EA042607B1 - TRIMER-STABILIZING MUTATIONS OF THE HIV ENVELOPE PROTEIN - Google Patents

Description

Background of the invention

The human immunodeficiency virus (HIV) affects millions of people around the world, and preventing HIV with an effective vaccine remains paramount even in the era of widespread antiretroviral therapy. The antigenic diversity between different strains and clades of the HIV virus makes it difficult to develop vaccines with a wide range of efficacy. HIV-1 is the most common and pathogenic strain of the virus, with more than 90% of HIV/AIDS cases due to infection with HIV-1 group M. Group M is further subdivided into clades or subtypes, of which clade C is the largest. the vaccine will be able to elicit both strong cellular responses and the production of broad-spectrum neutralizing antibodies capable of neutralizing HIV-1 strains from various clades.

The envelope styloid protein (Env) on the surface of HIV consists of a trimer of heterodimers from the glycoproteins gp120 and gp41 (Fig. 1A). The gp160 precursor protein is cleaved by furin into gp120, which is the head of the spike and contains the CD4 receptor binding site, as well as large hypervariable loops (V1-V5), and gp41, which is the membrane-anchored stalk of the coat spike protein. Like other class I fusogenic proteins, gp41 contains an N-terminal fusion peptide (FP), a C-terminal transmembrane (TM) domain, and a cytoplasmic domain. Membrane fusion between the membranes of HIV and the target cell requires a series of conformational changes in the envelope protein. HIV vaccines can be developed based on the envelope protein.

However, various factors make the development of an envelope protein-based HIV vaccine challenging, including the high genetic variability of HIV-1, the dense carbohydrate coating of the envelope protein, and the relatively dynamic and labile nature of the envelope spike protein structure. The wild-type coat protein is unstable due to its function. Therefore, stabilizing modifications are sometimes introduced into the shell structure in order to obtain candidate vaccines. The coat protein is a target for neutralizing antibodies and is highly glycosylated, which reduces immunogenicity by shielding protein epitopes. All known broad spectrum neutralizing antibodies (bNAbs) are indeed adapted to these glycans.

For vaccine development, it is preferable to use envelope proteins that can induce bNAb production. However, most bNAbs only recognize the native conformation of the envelope protein before it undergoes any conformational change. Thus, the development of a stable envelope protein in its similar native compact and closed conformation, while minimizing the presentation of non-native and therefore non-neutralizing epitopes, could increase the efficiency of production of such bNAbs. Previous efforts to develop an HIV vaccine have focused on the development of vaccines that contain the ectodomain of the trimeric HIV envelope protein, gp140, prior to fusion. gp140 lacks transmembrane (TM) and cytoplasmic domains, but, unlike gp120, it can form trimeric structures. Moreover, such previous attempts have focused mainly on the A clade. However, the width of the action spectrum in response with a neutralizing antibody that has been induced is still limited. Thus, it would also be preferable if stabilized trimers of the native envelope protein were available for use against several HIV clades.

For more than two decades, attempts have been made to develop a stable coat protein in its pre-fusion trimeric conformation, with only limited success in obtaining soluble, stable coat protein trimers capable of inducing a response with broad-spectrum neutralizing antibodies. For example, so-called sOsIP (501C, 605C and 559P) mutations have been introduced into the envelope protein sequence to improve the formation of a fraction of soluble gp140 trimers (Sanders et al., (2002), J. Virol. 76(17): 8875-89). The so-called SOSIP mutations include cysteine residues at positions 501 and 605 and a proline residue at position 559 according to the numbering in gp160 from the HIV-1 HXB2 isolate, which is the traditional numbering scheme used in the art. The introduction of two cysteine residues at positions 501 and 605, which are located close to each other in the three-dimensional structure of the protein, leads to the formation of a disulfide bridge. Coat proteins with SOSIP mutations such as BG505_SOSIP and B41_SOSIP (coat proteins from BG505 and B41 HIV strains (i.e. 9032-08.A1.4685) with SOSIP mutations) have been used in vaccine research and have been shown to induce the production of autologous neutralizing Category 2 Abs (Sanders et al., Science (2015), 349(6224):139-140).

However, even though so-called SOSIP mutations are capable of stabilizing the trimeric form of the envelope protein, the trimer fraction of such SOSIP mutants is typically less than 10%, while still producing a large amount of monomers and aggregates. Even the SOSIP mutant, BG505_SOSIP, which is one of the most promising envelope proteins currently known with SOSIP mutations in its ability to stabilize the trimeric form, typically produces only up to 25% of the trimeric form (Julien et al., Proc. Nat. Acad. Sci.

- 1 042607 (2015), 112(38), 11947-52). Moreover, in such a trimer fraction, the trimers are not completely stable, since their constituent parts do not fit tightly together at the top. Thus, to stabilize the apex and prevent loose fitting, several additional substitutions have been developed in addition to SOSIP mutations, such as E64K, A316W, and 201C-433C (de Taeye et al., Cell (2015), 163(7), 1702-15 ; Kwon et al., (2015) Nat Struct Mol Biol 22(7) 522-31).

Accordingly, there is a need for stabilized HIV envelope protein trimers that have an increased percentage of trimer formation, increased trimer yield, and/or improved trimer stability. Preferably, such stabilized HIV envelope protein trimers will also show good binding to broad spectrum neutralizing antibodies (bNAbs) and relatively limited binding to non-broad spectrum neutralizing Abs (non-bNAbs). It is an object of the present invention to provide HIV Env proteins that have increased trimer percentages and preferably also increased trimer yields.

Brief description of the invention

The present invention relates to recombinant HIV envelope proteins from various clades that are characterized by an increased percentage of trimer formation and/or increased trimer yields compared to previously described HIV envelope protein trimers. Using the mutations described herein, Env folding is optimized, strain-specific features are restored, and pre-fusion closed conformation regions important for the fusion process are stabilized. This provides a versatile approach to optimize the folding and stability of HIV-1 envelope protein trimers in a closed conformation prior to fusion. The resulting stable and properly folded HIV Env trimers are useful for immunization purposes, for example, to increase the likelihood of induction of broad spectrum neutralizing antibodies and to reduce the induction of non-neutralizing and weak neutralizing antibodies after administration of recombinant HIV Env trimers. The present invention also relates to isolated nucleic acid molecules and vectors encoding recombinant HIV envelope proteins, cells containing them, and compositions based on the recombinant HIV envelope protein, nucleic acid molecule, vector and/or cells.

In one general aspect, the present invention relates to recombinant human immunodeficiency virus (HIV) envelope proteins having specific amino acid residues at specified positions in the envelope protein sequence that provide stabilization during trimer formation.

In certain embodiments, the recombinant HIV envelope (Env) protein of the present invention comprises an amino acid sequence of the HIV Env protein having the indicated amino acid residues in at least two of the indicated positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp at position 651;

(ii) Phe, Ile, Met or Trp at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Trp at position 573;

(vi) Ile at position 204; and (vii) Phe, Met, or Ile at position 647, where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. In certain preferred embodiments, said amino acid residue at position 651 is Phe; said amino acid residue at position 655 is Ile; said amino acid residue at position 535 is Asn; and/or said amino acid residue at position 573 is Phe.

In certain embodiments, the recombinant HIV Env protein of the present invention comprises the amino acid sequence of the HIV Env protein and an amino acid substitution for the specified amino acid residue in at least one of the specified positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp at position 651;

(ii) Phe, Ile, Met or Trp at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Trp at position 573;

(vi) Ile at position 204; and (vii) Phe, Met, or Ile at position 647, wherein the HIV Env protein is selected from the group consisting of:

(1) an HIV Env consensus amino acid sequence, e.g., from clade C (e.g., containing the amino acid sequence of SEQ ID NO: 2 or 3) or from clade B (e.g.,

- 2 042607 containing the amino acid sequence under SEQ ID NO: 4 or 5);

(2) a synthetic HIV Env protein, for example, containing:

(a) the amino acid sequence under SEQ ID NO: 6, or (b) SEQ ID NO: 6 with a Glu to Arg type mutation at position 166, or (c) (a) or (b) with an amino acid substitution type mutation at positions 501 and 605 to Cys residues and an amino acid substitution mutation at position 559 to a Pro residue or (d) (a), (b) or (c) having an additional furin cleavage site mutation, e.g. amino acid substitution at positions 508 -511 on RRRRRR (SEQ ID NO: 10), or (e) SEQ ID NO: 7, or (f) an Env mosaic sequence such as Env containing the amino acid sequence under SEQ ID NO: 8 or 9; and (3) a parent HIV Env protein, which is preferably a wild-type HIV Env protein, preferably from clade C, containing at least one amino acid residue reducing mutation that occurs at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of HIV Env sequences in a population of at least 100, preferably at least 1000, preferably at least 10,000 wild-type HIV Env sequences, where the repair mutation is a substitution for an amino acid residue that occurs at the corresponding position with a frequency of at least 10% of the HIV Env sequences in said population, and preferably the repair mutation is a substitution for an amino acid residue that occurs most frequently at the corresponding position in said population;

and the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. In certain preferred embodiments, said amino acid residue at position 651 is Phe; said amino acid residue at position 655 is Ile; said amino acid residue at position 535 is Asn; and/or said amino acid residue at position 573 is Phe.

In certain embodiments, the recombinant HIV Env protein of the present invention comprises the amino acid sequence of the HIV Env protein and an amino acid substitution for the specified amino acid residue at at least one of the specified positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp at position 651;

(ii) Phe, Ile, Met or Trp at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Trp at position 573;

(vi) Ile at position 204; and (vii) Phe, Met or Ile at position 647, wherein the HIV Env protein is an HIV Env protein with SOSIP mutations containing at least one mutation resulting in the specified amino acid(s) residue(s) at the indicated positions selected from the group consisting of:

(a) Cys in regulations 501 and 605;

(b) Pro at position 559 and (c) Cys at positions 501 and 605 and Pro at position 559; and the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. In certain preferred embodiments, said amino acid residue at position 651 is Phe; said amino acid residue at position 655 is Ile; said amino acid residue at position 535 is Asn; and said amino acid residue at position 573 is Phe.

In other embodiments, the recombinant HIV Env protein of the present invention further comprises said amino acid residue at at least one of said positions selected from the group consisting of:

(viii) Gln, Glu, Ile, Met, Val, Trp or Phe, preferably Gln or Glu, at position 588;

(ix) Lys at position 64, or Arg at position 66, or Lys at position 64 and Arg at position 66;

(x) Trp at position 316;

(xi) Cys at both positions 201 and 433;

(xii) Pro at position 556, or Pro at position 558, or Pro at positions 556 and 558;

(xiii) replacing the loop at amino acid positions 548-568 (HR1 loop) with a loop having 710 amino acids, preferably an 8 amino acid loop, eg having a sequence selected from any of (SEQ ID NOs: 12-17);

(xiv) Gly at position 568, or Gly at position 569, or Gly at position 636, or Gly at both positions 568 and 636, or Gly at both positions 569 and 636; and/or (xv) Tyr at position 302 or Arg at position 519 or Arg at position 520 or Tyr at position 302 and Arg at position 519 or Tyr at position 302 and Arg at position 520 or Tyr at position

- 3 042607

302 and Arg at both positions 519 and 520.

In certain embodiments, the recombinant HIV Env protein of the present invention further comprises a mutation in the furin cleavage sequence of the HIV Env protein, such as a substitution at positions 508-511 on RRRRRR (SEQ ID NO: 10).

In one embodiment, the recombinant HIV Env protein is a gp140 protein.

In another embodiment, the recombinant HIV Env protein is a gp160 protein.

In certain embodiments, the implementation of the recombinant HIV Env protein is truncated in the cytoplasmic region, for example, after 7 amino acids of the cytoplasmic region.

In another general aspect, the present invention relates to a trimeric complex containing a non-covalent oligomer of any three recombinant HIV Env proteins described herein.

In another general aspect, the present invention provides a method for improving the folding and stability (measured as an increased percentage of trimers and/or trimer yield) of a parent HIV Env protein, the method comprising restoring the amino acid sequence of the parent HIV Env protein by introducing at least one repair mutation, preferably at least 3 repair mutations to the original HIV Env protein, where the repair mutation is an amino acid substitution of an amino acid residue that is present at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of the HIV Env sequences in a population of at least at least 100, preferably at least 500, preferably at least 1000, preferably at least 10,000 wild-type HIV Env sequences, where the substitution is a substitution for an amino acid residue that is present at the corresponding position with a frequency of at least 10% of the sequences Env HIV in said population, and preferably the substitution is for the amino acid residue that is most frequently present at the corresponding position in said population. The present invention also provides a reconstituted HIV Env protein that can be produced using said method of the present invention to improve folding and stability (measured as percentage of trimers and/or trimer yield) of the HIV Env protein. The present invention also provides a pharmaceutical composition containing said reconstituted HIV Env protein. The present invention also provides a method for producing an HIV Env protein, comprising a method for reconstituting the HIV Env protein described herein and expressing a nucleic acid encoding the reconstituted stabilized HIV Env protein in a recombinant host cell.

In another general aspect, the present invention provides a recombinant HIV Env protein comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, where positions 204, 535, 573 are preferred, 589, 647, 651 and 655 and preferably additional provisions 64, 66, 201, 316, 433, 501, 508-511, 556, 558, 559, 588, 548568 and 605 are not taken into account when determining the % identity, and where the numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. In certain embodiments thereof, the recombinant HIV Env protein contains an amino acid sequence that is at least 98%, 99%, or 100% identical to SEQ ID NO: 3, where positions 204, 535, 573, 589, 647, 651, and 655 are preferred and preferably additional provisions 64, 66, 201, 316, 433, 508-511, 556, 558, 588 and 548-568 are not taken into account when determining % identity, and where the numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate.

In another general aspect, the present invention provides a recombinant HIV Env protein comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, preferably positions 204, 535, 573, 589, 647, 651, and 655 and preferably additional provisions 64, 66, 201, 316, 433, 501, 508-511, 556, 558, 559, 588, 548568 and 605 are not taken into account when determining % identity, and where the numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. In certain embodiments thereof, the recombinant HIV Env protein comprises an amino acid sequence that is at least 98%, 99% or 100% identical to SEQ ID NO: 5, where positions 204, 535, 573, 589, 647, 651 and 655 are preferred and preferably additional provisions 64, 66, 201, 316, 433, 508-511, 556, 558, 588 and 548-568 are not taken into account when determining % identity, and where the numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate.

In these aspects and embodiments, one or more of the amino acids at the indicated positions, which are not taken into account when determining % identity, are preferably selected from the amino acids indicated as preferred herein, for example, Ile at position 204; Phe, Ala, Leu or Trp at position 651, etc. (see tables 1 and 2 below).

In another general aspect, the present invention provides a recombinant HIV Env protein comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 2, 3, 4, 5, 20 , 22, 24, 26, 27, 28, 29, 30, 31 or 32, where SEQ ID NO:

- 4 042607

20, 22, 24, 26, 27, 28, 29, 30, 31 or 32 are particularly preferred. In this aspect, preferably provisions 204, 535, 573, 589, 647, 651, 655 and 658 and preferably additional provisions 64, 66, 201, 316, 433, 508-511, 556, 558, 588 and 548-568 do not take into account attention when determining % identity, and where the numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. Also in this aspect, one or more of the amino acids at the indicated positions, which are not taken into account when determining the % identity, are preferably selected from the amino acids indicated as preferred herein in (i)-(vii) of the table. 1, (viii)-(xv) from Table. 2 and/or (xvi) from table. 1, for example Ile at position 204; Phe, Leu, Met or Trp at position 651, etc.

In another general aspect, the present invention relates to a particle, preferably a liposome or a nanoparticle, for example, a self-assembling nanoparticle, having on its surface the recombinant HIV Env protein of the present invention.

In another general aspect, the present invention relates to an isolated nucleic acid molecule encoding a recombinant HIV Env protein of the present invention, and vectors containing an isolated nucleic acid molecule operably linked to a promoter. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an expression vector. In one preferred embodiment, the viral vector is an adenoviral vector.

Another general aspect relates to a host cell containing an isolated nucleic acid molecule or vector encoding a recombinant HIV Env protein of the present invention. Such host cells can be used to produce a recombinant protein, express a recombinant protein, or produce viral particles.

Another general aspect relates to methods for producing a recombinant HIV Env protein, comprising growing a host cell containing an isolated nucleic acid molecule or vector encoding a recombinant HIV Env protein of the present invention under conditions suitable for producing a recombinant HIV Env protein.

Another general aspect relates to a composition comprising a recombinant HIV Env protein, a trimeric complex, an isolated nucleic acid molecule, a vector or host cell as described herein, and a pharmaceutically acceptable carrier.

Brief description of the figures

The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments shown in the drawings.

The figures show the following.

In FIG. 1A and 1B show a schematic representation of the structure of the envelope proteins (Env) of HIV.

In FIG. 1A shows the full-length HIV Env protein and FIG. 1B shows a soluble HIV Env protein containing the so-called SOSIP mutations and a truncation starting at residue 664 according to gp160 numbering from the HIV-1 HXB2 isolate (sequence SOSIP.644).

In FIG. 2A and 2B show percentage trimer formation (FIG. 2A) and trimer yield (FIG. 2B) for recombinant HIV Env proteins according to embodiments of the present invention as measured by the AlphaLISA assay described in Example 3; the HIV Env recombinant proteins tested contained a single, double or triple amino acid substitution introduced into the clade C HIV Env framework consensus sequence, ConC_SOSIP (SEQ ID NO: 3); trimer percentage and trimer yield were determined based on the binding of a trimer-specific monoclonal antibody (mAb), PGT145, to each of the recombinant HIV Env proteins; the trimer yield and percentage of trimer formation for each of the recombinant HIV Env proteins of the present invention is compared to trimer formation for an envelope protein having a ConC_SOSIP framework sequence without any of the additional trimer stabilizing mutations described herein.

In FIG. 3 shows chromatograms obtained by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) analysis of recombinant HIV Env proteins according to an embodiment of the present invention; The recombinant HIV Env proteins tested contained a single amino acid substitution introduced into the HIV clade C Env framework consensus sequence, ConC_SOSIP (SEQ ID NO: 3), and were purified by lectin affinity chromatography as described in Example 2; SEC-MALS analysis was performed as described in Example 3; the peak corresponding to the trimeric form is indicated on each of the chromatograms.

In FIG. 4 shows the thermal stability of recombinant HIV Env proteins according to embodiments of the present invention, described as the percentage of trimers remaining upon thermal exposure; the HIV Env recombinant proteins tested contained a single, double or triple amino acid substitution introduced into the clade C HIV Env framework consensus sequence, ConC_SOSIP (SEQ ID NO: 3); recombinant HIV Env proteins were thermally exposed and the percentage of trimers remaining on thermal exposure determined using AlphaLISA assay as described in Example 4; the thermal stability of an envelope protein having the framework sequence ConC_SOSIP without any trimer-stabilizing mutations is also shown.

In FIG. 5A-5B show percentage trimer formation (FIG. 5A) and trimer yield (FIG. 5B) for recombinant HIV Env proteins having a single amino acid substitution introduced into the clade B HIV Env framework consensus sequence, ConB_SOSIP (SEQ ID NO: 5) , according to embodiments of the present invention, compared to those of an envelope protein having a ConB_SOSIP framework sequence without any additional trimer-stabilizing mutations of the present invention described in Example 5; trimer yield and percentage trimer formation were measured by AlphaLISA assay.

In FIG. 6A-6B show percentage trimer formation and trimer yield for recombinant HIV Env proteins having amino acid substitutions introduced into the synthetic HIV envelope protein framework sequence, DS_sC4_SOSIP_E166R, according to embodiments of the present invention as described in Example 6; the percentage of trimer formation and trimer yield were measured by AlphaLISA assay.

In FIG. 7 shows chromatograms obtained by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) analysis of recombinant HIV Env proteins according to embodiments of the present invention; recombinant HIV Env proteins tested contained a single K655I mutation, with each subsequent variant introducing an additional mutation into the HIV clade C Env framework consensus sequence, ConC_SOSIP (SEQ ID NO: 3), and purified by lectin affinity chromatography as described in example 2; SEC-MALS analysis was performed as described in Example 3; the peak corresponding to gp140 monomers in ConC_SOSIP is indicated by a shaded box to the right of the trimer peak. The lower panel shows an increase in the lower part of the graph, whereby it can be seen that each additional mutation leads to an additional decrease in the peak height of the gp140 monomers.

In FIG. 8A-8B show the percentage of trimer formation (FIG. 8A) and trimer yield (FIG. 8B) for BG505_SOSIP (derived from a wild-type clade A strain) having single amino acid substitutions and combinations of substitutions introduced according to embodiments of the present invention, by compared to that of an envelope protein having the BG505_SOSIP framework sequence without any additional trimer stabilizing mutations of the present invention described in Example 9. Trimer yield and percentage of trimer formation were measured by AlphaLISA assay.

In FIG. 9 shows chromatograms obtained by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) analysis of recombinant HIV Env proteins according to embodiments of the present invention; SEC-MALS analysis was performed on the cell culture supernatant transfected with Env. The peak corresponding to the trimeric form elutes from 7 to 7.5 minutes. The dark gray line is BG505_SOSIP (derived from a wild-type clade A strain) and the light gray line is BG505_SOSIP with substitutions L556P, K655I, M535N, N651F, D589V, K588E.

In FIG. 10A-10B show the yield of trimers for the C97ZA_SOSIP variants described in Example 10. Yield of C97ZA trimers with three stabilizing substitutions (L556P, T651F and M535N) (FIGS. 10A and B). In FIG. 10B, the Env sequence was further optimized with additional mutations (21 additional mutations) that were added to restore the C97ZA Env sequence according to the conceptual backbone described in FIG. 12 and by introducing additional stabilizing substitutions (K655I, D589V, A204I and K588E). The yield of trimers and the percentage of formation of trimers was measured using analysis AlphaLISA. The signals were normalized to the ConC_SOSIP signal, which was set to 1. PNGS is a potential N-glycosylation site.

Fig. 11 Yield of HIV-1 strain DU422 Env trimers with four stabilizing substitutions (see Example 11 for details). All values were normalized to the ConC_SOSIP value (not shown), which was considered equal to 1.

Fig. 12 is a generic HIV-1 Env sequencing concept illustrated for the C97ZA strain. The highest frequency residue (herein referred to as the consensus residue) in the overall HIV-1 database (upper bars) and the residue in the C97ZA strain (lower bars) are sorted from low to high percentage of occurrence of the residue position in C97ZA. The positions in the C97ZA sequence to be replaced by the consensus residue were selected based on the following criteria. Residue positions in C97ZA that occur in less than 2% of sequences from the Env database (black bars). Residue positions in C97ZA that occur in 2-7.5% of Env database sequences and are immersed or partially immersed (dark gray bars). Positions that are accessible and hydrophobic in C97ZA and hydrophilic consensus residues (the two lightest gray bars), and

- 6 042607 position, which is the consensus residue (S234N) of the potential N-glycosylation site (PNGS).

Fig. 13-trimers of HIV Env_SOSIP in a closed conformation before fusion, obtained by resequencing and mutational stabilization. AlphaLISA signals for all SOSIP variants obtained from cell culture supernatant, normalized to ConC_SOSIP when using broad-spectrum neutralizing antibodies.

Fig. 14 is an analytical SEC profile of Env_SOSIP reference variants (SOSIP framework sequence), Env variants reconstructed according to the concept described in Example 12 and in FIG. 12, and Env options with additional stabilizing substitutions according to table. 3 using cell culture supernatants after transfection. The cell culture supernatant false signal was subtracted from all profiles. Trimer peaks are indicated as *.

Fig. 15 - output of trimers of variants ConC Env HIV-1 without stabilizing modifications of SOSIP.

Fig. 16 - yield of trimers (A) and percentage of trimers (B) of ConC_SOSIP with methionine substitution type mutations at positions 589, 647, 651 and 655. All values were normalized to the ConC SOSIP value (not shown), which was considered equal to 1. Planck errors are shown at the right end of the bars.

In FIG. 17A-17D show the percentage of trimer formation (Fig. 17A,B for various experiments) and trimer yield (Fig. 17C,D for various experiments) for recombinant HIV Env proteins with the indicated mutations described in Example 15 as measured by AlphaLISA analysis.

In FIG. 18 shows SEC-MALS chromatograms of recombinant HIV Env proteins with the indicated mutations described in Example 15.

Detailed description of the invention

Various publications, articles, and patents are cited or described in the Background section and throughout the description; and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, products, etc., which has been included in the present description, is intended to provide the context of the present invention. Such discussion is not an admission that any or all of these materials are part of the prior art with respect to any disclosed or claimed inventions.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as usually understood by an average person skilled in the art to which the present invention pertains. Otherwise, certain terms used in this document have the meanings set forth in the description. All patents, published patent applications and publications cited in this document are incorporated by reference as if they were fully set forth in this document. It should be noted that the singular forms used herein and in the appended claims include reference to the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values such as concentration or range of concentrations described herein should in all cases be understood as modified by the term approximately. Thus, the numerical value usually includes ±10% of the specified value. As used herein, the use of a numeric range unambiguously includes all possible subranges, all individual numeric values within that interval, including integers within such intervals, and fractional values unless the context explicitly states otherwise.

