WO2015127108A1 - Trimeric hiv-1 envelopes compositions and uses thereof - Google Patents

Abstract

The invention provides immunogenic compositions which comprise HIV-1 envelope trimers in and a liposome. In other aspects the invention provides method of using compositions HIV-1 envelope trimers to induce immune response in a subject. The invention further provides methods to make compositions which comprise HIV-1 envelope trimers and a liposome.

Description

TRIMERIC HIV-1 ENVELOPES COMPOSITIONS AND USES THEREOF

[0001 ] The application claims the benefit of U.S. Application Ser. No. 61/941 ,902 filed February 19, 2014 and U.S. Application Ser. No. 61/973,414 filed April 1 , 2014, the entire contents of which applications are herein incorporated by reference.

[0002] This invention was made with government support under Center for HIV/ AIDS Vaccine Immunology-Immunogen Design grant UM1 -All 00645 from the NIH, NIAID, Division of AIDS. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates in general, to a composition suitable for use in inducing anti-HIV-1 antibodies, and, in particular, to immunogenic compositions comprising envelope proteins to induce cross-reactive neutralizing antibodies with increased the breadth of coverage. The invention also relates to methods of inducing such broadly neutralizing anti-HIV-1 antibodies using such compositions.

BACKGROUND

[0004] One approach to designing HIV-1 immunogens is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Most approaches to making Env trimers involve truncating the gp41 component to remove the hydrophobic transmembrane region, yielding soluble gpl40 proteins containing three gpl20 and gp41 ectodomain (gp4l_EC_To) subunits. Soluble gpl40 trimers are highly unstable, perhaps because the inherently labile nature of the Env complex is exacerbated by the removal of the transmembrane region. Accordingly, g l40 trimers rapidly disintegrate into individual gpl20 and gp4l Ecro subunits unless preventative steps are taken. Two different methods have been used to stabilize gpl40 trimers. The most widely used involves eliminating the cleavage site between gpl20 and gp4lECTO and, in some cases, adding an additional trimer-stabilizing motif to the C-terminus of gp4lEcro, with or without other modifications. Trimer-forming constructs such as these are generally referred to as uncleaved gpl40s (gpl40uNc)- One alternative approach involves making fully cleaved trimers but stabilizing them by introducing specific mutations, namely a disulfide bond to covalently link gpl20 to gp4lECTO and an lie/Pro change at residue 559 to strengthen interactions between the gp41 subunits. The resulting trimers are designated SOSIP gpl40s. Cleaved and uncleaved trimers are known to be antigenically distinct, in that the latter consistently express the epitopes for various non-neutralizing antibodies (non-NAbs) that are occluded on cleaved trimers, irrespective of whether the Env proteins are soluble or expressed on the cell surface. See for example Sanders RW, Derking R, Cupo A, Julien J-P, Yasmeen A, et al. (2013) A Next- Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gpl 40, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies. PLoS Pathog 9(9): el 003618. doi: 10.1371/journal.ppat.l003618 and references therein; see Grundner C,

Mirzabekov T, Sodroski J, Wyatt R. Solid-phase proteoliposomes containing human

immunodeficiency virus envelope glycoproteins. J Virol. 2002;76:351 1-3521.

SUMMARY

[0005] In certain aspects the invention provides compositions comprising multimeric envelope complexes, for example envelope trimers, suitable for use as immongens; and methods for making such compositions. In certain embodiments the trimers are purified using 2G12 antibody. In certain aspects the invention provides compositions comprising multimeric envelope complexes suitable for use as immongens, wherein the envelope complexes are embedded in liposome. In certain aspects the invention provides methods to make the composition comprising such multimeric envelopes, and methods of using such compositions as immunogens.

[0006] In certain aspects the invention provides a modified HIV-1 envelope comprising consecutive amino acids of a gpl20 polypeptide portion, consecutive amino acids of a gp41 , including MPER domain and TM domain, a cleavage site mutation, Y712A mutation, wherein the envelope is expressed in a cell so as to allow cell surface membrane expression of the envelope as a multimeric complex, followed by purification and reconstitution of the multimeric complex in a synthetic liposome. In some embodiments, the envelope is a gpl40. In other embodiments, the envelope is a gpl 50. In other embodiments the envelope is a gpl 60.

[0007] In certain aspects the invention provides methods to stabilize HIV-1 trimer complexes which are embedded in synthetic liposomes. The trimer complexes comprise envelopes which do not include modifications conferring additional non-covalent links.

[0008] In certain aspects the invention provides an engineered or non-naturally occurring HIV-1 envelope comprising a modification which improves cell surface expression, purification, stability as a trimer in a cell membrane or in a liposome, binding to broadly neutralizing antibodies, and/or limits binding to non-neutralizing antibodies. In certain embodiments the engineered or non-naturally occurring HIV-1 envelope is recombinantly expressed as a cell surface membrane protein and purified as a trimer. In certain embodiments the engineered or non-naturally occurring HIV-1 envelope is purified from cell membranes and reconstituted as trimer in a liposome.

[0009] In certain aspects the modification includes amino acid changes which remove the envelope cleavage site, whereby the envelope is not cleaved. In some embodiments the modification is Y721 A mutation. In some embodiments the modification is Y712A mutation. In some embodiments the envelope is cleavage site deficient and also includes the Y- A mutation. The envelope does not comprise a SOSIP modification (See US 20140212458 Al).

[0010] In certain aspects the invention provides methods for producing a stabilized timeric envelope complex comprising: a) recombinantly expressing an engineered or non-naturally occurring HIV-1 envelope, b) purifying the engineered or non-naturally occurring HIV-1 envelope from the cell membrane under conditions which retain the envelope as a trimer, and c) reconstituting the trimer in a complex with a liposome.

[001 1 ] In certain aspects the invention provides a composition comprising an HIV-1 envelope polypeptide trimer complex in a liposome. In certain aspects the invention provides a composition comprising an HIV-1 envelope polypeptide trimer complex reconstituted in a liposome. In certain aspects, the invention provides a composition comprising a modified HIV-1 envelope polypeptide in a trimer complex stabilized in a liposome, wherein the modified envelope comprises amino acid changes which remove the envelope cleavage site, whereby the envelope is not cleaved, and the modified envelope comprises a Y721 A mutation.

