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WO2017055522A1 - Protéines env stabilisées du vih - Google Patents

Protéines env stabilisées du vih Download PDF

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Publication number
WO2017055522A1
WO2017055522A1 PCT/EP2016/073377 EP2016073377W WO2017055522A1 WO 2017055522 A1 WO2017055522 A1 WO 2017055522A1 EP 2016073377 W EP2016073377 W EP 2016073377W WO 2017055522 A1 WO2017055522 A1 WO 2017055522A1
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seq
sosip
amino acid
trimers
hiv
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PCT/EP2016/073377
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English (en)
Inventor
Alba Torrents DE LA PEÑA
Rogier Sanders
Andrew B. WARD
Ian Andrew Wilson
Jean-Philippe Julien
John P. Moore
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Academisch Medisch Centrum
Cornell University
The Scripps Research Institute
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Publication of WO2017055522A1 publication Critical patent/WO2017055522A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates generally to novel HIV envelope proteins and to methods, compositions and products related thereto. More particularly, the invention relates to methods and compositions for the preparation, production, and administration of isolated novel HIV envelope nucleic acid and protein sequences suitable, for example, as vaccines against HIV.
  • Env envelope glycoprotein trimer
  • Env HIV envelope glycoprotein
  • bNAbs broadly neutralizing antibodies
  • the inventors have recently described an Env spike mimic, BG505 SOSIP.664 gpl40, which is based on the above described SOS, and has in addition to the disulfide bond between gpl20 and gp41 a trimer stabilizing Ile-to-Pro substitution at position 559 in gp41.
  • Negative stain electron microscopy (EM) studies show that the protein resembles native spikes on virions.
  • the BG505 SOSIP.664 trimers induce NAbs to the neutralization-resistant (Tier 2) autologous virus in rabbits and macaques (Sanders et al., 2015).
  • Tier 2 neutralization-resistant
  • native-like SOSIP.664 trimers have also been produced from the B41, ZM197M and DU422 clade B or C env genes (Julien et al., 2015; Pugach et al., 2015).
  • BG505 SOSIP.664 gpl40 binds minimally but reproducibly to some non- NAbs, which might explain the induction of non-NAbs by BG505 SOSIP.664 trimers in vivo.
  • the native Env trimer and its SOSIP.664 mimics are conformationally flexible; they can "breathe” such that they alternate between closed and more open forms in a dynamic equilibrium (Guttman et al., 2014; Munro et al., 2014; Pugach et al., 2015). While unliganded BG505 SOSIP.664 trimers have a high propensity to remain in the closed, ground state conformation, the equilibrium for their B41 and
  • FIG. 1.1 shows the design of amino-acid substitutions to stabilize SOSIP.664 trimers.
  • A Crystal structure of BG505 SOSIP.664 trimer (Pancera et al., 2014), showing the locations of amino-acid substitutions relevant to this study. One protomer is colored according to sub-regions: gp41 in red; V1V2 in cyan; V3 in purple; gpl20 inner domain layer 1 in blue; layer 2 in yellow; layer 3 in orange; outer domain and N- and C- termini of g l20 in green.
  • B Detailed view of V3 and surrounding regions showing the A316W substitution, as well as residues V120, L122 and P206 with which W316 can now make hydrophobic interactions.
  • F Infection of TZM-bl cells by BG505.T332N Env- pseudoviruses with an A316W, E64K or H66R substitution
  • G Biolayer interferometry analysis of CD4 binding to AviB-tagged BG505 SOSIP trimers
  • H SPR analysis of CD4 binding (left panel) and sequential CD4 and 17b binding (right panel) to BG505 SOSIP.664-D7324 trimers and both versions of SOSIP .V4-D7324 trimers.
  • Figure 1.2 shows HD-X analysis of BG505 SOSIP.664 and SOSIP.v4 trimers.
  • Butterfly plots of PGT145-purified BG505 A) SOSIP.664, (B) SOSIP.v4.1, (C) SOSIP.v4.2 trimers, comparing deuterium exchange levels in the presence and absence of sCD4. Regions that are less (red) or more (blue) protected upon CD4-binding are mapped on the BG505 SOSIP.664 crystal structure (Pancera et al., 2014), to depict CD4-induced conformational changes.
  • Figure 1.4 shows the immunogenicity of AMC008 and BG505 SOSIP.v4 trimers in rabbits.
  • A Immunization schedule. Rabbits were immunized at week 0, 4 and 20 (green) and the Ab responses were analyzed at week 22 (magenta). Blue symbols represent BG505 trimer-immunized animals, red symbols AMC008.
  • the SOSIP.664 recipients are shown by closed circles, SOSIP.v4 by squares. Midpoint binding Ab titers were measured by D7324-capture ELISA against (B) SOSIP.664-D7324 or (C) SOSIP .v4- D7324 trimers.
  • the ratio of the SOSIP.664/SOSIP.v4 midpoint titers is plotted.
  • the V3 Ab response was determined by pre-incubating the sera with a cyclized V3 peptide (BG505 or AMC008 sequence, as appropriate, either unmodified or with the A316W change present in SOSIP .v4 trimers), before determining the remaining trimer binding titers by ELISA.
  • the relative V3 responses (as a % of the total anti-trimer responses) were calculated by comparing the midpoint titers in the absence and presence of the V3 peptide.
  • Figure LSI shows the screening of stabilizing mutations using unpurified AMC008, BG505 B41 and ZM197M SOSIP.664 proteins.
  • the A316W, I535M and L543N substitutions make a particularly substantial contribution to trimer formation, both individually and collectively.
  • introducing the reverse N543L substitution into BG505 SOSIP.664 reduced trimer formation (data not shown).
  • thermostability The unfolding pattern of the variant proteins was visualized by plotting the first derivative of the graph in (D) using Graphpad prism 5.
  • the T m values were derived from the peaks in the first derivative curves shown in (D). The T m values obtained in this assay are highly reproducible (standard deviation of ⁇ 0.3°C for BG505 SOSIP.664-D7324).
  • G Correlation plot between T m values obtained using unpurified BG505 SOSIP.664-D7324 proteins in the above thermostability assay and T m values obtained using PGT145-purified BG505 SOSIP.664-D7324 trimers in a DSC assay (Table 1.1 ; Fig.
  • H-L ELISA binding curves for bNAbs and non-NAbs to unpurified SOSIP.664 wild type and mutant proteins based on the following genotypes: (H) AMC008; (I) BG505; (J) B41 ; (K) ZM197M. (L) Competition ELISA using wild type and A316 W-substituted BG505-V3 peptides.
  • Wild type or position 316- mutated variant Env proteins in culture supernatants from transiently transfected 293T cells were captured via D7324.
  • the bound proteins were detected using either PGT145 (0.11 ⁇ g/ml; to detect native trimers) or 14e (0.5 ⁇ g/ml; to detect V3 exposure), and the ratios of the OD450 signals were plotted.
  • the wild type residue (alanine) is shown in grey, the designed mutant (tryptophan) in red and other substituted amino acids in black. Only bulky hydrophobic amino acids reduce V3 non-NAb binding.
  • N, O Spontaneous sampling of the CD4i conformation was assessed using a D7324-capture ELISA.
  • Exposure of CD4i epitopes was assessed by measuring the binding of high concentrations (10 ⁇ g/ml) of non-NAbs (N) 17b and (O) 412d.
  • the OD 45 o values obtained using the wild type BG505 SOSIP.664-D7324 protein were set at 100% and used to normalize the values for the E64K and H66R mutants. Mock indicates blank transfection supernatant.
  • Figure 1.S2 (A-N) which is related to figure 1.1, shows the effect of stabilizing mutations on the biochemical and biophysical properties of AMC008, BG505, B41 and ZM197M SOSIP.664 proteins.
  • Non-native forms are often elongated and no triangular center of density is visible. The classifications are quantified below each panel. The percentages of closed and partially open native-like trimers are in green, and of non-native forms in red. The total number of particles classified is defined as 100%.
  • D DSC analysis of PGT145-purified AMC008, BG505, B41 and ZM197M
  • the percentages of mannose-5 to mannose-9 glycans (M5-M9) derived from the glycan profiles are listed in Table 1.1
  • G-J ELISA binding of bNAbs and non-NAbs to D7324-captured, PGT145-purified (G) AMC008; (H) BG505; (I) B41 ; (J) ZM197M SOSIP.664-D7324 trimer variants.
  • K-M V3 Fab 19b binding to (K) AMC008, (L) BG505 and (M) B41 trimers was also assessed by ITC.
  • the enthalpy changes ( ⁇ ), dissociation constants ( ⁇ ⁇ ) and stoichiometrics of binding (molar ration; N) are listed in panel ( ⁇ ).
  • Figure 1.S3 (A-C), which is related to Figure 1.2, shows the effect of stabilizing mutations on HD-X profiles of BG505 SOSIP.664 trimers.
  • A, B Left panels: Butterfly plots of HD-X exchange levels in the absence of CD4.
  • A PGT145-purified BG505 SOSIP.664 vs. SOSIP.v4.1 trimers
  • B PGT145-purified BG505 SOSIP.664 vs. SOSIP.v4.2 trimers.
  • Right panels The differences in exchange patterns are mapped onto the BG505 SOSIP.664 structure (Pancera et al., 2014). Regions that are less or more protected from deuterium exchange are colored red and blue, respectively.
  • FIG. 2.3 shows the HIV- 1 SOSIP Env trimer structure at 3.0 A resolution.
  • A Ribbon representation of gp41 in the context of the BG505 SOSIP.664 trimer. The gpl20 subunits are shown in surface representation while gp41 is shown in ribbon representation.
  • B Ribbon representation of one protomer with the new region of 20 residues, located within the HR1 helix, and connecting the 1548 and 1568 residues, highlighted with a yellow surface. The position of the I559P mutation in the trimer is also indicated.
  • Figure 3.2 shows the dynamics of BG505 SOSIP.v5 trimers.
  • Butterfly plots comparing the HDX- MS profiles of wild-type (a) with A73C-A561 C (b), E64K A316W A73C-A561 C (c), and E64K A316W H72C-H564C (d) BG505 SOSIP.664 trimers upon sCD4 binding.
  • the percent exchange for each observable peptide is plotted at the position of the center of the peptide on the primary sequence for each time point (3 s to 20 h). Differences are mapped onto the trimer crystal structure by coloring regions with faster (red) or slower (blue) deuterium exchange upon sCD4 binding.
  • FIG. 3.3 shows the immunogenicity of BG505 SOSIP.v5 trimers in rabbits
  • the V3 antibody response was determined by incubating a V3 peptide with the sera before determining the anti- trimer binding titer.
  • the V3 peptide was used as in the BG505 SOSIP.664 and as in the BG505 SOSIP.v5 depending on the trimer used as an immunogen.
  • the relative V3 responses were calculated by comparing the midpoint titers with and without using the V3 peptide,
  • BG505 viruses was assessed by using the TZM-bl assay. The plots show the serum dilution at which 50% of the infectivity is inhibited.
  • Figure 3.4 shows the Immunogenicity of BG505 and ZM197M SOSIP.v5 trimers in rabbits, (a) Schematic representation of vaccination schedule, (b) Color coding for the various immunogens tested, (c and i) Midpoint Ab binding titers were measured by D7324-capture ELISA using sera dilutions for the trimer variants. Neutralization of HIV-1 viruses by animals immunized with BG505 (d-g, left panels) and ZM197M (j-m, right panels) (see bottom of figure for the specific immunogens).
  • Figure 3.S1(A-D) Introducing a novel disulfide bond between gpl20 and gp41. a, Five regions in gpl20 and gp41 that are in reasonably close contact are shown in the crystal structure of BG505 SOSIP.664.
  • Residues in gpl20 that might contact residues in gp41 are shown in blue spheres, b, A panel of unpurified His-tagged BG505 gpl40 proteins was screened in which the original disulfide bond was replaced by two new cysteine residues in the regions highlighted in panel A (i.e. the A501C-T605C "SOS" disulfide bond was absent during screening). Screening was based on recently published BG505 crystal structures. Trimerization efficiency was analyzed by BN- PAGE followed by western blotting. The majority of the mutants showed less efficient trimerization. The antigenic profile of the newly generated BG505 gpl40 trimers was determined by His-tag ELISA.
  • Figure 3.S2 shows the combining two disulfide bonds between gpl20 and gp41.
  • A)-Cleavage and trimerization efficiency of supernatant produced gpl40 proteins that incorporated the original disulfide bond at position 501-605, or a new disulfide bond at positions H72C-H564C or A73C-A561C, or both disulfide bonds, were determined by reducing SDS-PAGE (left panel) and BN-PAGE (right panel). All the mutants were properly cleaved, but showed slightly less efficient trimerization compared to original BG505 SOSIP.664 protein.
  • the double disulfide mutants adopted a combined phenotype.
  • binding of CD4i non-NAb 17b were abrogated, a property shared with the H72C-H564C and A73C-A561C mutants, and the binding of the gp41 non-NAb F240 was also very low, a property conferred by the original A501 C-T605C disulfide bond.
  • Additional Cys substitutions at residues 558-569 were screened for their ability to pair with 72C and 73C, and improve native like BG505 SOSIP.664 trimers that also contained the A501C-T605C disulfide bond.
  • S3 shows the biochemical, biophysical and antigenic characterization of PGT145-purified stabilized BG505 SOSIP.664 trimers.
  • a Cleavage efficiency and disulfide bond formation was determined by SDS-PAGE under reducing and non-reducing conditions, respectively. Under reducing conditions all proteins showed a gpl20 band, indicating that they were completely cleaved. Under non- reducing conditions the proteins all showed a gpl40 band, but they migrated differently through the gels, indicative of different levels of compactness (compact proteins take up less SDS and therefore migrate slower through SDS-PAGE gels).
  • the double disulfide variants all showed a slower migration pattern due to the presence of the extra disulfide bond, b, BG505 SOSIP.664, SOSIP.v4.1 and SOSIP.v5 gpl40 proteins purified by PGT145 affinity chromatography were exclusively trimeric as determined by BN- PAGE. c, The formation of native-like trimers was assessed by negative stain electron microscopy. The 2D reference free class averages of the two double disulfide bond proteins (BG505 SOSIP.v5.1 and BG505 SOSIP.V5.2) compared to BG505 SOSIP.664 and SOSIP.v4.1 are shown.
  • the percentage of closed native-like and open native-like trimers is shown in green and the percentage of non-native trimers in red.
  • d The thermal stability of BG505 SOSIP.664, BG505 SOSIP.v4.1 and BG505 SOSIP.v5 was measured by DSC.
  • the independent non-two state best fit curves are depicted in dashed red line and the T m values of each peak are given in the graphs.
  • Figure 3.S4 shows the HDX-MS profiles of PGT145 purified wild-type and stabilized BG505 SOSIP.664. Butterfly plots comparing the HDX-MS profiles of wild-type with A73C-A561C (A), BG505
  • Figure 3.S5D shows the antigenicity of AMC008, B41 and ZM197m SOSIPs was determined by D7324- ELISA. Representative curves of a panel of bNAbs and non-NAbs are shown. The plots are representative of two or three experiments.
  • Fig. 4.1 shows the design and biochemical characterization of BG505 SOSIP.664-ferritin nanoparticles.
  • Bottom the BG505 SOSIP.664-gpl40-ferritin construct.
  • the hexa-arginine furin cleavage site (R6), the SOS disulfide bond between gpl20 and gp41 (C501-C605), and the I559P substitution that facilitates trimerization are indicated on the SOSIP.664 component, to which the ferritin moiety is linked via a Gly-Ser-Gly (GSG) spacer.
  • GSG Gly-Ser-Gly
  • Fig. 4.2 shows the induction of increased antibody responses by BG505 SOSIP.664-ferritin in mice and rabbits
  • a Eight BALB/C mice were immunized three times (at weeks 0, 4 and 12) with either 2.8 ⁇ g of BG505 SOSIP.664 trimer or BG505 SOSIP.664-ferritin protein formulated with 25 ⁇ g MP LA adjuvant.
  • the midpoint binding (EC50) titers to BG505 SOSIP.664 trimer were determined at week 14 by NiNTA ELISA [2]; the median titers are denoted by horizontal lines.
  • Statistical analysis was performed using a two-tailed Mann- Whitney U test, b Two groups of five New Zealand White rabbits received
  • DNA plasmids encoded either the soluble BG505 SOSIP.664 gpl40 or the BG505 SOSIP.664 gpl40-ferritin nanoparticles; none of the plasmids encoded furin.
  • the protein boost was, correspondingly, either soluble SOSIP.664 trimers or SOSIP.664- ferritin particles, in both cases purified by a PGT145 bNAb column.
  • the four historic control rabbits (indicated by circles in panel b) received identical DNA priming, but were then boosted with
  • SEC size exclusion chromatography
  • Anti-trimer serum binding titers over the course of the experiment were tested in D7324-capture ELISA using 2G12/SEC purified D7324-tagged BG505 SOSIP.664 trimers (0.5 ⁇ g/ml), essentially as described before [2, 9]. The medians of the midpoint binding titers ( ⁇ error) are plotted.
  • the IC50 titers in c and d were determined using the TZM-bl neutralization assay.
  • the pre-bleed samples lacked neutralization activity (not shown).
  • Neutralization assays were performed either at the Academic Medical Center (SF162, 6535.3, ZM197M, HXB2, DJ268.3, BaL, ZM109F, 94UG103, 92RW020, Q23envl7 and MLV) or the Duke University Medical Center (DUMC) (BG505.T332 N, MN.3, MW965.26, Q259.d2.17, Cel 176_A3, Q769.d22, Q842.dl2, YU2, Q23envl7 and MLV).
  • DUMC Duke University Medical Center
  • the fold difference in median IC50 titer (horizontal lines) is depicted below the graphs.
  • the dotted horizontal lines in the BG505 SOSIP.664 group represent the median titers for the five animals from the current experiment, i.e. excluding the four control sera. The titers were very similar when the four control sera were included or excluded. Statistical differences between the nine trimer-immunized rabbits and the five nanoparticle-immunized rabbits were determined using a two-tailed Mann- Whitney U test
  • the invention is based on the surprising identification of a stabilizing disulfide bond resulting from a point mutation of a residue located within the alpha-0 and a residue located within the HR1 region of the Env protein of both HIV-1 and HIV-2.