Amino acids are mentioned throughout this disclosure. There are twenty naturally occurring amino acids, as well as many non-naturally occurring amino acids. Every known amino acid, including both natural and non-natural amino acids, has a full name, a one-letter abbreviated code, and a three-letter abbreviated code, all of which are well known to those of ordinary skill in the art. For example, for twenty naturally occurring amino acids, the following three- and one-letter abbreviated codes apply: alanine (Ala; A), arginine (Arg; R), aspartic acid (Asp; D), asparagine (Asn; N), cysteine (Cys; C), glycine (Gly; G), glutamic acid (Glu; E), glutamine (Gln; Q), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y ) and valine (Val; V). Amino acids may be referred to by their full name, a one-letter abbreviation code, or a three-letter abbreviation code.

Unless the context clearly dictates otherwise, the position numbering in the amino acid sequence of the HIV envelope protein used herein corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate, for example, as set forth in Korber et al. (Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber et al., Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mech.), which is incorporated herein document by reference in its entirety. Numbering according to HXB2 is generally accepted in

- 7 042607 HIV Env protein regions. gp160 from the HIV-1 HXB2 isolate has the amino acid sequence shown under SEQ ID NO: 1. Aligning the HIV Env sequence of interest with this sequence can be used to find the corresponding amino acid numbering in the sequence of interest.

The phrase contains the amino acid sequence of the HIV Env protein having the specified amino acid residue in at least one of the specified positions selected from the group consisting of and contains one or more of the following (amino acid residues) are used interchangeably herein.

The term percent (%) sequence identity or % identity describes the number of matches (hits) of identical amino acids in two or more aligned amino acid sequences relative to the number of amino acid residues that make up the total length of the amino acid sequences. In other words, by applying an alignment to two or more sequences, one can determine the percentage of amino acid residues that are the same (e.g., 95%, 97%, or 98% identity) when sequences are compared and aligned for maximum match, as measured using a sequence comparison algorithm, known from the prior art, or when they are manually aligned and checked visually. Thus, sequences that are compared to determine sequence identity may differ in amino acid substitution(s), addition(s) or deletion(s). Suitable programs for aligning protein sequences are known to those skilled in the art. Percent sequence identity in protein sequences can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, for example using the NCBI BLAST algorithm (Altschul SF, et al. (1997), Nucleic Acids Res. 25: 33893402).

As used herein, an 'HIV Env sequence set' is a collection of a typical number (e.g., at least 100, or 500, or 1,000 or more) random wild-type HIV Env protein sequences that may be from the same clade (e.g., from clade C) or from different clades (for example, from clade A, B, C, etc.). Suitable sets of such sequences are available from databases such as the HIV Sequence Database (Los Alamos National Laboratory) or smaller sets can be extracted from them. Such a population preferably contains at least 100 HIV Env protein sequences, 1000 HIV Env protein sequences, at least 10,000 HIV Env protein sequences, at least 50,000 HIV Env protein sequences, and may contain more than 90,000 HIV Env protein sequences.

The 'corresponding position' in the HIV Env protein refers to the position of the amino acid residue when at least two HIV Env sequences are aligned. Unless otherwise indicated, the amino acid position numbering for these purposes corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate, as is customary in the art.

Used in this document stabilizing mutation is a mutation described in this document in any of paragraphs. (i)-(vii) or (xvi) Table 1 or (viii)-(xv) tab. 2 that provides an increase in the percentage of trimers and/or trimer yield (which can be measured, for example, according to the AlphaLISA or SEC-MALS assays described herein) of the HIV Env protein compared to the parent molecule if the mutation is introduced by substitution of the corresponding amino acid in specified parent molecule. The amino acids generated by such stabilizing mutations are generally rare or non-existent in the Env proteins of wild-type HIV isolates.

As used herein, a repair mutation is a substitution of an amino acid residue in the original HIV Env protein if the amino acid residue is present less than 7.5%, preferably less than 2% of the time at the corresponding position in the population of HIV Env protein sequences, where the substitution is an amino acid substitution , which is present at the appropriate position in the specified population more frequently, for example, in at least 10% of the HIV Env proteins in the specified population, and preferably is an amino acid substitution that is present in the corresponding position in at least 20% of the HIV Env proteins of the specified population , or which is the most frequently occurring amino acid at the corresponding position in the specified population. Thus, the amino acids generated by such repair mutations are typically found in a relatively high percentage of Env proteins from wild-type HIV isolates, and in a few cases they may coincide with an amino acid at the appropriate position in the HIV Env consensus sequences.

As used herein, the repaired and stabilized HIV Env sequence typically contains at least one repair mutation and at least one stabilizing mutation, preferably multiple repair mutations and multiple stabilizing mutations, compared to the original HIV Env sequence.

The terms natural or wild-type are used interchangeably herein to refer to HIV strains (or Env proteins thereof) and refer to HIV strains (or Env proteins from

- 8 042607 them), occurring in nature, for example, such as in HIV-infected patients.

The present invention relates generally to recombinant HIV envelope (Env) proteins containing specific amino acid substitutions at specified positions in the envelope protein sequence that stabilize the trimeric form of the envelope protein. The introduction of one or more of said amino acid substitutions of the present invention into the HIV envelope protein sequence may result in an increased percentage of trimer formation and/or an increased trimer yield. They can be measured, for example, using trimer-specific antibodies, melting point, size exclusion chromatography, and binding to antibodies that bind to the correctly folded (stable trimeric) or alternatively misfolded (unstable or non-trimeric) Env protein, and increased percentage of trimers and /or trimer yields are considered to be indicative of a stable native correctly folded Env protein.

The human immunodeficiency virus (HIV) is a member of the genus Lentivirinae, which is part of the Retroviridae family. Humans are infected with two types of HIV: HIV-1 and HIV-2. HIV-1 is the most common strain of the HIV virus and is known to be more pathogenic than HIV-2. As used herein, the terms human immunodeficiency virus and HIV refer without limitation to HIV-1 and HIV-2. In preferred embodiments, HIV refers to HIV-1.

HIV is classified into several clades with a high degree of genetic divergence. As used herein, the terms HIV clade or HIV subtype refer to related human immunodeficiency viruses classified according to their degree of genetic similarity. The largest group of HIV-1 isolates is called the M group (major strains) and consists of at least ten clades, A through J.

In one general aspect, the present invention provides a recombinant envelope protein (Env) of HIV. When referring to a protein, the term recombinant refers to a protein that is produced by recombinant techniques or by in vitro chemical synthesis. According to embodiments of the present invention, the recombinant protein has an artificial amino acid sequence in which it contains at least one sequence element (e.g., amino acid substitution, deletion, addition, sequence substitution, etc.) that does not occur in the corresponding naturally occurring sequence . Preferably, the recombinant protein is a non-naturally occurring HIV envelope protein that is optimized to induce an immune response or provide immunity against one or more naturally occurring strains of HIV.

The terms HIV envelope protein, HIV Env, and HIV Env protein refer to a protein or fragment or derivative thereof that is naturally expressed on the envelope of the HIV virion and allows HIV to target and attach to the plasma membrane of HIV-infected cells. The terms shell and Env are used interchangeably throughout this disclosure. The HIV env gene encodes the precursor protein gp160, which undergoes proteolytic cleavage into two mature envelope glycoproteins gp120 and gp41. The cleavage reaction in the sequence motif, which is highly conserved among retrovirus envelope glycoprotein precursors, is mediated by the host cell protease, furin (or furin-like proteases). More specifically, gp160 trimerizes to (gp160) ₃ and then undergoes cleavage into two non-covalently linked mature glycoproteins gp120 and gp41. Virus entry into the cell is subsequently mediated by a trimer of the gp120/gp41 heterodimers. Gp120 is a receptor-binding fragment and it binds to the CD4 receptor (and co-receptor) on a target cell that has such a receptor, such as, for example, a T helper cell. Gp41, which is non-covalently linked to gp120, is a fusion fragment and provides the second step by which HIV enters the cell. Gp41 is initially immersed in the viral envelope, but when gp120 binds to the CD4 receptor and co-receptor, gp120 changes its conformation, which causes gp41 to become accessible and may facilitate fusion with the host cell. Gp140 is the ectodomain of gp160.

In embodiments of the present invention, the HIV envelope (Env) protein may be a gp160 or gp140 protein, or combinations, fusions, truncations, or derivatives thereof. For example, the HIV envelope protein may include the gp120 protein non-covalently linked to the gp41 protein. The HIV envelope protein can also be a truncated HIV envelope protein, including, but not limited to, envelope proteins containing a C-terminal truncation in the ectodomain (i.e., a domain that protrudes into the extracellular space), a truncation in gp41, such as a truncation in the ectodomain gp41, in the transmembrane domain of gp41, or a truncation in the cytoplasmic domain of gp41. The HIV envelope protein can also be gp140, corresponding to the gp160 ectodomain, or an extended or truncated version of gp140. The expression of gp140 proteins has been described in several publications (e.g., Zhang et al., 2001; Sanders et al., 2002; Harris et al., 2011), and different variants of this protein can also be ordered, for example, based on different strains of HIV, from service providers. The gp140 protein of the present invention may have a cleavage site mutation such that the gp120 domain and gp41 ectodomain are not cleaved and remain covalently linked, or alternatively

- 9 042607 gp120 domain and gp41 ectodomain can be cleaved, but still covalently linked, eg via a disulfide bridge (eg, such as in SOSIP variants). The HIV envelope protein may further be a derivative of the naturally occurring HIV envelope protein having sequence mutations, for example at furin cleavage sites and/or so-called SOSIP mutations. The HIV envelope protein of the present invention may also have a furin cleavage site, whereby the gp120 and gp41 ectodomains may be non-covalently linked.

In preferred embodiments of the present invention, the HIV Env protein is a gp140 protein or a gp160 protein, and more preferably a gp140 protein. In other preferred embodiments, the Env protein is truncated, eg, by deletion of residues after residue ⁷ of the cytoplasmic region, compared to the native Env protein.

According to embodiments of the present invention, the HIV envelope protein may be a trimer or a monomer, and preferably is a trimer. The trimer may be a homotrimer (eg, trimers containing three identical polypeptide units) or a heterotrimer (eg, trimers containing three polypeptide units that are not identical). Preferably the trimer is a homotrimer. In the case of cleaved gp140 or gp160, it is a trimer of polypeptide units that are dimers of gp120-gp41, and when all three of these dimers are the same, it is considered a homotrimer.

The HIV envelope protein can be a soluble protein or a membrane-bound protein. Membrane-bound envelope proteins typically contain a transmembrane domain, such as in the full-length HIV envelope protein containing a transmembrane domain (TM) shown in FIG. 1A. Membrane-bound proteins may have a cytoplasmic domain, but the presence of a cytoplasmic domain is not necessary to be membrane-bound. In the case of soluble envelope proteins, at least partial or complete deletion of the transmembrane domain is contemplated. For example, the C-terminal portion of the full-length HIV envelope protein can be truncated to remove the transmembrane domain, thereby providing the soluble protein shown in FIG. 1B. However, the HIV envelope protein can still be soluble at shorter truncations and truncations at alternative positions to those shown in FIG. 1b. Truncation can be performed at various positions, and non-limiting examples include truncation after amino acids 664, 655, 683, etc., all of which result in a soluble protein. The membrane-bound Env protein of the present invention may contain all or part of the C-terminal domain (e.g., by deletion of a portion of the C-terminal cytoplasmic domain, e.g., in some embodiments after the ^7th residue of the cytoplasmic region) compared to the native Env protein.

The signal peptide is typically present at the N-terminus of the HIV Env protein at the time of expression, but is cleaved off by the signal peptidase and thus is not present in the mature protein. The signal peptide may be substituted with other signal sequences, and some non-limiting examples of signal peptides are provided herein under SEQ ID NOS: 11, 18, 33, and 34.

According to embodiments of the present invention, an HIV envelope protein, e.g., gp160 or gp140, may be derived from an HIV envelope protein sequence from any HIV clade (or subtype), e.g., clade A, clade B, clade C, clade D, clade E, clade F , clade G, clade H, etc., or combinations thereof (such as circulating recombinant forms, or CRFs occurring by recombination between viruses of different subtypes, e.g. BC, AE, AG, BE, BF, ADG, etc.). d.). The HIV envelope protein sequence may be a naturally occurring sequence, a mosaic sequence, a consensus sequence, a synthetic sequence, or any derivative or fragment thereof. The mosaic sequence contains multiple epitopes derived from at least three HIV envelope protein sequences from one or more HIV clades and can be constructed with algorithms that optimize coverage of T cell epitopes. Examples of HIV mosaic envelope protein sequences include those described in, for example, Barouch et al, Nat Med 2010, 16:319-323 and WO 2010/059732, such as those shown under SEQ ID NOs: 8 and 9, for example. consensus sequence means an artificial amino acid sequence based on an alignment of amino acid sequences of homologous proteins, eg determined by alignment (eg using Clustal Omega) of amino acid sequences of homologous proteins. It represents the calculated order of the amino acid residues most frequently found at each position in a sequence alignment, based on Env sequences derived from at least 1000 natural HIV isolates. The synthetic sequence is a non-naturally occurring HIV envelope protein that is optimized to induce an immune response or provide immunity against more than one naturally occurring strain of HIV. Non-limiting examples of synthetic HIV envelope proteins are HIV mosaic envelope proteins. In preferred embodiments of the present invention, the HIV Env protein is an Env consensus protein or a synthetic Env protein having at least one of the specified amino acids at the specified positions (i)-(vii)

- 10 042607 according to the present invention. Particularly preferred are Env consensus proteins having at least one, preferably at least two of said amino acid residues at said positions (i)-(vii) according to the present invention, preferably further having SOSIP mutations and/or furin cleavage site mutations as described below.

In certain embodiments of the present invention, the HIV envelope protein, whether it be a naturally occurring sequence, a mosaic sequence, a consensus sequence, a synthetic sequence, etc., contains additional sequence mutations, for example, at furin cleavage sites and/or so-called SOSIP mutations.

In some embodiments, the HIV envelope protein is an HIV Env protein with SOSIP mutations. So-called SOSIP mutations are trimer-stabilizing mutations that include SOS mutations (Cys residues at positions 501 and 605, which lead to the introduction of a possible disulfide bridge between the newly formed cysteine residues) and IP mutation (Pro residue at position 559). According to embodiments of the present invention, the Env protein in SOSIP mutations contains at least one mutation selected from the group consisting of Cys at positions 501 and 605; Pro at position 559; and preferably Cys at positions 501 and 605 and Pro at position 559. The HIV Env protein with SOSIP mutations may further contain other sequence mutations, for example at the furin cleavage site. In addition, in certain embodiments, additional mutations may be added such that the Env protein contains Pro at position 556, or at position 558, or at positions 556 and 558, which, as found in this document, can function not only as an alternative to Pro at position 559 in the SOSIP variant, but also serve as additional mutations that can further increase trimer formation by the SOSIP variant that already has Pro at position 559.

In certain preferred embodiments of the present invention, the HIV Env protein with SOSIP mutations contains Cys at positions 501 and 605 and Pro at position 559.

In certain embodiments, the HIV envelope protein of the present invention further comprises a mutation at a furin cleavage site. A mutation in the furin cleavage sequence may be an amino acid substitution, deletion, insertion or substitution of one sequence for another, or substitution for a linker amino acid sequence. Preferably, in the present invention, mutation of the furin cleavage site can be used to optimize the cleavage site, whereby furin cleavage is improved over wild-type protein, for example, by replacing the sequence at residues 508-511 with RRRRRR (SEQ ID NO: 10) [i.e. . substitution of a typical amino acid sequence (e.g. EK) at positions 509-510 with four arginine residues (i.e. two substitutions and two additions), while at positions 508 and 511 most HIV Env proteins already have arginine residues, so they generally do not require substitution, but since the end result is often referred to in the literature as the amino acid sequence RRRRRR, the authors of the present invention have retained such nomenclature in this document]. Other mutations are also known that improve furin cleavage and can also be used. Alternatively, the furin cleavage site can be replaced by a linker, which eliminates the need for cleavage and the protein adopts a native-like conformation (e.g., described in (Sharma et al, 2015) and (Georgiev et al, 2015)).

In specific embodiments of the present invention, the HIV envelope protein of the present invention further comprises not only so-called SOSIP mutations (preferably Cys at positions 501 and 605 and Pro at position 559), but also a sequence mutation at the furin cleavage site, preferably a sequence substitution at residues 508 -511 on RRRRRR (SEQ ID NO: 10). In certain preferred embodiments, the HIV Env contains not only the indicated SOSIP mutations and the furin cleavage site mutation, but also additionally contains a Pro residue at position 556 or 558, most preferably at both positions 556 and 558.

In preferred embodiments of the present invention, the amino acid sequence of the HIV envelope protein is a consensus sequence, such as the clade C HIV envelope protein consensus sequence or the clade B HIV envelope protein consensus sequence. In a particularly preferred embodiment, the HIV envelope protein amino acid sequence is the envelope protein consensus sequence HIV clade C.

Exemplary HIV envelope proteins that can be used according to the present invention include the HIV clade C envelope protein consensus sequence (SEQ ID NO: 2) and the HIV clade B envelope protein consensus sequence (SEQ ID NO: 4). These clade C and clade B HIV envelope protein consensus sequences may contain additional mutations which, for example, increase stability and/or trimer formation, such as, for example, the so-called SOSIP mutations and/or sequence mutation at the furin cleavage site described

- 11 042607 above, such as, for example, in the ConC_SOSIP sequence shown under SEQ ID NO: 3 and in the ConB_SOSIP sequence shown under SEQ ID NO: 5.

Other non-limiting examples of preferred HIV envelope protein sequences that can be used in the present invention (as a background or parent molecule into which one or more of the mutations of the present invention are then introduced) include synthetic HIV Env proteins, for example, containing the amino acid sequence under SEQ ID NO: 6 or SEQ ID NO: 6 with a Glu to Arg mutation at position 166, or proteins optionally having additional SOSIP mutations and/or furin cleavage site mutations described above. Another non-limiting example is SEQ ID NO: 7. Additional non-limiting examples are HIV mosaic envelope proteins, as having the amino acid sequence under SEQ ID NO: 8 or 9.

In certain embodiments, the parent molecule is a wild-type HIV Env protein, wherein one or preferably several amino acids have been reduced according to the methods described herein. Such parent molecules contain at least one repair mutation at an amino acid residue that is present at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of the HIV Env sequences in a population of at least 100, preferably at least 500, preferably at least 1,000, preferably at least 10,000, preferably at least 20,000 wild-type HIV Env sequences, where the repair mutation is a substitution for an amino acid residue that is present at the corresponding position at a frequency of at least 10% of the HIV Env sequences in the given set. Preferably, said substitution is made with an amino acid residue that is present at the corresponding position at a frequency of at least 15%, at least 20%, at least 25% of the HIV Env sequences in said population. Preferably, said substitution is made with the amino acid residue most frequently present at the corresponding position in said population. In certain preferred embodiments, said parent molecules contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least at least 20 such repair mutations. Preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the amino acid residues, or all of them, that are present at the respective positions at a frequency of less than 2% of the HIV Env sequences in said population are recovered in the parent molecule compared to the wild-type Env protein. In certain embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the amino acid residues, or all of them, that are present at the respective positions at less than 7.5% of the HIV Env sequences in of the specified population, are recovered in the original molecule compared to the wild-type Env protein. In certain embodiments, the wild-type HIV Env protein is derived from a clade A, B, or C strain, preferably from a clade C strain. amino acid residue is sometimes referred to herein as consensus amino acid or consensus residue. The result of these recovery actions is a significant improvement in the properties of the resulting original molecule in terms of folding, trimerization, expression and/or stability, and the resulting molecule is referred to herein as a reduced protein Addition of stabilizing mutations of the present invention (for example, one or more of (i )-(vii) (Table 1), and/or (xvi) (Table 1), and/or optionally (viii)-(xv) (Table 2)) into such parent molecules leads to an even greater improvement in one or more of percentage trimer formation, trimer yield, stability, broad spectrum neutralizing antibody binding, folding, and resulting molecules that are derived from wild-type HIV Env proteins are referred to herein as a reduced and stabilized Env protein. One of skill in the art will appreciate that the introduction of stabilizing mutations actually deviates the resulting sequence slightly from the consensus sequence, so that the end result of significantly improving the properties of the reduced and stabilized HIV Env molecules is based on two completely different concepts.

Mutations resulting in the indicated amino acids at positions (i)-(vii) according to the present invention can also be used in HIV Env proteins in which SOSIP mutations are not present (for example, in Env consensus sequences or in Env proteins from wild-type HIV isolates) , and they also appear to improve their trimerization, since the mutations of the present invention are independent of SOSIP mutations and, in addition, they have been shown to work in several different framework sequences of the HIV Env proteins. Indeed, this document shows that the mutations of the present invention can work in the absence of SOS mutations as well as in the absence of IP mutation, leading to improved trimerization properties of the HIV Env.

A recombinant HIV envelope protein according to embodiments of the present invention provides an HIV envelope protein having specific amino acid(s) residue(s) at predetermined amino acid sequence positions of the HIV envelope protein. In particular, seven positions in the envelope protein have been identified, as well as specific amino acid residues that should optimally be located in each of the identified positions. Positions identified in the envelope protein sequence include (i) position 651, (ii) position 655, (iii) position 535, (iv) position 589, (v) position 573, (vi) position 204, and (vii) position 647, where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate. The HIV Env protein of the present invention has the predetermined amino acid(s) residue(s) at at least one of said positions (i)-(vii), preferably at least two of said positions (i)-(vii ), more preferably in at least three of said positions (i)-(vii). Specific amino acid residues that should optimally be in each of these positions according to the embodiments of the present invention, are shown in table. 1. The preferred positions of these options are (i), (ii), (iii), (iv), (vi) and/or (vii). Particularly preferred provisions of these options are (i), (ii), (iii), (iv) and/or (vii). An additional preferred option is (xvi), mentioned in somewhat more detail hereinafter.

Table 1. Optimal amino acids at indicated positions of recombinant HIV Env proteins according to embodiments of the present invention

No. Position ¹ Optimal amino acid residue (i) 651 Phe, Leu, Met or Trp (Phe preferred) (ii) 655 Phe, lie, Met or Trp (preferably lie) (iii) 535 Asn or Gin (preferably Asn) (iv) 589 Vai, lie or Ala (preferably Vai or lie, most preferably Vai) (v) 573 Phe or Trp (Phe preferred) (vi) 204 He (viii) 647 Phe, Met or He (preferably Phe) (xvi) 658 Vai, He, Phe, Met, Ala or Leu (preferably Vai or lie, most preferably Vai)

According to the numbering in gp160 from HIV-1 HXB2 isolate An HIV envelope protein amino acid sequence into which one or more optimal amino acid substitutions (or specified amino acids) are introduced at one or more of the specified positions is referred to as the HIV envelope protein framework sequence or the original HIV envelope protein sequence. . For example, if position 651 in the ConC_SOSIP sequence of SEQ ID NO: 3 is mutated to Phe, then the ConC_SOSIP sequence is considered a framework or parent sequence. Any HIV envelope protein can be used as a framework or parent sequence into which a new stabilizing mutation can be introduced according to an embodiment of the present invention, either alone or in combination with other mutations such as so-called SOSIP mutations and/or furin cleavage site mutations. . Non-limiting examples of an HIV Env protein that can be used as a framework sequence include an HIV Env protein from a natural HIV isolate, a synthetic HIV Env protein, or an HIV Env consensus protein, and in certain non-limiting examples, include proteins containing SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

According to embodiments of the present invention, the HIV envelope protein may have said amino acid residue at at least one of said positions selected from the group consisting of positions 651, 655, 535, 589, 573, 204, and 647, such as said amino acid residue in one, two, three, four, five, six or seven positions. Preferably, the HIV envelope protein is substituted at one, two, or three of the indicated positions, and more preferably, the HIV envelope protein is substituted at at least two of the indicated positions. Even more preferably, the HIV envelope protein is substituted at three of said positions, four of said positions, five of said positions, six of said positions, or all seven of said positions. Preferably, the HIV envelope protein contains said amino acid residues in at least two of the said positions. More preferably, the HIV envelope protein contains the indicated amino acid residues in three of the indicated positions. In other preferred embodiments, the HIV envelope protein contains the indicated amino acid residues at four, five, six or all seven of the indicated positions.

Embodiments of HIV Env proteins having the indicated amino acids at multiple positions (positions are numbered according to the numbering in gp160 from the HIV-1 HXB2 isolate, followed by a single letter amino acid code for the residue present at that position, positions within one embodiment of the HIV Env protein separated by commas [e.g., an Env protein embodiment having Phe at position 651 and Ile at position 655 is described as 651F, 655I], with different embodiments (i.e., different HIV Env proteins) separated by a semicolon) include the following.