[0012] In certain embodiments, the composition comprises an envelope which is recombinantly expressed on the cell surface, isolated from the membranes of the expression cell line, purified by antibody affinity chromatography, solubilized in detergent and reconstituted to be embedded in a liposome. In certain embodiments, the composition comprises a non-naturally occurring envelope embedded in a liposome. In certain embodiments, the composition mimics a naturally occurring envelope trimer presented in a lipid membrane. In certain embodiments, the composition is non-naturally occurring. In certain embodiments, the composition comprises about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% trimers. In certain embodiments, the composition comprises 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% trimers. In certain embodiments, the composition comprises at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% trimers. EM or any other suitable methods could be used to determine the % trimer in the composition. Certain non-limiting examples of envelope sequences which can be expressed and isolated by the methods of the invention so as to yield trimer envelopes reconstituted in a liposome are shown in Figure 30. It is readily appreciated that the envelopes reconstituted in liposomes do not include the N-terminal/signal peptide sequence used to facilitate the expression of the envelope.

[0013] In certain aspects the invention provides a trimer wherein the HIV-1 envelope polypeptide is a gpl60 or gpl 50 and comprises the MPER domain, or the TM domain, or both. In certain aspects the invention provides a trimer wherein the HIV-1 envelope polypeptide is a gpl 60 or gpl 50 and comprises the TM domain. The gpl50 protein includes amino acids 1 -808 of the gpl60 envelope (with reference to the amino acid sequence of JRFL). In certain embodiments, the HIV-1 envelope polypeptide complex is trimeric.

[0014] In certain embodiments the composition of the invention further comprise an adjuvant. In certain embodiments, the liposome further comprises an adjuvant.

[0015] In certain aspects the invention provides a trimer wherein the HIV-1 envelope polypeptide complex is purified by 2G12 affinity chromatography from HIV-1 envelope expressed on the cell surface, and wherein the membrane lipids are exchanged for a liposome comprising synthetic lipids. In certain embodiments, the liposome comprises synthetic lipids.

[0016] In certain aspects the HIV-1 envelope polypeptide trimer complex in a liposome shows preferential binding for bNabs versus non-neutralizing antibodies. [0017] In certain aspects the HIV-1 polypeptide is any other suitable envelope, wherein the envelope is cleavage site deficient, for example comprising SEKS sequence at the gpl20/gp41 envelope cleavage site, and comprises a mutation corresponding to JRFL Y721 A or Y712A. In non-limiting embodiments the envelope which can be expressed and purified by the methods of the invention is any one of the HIV-1 envelopes or selection of envelopes as described below: CH505 envelopes in Application WO2014042669 (PCT/US2013/000210), U.S. Application Ser. No. 61/955,402 ("Swarm Immunizaton with Envelopes form CH505" Examples 2-4, Figures 14- 19); US Application Ser. Nos. 61/972,531 and 62/027,427 (Examples 2-3, Figures 18-24);

CH848 Envs to induce V3 glycan Abs (See U.S. Application Ser. No. 61/972,649); VRC26 Envs to induce V1V2 glycan Abs (See Doria-rose NA et al. Nature 509: 55-62, 2014); CH01- CH04 bnAb heterologous Envs- V1V2 glycan (See Liao HX et al. Immunity 38: 176, 2013; Bonsignori M et al J. Virol 85: 9998, 201 1); CH1754 Envs to induce CD4 bs Abs (See U.S. Application^' Ser. No. 61/884,014); Group M B cell Mosaic Envs (See U.S. Patent Application Ser. No. 61/884,696. Series of CAP206 Envs to induce MPER Abs; mosaic envelopes (See e.g. sequences disclosed in US Patent No. 7951377), Con-S (See e,g. sequences disclosed in US Patent 8071107, Clade C envelope 1086.C, clade B envelope B.63521 (See e.g. US Pub

20120321699, Abrahams et al, J. Virol. 2009, 83(8):3556. DOI: 10.1 128/JVI.02132-08), the contents of all of these applications and publications are herein incorporated by reference in their entirety.

[0018] In certain aspects the invention provides a trimer comprising any one suitable HIV-1 polypeptide envelope, wherein the HIV-1 polypeptide is "JF-3", "JF-7", or "JF-8" variant as referenced for the JRFL envelope in Figure 30. [0019] In certain aspects the invention provides a vector comprising a nucleic acid encoding the HIV-1 envelope of the invention, wherein the vector is an expression vector and the nucleic acid is operably linked to a promoter. In certain aspects the vector comprises a nucleic acid wherein the nucleic acid comprises a signal peptide at the N-terminus of the HIV-1 envelope. In certain embodiments the nucleic acid is listed in Figure 30. The signal peptide is removed during expression and processing of the polypeptide in the cell, whereby the envelope expressed on the cell surface and which is purified as trimer does not have the signal peptide.

[0020] In certain aspects the invention provides a cell comprising a vector which comprises a nucleic acid encoding an HIV-1 envelope which is expressed on the cell surface, as a trimer.

[0021] In certain aspects the invention provides a method of inducing an immune response in a subject comprising administering a composition of the invention which comprises a trimeric HIV- 1 envelope complexed in a liposome. In certain aspects the composition is immunogenic. In certain aspect the composition administered in the methods further comprises an adjuvant. A method of inducing HIV-1 antibodies in a subject comprising administering to a subject in need thereof any one of the composition of the invention. Various immunization schedules and routes for administering immunogenic compositions are known in the art and contemplated by the methods. A skilled artisan can readily determine the dose, routes and frequency of administering of the compositions so as to induce HIV-1 envelope antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows a non-limiting embodiment of a process for purification of multimeric envelope complexes using 2G12 affinity purification followed by exchange of the lipid rafts for synthetic lipids. The multimeric complexes are not solubilized prior to binding of the 2G12 antibody. Multimeric envelope complexes are maintained in stable/native like conformation, i.e. aggregation of the monomers is reduced or prevented, due to their anchoring in the lipid rafts before and during the 2G12 affinity purification. After binding to the 2G12 antibody, the raft lipids are solubilized, and the multimeric complexes are maintained in stable conformation due to their binding to the 2G 12 antibody. Liposome containing multimeric complexes are reconstituted using synthetic lipids, and the interaction with 2G12 antibody is disrupted to form multimeric envelope liposomes comprising synthetic lipids and multimeric envelope complex.