  • the modified HIV envelope (Env) glycoprotein complexes of the invention are more structurally stable than native Env complexes, which are characteristically more labile or unstable in order to be capable of efficiently undergoing conformational changes during the process of virus-cell fusion.
  • the HIV envelope (Env) glycoprotein complexes of the invention display less reactivity to non-Nab 17b.
  • the Env proteins of the invention comprising the new disulfide bonds also stabilized and improved Env trimers from diverse virus isolates. These stabilized Env trimers allow for the design of multivalent vaccines aimed at inducing bNAbs.
  • the invention therefore provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus, the Env polypeptide comprising:
  • said gpl20 or gpl25 polypeptide portion comprises a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73
  • said gp41 or gp36 ectodomain polypeptide portion comprises a second cysteine residue at an amino acid position equivalent to an amino acid position selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 and 570, wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said first and said second cysteine residues form a disulfide bond between said gpl20 and said gp41 polypeptide portions, or between said gpl25 and said gp36 polypeptide portions.
  • disulfide bonds have several advantages.
  • An advantage is that these disulfide bond result in trimer variants that were stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes to more open forms.
  • These disulfide bonds also further increase stability and result in a higher melting temperature.
  • these disulfide bonds reduce or abolish non-neutralizing V3-directed antibody binding to stabilized trimers, while quaternary- dependent bNAb epitopes is maintained or improved. Consequently, the trimers stabilized with these disulfide bonds elicit reduced V3 -directed Tier 1 A NAb responses, while autologous Tier 2 or Tier IB NAb responses are maintained.
  • said first and second cysteinse are at the amino acid positions equivalent to a pair of amino acid positions selected from the group consisting of: a. position 49 and a position selected from the group consisting of positions 555, 556, and 560, and
  • position 72 and a position selected from the group consisting of positions 554, 555, 556, 558, 559, 560, 561, 562, 563, 564, 565, 566, and 567, and
  • position 73 e. position 73 and a position selected from the group consisting of positions 555, 556, 558, 559 ,560, 561, 562, 563, 565, 566, 567, 568, and 569 wherein said numbering is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.
  • said first cysteine is at a position selected from the group consisting of positions 49, 50 and 51 of SEQ ID NO:l, and wherein said second cysteine is at a position selected from the group consisting of positions 554, 555, 556 and 560 of SEQ ID NO: 1.
  • the inventors observed that after introduction of a disulfide bond between 555C and 49C, binding of the 17b non-bnAb to its CD4-induced epitope was decreased 5-fold (Figure 2.S10).
  • said first cysteine is at position equivalent to position 49 of SEQ ID NO:l and said second cysteine is present at position equivalent to position 555 of SEQ ID NO:l .
  • said isolated, recombinant or synthetic polypeptide has a high melting temperature.
  • said second cysteine residue is at an amino acid position equivalent to amino acid position of a position selected from the group consisting of: 558 (A558), 561 (A561), 564 (H564), or 568 (L568).
  • the amino acids between brackets refer to the amino acids in SEQ ID NO:l .
  • said first cysteine is at a position selected from the group consisting of positions 72 and 73 of SEQ ID NO:l
  • said second cysteine is at a position selected from the group consisting of positions 556, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568 of SEQ ID NO: 1.
  • said first and second cysteine are at the amino acid positions equivalent to amino acid positions of a pair of amino acid residues selected from the group consisting of: residues 72 and 564, residues 73 and 558, residues 73 and 561, residues 73 and 568 of SEQ ID NO: 1.
  • said first cysteine is at position equivalent ot position 72 or 73 of SEQ ID NO:l and said second cysteine is present at a position equivalent to position 561 or 564 of SEQ ID NO:l .
  • the fraction of closed native-like trimers was further increased when the 64K and 316W amino acid residues were also present see Table 3.1 and Fig. 3S3c). Therefore, in a preferred embodiment, said isolated, recombinant or synthetic polypeptide comprises the cystein residues at postions equivalent to postions72, 564, 501, 605, and a P at position equivalent to postion 559 of SEQ ID NO: 1.
  • said isolated, recombinant or synthetic polypeptide comprises the cystein residues at positions 73, 561, 501, 605, and a P at position 559, wherein said amino acid residues are at amino acid positions equivalent to the amino acid positions of SEQ ID NO: 1.
  • the invention is further based on the identification of a number of mutants which improve the performance of Env trimers and in particular BG505 SOSIP trimers as immunogens. These mutations have stabilizing effect on Env trimers higher global, improve affinity of neutralizing antibodies, improve and local stability and/or reduce the binding to non-NAb epitopes.
  • the invention therefore provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of a K at a postion equivalent to postion 64 of SEQ ID NO:l or an R at a position equivalent to position 66 in SEQ ID NO:l .
  • the invention further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315 W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, and 316V, wherein said amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1, and wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ
  • isolated, recombinant or synthetic polypeptide of the invention stabilizes the trimers in the closed, pre-fusion ground state, impeding or even preventing their spontaneous sampling of the CD4-induced conformation, and reducing the opening of their variable loops. Therefore, said isolated, recombinant or synthetic polypeptide comprises a W at a position equivalent to position 316 of SEQ ID NO:l A further advantage is that it increases
  • said at least one amino acid residue comprises 316W of SEQ ID NO: 1.
  • the invention further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide comprises at least one amino acid residue selected from the group consisting of 555K or 556K, wherein said amino acid residue is at amino acid position equivalent to the amino acid position of SEQ ID NO: 1, and wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.
  • the invention is further based on the finding that the affinity and stoichiometry of antibody-trimer binding was increased when the Nl 37 glycan of the HIV Env polypeptide was absent ( Figure 2.1 and Figure 2.S2).
  • the N137 glycan impedes Env binding of PGT121 bNAb antibody family members, implying that accommodating this glycan is the major driving force behind affinity maturation.
  • the Kd of 35 nM for binding of early precursor 3H+3L Ab to the SOSIP.664-N137A trimer represents an ⁇ 15-fold decrease vs.
  • SOSIP.664 (i.e., wild-type trimer) and is comparable to the Kd of 32 nM for the mature PGT124 bnAb and wild-type trimer ( Figure 1 and Figure S2).
  • the Kd of PGT124 for the SOSIP.664-N137A trimer was also decreased to 6nM ( Figure 2.1 and Figure 2.S2).
  • the invention therefore further provides an isolated, recombinant or synthetic polypeptide comprising an Env polypeptide of an HIV virus comprising a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion from HIV-1 or a gp 125 polypeptide portion and at least a gp36 ectodomain polypeptide portion from HIV-2, wherein said Env polypeptide wherein the N137 glycan of the HIV Env polypeptide was absent, preferably comprising 137A or wherein N137 is deleted, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1.
  • said gp41 ectodomain polypeptide portion comprises the amino acid sequence of the amino acids of positions 512-664 of SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto.
  • said 120 polypeptide portion comprises the amino acid sequence of the amino acids of positions 31-511 of SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto.
  • said isolated, recombinant or synthetic polypeptide which comprises at least one amino acid residue selected from the group consisting of:
  • said isolated, recombinant or synthetic polypeptide comprises the amino acid sequence according to any of the sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.
  • said isolated, recombinant or synthetic polypeptide is based on the envelope sequences from elite neutralizers. These envelope sequences have proven to be able to induce bNAb responses in patients. These sequences present multiple bNAb epitopes while occluding most non-NAb epitopes.
  • ACS Amsterdam Cohort Studies on HIV-1 and AIDS
  • the inventors identified three HIV-1 elite neutralizers. From those three patients, they obtained multiple full-length envelope sequences from viral DNA isolated from PBMCs, obtained at multiple timepoints after infection. These sequences are listed herein as SEQ ID NO: 10-99. These sequences can form the basis of envelope-based vaccines.
  • said isolated, recombinant or synthetic polypeptide has therefore at least the amino acid sequence corresponding to the gpl40 portion of any of the isolated, recombinant or synthetic polypeptides having the amino acid sequence of any of SEQ ID NO: 10-99.
  • said isolated, recombinant or synthetic polypeptide has at least the amino acid sequence corresponding to the gpl40 portion of any of the isolated, recombinant or synthetic polypeptides having the amino acid sequence of any of SEQ ID NO: 10-29.
  • Sera from these elite neutralizers very potently neutralized the parental BG505, 94UG103 and MGRM-C026 viruses (ID50 values of 484, 453 and 967, respectively, at 40 months post-SC).
  • said isolated, recombinant or synthetic polypeptide of the invention has a further disulfide bond present between cysteine residues at positions equivalent to the residues of 501 and 605 of SEQ ID NO 1, preferably by the point mutations A501C and T605C.
  • An advantage thereof is that these cysteine residues that form a further disulfide bond between gpl20 and gp41 and these isolated, recombinant or synthetic Env proteins showed higher percentages of closed trimers, which indicates further stabilization of the trimer.
  • Another advantage of this further disulfide bond is that these isolated, recombinant or synthetic Env protein display an increased binding of the bNAb 3BC315 to gp41 and quaternary bNAbs PGT145 and PG16 against the trimer apex, as well as quaternary bNAbs PGT151 and 35022 against the gpl20-gp41 interface, suggesting that these trimers have an improved quaternary native-like conformation.
  • said isolated, recombinant or synthetic polypeptide has the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.
  • the isolated, recombinant or synthetic polypeptide of the invention comprises further at least one amino acid residue selected from the group consisting: 25 W, 51C, 54C, 60E, 64K, 66N, 66R, 66A, 71C, 112A, 112S, 112R, 114C, 120C, 121 W, 121F, 121C, 122C, 125W, 127F, 127W, 128C, 129F, 129C, 134C, 136C, 136N, 139W,140I, 151C, 152C, 153C, E153F, 153W, 154C, 154F, 154W, 155C, 156C, 161C, 162F, 162W, 163F, 163W, 164F, 164W, 165F, 165W, 165Y, 166Y, 166F, 166W, 166C, 167C, 167F, 167E,
  • the isolated, recombinant or synthetic Env proteins comprise the amino acid residues 201C and 433 C, which also reduce binding of non-Nabs, increase thermostability and constrain conformational flexibility of the Env proteins.
  • said at least further at least one amino acid residue or mutation is selected from the group consisting of (N)302F, (N)302W, (R)304F, (R)304W, (R)306L, (I)307F, (I)307W, (R)308L, (R)315Y, (R)315K, (R)315I, (R)315W, (R)315F, (R)315V, (A)316Y, (A)316K, (A)316I, (A)316F, (A)316W, (A)316V, most preferably (A)316F, (A)316W, (R)315W, (R)315F, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ
  • said at least one amino acid residue is selected from the group consisting of: 64K, 66R, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1.
  • said isolated, recombinant or synthetic polypeptide has at least one amino acid residue selected from the group consisting of 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, 316V and at least one amino acid residue selected from the group consisting of: 64K, 66N, 66R, 66A, wherein numbering of said at least one amino acid residue is according to the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1 , and wherein said at least one amino acid residue is at an amino acid position equivalent to the amino acid position of SEQ ID NO: 1.
  • the combination of these mutations result in further improvement of the thermostability and an increase in compactness of the isolated, recombinant or synthetic polypeptide of the invention in comparison to unmodified counterparts (see Fig. 1.S1B-G).
  • it reduces the binding to non-NAbs 19b and 14e.
  • binding of non-NAbs to CD4i- epitopes (targets of for example the E64K or H66R substitutions) and V3 epitopes (targets of the A316W change and equivalents thereof) to the double mutants was diminished or abolished, while bNAb epitopes were unchanged (Fig. 1.S1H-K).
  • the isolated, recombinant or synthetic Env protein comprises E64 or H66, which appear to be critical for facilitating CD4-induced
  • said isolated, recombinant or synthetic polypeptide comprises the combination of amino acid residues selected from the group consisting of: 64K and 316W; 125W and 316W; 535M and 543Q; 535M and 543N; 64K, 316W and 543N; 535M, 543N, 316W and 64K;535M, 543N, 316W and 66R; 535M, 543N, 66R and 316W; 64K and 535M; 64K, 316W and 535M; 66R, 316W and 535M; 535M and 543Q; 535M and 543N; 316W, 535M and 543N; 64K, 316W, 535M and 543N, 66R, 316W, 535M and 543N, wherein numbering of said combination of amino acid
  • said isolated, recombinant or synthetic polypeptide has the amino acid sequence of SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:l l l, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:l l l, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:l l l, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:102, SEQ ID NO:103
  • the invention further provides a trimer comprising an isolated, recombinant or synthetic polypeptide according to the invention.
  • the invention further provides a virus like particle (VPLs) or pseudoparticle comprising the isolated, recombinant or synthetic polypeptide according to the invention or the trimer according to the invention.
  • VPLs virus like particle
  • pseudoparticle comprising the isolated, recombinant or synthetic polypeptide according to the invention or the trimer according to the invention.
  • the invention is further based on the finding that ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-b earing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-b earing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses, to several tier IB viruses and the autologous tier 2 virus than when the same trimers were delivered as soluble proteins. Nanoparticles that display hemagglutinin trimers from influenza virus on their surface are described in WO2013044203A2.
  • the invention further provides a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to a HIV Env protein, such that the nanoparticle comprises HIV Env trimers on its surface.
  • the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin, an insect ferritin, a fungal ferritin and a mammalian ferritin.
  • the monomeric ferritin subunit protein is a monomeric subunit of a Helicobacter pylori ferritin protein.
  • the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence of the isolated, recombinant or synthetic polypeptide as described above.
  • the monomeric ferritin subunit protein comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176
  • composition comprising the isolated, recombinant or synthetic polypeptide according to the invention, the trimer according to the invention, the VLP or the nanoparticle as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.
  • said pharmaceutical composition further comprises an anti- retroviral agent.
  • the invention provides the isolated, recombinant or synthetic polypeptide according to the invention, the trimer according to the invention, the VLP, the nanoparticle or the pharmaceutical composition according to the invention or for use as a medicament.
  • the invention further provides the isolated, recombinant or synthetic polypeptide according to the invention, the VLP, the nanoparticle or the pharmaceutical composition according to the invention or the trimer according to the invention for use in the treatment of an HIV infection or in the prevention of an HIV infection.
  • said HIV infection is a HIV- 1 infection.
  • the invention further provides a nucleic acid encoding the isolated, recombinant or synthetic polypeptide according to the invention.
  • the invention further provides a vector comprising the nucleic acid according to the invention.
  • said nucleic acid is a cDNA.
  • the invention further provides an eukaryotic or prokaryotic host cell comprising the vector of the invention.
  • HIV refers to the human immunodeficiency virus.
  • HIV includes, without limitation, HIV-1 and HIV-2.
  • the HIV-1 virus may represent any of the known major subtypes or clades (e.g., Classes A, B, C, D, E, F, G, J, and H) or outlying subtype (Group 0). Also encompassed are other HIV-1 subtypes or clades that may be isolated.
  • HIV-1 and HIV-2 are distinguished by their genomic organization and their evolution from other lentiviruses. Based on phylogenetic criteria (i.e., diversity due to evolution), HIV-1 can be grouped into three groups (M, N, and O). Group M is subdivided into 11 clades (A through K).
  • HIV-2 can be divided into six distinct phylogenetic lineages (clades A through F) (Human Retroviruses and AIDS 1998: A compilation and analysis of nucleic acid and amino acid sequences (Los Alamos National Laboratory, Los Alamos, NM, 1998, http://hiv- web.lanl.gov).
  • Env gene refers to the polynucleotide of the viral genome that encodes the envelope protein of HIV.
  • Env polypeptide “Env protein'Or “envelope polypeptide” refers to a molecule derived from an HIV envelope protein.
  • the HIV envelope glycoprotein complex (Env) is displayed on the surface of the virus and is the target of neutralizing antibodies.
  • Two different proteins comprise the Env complex:
  • the HIV-1 comprises gpl20, the surface component, and gp41, the transmembrane component.
  • the HIV-2 Env polypeptide comprises the Env gpl25 subunit, which is highly homologous to gpl20 of HIV-1 and the HIV-2 transmembrane subunit gp36, which is highly homologous to HIV-1 gp41.
  • Each functional Env complex consists of three copies of each of these two proteins in a trimer of heterodimers.
  • the glycoproteins are initially produced during virus infection as a polyprotein precursor, designated gpl60.
  • Cellular proteases cleave gpl60 into the two subunits, gpl20 and gp41, which remain non-covalently associated with each other in the Env complex.
  • the gpl40 is a modified gpl60, that lacks the segments of gp41 that normally are imbedded in the viral membrane (transmembrane or TM segment) or in the interior of the virus or cell (cytoplasmic tail, CT).
  • Env polypeptide encompasses without limitation a gpl40 envelope polypeptide, gpl45 envelope and gpl60 envelope polypeptide.
  • mature Env monomer refers to both a HIV-1 gpl60 Env glycoprotein, comprising the HIV-1 Env gpl20 subunit and the HIV-1 Env transmembrane subunit gp41, and to a HIV- 2 gpl40 glycoprotein, comprising the HIV-2 Env gpl25 subunit and the HIV-2 transmembrane subunit gp36.
  • soluble Env monomer refers to both the soluble HIV-1 Env glycoprotein (termed gpl40), comprising the HIV-1 Env gpl20 subunit and the extracellular region of the HIV-1 Env gp41 subunit, and the soluble HIV-2 Env glycoprotein, comprising the HIV-2 Env gpl25 subunit and the extracellular region of the HIV-2 Env gp36 subunit.
  • gpl40 soluble HIV-1 Env glycoprotein
  • soluble HIV-2 Env glycoprotein comprising the HIV-2 Env gpl25 subunit and the extracellular region of the HIV-2 Env gp36 subunit.
  • gpl40 envelope or "gpl40 envelope polypeptide” refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of the water-soluble portion of HIV gp41 glycoprotein ("gp41 portion").
  • HIV gpl40 protein includes, without limitation, proteins wherein the gp41 portion comprises a point mutation such as I559P.
  • a gpl40 envelope comprising such mutation is encompassed by the terms “HIV SOS gpl40", as well as “HIV gpl40 monomer” or "SOSIP gpl40".