In the case of Env proteins with the indicated amino acids in two positions: 651F, 655I; 651F, 655F; 651F, 655M; 651F, 655W; 651F, 535N; 651F, 535Q; 651F, 589V; 651F, 589I; 651F, 589A; 651F, 573F; 651F, 573W; 651F, 204I; 651F, 647F; 651F, 647I; 651F, 647M; 651L, 655I; 651L, 655F; 651L, 655M; 651L, 655W; 651L, 535N; 651L, 535Q; 651L, 589V; 651L, 5891; 651L, 589A; 651L, 573F; 651L, 573W; 651L, 204I; 651L, 647F; 651L, 647I; 651L, 647M; 651M, 655I; 651M, 655F; 651M, 655A; 651M, 655L; 651M, 655W; 651M, 535N; 651M, 535Q; 651M, 589V; 651M, 589I; 651M, 589A; 651M, 573F; 651M, 573W; 651M, 204I; 651M, 647F; 651M, 647I; 651M, 647M; 651W, 655I; 651W, 655F; 651W, 655M; 651W, 655W; 651W, 535N; 651W, 535Q; 651W, 589V; 651W, 589I; 651W, 589A; 651W, 573F; 651W, 573W; 651W, 204I; 651W, 647F; 651W, 647I; 651W, 647M; 655I, 535N; 655I, 535Q; 655I, 589V; 655I, 589I; 655I, 589A; 655I, 573F; 655I, 573W; 655I, 204I; 655I, 647F; 655I, 647I; 655I, 647M; 655F, 535N; 655F, 535Q; 655F, 589V; 655F, 589I; 655F, 589A; 655F, 573F; 655F, 573W; 655F, 204I; 655F, 647F; 655F, 647I; 655F, 647M; 655M, 535N; 655M, 535Q; 655M, 589V; 655M, 589I; 655M, 589A; 655M, 573F; 655M, 573W; 655M, 204I; 655M, 647F; 655M, 647I; 655M, 647M; 655W, 535N; 655W, 535Q; 655W, 589V; 655W, 589I; 655W, 589A; 655W, 573F; 655W, 573W; 655W, 204I; 655W, 647F; 655W, 647I; 655W, 647M; 535N, 589V; 535N, 589I; 535N, 589A; 535N, 573F; 535N, 573W; 535N, 204I; 535N, 647F; 535N, 647I; 535N, 647M; 535Q, 589V; 535Q, 589I; 535Q, 589A; 535Q, 573F; 535Q, 573W; 535Q, 204I; 535Q, 647F; 535Q, 647I; 535Q, 647M; 589V, 573F; 589V, 573W; 589V, 204I; 589V, 647F; 589V, 647I; 589V, 647M; 589I, 573F; 589I, 573W; 589I, 204I; 589I, 647F; 589I, 647I; 589I, 647M; 589A, 573F; 589A, 573W; 589A, 204I; 589A, 647F; 589A, 647I; 589A, 647M; 573F, 204I; 573F, 647F; 573F, 647I; 573F, 647M; 573W, 204I; 573W, 647F; 573W, 647I; 573W, 647M; 204I, 647F; 204I, 647I; 201I, 647M. According to the present invention, each of these embodiments may be present in any HIV Env sequence, such as a wild-type isolate, or an HIV Env protein with SOSIP mutations, or an HIV Env consensus protein, or a synthetic HIV Env protein. Each of these embodiments can be combined with one of the preferred amino acids of the present invention at a third position selected from one of the remaining positions (i)(vii) of the present invention. Such embodiments having preferred amino acid residues at three positions from said positions (i)-(vii) may be combined with one of the preferred amino acids at the fourth position selected from one of the remaining indicated positions (i)-(vii) according to the present invention. . Such embodiments having preferred amino acid residues at four positions of said positions (i)(vii) can be combined with one of the preferred amino acids at the fifth position selected from one of the remaining positions (i)-(vii) of the present invention. Such embodiments having preferred amino acid residues at five positions from said positions (i)-(vii) may be combined with one of the preferred sixth position amino acids selected from one of the remaining indicated positions (i)-(vii) according to the present invention. . Such embodiments having preferred amino acid residues at six positions from said positions (i)-(vii) can be combined with one of the preferred amino acids at the seventh position selected from one of the remaining indicated positions (i)-(vii) according to the present invention. , whereby the Env protein has a preferred amino acid according to the present invention in all seven positions (i)-(vii) according to the present invention. Any of these additional embodiments having the preferred amino acid of the present invention at three, four, five, six, or seven of positions (v)-(vii) of the present invention may be present in any HIV Env protein, such as a protein from a wild type, SOSIP variant, HIV Env consensus protein, synthetic HIV Env protein, and the like.

Preferred Env proteins according to the present invention with the indicated amino acids in two positions are: 651F, 655I; 651F, 535N; 651F, 589V; 651F, 589I; 651F, 573F; 651F, 204I; 651F, 647F; 655I, 535N; 655I, 589V; 655I, 589I; 655I, 573F; 655I, 204I; 655I, 647F; 535N, 589V; 535N, 589I; 535N, 573F; 535N, 204I; 535N, 647F; 589V, 573F; 589V, 204I; 589V, 647F; 589I, 573F; 589I, 204I; 589I, 647F; 573F, 204I; 573F, 647F; 204I, 647F. Particularly preferred Env proteins having preferred amino acids in at least two positions according to the present invention include 651F, 655I; 655I, 535N; 655I, 589V; 535N, 589V; 535N, 647F.

Some preferred HIV Env proteins having preferred amino acid residues at three positions are 651F, 655I, 535N; 651F, 589V, 535N; 651F, 589I, 535N; 651F, 573F, 535N; 651F, 204I, 535N; 651F, 647F, 535N; 655I, 589V, 535N; 655I, 589I, 535N; 655I, 573F, 535N;

- 14 042607

655I, 204I, 535N; 655I, 647F, 535N; 589V, 573F, 535N; 589V, 204I, 535N; 589V, 647F, 535N; 589I, 573F, 535N; 589I, 204I, 535N; 589I, 647F, 535N; 573F, 204I, 535N; 573F, 647F, 535N; 204I, 647F, 535N; 651F, 655I, 589V; 651F, 573F, 589V; 651F, 204I, 589V; 651F, 647F, 589V; 655I, 573F, 589V; 655I, 204I, 589V; 655I, 647F, 589V; 573F, 204I, 589V; 573F, 647F, 589V; 204I, 647F, 589V; 651F, 655I, 589I; 651F, 573F, 589I; 651F, 204I, 589I; 651F, 647F, 589I; 655I, 573F, 589I; 655I, 204I, 589I; 655I, 647F, 589I; 573F, 204I, 589I; 573F, 647F, 589I; 204I, 647F, 589I; 651F, 655I, 573F; 651F, 204I, 573F; 651F, 647F, 573F; 655I, 204I, 573F; 655I, 647F, 573F; 204I, 647F, 573F; 651F, 655I, 204I; 651F, 647F, 204I; 655I, 647F, 204I; 651F, 655I, 647F; 655I, 651F, 647F; 655I, 651F, 535N; 655I, 589V, 573F; 655I, 589V, 204I. Particularly preferred Env proteins having preferred amino acids in at least three positions according to the present invention include 651F, 655I, 535N; 655I, 589V, 535N; 655I, 573F, 589V; 655I, 204I, 589V; 651F, 655I, 647F. Some preferred HIV Env proteins having preferred amino acid residues at four positions are 651F, 655I, 535N, 589V; 651F, 655I, 535N, 573F; 651F, 655I, 589V, 573F; 651F, 535N, 589V, 573F; 655I, 535N, 589V, 573F; 651F, 655I, 535N, 204I; 651F, 655I, 589V, 204I; 651F, 535N, 589V, 204I; 655I, 535N, 589V, 204I; 651F, 655I, 573F, 204I; 651F, 535N, 573F, 204I; 655I, 535N, 573F, 204I; 651F, 589V, 573F, 204I; 655I, 589V, 573F, 204I; 535N, 589V, 573F, 204I; 651F, 655I, 535N, 647F; 651F, 655I, 589V, 647F; 651F, 535N, 589V, 647F; 655I, 535N, 589V, 647F; 651F, 655I, 573F, 647F; 651F, 535N, 573F, 647F; 655I, 535N, 573F, 647F; 651F, 589V, 573F, 647F; 655I, 589V, 573F, 647F; 535N, 589V, 573F, 647F; 651F, 655I, 204I, 647F; 651F, 535N, 204I, 647F; 655I, 535N, 204I, 647F; 651F, 589V, 204I, 647F; 655I, 589V, 204I, 647F; 535N, 589V, 204I, 647F; 651F, 573F, 204I, 647F; 655I, 573F, 204I, 647F; 535N, 573F, 204I, 647F; and 589V, 573F, 204I, 647F.

Some examples of preferred HIV Env proteins having preferred amino acid residues in at least four positions include 651F, 655I, 647F, I535N; 651F, 655I, 573F, 589V. A preferred example of an HIV Env protein containing the indicated amino acid residues in at least four positions includes 535N, 589V, 651F, 655I. Non-limiting examples of such HIV Env proteins are provided under SEQ ID NOs: 20, 22, 24, 26,27, 28, 29, 30, 31, and 32. Preferably, such an HIV Env protein is a clade C HIV Env protein or a clade A HIV Env protein. , most preferably the clade C HIV Env protein. In certain embodiments, said HIV Env protein further comprises 588E, ie. it contains at least 535N, 588E, 589V, 651F, 655I. Non-limiting examples of such an HIV Env protein are provided under SEQ ID NOs: 20, 24, 26, 27, 28, 29, 30, 31, and 32. In certain embodiments, said HIV Env further comprises 556P, i. it contains at least 535N, 556P, 589V, 651F, 655I or at least 535N, 556P, 588E, 589V, 651F, 655I. Non-limiting examples of such an HIV Env protein are provided under SEQ ID NOS: 22, 24, 26, 27, 29, 30, 31 and 32.

In one embodiment, the recombinant HIV Env protein of the present invention comprises an amino acid sequence of the HIV Env protein having the indicated amino acid residues in at least two of the indicated positions selected from the group consisting of:

(i) Phe, Leu, Met or Tip at position 651;

(ii) Phe, Ile, Met or Tip at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Tip at position 573;

(vi) Ile at position 204; and (vii) Phe, Met, or Ile at position 647.

For example, a recombinant HIV Env protein may have one of Phe, Leu, Met, or Tip residues at position 651, and Asn or Gln at position 535, optionally additional specified amino acid residues at additional specified positions. Preferably, at least one of the amino acids in (i)-(vii) is introduced into the recombinant HIV Env protein via an amino acid substitution. For example, a recombinant HIV Env protein can be obtained from an HIV Env protein that does not contain or contains only one of the amino acid residues in (i)-(vii) above, whereby all or one or more of the at least two of these amino acid residues is introduced into recombinant HIV Env protein by amino acid substitution.

In certain embodiments, the recombinant HIV Env protein of the present invention further comprises (viii) Gln, Glu, Ile, Met, Val, Tip, or Phe at position 588, where Gln or Glu are preferred.

The amino acid sequence of the HIV Env protein to which the above-described substitutions are made can be any HIV Env protein known in the art with regard to the present disclosure, such as, for example, the naturally occurring HIV sequence from clade A, clade B, clade C, and etc.; mosaic sequence; a consensus sequence, for example, a consensus sequence from clade B or clade C; a synthetic sequence or any derivative or fragment thereof. In certain embodiments of the present invention, the amino acid sequence of the HIV Env protein contains additional mutations, such as, for example, so-called SOSIP mutations and/or a furin cleavage site mutation.

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In one specific embodiment, the HIV Env framework protein is an HIV Env protein with SOSIP mutations containing at least one mutation selected from the group consisting of Cys at positions 501 and 605; Pro at position 559. In a preferred embodiment, the HIV Env protein with SOSIP mutations contains Cys at positions 501 and 605 and Pro at position 559. In this embodiment, the recombinant HIV Env protein contains the amino acid sequence of the HIV Env protein with SOSIP mutations and an amino acid substitution amino acid residue at at least one of said positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp at position 651;

(ii) Phe, Ile, Met or Trp at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Trp at position 573;

(vi) Ile at position 204; and (vii) Phe, Met, or Ile at position 647.

the HIV Env protein with SOSIP mutations may further comprise a furin cleavage site mutation, such as a substitution at positions 608-511 of SEQ ID NO: 10.

In another embodiment, the recombinant HIV Env protein of the present invention comprises the amino acid sequence of the HIV Env protein and an amino acid substitution for the specified amino acid residue in at least one of the specified positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp at position 651;

(ii) Phe, Ile, Met or Trp at position 655;

(iii) Asn or Gln at position 535;

(iv) Val, Ile or Ala at position 589;

(v) Phe or Trp at position 573;

(vi) Ile at position 204; and (vii) Phe, Met, or Ile at position 647, wherein the HIV Env protein is selected from the group consisting of:

(1) an HIV Env consensus sequence, such as a consensus sequence from clade C or clade B, for example, comprising the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5;

(2) a synthetic HIV Env protein, for example, comprising the amino acid sequence of (a) SEQ ID NO: 6; (b) SEQ ID NO: 6 with a Glu to Arg mutation at position 166, (c) SEQ ID NO: 7; (d) SEQ ID NO: 8 or 9, wherein (a), (b) or (d) optionally have additional SOSIP mutations and/or a furin cleavage site mutation as described above.

Preferably, the recombinant HIV Env protein comprises the amino acid sequence of the HIV Env protein and an amino acid substitution for said amino acid residue at at least two of said positions selected from the group consisting of (i)-(vii) above, such as, for example, two positions or three positions. However, the recombinant HIV Env protein may contain an amino acid substitution for the indicated amino acid residue at one or more of the indicated positions, for example, at one, two, three, four, five, six or seven of the indicated positions.

In one specific embodiment, the HIV Env framework protein is an HIV Env consensus sequence from clade C comprising an amino acid sequence that is at least 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence under SEQ ID NO: 2. Preferably, the HIV consensus sequence from clade C under SEQ ID NO: 2 further contains so-called SOSIP mutations, ie. Cys at positions 501 and 605 and Pro at position 559 and more preferably further comprises so-called SOSIP mutations and a furin cleavage site mutation such as, for example, a substitution at positions 508-511 of SEQ ID NO: 10. In a particularly preferred embodiment the HIV Env framework protein contains the sequence shown under SEQ ID NO: 3, or a sequence that is at least 95% identical to it, where preferably the amino acids at positions 501, 559, 605 and 508-511 are replaced by SEQ ID NO: 10 are not mutated compared to SEQ ID NO: 3.

In another specific embodiment, the HIV Env framework protein is an HIV Env consensus sequence from clade B containing an amino acid sequence that is at least 95%, 96, 97, 98, 99, or 100% identical to the amino acid sequence under SEQ ID NO: 4. Preferably, the HIV consensus sequence from clade B under SEQ ID NO: 4 further contains so-called SOSIP mutations, ie. Cys at positions 501 and 605 and Pro at position 559 and more preferably further comprises so-called SOSIP mutations and a furin cleavage site mutation such as, for example, a substitution at positions 508-511 of SEQ ID NO: 10. In a particularly preferred embodiment the HIV Env framework protein contains the sequence shown under SEQ ID NO: 5 or a sequence that is at least

95% identical to it, where preferably the amino acids at positions 501, 559, 605 and 508-511 substituted for SEQ ID NO: 10 are not mutated compared to SEQ ID NO: 5.

In yet another specific embodiment, the HIV Env framework protein is a synthetic HIV Env protein, for example, comprising the amino acid sequence of (a) SEQ ID NO: 6; (b) SEQ ID NO: 6 with a Glu to Arg mutation at position 166, (c) SEQ ID NO: 7; or (d) SEQ ID NO: 8 or 9, wherein (a), (b) or (d) optionally have additional SOSIP mutations (501C, 605C, 559P) and/or a furin cleavage site mutation (508-511RRRRRR), described above.

In still other specific embodiments, the HIV Env framework protein is an HIV Env protein from a wild-type HIV virus from clade A or clade C, optionally containing mutations to restore the sequence according to the methods described herein.

Exemplary combinations of two positions in the HIV Env protein at which substitutions can be made simultaneously include residues 535,589; 535.647; and 589.655; as, for example, in double mutants, I535N, D589V; I535N, E647F; and D589V, K655I. Other double mutants include K655I, I535N; N651F, K655I and K655I, I573F. An exemplary combination of three positions in the HIV Env protein at which substitutions can be made simultaneously includes 535,589,655, such as in the triple mutant, I535N, D589V, K655I. Other triple mutants include K655I, D589V, I573F and K655I, N651F, I535N.

In certain embodiments of the present invention, the recombinant HIV Env protein of the present invention may further comprise the indicated amino acid residue (e.g., obtained by substitution) at one or more additional indicated positions selected from the group consisting of positions (viii) 588, (ix) 64 or 66, (x) 316, (xi) 201/433, (xii) 556, or 558, or 556 and 558, (xiii) 548-568, (xiv) 568, 569 and 636, or (xv) 302 , 519 or 520 shown in table. 2 below. The present inventors have found that some of these amino acid substitutions (eg (viii)) combine very well with the mutations (mutation combinations) (i)-(vii) of the present invention described above. The rest of these amino acid substitutions have previously been described in the literature. For example, in De Taeye et al. (Cell (2015) 163(7), 1702-15) describe an HIV envelope protein having the E64K and T316W double mutation and an HIV Env protein having the 66R mutation; and in Kwon et al. (Nat. Struct. Mol. Biol. (2015) 22(7) 522-31) describes an HIV envelope protein having a disulfide substitution I204C, A433C; and in Guenaga et al. (Immunity (2017) 46, 792-803) describes an HIV envelope protein having L568G, T569G or N636G and a triple substitution N302Y, F519R, L520R. However, to the best of the present inventors' knowledge, these previously described mutations have not been described in combination with any of the novel substitutions described herein, such as those listed in Table 1. 1. These amino acid mutations, in combination with the amino acid substitutions of the present invention, can further increase trimer yield and/or percentage of trimer formation. These amino acid substitutions can be introduced into any of the recombinant HIV Env proteins described herein, in addition to a substitution for the specified amino acid residue at one or more of the indicated positions described in Table 1. 1.

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Table 2. Additional amino acid substitution positions and substitution residue

No. Position ¹ Specified amino acid remainder (viii) 588 Gin, Glu, He, Met, Vai, Trp or Phe (preferably Gin or Glu) (ix) 6 4 or 6 6 Lys at position 64 or Arg at position 66 (X) 316 trp (xi) 201 and 433 Cys in both positions (xi) 556 or 556 and 558 558 or Pro in any position or both (xiii) 548-568 a loop) (HR1- Substitution with a shorter and less flexible loop having 710 amino acids, preferably an 8 amino acid loop, e.g. having a sequence selected from any of (SEQ ID NOs: 12-17) (xiv) 568, 569, 636 Gly at any of these positions, or Gly at both positions 568 and 636, or Gly at both positions 569 and 636 (xv) 302, 519 520 Arg at position 302, or Arg at position 519, or Arg at position 520, or Arg at position 302 and Arg at position 519, or Arg at position 302 and Arg at position 520, or Arg at position 302 and Arg at position both provisions 519 and 520.

1According to numbering in gp160 from HIV-1 HXB2 isolate

Substitutions identified in these provisions of the present invention [(i)-(vii), see for example, table. 1], are not or rarely present in natural sequences, are not found in combination with previously described HIV Env protein sequences, and have not been previously thought to result in increased trimerization of the HIV Env protein, increased trimer yield, and/or improved trimer stability. All mutations (ix)-(xi) in Table. 2 (which have been previously described by other researchers) are in the region of gp120, which binds trimer-specific antibody PGT145. These mutations provide a closed trimer state at the apex (which is the top of the molecule). All substitutions (xii) and (xiii) are in HR1 gp41. With the exception of position 204, all mutations of the present invention in the table. 1 are located in the gp41 region (at the bottom of the molecule), but outside the HR1 region. It is clear that the previously described mutations did not give rise to any suggestion about the introduction of mutations of the present invention, not to mention their unexpected effects in relation to the formation of closed-top trimers, measured using PGT145 binding. In addition to point mutations (viii)-(xii) from Table. 2, it is also possible to replace the HR1 loop of the Env protein (amino acid residues 548-568 in wild-type sequence numbered according to gp160 from isolate HXB2) with a shorter and less flexible loop having 7-10 amino acids, preferably an 8 amino acid loop, e.g. a sequence selected from any of (SEQ ID NOs: 12-17), see e.g. Kong et al (Nat Commun. 2016 Jun 28; 7:12040. doi: 10.1038/ncomms12040) which describes such shorter loops replacing loop HR1. Such an Env variant further having said amino acid residues in at least one and preferably at least two of said positions (i)-(vii) according to the present invention is also an embodiment of the present invention. In certain embodiments of the present invention, the mutations listed in (viii)-(xiii) can be added to the HIV Env proteins of the present invention, i.e. having one or more of said amino acids at positions (i)-(vii). It is also possible to obtain combinations within groups (viii)-(xiii), with a non-limiting example being a combination of mutations (in addition to

- 18 042607 at least one mutation from (i)-(vii)) to (viii) and (xii) (eg I535N, A556P, K588E). Some non-limiting examples of double mutants that are derived from the HIV Env with SOSIP mutations and by combining mutations in at least one of positions (i)-(vii) and at least one of positions (viii)-(xiii), include: 535,588; 588.589; 655.588; 558.535 and 655.556; such as, for example, I535N, K588E; 5880, D589V; K655I, K588E; A558P, I535N and K655I, L556P. Some non-limiting examples of such triple mutants include 558,535,588; 558,535,589; 558,535,655 and 558,535,651 such as, for example, A558P, I535N, K588E; A558P, I535, D589V; A558P, I535N, K655I and A558P, I535N, N651F.

Additional non-limiting examples of combinations according to the present invention include 655I, 573F, 589V, 588E; 651F, 655I, 573F, 589V, 588E; 651F, 655I, 573F, 589V, 588E, 535N; 651F, 655I, 573F, 589V, 588E, 535N, 204I; 651F, 655I, 556P; 651F, 535N, 556P; 651F, 589V, 556P; 651F, 589I, 556P; 651F, 573F, 556P; 651F, 204I, 556P; 651F, 647F, 556P; 655I, 535N, 556P; 655I, 589V, 556P; 655I, 589I, 556P; 655I, 573F, 556P; 655I, 204I, 556P; 655I, 647F, 556P; 535N, 589V, 556P; 535N, 589I, 556P; 535N, 573F, 556P; 535N, 204I, 556P; 535N, 647F, 556P; 589V, 573F, 556P; 589V, 204I, 556P; 589V, 647F, 556P; 589I, 573F, 556P; 589I, 204I, 556P; 589I, 647F, 556P; 573F, 204I, 556P; 573F, 647F, 556P; 651F, 655I, 558P; 651F, 535N, 558P; 651F, 589V, 558P; 651F, 589I, 558P; 651F, 573F, 558P; 651F, 204I, 558P; 651F, 647F, 558P; 655I, 535N, 558P; 655I, 589V, 558P; 655I, 589I, 558P; 655I, 573F, 558P; 655I, 204I, 558P; 655I, 647F, 558P; 535N, 589V, 558P; 535N, 589I, 558P; 535N, 573F, 558P; 535N, 204I, 558P; 535N, 647F, 558P; 589V, 573F, 558P; 589V, 204I, 558P; 589V, 647F, 558P; 589I, 573F, 558P; 589I, 204I, 558P; 589I, 647F, 558P; 573F, 204I, 558P; 573F, 647F, 558P; 655I, 589V, 535N, 556P; 651F, 655I, 535N, 556P; 556P, 651F; 556P, 651F,

655I, 535N; 655I, 589V, 573F, 651F, 588E, 556P; 556P, 651F, 655I, 535N, 573F; 556P, 651F, 655I, 535N,

573F, 589V; 556P, 651F, 655I, 535N, 573F, 589V, 204I; 556P, 651F, 655I, 535N, 573F, 589V, 204I, 588Q;

556P, 651F, 655I, 535N, 573F, 589V, 204I, 588Q, 647F; 556P, 651F, 535N, 573F; 556P, 651F, 535N, 573F,

589V; 556P, 651F, 535N, 573F, 589V, 204I; 556P, 651F, 535N, 573F, 589V, 204I, 588Q; 556P, 651F, 535N, 573F, 589V, 204I, 588Q, 647F; 556P, 655I, 535N, 573F; 556P, 655I, 535N, 573F, 589V; 556P, 655I, 535N, 573F, 589V, 204I; 556P, 655I, 535N, 573F, 589V, 204I, 588Q; 556P, 655I, 535N, 573F, 589V, 204I, 588Q, 647F. In turn, any of these embodiments may be contained in any HIV Env protein, for example, a wild-type isolate, an Env consensus sequence, a synthetic Env protein, an Env protein with SOSIP mutations, a wild-type isolate containing repair mutations according to the concept described herein. document, etc. Some preferred combinations according to the present invention include 655I, 589V, 573F, 651F, 588E, 535N, 204I; 556P, 655I, 535N, 573F, 589V, 204I, 588Q; 204I, 535N, 556P, 588E, 589V, 651F, 655I; 535N, 556P, 589V, 651F, 655I and 535N, 556P, 588E, 589V, 651F, 655I.

In certain preferred embodiments, the HIV Env protein contains a sequence that is at least 95% identical, preferably at least 96%, 97, 98, 99% identical, preferably 100% identical to any of SEQ ID NOs: 20, 22, 24, 26, 27, 28, 29, 30, 31 and 32. Positions (i)(xv) of Tables 1 and 2 are preferably ignored for determining % identity, and positions 501, 559 and 605 are also preferred. Preferably amino acids residues at these positions are residues from the sequences under SEQ ID NO: 20, 22, 24, 26, 27, 28, 29, 30, 31 or 32, respectively.