[0023] Figure 2 shows isolation of detergent resistant membrane microdomains (DRM) with Env proteins. DRM was isolated by sucrose density centrifugation from cell membrane of Env- expressing CHO cells. 1 mL fractions were collected from the gradient run and analyzed by SDS-PAGE (non-reducing) and western blot analysis. Proteins enriched in DRM fractions (5-8) show Env proteins by western blot analysis with a gpl20 mAb (16H3). Lane 14 includes soluble gpl40 protein showing heterogeneous mixture of monomer, dimer (disulfide linked) and trimer.

[0024] Figure 3 shows Purification of gpl40 trimers by 2G12 affinity chromatography. A) Affinity chromatography profile of flow through fractions and eluted bound gpl40 protein following low pH gradient. B) BN-gel of gpl40 protein, flow-through wash from 2G12 column (wash 1 , wash 2) and eluent fractions. 2G12 eluent lane shows predominance of trimers with trace amount of dimer and no monomeric gpl40.

[0025] Figure 4 shows 2G12 purified Env trimers bind preferentially to broad neutralizing antibodies. SPR analysis show enhanced binding of 2G12 purified trimers to CD4 binding site BnAbs (A), and V3-glycan dependent BnAb (B, PGT 128). In contrast the non -neutralizing V3 mAb bound weakly to 2G12-purified trimers.

[0026] Figure 5 shows SDS-PAGE (A) and blue-native (B) gel analysis of DRM-associated Env proteins. Total proteins from DRM (lipid raft) fraction and DRM-associated ENv proteins following 2G12 affinity purification are shown in A. Western blot analysis were done using the gpl20 mAb 16H3 following both transfers from both SDS-PAGE and BN-gels.

[0027] Figure 6 shows Antigenicity analysis of DRM-associated Env trimer. DRM-associated Env trimer was captured on a hydrophobic LI sensor chip (BIAcore/GE Healthcare) as described previously (Alam et al., 2007) and each of the listed mAbs were injected to monitor binding by SPR analysis. Binding of 2G12 (B), CD4 binding site antibodies (bs) (C) and V3 mAbs (D) are shown. DRM-associated Env trimer bound strongly to 2G12, and showed preferential binding to CD4 bs BNAb CHI 06 and the V3/glycan BnAb PGT128.

[0028] Figure 7 shows that 2G12 purified gpl40 is primarily trimeric by BN gel analysis.

[0029] Figure 8 shows that 2G12 affinity selection enhances binding and improves on rate for CD4bs antibodies VRCOl (8A), CHI 03 (8B), and CH103UCA (8C).

[0030] Figure 9 shows that 2G12 selection selectively enhances V3 BnAb binding (9A)while reducing Non-neutralizing (nN) Ab binding (9B).

[0031 ] Figure lOA-C shows that 2G12 purification decreases binding to V2 non-neutralizing Abs.

[0032] Figure 1 1 A-C shows that 2G12 purification increases binding to open conformation dependent antibodies 17B and F105.

[0033] Figure 12 shows that BnAbs show higher binding than non BnAbs to CH505 gpl60.

[0034] Figure 13 shows that membrane bound gpl 60 shows preferential binding to BnAbs at the CD4 binding site.

[0035] Figure 14 shows that membrane bound gpl60 shows preferential binding to BnAbs at the V3 binding site. [0036] Figure 15 shows a non-limiting embodiment of a protocol for isolation and reconstitution of membrane bound Envelope. Any suitable cell line could be used for expression of membrane protein. Any suitable detergent, for example a non-ionic detergent, could be used instead of or in addition to 0rij 58 (polyoxyethylene acyl ether). The detergent could be anywhere from 0.01% to 1%, 0.1% to 1%, 0.5% to 1%. Any suitable bNAb could be used in the BnAb Affinity Chromatography step. Any suitable detergent could be used instead of or in addition to Cymal 5/Cymal 2) to solubilize the Env. Any suitable lipids could be used to

[0037] Figure 16 shows CH505 T/F membrane Env migrates as a trimeric gpl 50 protein in Blue Native gel and by size exclusion chromatography.

[0038] Figure 17 shows that Low constitutive 17b binding indicates that the membrane derived Env protein is in a pre-CD4 triggered conformation.

[0039] Figure 18 shows membrane derived CH505 Env gpl 50 binds to the glycan dependent quaternary V1V2 mAb PGT145.

[0040] Figure 19 shows Summary of Antigenicity of Env CH505 T/F gpl 50.

[0041 ] Figure 20 shows Liposome reconstitution of detergent solubilized CH505 gpl 50. A pictorial representation of methodology employed in reconstituting Cymal-2 solubilized CH505 g l 50 into liposomes is shown. Liposomes comprised of POPC, POPE, Sphingomyelin and Cholesterol at a 35:30:15:20 molar ratio.

[0042] Figure 21 shows liposome reconstituted CH505 gpl 50 retains antigenicity for key neutralizing antibodies. A: A schematic diagram of the Biolayer interferometry (BLI) assay used to study the binding of Env specific antibodies to CH505 gpl 50 liposomes reconstituted at a protein to total lipid ratio of 1 :3000 is shown. B-H: The BLI sensograms of key neutralizing antibodies (B, D, E, G and H), the unmutated common ancestor (CHI 03 UCA) of broadly neutralizing CD4 binding site specific mAb CH 106 (F) and a non-neutralizing CD4 binding site specific antibody binding to CH505 gpl 50 liposomes are shown. The data shown are specific binding of mAbs and were corrected for non-specific binding to lipids by subtracting the binding response of mAbs to mock reconstituted blank liposomes.