  • gpl60 envelope or "gpl60 envelope polypeptide” refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of the complete (thus including the transmembrane portion of gp41) HIV gp41 glycoprotein ("gp41 portion").
  • gpl45 envelope or "gpl45 envelope polypeptide” refers to a protein having two polypeptide chains, the first chain comprising the amino acid sequence of the HIV gpl20 glycoprotein and the second chain comprising the amino acid sequence of a truncated HIV gp41 glycoprotein ("gp41 portion"), wherein the cytoplasmic tail is deleted.
  • gp41 includes, without limitation, (a) the entire gp41 polypeptide including the
  • transmembrane and cytoplasmic domains also referred herein as "complete polypeptide portion”
  • gp41 ectodomain gp4lECTo
  • gp41 comprising the ectodomain and the transmembrane domains, but without the cytoplasmic tail
  • gp41 modified by deletion or insertion of one or more glycosylation sites gp41 modified so as to eliminate or mask the well-known immunodominant epitope
  • gp41 fusion protein g) gp41 labeled with an affinity ligand or other detectable marker.
  • ectodomain means the extracellular region of a transmembrane protein exclusive of the transmembrane spanning and cytoplasmic regions.
  • gp41 polypeptides or polypeptide portions also include "gp41 -derived molecules", which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native gpl20 structure, as well as protein sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp41, or combinations thereof.
  • gp41 portion encompasses any gp41 polypeptides as defined above, including consensus peptides, which are derived from the HR2 domain of gp41 from any HIV isolate. These peptides can include gp41 homo logs that have at least one amino acid substitution, deletion or insertion.
  • the term "g l20” as used herein refers to an envelope protein from HIV-1. The envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated as gpl60. gpl60 forms a homotrimer and undergoes glycosylation in the endoplasmic reticulum and within the Golgi apparatus.
  • Gp41 contains a transmembrane domain and remains in a trimeric configuration; it interacts with gp 120 in a non-covalent manner.
  • gpl20 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gpl20 which binds both to the cellular CD4 receptor and to the cellular chemokine receptors (such as CCR5).
  • the mature gpl20 wildtype polypeptides have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD.
  • Exemplary sequence of wt gpl60 polypeptides are shown on GENBANK®, for example accession numbers AAB05604 and AAD12142 incorporated herein by reference in their entirety as available on Feb. 25, 2009.
  • the gpl20 core has a unique molecular structure, which comprises two domains: an "inner” domain (which faces gp41) and an “outer” domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex).
  • the two gpl20 domains are separated by a "bridging sheet” that is not part of either domain.
  • the gpl20 core typically comprises 25 beta strands, 5 alpha helices, and 10 defined loop segments.
  • the 10 defined loop segments include five conserved regions (C1-C5) and five regions of high variability (VI -V5).
  • gpl20 polypeptide or “gpl20 polypeptide portion” encompasses peptides, including consensus peptides, which are derived from the HR2 domain of gpl20 from any HIV isolate. These peptides can include gpl20 homo logs that have at least one amino acid substitution, deletions or insertions.
  • the gpl20 polypeptide has the amino acid sequence corresponding to the amino acids of positions 31-511 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, or 99%, wherein the positions are numbered according to the HxB2 reference sequence.
  • said gpl20 polypeptide or gpl20 polypeptide portion refers to residues 31-511 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein numbering is according to SEQ ID NO:l .
  • gpl20 polypeptides and portions thereof also include "gpl20-derived molecules" which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native gpl20 structure, as well as protein sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gpl20, or combinations thereof.
  • analogs non-protein organic molecules
  • derivatives chemically functionalized protein molecules obtained starting with the disclosed protein sequences
  • mimetics three-dimensionally similar chemicals
  • a variant gpl20 polypeptide is a gpl20 polypeptide in which one or more amino acids have been altered (e.g., inserted, deleted or substituted).
  • a variant gpl20 polypeptide is a gpl20 polypeptide in which at least 8 consecutive residues, such as 9, 10, 11 or 12 consecutive residues, of the fourth conserved loop (C4) between residues 419 and 434 of gpl20 of SEQ ID NO:l have been deleted.
  • a variant gpl20 polypeptide includes a gpl20 polypeptide in which residues 424-432 are deleted.
  • Additional variant gpl20 polypeptides include deletions of INMWQKVGK (residues 434-442 of SEQ ID NO:l), INMWQKVGKA (residues 434-443 of SEQ ID NO:l),
  • INMWQKVGKAM (residues 434-444 of SEQ ID NO: 1), RIKQIINMWQKVGK (residues 429-442 of SEQ ID NO:l), IKQIINMWQKVGK (residues 430-442 of SEQ ID NO:l), KQIINMWQKVGK
  • variant gpl20 polypeptides include combinations of the amino and carboxyl ends between residues 429 and 444. Any of the disclosed variant gpl20 polypeptide including deletions in C4 can also include a deletion in the V1V2 loop region (with an amino acid sequence set forth in SEQ ID NO:l); see S R Pollard and DC Wiley, EMBO J. 11 :585-91, 1992 which is hereby incorporated by reference in its entirety.
  • amino acid numbering of gpl40/145/160/120/41/125/36 polypeptides disclosed herein is directly referred to a specific sequence, the numbering is relative to the HxB2 numbering scheme as shown for SEQ ID NO:l in this application.
  • fragment refers to a unique portion of the polynucleotide encoding the HIV-1 envelope polypeptide of the present invention shorter in length than the parent sequence.
  • fragment refers to an HIV-1 envelope polypeptide of the present invention comprising up to the entire length of the defined peptide sequence minus one amino acid residue and the coding nucleotide sequence thereof.
  • a fragment may comprise from 5 to 2500 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250, 500 or at least 700 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25 percent or 50 percent) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • truncated Env polypeptide refers to a fragment of the Env polypeptide, which comprises at least a gpl20 portion and an ectodomain portion of gp41.
  • the terms "heptad repeat 1 " and “HRl " are used indistinctly to refer to an heptad repeat region that is located at the amino terminus of wild- type gp41.
  • a heptad repeat is a motif in which a hydrophobic amino acid is repeated every seven residues; such motifs are designated a through g. See Lupas A, Trends Biochem. Sci. 1996; 21 :375-382.
  • Heptad repeats which contain hydrophobic or neutral residues at the a and d positions can form alpha helices and are able to interact with other heptad repeats by forming coiled coils. See Chambers P, et al., J. Gen. Virol. 1990; 71 :3075-3080; Lupas A, supra.
  • the gp41 HRl and HR2 sequences are well known in the art. See Miller M, et al., Proc. Natl. Acad. Sci. USA 2005; 102: 14759- 14764.
  • the HRl region corresponds to amino acids 542 to 591 of the polypeptide depicted in SEQ ID NO: l .
  • I559P refers to a point mutation wherein the isoleucine residue at position 559 of a polypeptide chain is replaced by a proline residue.
  • the invention encompasses an HIV- 1 isolate in which a proline residue replaces, or is substituted for, a non- proline (e.g., isoleucine) amino acid at an amino acid position equivalent to position 559 in the SEQ ID NO: l , for example.
  • a proline residue e.g., isoleucine
  • equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: 1.
  • the gpl40 of HIV-1 BG505 containing said I559P point mutation has the amino acid sequence of SEQ ID NO:2.
  • H564C refers to a point mutation wherein the histidine residue at position 564 of a polypeptide chain is replaced by a cysteine residue.
  • the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., histidine) amino acid at an amino acid position equivalent to position 564 in the SEQ ID NO: l , for example SEQ ID NO: 102, SEQ ID NO: l l l , SEQ ID NO: 1 13, SEQ ID NO: 160, SEQ ID NO: 165 and SEQ ID NO: 180.
  • a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., histidine) amino acid at an amino acid position equivalent to position 564 in the SEQ ID NO: l , for example SEQ ID NO: 102, SEQ ID NO: l l l , SEQ ID NO: 1 13, SEQ ID NO: 160,
  • isolated, recombinant or synthetic polypeptides according to the invention have the amino acid sequence of Illustratively, e.g., equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: l .
  • A73C refers to a point mutation wherein the alanine residue at position 73 of a polypeptide chain is replaced by a cysteine residue.
  • the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., alanine) amino acid at an amino acid position equivalent to position 73 in the SEQ ID NO: l , for example SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 1 12, SEQ ID NO: 1 14, 137- 150, SEQ ID NO: 161 , SEQ ID NO: 166 and SEQ ID NO: 181.
  • equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: l
  • H72C refers to a point mutation wherein the histidine residue at position 72 of a polypeptide chain is replaced by a cysteine residue.
  • the invention encompasses an HIV- 1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., histidine) amino acid at an amino acid position equivalent to position 72 in SEQ ID NO: l , example SEQ ID NO: 102, SEQ ID NO: l 1 1 , SEQ ID NO: 1 13, SEQ ID NO: 123-136, SEQ ID NO: 160, SEQ ID NO: 165 and SEQ ID NO: 180.
  • equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO:l.
  • A316 W refers to a point mutation wherein the alanine residue at position 316 of a polypeptide chain is replaced by a tryptophan residue.
  • the invention encompasses an HIV-1 isolate in which any hydrophobic amino acid residue (e.g.
  • equivalent amino acid position(s) in other HIV- 1 strains or clades may be determined by reference to SEQ ID NO: 1.
  • A561C refers to a point mutation wherein the alanine residue at position 561 of a polypeptide chain is replaced by a cysteine residue.
  • the invention encompasses an HIV-1 isolate in which a cysteine residue replaces, or is substituted for, a non- cysteine (e.g., alanine) amino acid at an amino acid position equivalent to position 561 in SEQ ID NO:l, for example SEQ ID NO:104, SEQ ID NO:l 12, SEQ ID NO:l 14, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:161, SEQ ID NO:166, and SEQ ID NO:181.
  • equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO:l .
  • E64K refers to a point mutation wherein the glutamic acid residue at position 64 of a polypeptide chain is replaced by a lysine residue.
  • the invention encompasses an HIV-1 isolate in which a lysine residue replaces, or is substituted for, a non- lysine (e.g., glutamic acid) amino acid at an amino acid position equivalent to position 64 in the SEQ ID NO:l, for example SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160-163, SEQ ID NO:168, SEQ ID NO:174, and SEQ ID NO:178.
  • equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: 1.
  • H66R refers to a point mutation wherein the histidine residue at position 66 of a polypeptide chain is replaced by an arginine residue.
  • the invention encompasses an HIV-1 isolate in which an arginine residue replaces, or is substituted for, a non- arginine (e.g., histidine) amino acid at an amino acid position equivalent to position 66 in the SEQ ID NO:l, for example.
  • equivalent amino acid position(s) in other HIV-1 strains or clades may be determined by reference to SEQ ID NO: l .
  • pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may include, but are not limited to, aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers, diluents and excipients include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate.
  • a nontoxic surfactant for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery.
  • Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like may also be included with all the above carriers.
  • Adjuvants are formulations and/or additives that are routinely combined with antigens to boost immune responses.
  • Suitable adjuvants for nucleic acid based vaccines include, but are not limited to, saponins, Quil A, imiquimod, resiquimod, interleukin-12 delivered in purified protein or nucleic acid form, short bacterial immunostimulatory nucleotide sequences such as CpG- containing motifs, interleukin-2/Ig fusion proteins delivered in purified protein or nucleic acid form, oil in water micro- emulsions such as MF59, polymeric microparticles, cationic liposomes, monophosphoryl lipid A, immunomodulators such as Ubenimex, and genetically detoxified toxins such as E. coli heat labile toxin and cholera toxin from Vibrio.
  • Such adjuvants and methods of combining adjuvants with antigens are well known to those skilled in the art.
  • Adjuvants suitable for use with protein immunization include, but are not limited to, alum; Freund' s incomplete adjuvant (FIA); saponin; Quil A; QS-21 ; Ribi Detox; monophosphoryl lipid A (MPL) adjuvants such as Enhanzyn(TM); nonionic block copolymers such as L- 121 (Pluronic; Syntex SAF); TiterMax Classic adjuvant (block copolymer, CRL89-41, squalene and microparticulate stabilizer;
  • TiterMax Gold Adjuvant new block copolymer, CRL- 8300, squalene and a sorbitan monooleate; Sigma- Aldrich
  • Ribi adjuvant system using one or more of the following: monophosphoryl lipid A, synthetic trehalose, dicorynomycolate, mycobacterial cell wall skeleton incorporated into squalene and polysorbate-80; Corixa); RC-552 (a small molecule synthetic adjuvant; Corixa) ; Montanide adjuvants (including Montanide IMS1I1X, Montanide IMS131x, Montanide IMS221x, Montanide IMS301x, Montanide ISA 26A, Montanide ISA206, Montanide ISA 207, Montanide ISA25, Montanide ISA27, Montanide ISA28, Montanide ISA35, Montanide ISA50V, Montanide ISA563, .
  • Methods of combining adjuvants with antigens are well known to those skilled in the art. Because current vaccines depend on generating antibody responses to injected antigens, commercially available adjuvants have been developed largely to enhance these antibody responses. To date, the only FDA-approved adjuvant for use with human vaccines is alum.
  • alum helps boost antibody responses to vaccine antigens, it does not enhance T cell immune responses.
  • adjuvants that are able to boost T cell immune responses after a vaccine is administered are also contemplated for use. It is also known to those skilled in the art that cytotoxic T lymphocyte and other cellular immune responses are elicited when protein-based immunogens are formulated and administered with appropriate adjuvants, such as ISCOMs and micron-sized polymeric or metal oxide particles. Certain microbial products also act as adjuvants by activating macrophages, lymphocytes and other cells within the immune system, and thereby stimulating a cascade of cytokines that regulate immune responses.
  • MPL monophosphoryl lipid A
  • Enhanzyn(TM) adjuvant (Corixa Corporation, Hamilton, MT) consists of MPL, mycobacterial cell wall skeleton and squalene.
  • Adjuvants may be in particulate form.
  • the antigen may be incorporated into biodegradable particles composed of poly- lactide-co-glycolide (PLG) or similar polymeric material.
  • PLG poly- lactide-co-glycolide
  • Such biodegradable particles are known to provide sustained release of the immunogen and thereby stimulate long-lasting immune responses to the immunogen.
  • Other particulate adjuvants include, but are not limited to, micellular particles comprising Quillaia saponins, cholesterol and phospholipids known as
  • Superparamagnetic microbeads include, but are not limited to, [mu]MACS(TM) Protein G and
  • [mu]MACS(TM) Protein A microbeads (Miltenyi Biotec) , Dynabeads(R) Protein G and Dynabeads(R) Protein A (Dynal Biotech) .
  • superparamagnetic particles such as [mu]MACS(TM) Protein G and Dynabeads(R) Protein G have the important advantage of enabling immunopurification of proteins.
  • a “prophylactically effective amount” is any amount of an agent which, when administered to a subject prone to suffer from a disease or disorder, inhibits or prevents the onset of the disorder.
  • prophylactically effective amount will vary with the subject being treated, the condition to be treated, the agent delivered and the route of delivery. A person of ordinary skill in the art can perform routine titration experiments to determine such an amount. Depending upon the agent delivered, the prophylactically effective amount of agent can be delivered continuously, such as by continuous pump, or at periodic intervals (for example, on one or more separate occasions) . Desired time intervals of multiple amounts of a particular agent can be determined without undue experimentation by one skilled in the art.
  • inhibiting the onset of a disorder means either lessening the likelihood of the disorder's onset, preventing the onset of the disorder entirely, or in some cases, reducing the severity of the disease or disorder after onset. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.
  • Reducing the likelihood of a subject's becoming infected with HIV means reducing the likelihood of the subject's becoming infected with HIV by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HIV, a twofold reduction in the likelihood of the subject becoming infected with HIV would result in the subject having a 0.5% chance of becoming infected with HIV. In the preferred embodiment of this invention, reducing the likelihood of the subject's becoming infected with HIV means reducing the likelihood of the subject's becoming infected with the virus by at least ten-fold.
  • Subject means any animal or artificially modified animal.
  • Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, goats, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds and fowl, such as chickens and turkeys.
  • Artificially modified animals include, but are not limited to, transgenic animals or SCID mice with human immune systems.
  • the subject is a human. "Exposed" to HIV means contact or association with HIV such that infection could result.
  • terapéuticaally effective amount is any amount of an agent which, when administered to a subject afflicted with a disorder against which the agent is effective, causes the subject to be treated. "Treating" a subject afflicted with a disorder shall mean causing the subject to experience a reduction, diminution, remission, suppression, or regression of the disorder and/or its symptoms. In one embodiment, recurrence of the disorder and/or its symptoms is prevented. Most preferably, the subject is cured of the disorder and/or its symptoms.
  • HIV infected means the introduction of viral components, virus particles, or viral genetic information into a cell, such as by fusion of cell membrane with HIV.
  • the cell may be a cell of a subject.
  • the cell is a cell in a human subject.
  • "Host cells” include, but are not limited to, prokaryotic cells, e.g., bacterial cells (including gram-positive cells), yeast cells, fungal cells, insect cells and animal cells.
  • Suitable animal cells include, but are not limited to HeLa cells, COS cells, CV1 cells and various primary mammalian cells.
  • mammalian cells can be used as hosts, including, but not limited to, mouse embryonic fibroblast NIH-3T3 cells, CHO cells, HeLa cells, L(tk-) cells, PER.C6 and COS cells. Mammalian cells can be transfected by methods well known in the art, such as calcium phosphate precipitation, electrop oration and microinjection.
  • Electroporation can also be performed in vivo as described previously (see, e.g., U.S. Patent Nos.
  • Immunizing means generating an immune response to an antigen in a subject. This can be
  • nucleic acid refers to any nucleic acid or polynucleotide, including, without limitation, DNA, RNA and hybrids thereof.
  • nucleic acid bases that form nucleic acid molecules can be the bases A, C, T, G and U, as well as derivatives thereof. Derivatives of these bases are well known in the art and are exemplified in PCR Systems, Reagents and Consumables (Perkin-Elmer Catalogue 1996- 1997, Roche Molecular Systems, Inc., Branchburg, NJ, USA) .
  • a “vector” refers to any nucleic acid vector known in the art. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors and bacteriophage vectors.