In certain embodiments, the HIV Env protein of the present invention further comprises: (xvi) an amino acid residue selected from Val, Ile, Phe, Met, Ala, or Leu at position 658. Preferably, the amino acid at position 658 is Val or Ile, most preferably Val . It was found to greatly increase the trimer percentage and trimer yield of the Env protein, either alone or in combination with mutations selected from (i)-(vii) Table. 1 and/or (viii)-(xv) tab. 2 described in this document.

According to embodiments of the present invention, the recombinant HIV Env protein is characterized by at least one of (a) an increased percentage of trimer formation and (b) an increased trimer yield compared to an HIV Env protein lacking said amino acid residues at one or more of the 651 positions. , 655, 535, 589, 573, 204 and 647, shown in the table. 1.

As used herein, an increased percentage of trimer formation means that a greater percentage of trimer is formed when the framework sequence of the HIV envelope protein contains one or more of the amino acid substitutions of the present invention, compared to the percentage of trimer obtained when the framework sequence of the HIV envelope protein The HIV envelope protein contains no such amino acid substitutions. As used herein, the increased yield of trimers means that more total trimer form is obtained when the framework sequence of the HIV envelope protein contains one or more of the amino acid substitutions of the present invention, compared to the total amount of trimer form of the envelope protein obtained when the framework sequence the sequence of the HIV envelope protein sequence does not contain such amino acid substitutions.

Trimer formation can be measured by antibody binding assay using

- 19 042607 antibodies that specifically bind to the trimeric form of the HIV Env protein. Examples of trimer-specific antibodies that can be used to detect the trimer form include, but are not limited to, the monoclonal antibodies (mAbs) PGT145, PGDM1400, PG16, and PGT151. Preferably, the trimer-specific antibody is a PGT145 mAb. Any antibody binding assay known in the art in view of the present disclosure can be used to measure the percentage trimer formation of the recombinant HIV Env protein of the present invention, such as ELISA, AlphaLISA, etc.

In a specific embodiment, trimer formation is measured using AlphaLISA. AlphaLISA is a bead-based proximity assay in which singlet oxygen molecules generated by high energy radiation from donor beads are transferred to acceptor beads that are located within a distance of approximately 200 nm from the donor beads. The transfer of singlet oxygen molecules to the acceptor beads initiates a cascade of a series of chemical reactions leading to the generation of a chemiluminescent signal that can then be detected (Eglen et al. Curr. Chem. Genomics, 2008, 25(1):2-10). For example, recombinant HIV envelope proteins labeled with a Flag-His tag can be incubated with a trimer-specific mAb, donor beads conjugated with an antibody that binds to the trimer-specific mAb, nickel-conjugated donor beads, acceptor beads conjugated with an antibody to His- tag, and acceptor beads conjugated with an antibody to the Flag tag. The amount of trimer formed can be determined by measuring the chemiluminescent signal generated by a pair of donor beads conjugated to an antibody that binds to a trimer-specific mAb and acceptor beads conjugated to an anti-His tag antibody. The total amount of HIV envelope protein expressed can be determined by measuring the chemiluminescent signal generated by a pair of nickel-conjugated donor beads and anti-Flag tag antibody-acceptor beads. For example, the amount of trimer and total coat protein expressed can be measured using the AlphaLISA assay, as detailed in Example 3. The percentage of trimer formation can be calculated by dividing the amount of trimer formed by the total amount of coat protein expressed.

The amount of trimer formed and the total amount of envelope protein expressed can also be determined using chromatographic techniques that are capable of separating the trimer form from other forms of the HIV envelope protein, such as the monomer form. Examples of such techniques that can be used include, but are not limited to, size exclusion chromatography, multi-angle light scattering (SEC-MALS). In certain embodiments, the percentage of trimer formation is determined using SEC-MALS. In certain embodiments, trimer yields are determined using SEC-MALS.

Nucleic acid, vectors and cells

In another general aspect, the present invention provides a nucleic acid molecule encoding a recombinant HIV Env protein according to the present invention, and a vector containing this nucleic acid molecule. The nucleic acid molecules of the present invention may be in the form of RNA or in the form of DNA obtained by cloning or obtained synthetically. DNA can be double stranded or single stranded. DNA, for example, may include cDNA, genomic DNA, or combinations thereof. Nucleic acid molecules and vectors can be used to generate recombinant proteins, express a protein in a host cell, or generate viral particles.

According to embodiments of the present invention, the nucleic acid encoding the recombinant HIV envelope protein is operably linked to a promoter, meaning that the nucleic acid is under the control of the promoter. The promoter may be a homologous promoter (ie, derived from the same genetic source as the vector) or a heterologous promoter (ie, derived from a different vector or genetic source). Examples of suitable promoters include the human cytomegalovirus immediate early promoter (hCMV IE, or CMV for short) and the Rous sarcoma virus (RSV) promoter. The promoter is preferably located 5' from the given nucleic acid in the expression cassette.

According to embodiments of the present invention, the vector may be an expression vector. Expression vectors include, but are not limited to, vectors for expressing a recombinant protein and vectors for delivering a nucleic acid to a subject's body for expression in the subject's tissue, such as a viral vector. Examples of viral vectors suitable for use in the present invention include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, modified vaccinia Ankara (MVA) vectors, enterovirus vectors, Venezuelan equine encephalitis virus vectors, Semliki forest virus vectors, tobacco mosaic virus vectors, lentiviral vectors, etc. The vector may also be a non-viral vector. Examples of non-viral vectors include, but are not limited to, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, and the like.

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In certain embodiments, the implementation of the present invention, the vector is an adenoviral vector, for example, a recombinant adenoviral vector. For example, the recombinant adenoviral vector may be derived from a human adenovirus (HAdV or AdHu) or a simian adenovirus such as a chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV) or a macacarhesus adenovirus (rhAd). Preferably, the adenoviral vector is a recombinant human adenovirus vector, for example, recombinant human adenovirus serotype 26 or any of the recombinant human adenovirus serotypes 5, 4, 35, 7, 48, etc. In other embodiments, the adenoviral vector is a rhAd vector, such as rhAd51, rhAd52, or rhAd53.

The production of recombinant adenoviral vectors is well known in the art. For example, the production of vectors based on recombinant adenovirus 26 is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol. 81(9): 4654-63. Exemplary adenovirus 26 genomic sequences are found in GenBank under accession number EF 153474 and under SEQ ID NO: 1 in WO 2007/104792. Illustrative genomic sequences of rhAd51, rhAd52 and rhAd53 are provided in US 2015/0291935.

According to embodiments of the present invention, any of the recombinant HIV Env proteins described herein may be expressed and/or encoded using any of the vectors described herein. Given the degeneracy of the genetic code, one skilled in the art is well aware that it is possible to construct multiple nucleic acid sequences that encode the same protein, according to methods that are fully consistent with established practice in this field. The nucleic acid encoding the recombinant HIV Env protein of the present invention may optionally be codon optimized to ensure proper expression in the host cell (eg, bacterial or mammalian cells). Codon optimization is a technology widely used in the art.

The present invention also provides cells, preferably isolated cells, containing any of the nucleic acid molecules and vectors described herein. For example, the cells can be used to produce a recombinant protein or to produce viral particles.

Thus, embodiments of the present invention also relate to a method for producing a recombinant HIV Env protein. The method involves transfecting a host cell with an expression vector containing a nucleic acid that encodes a recombinant HIV Env protein according to an embodiment of the present invention operably linked to a promoter, growing the transfected cell under conditions suitable for expressing the recombinant HIV Env protein, and optionally purifying or isolating the recombinant the HIV Env protein expressed in the cell. The recombinant HIV Env protein can be isolated or assembled from a cell using any method known in the art, including affinity chromatography, size exclusion chromatography, and the like. The techniques used to express the recombinant protein will be well known to those of ordinary skill in the art in light of the present disclosure. The expressed recombinant HIV Env protein can also be assayed without purification or isolation of the expressed protein, for example, by analyzing the supernatant of cells transfected with an expression vector encoding the recombinant HIV Env protein and grown under conditions suitable for expression of the HIV Env protein.

In a preferred embodiment, the expressed recombinant HIV Env protein is purified under conditions that assemble the protein such that a stabilized trimeric complex is formed. For example, mammalian cells transfected with an expression vector encoding a recombinant HIV Env protein operably linked to a promoter (e.g., the CMV promoter) can be cultured at 33-39°C, e.g., 37°C, and 2-12% CO ₂ , for example 8% CO ₂ . Expression can also be carried out in alternative expression systems such as insect cells or yeast cells, all of which are conventional in the art. The expressed HIV Env protein can then be isolated from the cell culture using, for example, affinity chromatography using a lectin that binds glycoproteins. The HIV Env protein bound to the column can be eluted with mannopyranoside. If necessary, the HIV Env protein eluted from the column can be subjected to additional purification steps, such as size exclusion chromatography, to remove any residual impurities, such as cellular impurities, as well as Env aggregates, gp140 monomers, and gp120 monomers. Alternative purification methods can also be used to isolate the expressed HIV Env protein, with non-limiting examples including antibody affinity chromatography, negative screening with antibodies other than bNAb, purification with a label antibody, or other chromatographic methods such as ion exchange chromatography and etc., as well as other methods known from the prior art.

Nucleic acid molecules and expression vectors encoding recombinant Env proteins

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The HIV of the present invention can be obtained using any method known in the art in view of the present disclosure. For example, a nucleic acid encoding a recombinant HIV Env protein can be obtained by introducing at least one of the amino acid substitutions at the indicated positions in the framework sequence of the HIV envelope protein using genetic engineering techniques and molecular biology techniques, for example, site-directed mutagenesis, polymerase chain reactions (PCR), etc., which are well known to those skilled in the art. The nucleic acid molecule can then be introduced or cloned into an expression vector, also using standard molecular biology techniques. The recombinant HIV envelope protein can then be expressed from the expression vector in the host cell and the expressed protein purified from the cell culture by any method known in the art given the present invention.

Trimer complex

In another general aspect, the present invention provides a trimeric complex comprising a non-covalently linked oligomer of three recombinant HIV Env proteins of the present invention. The trimeric complex may contain any of the recombinant HIV Env proteins described herein. Preferably, the trimeric complex contains three identical monomers (or three identical heterodimers if gp140 is cleaved) of the recombinant HIV Env proteins of the present invention. The trimeric complex may be separated from other forms of the HIV envelope protein, such as the monomeric form, or the trimeric complex may be present along with other forms of the HIV envelope protein, such as the monomeric form.

Compositions and Methods

In another general aspect, the present invention provides a composition comprising a recombinant HIV Env protein, a trimeric complex, an isolated nucleic acid, a host vector or cell, and a pharmaceutically acceptable carrier. The composition may contain any of the recombinant HIV Env proteins, trimeric complexes, isolated nucleic acid molecules, vectors, or host cells described herein.

The carrier may include one or more pharmaceutically acceptable excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavoring agents, sweeteners, preservatives, colorants, solubilizers, and coatings. The exact nature of the carrier or other material may depend on the route of administration, such as intramuscular, intradermal, subcutaneous, oral, intravenous, transdermal, transmucosal (eg, in the intestine), intranasal, or intraperitoneal routes. In the case of liquid injectables, such as suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents, and the like. In the case of solid preparations for oral administration, such as powders, capsules, caplets, gelatin capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants, and the like. In the case of nasal sprays/mixtures for inhalation, the aqueous solution/suspension may contain water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, fragrances, flavors, and the like. as suitable carriers and additives.

To facilitate administration and increase efficacy, the compositions of the present invention may be formulated in any form suitable for administration to a subject, including, without limitation, oral (enteral) and parenteral injection forms. Parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection and intramuscular injection. The compositions of the present invention may also be formulated for other routes of administration, including transmucosal, ocular, rectal, long-acting implant, sublingual, sublingual, transmucosal, bypassing the portal circulation, inhalation or intranasal introductions.

Embodiments of the present invention also relate to methods for preparing the composition. According to embodiments of the present invention, a method for preparing a composition comprises mixing a recombinant HIV Env protein, trimeric complex, isolated nucleic acid, vector, or host cell of the present invention with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be well aware of the conventional techniques used to prepare such compositions.

HIV antigens (e.g., proteins or fragments thereof derived from HIV gag, pol, and/or env gene products) and vectors, such as viral vectors, expressing HIV antigens have previously been used in immunogenic compositions and vaccines to vaccinate a subject against an infection caused by HIV, or to elicit an immune response in a subject to an infection caused by HIV. As used herein, a subject means any animal, preferably a mammal, most preferably a human, to which the immunogenic composition will be or has been administered.

- 22 042607 according to embodiments of the present invention. As used herein, the term mammal encompasses any mammal. Examples of mammals include, without limitation, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., preferably humans. The recombinant HIV Env proteins of the present invention can also be used as antigens to induce an immune response against human immunodeficiency virus (HIV) in a subject in need thereof. An immune response may be raised against one or more HIV clades, such as clade A, clade B, clade C, and so on. The compositions may contain a vector from which the recombinant HIV Env protein is expressed, or the composition may contain an isolated recombinant HIV Env protein according to an embodiment of the present invention.

For example, compositions containing a recombinant HIV protein or trimeric complex thereof can be administered to a subject in need thereof to induce an immune response in the subject against infection caused by HIV. A composition containing a vector, such as an adenoviral vector encoding a recombinant HIV Env protein of the present invention, wherein the recombinant HIV Env protein is expressed by the vector, can also be administered to a subject in need thereof to induce an immune response in the subject against infection caused by HIV. The methods described herein also include administering a composition of the present invention in combination with one or more additional HIV antigens (e.g., proteins or fragments thereof derived from HIV gag, pol, and/or env gene products) that are preferably expressed from one or more vectors, such as adenoviral or MVA-based vectors, including methods for priming and enhancing an immune response.

In certain embodiments, the HIV Env protein may be presented on a particle, such as a liposome, virus-like particle (VLP), nanoparticle, virosome, or exosome, optionally in combination with endogenous and/or exogenous adjuvants. Compared to the soluble or monomeric Env protein itself, such particles generally exhibit an increased efficiency of antigen presentation in vivo.

Examples of VLPs that represent the HIV Env protein can be generated, for example, by co-expressing the HIV Env protein with self-assembling viral proteins such as HIV core Gag proteins or Gag proteins of other retroviruses. VLPs are similar to viruses but are not infectious because they do not contain viral genetic material. Expression of viral structural proteins, such as the envelope or capsid, can lead to self-assembly of the VLP. VLPs are well known to the skilled person and their use in vaccines is described, for example, in (Kushnir et al, 2012).

In certain preferred embodiments, the particle is a liposome. A liposome is a spherical vesicle having at least one lipid bilayer. For example, HIV Env trimeric proteins can be non-covalently linked to such liposomes by electrostatic interactions, for example, by adding a His tag to the C-terminus of the HIV Env trimer and introducing a bivalent chelating atom, such as Ni ²⁺ or Co ²⁺ , into the head group. derivatized lipids in the liposome. In certain non-limiting and illustrative embodiments, the liposome contains 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dioleoyl-sn-glycero-3-[(N-(5 -amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA(Ni ²⁺ ) or DGS-NTA(Co ²⁺ )) in a molar ratio of 60:36:4. In preferred embodiments, the HIV Env trimeric proteins are covalently attached to the surface of the liposome, for example, via a maleimide functional group integrated into the surface of the liposome. In certain non-limiting exemplary embodiments, the liposome contains DSPC, cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexanecarboxamide]lipid in a molar ratio of 54:30:16. The HIV Env protein can be linked to it, for example, through an added C-terminal cysteine in the HIV Env protein. Covalently bound variants are more stable, produce high titers of antigen-specific IgG, and epitopes on the less antigenically relevant lower part of the Env trimer are masked. Methods for preparing liposome-bound HIV Env trimers and their characterization are known and have been described, for example, in (Bale et al, 2017), incorporated herein by reference. The present invention also provides the HIV Env protein of the present invention fused to and/or presented on a liposome.

In certain embodiments, the HIV Env protein of the present invention is fused to self-assembling particles or presented on nanoparticles. Antigenic nanoparticles are assemblies of polypeptides that present multiple copies of antigens, such as the HIV Env protein of the present invention, resulting in multiple binding sites (avidity) and may provide improved antigen stability and immunogenicity. The preparation and use of self-assembling protein nanoparticles for use in vaccines is well known to those skilled in the art, see for example (Zhao et al., 2014), (Lopez-Sagaseta et al., 2016). As non-limiting examples, self-assembling nanoparticles can be based on ferritin, bacterioferritin, or DPS. DPS-based nanoparticles presenting proteins on their surface are described, for example, in WO 2011/082087. The description of trimeric HIV-1 antigens on such particles has been described, for example, in (He et al., 2016).

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Other self-assembling protein nanoparticles, as well as their production, are disclosed, for example, in WO

2014/124301 and US 2016/0122392, incorporated herein by reference. The present invention also provides an HIV Env protein of the present invention fused to and/or presented on a self-assembling nanoparticle. The present invention also provides compositions containing VLPs, liposomes or self-assembling nanoparticles according to the present invention.

In certain embodiments, an adjuvant is included in a composition of the present invention or co-administered with a composition of the present invention. The use of an adjuvant is optional and may further enhance immune responses when the composition is used for vaccination purposes. Suitable adjuvants for co-administration or incorporation into the compositions of the present invention should preferably be those that are potentially safe, well tolerated and effective when used in humans. Such adjuvants are well known to the skilled artisan and non-limiting examples include QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, Valetin, MPC-026, Adjuvax, CpG ODN, betafectin, aluminum salts such as aluminum phosphate (eg AdjuPhos) or aluminum hydroxide, and MF59.

Other aspects of the present invention relate to recombinant HIV envelope proteins containing an amino acid sequence that is at least 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence under SEQ ID NO: 2 or SEQ ID NO: 4, which represent the HIV clade C envelope protein consensus sequence and the HIV clade B envelope protein consensus sequence, respectively. These consensus sequences have not been found in any naturally occurring sequences and thus are believed to be novel HIV envelope proteins. A recombinant HIV envelope protein containing an amino acid sequence that is at least 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence under SEQ ID NO: 2 or SEQ ID NO: 4 may optionally further contain so-called SOSIP mutations and/or a mutation at a furin cleavage site, such as, for example, in the sequences shown under SEQ ID NO: 3, or SEQ ID NO: 3, further containing Pro at position 558 and/or position 556; and SEQ ID NO: 5 or SEQ ID NO: 5 further comprising Pro at position 558 and/or position 556. When % identity is determined for these sequences, amino acids at the mutated furin cleavage site and at positions 501, 605, 559, 556 and 558 are preferably ignored. Surprisingly, it was found that such proteins are expressed at high levels and are characterized by a high level of stability and formation of trimers. In certain embodiments, such HIV Env proteins can be used as scaffold proteins that can be mutated as described above to produce a molecule of the present invention. Isolated nucleic acid molecules encoding these sequences, vectors containing these sequences operably linked to a promoter, and compositions containing a protein, an isolated nucleic acid molecule, or a vector are also contemplated by the present invention.

Embodiments

Embodiment 1 is a recombinant HIV Env protein comprising the amino acid sequence of the HIV Env protein having the indicated amino acid residues in at least two of the indicated positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp, preferably Phe, at position 651;

(ii) Phe, Ile, Met or Tip, preferably Ile, at position 655;

(iii) Asn or Gln, preferably Asn, at position 535;

(iv) Val, Ile or Ala, preferably Val or Ile, at position 589;

(v) Phe or Trp, preferably Phe, at position 573;

(vi) Ile at position 204; and (vii) Phe, Met or Ile, preferably Phe, at position 647, where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate.

Embodiment 2 is a recombinant HIV Env protein comprising the amino acid sequence of the HIV Env protein and an amino acid substitution for the specified amino acid residue at at least one of the specified positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp, preferably Phe, at position 651;

(ii) Phe, Ile, Met or Trp, preferably Ile, at position 655;

(iii) Asn or Gln, preferably Asn, at position 535;

(iv) Val, Ile or Ala, preferably Val or Ile, at position 589;

(v) Phe or Trp, preferably Phe, at position 573;

(vi) Ile at position 204; and (vii) Phe, Met or He, preferably Phe, at position 647, wherein the HIV Env protein is selected from the group consisting of:

(1) an HIV Env protein having a consensus sequence, eg, from clade C or from clade B, eg, having an amino acid sequence under (SEQ ID NO: 2, 3, 4, or 5); or

- 24 042607 (2) synthetic protein Env HIV, for example, containing the amino acid sequence under (a) SEQ ID NO: 6; (b) SEQ ID NO: 6 with Glu to Arg mutation at position 166, (c) SEQ

ID NO: 7; or (d) SEQ ID NO: 8 or SEQ ID NO: 9, where (a), (b) or (d) may optionally additionally have SOSIP mutations (Cys at positions 501 and 605 and Pro at position 559) and/or furin cleavage site mutations (eg, substitution for SEQ ID NO: 10 amino acids 508-511); or (3) a wild-type HIV Env protein, preferably from clade C, containing at least one amino acid residue reducing mutation that is present at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of the HIV Env sequences in the population of at least 100, preferably at least 1,000, preferably at least 10,000 wild-type HIV Env sequences, where the repair mutation is a substitution for an amino acid residue that is present at the corresponding position at a frequency of at least 10% of the HIV Env sequences in said population, and preferably the reducing mutation is a substitution for an amino acid residue that is most frequently present at the corresponding position in said population;

where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate.

Embodiment 3 is a recombinant HIV Env protein comprising the amino acid sequence of the HIV Env protein and an amino acid substitution for the specified amino acid residue at at least one of the specified positions selected from the group consisting of:

(i) Phe, Leu, Met or Trp, preferably Phe, at position 651;

(ii) Phe, Ile, Met or Trp, preferably Ile, at position 655;

(iii) Asn or Gln, preferably Asn, at position 535;

(iv) Val, Ile or Ala, preferably Val or Ile, at position 589;

(v) Phe or Trp, preferably Phe, at position 573;

(vi) Ile at position 204; and (vii) Phe, Met or Ile, preferably Phe, at position 647, wherein the HIV Env protein is an HIV Env protein with SOSIP mutations containing at least one mutation selected from the group consisting of:

(a) Cys in regulations 501 and 605;

(b) Pro at position 559;

(c) Cys at positions 501 and 605 and Pro at position 559, with the position numbering corresponding to that in gp160 from the HIV-1 HXB2 isolate.

Embodiment 4 is a recombinant HIV Env protein according to Embodiment 2, comprising the indicated amino acid residues in at least two of the indicated positions selected from the group consisting of (i)-(vii).

Embodiment 5 is a recombinant HIV Env protein according to Embodiment 3 comprising the indicated amino acid residues at at least two of the indicated positions selected from the group consisting of (i)-(vii).

Embodiment 6 is a recombinant HIV Env protein according to any one of Embodiments 1, 2 and 4 further comprising Cys at positions 501 and 605 or Pro at position 559, preferably Cys at positions 501 and 605 and Pro at position 559.

Embodiment 7 is a recombinant HIV Env protein according to any one of Embodiments 1-6, comprising said amino acid residues in at least three of said positions selected from the group consisting of (i)-(vii).

Embodiment 8 is a recombinant HIV Env protein according to any one of Embodiments 1-6, comprising said amino acid residues in at least four of said positions selected from the group consisting of (i)-(vii).

Embodiment 9 is a recombinant HIV Env protein according to any one of Embodiments 1-6, comprising said amino acid residues in at least five of said positions selected from the group consisting of (i)-(vii).

Embodiment 10 is a recombinant HIV Env protein according to any one of Embodiments 1-6, comprising the indicated amino acid residues in at least six of the indicated positions selected from the group consisting of (i)-(vii).

Embodiment 11 is a recombinant HIV Env protein according to any one of Embodiments 1-6, comprising the indicated amino acid residues at the indicated seven positions selected from the group consisting of (i)-(vii).

Embodiment 12 is a recombinant HIV Env protein according to any one of Embodiments 1, 4, and 5, wherein at least two of said positions and residues are a combination selected from the group consisting of 651F, 655I; 651F, 535N; 651F, 589V; 651F, 589I; 651F, 573F; 651F, 204I; 651F, 647F; 655I, 535N; 655I, 589V; 655I, 589I; 655I, 573F; 655I, 204I; 655I, 647F; 535N, 589V; 535N, 589I; 535N, 573F; 535N, 204I; 535N, 647F; 589V, 573F; 589V, 204I; 589V, 647F; 589I, 573F; 589I, 204I; 589I, 647F; 573F, 204I; 573F, 647F; and 204I, 647F.

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Embodiment 13 is a recombinant HIV Env protein according to Embodiment 7, wherein at least three of said positions and residues are a combination selected from the group consisting of 651F, 655I, 535N; 651F, 589V, 535N; 651F, 589I, 535N; 651F,

573F, 535N; 651F, 204I, 535N; 651F, 647F, 535N; 655I, 589V, 535N; 655I, 589I, 535N; 655I, 573F, 535N;

655I, 204I, 535N; 655I, 647F, 535N; 589V, 573F, 535N; 589V, 204I, 535N; 589V, 647F, 535N; 589I, 573F,

535N; 589I, 204I, 535N; 589I, 647F, 535N; 573F, 204I, 535N; 573F, 647F, 535N; 204I, 647F, 535N; 651F,

655I, 589V; 651F, 573F, 589V; 651F, 204I, 589V; 651F, 647F, 589V; 655I, 573F, 589V; 655I, 204I, 589V;

655I, 647F, 589V; 573F, 204I, 589V; 573F, 647F, 589V; 204I, 647F, 589V; 651F, 655I, 589I; 651F, 573F, 589I; 651F, 204I, 589I; 651F, 647F, 589I; 655I, 573F, 589I; 655I, 204I, 589I; 655I, 647F, 589I; 573F, 204I, 589I; 573F, 647F, 589I; 204I, 647F, 589I; 651F, 655I, 573F; 651F, 204I, 573F; 651F, 647F, 573F; 655I, 204I, 573F; 655I, 647F, 573F; 204I, 647F, 573F; 651F, 655I, 204I; 651F, 647F, 204I; 655I, 647F, 204I; 651F, 655I, 647F; 655I, 651F, 647F; 655I, 651F, 535N; 655I, 589V, 573F; and 655I, 589V, 204I.