[0043] Figure 22 shows set up for antigenicity determination of membrane-associated (DRM) Env.

[0044] Figure 23 shows preferential binding of V3 BnAb PGT 128 to membrane-associated CH505 Env.

[0045] Figure 24 shows Stronger binding of CD4 binding site BnAb CH31 than non-neutralizing mAb F105.

[0046] Figure 25 shows that both the mature CHI 06 and its UCA bind to membrane-associated Env.

[0047] Figure 26 shows binding of V1V2 Quaternary BnAb PGT 145 to membrane-associated Env.

[0048] Figure 27 shows CD4i mAb (17b) constitutive and sCD4-induced binding to membrane associated CH505 Env.

[0049] Figure 28. EM images of CH505 gpl40 show trimeric proteins.

[0050] Figure 29 shows set up of BLI antigenicity experiment.

[0051 ] Figure 30A shows sequences of HIV- 1 envelopes (JRFL unmodified and modified) which are expressed and purified as trimeric complex. SEQ ID NO: 1 (JRFL), SEQ ID NO: 2 (JF-3), SEQ ID NO: 3 (JF-7), and SEQ ID NO: 4 (JF-8) in order of appearance. Figure 30B shows CH505 envelope sequences which are not modified. SEQ ID NO: 5 (CH505 T/F aa), SEQ ID NO: 6, SEQ ID NO: 7 (CH505 w53 aa), SEQ ID NO: 8, SEQ ID NO: 9 (CH505 wlOO aa), SEQ ID NO: 10, SEQ ID NO: 1 1 (CH505 w78 aa), and SEQ ID NO: 12 in order of appearance. Specific modification as indicated for JRFL sequences in Figure 30A could also be introduced in other envelopes, for example the CH505 envelopes of Figure 30B. CH505TF JF-8 sequence is shown in Figure 30B (SEQ ID NO: 13 and 14).

DETAILED DESCRIPTION

[0052] Induction of high titer of broadly neutralizing antibodies can be protective against HIV-1 infection since passive immunization with select broadly neutralizing antibodies have been shown to protect non-human primates from HIV-1 infection at low titers (Hessel et al. 2009, PLoS Pathogen). However, only modest broadly neutralizing responses develop in about 20% of HIV-1 infected subjects but such antibodies appear after 1-2 years after transmission (Gray et al., 2009, J. Virol, 83:8925; Sather et al, 2009; J. Virol., 83:757; Walker et al., 2010, PloS

Pathogen, 6: 1 ; Shen et al. 2009; J. Virol., 83:3617). Current HIV-1 Envelope immunogens include monomeric gpl20 and oligomeric gpl40 proteins. To date, such immunogens when used in experimental animal studies or in human vaccine trials have failed to induce high-titer of protective antibody responses. Most antibodies that arise during infection and vaccination are strain-specific or non-neutralizing (Haynes et al, 2012, N. Engl. J. Med, 366: 1275; Montefiori et al, 2012; J. Infect. Dis., 206:431 ; Liao et al., 2013, Immunity, 38: 176) and therefore, are not able to protect against the highly mutating and diverse population of HIV-1 viruses. One explanation for the lack of the ability of current immunogens to induce neutralizing antibodies is that the recombinantly produced soluble Env proteins do not present the correct conformation of the epitopes recognized by broadly neutralizing antibodies.

[0053] Currently used immuogens include recombinantly produced soluble monomeric gpl20 and oligomeric gpl40 proteins, neither of which are close mimics of the trimeric Env expressed on the surface of HIV- 1 virions (Subramaniam, 2013, Trends Microbiol.)- In these immunogens, the membrane spanning and cytoplasmic domains are deleted, primarily to facilitate expression and purification. Although gpl40 proteins are oligomeric, the majority of gpl40 proteins is a heterogeneous mixture of monomers, dimers and trimers, and includes aberrant disulfide-linkage that can promote non-native folding of proteins and therefore, could alter antigenicity of the proteins. Thus a major hurdle in HIV-1 immunogen design is the preservation of structural properties in soluble version of Env proteins that mimic those on intact HIV-1 viruses. The recently described structure of the Env trimer (SOSIP) (Lyumkis et al., Science, 2013; Julien et al., Science, 2013) presents a significant advance in producing trimeric forms of soluble Env proteins, and a significant improvement upon the currently designed Env gpl20 and gpl40 proteins (Ringe et al., 2013, PNAS) . However, the SOSIP trimeric Env design does not present the fully intact Env and several non-native modifications were introduced to stabilize the Env trimer proteins. The SOSIP trimer lacks of transmembrane segment; also the MPER (membrane proximal external region) region, which is the site of several broadly neutralizing antibodies, was deleted; and non-native disulfide linkage was introduced to stabilize the trimeric structure.

[0054] The invention provides methods to isolate intact membrane bound trimeric HIV-1 Env from cell membrane and to develop non-naturally occurring membrane bound Env trimer as immunogens for the induction of broadly neutralizing antibodies against HIV- 1. Any HIV envelope could be isolated and reconstituted in liposomes by the methods of the invention.

[0055] Various strategies of the envelope isolation and reconstitution are illustrated in Figure 1 , Figure 15. In certain embodiments, the envelope is an envelope including various sequence designs and modification such as described in Figure 30A for JRFL sequences named JRFL, -3, - 7, -8 variants. In certain embodiments, these modifications include a cleavage site mutation(s), Y712A mutation, truncation of gp41 sequences, as described for JRFL in Figure 3 OA, amino acid sequence from position 1-808, or any combination thereof. In certain embodiments, the modification are introduced in a gpl60 envelopes, gp 50 envelope, gpl45, gpl40 or any other envelope fragment. A skilled artisan can readily determine the corresponding positions and changes in other envelopes, for e.g CH505 envelopes as described in Figure 30B.

[0056] It is understood that any suitable cell line could be used for expression of the envelopes of the invention. Non-limiting examples include CHO cells, HEK293T cells. Likewise any suitable promoter, vector or expression cassette could be used for the expression of envelopes of the invention. Various methods to design and optimize sequences for various expression systems are known in the art and are contemplated by the invention. Methods to design and target envelope for cell surface expression are known in the art.