  • plasmid vectors include, but are not limited to, plasmid vectors, cosmid vectors and bacteriophage vectors.
  • one class of vectors utilizes DNA elements which are derived from animal viruses such as animal papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTC or MoMLV) , Semliki Forest virus or SV40 virus.
  • the eukaryotic expression plasmid PPI4 and its derivatives are widely used in constructs described herein.
  • the invention is not limited to derivatives of the PPI4 plasmid and may include other plasmids known to those skilled in the art.
  • numerous vector systems for expression of recombinant proteins may be employed.
  • one class of vectors utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), Semliki Forest virus or SV40 virus.
  • cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells.
  • the marker may provide, for example, prototropy to an auxotrophic host, biocide (e.g., antibiotic) resistance, or resistance to heavy metals such as copper or the like.
  • biocide e.g., antibiotic
  • the selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.
  • the cDNA expression vectors incorporating such elements include those described by (Okayama and Berg, 1983).
  • Protein conformation refers to the characteristic 3 -dimensional shape of a protein, including the secondary (helices, sheet), supersecondary (motifs), tertiary (domains) and quaternary (multimeric proteins) structure of the peptide chain.
  • “native conformation” refers to the characteristic state, formation, shape or structure of a protein in the biologically active form in a living system in which it is folded to a global minimum of Gibbs free energy as defined by C.B. Anfinsen (Nobel Lecture, Dec. 1 1 , 1972).
  • the skilled person is capable of determining whether a protein comprising disulfide bonds is folded properly and in the native conformation. Such determinations may for instance comprise the measurement of the properly folded, oxidized and digested Lys-Arg-Insulin intermediate, by HPLC analysis. In embodiments, such determinations comprise X-ray crystallographic analyses as described herein.
  • a method of the present invention is used preferably for the isolation of recombinantly produced proteins that comprise disulfide-bonds in the native conformation. Recombinantly produced proteins can be either directly expressed or expressed as a fusion protein. Detection of the expressed protein is achieved by methods known in the art such as, for instance, radioimmunoassays, Western blotting techniques or immunoprecipitation.
  • the Env trimers may be classified as closed native-like, partially open native-like or non-native (Pugach et al., 2015).
  • Native-like trimers are regularly shaped and have the highest concentration of electron density at the particle center (usually shaped like a triangle because Env is trimeric). The absence or presence of additional density around this center of mass determines whether trimers are classified as closed native-like or partially open native-like, respectively.
  • Non-native forms are often elongated and no triangular center of density is visible.
  • closed native conformation refers to the visual shape of the Env trimer as previously described for BG505 SOSIP.664 (Sander et al. 2013, A next-generation cleaved, soluble HIV- 1 Env trimer, BG505 SOSIP. 664 gpl40, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.
  • an Env trimer has a closed native conformation if it has a compact triangular propeller shape with no additional density surrounding the trimer.
  • nucleic acid conformation refers to the visual shape of an Env trimer particle which does not clearly show a central, triangular mass.
  • a trimer having a nonnative conformation resembles previously described images of uncleaved, non-SOSIP gpl40 proteins (Ringe RPet al. 2013. Cleavage strongly influences whether soluble HIV- 1 envelope glycoprotein trimers adopt a native-like
  • the conformation as described above of the isolated, recombinant or synthetic Env protein may suitably be characterized by negative stain electron microscopy (EM) and Reducing SDS-PAGE as described herein.
  • EM negative stain electron microscopy
  • the isolated, recombinant or synthetic Env proteins of the invention may be made of any Env polypeptide and may be of any type or clade of HIV.
  • the HIV-1 Env polypeptide comprises a gpl20 polypeptide portion and at least a gp41 ectodomain polypeptide portion.
  • the Env polypeptides are modified to comprise a first cysteine residue at an amino acid position equivalent to amino acid position 49, 50, 51, 71, 72 or 73 and a second cysteine residue at an amino acid position equivalent to amino acid position of a residue selected from the group consisting of: 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570 of the HxB2 reference sequence having the amino acid sequence of SEQ ID NO: 1, resulting in an intermolecular disulfide bond between said first and said second cysteine residue.
  • the present invention encompasses HIV envelope (Env) glycoprotein complexes, which comprise covalently associated surface gpl20 and transmembrane gp41 glycoprotein subunits, and soluble forms thereof.
  • the HIV envelope (Env) glycoprotein complexes of the invention are more structurally stable than native Env complexes, which are characteristically more labile or unstable in order to be capable of efficiently undergoing conformational changes during the process of virus-cell fusion.
  • the HIV envelope (Env) glycoprotein complexes of the invention display less reactivity to non-Nab 17b.
  • the structural instability of the native HIV Env complex, or soluble forms thereof is overcome by the introduction of amino acid sequence changes designed to stabilize inter-subunit interactions between gpl20 and gp41 or between their HIV-2 equivalents gpl25 and gp36.
  • Such changes according to this invention include the introduction of a disulfide bond between gpl20 and gp41 or their HIV-2 equivalents, between a residue located within the alphaO and a residue located within the HR1 region of said Env polypeptide.
  • the introduction of the disulfide bond as described herein greatly enhances trimer stability, decreases conformational flexibility, and improves the presentation of bNAb epitopes, while diminishing the exposure of non-NAb epitopes.
  • the Env proteins of the invention comprising the new disulfide bonds also stabilized and improved Env trimers from diverse virus isolates. These stabilized Env trimers allow for the design of multivalent vaccines aimed at inducing bNAbs. The inventors believe that higher global and local stability and further occlusion of non-NAb epitopes might improve the performance of Env trimers as immunogens.
  • the gp41 portion encompasses any gp41 polypeptides or fragments or truncated forms thereof, including consensus peptides, which are derived from the HR2 domain of gp41 from any HIV isolate. These peptides can include gp41 homo logs that have at least one amino acid substitution, deletions or insertions.
  • Gp41 polypeptides or polypeptide portions also include "gp41 -derived molecules" which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three- dimensionally similar chemicals) of the native gpl20 structure, as well as proteins sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp41, or combinations thereof.
  • gp41 -derived molecules which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three- dimensionally similar chemicals) of the native gpl20 structure, as well as proteins sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp41, or combinations thereof.
  • the gp41 ectodomain polypeptide portion has the amino acid sequence of the amino acids of positions 512-664 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • said gp41 ectodomain polypeptide portion refers to residues 516-672 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • gpl20 polypeptide or "gpl20 polypeptide portion” encompasses peptides, including consensus peptides, which are derived from gpl20 from any HIV isolate. These peptides can include gpl20 homologs that have at least one amino acid substitution, deletions or insertions.
  • the gpl20 polypeptide has the amino acid sequence corresponding to the amino acids of positions 31-511 of HxB2 SEQ ID NO:l or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, wherein the positions are numbered according to the HxB2 reference sequence.
  • said gpl20 polypeptide or gpl20 polypeptide portion refers to residues 37-515 of BG505 SOSIP SEQ ID NO:2 or an amino acid sequence having sequence identity of at least 70% thereto, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • amino acid positions of the residues between which a disulfide bond of the invention may be formed may not be the same in all HIV isolates.
  • residue 73 and residue 561 in SEQ ID NO:l are located at different positions in SEQ ID NO:2. Therefore, as used herein the term "position equivalent to a certain residue of a certain sequence", refers to the position of the same residue a can be identified after alignment of the different sequences.
  • position equivalent to the amino acid 73 (which is an A) of SEQ ID NO 1 refers to the A in position 78 of SEQ ID NO:2.
  • the corresponding position in the gpl60 sequence of other HIV-1 or HIV-2 isolates can be easily identified by aligning the sequence of both gpl60 variants or by performing multiple alignment of the gpl60 wherein the position of the disulfide bond is to be identified within a plurality of other gpl60 sequences.
  • sequence comparison typically one sequence acts as a reference, to which the test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of HIV envelope glycoproteins fusion proteins comprising envelope glycoproteins and nucleic acid sequences encoding the same, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
  • comparison window includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith- Waterman local homology algorithm, by the
  • Needleman-Wunsch homology alignment algorithm by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection. See Smith T, Waterman M, Adv. Appl. Math. 1981 ; 2:482-489; Needleman S, Wunsch C, J. Mol. Biol. 1970; 48:443-453; Pearson W, Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448; the GAP,
  • the BLAST and BLAST 2.0 algorithms are suitable for determining percent sequence identity and sequence similarity. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402; Altschul S, et al., J. Mol. Biol. 1990; 215:403-410.
  • the BLAST and BLAST 2.0 programs are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W word length
  • E expectation
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the Env polypeptides according to the invention also encompass HIV-2 Env polypeptides.
  • HIV-2 Env proteins comprise a gpl25 polypeptide portion and at least a gp36 ectodomain polypeptide portion.
  • residues mentioned herein such as at residues 72 and 73 can be identified.
  • SEQ ID NO:9 which is an exemplary HIV-2 gpl60 protein
  • the equivalent residues are residues V and D of amino acid sequence RDTWGTVQCLPDNGDYTEIRLN of SEQ ID NO:9. These residues correspond to the H and A of the amino acid sequence
  • residues 561 and 564 can also be identified by alignment as residues R and E of amino acid sequence
  • the isolated, recombinant or synthetic polypeptide of the invention has an affinity for an bNAb including but not limited to 2G12, PGT145, PGT151, VRCOl, PGT121, PG16.
  • the isolated, recombinant or synthetic polypeptide is capable of forming homotrimers, preferably also of heterodimers.
  • the isolated, recombinant or synthetic polypeptide has a negligible affinity for antibodies directed against the V3 such as the antibodies 19b, 14E, 39F.
  • the isolated, recombinant or synthetic polypeptide has a negligible affinity forbinding to antibodies directed to the CD4-induced epitopes, such as the antibodiesantibody 17b, 48d and 412d.
  • the isolated, recombinant or synthetic polypeptide has a melting temperature higher than 60.0 °C, preferably higher than 63.0 °C, 63.7 °C; 65,0 °C, 68,0 °C, 68,3 °C, 68,5 °C, 69,0 °C, 70 °C , 75 °C, 75.2 °C, 80,0 °C, 85,0 °C, or 90,0 °C, preferably as determined by a DSC assay.
  • the melting temperature may be determined using a conventional DSC assay as previously described.
  • the isolated, recombinant or synthetic polypeptide is capable of folding a trimer in a closed native conformation.
  • At least 33%, 35%>, more preferably at least 40%>, 45%>, 50%>, 55%>, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95% is in a native conformation.
  • the isolated, recombinant or synthetic polypeptide having at least 33%> of homotrimers in closed native conformation.
  • the isolated, recombinant or synthetic Env proteins of the invention also encompass Env proteins which have the disulfide bond according to the invention and which have further modifications to the Env proteins. However, it will be understood that such modifications should preferably not have detrimental effects for the above mentioned functions of the isolated, recombinant or synthetic Env protein.
  • the isolated, recombinant or synthetic Env protein has also a negligible affinity to CD4i-Abs such as 48d, 412d.
  • the capacity for adhesion to CD4 can be determined by radio-immune precipitation, by ELISA, Octet, ITC or by surface plasmon resonance, the detail of these methods being set out in the remainder of the description. These methods can be modified within the limit of current knowledge, the objective being to simply make sure that the glycoprotein according to the invention indeed forms a complex with CD4.
  • the CD4 molecules can be prepared in all kinds of different ways, including purification from a natural source or using genetic recombination techniques. In this context, it is possible to use the CD4 molecules described in WO 89/03222, WO 89/02922, Smith et al. (Science, 238, 1704-1707, 1987) and Littman et al. (Nature, 325, 453-455, 1987), for example.
  • the company ERC BioServices Corporation, 649A Lofstrand Lane, Rockeville, Md. 20850, USA also sells a CD4 produced by CHO ST4.2 cells (In: Aids Research and Reference Reagent Program Catalog, the Nat. Inst. Health U.S.D.H.H.S.), for example.
  • the affinity (Kd) for an antibody can suitably be measured by surface plasmon resonance and is preferably be of the order of 10 ⁇ -4 >to 10 ⁇ -12 >M, preferably 10 ⁇ -9 >to 10 ⁇ -11 >M, which is in accordance with the affinities already measured for gpl20 molecules (Smith et al., Science, 238: 1704, 1987; Lasky et al., Cell, 50: 975, 1987), for example.
  • thermostability of the isolated, recombinant or synthetic Env proteins may suitably be determined using differential scanning calorimetry (DSC) as described herein.
  • the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:l, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:2, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the isolated, recombinant or synthetic Env protein of the invention has at least a homology of 70% to SEQ ID NO:9, more preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the isolated, recombinant or synthetic Env protein of the invention has a sequence homology of at least 70%> to a polypeptide having the amino acid sequence of any of SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, more preferably 71%, 72%, 7
  • the isolated, recombinant or synthetic Env protein of the invention comprises a polypeptide having the amino acid sequence having a sequence homology of at least 70%> to the amino acid sequence corresponding to the gpl40 portion of any of the Env polypeptides having the amino acid sequences of SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
  • the Env polypeptides contemplated for modification may also comprise modifications that do not substantially alter the function of the Env protein.
  • certain amino acids may be substituted for other amino acids in a polypeptide structure without appreciable loss of interactive binding capacity of the structure such as, for example, the epitope of an antigen that is recognized and bound by an antibody. Since it is the interactive capacity and nature of a polypeptide that defines its biological (e.g. immunological) functional activity, certain amino acid sequence substitutions can be made in an amino acid sequence (or its underlying DNA coding sequence) and nevertheless obtain a polypeptide with comparable properties. Various changes may be made to the amino acid sequences of the antigens of the present invention without appreciable loss of immunogenic activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. See Kyte J, Doolittle R, J. Mol. Biol. 1982; 15(1):105-132. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.
  • the substitution of amino acids whose hydropathic indices are within plus or minus 2 is preferred, those which are within plus or minus 1 are particularly preferred, and those within plus or minus 0.5 are even more particularly preferred.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics; these are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0 plus or minus 1), glutamate (+3.0 plus or minus 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 plus or minus 1), alanine (-0.5), histidine (- 0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5) and tryptophan (-3.4).
  • the isolated, recombinant or synthetic Env protein comprises a methionine at position equivalent to position 535 of SEQ ID NO:l(M535).
  • Residue 535 is positioned in the middle of the a6 helix, which might explain why a methionine here has a stabilizing effect; thus, Met has a higher helical propensity in the isolated, recombinant or synthetic Env (Fig. L IE, Table 1.S2).
  • the isolated, recombinant or synthetic Env protein comprises a glutamine, a methionine, or an asparagine at position equivalent to position 543 of SEQ ID NO:l .
  • the isolated, recombinant or synthetic Env protein comprises 535M together with 543N or 543Q.
  • NS-EM analysis showed an unchanged percentage of closed vs. open trimers (-15% closed; Table 1.1), but thermostability was increased (see Table 1.1).
  • Isolated, recombinant or synthetic Env proteins of the invention comprising 535M together with 543N or 543Q also have an improved binding of the quaternary structure-dependent bNAbs PGT145, 35022 and PGT151.
  • the isolated, recombinant or synthetic Env protein comprises an 316W, 316Y, 316V, or 3161.
  • the advatage thereof is that such residue strengthens hydrophobic interactions between V3 and the underlying gpl20 layer 2 (i.e., V120, L122 and P206), thereby decreasing the propensity for V3 to flip out of its ground-state location (Fig. 1.1 A,B).
  • the A316W, 316Y, 316V, or 3161 improves trimer formation, and it also increases the thermostability as assessed by a novel assay that can be used with unpurified Env proteins (Fig. 1.S1B-G).
  • V3 non-NAbs 447-52D, 39F, 14e and 19b each bound markedly less well to all the A316W variant trimers than to wild- type (Fig. 1.S1H-K).
  • 14e and 19b binding to PGT145 purified BG505 SOSIP.664 A316W trimers was reduced by ⁇ 80%> and ⁇ 50%>, respectively, and for the CD4i non-NAbs 17b and 412d binding was -50% lower (Table 1.2, Fig. 1.S2F).
  • 316W residue did not affect binding of the same non- NAbs to V3 peptides, we conclude that it works indirectly on the trimer, by impeding the exposure of V3 epitopes (Fig.
  • trimers based on the modified protein of the invention comprising 316W, 316Y, 316V, or 3161 have a high proportion in the closed conformation. Their thermostability was also improved see Table 1.1). Introduced into Env-pseudovirus, the 316W, 316Y, 316V, or 3161 residue reduced infectivity by -98%, implying either that conformational flexibility is important for co-receptor interactions during entry or that the 316 residue plays a direct role in co-receptor binding (Fig. 1.S1 L).
  • the isolated, recombinant or synthetic Env protein comprises the residue 64K or 66R. Both 64K and 66R residues substantially reduce, or even eliminate, the binding of the CD4i non-NAbs in the isolated, recombinant or synthetic Env proteins (Fig. l .SlH-O). After trimers of the isolated, recombinant or synthetic Env protein having the 64K residue were PGT145-purified and visualized by NS-EM, the percentage of closed trimers was substantially increased for the E64K mutant (-90%) closed vs. 35% for wild-type). Each residue had also a minor (improved) effect on thermostability. In an ELISA, bNAb binding was enhanced (e.g., for the trimer apex bNAbs PG16 and PGT145).
  • the isolated, recombinant or synthetic Env protein comprises the residues 316W, 316Y, 316V, or 3161 combined with any of the 64K or 66R residues.
  • These isolated, recombinant or synthetic Env proteins have acquired the beneficial properties associated with each individual residue.
  • Each double mutant was expressed efficiently and formed fully cleaved trimers, and thermostability was slightly increased over wild-type (Fig. 1.S1B-G).
  • SOSIP.v4 trimer refers to a trimer of the the isolated, recombinant or synthetic Env protein having the 559P amino acid residue comprising further the optimal amino acids at position 535M and 543N or 543Q, and in addition the 316W residue and either theE64K (designated SOSIP.v4.1) or the 66R (SOSIP.v4.2) residues (Table 1.S3).
  • SOSIP.v4 trimers induced weaker anti-V3 responses in rabbits and, as a result, lower titers of V3-dependent NAbs against Tier- IA virus SF162. This effect was observed without compromising the autologous NAb response.