Embodiment 14 is a recombinant HIV Env protein according to Embodiment 8, wherein at least four of said positions and residues are a combination selected from the group consisting of: 651F, 655I, 535N, 589V; 651F, 655I, 535N, 573F; 651F, 655I, 589V, 573F; 651F, 535N, 589V, 573F; 655I, 535N, 589V, 573F; 651F, 655I, 535N, 204I; 651F, 655I, 589V, 204I; 651F, 535N, 589V, 204I; 655I, 535N, 589V, 204I; 651F, 655I, 573F, 204I; 651F, 535N, 573F, 204I; 655I, 535N, 573F, 204I; 651F, 589V, 573F, 204I; 655I, 589V, 573F, 204I; 535N, 589V, 573F, 204I; 651F, 655I, 535N, 647F; 651F, 655I, 589V, 647F; 651F, 535N, 589V, 647F; 655I, 535N, 589V, 647F; 651F, 655I, 573F, 647F; 651F, 535N, 573F, 647F; 655I, 535N, 573F, 647F; 651F, 589V, 573F, 647F; 655I, 589V, 573F, 647F; 535N, 589V, 573F, 647F; 651F, 655I, 204I, 647F; 651F, 535N, 204I, 647F; 655I, 535N, 204I, 647F; 651F, 589V, 204I, 647F; 655I, 589V, 204I, 647F; 535N, 589V, 204I, 647F; 651F, 573F, 204I, 647F; 655I, 573F, 204I, 647F; 535N, 573F, 204I, 647F; and 589V, 573F, 204I, 647F.

Embodiment 15 is a recombinant HIV Env protein according to Embodiment 9, wherein at least five of the indicated positions and residues are a combination selected from the group consisting of 651F, 655I, 535N, 589V, 573F; 651F, 655I, 535N, 589V, 204I; 651F, 655I, 535N, 573F, 204I; 651F, 655I, 589V, 573F, 204I; 651F, 535N, 589V, 573F, 204I; 655I, 535N, 589V, 573F, 204I; 651F, 655I, 535N, 589V, 647F; 651F, 655I, 535N, 573F, 647F; 651F, 655I, 589V, 573F, 647F; 651F, 535N, 589V, 573F, 647F; 655I, 535N, 589V, 573F, 647F; 651F, 655I, 535N, 204I, 647F; 651F, 655I, 589V, 204I, 647F; 651F, 535N, 589V, 204I, 647F; 655I, 535N, 589V, 204I, 647F; 651F, 655I, 573F, 204I, 647F; 651F, 535N, 573F, 204I, 647F; 655I, 535N, 573F, 204I, 647F; 651F, 589V, 573F, 204I, 647F; 655I, 589V, 573F, 204I, 647F; and 535N, 589V, 573F, 204I, 647F.

Embodiment 16 is a recombinant HIV Env protein according to Embodiment 10, wherein at least six of the indicated positions and residues are a combination selected from the group consisting of: 651F, 655I, 535N, 589V, 573F, 204I; 651F, 655I, 535N, 589V, 573F, 647F; 651F, 655I, 535N, 589V, 204I, 647F; 651F, 655I, 535N, 573F, 204I, 647F; 651F, 655I, 589V, 573F, 204I, 647F; 651F, 535N, 589V, 573F, 204I, 647F and 655I, 535N, 589V, 573F, 204I, 647F.

Embodiment 17 is a recombinant HIV Env protein according to any one of Embodiments 1-16, further comprising an amino acid substitution for the specified amino acid residue at at least one of the specified positions selected from the group consisting of:

(yin) Gln, Glu, Ile, Met, Val, Trp or Phe, preferably Gln or Glu, at position 588;

(ix) Lys at position 64 or Arg at position 66, or both Lys at position 64 and Arg at position 66;

(x) Trp at position 316;

(xi) Cys at both positions 201 and 433;

(xii) Pro at position 556 or 558 or both positions 556 and 558 and (xiii) substitution of the loop at amino acid positions 548-568 (HR1-loop) with a loop having 7-10 amino acids, preferably an 8 amino acid loop, e.g. having a sequence selected from any of (SEQ ID NO: 12-17);

(xiv) Gly at position 568, or Gly at position 569, or Gly at position 636, or Gly at both positions 568 and 636, or Gly at both positions 569 and 636; and/or (xv) Tyr at position 302 or Arg at position 519 or Arg at position 520 or Tyr at position 302 and Arg at position 519 or Tyr at position 302 and Arg at position 520 or Tyr at position 302 and Arg at both positions 519 and 520.

where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate.

Embodiment 18 is a recombinant HIV Env protein according to any one of Embodiments 1-17, further comprising a mutation in the furin cleavage site sequence of the HIV Env protein.

Embodiment 19 is the recombinant HIV Env protein of Embodiment 18, wherein the mutation at the furin cleavage site is a substitution at positions 508-511 on RRRRRR (SEQ ID NO: 10).

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Embodiment 20 is a recombinant HIV Env protein according to any one of Embodiments 1-19, which is gp140 or gp160.

Embodiment 21 is a recombinant HIV Env protein according to any one of Embodiments 1-20, wherein the recombinant HIV Env protein is characterized by at least one of an increased percentage of trimer formation and an increased trimer yield compared to an HIV Env protein lacking one or more of said amino acid residues at said positions selected from the group consisting of (i)-(vii).

Embodiment 22 is the recombinant HIV Env protein of Embodiment 21, where trimer formation is measured by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS).

Embodiment 23 is a recombinant HIV Env protein according to any one of Embodiments 1-22, further comprising an amino acid residue selected from Val, Ile, Phe, Met, Ala, or Leu, preferably Val or Ile, most preferably Val, at position 658.

Embodiment 24 is a recombinant HIV Env protein according to any one of Embodiments 1-23, comprising an amino acid combination selected from the group consisting of:

(a) 655I, 589V, 573F, 651F, 588E, 535N, 204I;

(b) 556P, 655I, 535N, 573F, 589V, 204I, 588Q;

(c) 204I, 535N, 556P, 588E, 589V, 651F, 655I;

(d) 535N, 556P, 589V, 651F, 655I and (e) 535N, 556P, 588E, 589V, 651F, 655I.

Embodiment 25 is a recombinant HIV Env protein according to any one of Embodiments 1-24, comprising an amino acid sequence that is at least 95, 96, 97, 98, 99 identical or 100% identical to any of SEQ ID NO: 3, 5, 20, 22, 24, 26, 27, 28, 29, 30, 31, or 32, preferably at least 98% identical to any of SEQ ID NOs: 20, 22, 24, 26, 27, 28, 29 , 30, 31 or 32.

Embodiment 26 is a trimeric complex containing a non-covalently linked oligomer of three recombinant HIV Env proteins according to any one of Embodiments 1-25.

Embodiment 27 is a particle, such as a liposome or a nanoparticle, such as a self-assembling nanoparticle, representing the recombinant HIV Env protein of any of Embodiments 1-25 or the trimeric complex of Embodiment 26.

Embodiment 28 is an isolated nucleic acid molecule encoding a recombinant HIV Env protein according to any one of Embodiments 1-25.

Embodiment 29 is a vector containing the isolated nucleic acid molecule of Embodiment 28 operably linked to a promoter.

Embodiment 30 is the vector according to Embodiment 29, which is an adenoviral vector.

Embodiment 31 is a host cell containing an isolated nucleic acid molecule according to Embodiment 28 or a vector according to Embodiment 29 or 30.

Embodiment 32 is a method for producing a recombinant HIV Env protein, comprising growing a host cell according to Embodiment 31 under conditions suitable for producing a recombinant HIV Env protein.

Embodiment 33 is a method for producing a recombinant HIV Env protein, comprising obtaining an expression vector containing the isolated nucleic acid of Embodiment 28 operably linked to a promoter; transfecting the cell with an expression vector; growing the transfected cell under conditions suitable for expression of the recombinant HIV Env protein; and purifying the recombinant HIV Env protein under conditions that allow the formation of a stabilized trimeric complex.

Embodiment 34 is a method for producing a recombinant HIV Env protein according to any one of Embodiments 1-25, comprising introducing at least one amino acid substitution resulting in said amino acid residue at a position selected from the group consisting of (i)-(vii) , into the framework sequence of the HIV envelope protein.

Embodiment 35 is the method according to Embodiment 34, wherein a nucleotide sequence encoding an amino acid substitution is introduced into a nucleic acid encoding an HIV envelope protein backbone sequence.

Embodiment 36 is the method according to Embodiments 34 or 35, wherein the HIV envelope protein framework sequence is selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID

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NO: 9; SEQ ID NO: 6 having a Glu to Arg mutation at position 166; SEQ ID NO: 6 having Cys at positions 501 and 605 and Pro at position 559 and/or having a substitution on SEQ ID NO: 10 amino acids 508-511; SEQ ID NO: b having a Glu to Arg mutation at position 166, additionally having Cys at positions 501 and 605 and Pro at position 559 and/or having a substitution for SEQ ID NO: 10 amino acids 508-511; SEQ ID NO: 8 having Cys at positions 501 and 605 and Pro at position 559 and/or having a substitution on SEQ ID NO: 10 amino acids 508-511; SEQ ID NO: 9 having Cys at positions 501 and 605 and Pro at position 559 and/or having a substitution on SEQ ID NO: 10 amino acids 508-511; and a wild-type HIV Env protein having mutations that result in at least (a), (b) or (c), preferably at least two of (a), (b) and (c), most preferably to (a), (b) and (c) of the following: (a) Cys at positions 501 and 506 and Pro at position 559, (b) a substitution with SEQ ID NO: 10 amino acids 508-511, and/or ( c) at least one repair mutation at an amino acid residue that is present at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of the HIV Env sequences in a population of at least 100, preferably at least 1000, preferably at least 10,000 wild-type HIV Env sequences, where the repair mutation is a substitution for an amino acid residue that is present at the corresponding position at a frequency of at least 10% of the HIV Env sequences in said population, and preferably the repair mutation is a substitution for an amino acid residue , which is most often present at the corresponding position in the indicated population.

Embodiment 37 is a composition comprising a recombinant HIV Env protein according to any of Embodiments 1-25, a trimeric complex according to Embodiment 26, a particle according to Embodiment 27, an isolated nucleic acid molecule according to Embodiment 28, a vector according to Embodiments 29 or 30 or a host cell according to embodiment 31 and a pharmaceutically acceptable carrier.

Embodiment 38 is the composition of Embodiment 37 further comprising an adjuvant.

Embodiment 39 is a method for preparing the composition of Embodiment 37, comprising mixing a recombinant HIV Env protein, trimeric complex, particle, isolated nucleic acid, vector, or host cell with one or more pharmaceutically acceptable carriers.

Embodiment 40 is a method of vaccinating a subject against an infection caused by HIV, comprising administering to the subject a composition comprising a recombinant HIV envelope protein according to any one of Embodiments 1-25, a trimeric complex according to Embodiment 26, a particle according to Embodiment 27, or a vector according to Embodiments. 29 or 30.

Embodiment 41 is a method of generating an immune response against an HIV infection in a subject in need thereof, comprising administering to the subject a composition comprising a recombinant HIV envelope protein according to any one of Embodiments 1-25, a trimeric complex according to Embodiment 26, a particle according to embodiment 27 or the vector according to embodiments 29 or 30.

Embodiment 42 is a recombinant HIV Env protein containing the amino acid sequence of SEQ ID NO: 2, or a sequence that is at least 95% identical to it.

Embodiment 43 is a recombinant HIV Env protein containing the amino acid sequence of SEQ ID NO: 3, or a sequence that is at least 95% identical to it.

Embodiment 44 is a recombinant HIV Env protein containing the amino acid sequence of SEQ ID NO: 4, or a sequence that is at least 95% identical to it.

Embodiment 45 is a recombinant HIV Env protein containing the amino acid sequence of SEQ ID NO: 5, or a sequence that is at least 95% identical to it.

Embodiment 46 is an isolated nucleic acid molecule encoding a recombinant HIV Env protein according to any one of Embodiments 42-45.

Embodiment 47 is a vector containing the isolated nucleic acid molecule of Embodiment 46 operably linked to a promoter.

Embodiment 48 is the vector according to Embodiment 47, which is an adenoviral vector.

Embodiment 49 is a host cell containing an isolated nucleic acid molecule according to Embodiment 46 or a vector according to Embodiment 47 or 48.

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Embodiment 50 is a composition containing a recombinant protein

An HIV env according to any of Embodiments 42-45, an isolated nucleic acid molecule according to Embodiment 46, a vector according to Embodiments 47 or 48, or a host cell according to Embodiment 49, and a pharmaceutically acceptable carrier.

Embodiment 51 is a method for increasing the percentage of trimers and/or trimer yield (showing folding and stability) of a parent HIV Env protein, the method comprising restoring the amino acid sequence of the parent HIV Env protein by introducing at least one repair mutation, preferably at least at least 3 repair mutations to the original HIV Env protein, where the repair mutation is an amino acid substitution at an amino acid residue that occurs at the corresponding position at a frequency of less than 7.5%, preferably less than 2% of the HIV Env sequences in a population of at least 100 , preferably at least 500, preferably at least 1000, preferably at least 10,000 wild-type HIV Env sequences, where the substitution is a substitution for an amino acid residue that occurs at the corresponding position with a frequency of at least 10% of the HIV Env sequences in said population, and preferably the substitution is for the amino acid residue that occurs most frequently at the corresponding position in said population.

Embodiment 52 is the method of Embodiment 51 wherein at least 50%, preferably at least 80% of the amino acid residues in the original HIV Env protein that occur at the corresponding position at a frequency of less than 7.5% of the HIV Env sequences in specified collection are restored.

Embodiment 53 is the method of Embodiment 51 wherein at least 50%, preferably at least 80% of the amino acid residues in the original HIV Env protein that occur at the corresponding position at a frequency of less than 2% of the HIV Env sequences in said collection , are restored.

Embodiment 54 is a method according to any one of Embodiments 51-53, wherein the parent HIV Env protein is from clade C.

Embodiment 55 is a method according to any one of embodiments 51-54, wherein the parent HIV Env protein is a wild-type HIV Env protein.

Embodiment 56 is a method according to any one of Embodiments 51-54, wherein the original HIV Env protein contains one or more of the following:

(a) Cys at positions 501 and 506 and Pro at position 559;

(b) a mutation in the sequence of the furin cleavage site of the HIV Env protein, for example, having a substitution on SEQ ID NO: 10 amino acids 508-511;

(c) Phe at position 651;

(d) Ile at position 655;

(e) Asn at position 535;

(f) Val at position 589;

(g) Phe at position 573;

(h) Ile at position 204;

(i) Phe at position 647;

(j) Val at position 658;

(k) Gln or Glu at position 588; and/or (l) Pro at positions 556, 558, or 556 and 558. Embodiment 57 is a recombinant HIV Env protein produced by the method of any one of Embodiments 51-56.

Examples

Example 1 Derivation of Clade C and Clade B HIV Envelope Protein Consensus Clade C HIV Envelope Protein Consensus Sequence

The envelope protein (Env) consensus sequence of the HIV clade C was developed as a framework sequence to investigate the effects of various mutations on the formation of trimers of the HIV Env proteins. Sequence alignments for 3434 coat protein sequences derived from known HIV virus isolates were downloaded from the Los Alamos database (http://www.hiv.lanl.gov/content/index). Of the 3434 sequences, only 1252 clade C sequences were selected to obtain a clade C HIV Env protein consensus sequence. At positions for which a consensus residue could not be clearly identified based on alignment, the consensus sequence was used to identify the closest wild-type sequences by BLAST search. The consensus residue at these positions was then chosen as the amino acid in the closest wild-type sequences identified by the BLAST search. The consensus sequence of the HIV clade C Env protein is shown under SEQ ID NO: 2. The two sequences with the highest homology to SEQ ID NO: 2 were found

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BLASTs were sequences with Genbank accession numbers ADM30337.1 and

ADM30340.1, both of which had 90% sequence identity with SEQ ID

NO: 2.

The consensus sequence of the HIV clade C Env protein was further modified by introducing the so-called SOSIP mutations, which include cysteine residues at positions 501 and 605 and a proline residue at position 559, as well as by optimizing the furin cleavage site by replacing residues 508-511 in the furin cleavage site with b arginine residues. In addition, Val at position 295 was mutated to Asn (V295N) to create an N-linked glycosylation site present in most HIV strains and may improve binding to certain antibodies used in some experiments. In addition, the C-terminus was truncated at residue 664, resulting in a sequence encoding the soluble HIV gp140 protein. All substitution/modification positions described above are relative to gp160 numbering from HIV1 HXB2 isolate. The resulting HIV gp140 sequence, designated ConC_SOSIP, is shown under (SEQ ID NO: 3). The ConC_SOSIP sequence was used as a backbone or parent HIV envelope protein sequence that was further mutated, eg, single and double amino acid substitutions, to produce recombinant HIV Env proteins according to embodiments of the present invention.

HIV clade B envelope protein consensus sequence

The clade B HIV Env protein consensus sequence was generated using a procedure similar to that described above for the clade C HIV Env protein consensus sequence. HIV clade B is shown under SEQ ID NO: 4.

The HIV clade B Env consensus sequence was further modified by introducing so-called SOSIP mutations, optimizing the furin cleavage site by replacing the furin cleavage site with 6 arginine residues, and truncating the C-terminus at residue 664 as described above, resulting in a sequence encoding the consensus sequence soluble gpl40 HIV clade B. The resulting HIV gpl40 Env protein sequence, designated ConB_SOSIP, is shown under (SEQ ID NO: 5).

Surprisingly, it was found that molecules based on the consensus sequence had increased levels of expression compared to those based on natural isolates and, moreover, already had increased levels of trimerization. Therefore, molecules with SEQ ID NO: 2-5 have already been characterized by unexpectedly advantageous properties.

Example 2 Expression and Purification of Recombinant HIV Env Protein

Recombinant HIV Env proteins were expressed and purified as soluble gp140 proteins. Single mutations (amino acid substitutions) and combinations thereof (eg, double and triple mutations) were introduced into the framework consensus sequence ConC_SOSIP to generate a series of recombinant HIV Env protein variants.

Generation and Expression of HIV Env gpl40 Protein Constructs and Variants DNA encoding the HIV clade C Env protein consensus sequence, ConC_SOSIP, shown under SEQ ID NO: 3, was synthesized and subjected to codon optimization on GenScript (Piscataway, NJ 08854) or Gene Art ( Life Technologies, Carlsbad, California). The codon-optimized sequence was then cloned into the pcDNA2004 vector to generate the HIV clade C Env gp140 protein construct, which was used as the HIV envelope protein backbone sequence to introduce additional mutations. Mutations were introduced into the framework sequence ConC_SOSIP by site-directed mutagenesis and polymerase chain reaction (PCR) against the clade C HIV gp140 Env protein construct in pcDNA2004. HEK-Expi293F cells or HEK293F cells were transiently transfected with 90% pcDNA2004 vector encoding the ConC_SOSIP sequence or variant and 10% pcDNA2004 vector encoding furin protease (furinpcDNA2004) according to the manufacturer's instructions. Transfected cells were cultured for 5 days at 37°C and 10% CO ₂ . Culture supernatants were centrifuged for 10 minutes at 1250xg. Subsequently, the centrifuged supernatant was subjected to sterile filtration using a vacuum filter with a pore size of 0.22 μm and stored at 4°C until further use.

For expression procedures in 96-well format, cells were cultured for 3 days at 37°C and 10% CO ₂ . 4 µl of Optimem (culture medium) was mixed with 4 µl of 100 ng/µl DNA, 8 µl of Expi2 93F mixture (54 µl/ml in Optimem) was added and incubated for 20 min. Subsequently, 200 µl/well of Expi293F cells were added at a density of 2.5 x 10E6 cells/ml. The culture supernatant was collected and centrifuged for 5 min at 300 g to remove cells and cell debris. The centrifuged supernatant was subsequently subjected to sterile filtration using a vacuum filter with a pore size of 0.22 μm and stored at 4°C until further use.

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HIV Env gp140 protein purification

The HIV gpl40 Env protein expressed from the pcDNA2004 vector was purified according to a two-step purification protocol using a Galantus nivalis lectin column (Vectorlabs, AL-1243) for initial purification and a Superdex200 Increase (GE) column in the next step to remove residual contaminants. For the initial step using a Galantus nivalis lectin column, the culture supernatant was diluted with buffer (40 mM Tris, 500 mM NaCl, pH 7.5) and passed through a CV Tricorn 10-50 column containing 4 ml lectin agarose at a flow rate of 4 ml per minute. Subsequently, the column was washed with four column volumes of buffer (40 mM Tris, 500 mM NaCl, pH 7.5) and eluted with four column volumes of 40 mM Tris, 500 mM NaCl and 1 M mannopyranoside, pH 7.5 at an upflow rate of 1.6 ml/min., which means that the flow direction was changed from downward to upward to increase the elution rate of the coat protein and reduce the elution volume. The eluate was concentrated using a concentrator centrifuge (50K, Amicon Ultra, Millipore).

The HIV gp140 Env protein was further purified on a Superdex200 column with 50 mM Tris, 150 mM NaCl, pH 7.4 as running buffer. The second peak that eluted from the column contained the HIV Env gp140 protein. Fractions containing this peak were pooled and the identity of the peak as HIV Env gpl40 protein was confirmed using Western blot and SDS-PAGE and/or SECMALS analysis. The concentration of the purified HIV gp140 Env protein was determined by measuring the optical density at 280 nm, and the purified HIV gp140 Env protein was stored at 4° C. until further use.

SDS-PAGE analysis and Western blotting

Cell culture supernatants containing the expressed HIV gp140 Env protein and purified HIV Env gp140 protein samples were analyzed on 4-12% (w/v) Bis-Tris NuPAGE, 1X MOPS (Life Technologies) gels under reducing or non-reducing conditions and run blotting with iBlot technology (Life Technologies). All procedures were performed according to the manufacturer's instructions. For purity analysis, gels were stained with Krypton infrared protein stain (Thermo Scientific) or SYPRO Rubi protein stain (Bio-Rad). In the case of Western blot analysis, membranes were examined with an anti-bx-histidine tag antibody (anti-His antibody conjugated to HRP). Gels and blot membranes were scanned on an Odyssey instrument (Li-Cor) and images were analyzed using Odyssey 3.0 software (Li-Cor).

Visualization of Trimer Formation of the HIV Env Protein by Negative Staining Electron Microscopy

Negative staining electron microscopy (NS-EM) was used to visualize trimers of an envelope protein having the framework sequence ConC_SOSIP, which was purified with lectin from Galanthus nivalis followed by size exclusion chromatography and was performed as described in Julien et al. 2015 (Proc. Natl. Acad. Sci. (2015) 112(38) 11947-52). Trimer samples were diluted to a concentration of 0.01-0.5 mg/mL in Tris buffered saline (TBS), pH 7.4, and attached to a carbon-coated 200 mesh Cu grid (EMS CF200-Cu), which was exposed to a glow charge in air, 2*10 ^-1 mbar, 25 mA, 30 s, immediately before use. Subsequently, a 3 μl drop of the diluted trimer sample was applied to the grid for 1 min, and then blotted with filter paper (Whatman No. 1 or 4). The grids were dried for one minute, then stained with 3 µl of 2.3% uranyl acetate (UAc) for 60 seconds.

Data were collected using an FEI Tecnai F20 electron microscope operating at 120 keV with a magnification of 25,000x, resulting in a pixel size of 4.68 A in the preparation plane. The images were obtained using a Gatan BM ultrascanner.

Nearly all of the particles in the images (from this trimer-enriched material) were properly closed trimers (data not shown).

Example 3 Screening of Recombinant HIV Env gp140 Protein Variants for Trimer Yield and Percent Trimer Formation

The recombinant HIV Env protein variants produced in Example 2 were screened for trimer formation to identify those mutations that increased the percentage of trimer formed and/or increased trimer yields relative to the ConC_SOSIP framework sequence. High-throughput screening of percentage trimer formation and trimer yield rates was performed using the AlphaLISA assay to evaluate the binding of a panel of broad-spectrum HIV neutralizing antibodies (bNAbs) and non-bNAbs to recombinant HIV Env proteins. The results of the AlphaLISA assay were confirmed by size exclusion chromatography and multi-angle light scattering (SEC-MALS).

Examination with the AlphaLISA® Assay

Total HIV gp140 Env protein expression and total correctly folded native trimer in more than 200 HIV gp140 variants with single amino acid substitutions introduced into the ConC_SOSIP sequence prepared as described in Example 2 was measured in cell culture supernatant by AlphaLISA assay. HIV gp140 variants containing double and triple mutations were also tested. For comparison, we tested the Env HIV protein, which has the sequence

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ConC_SOSIP without any additional mutations.