[0057] The invention provides methods for production of native-like trimeric Env immunogen in which the Env is extracted from cell surface, and is fully intact and preserved in its membrane bound conformation. In some embodiments, the membrane bound Env trimer is purified as detergent resistant microdomains (DRM) and affinity purified using a monoclonal antibody that selectively binds to Env trimer.

[0058] The trimeric Env protein is reconstituted with synthetic lipids to form the equivalent of fully intact membrane bound HIV-1 Env trimer that mimics the antigenicity of pre-fusion state of the Env structure on HIV-1 virions. Various methods for Abs affinity measurement are known in the art and can be used to determine the properties of the reconstituted trimer. For example real time, probe free, in solution, low intensity spectroscopic methods which allow for accurate, quantitative, non-invasive measurements. Non-limiting examples include Bio-Layer

interferometry (BLI), Surface Plasmon resonance (SPR). [0059] The reconstituted membrane-like bound Env trimer will be a design of HIV- 1 vaccine that selectively presents the epitopes of sub-dominant neutralizing antibodies and hinder those of the non-neutralizing antibodies.

[0060] One approach to inducing broadly neutralizing antibodies (bNAbs) to HIV-1 is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible. The present invention provides methods to purify oligomeric, for example trimeric, envelope complexes for use as immunogens. These oligomeric envelope glycoproteins closely mimic trimers on the surface of the virus, or on the surface of virus expressing cells. In certain embodiments, these inventive envelope complexes are embedded in liposomes which comprise synthetic lipids. The synthetic lipids present the trimeric envelope complex in a native membrane bound environment.

[0061] Reconstitution of the Env trimers into the membrane bound form will be done using any suitable lipid compositions. The final choice of the lipid composition is based on Env stability and antigenicity.

[0062] Liposome compositions: contemplated for use in the inventive compositions are any suitable lipids, for example but not limited to any anionic lipids. Phospholipids such as but not limited to cholesterol, cardiolipin, phosphatidylserine, phosphatidylethanolamine,

phosphatidylcholine, phosphotidylinositol, sphingomyelin, and derivatives thereof, e.g., 1 - palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine](POPS), 1 -palmitoyl-2-oleoyl- phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine (DOPE), or any combination thereof. Use of hexagonal II phases of phospholipids can be advantageous and phospholipids that readily form hexagonally packed cylinders of the hexagonal II tubular phase (e.g., under physiological conditions) are preferred, as are phospholipids that can be stabilized in the hexagonal II phase. (See Rauch et al, Proc. Nati. Acad. Sci. USA 87:41 12-41 14 (1990); Aguilar et al et al, J. Biol. Chem. 274: 25193-25196 (1999)).

[0063] Liposomes suitable for use in the invention include, but are not limited to, those comprising POPC, POPE, DMPA (or sphingomyelin (SM)), lysophosphorylcholine,

phosphatidylserine, and 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 (l ,2-dimyristoyl-sn-glycero-3-phosphocholine) (or lysophosphorylcholine), cholesterol (Ch) and DMPG (l ,2-dimyristoyl-sn-glycero-3-phoshpho- rac-(l -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 2.sup.nd ed., vol. Ill 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 non-limiting embodiment, the compositions can comprise POPC:POPS:Ch at ratios of 60:30: 10 complexed with lipid A according to Schuster et al, J. Immunol. 122:900-905 (1979).

[0064] The composition of the synthetic liposomes can comprise the following phospholipids, POPC (l -Palmitoyl-2-01eoyl-sn-Glycero-3-Phosphocholine), POPE (l-Palmitoyl-2-Oleoyl-sn- Glycero-3-Phosphoethanolamine), DMPA (l ,2-Dimyristoyl-sn-Glycero-3 -Phosphate), and Cholesterol dissolved in chloroform (purchased from Avanti Polar Lipids (Alabaster, Ala.).).

[0065] Synthetic liposomes can be prepared by dispensing appropriate molar amounts of phospholipids (POPC:POPE:DMPA:Ch=45:25:20: 10) in chloroform resistant tubes. The phospholipids are mixed by vortexing and the mixture was dried in the fume hood under a gentle stream of nitrogen. Any residual chloroform is removed by storing the lipids under a high vacuum (15 h). Aqueous suspensions of phospholipids is prepared by adding PBS or TBS buffer, pH 7.4, and incubating at 37.degree. C. for 10-30 minutes, with intermittent, vigorous vortexing to resuspend the phospholipids. The milky, uniform suspension of phospholipids is then sonicated in a bath sonicator (Misonix Sonicator 3000, Misonix Inc., Farmingdale, N.Y.). The sonicator is programmed to run 3 consecutive cycles of 45 seconds of total sonication per cycle. Each cycle includes 5 seconds of sonication pulse (70 watts power output) followed by a pulse off period of 12 seconds. At the end of sonication, the suspension of lamellar liposomes is stored at 4. degree. C.

[0066] In some embodiments, solutions of lipids can be added to the envelope complexes, in the molar ratios of 45:25:20: 10 (POPC:POPE:DMPA:Cholesterol). In other embodiments the liposome composition is the following: POPC, POPE, Sphingomyelin and Cholesterol at a 35:30:15:20 molar ratio.

[0067] The composition of the invention can comprise a lipid, for example an anionic wherein the anionic lipid is phosphatidylserine. In other embodiments, the anionic lipid is phosphatidic acid. In other embodiments, the anionic lipid is cardiolipin.

[0068] In other embodiments, the composition comprises a liposome which comprises a viral membrane lipid. In some embodiments the liposome comprises a lipid such as sphingolipid, e.g. sphingomyelin, cholesterol, or a combination thereof, in any suitable ratio.