  • the inventors have surprisingly found that many bNAbs bound equally well to BG505 SOSIP.664-D7324 and SOSIP.v4-D7324 trimers in ELISA, indicating their epitopes are unaffected by the stabilizing substitutions. This outcome was confirmed by SPR for the quaternary structure-dependent bNAbs PG16, PGT145, 35022 and PGT151 (Fig. 1.1H).
  • the bNAb epitopes were generally unaffected, and the quaternary structure dependent bNAbs PGT145 and PGT151 actually more strongly in ELISA than to the wild type ZM197M SOSIP.664-D7324 trimers (Table 1.2, Fig. 1.S2J). Once more, the binding of non-NAbs to V3 and CD4i epitopes was greatly diminished (Table 1.2, Fig. 1.S2J).
  • the inventors have also found that SOSIP.v4 trimers have a native-like conformation.
  • SOSIP.v4 trimers have a native-like conformation.
  • they prepared complexes of the AMC008 SOSIP.V4 trimers with bNAbs PGV04 and 35022 (added as Fabs), and visualized them by NS- EM.
  • the stabilized trimers were all compact entities that were virtually indistinguishable from previously published low-resolution reconstructions of BG505 and B41 SOSIP.664 trimers (see Fig. 1.3). Therefore, in another preferred embodiment, the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, and 316W.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, and 316W.
  • An advantage of these 2 embodiments is that their trimers have retained the ability to bind the quaternary-dependent and various other bNAbs (2G12, PGT135, PGT121, PGT126, PG9, PG16, PGT145, PGT151, 35022, VRCOl and CHI 03).
  • their reactivity with non-NAbs was generally reduced.
  • they do not undergo CD4-induced conformational changes efficiently, as judged by the lack of induction of the 17b and 412d CD4i epitopes (Table 1.2, Fig. 1.S2G).
  • ITC Isothermal Titration Calorimetry
  • CD4-IgG2 binding to AMC008, B41 and ZM197 SOSIP.v4-D7324 trimers was slightly reduced compared to SOSIP.664-D7324, but more markedly so for the BG505 trimers.
  • CD4- IgG2 binding to BG505 SOSIP.664-D7324 trimers is defined as 100% (see Methods), binding to the
  • SOSIP.v4.1 and SOSIP.v4.2 variants was reduced to 27% and 32%, respectively (Table 1.2, Fig. 1.S3).
  • CD4-IgG2 association rates were comparable for the SOSIP.v4 and wild type trimers, but the ligand dissociated markedly faster from the stabilized trimers (Fig. 1.1H).
  • Biolayer interferometry data confirmed that the affinity of sCD4 for various AviB-tagged BG505 stabilized trimers was 2- to 4-fold lower in each case, with the greatest reduction (4-fold) seen with the A316 W single mutant and the SOSIP.v4.2 double mutant (Fig. 1.1G).
  • the stabilizing substitutions blocked the ability of CD4 to induce ordering of residues 370-382 (CD4bs), 245-256 and 476-483 (layer 3), 206-226 (layer 2), 53-92 (layer 1) and 566-592 (HR2; al) (Fig. 1.2B&C, Fig. 1.S3). Furthermore, the substitutions reduced the CD4-induced disorder of residues 165-181 (V2), 286-320 (V3), 520-537 (a6) and 593-628 (gp41 disulfide loop).
  • H66R+A316 W substitutions prevent the CD4-induced opening of the trimer apex, as well as the rigidification cascade that spreads from the CD4bs via layer 3, layer 2 and layer 1 to gp41 HR1 (Fig. 1.2B&C).
  • the inventors used a V3 peptide competition ELISA to assess the relative contribution of V3 Abs to the overall Ab response induced by the various trimers (Sanders et al., 2015). They estimated that the median anti-V3 response in the BG505 SOSIP.664 immunized rabbits was ⁇ 30%> of the total anti-Env response but only ⁇ 10%> in the SOSIP.v4 recipients (Fig. 1.4E). For the rabbits given AMC008-based trimers, the corresponding reduction in the contribution of the anti-V3 response was only minimal, however, i.e., from -50% to -40% (Fig. 1.4E).
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, 316W and 64K.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, 316W and 64K.
  • An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543N, 316W and 66R.
  • An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q, 316W and 66R.
  • An advantage of this the isolated, recombinant or synthetic Env protein is that a very high percentage of its trimers have of closed conformation. Trimers of these Env protein have no detectable binding to 19b IgG.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P, 535M, 543Q and 567K. These residues promote trimer formation.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 64K or 66R, 316W, 73C and 561C. These residues reduce binding to non-NAbs b6 and 14e and increase binding affinity with PGT145 and PGT151.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 64K and 316W or 66R and 316W, and more preferably combined with 73C and 561 C. These residues stabilize the proteins in their closed conformation, further occluding non-NAb epitopes and increasing their thermostability. In addition, the proteins show a high percentage of underprocessed oligomannose glycans, which is typical of virus-derived Envs and soluble native-like Env trimers.
  • the isolated, recombinant or synthetic Env protein comprises the amino acid residues 559P combined with the amino acid residues 125W and 316W; 535M and 543Q; 535M and 543N; 64K, 316W and 543N; 535M, 543N, 316W and 64K; 535M, 543N, 316W and 66R; 535M, 543N, 66R and 316W; 64K and 535M; 64K, 316W and 535M; 66R, 316W and 535M; 535M and 543Q; 535M and 543N; 316W, 535M and 543N; 64K, 316W, 535M and 543N, 66R, 316W, 535M and 543N; 559P, 316W, 64K, 73C, 561C, or 49C and 555C, wherein said residue is numbered by reference to SEQ ID NO:l .
  • the trimer of the invention is a SOSIP.v4 trimer as defined above.
  • SOSIP.v4 trimer as defined above.
  • An advantage of these trimers is that they are fully cleaved and highly homogeneous, as assessed by reducing and non-reducing SDS-PAGE and BN-PAGE analysis (Fig. 1.S2A-B). The proportion of closed trimers in each case. These trimers were more thermostable by 2-4°C, compared to wild-type (Table 1.1, Fig. 1.S2D). Their glycosylation profiles differed little if at all from their wild- type counterparts, with oligomannose glycans again predominating (Table 1.1 , Fig. 1.S2F). In addition, these trimers are all native-like, more thermostable and adopt the fully closed conformation more frequently.
  • Env trimers and VLPs encompasses envelope trimers for the production of virus like particles (VPLs) and pseudoparticles for use as VLP -based immunogens, to generate neutralizing antibodies, for example, and VLP -based vaccines against which a subject can mount a potent immune response against HIV.
  • Env trimers comprising the stabilizing disulfide bonds of the invention, as well as Env trimers comprising other stabilizing mutations in gpl20 and gp41 or equivalents thereof from HIV-2 as described herein, are used to generate VPLs and pseudovirions having reduced monomer, dimer and tetramer forms and enhanced trimer forms of gpl20/gp41 Env.
  • N- terminal stabilizing mutations in the context of HIV- 1 virus as described herein can yield trimer forms of Env (gpl20/gp41) on VLP and pseudovirions, to the virtual exclusion of monomer, dimer and tetramer forms, thus allowing for an immunogen that more closely resembles native HIV envelope trimers.
  • composition comprising the modified polypeptide of the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • This invention also provides a composition comprising the trimeric complex of the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the composition further comprises an adjuvant.
  • the composition further comprises an antiretroviral agent.
  • compositions and immunogenic preparations comprising the polypeptides of the present invention capable of inducing an immunological reaction (including protective immunity) in a suitably treated animal or human, and a suitable carrier therefore, are provided.
  • Immunogenic compositions are those which result in specific antibody production or in cellular immunity when injected into a human or an animal.
  • Such immunogenic compositions or vaccines are useful, for example, in immunizing an animal, including a human, against infection and/or damage caused by HIV.
  • the vaccine preparations comprise an immunogenic amount of one or more of the isolated, recombinant or synthetic polypeptides of the invention.
  • immunogenic amount is meant an amount capable of eliciting the production of antibodies directed against the retrovirus in a mammal into which the vaccine has been administered.
  • the route of administration and the immunogenic composition may be designed to optimize the immune response on mucosal surfaces, for example, using nasal administration (via an aerosol) of the immunogenic composition.
  • the methods and compositions of the invention also include use of another antiviral agent in addition to the one or more of the present Env polypeptides, or a combination of Env polypeptides as described herein.
  • antiretroviral agents or compounds which can be administered in addition to the polypeptides and compositions of the invention include, without limitation, protease inhibitors, retroviral polymerase inhibitors, azidothymidine (AZT) , didanoside (DDI), soluble CD4, a polysaccharide sulfates, T22, bicyclam, suramin, antisense ohogonulceotides, ribozymes, rev inhibitors, protease inhibitors, glycolation inhibitors, interferon and the like.
  • examples include acyclovir, 3-aminopyridine-2- carboxyaldehyde thiosemicarbazone (3-AP,
  • the pharmaceutical composition comprises a cocktail of different Env polypeptides.
  • said pharmaceutical composition comprises at least 2 different Env
  • said pharmaceutical composition comprises at least 2, 3 or 4 different Env polypeptides according to the invention.
  • An advantage thereof is that these compositions induce better heterologous responses against several Tier-2 viruses.
  • said pharmaceutical composition comprises at least 2, 3 or 4 different Env polypeptides of the invention.
  • the present invention provides novel Env-ferritin nanoparticle (np) vaccines.
  • Such nanoparticles comprise fusion proteins, each of which comprises a monomeric subunit of ferritin joined to an immunogenic portion of an Env protein. Because such nanoparticles display Env protein on their surface, they can be used to vaccinate an individual against HIV.
  • the various protein domains e.g., gpl40 protein, trimerization domain, etc.
  • linkers also referred to as a spacer sequences
  • linker sequence is designed to position the Env protein in such a way to that it maintains the ability to elicit an immune response to the HIV virus.
  • Linker sequences of the present invention comprise amino acids. Preferable amino acids to use are those having small side chains and/or those which are not charged. Such amino acids are less likely to interfere with proper folding and activity of the fusion protein. Accordingly, preferred amino acids to use in linker sequences, either alone or in combination are serine, glycine and alanine. Examples of such linker sequences include, but are not limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or subtracted as needed. Those skilled in the art are capable of determining appropriate linker sequences for proteins of the present invention.
  • This invention also provides a method for preventing a subject from becoming infected with HIV-1, comprising administering to the subject an amount of the composition of the invention effective to prevent the subject from becoming infected with HIV.
  • This invention further provides a method for reducing the likelihood of a subject becoming infected with HIV, comprising administering to the subject an amount of the composition of the invention effective to reduce the likelihood of the subject becoming infected with HIV.
  • This invention also provides a method for delaying the onset of, or slowing the rate of progression of, an HIV-related disease in an HIV-infected subject, which comprises administering to the subject an amount of the composition of the invention effective to delay the onset of, or slow the rate of progression of, the HIV- related disease in the subject.
  • This invention also provides a method for eliciting an immune response against HIV or an HIV infected cell in a subject comprising administering to the subject an amount of the composition of the invention effective to elicit the immune response in the subject.
  • This invention provides a method for eliciting an immune response against HIV or an HIV infected cell in a subject comprising administering to the subject an amount of the trimeric complex of the invention effective to elicit the immune response in the subject.
  • oligomannose glycoforms were predominant ( ⁇ 67%>), with a high Man9GlcNAc2 content ( ⁇ 34%>), properties that are hallmarks of native and native-like trimers (Table 1.1 ; Fig. l .S2F)(Bonomelli et al., 201 1 ; Pritchard et al., 2015).
  • B41 SOSIP.664 trimers were similar to their 2G12/SEC-purified counterparts in that they were partly open (45-60%) in both cases (Pritchard et al., 2015; Pugach et al., 2015). However, for ZM197M SOIP.664, the closed trimer content was reduced from 80% (2G12/SEC) to -15% (Table 1.1, Fig. 1.S2A-D) (Julien et al., 2015).
  • PGT145-purification has paradoxical effects: it can positively select native-like trimers from a mixture of native and non-native Env forms, but it can also open up previously closed, native-like trimers (at least for BG505 and ZM197M).
  • PGT145-affmity chromatography as both a purification strategy and a "stress-test"; i.e., to monitor whether stabilized trimers now resist PGT145-induced opening.
  • Residue 535 is positioned in the middle of the a6 helix, which might explain why a methionine here has a stabilizing effect; thus, Met has a higher helical propensity than the He present in AMC008 Env (Fig. L IE, Table 1.S2) (Levitt and Greer, 1977).
  • Position 543 is located at the end of the same helix; a Gin residue here has a lower helical propensity than the unfavorable Leu residue and might prevent the extension of the a6 helix and thereby the initiation of the fusion process (Levitt and Greer, 1977).
  • the BG505 sequence contains M535 and N543. Based on the high quality BG505 trimers we consider N543 to have the same stabilizing effect on the gp41 prefusion structure as Q543. In contrast, the AMC008 sequence contains neither M535 nor N/Q543, but instead 1535 and L543 (Fig. LIE, Table 1.S2). We therefore constructed AMC008 SOSIP.664 trimer mutants containing one or more of the I535M, L543N and L543Q substitutions (Fig. 1.S1A &1H). All three individual changes improved trimer formation and a similar effect was observed for L543N and L543Q (Fig. 1.S1A).
  • the B41 and ZM197M sequences also both lack one of the beneficial amino acids at position 535 or 543, so we introduced the L543N substitution into B41 and V535M into ZM197M. Both changes improved trimer formation (Fig. 1.S1J &1K).
  • SOSIP.664 proteins containing the optimal amino acids at position 535 (i.e., Met) and 543 (i.e., Asn/Glu) as SOSIP version 3 (SOSIP.v3).
  • SOSIP.v3.1 contains the 543Q change
  • SOSIP.v3.2 contains 543N.
  • Stabilizing the closed conformation of SOSIP.664 trimers requires the V3 region to be sequestered and a reduction in the exposure of non-NAb epitopes (for Tier-2 viruses), including CD4i epitopes.
  • the V3 region is tucked beneath the VI V2 domain in the BG505 SOSIP.664 cryo-EM and x-ray structures (Julien et al., 2013b; Lyumkis et al., 2013; Pancera et al., 2014), it can become exposed on a subset of trimers in vitro and is immunogenic in vivo (Sanders et al., 2013, 2015).
  • V3 exposure is MAb reactivity with SOSIP.664-D7324 trimers under ELISA conditions in which V3 epitopes become readily accessible (Sanders et al., 2013).
  • A316W variant trimers than to wild-type (Fig. 1.S1H-K).
  • 14e and 19b binding to PGT145 purified BG505 SOSIP.664 A316W trimers was reduced by -80% and ⁇ 50%>, respectively, and for the CD4i non-NAbs 17b and 412d binding was -50% lower (Table 1.2, Fig. 1.S2F).
  • the A316W substitution did not affect binding of the same non-NAbs to V3 peptides, we conclude that it works indirectly on the trimer, by impeding the exposure of V3 epitopes (Fig. 1.S1L).
  • the A316W substitution reduced infectivity by ⁇ 98%>, implying either that conformational flexibility is important for co-receptor interactions during entry or that A316 plays a direct role in co-receptor binding (Fig. S1L).
  • Fig. S1L Reducing spontaneous sampling of the CD4-bound conformation
  • two substitutions (E64K and H66R) in layer 1 of the g l20 inner domain that we had identified in a study of how HIV-1 escapes from the entry inhibitor VIR165 (Fig. 1A,C,D;).
  • the side chains of residues 64 and 66 are positioned to interact with HR1 of gp41 (Finzi et al., 2010; Do Kwon et al., 2015; Pancera et al., 2010), but the high crystallographic B-values around residues 64 and 66 imply there is local flexibility (Fig. 1.1C) (Pancera et al., 2014).
  • the multiply stabilized, PGT145-purified SOSIP.v4 trimers derived from 4 different env genes from 3 clades are all native-like. Moreover, compared to unmodified, earlier generation SOSIP.664 trimers, the SOSIP.v4 variants are more thermostable and adopt the fully closed conformation more frequently.
  • D7324-tagged versions of wild type i.e., SOSIP.664
  • stabilized trimer variants were PGT145 affinity-purified and studied by ELISA.
  • the AMC008 SOSIP.v4-D7324 trimers retained the ability to bind the quaternary-dependent and various other bNAbs (2G12, PGT135, PGT121, PGT126, PG9, PG16, PGT145, PGT151 , 35022, VRCOl and CH103).
  • their reactivity with non-NAbs was generally reduced compared to wild-type trimers, in some cases by >90%>.
  • the bNAb epitopes were generally unaffected, and the quaternary structure dependent bNAbs PGT145 and PGT151 actually more strongly in ELISA than to the wild type ZM197M SOSIP.664-D7324 trimers (Table 1.2, Fig. 1.S2J). Once more, the binding of non-NAbs to V3 and CD4i epitopes was greatly diminished (Table 1.2, Fig. 1.S2J).
  • CD4-binding rigidifies and/or buries these domains, as reported previously (Guttman et al., 2014). Conversely, CD4-binding led to decreased protection in the V2 and V3 loops as well as in several gp41 regions, which is consistent with a CD4-induced opening of the trimer apex that eventually increases the accessibility of the gp41 fusion machinery (Fig. 1.2A). Regions of the trimer that are less (red) or more (blue) protected upon CD4 binding are mapped onto the BG505 SOSIP.664 crystal structure (Fig. 1.2A-C).
  • the binding Ab titers in the SOSIP.664 trimer- immunized rabbits were higher when determined against the SOSIP.664-D7324 trimers than against the SOSIP.v4-D7324 variants (the titer ratios were -2 for BG505 and -3 for AMC008).
  • SOSIP.v4-D7324 trimers were similar for BG505 (ratio ⁇ 1) and higher to SOSIP.664-D7324 for
  • SOSIP.v4 trimers present neutralization-relevant epitopes more efficiently compared to SOSIP.664 trimers.
  • the AMC008 env gene is derived from a subtype B virus, isolated 8 months post-seroconversion from a participant in the Amsterdam Cohort studies on HIV/AIDS who later developed a bNAb response (patient H18818 in reference (Euler et al., 2010)).