The following monoclonal antibodies (mAb) were used for analysis, among others: mAb PGT145, mAb PGDM1400, mAb PG16, mAb PGT151, mAb 35022, mAb PGT128, mAb PG9, mAb F105, mAb B6, mAb 447-52d, mAb 14e and mAb 17b. MAb 447-52D (AB014), PG9 (AB015) and PG16 (AB016) were purchased from Polymun Scientific Immunbiologische Forschung GmbH (Klosterneuburg, Austria). Non-neutralizing antibody b6 was obtained from Dennis R. Burton (Scripps Research Institute, La Jolla, CA) and non-neutralizing antibody 14e was obtained from James E. Robinson (Tulane University, New Orleans, Louisiana). For mAb PGT145 (PDB: 3U1S), PGDM1400 (PDB: 4RQQ), PGT151 (PDB: 4NUG), 35022 (PDB: 4TVP), F105 (PDB: 1U6A), PGT128 (PDB: 3TYG) and 17b (PDB: 4RQS ) nucleic acids encoding published sequences were cloned into an expression vector and obtained for evaluation of HIV Env proteins. With the exception of mAb F105, B6, 447-52d, 14e and 17b, the antibodies used for the assay are broad spectrum neutralizing antibodies (bNAbs). BNAbs are capable of neutralizing several viral strains of HIV. Among bNAbs, PGT145, PGDM1400 and PG16 antibodies bind to the top of the trimer and are trimer specific. PGT151 is also trimer-specific, but binds at the interface of the two protomers gp120 and gp41 and is cleavage dependent. Binding of non-bNAb antibodies is indicative of a misfolded or open conformation of the trimer.

Protein folding was also tested by measuring the binding of soluble HIV gp140 Env protein variants to an antibody (mAb 17b) known to bind to the HIV envelope protein co-receptor binding site, which becomes accessible only after CD4 binding (data not shown). In particular, the soluble CD4 receptor (sCD4) in combination with mAb 17 was used to assess CD4-induced conformational change. Binding of mAb 17b to the HIV Env gp140 protein variant without prior binding of CD4 to the envelope protein is indicative of a partially unfolded or pre-activated envelope protein (i.e. e. an unstable Env that adopts an open conformation in the absence of binding to CD4).

For AlphaLISA analysis, Env gp140 HIV constructs were generated in a pcDNA2004 vector containing a linker followed by a sortase A tag followed by a Flag tag followed by a flexible (G ₄ S) ₇ linker and ending in a His tag (sequence the label that was placed at the C-terminus of the HIV Env protein is shown under SEQ ID NO: 19). The HIV gp140 Env constructs were expressed in HEK-Expi293 cells cultured for three days in 96-well plates (200 μl/well). Crude supernatants were diluted 120-fold in AlphaLISA buffer (PBS+0.05% Tween-20+0.5 mg/ml BSA). For assays based on mAb 17b, supernatants were diluted 12-fold. Then 10 μl of each dilution was transferred to a 96-well plate and mixed with 40 μl of acceptor beads, donor beads and one of the above mAbs. Donor beads were conjugated with ProtA (Cat. No.: AS102M, Lot No. 1831829, Perkin Elmer), which binds to the mAb. The acceptor beads were conjugated with an anti-His antibody (Cat. No.: AL128M, Perkin Elmer) that binds to the His tag of the construct. To quantify total protein yield, including all forms of envelope protein, a combination of nickel-conjugated donor beads (Cat. No.: AS101M, Perkin Elmer) was used for His-tag detection together with Anti-Flag conjugated acceptor beads (Cat. : AL112R, Perkin Elmer) to identify the Flag tag. For tests using mAb 17b in combination with sCD4-His, a combination of ProtA donor beads and anti-Flag acceptor beads was used (data not shown). One sample was mixed with donor and acceptor beads to detect trimer formation, and a second sample of the same Env variant was mixed with nickel-conjugated donor beads and an anti-Flag-conjugated acceptor bead to measure total protein expressed (i.e., total protein yield) .

A mixture of the supernatant containing the expressed HIV gp140 Env protein, mAb, donor beads and acceptor beads was incubated at room temperature for 2 hours without shaking. Subsequently, the chemiluminescent signal (BioTek) was measured using a Synergy NEO plate reader. The mean background signal attributable to the mock-transfected cells was subtracted from the AlphaLISA values measured for each of the Env gp140 HIV variants. The signals of the entire dataset were then divided by the signal measured for the HIV Env protein having the ConC_SOSIP framework sequence to normalize the signal for each of the HIV Env gp140 variants tested relative to the framework sequence. Binding data for each of the HIV Env gp140 variants to the trimer-specific mAb PGT145 was used to determine the percentage of trimer formation and trimer yield for each of the variants. Binding to other mAbs was used to evaluate the overall binding pattern of HIV Env variants to bNAb and non-bNAb antibodies (not shown).

The percentage of trimer formation for each of the HIV Env variants was calculated by dividing the normalized chemiluminescent signal obtained from the test mixture from the HIV Env variant, mAb PGT145, ProtA-conjugated donor beads and anti-His-conjugated acceptor beads, by the normalized chemiluminescent signal obtained from test mixture from HIV Env variant, anti-His conjugated donor beads and anti-Flag conjugated acceptor beads.

The trimer yield for each of the HIV Env variants was determined relative to the trimer yield for the HIV Env protein having the ConC_SOSIP framework sequence without any additional mutations. The normalized chemiluminescent signal obtained from the binding of the PGT145 mAb to the ConC_SOSIP envelope protein was considered to be 1, and the normalized chemiluminescent signal obtained from the binding of the PGT145 mAb to each of the HIV gp140 proteins was normalized to this value.

AlphaLISA Study Results - Percentage of Trimers and Trimer Yields

Percentage of trimer formation as determined by AlphaLISA analysis for some single, double and triple amino acid substitutions from list (i)-(vii) in Table. 1 above in the ConC_SOSIP framework sequence shown in FIG. 2A. Based on approximately 200 HIV Env gp140 variants tested containing single amino acid substitutions, seven substitution positions were identified in which the percentage of trimer formed increased by at least 25% relative to the percentage of trimer formed in the case of the ConC_SOSIP framework sequence without any additional amino acids. replacement

The results shown in FIG. 2A demonstrate that the seven preferred substitution positions at which a significant increase in trimer percentage was observed include N651, K655, I535, D589, I573, A204, and E647, as numbered in gp160 from the HIV-1 HXB2 isolate. In particular, the single amino acid substitutions that resulted in the largest percentage increase in trimer formation included N651F, K655I(/F/W) (however, there was also one experiment in which K655F did not appear to improve), I535N , D589V(/A), I573F, A204I, E647F. Some mutations that were tested in combination with some of these mutations included K588Q/E, I556P and A558P, and they further increased the percentage of trimer formation in mutants with preferred amino acids at the positions of the present invention ((i)-(vii) table. 1) in this experiment.

All double substitutions tested in this experiment had a higher percentage of trimer formation than the corresponding single substitutions, and all triple substitutions tested had a higher percentage of trimer formation than the corresponding single and double mutations (FIG. 2A). Such unexpected and unexpected results indicate that these mutations may have exhibited a form of synergy in these experiments in terms of coat protein trimerization.

In addition to an increased percentage of trimer formation, an increased trimer yield is also optimal. Thus, the yield of trimers of HIV gp140 variants containing single, double and triple mutations in the framework sequence ConC_SOSIP was also determined using the AlphaLISA assay. The results are shown in FIG. 2B. Most HIV gp140 variants containing single mutations (with the exception of I535N, D589A, and D589I) were characterized by a higher trimer yield than the ConC_SOSIP envelope protein. However, a more precise SECMALS analysis of the I535N mutant described below showed an increase in the yield of trimers. Moreover, additional mutations in combination with I535N, such as D589V, resulted in the same yield of trimers as was observed with an envelope protein having that particular additional substitution in the absence of the I535N mutation. The trimer yield of the double mutation variants also increased, with each of the single mutation variants having a higher trimer yield than the ConC_SOSIP envelope protein (FIG. 2B).

We also tested the percentage of trimer formation in HIV gp140 variants with double mutations in the framework sequence ConC_SOSIP that have previously been described in the literature, including the E64K, T316W double substitution described (De Taeye et al., supra), and the I204C disulfide double substitution. , A433C described (Kwon et al., supra). The double substitution of E64K, T316W resulted in a lower percentage of trimer formation than the ConC_SOSIP envelope protein; 15% (data not shown). Although the disulfide double substitution I204C, A433C increased the percentage of trimers to 43% (data not shown), new double substitutions described in this document such as I535N/K588E, K588Q/D589V, K655I/K588E, I535N/D589V, I535N/E647F , D589V/K655I and I535N/K655I (FIG. 2A) resulted in an even higher percentage of trimer formation in the AlphaLISA experiment.

Additional mutations were also introduced into the ConC_SOSIP framework sequence (proline at residues 558 and/or 556) and for these Env gp140 HIV proteins the percentage of trimer formation and trimer yield was measured. Both single substitutions to Pro at position 558 or 556 and double substitutions to Proline at both positions 556 and 558 in addition to the SOSIP mutations already contained in the ConC_SOSIP framework sequence (i.e. Cys at positions 501 and 605 and Pro at position 559) increased the percentage of trimer formation and trimer yield (data not shown). Indeed, the introduction of one or more of the novel stabilizing amino acid substitutions of the present invention into a ConC_SOSIP framework sequence further containing Pro residues at positions 558

- 33 042607 and / or 556, further increases the percentage of formation of trimers and / or the yield of trimers (for example, Fig. 2A, for example, A558P / I535N, K655I / L556P, and several triple mutants, including the A558P mutation).

Binding data of HIV gp140 Env variants to other bNAbs and non-bNAb antibodies demonstrated that most single, double, and triple mutation variants tested that increased trimer yield and percentage of trimer formation, such as those listed in FIG. 2A and 2B also showed increased binding to bNAb and similar or reduced binding to non-bNAb antibodies relative to the amount of binding observed with bNAb and non-bNAb antibodies in the case of the HIV envelope protein having the framework sequence ConC_SOSIP (data not shown). For vaccine development, increased binding to bNAb and reduced binding to non-bNAb antibodies are preferred. Thus, the data demonstrate that HIV envelope proteins containing amino acid substitutions at positions (i)-(vii) listed in Table. 1 above have optimal properties in terms of binding patterns with broad spectrum neutralizing antibodies and non-broad spectrum neutralizing antibodies.

SEC-MALS analysis

The SECMALS assay was also used to confirm trimer yield and percentage of trimer formation for HIV gp140 variants screened with the AlphaLISA assay. HIV gp140 variants were expressed in 30 ml cultures and purified by loading cell-free supernatants onto 200 µl Galanthus nivalis lectin beads (Vectorlab, cat. no. AL-1243) on Polyprep gravity current columns (Biorad, cat. no. 731-1550). The beads were washed with 2 ml of binding buffer (40 mM Tris, 500 mM NaCl, pH 7.4). Proteins were eluted using 250-500 μl of 40 mM Tris, 500 mM NaCl, 1 M mannopyranoside, pH 7.4. The SEC-MALS experiment was performed using a high performance liquid chromatography system (Agilent Technologies) and a MiniDAWN TREOS instrument (Wyatt) coupled with an Optilab T-rEX refractive index detector (Wyatt). In general, a TSK-Gel G3000SWxl column (Tosoh Bioscience) equilibrated with running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 1 ml/min was loaded with either 100 μl of eluate after lectin purification or approximately 30 micrograms of protein. The data was analyzed using the Astra 6 software package and molecular weight calculations were made based on the refractive index signal.

SEC-MALS chromatograms of the ConC_SOSIP envelope protein and HIV gp140 variants containing single mutations are shown in FIG. 3. Overall, the results obtained with the SECMALS assay were comparable and consistent with those obtained with the AlphaLISA assay. The chromatogram of the ConC_SOSIP coat protein has four major peaks, with the second peak, which elutes at approximately 7.3 minutes, being the trimer peak. The envelope protein ConC_SOSIP was determined to be approximately 27% trimeric. The formation of aggregates and monomers indicates that some degree of misfolding and instability is associated with the HIV Env gp140 protein having the ConC_SOSIP consensus sequence. As demonstrated by the chromatograms shown in FIG. 3, all single substitutions resulted in a relatively high trimer peak compared to the trimer peak for the ConC_SOSIP envelope protein, indicating that trimer yield was increased for each of the HIV gp140 variants.

Taken together, the results demonstrate that the amino acid substitutions identified in (i)-(vii) of Table. 1 herein provide recombinant HIV Env proteins with an increased percentage of trimer formation and/or an increased trimer yield. In particular, HIV Env protein variants having multiple substitutions at the identified positions (i)-(vii) of Table. 1, such as variants with combinations of two or more of the identified mutations, generally exhibited even more increased trimer yield and/or percentage of trimer formation compared to HIV Env protein variants having only a single mutation, indicating a possible synergistic effect of the combinations. mutations (i)-(vii) from the table. 1. To the best of the present inventors' knowledge, none of these combinations of amino acid substitutions have been described in naturally occurring sequences of the HIV envelope protein, and therefore all combinations ((i)-(vii)) are considered to be novel combinations of stabilizing mutation trimer. HIV envelope proteins having an increased percentage of trimer formation, such as the recombinant HIV envelope proteins of the present invention, are advantageous in terms of making e.g. conformations. Also, an increased overall trimer expression yield is advantageous for the manufacture of a vaccine product.

Example 4 Stability of Trimeric HIV Envelope Proteins

The thermal stability of recombinant HIV Env proteins according to embodiments of the present invention was tested using AlphaLISA and differential scanning calorimetry (DSC).

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Thermal stability measurements using AlphaLISA Thermal stability was tested by measuring the loss of intact trimer upon thermal exposure, based on binding to the trimer-specific mAb PGT145. The crude supernatant (20 µl) was heated at 60°C for 1 h.

The samples were then centrifuged at maximum speed for five minutes to remove aggregates. The AlphaLISA assay was performed as described in Example 3 above.

The results are shown in FIG. 4 and the data are presented as the percentage of trimers remaining intact after thermal treatment. Based on the results, it can be seen that most of the tested recombinant single mutation HIV Env gp140 proteins of the present invention had a higher heat tolerance than the ConC_SOSIP coat protein. It was also found that the HIV envelope proteins tested having the trimer-stabilizing double and triple substitutions identified herein were more thermally stable than the ConC_SOSIP envelope protein.

Thermal stability measurements using DSC

The melting point (T _m ) of HIV Env gp140 variants was determined by DSC using a DSC MicroCal capillary system. Each measurement was carried out at an initial temperature of 20°C and an end temperature of 110°C at a scan rate of 100°C/h. A 0.5 mg/ml (400 µl) protein sample was used for each measurement. Data was analyzed using Origin J. Software (VP analysis tool from MicroCal).

The melting point (T _m ) of the ConC_SOSIP coat protein, measured using DSC, was determined to be 69.8°C and the melting onset temperature was 60.1°C. The T _m measured for the ConC_SOSIP envelope protein was higher than the temperature for the BG505_SOSIP envelope protein (an HIV envelope protein from a BG505 virus isolate having so-called SOSIP mutations), which was reported to have a T _m of 67.0°C ( Kwon et al., 2015). This indicates that the HIV envelope protein having the framework sequence ConC_SOSIP has more favorable thermal stability properties than other known HIV envelope sequences with trimer stabilizing mutations.

As measured, the T _m of ConC_SOSIP with the K655I mutation was 72.3° C. and the melting point was 63.7° C., which is even higher than the T _{m of} the ConC_SOSIP coat protein. As measured, T _m ConC_SOSIP with mutations A558P, N651F, I535N was 77.29°C at a melting point of 74.87°C. Thus, the DSC results confirm the thermal stability results determined by the AlphaLISA assay.

Taken together, the results demonstrate that HIV Env proteins containing at least one of the amino acid substitutions described herein generally have higher heat tolerance than envelope proteins without such mutations. The results also demonstrate that all double substitution variants of the HIV Env protein were more heat tolerant than the ConC_SOSIP envelope protein. Triple substitution variants of the HIV Env protein were also more stable than the ConC_SOSIP envelope protein.

Example 5 Variants of Recombinant HIV Envelope Proteins Based on Consensus Clade B Envelope Protein Sequence

Recombinant HIV Env proteins according to embodiments of the present invention containing a single amino acid substitution (I535N, D589V, N651F, or K655I) introduced into the clade B consensus sequence, ConB_SOSIP (SEQ ID NO: 5) were generated and purified as described in Example 2. Yield trimers and the percentage of trimer formation were measured using the AlphaLISA assay as described in Example 3.

The results are shown in FIG. 5A (percentage of trimer formation) and FIG. 5V (trimer output). Values are presented relative to the value measured for the envelope protein ConB_SOSIP, which was considered equal to 1 for both the percentage of trimer formation and trimer release. The results show that all mutations tested increased the percentage of trimer formation. The trimer yield was nearly the same or improved compared to the ConB_SOSIP envelope protein for all mutations tested.

These results demonstrate that these mutations also had a stabilizing effect on the envelope protein, such as increased trimer yield, increased percentage of trimer formation, etc., when introduced into another HIV envelope protein framework sequence, in this case the consensus sequence derived from proteins of clade B.

Example 6 Variants of Recombinant HIV Envelope Proteins Based on Synthetic Envelope Protein Sequence

Recombinant HIV Env proteins according to embodiments of the present invention containing amino acid substitutions introduced into a synthetic HIV envelope protein (referred to as 'DS_sC4_SOSIP_E166R') having the sequence shown under SEQ ID NO: 7 were prepared and purified as described in Example 2. Synthetic protein The HIV envelope, DS_sC4_SOSIP_E166R, has the so-called SOSIP mutations (Cys at residues 501 and 605 and Pro at residue 559), Cys at residues 201 and 433, leading to the introduction of a disulfide (DS) bond, and Arg at position 166 to stabilize the apex.

- 35 042607

In addition, the protein is truncated at position 655. The percentage of trimer formation and trimer yield was measured using the AlphaLISA assay described in Example 3.

The results are shown in FIG. 6, in which the percentage of trimer formation for each of the variants tested is compared to the percentage of trimer formation (FIG. 6A) and trimer yield (FIG. 6B) for the DS_sC4_SOSIP_E166R framework sequence. For each of the variants tested, a higher percentage of trimer formation was observed compared to the framework sequence.

In addition to E166R, several other rarer amino acids were changed to more common ones at the appropriate position in the population of wild-type HIV Env proteins (A114Q, E117K, T375S, and I434M) to reconstitute the protein according to the backbone described in more detail in Example 12 below and in FIG. 12. In this reduced protein, the A204I and K655I stabilizing mutations further improved sC4_SOSIP (FIG. 13).

The results of this example are consistent with the results from example 5, demonstrating that the mutations described herein also have a stabilizing effect on the coat protein, for example, lead to an increased percentage of trimer formation and/or an increased yield of trimers, etc., if they were introduced into other framework sequences of the HIV envelope proteins, in this case into the non-consensus synthetic Env sequence.

Example 7 Additional HIV Env Mutation Combinations

Recombinant HIV Env proteins according to embodiments of the present invention containing amino acid substitutions introduced in ConC_SOSIP (having the sequence shown under SEQ ID NO: 3) were generated and purified as described in Example 2. The percentage of trimer formation was measured by AlphaLISA assay, as described in Example 3. Subsequently, a small sample of combinations (combinations shown in italics below and additionally K655I; I535N, D589V; I535N, K655I; D589V, K655I) were purified using Galanthus nivalis lectin and analyzed for trimer content using SEC-MALS, as described in example 3. Stability was measured as described in example 4.

For this experiment, the following mutants were obtained:

K655I, N651F;

K655I, N651F, E647F;

K655I, N651F, E647F, I535N;

K655I, N651F, I535N;

K655I, I573F;

K655I, D589V, I573F;

K655I, D589V, I573F, N651F;

K655I, D589V, I573F, K588E;

K655I, D589V, I573F, N651F, K588E;

K655I, D589V, I573F, N651F, K588E, I535N;

K655I, D589V, I573F, N651F, K588E, I535N, A204I;

K655I, D589V, I535N, L556P;

K655I, D589V, I573F, N651F, K588E, L556P;

K655I, D589V, A204I;

L556P, N651F;

L556P, N651F, K655I;

L556P, N651F, K655I, I535N;

L556P, N651F, K655I, I535N, I573F;

L556P, N651F, K655I, I535N, I573F, D589V;

L556P, N651F, K655I, I535N, I573F, D589V, A204I;

L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q;

L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q, E647F;

L556P, N651F, I535N;

L556P, N651F, I535N, I573F;

L556P, N651F, I535N, I573F, D589V;

L556P, N651F, I535N, I573F, D589V, A204I;

L556P, N651F, I535N, I573F, D589V, A204I, K588Q;

L556P, N651F, I535N, I573F, D589V, A204I, K588Q, E647F;

L556P, K655I, I535N;

L556P, K655I, I535N, I573F;

L556P, K655I, I535N, I573F, D589V;

L556P, K655I, I535N, I573F, D589V, A204I;

L556P, K655I, I535N, I573F, D589V, A204I, K588Q;

L556P, K655I, I535N, I573F, D589V, A204I, K588Q, E647F;

L556P, N651F, I535N, I573F, D589V, A204I, K588Q with SOS mutation removed.

- 36 042607

All tested combinations of substitutions in the framework sequence ConC_SOSIP showed a higher percentage of trimers, higher trimer yield and higher trimer stability at 60°C compared to the framework sequence in the AlphaLISA assay (data not shown). SEC_MALS confirmed the increased percentage of trimers for all tested mutations in the framework sequence (data not shown).

For a set of proteins containing up to nine additional mutations in order, it was demonstrated using SEC-MALS that each subsequent mutation increased the trimer/monomer ratio, while the monomer peak height on the SEC plot decreased, while the trimer peak height remained the same. the same (Fig. 7). Of all variants tested with SEC-MALS, the L556P, N651F, K655I, I535N, I573F, D589V, A204I, K588Q, E647F substitution variant showed the highest percentage of trimers (least gp140 monomers and least gp120 monomers), the highest total protein yield and one of the highest indicators of temperature stability. This means that these mutations can be combined without loss of trimer compared to the framework sequence. In addition, this suggests, in general, that the addition of the mutations described in (i)-(vii) of Table. 1, optionally in combination with the mutations described in table. 2 leads to further increased trimerization.

We also tested a construct with mutations L556P, N651F, I535N, I573F, D589V, A204I, K588Q, in which SOS mutations were removed (i.e., two cysteine residues at positions 501 and 605 were converted to amino acid residues that were originally present in the consensus sequence of the clade WITH). Even though its temperature stability was lower, the mutant had a percentage and yield of trimers comparable to those of its corresponding mutant that did not contain the SOS mutation. The mutant in which the SOS mutation was deleted even had the advantage of binding to fewer non-bNAb antibodies than the corresponding SOS-containing counterpart (having mutations L556P, N651F, I535N, I573F, D589V, A204I, K588Q). This shows that the advantageous properties of the present invention, such as a high percentage of trimerization, can also be obtained in HIV Env proteins that do not contain all SOSIP mutations.

One mutant (tested based on the framework sequence ConC_SOSIP), based on a combination of favorable properties in terms of expression level, trimer formation and binding to the broad spectrum neutralizing antibody PGT151, has the following mutations: L556P, N651F, I535N, I573F, D589V, A204I, K588Q .

The nine most successful ConC_SOSIP-based substitutions were L556P, E647F, N651F, K655I, I535N, D589V, I573F and K588E in gp41 and A204I in gp120. The combination of all these 9 substitutions resulted in increased stability, trimer content and trimer yield. Because the addition of L556P to this 9-change variant had a relatively limited effect on increasing the percentage of trimers, and the E647F substitution in this context appeared to interfere with PGT151 binding, these two mutations were not always used in additional variants, and it was found that the variant with 7 substitutions (referred to as ConC_SOSIP_7mut, also sometimes referred to herein as stabilized ConC_SOSIP or ConC_base; including N651F, K655I, I535N, D589V, I573F, K588E and A204I) was slightly more stable (increased melting point) than the 9 substitution variant indicated higher. The complete sequence of this variant (stabilized protein Env ConC_SOSIP, HIV 160544) is shown under SEQ ID NO: 20.

At the moment, a particularly preferred mutant [tested based on the ConC_SOSIP framework sequence with the following additional mutations: (a) D279N, A281V, A362Q (increase similarity to transmitted founder viruses as described by other authors); (b) Del139-152 (removal of the variable loop to reduce the chance of inducing antibodies to this loop) and (c) V295N (introduction of a glycan site that is present in most strains of HIV)], based on a combination of favorable properties in terms of expression level, formation of trimers and binding to a broad spectrum neutralizing antibody has the following stabilizing mutations of the present invention: N651F, K655I, I535N, I573F, D589V, A204I, K588E. The complete sequence of this variant (ConC_SOSIP.v3 stabilized Env protein (HIV170654, ConC_SOSIP.v3)) is shown under SEQ ID NO: 28.

In an additional variant, the K658V mutation was added to this construct (see also Example 15 below), which further improved the results.