[0069] In other embodiments, the liposome comprises POPC (l-Palmitoyl-2-Oleoyl-sn-Glycero- 3-Phosphocholine), POPE (l-Palmitoyl-2-01eoyl-sn-Glycero-3-Phosphoethanolamine), DMPA (l ,2-Dimyristoyl-sn-Glycero-3-Phosphate), or Cholesterol, or any combination thereof. In other embodiments, the liposome comprises POPC and l-palmitoyl-2-oleoyl-sn-glycero-3-[phospho- L-serine] (POPS). In other embodiments, the liposome comprises POPC and POPS and lysophosphorylcholine (lysoPC). In other embodiments, the liposome comprises POPC and POPE and Sphingomyelin and Cholesterol. In other embodiments, the liposome comprises Sphingomyelin and Cholesterol. In other embodiments, the liposome comprises Cholesterol. In other embodiments, the liposome comprises Cholesterol. In other embodiments, the liposome comprises POPC and cholesterol. In other embodiments, the liposome comprises DOPC and cholesterol. In other embodiments, the liposome comprises DPPC, DPPG and cholesterol.

[0070] The composition optionally further comprise any suitable adjuvant or any combination thereof. Non-limiting examples as described herein include TLR agonists— TLR4, TLR9, TLR7/8 ligands. Non-limiting examples of such ligands include Lipid A, oCpG, R848. The compositions of the invention can be formulated with, and/or administered with, adjuvants such as lipid A, oCpGs, TRL4 agonists or TLR 7 agonists that facilitate robust antibody responses (Rao et al, Immunobiol. Cell Biol. 82(5):523 (2004)). In certain embodiments, the adjuvant is Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, or GLA-SE. Other adjuvants that can be used include alum and Q521 (which do not break existing B cell tolerance). Preferred 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 certain embodiment, the composition comprises a combination of adjuvants. In certain embodiments the combination is a TLR4 agonist, eg. GLA, and alum. Additional suitable adjuvants include those described in Ser. No. 1 1/302,505, filed Dec. 14, 2005, including the TRL agonists disclosed therein. The compositions of the invention could further comprise or be administered with any agent that breaks immune tolerance. [0071 ] In certain embodiments the compositions comprise envelope proteins which are not truncated, do not include any amino acid changes to form non-naturally present cystein bond, include an intact MPER region, and transmembrane domain such that the envelope is cell membrane expressed, and the multimeric envelope complexes formed on the cell membrane are stable via the natural transmembrane domain. Certain embodiments provide compositions which comprise uncleaved envelope sequences. Certain embodiments provide compositions which comprise cleaved envelope sequences.

[0072] The non-limiting examples show methods according to which CHO cell surface expressed CH505 Env can be isolated in a stable form following detergent resistant microdomain and BnAb affinity purification; Env protein isolated from cell membrane migrates as a trimer as analyzed by BN gel and by size-exclusion chromatography; Env protein isolated from cell membrane can be reconstituted into liposomes, for example synthetic liposomes of a select composition.

[0073] The non-limiting examples show that CH505 Env gpl 50 trimer reconstituted in a liposome has antigenicity that showed several characteristics of a near-native Env trimer. These include low constitutive binding to CD4i mAb (17b); strong CD4i mAb binding induced by sCD4; Stronger binding to N³³²-glycan dependent V3 BnAbs (PGT128, PGT125) than to V3 non-BnAb (19b); binding to quaternary/glycan dependent BnAb PGT145; binding to both mature and unmutated common ancestor mAbs of the CH103/CH106 lineage antibodies derived from the CH505 subject .

[0074] In certain aspects the compositions of the invention can be administered to a mammalian subject, including a human, as immunogens so as to induce an immune response to HIV-1. In certain embodiments the compositions of the invention are co-administered to a mammalian subject, including a human, with any other suitable immunogenic composition so as to induce an immune response to HIV-1. The immune response could include B-cell or T-cell response. In certain embodiments the immune response comprises induction of antibodies to the HIV-1 envelope. In certain aspects the antibodies are bNAbs. Such methods of inducing an immune response are expected to provide therapeutic protection for the treatment and/or prevention of HIV-1 infection.

[0075] The compositions of the invention can be formulated as pharmaceutical composition and can be administered in any suitable dose, by any suitable mode of administering. The amount administered, as well as the route and frequency of administering could be readily determined.

[0076] The non-limiting examples and figures show methods for purification and

characterization of multimeric HIV-1 Envelope complexes, for example a trimeric envelope complex isolated from cell membrane and reconstituted in a liposome. Also provided are methods which comprise administering of such complexes as an immunogen to induce immune response in a host animal including human subjects.

[0077] Example 1: Membrane bound trimeric HIV-1 Envelope

[0078] Isolation and reconstitution of intact HIV-1 Envelope

[0079] One strategy of the immunogen design is illustrated in Figure 1. The procedure involves isolation of total membrane from CHO- cells expressing Env proteins, followed by solubilization of membrane in non-ionic detergent. The detergent treatment followed by sucrose density gradient centrifugation allows the isolation of detergent-resistant microdomains (DRM)

(commonly known as "lipid raft" that are rich in sphingomyelin and cholesterol) (Simons & Ikonen, 1997; London & Brown, 2000). During HIV-1 assembly, Gag and Env segregates into DRM on cell surface (Nguyen & Hildreth, 2000; Ono & Freed, 2001) and have lipid composition enriched in sphingomyelin and cholesterol (Bruegger et al., 2007), the signature proteins of lipid raft domains (DRM). As shown in Figure 2, the Env protein was enriched in fractions isolated from sucrose density gradient where the DRM bands. We have purified the DRM fraction that is enriched in Env proteins by affinity chromatography using the conformational and glycan- dependent broadly neutralizing mAb 2G12. The 2G12 mAb preferentially bind to trimeric Env protein and can be used to selectively purify membrane bound trimeric Env proteins (Figure 3). Antigenicity of 2G12- affinity purified trimeric Env showed enhanced binding of CD4 binding site broadly neutralizing antibodies (BnAb) VRCOl and CHI 03 (Figure 4). Furthermore, affinity purified Env trimer bound to the CHI 03 unmutated common ancestor (UCA) (Figure 4), an antigenicity trait that is required for triggering of naive B cell receptor.