  • the design of AMC008 SOSIP.664 trimers is identical to the BG505, B41, and ZM197M SOSIP.664 constructs described elsewhere (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013).
  • Env proteins were expressed by PEI transient transfection in adherent HEK293T cells or suspension cultures of HEK293F or HEK293S (Gn /_ ), as described previously (Julien et al., 2013c; Sanders et al., 2013). Env proteins were purified from transfection supernatants by affinity chromatography using a PGT145- or 2G12-column (Julien et al., 2013c; Pugach et al., 2015; Sanders et al., 2013). 2G12-purified Env proteins were further fractionated by SEC, whereas the PGT145 column yielded pure trimers without the need for SEC. SDS-PAGE and BN-PAGE assays were used to assess trimer cleavage and purity (Sanders et al., 2002; Schiilke et al., 2002).
  • Antigenicity was determined using SOSIP.664-D7324 trimers for SPR or D7324-capture ELISA, as described elsewhere (Derking et al., 2015; Sanders et al., 2013; Yasmeen et al., 2014).
  • the D7324 antibody was attached to the solid phase and used to capture the epitope-tagged trimers, which were then recognized by various solution-phase MAbs.
  • ITC was used to generate data on MAb-trimer interactions in solution.
  • SOSIP.664 trimers either alone or as Fab complexes, were generated by NS-EM as previously described. Rabbit immunization and analysis
  • Rabbits (5 per group) were immunized with 22 ⁇ g of PGT145-purified trimers at week 0, 4 and 20, and NAb responses were assessed at week 22.
  • the rabbit sera were assayed for autologous and cross-reactive NAbs using Env-pseudoviruses in the TZM-bl cell assay, and for trimer-binding antibodies by D7324- capture ELISA (Sanders et al., 2013).
  • the glycan shield of HIV is predominantly oligomannose independently of production system or viral clade.
  • N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus. Virology 372, 187-200.
  • HIV A stamp on the envelope. Nature 514, 437-438.
  • Recombinant HIV envelope trimer selects for quaternary -dependent antibodies targeting the trimer apex. Proc. Natl. Acad. Sci. Ill, 17624-17629.
  • the DSC data were fitted using both two state and non-two state models (Fig. 1.S2D).
  • the T m values based on two-state model fitting are listed here, while values based on the alternative model fitting are in Table LSI .
  • Binding of bNAbs and non-NAbs to PGT145-purified SOSIP.v4 and SOSIP.664 trimers was assessed by D7324-capture ELISA (Fig. 1.S2).
  • the BG505, B41, and ZM197M SOSIP.664 constructs have been described elsewhere (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013).
  • the AMC008 env gene is derived from a subtype B virus, isolated 8 months post-seroconversion from a participant in the Amsterdam Cohort studies on HIV/AIDS that later developed a bNAb response (patient H18818 in reference (Euler et al., 2010)).
  • the AMC008 SOSIP.664 gpl40 construct was designed as previously described (Julien et al., 2015; Pugach et al., 2015; Sanders et al., 2013), by introducing the following sequence changes: A501C and T605C (gpl20-gp4lECTO disulfide bond; (Binley et al., 2000)); I559P in gp41 EC TO (trimer-stabilizing; (Sanders et al., 2002)); REKR to R R R (SEQ ID NO: 186) in gpl20 (cleavage enhancement; (Binley et al., 2002)); a stop codon at gp4lECTO residue 664 (improvement of homogeneity and solubility; (Klasse et al., 2013)).
  • SOSIP.664-D7324 trimers contain a D7324 epitope-tag sequence at the C-terminus of gp4lECTo; they were made by adding the amino-acid sequence GSAPTKAKRRWQREKR (SEQ ID NO: 187) after residue 664 in gp41 EC TO and preceding the stop codon (Sanders et al., 2013).
  • biotinylated SOSIP.664-aviB trimers were generated by adding the avindin-B (aviB) sequence GLNDIFEAQKIEWHE (SEQ ID NO: 188) after residue 664, followed by biotininylation as described (Sok et al., 2014).
  • PEI- MAX 1.0 mg/ml
  • OPTI-MEM OPTI-MEM
  • Env proteins were produced in 293F cells using a protocol similar to that described previously (Julien et al., 2013; Sanders et al., 2013). Briefly, PEI-MAX (1.0 mg/ml) in water was mixed with expression plasmids for Env and Furin in OPTI-MEM. For cultures in a 2L disposable Nalgene flask (VWR), 250 ⁇ g of Env plasmid, 62.5 ⁇ g of Furin plasmid and 0.94 mg of PEI-MAX were added to 1L of pre-warmed Free-style 293 expression medium (Life Technologies). 293F cells were cultured for 6-7 days at 37°C, in an atmosphere containing 8%> CO2 and at a rotation speed of 125 rpm. Trimer purification
  • Env proteins were purified from transfection supernatants by affinity chromatography using a PGT145- or 2G12-column, essentially as described (Julien et al., 2013; Pugach et al., 2015; Sanders et al., 2013). The columns were made by coupling PGT145 or 2G12 to CNBr-activated Sepharose 4B beads (GE Healthcare). Briefly, supernatants were vacuum filtered through 0.2- ⁇ filters and then passed (0.5-1 ml/min flow rate) through a column, which was then washed with 2 column volumes of buffer (0.5 M NaCl, 20 mM Tris, pH 8.0). Bound Env proteins were eluted using 1 column volume of 3 M MgCh.
  • Env proteins were analyzed using SDS-PAGE followed by western blotting or Coomassie blue dye staining (Sanders et al., 2002; Schulke et al., 2002).
  • the input material was mixed with loading dye (25 mM Tris, 192 mM Glycine, 20% v/v glycerol, 4% m/v SDS, 0.1 % v/v bromophenol blue in water) and incubated at 95°C for 5 min prior to loading on a 4-12%> or 8% Tris-Glycine gel (Invitrogen).
  • DTT dithiothreitol
  • the input Env proteins were mixed with loading dye (500 ⁇ 1 20x MOPS Running Buffer + ⁇ 100% Ultrapure Glycerol (Invitrogen cat#15514-011) + 50 ⁇ 1 5% Coomassie Brilliant Blue G-250 + 600 ⁇ 1 ddH20) and directly loaded onto a 4-12% Bis-Tris NuPAGE gel.
  • loading dye 500 ⁇ 1 20x MOPS Running Buffer + ⁇ 100% Ultrapure Glycerol (Invitrogen cat#15514-011) + 50 ⁇ 1 5% Coomassie Brilliant Blue G-250 + 600 ⁇ 1 ddH20
  • BN-PAGE gels were run for 1.5 h at 200 V (0.07 A) using Anode -Buffer (20x NativePAGE Running Buffer (Invitrogen) in dd H20) and Cathode-Buffer (1% NativePAGE Cathode-Buffer Additive in Anode-Buffer; both from Invitrogen).
  • Western blot analysis of BN-PAGE gels was carried out using human MAb 2G12 (0.1 ⁇ g/ml), followed by HRP-labeled goat anti-human IgG (1 :5,000 dilution Jackson Immunoresearch, Suffolk, England) and the Western Lightning ECL system (PerkinElmer Life Sciences), essentially as described previously (Sanders et al., 2002).
  • BN-PAGE gels were stained using the Colloidal Blue Staining Kit (Life Technologies).
  • MAb or CD4-IgG2 (500 nM, diluted in HBS-EP), was allowed to associate with the trimers for 5 min before dissociation was recorded for 10 min.
  • the flow rate was 50 ⁇ /min throughout each run.
  • the surface was regenerated by a 60 s injection of lOmM Glycine [pH 2.0], at a flow rate of 30 ⁇ /min.
  • Induction of conformational changes by CD4 was studied in the SPR format by injecting two analytes in a single cycle.
  • the first analyte (CD4-IgG2; 500 nM) was injected for 200 s followed by the second (17b; 500 nM) for a further 200 s, both at a flow rate of 30 ⁇ /min. After each cycle, the surface was regenerated as described above, before fresh trimer was captured for the next run. D7324-capture ELISA
  • Unbound trimers were removed by 2 wash steps with TBS before various concentrations of test Abs were added for 2 h. After 3 washes with TBS, HRP-labeled Goat anti-Human IgG (Jackson Immunoresearch, Suffolk, England) was added at a 1 :3000 dilution in TBS/2% skimmed milk for 1 h, followed by 5 washes with TBS/0.05% Tween-20. Colorimetric detection was performed using a solution containing 1% 3,3',5,5'- tetramethylbenzidine ( Sigma- Aldrich, Zwijndrecht, The Netherlands), 0.01% H2O2, 100 mM sodium acetate and 100 mM citric acid.
  • CTRPNNNTRKSIRIGPGQWFYATGDIIGDIRQAHC (SEQ ID NO: 183)) were cyclized by a disulfide bond between residues 1 and 35.
  • sera from week-22 were serially diluted in 3-fold steps from a 1 : 100 start point, using 40% sheep serum (Biotrading), 2% milk powder in TBS as the buffer.
  • V3 -directed Ab responses were analyzed, the sera were incubated in solution with a V3 peptide (1 ⁇ g/ml) for 1 h prior to adding the mixture to the test wells.
  • the peptides were identical to the V3 region of the immunogen.
  • the BG505 V3 peptides described above the peptides were cyclized by a disulfide bond between residues 1 and 35.
  • the sequences of the AMC008 peptides were (wild-type:
  • CTRPNNNTRKSINIGPGRAFYTTGEIIGDIRQAHC (SEQ ID NO: 184); A316W:
  • CTRPNNNTRKSINIGPGRWFYTTGEIIGDIRQAHC (SEQ ID NO: 185).
  • the secondary antibody was Goat anti-Rabbit IgG (Jackson Immunoresearch, Suffolk, England), and the color development procedures were as described above.
  • the 2G12 detection MAb was used at 0.1 ⁇ g/ml, a concentration that gives -75% of the maximal signal in this ELISA format and hence allows any temperature-dependent loss of 2G12 reactivity to be quantified.
  • the results from this screening assay correlated well with the outcome of DSC experiments using the same, but purified, trimers (Fig. 1.S1G).
  • the midpoints of thermal denaturation (T m ) in the present assay were consistently 3-5°C lower than obtained via DSC, probably because the 2G12 epitope unfolds or is perturbed at a lower temperature than the bulk of the trimer.
  • DSC Differential scanning calorimetr
  • Thermal denaturation was studied using a nano-DSC calorimeter (TA instruments). All Env protein samples were first extensively dialyzed against PBS, and the protein concentration then adjusted to 0.1-0.3 mg/ml. After loading the sample into the cell, thermal denaturation was probed at a scan rate of 60°C/h. Buffer correction, normalization and baseline subtraction procedures were applied before the data were analyzed using NanoAnalyze Software v.3.3.0 (TA Instruments). The data were fitted using a two-state model as well as a non-two-state model, as the asymmetry of some of the peaks suggested that unfolding intermediates were present.
  • Rh values derived from this method thus represent the mean and distribution of diffusion constants. For a protein sample containing a small amount of polydispersity that is attributable to the presence of larger species, the resulting Rh value can be artificially elevated. As some preparations of BG505
  • SOSIP.664 trimers can contain up to -5% of higher-order aggregates that were not completely separated from the trimer by SEC, their presence may account for the higher Rh values (i.e. 8.1 nm for the BG505 SOSIP.664 trimer (Julien et al., 2013) than are presented here. Overall, because DLS measurements allow for multi-modal fitting models, we considered them to be a better way to measure Rh values for the wild type and stabilized SOSIP.664 trimers.
  • SAXS Small angle X-ray scattering
  • a 50 ⁇ sample of various PGT145-purified BG505 SOSIP.664 trimer variants (1 - 2 mg/ml) were injected onto a high resolution Sepharose 200 column (GE Healthcare) with a flow rate of 50 ⁇ /min in 20mM Na 3 P0 4 pH 7.4, 150mM NaCl, 0.02% NaN 3 , ImM EDTA.
  • the column eluate passed through a UV detector cell and into the quartz capillary cell. 1 second X-ray exposures were collected every 5 s throughout the run, during which a circulating water bath maintained the capillary cell temperature at 8°C.
  • a background scattering curve was obtained from the first 100 exposures (before the void volume), and was subtracted from all subsequent exposures during generation of the sample elution profile.
  • the radius of gyration values (R g ) and 1(0) for each frame were batch-analyzed using AutoRg, and frames with stable R g values were merged using Primus (Petoukhov et al., 2007) for the final scattering curve .
  • the real space distance distribution functions were calculated from the merged data sets by indirect transformation using the program GNOM (Svergun, 1992).
  • Soluble two-domain CD4 (Garlick et al., 1990) was obtained from the NIH AIDS reagents program. Immediately before HDX-MS analysis, all proteins were SEC-purified using a Superdex S200 column (GE Healthcare) and a PBS-based elution buffer (20 mM sodium phosphate pH 7.4, 150 mM NaCl with 1 mM EDTA and 0.02 % sodium azide). Complexes were formed by overnight incubation at 4°C with a 3-fold molar excess of sCD4 (relative to each protomer). Native gels were run for each sample to assess complex formation (Fig. 1.S3C).
  • ITC Isothermal titration calorimetry
  • ITC was performed using an Auto-iTC 200 instrument (GE Healthcare) using a protocol similar to one described previously (Julien et al., 2013; Sanders et al., 2013). Briefly, prior to conducting the titrations, proteins were dialyzed against Tris-saline buffer (150 mM NaCl, 20 mM Tris, pH 8.0). Absorbance at 280 nm using calculated extinction coefficients served to determine and adjust protein concentrations. The ligand present in the syringe was 19b Fab at a concentration of 10-20 ⁇ , while the BG505, B41 or AMC008 SOSIP.664 trimer was present in the cell at a concentration of 4-6 ⁇ .
  • Env trimers (10 ⁇ g) were resolved by SDS-PAGE under non-reducing conditions, followed by staining with Coomassie blue. Bands corresponding to gpl40 were excised from the gels and washed alternately with acetonitrile and water, five times. N-linked glycans were then released by addition of protein N-glycosidase F (PNGase F) at 5000 U/ml and incubation at 37°C for 16 h, according to the manufacturer's instructions (NEB). The released glycans were subsequently eluted from gel bands by extensive washing with water, and then dried using a SpeedVac concentrator.
  • PNGase F protein N-glycosidase F
  • glycans were labelled with 2-aminobenzoic acid (2-AA) as previously described (Neville et al., 2009). Briefly, glycans were resuspended in 30 ⁇ of water followed by addition of 80 ⁇ of labelling mixture (comprising 30 mg/ml 2-AA and 45 mg/ml sodium cyanoborohydride in a solution of sodium acetate trihydrate [4% w/v] and boric acid [2% w/v] in methanol). Samples were then incubated at 80°C for 1 h. Excess label was removed using Spe-ed Amide -2 cartridges, as previously described (Neville et al., 2009).
  • labelling mixture comprising 30 mg/ml 2-AA and 45 mg/ml sodium cyanoborohydride in a solution of sodium acetate trihydrate [4% w/v] and boric acid [2% w/v] in methanol. Samples were then incubated at 80°C for 1 h. Ex
  • Fluorescently labelled glycans were resolved by Hydrophilic interaction liquid chromatography-ultra performance liquid chromatography (HILIC-UPLC) using a 2.1 mm x 10 mm Acquity BEH Amide Column (1.7 ⁇ particle size) (Waters, Elstree, UK).
  • HILIC-UPLC Hydrophilic interaction liquid chromatography-ultra performance liquid chromatography
  • the percentage abundance of oligomannose-type glycans was calculated by integration of the relevant peak areas before and after Endoglycosidase H digestion, following normalization. Digestions were performed on free glycans at 37°C for 16 h. The digested glycans were purified using a PVDF protein-binding membrane plate (Millipore) prior to HILIC-UPLC analysis.
  • Rabbit immunizations and blood sampling were carried out under subcontract at Covance (Denver, PA) according to the schedule presented in fig. 1.4A. The protocol was broadly the same as previously described.
  • Female New Zealand White rabbits (5 per group) were immunized intramuscularly with 40 ⁇ g of the various Env trimers.
  • the proteins were formulated in 75 Units of ISCOMATRIXTM, a saponin-based adjuvant obtained from CSL Ltd. (Parkville, Victoria, Australia) (Maraskovsky et al., 2009).
  • the TZM-bl reporter cell line which stably expresses high levels of CD4 and the co-receptors CCR5 and
  • CXCR4 contains the luciferase and ⁇ -galactosidase genes under the control of the HIV-1 long-terminal- repeat promoter, was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc. Durham, NC).
  • TZM-bl cell neutralization assays using Env-pseudotyped viruses were performed at two different sites:
  • HEK293T cells (2* 10 5 ) were seeded in a 6-well tissue culture plate (Corning) in 3 ml DMEM (Gibco) containing 10%> FCS, penicillin (Sigma) and streptomycin (Sulphate-Gibco) (both at 100 U/ml) per well.
  • the culture was refreshed after 1 d by adding 3 ml culture medium when the cells had reached a confluence of 90-95%>, and the cells were then transfected using Lipofectamine 2000 (Invitrogen).
  • 1.6 ⁇ g of BG505.T332N plasmid and 2.4 ⁇ g of pSG3AEnv plasmid were mixed in 250 ⁇ of OPTI-MEM (Gibco).
  • a 10- ⁇ 1 aliquot of lipofectamine 2000 was mixed with 240 ⁇ of OPTI-MEM immediately before addition to the solution containing the expression plasmids. After incubation for 20 min at room temperature, the transfection mixture was added to the cells, and the culture supematants were harvested 48 h later as the source of Env-pseudotyped viruses for infection/neutralization experiments.
  • 1.8x l0 4 TZM-bl cells per well were seeded in a 96-well plate in DMEM containing 10% FCS, l x MEM nonessential amino acids, penicillin and streptomycin (both at 100 U/ml), and incubated at 37°C for 24h in an atmosphere containing 5% CO2.
  • a fixed amount of virus 500pg of p24-antigen equivalent was incubated for one hour at RT with 1 :3 serial diluted heat-inactivated sera, starting with a serum dilution of 1 :20.
