 6, pp. 3030-3041 (Mar. 2006). |
| Zhang, H., et al., “Induction of Mucosal and Systemic Neutralizing Antibodies against Human Immunodeficiency Virus Type 1 (HIV-1) by Oral Immunization with Bovine Papillomavirus-HIV-1 gp41 Chimeric Virus-Like Particles,” Journal of Virology, vol. 78, No. 15, pp. 8342-8348 (Aug. 2004). |
| Zolla-Pazner, S., et al., “The Cross-Clade Neutralizing Activity of a Human Monoclonal Antibody Is Determined by the GPGR V3 Motif of HIV Type 1,” Zolla-Pazner et al., AIDS Res. Hum. Retrovirol., vol. 20, No. 11, pp. 1254-1258 (2004). |
| Zwick, M. B., et al., “Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human ImmunodeficiencyVirus Type 1 gp120,” Journal of Virology, vol. 77, No. 10, pp. 5863-5876 (May 2003). |
| Albert, J., et al, “Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera,” AIDS, vol. 4, No. 2, pp. 107-112, 8 pages in total (Feb. 1990). |
| Alving, C. R. and Rao, M., “Lipid A and liposomes containing lipid A as antigens and adjuvants,” Vaccine, vol. 26, pp. 3036-3045 (2008). |
| Baba, T. W., et al, “Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection,” Nat. Med., vol. 6, No. 2, pp. 200-206 (Feb. 2000). |
| Beck, Z., et al, “Membrane-specific antibodies induced by liposomes can simultaneously bind to HIV-1 protein, peptide, and membrane lipid epitopes,” J. Drug Target, vol. 16, Nos. 7-8, pp. 535-542 (Aug.-Sep. 2008). |
| Buchacher, A., et al, “Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization,” AIDS Res. Hum. Retroviruses, vol. 10, No. 4, pp. 359-369, 13 pages in total (Apr. 1994). |
| Decker, J.M., et al, “Failure of the HIV-1 Membrane Proximal External Region (MPER) of HIV-1 gp41 to Elicit 4E10 or 2F5-like Neutralizing Antibodies in Natural Human Infection,” presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, Colo., 1 page in total (2006). |
| Douvas, A. and Takehana, Y., “Cross-Reactivity between Autoimmune Anti-U1 snRNP Antibodies and Neutralizing Epitopes of HIV-1 gp120/41”, AIDS Research and Human Retroviruses, vol. 10, No. 3, pp. 253-262, 12 pages in total (Mar. 1994). |
| Douvas, A., et al, “Neutralization of HIV Type 1 Infectivity by Serum Antibodies from a Subset of Autoimmune Patients with Mixed Connective Tissue Disease”, AIDS Research and Human Retroviruses, vol. 12, No. 16, pp. 1509-1517, 11 pages in total (Nov. 1, 1996). |
| Hemmi, H., et al, “Small anti-viral compounds activate immune cells via the TLR7 MyD88 dependent signaling pathway,” Nat. Immunol., vol. 3, No. 2, pp. 196-200 (Feb. 2002). |
| Hemmi, H., et al, “A Toll-like receptor recognizes bacterial DNA”, Letters to Nature, vol. 408, pp. 740-745 (Dec. 2000). |
| Leadbetter, E.A., et al, “Chromatin—IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors,” Nature, vol. 416, pp. 603-607 (Apr. 11, 2002). |
| Matyas, G.R., et al, “Neutralizing antibodies induced liposomal HIV-1 glycoprotein 41 peptide simultaneously bind to both the 2F5 or 4E10 epitope and lipid epitopes,” AIDS, vol. 23, No. 16, pp. 2069-2077 (2009). |
| Ruprecht, R.M., et al, “Antibody protection: passive immunization of neonates against oral AIDS virus challenge,” Vaccine, vol. 21, pp. 3370-3373 (2003). |
| Sanchez-Martinez, S., et al, “Membrane Association and Epitope Recognition by HIV-1 Neutralizing Anti-gp41 2F5 and 4E10 Antibodies,” AIDS Res. And Human Retrovir., vol. 22, No. 10, pp. 998-1006 (2006). |
| Schuster, B.G., et al, “Production of Antibodies against Phosphocholine, Phosphatidylcholine, Sphingomyelin, and Lipid A by Injection of Liposomes Containing Lipid A,” J. Immunol., vol. 122, No. 3, pp. 900-905 (Mar. 1979). |
| Takeda, K., et al, “Toll-like Receptors,” Annu. Rev. Immunol., vol. 21, pp. 335-376, 44 pages in total (2003). |
| Tran, T.T., et al, “Specificity and Immunochemical Properties of anti-DNA Antibodies Induced in Normal Mice by Immunization with Mammalian DNA with a CpG Oligonucleotide as Adjuvant,” Clin. Immunol., vol. 109, pp. 278-287 (2003). |
| Trkola, A., et al, “Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies”, Nature Medicine, vol. 11, No. 6, pp. 615-622 (Jun. 2005). |
| Weill, J.-C., et al, “Human Marginal Zone B Cells,” Annu. Rev. Immunol., vol. 27, pp. 267-285 (2009). |
| Number | Date | Country | |
|---|---|---|---|
| 20170014503 A1 | Jan 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61883306 | Sep 2013 | US |