[0080] Biochemical analysis of Env trimers isolated from 2G12-affmity purified DRM showed that the Env proteins migrate as monomeric Env gpl60 under denaturing condition (Figure 5 A, SDS-PAGE analysis), which is expected of Env trimer in which the monomeric protomers are non-covalently associated. The identity of the Env protein was confirmed by western blot analysis under both reducing and non-reducing SDS-PAGE analysis (Figure 5A). The DRM- isolated Env migrated as trimeric Env protein by native gel analysis (BN-gel, Figure 5B). Thus the isolation of DRM from cell expressing Env protein followed by affinity capture on 2G12- beads allowed for purification of membrane bound trimeric Env protein.

[0081 ] Antigenicity studies of DRM-associated Env showed strong binding of 2G12 mAb (Figure 6B), which was expected as the DRM was affinity purified. However, strong 2G12 binding showed that the purification steps utilized in the extraction and purification of Env associated DRM did not impair the antigenicity of the membrane bound Env. Comparison of binding of BNAb binding to non-neutralizing mAbs showed enhanced binding of the BNAbs to the DRM-associated Env trimers. The integrity of the membrane bound state of the Env trimer was maintained by capturing the DRM-associated Env trimer on a hydrophobic sensor and mAb binding was studied by SPR analysis. The DRM-associated Env trimer showed much stronger binding to the BnAb that bind to CD4 binding site (CHI 06) than the non-neutralizing mAb F105 (Figure 6C). Similarly, the binding of the V3/glycan BnAb PGT128 was stronger than the non- neutralizing V3 mAb 19b (Figure 6D). Together, these data show that the DRM-associated Env trimer preferentially presents the epitope of BnAbs while hinders the binding of non-neutralizing mAbs that target the same site.

[0082] Figure 8-11 , 12-14 show that multimeric complexes purified by 2G12 affinity

chromatography show increased affinity for bNabs. Figure 13 for example shows that lipid rafts containing multimeric envelopes are produced by the inventive methods show different antigenicity to CHI 06 and F105 antibodies.

[0083] Reconstitution of Env trimers with synthetic lipids will be carried out as outlined in Figure 1. 2G12 bound DRM will be solubilized to remove non-Env proteins and DRM membrane and the mAb bound Env protein will be stabilized with detergents (Cymal-5, Cymal - 6, Brij-58).

[0084] Reconstitution of the Env trimers into the membrane bound form will be done using the following lipid compositions. Lipid composition 1 has been used previously in anchoring MPER peptide (Alam et al., 2007 J Immunol. 2007 April 1 ; 178(7): 4424-4435, the contents of which is hereby incorporated by reference in its entirety); while lipid 2 will allow phase separation using a simple binary lipid composition. Lipid 3 is a mimic of the major lipid components in the viral membrane (composition derived and modified from Bruegger et al., 2007; the contents of which is hereby incorporated by reference in its entirety). The final choice of the lipid composition will be based on Env stability and antigenicity.

1. PC-PE liposomes - PC:PE:DMPA:Chol

2. Phosphatidylserine liposomes - PC holesterol liposomes

3. Viral liposomes - PC:PE:PS:Chol:SM

4. POPC:POPE:SM:Cholesterol (35:30: 15:20)

[0085] Example 2: Purification and characterization of trimeric HIV-1 Envelope CH505 (Figures 23-27)

[0086] This example provides strategies for Env purification as trimers and methodologies for incorporating these trimers in liposome formulations. This example provides methods to characterize antigenicity and structure of Env trimers in liposomes, and methods to produce liposome-bound trimeric forms of the CH505, T/F, week 53, week 78 and week 100 Envs.

[0087] CH505 T/F (Liao et al., Nature 2013) Env gpl 50 (residues 1 -808, cleavage site deficient) was expressed as membrane anchored Env in CHO cells using lentiviral vectors. Env expression on cell surface was doxycycline inducible with selectable marker (GFP). Cell surface Env expression was confirmed by flow cytometric analysis using gpl20 mAbs.

[0088] Figure 23 shows that Membrane bound Env (DRM) bound preferentially to the broadly neutralizing antibodies glycan dependent BnAbs PGT 128 and PGT 125 and bound relatively weakly to the non-neutralizing V3 mAb 19b. The preferential binding of the N332-glycan dependent V3 bNAbs suggest that the bnAb epitope in the envelope bound trimer is exposed favorably.

[0089] Figure 24 shows that The CD4 binding site broadly neutralizing mAb CH31 bound more strongly to the membrane bound Env trimer than the non-neutralizing mAb F105. The trimer configuration hinders the binding of the non-neutralizing CD4 binding site mabs but not the neutralizing mAb CH31.

[0090] Figure 25 shows that The mature CHI 06 mAb and the unmutated common ancestor mAb both bound to the membrane bound Env. CHI 06 lineage antibodies were induced in the CH505 subject (Liao et al., 2013) and the binding of the UCA suggests that the CD4 binding site for BnAbs is exposed on the membrane bound autologous Env.

[0091 ] Figure 26 shows that The quaternary V1V2 BnAb PGT 145 bound to the membrane bound Env trimer (right panel) but not to soluble gpl20 (left panel). PGT 145 has been reported to bind exclusively to quaternary V1V2 epitopes presented on native-like Env trimer (Sanders et al., 2013; Ringe et al., 2013). The binding of mAb PGT 145 to the membrane bound Env shows that the V1V2 conformation is correctly configured for the binding of VI V2 BnAb.

[0092] In Figures 23-25 the experiments were done as follows: On the OctetRed 96 (ForteBio) Membrane bound CH505 (JF-8) DRM purified 2G12 purified was diluted 1/20 was incubated with Aminopropylsilane (APS) Octet Red96 tips (~1.85nm response)(600s), o PBS. Following a wash in PBS APS tips were then coated with 1%BSA solution to reduce noise due to nonspecific binding(600s). Tips were then washed and equilibrated and added consecutively to 4 different wells each with increasing antibody cone. (5ug/ml, 20ug/ml, 40ug/ml 80ug/ml) for 120s for all but the 80ug/ml concentration which had 1000s incubation with tips. Tips were then moved to a plate with only PBS and the dissociation recorded. Results shown have been Synagis (mAb) subtracted. The scatter graph on the right displays the height of the curve at the end of each injection period.