Example 8 Self-Assembling Particles Presenting a Stabilized HIV Env Protein

Self-assembling particles based on ferritin and DPS were obtained, which presented stabilized Env proteins in the same way as described in (He et al., 2016). To achieve this, the gp140 protein was fused to the N-terminus of the particles via a short amino acid linker (e.g. GSG or AAAGS, but other linkers can also be used, see e.g. He et al., 2016) at the DNA level and expressed the fusion protein at Expi293F cells. One example of a particle thus prepared was based on ferritin fused to the HIV Env protein with ConC SOSIP (SEQ ID NO: 3) with the following mutations: I535N, A558P, D589V, K655I. Ferritin-based particles with the given

- 37 042607 Env protein having an additional V570D mutation that has been reported to increase trimerization (Kesavardhana et al, 2014), but this mutation has also been observed to result in a strong increase in non-neutralizing antibody binding (17b), which is undesirable. Env with these five mutations were also fused with two types of DPS-based particles from Helicobacter pylori and from Mycobacterium smegmatis (see, for example, WO 2011/082087 regarding the preparation of DPS-based particles). Env with these five mutations and, in addition, with the double mutation I201C-A433C allowing the introduction of a disulfide bridge, were also fused with ferritin.

Particles were purified from cell-free supernatant with PGDM140 affinity beads and the particles were analyzed using SEC-MALS with a G6000PWCL TSKgel column. Using SECMALS, as well as Native PAGE (3-12%), it was confirmed that particles of approximately the expected sizes were formed.

Similarly, ferritin and DPS based self-assembling nanoparticles were also prepared representing the HIV Env protein having the ConC_SOSIP sequence with the following mutation combinations: (L556P, N651F, I535N, I573F, D589V, A204I, K588Q).

Additional liposomes and/or self-assembling nanoparticles representing the HIV Env variants described herein, such as HIV Env having SEQ ID NOs: 20, 22, 24, 26, 27, 28, 29, 30, 31, or 32, were also prepared.

Example 9 Recombinant HIV Envelope Protein Variants Based on Clade A Envelope Protein Sequence

Recombinant HIV Env proteins according to embodiments of the present invention containing single amino acid substitutions (I535N, D589V, N651F, K655I, I573F, A204I, or E647F) were introduced into the wild-type HIV clade A envelope protein with the modification SOSIP (referred to as 'BG505_SOSIP') described in Example 2. The HIV envelope protein, BG505_SOSIP, has the so-called SOSIP mutations (Cys at residues 501 and 605 and Pro at residue 559), as well as additional Cys at residues 201 and 433, resulting in the introduction of a disulfide (DS) bond, and potential N-glycosylation site at position 332 (T332N mutation). The protein is truncated at position 664. The BG505_SOSIP sequence is shown under SEQ ID NO: 21.

Percent trimer formation and trimer yield were measured using the AlphaLISA assay described in Example 3. Percent trimer formation and trimer yield for each of the variants tested was compared to BG505_SOSIP. A higher percentage of trimer formation was observed with the M535N, D589V, N651F, or K655I substitutions compared to the framework sequence (eg, FIG. 8A). The combination of eg L556P, K655I and M535N showed an even higher yield and trimer percentage (eg, FIGS. 8A and 8B). The combination of N651F and D589V further increased the yield and percentage of trimers (data not shown). The results from this example for clade A virus are consistent with those of examples 10 and 11 (clade C) below and example 5 (clade B) in which mutations I535N, D589V, N651F and K655I also had a stabilizing effect on the envelope protein derived from wild-type strains, for example, resulted in an increased percentage of trimer formation and/or an increased trimer yield. It appears that these mutations of the present invention also improve the trimerization of HIV Env derived from a wild-type clade A strain.

At the moment, a particularly preferred mutant (tested based on the BG505_SOSIP framework sequence), based on a combination of favorable properties in terms of expression level, trimer formation and binding to a broad spectrum neutralizing antibody, is a mutant having the following mutations: L556P, K655I, M535N, N651F , D589V (see, for example, Fig. 9, which shows the greatly increased trimer formation of such a mutant in SEC-MALS analysis, and Fig. 13, which shows the clearly improved binding of such a mutant by broad-spectrum neutralizing antibodies). The sequence of this stabilized Env BG505_SOSIP (HIV170863) protein is shown under SEQ ID NO: 22.

The addition of the Q658V mutation provided a slight additional improvement.

An additional preferred construct contains mutations L556P, K655I, M535N, N651F, D589V, as well as mutations 'DS' (Cys at positions 201 and 433, which leads to the introduction of a disulfide bond), R588E and Q658V. The sequence of such a variant (Env protein with BG505_SOSIP.v2, HIV171814) is shown under SEQ ID NO: 29.

Example 10 Recombinant HIV Envelope Protein Variants Based on Clade C Wild Type Envelope Protein Sequence

Recombinant HIV Env proteins according to embodiments of the present invention, comprising single amino acid substitution T651F, double amino acid substitution T651F, M535N introduced into Env WT sequence, C97ZA_SOSIP (SEQ ID NO: 23) with additional substitution L556P (C97ZA_SOSIP_L556P), were generated and expressed as described in Example 2. Trimer yield and percentage of trimer formation were measured using the AlphaLISA assay as described in Example 3.

The results are shown in FIG. 10A and B. C97ZA_SOSIP_L556P_T651F_M535N trimer output at five

- 38 042607 times greater than that in the case of the C97ZA_SOSIP framework sequence.

Thus, the L556P, T651F, and M535N substitutions provided a large improvement in C97ZA_SOSIP, but the bNAb binding and percentage of trimers for this variant, derived from the clade C wild-type sequence, were still much lower than for the ConC_SOSIP framework sequence. Since Env wt can be adapted to its host, possibly reducing its overall fitness, and as a result folding may be impaired, the Env sequence was reconstructed according to the conceptual framework described below in Example 12 and in FIG. 12. To restore the sequence, a total of 21 residues were changed and three potential N-glycosylation sites (PNGS) were added to fill the so-called glycan holes (positions where at least 50% of the Env proteins of HIV wild-type strains have a potential N-glycosylation site ). Mutations introduced into C97ZA_SOSIP according to this backbone are listed in Table 1. 3 in the Repair Mutations column. The addition of the stabilizing mutation K655I disclosed herein increased trimer percentage and yield even more than D589V, A204I and K588E.

These results demonstrate that the T651F, M535N and K655I, D589V, A204I and K588E mutations described herein also had a stabilizing effect on the coat protein, e.g. from the Env protein of the wild-type strain of clade C) and its variants.

Currently, a particularly preferred variant (tested based on the C97ZA_SOSIP framework sequence), based on a combination of favorable properties in terms of expression level, trimer formation and binding to a broad spectrum neutralizing antibody, is the variant having the following mutations: Q567K (described by other authors previously) ; A198T, S243N, K236T, V295N (for filling glycan holes); M34L, T46K, T58A, Q171K, G172V, P179L, L183Q, I192R, N209T, M307I, Q350R, N352H, Y353F, D412N, G429E, V455T, I489V, L491I, G500K, S547G, T578A, T65; V505N, E507T, T663N (with the addition of potential N-glycosylation sites at the base of the molecule) and A204I, M535N, L556P, K588E, D589V, T651F, K655I (stabilizing mutations of the present invention). The data for this variant is shown, for example, in Fig. 13, (see, in particular, stabilized and reduced C97ZA on it), which shows a huge increase in binding to broad-spectrum neutralizing antibodies compared to the original Env C97ZA wt molecule. The sequence of this variant (stabilized and reduced protein Env C97ZA_SOSIP (HIV170690)) is given under SEQ ID NO: 24.

The addition of the K658V mutation further stabilized this protein.

An additional preferred variant contains the DS mutation and K658V, and the sequence of this variant (Env protein with C97ZA_SOSIP.v2, HIV171810) is shown under SEQ ID NO: 30.

Example 11 Variants of Recombinant HIV Envelope Proteins Based on a Different Clade C Wild-Type Envelope Protein Sequence

SOSIP mutations were introduced into the Env protein from strain Du422 of clade C, and two glycan holes at positions 295 and 386 were filled with K295N and D386N mutations. In addition, some remnants were reconstructed according to the conceptual framework described in Example 12 and in FIG. 12 (V272I, W456R, G466E and F643Y) and introduced the stabilizing substitutions L556P, I535N, N651F and D589V. All additional substitutions resulted in higher trimer yields and trimer percentages (eg, FIG. 11).

In the specific variant tested with these four stabilizing mutations (SEQ ID NO: 25), the additional K655I substitution further increased trimer yield and trimer percentage by 1.3-fold and 1.4-fold, respectively (data not shown).

Currently, a particularly preferred Env Du422_SOSIP variant, based on the combination of favorable properties in terms of expression level, trimer formation and binding to a broad spectrum neutralizing antibody, is the variant having the following mutations: L556P, K655I, M535N, N651F, D589V, K588E, I201C , A433C, V272I, W456R, G466E, F643Y, D386N, and K295N. The sequence of this variant (Stabilized and reduced Env protein Du422_SOSIP (HIV170859) is shown under SEQ ID NO: 26. Data for this variant is shown, for example, in Fig. 13 (see stabilized and reduced Du422 on it), which shows a huge increase in binding with broad-spectrum neutralizing antibodies compared to the original Env Du422 wt molecule.

An additional preferred variant further contains the 'DS' and K658V mutation and the sequence of this variant (Env Du422_SOSIP.v1 protein, HIV171812) is shown under SEQ ID NO: 31.

Example 12 Recovery and Stabilization of Various HIV-1 Env Sequences

Because wt sequences from viruses isolated from infected patients may have acquired destabilizing mutations that make proper folding difficult, Env wt sequences from clade C, C97ZA, DU422, and the sC4 mosaic sequence are initially under

- 39 042607 to restore.

To search for suboptimal mutations in wild-type sequences, all HIV-1 Env sequences were aligned in the UniProt database and the HIV Los Alamos database (~90,000 sequences) and the distribution of amino acids was calculated for each amino acid. In general, a number of relatively rare amino acids in the Env wt sequences were replaced with more common amino acids (based on the frequency at the corresponding position in the database) according to the conceptual framework described in FIG. 12.

In addition, two additional substitutions Y353F and Q171K were introduced at the C97ZA_SOSIP apex to possibly improve binding to apex-targeting antibodies, and additional glycan sites were introduced by substitution D411N, K236T and V295N as these potential N-glycosylation sites (PNGS) were conserved. by > 50%. The next step was to transfer the stabilizing substitutions described in the previous examples into the restored sequence.

Stabilized ConC_SOSIP contains replacements A204I, I535N, I573F, K588E, D589V, N651F and K655I (stabilized ConC_SOSIP). The complete sequence of the stabilized ConC_SOSIP is shown under SEQ ID NO: 20.

An overview of some of the Env protein variants and their mutations is presented in Table 1. 3.

Table 3. HIV Env protein variants

Protein Mutations described in the literature Added PNGS Leader Sequence (SEQ ID NO: ) Restore infusion mutations We put Circulation mutation Other mutations End ConC_SOSIP V2 9 5N eleven 664 We put Zirovanny ConC_SOSIP V2 9 5N eleven A204I, I535N, I573F, K588E, D589V, N651F, K655I 664 BG505_SOSIP T332N 34 664 Installed Zirovanny BG505_SOSIP T332N 34 M535N, L556P, D589V, N651F, K655I 664 C97ZA_SOSIP L535M, Q567K Native 664 Restored C97ZA_SOSIP L535M, Q567K A198T, S243N, K236T, V2 9 5N eleven M34L, T46K, T58A, Q171K, G172V, P179L, L183Q, I192R, N209T, M307I, Q350R, N352H, Y353F, D412N, G429E, V455T, I489V, L491I, G500K, S547G, V505N, E507T, T663N 664

- 40 042607

T578A, T651N Refurbished and stabilized C97ZA_SOSIP Q567K A198T, S243N, K236T, V2 95N eleven M34L, T46K, T58A, Q171K, G172V, P179L, L183Q, I192R, N209T, M307I, Q350R, N352H, Y353F, D412N, G429E, V455T, I489V, L491I, G500K, S547G, T578A, T651N A204I, M535N, L556P, K588E, D589V, T651F, K655I V505N, E507T, T663N 664 Du422_SOSIP D386N, K295N eleven 664 Refurbished Du422_SOSIP D386N, K295N eleven V272I, W456R, G466E, F643Y 664 Remanufactured and stabilized Du422_SOSIP D386N, K295N eleven V272I, W456R, G466E, F643Y M535N, L556P, K588E, D589V, N651F, K655I 664 DS_sC4_SOSIP I201C-A433C V2 95N 33 655 Restore lazy I201C-A433C V2 95N 33 A114Q, E117K, 655 DS_sC4_SOSIP E166R, T375S, I434M Restored and stabilized DS_sC4_SOSIP I201C-A433C V2 95N 33 A114Q, E117K, E166R, T375S, I434M A204I, I535N, L556P, Q588E, D589V, N651F, K655I delta13 8-152 (SSNGTYN IIHNETYK ) , delta 19 1 (SEKSSEN SSE) , delta 463 (GVP) 655

Table 3. Several variants of HIV Env proteins described in this document. The Mutations Described in the Literature column describes mutations that have been used in these constructs and have been previously described by other authors. The Added PNGS column describes mutations that add a potential N-glycosylation site (at positions where many wild-type Env proteins contain such a site). The Leader Sequence column describes which leader sequence was used for expression if it was not the original (native) leader sequence. The Repair Mutations column describes mutations that improve folding and stability (measured as yield and percentage of trimers based on bNAb binding) of some wild-type Env proteins, as described in Example 12 and FIG. 12. The Stabilizing Mutations column describes mutations of the present invention that stabilize the protein and improve trimerization as disclosed herein. The Additional Mutations column describes additional mutations that have been made on some of the constructs. The End column describes the position of the last amino acid (numbering throughout the table corresponds to the HXB2 Env sequence).

Supernatants of cells transiently transfected with wild-type (wt), reconstituted and stabilized Env variants were tested for binding to several trimer-specific, broad-spectrum, apex-targeting neutralizing antibodies. Restorative substitutions, and especially stabilizing substitutions, had a significant effect on trimer content (FIGS. 13 and 14) as determined by AlphaLISA (FIG. 13) and SEC-MALS (FIG. 14).

The sequence of a preferred variant of the reduced and stabilized Env DS_sC4 protein (reduced and stabilized Env DS_sC4_SOSIP (HIV170686)) is shown under SEQ ID NO: 27.

Another preferred protein variant is shown under SEQ ID NO: 32 (reduced and stabilized Env sC4_SOSIP.v4).

Example 13 Functioning of the Stabilizing Mutations of the Invention in the Absence of SOSIP Mutations

As shown in the previous examples, 7 mutations (A204I, I535N, I573F, K588E, D589V, N651F, and K655I) increased the yield and trimer percentage of ConC_SOSIP (resulting in ConC_base, or stabilized ConC_SOSIP, or ConC_SOSIP 7mut) (e.g., FIG. 13 and 15).

This example demonstrates that various SOSIP mutations (i.e. SOS mutation: 2 substitutions for Cys residues at positions 501 and 605 and IP mutation: substitution for Pro residue at position 559) contribute additionally to stabilization but are not required to obtain benefits from the mutations of the present invention.

The 7 mutations have also been shown to increase trimer yield in a termed ConC_SOS that does not contain the I559P stabilizing mutation (IP mutation) as shown in FIG. 15 (cf. ConC_SOS vs. ConC_SOS, 7mut). Therefore, an IP mutation is not necessary to benefit from the mutations described herein. The addition of the I559P mutation resulted in a large increase, indicating that in this construct the IP mutation is predominant in addition to the 7 mutations of the present invention. The stabilizing IP mutation (I559P) could also be replaced by A558P or L556P, both of which also result in a large increase over the variant lacking the I559P mutation.

Also, ConC_IP, 7 mut, which contains the 7 mutations described above of the present invention, but which lacks SOS mutations, still showed a very high yield of trimers, which also indicates that SOS mutations are not necessary to benefit from the mutations described in this document (eg, compare ConC_SOSIP versus ConC_IP, 7 mut), along with the observations in Example 7. The addition of the SOS mutation did not result in an additional increase in trimer yield.

Thus, while Env trimers containing the stabilizing mutations described herein may benefit from stabilization by SOSIP mutations, none of the 3 SOSIP mutations are required to gain benefits (e.g., increased trimer yield) from stabilizing mutations. described in this document.

Example 14 Methionine Substitution at Positions 647, 651, or 655 Improves Trimer Quality

In addition to the mutations described in Example 2, positions 589, 647, 651 and 655 were individually changed to a Met residue in the framework sequence ConC_SOSIP (SEQ ID NO: 3) and tested for percentage trimerization and trimer yield using the methods described higher. Met at positions 647, 651 or 655, like the mutations described in Example 2, was shown to improve trimer quality (higher percentage and yield of trimers, increased binding to bNAb), as can be seen in FIG. 16.

Thus, in addition to changing to Phe, Ala, or Trp at position 651, changing to Met at position 651 also improves trimer formation; besides changing to Phe, Ile, or Trp at position 655, changing to Met at position 655 also improves trimer formation; and, in addition to changing to Phe or Ile at position 647, changing to Met at position 647 also improves trimer formation.

Example 15 HIV Env Protein with Trimer Stabilizing Mutation at Position 658

Recombinant HIV Env proteins with substitution type mutations at position 658 (numbered according to gp160 from HIV-1 HXB2 isolate) were generated using the ConC_SOSIP framework sequence (SEQ ID NO: 3). K658 was mutated to Val, Ile, Phe, Leu, Met, or Ala. In addition, several double mutants were generated in which these mutations were combined with one of the stabilizing mutations described above, K655I. The percentage of trimer formation was determined using the analysis

AlphaLISA as described in Example 3.

The results are shown in FIG. 17A and B (percentage of trimers measured in different experiments, hence two panels) and FIG. 17C and D (trimer yield measured in different experiments, hence two panels). Using these results, it was demonstrated that a change at position 658 to Ile, Phe, Met, Leu, Ala, or Val resulted in an increased percentage of trimer formation and increased trimer yield. A change to Ile at position 658 resulted in an increase that was approximately in the same range as that for the K655I mutation (FIG. 17A, C), which was the single best-performing mutation among mutations (i)- (vii) in Table. 1 described above (see, for example, FIG. 2A). The change to Val at position 658 resulted in an even greater improvement (FIGS. 17A, C).

The results also show that a change at position 658 to Ile or Val can be combined with the K655I mutation described above and that this results in an additional improvement over each of the respective single mutations (FIG. 17A, C).

The K658V mutation was also tested using SEC-MALS. Cultures were grown in 96 well plates for three days as in the case of AlphaLISA. The supernatant was directly loaded onto the SEC-MALS column. The chromatograms obtained for the false supernatant (with furin expression) were subtracted from the chromatograms of the supernatant with Env proteins. The trimeric protein was eluted from the column within 7-8 minutes. The results are shown in FIG. 18 and they confirm that the K658V mutated protein showed improved trimerization compared to the background Env protein and compared to the K655I mutated Env protein.

This example demonstrates that changing the amino acid at position 658 of the HIV Env protein to Val, Ile, Phe, Met, Leu, or Ala results in an increased trimer percentage and trimer yield.

Additional experiments to measure trimer formation by variants using AlphaLISA and/or SEC-MALS are performed for HIV Env variants in which the K658V mutation is present in combination with other mutations from Table 1. 1 and/or 2 described herein, as well as for strains of HIV from clade A and B. For example, the 658V mutation has already been shown to improve the ConC_SOSIP,7mut variant described above (example 7), as well as BG505_SOSIP with L556P, K655I, M535N, N651F, D589V, K588E (example 9), as well as restored and stabilized C97ZA_SOSIP (example 10).

Based on the results described above, a mutation in the amino acid position 658 to valine, isoleucine, phenylalanine, leucine, methionine, or alanine, preferably on a valine residue, is expected to increase trimer formation and/or trimer yield in various HIV Env background proteins. .

Example 16 Immunization with Stabilized HIV Env Proteins

The rabbit immunization study was performed with soluble Env protein and liposome-bound Env proteins. Priming was done with stabilized ConC_SOSIP.v3 (SEQ ID NO: 28) and continued with four boosters each using a different protein, ie. using:

1) restored and stabilized sC4_SOSIP.v4 (SEQ ID NO: 32);

2) restored and stabilized C97ZA_SOSIP.v2 (SEQ ID NO: 30);

3) restored and stabilized Du422_SOSIP.v1 (SEQ ID NO: 31) and

4) stabilized BG505_s0sIP.v2 (SEQ ID NO: 29).

After successful immunizations, sera are isolated and analyzed for induced antibodies, which in particular bind to the stable Env closed conformation prior to fusion (using ELISA) as well as for bNAb induction (using virus neutralization assays).

The above examples demonstrate that the present invention provides a versatile approach for optimizing the folding and stability of closed trimeric HIV envelope proteins prior to fusion.

It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that changes may be made to the above described embodiments without departing from their general inventive intent. Thus, it should be understood that the present invention is not limited to the specific disclosed embodiments, but is intended to cover modifications within the spirit and scope of the present invention as defined in the appended claims.

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Sequence listing

SEQ ID NO: 1, gpl60 from HIV-1 HXB2 isolate (signal sequence in italics; amino acids at positions (i)-(vii) for mutations according to the present invention in grey)

MRVKEK YQHihZRhZGhZRhZGTMiiGMDMYCSATEKLWVTVYYGVPVWKEAT Т TL EGAS DAK AYDTEVHNVWATHACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLT PLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDND TTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGI RPWSTQLLLNGSLAEEEWIRSVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGR AFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKT11FKQS SGGDPEIVTHS FNCG GEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQI RCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQ REKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVW GIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREI NNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVF AVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSL CLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTD RVIEWQGACRAIRHIPRRIRQGLERILL

SEQ ID NO: 2, HIV clade C Env consensus sequence (consensus sequence only, not including any signal sequence, transmembrane domain (last amino acid 664), SOSIP mutations, and/or furin cleavage site mutations; amino acids at positions (i) to (vii ) for mutations according to the present invention are highlighted in gray)

NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNT TTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQACPKVSFDPIPIHYCAPAGYAI LKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTDNAKTIIVHLN ESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFP NKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQE VGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKWEIKP LGIAPTKAKRRWEREKRRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLL RAIEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQE DIWDNMTWMQWDREISNYTDTIYRLLEESQNQQEKNEKDLLALD

SEQ ID NO: 3, ConC_SOSIP (Mature Consensus

- 44 042607 sequence of clade C proteins with SOSIP mutations, as well as a furin cleavage site (italic) and C-terminal truncation; amino acids at positions (i) - (vii) for mutations according to the present invention are highlighted in gray)

NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNT TTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQACPKVSFDPIPIHYCAPAGYAI LKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTDNAKTIIVHLN ESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFP NKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQE VGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKWEIKP LGIAPTKCKRRWERRRRRRAVGIGAVFLGFLGAAGSTMGAASITLTVOAROLLSGIVOOOSNL LRAPEAQQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGIWGCSGKLICCTAVPWNSSWSNKSQ EDIWDNMTWMQWDREISNYTDTIYRLLEESQNQQEKNEKDLLALD

SEQ ID NO: 4, HIV clade B Env consensus sequence (consensus sequence only, not including any signal sequence, transmembrane domain (last amino acid 664), SOSIP mutations, and/or furin cleavage site mutations; amino acids at positions (i) to (vii ) for mutations according to the present invention are highlighted in gray)

AEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWLENV TENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLNNNTTNNNSSSEKMEKGEI KNCSFNITTSIRDKVQKEYALFYKLDWPIDNNNTSYRLISCNTSVITQACPKVSFEPIPIHYC APAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSENFTDNAK TIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNISRTKWNNTLKQIVK KLREQFGNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNSNGTWNNTTGNDTITL PCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNNNNTTETFRPGGGDMRDNW RS Е L YK YKWKIЕ Р L GVAP Т KCKRRWQRRRRRRAVGI GAM FL G FL GAAG S TMGAASIT L T VQA RQLLSGIVQQQNNLLRAPEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICC TAVPWNTSWSNKSLDEIWDNMTWMQWEREIDNYTGLIYTLIEESQNQQEKNEQELLELD

SEQ ID NO: 5, ConB_SOSIP (mature consensus sequence of clade B proteins with mutations in SOSIP and at the furin cleavage site (in italics), also C-terminal truncation; amino acids at positions (i) to (vii) for mutations according to the present invention are highlighted in gray )

AEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWLENV

- 45 042607

TENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDLNNNTTNNNSSSEKMEKGEI KNCSFNITTSIRDKVQKEYALFYKLDWPIDNNNTSYRLISCNTSVITQACPKVSFEPIPIHYC APAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSENFTDNAK TIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHCNISRTKWNNTLKQIVK KLREQFGNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNSNGTWNNTTGNDTITL PCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNNNNTTETFRPGGGDMRDNW RS E L YK YKWKIE P L GVAP T К CKRRWQRRRRRRAVGI GAM FL G FL GAAG S TMGAASIT L T VQA RQLLSGIVQQQNNLLRAFEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICC TAVPWNTSWSNKSLDEIWDNMTWMQWEREIDNYTGLIYTLIEESQNQQEKNEQELLELD

SEQ ID NO: 6, fragment C4 of the synthetic coat protein Env HIV Mos2S; amino acids at positions (i) - (vii) for mutations according to the present invention are highlighted in gray)

MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNV TENFNMWKNDMVDQMHEDIISLWDASLEPCVKLTPLCVTLNCRNVRNVSSNGTYNIIHNETYKE MKNCSFNATTWEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPK VS FDPIРIHYCAPAGYAILKCNNKT FNGTGPCNNVS TVQCTHGIKPWS TQLLLNGS LAEEE11 IRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRD GWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHTFNCRGEFFYCNTSNLFNESNIERNDS IITLPCRIKQIINMWQEVGRAIYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGG DMRNNWRS Е LYK YKWE VKP LGVAP ТЕAKRRWEREKRAVGI GAVELGILGAAGS TMGAAS IТ L TVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGK LICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEK

SEQ ID NO: 7 (sequence DS_sC4_SOSIP_E166R; amino acids at positions (i) - (vii) for mutations according to the present invention are in gray)

MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNV TENFNMWKNDMVDQMHEDIISLWDASLEPCVKLTPLCVTLNCRNVRNVSSNGTYNIIHNETYKE MKNCSFNATTWRDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSACTQACPK VS FDPIРIHYCAPAGYAILKCNNKT FNGTGPCNNVS TVQCTHGIKPWS TQLLLNGS LAEEE11 IRSENLTNNAKTIIVHLNETVNINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRD GWNKTLQGVKKKLAEHFPNKTIKFAPHSGGDLEITTHTFNCRGEFFYCNTSNLFNESNIERNDS IITLPCRIKQIINMWQEVGRCIYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGG DMRNNWRS E LYK YKWE VKP L GVAP T E CKRRWE RRRRRRAVGIGAV FL GIL GAAG S TMGAAS I TLTVQARQLLSGIVQQQSNLLRAPEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCS GKLICCTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEK

SEQ ID NO: 8 (Mosl.Env, mosaic protein sequence

- 46 042607 HIV sheaths; amino acids at positions (i) - (vii) for mutations according to the present invention are highlighted in gray)

AGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWLENV TENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPM EKGEIKNCSFNITTSIRNKVQKQYALFYKLDWPIDNDSNNTNYRLISCNTSVITQACPKVSFE PIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSE NFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNN TLRQIVEKLGKQFGNNKTIVFNHS S GGDPEIVMHS FNCGGE FFYCNS TKL FNS TWTWNNS TWNN TKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEI FRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQSEKSAVGIGAVFLGFLGAAGSTMG AASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGI WGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEK

SEQ ID NO: 9 (Mos2.Env, HIV mosaic envelope protein sequence; amino acids at positions (i)-(vii) for mutations according to the present invention are in gray)

MGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENV TENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKE MKNCSFNATTWEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPK VS FDPIРIHYCAPAGYAILKCNNKT FNGTGPCNNVS TVQCTHGIKPWS TQLLLNGS LAEEE11 IRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRD GWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTYMPNGTN SNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETF RPGGGDMRNNWRSELYKYKWEVKPLGVAPTEAKRRWEREKRAVGIGAVFLGILGAAGSTMGA ASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLW GCSGKLICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEK

SEQ ID NO: 10 (furin cleavage site mutant sequence)

RRRRRR

SEQ ID NO: 11 (example of signal sequence (e.g. used in case of ConC_SOSIP and some variants derived from wild-type sequence))

MRVRGILRNWQQWWIWGILGFWMLMICNWG (note: the last VGs may be the beginning of the mature protein or the end of the signal sequence)

SEQ ID NO: 12 (Example of an 8 amino acid sequence that can replace the HR1 loop)

- 47 042607

NPDWLPDM

SEQ ID NO: 13 (Example of an 8 amino acid sequence that can replace the HR1 loop)

GSGSGSGS

SEQ ID NO: 14 (Example of an 8 amino acid sequence that can replace the HR1 loop)

DDVHPDWD

SEQ ID NO: 15 (Example of an 8 amino acid sequence that can replace the HR1 loop)

RDTFALMM

SEQ ID NO: 16 (Example of an 8 amino acid sequence that can replace the HR1 loop)

DEEKVMDF

SEQ ID NO: 17 (Example of an 8 amino acid sequence that can replace the HR1 loop)

DEDPHWDP

SEQ ID NO: 18 (example of signaling sequence (e.g. used in case of ConB_SOSIP)

MRVKGIRKNYQHLWRWGTMLLGMLMICSA

SEQ ID NO: 19 (label used for HIV gpl40 constructs in AlphaLISA assay)

AAALPETGGGSDYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHHHH HH

SEQ ID NO: 20 (stabilized ConC_SOSIP, 'ConC_SOSIP_7mut' (HIV160544))

NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTNNNNMKEEMKNCSFNT TTEIRDKKQKEYALFYRLDIVPLNENSSEYRLINCNTSTITQiCPKVSFDPIPIHYCAPAGYAI LKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTDNAKTIIVHLN ESVEINCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFP NKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQE VGRAMYAPPIAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKVVEIKP LGIAPTKCKRRWERRRRRRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSGIVQQQSNL LRAPEAQQHMLQLTVWGfKQLQARVLAIERYLevQQLLGIWGCSGKLICCTAVPWNSSWSNKSQ EDIWDNMTWMQWDREISNYTDTIYRLLEESQfQQEiNEKDLLALD

SEQ ID NO: 21 (Protein Env BG505_SOSIP (HIV150673))

- 48 042607

AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENV TEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNM TTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHY CAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNA KNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVV KQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSN DSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDM RDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTL TVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGK LICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

SEQ ID NO: 22 (Env protein stabilized BG505_SOSIP (HIV170863))

AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENV TEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNM TTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHY CAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNA KNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVV KQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSN DSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDM RDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASnTL TVQARNLLSGIVQQQSNLpRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRvQQLLGIWGCSGK LICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIlYGLLEESQfQQEiNEQDLLALD

SEQ ID NO: 23 (Env C97ZA_SOSIP wt protein c L535M and Q567K (HIV150673))

NMWVTVYYGVPVWTDAKTTLFCASDTKAYDREVHNVWATHACVPTDPNPQEIVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKEIRNCSFNT TTEIRDKKQQGYALFYRPDIVLLKENRNNSNNSEYILINCNASTITQACPKVNFDPIPIHYCAP AGYAILKCNNKTFSGKGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEKEIIIRSENLTDNVKTI IVHLNKSVEIVCTRPNNNTRKSMRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKL QENYNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEDETITLPCRIKQIINM WQGVGRAMYAPPIAGNITCKSNITGLLLVRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVIE LKPLGIAPTGcKRRWERrrrrRAVGIGAVFLGFLGAAGSTMGAASmTLTVQARQLLSSIVQQQ SNLLRApEAQQHMLkLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICcTNVPWNSSWSN KSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQTQQEKNEKDLLALD

SEQ ID NO: 24 (reduced and stabilized protein Env C97ZA_SOSIP (HIV170690))

-49042607

NlWVTVYYGVPVWkDAKTTLFCASDaKAYDREVHNVWATHACVPTDPNPQEIVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKEIRNCSFNT TTEIRDKKQkvYALFYRlDIVqLKENRNNSNNSEYrLINCNtSTITQiCPKVtFDPIPIHYCAP AGYAILKCNNKTFnGtGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEKEIIIRSENLTDNVKTI IVHLNKSVEInCTRPNNNTRKSiRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKL rEhfNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEnETITLPCRIKQIINM WQeVGRAMYAPPIAGNITCKSNITGLLLtRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVvE iKPLGIAPTkcKRRnVtRrrrrRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSgIVQQQ SNLpRApEAQQHMLkLTVWGIKQLQaRVLAIERYLevQQLLGIWGCSGKLICcTNVPWNSSWSN KSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQfQQEiNEKDLLAnD

SEQ ID NO: 25 (Remanufactured and Stabilized Design Variant Du422 (HIV161818))

NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIK NCSFNTTTELRDKKQKVYALFYKPDWPLNGGEHNETGEYILINCNSSTcTQACPKVSFDPIPI HYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTN NIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQ VKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIIL PCRIKQIINMWQEVGRcMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRS ELYKYKWEIKPLGVAPTKCKRKnVtRRRRRRAVGLGAVLLGFLGAAGSTMGAASnTLTVQARQ LLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLKvQQLLGLWGCSGKLICCTA VPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEKNEKDLLAnD

SEQ ID NO: 26 (Remanufactured and Stabilized Du422_SOSIP (HIV170859))

NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIK NCSFNTTTELRDKKQKVYALFYKPDWPLNGGEHNETGEYILINCNSSTITQACPKVSFDPIPI HYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTN NIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQ VKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIIL PCRIKQIINMWQEVGRAMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRS ELYKYKWEIKPLGVAPTKCKRKWGRRRRRRAVGLGAVLLGFLGAAGSTMGAASnTLTVQARQ LLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTA VPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEiNEKDLLALD

SEQ ID NO: 27 (rebuilt and stabilized ^D S_sC4_SOSIP (HIV170686))

- 50 042607

MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNV TENFNMWKNDMVDQMHEDIISLWDqSLkPCVKLTPLCVTLNCRNVRNVEMKNCSFNATTVVrDR KQKVHALFYRLDIVPLDENNSSYRLINCNTSAcTQiCPKVSFDPIPIHYCAPAGYAILKCNNKT FNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNINC TRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFA PHSGGDLEITTHsFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRcmYAP PIAGNITCRSNITGLLLTRDGGSNNNDTETFRPGGGDMRNNWRSELYKYKWEVKPLGVAPTEC KRRWERRRRRRAVGIGAVFLGILGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQ HMLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTAVPWNTSWSNKSQTDIWDNMT WMQWDKEIGNYTGEIYRLLEESQfQQEi

SEQ ID NO: 28 (stabilized ConC_SOSIP.v3 (HIV170654))

NLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTEMKNCSFNTTTEIRDKKQ KEYALFYRLDIVPLNENSSEYRLINCNTSTITQICPKVSFDPIPIHYCAPAGYAILKCNNKTFN GTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTNNVKTIIVHLNESVEIVCTR PNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNISEAKWNKTLQRVKKKLKEHFPNKTIKFQPS SGGDLEITTHSFNCRGEFFYCNTSKLFNSTYNNTTSNSTITLPCRIKQIINMWQEVGRAMYAPP IAGNITCKSNITGLLLTRDGGNNNNNTETFRPGGGDMRDNWRSELYKYKWEIKPLGIAPTKCK RRWERRRRRRAVGIGAVFLGFLGAAGSTMGAASNTLTVQARQLLSGIVQQQSNLLRAPEAQQH MLQLTVWGFKQLQARVLAIERYLEVQQLLGIWGCSGKLICCTAVPWNSSWSNKSQEDIWDNMTW MQWDREISNYTDTIYRLLEESQFQQEINEKDLLALD

SEQ ID NO: 29 (stabilized BG505_SOSIP.v2 (HIV171814))

AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENV TEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNM TTELRDKKQKVYSLFYRLDWQINENQGNRSNNSNKEYRLINCNTSAcTQACPKVSFEPIPIHY CAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNA KNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKW KQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSN DSITLPCRIKQIINMWQRIGQcMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDM RDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGIGAVFLGFLGAAGSTMGAASnTL TVQARNLLSGIVQQQSNLpRAPEAQQHLLKLTVWGIKQLQARVLAVERYLevQQLLGIWGCSGK LICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIlYGLLEESQfQQEiNEvDLLALD

SEQ ID NO: 30 (restored and stabilized C97ZA_SOSIP .v2 (HIV171810))

NlWVTVYYGVPVWkDAKTTLFCASDaKAYDREVHNVWATHACVPTDPNPQEIVLENVTE

- 51 042607

NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNATFKNNVTNDMNKEIRNCSFNT TTEIRDKKQkvYALFYRlDIVqLKENRNNSNNSEYrLINCNtSTcTQiCPKVtFDPIPIHYCAP AGYAILKCNNKTFnGtGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEKEIIIRSENLTDNVKTI IVHLNKSVEInCTRPNNNTRKSiRIGPGQTFYATGDIIGDIRQAYCNISGSKWNETLKRVKEKL rEhfNNNKTIKFAPSSGGDLEITTHSFNCRGEFFYCNTTRLFNNNATEnETITLPCRIKQIINM WQeVGRcMYAPPIAGNITCKSNITGLLLtRDGGEDNKTEEIFRPGGGNMKDNWRSELYKYKVvE iKPLGIAPTkcKRRnVtRrrrrRAVGIGAVFLGFLGAAGSTMGAASnTLTVQARQLLSglVQQQ SNLpRApEAQQHMLkLTVWGIKQLQaRVLAIERYLevQQLLGIWGCSGKLICcTNVPWNSSWSN KSQTDIWNNMTWMEWDREISNYTDTIYRLLEDSQfQQEiNEvDLLAnD

SEQ ID NO: 31 (refurbished and stabilized Du422_SOSIP.vl (HIV171812))

NLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTE NFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIK NCSFNTTTELRDKKQKVYALFYKPDWPLNGGEHNETGEYILINCNSSTcTQACPKVSFDPIPI HYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTN NIKTIIVHLNKSVEINCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQ VKEKLREHYNKTIKFEPSSGGDLEVTTHSFNCRGEFFYCNTTKLFNETKLFNESEYVDNKTIIL PCRIKQIINMWQEVGRcMYAPPIEGNITCKSNITGLLLTRDGGENSTEEVFRPGGGNMKDNWRS E LYKYKVVEIKPLGVAP TKCKRKWGRRRRRRAVGLGAVLLG FLGAAGS TMGAAS ηT L TVQARQ LLSGIVQQQSNLpRAPEAQQHLLQLTVWGIKQLQTRVLAIERYLevQQLLGLWGCSGKLICCTA VPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIYRLLEDSQfQQEiNEvDLLALD

SEQ ID NO: 32 (stabilized and restored sC4_SOSIP.v4)

MGNLWVTVYYGVPVWKDAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIVLGNV TENFNMWKNDMVDQMHEDIISLWDqSLkPCVKLTPLCVTLNCRNVRNVEMKNCSFNATTVVrDR KQKVHALFYRLDIVPLDENNSSYRLINCNTSAcTQiCPKVSFDPIPIHYCAPAGYAILKCNNKT FNGTGPCNNVSTVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNIVC TRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTIKFA PHSGGDLEITTHsFNCRGEFFYCNTSNLFNESNIERNDSIITLPCRIKQIINMWQEVGRcmYAP PIAGNITCRSNITGLLLTRDGGSNNNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTEC KRRnVtRRRRRRAVGIGAVFLGILGAAGSTMGAASnTLTVQARQLLSGIVQQQSNLpRAPEAQQ HMLQLTVWGIKQLQTRVLAIERYLeDQQLLGLWGCSGKLICCTAVPWNTSWSNKSQTDIWDNMT WMQWDKEIGNYTGEIYRLLEESQfQQEi

SEQ ID NO: 33 (example of signaling sequence (e.g. used in case of DS_sC4_SOSIP variants)

MRVRGMLRNWQQWWIWSSLGFWMLMIYSV

SEQ ID NO: 34 (example of signaling sequence (e.g. used in case of BG505_SOSIP variants)

MRVMGIQRNCQHLFRWGTMILGMIIICSA

- 52 042607

1 . Sanders et al. Literary sources J. Virol. (2002) 76(17), 8875-89 2 . Sanders et al. Science (2015) 349(6224), 139-140 3 . Julien et al . Proc. Nat. Acad. sci. (2015) 112 (38) 11947-. 52 4 . de Taeye et al . Cell (2015) 163(7), 1702-15 5 . Kwon and al. (2015) Nat. Struct. Mol. Biol. 22(7) 522-31 6 . Eglen and al. Curr. Chem. Genomics, (2008) 25(1), 2-10

7. Kong et al, Nat Commun. 2016 Jun 28,-7:12040. doi

10.1038/ncommsl2040

8 Julien et al. Proc. Natl. Acad. sci. (2015) 112(38)

11947-52

9. Barouch et al, Nat Med 2010, 16:319-323

10. WO 2010/059732

11. European Patent Application EP15200138.4

12. Sharma SK, et al. Cell Rep. (2015) 11(4):539-50. doi: 10.1016/j.celrep.2 015.03.047.

13 Georgiev IS, et al. J Virol. (2015) 89(10):5318-29.

doi: 10.1128/JVI.03451-14.

14. Lopez-Sagaseta J, et al (2016) Computational and Struct Biotechnol J 14: 58-68.

15. Zhao L, et al (2014) Vaccine 32: 327-337

16. He L, et al (2016) Nat Commun. 2016 Jun 28,-7:12041. doi:10.1038/ncomms12041

17. WO 2011/082087

18. Kesavardhana A and Varadarajan R (2014) J Virol 88: 9590-9604

19. Guenaga J, et al (2015) Immunity 46: 792-803

20. Bale S, et al (2017) J. Virol, doi:10.1128/JVI.00443-17

21. Abbink et al (2007) Virol. 81(9): 4654-64

22. Altschul SF et al (1997) Nucleic Acid Res. 25:33893402

23. Harris et al (2011) PNAS 108(28): 11440-11445

24 Kushnir et al (2012) Vaccine (31): 58-83

25. WO 2007/104792

26. WO 2014/124301

27 US 2016/0122392

Claims (32)

(1) HIV Env consensus sequence; 1. Recombinant human immunodeficiency virus (HIV) envelope protein (Env) containing two or more of the following amino acid residues: (i) Phe, Leu, Met or Trp at position 651; (ii) Phe, Ile, Met or Trp at position 655; (iii) Asn or Gln at position 535; (iv) Val, Ile or Ala at position 589; (v) Phe or Trp at position 573; (vi) Ile at position 204 and/or (vii) Phe, Met or Ile at position 647, where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate having the amino acid sequence of SEQ ID NO: 1. (2) a synthetic HIV Env protein; and (3) a parent HIV Env protein containing at least one amino acid residue repair mutation that is present at the corresponding position at a frequency of less than 7.5% of the HIV Env sequences, in the aggregate of at least 1000 wild-type HIV Env, where the repair mutation is a substitution for an amino acid residue that is present at the corresponding position with a frequency of at least 10% of the HIV Env sequences in the specified population, and where the position numbering corresponds to the numbering in gp160 from the HXB2 isolate of HIV- 1 having the amino acid sequence of SEQ ID NO: 1. 2. The recombinant envelope protein (Env) of the human immunodeficiency virus (HIV) according to claim 1, containing two or more of the following amino acid residues: (i) Phe at position 651; (ii) Ile at position 655; (iii) Asn at position 535; (iv) Val at position 589; (v) Phe at position 573; (vi) Ile at position 204 and/or (vii) Phe at position 647. 3. Recombinant HIV Env protein containing one or more of the following amino acids - 53 042607 residues: (i) Phe, Leu, Met or Trp at position 651; (ii) Phe, Ile, Met or Trp at position 655; (iii) Asn or Gln at position 535; (iv) Val, Ile or Ala at position 589; (v) Phe or Trp at position 573; (vi) Ile at position 204 and/or (vii) Phe, Met or Ile at position 647, wherein the HIV Env protein is selected from the group consisting of: 4. Recombinant HIV Env protein according to claim 3, containing one or more of the following amino acid residues: (i) Phe at position 651; (ii) Ile at position 655; (iii) Asn at position 535; (iv) Val at position 589; (v) Phe at position 573; (vi) Ile at position 204 and/or (vii) Phe at position 647. 5. Recombinant HIV Env protein containing one or more of the following amino acid residues: (i) Phe, Leu, Met or Trp at position 651; (ii) Phe, Ile, Met or Trp at position 655; (iii) Asn or Gln at position 535; (iv) Val, Ile or Ala at position 589; (v) Phe or Trp at position 573; (vi) Ile at position 204 and/or (vii) Phe, Met or Ile at position 647, wherein the HIV Env protein is an HIV Env protein containing at least one of the following: Cys at positions 501 and 605; Pro at position 559; Cys at positions 501 and 605 and Pro at position 559, and the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate having the amino acid sequence of SEQ ID NO: 1. 6. Recombinant HIV Env protein according to claim 5, containing one or more of the following amino acid residues: (i) Phe at position 651; (ii) Ile at position 655; (iii) Asn at position 535; (iv) Val at position 589; (v) Phe at position 573; (vi) Ile at position 204 and/or (vii) Phe at position 647. 7. Recombinant HIV Env protein according to any one of claims 3 to 6, containing two or more of the amino acid residues specified in (i)-(vii). 8. Recombinant HIV Env protein according to any one of claims 1, 3 and 7, containing Cys at positions 501 and 605 or Pro at position 559. 9. Recombinant HIV Env protein according to any one of claims 1 to 8, containing three or more of the amino acid residues specified in (i)-(vii). 10. Recombinant HIV Env protein according to any one of claims 1 to 8, containing four or more of the amino acid residues specified in (i)-(vii). 11. Recombinant HIV Env protein according to any one of claims 1-10, containing Phe at position 651, Not at - 54 042607 at position 655, Asn at position 535 and Val at position 589. 12. Recombinant HIV Env protein according to any one of claims 1 to 11, containing five or more of the amino acid residues specified in (i)-(vii). 13. Recombinant HIV Env protein according to any one of claims 1-12, further comprising one or more of the following: (viii) Gln, Glu, Ile, Met, Val, Trp or Phe at position 588; (ix) Lys at position 64, or Arg at position 66, or Lys at position 64 and Arg at position 66; (x) Trp at position 316; (xi) Cys at both positions 201 and 433; (xii) Pro at position 556 or 558 or both positions 556 and 558; (xiii) replacing the loop at amino acid positions 548-568 (HR1-loop) with a loop having 7-10 amino acids; (xiv) Gly at position 568, or Gly at position 569, or Gly at position 636, or Gly at both positions 568 and 636, or Gly at both positions 569 and 636; and/or (xv) Tyr at position 302 or Arg at position 519 or Arg at position 520 or Tyr at position 302 and Arg at position 519 or Tyr at position 302 and Arg at position 520 or Tyr at position 302 and Arg at both positions 519 and 520. 14. The recombinant HIV Env protein of any one of claims 1 to 13, further comprising a mutation in the furin cleavage sequence of the HIV Env protein. 15. Recombinant HIV Env protein according to any one of claims 1-14, containing an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 3, 5, 20, 22, 24, 26, 27, 28, 29 , 30, 31 or 32. 16. Recombinant HIV Env protein according to any one of claims 1-15, further comprising: (xvi) an amino acid residue selected from Val, Ile, Phe, Met, Ala, or Leu at position 658. 17. Recombinant HIV Env protein according to any one of claims 1 to 16, which is a gp140 or gp160 protein. 18. A recombinant HIV Env protein according to any one of claims 1 to 17, which is derived from clade C or clade A. 19. A trimeric complex containing a non-covalently linked oligomer of three recombinant HIV Env proteins according to any one of claims 1-18. 20. A particle representing on its surface a recombinant HIV Env protein according to any one of claims 1-18 or a trimeric complex according to claim 19. 21. An isolated nucleic acid molecule encoding a recombinant HIV Env protein according to any one of claims 1-13. 22. An expression vector containing the isolated nucleic acid molecule of claim 21 operably linked to a promoter. 23. The vector of claim 22, which is an adenoviral vector. 24. A host cell for producing a recombinant HIV Env protein, comprising the isolated nucleic acid molecule of claim 21 or the vector of claim 22 or 23. 25. A method for producing a recombinant HIV Env protein, comprising growing a host cell according to claim 24 under conditions suitable for producing a recombinant HIV Env protein. 26. Composition for obtaining an immune response against an infection caused by HIV, containing a recombinant HIV Env protein according to any one of claims 1-18 and a pharmaceutically acceptable carrier. 27. Composition for obtaining an immune response against an infection caused by HIV, containing a trimeric complex according to claim 19 and a pharmaceutically acceptable carrier. 28. Composition for obtaining an immune response against an infection caused by HIV, containing a particle according to claim 20 and a pharmaceutically acceptable carrier. 29. Composition for obtaining an immune response against an infection caused by HIV, containing an isolated nucleic acid molecule according to claim 21 and a pharmaceutically acceptable carrier. 30. Composition for obtaining an immune response against an infection caused by HIV, containing a vector according to claim 22 or 23 and a pharmaceutically acceptable carrier. 31. A method for improving trimer formation of an HIV Env protein, the method comprising replacing one or more amino acid residues in the original HIV Env protein, wherein the one or more substitutions result in one or more of the following amino acids: (i) Phe, Leu, Met or Trp at position 651; (ii) Phe, Ile, Met or Trp at position 655; (iii) Asn or Gln at position 535; (iv) Val, Ile or Ala at position 589; (v) Phe or Trp at position 573; (vi) Ile at position 204 and/or (vii) Phe, Met or Ile at position 647, where the position numbering corresponds to the numbering in gp160 from an HIV-1 HXB2 isolate having - 55 042607 amino acid sequence SEQ ID NO: 1. 32. The method of improving trimer formation of the HIV Env protein of claim 31, wherein the method comprises replacing one or more amino acid residues in the original HIV Env protein, wherein the one or more substitutions result in one or more of the following amino acids: (i) Phe at position 651; (ii) Ile at position 655; (in) Asn at position 535; (iv) Val, Ile or Ala, preferably Val, at position 589; (v) Phe at position 573; (vi) Ile at position 204 and/or (vii) Phe at position 647, where the position numbering corresponds to the numbering in gp160 from the HIV-1 HXB2 isolate having the amino acid sequence of SEQ ID NO: 1.