  • Uninfected cells were used to correct for background luciferase activity. The infectivity of each mutant without inhibitor was set at 100%). Nonlinear regression curves were determined and 50% inhibitory serum dose (ID50) were calculated using a sigmoid function in Prism software version 5.0.
  • Biolayer interferometry (BLI) assays were performed using the Octet Red96 instrument (Pall ForteBio).
  • C- terminal His-tagged sCD4 domains 1 and 2, expressed in HEK293F and purified via Ni-NTA affinity and size-exclusion chromatography
  • IX kinetics buffer phosphate-buffered saline pH 7.2 supplemented with 0.01% w/v bovine serum albumin and 0.002%> v/v Tween 20.
  • the probes were then dipped for 300 s into wells containing 200 nM of BG505 SOSIP.664 WT or BG505 SOSIP.664 containing single or double stabilizing mutations (E64K, H66R, A316W, E64K/A316W, or H66R/A316W).
  • the probes were placed into wells containing IX kinetics buffer for 600 s to measure dissociation.
  • An inter-step correction was applied to align the end of the association curve to the begging of the dissociation curve, and a single binding site model was fit to the aligned curves to determine on- and off-rates.
  • HIV-1 human immunodeficiency virus type 1 envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent.
  • Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex. Proc. Natl. Acad. Sci. Ill, 17624-17629.
  • Table 1.S3, related to Table 1.1 Nomenclature of new stabilized SOSIP.664 trimers. An overview of the modifications present in the stabilized SOSIP.664 trimer versions. Horizontal shading: present, vertical shading: not present, black: not systematically present.
  • I535M-L543N-A316W-E64K no detectable binding 0 I535M-L543N-A316W-H66 * ⁇ 0.1
  • Binding isotherms do not allow to confidently determining thermodynamic parameters of binding.
  • the L555K substitution was designed to stabilize the trimer by introducing a hydrogen bond between gp41 in one protomer with gpl20 from an adjacent protomer, and an L556K substitution was designed to create a hydrogen bond between HR1 in gp41 and a-helix 0 in gpl20 within a protomer.
  • an L555K substitution was designed to create a hydrogen bond between HR1 in gp41 and a-helix 0 in gpl20 within a protomer.
  • the resulting L555K and L556K trimers were fully cleaved as shown by the conversion of gpl40 bands to gpl20 and gp41 on a reducing SDS-PAGE gel. Hence the point substitutions do not adversely affect the overall trimer conformation.
  • a SOSIP.664-L555C/Q49C double mutant was then designed to introduce a new disulfide bond to covalently link gp41 with the gpl20 N-terminus of the adjacent protomer ( Figure 2.3C).Its melting temperature of 75.2° is the highest that we have observed to date for a SOSIP.664 trimer variant.
  • binding of the 17b non-bnAb to its CD4-induced epitope was decreased 5-fold.
  • BG505 SOSIP.664 wild-type and mutant trimers (SOSIP- 137A and SOSIP-N332A) cloned in a phMCV3 vector, were expressed in FreeStyleTM 293F or 293S cells and purified using a 2G12-coupled affinity matrix followed by SEC, with exception for the BG505 SOSIP.664 N332A mutant for which a GN lectin affinity column was used in the initial stage.
  • the structure-based SOSIP.664 L555K, L556K and L555C-Q49C mutants were purified using a PGT145- coupled affinity matrix and then by SEC.
  • Electron microscopy All complexes were analyzed by negative-stain EM.
  • the 3H+3L Fab complex was formed with the BG505 SOSIP.664-N137A mutant, while 32H+3L Fab was in complex with the wild type BG505 SOSIP.664.
  • Both Env proteins were produced in HEK 293S cells.
  • the BG505 SOSIP.664 and BG505 SOSIP.664-N137A trimers were produced in HEK 293F cells.
  • a 3 ⁇ aliquot of 10 ⁇ g/ml of the complex was applied for 15s onto a glow discharged, carbon coated 400 Cu mesh grid and stained with 2% uranyl formate for 20s.
  • Grids were imaged using a FEI Tecnai T12 electron microscope operating at 120 kV using 52,000 x magnification and electron dose of 25 e " /A 2 , resulting in a pixel size of 2.05 A at the specimen plane. Images were acquired with a Tietz 4k x 4k CCD camera in 5° tilt increments from 0° to 55° at a defocus of 1000 nm using LEGINON.
  • the resolution of the final reconstruction for 3H+3L with BG505 SOSIP.664-N137A was calculated to be 17A, for 9H+3L with BG505 SOSIP.664 was 21 A, for 9H+3L with BG505 SOSIP-N137A was 19 A and for 32H+109L with BG505 SOSIP.664 was 22 A using an FSC cut-off of 0.5.
  • BG505 SOSIP.664 trimers we screened for positions in gpl20 and gp41 that would accommodate the introduction of an alternative or additional intermolecular disulfide bond between the gpl20 and gp41 subunits. Since the original disulfide bond in SOSIP gpl40 between residues 501 and 605 is located at the membrane proximal side of the trimer, we focused on potential locations nearer to the trimer apex. Based on favorable biochemical and antigenic properties, we selected two variants, BG505 SOSIP.664 H72C-H564C and SOSIP.664 A73C-A561C, for further analyses (Fig. 3.1 A; 3. Figs 3.
  • BG505 SOSIP.664 stability A316W, which fills a hydrophobic pocket and prevents the V3 from popping out of its hidden location underneath VI V2, and E64K, which prevents spontaneous sampling of the CD4-bound conformation (Fig. 3.1A;).
  • BG505 SOSIP.664 trimers containing the additional H72C- H564C or A73C-A561C are defined as BG505 SOSIP.v5.1 and BG505 SOSIP.v5.2 respectively (Fig. 3.1A).
  • BG505 SOSIP.v5 trimers were purified via PGT145-affinity chromatography. PGT145-purification yielded higher amounts of pure BG505 SOSIP.v5 trimers compared to BG505 SOSIP.664 (2.9 and 2.9 mg/1 for SOSIP.v5.1 and SOSIP.v5.2 respectively, vs. 1.6 mg/1 for SOSIP.664; Table 3.1, Fig. 3.S3a, Fig 3. IB).
  • Non-reducing SDS-PAGE analysis i.e.
  • BG505 SOSIP.664 H72C-H564C and A73C-A561C by negative stain electron microscopy (EM) revealed that the PGT145-purified trimers retained a native-like conformation (>98 % native-like trimers; Table 3.1 ; Fig. 3.S3c). While BG505 SOSIP.664 was predominantly in a more open conformation after PGT145-purification (33% closed trimers), BG505 SOSIP.664 H72C-H564C and A73C-A561C proteins showed higher percentages of closed trimers (69%> and 67%>, respectively).
  • thermostability of the mutants by differential scanning calorimetry (DSC).
  • the midpoint of thermal denaturation T m ) of BG505 SOSIP.664 H72C-H564C and A73C-A561C were 72.0°C and 72.5°C, respectively, i.e. 4.4°C and 4.9°C higher than that of the original BG505 SOSIP.664 trimer (Table 1).
  • the thermostability was further increased when the E64K and A316W substitutions were incorporated:
  • the T m values were 75.0°C and 75.3°C, for SOSIP.v5.1 and SOSIP.v.5.2, respectively, i.e. 7.4°C and 7.7°C higher than that of the original BG505 SOSIP.664 trimer; Table 3.1, Fig. 3.S3e).
  • the SOSIP.664 H72C-H564C trimer was expressed in 293 S cells and purified using 2G12-affmity chromatography followed by size exclusion chromatography (SEC) and its structure in complex with bNAbs PGT122, 35022 and NIH45-46 was solved by X-ray crystallography (Fig. 3.1).
  • SEC size exclusion chromatography
  • Fig. 3.1 X-ray crystallography
  • BG505 SOSIP.v5.2 showed nearly identical exchange kinetics compared to the wild-type protein indicating that the structure and conformational dynamics were very similar.
  • the other two variants showed subtly increased dynamics in regions close to the new disulfide bond, while the BG505 SOSIP.v5.1 variant also showed reduced dynamics in C- terminal half of al (Fig. 3.S4). Overall, the substitutions did not dramatically alter the conformational dynamics of the unliganded trimer.
  • the AMC008, B41 and ZM197M SOSIP.664 and SOSIP.v5 variants were purified using PGT145- affinity chromatography and analyzed by SDS-PAGE and BN-PAGE analysis. While AMC008 SOSIP.v5 yielded similar amounts of pure trimers compared to AMC008 SOSIP.664, ZM197M yielded considerably higher amounts of pure trimers compared to ZM197M SOSIP.664 (1.0 mg/1 for both SOSIP.V5.1 and SOSIP.v5.2, compared to 0.2 mg/1 for SOSIP.664; Table 3.1).
  • B41 trimers yielded higher or lower amounts of pure trimers depending on the SOSIP.v5 used (0.2 mg/1 and 2.9 mg/1 for SOSIP.v5.1 and SOSIP.v5.2 respectively) compared to B41 SOSIP.664, which yielded XXX mg/1 (Table 3.1). All variants formed pure trimers that were cleaved efficiently (Fig. 3.S5). In non- reducing SDS-PAGE, the variants with the extra disulfide bonds ran slower, consistent with the results obtained with BG505 SOSIP.664 and in line with a more compact structure caused by the formation of the two disulfide bonds between gpl20 and gp41 (Fig. 3.S5a).
  • glycan structures present AMC008 and B41 SOSIP variants compared to their wild-type counterparts.
  • BG505 oligomannose glycoforms dominated (63-69%>), in particular Man 8 GlcNAc2 and Man 9 GlcNAc2 glycans (Table 3.1).
  • V3 peptide competition ELISA was performed in order to determine the proportion of the response directed to the V3.
  • V3 responses accounted for -30% of the ELISA binding signal in BG505 SOSIP.664 immunized.
  • Rabbits immunized with BG505 SOSIP.v5, in particular SOSIP.v5.2 trimers showed a significantly lower proportion of V3 directed response compared to BG505 SOSIP.664 immunized rabbits (Fig. 3.3B), consistent with the reduced presentation of V3-epitopes (Table 3.2a and table 3.2b).
  • BG505 SOSIP.664 induces NAbs against the autologous neutralization resistant (Tier 2) BG505.T332N virus, something that has not been achieved by other gpl40 immunogens.
  • the sera from BG505 are the autologous neutralization resistant (Tier 2) BG505.T332N virus, something that has not been achieved by other gpl40 immunogens.
  • SOSIP.v5 recipient animals in particular from those receiving SOSIP.v5.2, were at least as efficient at neutralizing BG505.T332N as the sera from BG505 SOSIP.664 immunized animals (Fig. 3.3C).
  • the substitutions present in the SOSIP.v5 trimers do not compromise the autologous Tier 2 NAb response.
  • NAbs against neutralization-sensitive (Tier 1) isolates are dominated by V3- directed antibodies that cannot neutralize Tier 2 viruses, and might potentially distract from bNAb responses. Since the percentage of the V3 response is lower in the animals that received SOSIP.v5 trimers.
  • the autologous NAb responses (median IC50 values) elicited by the different trimers were generally comparable (SOSIP.664, 4432; SOSIP.v4.1, 4503; SOSIP.v5.1 + SOSIP.v5.2 pooled, 3457; SOSIP.v6, 7798), except for the SOSIP.v5.2 201C-433C hyper-stabilized trimer (median IC50, 226; not significant compared to SOSIP.664) ( Figure 3 4!).
  • the modifications used to create new, more stable trimers do not impair the induction of the autologous BG505.T332N NAb response.
  • trimer-stabilization projects are to reduce the antigenicity and immunogenicity of epitopes for non-NAbs and Tier-1 NAbs, the latter dominated by V3 -directed antibodies, and thereby focus the immune response on more productive targets.
  • the clade A virus 92RW and the clade B virus SHIV162P3 were occasionally neutralized in the titer range 40-200 ( Figures 3.41 and 3.4K).
  • BG505 SOSIP.664 and SOSIP.v5.2 were introduced in BG505 SOSIP.664 and SOSIP.v5.2 and determined the antigenic profile and thermal stability of the proteins.
  • the 4 trimers were purified to high homogeneity using PGT145-affinity chromatography. However, the yields differed substantially.
  • the BG505 SOSIP.664 I201C-A433C and E49C-L555C yielded 0.1 and 0.2 mg/1, substantially lower than BG505 SOSIP.664 (1.6 mg/1) and SOSIP.v5.2 (2.9 mg/1).
  • Table 3.1 shows the biophysical characterization of SOSIP.v5 trimers from isolates BG505, AMC008, B41 and ZM197M. The percentage of native-like and closed native-like trimers based negative stain EM are listed. The 2D class averages are shown in Supplementary Fig. 3.3c. The T m values for each construct were obtained by DSC using a two state model. The percentages of MangGlcNAc2 and Man9GlcNAc2 glycans, as well as the total percentage of oligomannose glycans are given for each trimer.
  • Table 3.2a and table 3.2b show the antigenic characterization of SOSIP.v5 trimers from isolates BG505, AMC008, B41 and ZM197M.
  • Half-maximal binding concentrations (EC50, in ⁇ g/ml) were derived from Ni-NTA ELISAs. The values are representative of at least 2 independent experiments.
  • the new trimers described here provide new insights into Env dynamics, biophysics and structure.
  • the improved presentation of bNAb epitopes and reduced exposure of potentially interfering non-NAb epitopes should improve the induction of the desirable bNAbs. Furthermore, the improved stability and hyperstability might enhance the in vivo half- life and prolong the presentation of bNAb epitopes to naive B cells.
  • An HIV-1 subunit vaccine should induce a broad and potent neutralizing antibody (NAb) response against the envelope glycoprotein spike (Env) [1].
  • NAb neutralizing antibody
  • These trimers bind virtually all known broadly neutralizing antibodies (bNAbs) but almost no non-neutralizing antibodies (non-NAbs), and adopt a native-like conformation with a well-defined structure [2, 6-8].
  • soluble, adjuvanted BG505 SOSIP.664 trimers induce NAbs against the autologous, neutralization-resistant (tier 2) virus efficiently in animals [9] .
  • Licensed subunit vaccines against viral pathogens, such as hepatitis B and human papillomavirus, are however particulate antigens [10].
  • the greater size and the capacity for multivalent antigen presentation and B cell receptor cross-linking provide such particulate vaccines with advantages over soluble proteins for inducing antibody responses [11].
  • ferritin H. Pylori ferritin (GenBank accession no. NP 223316)
  • SOSIP.664 C-terminus starting from Asp5
  • GSG Gly-Ser-Gly linker
  • the secreted nanoparticles and control trimers were purified using PGT145 bNAb- affinity chromatography [15].
  • Judged by BN-PAGE and SDS-PAGE analysis followed by Coomassie staining this purification method yielded highly pure (>95 % purity) SOSIP.664 trimer and SOSIP.664- ferritin protein preparations (Fig. 4.1b).
  • SDSPAGE also confirmed that the SOSIP.664 component of the nanoparticles was cleaved efficiently between gpl20 and gp41 (Fig. 4.1b, left panel).
  • NS-EM negative stain electron microscopy
  • tier IB viruses HxB2, Q23envl7, ZM109F and ZM197M and the tier 2 viruses 94UG103, 92RW020, Q259.d2.17, Q769. d22, Q842.dl2 (all clade A), YU2 (clade B) and Cel l76_ A3 (clade C) were not neutralized by any rabbit sera.
  • a recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gpl20 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol. 2000;74:627-43.
  • Monomeric gpl20 immunogens and trimers that do not mimic the native Env protein have not been successful at inducing NAbs against relatively neutralization-resistant (Tier-2) primary viruses.
  • SOS intermolecular disulfide bond between gpl20 and gp41
  • IP trimer stabilizing mutation in gp41
  • SOSIP.664 gpl40 proteins over the last few years, SOSIP.664 proteins from multiple virus isolates from different HIV-1 subtypes have been described.
  • SOSIP.664 proteins based on the BG505 (subtype A) and B41 (subtype B) strains developed Tier-2 autologous NAbs in rabbits and macaques, something not achieved previously with other types of gpl40 proteins. However, only sporadic and very weak heterologous Tier-2 NAb responses were observed.
  • SOSIP.v4 SOSIP version 4
  • trimerization and trimer stability reduced the exposure of non-NAb V3- and CD4i-epitopes.
  • introduction of a second dilsufide bond connecting CI of gpl20 with the HR1 region of gp41, further increased trimer stability.
  • these modifications allowed the generation of SOSIP proteins from virus strains that do not form well- behaved native-like trimers otherwise.
  • ACS HIV-1 infection and AIDS
  • the AMC009 SOSIP is based on HTV-1 variants isolated two month after seroconversion.
  • the recombinant protein formed stable trimers with a native-like conformation, exposing bNAb epitopes while occluding most non-NAb epitopes.
  • Rabbit immunizations with the monovalent AMC009 induced neither NAbs against Tier 1 viruses nor against heterologous Tier-2 viruses.
  • a sequential immunization using AMC008, AMC009 and AMCOl 1 resulted in weak and sporadic heterologous Tier-2 neutralisation.
  • a trivalent cocktail of these proteins yielded similar results.
  • a tetravalent cocktail consisting of AMC008, AMC009, AMCOl 1 and B41 SOSIP proteins, induced consistent heterologous responses against several Tier-2 viruses.
  • the env gene used here is derived from early clonal viruses isolated from HIV-1 subtype B infected ACS participant, infected via male homosexual contact (men who have sex with men; MSM).
  • MSM men who have sex with men
  • the rationale to use early sequences is based on the observation that the development of bNAbs might be driven by epitopes exposed on early viruses
  • HI 8877 env genes from five clonal viral isolates (HI 8877.2m.1B5 (SEQ ID NO: 53), HI 8877.2m.1C3 (SEQ ID NO: 54), HI 8877.2m.1F9 (SEQ ID NO: 55), HI 8877.2m.1G1 (SEQ ID NO: 56) and H18877.2m.2Bl (SEQ ID NO: 57)) from 2 months post-SC were used to obtain a consensus sequence, using a cutoff >60% for an amino acid at a specific position (i.e. only amino acids that occurred in 3 out of 5 sequences were retained in the consensus sequence). This consensus sequence was used to generate the expression vector to make the AMC009 SOSIP protein.