[0093] In Figure 26 the experiment was done as follows: On the left Curves were generated by SPR, BIACORE 3000, in short -9000 RU of antihuman Fc antibodies were amine coupled to the ship using NHS/EDC (N-hydroxysuccinimide/ ethyl(dimethylaminopropyl)Carbomide). 1 Oug/ml ( 5ul injection) of mAbs of choice were then captured on the CM5chip, CH505 gpl20 was then flowed over the chip for binding analysis at 1 OOug/ml (40ul). Curves are double subtracted for buffer and synagis.

[0094] Figure 29 shows the following experimental set up: On the OctetRed 96 (ForteBio) Membrane bound CH505 (JF-8) DRM purified 2G12 purified was diluted 1/20 was incubated with Aminopropylsilane (APS) Octet Red96 tips (~1.85nm response)(600s), o PBS. Following a wash in PBS APS tips were then coated with 1%BSA solution to reduce noise due to nonspecific binding(600s). Tips were then washed and equilibrated and added consecutively to 4 different wells each with increasing antibody cone. (5ug/ml, 20ug/ml, 40ug/ml 80ug/ml) for 120s for all but the 80ug/ml concentration which had 1000s incubation with tips. Tips were then moved to a plate with only PBS and the dissociation recorded. Results shown have been Synagis (mAb) subtracted.

[0095] In summary, CHO cell surface expressed CH505 T/F Env segregate into detergent- resistant microdomains (DRM). Membrane bound Env can be isolated in a stable form following DRM and 2G12 affinity purification. Env protein isolated from DRM migrate as a trimer in BN gel. Membrane bound Env antigenicity showed several characteristics of a near- native Env trimer: no constitutive binding to CD4i mAb (17b); CD4i epitope induced by sCD4; Preferential binding to glycan dependent BnAbs (2G12, PGT128) than to non-neutralizing V3 mAb (19b); Stonger binding to CD4 bs bNAb (CH31) than to non-neutralizing mAb F105; Bound to both mature CHI 06 and its UCA; Bound to the quaternary V1V2 BnAb PGT 145. [0096] EM negative staining of Env preparations were made to assess the overall morphology of the proteins. EM staining can also be used to determine the proportion of trimers after purification and liposome reconstitution.

[0097] Example 3: CH505 T/F Env expression in CHO cells, purification and liposome reconstitution (Figures 15-21)

[0098] CH505 T/F (Liao et al, 2013) Env gpl 50 (residues 1-808, cleavage site deficient, Y721 A) was expressed as membrane anchored Env in CHO cells using lenti viral vectors. Env expression on cell surface was doxycycline inducible with selectable marker (GFP). Cell surface Env expression confirmed by flow cytometric analysis using gpl20 mAbs

[0099] Methods for isolation and reconstitution of membrane bound Env

[0100] CHO cells expressing membrane bound Env proteins are homogenized and membrane fraction was subjected to non-ionic detergent (Brij 58) solubilization to extract Env enriched detergent resistant microdomains (DRM).

[0101] A detergent solubilized fraction of Env proteins prepared in Cymal-5 detergent was passed through an HIV-1 bnAb affinity column (e.g., 2G12) and antibody bound protein fraction eluted with high salt (2M MgCl₂).

[0102] Aggregates of Env proteins were removed by size exclusion chromatography and the trimeric Env fraction was collected for -liposome reconstitution.

[0103] A second step of affinity chromatography using non-bnAbs (V3 loop mab 19b, CD4 binding site F105, or V2 mAb CH58) may be used to remove non-native fraction of the Env proteins. [0104] Liposome reconstitution was done by dialyzing detergent solubilized Env proteins into the high CMC detergent Cymal-2 and using detergent destabilized preformed liposomes of a select composition.

[0105] Removal of detergent by dialysis results in the final reconstitution of the membrane derived Env proteins as trimers into synthetic liposomes

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a modified HIV-1 envelope polypeptide in a trimer complex in a liposome, wherein the modified envelope comprises amino acid changes which remove the envelope cleavage site, whereby the envelope is not cleaved, and the modified envelope comprises a Y721A mutation.

2. The composition of claim 1 wherein the HIV-1 envelope polypeptide is a gpl60 or gpl 50 and comprises the MPER domain, or the TM domain, or both.

3. The composition of claim 1 or 2 further comprising an adjuvant.

4. The composition of claim 1 , 2, or 3, wherein the HIV-1 envelope polypeptide trimer complex is purified by antibody affinity chromatography from an HIV-1 envelope expressed on the cell surface of an expression cell.

5. The composition of any one of claim 1- 4, wherein the HIV-1 envelope polypeptide trimer complex in the liposome shows preferential binding for bNabs versus non-neutralizing antibodies.

6. The composition of any one of claim 1- 5, wherein the liposome comprises cholesterol, sphingomyelin, or both.

7. The composition of any one of claims 1-6 wherein the envelope is JF-8 variant of CH505 T/F.

8. A vector comprising a nucleic acid encoding the HIV-1 envelope of claim 1 or 2, wherein the vector is an expression vector and the nucleic acid is operably linked to a promoter.

9. A cell comprising the vector of claim 8.

10. A method of inducing an immune response in a subject comprising administering to the subject the composition of any one of claims 1-7 in an amount and manner sufficient to effect such induction.

1 1. A method for producing an HIV-1 envelope polypeptide in a trimer complex stabilized in a liposome comprising: a) recombinantly expressing an engineered or non-naturally occurring HIV-1 envelope on the cell surface of a cell, b) purifying the engineered or non-naturally occurring HIV-1 envelope from the cell membrane under conditions which retain the envelope as a trimer, and c) reconstituting the trimer in a complex with a liposome.

12. The method of claim 1 1 , wherein step b) comprises bNAb affinity chromatography.

13. The method of claim 1 1 , wherein step b) further comprises a non-neutralizing antibody affinity chromatography step.