  • the genes encoding the gpl40 SOSIP constructs were designed as described previously. In short, we introduced the A501C and T605C substitutions, resulting in a stabilizing intermolecular disulfide bond between gpl20 and gp41 ectodomain (gp41ECTO); the trimer-promoting I559P, V/T535M, L543Q and Q567K substitutions in gp41ECTO; the hexa-arginine furin cleavage site at the C-terminus of gpl20 to enhance cleavage ; a tissue plasminogen activator (tPA) signal peptide replacing the natural Env signal peptide to improve secretion; a stop codon at position 664 to prevent aggregation (HXB2 numbering system).
  • tPA tissue plasminogen activator
  • the resulting gpl40 SOSIP proteins based on the env genes of individuals HI 8877 was designated AMC009 SOSIP version 3.1 (SOSIP.v3.1).
  • the resulting codon-optimized env genes were obtained from Genscript (Piscataway, NJ) and cloned into the pPPI4 expression vector.
  • Genscript Procataway, NJ
  • SOSIP.v4 and SOSIP.v5 was further improved.
  • the SOSIP protein was expressed as described earlier herein.
  • Purified SOSIP protein was analysed by NS-EM. A 3 ⁇ aliquot containing ⁇ 5.5 ⁇ g/ml of a protein was applied for 5 s onto a carbon-coated 400 Cu mesh grid that had been glow discharged at 20 mA for 30 s, then negatively stained with 2% (w/v) uranyl formate for 60 s. Images were collected on either a Tietz
  • the nominal defocus range for all data negative stain data sets was -1.5x10-6 to - 2.0xl0-6m..
  • Glycan profiling was done as described previously. Briefly, the Env protein (5.5 ⁇ g) was resolved by SDS-PAGE under non-reducing conditions, followed by Coomassie blue staining. Bands corresponding to gpl40 were excised from the gels, washed and N- linked glycans were then released according to the manufacturer's instructions (NEB). The released glycans were subsequently eluted from gel bands, then dried using a SpeedVac concentrator and labelled with 2-aminobenzoic acid (2-AA) as previously described. Fluorescently labelled glycans were resolved by Hydrophilic interaction liquid
  • the percentage abundance of oligomannose-type glycans was calculated by integration of the relevant peak areas before and after Endoglycosidase H digestion, following normalization. Digestions were performed on free glycans at 37°C for 16 h. The digested glycans were purified using a PVDF protein- binding membrane plate (Millipore) prior to HILIC-UPLC analysis.
  • Microlon 96-wells plates (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) were coated overnight with mAb D7324 (Alto Bioreagents, Dublin, Ireland) at 10 ⁇ in 0.1 M NaHC03, pH 8.6 (50 ⁇ /well).
  • mAb D7324 Alto Bioreagents, Dublin, Ireland
  • purified D7324-tagged SOSIP protein (2.75 ⁇ g/ml) was added in TBS/10% FCS for 2 h. Unbound proteins were washed away by 2 wash steps, and TBS (150 mM NaCl, 20 mM Tris) plus 2% skimmed milk was added to further block non-specific protein-binding sites.
  • His-tagged proteins were captured at RL ⁇ 500-530 RU on anti-His CM5 sensor chips.
  • IgGs of VRCOl, 3BNC60, PG16, PGT145, PGT121, PGT151, 35022, F105 and 19b were injected individually at 500 nM for 300 s and allowed to dissociate for 600s.
  • Rabbit immunizations with AMC009 SOSIP protein and blood sampling were carried out under subcontract at Covance (Denver, PA).
  • rabbit immunizations were performed with a cocktail of AMC008, AMC009 and AMCOl 1 SOSIP proteins.
  • One group of animals was immunized with a total of 22 ⁇ g of the SOSIP proteins per animal per dose (low dose), 7.3 ⁇ g per individual SOSIP protein, while the other group received a total of 66 ⁇ g of the SOSIP proteins per animal per dose (high dose), 22 ⁇ g per SOSIP protein, formulated in 75 Units of ISCOMATRIXTM.
  • Each group was immunized four times (at week 0, 4, 20 and 36) with the corresponding SOSIP.v4.2 protein, except the first, fourth and fifth groups which received the corresponding SOSIP.v5.2 proteins during the fourth immunization.
  • AMC008 v5.2, AMC009 v5.2, AMCOl 1 v5.2 and B41 v4.1 were administered sequentially at week 0, 4, 20 and 36, each of the five animals received 22 ⁇ g of protein.
  • the five animals of the tetravalent cocktail group received a cocktail of the above mentioned proteins and week 0, 4, 20 and 38, with a total of 22 ⁇ g of SOSIP protein per animal.
  • the TZM-bl reporter cell line stably expresses high levels of CD4 and the co-receptors CCR5 and CXCR4 and contains the luciferase and ⁇ -galactosidase genes under the control of the HIV-1 long- terminal-repeat promoter, and was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (John C. Kappes, Xiaoyun Wu, and Tranzyme Inc., Durham, NC). TZM-bl cell neutralization assays using viruses were performed as described previously.
  • HEK293T cells (2x 105) were seeded in a 6-well tissue culture plate (Corning) in 3 ml DMEM (Gibco) containing 10% FCS, penicillin (Sigma) and streptomycin (Sulphate-Gibco) (both at 100 U/ml) per well.
  • Lipofectamine 2000 was mixed with 240 ⁇ of OPTI-MEM immediately before addition to the solution containing the expression plasmids and incubated for 20 min at RT. Then the transfection mixture was added to the cells, and the culture supematants were harvested after 48 h for infection and neutralization experiments. The molecular clone based on the AMC009 Env was not infectious and could not be used for neutralization experiments.
  • TZM-bl cells were seeded in a 96-well plate in DMEM containing 10%> FCS, l x MEM nonessential amino acids, penicillin and streptomycin (both at 100 U/ml), and incubated at 37°C for 24 h in an atmosphere containing 5% C02.
  • DMEM fetal calf serum
  • penicillin and streptomycin both at 100 U/ml
  • an A316W substitution in combination with either E64K or H664 improved trimerization and trimer stability, and reduced exposure and immunogenicity of non-NAb epitopes.
  • an additional disulfide bond between gpl20 and gp41 residues A73C and A561C provided additional stability. This disulfide bond was added to AMC009 SOSIP to create AMC009 SOSIP.v5.2. The same mutations were applied to AMCOl 1 v4.2 SOSIP, creating AMCOl 1 v5.2.
  • the modified env genes were transiently transfected into 293F cells, together with the furin gene to enhance the cleavage between gpl20 and gp41ECTO.
  • the secreted Env proteins were affinity-purified using bNAb PGT145.
  • SDS-PAGE analyses performed under reducing and non-reducing conditions and stained with Coomassie blue showed that the purified SOSIP proteins were fully and efficiently cleaved into their gpl20 and gp41ECTO subunits (+DTT vs -DTT).
  • Single gpl40 bands were observed under non-reducing conditions, showing that there were no inter-protomer disulfide -bonds.
  • a Coomassie blue stained BN-PAGE showed trimers only, without any amounts of monomers, dimers or higher aggregates.
  • AMC009 SOSIP protein resembles the native Env trimer
  • NS-EM negative stain electron microscopy
  • Native virion-derived Env contains a large number of underprocessed oligomannose glycans. While uncleaved gpl40 proteins have a high percentage of processed glycans, native-like SOSIP trimers usually have a high oligomannose content, resembling the glycan composition of native Env.
  • the underprocessed glycans on native virion-derived Envs are probably a consequence of the protection from processing enzymes, partially dependent on its tight and compact conformation.
  • the differential scanning calorimetry (DSC) was used to measure the thermal stability of the PGT145- purified SOSIP proteins.
  • DSC differential scanning calorimetry
  • AMC009 SOSIP.v4.2 the DSC profile, obtained with a two-state model, showed a thermal denaturation midpoint (Tm) of 67.6°C.
  • the proteins were also analyzed using a non- two state model, revealing three individual unfolding peaks.
  • the Tm value of AMC009 is comparable with the BG505 SOSIP protein.
  • SOSIP.v4.1 and SOSIP.v5.2 variants and the results obtained were comparable with those derived from SOSIP.v4.2 proteins. If they were not, they were consistent with the effect of the amino acid
  • Env protein that resembles the native-like spike should bind most bNAbs, but not non-NAbs.
  • bNAbs that recognize the apex of the trimer and depend on the proper quaternary conformation of the protein.
  • PG16 and PGT145 bound relatively weakly to AMC009 SOSIP.
  • CD4bs bNAbs VRCOl and 3BNC117 interacted similarly efficiently with AMC009 SOSIP protein.
  • CD4 IgG2 bound strongly AMC009 SOSIP.
  • PGT 121 and PGT128 are antibodies that bind to the N332-glycan V3 cluster. Binding of PGT128 was the same for all the SOSIP proteins, but the binding affinity of PGT121 to AMC009 SOSIP was higher compared to AMC016 and AMC018. Next we tested the binding affinity of 2G12 and PGT135, two antibodies that bind to the glycan outer domain cluster, also involving the N332 glycan. The binding affinity for 2G12 was almost the same for the SOSIP proteins. The binding affinity of PGT135 was the highest for
  • AMC009 SOSIP substantially decreased for AMC016 SOSIP and more so for AMC018 SOSIP.
  • bNAbs PGT151 and 35022 bind to the interface of gpl20 and gp41 and require proper quaternary structure.
  • the AMC016 and AMC018 SOSIP proteins bound similarly to PGT151, while the binding of AMC009 SOSIP was decreased.
  • the maximum ELISA signal obtained with PGT151 and AMC009 was low, probably related to fast dissociation (see below), and loss of signal during washing steps.
  • AMC018 SOSIP bound relatively weak compared to the AMC009 and AMC016 SOSIP proteins.
  • SOSIP.v3.1 and v4.1 proteins bound bNAbs with similar affinities to their SOSIP.4.2, as observed previously for the comparison of SOSIP.v3.1 and SOSIP.v4 proteins based on other virus isolates.
  • a notable exceptions was the improved binding of PGT 145 to AMC016 SOSIP.v4.2 vs.
  • non-NAbs against the CD4bs and V3 do not interact efficiently with SOSIP trimers.
  • binding of these non-NAbs to D7324-tagged SOSIP trimers can be readily detected in ELISA, although the reason remains to be understood. It is probable that the antigen capture in ELISA induces some local protein unfolding, resulting in the presentation of on a subset of proteins of some non-NAb epitopes. Alternatively, proteins captured on an ELISA plate might be more or flexible, allowing different conformations to be sampled over time in a way that registers more strongly in an ELISA than in other binding assays.
  • PGT121 a bNAbs against the V3 and outer domain glycan cluster, and VRCOl and 3BNC60, two CD4bs-directed bNAbs, bound very efficiently.
  • the bNAbs PGT151 and 35022 both directed against the gpl20-gp41 interface and dependent on proper quaternary structure, both reacted very efficiently with AMC009 SOSIP.v4.2, although both the association and dissociation kinetics for PGT151 were rather high.
  • the non-NAbs F105 and 19b were not reactive with AMC009 SOSIP.v4.2 in SPR assays.
  • AMC009, AMC016 and AMC018 SOSIP.v4.2 proteins display most bNAb epitopes.
  • the clonal AMC009 viruses are relatively neutralization-resistant (Tier-2)
  • AMC009 was classified as relatively neutralization-resistant (Tier-2) viruses .
  • Heterologous NAbs responses were observed in almost all animals against the four easy-to-neutralize (Tier-1) viruses tested: namely SF162, BaL, AMC008 and ZM197M .
  • Median NAb titers against the subtype B viruses SF162 and BaL were lower for AMC016 SOSIP - immunized animals (median ID50 values of 73 and 80) compared to AMC009 and AMCOl 8 SOSIP recipients (median ID50 values of 364 and 135 for AMC009, and 998 and 136 for AMC018, for SF162 and BaL, respectively).
  • NAb titers against AMC008 (Tier-IB), a virus based on the Env sequence of another subtype B infected participant from the ACS.
  • the NAb responses against AMC008 were generally higher for animals receiving AMC009 and AMCOl 6 immunogens (median ID50 values of 122 and 57) compared to AMCOl 8 SOSIP vaccinees (median ID50 of 47).
  • Intersubtype neutralization of ZM109f (Tier-IB; subtype C) was weak in animals immunized with AMC016 and AMCOl 8 immunogens and virtually absent in AMC009 SOSIP recipients.
  • a trivalent cocktail of AMC008, AMC009 and AMCOl 1 SOSIP trimers induces Tier-2 NAbs in rabbits
  • the second rabbit immunization study was performed to assess whether a cocktail of SOSIP proteins (AMC008, AMC009, and AMCOl 1) would induce better antibody response compared to monovalent immunizations.
  • SOSIP proteins derived from ACS individuals who developed elite bNAbs (AMC009 and AMCOl 1) and bNAbs (AMC008).
  • the immunization regimen was similar as for the first study, but SOSIP.v5.2 proteins were used for all three strains in the fourth immunization.
  • bNAbs One approach to induce bNAbs is based on the hypothesis that an HIV-1 immunogen that is a stable antigenic mimic of the native trimeric envelope spike, is better at inducing NAbs than other Env forms. Native-like trimers present most bNAb epitopes, and few potentially distractive non-NAb epitopes.
  • native-like trimers pose the appropriate constraints on the NAb approach angles. Over the last two years, multiple native-like proteins based upon different subtypes have been described, and here we report on three novel SOSIP trimers derived from early clade B isolates to add to this arsenal.
  • SOSIP.v4.2 proteins and subsequently the A73C and A561C substitutions, creating SOSIP.v5.2.
  • substitutions stabilized the proteins in their closed conformation, further occluding non-NAb epitopes and increasing their thermostability.
  • the proteins showed a high percentage of underprocessed oligomannose glycans, which is typical of virus-derived Envs and soluble native-like Env trimers.
  • NAb responses against Tier-1 A viruses are dominated by V3 specificities and are not effective against Tier-2 viruses. It is possible that responses against the highly immunodominant V3 might distract from bNAb responses.
  • the responses against such immunological decoys can be reduced by using SOSIP.v4 proteins instead of SOSIP.664 proteins.
  • SOSIP.v4.2 immunogens or SOSIP.v5.2 in one case, which indeed minimized the exposure of the V3 epitopes in vitro.
  • AMC008 and AMCOl 1 are immunogens also derived from subtype B ACS participants, who developed bNAbs and elite bNAbs, respectively.
  • the trivalent vaccine induced heterologous NAb responses against the Tier-2 subtype B viruses REJO, SHIV162p3 and WITO. These responses were significantly stronger than those induced by monovalent AMC008 or AMC009 SOSIP vaccination, although we note that monovalent AMCOl 1 SOSIP vaccination also resulted in some heterologous Tier-2 NAb induction.
  • heterologous Tier-2 NAb response can be increased by using cocktail immunogens.
  • cocktail immunogens We choose to formulate a trivalent cocktail based on three subtype B Env proteins and we observed cross- neutralization of subtype B viruses.
  • heterologous Tier-2 NAb responses were more frequent and stronger than those induced by BG505 and B41 SOSIP trimers.

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Abstract

L'invention concerne un polypeptide isolé, recombinant ou synthétique comprenant un polypeptide Env d'un virus du VIH comprenant une partie polypeptidique gp120 et au moins une partie polypeptidique de l'ectodomaine de la gp41 du VIH-1 ou une partie polypeptidique gp125 et au moins une partie polypeptidique de l'ectodomaine de la gp36 du VIH-2, ladite partie polypeptidique gp120 ou gp125 et ladite partie polypeptidique de l'ectodomaine de la gp41 ou gp36 comprenant un premier résidu cystéine au niveau d'une position d'acide aminé équivalente à la position d'acide aminé 49, 50, 51, 71, 72 ou 73 et un second résidu de cystéine au niveau d'une position d'acide aminé équivalente à la position d'acide aminé d'un résidu choisi dans le groupe constitué par : 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 et 570, la numérotation étant conforme à la séquence de référence HxB2 ayant la séquence d'acides aminés de SEQ ID NO: 1, et lesdits premier et second résidus de cystéine formant une liaison disulfure intermoléculaire. L'invention concerne en outre un polypeptide isolé, recombinant ou synthétique comprenant un polypeptide Env d'un virus du VIH comprenant une partie polypeptidique gp120 et au moins une partie polypeptidique de l'ectodomaine de la gp41 du VIH-1 ou une partie polypeptidique gp125 et au moins une partie polypeptidique de l'ectodomaine de la gp36 du VIH-2, ledit polypeptide Env comprenant au moins un résidu d'acide aminé choisi dans le groupe constitué de : 64K et 66R ; et 302F, 302W, 304F, 304W, 306L, 307F, 307W, 308L, 315Y, 315K, 3151, 315W, 315F, 315V, 316Y, 316K, 3161, 316F, 316W, et 316V ; et 555K ou 556K ; et 137A, le numérotage dudit au moins un résidu d'acide aminé étant conforme à la séquence de référence HxB2 ayant la séquence d'acides aminés de SEQ ID NO: 1, et ledit au moins un résidu d'acide aminé étant au niveau d'une position d'acide aminé équivalente à la position d'acide aminé de SEQ ID NO: 1.
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WO2020117590A1 (fr) * 2018-12-04 2020-06-11 The Rockefeller University Immunogènes de vaccin contre le vih
CN114478796A (zh) * 2020-11-12 2022-05-13 厦门大学 经改造的人免疫缺陷病毒膜蛋白及其应用
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WO2019042950A1 (fr) 2017-08-26 2019-03-07 Academisch Medisch Centrum Immunogènes glycoprotéiques d'enveloppe de vih améliorés
WO2020117590A1 (fr) * 2018-12-04 2020-06-11 The Rockefeller University Immunogènes de vaccin contre le vih
CN113454100A (zh) * 2018-12-04 2021-09-28 洛克菲勒大学 Hiv疫苗免疫原
CN114478796A (zh) * 2020-11-12 2022-05-13 厦门大学 经改造的人免疫缺陷病毒膜蛋白及其应用
WO2024091921A1 (fr) * 2022-10-24 2024-05-02 Duke University Stabilisation d'enveloppes du virus de l'immunodéficience humaine (vih)

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